Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P23/00—Anaesthetics
  • A61P23/02—Local anaesthetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention is directed to the use of ⁇ O-conopeptides as a local anesthetic for treating pain.
• the ⁇ O-conopeptides have long-lasting anesthetic activity and are particularly useful for spinal anesthesia, administered either acutely for post-operative pain or via an intrathecal pump for severe chronic pain situations.
• the present invention is further directed to new ⁇ O- conopeptides, their coding sequences and their propeptides.
• Conus is a genus of predatory marine gastropods (snails) which envenomate their prey.
• Venomous cone snails use a highly developed projectile apparatus to deliver their cocktail of toxic conotoxins into their prey.
• the cone detects the presence of the fish using chemosensors in its siphon and when close enough extends its proboscis and fires a hollow harpoon-like tooth containing venom into the fish. This immobilizes the fish and enables the cone snail to wind it into its mouth via an attached filament.
• Conus and their venom For general information on Conus and their venom see the website address http://grimwade.biochem.unimelb.edu.au/cone/ referenc.html. Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes.
• Each Conus species venom appears to contain a unique set of 50-200 peptides.
• the composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyse it's prey.
• the active components of the venom are small peptides toxins, typically 12-30 amino acid residues in length and are typically highly constrained peptides due to their high density of disulphide bonds.
• the venoms consist of a large number of different peptide components that when separated exhibit a range of biological activities: when injected into mice they elicit a range of physiological responses from shaking to depression.
• the paralytic components of the venom that have been the focus of recent investigation are the -, ⁇ - and ⁇ -conotoxins. All of these conotoxins act by preventing neuronal communication, but each targets a different aspect of the process to achieve this.
• the -conotoxins target nicotinic ligand gated channels
• the ⁇ -conotoxins target the voltage- gated sodium channels
• the ⁇ -conotoxins target the voltage-gated calcium channels (Olivera et al., 1985).
• a linkage has been established between -, A- & ⁇ -conotoxins and the nicotinic ligand-gated ion channel; ⁇ -conotoxins and the voltage-gated calcium channel; ⁇ - conotoxins and the voltage-gated sodium channel; ⁇ -conotoxins and the voltage-gated sodium channel; K-conotoxins and the voltage-gated potassium channel; conantokins and the ligand-gated glutamate (NMD A) channel.
• NMD A ligand-gated glutamate
• peptides where function has been determined three classes of targets have been elucidated: voltage-gated ion channels; ligand-gated ion channels, and G-protein-linked receptors.
• Conus peptides which target voltage-gated ion channels include those that delay the inactivation of sodium channels, as well as blockers specific for sodium channels, calcium channels and potassium channels.
• Peptides that target ligand-gated ion channels include antagonists of NMDA and serotonin receptors, as well as competitive and noncompetitive nicotinic receptor antagonists.
• Peptides which act on G-protein receptors include neurotensin and vasopressin receptor agonists.
• the unprecedented pharmaceutical selectivity of conotoxins is at least in part defined by a specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (for a review see Mclntosh et al., 1998).
• the pain response is a protective reflex system warning an individual of hostile situations and tissue injury.
• the origins of clinically significant acute and chronic pain in a mammal are different, but the biochemical and neurological pathways are similar.
• the focus is primarily on humans, however, it should be understood that the concepts of pain are applicable to mammalian animals and the management of such pain is applicable to veterinary medicine.
• Acute pain is often associated with surgery and with trauma.
• the intensity of acute postoperative pain varies considerably depending on the extent of the surgical procedure performed, on the individual's pain sensitivity, and on the type of anesthetic management employed during surgery.
• major operations on the thorax and the upper abdominal region induce the most intensive postoperative pain.
• Extensive orthopedic operations also produce strong postoperative pain.
• Chronic pain is persistent pain which has long outlasted the onset of any known or suspected physical cause. It can occur after a known injury or disease, or it can occur without any known physical cause whatsoever. Moreover, it can be accompanied by known tissue pathology, such as chronic inflammation that occurs in some types of arthritis, or it can occur long after the healing of the injured tissue which is suspected or known to be the cause of chronic pain. Chronic pain is a very general concept and there are several varieties of chronic pain related to the musculoskeletal system, visceral organs, skin, and nervous system.
• Neuropathic pain can occur as a form of chronic pain and can also occur under acute conditions such as those following surgery or accidental trauma.
• Neuropathic pain can be defined as pain that results from an abnormal functioning of the peripheral and/or central nervous system.
• a critical component of this abnormal functioning is an exaggerated response of pain-related nerve cells either in the peripheral or in the central nervous system. This exaggerated responsiveness is manifested behaviorally as increased sensitivity to pain, i.e., as hyperalgesia or allodynia, both of which can occur in chronic neuropathic and acute inflammatory pains.
• An example is the pain from causalgia wherein even a light touch to the skin is felt as an excruciating burning pain (allodynia) or a normally mild pain is experienced as an excruciating one (hyperalgesia).
• Neuropathic pain is thought to be a consequence of damage to peripheral nerves or to regions of the central nervous system.
• abnormal functioning of pain-related regions of the nervous system call also occur with chronic inflammatory conditions such as certain types of arthritis and metabolic disorders such as diabetes as well as with acute inflammatory conditions.
• chronic pains that are related to inflammation as well as acute pains that are related to inflammation can be considered to be at least partly neuropathic pains.
• the modern concept of pain treatment emphasizes the significance of prophylactic prevention of pain, as pain is more easily prevented than relieved. Additionally, the hormonal stress responses associated with pain are considered harmful to the patient, impair the healing process and overall recovery, and generally are to be avoided.
• Currently used local anesthetics have durations of action lasting only several hours. While this length of duration meets many needs, particularly the control of acute pain, local anesthetic agents with longer duration of action would have broad clinical application for the treatment of postoperative and chronic pain (Kuzma et al., 1997).
• the duration of action of a local anesthetics is proportional to the time during which it is in actual contact with the nervous tissues.
• procedures or formulations that maintain localization of the drug at the nerve greatly prolong anesthesia.
• All local anesthetics are potentially toxic, and therefore it is of great importance that the choice of drug, concentration, rate and site of administration, as well as other actors, be considered in their use.
• a local anesthetic must remain at the site long enough to allow sufficient time for the localized pain to subside.
• Different devices and formulations are known in the art for administration of local anesthetics. See U.S. Patent No. 5,747,060, which discloses such devices and formulations.
• an object of the invention is to provide methods and compositions for the treatment of acute or chronic pain which provide effective control of pain with longer duration of action and reduced side effects associated with traditional analgesics.
• the present invention is directed to the new ⁇ O-conopeptides, their coding sequences and their propeptides and to the use of ⁇ O-conopeptides as a local anesthetic for treating pain.
• the ⁇ O- conopeptides have long lasting anesthetic activity and are particularly useful for spinal anesthesia, either administered acutely for post-operative pain or via an intrathecal pump for severe chronic pain situations or for treatment of pain in epithelial tissue.
• Xaa 27 -Xaa 28 -Xaa 29 -Xaa 30 (SEQ ID NO: l), wherein Xaa, is des-Xaa,, Pro, hydroxy-Pro (Hyp), Arg, Lys, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys or any synthetic basic amino acid; Xaa 2 is des-Xaa 2 , Ala, Gly, Asp, Glu, ⁇ -carboxy-glutamate (Gla), any synthetic acidic amino acid, Thr, Ser, g-Thr (where g is glycosylation), g-Ser, Tip (D or L), neo-Trp or halo-
• Trp (D or L) or Xaa 2 may be pyroglutamate if Xaa, is des-Xaa,;
• Xaa 3 is Arg, Lys, ornithine, homo- Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys, any synthetic basic amino acid, Ser, Thr, g-Ser, g-Thr, Ala, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), He and Val or non-natural derivatives of the aliphatic amino acid, His, Glu, Gin, Gla, Asp, Asn or any synthetic acidic amino acid;
• Xaa 4 is Glu, Gla, Gin, Asp, Asn, any synthetic acidic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Ly
• Xaa 5 is Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl- Lys, any synthetic basic amino acid, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), He and Val or non-natural derivatives of the aliphatic amino acid, Ser, Thr, Pro, Hyp, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid;
• Xaa 5 is Lys, Arg, ornithine, homo-Lys, homoarginine, nor-L
• Glu, Gla, Gin, Asp, Asn any synthetic acidic amino acid, Pro or Hyp
• Xaa 6 is Trp (D or L), neo-Trp, halo-Trp (D or L), Gly, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Glu, Gla, Gin, Asp, Asn, any synthetic acidic amino acid;
• Xaa 7 is Glu, Gla, Gin, Asp, Asn, any synthetic acidic amino acid, Met, norleucine (Nle), Ala, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), He and Val or non-natural derivatives of the aliphatic amino acid, Tyr, meta-Tyr, ortho- Tyr, nor-Tyr, mono-halo-T
• Pro, Hyp an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), He and Val or non-natural derivatives of the aliphatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys or any synthetic basic amino acid;
• Xaa, 4 is Gly, His, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys or any synthetic basic amino acid;
• Xaa 15 is des-Xaa, 5 , Ser, Thr, g-Ser, g-Thr, Val, Asn, Phe, Tyr, meta-Tyr, ortho
• Xaa, 7 is Pro, Hyp, Ser, Thr, g-Hyp, g-Ser, g-Thr, any hydroxylated amino acid, Ala, Glu, Gla, Gin, Asp, Asn, any synthetic acidic amino acid, His or Gly;
• Xaa, 8 is Gly, Asn or Gin;
• Xaa, 9 is Leu, T ⁇ (D or L), neo-T ⁇ or halo-Trp (D or L);
• Xaa 20 is des- Xaa 20 , Leu or T ⁇ (D or L), neo-T ⁇ or halo-T ⁇ (D or L);
• Xaa 2 is des-Xaa,, or an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), He and Val or non-natural derivatives of the aliphatic amino acid;
• Xaa 22 is des-Xaa 22 , Gly, Met,
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O- phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
• the halogen is iodo, chloro, fluoro or bromo; preferably iodo for halogen substituted-Tyr and bromo for halogen-substituted T ⁇ .
• MrVIA/B has the sequence: Ala-Cys-Xaa 3 ,-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Xaa 32 -Ile- Xaa 33 -Gly-Phe-Xaa 34 -Tyr-Cys-Cys-Xaa 32 -Gly-Leu-Ile-Cys-Gly-Xaa 32 -Phe-Val-Cys-Val, wherein Xaa 3 , is Arg or Ser, Xaa 32 is Pro or hydroxy-Pro, Xaa 33 is He or Leu and Xaa 34 is He or Val (SEQ
• the present invention is also directed to novel specific conotoxin peptides within general formula I having the formulas:
• the halo is preferably chlorine or iodine, more preferably iodine.
• the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl- Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid;
• the Xaa, residues may be substituted by Arg, ornithine, homoargine, nor-Lys, or any synthetic basic amino acid;
• the Tyr residues may be substituted with any synthetic hydroxy containing amino acid;
• the Ser residues may be substituted with Thr or any synthetic hydroxylated amino acid;
• the Thr residues may be substituted with Ser or any synthetic hydroxylated amino acid;
• the Phe and T ⁇ residues may be substituted with any synthetic aromatic amino acid; and
• the Asn, Ser, Thr or Hyp residues may be glycosylated.
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may also be substituted with the 3-hydroxyl or 2- hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O- phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
• MrVIA SEQ ID NO:2, whererin Xaa 30 is Arg, Xaa 3 , is He and Xaa 32 is He;
• MrVIB SEQ ID NO:2, wherein Xaa 30 is Ser, Xaa 3 , is Leu and Xaa 32 is Val;
• A657 SEQ ID NO:3, wherein Xaa, is Lys, Xaa 2 is Tyr, Xaa 3 is Glu and Xaa 4 is Pro;
• F079 SEQ ID NO:4, wherein Xaa, is Lys, Xaa 2 is Tyr and Xaa 4 is Pro;
• Ca6.1 SEQ ID NO:5, wherein Xaa, is Lys, Xaa 2 is Tyr, Xaa 3 is Glu, Xaa 4 is Pro and
• Xaa 5 is Trp; Tx6.12 : SEQ ID NO:6, wherein Xaa, is Tyr, Xaa 3 is Glu, Xaa 4 is Pro and Xaa 5 is T ⁇ ;
• Tx6.13 SEQ ID NO:7, wherein Xaa, is Lys, Xaa, is Tyr, Xaa 3 is Glu, Xaa 4 is Pro and Xaa 5 is T ⁇ ;
• G28 SEQ ID NO: 8, wherein Xaa, is Tyr and Xaa 4 is Pro;
• F763 SEQ ID NO:9, wherein Xaa, is Lys, Xaa, is Tyr, Xaa 4 is Pro and Xaa 5 is T ⁇ ;
• F080 SEQ ID NO: 10, wherein Xaa, is Tyr, Xaa 3 is Glu, Xaa 4 is Pro and Xaa 5 is
• Trp Trp; and Gl 8: SEQ ID NO: 12, wherein Xaa, is Tyr, Xaa 3 is Glu and Xaa 4 is Pro. 10
• Examples of synthetic aromatic amino acid include, but are not limited to, such as nitro-Phe, 4-substituted-Phe wherein the substituent is C,-C 3 alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, -CHO, -CN, -SO 3 H and -NHAc.
• Examples of synthetic hydroxy containing amino acid include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6- dimethyl-Tyr and 5-amino-Tyr.
• Examples of synthetic basic amino acids include, but are not limited to, N-l-(2-pyrazolinyl)- Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)- Gly and 2-[3-(2S)pyrrolininyl)-Ala.
• the Asn residues may be modified to contain an N-glycan and the Ser, Thr and Hyp residues may be modified to contain an O-glycan (e.g., g-N, g-S, g-T and g-Hyp).
• a glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art.
• the monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose.
• These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof.
• the gylcan may also include similar polyhydroxy groups, such as D- penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives.
• the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3.
• the linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-.
• Core O-glycans have been described by Van de Steen et al. (1998), incorporated herein by reference.
• Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue.
• the monosaccharide building blocks and the linkage attached to this first GalNAc residue define the "core glycans," of which eight have been 1 1 identified.
• the type of glycosidic linkage (orientation and connectivities) are defined for each core glycan.
• Suitable glycans and glycan analogs are described further in U.S. Serial No. 09/420,797 filed 19 October 1999 and in PCT Application No. PCT/US99/24380 filed 19 October 1999 (PCT Published Application No. WO 00/23092), each incorporated herein by reference.
• a preferred glycan is Gal( ⁇ l -3)GalNAc( ⁇ l-).
• pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp) or Cys/Ala combinations.
• isoteric lactam or ester-thioether replacements such as Ser/(Glu or Asp), Lys/(Glu or Asp) or Cys/Ala combinations.
• Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges.
• Thioether analogs may be readily synthesized using halo-
• the present invention is also directed to the identification of the nucleic acid sequences encoding these peptides and their propeptides and the identication of nucleic acid sequences of additional related ⁇ O-conopeptides.
• the present invention is further directed to a method of reducing/alleviating/decreasing the perception of pain by a subject or for inducing analgesia, particularly local analgesia, in a subject comprising administering to the subject an effective amount of the pharmaceutical composition comprising a therapeutically effective amount of a ⁇ O-conotoxin peptide described herein or a pharmaceutically acceptable salt or solvate thereof, including MrVIA and MrVIB.
• the present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a ⁇ O-conotoxin peptide described herein or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.
• FIGURES Figure 1 shows ⁇ O-conopeptide MrVIB inhibits skin flinch sensitivity in the Guinea pig intracutaneous wheal assay with greater potency than lidocaine or bupivacaine. Data represent the number of flinches observed after 36 pin pricks in a 30 minutes test period. Each point represents the mean of at least three observations.
• Figure 2 shows ⁇ O-conopeptide MrVIB produces a long-lasting inhibition of skin flinch sensitivity relative to either lidocaine or bupivacaine in the Guinea pig intracutaneous wheal assay.
• SEQ ID NO:l is a generic formula for ⁇ O-conopeptides.
• SEQ ID NO:2 is a generic formula for ⁇ O-conopeptides MrVIA and MrVIB.
• SEQ ID NO:3 is a generic formula for ⁇ O-conopeptide A657.
• SEQ ID NO:4 is a generic formula for ⁇ O-conopeptide F079.
• SEQ ID NO:5 is a generic formula for ⁇ O-conopeptide Ca6.1.
• SEQ ID NO:6 is a generic formula for ⁇ O-conopeptide Tx6.12.
• SEQ ID NO:7 is a generic formula for ⁇ O-conopeptide Tx6.13.
• SEQ ID NO:8 is a generic formula for the ⁇ O-conopeptide G28.
• SEQ ID NO:9 is a generic formula for the ⁇ O-conopeptide F763.
• SEQ ID NO: 10 is a generic formula for the ⁇ O-conopeptide F080.
• SEQ ID NO:l 1 is a generic formula for the ⁇ O-conopeptide F008.
• SEQ ID NO: 12 is a generic formula for the ⁇ O-conopeptide G18.
• SEQ ID NO: 13 is a primer for amplifying "O-Superfamily" conotoxins.
• SEQ ID NO:14 is a primer for amplifying "O-Superfamily" conotoxins.
• SEQ ID NO: 15 is a nucleotide sequence for the gene coding for the A657 propeptide.
• SEQ ID NO: 16 is an amino acid sequence of the A657 propeptide.
• SEQ ID NO: 17 is a nucleotide sequence for the gene coding for the F079 propeptide.
• SEQ ID NO:18 is an amino acid sequence of the F079 propeptide.
• SEQ ID NO:19 is a nucleotide sequence for the gene coding for the Ca6.1 propeptide.
• SEQ ID NO:20 is an amino acid sequence of the Ca6.1 propeptide.
• SEQ ID NO:21 is a nucleotide sequence for a portion of the gene coding for the Tx6.12 propeptide.
• SEQ ID NO:22 is an amino acid sequence of a portion of the Tx6.12 propeptide.
• SEQ ID NO:23 is a nucleotide sequence for a portion of the gene coding for the Tx6.13 propeptide.
• SEQ ID NO:24 is an amino acid sequence of a portion of the Tx6.13 propeptide.
• SEQ ID NO:25 is a nucleotide sequence for the gene coding for the G28 propeptide.
• SEQ ID NO:26 is an amino acid sequence of the G28 propeptide.
• SEQ ID NO:27 is a nucleotide sequence for the gene coding for the F763 propeptide.
• SEQ ID NO:28 is an amino acid sequence of the F763 propeptide.
• SEQ ID NO:29 is a nucleotide sequence for the gene coding for the F080 propeptide.
• SEQ ID NO:30 is an amino acid sequence of the F080 propeptide.
• SEQ ID NO:31 is a nucleotide sequence for the gene coding for the F008 propeptide.
• SEQ ID NO:32 is an amino acid sequence of the F008 propeptide.
• SEQ ID NO:33 is a nucleotide sequence for the gene coding for the G18 propeptide.
• SEQ ID NO:34 is an amino acid sequence of the G18 propeptide.
• the present invention is directed to the new ⁇ O-conopeptides, their coding sequences and their propeptides and to the use of ⁇ O-conopeptides as a local anesthetic for treating pain.
• the ⁇ O- conopeptides have long lasting anesthetic activity and are particularly useful for spinal anesthesia, 13 either administered acutely for post-operative pain or via an intrathecal pump for severe chronic pain situations or for treatment of pain in epithelial tissue.
• the present invention in another aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of a conotoxin peptide described herein or a pharmaceutically acceptable salt or solvate thereof.
• Such a pharmaceutical composition has the capability of acting as analgesic agents.
• the present invention also provides for a method provides local anesthesia to a patient having pain.
• the pain results from surgical or medical procedures, and the compounds are administered to the central nervous system (CNS), e.g. to the spine for spinal analgesia.
• the pain is in an epithelial tissue region associated with damage or loss of epithelial tissue as a result of, for example, plastic surgery, canker sores, burns, sore throats, genital lesions, upper or lower gastrointestinal bronchoscopy or endoscopy, intubation, dermatologic abrasions or chemical skin peels, and the compounds are administered to alleviate the associated pain.
• the conotoxin peptides described herein are sufficiently small to be chemically synthesized.
• conotoxin peptides of the present invention can be obtained by purification from cone snails, because the amounts of conotoxin peptides obtainable from individual snails are very small, the desired substantially pure conotoxin peptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy.
• the yield from a single cone snail may be about 10 micrograms or less of conotoxin peptide.
• substantially pure is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95%) purity.
• Chemical synthesis of biologically active conotoxin peptides depends of course upon correct determination of the amino acid sequence.
• conotoxin peptides can also be produced by recombinant DNA techniques well known in the art. Such teclmiques are described by Sambrook et al. (1989). The peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds. 14
• One method of forming disulfide bonds in the peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides.
• the oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
• the peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
• the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence.
• various coupling reagents e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole
• various active esters e.g., esters of N-hydroxyphthalimide or N- hydroxy-succinimide
• the various cleavage reagents to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology.
• the protecting group preferably retains its protecting properties and is not split off under coupling conditions
• the protecting group should be stable under the reaction conditions selected for removing the ⁇ -amino protecting group at each step of the synthesis
• the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
• Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin.
• a suitable resin can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or para- methylbenzhydrylamine (MB HA) resin.
• BHA benzhydrylamine
• MB HA para- methylbenzhydrylamine
• Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, CA) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart and Young (1969).
• BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus.
• solid resin supports may be any of those known in the art, such as one having the formulae -O-CH,-resin support, -NH BHA resin support, or -NH-MBHA resin support.
• use of a BHA or MBHA resin is preferred, because cleavage directly gives the amide.
• N-methyl amide In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Patent No. 4,569,967 (Kornheim et al, 1986) can be used, or should still other groups than 16 the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text (1974).
• the C-terminal amino acid protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Horiki et al. (1978), using KF in DMF at about 60°C for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized.
• the ⁇ -amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.
• TFA trifluoroacetic acid
• the deprotection is carried out at a temperature between about 0°C and room temperature.
• Other standard cleaving reagents, such as HC1 in dioxane, and conditions for removal of specific ⁇ -amino protecting groups may be used as described in Schroder & Lubke (1965).
• the remaining ⁇ -amino- and side chain- protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor.
• coupling reagent selection of an appropriate coupling reagent is within the skill of the art.
• Particularly suitable as a coupling reagent is N,N'-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).
• activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
• suitable activating reagents are carbodiimides, such as N,N'- diisopropylcarbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
• Other activating reagents and their use in peptide coupling are described by Schroder & Lubke (1965) and Kapoor (1970).
• Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH 2 C1, (1 :1) or in DMF or CH,C1, alone.
• DMF dimethylformamide
• the coupling procedure is repeated before removal of the ⁇ -amino protecting group prior to the coupling of the next amino acid.
• the success of the coupling reaction at each stage of the synthesis if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970).
• Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978). 17
• the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the ⁇ -amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid.
• a reagent such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry)
• TFA liquid hydrogen fluoride
• the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S- alkylation.
• one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel. Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the pepti do-resin, to create bonds between Cys residues.
• fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected.
• deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0°C with hydrofluoric acid (HF) or TFA, followed by oxidation as described above.
• the peptides are also synthesized using an automatic synthesizer.
• Amino acids are sequentially coupled to an MBHA Rink resin (typically 100 mg of resin) beginning at the C- terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings are carried out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-(lH-benzotriazole- l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro- pylethylamine (DIEA).
• NMP N-methylpyrrolidinone
• HBTU 2-(lH-benzotriazole- l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
• DIEA diethylisopro- pylethylamine
• RT-PCR reverse transcription-polymerase chain reaction
• the PCR primers are based on the DNA sequences coding for the precursor peptides of the "O-Superfamily" as described herein.
• RT-PCR of venom duct mRNA produces a product of about 250-300 nucleotides in Conus species that express conotoxin genes.
• the PCR product is then cloned into a plasmid vector and individual clones are sequenced to determine the sequence of various conotoxin genes.
• cDNA libraries are prepared from Conus venom duct using conventional techniques.
• DNA from single 18 clones is amplified by conventional techniques using primers which correspond approximately to the Ml 3 universal priming site and the Ml 3 reverse universal priming site.
• Clones having a size of approximately 250 nucleotides are sequenced and screened for similarity in sequence to the propeptide described herein. In this manner, conotoxins having the basic structure and activity described herein are cloned from many Conus species.
• Muteins, analogs or active fragments (collectively referred to herein as derivatives) of ⁇ O- conopeptides are also contemplated for use as local anesthetics. See, e.g., Hammerland et al. (1992).
• Derivative muteins, analogs or active fragments of ⁇ O-conopeptides may be synthesized according to known techniques, including conservative amino acid substitutions, such as outlined in U.S. Patent Nos. 5,545,723; 5,534,615 and 5,364,769.
• the derivative muteins, analogs or active fragments may be conveniently assayed for activity by using a hindlimb paralysis test such as described in Example 2 or a local anesthetic test such as described in Example 3.
• ⁇ -Conopeptides i.e., GVIA
• GVIA block sodium channels expressed by muscle cells
• ⁇ -Conopeptides i.e., GmVIA
• delay the inactivation of neuronal sodium channels Olivera et al., 1990).
• ⁇ -PnIVA and ⁇ -PnIVB Another class of conopeptide (i.e., ⁇ -PnIVA and ⁇ -PnIVB; unfortunately also called ⁇ but having a distinct cysteine framework from that which is considered a ⁇ -conopeptide) blocks sodium channels in molluscan neurons, but has no effect on sodium currents in bovine chromaffin cells or in rat brain synaptosomes (Fainzilber et al., 1995). Finally, the ⁇ O-conopeptides (MrVIA and MrVIB) block mammalian sodium channels (Mclntosh et al., 1995).
• the present invention examined whether the ⁇ O-conopeptides might represent a candidate for a long-lasting local anesthetic.
• the present invention is directed to a method for inducing local analgesia by administering the ⁇ O-conopeptides described herein.
• ⁇ O-conopeptides are used to provide local anesthesia for pain associated with any epithelial tissue region in a subject, for example, pain associated with epithelial ulcers, such as a canker sore or genital lesions.
• Canker sores can occur alone or in groups on the inside of the cheek or lip or underneath the tongue.
• Severely affected people have continuously recurring ulcers which last for one to two weeks (Clayman).
• Genital ulcers are usually caused by sexually transmitted diseases, including he ⁇ es and syphilis. The early stages of syphilis are 19 characterized by a hard chancre, a painful ulcer where bacteria has penetrated the skin.
• ulcers are painful.
• Genital ulceration may also be a side effect of drugs taken orally or caused by solutions applied to genital warts. Pain in epithelial tissue is also caused by burns. Burns affecting the epidermal layer are usually associated with pain, restlessness and fever. Treatment of such a burn in accordance with the method of the invention can provide relief from the attendant pain.
• Pain as a result of damage to or loss of epithelial tissue is also associated with other conditions and procedures, such as sore throats and plastic surgery, for example carbon dioxide laser surgery to remove for skin resurfacing and removal of wrinkles (Rosenberg et al., 1996), burns, genital lesions, upper or lower gastrointestinal bronchoscopy or endoscopy, intubation, dermatologic abrasions or chemical skin peels.
• the ⁇ O-conopeptides administered in accordance with the method of the invention is beneficial in relieving pain associated with such damaged tissues.
• compositions containing a ⁇ O-conopeptide or pharmaceutically acceptable salts thereof as the active ingredient (agent) can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remingto 's Pharmaceutical Sciences,
• compositions may further contain antioxidizing agents, stabilizing agents, preservatives and the like.
• “Pharmaceutical composition” means physically discrete coherent portions suitable for medical administration.
• “Pharmaceutical composition in dosage unit form” means physically discrete coherent units suitable for medical administration, each containing a daily dose or a multiple (up to four times) or a sub-multiple (down to a fortieth) of a daily dose of the active compound in association with a carrier and/or enclosed within an envelope. Whether the composition contains a daily dose, or for example, a half, a third or a quarter of a daily dose, will depend on whether the pharmaceutical composition is to be administered once or, for example, twice, three times or four times a day, respectively.
• salt denotes acidic and/or basic salts, formed with inorganic or organic acids and/or bases, preferably basic salts. While pharmaceutically acceptable salts are preferred, particularly when employing the compounds of the invention as medicaments, other salts 20 find utility, for example, in processing these compounds, or where non-medicament-type uses are contemplated. Salts of these compounds may be prepared by art-recognized techniques.
• salts include, but are not limited to, inorganic and organic addition salts, such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or the like. Lower alkyl quaternary ammonium salts and the like are suitable, as well.
• inorganic and organic addition salts such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or
• the term "pharmaceutically acceptable" carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
• sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
• antioxidants examples include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like
• oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (B
• the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, 21 suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
• tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
• the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/1 1698.
• the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
• suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
• the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
• the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
• the compound may be formulated as an ointment, cream, gel or paste comprising the compound to be administered in a pharmaceutical acceptable carrier.
• a transdermal patch containing the compound to be administered.
• a variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the disease state being treated and the dosage required for therapeutic efficacy.
• the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
• modes of administration include oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes.
• parenteral includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion.
• administration of the active agent according to this invention may be achieved using any suitable delivery means, including:
• an active agent is delivered directly into the CNS, preferably to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS location, most preferably intrathecally.
• targeting therapies may be used to deliver the active agent more specifically to certain types of cells, by the use of targeting systems such as antibodies or cell-specific ligands.
• Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, if it would otherwise require too high a dosage, or if it would not otherwise be able to enter target cells.
• the active agents which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• Suitable delivery systems are described in U.S. Patent No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO
• Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the developed sequences and the known genetic code.
• the active agent is preferably administered in an therapeutically effective amount.
• a “therapeutically effective amount” or simply “effective amount” of an active compound is meant a sufficient amount of the compound to treat or alleviate pain or to induce analgesia at a reasonable benefit/risk ratio applicable to any medical treatment.
• the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or spealists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington 's Parmaceutical Sciences. 23
• the dosage contemplated is from about 1 ng to about 100 mg per day, preferably from about 100 ng to about 10 mg per day, more preferably from about 1 ⁇ g to about 100 ⁇ g per day. If administered peripherally, the dosage contemplated is somewhat higher, from about 100 ng to about 1000 mg per day, preferably from about 10 ⁇ g to about 100 mg per day, more preferably from about 100 ⁇ g to about 10 mg per day.
• ⁇ O-conopeptide is delivered by continuous infusion (e.g., by pump delivery, biodegradable polymer delivery or cell-based delivery), then a lower dosage is contemplated than for bolus delivery.
• the amount of the active compound actually administered will be determined by a physician, in the light of the relevant circumstances including the condition to be treated, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way.
• compositions and “pharmaceutically acceptable” include compositions and ingredients for both human and veterinary use.
• ⁇ O-conopeptides are extremely potent and long-lasting local anesthetic agents, most likely due to their ability to block neuronal sodium channels. Moreover, since ⁇ O-conopeptides probably act at a site on sodium channels distinct from other local anesthetics or guanidinium toxins like tetrodotoxin (since they are likely to act at an extracellular target, but do compete for [ 3 H]saxitoxin at site I) (Terlau et al., 1996), and probably do not affect sodium channels in the muscles or heart (since i.p. injection of 10 nmol is without effect in mice (Mclntosh et al., 1995), these peptides lack the untoward side effects of clinically used local anesthetics.
• Amplification products in the appropriate size range were cloned and sequenced.
• a range of "O- Superfamily" gene sequences were identified.
• the novel genes, A657 from C. skinneri, F079, F080 and G28 from C. tessulatus, F763 from C. atlanticus, F008 from C. arenatus, Tx6.12 and Tx6.13 from C. textile and G18 from C. generalis, were identified as ⁇ O-conopeptides on the basis of their similarity to the ⁇ -O conopeptides MrVIA and MrVIB. This similarity was much greater than the similarity with any of the ⁇ -, K- or ⁇ -conopeptides that comprise the "O Superfamily" peptides.
• the cDNA and amino acid sequence for the A657, F079, Ca ⁇ .l, Tx6.12 (portion), Tx6.13 (portion), G28, F763, F080, F008 and G18 propeptides are set forth in Tables 1-10, respectively.
• the amino acid sequences of the mature ⁇ O-conopeptides are as shown above.
• EXAMPLE 2 Effect of Intrathecal Administration of MrVIB
• Male C57 black mice (20-25g) were obtained from Charles River Laboratories. These mice and the animals used in the other examples were housed in a temperature controlled (23 ° ⁇ 3 ° C) room with a 12 hour light-dark cycle with free access to food and water. All animals were euthanized in accordance with Public Health Service policies on the humane care of laboratory animals.
• mice Male Hartley guinea pigs (retired breeders) were obtained form Charles River Laboratories. The local anesthetic test was performed essentially as described (Bulbring and Wajda, 1945). On the day prior to test day, a patch on the back of the guinea pig was denuded of hair, first by shaving with electric clippers and subsequently with depilatory cream (Nair®). Depilatory cream was applied for five minutes and removed with a warm washcloth. The guinea pigs were dried and returned to their cages.
• guinea pigs typically, four injections were made on the back of each guinea pig. In some cases, guinea pigs were reused following at least one week of recovery and injecting into an unused portion of the skin.
• the stimulus consisted of mild pin pricks (not hard enough to break the skin) with a 26G needle.
• the response is a localized skin twitch caused by contraction of cutaneous muscles.
• a unit test consisted of six uniform pin pricks, 3-5 seconds apart, within the injected area. Unit scores ranged from 0 (complete anesthesia) to 6 (no anesthesia). For potency experiments, the unit test was 30 repeated at each site at five minute intervals for 30 minutes, and unit test scores summed (with 36 representing no anesthesia to 0 representing complete anesthesia. For duration experiments, unit tests were performed as described over the course of several hours to days.
• MrVIB produced a potent ( Figure 1) and long lasting (Figure 2) local anesthetic effect in the intracutaneous wheal test in the guinea pig.
• bupivacaine ahd a slightly longer duration thatn lidocaine, consistent with clinical observations.
• MrVIA A657, F079, Ca ⁇ .l, Tx6.12, Tx6.13, G28, F763 and F080.

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