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WO2004060490A1 - Composition comprenant un complexe desferrioxamine-metal, et son utilisation pour le traitement de dommages causes aux tissus consecutivement a une exposition a des agents chimiques de combat - Google Patents

Composition comprenant un complexe desferrioxamine-metal, et son utilisation pour le traitement de dommages causes aux tissus consecutivement a une exposition a des agents chimiques de combat Download PDF

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Publication number
WO2004060490A1
WO2004060490A1 PCT/IL2003/001122 IL0301122W WO2004060490A1 WO 2004060490 A1 WO2004060490 A1 WO 2004060490A1 IL 0301122 W IL0301122 W IL 0301122W WO 2004060490 A1 WO2004060490 A1 WO 2004060490A1
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Prior art keywords
mustard
dfo
desferrioxamine
treatment
pharmaceutical composition
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English (en)
Inventor
Mordechai Chevion
Eduard Berenshtein
Eyal Banin
Alexey Obolensky
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Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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Priority to AU2003290393A priority Critical patent/AU2003290393A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds 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
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • 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

Definitions

  • the invention relates to the topical use of desferrioxamine-metal complexes, particularly zinc and gallium complexes of desferrioxamine (DFO), in the treatment of damage to tissues exposed to mustard gas and other warfare agents.
  • DFO desferrioxamine
  • the invention is particularly directed, but not limited to, the use of such DFO complexes in the treatment of ocular tissues and skin burns.
  • Free radicals can react in a variety of mechanisms, one of which being the formation of hydroxyl radicals through catalysis of transition metal ions.
  • the most widely accepted mechanism of antioxidants activity is based on their scavenging capacity of free radicals.
  • the DFO complexes use of which is an object of this invention, are expected to exert their protection primarily by means of inhibition of the formation of hydroxyl radicals via transition metal- catalyzed reaction/s, and not only by scavenging of the already formed free radicals, or some other pathway. This mechanism of protection by the chelators used in the invention has not been taught neither suggested by the prior art, and its success was unexpected.
  • Zinc desferrioxamine (Zn/DFO) and gallium desferrioxamine (Ga/DFO) are known metal complexes, which inhibit the catalysis of iron (and copper) in the formation of free radicals. Their protective activity can be visualized through the "pulling" out of redox active iron that is responsible for the production of the hydroxyl radicals via chelation by the DFO component.
  • the relatively inert zinc (or gallium) ion that is liberated during the exchange of iron within the complex, further acts as a secondary antioxidant, by "pushing" out an additional iron ion from its binding site [Chevion, M. (1988) Free Radic Biol Med 5, 27-37; Chevion, M.
  • the present invention relates to a desfemoxamine-metal complex for use in the topical or combined topical/systemic treatment and/or prevention of damage to a tissue exposed to a warfare agent, particularly mustard gas, nitrogen mustard, sulfur mustard, chlorine, phosgene oximine, lewisite, Tabun, Sarin or Soman, and most particularly nitrogen mustard.
  • a warfare agent particularly mustard gas, nitrogen mustard, sulfur mustard, chlorine, phosgene oximine, lewisite, Tabun, Sarin or Soman, and most particularly nitrogen mustard.
  • the desferrioxamine complexes for use in this invention are particularly zinc, gallium or manganese complexes, or their combination, preferably zinc and gallium desferrioxamine complexes.
  • the desferrioxamine complex may be used in the topical treatment of acute or chronic injury to an ocular tissue following exposure to said warfare agent, particularly damage to the conjunctiva, cornea, iris and/or the eye anterior chamber.
  • the desferrioxamine complex may also be use in the topical or combined topical/systemic treatment of skin burns and damage, particularly skin burns induced by any one of said warfare agents.
  • the desferrioxamine complex may be used in the topical treatment of lung and airway damage, particularly via aerosol.
  • the invention in a second aspect, relates to a pharmaceutical composition for the topical treatment and/or prevention of damage to tissues exposed to a warfare agent, comprising as active ingredient at least one desferrioxamine metal complex, optionally further comprising a pharmaceutically acceptable carrier, additive, excipient and/or diluent.
  • the pharmaceutical composition is particularly useful in the treatment of damage induced by mustard gas, nitrogen mustard, sulfur mustard, chlorine, phosgene oximine, lewisite, Tabun, Sarin and/or Soman, particularly mustard gas and most particularly nitrogen mustard.
  • the desferrioxamine metal complex comprised in the pharmaceutical composition of the invention can be a zinc, gallium or manganese complex, and their combinations can also be comprised in the composition. Most preferred are zinc and gallium complexes.
  • the pharmaceutical composition of the invention is particularly intended for the topical treatment of acute or chronic injury to an ocular tissue following exposure to a mustard gas, particularly the conjunctiva, cornea, iris and/or the eye anterior chamber.
  • the pharmaceutical composition of the invention may be used in the topical treatment of warfare agent-induced skin burns and damage.
  • the invention further relates to a method for the topical treatment and/or prevention of damage to tissues exposed to a warfare agent, particularly mustard gas, comprising administering to a subject in need thereof a therapeutically effective amount of any of the above desferrioxamine-metal complexes or of a pharmaceutical composition comprising the same.
  • the invention relates to the use of any of the above desferrioxamine-metal complexes, or their combinations, in the preparation of a pharmaceutical composition for the topical treatment and/or prevention of damage to tissues exposed to a mustard gas.
  • the invention will be described in more details on hand of the following Figures.
  • Figure 1A-F Corneal and conjunctival injury in eyes after exposure to 2%
  • Nitrogen mustard is N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N
  • Fig. 1A Slit-lamp photograph of anterior segment at four weeks post-injury in saline administered eyes, note marked conjunctival edema and hemorrhages.
  • Fig. IB Slit-lamp photograph of anterior segment at four weeks post-injury in saline-administered eyes, note corneal neovascularization and opacity.
  • Fig. IC Slit-lamp photograph of anterior segment at four weeks post- injury in eyes treated with Ga/DFO, note that the changes were much less severe.
  • Fig. ID Corneal histology of the normal cornea in a non-exposed rabbit eye.
  • Fig. IE Corneal histology of a mustard-exposed eye, note epidermalization of the corneal epithelium and infiltration of the corneal stroma with inflammatory cells, blood vessels and hemorrhages.
  • Fig. IF Corneal histology of a mustard-exposed Ga/DFO -treated eye, note markedly milder changes, as compared to Fig. IE.
  • Figure 2 Degree of corneal opacity following exposure to 1% Nitrogen mustard. Initial injury and opacity were similar among the different experimental groups one day after injury. However, four weeks later corneal opacity was markedly reduced in Zn/DFO- and DFO-alone treated eyes as compared with saline-administered eyes (*p ⁇ 0.05, Pearson Chi-square test). Eyes treated with ZnCl 2 showed a similar trend. Observers blinded to the treatment regimen performed grading of corneal opacity according to a 4-step severity scale. Each bar represents mean score of 6 eyes (see Experimental Procedures for details).
  • Fig. 1A NM-exposed and saline- administered eyes, note areas of iris pigmentation as well as areas of atrophy.
  • Fig. IB Non-injured fellow eyes, the areas shown in (A) are not present in non-injured fellow eyes.
  • Fig. IC NM-exposed and Zn/DFO-treated eyes, the injured areas shown in (A) are markedly reduced.
  • Fig. ID Mean iris pigmentation score at 4-5 weeks post-injury was significantly reduced in Zn/DFO treated eyes as compared to saline- administered, representing a 64% protective effect (p ⁇ 0.05, Pearson Chi- square test).
  • Figure 4A-E Iris fluorescein angiography at 4-5 weeks after exposure to 1%
  • Nitrogen mustard shows that iris pigmentation and atrophy differed among the treatment groups. Integrity of iris vessels was not compromised and no leakage occurred at this time.
  • Fig. 1A Control (no exposure to NM).
  • Fig. IB 1%NM + Saline.
  • Fig. IC 1%NM + Zn/DFO.
  • Fig. ID 1%NM + ZnCl 2 .
  • Fig. IE 1%NM + DFO.
  • Figure 5A-I Iris and lens injury following exposure to Nitrogen mustard.
  • Fig. 5 A Control (no exposure to NM).
  • Fig. 5B Control (no exposure to NM).
  • Fig. 5C Control (no exposure to NM).
  • Fig. 5D Eyes exposed to 2% NM followed by saline-administration.
  • Fig. 5E Eyes exposed to 2% NM followed by saline-administration.
  • Fig. 5F Eyes exposed to 2% NM followed by sahne-administration.
  • Fig. 5G Eyes exposed to 2% NM followed by treatment with Ga/DFO.
  • Fig. 5H Eyes exposed to 2% NM followed by treatment with Ga/DFO.
  • Fig. 51 Eyes exposed to 2% NM followed by treatment with Ga/DFO.
  • IOP Intraocular pressure
  • Nitrogen mustard 2% Nitrogen mustard.
  • IOP peaks at day 4 post-injury, and from one week onwards does not significantly differ from control, non-exposed eyes.
  • Zn/DFO and Ga/DFO significantly attenuate the IOP rise at day 4, representing a 57% and 53% protective effect, respectively (insert; p ⁇ 0.05, Mann- Whitney test).
  • Each data point represents mean IOP + SEM of 7-8 eyes at days 0, 1, 4, 7, 10 and 4-8 eyes at days 14, 21 and 28.
  • T. time; Da. Po. Inj., Days Post-Injury; Sal., saline; Norm., Normal; Cont., control.
  • FIG. 7 Electroretinography (ERG) six weeks after exposure to 1% Nitrogen mustard. Retinal function as measured by ERG was not affected by NM -injury to the anterior segment. Rod and cone function in the experimental NM- exposed, saline-administered eye (RE) did not differ from the fellow, non- exposed control eye (LE).
  • FIG. 8 Systemic Oxidative Stress as assessed by Ascorbic Acid (AA) and its oxidized form, dehydroascorbate (DHAA).
  • OSAA [DHAA] x 100/ ⁇ [AA] + [DHAA] ⁇ .
  • Fig. 9A Corneal neovascularization score following exposure to NM (2%).
  • Fig. 9B Average corneal opacity score following exposure to NM (2%).
  • Fig. 9C Intra-ocular pressure (IOP) following exposure to NM (2%).
  • Fig. 9D Activity of serum Methionine Sulfoxide reductase (Msr) of NM- exposed rabbits.
  • Msr Methionine Sulfoxide reductase
  • Fig. 10A Levels of 2,3-DHBA in skin punches of the exposed area (data is presented as normalized values [2,3-DHBA]/[SAL]).
  • Fig. 10B Levels of 2,5 -DHB A/SAL in the skin punches.
  • Fig. 10C UA/protein ( ⁇ g/mg).
  • Fig. 10D Value of OSAA (%), as an indicator for oxidative stress in the exposed skin.
  • Fig. 10E Area of the wound of NM-exposed skin (cm 2 ).
  • Fig. 10F Histology scores of exposed skin punches.
  • Fig. 11A Values of 2,5-DHBA SAL in skin punches.
  • Fig. 11B Values of 2,3-DHBA/SAL in skin punches.
  • Fig. 11C UA values (ng/ml).
  • Fig. llD OSAA (%).
  • Fig. HE Skin wound area (cm 2 ).
  • Figure 12A-F Protection skin against NM damage (Experiment C).
  • Fig. 12A 2,5-DHBA/SAL (ng/ ⁇ gr).
  • Fig. 12B 2,3-DHBA+Catechol7SAL (ng/ ⁇ gr).
  • Fig. 12C UA ( ⁇ g/ml).
  • Fig. 12D OSAA (%).
  • Fig. 12E Burn area (cm2).
  • Fig. 12F Histology scores.
  • GMC or Ga/DFO Gallium desferrioxamine complex
  • HPLC-ECD HPLC coupled to electrochemical detector
  • IOP Intraocular pressure i.p.: Intra-peritoneal
  • OSAA Oxidative stress as determined by the dehydro- ascorbate/total ascorbate ratio
  • ROS Reactive oxygen species
  • the invention therefore relates to the use of a DFO-metal complex as a topical agent that alleviates the symptoms of exposure to mustard and other chemical warfare agents such as chlorine, phosgene oximine, lewisite, Tabun, Sarin or Soman, which act by mechanisms similar to that of mustard.
  • mustard and other chemical warfare agents such as chlorine, phosgene oximine, lewisite, Tabun, Sarin or Soman, which act by mechanisms similar to that of mustard.
  • the invention further relates to a method of treating tissue damage caused by exposure to mustard, by topically applying to the damaged tissue a metal complex of DFO.
  • Preferred DFO metal complexes are zinc, gallium and manganese complexes, and/or their combinations, with zinc and gallium complexes being most preferred.
  • the use and methods of the invention are particularly suitable for the treatment of skin and ocular tissues, as well as for the treatment of injuries to the lungs and airways, by topical application by virtue of a spray.
  • NM injury is mediated by the formation of ROS, in addition to its action as an alkylating agent.
  • Oxidative stress following ocular exposure to mustard has also been observed in other clinical studies [McGahan, M. C. & Bito, L. Z. (1982) id ibid.] and in a mouse model: subcutaneous injection of a sulfur mustard analog increased the activity of several enzymes including glucose-6-phosphate dehydrogenase and glutathione S-transferases [Elsayed, N. et al. (1992) id ibid.].
  • the protective effect of the complexes used in the present invention is the result of suppressed formation of ROS.
  • the ability of the DFO-metal complexes to act via a combined "push- pull" mechanism to achieve such a reduction in free radical formation is supported by both theoretical considerations and previously reported experimental findings.
  • the conversion of low reactive species to the highly reactive hydroxyl radicals apparently depends on the availability of trace amounts of the redox-active and labile iron or copper ions which serve as essential catalysts [Chevion, M. (1988) id ibid.; Chevion, M. et al.
  • Effectiveness might be improved by using a preparation that would allow increased residence time of the active complexes on the ocular surface by using topical preparations, preferably gel-containing preparations.
  • topical preparations preferably gel-containing preparations.
  • the DFO/metal complex preferably Zn/DFO or Ga/DFO, or a composition comprising thereof can be applied to a subject in need as eye drops, ophthalmic gel, ophthalmic ointment, spray or patches.
  • such a preparation may also be useful in the treatment of exposed skin.
  • the preparations may be used in the treatment of heat burns.
  • Topical treatment of skin damages may be combined with systemic treatment, e.g. injection of the DFO/metal complex. Injection may be intra- peritoneal, subcutaneous, intra-lesional, intra-osseous and other suitable modes of administration.
  • the DFO/metal complex preferably Zn DFO or Ga/DFO, or compositions comprising thereof, may be applied as a cream, an ointment, a liquid, or even as sustained-release patches, all of which said DFO/metal or said composition shall be a component thereof. While the use of either systemic or topical treatments (i.p injection or applying ointment containing 2.5% of either complex in a fatty carrier) proved protective, the application of the combination of both modes was found more effective than either treatment alone.
  • pre-conditioning initiating treatment shortly before potential exposure may provide further benefit.
  • the invention further relates to use of the above desferrioxamine complexes in combination with steroids.
  • Suitable steroids are, for example, but not limited to dexamethasone, cortisone or prednisone.
  • the inventors have shown that the use of metal/DFO together with a steroid drug gives even improved results.
  • the invention also relates to a pharmaceutical composition for the prevention, by pre-treatment, and/or treatment of tissue damage caused by exposure to mustard, which comprises as active ingredient a therapeutically effective amount of at least one DFO-metal complex.
  • the metal is zinc, gallium or manganese.
  • the compositions of the invention are particularly intended for the treatment of skin burns and damages and injured eye tissues.
  • topical application of active agents delivers the drug to the affected site, thus minimizing drug levels in the circulatory and gastrointestinal systems.
  • Undesirable side effects occurring from systemic administration of such drugs can thus be considerably reduced when using topical treatment, while still providing therapeutically sufficient and effective levels of the drug.
  • compositions for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays, powders or granules, emulsions, suspensions or solutions in water or non-aqueous media, sachets and dissolvable capsules or tablets.
  • Conventional pharmaceutically acceptable carriers, aqueous, powder or oily bases, diluents, thickeners, flavorings, dispersing agents, emulsifiers or binders may be desirable.
  • pharmaceutically acceptable as used herein is to be taken to mean any additive, carrier, base, diluent or thickener that is non-therapeutic and non- toxic to the recipient at the dosages and concentrations employed, and that does not markedly affect the pharmacological activity of the active agent.
  • the term "effective amount" as used herein is that determined by such considerations as are known to the man of skill in the art. The amount must be sufficient to prevent or ameliorate tissue damage caused by exposure to mustard. Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the active drug. Medically trained professionals can easily determine the optimum dosage, dosing methodology and repetition rates. In any case, the attending physician, taking into consideration the age, sex, weight and state of the disease of the subject to be treated, will determine the dose.
  • the concentration of the active metal/DFO complex in the composition is from 0.05 %w/v to 5 %w/v, more preferably from 0.1 %w/v to 1.0 %w/v, and particularly 0.25 %w/v.
  • eye drops or gels may be preferred.
  • gels, ointments and aerosols may be preferred.
  • the DFO complexes and the compositions of the invention may be applied topically from thrice weekly to 12 times daily. In preferred embodiments, the compositions are applied 7 times daily. Systemic administration of the DFO complexes or their combination is limited to once daily, and preferably two- three times per week.
  • Animal model of ocular NM injury New Zealand Albino rabbits weighing 2.5 to 3.5 kg were used. All animal experiments were conducted in compliance with the ARNO Statement for the Use of Animals in Ophthalmic and Nision Research. Animals were anesthetized with Ketamine HC1 (Ketalar, Parke Davis, UK, 50mg/kg), injected intramuscularly in combination with the relaxing agent Xylazine (5.0mg/kg). Local anesthetic drops (Benoxinate HC1 0.4%, Fischer Pharmaceuticals, Israel) were administered.
  • Ketamine HC1 Ketalar, Parke Davis, UK, 50mg/kg
  • Xylazine 5.0mg/kg
  • Local anesthetic drops Benoxinate HC1 0.4%, Fischer Pharmaceuticals, Israel
  • Nitrogen mustard (NM, mechlorethamine) at concentration of 1% w/v (48 rabbits) or 2% w/v (24 rabbits) was applied to the cornea of one eye of each animal (the experimental eye) for 5 minutes within a trephine.
  • the vacuum trephine was used to limit the area of application to a circle 4mm in diameter in the center of the cornea.
  • NM was quickly absorbed from within the trephine using small Week-cell sponges, followed by washing of the eye within the trephine with copious amounts of normal saline. The trephine was then removed, and the eye was again washed intensively with additional normal saline.
  • saline solution instead of NM was applied to the cornea for 5 minutes using the trephine.
  • vehicle saline
  • intra-muscular Dipyrone injections (lOmg/kg) were given to animals showing pain or distress. This was necessary mainly during the first 5-10 days after injury.
  • Groups 1 and 5 as well as the fellow uninjured eye in each animal, served as controls.
  • group 1 examining the effect of placebo drops (saline administration) on injured eyes effectively served as the model for exposed but not treated eyes.
  • group 5 normal saline was applied in the trephine (no exposure to mustard); in this group both eyes of each animal served as "experimental" eyes.
  • the fellow control eye in each animal served to assess the possible toxicity of the treatment drops used in that animal, when the drops were applied to practically healthy eyes.
  • the chloride salt of the metal was dissolved in doubly distilled water, whilst maintaining the pH at 3, and adding DFO at equivalent concentration, followed by titration to pH 7.4 with sodium bi-carbonate.
  • the stock was diluted in saline to a final concentration of 3.5 mM, based on DFO concentration.
  • Magnitude of ocular injury and response to treatment were assessed by examiners masked to the treatment groups. Repeated slit-lamp examinations with scoring of anterior segment injury, measurements of intraocular pressure (IOP), color photos of anterior segment, fluorescein angiography (FA), electroretinography (ERG), and blood test for anti-oxidant status were performed according to the timetable and guidelines described below.
  • IOP intraocular pressure
  • FA fluorescein angiography
  • ERP electroretinography
  • blood test for anti-oxidant status were performed according to the timetable and guidelines described below.
  • corneal epithelial loss (corneal erosion): The average horizontal and vertical linear dimensions of the epithelial defect as stained by locally applied fluorescein were measured using the adjustable slit-lamp beam, and the area computed in mm 2 .
  • Intraocular pressure (IOP): Repeated IOP measurements were performed in 8 eyes each from the following groups: group lb (2%NM+saline), group 2b (2%NM+Zn/DFO), group 6 (2%NM+Ga/DFO) and group 5 (Saline "injury"). Baseline IOP was measured before NM (or saline) exposure and re- measured on days 1, 3, 7, 10, 14, 21, and 28 post-injury. A hand-held automated tonometer (Tonopen, Mentor, Norwell, MA) was used.
  • Electroretinography To examine possible involvement of the posterior segment in ⁇ M-induced injury, as well as possible toxicity of the Zn/DFO complex in itself, retinal function was assessed by performing ERG recordings in 4 animals from groups 1 and 2 at 6-7 weeks after injury. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine. Anesthetized animals were dark adapted for at least 60 minutes. Following application of local anesthetic drops, a Burian-Allen contact lens electrode was inserted in both eyes with a clip electrode placed on one ear serving as ground.
  • the dark- adapted rod response, dark-adapted mixed cone-rod response and light- adapted 1Hz and 30Hz cone responses were recorded according to ISCEN standards using a computerized ERG system and a Ganzfeld bowl (UTAS 3000, LKC instruments, MD, USA). All ERG responses were filtered at 0.3-500 Hz and signal averaging was used. The fellow (uninjured) eye in each animal served as control.
  • AA Ascorbic acid
  • DHAA dehydro-ascorbate
  • Histology of ocular structures At 4-8 weeks after NM injury, 2-4 animals from each group were sacrificed for histological examination. Eyes were enucleated and fixed in 4% paraformaldehyde. Following embedding in paraffin, 4 ⁇ m sections were cut and stained with hematoxylin and eosin for histological evaluation of injury.
  • Animal model for NM skin damage Female Hartley guinea pigs (350 ⁇ 50 g) were used in this study. They were acclimatized in separate cages, two animals per cage, and received food and water ad libitum. The study was approved by the Institution Animal Care and Use Committee. The animals were anesthetized by intramuscular injection of 12 mg/kg Ketamine (Parke- Davis Medical, Southampton, UK) and 1.5 mg/kg Droperidol (Abie Ltd, Ramat- Gan, Israel). Back hair were clipped and subsequently chemically depilated. Two target areas, each 3 cm in diameter were marked on each side of the back midline, 4 cm apart.
  • the column used for separation of salicylate and DHBAs was a 250x4 mm LiChrospher. lOORP- 18, 5 ⁇ m (Merck, Darmstadt, Germany).
  • the chromatograms were recorded using a PC-based data acquisition and processing system (EZChrom Elite, San-Ramon, CA, USA).
  • Ascorbate and urate determination Ascorbate and urate were quantified by a modification of the HPLC-ECD method as previously described (17, 30) [Motchnik, P. et al. (1994) Methods Enzymol 234, 269-79; Chevion S. et al. (1997) Free Radic. Biol. Med. 22(3), 411-21].
  • UA and AA ascorbic acid were prepared in a solution of metaphosphoric acid (MPA) (5% w/v; Aldrich Chem. Co.) containing 0.54mM Na 2 EDTA (BDH). All MPA solutions were freshly prepared each day.
  • MPA metaphosphoric acid
  • UA standards (4.6 ⁇ g/ml) were prepared from uric acid sodium salt and either used immediately, or stored at -80°C for several weeks.
  • AA standards were prepared from the sodium salt in the same solution as for UA.
  • Stock solutions were diluted with the mobile phase prior to injection. 60 ⁇ l of plasma were added to an equal volume of 10% (w/v) MPA (containing Na 2 EDTA), mixed on a vortex mixer and centrifuged for 3 minutes to precipitate plasma proteins.
  • the separations were performed at a flow rate of 1.1 ml/min with a backpressure of 100 torr.
  • the chromatograms were recorded on a PC-based data acquisition and processing system (Chrom-A-Set, Barspec, Israel).
  • the sensitivity for AA was 20 pg in a loop of 20 ⁇ l.
  • Msr Activity Quantification of the activity of methionine-sulfoxide reductase (Msr) was carried out by incubation the tissue lysates with dabsyl-methionine sulfoxide for 30 min at 37°C, followed by analysis of the reduced product (dabsyl methionine) by HPLC-spectrophotometric detection at 436 nm [Moskovitz, J. et al. (2001) Proc Natl Acad Sci U S A, 98(23): p. 12920-5; Moskowitz et al. (1997) Proc Natl Acad Sci U S A, 94(18): p. 9585-93].
  • Assay total volume lOO ⁇ l, including 200 ⁇ M Dabsyl-met(O) (as a substrate).
  • the reaction mixture contained also 20mM DTT, buffer, ⁇ 100 ⁇ g protein.
  • the incubation (reaction) was stopped by adding lOO ⁇ l of acetonitrile, spinning down, and discarding the protein fraction.
  • the substrate, dabsyl-Met(O) was prepared as described [Moskowitz et al. (1997) id ibid.].
  • Fig. 1A Conjunctival injury and scarring persisted for 4 weeks after exposure
  • Fig. IB Conjunctival and corneal injury was markedly reduced in eyes treated with Zn/DFO (group 2) and Ga/DFO (group 6), although some residual corneal opacity persisted (Fig. IC, Ga/DFO treated eye). Histological examination corroborated the clinical findings: as opposed to the normal corneal structure in the fellow uninjured eye (Fig. ID), epidermalization of the corneal epithelium and severe inflammation within the corneal stroma were seen in the eye exposed to 2% NM and treated by administration of saline only (Fig. IE).
  • the corneal epithelial erosion caused by NM was similar in size in all experimental groups at day one after exposure, but was significantly reduced in Zn/DFO-treated eyes at day 3 (Table 2).
  • Table 2 Area of corneal epithelial damage (mm 2 ) following exposure to 1% NM on day 0
  • Iris pigmentation score at 4 weeks post- injury was significantly reduced (by >60%) in Zn/DFO treated eyes as compared to saline-administered eyes (Fig. 3D).
  • treatment with each component of the complex separately i.e., ZnCl 2 or DFO
  • Fig. 3D exemplified also in Fig. 4
  • iris fluorescein angiography FA was performed at 4-5 weeks post-exposure.
  • IOP intraocular pressure
  • AA ascorbic acid
  • Ocular NM injury was performed as described in Experimental Procedures, using 2% NM. A combination of Zn/DFO and the steroid dexamethasone (0.1%) was used, and dexamethasone alone as a control. The dexamethasone used was in soluble form (dexamethasone sodium phosphate, Dexacort Forte, Teva Pharmaceuticals, Israel). The protocol for injury and treatment are as detailed in Table 1. In addition, the eyes were treated once daily with Gentamycin 0.3% (Gentamycin-IKA, Teva Pharmaceuticals, Israel), and antibiotic ointment.
  • Methionine is a sulfur containing amino acid. Methionine residues are known for their high susceptibility to oxidation, yielding methionine sulfoxide. Methionine sulfoxide reductase (Msr) is an enzyme, which reduces either the free or the protein-bound methionine sulfoxide back to methionine. Thus, methionine residues are considered the cell "last chance" antioxidant defense system for proteins. Msr activity is therefore an indicator for systemic antioxidants serum level.
  • Msr Methionine sulfoxide reductase
  • the present results show that exposure to NM followed by administration of the carrier alone (saline) caused severe and long-lasting injury to ocular anterior segment structures.
  • Treatment with the combination of Zn/DFO and dexamethasone was significantly more effective than dexamethasone or Zn/DFO alone. Corneal re-epithelization was faster, and corneal neovascularization was less severe, while intraocular pressure rise and depletion in serum anti-oxidants was lower.
  • GMC Ga/DFO
  • the level of UA within the exposed skin punches was consistently higher in NM-exposed and saline treated animals, than in exposed animals treated with DFO-metal complex. Elevated UA levels reflects higher levels of tissue catabolism and breakdown of purine nucleotides in the NM-exposed tissue, when compared to non exposed animals. Treatment with either DFO-complex markedly protected the skin (Fig. 10C).
  • FIG 10D clearly shows that OSAA values are consistently lower in the DFO- complex-treated animals than in the exposed controls (saline administered), indicating that the tissue is better protected, from day 4 to day 14, in both treated groups.
  • the normal value of OSAA in un- injured skin is 11 ⁇ 3%.
  • FIGs. HA to HE The results are shown in Figs. HA to HE.
  • Figure HA and HB show that while the skin from the saline administered group is characterized by a marked elevation in production of free radicals, skin punch from the three treated groups showed significantly lower oxidative stress, similar to normal skin.
  • the levels of uric acid (UA) in the skin are shown in Figure 11 C.
  • the elevated level of UA in the saline administered group reflects the excessive oxidative stress in the exposed skin.
  • the treated groups show markedly lower elevation during the first 14 days of the experiment.
  • the level of oxidative stress is elevated during the entire experiment in both exposed and saline administered group.
  • Group 1 treated with GMC (Ga/DFO), i.p., 2.5 mg/kg, twice weekly, starting
  • Group 3 treated with a combination of GMC, 2.5 (mg/kg), i.p., and AA,
  • Group 4 shows the protection provided by the topical application of carrier ointment (without the DFO- complex).
  • topical application of active ointment alone resulted in 24-51% of the protection accomplished by the combination of topical and i.p. administrations.

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Abstract

Parmi les armes chimiques tactiques existant actuellement, les agents moutarde sont les plus puissants, et aucune antidote spécifique ou traitement hautement efficace contre la toxicité moutarde n'est couramment disponible. L'invention a pour but de fournir un tel antidote, lequel se présente sous la forme d'un complexe desferrioxamine (DFO)-métal, destiné à l'utilisation pour le traitement topique et/ou la prévention des dommages causés à un tissu exposé à de telles armes chimiques. Plus spécifiquement, ledit complexe DFO-métal contient du zinc ou du gallium et est le composé agissant le plus efficacement à l'encontre de dommages causés par l'agent moutarde azoté ou soufré. L'invention concerne également des compositions pharmaceutiques pour le traitement topique et/ou la prévention de dommages causés aux tissus par des armes chimiques, ces compositions comprenant, comme agent actif, ledit complexe DFO-métal selon l'invention. L'invention concerne en outre des procédés de traitement utilisant ledit complexe DFO-métal, ainsi que l'utilisation de ces procédés.
PCT/IL2003/001122 2003-01-07 2003-12-31 Composition comprenant un complexe desferrioxamine-metal, et son utilisation pour le traitement de dommages causes aux tissus consecutivement a une exposition a des agents chimiques de combat Ceased WO2004060490A1 (fr)

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US8680148B2 (en) 2006-08-11 2014-03-25 University Of Washington Metallo-desferrioxamine complexes and their use in the treatment of bacterial infections
WO2008091829A1 (fr) * 2007-01-23 2008-07-31 Lawrence Bernstein Compositions ophtalmiques a base de gallium et methodes d'utilisation associees
US9604085B2 (en) 2008-01-22 2017-03-28 Emergent Protective Products Canada Ulc Method and formulation for neutralizing toxic chemicals and materials
US9770430B2 (en) 2009-08-19 2017-09-26 Mordechai Chevion Desferrioxamine-metal complexes for the treatment of immune-related disorders
EP3189836A3 (fr) * 2009-08-19 2017-09-27 Mordechai Chevion Complexes desferrioxamine-métal pour le traitement de troubles liés à l'immunité
US8975294B2 (en) 2009-08-19 2015-03-10 Hadasit Medical Research Services And Development Ltd. Desferrioxamine-metal complexes for the treatment of immune-related disorders
AU2016244189B2 (en) * 2009-08-19 2018-06-28 Mordechai Chevion Desferrioxamine-metal complexes for the treatment of immune-related disorders
AU2010286054B2 (en) * 2009-08-19 2016-10-27 Mordechai Chevion Desferrioxamine-metal complexes for the treatment of immune-related disorders
WO2011021203A3 (fr) * 2009-08-19 2011-04-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Complexes desferrioxamine-métal pour le traitement de troubles liés à l'immunité
EP3189836A2 (fr) 2009-08-19 2017-07-12 Mordechai Chevion Complexes desferrioxamine-métal pour le traitement de troubles liés à l'immunité
WO2011021203A2 (fr) 2009-08-19 2011-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Complexes desferrioxamine-métal pour le traitement de troubles liés à l'immunité
CN102573831A (zh) * 2009-08-19 2012-07-11 耶路撒冷希伯来大学伊森姆研究发展公司 用于治疗免疫相关病症的去铁胺-金属络合物
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WO2016103260A1 (fr) 2014-12-22 2016-06-30 Mordechai Chevion Nouveaux complexes métalliques de nocardamine et leur utilisation dans des compositions pharmaceutiques
AU2015369552B2 (en) * 2014-12-22 2020-11-19 Eduard Berenshtein New metal complexes of nocardamine and their use in pharmaceutical compositions
CN108601750A (zh) * 2016-02-11 2018-09-28 莫迪凯·舍维龙 用于治疗神经变性的方法和药物组合物
JP2019519465A (ja) * 2016-02-11 2019-07-11 モデハイ シェヴィオンCHEVION, Mordechai 神経変性の治療のための方法及び医薬組成物
EP3413882A4 (fr) * 2016-02-11 2019-10-09 Mordechai Chevion Méthode et composition pharmaceutique pour le traitement de la neurodégénérescence
JP7003045B2 (ja) 2016-02-11 2022-02-04 シェヴィオン モデハイ 神経変性の治療のための方法及び医薬組成物
CN117065087A (zh) * 2023-10-16 2023-11-17 中国人民解放军总医院第四医学中心 一种黑磷壳聚糖dfo温敏水凝胶的制备方法及应用

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