WO2005060437A2 - Composition combinations and methods for treating, preventing, reversing and inhibiting pain - Google Patents

Composition combinations and methods for treating, preventing, reversing and inhibiting pain Download PDF

Info

Publication number
WO2005060437A2
WO2005060437A2 PCT/US2004/036187 US2004036187W WO2005060437A2 WO 2005060437 A2 WO2005060437 A2 WO 2005060437A2 US 2004036187 W US2004036187 W US 2004036187W WO 2005060437 A2 WO2005060437 A2 WO 2005060437A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
aryl
group
acid
independently selected
Prior art date
Application number
PCT/US2004/036187
Other languages
French (fr)
Other versions
WO2005060437A3 (en
Inventor
Daniela Salvemini
Original Assignee
Metaphore Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metaphore Pharmaceuticals, Inc. filed Critical Metaphore Pharmaceuticals, Inc.
Publication of WO2005060437A2 publication Critical patent/WO2005060437A2/en
Publication of WO2005060437A3 publication Critical patent/WO2005060437A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/32—Tin compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins

Definitions

  • the present invention relates to methods of preventing nitration, whether in vitro or in vivo, and compositions and composition combinations which inhibit nitration.
  • NSAIDs nonsteroidal analgesic/anti-inflammatory drugs
  • opioids opioids
  • NSAIDs operate by inhibiting cyclooxygenase enzymes and thereby the synthesis of prostaglandins.
  • Prostaglandins sensitize pain receptors which lower the pain threshold perceived by subjects making normal stimuli, such as touch and stretch sensations, painful.
  • NSAIDs can be quite effective at returning the lowered pain threshold to normal but do not elevate the pain threshold.
  • NSAIDs available over-the-counter include: ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), and aspirin (Bayer®).
  • Prescription NSAIDs include: celecoxib —Celebrex®, diclofenac -- Voltaren®, etodolac ⁇ Lodine®, fen ⁇ profen - Nalfon®, indomethacin - Indocin®, ketoprofen - Orudis®, Oruvail®, ketoralac -Toradol®, oxaprozin - Daypro®, nabumetone - Relafen®, sulindac - Clinoril®, tolmetin - Tolectin®, and rofecoxib - Vioxx®.
  • a second class of pain relievers operate by mimicking natural peptides such as enkephalins and endorphins to stimulate one or more of the ⁇ , ⁇ - and /c-receptor systems in the nervous system.
  • Opioids elevate the pain threshold so that normally painful stimuli are perceived as less painful or even euphoric.
  • Opioids are commonly used in the clinical management of severe pain, including chronic severe pain of the kind experienced by cancer patients.
  • Each of these classes of compounds has inherent problems and limitations.
  • NSAIDs that are nonselective for the cyclooxygenase 2 (COX-2) produced in inflammation also inhibit constitutive cyclooxygenase 1 (COX-1), causing undesirable damage to the gastric mucosa.
  • opioids have limited effectiveness as analgesics in lowering an elevated threshold to normal and are generally used for mild to moderate pain. They are also ineffective drugs for elevation of the pain threshold above normal levels, which prevents their use in pain such as surgical pain where an underlying pathological condition has not elevated the pain threshold.
  • Opioid analgesics are antagonized by analogous N-allyl compounds such as naloxone.
  • opioids have problems with tolerance and dependency, so that over a course of therapy increasing dosages of compound are required to achieve the same level of analgesia, and cessation of opioid administration when analgesia is no longer needed elicits a withdrawal syndrome with unpleasant and potentially serious symptoms.
  • NASH Narcotic induced hyperalgesia
  • NMDA N-methyl-D-aspartate
  • NOS nitric oxide synthase
  • Non- selective NOS inhibitors such as NG-nitroarginine prevent and reverse morphine tolerance.
  • compositions, composition combinations and methods to treat pain by administering superoxide- and peroxynitrite-mediated nitration inhibitors to a subject in need thereof.
  • the compositions may be administered to a subject in combination with a known nitration, pain or inflammation inhibitor to enhance the effect of such known inhibitors to extend the potency and efficacy of the pain inhibitor.
  • This invention provides a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier.
  • the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
  • the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
  • the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof;
  • the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-omithine (L-NIL), aminoguanidine, 1400W (N-3- aminomethyl-benzylacetamidine), and any combination thereof;
  • the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6- nitroindazole, Ng-propyl-L-arginine, and any combination thereof.
  • the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
  • the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said combination optionally admixed with a pharmaceutically acceptable salt or carrier.
  • the pain inhibitor is an opioid.
  • the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
  • the SO generating system inhibitor is a xanthine oxidase inhibitor.
  • the SO scavenger is represented by the following formula: wherein (a) one or more of R, R', R 1 f R' 1 ( R 2 , R' 2> R 3 , R' 3> R , R' , Re, R' ⁇ , Re, R' ⁇ , R7, RV, R 8 , R' 8) R 9 , and R' 9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR 10 ,
  • R's, Rg, and R' 9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH 2 ), -- Q - (CH 2 )j - R - (CH 2 ) K -- S - (CH 2 ) L -- wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes,
  • R' 7 , R 8 , R's, Rg, and R'g may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH 2 ), - Q - (CH 2 )j - R - (CH 2 ) K - S - (CH 2 ) L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes,
  • U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings. In alternatives referring to a W moiety, W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
  • transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V.
  • the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
  • R 3 , R 6 , R 9 and R 12 are independently selected from the group consisting of H, alkyl, alkenyl, CH 2 , COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
  • N-Alkylpyridyl which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a
  • R' is CH or N
  • R-i, R 2 , R 3 , R , Rs, Re, R7, Rs, R9, R10, R11, ⁇ 2 , R13, R ⁇ 4 , R15, and R16 are independently selected from the group consisting of H, S0 3 H, COOH, N0 2 , NH 2 , and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
  • R ⁇ R 5 , R 9 , and R 13 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R'2, R 4 , R' 4 , Re, R'e, Rs, R's, R10, R'10, R12, R'12, R ⁇ 4 , R' ⁇ 4 , R16, and R'1,6 are independently selected from the group consisting of H and alkyl;
  • R 3 , R7, R 11 , and R15 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • B wherein R ⁇ R 5 , R 8 , and R 12 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 4 , R' 4 , R 6 , R' 6 , R7, R9, R'g, Ru, R'n, R13, R' ⁇ 3 , and R 1 are
  • R-,, R 4 , R 8 , and R 12 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R'2, R3, Rs, R's, R7, Rg, R'9, R11, R'11, R13, R'13 and R 14 are independently selected from the group consisting of H and alkyl;
  • R 10 is H or alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R ⁇ R , R 7 and R 10 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R's, Re, Rs, R's, g, Rn, R'n and R 12 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R-i, R 4 , R 8 and R-n are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R3, R'3, Rs, R's, R7, R'7, R9, R10, R'10, R12, R'12 and R 13 are independently selected from the group consisting of H and alkyl;
  • R 6 is hydrogen or alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R and R ⁇ 0 are independently selected from the group consisting of H and alkyl;
  • R 2 , R 3 , R' 3 , R 5 , R' 5 , Re, Rs, Rg, R'g, Ru, R'n and R 12 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R ⁇ R 3 , R , and R 6 are independently selected from the group consisting of H and alkyl;
  • R 2 and R 5 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and
  • X, Y, Z, M and n are as defined above;
  • R 1 ? R 2> R 3 and R are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
  • R ⁇ R'-,, R 2 , R' 2 , R 3 , R' 3 , R 4 , R' 4 , R 5 , R' 5 , R 6 , R'e, R 7 and R' 7 are independently selected from the group consisting of H, alkyl, alkoxy, N0 2 , aryl, halogen, NH 2 and S0 3 H, further wherein R 6 , R' 6> R7 and R' 7 together with one other of R 6 , R' ⁇ , R 7 and R' 7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M 1 is selected from the group consisting of Fe, Ni or V; and , X, Y, Z and n are as defined above.
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the combination provides a pain inhibitor which is an opioid and a nitration inhibitor selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compoound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
  • the NMDA receptor antagonist is MK801.
  • This invention also provides a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier.
  • the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
  • the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
  • the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof;
  • the iNOS inhibitor is selected from the group consisting of L-N5-(1- iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3-aminomethyl- benzylacetamidine), and any combination thereof;
  • the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng- propyl-L-arginine, and any combination thereof.
  • the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
  • the pain is initiated in a central sensitization pathway of the subject.
  • the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier.
  • the pain inhibitor is an opioid.
  • the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
  • the SO generating system inhibitor is a xanthine oxidase inhibitor.
  • the SO scavenger is represented by the following formula:
  • R, R', R n , R' ⁇ , R 2 , R' 2 , R 3 , R's, R 4 , R' 4 , Rs, R's, Re, R'e, R , R'7, R 8 , R's, Rg, and R' 9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR 10 , -NR ⁇ oR ⁇ > -COR10,
  • R' 7 , R 8 , R's, Rg, and R' 9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH 2 ), - Q - (CH 2 )j - R -- (CH 2 ) K - S - (CH 2 ) L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, bor
  • R 10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the o/-carbon of -amino acids; and (d) optionally, one or more of R 2 or R' 2 and R 3 , R 4 and R 5 or R' 5 , R 6 or R' 6 and R 7 , or R 8 and R 9 or R' 9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached
  • U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings.
  • W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
  • transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V.
  • the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
  • R 3 , R 6 , R 9 and R 12 are independently selected from the group consisting of H, alkyl, alkenyl, CH 2 , COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
  • N-Alkylpyridyl which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a
  • R' is CH or N
  • Ri, R 2 , R 3 , R 4 , Rs, R ⁇ , R7, Rs, Rg, R10, Ru, R12, Ri3, R ⁇ 4 , R15, and R16 are independently selected from the group consisting of H, S0 3 H, COOH, N0 2 , NH 2 , and N- alkylamino
  • X, Y, Z, M and n are as defined above; Structure III
  • R-,, R 5 , R 9 , and R 13 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R2, R'2, R 4 , R' 4 , Re, R' ⁇ , Re, R's, R10, R'10, R12, R'12, R ⁇ 4 , R' ⁇ 4 , R16, and R' ⁇ 6 are independently selected from the group consisting of H and alkyl;
  • R 3 , R 7 , Ru, and R 1 5 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 5 , R 8 , and R ⁇ 2 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2l R 4 , R' 4 , R 6 , R'e, R 7 , R 9 , R' 9 , Rn, R'n, Ris, R' ⁇ 3 , and R 14 are independently selected from the group consisting of H and alkyl;
  • R 3 and Rio are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R , R 8 , and R ⁇ 2 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R' 5 , R 7 , R g , R'g, Ru, R'n, R ⁇ 3 , R' ⁇ 3 and R are independently selected from the group consisting of H and alkyl;
  • Rio is H or alkyl;
  • X, Y, Z, M and n are as defined above;
  • R 1 t R 4 , R 7 and R ⁇ 0 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R2, R'2, R 3 , R5, R's, Re, Rs, R's, Rg, R11, R'11 and R ⁇ 2 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R 8 and Ru are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , Rs, R'3, Rs, R's, R7, R'7, Rg, R10, R'10, R12, R'12 and R ⁇ 3 are independently selected from the group consisting of H and alkyl;
  • R 6 is hydrogen or alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R 1 ? R 4 , R 7 and R 0 are independently selected from the group consisting of H and alkyl;
  • R 2 , R3, R'3, Rs, R's, Re, Rs, Rg, R'g, Ru, R'11 and R 2 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 3 , R 4 , and R 6 are independently selected from the group consisting of H and alkyl;
  • R 2 and R 5 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and
  • X, Y, Z, M and n are as defined above;
  • R-,, R 2 , R 3 , and R 4 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
  • R,, R' 1 f R 2 , R' 2 , R 3 , R' 3 , *, R' 4 , R 5 , R' 5 , R 6 , R'e, R7 and R' 7 are independently selected from the group consisting of H, alkyl, alkoxy, N0 2 , aryl, halogen, NH 2 and S0 3 H, further wherein R 6 , R' 6 , R and R' 7 together with one other of R 6 , R' ⁇ , R 7 and R' 7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M 1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above.
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
  • the NMDA receptor antagonist is MK801.
  • the subject is a mammal.
  • the mammal is a human.
  • Figure 1 Structures of synthetic SODm M40403 and M40404.
  • Figure 2. Decay of superoxide (top line) uncatalyzed and in the presence of an active SOD mimetic (M40403, 5 ⁇ M; bottom line) and an inactive analog (M40404, 5 ⁇ M; middle line).
  • the active catalyst affords a pure 1st-order decay of superoxide while the uncatalyzed and M40404 decays remain pure 2nd-order decays.
  • Figure 3
  • Intrathecal injection of NMDA (2 nmol, A) causes thermal hyperalgesia (when compared to vehicle, ⁇ ) and this response is blocked by subcutaneous injection of M40403 (10 mg/kg, given subcutaneously 30 min before NMDA, •).
  • Each point represents the mean ⁇ s.e.m. for 6 rats.
  • Figure 5 Inhibition of NMDA-induced hyperalgesia by M40403 is associated with inhibition of spinal manganese superoxide dismutase (MnSOD).
  • NMDA-induced hyperalgesia seen at time of peak nociception (20 minutes, B) is associated with nitration of the endogenous MnSOD at the level of the spinal cord (A).
  • Nitrated MnSOD is deactivated as shown by the reduced capacity of the enzyme to dismute superoxide (C, p ⁇ 0.05; unpaired Students t-test was employed as statistical analysis test for Fig. 5C).
  • M40403 (10 mg/kg, n 6) attenuates spinal MnSOD nitration (A) reducing the hyperalgesic response (B).
  • Immunoprecipitation data shown in panel A is representative of 6 experiments. Rat brain treated with peroxynitrite was used as positive control.
  • M40404 did not inhibit cytokine release (b-d). * P ⁇ 0.05 and ** P ⁇ 0.001 (compared to carrageenan alone).
  • PARP poly-ADP-ribose- polymerase
  • FeTMPS when given prophylactically 30 minutes before carrageenan injection inhibited edema and hyperalgesia in a dose-dependent manner as measured at 5 hours (n 6).
  • Figure 10. M40403 when given at a time of maximal hyperalgesia blocks nociception.
  • the anti-nociceptive effect of M40403 includes overall inhibition of the effects of SO exerted peripherally and centrally.
  • Controlled-Release Component refers to a composition or compound, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the controlled-release of a composition or composition combination.
  • Pharmaceutically Acceptable As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • compositions for use in animals, and more particularly in humans.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered.
  • Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when a composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. This term expressly incorporates additional carriers provided in the Formulations and Use section provided below.
  • compositions include those salts of a pharmaceutically acceptable composition formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ⁇ ethylamino ethanol, histidine, and procaine. If the composition is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Such acids include acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like. Particularly preferred are besylate, hydrobromic, hydrochloric, phosphoric and sulfuric acids. If the composition is acidic, salts may be prepared from pharmaceutically acceptable organic and inorganic bases.
  • Suitable organic bases include, but are not limited to, lysine, N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Suitable inorganic bases include, but are not limited to, alkaline and earth-alkaline metals such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. This term expressly incorporates additional salts provided in the Formulations and Use section provided below.
  • Pro-drug As used herein, the term "pro-drug" refers to any composition which releases an active drug in vivo when such a composition is administered to a mammalian subject.
  • Pro-drugs can be prepared, for example, by functional group modification of a parent drug.
  • the functional group may be cleaved in vivo to release the active parent drug compound.
  • Pro-drugs include, for example, compounds in which a group that may be cleaved in vivo is attached to a hydroxy, amino or carboxyl group in the active drug.
  • pro-drugs include, but are not limited to esters (e.g., acetate, methyl, ethyl, formate, and benzoate derivatives), carbamates, amides and ethers. Methods for synthesizing such pro-drugs are known to those of skill in the art.
  • Therapeutically Effective Amount refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject's disease or condition, will have a desired therapeutic effect, e.g., an amount which will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition, including pain. More specific embodiments are included in the Pharmaceutical Preparations and Methods of Administration section below.
  • NMDA N-methyl-D- aspartate
  • SO superoxide
  • PN peroxynitrite
  • An important mechanism of SO and PN in disease states is nitration of tyrosine residues in various receptors , enzymes, co-factors, cytokines and other compounds in a subject.
  • nitration of receptors, enzymes and other compounds has not been implicated in the development and maintenance of pain or inflammation.
  • the present invention provides novel compounds, compositions, and combinations of nitration inhibitors and pain inhibitors.
  • methods of administering these compounds, compositions and combinations for the treatment of pain and nitration are aspects of the present invention.
  • Applicants invention provides SO-driven and PN-driven nitration as a target for the development and maintenance of central sensitization and hyperalgesia provides a novel route of administration for the treatment of pain and inflammation in subjects suffering from acute or chronic pain. Inflammation is also a key component of pain.
  • Superoxide is a potent pro-inflammatory mediator in, e.g., the release of various cytokines, recruitment of neutrophils at sites of inflammation, poly-ADP-ribose-polymerase (PARP) activation, formation of peroxynitrite (PN) and nitration with deactivation of key proteins including endogenous superoxide dismutase, the enzyme that keeps SO at low levels in vivo.
  • PARP poly-ADP-ribose-polymerase
  • PN peroxynitrite
  • MnSOD, GS and NMDA receptor subunits are examples of key targets in this pathway.
  • compositions which inhibit nitration of SO- mediated PN formation and subsequent nitration are key in the development and maintenance of central sensitization, and when combined with other nitration, pain and/or inflammation inhibitors provides new analgesics and analgesic combinations which overcome the problems associated with NSAID or opioid administration while at the same time enhance the effect of the such known inhibitors to extend the potency and efficacy of the nitration, pain or inflammation inhibitor.
  • this invention provides a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof.
  • Nitration inhibitors are commercially available and methods for determining whether a compound or composition is an active nitration inhibitor are provided in the Examples below.
  • the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
  • the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof. Preferred examples of such NOS inhibitors will be recognized by those of skill in the art.
  • the NOS inhibitor is selected from the group consisting of NG- monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof;
  • the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3- aminomethyl-benzylacetamidine), and any combination thereof;
  • the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6- nitroindazole, Ng-propyl-L-arginine, and any combination thereof.
  • the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
  • the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof.
  • the pain inhibitor is an opioid.
  • the opioid is morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • the NSAID is one of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
  • the SO generating system inhibitor is a xanthine oxidase inhibitor.
  • the complexed metal of the macrocycle provides for catalytic dismutation (or "scavenging") of superoxide.
  • SO scavenger is represented by the following formula:
  • Rs, R's, Rg, and R' 9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR ⁇ 0 , -NRioRn, -COR 10 , -CO 2 R ⁇ 0 , -CONR10R11, -O-(-(CH 2 ) a -O) b -R 10 , -SR 10 , -SOR 10 , -SO 2 R ⁇ 0 ,
  • the SO scavenger is represented by the following formula:
  • a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, R 1 t R 2 , R' 2 , R 3 , R's, R , R' 4 , R 5 , R's, Re, R'e, R7,
  • R' 7 , R 8 , R's, Rg, and R' 9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR ⁇ 0 , -NR ⁇ 0 Rn, -COR 1 0, -CO 2 R ⁇ 0 , -CONR 10 Rn, -O-(-(CH 2 ) a -O) b -R ⁇ 0> -SRi 0 , -SOR 10 ,
  • R' 7> R 8 , R's, R 9 , and R' 9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH 2 ), - Q - (CH 2 )j - R - (CH 2 ) K ⁇ S -- (CH 2 ) L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, bor
  • U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings.
  • W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
  • transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V. M40403, which is within the scope of the description above, is described in Fig.
  • the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
  • R 3 , R 6 , Rg and R 12 are independently selected from the group consisting of H, alkyl, alkenyl, CH 2 , COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
  • N-Alkylpyridyl which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH 2 , S0 3 H, N0 2 , NH 2 , N(R) 3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a
  • R' is CH or N
  • R-i, R 2 , R3, R 4 > Rs, Re, R7, Ra, Rg, R10, Rft, R- ⁇ 2 , R13, R- ⁇ 4 , R15, and R16 are independently selected from the group consisting of H, S0 3 H, COOH, N0 2 , NH 2 , and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
  • Ri, R 5 , Rg, and R ⁇ 3 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R , R 4 , Re, R' ⁇ , R 8 , R's, Rio, R'10, R ⁇ 2 , R'12, ⁇ 4 , R' ⁇ 4 , R16, and R'16 are independently selected from the group consisting of H and alkyl;
  • R3, R 7 , R 11 , and R 1 5 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 4 , R 7 and R 10 are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R' 2 , R 3 , R 5 , R' 5 , R 6 , R 8 , R's, Rg, Ru, R'n and R ⁇ 2 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R 1 f R 4 , R 8 and Ru are independently selected from the group consisting of a direct bond and CH 2 ;
  • R 2 , R 3 , R' 3 , R5, R's, R7, R'7, Rg, R10, R'10, R12, R'12 and R 3 are independently selected from the group consisting of H and alkyl;
  • R 6 is hydrogen or alkyl; and
  • X, Y, Z, M and n are as defined above;
  • R-i, R , R 7 and R 10 are independently selected from the group consisting of H and alkyl;
  • R 2 , R 3 , R'3, R5, R' ⁇ , Re, Rs, Rg, R'g, Ru, R'11 and R 12 are independently selected from the group consisting of H and alkyl; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 3 , R 4 , and R 6 are independently selected from the group consisting of H and alkyl;
  • R 2 and R 5 are independently selected from the group consisting of H, alkyl, S0 3 H, N0 2 , NH 2 , halogen, COOH and N(R) 3+ wherein R is as defined above; and
  • X, Y, Z, M and n are as defined above;
  • Ri, R 2 , R 3 , and R 4 are independently selected from the group consisting of
  • R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV wherein Ri, R' ⁇ , R 2 , R' 2 , R 3 , R' 3 , R 4 , R' 4 , R 5 , R' 5 , Re, R'e, R7 and R' 7 are independently selected from the group consisting of H, alkyl, alkoxy, N0 2 , aryl, halogen, NH 2 and S0 3 H, further wherein R 6 , R'e, R 7 and R' 7 together with one other of R 6 , R' 6 , R 7 and R' 7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M 1 is selected from the group consisting of Fe, Ni or V; and X,
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the combination provides a pain inhibitor which is an opioid and a nitration inhibitor selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • a pain inhibitor which is an opioid and a nitration inhibitor selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compoound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
  • the NMDA receptor antagonist is MK801.
  • This invention also provides a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination comprises a nitration inhibitor.
  • the nitration inhibitor is described as above.
  • this method also provides a nitration inhibitor admixed with a pain inhibitor.
  • a pain inhibitor is an opioid.
  • the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
  • the pain inhibitor is an NSAID.
  • the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof. Such compositions are described above and may be used in the present method. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Such compositions are described above and may be used in the present method. Those of skill in the art will recognize multiple combinations may be used in the present methods.
  • the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
  • nitration inhibitors may be used in the present invention to treat pain.
  • a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite- driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
  • the NMDA receptor antagonist is MK801.
  • the subject may be a mammal.
  • the mammal is a human.
  • NMDA N-methyl-D- aspartate
  • NMDA-receptor activation releases superoxide the formation of which has been closely associated with numerous glutamate-mediated actions including neurotoxicity and stroke.
  • MnSOD manganese superoxide dismutase
  • SOD2 endogenous manganese superoxide dismutase
  • Nitration of the endogenous SOD enzyme is carried out by peroxynitrite, the reaction product of superoxide and nitric oxide.
  • NMDA-receptor activation favors the accumulation of peroxynitrite by forming superoxide and nitric oxide simultaneously.
  • nitration destroys the enzymatic activity of MnSOD, an event favoring the accumulation of superoxide (the enzyme can no longer dismute and hence remove superoxide) and superoxide-driven damage.
  • the associations of many glutamate actions with the formation of superoxide suggest that superoxide is an important mediator of NMDA-mediated hyperalgesia and subsequently that nitration of the endogenous MnSOD enzyme is an important pathway by which superoxide modulates the hyperalgesic response to NMDA.
  • M40403 (Fig. 1A), a synthetic and selective superoxide dismutase mimetic.
  • M40403 is a stable, low molecular weight, manganese-containing, non-peptidic molecule possessing the function and catalytic rate of native superoxide dismutase enzymes, but with the advantage of being a much smaller molecule (MW 483 vs MW 30,000 for the mimetic and native enzyme, respectively) was used to elucidate this novel pathway. Furthermore, M40403 is stable in vivo, penetrates cells readily, has wide tissue distribution in rats, is excreted intact with no detectable dissociation and is recovered in urine and feces intact. M40404 (Fig. 1 B) the inactive congener of M40403 has no SOD catalytic activity and is pharmacologically inactive in vivo in models of inflammation.
  • Important pro-inflammatory roles for superoxide include: endothelial cell damage and increased microvascular permeability, release of cytokines, recruitment of neutrophils at sites of inflammation, single-strand DNA damage and poly-ADP-ribose- polymerase (PARP) activation.
  • PARP poly-ADP-ribose- polymerase
  • the present invention provides that superoxide rapidly combines with nitric oxide removing an important homeostatic signaling molecule and at the same time forming peroxynitrite (PN), a potent cytotoxic and pro-inflammatory agent.
  • PN peroxynitrite
  • the invention provides that removal of superoxide by synthetic superoxide dismutase mimetics such as Mn(lll)tetrakis (4-benzoic acid) porphyrin (MnTBAP), M40403 and peroxynitrite by decomposition catalysts such as 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron III (FeTMPS) are shown to be affective in treating, preventing, reversing or inhibiting pain, and in particular acute inflammatory pain.
  • synthetic superoxide dismutase mimetics such as Mn(lll)tetrakis (4-benzoic acid) porphyrin (MnTBAP), M40403
  • peroxynitrite such as 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron III (FeTMPS) are shown
  • compositions treat, prevent, reverse, inhibit, control, or at least partially arrest or partially prevent, pain associated with superoxide-induced nitration of enzymes, receptors or other compounds in a subject and can be administered to a subject at therapeutically effective doses for the inhibition, prevention, prophylaxis or therapy for such pain caused by superoxide-induced nitration.
  • the compositions of the present invention comprise a therapeutically effective dosage of a nitration inhibitor which includes, e.g., nitration inhibitors, alone or in combination with another or a known nitration, pain or inflammation inhibitors.
  • compositions of the present invention which includes therapeutically, inhibitory, preventive and prophylactically effective doses of the compositions of the present invention and is more particularly defined below.
  • the subject is preferably an animal, including, but not limited to, mammals, reptiles and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.
  • compositions that exhibit large therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compositions to the site affected by the disease or disorder in order to minimize potential damage to unaffected cells and reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans and other mammals.
  • the dosage of such compositions lies preferably within a range of circulating plasma or other bodily fluid concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans and other mammals.
  • composition levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the amount of a composition that may be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of a composition contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.
  • the dosage regime for treating a disease or condition with the compositions and/or composition combinations of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profiles of the particular composition employed, whether a composition delivery system is utilized and whether the composition is administered as a pro-drug or part of a drug combination.
  • the dosage regime actually employed may vary widely from subject to subject.
  • compositions or composition combinations may be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and/or excipients.
  • the compositions and their pharmaceutically acceptable salts and solvates may be specifically formulated for administration, e.g., by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the composition or composition combinations may take the form of charged, neutral and/or other pharmaceutically acceptable salt forms.
  • pharmaceutically acceptable carriers include, but are not limited to, those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A.R.
  • compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, controlled- or sustained-release formulations and the like.
  • Such compositions will contain a therapeutically effective amount of the composition, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • composition or composition combination may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or the like.
  • the composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • a parenteral preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as a solution in 1 ,3- butanediol).
  • a nontoxic parenterally acceptable diluent or solvent e.g., as a solution in 1 ,3- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may be used in the parenteral preparation.
  • the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.
  • microcrystalline cellulose 2208, 2906, 2910
  • microcrystalline cellulose and mixtures thereof.
  • Suitable forms of microcrystalline cellulose include, for example, the materials sold as AVICEL-PH-101 , AVICEL-PH- 103 and AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pennsylvania, USA).
  • An exemplary suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581 by FMC Corporation.
  • Fillers include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), lactose, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • Lubricants include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium Iauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co.
  • Disintegrants include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
  • the tablets or capsules may optionally be coated by methods well known in the art. If binders and/or fillers are used with the compositions of the invention, they are typically formulated as about 50 to about 99 weight percent of the composition. Preferably, about 0.5 to about 15 weight percent of disintegrant, preferably about 1 to about 5 weight percent of disintegrant, may be used in the composition. A lubricant may optionally be added, typically in an amount of less than about 1 weight percent of the composition. Techniques and pharmaceutically acceptable additives for making solid oral dosage forms are described in Marshall, SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker and Rhodes, Eds.), 7:359-427 (1979). Other less typical formulations are known in the art.
  • Liquid preparations for oral administration may take the form of solutions, syrups or suspensions. Alternatively, the liquid preparations may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, eth
  • compositions of the present invention may also contain buffer salts, flavoring, coloring, perfuming and sweetening agents as appropriate.
  • Preparations for oral administration may also be formulated to achieve controlled release of the composition.
  • Oral formulations preferably contain 10% to 95% composition.
  • the compositions of the present invention may be formulated for buccal administration in the form of tablets or lozenges formulated in a conventional manner. Other methods of oral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • Controlled-Release Administration may be formulated to extend the activity of the composition or composition combination and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the composition, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of a composition that produces the desired therapeutic effect, and gradually and continually release other amounts of the composition to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of a composition in the body, the composition could be released from the dosage form at a rate that will replace the amount of composition being metabolized and/or excreted from the body.
  • Controlled-release of a composition may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Controlled-release systems may include, for example, an infusion pump which may be used to administer the composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
  • the composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the composition over a controlled period of time at a selected site.
  • polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and blends thereof.
  • compositions of the invention may be administered by other controlled-release means or delivery devices that are well known to those of ordinary skill in the art. These include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination of any of the above to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • compositions or composition combination may also be administered directly to the lung by inhalation.
  • a composition may be conveniently delivered to the lung by a number of different devices.
  • a Metered Dose Inhaler which utilizes canisters that contain a suitable low boiling point propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas may be used to deliver a composition directly to the lung.
  • MDI devices are available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura.
  • a Dry Powder Inhaler (DPI) device may be used to administer a composition to the lung.
  • DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient.
  • DPI devices are also well known in the art and may be purchased from a number of vendors which include, for example, Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura.
  • MDDPI multiple dose DPI
  • MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering
  • capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch for these systems.
  • a liquid spray device supplied, for example, by Aradigm Corporation.
  • Liquid spray systems use extremely small nozzle holes to aerosolize liquid composition formulations that may then be directly inhaled into the lung.
  • a nebulizer device may be used to deliver a composition to the lung. Nebulizers create aerosols from liquid composition formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled.
  • nebulizers examples include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd., Aventis and Batelle Pulmonary Therapeutics.
  • an electrohydrodynamic (“EHD") aerosol device may be used to deliver a composition to the lung.
  • EHD aerosol devices use electrical energy to aerosolize liquid composition solutions or suspensions.
  • the electrochemical properties of the composition formulation are important parameters to optimize when delivering this composition to the lung with an EHD aerosol device. Such optimization is routinely performed by one of skill in the art.
  • Other methods of intra-pulmonary delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • Liquid composition formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include the composition with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon.
  • a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon.
  • another material may be added to alter the aerosol properties of the solution or suspension of the composition.
  • this material may be a liquid such as an alcohol, glycol, polyglycol or a fatty acid.
  • Other methods of formulating liquid composition solutions or suspensions suitable for use in aerosol devices are known to those of skill in the art.
  • compositions or composition combination may also be formulated as a depot preparation.
  • Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt.
  • Other methods of depot delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • composition or composition combination may be combined with a carrier so that an effective dosage is delivered, based on the desired activity ranging from an effective dosage, for example, of 1.0 ⁇ M to 1.0 mM.
  • a topical composition is applied to the skin.
  • the carrier may be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.
  • a topical formulation may also consist of a therapeutically effective amount of the composition in an ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • an ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
  • Any of these compositions may also include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically or pharmaceutically effective agents which do not exert a detrimental effect on the composition.
  • composition or composition combination may also be formulated in rectal formulations such as suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides and binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • Suppositories may contain the composition in the range of 0.5% to 10% by weight.
  • Other methods of suppository delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
  • compositions of the invention are known in the art and can be used to administer the compositions of the invention. Moreover, these and other delivery systems may be combined and/or modified to optimize the administration of the compositions of the present invention. Exemplary formulations using the compositions of the present invention are described below in Example 4 (the compositions of the present invention are indicated as the active ingredient, but those of skill in the art will recognize that pro-drugs and composition combinations are also meant to be encompassed by this term).
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Example 1 Treatments for Pain Male Sprague-Dawley rats (250-260 g, Raleigh, NC, USA) were purchased with intrathecally-implanted cannulas (32 gauge, polyurethane) and used for these studies. All animals were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee and in accordance with NIH guidelines on laboratory animal welfare.
  • the active SOD mimetic M40403 and its inactive congener, M40404 were synthesized as previously described (Salvemini D, Wang Z-Q, Zweier JL, Samouilov A, Macarthur H, Misko TP, Currie MG, Cuzzocrea S, Sikorski JA and Riley DP (1999). "A non- peptidyl mimic of superoxide dismutase with therapeutic activity in rats.” Science 286: 304- 306.). Unless specified, all materials were purchased from Sigma-Aldrich Corp. (St Louis, MO).
  • the dose used was selected from previous studies where it was shown that at this dose and in vivo superoxide-mediated damage in rats was completely blocked by M40403 and not by M40404 (Coppey LJ, Gellett JS, Davidson EP, Dunlap JA, Lund DD, Salvemini D and Yorek MA (2001).
  • Lumbar spinal cord enlargements (L4/L6) were homogenized with lysis buffer with a 1 :3 w/v ratio.
  • the lysis buffer (20 mM Tris-base, 150 mM NaCl, 10 % glycerol, 0.1% Triton- X-100, 1% Chaps, 2 mM EGTA) contained 1% protease inhibitor cocktail (v/v). Solubilized extracts were sonicated (5 min.) using a Sonicator (Fisher Scientific) and after 10 min of incubation in ice the lysates were centrifuged (12500 g, 30 min at 4 °C). These supematants were stored immediately at -80 °C. Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce).
  • BCA Bicinchoninic Acid
  • the mixture of the beads-antibody and binding proteins were resuspended in 50 ⁇ l of sample buffer [2x, 0.5 M Tris-HCl, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue], heated to 95 °C (5 min).
  • sample buffer [2x, 0.5 M Tris-HCl, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue]
  • the samples were then loaded in 12% SDS-PAGE mini-gels (Bio-Rad). Rat brain treated with peroxynitrite (Upstate Biotechnology) was used as positive control.
  • the membranes were incubated with anti-rabbit horseradish peroxidase- conjugated secondary antibody (1 :10,000 dilution; Amersham) and the specific complex was detected by an enhanced chemiluminescence detection system (ECL, Amersham).
  • ECL enhanced chemiluminescence detection system
  • a competitive inhibition assay was performed which used xanthine-xanthine oxidase- generated superoxide to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt.
  • the reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 ⁇ M; Sigma-Aldrich Corp., Milan, Italy), xanthine and xanthine- oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany).
  • the rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm.
  • the amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity.
  • Cu,Zn-SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre-incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein
  • an important pathway through which superoxide modulates disease is by forming peroxynitrite, which in turn nitrates and deactivates endogenous MnSOD, an event critical in maintaining and in sustaining high levels of superoxide.
  • this pathway operates in central sensitization (as stated earlier NMDA-receptor activation releases both nitric oxide and superoxide, providing the source of peroxynitrite).
  • MnSOD was found to be nitrated in the spinal cord as shown by immunoprecipitation (Fig. 5, panel A). Furthermore, once nitrated, endogenous MnSOD is deactivated losing its ability to remove superoxide (Fig. 5, panel C).
  • excitatory amino acids including glutamate
  • stimulation of the nociceptors induces the firing of the C-fibers (unmyelinated slowly- conducting afferent) leading to the release of excitatory amino acids, including glutamate, that in turn act on the NMDA-receptor in the spinal cord.
  • C-fibers unmyelinated slowly- conducting afferent
  • NMDA receptor activation is believed to induce the spinal cord neurons to become more sensitive to all of their inputs, resulting in central sensitization seen in acute inflammatory pain and neuropathic pain. Similar mechanisms may be an important part of the development of opioid tolerance that is also blocked by NMDA-receptor antagonists.
  • the present invention provides, for the first time, that superoxide is an important mediator of spinal (i.e., central nervous system or "CNS")hyperalgesia.
  • CNS central nervous system
  • synthetic SOD mimetics and other compounds and compositions are useful agents for the management of pain of various etiologies.
  • the present invention reveals a mechanism by which superoxide modulates NMDA- mediated hyperalgesia (Figs. 6 and 12).
  • MnSOD hyperoxide dismutase mimetic
  • the present invention also provides that nitration of this enzyme is also important in central sensitization in addition to being associated closely to those disease states driven by overt production of superoxide (as the enzyme is now no longer able to dismute superoxide), for example, ischemia and reperfusion, organ transplantation, shock and inflammation. .
  • the present invention provides at least two pathways which can lead to protein nitration. One uses peroxynitrite, (the reaction product of superoxide and nitric oxide) and the other uses hydrogen peroxide and myeloperoxidase. Without being bound to a particular theory, the involvement of these pathways in a particular setting can be dissected pharmacologically by the use of agents which remove superoxide or nitric oxide.
  • nitric oxide inhibits as shown by numerous investigators the formation of peroxynitrite and peroxynitrite- mediated protein nitration.
  • MnSOD nitration was blocked by M40403 supports the role of peroxynitrite and not hydrogen peroxide/myeloperoxidase in this process.
  • nitric oxide has been implicated as a central mediator of NMDA-driven central sensitization.
  • NMDA-mediated hyperalgesia is blocked by nitric oxide synthase inhibitors. Based on the data provided in the present invention, it is believed that inhibition of NMDA-mediated hyperalgesia by various nitric oxide synthase inhibitors is in fact due to inhibition of peroxynitrite-mediated MnSOD nitration. Thus, formation of peroxynitrite and subsequent nitration of spinal MnSOD is a novel pathway of NMDA-mediated hyperalgesia and a new target which can be affected by the compounds and compositions of the present invention. The broader implication of our findings is that superoxide may contribute to various forms of centrally induced hyperalgesia that are driven by NMDA-receptor activation. Thus, superoxide is a therapeutic target for the development of novel analgesics and analgesic combinations (e.g., additive and synergistic combinations).
  • analgesics and analgesic combinations e.g., additive and synergistic combinations.
  • Example 2 Treatments of Pain Due to Inflammation
  • Tail flick and hot plate tests Nociceptive testing was performed by placing the distal third of the tail of a rat in a water bath maintained at 52 °C (tail flick) or by placing rats on a metal surface maintained at 52 °C.
  • the latency to withdrawal (tail flick) or to jumping/licking of a hindpaw (hot plate) was measured once before (control) and at selected time intervals after drug injection.
  • a cut-off latency of 10 seconds is employed to prevent tissue injury.
  • Groups of at least 6 rats each received a subcutaneous injection of SODm (10 mg/kg) or an equivalent volume of vehicle and testing was performed 15, 30, 45 and 60 minutes after injection of the drug or vehicle. Determination of anti-nociception was assessed between 7:00 and 10:00 AM.
  • Decomposed peroxynitrite was obtained by exposing the stock solution of peroxynitrite to air for at least 30 minutes. Drugs or vehicle were given intravenously (2ml/kg) 15 minutes before injection of superoxide and peroxynitrite. Hyperalgesic responses to heat were measured at time of maximal hyperalgesic effect. Time course studies revealed that maximal hyperalgesia was reached at 20 and 30 minutes respectively, for superoxide and peroxynitrite.
  • Drugs or vehicle were administered intravenously (2 ml/kg), subcutaneously (1 ml/kg) or intrathecally (in 10 ⁇ l) at 2 hours post-carrageenan injection (therapeutic treatment) and testing performed 0.25, 0.5, 0.75, 1, 2 and 3 hours after drug injection. In some experiments, drugs or vehicle were also given 30 minutes before carrageenan (prophylactic treatment) and testing performed every hour up to 5 hours post carrageenan. Changes in paw-volume were measured as previously described. Briefly, paw-volume was measured with a plethysmometer (Ugo-Basile, Varese, Italy) immediately prior to the injection of carrageenan and thereafter at hourly intervals for 5 hours. Edema was expressed as the increase in paw-volume (ml) after carrageenan injection relative to the pre-injection value for each animal. Results are expressed as Paw-volume change (ml).
  • Cytokines (TNF- ⁇ , IL-1 ?, IL-6) released in the paw exudates were measured by ELISA as described previously. Briefly, at 5 hours following the intraplantar injection of carrageenan, rats were sacrificed, and each paw was cut at the level of the calcaneus bone. Paws were gently centrifuged at 250 x g for 20 min in order to recover a sample of the edematous fluid. The volume of fluid recovered from each paw was measured. Cytokines were measured by ELISA (R&D systems, Minneapolis, MN) and results expressed in pg/paw, normalizing values to the amount of exudates recovered from each paw. All determinations were performed in duplicate. Previous studies indicated that maximal levels of these cytokines were seen at 5 hours post carrageenan. Therefore, animals were sacrificed at these time points.
  • Sections were incubated overnight with 1) anti-nitrotyrosine rabbit polyclonal antibody (1 :500 in PBS) and with anti- poly (ADP-Ribose) goat policlonal antibody rat (1:500 in PBS). Sections were washed with PBS, and incubated with secondary antibody (FITC-conjugated anti-rabbit and with TRIC- conjugated anti-goat; Jackson, West Grove, PA) for 2 h at RT. Sections were washed as before, mounted with 90% glycerol in PBS, and observed with a Nikon RCM8000 confocal microscope equipped with a 40X oil objective.
  • Sections were blocked for 1 hr with 100% normal horse serum (Vector), followed by application of monoclonal anti-nitrotyrosine antibody (1 :100 in 100% normal horse serum) at 4 °C overnight in a moist chamber. Treatment with secondary antibody, A/B complex, and DAB were performed by the manufacturer's instructions (Vector ABC Elite Kit, Vector Laboratories).
  • Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce). 4 mg of the solubilized proteins in 500 ⁇ l lysate buffer were incubated with 10 ⁇ g of agarose-conjugated anti-nitrotyrosine antibody (Upstate Biotechnology) for 2 h at room temperature (RT). Agarose beads were collected by centrifugation (1 min at 12000 x g at 4 °C) and washed in PBS (pH 7.4) three times.
  • BCA Bicinchoninic Acid
  • blots were incubated with rabbit polyclonal anti-Mn-SOD (2 hr, RT, 1 :1 ,000 dilution; Upstate Biotechnology). After washing with TBS/T, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 :10,000 dilution; Amersham) and the specific complex was detected by an enhanced chemiluminescence detection system (ECL, Amersham).
  • ECL enhanced chemiluminescence detection system
  • a competitive inhibition assay was performed which used xanthine-xanthine oxidase-generated SO to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt.
  • the reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 ⁇ M; Sigma, Milan, Italy), xanthine and xanthine-oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany).
  • the rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm.
  • the amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity.
  • Cu,Zn- SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre- incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein.
  • Rotarod Test Rats were placed on a rotating rod (7 cm diameter) turning at 10 rpm. The animals were exposed to the rotarod for one session of 180 seconds each day for 3 days to adapt the rats to the apparatus. The rats were then injected subcutaneously with SODm (100 mg/kg) and tested again on the rotarod for periods of approximately 180 sec at selected time intervals.
  • Example 2 Results Utilizing a well-established model of acute inflammatory hyperalgesia, (carrageenan- induced inflammation and hyperalgesia) the role of superoxide in nociception associated with acute inflammation was investigated.
  • Hyperalgesic responses defined as augmented pain intensity in response to painful stimuli seen upon intraplantar injection of carrageenan involve central and peripheral sensitization.
  • Intraplantar injection of carrageenan leads to a time dependent development of inflammation and hyperalgesia, which peaks within 2-3 hours and lasts for a subsequent 6-8 hours.
  • Administration of M40403 (1-10 mg/kg, n 6) prior to carrageenan administration inhibited in a dose-dependent manner the development of edema and thermal hyperalgesia at all time points.
  • Dose-dependent inhibition curves for inflammation and pain taken at 5 h after carrageenan are shown in Fig. 7a.
  • M40403 prevented carrageenan-induced edema and hyperalgesia, in part by inhibiting superoxide- driven cytokine production.
  • the potential participation of peroxynitrite in superoxide-mediated nociception was evaluated initially by immunohistochemical detection of nitrated proteins and PARP activation and subsequently by evaluating whether peroxynitrite promotes hyperalgesia and whether it's removal inhibits acute inflammatory hyperalgesia.
  • nitrated proteins are readily detected in the inflamed paw and are associated with PARP activation (Fig. 8a).
  • the formation of nitrated proteins and PARP activation were blocked by M40403 (Fig. 8a).
  • FIG. 11b reveal that the endogenous mitochondrial form of SOD, MnSOD, is nitrated at the time of near-to-maximal hyperalgesia (Fig. 11c). Once nitrated, endogenous MnSOD is deactivated losing its ability to remove superoxide (Fig. 11 of).
  • Pre-treatment of rats with M40403 (10 mg/kg, given s.c 30 minutes before carrageenan, n 6), prevented MnSOD nitration (Fig. 11b) and inhibited hyperalgesia (Fig. 11 c). Lack of effect of M40403 on basal acute nociception in naive animals.
  • M40403 when given forty minutes prior to formalin at doses failed to inhibit this phase failed to inhibit this phase (not shown).
  • paw licking time (sec) evoked by formalin in control mice was reduced from 63 ⁇ 5 to 45 ⁇ 2, 15 ⁇ 3, 6 ⁇ 2 and 2 ⁇ 0 in the presence of 0.3, , 3 and 10 mg/kg M40403 respectively.
  • Example 2 Discussion The present invention provides that superoxide is a newly identified mediator of pain, providing an opportunity for novel pain management.
  • the picture depicted in Fig. 12 summarizes the key findings of this investigation. It is well appreciated that during tissue injury and inflammation, hyperalgesia results from a persistent state of peripheral afferent sensitization that subsequently initiates spinal sensitization through the release of the excitatory amino acid, glutamate.
  • the mechanism(s) are complex in nature and involve peripherally and spinally formed inflammatory mediators including peptides, prostanoids, nitric oxide, cytokines, excitatory amino acids (glutamate) as well as spinal cord glial cells activation.
  • the present invention provides that superoxide is formed and plays a major role in the development of pain through direct peripheral sensitization, by promoting inflammation, and by favouring nitration of endogenous MnSOD in the spinal cord.
  • anti-nociceptive effect of M40403, and like compounds includes overall inhibition of the effects of superoxide exerted peripherally and centrally.
  • M40403 exerted a profound anti-inflammatory effect which included inhibition of edema, cytokine release, formation of peroxynitrite and PARP activation. Furthermore, it was noted that by inhibiting inflammation, M40403 inhibited hyperalgesia.
  • a mechanism by which M40403 blocks cytokine release is by preventing the activation of REDOX sensitive transcription factors including NF-/cB and AP-1 by superoxide which in turn regulates the genes that encode (amongst numerous things) various pro-inflammatory and pronociceptive cytokines.
  • REDOX sensitive transcription factors including NF-/cB and AP-1 by superoxide which in turn regulates the genes that encode (amongst numerous things) various pro-inflammatory and pronociceptive cytokines.
  • the same inflammatory molecules play a role in pain as shown by their ability to directly induce hyperalgesia.
  • these cytokines participate in the induction of pain by peripheral nerve inflammation and trauma, subcutaneous inflammation and spinal dynorphin.
  • NAD + functions as a cofactor in glycolysis and the tricarboxylic acid cycle
  • NAD + depletion leads to a rapid fall in intracellular ATP and, ultimately, cell injury.
  • PARP activation has been implicated for instance in the development of hyperalgesia seen upon chronic use of opioids. Furthermore, substantial evidence provided by the utility of PARP inhibitors exists to support that PARP activation is important in inflammation. SOD mimetics of the M40403 class reduce PARP activation in models of acute and chronic inflammation supporting the role of superoxide in this pathway.
  • NMDA N-methyl- D-aspartate
  • MnSOD is a newly identified mediator of pain (Fig. 12) and that its removal by low molecular weight synthetic enzymes of superoxide dismutase represents a therapeutic target for the development of novel non- narcotic analgesics including those compounds and compositions of the present invention.
  • compositions can be provided in combination with sufficient amounts of known nitration, pain or inflammation inhibitors to elicit a synergistic response in a subject.
  • Synergy may be measured by comparing the baseline pain inhibition by the rat model above and comparing composition combinations.
  • Thermal hyperalgesia and antinociception were assessed in the testing of SC- 72325A for treatment of pain.
  • Thermal hyperalgesia was determined by the Hargreaves method (Id.). A radiant heat source was focused onto the plantar surface of the affected paw of nerve-injured or carageenan-injected rats. When the animal withdrew its paw, a motion sensor halted the stimulus and timer. A maximal cut-off of 40 seconds was utilized to prevent tissue damage. Paw withdrawal latencies were thus determined to the nearest 0.1 seconds.
  • Reversal of thermal hyperalgesia was indicated by a return of the paw withdrawal latencies to the pre-tremeant baseline latencies (i.e., 21 seconds). Antinociception was indicated by a significant (p ⁇ 0.05) increase in paw withdrawal latency above this baseline. Data were converted to % antihyperalgesia or % antinociception by the formula: 100 x (test latency - baseline)/(cut-off baseline) where cut-off was 21 seconds for determining antihyperalgesia and 40 seconds for determining antinociception. Dose response curves were generated for each drug and drug combination for data obtained at the time of peak effect, which was consistently at the 30 minute time point.
  • the amount of synergy of a combination of compounds or compositions can be determined.
  • the preferred combinations of the .present invention treat pain using a smaller dose of an analgesic, such as an NSAID or opioid, when compared to administering the analgesic alone.
  • an analgesic such as an NSAID or opioid
  • a preferred combination will result, for example, in the same amount of pain relief after administering 50 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 500 mg of an NSAID or opioid alone or 500 mg of a synthetic superoxide dismutase catalyst alone.
  • the preferred combinations of the present invention treat pain to a greater extent when compared to treating pain with an analgesic alone or a synthetic superoxide dismutase catalyst alone.
  • a preferred combination will result, for example, in an equivalent amount of pain relief after administering 500 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 1 ,000 mg of the NSAID or opioid or 1 ,000 mg of a synthetic superoxide dismutase catalyst alone.
  • preferred combinations result in additive or synergistic antinociceptive effects allowing an NSAID or opioid to be administered in a dosage that is at least 50% less than the same NSAID or opioid administered alone.
  • the NSAID or opioid combination may be administered in a dosage that is at least 25% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. Still more preferably, the NSAID or opioid may be administered in a dosage that is at least 10% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. And still more preferably, the NSAID or opioid may be administered in a dosage that is at least 1% less than the same NSAID or opioid administered alone to achieve said therapeutic effect.
  • the A 50 for the log dose-response curve of a drug mixture at a fixed ratio was calculated in terms of "total dose" administered.
  • a 50 add A 50 drug ⁇ x (pi + Rp 2 ) where R is the potency ratio of drug 1 to drug 2, pi is the proportion of drug 1 in the mixture and p 2 is the proportion of drug 2.
  • Variances and 95% CL. for the theoretical additive A 50 are derived from the variances of each drug administered alone.
  • a t-test is employed to compare the theoretical additive A 50 and 95% CL. to that obtained for the mixture.
  • a significantly ((p ⁇ 0.05); t-test) lower experimental value compared to theoretical value denotes a synergistic interaction. See Table 1 below.
  • Example 4 - SC-72325A Treats Pain Analgesic effects provided by subcutaneous injection of SC-72325A was studied by formalin-induced hind paw licking response.
  • Male CD-1 mice (Charles River, 28-35 gm) were allowed to feed ad libitum. Mice were housed 5-7 per cage in a temperature controlled room with a twelve hour light-dark cycle. Determination of antinociception was assessed between 7:00 and 10:00 AM. Groups consisted of 7-14 mice, and each animal was used for one experimental condition. The antinociceptive effects of SC-72325A were tested in the formalin-induced hind paw licking procedure (Hunskaar et al., Pain, 30: 103-114, 1987).
  • mice were injected with by sub-plantar administration with formalin (20 ⁇ g of a 1% stock solution) and the duration of paw licking was monitored in the periods of 5-10 minutes (Phase I) and 15-30 minutes (Phase II) thereafter.
  • SC-72325A (10 mg/kg) was given s.c. 10 minutes prior to formalin. At 10 mg/kg, the s.c. injection of SC-72325A had a small inhibitory effect on phase 1 of the response but nearly completely abolished Phase II of the response.
  • Example 5 Antihyperalgesia and Antinociception Synergy using SC-72325A and Morphine Combination
  • Carrageenan-induced inflammation is a well characterized and commonly employed model of peripheral inflammation. This procedure reliably produces a marked inflammatory response within 3 hours of injection which is indicated by swelling of the hind paw, edema, rubor and hyperalgesia and allodynia.
  • Peripheral inflammation was induced in the hind paw of male Sprague-Dawley rats by injecting 0.1 ml of a 2% ⁇ -carrageenan suspension into the subplanar surface of the hind paw of lightly ether-anesthetized rats. Testing was performed 15, 30, 45, 60, 120 and 180 minutes after drug injections.
  • the s.c. injection of SC-72325A produced time-related and dose-dependant antihyperalgesia over the dose range of 0.3 to 30 mg/kg.
  • morphine also produced time-related and dose-dependent antihyperalgesia and antinociception over the dose range of 0.03 to 10 mg/kg.
  • the 1 :1 combination of SC-72325A with morphine produced antihyperalgesia activities at much lower doses than either drug alone. Isobolographic analyses revealed that the combination of SC-72325A with morphine resulted in a definitive synergistic interaction against hyperalgesia; the A 50 values with confidence intervals are presented in Table 1 , below.
  • the antihyperalgesic A 50 value for the 1:1 combination of SC-72325A plus morphine was 0.046 mg/kg, s.c, which was significantly (p ⁇ 0.05) less than the calculated theoretical A 50 value for the combination if the activity was additive. See Table 1. SC-72325A also exhibited a slight antinociceptive effect. TABLE 1 Antihyperalgesia Aso (mg/kg, s.c.) SC-72325A 1.34 Morphine 0.22 Morphine + SC-72325A 0.046 Theoretical Additive Curve 0.380
  • Example 6 Formulations of Compositions The following formulations are provided as examples of composition preparations according to the present invention:
  • Hard gelatin capsules are prepared using the following ingredients:
  • the above ingredients are mixed and filled into hard gelatin capsules in 560 mg quantities.
  • Formulation 2 A tablet formula is prepared using the following ingredients:
  • the components are blended and compressed to form tablets, each weighing 665 mg.
  • Formulation 3 A dry powder inhaler formulation is prepared containing the following components:
  • the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • Formulation 4 Tablets each containing 60 mg of active ingredient, are prepared as follows: TABLE 5 Ingredients milligrams Active ingredient 60.0 Starch 45.0 Microcrystalline cellulose 35.0 Polyvinylpyrrolidone (as 10% solution in water) 4.0 Sodium carboxymethyl starch 4.5 Magnesium stearate 0.5 Talc 1.0 Total 150.0
  • the active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a 16 mesh U.S. sieve.
  • the granules as produced are dried at 50-60 °C and passed through a 16 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 190 mg quantities.
  • Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
  • Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5.0 ml dose are made as follows:
  • the active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.
  • the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
  • Capsules each containing 150 mg of active ingredient, are made as follows:
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 560 mg quantities.

Abstract

The invention provides a combination of compositions for treating, preventing, reversing or inhibiting pain, wherein at least one composition comprises a nitration inhibitor. Further provided is a method wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Preferably, the nitration inhibitor is combined with a pain inhibitor. The pain inhibitor is still more preferably an opioid.

Description

TITLE OF THE INVENTION Composition Combinations and Methods for Treating, Preventing, Reversing and Inhibiting Pain CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application Serial No. 60/516,165 filed on October 31 , 2003, which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not Applicable.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC Not Applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates to methods of preventing nitration, whether in vitro or in vivo, and compositions and composition combinations which inhibit nitration.
2. Description of the Related Art Numerous analgesics are known to medical science. Many analgesics fall into one of two large categories - nonsteroidal analgesic/anti-inflammatory drugs (NSAIDs) and opioids. NSAIDs operate by inhibiting cyclooxygenase enzymes and thereby the synthesis of prostaglandins. Prostaglandins, in turn, sensitize pain receptors which lower the pain threshold perceived by subjects making normal stimuli, such as touch and stretch sensations, painful. NSAIDs can be quite effective at returning the lowered pain threshold to normal but do not elevate the pain threshold. Common NSAIDs available over-the-counter include: ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), and aspirin (Bayer®). Prescription NSAIDs include: celecoxib —Celebrex®, diclofenac -- Voltaren®, etodolac ~ Lodine®, fenøprofen - Nalfon®, indomethacin - Indocin®, ketoprofen - Orudis®, Oruvail®, ketoralac -Toradol®, oxaprozin - Daypro®, nabumetone - Relafen®, sulindac - Clinoril®, tolmetin - Tolectin®, and rofecoxib - Vioxx®. A second class of pain relievers, opioids, operate by mimicking natural peptides such as enkephalins and endorphins to stimulate one or more of the μ~, δ- and /c-receptor systems in the nervous system. Opioids elevate the pain threshold so that normally painful stimuli are perceived as less painful or even euphoric. Opioids are commonly used in the clinical management of severe pain, including chronic severe pain of the kind experienced by cancer patients. Each of these classes of compounds has inherent problems and limitations. NSAIDs that are nonselective for the cyclooxygenase 2 (COX-2) produced in inflammation also inhibit constitutive cyclooxygenase 1 (COX-1), causing undesirable damage to the gastric mucosa. They have limited effectiveness as analgesics in lowering an elevated threshold to normal and are generally used for mild to moderate pain. They are also ineffective drugs for elevation of the pain threshold above normal levels, which prevents their use in pain such as surgical pain where an underlying pathological condition has not elevated the pain threshold. Opioid analgesics are antagonized by analogous N-allyl compounds such as naloxone. In addition, opioids have problems with tolerance and dependency, so that over a course of therapy increasing dosages of compound are required to achieve the same level of analgesia, and cessation of opioid administration when analgesia is no longer needed elicits a withdrawal syndrome with unpleasant and potentially serious symptoms. The dependency and withdrawal syndrome both make it difficult for the clinician to discontinue opioid therapy even when the opioids are no longer effective in relieving pain because of the development of tolerance. Narcotic induced hyperalgesia (NIH) can also develop in association with tolerance to the opioids. All of these factors limit the usefulness of opioids in the management of chronic severe pain, despite their potency. No adequate strategy has been devised to overcome the problems associated with
NSAID and opioid analgesics to manage pain. Mechanisms of tolerance are not well understood but are known to involve the N-methyl-D-aspartate (NMDA) receptor. NMDA stimulates nitric oxide synthase (NOS) and NOS has been observed histochemically in tissues that contain opioid receptors and are important in the pain response, such as the amygdala, cortical gray matter, and the substantia gelatinosa of the spinal cord. Non- selective NOS inhibitors such as NG-nitroarginine prevent and reverse morphine tolerance. However, nonselective inhibition of NOS is associated with a vast array of undesirable side effects, including hypertension, increased platelet and white blood cell reactivity, decreased cerebral blood flow, and gastrointestinal and renal toxicity. For these and other reasons, a continuing need exists for new analgesics which overcome the problems associated with NSAID or opioid administration. A need also exists for methods for treating pain using analgesics which do not have these problems.
BRIEF SUMMARY OF THE INVENTION Accordingly, it is an object of the invention to overcome these and other problems associated with the related art. These and other objects, features and technical advantages are achieved by providing compositions, composition combinations and methods to treat pain by administering superoxide- and peroxynitrite-mediated nitration inhibitors to a subject in need thereof. The compositions may be administered to a subject in combination with a known nitration, pain or inflammation inhibitor to enhance the effect of such known inhibitors to extend the potency and efficacy of the pain inhibitor. This invention provides a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier. Preferably, the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof. In accordance with a further aspect of the invention, the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof. Preferably, the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof; the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-omithine (L-NIL), aminoguanidine, 1400W (N-3- aminomethyl-benzylacetamidine), and any combination thereof; and the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6- nitroindazole, Ng-propyl-L-arginine, and any combination thereof. In accordance with a further aspect of the invention, the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof. In accordance with yet another aspect of the invention, the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said combination optionally admixed with a pharmaceutically acceptable salt or carrier. In one alternative, the pain inhibitor is an opioid. Preferably, the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof. In a second alternative, the pain inhibitor is an NSAID. Preferably, the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®). In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof. Preferably, the SO generating system inhibitor is a xanthine oxidase inhibitor. In one alternative, the SO scavenger is represented by the following formula:
Figure imgf000006_0001
wherein (a) one or more of R, R', R1 f R'1 ( R2, R'2> R3, R'3> R , R' , Re, R'β, Re, R'β, R7, RV, R8, R'8) R9, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10Rιι, -COR10, -CO2R10, -CONR10R11, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -SO2R10, -SO2NR10Rn, -N(OR10)(R11), -P(O)(OR10)(OR11), -P(O)(OR10)(Rn) and -OP(O)(OR10)(OR11) substituents (in which case R10 and R-n are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the α-carbon of σ-amino acids; and (b) optionally, one or more of R or R' and R^ or R'.|, R2 or R'2 and R3 or R'3, R4 or R4 and R5 or R'5) R6 or R'6 and R7 or R'7, or R8 or R'8 and R9 or R'g together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (c) optionally, one or more of ^ or R and R2 or R'2, R3 or R'3 and R4 or R4, R5 or R'5 and R6 or R'6) R7 or R'7 and R8 or R'8, R9 or R'9 and R or R', together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (d) optionally, one or more of R and R', R-, and R^, R2 and R'2, R3 and R'3, R4 and
R4, R5 and R'5, R6 and R'6, R7 and R , R8 and R'8> and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R1 f R^, R2, R'2, R3, R's, R4, R4, R5, R5, Re, R'6, R7, R7, Rg, R's, R9, and R9 together with a different one of R, R', R^ R'^ R2, R'2, R3, R'3, R4, R'4) R5, R'5> R6, R'e, R7, RV. a, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), -- Q - (CH2)j - R - (CH2)K -- S - (CH2)L -- wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (f) combinations of any of (a) through (e) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R', Ri, R'-i, R2, R'2l R3, R'3, R4, R'4, R5, R'5, Re, R'β, R7, RV, Rδ, R's, Rg, and R'g; and M is a transition metal; and n is an integer from 0 to 3. In a second alternative, the SO scavenger is represented by the following formula:
Figure imgf000008_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, R^ R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'6, R7, R'7, Rs, R'β, R9, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR^Rn, -COR10, -CO2R10, -CONR10Rn, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -SO2R10, -SO2NR10Rn, -N(OR10)(Rn), -P(O)(OR10)(OR11), -P(O)(OR10)(Rn) and -OP(O)(OR10)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the -carbon of -amino acids; and (c) optionally, one or more of R2 or R'2 and R3 or R'3> R4 or R'4 and R5 or R'5, R6 or R'6 and R7 or R'7, or R8 or R'8 and Rg or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (d) optionally, one or more of R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (e) optionally, one or more of R2 and R'2> R3 and R'3, R4 and R'4, R5 and R'5) R6 and R'6, R and R'7, R8 and R'8, and Rg and R'g, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (f) optionally, one or more of R, R1 f R2, R'2, R3, R'3, R , R'4, R5, R's, Re, R'e, Ry, RV, Rs, R's, RΘ, and R'9 together with a different one of R, R1 ( R2, R2, R3, R's, R4, R'4, Rs, R's, R6, R'6, R7, R'7, R8, R'8, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (g) optionally, one or more of R, R1 ; R2, R'2, R3, R'3, R4, R'4, R5, R's, R6, R'e, R?, R'7, R8, R's, Rg, and R'g may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) combinations of any of (a) through (g) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate.or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R1t R2, R'2, R3, R'3, R , R' , R5, R'5, Rβ, R'ε, R7> R'7, Rs, R's, R9, arid R'g; and M is a transition metal; and n is an integer from 0 to 3. In a third alternative, the SO scavenger is represented by the following formula:
Figure imgf000011_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, in the case which W is an aromatic heterocycle, the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) two sets of two adjacent carbon atoms of the macrocycle independently form substituted or unsubstituted, saturated or unsaturated, heterocycles U and V having 3 to 20 carbon atoms; and (c) optionally, one or more of R, R1; R2, R'2, R3, R4, Rs, R's, Re, R'e, R , Rs, g, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10R11, -COR10, -C02Rιo, -CONR10R11, -0-(-(CH2)a-0)b-RiQ, -SR10, -SOR10, -S02R1o, -SO2NR10Rιι, -N(OR10)(Rιι), -P(O)(OR10)(OR11), -P(O)(OR10)(Rn) and -OP(O)(OR10)(OR11) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the -carbon of σ-amino acids; and (d) optionally, one or more of R2 or R'2 and R3, R4 and R5 or R'5, R6 or R'6 and R7, or R8 and Rg or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (e) optionally, R5 or R'5 and R6 or R'6 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (f) optionally, one or more of R2 and R'2, R5 and R'5, R6 and R'6, and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (g) optionally, one or more of R, R^ R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, and R'9 together with a different one of R, R-,, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, R1 ( R2, R'2, R3, R4, R5, R'5, Re, R'e, R7, Rs, Rg, and R'g may be bound to an atom of at least one of heterocycle U, V or W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (i) combinations of any of (a) through (h) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R ; R2, R'2, R3) R4, R5,
R's, Re, Re, R7, Rs, R9, and R'9; and M is a transition metal; and n is an integer from 0 to 3. Preferably, U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings. In alternatives referring to a W moiety, W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety. In accordance with yet another aspect of the invention, transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V. In accordance with another aspect of the invention, the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Preferably, the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
Figure imgf000014_0001
wherein R3, R6, R9 and R12 are independently selected from the group consisting of H, alkyl, alkenyl, CH2, COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
Phenyl
Pyridyl
N-Alkylpyridyl
Figure imgf000014_0002
which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein R-i, R2, R4, R5, R7, R8, R10, or Ru are independently selected from the group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, N02, hydroxyalkyl, and S03H; and further wherein R1 and R2 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins; with the proviso that when the X and Y containing complex has a net positive charge then Z is a counter ion which is independently selected from the group consisting of X and Y, or when the X and Y containing complex has net negative charge then Z is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V; and n is an integer from 0 to 4. Structure il
Figure imgf000016_0001
wherein R' is CH or N; R-i, R2, R3, R , Rs, Re, R7, Rs, R9, R10, R11, ι2, R13, Rι4, R15, and R16 are independently selected from the group consisting of H, S03H, COOH, N02, NH2, and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
Figure imgf000016_0002
A wherein R^ R5, R9, and R13 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, Re, R'e, Rs, R's, R10, R'10, R12, R'12, Rι4, R'ι4, R16, and R'1,6 are independently selected from the group consisting of H and alkyl; R3, R7, R11, and R15 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000017_0001
B wherein R^ R5, R8, and R12 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, R6, R'6, R7, R9, R'g, Ru, R'n, R13, R'ι3, and R1 are independently selected from the group consisting of H and alkyl; R3 and R10 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000017_0002
wherein R-,, R4, R8, and R12 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, Rs, R's, R7, Rg, R'9, R11, R'11, R13, R'13 and R14 are independently selected from the group consisting of H and alkyl; R10 is H or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000018_0001
D wherein R^ R , R7 and R10 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R's, Re, Rs, R's, g, Rn, R'n and R12 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000018_0002
wherein R-i, R4, R8 and R-n are independently selected from the group consisting of a direct bond and CH2; R2, R3, R'3, Rs, R's, R7, R'7, R9, R10, R'10, R12, R'12 and R13 are independently selected from the group consisting of H and alkyl; R6 is hydrogen or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000019_0001
wherein Ri, R4, R and Rι0 are independently selected from the group consisting of H and alkyl; R2, R3, R'3, R5, R'5, Re, Rs, Rg, R'g, Ru, R'n and R12 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000019_0002
wherein R^ R3, R , and R6 are independently selected from the group consisting of H and alkyl; R2 and R5 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above;
Figure imgf000020_0001
H wherein R1 ? R2> R3, and R are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
Figure imgf000020_0002
wherein R^ R'-,, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'e, R7 and R'7 are independently selected from the group consisting of H, alkyl, alkoxy, N02, aryl, halogen, NH2 and S03H, further wherein R6, R'6> R7 and R'7 together with one other of R6, R'β, R7 and R'7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M1 is selected from the group consisting of Fe, Ni or V; and , X, Y, Z and n are as defined above. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with another aspect of the invention, the combination provides a pain inhibitor which is an opioid and a nitration inhibitor selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with yet another aspect of the invention, a combination of compounds is provided for treating, preventing, reversing or inhibiting pain, wherein at least one compoound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof. Preferably, the NMDA receptor antagonist is MK801. This invention also provides a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier. Preferably, the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof. Preferably, the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof. In one alternative, the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof; the iNOS inhibitor is selected from the group consisting of L-N5-(1- iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3-aminomethyl- benzylacetamidine), and any combination thereof; and the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng- propyl-L-arginine, and any combination thereof. In accordance with a further aspect of the invention, the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof. In accordance with yet another aspect of the invention, the pain is initiated in a central sensitization pathway of the subject. In accordance with another aspect of the invention, the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier. In one alternative, the pain inhibitor is an opioid. Preferably, the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof. In a second alternative, the pain inhibitor is an NSAID. Preferably, the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®). In accordance with a further aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof. In another aspect of the invention, the SO generating system inhibitor is a xanthine oxidase inhibitor. In" one alternative, the SO scavenger is represented by the following formula:
Figure imgf000022_0001
wherein (a) one or more of R, R', Rn, R'ι, R2, R'2, R3, R's, R4, R'4, Rs, R's, Re, R'e, R , R'7, R8, R's, Rg, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NRιoRιι> -COR10, -C02Rιo, -CONR10R11, -0-(-(CH2)a-0)b-R-ιo, -SR-io, -SOR10, -S02Rιo, -SO2NR10Rn, -N(OR10)(Rn), -P(O)(OR10)(OR11), -P(O)(OR10)(Rn) and -OP(O)(OR10)(OR11) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of σ-amino acids; and (b) optionally, one or more of R or R' and R-i or R'1, R2 or R'2 and R3 or R'3) R or R'4 and R5 or R'5) R6 or R'6 and R7 or R'7, or R8 or R'8 and Rg or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (c) optionally, one or more of R^ or R'ι and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8> R9 or R'9 and R or R', together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (d) optionally, one or more of R and R', R^ and R'ι, R2 and R'2, R3 and R3, R4 and R' , Rs and R'5) R6 and R'6, R7 and R'7, R8 and R'8, and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R1 ? R',, R2, R'2, R3, R'3, R , R' , Rs, R's, Re, R'e, R7, R'7, Rs, R's, Rg, and R'9 together with a different one of R, R', R,, R , R2, R'2) R3, R's, R4, R 4, R5, R'5, R6, R'e, R7, R'7, Rs, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R -- (CH2)K - S - (CH2)L ~ wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (f) combinations of any of (a) through (e) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R', Ri, R'-i, R2, R'2, R3, R'3, R > R' > Rs, R's, Re, 'δ, R7, R'7, Rs, R's, R9, and R'9; and M is a transition metal; and n is an integer from 0 to 3. In a second alternative, the SO scavenger is represented by the following formula:
Figure imgf000024_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W haying 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, R-i, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'e, R7, R'7, R8, R's, Rg, and R'g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10Rn, -COR10, -CO2Rι0, -CONR10Rn, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -SO2R10, -SO2NR10Rn, -N(OR10)(Rn), -P(O)(OR10)(OR11), -P(O)(OR10)(Rn) and -OP(O)(OR10)(ORn) substituents (in which case R10 and R^ are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of σ-amino acids; and (c) optionally, one or more of R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R '5, R6 or R'e and R7 or R'7, or R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached- to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (d) optionally, one or more of R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (e) optionally, one or more of R2 and R'2, R3 and R'3, R4 and R4, R5 and R'5, R6 and R'6, R7 and R'7, R8 and R'8) and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (f) optionally, one or more of R, R1 f R2, R'2, R3, R'3, R4, R'4, R5, R'5, Re, R'e, R7, R'7, R8, R's, Rg, and R'9 together with a different one of R, R R2, R'2, R3, R'3^ R4, R'4, R5, R'5> Re, R'e, R7, R'7, Re, R's, R9, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (g) optionally, one or more of R, R1 t R2, R'2, R3, R's, R , R' , R5, R's, Re, R'e, R7,
R'7, R8, R's, Rg, and R'9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R -- (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) combinations of any of (a) through (g) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R1 ( R2, R'2, R3, R'3, R4, R' , R5, R's, Re, R'e, R7, R'7, s, R's, 9, and R'9; and M is a transition metal; and n is an integer from 0 to 3. In a third alternative, the SO scavenger is represented by the following formula:
Figure imgf000027_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, in the case which W is an aromatic heterocycle, the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) two sets of two adjacent carbon atoms of the macrocycle independently form substituted or unsubstituted, saturated or unsaturated, heterocycles U and V having 3 to 20 carbon atoms; and (c) optionally, one or more of R, R1 f R2, R'2, R3, R4, Rg, R'5, R6, R'6, R7, Rs, Rg, and R'g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10R11, -COR10, -CO2R10, -CONR10Rιι, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -SO2R10,
-SO2NR10Rn, -N(OR10)(Rn), -PfOXORuXORn), -P(O)(OR10)(Rn) and -OP(O)(OR10)(OR11) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the o/-carbon of -amino acids; and (d) optionally, one or more of R2 or R'2 and R3, R4 and R5 or R'5, R6 or R'6 and R7, or R8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (e) optionally, R5 or R'5 and R6 or R'6 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (f) optionally, one or more of R2 and R'2, R5 and R'5, R6 and R'6, and R9 and R'9) together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (g) optionally, one or more of R, R ; R2, R'2, R3, R4, Rs, R's, Re, R'e, R7, Rs, Rg, and R'g together with a different one of R, R1t R2, R'2, R3, R4, R5, R'5, R6, R'e, R7, R8, R9, and R'g which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, R1 ( R2, R'2, R3, R4, R5, R'5, R6, R'e, R7, Rs, Rg, and R'g may be bound to an atom of at least one of heterocycle U, V or W to form a strap represented by the formula: - (CH2), -- Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (i) combinations of any of (a) through (h) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R^ R2, R'2, R3, R4, R5, R's, e, e, R7, s, Rg, and Rg; and M is a transition metal; and n is an integer from 0 to 3. Preferably, U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings. In alternatives referring to a W moiety, W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety. In accordance with yet another aspect of the invention, transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with another aspect of the invention, the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
Figure imgf000030_0001
wherein R3, R6, R9 and R12 are independently selected from the group consisting of H, alkyl, alkenyl, CH2, COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
Phenyl
Pyridyl
N-Alkylpyridyl
Figure imgf000030_0002
which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, SOsH, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein R-i, R2, R , Rs, R7, Rs, R10, or Ru are independently selected from the group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, N02, hydroxyalkyl, and S03H; and further wherein R1 and R2 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins; with the proviso that when the X and Y containing complex has a net positive charge then Z is a counter ion which is independently selected from the group consisting of X and Y, or when the X and Y containing complex has net negative charge then Z is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V; and n is an integer from 0 to 4. Structure II
Figure imgf000032_0001
wherein R' is CH or N; Ri, R2, R3, R4, Rs, Rε, R7, Rs, Rg, R10, Ru, R12, Ri3, Rι4, R15, and R16 are independently selected from the group consisting of H, S03H, COOH, N02, NH2, and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
Figure imgf000032_0002
A wherein R-,, R5, R9, and R13 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, Re, R'ε, Re, R's, R10, R'10, R12, R'12, Rι4, R'ι4, R16, and R'ι6 are independently selected from the group consisting of H and alkyl; R3, R7, Ru, and R15 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000033_0001
B wherein Ri, R5, R8, and Rι2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2l R4, R'4, R6, R'e, R7, R9, R'9, Rn, R'n, Ris, R'ι3, and R14 are independently selected from the group consisting of H and alkyl; R3 and Rio are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000033_0002
wherein Ri, R , R8, and Rι2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R'5, R7, Rg, R'g, Ru, R'n, Rι3, R'ι3 and R are independently selected from the group consisting of H and alkyl; Rio is H or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000034_0001
D wherein R1 t R4, R7 and Rι0 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R's, Re, Rs, R's, Rg, R11, R'11 and Rι2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000034_0002
wherein Ri, R4, R8 and Ru are independently selected from the group consisting of a direct bond and CH2; R2, Rs, R'3, Rs, R's, R7, R'7, Rg, R10, R'10, R12, R'12 and Rι3 are independently selected from the group consisting of H and alkyl; R6 is hydrogen or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000035_0001
wherein R1 ? R4, R7 and R 0 are independently selected from the group consisting of H and alkyl; R2, R3, R'3, Rs, R's, Re, Rs, Rg, R'g, Ru, R'11 and R 2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000035_0002
wherein Ri, R3, R4, and R6 are independently selected from the group consisting of H and alkyl; R2 and R5 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above;
Figure imgf000036_0001
H wherein R-,, R2, R3, and R4 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
Figure imgf000036_0002
wherein R,, R'1 f R2, R'2, R3, R'3, *, R'4, R5, R'5, R6, R'e, R7 and R'7 are independently selected from the group consisting of H, alkyl, alkoxy, N02, aryl, halogen, NH2 and S03H, further wherein R6, R'6, R and R'7 together with one other of R6, R'β, R7 and R'7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with another aspect of the invention, the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with a further aspect of the invention, a method is provided for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof. Preferably, the NMDA receptor antagonist is MK801. In accordance with yet another aspect of the invention, the subject is a mammal. Preferably, the mammal is a human. These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, examples and appended claims. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Figure 1. Structures of synthetic SODm M40403 and M40404. Figure 2. Decay of superoxide (top line) uncatalyzed and in the presence of an active SOD mimetic (M40403, 5 μM; bottom line) and an inactive analog (M40404, 5 μM; middle line). The active catalyst affords a pure 1st-order decay of superoxide while the uncatalyzed and M40404 decays remain pure 2nd-order decays. Figure 3. Decomposition of peroxynitrite (top line) uncatalyzed and in the presence of the peroxynitrite decomposition catalyst (FeTMPyP, 5 μM; bottom line) and of an active SOD mimetic (M40403, 5 μM; middle line (interspersed withing top line)). M40403 does not interact with peroxynitrite, while the synthetic iron porphyrin complex FeTMPyP catalyzes the peroxynitrite decomposition. Figure 4. Thermal hyperalgesia induced by intrathecal NMDA is driven by spinally released superoxide. Intrathecal injection of NMDA (2 nmol, A) causes thermal hyperalgesia (when compared to vehicle, ■) and this response is blocked by subcutaneous injection of M40403 (10 mg/kg, given subcutaneously 30 min before NMDA, •). * p < 0.05, ** p < 0.01 (compared to vehicle) and f p < 0.05 (compared to NMDA alone). Each point represents the mean ± s.e.m. for 6 rats. Figure 5. Inhibition of NMDA-induced hyperalgesia by M40403 is associated with inhibition of spinal manganese superoxide dismutase (MnSOD). NMDA-induced hyperalgesia seen at time of peak nociception (20 minutes, B) is associated with nitration of the endogenous MnSOD at the level of the spinal cord (A). Nitrated MnSOD is deactivated as shown by the reduced capacity of the enzyme to dismute superoxide (C, p < 0.05; unpaired Students t-test was employed as statistical analysis test for Fig. 5C). M40403 (10 mg/kg, n=6) attenuates spinal MnSOD nitration (A) reducing the hyperalgesic response (B). Immunoprecipitation data shown in panel A is representative of 6 experiments. Rat brain treated with peroxynitrite was used as positive control. * p < 0.01 (compared to vehicle) and f p < 0.05 (compared to NMDA alone). Each point represents the mean ± s.e.m. for 6 rats. Figure 6. Superoxide-mediated nitration and deactivation of spinal MnSOD: a novel pathway of NMDA-mediated central sensitization. NMDA receptor activation increases intracellular calcium influx and leads to the production of superoxide which in turn contributes to the state of hyperalgesia by nitrating and subsequently deactivating spinal endogenous manganese SOD. MnSOD nitration is important in the hyperalgesic response since it helps to maintain high levels of superoxide that in turn maintains nociceptive signaling. Removal of superoxide with M40403 abolished NMDA-mediated hyperalgesia by preventing nitration and deactivation of the endogenous enzyme. Figure 7. Anti-hyperalgesic profile of the superoxide dismutase mimetic, M40403. a M40403 when given prophylactically 30 minutes before carrageenan injection inhibited edema and hyperalgesia in a dose-dependent manner as measured at 5 hours (n=6). At the same time point M40403 inhibited in a dose-dependent manner (1-10 mg/kg, (n=6) the following cytokines: b tumor necrosis factor-σ (TNF-α), c interleukin-1β (IL-1 ?) and d interleukin-6. The inactive SOD mimetic M40404 did not inhibit cytokine release (b-d). * P < 0.05 and ** P < 0.001 (compared to carrageenan alone). Figure 8. M40403 inhibits nitrotyrosine formation and poly-ADP-ribose- polymerase (PARP) activation in the carrageenan-inflamed rat hindpaw. a No positive staining for nitrotyrosine and for PAR was found in the paw section from control animals. At 2 and 5 hours after carrageenan, immunohistochemical analysis for nitrotyrosine and for PAR shows positive staining mainly localized to the vessels (arrows) and in the infiltrated inflammatory cells (arrowhead) in the tissue section collected at 2 h and 5h after carrageenan administration. The intensity of the positive staining for nitrotyrosine and for PAR was significantly reduced by M40403. Original magnification: x 145. This figure is representative of at least 3 experiments performed on different experimental days, b Overall protective effect of M40403 as determined by histological examination. No histological modification was observed in paw tissue from sham-treated rats (a). Representative paw tissues sections collected at 2h (b,b1) and 5h (c,c1) after carrageenan administration demonstrate marked inflammatory changes including pronounced cellular infiltration (see arrows). These events were reduced by M40403 (d,d1). Original magnification: x 195. This figure is representative of at least 3 experiments performed on different experimental days. Figure 9. Direct hyperalgesic effects of superoxide and peroxynitrite. a Superoxide (SO) when given by intraplantar injection, causes hyperalgesia, and this is blocked by M40403 (3 mg/kg, n=6) but not by the same dose of the inactive SODm, M40404 (n=6). *P < 0.001 (compared to control), f P < 0.001 (compared to SO), b Peroxynitrite (PN, 1μM) when given by intraplantar injection, causes hyperalgesia, and this is blocked by the active but not inactive PN decomposition catalyst (FeTMPS and H2TMPS respectively, 30 mg/kg, n=6). Decomposed SO or PN (dec SO or decPN) had no hyperalgesic activity. *P < 0.001 (compared to control), f P < 0.001 (compared to PN). c Carrageenan-induced edema and hyperalgesia is blocked by the PN decomposition catalyst, FeTMPS. FeTMPS when given prophylactically 30 minutes before carrageenan injection inhibited edema and hyperalgesia in a dose-dependent manner as measured at 5 hours (n=6). Figure 10. M40403 when given at a time of maximal hyperalgesia blocks nociception. a M40403 when given intravenously 2h after carrageenan, at 0.3 mg/kg (Δ, n=4), 1 mg/kg (•, n=11) or 3 mg/kg (■, n=16) inhibits carrageenan (♦, n=20) induced hyperalgesia with a rapid onset of action. *P < 0.001 (compared to carrageenan alone), b When tested at the highest dose (3 mg/kg), M40403 (given at 2h post carrageenan) inhibited edema, but the time course of inhibition was different to the one seen for anti-hyperalgesic responses *P < 0.05, **P < 0.01 and ***P < 0.001 (when compared to the value obtained at 2 h post carrageenan before drug injection; statistical analysis was done using unpaired Students t-test). c M40403 when given intrathecally (2 nmol) 2 hours after carrageenan inhibits carrageenan (♦) induced hyperalgesia with a rapid onset of action (n=6). *P < 0.05 and **P < 0.01 (compared to carrageenan alone; statistical analysis was done using unpaired Student's t-test). Figure 11. Inhibition of carrageenan-induced hyperalgesia by M40403 is associated with nitration and subsequent deactivation of spinal cord (L4/L6) manganese superoxide dismutase (MnSOD). a Spinal cord sections at the L4-L6 were immunostained with affinity purified monoclonal anti-nitrotyrosine antibodies. Labeling was observes in the motor neurons in the spinal cord layers both at 2 and 5 hours post carageenan injection. There was no appreciable staining in the saline control sections. The immunostaining of the motor neurons seen after the carrageenan challenge was significantly reduced by M40403. c Carrageenan-induced hyperalgesia seen at time of peak nociception (2-5h) is associated with b nitration of the endogenous MnSOD at the level of the spinal cord. M40403 (10 mg/kg, n=6) attenuates spinal MnSOD nitration (b) reducing the hyperalgesic response (c). Immunoprecipation data shown in panel b is representative of 6 experiments. Rat brain treated with PN was used as positive control (data not shown). * P < 0.001 (compared to control) and f P < 0.001 (compared to carrageenan alone), d At 2 and 5 hours after carrageenan, nitrated MnSOD is deactivated as shown by the reduced capacity of the enzyme to dismute SO. Figure 12. Role(s) of superoxide in pain. Acute inflammatory hyperalgesia
(evoked by an inflammatory stimuli, in this case carrageenan) releases a variety of pro- inflammatory and pro-nociceptive mediators such as bradykinin, (BK), serotonin (5-HT), histamine, nitric oxide (NO) prostaglandins (PGs) and cytokines. As shown in our study, SO is also formed and plays a major role in the development of pain through direct peripheral sensitization, through the release of various cytokines (TNFσ, \ -lβ, IL-6), through the formation of peroxynitrite (ONOO") and through PARP activation. Furthermore, when released at the level of the spinal cord in response to presumably glutamate (see discussion) SO in turn interacts with NO to form ONOO". The latter in turn nitrates and subsequently deactivates MnSOD in the spinal cord. As a consequence, levels of SO remain elevated favoring the maintenance of nociceptive signaling. The anti-nociceptive effect of M40403 includes overall inhibition of the effects of SO exerted peripherally and centrally.
DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions To facilitate the understanding of the invention, a number of terms and abbreviations as used herein are defined below and have the following meanings: Controlled-Release Component: As used herein, the term "controlled-release component" refers to a composition or compound, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the controlled-release of a composition or composition combination. Pharmaceutically Acceptable: As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Pharmaceutically Acceptable Carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when a composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. This term expressly incorporates additional carriers provided in the Formulations and Use section provided below. Pharmaceutically Acceptable Salt: As used herein, the term "pharmaceutically acceptable salt" includes those salts of a pharmaceutically acceptable composition formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-< ethylamino ethanol, histidine, and procaine. If the composition is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Such acids include acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like. Particularly preferred are besylate, hydrobromic, hydrochloric, phosphoric and sulfuric acids. If the composition is acidic, salts may be prepared from pharmaceutically acceptable organic and inorganic bases. Suitable organic bases include, but are not limited to, lysine, N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable inorganic bases include, but are not limited to, alkaline and earth-alkaline metals such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. This term expressly incorporates additional salts provided in the Formulations and Use section provided below. Pro-drug: As used herein, the term "pro-drug" refers to any composition which releases an active drug in vivo when such a composition is administered to a mammalian subject. Pro-drugs can be prepared, for example, by functional group modification of a parent drug. The functional group may be cleaved in vivo to release the active parent drug compound. Pro-drugs include, for example, compounds in which a group that may be cleaved in vivo is attached to a hydroxy, amino or carboxyl group in the active drug.
Examples of pro-drugs include, but are not limited to esters (e.g., acetate, methyl, ethyl, formate, and benzoate derivatives), carbamates, amides and ethers. Methods for synthesizing such pro-drugs are known to those of skill in the art. Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject's disease or condition, will have a desired therapeutic effect, e.g., an amount which will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition, including pain. More specific embodiments are included in the Pharmaceutical Preparations and Methods of Administration section below.
A Novel Nitration Pathway Provides a New Target for Pain and Inflammation Treatment Numerous studies have shown that glutamate-mediated activation of the N-methyl-D- aspartate (NMDA) receptor is fundamental in the development of hyperalgesia. Overt activation of this receptor release superoxide (SO) and nitric oxide that in turn form peroxynitrite (PN). An important mechanism of SO and PN in disease states is nitration of tyrosine residues in various receptors , enzymes, co-factors, cytokines and other compounds in a subject. However, to date, nitration of receptors, enzymes and other compounds has not been implicated in the development and maintenance of pain or inflammation. The present invention provides novel compounds, compositions, and combinations of nitration inhibitors and pain inhibitors. In addition, methods of administering these compounds, compositions and combinations for the treatment of pain and nitration are aspects of the present invention. Thus, Applicants invention provides SO-driven and PN-driven nitration as a target for the development and maintenance of central sensitization and hyperalgesia provides a novel route of administration for the treatment of pain and inflammation in subjects suffering from acute or chronic pain. Inflammation is also a key component of pain. Superoxide (SO) is a potent pro-inflammatory mediator in, e.g., the release of various cytokines, recruitment of neutrophils at sites of inflammation, poly-ADP-ribose-polymerase (PARP) activation, formation of peroxynitrite (PN) and nitration with deactivation of key proteins including endogenous superoxide dismutase, the enzyme that keeps SO at low levels in vivo. MnSOD, GS and NMDA receptor subunits are examples of key targets in this pathway. Accordingly, the discovery of the novel mechanism and pathway for SO in the nociceptive signaling cascade provides a new mode of action for SO dismutase mimetics ("SODm") and PN decomposition catalysts, either alone or in combination with other compositions known to inhibit nitration, pain, and/or inflammation, as therapeutics in the management of pain and inflammation. Such compositions which inhibit nitration of SO- mediated PN formation and subsequent nitration are key in the development and maintenance of central sensitization, and when combined with other nitration, pain and/or inflammation inhibitors provides new analgesics and analgesic combinations which overcome the problems associated with NSAID or opioid administration while at the same time enhance the effect of the such known inhibitors to extend the potency and efficacy of the nitration, pain or inflammation inhibitor. U.S. Patent Nos. 5,610,293, 5,637,578, 5,874,421 , 5,976,498, 6,084,093, 6,180,620, 6,204,259, 6,214,817, 6,395,725, and 6,525,041, each of which is incorporated herein by reference in its entirety, describe particular SODm and methods of making the same which are useful in the present invention. Accordingly, this invention provides a combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof. Nitration inhibitors are commercially available and methods for determining whether a compound or composition is an active nitration inhibitor are provided in the Examples below. Several classes of potential nitration inhibitors exist, but preferably, the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof. In accordance with a further aspect of the invention, the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof. Preferred examples of such NOS inhibitors will be recognized by those of skill in the art. Preferably, the NOS inhibitor is selected from the group consisting of NG- monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof; the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3- aminomethyl-benzylacetamidine), and any combination thereof; and the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6- nitroindazole, Ng-propyl-L-arginine, and any combination thereof. Those of skill in the art will also recognize several NO scavengers. Thus, in accordance with a further aspect of the invention, the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof. In accordance with yet another aspect of the invention, the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof. In one alternative, the pain inhibitor is an opioid. Those of skill in the art will recognize many opioids, both synthetic and organic. Preferably, the opioid is morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof. In a second alternative, the pain inhibitor is an NSAID. Those of skill in the art will recognize many classes and individual examples of NSAIDs. Preferably, the NSAID is one of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®). In several examples, methods are provided for determining whether such combinations of nitration inhibitors and pain inhibitors may be found to be either additive or synergistic. Such combinations are preferred in the present invention. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof. Preferably, the SO generating system inhibitor is a xanthine oxidase inhibitor. Those of skill in the art will recognize other SO generating system inhibitors, which are meant to be within the scope of the present invention. As described in U.S. Patent Nos. 5,610,293, 5,637,578, 5,874,421 , 5,976,498, 6,084,093, 6,180,620, 6,204,259, 6,214,817, 6,395,725, and 6,525,041 , the complexed metal of the macrocycle provides for catalytic dismutation (or "scavenging") of superoxide. The Examples below and the methods provided in these patents provide those of skill in the art the ability to determine which SO scavengers are preferred. Thus, a broad class of such SO scavengers are withing the scope of the present invention. In one alternative, the SO scavenger is represented by the following formula:
Figure imgf000044_0001
wherein (a) one or more of R, R', Ri, R'1 f R2, R'2, R3, R'3, R4, R'4, R5, R's, Re, R'e, R7, R'7,
Rs, R's, Rg, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -ORι0, -NRioRn, -COR10, -CO2Rι0, -CONR10R11, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -SO2Rι0,
-SO2NRi0Rii, -N(OR10)(Rn), -P(O)(OR10)(ORn), -P(O)(OR10)(Rn) and -OP(O)(OR10)(ORn) substituents (in which case Rio and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the α-carbon of σ-amino acids; and (b) optionally, one or more of R or R' and Ri or R'ι, R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R'5, R6 or R'6 and R7 or R'7, or R8 or R'8 and R9 or R'g together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (c) optionally, one or more of Ri or R'ι and R2 or R'2, R3 or R'3 and R or R4, R5 or R'5 and R6 or R'6) R7 or R'7 and R8 or R'8, R9 or R'9 and R or R', together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (d) optionally, one or more of R and R', R-t and R , R2 and R'2, R3 and R'3, R4 and R'4, R5 and R'5, R6 and R'6, R7 and R'7, R8 and R'8, and R9 and R'9> together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R^, R , R2, R'2> R3, R'3, R4, R' , R5, R's, Re, R'e, R , RV, Rs, R's, Rg, and R'9 together with a different one of R, R', R^ R'ι, R2, R'2, R3, R'3, R , R'4, R5, R'5, R6, R'e, R7, R'7, R8, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (f) combinations of any of (a) through (e) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R', R1 f R , R2, R'2, R3,
R'3, R4, R4, s, s, Re, ε, Rr, RV> s, Rs, Rg, and R9; and M is a transition metal; and n is an integer from 0 to 3. In a second alternative, the SO scavenger is represented by the following formula:
Figure imgf000046_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, R1 t R2, R'2, R3, R's, R , R'4, R5, R's, Re, R'e, R7,
R'7, R8, R's, Rg, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -ORι0, -NRι0Rn, -COR10, -CO2Rι0, -CONR10Rn, -O-(-(CH2)a-O)b-Rι0> -SRi0, -SOR10, -S02Rιo, -SO2NR10Rn, -N(ORι0)(Rn), -P(O)(ORι0)(OR1ι), -P(O)(ORι0)(Rn) and -OP(0)(ORio)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the α-carbon of σ-amino acids; and (c) optionally, one or more of R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R'5, R6 or R'e and R7 or R'7, or R8 or R'8 and R9 or R'g together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (d) optionally, one or more of R3 or R'3 and R or R' , R5 or R'5 and R6 or R'6) R7 or R'7 and R8 or R'8 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (e) optionally, one or more of R2 and R'2, R3 and R'3, R and R'4, R5 and R'5, R6 and R'6, R7 and R'7, R8 and R'8, and R9 and R'g, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (f) optionally, one or more of R, R1 ; R2, R'2, R3, R'3, R4, R'4, R5, R's, Re, R'e, R7, R'7, Rs, R's, Rg, and R'g together with a different one of R, R,, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'e, R7, R'7, Rs, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j -- R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (g) optionally, one or more of R, R.,, R2, R'2, R3, R'3, R4, R'4, R5, R's, Re, R'e, R7,
R'7> R8, R's, R9, and R'9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K ~ S -- (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) combinations of any of (a) through (g) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, Ri, R2, R'2, R3, R'3, R , R'4, Rs, R's, Re, R'β, R7, R'7, Rs, R's, Rg, and R'9; and M is a transition metal; and n is an integer from 0 to 3. In a third alternative, the SO scavenger is represented by the following formula:
Figure imgf000049_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, in the case which W is an aromatic heterocycle, the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) two sets of two adjacent carbon atoms of the macrocycle independently form substituted or unsubstituted, saturated or unsaturated, heterocycles U and V having 3 to 20 carbon atoms; and (c) optionally, one or more of R, Ri, R2, R'2, R3, R4, R5, R'5, R6, R'e, R7, Rs, Rg, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NRioRn, -COR10, -CO2R10, -CONRι0Rn, -O-(-(CH2)a-O)b-Rι0, -SR10, -SOR10, -SO2Rι0, -SO2NR10Rn, -N(OR10)(Rn), -P(O)(OR10)(ORn), -P(O)(ORι0)(Rn) and -OP(O)(ORι0)(ORn) substituents (in which case Rio and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the α-carbon of σ-amino acids; and (d) optionally, one or more of R2 or R'2 and R3, R4 and R5 or R'5, R6 or R'6 and R7, or R8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (e) optionally, R5 or R'5 and R6 or R'6 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (f) optionally, one or more of R2 and R'2, R5 and R'5, R6 and R'6, and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (g) optionally, one or more of R, R1 ( R2, R'2, R3, R4, R5, R'5, Re, R'e, R7, Rs, Rg, and R'g together with a different one of R, Ri, R2, R'2, R3, R , R5, R's, Re, R'e, R7, Rs, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, Ri, R2, R'2, R3, R4, R5, R's, Re, R'e, R7, Rs, Rg, and R'9 may be bound to an atom of at least one of heterocycle U, V or W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)κ - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (i) combinations of any of (a) through (h) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R.,, R2, R'2, R3, R4, R5, R's, Re, R'e, Ry, Rs, Rg, and R'9; and M is a transition metal; and n is an integer from 0 to 3. Preferably, U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms. More preferably, U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms. Still more preferably, U and V are trans-cyclohexanyl fused rings. In alternatives referring to a W moiety, W is preferably a substituted pyridino moiety. More preferably, U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety. In accordance with yet another aspect of the invention, transition metal M is preferably selected from the group consisting of Mn, Fe, Ni and V. M40403, which is within the scope of the description above, is described in Fig. 1 and in the Examples below. Those of skill in the art will also recognize that other nitration inhibitors are part of the present invention. Thus, in accordance with another aspect of the invention, the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Preferably, the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
Figure imgf000052_0001
wherein R3, R6, Rg and R12 are independently selected from the group consisting of H, alkyl, alkenyl, CH2, COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
Phenyl
Pyridyl
N-Alkylpyridyl
Figure imgf000052_0002
which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein R^ R2, R , R5, R7, R8, Rio, or Ru are independently selected from the group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, N02, hydroxyalkyl, and S03H; and further wherein Ri and R2 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins; with the proviso that when the X and Y containing complex has a net positive charge then Z is a counter ion which is independently selected from the group consisting of X and Y, or when the X and Y containing complex has net negative charge then Z is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V; and n is an integer from 0 to 4. Structure II
Figure imgf000054_0001
wherein R' is CH or N; R-i, R2, R3, R4> Rs, Re, R7, Ra, Rg, R10, Rft, R-ι2, R13, R-ι4, R15, and R16 are independently selected from the group consisting of H, S03H, COOH, N02, NH2, and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
Figure imgf000055_0001
A wherein Ri, R5, Rg, and Rι3 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R , R4, Re, R'β, R8, R's, Rio, R'10, Rι2, R'12, ι4, R'ι4, R16, and R'16 are independently selected from the group consisting of H and alkyl; R3, R7, R11, and R15 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000055_0002
B wherein R1 f R5, R8, and R12 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, R6, R'e, R7, Rg, R'g, R11, R'n, R13, R'ιs, and R14 are independently selected from the group consisting of H and alkyl; R3 and R10 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000056_0001
wherein Ri, R4, R8, and Rι2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R'5, R7, R9, R'g, Ru, R'n, R13, R'13 and Rι4 are independently selected from the group consisting of H and alkyl; R10 is H or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000056_0002
D wherein Ri, R4, R7 and R10 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R'5, R6, R8, R's, Rg, Ru, R'n and Rι2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000057_0001
wherein R1 f R4, R8 and Ru are independently selected from the group consisting of a direct bond and CH2; R2, R3, R'3, R5, R's, R7, R'7, Rg, R10, R'10, R12, R'12 and R 3 are independently selected from the group consisting of H and alkyl; R6 is hydrogen or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000057_0002
wherein R-i, R , R7 and R10 are independently selected from the group consisting of H and alkyl; R2, R3, R'3, R5, R'δ, Re, Rs, Rg, R'g, Ru, R'11 and R12 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000058_0001
wherein Ri, R3, R4, and R6 are independently selected from the group consisting of H and alkyl; R2 and R5 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above;
Figure imgf000058_0002
H wherein Ri, R2, R3, and R4 are independently selected from the group consisting of
H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
Figure imgf000059_0001
wherein Ri, R'ι, R2, R'2, R3, R'3, R4, R'4, R5, R'5, Re, R'e, R7 and R'7 are independently selected from the group consisting of H, alkyl, alkoxy, N02, aryl, halogen, NH2 and S03H, further wherein R6, R'e, R7 and R'7 together with one other of R6, R'6, R7 and R'7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above. Those of skill in the art will also recognize that several combinations, more particularly laid out, are part of the present invention. Thus, in accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with another aspect of the invention, the combination provides a pain inhibitor which is an opioid and a nitration inhibitor selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Those of skill in the art will also recognize that known compounds which have been previously described to affect nitration are also encompassed by the present invention to affect pain. Thus, in accordance with yet another aspect of the invention, a combination of compounds is provided for treating, preventing, reversing or inhibiting pain, wherein at least one compoound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof. Preferably, the NMDA receptor antagonist is MK801. This invention also provides a method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination comprises a nitration inhibitor. Preferably, the nitration inhibitor is described as above. In accordance with another aspect of the invention, this method also provides a nitration inhibitor admixed with a pain inhibitor. As mentioned above and in the Examples below, preferred mixtures are additive or synergistic when used in the present methods to treat pain. In one alternative, the pain inhibitor is an opioid. Preferably, the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof. In a second alternative, the pain inhibitor is an NSAID. Preferably, the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®). In accordance with a further aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof. Such compositions are described above and may be used in the present method. In accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Such compositions are described above and may be used in the present method. Those of skill in the art will recognize multiple combinations may be used in the present methods. Thus, in accordance with yet another aspect of the invention, the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. In accordance with another aspect of the invention, the pain inhibitor is an opioid or NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof. Again, known nitration inhibitors may be used in the present invention to treat pain. Thus, in accordance with a further aspect of the invention, a method is provided for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite- driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof. Preferably, the NMDA receptor antagonist is MK801. Those of skill in the art will also recognize that the Examples provided below may be performed using other mammals, including humans. The data in the Examples provided below are expected by those of skill in the art to correlate with data obtained from humans and other mammals. Thus, in accordance with yet another aspect of the invention, the subject may be a mammal. Preferably, the mammal is a human.
The Effect of Nitration in Pain Production and Management Numerous studies have shown that glutamate-mediated activation of the N-methyl-D- aspartate (NMDA) receptor, a subclass of excitatory amino acid receptor, is fundamental in the development of hyperalgesic responses (defined as augmented pain intensity in response to painful stimuli) associated with pain of various etiologies. Thus, intrathecal injection of NMDA results in hyperalgesic responses in experimental models of acute inflammatory and neuropathic pain that are blocked by intrathecal delivery of NMDA-receptor antagonists. NMDA-receptor activation releases superoxide the formation of which has been closely associated with numerous glutamate-mediated actions including neurotoxicity and stroke. Once superoxide is released, a key mechanism that is critical in maintaining and in sustaining high levels of superoxide at sites of action is nitration of endogenous manganese superoxide dismutase (MnSOD; SOD2), the enzyme that normally keeps superoxide under tight control. Nitration of the endogenous SOD enzyme is carried out by peroxynitrite, the reaction product of superoxide and nitric oxide. NMDA-receptor activation favors the accumulation of peroxynitrite by forming superoxide and nitric oxide simultaneously. It is now established by the present invention that nitration destroys the enzymatic activity of MnSOD, an event favoring the accumulation of superoxide (the enzyme can no longer dismute and hence remove superoxide) and superoxide-driven damage. Without being bound to a particular theory, the associations of many glutamate actions with the formation of superoxide suggest that superoxide is an important mediator of NMDA-mediated hyperalgesia and subsequently that nitration of the endogenous MnSOD enzyme is an important pathway by which superoxide modulates the hyperalgesic response to NMDA. M40403 (Fig. 1A), a synthetic and selective superoxide dismutase mimetic. M40403 is a stable, low molecular weight, manganese-containing, non-peptidic molecule possessing the function and catalytic rate of native superoxide dismutase enzymes, but with the advantage of being a much smaller molecule (MW 483 vs MW 30,000 for the mimetic and native enzyme, respectively) was used to elucidate this novel pathway. Furthermore, M40403 is stable in vivo, penetrates cells readily, has wide tissue distribution in rats, is excreted intact with no detectable dissociation and is recovered in urine and feces intact. M40404 (Fig. 1 B) the inactive congener of M40403 has no SOD catalytic activity and is pharmacologically inactive in vivo in models of inflammation. Hence, the present invention provides that superoxide formation is a critical step in NMDA-mediated hyperalgesia and that superoxide-driven nitration of endogenous MnSOD is a critical pathway in the response. Such superoxide-driven nitration when targeted by the compounds of the present invention may be used to treat, prevent, reverse and inhibit such hyperalgesia.
The Effect of Nitration in Inflammation Production and Management Without being bound by a particular theory, it is generally understood that under physiological circumstances the biological reactivity of superoxide (SO) is kept under the control of superoxide dismutase (SOD) enzymes. These include: the Mn enzyme in mitochondria (SOD2) and the Cu/Zn enzyme present in the cytosol (SOD1 ) or extracellular surfaces (SOD3). In acute and chronic inflammation, superoxide is produced at a rate that overwhelms the capacity of the endogenous SOD enzyme defense system to remove it. Such an imbalance results in superoxide-mediated injury as shown in numerous animal models of disease. Important pro-inflammatory roles for superoxide include: endothelial cell damage and increased microvascular permeability, release of cytokines, recruitment of neutrophils at sites of inflammation, single-strand DNA damage and poly-ADP-ribose- polymerase (PARP) activation. Furthermore, the present invention provides that superoxide rapidly combines with nitric oxide removing an important homeostatic signaling molecule and at the same time forming peroxynitrite (PN), a potent cytotoxic and pro-inflammatory agent. Without being bound to a particular theory, it is believed that a detrimental action of peroxynitrite is the nitration and oxidation of proteins such as MnSOD resulting in loss of function, which subsequently elevates the levels of superoxide. Thus, the invention provides that removal of superoxide by synthetic superoxide dismutase mimetics such as Mn(lll)tetrakis (4-benzoic acid) porphyrin (MnTBAP), M40403 and peroxynitrite by decomposition catalysts such as 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron III (FeTMPS) are shown to be affective in treating, preventing, reversing or inhibiting pain, and in particular acute inflammatory pain.
Pharmaceutical Preparations and Methods of Administration The identified compositions treat, prevent, reverse, inhibit, control, or at least partially arrest or partially prevent, pain associated with superoxide-induced nitration of enzymes, receptors or other compounds in a subject and can be administered to a subject at therapeutically effective doses for the inhibition, prevention, prophylaxis or therapy for such pain caused by superoxide-induced nitration. The compositions of the present invention comprise a therapeutically effective dosage of a nitration inhibitor which includes, e.g., nitration inhibitors, alone or in combination with another or a known nitration, pain or inflammation inhibitors. This term which includes therapeutically, inhibitory, preventive and prophylactically effective doses of the compositions of the present invention and is more particularly defined below. Without being bound to any particular theory, applicants surmise that these pharmaceutical compositions prevent pain when administered to a subject suffering from a related condition by inhibiting superoxide-induced nitration in a subject. The subject is preferably an animal, including, but not limited to, mammals, reptiles and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.
Therapeutically Effective Dosage Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compositions to the site affected by the disease or disorder in order to minimize potential damage to unaffected cells and reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans and other mammals. The dosage of such compositions lies preferably within a range of circulating plasma or other bodily fluid concentrations that include the ED50with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans and other mammals. Composition levels in plasma may be measured, for example, by high performance liquid chromatography. The amount of a composition that may be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of a composition contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art. The dosage regime for treating a disease or condition with the compositions and/or composition combinations of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profiles of the particular composition employed, whether a composition delivery system is utilized and whether the composition is administered as a pro-drug or part of a drug combination. Thus, the dosage regime actually employed may vary widely from subject to subject.
Formulations and Use The compositions of the present invention may be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and ophthalmic routes. The individual compositions may also be administered in combination with one or more additional compositions of the present invention and/or together with other biologically active or biologically inert agents ("composition combinations"). Such biologically active or inert agents may be in fluid or mechanical communication with the composition(s) or attached to the composition(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces. It is preferred that administration is localized in a subject, but administration may also be systemic. The compositions or composition combinations may be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and/or excipients. Thus, the compositions and their pharmaceutically acceptable salts and solvates may be specifically formulated for administration, e.g., by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. The composition or composition combinations may take the form of charged, neutral and/or other pharmaceutically acceptable salt forms. Examples of pharmaceutically acceptable carriers include, but are not limited to, those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000). The compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, controlled- or sustained-release formulations and the like. Such compositions will contain a therapeutically effective amount of the composition, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
Parenteral Administration The composition or composition combination may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or the like. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For example, a parenteral preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as a solution in 1 ,3- butanediol). Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the parenteral preparation. Alternatively, the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. For example, a composition suitable for parenteral administration may comprise a sterile isotonic saline solution containing between 0.1 percent and 90 percent weight per volume of the composition or composition combination. By way of example, a solution may contain from about 5 percent to about 20 percent, more preferably from about 5 percent to about 17 percent, more preferably from about 8 to about 14 percent, and still more preferably about 10 percent of the composition. The solution or powder preparation may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Other methods of parenteral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Oral Administration For oral administration, the composition or composition combination may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants and disintegrants: A. Binding Agents Binding agents include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre- gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Suitable forms of microcrystalline cellulose include, for example, the materials sold as AVICEL-PH-101 , AVICEL-PH- 103 and AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pennsylvania, USA). An exemplary suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581 by FMC Corporation.
B. Fillers Fillers include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), lactose, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
C. Lubricants Lubricants include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium Iauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Maryland, USA), a coagulated aerosol of synthetic silica (marketed by Deaussa Co. of Piano, Texas, USA), CAB-O- SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Massachusetts, USA), and mixtures thereof.
D. Disintegrants Disintegrants include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
The tablets or capsules may optionally be coated by methods well known in the art. If binders and/or fillers are used with the compositions of the invention, they are typically formulated as about 50 to about 99 weight percent of the composition. Preferably, about 0.5 to about 15 weight percent of disintegrant, preferably about 1 to about 5 weight percent of disintegrant, may be used in the composition. A lubricant may optionally be added, typically in an amount of less than about 1 weight percent of the composition. Techniques and pharmaceutically acceptable additives for making solid oral dosage forms are described in Marshall, SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker and Rhodes, Eds.), 7:359-427 (1979). Other less typical formulations are known in the art. Liquid preparations for oral administration may take the form of solutions, syrups or suspensions. Alternatively, the liquid preparations may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, perfuming and sweetening agents as appropriate. Preparations for oral administration may also be formulated to achieve controlled release of the composition. Oral formulations preferably contain 10% to 95% composition. In addition, the compositions of the present invention may be formulated for buccal administration in the form of tablets or lozenges formulated in a conventional manner. Other methods of oral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Controlled-Release Administration Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the composition or composition combination and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the composition, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of a composition that produces the desired therapeutic effect, and gradually and continually release other amounts of the composition to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of a composition in the body, the composition could be released from the dosage form at a rate that will replace the amount of composition being metabolized and/or excreted from the body. The controlled- release of a composition may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules. Controlled-release systems may include, for example, an infusion pump which may be used to administer the composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, the composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the composition over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and blends thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage. The compositions of the invention may be administered by other controlled-release means or delivery devices that are well known to those of ordinary skill in the art. These include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination of any of the above to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Inhalation Administration The composition or composition combination may also be administered directly to the lung by inhalation. For administration by inhalation, a composition may be conveniently delivered to the lung by a number of different devices. For example, a Metered Dose Inhaler ("MDI") which utilizes canisters that contain a suitable low boiling point propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas may be used to deliver a composition directly to the lung. MDI devices are available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura. Alternatively, a Dry Powder Inhaler (DPI) device may be used to administer a composition to the lung. DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient. DPI devices are also well known in the art and may be purchased from a number of vendors which include, for example, Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. A popular variation is the multiple dose DPI ("MDDPI") system, which allows for the delivery of more than one therapeutic dose. MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering
Plough, SkyePharma and Vectura. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch for these systems. Another type of device that may be used to deliver a composition to the lung is a liquid spray device supplied, for example, by Aradigm Corporation. Liquid spray systems use extremely small nozzle holes to aerosolize liquid composition formulations that may then be directly inhaled into the lung. For example, a nebulizer device may be used to deliver a composition to the lung. Nebulizers create aerosols from liquid composition formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled. Examples of nebulizers include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd., Aventis and Batelle Pulmonary Therapeutics. In another example, an electrohydrodynamic ("EHD") aerosol device may be used to deliver a composition to the lung. EHD aerosol devices use electrical energy to aerosolize liquid composition solutions or suspensions. The electrochemical properties of the composition formulation are important parameters to optimize when delivering this composition to the lung with an EHD aerosol device. Such optimization is routinely performed by one of skill in the art. Other methods of intra-pulmonary delivery of compositions will be known to the skilled artisan and are within the scope of the invention. Liquid composition formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include the composition with a pharmaceutically acceptable carrier. In one exemplary embodiment, the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally, another material may be added to alter the aerosol properties of the solution or suspension of the composition. For example, this material may be a liquid such as an alcohol, glycol, polyglycol or a fatty acid. Other methods of formulating liquid composition solutions or suspensions suitable for use in aerosol devices are known to those of skill in the art.
Depot Administration The composition or composition combination may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Accordingly, the compositions may be formulated with suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt. Other methods of depot delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Topical Administration For topical application, the composition or composition combination may be combined with a carrier so that an effective dosage is delivered, based on the desired activity ranging from an effective dosage, for example, of 1.0 μM to 1.0 mM. In one embodiment, a topical composition is applied to the skin. The carrier may be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick. A topical formulation may also consist of a therapeutically effective amount of the composition in an ophthalmologically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products. Any of these compositions may also include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically or pharmaceutically effective agents which do not exert a detrimental effect on the composition. Other methods of topical delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Suppository Administration The composition or composition combination may also be formulated in rectal formulations such as suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides and binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Suppositories may contain the composition in the range of 0.5% to 10% by weight. Other methods of suppository delivery of compositions will be known to the skilled artisan and are within the scope of the invention.
Other Systems of Administration Various other delivery systems are known in the art and can be used to administer the compositions of the invention. Moreover, these and other delivery systems may be combined and/or modified to optimize the administration of the compositions of the present invention. Exemplary formulations using the compositions of the present invention are described below in Example 4 (the compositions of the present invention are indicated as the active ingredient, but those of skill in the art will recognize that pro-drugs and composition combinations are also meant to be encompassed by this term).
Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. EXAMPLES Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following specific examples are offered by way of illustration and not by way of limiting the remaining disclosure.
Example 1 - Treatments for Pain Male Sprague-Dawley rats (250-260 g, Raleigh, NC, USA) were purchased with intrathecally-implanted cannulas (32 gauge, polyurethane) and used for these studies. All animals were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee and in accordance with NIH guidelines on laboratory animal welfare. The active SOD mimetic M40403 and its inactive congener, M40404 were synthesized as previously described (Salvemini D, Wang Z-Q, Zweier JL, Samouilov A, Macarthur H, Misko TP, Currie MG, Cuzzocrea S, Sikorski JA and Riley DP (1999). "A non- peptidyl mimic of superoxide dismutase with therapeutic activity in rats." Science 286: 304- 306.). Unless specified, all materials were purchased from Sigma-Aldrich Corp. (St Louis, MO).
A. Stopped-flow kinetic analysis for the dismutation of superoxide The self-dismutation and catalyzed decay of superoxide in aqueous buffers were measured by stopped-flow spectrophotometer, as previously described (Riley DP, Rivers WJ and Weiss RH (1991 ) "Stopped-Flow Kinetic Analysis for Monitoring Superoxide Decay in Aqueous Systems." Analytical Biochemistry 196: 344-49). Briefly, a dimethyl sulfoxide solution of superoxide (ca. 2mM, 7.5 μL) was mixed in less then 2 milliseconds with an hepes buffer (60 mM, pH 7.8, 150 μL) and the subsequent (self-dismutation) decay of superoxide was monitored spectrophotometrically at 245 nm. Superoxide dismutase mimetic M40403 (5 μM) or its inactive congener M40404 (5 μM) were added to the aqueous buffer and the reaction monitored spectrophotometrically at 245 nm. The kinetics of the two decay curves were processed by computer, and the analysis mathematically identifies the former as uncatalyzed self-dismutation and the latter as catalyzed dismutation.
β. Stopped-flow kinetic analysis for the decomposition of peroxynitrite A solution of sodium peroxynitrite (ca. 2 mM in 27 mM NaOH, 7.5 μL, from Cayman
Chemical Co., quantitated using a 1 cm quartz cell with a DU-650 UV Spectrophotometer from Beckman Coulter) was mixed in less than 2 milliseconds with phosphate buffer (100 mM, pH 7.6, 150 μL) at room temperature (22 °C), and the absorbance (λ=302 nm) of the mixture was recorded from 2 milliseconds to 16 seconds. The experiment was repeated with superoxide dismutase mimetic M40403 or FeTMPyP (Iron (III), [[4, 4', 4", 4"'-(21 H, 23H- porphine-5, 10, 15, 20-tetrayl)tetrakis[1-methylpyridiniumato]](2-)-N21 , N22, N23, N24]- pentachloride salt) (Cayman Chemicals, Co) present in the buffer at 5 μM.
C. NMDA-induced hyperalgesia Hyperalgesic responses to heat were determined by the Hargreaves method
(Hargreaves K, Dubner R, Brown F, Flores C and Joris J (1988). "A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia." Pain 32: 77-88.) as described above. Animals received intrathecal injection of NMDA (2nmol in 10 μl, n=6; this dose was taken from previous studies) or vehicle (saline), followed by a 10 μl flush. The thermal stimulus was applied separately to the right and left hind paw and paw withdrawal latencies were assessed immediately before and subsequently at 10, 20 and 40 minutes post-NMDA. M40403 (10 mg/kg, n=6) and M40404 (10 mg/kg, n=3) or vehicle (26 mM sodium bicarbonate buffer, pH=8.1-8.3) were given subcutaneously (0.2 ml) 30 minutes before the intrathecal administration of NMDA. The dose used was selected from previous studies where it was shown that at this dose and in vivo superoxide-mediated damage in rats was completely blocked by M40403 and not by M40404 (Coppey LJ, Gellett JS, Davidson EP, Dunlap JA, Lund DD, Salvemini D and Yorek MA (2001). "Effect ofM40403 treatment of diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular function ofepineurial arterioles of the sciatic nerve." Br. J. Pharmacol. 134: 21-9). Results are expressed as paw-withdrawal latency (sec); a decrease in paw withdrawal latency relative to baseline is indicative of hyperalgesia.
D. Tail flick and hot plate tests Nociceptive testing was performed by placing the distal third of the tail of a rat in a water bath maintained at 52 °C (tail flick) or by placing rats on a metal surface maintained at 52 °C. The latency to withdrawal (tail flick) or to jumping/licking of a hind paw (hot plate) was measured once before (control) and at selected time intervals after drug injection. A cut-off latency of 10 seconds is employed to prevent tissue injury. Groups of at least 6 rats each received a subcutaneous injection of M40403 (10 mg/kg) or vehicle (26 mM sodium bicarbonate buffer, pH = 8.1-8.3) and testing was performed at 15, 30, 45 and 60 minutes after injection of the drug. Determination of anti-nociception was assessed between 7:00 and 10:00 AM.
E. Spinal cord protein determination Lumbar spinal cord enlargements (L4/L6) were homogenized with lysis buffer with a 1 :3 w/v ratio. The lysis buffer (20 mM Tris-base, 150 mM NaCl, 10 % glycerol, 0.1% Triton- X-100, 1% Chaps, 2 mM EGTA) contained 1% protease inhibitor cocktail (v/v). Solubilized extracts were sonicated (5 min.) using a Sonicator (Fisher Scientific) and after 10 min of incubation in ice the lysates were centrifuged (12500 g, 30 min at 4 °C). These supematants were stored immediately at -80 °C. Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce).
F. Immunoprecipitation assay 4 mg of the solubilized proteins in 500 μl lysate buffer were incubated with 10 μg of agarose-conjugated anti-nitrotyrosine antibody (Upstate Biotechnology) for 2 h at room temperature (RT). Agarose beads were collected by centrifugation (1 min at 12000 x g at 4 °C) and washed in PBS (pH 7.4) three times. The mixture of the beads-antibody and binding proteins were resuspended in 50 μl of sample buffer [2x, 0.5 M Tris-HCl, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue], heated to 95 °C (5 min). The samples were then loaded in 12% SDS-PAGE mini-gels (Bio-Rad). Rat brain treated with peroxynitrite (Upstate Biotechnology) was used as positive control.
G. Western blot analysis After separation by SDS/PAGE, proteins were transferred electrophoretically (200 V, 1.5 hr) to nitrocellulose membranes (Bio-Rad). Membranes were blocked (1 hr, RT) with 1% Bovine Serum Albumin (BSA)/ 0.1% Thimerosal in 50 mM Tris-HCl, (pH 7.4) /150 mM NaCI/0.01 % Tween 20 (TBS/T). For detection of Mn-SOD, blots were incubated with rabbit polyclonal anti-Mn-SOD (2 hr, RT, 1 :1000 dilution; Upstate Biotechnology). After washing with TBS/T, the membranes were incubated with anti-rabbit horseradish peroxidase- conjugated secondary antibody (1 :10,000 dilution; Amersham) and the specific complex was detected by an enhanced chemiluminescence detection system (ECL, Amersham).
H. Determination of MnSOD activity in the spinal cord Animals were anesthetized and killed by decapitation and exanguined, the vertebral column was opened and spinal cord from L4-L6 removed, cut in slices and immediately frozen in N2 and stored at -80 °C for subsequent determination of SOD activity. Tissue samples were homogenized with 10 mM phosphate buffered saline (pH 7.4) in a Polytron homogenizer and then sonicated on ice for 1 min. (20 s, 3 times). The sonicated samples were subsequently centrifuged at 1 ,100 g for 10 min. SOD activity was measured in the supematants as described (Beauchamp C and Fridovich I (1971). "Superoxide dismutase: improved assays and an assay applicable to acrylamide gels." Analytical Biochemistry 44: 276-287; and Nishida S, Teramoto K, Kimoto-Kinoshita S, Tohda Y, Nakajima S, Tomura TT and Irimajiri K (2002). "Changes of Cu,Zn-superoxide Dimutase Activity of Guinea Pig Lung in Experimental Asthma." Free Radical Reasearch 36: 601-606) with some modifications. In brief, a competitive inhibition assay was performed which used xanthine-xanthine oxidase- generated superoxide to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt. The reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 μM; Sigma-Aldrich Corp., Milan, Italy), xanthine and xanthine- oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany). The rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm. The amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity. Cu,Zn-SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre-incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein
/. Rotarod Test Rats were placed on a rotating rod (7 cm diameter) turning at 10 RPM. The animals were exposed to the rotarod for one session of 180 seconds each day for 3 days to adapt the rats to the apparatus. The rats were then injected subcutaneously with the superoxide dismutase mimetic (10-100 mg/kg, n = 6) and tested again on the rotarod for periods of 180 sec at selected time intervals.
J. Statistical analysis Results are given as mean ± s.e.m. Unless specified, statistical analysis was performed using ANOVA followed by post hoc Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
Example 1 Results
Chemical characterization of superoxide removal by M40403 The kinetics of the reaction between superoxide with the superoxide dismutase catalyst, M40403, is shown in the Fig. 2. Utilizing a stopped-flow instrument with 2 ms mixing time the decomposition of superoxide is followed at 245 nm in the absence or presence of M40403. The self-decay of superoxide in buffer follows typical second order reaction rate (Fig. 2, top line). However in the presence of catalytic amount (i.e.; [SO]/[catalysts] ~ 20) of M40403 (bottom line), a very rapid and complete removal of superoxide is accomplished. The decomposition of superoxide in the presence of the SOD mimetic appears to follow first order kinetics as seen in Fig. 2. The kinetics of the superoxide decay are not altered by the presence of a catalytically inactive Mn(ll) complex with no SOD activity (M40404, Fig. 2, middle line). As shown in Fig. 3, M40403 had no effect on the rate of peroxynitrite decomposition when present in the same relative catalytic amount (both lines interspersed among overlapping top lines), whereas the same catalytic quantity of the synthetic iron porphyrin complex FeTMPyP effected complete peroxynitrite decay in less than one second under the conditions of these measurements (Fig. 3 bottom line).
Superoxide plays a role in NMDA mediated nociception Intrathecal injection of NMDA in rats (2 nmol) produces a time-dependent development of thermal hyperalgesia (Fig. 4). Prophylactic administration of M40403 (10 mg/kg, given subcutaneously 30 minutes before NMDA) reduces NMDA-mediated thermal hyperalgesia (Fig. 4) whereas the catalytically inactive SODm, M40404 has no effect (n=3, not shown). These results support the notion that superoxide is important in NMDA- mediated hyperalgesia. In the absence of hyperalgesia (i.e., in naϊve animals), M40403 (10 mg/kg, n=6, not shown) was found to have no analgesic or hyperalgesic activity as measured in the hot plate/tail flick assays suggesting that the production of superoxide does not occur during synaptic activation in the absence of a hyperalgesic state.
Superoxide plays a role in NMDA mediated nociception by nitrating spinal MnSOD Having shown that superoxide mediates NMDA-mediated hyperalgesia we next addressed what pathway was used by this free radical to modulate the response. As indicated earlier, an important pathway through which superoxide modulates disease is by forming peroxynitrite, which in turn nitrates and deactivates endogenous MnSOD, an event critical in maintaining and in sustaining high levels of superoxide. We hypothesized that this pathway operates in central sensitization (as stated earlier NMDA-receptor activation releases both nitric oxide and superoxide, providing the source of peroxynitrite). At time of near-to-maximal hyperalgesia (20 minutes, Fig. 5, panel B), MnSOD was found to be nitrated in the spinal cord as shown by immunoprecipitation (Fig. 5, panel A). Furthermore, once nitrated, endogenous MnSOD is deactivated losing its ability to remove superoxide (Fig. 5, panel C). Pre-treatment of rats with M40403 (10 mg/kg, given subcutaneously 30 minutes before NMDA), prevented MnSOD nitration (Fig. 5, panel A) and inhibited hyperalgesia (Fig. 5, panel B).
Lack of effect ofM40403 in the rotarod test M40403 when given subcutaneously at doses as high as 100 mg/kg, demonstrated no evidence of loss of motor activity as determined by the rotarod test (n=6, not shown).
Example 1 Discussion Without being bound to a particular theory, excitatory amino acids, including glutamate, are believed to be key mediators involved in central sensitization. In particular, stimulation of the nociceptors induces the firing of the C-fibers (unmyelinated slowly- conducting afferent) leading to the release of excitatory amino acids, including glutamate, that in turn act on the NMDA-receptor in the spinal cord. Then, NMDA receptor activation is believed to induce the spinal cord neurons to become more sensitive to all of their inputs, resulting in central sensitization seen in acute inflammatory pain and neuropathic pain. Similar mechanisms may be an important part of the development of opioid tolerance that is also blocked by NMDA-receptor antagonists. There is general agreement that inhibiting the actions of glutamate by inhibiting the receptor (for example NMDA-receptor antagonists) or by inhibiting downstream pathways that are engaged upon receptor activation (for example the nitric oxide pathway) is a viable strategy for drug development in the pain area. The present invention provides, for the first time, that superoxide is an important mediator of spinal (i.e., central nervous system or "CNS")hyperalgesia. Thus, synthetic SOD mimetics and other compounds and compositions are useful agents for the management of pain of various etiologies. The present invention reveals a mechanism by which superoxide modulates NMDA- mediated hyperalgesia (Figs. 6 and 12). This mechanism suggests that upon NMDA activation, there are increases intracellular calcium influx which leads to the production of superoxide which in turn contributes to the state of hyperalgesia by nitrating and subsequently deactivating spinal endogenous manganese SOD, the enzyme that normally keeps superoxide under well-controlled condition. MnSOD nitration is important in the hyperalgesic response since it-helps to maintain high levels of superoxide that in turn maintains nociceptive signaling. Indeed, at time of maximal hyperlagesia MnSOD was found to be nitrated and deactivated at the level of the spinal cord and removal of superoxide with M40403 (but not with its inactive congener, M40404) attenuated MnSOD nitration and hyperalgesia. In addition, the anti-hyperalgesic effects of the superoxide dismutase mimetic were observed at doses devoid of behavioral side effects. For example, when M40403 was given subcutaneously at a dose at least 10-fold higher than the one shown to block NMDA- mediated hyperalgesia, there was no evidence of loss of motor activity in the rotarod test in rats. The present invention also provides that nitration of this enzyme is also important in central sensitization in addition to being associated closely to those disease states driven by overt production of superoxide (as the enzyme is now no longer able to dismute superoxide), for example, ischemia and reperfusion, organ transplantation, shock and inflammation. . The present invention provides at least two pathways which can lead to protein nitration. One uses peroxynitrite, (the reaction product of superoxide and nitric oxide) and the other uses hydrogen peroxide and myeloperoxidase. Without being bound to a particular theory, the involvement of these pathways in a particular setting can be dissected pharmacologically by the use of agents which remove superoxide or nitric oxide. Indeed, removal of superoxide (with for instance M40403 or superoxide dismutase) or nitric oxide (with for instance a nitric oxide synthase inhibitor, e.g., iNOS, eNOS or nNOS inhibitors) inhibits as shown by numerous investigators the formation of peroxynitrite and peroxynitrite- mediated protein nitration. The finding that in our study MnSOD nitration was blocked by M40403 supports the role of peroxynitrite and not hydrogen peroxide/myeloperoxidase in this process. To date nitric oxide has been implicated as a central mediator of NMDA-driven central sensitization. NMDA-mediated hyperalgesia is blocked by nitric oxide synthase inhibitors. Based on the data provided in the present invention, it is believed that inhibition of NMDA-mediated hyperalgesia by various nitric oxide synthase inhibitors is in fact due to inhibition of peroxynitrite-mediated MnSOD nitration. Thus, formation of peroxynitrite and subsequent nitration of spinal MnSOD is a novel pathway of NMDA-mediated hyperalgesia and a new target which can be affected by the compounds and compositions of the present invention. The broader implication of our findings is that superoxide may contribute to various forms of centrally induced hyperalgesia that are driven by NMDA-receptor activation. Thus, superoxide is a therapeutic target for the development of novel analgesics and analgesic combinations (e.g., additive and synergistic combinations).
Example 2 - Treatments of Pain Due to Inflammation Male Sprague-Dawley rats (175-200 g, Harlan, Indianapolis, IN, USA) and male CD- 1 mice (28-35 g, Charles River, USA) were used for these studies. All animals were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee and in accordance with NIH guidelines on laboratory animal welfare. The SOD mimetics, peroxynitrite decomposition catalysts and peroxynitrite used in this study were synthesized as previously described. Unless specified, all materials were purchased from Sigma-Aldrich Corp. (St Louis, MO). The SOD mimetics were dissolved in the following vehicle: 26 mM sodium bicarbonate buffered saline, pH=8.1-8.3. All other drugs were dissolved in saline.
A. Tail flick and hot plate tests Nociceptive testing was performed by placing the distal third of the tail of a rat in a water bath maintained at 52 °C (tail flick) or by placing rats on a metal surface maintained at 52 °C. The latency to withdrawal (tail flick) or to jumping/licking of a hindpaw (hot plate) was measured once before (control) and at selected time intervals after drug injection. A cut-off latency of 10 seconds is employed to prevent tissue injury. Groups of at least 6 rats each received a subcutaneous injection of SODm (10 mg/kg) or an equivalent volume of vehicle and testing was performed 15, 30, 45 and 60 minutes after injection of the drug or vehicle. Determination of anti-nociception was assessed between 7:00 and 10:00 AM.
S. Formalin-induced nociception Mice were allowed to feed ad libitum and housed 5-7 per cage in a temperature- controlled room with a 12-hr light-dark cycle. Determination of anti-nociception was assessed between 7:00 and 10:00 AM. Groups consisted of 8 mice, and each animal was used for one experimental condition. The anti-nociceptive effects of the SODm were tested in the formalin-induced hindpaw licking procedure as described by Hunskaar & Hole. Formalin (20 μl of a 1% stock solution) was injected into the subplantar region of one hindpaw and the duration of paw-licking (an index of nociception) was monitored over the period of 0-5 (early phase) and 10-30 minutes (late phase) thereafter. M40403 (0.3-10 mg/kg, n=8) or an equivalent volume of vehicle was given subcutaneously 40 minutes before formalin. Results are expressed as Paw-licking time (sec).
C. Superoxide and peroxynitrite induced hyperalgesia Fifty microliters of superoxide (100 nmol) [or of a decomposed solution of superoxide solution (using a dimethyl sulfoxide solution of potassium superoxide at 2mM exposed to air for 30 minutes)] or peroxynitrite (PN, 1 μM) were given by intraplantar injection.
Decomposed peroxynitrite was obtained by exposing the stock solution of peroxynitrite to air for at least 30 minutes. Drugs or vehicle were given intravenously (2ml/kg) 15 minutes before injection of superoxide and peroxynitrite. Hyperalgesic responses to heat were measured at time of maximal hyperalgesic effect. Time course studies revealed that maximal hyperalgesia was reached at 20 and 30 minutes respectively, for superoxide and peroxynitrite. D. Carrageenan-induced edema and hyperalgesia Rats received a subplantar injection of carrageenan (0.1 ml of a 1% suspension in 0.85% saline) into the right hindpaw of lightly anesthetized rats [C02 (80%) 02 (20%)]. Drugs or vehicle were administered intravenously (2 ml/kg), subcutaneously (1 ml/kg) or intrathecally (in 10 μl) at 2 hours post-carrageenan injection (therapeutic treatment) and testing performed 0.25, 0.5, 0.75, 1, 2 and 3 hours after drug injection. In some experiments, drugs or vehicle were also given 30 minutes before carrageenan (prophylactic treatment) and testing performed every hour up to 5 hours post carrageenan. Changes in paw-volume were measured as previously described. Briefly, paw-volume was measured with a plethysmometer (Ugo-Basile, Varese, Italy) immediately prior to the injection of carrageenan and thereafter at hourly intervals for 5 hours. Edema was expressed as the increase in paw-volume (ml) after carrageenan injection relative to the pre-injection value for each animal. Results are expressed as Paw-volume change (ml).
E. Measurements of thermal hyperalgesia Hyperalgesic responses to heat were determined as described by Hargreaves (Id.) and a cut off latency of 20 sec was employed to prevent tissue damage in non-responsive animals. Rats were individually confined to plexiglass chambers. A mobile unit consisting of a high intensity projector bulb was positioned to deliver a thermal stimulus directly to an individual hindpaw from beneath the chamber. The withdrawal latency period of injected and contralateral paws was determined to the nearest 0.1 sec with an electronic clock circuit and thermocouple. If the animal failed to respond by 20 sec the test was terminated. Each point represents the delta change (sec) in withdrawal latency (withdrawal latency of controlateral minus withdrawal latency of injected paw) at each time point. Results are expressed as Paw-withdrawal latency changes (sec).
F. Determinations of cytokine levels in paw exudates Cytokines (TNF-α, IL-1 ?, IL-6) released in the paw exudates were measured by ELISA as described previously. Briefly, at 5 hours following the intraplantar injection of carrageenan, rats were sacrificed, and each paw was cut at the level of the calcaneus bone. Paws were gently centrifuged at 250 x g for 20 min in order to recover a sample of the edematous fluid. The volume of fluid recovered from each paw was measured. Cytokines were measured by ELISA (R&D systems, Minneapolis, MN) and results expressed in pg/paw, normalizing values to the amount of exudates recovered from each paw. All determinations were performed in duplicate. Previous studies indicated that maximal levels of these cytokines were seen at 5 hours post carrageenan. Therefore, animals were sacrificed at these time points.
G. Immunohistochemical localization of nitrotyrosine and poly-ADP-ribose (PAR) in the carrageenan-inflamed rat hindpaw Indirect imrnunofluorescence staining was performed on 7 μm thick sections of rat paw tissues embedded in Paraplast. After deparaffinization, endogenous peroxidase was quenched with 0.3% H202 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. Sections were incubated overnight with 1) anti-nitrotyrosine rabbit polyclonal antibody (1 :500 in PBS) and with anti- poly (ADP-Ribose) goat policlonal antibody rat (1:500 in PBS). Sections were washed with PBS, and incubated with secondary antibody (FITC-conjugated anti-rabbit and with TRIC- conjugated anti-goat; Jackson, West Grove, PA) for 2 h at RT. Sections were washed as before, mounted with 90% glycerol in PBS, and observed with a Nikon RCM8000 confocal microscope equipped with a 40X oil objective. In order to confirm that the immunoreaction for the nitrotyrosine was specific some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PAR, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections indicating that the immunoreaction was positive in all the experiments carried out.
H. Histological examination of the carrageenan-inflamed rat hindpaw For histopathological examination, biopsies of paws were taken 2 and 5 hours following the intraplantar injection of carrageenan. Tissue from the pads of the rats hindpaw was removed with a scalpel. Tissue samples were fixed in Dietric solution (14.25% ethanol, 1.85% formaldehyde, 1% acetic acid) for 1 week at room-temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical). Tissue sections (thickness 7 μm) were deparaffinized with xylene, stained with haematoxylin/eosin and studied using light microscopy (Dialux 22 Leitz). All the histological studies were performed in a blinded fashion.
/. Immunohistochemical detection of nitrated proteins in the spinal cord Central modulation of the nociceptive signal takes place at the lumbar tract of the spinal cord. At that level there is the first synapse where peripheral sensitization transmits the signal to the central nervous system. Paraffin-embedded, formaldehyde-fixed rat spinal cord sections (L4/L6) were de-paraffinized and re-hydrated by standard methods. Following re-hydration and immersion in PBS/0.3% Triton X100 (2 x 5min), sections were treated for microwave antigen retrieval (2 x 5min, 1mM EDTA, 4.5mM Tris, pH 8.0). Sections were blocked for 1 hr with 100% normal horse serum (Vector), followed by application of monoclonal anti-nitrotyrosine antibody (1 :100 in 100% normal horse serum) at 4 °C overnight in a moist chamber. Treatment with secondary antibody, A/B complex, and DAB were performed by the manufacturer's instructions (Vector ABC Elite Kit, Vector Laboratories).
J. Immunoprecipitation assay and Western blot analysis Lumbar spinal cord enlargements were homogenized in Lysis buffer (20 mM Tris- base, 150 mM NaCl, 10 % glycerol, 0.1% Triton-X-100, 1% Chaps, 2 mM EGTA, 1 % protease inhibitor cocktail) with 1 :3 w/v ratio. Solubilized extracts were sonicated (5 min.) using a Sonicator (Fisher Scientific) and after 10 min of incubation in ice the lysates were centrifuged (12500 g, 30 min at 4 °C). These lysate samples were stored at -80 °C immediately. Protein concentration was determined using the Bicinchoninic Acid (BCA) protein assay (Pierce). 4 mg of the solubilized proteins in 500 μl lysate buffer were incubated with 10 μg of agarose-conjugated anti-nitrotyrosine antibody (Upstate Biotechnology) for 2 h at room temperature (RT). Agarose beads were collected by centrifugation (1 min at 12000 x g at 4 °C) and washed in PBS (pH 7.4) three times. The mixture of the beads-antibody and binding proteins were resuspended in 25 μl of sample buffer (2x, 0.5 M Tris-HCl, (pH 6.8) 2.5% glycerol/0.5%SDS/200 mM 2- mercaptoethanol/0.001% bromophenol blue), heated to 95 °C (5 min). The samples (50 μl) were then loaded in 12% SDS-PAGE mini-gels (Bio-Rad). Membranes were blocked (1 hr, RT) with 1% Bovine Serum Albumin (BSA)/ 0.1% Thimerosal in 50 mM Tris-HCl, (pH 7.4)/150 mM NaCI/0.01 % Tween 20 (TBS/T). For detection of Mn-SOD, blots were incubated with rabbit polyclonal anti-Mn-SOD (2 hr, RT, 1 :1 ,000 dilution; Upstate Biotechnology). After washing with TBS/T, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 :10,000 dilution; Amersham) and the specific complex was detected by an enhanced chemiluminescence detection system (ECL, Amersham).
K. Determination of MnSOD activity in the spinal cord Animals were anesthetized and killed by decapitation and exanguined, the vertebral column was opened and spinal cord from L4-L6 removed, cut in slices and immediately frozen in N2 and stored at -80 °C for subsequent determination of SOD activity. Tissue samples were homogenized with 10 mM phosphate buffered saline (pH 7.4) in a Polytron homogenizer and then sonicated on ice for 1 min. (20 s, 3 times). The sonicated samples were subsequently centrifuged at 1 ,100 g for 10 min. SOD activity was measured in the supematants as described with some modifications. In brief, a competitive inhibition assay was performed which used xanthine-xanthine oxidase-generated SO to reduce nitroblue tetrazolium (NBT) to blue tetrazolium salt. The reaction was performed in sodium carbonate buffer (50 mM, pH 10.1) containing EDTA (0.1 mM), nitroblue tetrazolium (25 μM; Sigma, Milan, Italy), xanthine and xanthine-oxidase (0.1 mM and 2 nM respectively; Boehringer, Germany). The rate of NBT reduction was monitored spectrophotometrically (Perkin Elmer Lambda 5 Spectrophotometer, Milan, Italy) at 560 nm. The amount of protein required to inhibit the rate of NTB reduction by 50% was defined as one unit of enzyme activity. Cu,Zn- SOD activity was inhibited by performing the assay in the presence of 2 mM NaCN after pre- incubation for 30 min. Enzymatic activity was expressed in units per milligram of protein.
L. Rotarod Test Rats were placed on a rotating rod (7 cm diameter) turning at 10 rpm. The animals were exposed to the rotarod for one session of 180 seconds each day for 3 days to adapt the rats to the apparatus. The rats were then injected subcutaneously with SODm (100 mg/kg) and tested again on the rotarod for periods of approximately 180 sec at selected time intervals.
M. Lack of effect of M40403 in the rotarod test M40403 when given subcutaneously at doses as high as 100 mg/kg, demonstrated no evidence of loss of motor activity as determined by the rotarod test. Thus the time for the rats to remain on the rotarod (sec) was 182+7, 185+4, 176+5, 180+6 177+7 seconds when measured just before drug injection and subsequently at times 15, 20, 25 and 40 minutes • post drug injection (n=6).
N. Statistical analysis Results are shown as mean ± s.e.m. for n animals. Unless specified, statistical analysis was done using ANOVA followed by Student-Newman-Keuls test.
Example 2 Results Utilizing a well-established model of acute inflammatory hyperalgesia, (carrageenan- induced inflammation and hyperalgesia) the role of superoxide in nociception associated with acute inflammation was investigated. Hyperalgesic responses defined as augmented pain intensity in response to painful stimuli seen upon intraplantar injection of carrageenan involve central and peripheral sensitization. Intraplantar injection of carrageenan leads to a time dependent development of inflammation and hyperalgesia, which peaks within 2-3 hours and lasts for a subsequent 6-8 hours. Administration of M40403 (1-10 mg/kg, n=6) prior to carrageenan administration inhibited in a dose-dependent manner the development of edema and thermal hyperalgesia at all time points. Dose-dependent inhibition curves for inflammation and pain taken at 5 h after carrageenan are shown in Fig. 7a. Dose-dependent inhibition of edema and hyperalgesia were associated with dose-dependent inhibition of the known pro-inflammatory and pro-nociceptive cytokines, tumor necrosis factor-σ, interleukin- ■\β, and interleukin-6 (n=6, Figs. 7b-d). When tested at the same time point and at the highest dose used (10 mg/kg) the inactive SOD catalyst M40404 did not inhibit edema, thermal hyperalgesia (n=6, data not shown) or cytokine release (Figs. 7b-d). Thus, M40403 prevented carrageenan-induced edema and hyperalgesia, in part by inhibiting superoxide- driven cytokine production. The potential participation of peroxynitrite in superoxide-mediated nociception was evaluated initially by immunohistochemical detection of nitrated proteins and PARP activation and subsequently by evaluating whether peroxynitrite promotes hyperalgesia and whether it's removal inhibits acute inflammatory hyperalgesia. At the time of maximal inflammation and hyperalgesia (5 h), nitrated proteins are readily detected in the inflamed paw and are associated with PARP activation (Fig. 8a). The formation of nitrated proteins and PARP activation were blocked by M40403 (Fig. 8a). Furthermore, the degree of inflammation as determined histologically was reduced by M40403 (Fig. 8b). The role of superoxide in inducing a hyperalgesic response was further confirmed by showing that intraplantar injection of exogenous superoxide causes pain. Injection of potassium superoxide (100 nmol, n=6) but not decomposed superoxide (n=6) evoked an immediate hyperalgesic response (within 5 min) to noxious heat that peaked at approximately 20 min and then subsided (not shown). The development of thermal hyperalgesia seen after superoxide injection at time of peak hyperalgesia was inhibited by M40403 (3 mg/kg, n=6) (Fig. 9a) but not by the catalytically inactive superoxide dismutase mimetic M40404 (3 mg/kg, n=6) (Fig. 9a). Moreover, intraplantar injection of peroxynitrite (1 μM, n=6) but not of decomposed peroxynitrite (n=6) evoked a hyperalgesic response to noxious heat that peaked at approximately 30 min. The response was inhibited by prophylactic administration of the active (FeTMPS) but not inactive peroxynitrite decomposition catalyst (H2TMPS) (30 mg/kg, n=6, Fig. 9b). As expected, M40403 (3 mg/kg, n=6) did not inhibit the response to peroxynitrite (Fig. 9b). Taken together these results suggest that inhibition of the formation of peroxynitrite accounts at least in part for the beneficial anti-nociceptive action of M40403. These findings were further confirmed by the fact that prophylactic administration of FeTMPS (1-30 mg/kg, n=6) blocked in a dose- dependent manner carrageenan-induced hyperalgesia (Fig. 9c). On the other hand, the inactive peroxynitrite decomposition catalyst H2TMPS (30 mg/kg) did not inhibit pain (paw- withdrawal latency changes at 5 hours post carrageenan were 11±0.5 and 12±0.05 seconds for carrageenan and carrageenan+H2TMPS, respectively, n=6). The demonstration that FeTMPS (Fig. 9c) inhibited edema is consistent with previous findings. Release of superoxide in the spinal cord contributes to carrageenan-induced hyperalgesic responses. The central involvement of superoxide in the induction of hyperalgesic responses was defined by administering M40403 after the inflammatory response had developed. Results shown in Fig. 10a show that when given 2 h after carrageenan, M40403 (0.3-3 mg/kg) produced a time-related and dose-dependent inhibition of hyperalgesia, which was rapid in onset (70-80% inhibition within 15 minutes), maximal at 1 hour (IC50=2.6 mg/kg), and sustained for 3 hours (Fig. 10a). The slopes for the time- course of reversal of hyperalgesia and inflammation were dissociated. Thus, inhibition of hyperalgesia was rapid in onset, whereas inhibition of inflammation was gradual (Fig. 10b). Based on the observation that the onset of reversal of established hyperalgesia was quick in onset, it was hypothesized that once the hyperalgesic response had initiated, spinal release of superoxide maintains the nociceptive input. This hypothesis was tested by determining whether carrageenan-induced hyperalgesia was inhibited by intrathecal injection of M40403. When given at 2 h after carrageenan, intrathecal injection of M40403 (2 nmol, n=6) reversed hyperalgesia with a rapid onset of action (Fig. 10c) verifying our hypothesis. The anti- hyperalgesic effects of M40403 were not affected by naloxone (given at 1 mg/kg sc, 30 min prior to M40403, n=6; not shown) excluding potential involvement of an opiate pathway. Nitration of spinal endogenous manganese superoxide dismutase: an important event in maintaining hyperalgesic responses. Immunohistochemical evaluation of the spinal cords using affinity purified monoclonal anti-nitrotyrosine antibodies revealed the presence of nitrated proteins in motor neurons in the spinal cord layers (Fig. 11a). The staining for nitrated proteins was nearly undetectable in saline-treated animals and was significantly diminished upon M40403 treatment (Fig. 11a). Data in Fig. 11b reveal that the endogenous mitochondrial form of SOD, MnSOD, is nitrated at the time of near-to-maximal hyperalgesia (Fig. 11c). Once nitrated, endogenous MnSOD is deactivated losing its ability to remove superoxide (Fig. 11 of). Pre-treatment of rats with M40403 (10 mg/kg, given s.c 30 minutes before carrageenan, n=6), prevented MnSOD nitration (Fig. 11b) and inhibited hyperalgesia (Fig. 11 c). Lack of effect of M40403 on basal acute nociception in naive animals. In rats or mice, M40403 (up to 10 mg/k, n=6, not shown) has no analgesic activity as measured in the hot plate/tail flick assays suggesting that superoxide does not play a role in normal nociception. This concept was confirmed using the formalin test. Subplantar injection of formalin in mice results in an early response (Phase I) and a late response (Phase II, seen between 10-20 minutes post-formalin). The early response, which usually lasts <5 minutes, occurs a few seconds after the formalin injection and is characterized by intense licking or lifting of the injected paw. This phase is believed to represent a direct irritant effect of formalin on sensory C-fibers and is blocked by opioids but not by anti-inflammatory agents (22). M40403 when given forty minutes prior to formalin at doses failed to inhibit this phase (not shown). Phase I was as expected blocked by morphine but not by the non-steroidal anti-inflammatory drug indomethacin (both at 10 mg/kg, n= 8, not shown). On the other hand and as expected from the data gathered in this study, the second phase of the formalin response (known to involve central and peripheral sensitization associated with inflammation (28) is blocked in a dose-dependent manner by M40403 (0.3-10 mg/kg, n=8). Thus, paw licking time (sec) evoked by formalin in control mice (n=8) was reduced from 63±5 to 45±2, 15±3, 6±2 and 2±0 in the presence of 0.3, , 3 and 10 mg/kg M40403 respectively. Phase II was also blocked by morphine or indomethacin (10 mg/kg, n=8, not shown).
Example 2 Discussion The present invention provides that superoxide is a newly identified mediator of pain, providing an opportunity for novel pain management. The picture depicted in Fig. 12 summarizes the key findings of this investigation. It is well appreciated that during tissue injury and inflammation, hyperalgesia results from a persistent state of peripheral afferent sensitization that subsequently initiates spinal sensitization through the release of the excitatory amino acid, glutamate. The mechanism(s) are complex in nature and involve peripherally and spinally formed inflammatory mediators including peptides, prostanoids, nitric oxide, cytokines, excitatory amino acids (glutamate) as well as spinal cord glial cells activation. The present invention provides that superoxide is formed and plays a major role in the development of pain through direct peripheral sensitization, by promoting inflammation, and by favouring nitration of endogenous MnSOD in the spinal cord. Thus anti-nociceptive effect of M40403, and like compounds, includes overall inhibition of the effects of superoxide exerted peripherally and centrally. M40403 exerted a profound anti-inflammatory effect which included inhibition of edema, cytokine release, formation of peroxynitrite and PARP activation. Furthermore, it was noted that by inhibiting inflammation, M40403 inhibited hyperalgesia. A mechanism by which M40403 blocks cytokine release is by preventing the activation of REDOX sensitive transcription factors including NF-/cB and AP-1 by superoxide which in turn regulates the genes that encode (amongst numerous things) various pro-inflammatory and pronociceptive cytokines. Other than the well-recognized role of these cytokines in inflammation, the same inflammatory molecules play a role in pain as shown by their ability to directly induce hyperalgesia. Furthermore, when released from activated spinal cord glial cells and other inflammatory cells these cytokines participate in the induction of pain by peripheral nerve inflammation and trauma, subcutaneous inflammation and spinal dynorphin. Another important mechanism by which superoxide dismutase mimetics attenuate inflammation and hyperalgesia is by reducing peroxynitrite formation by removing superoxide before it can react with nitric oxide. This is important since the pro-inflammatory and cytotoxic effects of peroxynitrite are numerous. Removal of peroxynitrite by agents such as FeTMPS, a porphyrin-containing molecule that increases the rate of isomerization of peroxynitrite to nitrate is cytoprotective and anti-inflammatory. The present invention provides that a mechanism by which superoxide modulates hyperalgesia is through the formation of peroxynitrite. Thus, carrageenan-induced hyperalgesia was blocked by the peroxynitrite decomposition catalyst, FeTMPS. Furthermore, peroxynitrite directly evokes hyperalgesia and leads to nitration of proteins as detected in the periphery and in the spinal cord. Protein nitration was blocked by M40403. There are at least two pathways which can lead to protein nitration. One uses peroxynitrite, (the reaction product of superoxide and nitric oxide) the other uses hydrogen peroxide and myeloperoxidase. The involvement of these pathways in a particular setting can be dissected pharmacologically by the use of agents which remove superoxide or nitric oxide. Removal of superoxide (with for instance M40403 or superoxide dismutase) or of nitric oxide (with for instance a nitric oxide synthase inhibitor) inhibits as shown by numerous investigators the formation of peroxynitrite and indirectly peroxynitrite-mediated protein nitration. The finding that in our study nitration was blocked by M40403 support the role of peroxynitrite and not hydrogen peroxide/myeloperoxidase in this process. Superoxide and peroxynitrite have been shown to induce DNA single-strand damage, which has been associated with PARP activation resulting in the depletion of its substrate NAD+ in vitro and a reduction in the rate of glycolysis. As NAD+ functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD+ depletion leads to a rapid fall in intracellular ATP and, ultimately, cell injury. PARP activation has been implicated for instance in the development of hyperalgesia seen upon chronic use of opioids. Furthermore, substantial evidence provided by the utility of PARP inhibitors exists to support that PARP activation is important in inflammation. SOD mimetics of the M40403 class reduce PARP activation in models of acute and chronic inflammation supporting the role of superoxide in this pathway. Here we have found that PARP was in fact activated in the paw at time of maximal hyperalgesia and this was blocked by M40403 suggesting that the anti-hyperalgesic effect of M40403 is derived in part by the inhibition of superoxide driven PARP activation. Taken together our results have shown that by blocking inflammation, a key component of pain, M40403 attenuated hyperalgesia. Besides the peripheral role of superoxide, described above, the present invention provides that superoxide released centrally in response to carrageenan is a key event in the maintenance of nociception. Without being bound to a particular theory, it is believed that glutamate released at the level of the spinal cord and subsequent activation of the N-methyl- D-aspartate (NMDA) receptor, a subclass of excitatory amino acid receptor, is fundamental in the development of hyperalgesic responses associated with pain of various etiologies. NMDA-receptor activation releases superoxide and nitric oxide, which in turn interacts to form peroxynitrite which, as discussed, in turn nitrates and deactivates key enzymes including endogenous superoxide dismutase. Removal of superoxide by M40403 once the hyperalgesic response has been initiated, leads to a rapid reversal of hyperalgesia. Thus, once the hyperalgesic response had initiated, spinal release of superoxide maintains the nociceptive input. Indeed, intrathecal injection of M40403 led to a rapid reversal of hyperalgesia. As shown in several studies, an important mechanism in maintaining high levels of superoxide and in sustaining superoxide-driven pathological effects is nitration and deactivation of endogenous superoxide dismutase, the enzyme that normally lowers the levels of superoxide. Nitration of the enzyme is in fact closely linked to those disease states driven by over production of superoxide, for example, ischemia and reperfusion, organ transplantation, shock and inflammation. Thus, the present invention provides that nitration of MnSOD is associated with the development of hyperalgesia. At the time of near-to-maximal hyperalgesia MnSOD was found to be nitrated in the lumbar spinal cord. In addition, M40403 blocked MnSOD nitration and inhibiting the hyperalgesic response. Taken together these results support the concept that MnSOD nitration and as shown subsequent inactivation is a critical event in the hyperalgesic response possibly by allowing the levels of superoxide to remain elevated and in turn maintain nociceptive signaling. Overall, the present invention provides that superoxide is a newly identified mediator of pain (Fig. 12) and that its removal by low molecular weight synthetic enzymes of superoxide dismutase represents a therapeutic target for the development of novel non- narcotic analgesics including those compounds and compositions of the present invention.
Example 3 - Synergy The compositions can be provided in combination with sufficient amounts of known nitration, pain or inflammation inhibitors to elicit a synergistic response in a subject. Synergy may be measured by comparing the baseline pain inhibition by the rat model above and comparing composition combinations. For example, a protocol to determine synergistic combinations includes: The protocol uses male Sprague-Dawley rats and all drugs were dissolved in 26 mM NaHC03 buffer (0.218 g NaHC03 in 100 ml dH20; pH = 8.1 to 8.3) and injections were given subcutaneously (hereinafter "s.c"). When drug combinations were employed, each drug was injected separately, but concurrently. Drugs employed morphine sulfate and SC- 72325A, an enantiomer of M40403. In some studies ketorolac was also used and was given by s.c. injection. Thermal hyperalgesia and antinociception were assessed in the testing of SC- 72325A for treatment of pain. Thermal hyperalgesia was determined by the Hargreaves method (Id.). A radiant heat source was focused onto the plantar surface of the affected paw of nerve-injured or carageenan-injected rats. When the animal withdrew its paw, a motion sensor halted the stimulus and timer. A maximal cut-off of 40 seconds was utilized to prevent tissue damage. Paw withdrawal latencies were thus determined to the nearest 0.1 seconds. Reversal of thermal hyperalgesia was indicated by a return of the paw withdrawal latencies to the pre-tremeant baseline latencies (i.e., 21 seconds). Antinociception was indicated by a significant (p ≤ 0.05) increase in paw withdrawal latency above this baseline. Data were converted to % antihyperalgesia or % antinociception by the formula: 100 x (test latency - baseline)/(cut-off baseline) where cut-off was 21 seconds for determining antihyperalgesia and 40 seconds for determining antinociception. Dose response curves were generated for each drug and drug combination for data obtained at the time of peak effect, which was consistently at the 30 minute time point. Studies employing combinations of drugs were analyzed for additive or synergistic interactions by isobolographic analysis as described by Tallarida (Tallarida et al., Life Sciences, 45:947-61 , 1987) and employed by other (Ossipov et al., J. Pharmacol. Exp. Ther., 255:1107-1116, 1990; Porreca et al., Euro. J. Pharm., 179: 463-468, 1990) by means of a customized Visual Basic computer program (Ossipov, personal communication). Log dose-response curves for each component administered alone were established and the A50 (95% CL.) were calculated. Using these methods, the amount of synergy of a combination of compounds or compositions can be determined. The preferred combinations of the .present invention treat pain using a smaller dose of an analgesic, such as an NSAID or opioid, when compared to administering the analgesic alone. In other words, a preferred combination will result, for example, in the same amount of pain relief after administering 50 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 500 mg of an NSAID or opioid alone or 500 mg of a synthetic superoxide dismutase catalyst alone. Conversely, the preferred combinations of the present invention treat pain to a greater extent when compared to treating pain with an analgesic alone or a synthetic superoxide dismutase catalyst alone. In other words, a preferred combination will result, for example, in an equivalent amount of pain relief after administering 500 mg of an NSAID or opioid in combination with 50 mg of a synthetic superoxide dismutase catalyst as would normally result from administering 1 ,000 mg of the NSAID or opioid or 1 ,000 mg of a synthetic superoxide dismutase catalyst alone. Thus, preferred combinations result in additive or synergistic antinociceptive effects allowing an NSAID or opioid to be administered in a dosage that is at least 50% less than the same NSAID or opioid administered alone. More preferably, the NSAID or opioid combination may be administered in a dosage that is at least 25% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. Still more preferably, the NSAID or opioid may be administered in a dosage that is at least 10% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. And still more preferably, the NSAID or opioid may be administered in a dosage that is at least 1% less than the same NSAID or opioid administered alone to achieve said therapeutic effect. The A50 for the log dose-response curve of a drug mixture at a fixed ratio was calculated in terms of "total dose" administered. For a given drug combination a theoretical A50 exists such that A50 add = A50 drugι x (pi + Rp2) where R is the potency ratio of drug 1 to drug 2, pi is the proportion of drug 1 in the mixture and p2 is the proportion of drug 2.
Variances and 95% CL. for the theoretical additive A50 are derived from the variances of each drug administered alone. A t-test is employed to compare the theoretical additive A50 and 95% CL. to that obtained for the mixture. A significantly ((p <0.05); t-test) lower experimental value compared to theoretical value denotes a synergistic interaction. See Table 1 below.
Example 4 - SC-72325A Treats Pain Analgesic effects provided by subcutaneous injection of SC-72325A was studied by formalin-induced hind paw licking response. Male CD-1 mice (Charles River, 28-35 gm) were allowed to feed ad libitum. Mice were housed 5-7 per cage in a temperature controlled room with a twelve hour light-dark cycle. Determination of antinociception was assessed between 7:00 and 10:00 AM. Groups consisted of 7-14 mice, and each animal was used for one experimental condition. The antinociceptive effects of SC-72325A were tested in the formalin-induced hind paw licking procedure (Hunskaar et al., Pain, 30: 103-114, 1987). Mice were injected with by sub-plantar administration with formalin (20 μg of a 1% stock solution) and the duration of paw licking was monitored in the periods of 5-10 minutes (Phase I) and 15-30 minutes (Phase II) thereafter. SC-72325A (10 mg/kg) was given s.c. 10 minutes prior to formalin. At 10 mg/kg, the s.c. injection of SC-72325A had a small inhibitory effect on phase 1 of the response but nearly completely abolished Phase II of the response.
Example 5 - Antihyperalgesia and Antinociception Synergy using SC-72325A and Morphine Combination Carrageenan-induced inflammation is a well characterized and commonly employed model of peripheral inflammation. This procedure reliably produces a marked inflammatory response within 3 hours of injection which is indicated by swelling of the hind paw, edema, rubor and hyperalgesia and allodynia. Peripheral inflammation was induced in the hind paw of male Sprague-Dawley rats by injecting 0.1 ml of a 2% ^-carrageenan suspension into the subplanar surface of the hind paw of lightly ether-anesthetized rats. Testing was performed 15, 30, 45, 60, 120 and 180 minutes after drug injections. The s.c. injection of SC-72325A produced time-related and dose-dependant antihyperalgesia over the dose range of 0.3 to 30 mg/kg. Similarly, morphine also produced time-related and dose-dependent antihyperalgesia and antinociception over the dose range of 0.03 to 10 mg/kg. The 1 :1 combination of SC-72325A with morphine produced antihyperalgesia activities at much lower doses than either drug alone. Isobolographic analyses revealed that the combination of SC-72325A with morphine resulted in a definitive synergistic interaction against hyperalgesia; the A50 values with confidence intervals are presented in Table 1 , below. The antihyperalgesic A50 value for the 1:1 combination of SC-72325A plus morphine was 0.046 mg/kg, s.c, which was significantly (p <0.05) less than the calculated theoretical A50 value for the combination if the activity was additive. See Table 1. SC-72325A also exhibited a slight antinociceptive effect. TABLE 1 Antihyperalgesia Aso (mg/kg, s.c.) SC-72325A 1.34 Morphine 0.22 Morphine + SC-72325A 0.046 Theoretical Additive Curve 0.380
Example 6 - Formulations of Compositions The following formulations are provided as examples of composition preparations according to the present invention:
Formulation 1 Hard gelatin capsules are prepared using the following ingredients:
TABLE 2 Ingredients (mg/capsule) Active Ingredient 250.0 Starch 305.0 Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin capsules in 560 mg quantities.
Formulation 2 A tablet formula is prepared using the following ingredients:
TABLE 3 Ingredients (mg/tablet) Active Ingredient 250.0 Cellulose, microcrystalline 400.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0
The components are blended and compressed to form tablets, each weighing 665 mg.
Formulation 3 A dry powder inhaler formulation is prepared containing the following components:
Ingredients Weight % Active ingredient 5 Lactose 95
The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
Formulation 4 Tablets, each containing 60 mg of active ingredient, are prepared as follows: TABLE 5 Ingredients milligrams Active ingredient 60.0 Starch 45.0 Microcrystalline cellulose 35.0 Polyvinylpyrrolidone (as 10% solution in water) 4.0 Sodium carboxymethyl starch 4.5 Magnesium stearate 0.5 Talc 1.0 Total 150.0
The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a 16 mesh U.S. sieve. The granules as produced are dried at 50-60 °C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
Formulation 5 Capsules, each containing 80 mg of active ingredient are made as follows:
1 ΓABLE 6 Ingredients milligrams Active ingredient 80.0 Starch 109.0 Magnesium stearate 1.0 Total 190.0
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 190 mg quantities.
Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows:
TABLE 7 Ingredients milligrams Active Ingredient 225 Saturated fatty acid glycerides to 2000
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5.0 ml dose are made as follows:
TABLE 8 Ingredients Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor q.v. Color q.v. Purified water to 5.0 ml
The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
Formulation 8 Capsules, each containing 150 mg of active ingredient, are made as follows:
TABLE 9 Ingredients milligrams Active ingredient 150.0 Starch 407.0 Magnesium stearate 3.0 Total 560.0
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 560 mg quantities.
Other Embodiments The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.
References Cited All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention. Such references include: Carolina Muscolia, Vincenzo Mollaceb, James Wheatleya, Emanuela Masinic, Michael Ndengelea, Zhi-Qiang Wanga and Daniela Salvemini, "Superoxide-mediated nitration of spinal manganese superoxide dismutase: a novel pathway in N-methyl- aspartate-mediated hyperalgesia." Pain, Volume 111 , Issues 1-2, September 2004, Pages 96-103 (Available online 28 July 2004), incorporated herein by reference in its entirety.

Claims

CLAIMS What is claimed is: 1. A combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor optionally admixed with a pharmaceutically acceptable salt or carrier.
2. A combination according to claim 1, wherein the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
3. A combination according to claim 2, wherein the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
4. A combination according to claim 3, wherein the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-
NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof.
5. A combination according to claim 3, wherein the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-omithine (L-NIL), aminoguanidine, 1400W (N-3-aminomethyl-benzylacetamidine), and any combination thereof.
6. A combination according to claim 3, wherein the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng-propyl-L-arginine, and any combination thereof.
7. A combination according to claim 2, wherein the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
8. A combination according to claim 7, wherein the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said combination optionally admixed with a pharmaceutically acceptable salt or carrier.
9. A combination according to claim 8, wherein the pain inhibitor is an opioid.
10. A combination according to claim 9, wherein the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
11. A method according to claim 8, wherein the pain inhibitor is an NSAID.
12. A method according to claim 11 , wherein the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
13. A combination according to claim 1 , wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
14. A combination according to claim 13, wherein the SO generating system inhibitor is a xanthine oxidase inhibitor.
15. A combination according to claim 13, wherein the SO scavenger is represented by the following formula:
Figure imgf000096_0001
wherein (a) one or more of R, R\ R1 f R'ι, R2, R'2) R3, R'3, R4, R'4, R5, R'5, Re, R'e, R , R'7.
Re, R'e, Rg, and R'g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR 0, -NR10Rn, -COR10, -C02Rιo, -CONR10Rn, -O-(-(CH2)a-O)b-Rι0, -SR10, -SOR10, -SO2Rι0, -SO2NRi0Rii, -N(ORio)(Rn), -P(O)(ORι0)(ORn), -P(O)(ORι0)(Rn) and -OP(O)(ORι0)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the α-carbon of σ-amino acids; and (b) optionally, one or more of R or R' and Ri or R'1 ? R2 or R'2 and R3 or R'3, R4 or R4 and R5 or R'5, R6 or R'6 and R7 or R'7, or R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (c) optionally, one or more of Ri or R'ι and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8, Rg or R'9 and R or R', together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (d) optionally, one or more of R and R', Ri and R'ι, R2 and R'2, R3 and R'3, R4 and R' , R5 and R'5, R6 and R'6, R and R'7, R8 and R'8, and Rg and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R^ R'ι, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'e, R7, R'7, R8, R's, Rg, and R'9 together with a different one of R, R', R1 f R'1 f R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'e, R7, R'7, Rs, R's, R9, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (f) combinations of any of (a) through (e) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R', R-i, R'ι, R2, R'2, R3,
Rs, R4> R4, Rs, δ, Re, Re, Ry, R7, Re, Rs, Rg, and Rg; and M is a transition metal; and n is an integer from 0 to 3.
16. A combination according to claim 13, wherein the SO scavenger is represented by the following formula:
Figure imgf000099_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, Ri, R2, R'2, R3, R'3, R , R' , R5, R's, Re, R'e, R7, R'7, R8, R's, R9, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10Rn, -COR10, -CO2R10, -CONR10Rn, -O-(-(CH2)a-O)b-R10, -SR10, -SOR10, -S02Rιo, -SO2NRi0Rii, -N(OR10)(Rn), -P(O)(OR10)(OR11), -P(O)(ORι0)(Rn) and -OP(0)(ORio)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of σ-amino acids; and (c) optionally, one or more of R2 or R'2 and R3 or R'3, R4 or R 4 and R5 or R'5, R6 or R'e and R7 or R'7, or R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (d) optionally, one or more of R3 or R'3 and R or R'4, R5 or R'5 and R6 or R'e, R7 or R'7 and R8 or R'8 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (e) optionally, one or more of R2 and R'2, R3 and R'3, R4 and R'4, R5 and R'5, R6 and R'e, R7 and R'7l R8 and R'8, and Rg and R'9) together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (f) optionally, one or more of R, R^ R2, R'2, R3, R'3, R4, R'4, R5, R'5, Re, R'e, R7, RV, Rs, R's, Rg, and R'9 together with a different one of R, R1 f R2, R'2, R3, R's, R4, R'4, R5, R'δ, Re, R'e, R7, R'7, Rs, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (g) optionally, one or more of R, Ri, R2, R'2, R3, R'3, R4, R'4, R5, R'5, Re, R'e, R7, R'7, R8, R's, R9, and R'9 may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) combinations of any of (a) through (g) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, ■ pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R1 ( R2, R'2, R3, R'3, R4, R4, R5, R5, Re, R'β, R7, R'7, Re 1 Rs, Rg, and R9; and M is a transition metal; and n is an integer from 0 to 3.
17. A combination according to claim 13, wherein the SO scavenger is represented by the following formula:
Figure imgf000101_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, in the case which W is an aromatic heterocycle, the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) two sets of two adjacent carbon atoms of the macrocycle independently form substituted or unsubstituted, saturated or unsaturated, heterocycles U and V having 3 to 20 carbon atoms; and (c) optionally, one or more of R, Ri, R2, R'2, R3, R4, R5, R'5) R6, R'6, R7, R8, Rg, and R'g are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NRIQRH, -COR10, -CO2R10, -CONR10Rn, -O-(-(CH2)a-O)fa-R10, -SRι0, -SOR10, -S02Rιo,
-SO2NR10Rn, -N(OR10)(Rn), -P(O)(ORι0)(ORn), -P(O)(OR10)(Rn) and -OP(O)(ORι0)(ORn) substituents (in which case Rι0 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the αr-carbon of σ-amino acids; and (d) optionally, one or more of R2 or R'2 and R3, R4 and R5 or R'5, R6 or R'6 and R7, or R8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (e) optionally, R5 or R'5 and R6 or R'e together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (f) optionally, one or more of R2 and R'2, R5 and R'5, R6 and R'6, and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (g) optionally, one or more of R, R.,, R2, R'2, R3, R4, Rs, R'5, R6, R'6> R7, R8, Rg, and R'9 together with a different one of R, R1 f R2, R2, R3> R4, R5, R's, Re, R'e, R7, Rs, Rg, and R'g which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)κ - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, Ri, R2, R'2, R3, R , R5, R's, Re, R'e, R7, Rs, Rg, and R'g may be bound to an atom of at least one of heterocycle U, V or W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (i) combinations of any of (a) through (h) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R-i, R2, R'2, R3, R4, R5, R's, R6, R'e, R7, Rs, Rg, and R'9; and M is a transition metal; and n is an integer from 0 to 3.
18. A combination according to claim 17, wherein U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms.
19. A combination according to claim 18, wherein U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms.
20. A combination according to claim 19, wherein U and V are trans-cyclohexanyl fused rings.
21. A combination according to claims 16 and 17, wherein W is a substituted pyridino moiety.
22. A combination according to claim 19, wherein U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
23. A combination according to claims 15, 16 or 17, wherein transition metal M is selected from the group consisting of Mn, Fe, Ni and V.
24. A combination according to claim 1 , wherein the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
25. A combination according to claim 24, wherein the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure
Figure imgf000105_0001
wherein R3, R6, R9 and Rι2 are independently selected from the group consisting of H, alkyl, alkenyl, CH2, COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
Phenyl
Pyridyl
N-Alkylpyridyl
Figure imgf000105_0002
which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and
NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and
NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein Ri, R2, R4, R5, R7, R8, R10, or Ru are independently selected from the group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, N02, hydroxyalkyl, and S03H; and further wherein Ri and R2 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atorhs of the macrocycle to which they are attached; X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperox f, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins; with the proviso that when the X and Y containing complex has a net positive charge then Z is a counter ion which is independently selected from the group consisting of X and Y, or when the X and Y containing complex has net negative charge then Z is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V; and n is an integer from 0 to 4. Structure II
Figure imgf000107_0001
wherein R' is CH or N; R-i, R2, R3, R4, Rδ, Re, R7, Rs, Rg, R10, Ru, R-ι2, Ris, Rι4, iδ, and R 6 are independently selected from the group consisting of H, S03H, COOH, N02, NH2, and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure 111
Figure imgf000107_0002
wherein R1; R5, Rg, and R13 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, Re, R'e, Rs, R's, R10, R'10, Rι2, R'12, Rι4, R'ι4, R16, and R'ι6 are independently selected from the group consisting of H and alkyl; R3, R7, R11, and R15 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000108_0001
B ' wherein Ri, R5, R8, and Rι2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, R6, R'6, R7, Rg, R'g, Ru, R'n, R13, R'ιs, and R14 are independently selected from the group consisting of H and alkyl; R3 and R10 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000108_0002
wherein R1 f R4, R8, and R 2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, R5, R'5l R7, Rg, R'g, Ru, R'n, R13, R'13 and R14 are independently selected from the group consisting of H and alkyl; R10 is H or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000109_0001
D wherein R1 ? R , R7 and Rι0 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, Rs, R's, Re, Rs, R's, Rg, R11, R'11 and Rι2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000109_0002
wherein Ri, R4, R8 and Ru are independently selected from the group consisting of a direct bond and CH2; R2, R3, R'3, Rδ, R'δ, R7, R'7, Rg, R10, R'10, R12, R'12 and Ri3 are independently selected from the group consisting of H and alkyl; R6 is hydrogen or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000110_0001
wherein Ri, R4, R7 and Rι0 are independently selected from the group consisting of H and alkyl; R2, R3, R'3, R5, R'5, R6, R8, R9, R'g, Ru, R'n and Ri2 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000110_0002
wherein R1 f R3, R , and R6 are independently selected from the group consisting of H and alkyl; R2 and R5 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above;
Figure imgf000111_0001
H wherein Ri, R2, R3, and R are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
Figure imgf000111_0002
wherein R1 ( R'.,, R2, R2, R3, R'3, R4, R'4, R5, R'5, R6, R'6, R7 and R'7 are independently selected from the group consisting of H, alkyl, alkoxy, N02, aryl, halogen, NH2 and S03H, further wherein R6, R'6, R7 and R'7 together with one other of R6, R'6, R7 and R'7 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above.
26. A combination according to claim 1, wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
27. A combination according to claim 8, wherein the pain inhibitor is an opioid and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
28. A combination of compounds for treating, preventing, reversing or inhibiting pain, wherein at least one compoound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
29. A combination according to claim 26, wherein the NMDA receptor antagonist is MK801.
30. A method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-d riven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination comprises a nitration inhibitor or pro-drug thereof, said nitration inhibitor and pro-drug optionally admixed with a pharmaceutically acceptable salt or carrier.
31. A method according to claim 30, wherein the nitration inhibitor is selected from the group consisting of a NOS inhibitor, NO generating system inhibitor, NO scavenger, and any combination thereof.
32. A method according to claim 31 , wherein the NOS inhibitor is selected from the group consisting of an eNOS inhibitor, iNOS inhibitor, nNOS inhibitor, and any combination thereof.
33. A method according to claim 32, wherein the eNOS inhibitor is selected from the group consisting of NG-monomethyl-L-arginine (LNMMA), NG nitro-L-arginine (L-NA), NG-nitro-L-arginine methyl ester (L-NAME), and any combination thereof.
34. A method according to claim 32, wherein the iNOS inhibitor is selected from the group consisting of L-N5-(1-iminoethyl)-ornithine (L-NIL), aminoguanidine, 1400W (N-3- aminomethyl-benzylacetamidine), and any combination thereof.
35. A method according to claim 32, wherein the nNOS inhibitor is selected from the group consisting of 7-nitroindazole (7-NI), 3-bromo-7-nitroindazole, 6-nitroindazole, Ng- propyl-L-arginine, and any combination thereof.
36. A method according to claim 31 , wherein the NO scavenger is selected from the group consisting of hemoglobin, PTIO, NPO, and any combination thereof.
37. A method according to claim 30, wherein the pain is initiated in a central sensitization pathway of the subject.
38. A method according to claim 30, wherein the nitration inhibitor is admixed with a pain inhibitor or pro-drug thereof, said combination optionally admixed with a pharmaceutically acceptable salt or carrier.
39. A method according to claim 38, wherein the pain inhibitor is an opioid.
40. A method according to claim 39, wherein the opioid is selected from the group consisting of morphine, oxycontin, oxycodone, codeine, fentanyl and any combination thereof.
41. A method according to claims 38, wherein the pain inhibitor is an NSAID.
42. A method according to claim 41 , wherein the NSAID is selected from the group consisting of ibuprofen (Advil®), naproxen (Aleve® or Naprosyn®), aspirin (Bayer®), celecoxib (Celebrex®), diclofenac (Voltaren®), etodolac (Lodine®), fenoprofen (Nalfon®), indomethacin (Indocin®), ketoprofen (Orudis® or Oruvail®), ketoralac (Toradol®), oxaprozin (Daypro®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), and rofecoxib (Vioxx®).
43. A method according to claim 30, wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, and any combination thereof.
Ill
44. A method according to claim 43, wherein the SO generating system inhibitor is a xanthine oxidase inhibitor.
45. A method according to claim 43, wherein the SO scavenger is represented by the following formula:
Figure imgf000114_0001
wherein (a) one or more of R, R', R1 f R'ι, R2, R'2, R3, R'3, R4, R'4, R5, R'δ, Re, R'e, R7, R'7, R8, R'8, R9, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10Rιι, -COR10, -CO2R10, -CONRi0Rii, -O-(-(CH2)a-O)b-Rι0, -SR10, -SOR10, -SO2Rι0, -SO2NRi0Rii, -N(ORιo)(Rιι), -P(O)(ORι0)(ORn), -P(0)(ORio)(Rn) and -OP(O)(ORι0)(ORn) substituents (in which case R10 and Rn are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of σ-amino acids; and (b) optionally, one or more of R or R' and Ri or R , R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R'5, R6 or R'6 and R7 or R'7, or R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (c) optionally, one or more of Ri or R'1 and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8, R9 or R'g and R or R', together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (d) optionally, one or more of R and R', R^ and R , R2 and R'2, R3 and R'3, R and R'4, Rs and R'5, R6 and R'6, R7 and R'7, R8 and R 8, and Rg and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (e) optionally, one or more of R, R', R1 f R'1 f R2, R'2, R3, R's, R4, R'4, R5, R's, Re, R'e, R7, R'7, R8, R's, Rg, and R'9 together with a different one of R, R', R1t R'1, R2, R'2, R3, R'3, R4, R' , Rs, R'δ, Re, R'e, R7, R'7, Rs, R's, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K -- S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (f) combinations of any of (a) through (e) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R', Ri, R'ι, R2, R'2, R3, R'3, R*, R'4, R5, R's, Re, R'e, R7, R'7, Rs, R's, Rg, and R'9; and M is a transition metal; and n is an integer from 0 to 3.
46. A method according to claim 43, wherein the SO scavenger is represented by the following formula:
Figure imgf000116_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) optionally, one or more of R, Ri, R2, R2, R3, R'3, R , R' , Rg, R'δ, Re, R'e, R7, R'7, R8, R's, R9, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -OR10, -NR10R11, -COR10, -CO2Rι0, -CONR10R11, -O-(-(CH2)a-O)b-R10> -SR10, -SORio, -S02Rιo, -SO2NRi0Rn, -N(OR10)(Rn), -P(O)(ORι0)(OR11), -P(O)(ORι0)(Rn) and -OP(O)(OR10)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of -amino acids; and (c) optionally, one or more of R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R'5, R6 or R'6 and R7 or R'7, or R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (d) optionally, one or more of R3 or R'3 and R4 or R' , R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (e) optionally, one or more of R2 and R'2, R3 and R'3, R4 and R'4, R5 and R'5, R6 and R'e, R7 and R'7, R8 and R'8, and R9 and R'9, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (f) optionally, one or more of R, Ri, R2, R'2, R3, R'3, R , R' , R5, R's, Re, R'e, R7, R'7, Rs, R's, R9, and R'g together with a different one of R, R.,, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'6> R7, R'7, R8, R'e, Rg, and R'g which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (g) optionally, one or more of R, R,, R2, R'2, R3, R'3, R4, R'4, R5, R's, Re, R'e, R7, R'7, R8, R's, R9, and R'g may be bound to an atom of heterocycle W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L -- wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) combinations of any of (a) through (g) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, R^ R2, R'2, R3, R'3> R4, R'4, Rs, R's, Re, R'β, R7, R7, Re, Re, Rg, and R'g; and M is a transition metal; and n is an integer from 0 to 3.
47. A method according to claim 43, wherein the SO scavenger is represented by the following formula:
Figure imgf000119_0001
wherein (a) a nitrogen of the macrocycle and two adjacent carbon atoms to which the nitrogen is attached independently form a substituted or unsubstituted, saturated or unsaturated, nitrogen-containing heterocycle W having 2 to 20 carbon atoms, in the case which W is an aromatic heterocycle, the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (b) two sets of two adjacent carbon atoms of the macrocycle independently form substituted or unsubstituted, saturated or unsaturated, heterocycles U and V having 3 to 20 carbon atoms; and (c) optionally, one or more of R, Ri, R2, R'2, R3, R4, R5, R'5, Re, R'e, R7, Rs, Rg, and R'9 are independently selected from the group consisting of hydrogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, alkenylcycloalkenyl, heterocyclic, aryl, aralkyl, hyaluronic acid, polyethyleneglycol, -ORι0, -NR10R11, -COR10, -C02R o, -CONR10R11, -0-(-(CH2)a-0)b-Rιo, -SR10, -SOR10, -S02Rιo, -SO2NRi0Rii, -N(ORio)(Rn), -P(O)(ORι0)(ORn), -P(O)(ORι0)(Rn) and -OP(O)(ORι0)(ORn) substituents (in which case R10 and Ru are independently selected from the group consisting of hydrogen and alkyl groups, and a and b are integers independently selected from 1 to 6), and substituents attached to the σ-carbon of -amino acids; and (d) optionally, one or more of R2 or R'2 and R3, R4 and R5 or R'5, R6 or R'6 and R , or R8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted nitrogen containing heterocycle having 2 to 20 carbon atoms, which may be an aromatic heterocycle in which case the hydrogen attached to the nitrogen which is both part of the heterocycle and the macrocycle and the R groups attached to the carbon atoms which are both part of the heterocycle and the macrocycle are absent; and (e) optionally, R5 or R'5 and R6 or R'6 together with the carbon atoms to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 2 to 20 carbon atoms; and (f) optionally, one or more of R2 and R'2, R5 and R'5, R6 and R'6l and R9 and R'g, together with the carbon atom to which they are attached independently form a substituted or unsubstituted and saturated, partially saturated, or unsaturated cyclic or heterocyclic having 3 to 20 carbon atoms; and (g) optionally, one or more of R, Ri, R2, R'2, R3, R4, Rs, R's, Re, R'e, R7, Rs, Rg, and R'g together with a different one of R, Rn, R2, R'2, R3, R , R5, R's, Re, R'e, R7, Rs, Rg, and R'9 which is attached to a different carbon atom in the macrocyclic ligand may be bound to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from 0 to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (h) optionally, one or more of R, R1 ? R2, R'2, R3, R4, R5, R'5) R6, R'e, R7, Rs, Rg, and R'g may be bound to an atom of at least one of heterocycle U, V or W to form a strap represented by the formula: - (CH2), - Q - (CH2)j - R - (CH2)K - S - (CH2)L - wherein I, J, K and L independently are integers from O to 10 and Q, R and S are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, alkaryl, alkheteroaryl, aza, amide, ammonium, oxa, thia, sulfonyl, sulfinyl, sulfonamide, phosphoryl, phosphinyl, phosphino, phosphonium, keto, ester, alcohol, carbamate, urea, thiocarbonyl, borates, boranes, boraza, silyl, siloxy, silaza and combinations thereof; and (i) combinations of any of (a) through (h) above; and wherein X, Y and Z are independently selected from the group consisting of suitable ligands, including halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkylaryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or the corresponding anions thereof; or X, Y and Z are independently selected from charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system and the corresponding anion thereof; or X, Y and Z are independently attached to one or more of R, Ri, R2, R'2, R3, R4, R5, R5, e, R'β, R7, s, R9, and Rg; and M is a transition metal; and n is an integer from 0 to 3.
48. A method according to claim 47, wherein U and V are saturated cycloalkyl heterocycles having 3 to 20 carbon atoms.
49. A method according to claim 48, wherein U and V are saturated cycloalkyl heterocycles having 4 to 10 carbon atoms.
50. A method according to claim 49, wherein U and V are trans-cyclohexanyl fused rings.
51. A method according to claims 46 and 47, wherein W is a substituted pyridino moiety.
52. A method according to claim 49, wherein U and V are trans-cyclohexanyl fused rings and W is a substituted pyridino moiety.
53. A method according to claims 45, 46 or 47, wherein transition metal M is selected from the group consisting of Mn, Fe, Ni and V.
54. A method according to claim 30, wherein the nitration inhibitor is selected from the group consisting of a peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
55. A method according to claim 54, wherein the peroxynitrite scavenger is represented by a formula selected from the group of formulas consisting of: Structure I
Figure imgf000122_0001
wherein R3, R6, R9 and Rι2 are independently selected from the group consisting of H, alkyl, alkenyl, CH2, COOH, phenyl, pyridyl, and N-alkylpyridyl, such that phenyl, pyridyl and N-alkylpyridyl are:
Phenyl
Pyridyl
N-Alkylpyridyl
Figure imgf000122_0002
which are attached at a carbon atom; and wherein Phenyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R is selected from the group consisting of hydrogen, alkyl, aryl and alkaryl, and R' is alkyl; wherein Pyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, SOsH, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein N-Alkylpyridyl is optionally substituted by a substituent selected from the group consisting of a halogen, alkyl, aryl, benzyl, COOH, CONH2, S03H, N02, NH2, N(R)3+ and NHCOR', wherein R and R' are as defined above; and wherein Ri, R2, R4, R5, R7, R8, Rι0, or Ru are independently selected from the group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, N02, hydroxyalkyl, and S03H; and further wherein Ri and R2 optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins; with the proviso that when the X and Y containing complex has a net positive charge then Z is a counter ion which is independently selected from the group consisting of X and Y, or when the X and Y containing complex has net negative charge then Z is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V; and n is an integer from 0 to 4. Structure II
Figure imgf000124_0001
wherein R' is CH or N; Riι R2, R3, R , Re, Re, R7, Rs, Rg, R10, Ru, R12, R13, Rι4, Ris, and R16 are independently selected from the group consisting of H, S03H, COOH, N02, NH2, and N- alkylamino; and X, Y, Z, M and n are as defined above; Structure III
Figure imgf000125_0001
A wherein Ri, R5, R9, and R13 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, Re, R'e, Rs, R's, R10, R'10, R12, R'12, Rι , R'ι4, Rιβ, and R'ιβ are independently selected from the group consisting of H and alkyl; R3, R7, Ru, and R15 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000125_0002
B wherein R ? R5, R8, and Rι2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R4, R'4, R6, R'6, R7, Rg, R'g, Ru, R'n, Ris, R'13, and R are independently selected from the group consisting of H and alkyl; R3 and R10 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000126_0001
wherein Ri, R4, R8, and R 2 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, Rδ, R'δ, R7, Rg, R'g, Rn, R'n, R13, R'13 and R14 are independently selected from the group consisting of H and alkyl; R10 is H or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000126_0002
D wherein Ri, R4, R7 and R10 are independently selected from the group consisting of a direct bond and CH2; R2, R'2, R3, Rδ, R's, Re, Rs, R's, Rg, R11, R'11 and R12 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000127_0001
wherein Ri, R4, R8 and Rn are independently selected from the group consisting of a direct bond and CH2; R2, Rs, R's, Rδ, R's, R7, R'7, Rg, R10, R'10, R12, R'12 and R13 are independently selected from the group consisting of H and alkyl; R6 is hydrogen or alkyl; and X, Y, Z, M and n are as defined above;
Figure imgf000127_0002
wherein R-,, R , R and R10 are independently selected from the group consisting of H and alkyl; R2, Rs, R'3, Rs, R's, Re, Rs, Rg, R'g, R11, R'11 and R12 are independently selected from the group consisting of H and alkyl; and X, Y, Z, M and n are as defined above;
wherein R-,, R3, R4, and R6 are independently selected from the group consisting of H and alkyl; R2 and R5 are independently selected from the group consisting of H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above;
Figure imgf000128_0002
H wherein R1 f R2, R3, and R4 are independently selected from the group consisting of
H, alkyl, S03H, N02, NH2, halogen, COOH and N(R)3+ wherein R is as defined above; and X, Y, Z, M and n are as defined above; and Structure IV
Figure imgf000129_0001
wherein R1 f R'ι, R2, R'2, R3, R' , R , R'4, R5, R'δ, Re, R'e, R7 and R'7 are independently selected from the group consisting of H, alkyl, alkoxy, N02, aryl, halogen, NH2 and S03H, further wherein R6, R'e, R and R'7 together with one other of R6, R'β, R7 and R' optionally form a heterocycle having 5 to 8 carbon atoms and form a ring with the carbon atoms of the macrocycle to which they are attached; M1 is selected from the group consisting of Fe, Ni or V; and X, Y, Z and n are as defined above.
56. A method according to claim 30, wherein the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
57. A method according to claim 38, wherein the pain inhibitor is an opioid or
NSAID and the nitration inhibitor is selected from the group consisting of a SO generating system inhibitor, SO scavenger, NOS inhibitor, NO generating system inhibitor, NO scavenger, peroxynitrite generating system inhibitor, peroxynitrite scavenger, and any combination thereof.
58. A method for treating, preventing, reversing or inhibiting pain which is mediated, at least in part, by peroxynitrite-driven nitration comprising administering to a subject in need thereof a therapeutically effective amount of a combination of compounds, wherein at least one compound of the combination is selected from the group consisting of a NMDA receptor antagonist, a glutamate receptor antagonist, and any combination thereof.
59. A method according to claim 58, wherein the NMDA receptor antagonist is
MK801.
60. A method according to claim 30, wherein the subject is a mammal.
61. A method according to claim 60, wherein the mammal is a human.
PCT/US2004/036187 2003-10-31 2004-10-31 Composition combinations and methods for treating, preventing, reversing and inhibiting pain WO2005060437A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US51616503P 2003-10-31 2003-10-31
US60/516,165 2003-10-31

Publications (2)

Publication Number Publication Date
WO2005060437A2 true WO2005060437A2 (en) 2005-07-07
WO2005060437A3 WO2005060437A3 (en) 2013-07-11

Family

ID=34710033

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/036187 WO2005060437A2 (en) 2003-10-31 2004-10-31 Composition combinations and methods for treating, preventing, reversing and inhibiting pain

Country Status (1)

Country Link
WO (1) WO2005060437A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2544542A1 (en) * 2010-03-10 2013-01-16 Galleon Pharmaceuticals, Inc. Analgesic compounds, compositions, and uses thereof
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
EP2544542A1 (en) * 2010-03-10 2013-01-16 Galleon Pharmaceuticals, Inc. Analgesic compounds, compositions, and uses thereof
EP2544542A4 (en) * 2010-03-10 2013-07-24 Galleon Pharmaceuticals Inc Analgesic compounds, compositions, and uses thereof

Also Published As

Publication number Publication date
WO2005060437A3 (en) 2013-07-11

Similar Documents

Publication Publication Date Title
ES2215305T3 (en) ANALGESIC METHODS USING THE SYNTHETIC CATALYSTS FOR THE DISMUTATION OF SUPEROXIDE RADICALS.
US6596733B2 (en) Use of nitric oxide scavengers to treat side effects caused by therapeutic administration of sources of nitric oxide
Aydogan et al. Melatonin and nitric oxide
EP2182961B1 (en) Prevention of gastric ulcery by carbon monoxide
Sheng et al. Metalloporphyrins as therapeutic catalytic oxidoreductants in central nervous system disorders
JP2003535034A (en) Dipeptidyl peptidase IV inhibitors and methods for producing and using dipeptidyl peptidase IV inhibitors
EP2772136A1 (en) Methods for treating a variety of diseases and conditions, and compounds useful therefor
KR20030096227A (en) Method for the treatment of neurological and neuropsychological disorders
WO2009035534A2 (en) Treatment of ischemic eye disease by the systematic pharmaceutical activation of hypoxia inducible factor (hif)
US20200179417A1 (en) Combination chemotherapies
JP2008515800A (en) Polyamine analogs as therapeutic agents for ophthalmic diseases
US20060147548A1 (en) Therapeutic delivery of carbon monoxide
ES2346859T3 (en) GLUTATION PRECURSORS FOR THE TREATMENT OF NEUROSIQUIATRIC DISORDERS.
WO2005041886A2 (en) Methods for generating or increasing revenues related to pain inhibitor commerce
US20040219138A1 (en) Combinations of superoxide dismutase mimetics and opioids
WO2005042718A2 (en) Compositions and methods for treating, preventing, reversing and inhibiting pain
WO2006078713A2 (en) Methotrexate combinations for treating inflammatory diseases
Genovese et al. Beneficial effects of FeTSPP, a peroxynitrite decomposition catalyst, in a mouse model of spinal cord injury
WO2005060437A2 (en) Composition combinations and methods for treating, preventing, reversing and inhibiting pain
WO2005041885A2 (en) Compositions and methods for treating, preventing, reversing and inhibiting pain
WO2005041894A2 (en) Compositions and methods for treating, preventing, reversing and inhibiting pain
JP2007523148A (en) Pain relief method
Gan et al. Induction of heme oxygenase-1 improves impaired intestinal transit after burn injury
US20050171198A1 (en) SODm therapy for treatment, prevention, inhibition and reversal of inflammatory disease
US20160166577A1 (en) Treatment of pulmonary fibrosis using an inhibitor of cbp/catenin

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
122 Ep: pct application non-entry in european phase