EP1189604A2 - Ionophores de zinc utilises comme agents anti-apoptose - Google Patents

Ionophores de zinc utilises comme agents anti-apoptose

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Publication number
EP1189604A2
EP1189604A2 EP00979231A EP00979231A EP1189604A2 EP 1189604 A2 EP1189604 A2 EP 1189604A2 EP 00979231 A EP00979231 A EP 00979231A EP 00979231 A EP00979231 A EP 00979231A EP 1189604 A2 EP1189604 A2 EP 1189604A2
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EP
European Patent Office
Prior art keywords
zinc
pyrithione
ionophore
group
body weight
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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EP00979231A
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German (de)
English (en)
Inventor
Henry Fliss
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Zinc Therapeutics Canada Inc
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Zinc Therapeutics Canada Inc
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Publication of EP1189604A2 publication Critical patent/EP1189604A2/fr
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/325—Carbamic acids; Thiocarbamic acids; Anhydrides or salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

  • the present invention relates to a method of preventing or treating apoptosis using a zinc ionophore.
  • the present invention also relates to a method of protecting cells against the harmful effects of injurious agents, for example, oxidants, TNF ⁇ , neurotoxins, ischemia and radiation.
  • Zinc plays a critical role in cellular biology, and is involved in virtually every important cellular process such as transcription, translation, ion transport, and others (O'Halloran, T.V. (1993) Science 261:715-725; Cousins, R.J. (1994) Annu .Rev. Nutr. 14:449-469; Harrison, ⁇ .L. et al . (1994) Neuropharmacology- 33 :935-952; Berg, J.M. et al . (1996) Science 271:1081-1085).
  • the involvement of cellular zinc in apoptosis has been recognized for close to 20 years (Sunderman, F.W.,Jr. (1995) Ann . Clin. Lab. Sci .
  • Apoptosis is a form of cell death normally activated under physiological conditions, such as involution in tissue remodeling during morphogenesis, and several immunological
  • the apoptotic process is characterized by cell shrinkage, chromatin condensation, and internucleosomal degradation of the cell's DNA (Verh ' aegen et al . (1995) Biochem. Pharmacol. 50(7):1021- 1029) .
  • Zinc-pyrithione (zinc pyridinethione, C 10 H 8 N 2 O 2 S 2 Zn, MW 317.75, commercially available from Sigma) is the active ingredient in the anti-dandruff shampoo Head & Shoulders® (U.S.
  • a method to protect against apoptosis using one or more zinc ionophores In one embodiment of the present invention there is provided a method of treating or preventing apoptosis by administering to a patient in need thereof a pharmaceutically effective amount of a zinc ionophore.
  • a pharmaceutical composition comprising a zinc ionophore and a pharmaceutically acceptable carrier.
  • a method of protecting against the harmful effects of injurious agents selected from the group consisting of oxidants, TNF , neurotoxins, ischemia and radiation by administering to a patient in need of such protection an effective amount of a zinc ionophore.
  • FIGURE 1 shows the effect of zinc pyrithione on rat cardiac apoptosis.
  • FIGURE 2 shows the effects of zinc pyrithione on HSP-70 in heart.
  • FIGURE 3 shows the anti -apoptotic effect of zinc pyrithione on Spl in brain.
  • FIGURE 4 shows the effects of zinc pyrithione on kainic acid induced damage in rat brain areas.
  • FIGURE 5 shows the effects of zinc pyrithione on the severity of kainic acid induced seizures in rats.
  • FIGURE 6 shows the protective effects of zinc pyrithione in PC12 cells subjected to oxidative stress.
  • FIGURE 7 shows the protective effects of zinc pyrithione in PC12 cells.
  • FIGURE 8 shows the anti -apoptotic effect of zinc-pyrithione in irradiated human primary endothelial cells .
  • FIGURE 9 shows the effects of zinc pyrithione on transcription factor binding activity in human primary endothelial cells.
  • FIGURE 10 shows the effects of zinc pyrithione on the TNF -induced transcription factor binding activity in human primary endothelial cells.
  • FIGURE 11 shows the effects of zinc pyrithione on cytosolic Ikappa B protein levels in human primary endothelial cells.
  • FIGURE 12 shows photomicrographs depicting histological evidence of protection by zinc-pyrithione in 4 vessel occlusion stroke model in rats.
  • FIGURE 13 shows the effect of zinc pyrithione on neuronal survival in 4 vessel occlusion stroke model in rats.
  • FIGURE 13A shows the effect of zinc- diethyldithiocarbamate on neuronal survival in 4 vessel occlusion stroke model in rats.
  • FIGURE 14 shows the effect of zinc pyrithione in rats and a 4x reduction in the number of apoptotic nuclei.
  • FIGURE 15 shows middle cerebral artery occlusion caused infarcts in the left hemisphere of mouse brain. Infarcts are visible as white regions in the left hemisphere.
  • FIGURE 16 shows the reduction in infarct area upon treatment with zinc-pyrithione in mice with middle cerebral artery occlusion stroke model.
  • FIGURE 16A shows the reduction in infarct area upon treatment with zinc-diethyldithiocarbamate in mice with middle cerebral artery occlusion stroke model.
  • FIGURE 17 shows the administration of zinc- pyrithione decreased infarct volumes in mouse brain.
  • FIGURE 17A shows the administration of zinc- diethyldithiocarbamate decreased infarct volumes in mouse brain.
  • FIGURE 18 shows the effect of zinc-pyrithione administration on neurological score in mice with middle cerebral artery occlusion stroke model.
  • the present invention is directed to a method of blocking apoptosis.
  • blocking includes treating, preventing, inhibiting, protecting against and reducing the occurrence of apoptosis.
  • treating includes blocking, preventing, inhibiting, protecting against and reducing the occurrence of apoptosis
  • the concentration of zinc ionophore used to block apoptosis ranges from about .005 ⁇ g zinc ionophore per kg of body weight to about 2 mg zinc ionophore per kg of body weight (i.e. about 600pM zinc ionophore to about 6 uM zinc ionophore) .
  • the concentration of zinc ionophore used to block apoptosis ranges from about 1.0 ⁇ g zinc ionophore per kg of body weight to about 800 ⁇ g zinc ionophore per kg of body weight.
  • the concentration of zinc ionophore used to block apoptosis ranges from about 0.2 ⁇ g zinc ionophore per kg of body weight to about 600 ⁇ g zinc ionophore per kg of body weight.
  • the concentration of zinc ionophore used to block apoptosis is about 0.9 mg/kg body weight, or about 0.18mg zinc/kg body weight.
  • any non- toxic compound capable of binding zinc with moderate affinity and having sufficient lipophilic properties to penetrate cell membranes will be capable of effecting the protection demonstrated in the present invention with zinc-pyrithione.
  • zinc- pyrithione was shown to operate at the cell signalling level, as demonstrated by its ability to alter cytosolic PKC- ⁇ content. Further, according to the present invention, the zinc-pyrithione was shown to operate at the transcriptional level, as demonstrated by its ability to alter the nuclear activity of transcription factors NF-kB, AP-1 and Spl . Still further, according to the present invention the zinc-pyrithione was shown to upregulate cytoprotective proteins, for example HSP70. In accordance with the present invention the zinc ionophores protect against neuronal cell loss in stroke patients.
  • zinc pyrithione demonstrates neuroprotective properties, showing protection against cell loss in the selectively vulnerable zone of the CAI region of the hippocampus in a rat model of severe global ischemia. With the mouse model of severe focal ischemia, zinc pyrithione demonstrates neuroprotective properties, significantly decreasing brain infarct volume and neurological deficit.
  • the zinc ionophores are administered in a pharmaceutically effective amount to a patient in need thereof in a pharmaceutical carrier by intravenous, intramuscular, subcutaneous, or intracerebroventricular injection or by oral administration or topical application.
  • one zinc ionophore may be administered, preferably by the intravenous injection route, alone or in conjunction with a second, different zinc ionophore.
  • conjunction with is meant together, substantially simultaneously or sequentially.
  • the zinc ionophores of the present invention are administered acutely, such as, for example, substantially immediately following an injury that results in apoptosis, such as a stroke.
  • the zinc ionophores may therefore be administered for a short course of treatment, such as for about 1 day to about 1 week.
  • the zinc ionophores of the present invention may be administered over a longer period of time to ameliorate chronic apoptotic episodes, such as, for example, for about one week to several months depending upon the condition to be treated.
  • a pharmaceutically effective amount as used herein is meant an amount of zinc ionophore, e.g., zinc- pyrithione, high enough to significantly positively modify the condition to be treated but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) , within the scope of sound medical judgment.
  • a pharmaceutically effective amount of zinc ionophore will vary with the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disease, the duration of treatment, the nature of concurrent therapy and the specific zinc ionophore employed. For example, a therapeutically effective amount of a zinc ionophore administered to a child or a neonate will be reduced proportionately in accordance with sound medical judgment. The effective amount of zinc ionophore will thus be the minimum amount which will provide the- desired anti -apoptotic effect.
  • the zinc ionophore e.g. zinc-pyrithione
  • the active ingredients which comprise zinc ionophores may be required to be coated in a material to protect said zinc ionophores from the action of enzymes, acids and other natural conditions which may inactivate said zinc ionophores.
  • they should be coated by, or administered with, a material to prevent inactivation.
  • zinc ionophores may be co- administered with enzyme inhibitors or in liposomes.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, and trasylol .
  • Liposomes include water- in-oil - in-water P40 emulsions as well as conventional and specifically designed liposomes.
  • the zinc ionophores may be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared, for example, in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, DMSO, ethanol , polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) , suitable mixtures thereof and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion.
  • a coating such as lecithin
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the zinc ionophore in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized zinc ionophores into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
  • the zinc ionophores may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains a zinc ionophore concentration sufficient to treat or block apoptosis in a patient.
  • the tablets, troches, pills, capsules, and the like may contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil or wintergreen or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid, and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as pepper
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non- toxic in the amounts employed.
  • the zinc ionophore may be incorporated into sustained- release preparations and formulations .
  • pharmaceutically-acceptable carrier as used herein is meant one or more compatible solid or liquid filler diluents or encapsulating substances.
  • compatible as used herein is meant that the components of the composition are capable of being comingled without interacting in a manner which would substantially decrease the pharmaceutical efficacy of the total composition under ordinary use situations.
  • substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; and phosphate buffer solution; skim milk powder; as well as other non- toxic compatible substances used in pharmaceutical formulations such as Vitamin C, estrogen and echinacea, for example.
  • Wetting agents and lubricants such as sodium lauryl
  • the patient is administered a therapeutically effective amount of at least one zinc ionophore and a pharmaceutically acceptable carrier in accordance with the present invention.
  • a preferred zinc ionophore is zinc pyrithione.
  • Another preferred zinc ionophore is zinc diethyldithiocarbamate.
  • the zinc ionophores of the present invention are effective against a wide range of injurious agents, for example, but not limited to: oxidants, TNF ⁇ , neurotoxins, or radiation.
  • the zinc ionophores of the present invention are also effective in treating ischemia. Therefore, in a preferred form of treating ischemia the patient is administered a therapeutically effective amount of at least one zinc ionophore and a pharmaceutically acceptable carrier.
  • a preferred zinc ionophore is zinc pyrithione.
  • Another preferred zinc ionophore is zinc diethyldithiocarbamate.
  • compositions suitable for blocking apoptosis which comprise one or more zinc ionophores and a pharmaceutically acceptable carrier.
  • Human umbilical vein endothelial cells were purchased from Clonetics (San Diego, California) and passages 2-4 were used for these studies. Cells were cultured on flame- sterilzed glass coverslips in Endothelial Basal Medium (Clonetics) supplemented with lOng/ml human recombinant epidermal growth factor, 1.0 ug/ml hydrocortisone, 50ug/ml gentamicin, 50ng/ml amphotetericin B, 12ug/ml bovine brain extract and 2%v/v fetal bovine serum (all from Clonetics) , in a humidified chamber at 37°C and 5% C0 2 .
  • Clonetics Endothelial Basal Medium
  • HUVEC proliferating HUVEC were passaged at 80-90% confluency.
  • Cardiac myocytes were isolated from the ventricular septum of adult rabbit hearts, following collagenase digestion, in a manner similar to that described previously (Turan, B. et al., (1997) Am. J. Physiol. 272 :H2095-H2106) .
  • the modification consisted of introducing low concentrations of CaCl 2 during the perfusion with collagenase and the dispersion of the myocytes.
  • Hearts were perfused for about 2 min by gravity under a hydrostatic pressure of 1 m, with a nominally Ca 2+ -free solution containing (in mM) : NaCl, 145; KC1, 5; MgS0 4 , 1.2; Na 2 HP0 4 , 1.8; HEPES, 5; glucose, 10; pH adjusted to 7.4 with NaOH. Forty ml of this perfusate were then supplemented with collagenase (1 mg/ml) and perfusion was continued with recirculation. Within 2-3 min, this treatment resulted in a complete loss of ventricular pressure. The flow rate was then adjusted to 15 ml/min and 50 uM CaCl 2 was added to the collagenase solution.
  • mice Primary cultures of mouse cerebellar granule neurons were obtained from dissociated cerebella of postnatal day 8 or 9 mice according to the following protocol (Cregan et al . , (1999) J. Neurosci . 19:7860- 7869, incorporated herein by reference). Brains were removed and placed into separate dishes containing solution A (124 mM NaCl, 5.37 mM KC1, 1 mM NaH2 P04 , 1.2 mM MgS04 , 14.5 mM D- (1) -glucose, 25 mM HEPES, 3 mg/ml BSA, pH 7.4) in which the cerebella were dissected, meninges removed, and tissue sliced into small pieces.
  • solution A 124 mM NaCl, 5.37 mM KC1, 1 mM NaH2 P04 , 1.2 mM MgS04 , 14.5 mM D- (1) -glucose, 25 mM HEPES, 3 mg/
  • the tissue was briefly centrifuged and transferred to solution A containing 0.25 mg/ml trypsin, then incubated at 37°C for 18 min. After the addition of 0.082 mg/ml trypsin inhibitor (Boehringer Mannheim, Indianapolis, I N) and 0.25 mg/ml DNase I (Boehringer Mannheim), the tissue was incubated at 25°C for 2 min. After a brief centrifugation, the resulting pellet was gently titrated in solution A yielding suspension that was further incubated for 10 min at 25°C in solution A containing 2.7 mM MgS04 and 0.03 mM CaC12. After a final centrifugation the pellet was resuspended in EMEM media (Sigma, St.
  • test compounds with potential zinc- ionophore activity were screened for their ability to transport zinc into selected target cells.
  • the test compounds were first complexed with zinc.
  • the zinc-complexed ionophores holo- ionophores
  • the zinc- free forms of these compounds apo- ionophores
  • purified holo- ionophores were purchased commercially (e.g. zinc-diethyldithiocarbamate, Sigma- Aldrich) . However, in most cases only the apo-ionophores were available commercially.
  • the holo-ionophores were therefore prepared in our laboratory. Since zinc ionophores (e.g. pyrithione, diethyldithiocarbamate, 8- hydroxyquinoline) complex with zinc in a 2:1 molar ratio (ionophore: zinc) , stock solutions (generally 15.7 mM) of holo-ionophores were prepared by combining the apo- ionophore with ZnCl 2 in a 2:1 molar ratio either in water or DMSO, depending on the solubility of the reactants, and incubating at room temperature for 15 min. The holo- ionophores were then stored at -20°C.
  • zinc ionophores e.g. pyrithione, diethyldithiocarbamate, 8- hydroxyquinoline
  • stock solutions generally 15.7 mM
  • test compounds were performed with cultured HUVEC, isolated cardiac myocytes, and cultured cerebellar neurons following an approach described previously (Turan et al . , (1997) Am. J. Physiol. 272 :H2095 -H2106) .
  • the cells were loaded with Fura-2, a zinc and calcium- sensitive indicator, by incubating the cells for 30 min in medium containing 4 uM Fura-2 -am (Molecular Probes) .
  • Glass coverslips bearing HUVEC or cerebellar cells were placed directly in a superfusion chamber on the stage of an epifluorescence inverted microscope (Nikon Diaphot-DM) .
  • the cells were then superfused with superfusion buffer containing a test compound and the fluorescence at 505 nm was recorded in response to excitation at 340 nm and 380 nm.
  • the slope of the fluorescence intensity ratio in response to excitation at 340 and 380 nm was used to determine - ionophore activity.
  • the membrane-permeant heavy metal chelator N,N,N' ,N' , - tetrakis (2 - pyridylmethyl) ethylenediamine (TPEN, 30 uM) was added to the superfusate at the end of the run.
  • Vitamin A (all - trans -retinol) ⁇
  • Vitamin E (alpha- tocopherol) ⁇
  • EXAMPLE 2 In vivo heart model - ischemic injury.
  • the sections were then incubated in 75 ml of a buffer solution containing 200 mmol/L potassium cacodylate, 2 mmol/L CoCl 2 , 0.25 mg/ml bovine serum albumin, 25 mmol/L Tris-HCl, pH 6.6, 10 mmol/L biotin-16- dUTP (Boehringer Mannheim Canada, Laval, Quebec) , and 25 units of terminal transferase (Boehringer) , for 1 h at 37°C in a humidified chamber. The reaction was terminated by washing the sections 3 times (1 min each) with PBS at RT.
  • a buffer solution containing 200 mmol/L potassium cacodylate, 2 mmol/L CoCl 2 , 0.25 mg/ml bovine serum albumin, 25 mmol/L Tris-HCl, pH 6.6, 10 mmol/L biotin-16- dUTP (Boehringer Mannheim Canada, Laval, Quebec) , and 25 units of terminal transfera
  • the sections were then incubated with 1 ml of a staining solution containing 2.5 mg/ml fluorescein isothiocyanate-avidin (avidin-FITC) , 4X saline- sodium citrate buffer, 0.1% Triton X-100, and 5% powdered milk, for 30 min at RT, protected from light.
  • the sections were washed 3 times with PBS, were coverslipped in "anti -fade” solution containing 1 mg/ml p-phenylenediamine, 90% glycerol, in PBS, and histofluorescence was monitored with a Zeiss Axiophot microscope.
  • Positive control samples were prepared by incubating sections with 10 units/ml DNAse I for 20 min at 37°C prior to treatment with terminal transferase.
  • the heart tissue was homogenized on ice for 45 s using a Polytron homogenizer at 10,000 rpm in 8 volumes of 10 mM HEPES (pH 7.9), containing 10 mM KC1, 1.5 mM MgCl 2 , 0.1% Nonidet P-40, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM spermine, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 5 mg/ml pepstatin.
  • the homogenate was incubated on ice for 15 minutes and centrifuged at 35,000xg at 4°C for 15 min.
  • PVDF polyvinylidene difluoride
  • HSP70 Since HSP70 has been shown to protect against apoptosis (Wong, H.R., et al . (1996) Am . J. Respir. Cell Mol . Biol . 15:745-751), and to be induced by zinc (Klosterhalfen, B., et al . , (1997) Shock 7:254-262), the data suggest that zinc-pyrithione may exerts its protective effect by upregulating HSP70 synthesis. Zinc-pyrithione also caused a statistically significant decrease in the cytosolic concentration of PKC- ⁇ (data not shown) , using the same method as that described for HSP70, with the exception that instead of using a polyclonal rabbit antibody against HSP70, we used an antibody against PKC- ⁇ . As PKC- ⁇ is a well known mtracellular signalling agent, it was therefore concluded that Zn 2+ is capable of modulating mtracellular signalling.
  • EMSA Electrophoretic Mobility Shift Assay
  • EDTA EDTA
  • 10% glycerol The homogenate was centrifuged at 100,000 xg at 4°C for 1 h, the supernatant was discarded, and the pelleted nuclei were gently resuspended in 40 ml of a lysis buffer containing 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM spermine, and 5 mg/ml each of aprotinin, leupeptin and pepstatin.
  • the suspension was incubated on ice for 45 min and centrifuged at 20,000xg at 4°C for 10 min.
  • the supernatant containing nuclear protein was collected and diluted 1:1 with a buffer containing 20 mM HEPES, pH 7.9, 50 mM KC1, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM spermine, and 5 mg/ml each of aprotinin, leupeptin and pepstatin. Protein concentrations were determined using the Bio Rad protein assay.
  • double- stranded consensus oligonucleotides for NF-kB, AP-1 and Spl were radiolabelled with g[ 32 P]ATP (Amersham, Arlington Heights, Illinois) .
  • Five mg of nuclear protein were first incubated for 10 min at room temperature with 5 mg poly-d[I-C] (Boehringer Manheim, Montreal, Quebec) in DNA binding buffer (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KC1, 5% glycerol, and 2 mM DTT) .
  • Labeled probe (0.2 ng) was then added and the reaction mix incubated for a an additional 20 min in a final volume of 20 ml.
  • the reaction mixture was subjected to electrophoresis on 5% polyacrylamide gel, and the dried gel was exposed to X-ray film. The intensity of the bands was quantitated with a densitometer and commercially available software (Molecular Analyst, Bio-Rad Laboratories, Hercules, California) .
  • the subunit composition of NF-kB was determined with supershift assays.
  • Antibodies (2 mg) to either p50 or p65 (Santa Cruz Biotechnologies, Inc.
  • Glutamate is the principal excitatory neurotransmitter in the brain, and plays a critical role in the etiology of different major brain pathologies such as cerebral ischemia, neurodegeneration, epilepsy, etc (Coyle, J.T. (1993) Science 262 : 689 - 695) . Compounds which interact with the glutamate receptors are therefore important tools in the investigation of these diseases.
  • Kainate is an excitotoxic glutamate analog that produces excessive neuronal excitation and seizures within hours following its intraperitoneal injection into adult rats. At 2-3 days after treatment, neurodegeneration can be observed in the limbic system in the form of apoptosis
  • the neurotoxic effect of kainate in the rat hippocampal subregions involves a direct effect on presynaptic kainate receptors and an indirect effect on postsynaptic glutamate receptors due to the enhanced release of glutamate (Malva, J.O., et al . , (1998)
  • Zinc pyrithione was injected 15 min after KA administration. Sham operated rats received 1.2% DMSO in the lateral ventricles and isotonic saline mtraperitoneally. ' The volume of substances injected into ventricles was 1.5 uL. Seizure activity in the rats was followed during first 4-6 h after KA administration.
  • Results Brains of sham-operated rats and control animals injected icv with zinc-pyrithione did not show any obvious damage in any brain region. However, administration of KA caused neuronal degeneration and cell loss in a number of brain regions, with injury reaching maximal levels within the first day after KA treatment. Preliminary data suggest that the brain damage is attributable to apoptosis, as detected by the TUNEL stain (not shown) . Twelve rats were subjected to KA treatment alone and 12 were treated with KA followed by zinc-pyrithione. Figure 6 shows the number of rats (out of 12) that displayed detectable signs of injury in each group.
  • Zinc-pyrithione also changed the pattern of KA- induced seizures in the rats.
  • the seizure study was performed with 57 rats in the KA- alone group, and 58 in the KA plus zinc-pyrithione group.
  • the data presented in Figure 5 show the number of rats in which a given seizure severity was the final stage of severity observed.
  • zinc-pyrithione caused a statistically significant 3.2 -fold decrease in the incidence of the most severe and irreversible stage of seizures
  • stage 6 In other words, of the 57 rats treated with KA alone, fully 18 reached the lethal stage 6, as compared with only 5 out of 58 in the KA plus zinc- pyrithione group.
  • the data further show that the final stage of severity tended to be much lower in the zinc- pyrithione group, as illustrated by the 3.2-fold increase in the number of zinc-pyrithione treated rats at level 2 ("wet dog shake” stage). (*, P ⁇ 0.01, Fisher's exact test) .
  • zinc-pyrithione significantly decreased KA- induced cell death in a number of brain regions, and significantly lowered the severity of KA- induced seizures in rats.
  • Oxidative stress is believed to play an important role in the apoptotic neuronal cell death associated with many different neurodegenerative conditions (e.g., Alzheimer's disease, Parkinson's disease, cerebral ischemia, etc.) (Jenner, P. (1994) Lancet 344:796-798).
  • the non-differentiated rat pheochromocytoma PC12 cells are a cell line which differentiates to a neuronal cell type in the presence of Nerve Growth Factor (NGF) , but undergoes apoptotic cell death when deprived of NGF. These cells also undergo apoptotic cell death when exposed to oxidants such as hydrogen peroxide (Satoh, T., et al . , (1997) J. Neurosci . Res . 50:413-420; Maroto, R. et al . , (1997)
  • PC12 cells are therefore a useful cell model with which to analyze the molecular mechanisms of apoptosis induced by oxidative stress and other stimuli in neuronal cells (Kubo, T.,et al . , (1996) Brain Res . 733:175-183, incorporated herein by reference).
  • PC12 cells were seeded on coverslips covered with poly-L- lysine in 24 -well plates, and were grown in RPMI-1640 containing 10% FCS, 5% horse serum, 2 mM glutamine, and 40 mg/kg gentamicin in 5% C0 2 at 37°C.
  • RPMI-1640 medium containing 1% serum (embryonal calf and horse serum, 2:1) and 50 ng/ml NGF was used to induce differentiation.
  • Differentiated PC12 monolayers were washed and were induced to undergo apoptosis in two ways: a) incubation for 4 h with normal growth medium without serum or NGF, and b) incubation for 4 h with normal growth medium (plus serum and NGF) containing 20 ⁇ M hydrogen peroxide (H 2 0 2 ) .
  • the protective effect of zinc- pyrithione against both types of apoptosis was tested by preincubating the cells with this compound for only 5 minutes immediately prior to initiating apoptosis - inducing treatments a or b. Control cells were pretreated with the carrier DMSO alone.
  • Pretreatment with zinc-pyrithione rather than the more relevant post- treatment approach, was used in this model because of practical experimental considerations.
  • the close temporal proximity of the pretreatment to the initiation of the injurious treatment is more representative of a concomitant exposure of the cells to both zinc-pyrithione and the injurious agent, rather than an authentic pretreatment.
  • the cells were fixed with methanol : acetone (1:1) at -20°C and were stained with Hoechst 33258 to> visualize the nuclei.
  • ionizing radiation has been shown to induce the activation of the transcription factor NF-kB in several cell types (Valerie, K. , et al . , (1995) Biochemistry 34:15768-15776), including endothelial cells (Hallahan, D. et al . , (1995) Biochem . Biophys . Res . Commun . 217:784-795).
  • the cytokine TNF ⁇ is also capable of causing apoptosis in endothelial cells either alone
  • the studies performed with this model were designed to examine the ability of zinc- pyrithione to block radiation- induced apoptosis, and to elucidate the effect of zinc-pyrithione at the transcriptional level in response to either ionizing radiation or TNF ⁇ .
  • HUVEC Human umbilical vein endothelial cells
  • Clonetics San Diego, California
  • Cells were cultured on gelatin- coated culture dishes in Endothelial Basal Medium (Clonetics) supplemented with lOng/ml human recombinant epidermal growth factor, 1.0 ug/ml hydrocortisone, 50ug/ml gentamicin, 50ng/ml amphotetericin B, 12ug/ml bovine brain extract and 2%v/v fetal bovine serum, in a humidified chamber at 37°C and 5% C0 2 . To maintain cell populations, proliferating HUVEC were passaged at 80-90% confluency.
  • HUVEC HUVEC were grown to confluency, and then given an additional 24 hours to achieve quiescence prior to experimental treatment. The following treatments were performed:
  • TNF ⁇ The cells were washed twice with 37°C D-PBS and then irradiated in fresh media. Irradiated cells received a dose of 1000 Rads of gamma- irradiation from a 137 Cesium source (Atomic Energy of Canada) . The cells were then incubated for 2 hours (cytosolic and nuclear protein extraction) or 8 hours (Hoechst staining and DNA electrophoresis) .
  • TNF ⁇ The cells were washed twice with 37°C D-PBS and then incubated in media containing the TNF ⁇ (20ng/ml, from a stock of lOug/ml prepared in phosphate buffered saline (PBS) - l%bovine serum albumin. Control cells received fresh media alone. The cells were then incubated for 2 hours (cytosolic and nuclear protein extraction) or 8 hours (Hoechst staining and DNA electrophoresis) .
  • Hoechst Staining Cells were grown on round, gelatin coated 12 mm glass coverslips, and following treatment, were fixed with 0.5 ml of 1% glutaraldehyde in PBS for 10 minutes at room temperature (RT) . The cells were then washed twice with PBS for 5 minutes, and permeabilized with 0.5 ml of 1:1 methanol/acetone for 10 minutes at RT, followed by two five minute PBS washes. The cells were then incubated with Hoechst 33258 (bis-benzimide, 0.05mg/ml in H 2 0) , a fluorescent DNA binding dye, for 30 minutes at room temperature, in the dark. The nuclear morphology of the cells was then visualized under a Zeiss Axiophot fluorescence microscope.
  • the supernatant containing residual cytosolic proteins was discarded and the pelleted nuclei were resuspended in 35ul of Buffer B (20mM HEPES, 420mM NaCl, 1.5mM MgCl 2 , 0.2mM EDTA, 25% glycerol, pH 7.9, 0.5mM DTT, 0.5mM PMSF, and the protease inhibitors spermidine, spermine, aprotinin, leupeptin and pepstatin) for 45 minutes on ice in order to extract the nuclear proteins.
  • Buffer B 20mM HEPES, 420mM NaCl, 1.5mM MgCl 2 , 0.2mM EDTA, 25% glycerol, pH 7.9, 0.5mM DTT, 0.5mM PMSF, and the protease inhibitors spermidine, spermine, aprotinin, leupeptin and pepstatin
  • the protein concentration in the nuclear and cytosolic extracts was determined using the Bradford Assay (Biorad) using bovine serum albumin as the standard.
  • Electrophoretic Mobility Shift Assay Equal amounts of nuclear protein (5ug) were incubated with poly dl-dC (5ug from a stock of 2.5ug/ul in TE buffer) for 10 minutes at RT. This reaction mixture was then incubated with 0.2ng of 5' end- 32 phosphorus - labelled double stranded oligonucleotide probe for 20 minutes at RT to allow the binding of nuclear proteins with the labeled probe.
  • Loading buffer (5ul of a mixture containing 20mM HEPES, lOOmM KC1, 60% glycerol, 0.5mM EDTA, 0.5mM EGTA and 0.125% bromophenol blue) was added to the reaction mixture prior to the electrophoresis on a 5% native polyacrylamide gel.
  • the gels were run in Tris-Glycine solution for 1.5 hours at 200V and were then dried between filter paper and cellophane for 1.5 hours at 80°C under vacuum. The dried gels were exposed to X-ray film (Cronex) for up to 2 days at -80°C.
  • reaction mixture was incubated with a 125- fold excess of unlabeled probe for 20 minutes at RT prior to the addition of the labeled probe.
  • reaction mixture was incubated with 2mg of rabbit polyclonal anti-NFkB p50 or p65 antibody (Santa Cruz Biotechnology) for 20 minutes at RT immediately subsequent to the addition of the labeled probe. The bound antibody retards the mobility of the protein-DNA complex, resulting in a shifted band.
  • the consensus oligonucleotides for the transcription factors NFkB (5' - ACT TGA GGG GAC TTT CCC AGG C - 3 ' ) , AP - 1 ( 5 ' - CGC TTG ATG AGT CAG CCG GAA-3') and Spl (5' -ATT CGA TCG GGG CGG GGC GAG C-3') (Promega) and were labeled as suggested by Promega with minor modifications.
  • oligonucleotides (20ng) , T4 Polynucleotide kinase and [g32P]ATP (60uCi) were mixed in kinase buffer (50mM Tris- HCl, pH 7.6, lOmM MgCl 2 , 5% glycerol and 5mM DTT) and incubated at 37°C for 1 hour. Labeled oligonucleotides were removed by centrifugation through a G-25 Sephadex Column at 8500rpm for 20 minutes. The labeled oligonucleotides were then diluted such that 2ul of the probe mixture contained approximately 50000 - lOOOOOcpm.
  • the blots were then washed with TBS-T and incubated for 1.5 hours in primary antibody (anti-IkBa, Santa Cruz Biotechnology) diluted 1:1000 in 2% skimmed milk in TBS-T and sodium azide at RT with constant shaking. The blots were then washed with TBS-T and incubated for 30 minutes in horseradish peroxidase labeled goat anti -rabbit IgG diluted 1:10000 in 2% skimmed milk in TBS-T at RT with constant shaking. Following treatment with the secondary antibody, the blots were extensively washed with TBS-T and incubated for 1 minute with chemiluminescent substrate. The blots were then exposed to X-ray film for 1-5 minutes.
  • primary antibody anti-IkBa, Santa Cruz Biotechnology
  • Results The data show protection by zinc-pyrithione against endothelial apoptosis, and also show that this protective effect is associated with transcriptional modulation.
  • the data also show that zinc is required for this effect since the sodium salt of pyrithione was not effective in preventing apoptosis.
  • DMSO alone was also ineffective. No apoptosis was caused in control cultures by zinc-pyrithione, sodium-pyrithione, or DMSO alone ( Figure 8).
  • (*, P ⁇ 0.05 vs. Control; °, P ⁇ 0.05 vs. irradiated cells, n 4).
  • EMSA tests showed that irradiation- induced apoptosis is associated with a significant increase in nuclear NF-kB content, and that zinc-pyrithione, but not sodium-pyrithione or DMSO alone, blocked this increase (Figure 9) .
  • Zinc-pyrithione had a very similar effect in TNF ⁇ -treated HUVEC ( Figure 10), and was particularly potent at blocking the TNF -induced increase in NF-kB content.
  • Stroke is an extremely variable clinical condition which reflects the variability of the underlying disease process.
  • the vascular occlusion can occur at many different sites in the brain and the cause of the occlusion, the severity of the problem, and the degree of reversibility can all contribute to the variability of outcome.
  • most of these variables can be controlled or eliminated, enabling a meaningful interpretation of the results.
  • stroke models are grouped into those producing either global or focal ischemia
  • ZP zinc pyrithione
  • 4VO a global ischemia -reperfusion model of 4 vessel occlusion in rats
  • MCAO middle cerebral artery occlusion in mice
  • the 4VO approach sealed off the two carotid and two vertebral arteries which carry all the blood to the brain. This approach permitted severe forebrain ischemia to be produced in awake and freely moving rats, and produced reproducible neuropathology.
  • the 4VO is a two-stage operative procedure (Pulsinelli, et al . , (1979) Stroke, 10: 267-272, incorporated herein by reference) .
  • mice the 4VO is a favorite stroke model because it results in highly reproducible damage in the CAI region of the hippocampus, as well as in some other brain regions.
  • MCAO in mice is one of the most clinically relevant stroke models. It shuts off blood flow to only a portion of the brain, producing a focal injury which closely resembles the clinical situation with stroke patients. This procedure was performed with an intraluminal thread. A nylon suture was introduced into the external carotid artery and was gently advanced into the internal carotid artery. The diameter of the suture was such that it lodged in the anterior cerebral artery, occluding the medial cerebral artery at its origin.
  • Brain damage in this model was observed as early as several hours after the ischemic episode, with optimal injury occurring at 24 h.
  • the injury occupied a large part of the hemisphere including the cerebral cortex and subcortical structures, and its severity depended on the duration of ischemia and the strain of mice used.
  • the 4VO procedure was performed on male Wistar rats weighing 240-300 g.
  • the rats were housed in groups of 5 in plastic cages with free access to food and water.
  • the MCAO protocol was performed on male C57BL/6 mice weighing 20-28 g which were housed in groups of 10 in plastic cages with free access to food and water. All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
  • Rats under chloral hydrate anesthesia (325 mg/kg) were positioned in a stereotaxic frame.
  • the vertebral arteries were exposed and permanently occluded by electrocautery at the first cervical vertebra.
  • Snares surgical silk strings
  • the animals were then allowed to recover for 24 hours with free access to water.
  • the rats were lightly anesthetized with ether, were secured to surgical boards, ventral side up, and their common carotid arteries were exposed.
  • Forebrain ischemia was initiated by tightening the snares around the carotid arteries for 10 min.
  • the body temperature of the rats was carefully maintained at ⁇ 37-37.5°C, both before and during the ischemic insult, using a feedback- controlled heating pad and a rectal thermistor (Homeothermic Blanket System, Harvard Apparatus LTD, England) .
  • the initial (1 st min of ischemia) and final (10 th minute of ischemia) temperature did not differ among all groups of rats studied.
  • the temperature was maintained in similar fashion at 37°C for at least 4 h. Only rats that showed signs of severe neurological injury, such as a loss of the righting reflex, pupil dilation, etc., were included in this study.
  • the vertebral and carotid arteries were exposed, but were not occluded.
  • Evaluations of neurological deficit were performed at 24 and 96 h after ischemia, and were based on a scoring system which recorded activity level, motility, pain reflex, grabbing reflex, and the ability to see and hear (Miljkovic, L.M., et al . , (1997) Ann. Emerg. Med. 29, 758-765, incorporated herein by reference).
  • mice C57BL/6 were anesthetized with an intraperitoneal injection of chloral hydrate (350 mg/kg) and xylasine (4 mg/kg) .
  • Focal cerebral ischemia was produced by occlusion of the MCA using the intraluminal filament technique (Longa, Z.E., et al . , (1989) Stroke 20 : 84 -91, incorporated herein by reference).
  • a 8.0 nylon microfilament coated with a silicon resin (Xantopren) - hardener mixture Hara, H., et al . , (1996) J. Cereb. Blood Flow Metab.
  • Stroke 17:472-476 incorporated herein by reference: 0, no observable neurological deficit (normal); 1, failure to extend the right forepaw (mild); 2, circling to the contralateral side (moderate); 3, falling to the right (severe); 4, inability to walk spontaneously (most severe) .
  • ZP zinc pyrithione
  • ZnDDC zinc- diethyldithiocarbamate
  • control and experimental animals were deeply anaesthetized with sodium thiopental (60 mg/kg, mtraperitoneally) and were perfused transcardially with 250 ml of AFA fixative (96% alcohol, 39 % formalin, glacial acetic acid , 7:2:1). After the AFA perfusion the heads were collected intact and were kept at 4°C for 4-5 h. The brains were then removed and immersed in the same fixative for 1 h, and were then stored in 70% alcohol. Each forebrain was cut into three frontal blocks and imbedded in paraffin. Ten ⁇ m thick sections were cut from a region 3.0-4.0 mm posterior to the bregma. The sections were stained with cresyl violet (Nissl) .
  • ISEL In Situ End Labeling
  • MCAO Mice were killed 24 h after reperfusion with an overdose of sodium thiopental (60 mg/kg, mtraperitoneally) , and the brain was rapidly removed and sectioned coronally into five 1.7 mm slices. The slices were then placed in 2% (wt/vol) 2,3,5- triphenyltetrazolium chloride solution (TTC) in PBS (pH 7.4) for 20 min at 37°C. This procedure, which tests mitochondrial activity, stains viable tissue a bright red, while the infarcted regions remain white. Following TTC staining the sections were fixed in 10% formalin overnight. The area of infarct in each section was determined using an image-analysis system.
  • TTC 2,3,5- triphenyltetrazolium chloride solution
  • the infarct volume was subsequently calculated by summing the infarct areas in the sequential 1.7 mm- thick sections with correction for edema.
  • the person measuring infarct volumes was blinded as to the identity of the experimental groups.
  • ISEL staining the brains were removed, were frozen rapidly, and were sectioned with a cryostat into 20 urn sections from the anterior side to the posterior side at 500 urn intervals. ISEL was performed as described above. Adjacent sections were stained with cresyl violet (Nissl) .
  • the ApopTag® Peroxidase In situ Apoptosis detection Kit (Intergen) was also used to detect apoptosis in mouse brain sections .
  • the neurological deficit data in 4VO rats are presented in Table 2.
  • a method of scoring in which increasing neurological scores are indicative of decreasing neurological function compared to a perfect score of 0 for the shams was employed.
  • a large number of the 4VO rats did not show any neurological deficit (score 0) , despite the fact that they sustained an almost complete loss of cells in the CAl region. Therefore, the administration of ZP did not influence the neurological deficit in the 4VO rats despite clear evidence of histological protection.
  • the neurological deficit data for this ZP administration schedule are presented in Table 2.
  • the neurological score in "3, 4, 6 h schedule" rats did not differ from that in the other regimen (10 min, 1 h, 2 h after ischemic episode) .
  • These data therefore indicated that delaying the first administration of ZP by 3 hours did not significantly change its neuroprotective effect at the dose of 3x6 ⁇ g/kg, indicating a possible wide therapeutic window for ZP in the 4VO stroke model.
  • MCAO MCAO for 1 h produced significant infarcts in the left hemisphere of mouse brain (Figure 15) .
  • Three doses of ZP were used (3x1.2 ⁇ g/kg, 3x6 ⁇ g/kg, and 3x30 ⁇ g/kg) and infarct areas, infarct volumes and neurological scores were measured.
  • ZP at all three doses significantly decreased the infarct area at a distance of 3.4-5.8 mm from the frontal pole ( Figure 16) .
  • Table 3 presents data on the absolute and relative (% of the contralateral hemisphere) infarct volumes in the mouse MCAO model in response to zinc pyrithione treatment.
  • the calculated infarct volumes in the ischemic control mice were similar to those reported previously for this strain of mouse (Hara, H. , et al . (1996) J. Cereb. Blood Flow Metab. 16:605-611; MacManus, J.P., et al., (1999) NeuroReport 10:2711-2714; Nagayama, M., et al., (1999) J Cereb. Blood Flow Metab. 11:1213- 1219; Nogawa, S., et al . , (1998) Proc . Natl .
  • the pericardium will be opened and fixed to the border of the sternum.
  • a left atrial pressure (LAP) catheter will be introduced.
  • Coronary occlusion by a snare will be applied by tunneling the left anterior descending (LAD) coronary artery with a monofil suture between the proximal and medial third behind the first diagonal branch.
  • LAD left anterior descending coronary artery
  • the vena cordis magna will be cannulated for blood analysis with a small catheter.
  • a myocardial P0 2 probe will then be implanted into the expected center of the area at risk.
  • a temperature probe will be positioned next to the P0 2 sensor. Baseline values will be acquired during a 1-hour preoperative period.
  • Coronary occlusion will be achieved by tightening the snare around the LAD for 60 min. The snare will then be loosened to initiate reperfusion.
  • Two models will be tested: I. An acute non- survival model with a total of 6 h reperfusion, and II. A recovery model with 7 days of reperfusion. Each of the two models will consist of 5 groups: Sham operated (Sham), Ischemia/reperfusion alone (I/R) , I/R plus vehicle alone (I/R + DMSO) , I/R plus zinc pyrithione (I/R + ZP) , and I/R plus Zinc-diethyldithiocarbamate (I/R + ZD) .
  • the zinc ionophores will be infused into the LAD in three boluses of 5ml saline, at 0, 1, and 2h after the initiation of reperfusion to give a cumulative dose of 1 ⁇ g/kg body weight.
  • Vehicle will be administered in a similar and blinded fasion.
  • Hemodynamic and P0 2 measurements and blood samples will be obtained at 5 , 10, 20, 30, and 60 minutes after coronary occlusion; and after 5, 10, 20, 30, 60, 90, and 120 minutes of coronary reperfusion.
  • Global and regional contractility will be recorded by regional 2Dultrasound before, after 15 and 60 minutes of coronary occlusion, and after 15, 60, and 120 minutes of reperfusion.
  • the pigs will be killed, and their hearts will be recovered for further analysis .
  • ECG, right atrial, pulmonary artery, and arterial pressure and LAP will be recorded on a Siemens Sirecust 404-1 at different time points.
  • Cardiac output will be determined by thermodilution (5 mL NaCl 0.9%, room temperature) and by continuous measurement with a Baxter Vigilance monitor.
  • the Licox catheter probe measurement system will be used in which the flexible
  • Licox catheter P0 2 microprobe is in direct contact with the myocardium.
  • One milliliter of heparinized venous and arterial blood will be drawn with polypropylene syringes from the femoral artery and the vena cordis magna. Lactate and arteriovenous 0 2 differences will be determined before, after 60 minutes of coronary occlusion, and after 10 and 120 minutes of reperfusion.
  • Blood gas analysis will be performed with the Radiometer Copenhagen Arterial Bloodgas Laboratory 3.
  • samples will be centrifuged at 2000g (10 minutes at 4°C) , plasma will be decanted, and lactate will be measured with the Lactate Analyzer model 23L from Yellow Springs Instrument Co Inc.
  • Venous blood samples (3 ml) will be collected in polypropylene tubes containing citrate and will be centrifuged at 2000g for 15 minutes at 4°C. Plasma creatine kinase activity will be determined and expressed as international units per milliliter. Troponin-T will be measured.
  • Unstained myocardium will be defined as the area at risk. After cardioplegia with 20 mL potassium chloride IV (20%) , the heart will be excised. The right ventricle, the large vessels, and fat tissue will be removed. The left ventricle will then be sliced perpendicular to the axis of the left side of the heart from the apex to the AV groove in 4 -mm slices.
  • the unstained part of the left ventricular myocardium will be separated from the Evan's blue-stained portion and immersed in a 0.09-mol/L sodium phosphate buffer, pH 7.4, containing 1% triphenyltetrazolium chloride (TTC, Sigma) and 8% dextran (molecular weight, 77.800) for 20 minutes at 37°C.
  • TTC triphenyltetrazolium chloride
  • the TTC dye will form a dark-red formazan complex in the presence of viable myocardial cells that contain active dehydrogenases and cofactors. Dead cells will remain unstained.
  • the ischemic but non-necrotic, red- stained tissue will be separated from the unstained, infarcted tissue.
  • the three tissue sections—nonischemic (area not at risk) , ischemic non-necrotic, and ischemic necrotic tissue will be weighed.
  • the arterial pressure-rate product (mean arterial pressure times heart rate) will be taken as a global parameter of myocardial contractility.
  • TUNEL staining (Fliss et al . (1996) supra) , as described above, will be used to identify the percent of apoptotic myocytes in the myocardial tissue.

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Abstract

La présente invention concerne des procédés et des compositions comprenant un ou plusieurs ionophores de zinc destinés à inhiber l'apoptose chez des patients nécessitant un tel traitement. Des concentrations de pyrithione de zinc et de diéthyl-dithiocarbamate dans la plage picomolaire-nanomolaire produisent un effet protecteur important contre l'aopotose.
EP00979231A 1999-06-23 2000-06-23 Ionophores de zinc utilises comme agents anti-apoptose Ceased EP1189604A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14063299P 1999-06-23 1999-06-23
US140632P 1999-06-23
PCT/IB2000/001005 WO2001000193A2 (fr) 1999-06-23 2000-06-23 Ionophores de zinc utilises comme agents anti-apoptose

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EP1189604A2 true EP1189604A2 (fr) 2002-03-27

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EP00979231A Ceased EP1189604A2 (fr) 1999-06-23 2000-06-23 Ionophores de zinc utilises comme agents anti-apoptose

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EP (1) EP1189604A2 (fr)
JP (1) JP2003503346A (fr)
AU (1) AU1839601A (fr)
CA (1) CA2375852A1 (fr)
WO (1) WO2001000193A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1377300A1 (fr) * 2001-04-04 2004-01-07 Henry Fliss Ionophores de zinc utilises comme agents anti-stress
WO2002089919A2 (fr) * 2001-05-08 2002-11-14 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Utilisation du zinc dans la prevention d'une pathologie induite par tnf pendant une therapie contre le cancer
EP1585474B1 (fr) * 2003-01-15 2010-11-10 Stryker Corporation Dispositif de chargement et de dechargement de civiere d'ambulance
JP2006206538A (ja) * 2005-01-31 2006-08-10 Institute Of Physical & Chemical Research 抗原提示細胞の機能制御剤
WO2014152207A1 (fr) 2013-03-15 2014-09-25 Mylan Laboratories, Inc. Formulations de granulation par fusion de principes actifs faiblement hydrosolubles
AU2018260927B1 (en) * 2018-05-25 2019-02-21 Graham Sherard Baldwin Methods and compositions for inhibiting a noxious insult
WO2021035060A1 (fr) * 2019-08-21 2021-02-25 Brain Chemistry Labs Compositions comprenant du métal et de la l-serine, et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0100193A2 *

Also Published As

Publication number Publication date
WO2001000193A3 (fr) 2001-12-06
AU1839601A (en) 2001-01-31
CA2375852A1 (fr) 2001-01-04
JP2003503346A (ja) 2003-01-28
WO2001000193A2 (fr) 2001-01-04

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