WO2002064620A2 - Composes de liaison au metal et utilisations - Google Patents

Composes de liaison au metal et utilisations Download PDF

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Publication number
WO2002064620A2
WO2002064620A2 PCT/US2002/004275 US0204275W WO02064620A2 WO 2002064620 A2 WO2002064620 A2 WO 2002064620A2 US 0204275 W US0204275 W US 0204275W WO 02064620 A2 WO02064620 A2 WO 02064620A2
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WIPO (PCT)
Prior art keywords
xaa
cys
peptide
alanine
metal
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PCT/US2002/004275
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English (en)
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WO2002064620A3 (fr
Inventor
David Bar-Or
C. Gerald Curtis
Edward Lau
Nagaraja K. R. Rao
James V. Winkler
Wannell M. Crook
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Dmi Biosciences, Inc.
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Priority to AU2002255540A priority Critical patent/AU2002255540A1/en
Publication of WO2002064620A2 publication Critical patent/WO2002064620A2/fr
Publication of WO2002064620A3 publication Critical patent/WO2002064620A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • 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 invention relates to a method of reducing the molecular, cellular and tissue damage done by reactive oxygen species (ROS).
  • the invention also relates to certain compounds, especially certain peptides and peptide derivatives, that bind metal ions, particularly Cu(H).
  • the binding of metal ions by the compounds of the invention inhibits the formation and/or accumulation of ROS and/or targets the damage done by ROS to the compounds themselves (i.e., the compounds of the invention may act as sacrificial antioxidants).
  • the compounds of the invention can also be used to reduce the concentration of a metal in an animal in need thereof.
  • ROS Reactive oxygen species
  • free radicals e.g., superoxide anion and hydroxyl, peroxyl, and alkoxyl radicals
  • non-radical species e.g., singlet oxygen and hydrogen peroxide
  • ROS are capable of causing extensive cellular and tissue damage, and they have been reported to play a major role in a variety of diseases and conditions. Indeed, ROS have been implicated in over 100 diseases and pathogenic conditions, and it has been speculated that ROS may constitute a common pathogenic mechanism involved in all human diseases. Stohs, J. Basic Clin. Physiol. Pharmacol, 6, 205-228 (1995).
  • Ischemia/reperfusion is the leading cause of illness and disability in the world.
  • Cardiovascular ischemia in which the body's capacity to provide oxygen to the heart is diminished, is the leading cause of illness and death in the United States.
  • Cerebral ischemia is a precursor to cerebrovascular accident (stroke), which is the third leading cause of death in the United States.
  • Ischemia also occurs in other organs (e.g., kidney, liver, lung, and the intestinal tract), in harvested organs (e.g. , organs harvested for transplantation or for research (e.g., perfused organ models)), and as a result of surgery where blood flow is interrupted (e.g., open heart surgery and coronary bypass surgery).
  • Ischemia need not be limited to one organ; it can also be more generalized (e.g., in hemorrhagic shock).
  • ROS have been reported to be responsible for the severe damage caused by reperfusion of ischemic tissues and organs.
  • Metal ions can cause the production and accumulation of ROS.
  • copper and iron ions released from storage sites are one of the main causes of the production of ROS following injury, including ischemia/reperfusion injury and injury due to heat, cold, trauma, excess exercise, toxins, radiation, and infection. Roth, ⁇ 4ct ⁇ Chir. Hung., 36, 302-305 (1997).
  • Copper and iron ions, as well as other transition metal ions have been reported to catalyze the production of ROS. See, e.g., Stohs, . Basic Clin. Physiol.
  • albumin The antioxidant character of albumin has been attributed to several of albumin's many physiological functions, including albumin's ability to bind metals (particularly copper ions), to bind fatty acids, to bind and transport steroids, to bind and transport bilirubin, to scavenge HOC1, and others.
  • Albumin contains several metal binding sites, including one at the N-terminus.
  • N-terminal metal-binding sites of several albumins exhibit high- affinity for CU( ⁇ T) and Ni(H), and the amino acids involved in the high-affinity binding of these metal ions have been identified. See Laussac et al., Biochem., 23, 2832-2838 (1984); Predki et al., Biochem. J., 287, 211-215 (1992); Masuoka et al., J. Biol. Chem., 268, 21533- 21537 (1993).
  • albumin is, and is not, neuroprotective in animal models of cerebral ischemia. Compare Huh et ⁇ ., Brain Res., 804, 105-113 (1998) andRemmers 827, 237-242 (1999), with Little 9, 552- 558 (1981) and Beaulieu et al., J. Cereb. Blood Flow. Metab., 18, 1022-1031 (1998).
  • albumin had not been shown to be effective for cardioprotection. They further noted that the use of albumin in cardioplegia solutions could be unsafe due to possible allergic reactions and the risks associated with the use of blood products. Finally, although albumin has been characterized as an antioxidant, it has also been reported to enhance superoxide anion production by microglia (Si et al., GLIA, 21, 413-418 (1997)). This result led the authors to speculate that albumin leaking through the disrupted blood brain barrier in certain disorders potentiates the production of superoxide anion by microglia, and that this increased production of superoxide anion is responsible for the pathogenesis of neuronal damage in cerebral ischemia/reperfusion and some neurodegenerative diseases.
  • the N-terminal metal-binding sites of several albumins exhibit high- affinity for Cu(H) and Ni(lT). These sites have been studied extensively, and a general amino terminal Cu(H)- and Ni(H)-binding (ATCUN) motif has been identified. See, e.g., Harford and Sarkar, Ace. Chem. Res, 30, 123-130 (1997).
  • the ATCUN motif can be defined as being present in a protein or peptide which has a free -NH 2 at the N-terminus, a histidine residue in the third position, and two intervening peptide nitrogens. See, e.g., Harford and Sarkar, Ace. Chem. Res, 30, 123-130 (1997).
  • the ATCUN motif is provided by the peptide sequence Xaa Xaa His, where Xaa is any amino acid except proline. See, e.g., Harford and Sarkar, Ace. Chem. Res, 30, 123-130 (1997).
  • the Cu(H) and Ni(H) are bound by four nitrogens provided by the three amino acids of the ATCUN motif (the nitrogen of the free -NH 2 , the two peptide nitrogens, and an imidazole nitrogen of histidine) in a slightly distorted square planar configuration. See, e.g., Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997).
  • the sequence of the N-terminal metal-binding site of human serum albumin is Asp Ala His Lys [SEQ ID NO:l], and the free side-chain carboxyl of the N-terminal Asp and the Lys residue have been reported to be involved in the binding of Cu(ll) and Ni(II), in addition to the four nitrogens provided by Asp Ala His. See Harford and Sarkar, Ace. Chem. Res.,30, 123-130 (1997); Laussac etd ⁇ .,Biochem., 23, 2832- 2838 (1984); and Sadler et al., Eur. J. Biochem., 220, 193-200 (1994).
  • the ATCUN motif has been found in other naturally-occurring proteins besides albumins, and non-naturally-occurring peptides and proteins comprising the ATCUN motif have been synthesized. See, e.g., Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997); Bal et al., Chem. Res. Toxicol, 10, 906-914 (1997); Mlynarz, et al., Speciation 98: Abstracts, http://www.jate.u-szeged.hu/ ⁇ spec98/abstr/mlynar.html.
  • the invention provides a method of reducing the damage done by reactive oxygen species (ROS) in an animal.
  • the method comprises administering to the animal an effective amount of a metal-binding peptide having the formula P, - P 2 or a physiologically-acceptable salt thereof.
  • the invention further provides a method of reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • This method comprises contacting the cell, tissue or organ with a solution containing an effective amount of the peptide P, - P 2 or a physiologically-acceptable salt thereof.
  • the invention also provides a method of reducing the concentration of a metal in an animal in need thereof.
  • the method comprises administering to the animal an effective amount of a metal-binding peptide having the formula P , - P 2 or a physiologically-acceptable salt thereof.
  • the invention also provides a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and the peptide P ; - P 2 or a physiologically-acceptable salt thereof.
  • the invention provides a kit for reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • the kit comprises a container holding the peptide Pj - P 2 .
  • P, - P 2 In the formula P, - P 2 :
  • P is Xaa ! Xaa ⁇ is or Xaa, Xaa 2 His Xaa 3 ; and P 2 is (Xaa 4 ) n .
  • Xaa is glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), serine (Ser), threonine (Thr), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gin), lysine (Lys), hydroxylysine (Hylys), histidine (His), arginine (Arg), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine (Met) or ⁇ -hydroxymethylserine (HMS).
  • Xaa can be an amino acid which comprises a ⁇ -amino group (e.g., Orn, Lys) having another amino acid or a peptide attached to it (e.g., Gly ( ⁇ )-Orn).
  • Xaa is preferably Asp, Glu, Arg, Thr, or HMS. More preferably, Xaa, is Asp or Glu. Most preferably Xaa, is Asp.
  • Xaa 2 is Gly, Ala, ⁇ -Ala, Nal, Leu, He, Ser, Thr, Asp, Asn, Glu, Gin, Lys, Hylys, His, Arg, Orn, Phe, Tyr, Trp, Cys, Met or HMS.
  • Xaa 2 is preferably Gly, Ala, Nal, Leu, lie, Thr, Ser, Asn, Met, His or HMS. More preferably Xaa 2 is Ala, Nal, Thr, Ser, Leu, or HMS. Even more preferably Xaa 2 is Ala, Thr, Leu, or HMS. Most preferably Xaaj is Ala.
  • Xaa 3 is Gly, Ala, Nal, Lys, Arg, Orn, Asp, Glu, Asn, Gin, or Trp, preferably Lys.
  • Xaa 4 is any amino acid.
  • n is 0-100, preferably 0-10, more preferably 0-5, and most preferably 0.
  • at least one of the amino acids of P concentrate other than ⁇ -Ala when it is present is a D-amino acid.
  • the D-amino acid is Xaa,, His, or both.
  • Most preferably all of the amino acids of P concentrate other than ⁇ -Ala when it is present, are D- amino acids.
  • at least one of the amino acids of P concentrate other than ⁇ -Ala when it is present is a D-amino acid, and at least 50% of the amino acids of P 2 are also D-amino acids.
  • Most preferably all of the amino acids of P 2 are D-amino acids.
  • At least one amino acid of P , and/or P 2 is substituted with (a) a substituent that increases the lipophilicity of the peptide without altering the ability of P, to bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of P, to bind metal ions, or (c) a substituent which is a non-peptide, metal-binding functional group that improves the ability of the peptide to bind metal ions.
  • the invention provides another method of reducing the damage done by ROS in an animal.
  • the method comprises administering to the animal an effective amount of a metal- binding peptide (MBP) having attached thereto a non-peptide, metal-binding functional group.
  • MBP metal-binding peptide
  • the metal-binding peptide MBP may be any metal-binding peptide, not just P, - P 2 .
  • the invention further provides another method of reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • This method comprises contacting the cell, tissue or organ with a solution containing an effective amount of a metal-binding peptide MBP having attached thereto a non-peptide, metal-binding functional group.
  • the invention provides another method of reducing the concentration of a metal in an animal in need thereof.
  • the method comprises administering to the animal an effective amount of a metal-binding peptide MBP having attached thereto a non-peptide, metal- binding functional group.
  • the invention also provides a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a metal-binding peptide MBP having attached thereto a non-peptide, metal-binding functional group.
  • the invention also provides a kit for reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • the kit comprises a container holding a metal-binding peptide MBP having attached thereto a non-peptide, metal-binding functional group.
  • the invention provides yet another method of reducing the damage done by reactive oxygen species (ROS) in an animal.
  • the method comprises administering to the animal an effective amount of a metal-binding peptide dimer of the formula P 3 - L - P 3 , wherein each P 3 may be the same or different and is a peptide which is capable of binding a metal ion, and L is a chemical group which connects the two P 3 peptides through their C-terminal amino acids.
  • one or both of the two P 3 peptides is P,.
  • the invention further provides a method of reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • This method comprises contacting the cell, tissue or organ with a solution containing an effective amount of the metal-binding peptide dimer of the formula P 3 - L - P 3 .
  • the invention also provides a method of reducing the concentration of a metal in an animal in need thereof.
  • the method comprises administering to the animal an effective amount of the metal-binding peptide dimer of the formula P 3 - L - P 3 .
  • the invention also provides a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and the metal-binding peptide dimer of the formula P 3 - L - P 3 .
  • the invention provides a kit for reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal.
  • the kit comprises a container holding the metal-binding peptide dimer of the formula P 3 - L - P 3 .
  • the invention provides a peptide having the formula P, - P 2 , or a physiologically-acceptable salt thereof, wherein at least one amino acid of P concentrate other than ⁇ - Ala when it is present, is a D-amino acid.
  • the invention provides a peptide having the formula P, - P 2 , wherein P, is defined above, and P 2 is a peptide sequence which comprises the sequence of a metal- binding site.
  • the invention also provides a metal-binding peptide MBP having attached thereto a non-peptide, metal-binding functional group.
  • the invention provides the metal-binding peptide dimer of the formula P 3 - L - P 3 .
  • Figures 2A-B Schematic diagrams of the synthesis of derivatives of the tetrapeptide Asp Ala His Lys [SEQ ID NO:l] coming within the formula of Figure 1C ( Figure 2 A) and Figure IB ( Figure 2B).
  • Figure 3A-B Formulas of cyclohexane diamine derivatives.
  • Figures 3C-D Schematic diagrams of syntheses of cyclohexane diamine derivatives of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1].
  • Figure 4 Formula of a tetraacetic acid derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO:l].
  • Figure 5 Formula ofabispyridylethylamine derivative of the tetrapeptide Asp Ala
  • Figures 6A-B Formulas of mesoporphyrin IX with ( Figure 6B) and without ( Figure 6A) a bound metal ion M.
  • Figure 6C Formula of mesoporphyrin LX derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1].
  • Figure 7 Formulas of monosaccharides.
  • Figure 8 Diagram of a parabiotic blood perfusion system in which an isolated heart is perfused in the Langendorff mode with blood at 37°C from a support animal of the same species.
  • Figure 9 Diagram of the treatments of isolated perfused hearts with drug and saline in the parabiotic blood perfusion system illustrated in Figure 8.
  • Figure 10 Graph of contracture versus duration of ischemia showing the effect of a drug (D-Asp D-Ala D-His D-Lys) on contracture during ischemia in the blood-perfused rat heart model illustrated in Figures 8 and 9.
  • a drug D-Asp D-Ala D-His D-Lys
  • - ⁇ - is saline control
  • -o- is drug.
  • Figure 11 Graph of left ventricle diastolic pressure (LNDP; expressed as a percentage of the 20-minute pre-intervention baseline value) versus duration of reperfusion showing the effect of the drug D-Asp D-Ala D-His D-Lys on post-ischemic recovery of
  • - ⁇ - is saline control
  • -o- is drug.
  • Figure 12 Graph of left ventricle end diastolic pressure (LNEDP) versus duration of reperfusion showing the effect of the drug D-Asp D-Ala D-His D-Lys on post-ischemic recovery of LVEDP in the blood-perfused rat heart model illustrated in Figures 8 and 9. * indicates p ⁇ _ 0.05.
  • - ⁇ - is saline control
  • -o- is drug.
  • Figure 13 Graph of heart rate (expressed as a percentage of the 20-minute pre- intervention baseline value) versus duration of reperfusion showing the effect of the drug D- Asp D-Ala D-His D-Lys on post-ischemic recovery of heart rate in the blood-perfused rat heart model illustrated in Figures 8 and 9. * indicates p ⁇ _ 0.05.
  • - ⁇ - is saline control
  • -o- is drug.
  • Figure 14 Graph of perfusion pressure (expressed as a percentage of the 20-minute pre-intervention baseline value) versus duration of reperfusion showing the effect of the drug D-Asp D-Ala D-His D-Lys on post-ischemic recovery of perfusion pressure in the blood- perfused rat heart model illustrated in Figures 8 and 9.
  • - ⁇ - is saline control
  • -o- is drug.
  • Figure 15 A-B Graphs of absorbance at 532 nm (A532) versus incubation time in an assay for the production of hydroxyl radicals.
  • ⁇ ascorbate only
  • ⁇ copper and ascorbate
  • A tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]), copper and ascorbate (tetrapeptide/copper ratio of 1:1)
  • X tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1).
  • Figure 16 Graph of % inhibition versus concentration tetrapeptide (L-Asp L-Ala L- His L-Lys [SEQ ID NO:l])-copper complex at a tetrapeptide/copper ratio of 1:1 in the xanthine oxidase assay for superoxide dismutase activity.
  • Figure 17 Graph of absorbance at 560 nm (A560) versus time in an assay for superoxide radical production.
  • ⁇ ascorbate only
  • ⁇ copper and ascorbate
  • ⁇ tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l])
  • copper and ascorbate tetrapeptide/copper ratio of 1 : 1
  • X tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1).
  • Figure 18 Gel after electrophoresis of DNA treated in various ways.
  • Lane 1 - 17 ⁇ g/ml plasmid DNA (untreated control); Lane 2 - 17 ⁇ g/ml plasmid DNA and 50 ⁇ M CuCl 2 ; Lane 3 - 17 ⁇ g/ml plasmid DNA and 2.5 mM ascorbate; Lane 4 - 17 ⁇ g/ml plasmid DNA, 2.5 mM ascorbate, 50 ⁇ M CuCl 2 , and 200 ⁇ M tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]) (4:1 ratio tetrapeptide/copper); Lane 5 - 17 ⁇ g/ml plasmid DNA, 2.5 mM ascorbate, 50 ⁇ M CuCl 2 , and 100 ⁇ M tetrapeptide (2: 1 ratio tetrapeptide/copper); Lane 6-17 ⁇ g/ml plasmi
  • Figure 19 A Formulas of peptide dimers according to the invention.
  • Figures 19B-C Diagrams illustrating the synthesis of peptide dimers according to the invention.
  • Figure 20 TAE (tris acetic acid EDTA (ethylenediamine tetracetic acid)) agarose gel visualized with ethidium bromide showing attenuation of ROS -induced DNA double strand breaks in genomic DNA by D-Asp Ala His Lys.
  • TAE tris acetic acid EDTA (ethylenediamine tetracetic acid)
  • Figure 21 TAE agarose gel visualized with ethidium bromide showing attenuation of ROS-induced DNA double strand breaks in genomic DNA by D-Asp Ala His Lys.
  • Lane 1 no treatment; Lane 2 - CuCl 2 , 50 ⁇ M; Lane 3 - ascorbic acid, 500 ⁇ M; Lane 4 - D-Asp Ala His Lys, 200 ⁇ M; Lane 5 - CuCl 2 , 10 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 6 - CuCl 2 , 25 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 7 - CuCl 2 , 50 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 8 - CuCl 2 , 50 ⁇ M + ascorbic acid, 100 ⁇ M; Lane 9 - CuCl 2 , 50 ⁇ M + ascorbic acid, 250 ⁇ M; Lane 10 - CuCl 2 , 50 ⁇ M + ascorbic acid, 500 ⁇ M +
  • Figure 22 Southern Blot showing attenuation of ROS-induced DNA double strand breaks in telomere DNA by D-Asp Ala His Lys.
  • Lane 1 no treatment
  • Lane 2 CuCl 2 , 50 ⁇ M
  • Lane 3 ascorbic acid, 100 ⁇ M
  • Lane 4 D-Asp Ala His Lys, 200 ⁇ M
  • Lane 5 - CuCl 2 , 50 ⁇ M + ascorbic acid, 100 ⁇ M
  • Figure 23 Southern Blot showing attenuation of ROS-induced DNA double strand breaks in telomere DNA by D-Asp Ala His Lys.
  • Lane 1 no treatment
  • Lane 3 ascorbic acid, 500 ⁇ M
  • Lane 4 D-Asp Ala His Lys, 200 ⁇ M
  • Lane 5 - CuCl 2 , 50 ⁇ M + ascorbic acid, 100 ⁇ M
  • Lane 9 - CuCl 2 , 50 ⁇ M + ascorbic acid, 500 ⁇ M + D-Asp Ala His Lys, 100 ⁇ M.
  • the invention provides a peptide of the formula P, - P 2 P, is Xaa, Xaa j His or is Xaa,
  • P is a metal-binding peptide sequence that binds transition metal ions of Groups lb-7b or 8 of the Periodic Table of elements (including V, Co, Cr, Mo, Mn, Ba, Zn, Hg, Cd, Au, Ag, Co, Fe, Ni, and Cu) and other metal ions (including As, Sb and Pb).
  • the binding of metal ions by P inhibits (i.e., reduces or prevents) the production of ROS and/or the accumulation of ROS by these metal ions and/or targets the damage done by ROS that may still be produced by the bound metal ions to the peptide itself.
  • the damage that can be caused by ROS in the absence of the binding of the metal ions to P is reduced.
  • P binds Cu(H), Ni(H), Co(II), and Mn(II) with high affinity. It should, therefore, be particularly effective in reducing the damage caused by the production and accumulation of ROS by copper and nickel.
  • Xaa is most preferably Asp
  • Xaa 2 is most preferably Ala
  • Xaa 3 is most preferably Lys (see above).
  • the preferred sequences of P are Asp Ala His and Asp Ala His Lys [SEQ ID NO: I].
  • Most preferably the sequence of P, is Asp Ala His Lys [SEQ ID NO: 1].
  • Asp Ala His is the minimum sequence of the N-terminal metal-binding site of human serum albumin necessary for the high-affinity binding of Cu(H) and Ni(H), and Lys has been reported to contribute to the binding of these metal ions to this site.
  • Asp Ala His Lys [SEQ ID NO: 1] has been found by mass spectometry to bind Fe( ⁇ ) and to pass through a model of the blood brain barrier.
  • Other preferred sequences for P include Thr Leu His (the N-terminal sequence of human ⁇ -fetoprotein), Arg Thr His (the N-terminal sequence of human sperm protamin HP2) and HMS HMS His (a synthetic peptide reported to form extremely stable complexes with copper; see Mlynarz et al., Speciation 98: Abstracts, http://www.jate.u-szeged.hu/spec98/abstr/mlynar.html 4/21/98).
  • P 2 is (Xaa 4 ) n , wherein Xaa 4 is any amino acid and n is 0-100.
  • n is large (n > about 20)
  • the peptides will reduce the damage done by ROS extracellularly. Smaller peptides are better able to enter cells, and smaller peptides can, therefore, be used to reduce the damage done by ROS both intracellularly and extracellularly. Smaller peptides are also less subject to proteolysis. Therefore, in P 2 , preferably n is 0-10, more preferably n is 0-5, and most preferably n is 0.
  • P 2 may have any sequence
  • P 2 preferably comprises a sequence which (1) binds a transition metal, (2) enhances the ability of the peptide to penetrate cell membranes and/or reach target tissues (e.g., to be able to cross the blood brain barrier), or (3) otherwise stabilizes or enhances the performance of the peptide.
  • P 2 may comprise the sequence of one or more of the metal-binding sites of these peptides.
  • P 2 comprises a metal-binding site
  • it preferably has a sequence which includes a short spacer sequence between P, and the metal binding site of P 2 , so that the metal-binding sites of P, and P 2 may potentially cooperatively bind metal ions (similar to a 2:1 peptide:metal complex; see Example 10).
  • the spacer sequence is composed of 1-5, preferably 1 -3, neutral amino acids.
  • the spacer sequence may be Gly, Gly Gly, Gly Ala Gly, Pro, Gly Pro Gly, etc.
  • P 2 when P 2 comprises a metal-binding site, it preferably comprises one of the following sequences: (Xaa 4 ) m Xaa 5 Xaa 2 His Xaa 3 or (Xaa 4 ) m Xaa 5 Xaa 2 His.
  • Xaa ⁇ Xaa 3 and Xaa 4 are defined above, and m is 0-5, preferably 1 -3.
  • Xaa 4 is preferably a neutral amino acid (see the previous paragraph).
  • Xaa 5 is an amino acid which comprises a ⁇ -amino group (preferably Orn or Lys, more preferably Orn) having the Xaa 4 amino acid(s), if present, or P, attached to it by means of the ⁇ -amino group.
  • a ⁇ -amino group preferably Orn or Lys, more preferably Orn
  • P, - P 2 could be Asp Ala His Gly Gly ( ⁇ )-Orn Ala His [SEQ ID NO:2].
  • P 2 may comprise one of the following sequences : [(Xaa 4 ) m Xaa 5 Xaa j His Xaa 3 ] r , [(Xaa 4 ) m Xaa 5 Xaa 2 His] r , [(Xaa 4 ) m Xaa 5 Xaa 2 His Xaa 3 (Xaa 4 ) m Xaa 5 Xaa 2 His] flesh and [(Xaa 4 ) m Xaa 5 Xaa 2 His(Xaa 4 ) m Xaa 5 Xaa 2 His Xaa 3 ] r , wherein Xaa 2 , Xaa 3 , Xaa 4 , Xaa 5 and m are defined and described above, and r is 2- 100. In this manner metal-binding polymers may be formed.
  • P 2 comprises a peptide sequence that can bind Cu(I).
  • Cu(H) is converted to Cu(I) in the presence of ascorbic acid or other reducing agents, and the Cu(I) reacts with oxygen to produce ROS (see equations in Examples 10 and 11).
  • P can bind Cu( ⁇ ) tightly (see above) and is very effective by itself in inhibiting the production of ROS by copper (see Examples 7-11).
  • Peptide sequences which can bind Cu(I) are known in the art. See, e.g, Pickering et al., J. Am. Chem.
  • the Cu(I)-binding peptide comprises the sequence Cys Xaa 4 Xaa 4 Cys (e.g., Gly Met Xaa 4 Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9]).
  • Cys Xaa 4 Xaa 4 Cys e.g., Gly Met Xaa 4 Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9]
  • P 2 is preferably hydrophobic or an arginine oligomer (see Rouhi, Chem. & Eng. News, 49-50 (January 15, 2001)).
  • P 2 is hydrophobic, it preferably contains 1-3 hydrophobic amino acids (e.g. , Gly Gly), preferably D-amino acids.
  • a hydrophobic P 2 may be particularly desirable for uses of P, - P 2 where P, - P 2 must cross the blood brain barrier.
  • the arginine oligomer preferably contains 6-9 Arg residues, most preferably 6-9 D-Arg residues (see Rouhi, Chem. & Eng. News, 49-50 (January 15, 2001).
  • the use of a P 2 which is an arginine oligomer may be particularly desirable when P, - P 2 is to be administered topically or transdermally.
  • the amino acids of the peptide may be L-amino acids, D-amino acids, or a combination thereof.
  • at least one of the amino acids of P is a D-amino acid (preferably Xaa, and/or His), except for ⁇ -Ala, when present.
  • all of the amino acids of P, , other than ⁇ -Ala, when present, are D-amino acids.
  • preferably about 50% of the amino acids of P 2 are D-amino acids, and most preferably all of the amino acids of P 2 are D-amino acids.
  • D-amino acids are preferred because peptides containing D-amino acids are resistant to proteolytic enzymes, such as those that would be encountered upon administration of the peptide to an animal (including humans) or would be present in an excised organ perfused with a solution containing the peptide. Also, the use of D-amino acids would not alter the ability of the peptide to bind metal ions, including the ability of the peptide to bind copper with high affinity.
  • the peptides of the invention may be made by methods well known in the art.
  • the peptides whether containing L-amino acids, D-amino acids, or a combination of L- and D-amino acids, may be synthesized by standard solid-phase peptide synthesis methods. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polypeptides. pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc. 85, 2149 (1963); Davis et al., Biochem. Int'l.
  • the peptides may be synthesized by recombinant DNA techniques if they contain only L-amino acids.
  • Recombinant DNA methods and suitable host cells, vectors and other reagents for use therein, are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).
  • the invention further comprises derivatives of the peptide P, - P 2 , whether composed of L-amino acids, D-amino acids, or a combination of L- and D-amino acids, which are more resistant to proteolytic enzymes, more lipid soluble (to allow the peptides to more readily penetrate cell membranes and/or reach target organs, such as the brain), or both.
  • P can be modified in the regions indicated by the arrows without altering the metal binding function of P,.
  • P can be substituted at carbons 1 or 2 with R, , and the terminal -COOH of P , can be substituted with protecting group R 2 ( Figures 1B-D).
  • P 2 can be modified in ways similar to those described for P, to make P 2 more resistant to proteolytic enzymes, more lipid soluble, or both.
  • R can be a straight-chain or branched-chain alkyl containing from 1 to 16 carbon atoms, and the term “alkyl” includes the R and S isomers.
  • R can also be an aryl or heteroaryl containing 1 or 2 rings.
  • aryl means a compound containing at least one aromatic ring (e.g., phenyl, naphthyl, and diphenyl).
  • heteroaryl means an aryl wherein at least one of the rings contains one or more atoms of S, N or O.
  • substituents such as a n-butyl attached to carbon 2 (see Figure IC, R, is n-butyl) should increase the affinity of the peptide for metal ions, such as copper, due to the inductive effect of the alkyl group.
  • substituents such as a n-butyl attached to carbon 2 (see Figure IC, R, is n-butyl) should increase the affinity of the peptide for metal ions, such as copper, due to the inductive effect of the alkyl group.
  • Substitution of carbon 2 ( Figure IC) with an aryl, heteroaryl, or a long chain alkyl (about 6-16 carbon atoms) should enhance transport of the peptide across lipid membranes.
  • the derivative of P illustrated in Figure IC, wherein R, is octyl
  • Figure 2A the elliptical element represents the polymer resin and R p is a standard carboxyl protecting group.
  • octanoic acid freshly distilled
  • the mixture is heated to about 100°C and kept at that temperature for 4 hours.
  • ⁇ -Bromooctanoic acid is obtained as a colorless liquid upon distillation.
  • Amination of the bromoacid is achieved by allowing the acid and an ammonia solution to stand at 40-50° C for 30 hours.
  • the octyl derivative of the amino acid is obtained by removing ammonium bromide with methanol washes.
  • Classical resolution methods give the desired optically-pure D-form.
  • Other derivatives wherein R, is an alkyl, aryl or heteroaryl can be prepared in the manner illustrated in Figure 2 A.
  • the derivative of P, illustrated in Figure IB, wherein R, is phenyl can be prepared as illustrated in Figure 2B.
  • Polymer is the resin
  • t-Bu is t-butyl
  • Bz is benzyl.
  • R 2 can be -NH 2 , -NHR réelle -N(R,) 2 , -OR,, or R, (see Figure ID), wherein R, is defined above.
  • R 2 can be prepared as the last step of a solid-phase peptide synthesis before the peptide is removed from the resin by methods well known in the art. Substitutions with R 2 do not substantially decrease the ability of P, to bind metal ions.
  • P , and P 2 can be substituted with non-peptide functional groups that bind metal ions.
  • These metal-binding functional groups can be attached to one or more pendent groups of the peptide, and the resulting peptide derivatives will possess one or more sites that are capable of binding metal ions, in addition to the binding site provided by P, and, optionally, the binding site provided by P 2 .
  • the ability of such peptide derivatives to bind metal ions is improved as compared to the corresponding unmodified peptide.
  • the peptide derivative can bind two of the same type of metal ion instead of one (e.g., two Cu(II)), the peptide derivative can bind two different metal ions instead of one type of metal ion (e.g., one Cu(H) and one Fe(IT ⁇ )), or the peptide derivative can bind one metal ion better (e.g. , with greater affinity) than the corresponding unmodified peptide.
  • Metal-binding functional groups include polyamines (e.g. , diamines, triamines, etc.).
  • a particularly preferred diamine is 1,2-diaminocyclohexane ( Figures 3A-B).
  • Previous studies carried out by Rao & P. Williams have shown that a cyclohexane diamine derivative ( Figure 3 A, where PYR is pyridine) binds to a variety of metal ions.
  • the resulting metal chelator has been successfully used to resolve amino acids and peptides, showing that the molecule has a very high affinity for ⁇ -amino acids, forming a very stable coordination complex, which is unique in many respects.
  • 1,2- Diaminocyclohexane possesses a reactive amino functional group to which a peptide of the invention can be attached.
  • R 4 is -alkyl-CO-peptide, -aryl-CO-peptide, -aryl-alkyl-CO-peptide, or -alkyl-aryl-CO-peptide (see also Figures 3C-D).
  • the other R 4 may be the same or may be -alkyl-COOH, -aryl-COOH, -aryl-alkyl-COOH, or alkyl-aryl-COOH.
  • Derivatives of the type shown in Figure 3B will have several metal-binding sites and can, therefore, be expected to bind metal ions more readily than the unsubstituted peptide. Further, due to the presence of the cyclohexane functionality, the compound will possess lipid-like characteristic which will aid its transport across lipid membranes.
  • Cyclohexane diamine derivatives of the peptides of the invention can be prepared by two distinct routes. The first involves initial condensation with an aldehyde followed by reduction (see Figure 3C; in Figure 3C Bz is benzyl). A number of aldehydes (alkyl and aryl) react readily with cyclohexane diamine at room temperature, forming an oxime. The oxime can be reduced with sodium borohydride under anaerobic conditions to give the diacid derivative. The carboxyl moieties are then reacted with the free amino groups present in carboxy-protected P, to give the cyclohexane diamine derivative of the peptide.
  • the second route is a direct alkylation process which is illustrated in Figure 3D.
  • cyclohexane diamine is treated with bromoacetic acid to give the diacetic acid derivative.
  • the carboxyl moieties are then reacted with the free amino groups present in carboxy- protected P, to give the derivative.
  • R 5 is H or another peptide.
  • the derivative can be further reacted to produce typical carboxylic acid derivatives, such as esters, by methods well known in the art. Metal binding experiments have indicated that the presence or absence of this group does not have a bearing on the metal binding capacity of the whole molecule.
  • vicinal diacids bind to metal ions, and the affinity for copper is particularly high. It is therefore envisaged that a peptide having a vicinal diacid functional group will be extremely effective in metal binding.
  • Suitable vicinal diacids include any 1 ,2- alkyldiacid, such as diacetic acid (succinic acid), and any 1,2-aryldiacid.
  • the amino groups of the peptide can be reacted with diacetic acid to produce a diacid derivative (see Figure 4).
  • This can be conveniently accomplished by reacting the amino groups of the resin-bound peptide with a halogenated acetic acid (e.g., bromoacetic acid or chloroacetic acid) or a halogenated acetic acid derivative (e.g., benzyloxy ester).
  • a halogenated acetic acid e.g., bromoacetic acid or chloroacetic acid
  • a halogenated acetic acid derivative e.g., benzyloxy ester
  • Polyaminopolycarboxylic acids are known to bind metals, such as copper and iron. Suitable polyaminopolycarboxylic acids for making derivatives of the peptides of the invention and methods of attaching them to peptides are described in U.S. Patents Nos. 5,807,535 and 5,650,134, and PCT application WO 93/23425, the complete disclosures of which are incorporated herein by reference. See also, U.S. Patent No. 5,739,395.
  • Vicinal polyhydroxyl derivatives are also included in the invention. Suitable vicinal polyhydroxyls include monosaccharides and polysaccharides (i.e., disaccharide,trisaccharide, etc.). Presently preferred are monosaccharides. See Figure 7. The monosaccharides fall into two major categories - furanoses and pyranoses. One of the prime examples of a furanose ring system is glucose. The hydroxyl groups of glucose can be protected as benzyl or labile t-butyloxy functional groups, while leaving the aldehyde free to react with an amine group (e.g., that of lysine) of the tetrapeptide.
  • an amine group e.g., that of lysine
  • Porphyrins are a group of compounds found in all living matter and contain a tetrapyrrolic macrocycle capable of binding to metals. Heme, chlorophyll and corrins are prime examples of this class of compounds containing iron, magnesium and cobalt, respectively.
  • Mesoporphyrin LX ( Figure 6A-B, where M is a metal ion) is derived from heme and has been observed to possess specific affinity for copper. Addition of this structure to a peptide of the invention would produce a porphyrin-peptide derivative possessing several sites for binding of copper (see Figure 6C).
  • the imidazole residues at positions 3 and 3' of the tetrapeptide shown in Figure 6C may provide a binding site for metals other than copper, thereby stabilizing the po ⁇ hyrin-metal complex.
  • cyanocobalamine vitamin B-12
  • the porphyrin-tetrapeptide derivative would bind cobalt (or other metals) at normal physiological conditions in the prophyrin nucleus and that the complex would be stabilized by the His imidazole groups.
  • the carboxyl groups of mesoporphyrin LX can be activated and coupled with the amino groups of the peptide employing standard solid-phase peptide synthesis.
  • the free amino group of the lysine residue of the resin-bound peptide can be coupled with carboxy activated porphyrin nucleus.
  • the condensation product can be cleaved off the resin using standard methods. This method can be used to synthesize other po ⁇ hyrin derivatives of peptides of the invention.
  • Dithiocarbamates are known to bind metals, including iron. Suitable dithiocarbamates for making derivatives of the peptides of the invention are described in U.S.
  • Patents Nos. 5,380,747 and 5,922,761 the complete disclosures of which are inco ⁇ orated herein by reference.
  • Hydroxypyridones are also known to be iron chelators. Suitable hydroxypyridones for making derivatives of the peptides of the invention are described in U.S. Patents Nos.
  • MBP contains from 2-10, more preferably 3-5, amino acids.
  • MBP contains one or more D-amino acids; most preferably all of the amino acids of MBP are D-amino acids.
  • sequences of many metal-binding peptides are known. These peptides and peptides comprising the metal-binding sites of these peptides can be prepared in the same ways as described above for peptide P, - P 2 . Derivatives of these peptides having one or more metal- binding functional group attached to the peptide can be prepared in the same ways as described above for derivatives of peptide P, - P 2 .
  • the invention also provides metal-binding peptide dimers of the formula: P 3 - - P 3 .
  • P 3 is any peptide capable of binding a metal ion, and each P 3 may be the same or different. Each P 3 preferably contains 2- 10, more preferably 3-5, amino acids. As described above, metal-binding peptides are known, and each P 3 may comprise the sequence of one or more of the metal-binding sites of these peptides. Although each P 3 may be substituted as described above for P, and P 2 , including with a non-peptide, metal-binding functional group, both P 3 peptides are preferably unsubstituted. P 3 may also comprise any amino acid sequence substituted with a non-peptide, metal-binding functional group as described above to provide the metal-binding capability of P 3 .
  • each P 3 is an unsubstituted metal-binding peptide (i.e., an unsubstituted peptide comprising a peptide sequence which binds metal ions).
  • P is an unsubstituted metal-binding peptide
  • the dimers have the sequence P 3 - L - P réelle P, - L - P 3 or, most preferably, P, - L - P,). P, is defined above.
  • L is a linker which is attached to the C-terminal amino acid of each P 3 .
  • L may be any physiologically-acceptable chemical group which can connect the two P 3 peptides through their C-terminal amino acids.
  • physiologically-acceptable is meant that a peptide dimer containing the linker L is not toxic to an animal (including a human) or an organ to which the peptide dimer is administered as a result of the inclusion of the linker L in the peptide dimer.
  • L links the two P 3 groups so that they can cooperatively bind metal ions (similar to a 2:1 peptide:metal complex; see Example 10).
  • L is also preferably neutral.
  • L is a straight-chain or branched-chain alkane or alkene residue containing from 1-18, preferably from 2-8, carbon atoms (e.g., -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 (CH 3 )CH 2 -, -CHCH-, etc.) or a cyclic alkane or alkene residue containing from 3-8, preferably from 5-6, carbon atoms (see Figure 19A, compound D,), preferably attached to a P 3 by means of an amide linkage.
  • Such linkers are particularly preferred because they impart hydrophobicity to the peptide dimers.
  • L is a nitrogen- containing heterocyclic alkane residue (see Figure 19 A, compounds D 2 , D 3 and D 4 ), preferably a piperazide (see Figure 19A, compound D 2 ).
  • L is a glyceryl ester (see Figure 19 A, compound D 5 ; in formula D 5 , R is an alkyl or aryl containing, preferably containing 1-6 carbon atoms).
  • L could be a metal-binding po ⁇ hyrin (see Figure 6C).
  • a peptide dimer where each peptide has the sequence Asp Ala His Lys, [SEQ ID NO: 1] can be synthesized by coupling protected lysines to a free diamine functional group, either as an acid chloride or by using standard coupling agents used in peptide synthesis (see Figures 19B-C).
  • a free diamine functional group either as an acid chloride or by using standard coupling agents used in peptide synthesis (see Figures 19B-C).
  • Many suitable diamines are available commercially or suitable diamines can be readily synthesized by methods known in the art.
  • the lysine dimer 2 ( Figure 19B) can be prepared as follows. To a stirred solution of 9-fluorenylmethyloxycarbonyl (Fmoc)- andt-benzyloxycarbonyl(Boc)- protected D-Lys (Fmoc-D-Lys(Boc)-OH) (20 mmole) in dry dimethylformamide (DMF; 100 mL; dry argon flushed) are added butane- 1,4-diamine 1 and 2-(lH-benzotriazole-l-yl)-l,2,3,3- tetramethyluroniumtetrafluoroborate (TBTU; 0.5 mmole).
  • DMF dry dimethylformamide
  • TBTU 2-(lH-benzotriazole-l-yl)-l,2,3,3- tetramethyluroniumtetrafluoroborate
  • the solution is stirred for 36 hours at room temperature.
  • the bis-protected lysine 2 is isolated by flash chromatography over silica and elution with mixtures of ethyl acetate/methanol.
  • the peptide dimer 3 is then prepared from the protected lysine dimer 2 employing classical peptide synthesis methodology (see Figure 19B).
  • Another peptide dimer where each peptide has the sequence Asp Ala His Lys [SEQ ID NO:l], can be synthesized as follows. First, a different protected lysine dimer 4 is synthesized by acylating the two amino centers of a piperazine 5 (see Figure 19C; see also Chambrieretal., Proc. Natl Acad. Sci., 96, 10824-10829 (1999)). Then, the remainder of the amino acid residues are added employing standard peptide synthesis methodology to give the peptide dimer 6 (see Figure 19C).
  • Peptide dimers where each peptide has the sequence Asp Ala His Lys [SEQ ID NO: 1 ] and where L is a glyceryl ester, can be synthesized as follows.
  • a lysine diester 8, wherein R is methyl, can be prepared as follows (see Figure 19D).
  • the bis-protected lysine 8 is isolated by flash chromatography over silica and elution with mixtures of ethyl acetate/methanol.
  • the peptide dimer 9 is then prepared from the protected lysine dimer 8 employing classical peptide synthesis methodology (see Figure 19D).
  • physiologically-acceptable salts of the metal-binding compounds are also included in the invention.
  • Physiologically-acceptable salts include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids (such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, glutamic, benzoic, salicylic, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation).
  • the salts are prepared in a conventional manner, e.g., by neutralizing the free base form of the compound with an acid.
  • a metal-binding compound of the invention can be used to reduce the damage done by ROS or to reduce the metal ion concentration in an animal in need thereof.
  • a metal-binding compound of the invention is administered to the animal.
  • the animal is a mammal, such as a rabbit, goat, dog, cat, horse or human.
  • Effective dosage forms, modes of administration and dosage amounts for the various compounds of the invention may be determined empirically, and making such determinations is within the skill of the art. It has been found that an effective dosage is from about 2 to about 200 mg/kg, preferably from about 10 to about 40 mg kg, most preferably about 20 mg/kg.
  • the dosage amount will vary with the particular metal-binding compound employed, the disease or condition to be treated, the severity of the disease or condition, the route(s) of administration, the rate of excretion of the compound, the duration of the treatment, the identify of any other drugs being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts.
  • a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • the daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment.
  • the effective daily dose may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Administration of the compound should be continued until an acceptable response is achieved.
  • the compounds of the present invention maybe administered to an animal patient for therapy by any suitable route of administration, including orally, nasally, rectally, vaginally, parenterally (e.g., intravenously, intraspinally, intraperitoneally, subcutaneously, or intramuscularly), intracisternally, transdermally, transmucosally, intracranially, intracerebrally, and topically (including buccally and sublingually).
  • suitable routes of administration are orally, intravenously, and topically.
  • compositions of the invention comprise a metal-binding compound or compounds of the invention as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, with one or more other compounds, drugs or other materials.
  • Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the animal.
  • Pharmaceutically-acceptable carriers are well known in the art. Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington's Pharmaceutical Sciences.
  • Formulations of the invention suitable for oral administration maybe in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in- water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds of the present invention as an active ingredient.
  • a compound or compounds of the present invention may also be administered as bolus, electuary or paste.
  • h solid dosage forms of the invention for oral administration capsules, tablets, pills, dragees, powders, granules and the like
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) abso ⁇ tion accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding optionally with one or more accessory ingredients.
  • Compressed tablets maybe prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent.
  • Molded tablets maybe made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • opacifying agents include polymeric substances and waxes.
  • the active ingredient can also be in microencapsulated form.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents,
  • Suspensions in addition to the active compound(s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical, transdermal or transmucosal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active compound(s) maybe mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a compound or compound(s) of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound or compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • the active ingredient i e., a metal-binding compound or compounds of the invention
  • the active ingredient may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal patches, wherein the active ingredient is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin.
  • the active ingredient is typically contained in a layer, or "reservoir," underlying an upper backing layer.
  • the laminated device may contain a single reservoir, or it may contain multiple reservoirs.
  • the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
  • the backing layer in these laminates which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility.
  • the material selected for the backing material should be selected so that it is substantially impermeable to the active ingredient and any other materials that are present.
  • the backing layer may be either occlusive or nonocclusive, depending on whether it is desired that the skin become hydrated during drug delivery.
  • the backing is preferably made of a sheet or film of a preferably flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, polyesters, and the like.
  • the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the drug reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin.
  • the release liner should be made from a drug/vehicle impermeable material.
  • Transdermal drug delivery devices maybe fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, active ingredient and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer. Alternatively, the drug reservoir may be prepared in the absence of active ingredient or excipient, and then loaded by "soaking" in a drug/vehicle mixture.
  • the laminated transdermal drug delivery systems may, in addition, contain a skin permeation enhancer. That is, because the inherent permeability of the skin to some active ingredients maybe too low to allow therapeutic levels of the drug to pass through a reasonably sized area of unbroken skin, it is necessary to coadminister a skin permeation enhancer with such drugs. Suitable enhancers are well known in the art.
  • compositions of the invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known intheartofpharmaceuticalformulationandmaybepreparedas solutions in saline, employing benzyl alcohol or other suitable preservatives, abso ⁇ tion promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and/or other conventional solubilizing or dispersing agents.
  • Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives.
  • Creams containing the selected active agent are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil.
  • Cream bases are water- washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
  • the specific ointment or cream base to be used is one that will provide for optimum drug delivery.
  • an ointment base should be inert, stable, nonirritating and nonsensitizing.
  • Formulations for buccal administration include tablets, lozenges, gels and the like.
  • buccal administration can be effected using a transmucosal delivery system as known to those skilled in the art.
  • compositions of this invention suitable for parenteral administrations comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which maybe reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions.
  • prolonged abso ⁇ tion of the inj ectable pharmaceutical form may be brought about by the inclusion of agents which delay abso ⁇ tion such as aluminum monosterate and gelatin.
  • agents which delay abso ⁇ tion such as aluminum monosterate and gelatin.
  • the rate of abso ⁇ tion of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed abso ⁇ tion of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable fo ⁇ nulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets of the type described above.
  • ROS have been reported to play a major role in a variety of diseases and conditions. See Manso, Rev. Port.
  • the metal-binding compounds of the invention can be used to treat any of these diseases and conditions and other diseases and conditions in which ROS or metal ions play a role by administering a metal-binding compound of the invention as described above.
  • Treatment and variations thereof are used herein to mean to cure, prevent, ameliorate, alleviate, inhibit, or reduce the severity of a disease or condition or of at least some of the symptoms or effects thereof.
  • Specific diseases and conditions that are treatable with the metal-binding compounds of the invention include adult respiratory distress syndrome, aging, AIDS, angiogenic diseases, artherosclerosis (hypertension, senility and impotence), arthritis, asthma, autoimmune diseases, cancer (e.g., kidney, liver, colon, breast, gastrointestinal and brain), carcinogenesis, cellular damage caused by ionizing radiation (e.g., radiation of tumors), chronic granulomatous disease, cirrhosis, colitis, Crohn's disease, cystic fibrosis, degenerative diseases of aging, diabetes (diabetic retinopathy, renal disease, impotence and peripheral vascular disease), eye diseases (e.g., cataracts, central artery occlusion, benign monoclonal gammopathy, and macular degeneration), em
  • ischemic conditions and diseases treatable with the metal-binding compounds of the invention include: Central nervous system ischemia -
  • Stroke thrombotic, embolic or hemorrhagic cerebrovascular accident
  • Transplantation surgery both the donor organ and the recipient of the organ
  • Surgical ischemia of the limbs Surgical ischemia of the limbs (tourniquet injury).
  • An angiogenic disease or condition is a disease or condition involving, caused by, exacerbated by, or dependent on angiogenesis.
  • Angiogenesis is the process of new blood vessel formation in the body. Copper is required for angiogenesis . See PCT application WO 00/21941 and "The Role Of Copper In The Angiogenesis Process (http://www.cance ⁇ rotocol.com/role_of_copper.html. 1/28/02), and references cited in both of them.
  • copper is involved in the activation of growth factors (such as the dimerization of b-FGF and serum Cu 2+ -GHK), activation of angiogenic factors (such as Cu 2+ - (K)GHK derived from SPARC), cross-linking of the transitional matrix (e.g., collagens NIH and I by Cu 2+ -dependent lysyl oxidase), and formation of basement membrane (e.g. , collagens IN and elastin by Cu 2+ -dependent lysyl oxidase).
  • growth factors such as the dimerization of b-FGF and serum Cu 2+ -GHK
  • activation of angiogenic factors such as Cu 2+ - (K)GHK derived from SPARC
  • cross-linking of the transitional matrix e.g., collagens NIH and I by Cu 2+ -dependent lysyl oxidase
  • basement membrane e.g. , collagens IN and elastin by Cu 2+
  • angiogenic diseases and conditions treatable with the metal-binding compounds of the invention include neoplastic diseases (e.g., tumors (e.g., tumors of the bladder, brain, breast, cervix, colon, rectum, kidney, lung, ovary, pancreas, prostate, stomach and uterus) and tumor metastasis), benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyrogenic granulomas), hypertrophy (e.g.
  • tumors e.g., tumors of the bladder, brain, breast, cervix, colon, rectum, kidney, lung, ovary, pancreas, prostate, stomach and uterus
  • benign tumors e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyrogenic granulomas
  • hypertrophy e.g.
  • cardiac hypertrophy induced by thyroid hormone connective tissue disorders (e.g., rheumatoid arthritis and atherosclerosis), psoriasis, ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, and rabeosis), cardiovascular diseases, cerebral vascular diseases, endometriosis, polyposis, obesity, diabetes-associated diseases, hemophiliac joints, and immune disorders (e.g., chronic inflammation and autoimmunity).
  • the metal-binding compounds of the invention can also be used to inhibit the vascularization required for embryo implantation, thereby providing a method of birth control.
  • the metal-binding compounds of the invention may be used to reduce copper levels in cancer patients.
  • the metal-binding compounds of the invention may also be used to inhibit (reduce or prevent) carcinogenesis in individuals at risk (e.g., metal-exposed individuals, such as welders, machinists, autobody repairmen, etc.).
  • Specific inflammatory diseases and conditions treatable with the metal-binding compounds of the invention include acute respiratory distress syndrome, allergies, arthritis, asthma, autoimmune diseases, bronchitis, cancer, Crohn's disease, cystic fibrosis, emphysema, endocarditis, gastritis, inflammatory bowel disease, ischemia reperfusion, multiple organ dysfunction syndrome, nephritis, pancreatitis, respiratory viral infections, sepsis, shock, ulcerative colitis and other inflammatory disorders.
  • Acidosis is present in, or plays a role in, a number of diseases and conditions, including hypoventilation, hypoxia, ischemia, prolonged lack of oxygen, severe dehydration, diarrhea, vomiting, starvation, AIDS, sepsis, kidney disease, liver disease, metabolic diseases (e.g., advanced stages of diabetes mellitus), and neurodegenerative diseases (e.g., Alzheimer's). Acidosis is also caused by certain medications (e.g., large amounts of aspirin and oral medications used to treat diabetes), and instances of mild acidosis have been reported to increase with age (Knight,”Metal Heads," New Scientist, 8/26/00, http://www.purdeyenvironment.com/Full%20New%20scientistOO 1.htm).
  • the metal-binding compounds of the invention may be used to treat diseases or conditions involving acidosis to prevent damage due to ROS, to prevent other deleterious effects of free copper, or both. Sepsis can also be treated using the metal-binding compounds of the invention. Sepsis is a systemic inflammatory response to infection. Sepsis is also characterized by ischemia (caused by coagulopathy and suppressed fibrinolysis) and acidosis.
  • a compound of the invention is preferably administered prophylactically.
  • a compound of the invention is preferably administered prior to and/or simultaneously with reperfusion of an ischemic tissue or organ (e.g., prior to and/or simultaneously with angioplasty or treatment with clot dissolving drugs, such as tissue plasminogen activators).
  • administration of a compound of the invention should be continued for a period of time after reperfusion has been achieved.
  • a compound of the invention should be administered prior to and/or during surgery (e.g., open-heart surgery or surgery to transplant an organ into an animal), and administration of the compound should be continued for a period of time after the surgery.
  • a compound of the invention can be administered to a patient presenting symptoms of a serious condition (e.g., cerebrovascular ischemia or cardiovascular ischemia) while the patient is tested to diagnose the condition.
  • a serious condition e.g., cerebrovascular ischemia or cardiovascular ischemia
  • treatment with the metal-binding compounds of the invention may also prolong the time during which other therapies (e.g., administration of tissue plasminogen activator for cerebrovascular ischemia) can be administered.
  • a compound of the invention can be administered at the time a patient is to undergo radiation therapy (e.g. , radiation for a tumor or prior to a bone marrow transplant).
  • a compound of the invention can also be used to treat patients who have suffered blunt trauma.
  • a compound of the present invention may be very beneficial in treating patients suffering from multiple blunt trauma who have a low albumin level, since it has been found that a low albumin level is a predictor of mortality in such patients. More specifically, 34 patients suffering from multiple blunt trauma were studied. These patients were admitted to the intensive care unit of Swedish Hospital, Denver, CO in 1998. Two groups of patients were matched by a trauma surgeon by age, injury severity score (ISS), and type and area of injury without knowledge of the albumin levels of the patients. One group was composed of the patients who died, and the other group was composed of survivors.
  • ISS injury severity score
  • the admission albumin levels were retrieved from the medical records by an independent observer, and the albumin levels of the two groups were compared.
  • the mean albumin level was 3.50 ; .OO g/dl.
  • the mean albumin level was 2.52 + 0.73 g/dl.
  • the % variance was 28.6 and 28.9, respectively, and the p-value was 0.0026 (95% confidence interval 0.3462 - 0.4771).
  • the compounds of the invention may be given alone to reduce the damage done by
  • Free radical scavengers include superoxide dismutase, catalase, glutathione peroxidase, ebselen, glutathione, cysteine, N-acetyl cysteine, penicillamine, allopurinol, oxypurinol, ascorbic acid, ⁇ -tocopherol, Trolox (water-soluble ⁇ -tocopherol), ⁇ - carotene, fatty-acid binding protein, fenozan, probucol, cyanidanol-3, dimercaptopropanol, indapamide, emoxipine, dimethyl sulfoxide, and others.
  • the compounds of the invention can also be given in combination with another metal-binding peptide or non-peptide chelator (suitable metal-binding peptides and non-peptide chelators are described above and others are known in the art). For instance, a peptide P, ( .
  • a peptide P could be given in combination with a separate peptide or non-peptide chelator capable of binding iron. Suitable iron-binding peptides and non-peptide chelators are described above and others are known in the art (e.g., deferoxamine mesylate).
  • the compounds of the invention can, of course, also be given along with standard therapies for a given conditions (e.g., insulin to treat diabetes).
  • the metal-binding compounds of the invention can also be used to reduce the damage done by ROS to a cell, a tissue or organ that has been removed from an animal.
  • a solution containing an effective amount of a metal-binding compound of the invention is contacted with a solution containing an effective amount of a metal-binding compound of the invention.
  • Many suitable solutions are known. See, e.g., Dunphy et al., Am. J. Physiol, 276, H1591-H1598 (1999); Suzer et al., Pharmacol Res., 37, 97-101 (1998); Hisatomi et al., Transplantation, 52, 754- 755 (1991); U.S. Patent No. 5,710,172.
  • Effective amounts of the metal-binding compound to include in such solutions can be determined empirically, and doing so is within the skill in the art.
  • the harvested tissue or organ may subsequently be used for transplantation into a recipient or for research pu ⁇ oses (e.g. , using a perfused liver to screen drugs).
  • the metal- binding compounds of the invention can be used alone or can be used in combination with a free radical scavenger or another metal-binding compound (see above).
  • Cells isolated from an animal can be stored or cultured in a medium containing an effective amount of a metal-binding compound of the invention. Many suitable media are known. Effective amounts of the metal-binding compound to include in the medium can be determined empirically, and doing so is within the skill in the art.
  • the cells may be administered to a recipient in need thereof (e.g., for cellular immunotherapy or gene therapy) or may be used for research pu ⁇ oses.
  • media containing an effective amount of a metal-binding compound of the invention can be used in in vitro fertilization (INF) procedures for reducing the damage done by ROS to gametes (sperm and/or ova), zygotes, and blastocysts during collection, storage and/or culture.
  • INF in vitro fertilization
  • seminal fluid is known to contain substantial amounts of copper and fructose, conditions suitable for the production of ROS.
  • Many suitable media for use in INF procedures are known (e.g., Gardner media Gl, G2, etc.). Effective amounts of the metal-binding compound to include in the media can be determined empirically, and doing so is within the skill in the art.
  • the invention further provides a kit for reducing the damage done by ROS to a cell, a tissue or organ that has been removed from an animal.
  • the kit is a packaged combination of one or more containers holding reagents and other items useful for preserving harvested cells, tissues or organs.
  • the kit comprises a container holding a metal-binding compound of the invention. Suitable containers include bottles, bags, vials, test tubes, syringes, and other containers known in the art
  • the kit may also contain other items which are known in the art and which may be desirable from a commercial and user standpoint, such as a container for the cells, tissue or organ, diluents, buffers, empty syringes, tubing, gauze pads, disinfectant solution, etc.
  • a or “an” entity refers to one or more of that entity.
  • a cell refers to one or more cells.
  • This example describes the synthesis of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] composed of all L-amino acids using standard solid-phase synthesis techniques.
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • DMF dimethylformamide
  • a ninhydrin test was used to monitor the reaction.
  • the resin was swollen with DMF ( ⁇ 1 ml).
  • the C-protected t-benzyloxycarbonyl (Boc) ester of alanine in DMF was added, followed by a mixture of diisopropylamine (8 equivalent) and 2-(lH-benzotriazole-l-yl)-l,2,3,3-tetramethyluroniumtetrafluoroborate (TBTU-) (4 equivalents).
  • the resin was shaken for about 24 hours, and the reaction was monitored by the ninhydrin test. At the end of this period, DMF was drained, and the resin was washed with DMF and DCM.
  • the solution was drained, and the beads were washed with DCM (3 2 ml).
  • the protecting group of the dipeptide-resin was removed, and the beads were suspended in DMF.
  • Amino protected (benzyloxy) derivative of histidine (4 mmole) was added, followed by mixture of diisopropylamine (8 equivalent) and TBTU- (4 equivalent).
  • the resin was shaken for about 24 hours, and the reaction monitored by ninhydrin test. At the end of this period, DMF was drained, and the resin was washed with DMF and DCM.
  • the tripeptide-resin was briefly dried in a gentle stream of nitrogen and suspended in nitrogen- saturated DMF.
  • the ninhydrin test gave a blue color, indicating the release of the tetrapeptide from the resin.
  • addition of 5%( V) of DMF to TFA accelerated the rate of release of the peptide from the resin.
  • Removal of TFA at reduced pressure gave the tetrapeptide (all D) as TFA salt and was dried under vacuum at 5°C for 24 hours. The residue was a white powder and was characterized by spectrometric methods.
  • a number of enantiomers of the tetrapeptide can be prepared in this manner.
  • use of D-amino acids in the peptide synthesis forms the tetrapeptide containing all D-amino acids.
  • combinations of L-amino acids and D-amino acids can be used.
  • Trans-diaminocyclohexane was prepared by resolving cis/trans 1,2- diaminocyclohexane (Aldrich-Sigma) as the tartaric acid salt.
  • the R-trans isomer melts at 75°C and the S-trans isomer melts between 43-45°C (Ph.D. Thesis, P.D. Newman, University College, Edinburgh, U.K., 1994).
  • the trans-diaminocyclohexane (10 gm) was then suspended in anhydrous toluene (30mL) and cooled to 5°C in an ice bath, and bromoacetic acid (8 gm) in toluene (25 mL) was added dropwise. At the end of the addition, the reaction temperature was raised to 30°C and kept at that temperature for a further 5 hours. Toluene was evaporated, and the R-trans 1 ,2-diaminocyclohexane diacetic acid was crystallized from hexane/toluene to give a white solid (yield 70%). The product was characterized by spectroscopic methods.
  • the resin-bound tetrapeptide prepared in Example 1 (20mg) was suspended in DMF (5 mL) and was treated with the R-trans 1,2-diaminocyclohexanediacetic acid (20 mg) followed by addition of a mixture of diisopropylamine (8 equivalent) and TBTU-(4 equivalent). The resin was shaken for about 24 h on a roller. Then, the resin was washed with DMF followed by DCM (5x3mL) and partially dried. Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). The resin was separated and washed with DCM. The washings were combined with TFA and concentrated under vacuum. The residue (cyclohexanediamine tetrapeptide; formula given in Figure 3D where R 5 is H) was characterized by spectrometric analysis.
  • the resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with mesopo ⁇ hyrin LX dicarboxylic acid (10 ⁇ mole; formula given in Figure 6A), followed by addition of a mixture of diisopropylamine (8 equivalent) and TBTU- (4 equivalent). The resin was shaken for about 24 hours on a roller kept in a dark chamber. The resin was washed with DMF followed by DCM (5x3 mL) and partially dried. Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). Theresinwas separatedandwashedwithDCM/TFAmixture (l:1.5mL). Thewashings were combined and concentrated under vacuum. The po ⁇ hyrin tetrapeptide (formula given in Figure 6C) was purified by semi-preparative HPLC (yield 60%). The structure was confirmed by spectrometric methods.
  • This procedure can be used to synthesize other po ⁇ hyrin-peptides, such as mesopo ⁇ hyrin I and related molecules.
  • the resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with bromoethylpyridine (20 ⁇ mole). This was followed by the addition of pyridine (0.5 mL). The resin was shaken for about 48 hours on a roller. The resin was washed with DMF, followed by DCM (5x3mL) to remove all of the unreacted monomers, and then dried under vacuum for 30 minutes. Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). The resin was separated and washed with DCM/TFA mixture (1:1.5mL). The washings were combined and concentrated under vacuum. The pyridylethyl tetrapeptide derivative (formula given in Figure 5) was purified by semi-preparative HPLC (yield 50 %). The structure was confirmed by spectrometric methods.
  • This procedure can be applied to other heterocycles, such as phenanthroline and related molecules.
  • the crude product was hydrolyzed with hydrochloric acid ( 100 mL) and decarboxylated to give phenyl substituted aspartic acid (10 gm).
  • the N-benzoyloxy t-butyl derivative was prepared using a standard reaction sequence.
  • the resin was shaken for about 24 h, and the reaction monitored by the ninhydrin test.
  • EXAMPLE 7 Inhibition Of The Generation Of ROS By The Tetrapeptide Asp Ala His Lvs [SEQ ID NO: ⁇
  • a tetrapeptide having the sequence L-Asp L-Ala L-His L-Lys [SEQ ID NO: 1 ] was obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services, QCB, Genosys and Bowman Research.
  • the peptide was prepared by standard solid phase synthesis methods (see also Example 1). The ability of the L-tetrapeptide to inhibit the generation of ROS was tested as described in Gutteridge and Wilkins, Biochim. Biophys. Acta, 759, 38-41 (1983) and
  • the assay was performed with and without the L-tetrapeptide.
  • the results are summarized in Table 1.
  • Table 1 As can be seen from Table 1, when the L-tetrapeptide was present at Cu( ⁇ ):tetrapeptide ratios of 1:1.2 and 1:2, the degradation of 2-deoxy-D-ribose was inhibited by 38% and 73%, respectively.
  • the L-tetrapeptide inhibited the degradation of 2-deoxy-D-ribose by hydroxyl radicals.
  • D-tetrapeptide having the sequence Asp Ala His Lys composed of all D-amino acids.
  • the D-tetrapeptide was obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services and QCB.
  • the peptide was prepared by standard solid phase synthesis methods (see Example 1)
  • Support rats 300-400g were anesthetized with sodium pentobarbitone (60 mg/kg, intraperitoneally) and anticoagulated with heparin (1000 IU/kg intravenously).
  • the right femoral vein and left femoral artery were exposed by blunt dissection and cannulated (18G and 22G Abbocath-T catheters respectively) for the return and supply of blood to the perfused heart.
  • An extraco ⁇ oreal circuit was established, primed with Gelofusine® plasma substitute (B. Braun Medical Ltd., Aylesbury, UK) and was maintained for 15 minutes (min) before connection to the isolated heart. This period was to ensure that the priming solution was adequately mixed with the blood of the support rat and that the entire preparation was stable.
  • Each 500 ml of Gelofusine® contains 20.00 g succinated gelatin (average molecular weight 30,000), 3.65 g. sodium chloride, water for injection to 500 ml (electrolytes mmol/500 ml: cations Na 77, anions Cl 62.5, pH 7.4).
  • succinated gelatin average molecular weight 30,000
  • 3.65 g. sodium chloride water for injection to 500 ml
  • electrolytes mmol/500 ml electrostatic mmol/500 ml: cations Na 77, anions Cl 62.5, pH 7.4
  • an additional 7-8 ml of blood from a rat of the same strain was added to the central reservoir. This was to ensure that the support rat had an adequate supply of blood during the experiment when blood was not recirculated but instead collected for a 2 min period.
  • a peristaltic pump (Gilson Minipuls 3) was located on the arterial outflow of the support rat and flow through the extraco ⁇ oreal circuit was increased gradually over 10 min to a value of 2.5 ml/min. This gradual increase prevented the drop in arterial pressure that would have occurred if a flow rate of 2.5 ml/min had been established immediately.
  • the blood was pumped through a cannula (to which the aorta of the perfused heart would subsequently be attached) and returned, by gravity, via a reservoir and filter to the venous inflow line of the support animal.
  • An air-filled syringe above the perfusion cannula acted as a compliance chamber, which served to dampen oscillations in perfusion pressure which occurred as a consequence of the contraction of the isolated heart and the peristaltic action of the pump.
  • the support animal was allowed to breathe a mixture of 95% O 2 + 5% CO 2 through a 35% Nenturi face mask.
  • the flow rate was adjusted to maintain blood pO 2 and pCO 2 within the physiological range.
  • Body temperature was stabilized at 37.0 ( ⁇ 0.5) ° C by means of a thermostatically-controlled heating pad and was monitored by a rectal thermometer. Blood pressure was monitored by means of a pressure transducer attached to the arterial line.
  • each rat (270-350 g) was anesthetized with diethyl ether and anticoagulated with heparin (1000 IU/kg intravenously). The heart was then immediately excised and immersed in cold (4°C) Gelofusine®. The aorta was rapidly cannulated and perfused in the Langendorff mode, (Langendorff, Pflugers Archives fur die Gestamte Physiologie desowned and der Tiere, 61:291-332 (1895)) with arterial blood from the support animal, at a constant flow rate of 2.5 ml/min.
  • a fluid-filled balloon catheter (for the measurements of left ventricular systolic and diastolic pressures, and, by difference, left ventricular developed pressure), attached to a pressure transducer, was introduced into the left ventricle via the mitral valve.
  • the balloon was inflated with water until a left ventricular end diastolic pressure (LNEDP) of between 4-8 mmHg was obtained.
  • LNEDP left ventricular end diastolic pressure
  • Heart rate was calculated from the pressure trace and expressed as beats per minute (bpm).
  • Perfusion pressure was measured via a sidearm of the aortic cannula. All pressure transducers were connected to a MacLab, which was run continuously through the experiment.
  • Excised hearts were randomly assigned to two treatment groups (see Figure 9; 6 hearts/group) and aerobically perfused for 20 min prior to (i) saline control with 2 min saline infusion immediately prior to a 30 min period of ischemia plus 2 min saline infusion at the onset of reperfusion and (ii) drug with 2 min drug infusion immediately prior to a 30 min period of ischemia (the drug was therefore trapped in the vasculature for the duration of the ensuing ischemic period), plus a 2 min drug infusion at the onset of reperfusion. Hearts were then subjected to 30 min of global, zero-flow ischemia, during which time they were immersed in saline at 37.0°C.
  • Ischemia was initiated by clamping the line leading from the pump to the aortic cannula, thus diverting the flow away from the isolated heart back to the support animal, via the bypass line. Hearts were then reperfused for 40 min, during which time contractile function was continuously measured.
  • the drug whose identity was unknown to the researchers performing the experiments, was the tetrapeptide D-Asp D-Ala D-His D-Lys.
  • the tetrapeptide was supplied to the researchers by Bowman Research, UK, dissolved in saline at a concentration of 16.7 mg/mL. It was infused as supplied without any dilution or modification.
  • Physiological saline was supplied by Baxter, UK and used in controls. Fresh solutions of saline and the drug were used daily.
  • Drug or vehicle was infused into a sidearm of the aortic cannula by means of a peristaltic pump (Gilson Minipuls 3), set at a constant flow of 0.25 ml/min.
  • Predefined exclusion criteria stated that: (i) support animals would be excluded from the study if they did not attain a stable systolic blood pressure ⁇ 80 mm Hg before cannulation of the donor heart, (ii) donor hearts would be excluded from the study if, at the 20 min baseline pre-intervention reading, LNDP ⁇ 100 mm Hg or (iii) blood chemistry values were outside the normal range.
  • Results are expressed as mean ⁇ SEM.. All recovery values are expressed as a percent of the pre-intervention baseline value (measured 20 min after the onset of the experiment) for each individual heart. The two-tailed unpaired Student's t test was used for the comparison of two means between groups. A difference was considered statistically significant when p ⁇ 0.05.
  • the stability and reproducibility of the system were monitored by measuring the blood chemistry (pH, pO 2 , pCO 2 , haematocrit, Na + , K + and Ca 2+ , glucose) and baseline contractile function of each support animal (immediately before perfusing a donor heart and at the end of each experiment) and each perfused heart.
  • Table 3 reveals that there were only minor changes in each index measured, confirming that similar perfusion conditions applied in both study groups and that all values were within the acceptable physiological range.
  • the systolic pressure and heart rate of the support rats are shown in Table 4. As can be seen, there were no significant differences between the two study groups at the 15 min baseline reading.
  • Table 5 shows that there were no significant differences between groups at the end of the 20 min aerobic perfusion period (i e. just prior to the infusion of drug or vehicle) in LNDP, heart rate and perfusion pressure.
  • the mean values were 177.3 ⁇ 10.6 mmHg and 177.2 ⁇ 5.6 mmHg for the groups that were to become saline control and drug treated.
  • Figure 11 shows the profiles for the mean recovery of LNDP (expressed as a percent of baseline pre-intervention values) in both study groups. It is evident that hearts in the saline control group recovered slowly and poorly, such that by the end of the 40 min reperfusion period, LNDP was only 15.3 ⁇ 3.2% of the pre-intervention control. By contrast, hearts in the drug group recovered more rapidly and to a greater extent (50.5 ⁇ 9.3%).
  • Figure 12 shows the absolute values for the left ventricular end diastolic pressure in both study groups during the 40 min period of reperfusion.
  • the high levels of LNEDP resulting from the contracture which developed during ischaemia fell with time towards the pre-intervention control value.
  • the drug group normalized their LNEDP more quickly and more completely than that seen in the saline control group, the difference being significant at every time point studied. This would be consistent with the enhanced recovery seen during reperfusion in the drug group.
  • a comparison of the heart rates obtained in the saline control group and drug, as shown in Figure 13 reveals that these two groups were essentially identical (115.0 ⁇ 3.8 versus 127.2 ⁇ 22.8%).
  • Asp Ala His Lys appears to have significant and substantial protective properties as assessed by an approximately three and a half (3.5) fold (15.3 ⁇ 3.2 % to 50.5 ⁇ 9.3%) enhancement of post-ischemic functional recovery.
  • the magnitude of protection is equal to some of the most powerful interventions studied.
  • LVDP left ventricular developed pressure
  • animals received an intravenous infusion of vehicle alone (control) or drug (D-Asp D-Ala D-His D-Lys) in vehicle over one minute.
  • the identity of the drug was unknown to the researchers performing the experiments. It was supplied to the researchers as a concentrated stock (16.67 mg/ml) in phosphate buffered saline, pH 7.4, and was stored it at -80°C.
  • the drug was determined to be biologically active prior to use by determining its ability to reduce free radical formation in vitro as described in Example 7. The stock solution was thawed just prior to use, and a sufficient quantity was administered to give a dose of 20 mg/kg.
  • the nylon suture was immediately advanced to occlude the MCA. Following the 2 hours of occlusion of the MCA, the animals received a repeat intravenous infusion of drug or vehicle over one minute. At the end of the second infusion, the nylon suture was immediately pulled back from occluding the MCA to allow for reperfusion. Also, after the second infusion, the animals were re-anesthetized, and the cannulae were removed. The animals were returned to their home cages, where they were allowed free access to food and water.
  • mice Twenty-four hours after MCA occlusion, animals were anesthetized with ketamine (44 mg/kg) and xylazine (13 mg/kg), both given intramuscularly, and perfused transcardially with heparinized saline, followed by 10% buffered formalin.
  • the brains were removed and cut into 2-mm coronal slices using a rat brain matrix (Activational System, Inc., Warren, MI; a total of 7 slices). The slices were then embedded in paraffin, and 6-mm sections were cut from the anterior surface of each slice and stained with hematoxylin and eosin (H and E).
  • Infarct volume was determined using a computer-interfaced image analysis system (Global Lab Image system, Data Translation, Marlboro, MA), using the "indirect” method (Swanson et al.., J. Cerebral Blood Flow Metabol, 10:290-293 (1990)): the area of intact regions of the ipsilateral (right) hemisphere and area of the intact contralateral (left) hemisphere were determined for each slice, the former was subsfracted from the latter to calculate infract area per slice. Infarct areas were then summed and multiplied by slice thickness to yield infarct volume per brain (in mm 3 ).
  • EXAMPLE 10 Inhibition Of The Generation Of ROS The ability of the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO: 1] and other peptides and compounds to inhibit the production of ROS was tested.
  • the other peptides tested were: L-Asp L-Ala L-His L-Lys L-Ser L-Glu L-Nal L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID ⁇ O:3]; L-Ala L-His L-Lys L-Ser L-Glu L-Nal L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:4]; L-His L-Lys L-Ser L-Glu L-Nal L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:5]; and Acetylated-L-Asp L-Ala L-His L-Lys L-Ser L-Glu L-Nal L-Ala L-His L-Arg L- Phe L-Lys [SEQ ID ⁇ O:6].
  • the peptides were obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services, QCB, Genosys and Bowman Research.
  • the other compounds tested were histidine (Sigma Chemical Co.), catalase (Sigma Chemical Co.), and superoxide dismutase (Sigma Chemical Co.).
  • the hydroxyl radical is probably the most reactive oxygen-derived species.
  • the hydroxyl free radical is very energetic, short-lived and toxic. Some researchers suggest that the toxicity of hydrogen peroxide and superoxide radical may be due to their conversion to the hydroxyl free radical.
  • the superoxide radical can be directly converted to the hydroxyl radical via the Haber-Weiss reaction. Alternatively, it can be converted to hydrogen peroxide which, in turn, is converted into the hydroxyl radical via the Fenton reaction. Both pathways require a transition metal, such as copper (Acworth and Bailey, The Handbook OfOxidative Metabolism (ESA, Inc. 1997)).
  • absorbance at 532 nm is a measure of the damage to deoxyribose and, therefore, of hydroxyl radical formation.
  • CuCl 2 in buffer (20 mM KH 2 PO 4 buffer, pH 7.4) and either one of the test compounds in buffer or buffer alone were added to test tubes (final concentration of CuCl 2 was lO ⁇ M).
  • the test tubes were incubated for 15 minutes at room temperature.
  • 0.5 mM ascorbic acid in buffer and 1.9 mM 2-deoxy-D-ribose in buffer were added to each test tube, and the test tubes were incubated for 1 hour at 37°C.
  • Histidine and several peptides with histidine in different positions were tested at 1 : 1 and 2:1 peptide:copper ratios for their ability to inhibit the production of hydroxyl radicals. Also, a peptide having an acetylated aspartic acid (Ac- Asp) as the N-terminal amino acid was also tested. The results are presented in Table 11. In Table 11 , the % inhibition is the percent decrease in absorbance compared to buffer alone divided by the absorbance of the buffer alone.
  • the peptides with histidine in the second and third positions gave >95% inhibition at a 2: 1 peptide: copper ratio, while these peptides at a 1 : 1 peptide opper ratio were ineffective.
  • the peptide with histidine in the first position and the peptide with acetylated aspartic acid as the N-terminal amino acid provided some protection (about 47% and about 28% inhibition, respectively), although this protection might be attributable to the histidine in the seventh and ninth positions, respectively, of these peptides.
  • Histidine alone at a 2: 1 histidine: copper ratio provided some protection (about 20% inhibition).
  • Catalase has been shown to prevent hydroxyl radical formation. Gutteridge and Wilkins, Biochim. Biophys. Acta, 759:38-41 (1983); Facchinetti et al., Cell Molec. Neurobiol, 18(6):667-682 (1998); Samuni et al., Eur. J. Biochem., 137:119-124 (1983). Catalase (0-80 nM) was, therefore, tested in this assay, and it was found to prevent the formation of the pink chromogen (data not shown). This finding suggests that hydrogen peroxide is formed in this assay, since catalase breaks down hydrogen peroxide to water and agrees with Equations 3 and 4 above.
  • Catalase also prevents the formation of the pink chromogen when the L-Asp L-Ala L-His L-Lys [SEQ ID NO:l] tetrapeptide at a tetrapeptide/copper ratio of 1 : 1 is present (data not shown). As shown above, at this ratio, the copper is still able to participate in the redox reactions to produce hydroxyl radicals. These experiments show that hydrogen peroxide is an important precursor to the formation of the hydroxyl radical.
  • SOD superoxide dismutase
  • the enzyme superoxide dismutase (SOD) is a naturally-occurring enzyme which is responsible for the breakdown in the body of superoxide to hydrogen peroxide (similar to Equation 3). Hydrogen peroxide can then be detoxified by catalase. SOD was assayed for activity in the assay described in the previous section and was found to have none (data not shown). This result is not su ⁇ rising since SOD actually converts superoxide radical into hydrogen peroxide. Hydrogen peroxide can then be converted into the hydroxyl radical by reduced copper.
  • the reaction was started by the addition of various amounts of a tefrapeptide-copper complex (tetrapeptide/copper ratios of 1 :1 and 2: 1 ) and 20 nM xanthine oxidase (Sigma Chemical Co.).
  • the tetrapeptide-copper complex was prepared by mixing the tetrapeptide and copper (as CuCl 2 ) and allowing the mixture to incubate for 15 minutes at room temperature immediately before addition to the cuvette. The samples were read at time 0 and every 60 seconds for five minutes at 560 nm.
  • the complex of the tetrapeptide with copper at a ratio of 1 :1 was shown to have SOD activity, as evidenced by inhibition of NBT reduction (see Figure 16). However, the complex was about 500 times less effective than SOD itself, based on IC 50 values (amount that gives
  • uric acid production was measured at 295 nm. Athar et al., Biochem. Mol. Biol. Int., 39(4):813-821 (1996); Ciuffi et al., Pharmacol Res, 38(4):279-287 (1998).
  • This assay is similar to the SOD assay, except that NBT is not present. Instead, uric acid is assayed at 295 mn every 60 seconds for 5 minutes. It was found that the 1:1 tefrapeptide-copper complex only inhibited uric acid production by 11% at a concentration of 600 nM (data not shown). Therefore, the 1:1 tefrapeptide-copper complex has true SOD activity. Since superoxide is converted to hydrogen peroxide by the complex, this could help to explain why it is not effective at preventing hydroxyl radical production.
  • the sample containing the 1 : 1 tefrapeptide-copper complex also showed an increase in NBT reduction, with a decreased maximum reached at 60 minutes. These data suggest that superoxide accumulates in the sample containing the 2: 1 tefrapeptide-copper complex, while the 1 : 1 tefrapeptide-copper complex mimics superoxide dismutase.
  • the 2:1 tefrapeptide-copper complex is so effective because it inhibits the formation of hydrogen peroxide, which could in turn react with reduced copper to produce hydroxyl radicals via the Fenton reaction.
  • the 1 : 1 tefrapeptide-copper complex also provides a valuable service by eliminating the superoxide radical. Even though it produces hydrogen peroxide, most compartments of the human body have sufficient quantities of the enzyme catalase that can eliminate hydrogen peroxide. In the brain, however, catalase activity is reported to be minimal. Halliwell et al., Methods in Enzymol, 186:1-85 (1990). Therefore, the brain is a particularly vulnerable organ during periods of ischemia, since copper is released due to the acidosis that accompanies ischemia.
  • DNA strand breaks were measured according to the method of Asaumi et al., Biochem. Mol. Biol. Int., 39(l):77-86 (1996). Briefly, 17 ⁇ g/ml of plasmid pBR322 DNA was allowed to pre-incubate for 15 minutes at room temperature with 50 ⁇ M CuCl 2 and concentrations of the tetrapeptide of 0-200 ⁇ M. Then, 2.5 mM ascorbate was added to each reaction, and the mixture was incubated for 1 hour at 37°C. The total volume of the mixture was 16 ⁇ L.
  • ROS damages DNA by causing strand breaks, base modifications, point mutations, altered methylation patterns, and DNA-protein cross linking (Marnett, Carcinogenesis 21:361- 370 (2000); Cerda et al., Mutat. Res. 386:141-152 (1997)).
  • OH* is considered the most reactive and damaging ROS and is capable of producing all the above DNA lesions (Marnett, Carcinogenesis 21:361-370 (2000)).
  • Previous investigations have reported that OH» induced, single- and double-strand DNA breaks occur during site-specific copper ion reactions in vitro and during excessive copper exposure in vivo (Chiu et al.,
  • Telomeres which are repeats of the hexanucleotide TTAGGG, exist at the ends of DNA to form a "protective cap” against degradation, chromosomal rearrangement, and allow the replication of DNA without the loss of genetic information (Reddel, Carcinogenesis 21:477-484 (2000)).
  • DNA polymerase is unable to replicate the terminal end of the lagging strand during DNA replication resulting in the loss of 30-500 base pairs (Harley et al., Nature 345:458-460 (1990); von Zglinicki et al., Exp. Cell Res.220: 186- 193, doi: 10.1006/excr.1995.1305 (1995)). Somatic cells are unable to replace these lost telomeric repeats, leading to progressive telomere shortening during a cell's replicative life. Senescence is manifested when telomere length reaches a critical threshold (Reddel, Carcinogenesis 21:477-484 (2000)).
  • D-Asp Ala His Lys The synthetic D-analog of Asp Ala His Lys (D-Asp Ala His Lys) was obtained from Bowman Research Ltd. (Newport, Wales, UK). TeloTAGG Telomere Length Assay and X-ray film were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DNeasy genomic isolation kits were purchased from Qiagen (Valencia, CA). Hybond-N+ nylon membrane was ordered from Amersham Pharmacia Biotech (Piscataway, NJ). All other chemicals were obtained from Sigma (St. Louis, MO). DNA treatments: DNA strand breaks were measured using a modified method of
  • Raji cells a Burkitt lymphoma derived cell line (obtained from American Type Culture Collection (ATCC), Rockville, MD, ascension number CCL-86), were grown in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal calf serum (FCS) at 10% CO 2 and 37 °C. Genomic DNA was isolated using DNeasy spin columns (Qiagen) following the manufacturer's protocol.
  • IMDM Iscove's modified Dulbecco's medium
  • FCS fetal calf serum
  • strand breaks were visualized by immediately adding 5 ⁇ l of loading dye [0.25% (w/v) bromophenol blue and 40% (w/v) sucrose] and loading on a 0.5% tris acetic acid EDTA (TAE) agarose gel. Gels were then run at 70N for 90 min and stained using 2 ⁇ g/ml ethidium bromide for 30 minutes. Prior to photographing, gels were rinsed in TAE for 10 minutes.
  • TAE tris acetic acid EDTA
  • Telomere Length Assay To examine telomere damage, the TeloTAGG Telomere Length Assay (Roche) was used according to manufacturer's recommendations: digesting 1 ⁇ g of genomic DNA per reaction using Hinfl and RSA I. Samples were then run on a 0.8% TAE agarose gel at 70V for 2 hours. Southern blots were performed and probed using a digoxigenin (DIG) labeled telomere specific oligonucleotide. For cell treated samples, genomic DNA was used as described above. For DNA treated samples, reactions were setup as above, brought to 200 ⁇ l with PBS, and isolated using DNeasy columns prior to restriction digestions.
  • DIG digoxigenin
  • OH» scavengers do not prevent copper-mediated oxidative damage suggesting that oxidative DNA damage occurs in close proximity to the copper ions (Oikawa etal., Biochim. Biophys. Acta 1399: 19-30 (1998)).
  • the reactivity of OH» is so great that, presumably, OH» interactions only occur at or near the site of OH « production (Marnett, Carcinogenesis, 21, 361-370 (2000)).
  • Oikawa, et. al. (Oikawa et al., Biochim. Biophys. Acta 1399:19-30 (1998)) have shown that the following copper-mediated ROS reaction also occurs, and that the resulting DNA-copper-peroxide complex may be even more damaging to DNA than OH»:
  • Another mode of protection may be the formation of D-Asp Ala His Lys-copper- peroxide complexes which would absorb the OH « damage rather than DNA, "mop-up" peroxides, and perhaps, in cell samples, keep H 2 O 2 outside the cell.
  • telomere in the genomic DNA samples in the present study showed double strand breaks in response to oxidative stress.
  • DNA samples examined by Southern blot showed severely depleted and shortened telomere sequences (Fig. 22).
  • Cell treatments showed damage to the telomere with some conservation of the sequence, even at the highest levels of copper and ascorbic acid used (Fig.23), which maybe attributed to ROS production outside the cells with the DNA sheltered inside the nucleus.
  • D-Asp Ala His Lys protected the telomere from copper-mediated damage in these samples.
  • 8-Oxo-deoxyguanosine (8-oxo-dG) is a common DNA adduct produced by ROS, which can result in G -> • T point mutations widely seen in mutated oncogenes (Marnett, Carcinogenesis 21:361-370 (2000)).
  • Conditions such as acidosis occurring during myocardial ischemia or alterations of ceruloplasmin have been shown to mobilize free copper to catalyze local oxidative tissue and DNA damage (Kim et al., Free Radic. Res. 33:81-89 (2000); Chevion et al., Proc. Natl. Acad. Sci. USA 90:1102-1106 (1993)).
  • Nitric oxide and superoxide released from activated leukocytes can lead to the production of peroxynitrite, which is more reactive with 8-oxo-dG thanunmodifiedbases and possibly exacerbates the damage (Marnett, Carcinogenesis 21:361- 370 (2000)).
  • the D-tetrapeptide at a ratio of 4: 1 (peptide:Cu), provided complete protection of isolated DNA and, at a ratio of 2: 1 (peptide:Cu), completely protected Raji Burkitt cells' DNA exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres with some conservation of telomere sequences.
  • the D-tetrapeptide provided complete telomere length protection at a ratio of 2:1 (peptide:Cu). While the exact mechanisms for ROS DNA damage have yet to be fully elucidated, D-Asp Ala His Lys inhibited copper-induced DNA double-strand breaks by ROS in both genomic DNA and in the telomere sequence.
  • SPF research-grade fertilized eggs were obtained from Charles River Laboratories (800-772-3721).
  • the eggs were candled to determine the position of the yolk and to mark the air cell with a pencil. Under sterile conditions, a small hole was drilled in the shell using a micro hand drill.
  • the eggs were divided randomly into four groups, six eggs per group: A - no injection;
  • Interleukin 8 is a pro-inflammatory cytokine and a potent chemoattractant and activator of neutrophils. It has also been reported to be a chemoattractant and activator of T- lymphocytes and eosinophils. IL-8 is produced by immune cells (including lymphocytes, neutrophils, monocytes and macrophages), fibroblasts and epithelial cells. Reports indicate an important role for IL-8 in the pathogenesis of respiratory viral infections, asthma, bronchitis, emphysema, cystic fibrosis, acute respiratory distress syndrome, sepsis, multiple organ dysfunction syndrome, and other inflammatory disorders. The IL-8 release by Jurkat cells (American Type Culture Collection (ATCC),
  • Experiment 1 a. None (control); b. Asp Ala His Lys [SEQ ID NO: 1] ("DAHK”) - 200 ⁇ M and ascorbic acid - 500 ⁇ M; c. CuCl 2 - 10 ⁇ M and ascorbic acid - 500 ⁇ M; d. CuCl 2 - 25 ⁇ M and ascorbic acid - 500 ⁇ M; e. CuCl 2 - 50 ⁇ M and ascorbic acid - 500 ⁇ M; f. CuCl 2 - 100 ⁇ M and ascorbic acid - 500 ⁇ M; g. CuCl 2 - 50 ⁇ M and DAHK - 50 ⁇ M and ascorbic acid - 500 ⁇ M; h.
  • DAHK Asp Ala His Lys
  • Experiment 2 a. None (confrol); b. CuCl 2 - 100 ⁇ M; c. DAHK - 200 ⁇ M and ascorbic acid - 500 ⁇ M; d. CuCl 2 - 25 ⁇ M and ascorbic acid - 500 ⁇ M; e. CuCl 2 - 50 ⁇ M and ascorbic acid - 500 ⁇ M; f.
  • Experiment 3 a. None (confrol); b. CuCl 2 - 100 ⁇ M; c. DAHK - 400 ⁇ M and ascorbic acid - 250 ⁇ M; d.
  • Coenzyme A is essential for acetylation reactions in the body and, as a consequence, plays a critical role in the metabolism of carbohydrates and fatty acids. CoA can be oxidized to a disulfide which cannot participate in acetylation reactions. As a result, metabolism and energy utilization are inhibited. h this example, it was investigated whether Cu(H) could oxidize CoA and, if so, whether the tetrapeptide Asp Ala His Lys [SEQ ID NO:l] (Bowman Research, United
  • the tetrapeptide at a 1 : 1 tetrapeptide: Cu(II) ratio provided some protection of CoA
  • the tetrapeptide at a 2.T tefrapeptide :Cu( ⁇ ) ratio provided 100% protection.

Abstract

L'invention concerne un procédé de réduction des dommages causés par des dérivés d'oxygène réactifs (ROS) chez un animal. L'invention concerne également un procédé de réduction de la concentration d'un métal chez un animal. Ces procédés consistent à administrer à l'animal une quantité efficace de composés de liaison au métal, tel que décrit dans la description. L'invention concerne, de plus, un procédé de réduction des dommages causés par les ROS à une cellule, un tissu ou un organe ayant été retiré d'un animal. Ce procédé consiste à mettre en contact la cellule, le tissu ou l'organe avec une solution ou un milieu contenant une quantité efficace d'un composé de fixation au métal de l'invention. L'invention concerne enfin de nouveaux composés de liaison au métal, des compositions pharmaceutiques comprenant ces composés de liaison au métal, et des kits comprenant un récipient renfermant un composé de liaison au métal de l'invention.
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WO2004050023A2 (fr) 2002-11-27 2004-06-17 Dmi Biosciences, Inc. Traitement de maladies et d'etats a mediation de phosphorylation accrue
EP1482960A2 (fr) * 2001-11-20 2004-12-08 DMI Biosciences, Inc. Procedes et produits de soins bucco-dentaires
US7141589B2 (en) 2001-08-23 2006-11-28 The United States Of America As Represented By The Department Of Health And Human Services Methods of inhibiting formation of vascular channels and methods of inhibiting proliferation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7141589B2 (en) 2001-08-23 2006-11-28 The United States Of America As Represented By The Department Of Health And Human Services Methods of inhibiting formation of vascular channels and methods of inhibiting proliferation
EP1482960A2 (fr) * 2001-11-20 2004-12-08 DMI Biosciences, Inc. Procedes et produits de soins bucco-dentaires
EP1482960A4 (fr) * 2001-11-20 2009-04-08 Dmi Biosciences Inc Procedes et produits de soins bucco-dentaires
WO2004050023A2 (fr) 2002-11-27 2004-06-17 Dmi Biosciences, Inc. Traitement de maladies et d'etats a mediation de phosphorylation accrue

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