WO1996040149A1 - Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease - Google Patents
Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease Download PDFInfo
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- WO1996040149A1 WO1996040149A1 PCT/US1996/010267 US9610267W WO9640149A1 WO 1996040149 A1 WO1996040149 A1 WO 1996040149A1 US 9610267 W US9610267 W US 9610267W WO 9640149 A1 WO9640149 A1 WO 9640149A1
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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
- A61K31/28—Compounds containing heavy metals
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
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- 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
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- 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
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
- C07C251/24—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to carbon atoms of six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic System
- C07F13/005—Compounds without a metal-carbon linkage
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/58—Metal complex; Coordination compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
Definitions
- the invention provides antioxidant compositions, including pharmaceutical compositions, of synthetic catalytic small molecule antioxidants and free radical scavengers for therapy and prophylaxis of disease and prevention of
- compositions and methods of the invention are also used for preventing oxidative damage to individuals exposed to irritating oxidants or other sources of oxidative damage, particularly oxygen-derived oxidative species such as superoxide radical.
- the compositions and methods of the invention are also used for preventing oxidative damage to individuals exposed to irritating oxidants or other sources of oxidative damage, particularly oxygen-derived oxidative species such as superoxide radical.
- compositions and methods of the invention are also useful for chemoprevention of chemical carcinogenesis and alteration of drug metabolism involving epoxide or free oxygen radical intermediates.
- the invention also provides novel compounds having therapeutically useful catalytic properties, and compositions containing said novel compounds.
- Oxygen is an essential nutrient for nonfacultative aerobic organisms, including, of course, humans. Oxygen is used in many important ways, namely, as the terminal electronic acceptor in oxidative phosphorylation, in many dioxygenase reactions, including the synthesis of
- prostaglandins and of vitamin A from carotenoids in a host of hydroxylase reactions, including the formation and modification of steroid hormones, and in both the activation and the inactivation of xenobiotics, including carcinogens.
- the extensive P-450 system uses molecular oxygen in a host of important cellular reactions.
- nature employs free radicals in a large variety of enzymic reactions.
- Biological antioxidants include well-defined
- enzymes such as superoxide dismutase, catalase, selenium glutathione peroxidase, and phospholipid hydroperoxide
- Nonenzymatic biological antioxidants include tocopherols and tocotrienols, carotenoids, guinones, bilirubin, ascorbic acid, uric acid, and metal-binding
- antioxidants being both lipid and water soluble, are found in all parts of cells and tissues, although each specific antioxidant often shows a characteristic
- ovothiols which are mercaptohistidine derivatives, also decompose peroxides nonenzymatically.
- Oxyradical oxygen radical
- Oxyradical injury has been implicated in the pathogenesis of pulmonary oxygen toxicity, adult respiratory distress syndrome (ARDS), bronchopulmonary dysplasia, sepsis syndrome, and a variety of ischemia-reperfusion syndromes, including
- Oxyradicals can react with proteins, nucleic acids, lipids, and other biological macromolecules producing damage to cells and tissues,
- Free radicals are atoms, ions, or molecules that contain an unpaired electron (Pryor, WA (1976) Free Radicals in Biol. 1: 1). Free radicals are usually unstable and exhibit short half-lives. Elemental oxygen is highly
- Free radicals can originate from many sources, including aerobic respiration, ⁇ ytochrome P-450-catalyzed monooxygenation reactions of drugs and xenobiotics (e.g., trichloromethyl radicals, CCl 3 •, formed from oxidation of carbon tetrachloride), and ionizing radiation.
- trichloromethyl radicals CCl 3 •, formed from oxidation of carbon tetrachloride
- ionizing radiation e.g., when tissues are exposed to gamma radiation, most of the energy deposited in the cells is absorbed by water and results in scission of the oxygen-hydrogen covalent bonds in water, leaving a single electron on hydrogen and one on oxygen creating two radicals H• and •OH.
- the hydroxyl radical, •OH is the most reactive radical known in chemistry. It reacts with biomolecules and sets off chain reactions and can
- neutrophils produces abundant superoxide radical, which is believed to be an essential factor in producing the cytotoxic effect of activated neutrophils.
- Reperfusion of ischemic tissues also produces large concentrations of oxyradicals, typically superoxide (Gutteridge JMC and Halliwell B (1990) Arch. Biochem. Biophys. 283: 223).
- superoxide may be produced physiologically by endothelial cells for reaction with nitric oxide, a physiological regulator, forming peroxynitrite, ONOO- which may decay and give rise to hydroxyl radical, •OH (Marietta MA (1989) Trends Biochem. Sci. 14: 488; Moncada et al. (1989) Biochem. Pharmacol. 38: 1709; Saran et al. (1990) Free Rad. Res. Commun. 10: 221; Beckman et al.
- Oxygen though essential for aerobic metabolism, can be converted to poisonous metabolites, such as the superoxide anion and hydrogen peroxide, collectively known as reactive oxygen species (ROS). Increased ROS formation under
- ROS pathological conditions
- ROS are generated by activated phagocytic "leukocytes; for example, during the neutrophil "respiratory burst", superoxide anion is generated by the membrane-bound NADPH oxidase.
- ROS are also believed to accumulate when tissues are subjected to ischemia followed by reperfusion.
- malondialdehyde is a reaction product of peroxidized lipids that reacts with virtually any amine-containing
- Oxygen free radicals also cause oxidative damage
- Aerobic cells generally contain a number of defenses against the deleterious effects of oxyradicals and their reaction products.
- Superoxide dismutases catalyze the reaction: 2 •O 2 - + 2 H + ---- > O 2 + H 2 O 2
- H 2 O 2 is not a radical, but it is toxic to cells; it is removed by the enzymatic activities of catalase and glutathione peroxidase (GSH-Px).
- Catalase catalyzes the reaction:
- GSH-Px removes hydrogen peroxide by using it to oxidize reduced glutathione (GSH) into oxidized glutathione (GSSG) according to the following reaction:
- PLOOH-GSH-Px phospholipid hydroperoxide glutathione peroxidase
- Glutathione S-transferases also participate in detoxifying organic peroxides. In the absence of these enzymes and in presence of transition metals, such as iron or copper, superoxide and hydrogen peroxide can participate in the following reactions which generate the extremely reactive hydroxyl radical •OH-:
- tocopherol, ubiquinone, bilirubin, and uric acid serve as naturally-occurring physiological antioxidants (Krinsky NI (1992) Proc. Soc. EXP. Biol. Med. 200:248-54).
- Carotenoids are another class of small molecule antioxidants and have been implicated as protective agents against oxidative stress and chronic diseases. Canfield et al. (1992) Proc. Soc. Exp.
- Biol. Med. 200: 260 summarize reported relationships between carotenoids and various chronic diseases, including coronary heart disease, cataract, and cancer. Carotenoids dramatically reduce the incidence of certain premalignant conditions, such as leukoplakia, in some patients.
- One strategy for preventing oxyradical-induced damage is to inhibit the formation of oxyradicals such as superoxide.
- Iron ion chelators such as desferrioxamine (also called deferoxamine or Desferol) and others, inhibit iron ion- dependent •OH generation and thus act as inhibitors of free radical formation (Gutteridge et al. (1979) Biochem. J. 184 : 469; Halliwell B (1989) Free Radical Biol. Med. 2: 645; Van der Kraaij et al. (1989) Circulation 80: 158). Amino-steroid- based antioxidants such as the 21-aminosteroids termed
- allopurinol, and other pyrazolopyrimidines such as oxypurinol have also been tested for preventing oxyradical formation in a myocardial stunning model system (Bolli et al. (1989) Circ. Res. 65: 607) and following hemorrhagic and endotoxic shock (DeGaravilla et al. (1992) Dru ⁇ Devel. Res. 25: 139).
- deferoxamine is not an ideal iron chelator and its cellular penetration is quite limited.
- Another strategy for preventing oxyradical-induced damage is to catalytically remove oxyradicals such as
- An alternative strategy for preventing oxyradical- induced damage is to scavenge oxyradicals such as superoxide once these have been formed, typically by employing small molecule scavengers which act stoichiometrically rather than catalytically.
- Congeners of glutathione have been used in various animal models to attenuate oxyradical injury.
- N-2-mercaptopropionylglycine has been found to confer protective effects in a canine model of myocardial ischemia and reperfusion (Mitsos et al.
- inhibitors of oxyradical formation and/or enzymes that remove superoxide and hydrogen peroxide and/or small molecule oxyradical scavengers have all shown promise for preventing reoxygenation damage present in a variety of ischemic pathological states and for treating or preventing various disease states associated with free
- inhibitors of oxyradical formation typically chelate transition metals which are used in
- inhibitors do not completely prevent oxyradical formation.
- Superoxide dismutases and catalase are large polypeptides which are expensive to manufacture, do not penetrate cells or the blood-brain barrier, and generally require parenteral routes of administration.
- Free radical scavengers act
- desferroxamine and manganese has SOD activity and has shown some activity in biological models but the instability of the metal ligand complex apparently precludes its pharmaceutical use. Porphyrin-manganese complexes have been shown to protect bacteria from paraquat toxicity and to promote the aerobic survival of SOD-deficient E. coli mutants.
- antioxidant agents which are efficient at removing dangerous oxyradicals, particularly superoxide and hydrogen peroxide, and which are inexpensive to manufacture, stable, and possess advantageous pharmacokinetic properties, such as the ability to cross the blood-brain barrier and penetrate tissues.
- Such versatile antioxidants would find use as pharmaceuticals, chemoprotectants, and possibly as dietary supplements. It is one object of the invention to provide a class of novel antioxidants which possess advantageous
- hydrocarbons including plastics, nitrile rubbers, chloroprene rubbers, silicone rubber, isoprene rubbers, other rubber analogs, oils and waxes, cosmetic bases, animal fats,
- the polymers are usually formed by reactions of unsaturated hydrocarbons, although any hydrocarbon can polymerize. Generally, olefins tend to polymerize more readily than aromatics, which in turn
- hetero atoms such as nitrogen, oxygen and sulfur also contribute to polymerization, as does molecular oxygen, oxyradicals (e.g., superoxide, peroxides, hydroxyl radical), and other free radicals.
- Polymers are generally formed by free radical chain reactions. These reactions, typically consist of two phases, an initiation phase and a propagation phase. Free radicals, which have an odd
- antioxidant antifoulants have been developed to prevent oxygen from initiating polymerization, such as in petroleum refining apparatus. Antioxidants act as chain-stoppers by forming inert molecules with the oxidized free radical hydrocarbons.
- This system comprises a combination of an antioxidant such as a phenylenediamine and a chelating agent or metal deactivator such as a polyamine.
- an antioxidant such as a phenylenediamine
- a chelating agent or metal deactivator such as a polyamine.
- compositions and methods for inhibiting the polymerization of vinyl aromatic monomers during the preparation of monomers and the storage and shipment of products containing such monomers are needed in the art, particularly for use in aqueous or mixed aqueous/organic systems.
- the present invention fulfills these and other needs.
- compositions which have potent antioxidant and/or free radical scavenging properties and function as in vivo antioxidants.
- the pharmaceutical compositions of the invention comprise an efficacious dosage of at least one species of salen-transition metal complex, typically a salen-manganese complex such as a salen-Mn(III) complex.
- the pharmaceutical composition comprises a salen-Mn complex which is a chelate of Mn(III) with a diamine derivative, such as ethylenediamine linked to two substituted salicylaldehydes.
- compositions possess the activity of
- compositions are effective at reducing pathological damage related to formation of oxyradicals such as superoxide and peroxides and other free radical species.
- the invention also provides methods for treating and preventing pathological conditions by applying or
- Salen-transition metal complexes used in the methods of the invention are typically salen-manganese complexes, such as Mn(III) -salen complexes.
- the invention provides methods for preventing or reducing ischemic/reperfusion damage to critical tissues such as the myocardium and central nervous system.
- the invention also provides methods for preventing or reducing cellular damage resulting from exposure to various chemical compounds which produce potentially damaging free radical species, comprising administering a therapeutically or prophylactically efficacious dosage of at least one species of salen-transition metal complex, preferably a salen-manganese complex having detectable SOD activity and preferably also having detectable catalase activity.
- the antioxidant salen-transition metal complexes of the invention are administered by a variety of routes, including parenterally, topically, and orally.
- a therapeutic or prophylactic dosage of a salen-transition metal complex of the present invention is administered alone or combined with (1) one or more antioxidant enzymes, such as a Mn-SOD, a Cu,Zn- SOD, or catalase, and/or (2) one or more free radicals
- scavengers such as tocopherol, ascorbate, glutathione, DMTU, N-acetylcysteine, or N-2-mercaptopropionylglycine and/or (3) one or more oxyradical inhibitors, such as desferrioxamine or allopurinol, and/or one or more biological modifier agents, such as calpain inhibitors.
- oxyradical inhibitors such as desferrioxamine or allopurinol
- biological modifier agents such as calpain inhibitors.
- compositions is dependent upon the specific pathological condition sought to be treated or prevented, the route and form of administration, and the age, sex, and condition of the patient. These compositions are administered for various indications, including: (1) for preventing ischemic/reoxygenation injury in a patient, (2) for preserving organs for transplant in an anoxic, hypoxic, or hyperoxic state prior to transplant, (3) for protecting normal tissues from free radical-induced damage consequent to exposure to ionizing radiation and/or chemotherapy, as with bleomycin, (4) for protecting cells and tissues from free radical-induced injury consequent to exposure to xenobiotic compounds which form free radicals, either directly or as a consequence of monooxygenation through the cytochrome P-450 system, (5) for enhancing cryopreservation of cells, tissues, organs, and organisms by increasing viability of recovered specimens, and (6) for prophylactic administration to prevent:
- carcinogenesis cellular senescence, cataract formation, formation of malondialdehyde adducts, HIV pathology and macromolecular crosslinking, such as collagen crosslinking.
- salen-transition metal complexes are formulated for administration by the oral route by forming a pharmaceutical dosage form comprising an excipient and not less than 1 ⁇ g nor more than about 10 grams of at least one antioxidant salen-transition metal complex of the invention. Dietary formulations are administered for therapy of free radical-induced diseases and/or for the chemoprevention of neoplasia and/or oxidative damage
- compositions generally comprise at least one species of a salen-metal complex having SOD activity, catalase activity, and/or peroxidase activity; such species can be obtained from the disclosed generic formulae, general synthesis methods, and exemplified species, typically in conjuction with a routine determination of the various activities, such as to calibrate dosage levels for efficacy, and the like.
- the salen-metal complex species is selected from the group consisting of: C7, C31, C32, C36, C37, C38, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, C50, C51, C54 , C55, C56, C58, C67, C68, C71, C72, C73, C74, C76, C79, C80, C81, C82, C83, C84 , C85, C86, and C87.
- aqueous solutions comprising at least one antioxidant salen-transition metal complex of the invention at a concentration of at least 1 nM but not more than about 100 mM is formulated for
- an ischemic episode such as a myocardial
- antineoplastic or antihelminthic chemotherapy employing a chemotherapeutic agent which generates free radicals, (3) endotoxic shock or sepsis, (4) exposure to ionizing radiation, (5) exposure to exogenous chemical compounds which are free radicals or produce free radicals, (6) thermal or chemical burns or ulcerations, (7) hyperbaric oxygen, or (8) apoptosis of a predetermined cell population (e.g., lymphocyte
- aqueous solutions of the invention may also be used, typically in conjunction with other established methods, for organ culture, cell culture, transplant organ maintenance, and myocardial irrigation.
- compositions such as lipid-based formulations are also provided, including stabilized emulsions.
- the antioxidant salen-metal compositions are administered by various routes, including intravenous injection, intramuscular injection, subdermal injection, intrapericardial injection, surgical irrigation, topical application, ophthalmologic application, lavage, gavage, enema, intraperitoneal infusion, mist
- antioxidant salen-transition metal complexes of the invention are employed to modulate the expression of naturally-occurring genes or other polynucleotide sequences under the transcriptional control of an oxidative stress response element (e.g., an antioxidant responsive element, ARE) , such as an antioxidant response element of a glutathione S-transferase gene or a NAD(P)H:quinone reductase gene.
- ARE antioxidant responsive element
- the antioxidant salen-metal complexes may be used to modulate the transcription of ARE- regulated polynucleotide sequences in cell cultures (e.g., ES cells) and in intact animals, particularly in transgenic animals wherein a transgene comprises one or more AREs as transcriptional regulatory sequences.
- the present invention also encompasses
- compositions of antioxidant salen-manganese complexes therapeutic uses of such antioxidant salen- manganese complexes, methods and compositions for using antioxidant salen-manganese complexes in diagnostic,
- the invention also provides methods for preventing food spoilage and oxidation by applying to foodstuffs an effective amount of at least one antioxidant salen-metal complex species.
- the invention also provides compositions for preventing food spoilage comprising an effective amount of at least one species of antioxidant salen-metal complex,
- an antioxidant salen-metal complex is
- the invention relates to antioxidant compositions and methods of use in inhibiting formation of undesired hydrocarbon polymers generated via free radical- mediated polymerization mechanisms, especially oxyradical- mediated polymerization and/or oxyradical-mediated
- the antioxidant salen-metal complexes of the invention can be applied to a variety of hydrocarbons to reduce undesired oxidation and/or polymerization, or to quench a polymerization reaction at a desired state of polymer formation (e.g., at a desired average chain length).
- hydrocarbons include: petroleum distillates and petrochemicals, turpentine, paint, synthetic and natural rubber, vegetable oils and waxes, animal fats, polymerizable resins, polyolefin, and the like.
- the invention relates to antioxidant compositions and methods of use in hydrocarbon compositions to reduce and/or control the formation of undesired polymers which comtaminate such hydrocarbon compositions, including
- This invention relates to a method and composition for controlling the formation of polymers in such systems which comprises an antioxidant composition comprising an antioxidant salen-metal compound, optionally in combination with an antioxidant or stabilizer other than a salen-metal compound (e.g., BHT, BHA, catechol, tocopherol, hydroquinone, etc.). More particularly, this invention relates to a method and composition for
- polymers which comprises an antioxidant composition comprising an antioxidant salen-metal complex.
- the amount of the individual ingredients of the antioxidant composition will vary depending upon the severity of the undesirable polymer formation encountered due to free radical polymerization as well as the activity of the salen- metal compound utilized.
- the invention provides methods for enhancing the recovery of skin of a warm-blooded animal from wounds, such as surgical incisions, burns, inflammation or minor irritation due to oxidative damage, etc.
- the methods comprise administering to the skin wound or irritation a therapeutically or, in some cases a prophylactically effective amount of a composition which comprises an antioxidant salen- metal complex.
- the present invention also provides compounds having peroxidase activity and, therefore, capable of serving as effective peroxidase replacements. These compounds are useful as drugs for the prevention of many pathological conditions, including but not limited to neoplasia, apoptosis of somatic cells, skin aging, cataracts, and the like; and as
- the present invention also provides methods and pharmaceutical compositions of these compounds.
- the present invention also concerns a method of reducing H 2 O 2 and/or other peroxides which comprises contacting H 2 O 2 and/or other peroxides with a suitable amount of any of the compounds of the invention effective to reduce H 2 O 2 and/or other peroxides. Additionally, the invention provides a method of treating a peroxide-induced condition in a subject which comprises administering to the subject an amount of any of the compounds of the invention effective to reduce peroxide in a subject and thereby treat the peroxide-induced condition. Further, the invention provides a pharmaceutical composition which comprises an amount of any of the compounds of the invention effective to reduce peroxide in a subject with a peroxide-induced condition and a pharmaceutically acceptable carrier.
- the invention provides a method of treating a peroxide-induced condition in a subject, e.g. a human subject, which comprises administering, e.g. by topical, oral, intravenous, intraperitoneal, intramuscular, intradermal, or subcutaneous administration, to the subject an amount of an antioxidant salen-metal compound effective to reduce peroxide in the subject and thereby treat the peroxide-induced
- peroxide-induced condition may involve cataracts, inflammation of a tissue, ischemia, an allergic reaction, or pathology caused by oxidative stress.
- composition involves cataracts
- administration is effected by, but is not limited to, topical contact to the surface of an eye.
- Fig. 1 shows the general structure of salen deriviatives of the invention.
- Fig. 2 shows a salen derivative according to the structure shown in Figure 1, wherein n is 0.
- Fig. 3 shows structures of preferred compounds of the invention.
- Fig. 4 shows schematically the effect of an ischemic/reoxygenation episode on synaptic transmission in isolated brain slices.
- Fig. 5 shows the effect of a salen-Mn complex on
- EPSP amplitude following an episode of ischemia/reoxygenation.
- Fig. 6 shows the effect of a salen-Mn complex on EPSP initial slope following an episode of
- Fig. 7 shows the effect of a salen-Mn complex on brain slice viability following repeated episodes of
- Fig. 8A and 8B show the protective effect of a salen-Mn complex in an animals model of iatrogenic Parkinson's disease.
- Fig. 9 shows that C7 protects hippocampal slices from lactic acid-induced lipid peroxidation.
- Fig 10 shows C7 protects dopaminergic neurons in mouse striatum from 6-OHDA-induced degeneration.
- Fig. 11 shows a generic structural formula of preferred salen-metal complexes of the invention.
- Panel (A) shows the generic structural formula.
- Panel (B) shows some preferred substituents.
- Fig. 12 shows examples of structures of antioxidant salen-metal complexes.
- Fig. 13A and 13B shows that C7 inhibits NBT
- Fig. 14 shows that C7 exhibits catalase activity.
- C7 was assayed for catalase activity as described in Example 2.
- the concentration of C7 was 10 ⁇ M and the concentration of H 2 O 2 was as indicated: (solid circle), 0.6 mM; (open circle), 1.2 mM; (solid square), 2.3 mM; (solid triangle), 4.6 mM;
- Fig. 15A and 15B show that C7 exhibits peroxidase activity toward the substrate ABTS. C7 was assayed for peroxidase activity as described in Example 2. The
- Fig. 15A pH 8.1, H 2 O 2 concentration of: (solid circle), 0.1 mM; (open circle), 1 mM; (solid triangle), 10.mM; Fig. 15B: 10 mM H 2 O 2 , pH was: (solid circle), 6.0; (open circle) 7.1; (solid triangle), 8.1.
- Fig. 16A and 16B show inactivation of C7 in the presence of H 2 O 2 .
- C7 was incubated with H 2 O 2 as described in Example 2 with aliquots removed and analyzed by HPLC.
- Fig. 16A Time-dependent changes in levels of C7 (solid circle), salicylaldehyde (X), and an unidentified substance (open triangles) in incubation mixtures lacking ABTS.
- Fig. 16B The percent of initial C7 remaining in incubations conducted in the absence (solid circle) and presence (open circle) of 1 mM ABTS.
- Fig. 17 shows a comparison of the catalase activities of C7 and C40.
- Catalase assays were performed as described for Example 2, using C7 (solid circle) or C40 (open circle).
- Fig. 18 shows protection against glucose and glucose-oxidase induced cytotoxicity by salen manganese complexes. Cytotoxicity studies were performed as described in Example 2. Absorbance values, corrected by subtracting the blank signal of 0.17 OD units, are the means + sd of
- Control cells received no glucose oxidase.
- Catalase-treated (solid circle) cells received glucose oxidase (0.019) units/ml) as well as bovine liver catalase (290 units/ml).
- Other samples received the same dose of glucose oxidase and the indicated concentrations of salen manganese complex.
- C40 open triangle
- C32 solid triangle
- C41 open square
- C38 solid square
- C7 open diamond
- C35 solid diamond
- Figure 19A shows structures of salen-manganese complexes.
- Figure 19B shows the catalase rate, catalase endpoint, peroxidase rate, and SOD activity of these compounds relative to C7.
- FIG. 20 Time-dependent generation of oxygen in the catalase assay.
- Catalase was assayed with a polarographic oxygen electrode as described in Example 2. Each compound was present at 10 ⁇ M. Hydrogen peroxide was added at a final concentration of 10 mM at the indicated times (arrows).
- Cytotoxicity by glucose and glucose oxidase were performed using human dermal fibroblasts as for Fig. 18.
- Panel (A) shows a structural formula, wherein: M is a transition metal such as Mn, Mg, Co, Fe, Cu, Ni, V, Cr, and Ni; A is an axial ligand composed of a halide, acetate, formate, FF 6 , triflate, tosylate, or is an oxygen atom typically bound via a double bond to the metal (M) ; R 1 through R 4 are independently H, optionally substituted hydrocarbyl, CH 3 , C 2 H 5 , C 5 H 5 , O-benzyl, primary alkyls, fatty acid esters, substituted alkoxyaryls, heteroatom-bearing aromatic groups, arylalkyls, secondary alkyls, or tertiary alkyls.
- M is a transition metal such as Mn, Mg, Co, Fe, Cu, Ni, V, Cr, and Ni
- A is an axial ligand composed of a halide, acetate, formate, FF 6
- R 5 is an optionally substituted hydrocarbyl, typically -(CH 2 )n-, where n is generally 1, 2, 3, 4, 5, 6, 7 or 8, often 2 or 6, and when 6, often R 5 is a benzene ring.
- the portion of the molecule designated "bridge" indicates that R 5 or an equivalent covalent moiety, serves to link the nitrogens which are bound to M, preferably in a planar
- Panel (B) shows an embodiment wherein there is no covalent bridge structure: R 1 through R 4 are independently H, optionally substituted hydrocarbyl, CH 3 , C 2 H 5 , C 5 H 5 , O-benzyl, primary alkyls, fatty acid esters, substituted alkoxyaryls,
- R 5 and R 5 ' are independently selected and are each optionally substituted hydrocarbyls.
- Panel (C) shows a preferred class of structures wherein R 1 , R 2 , and the nitrogens conjugated to the transition metal (M) are in the same geometric plane.
- Panel (D) shows a preferred class of structures wherein the oxygens and the nitrogens conjugated to the transition metal (M) are in the same geometric plane; generally the axial ligand (A) is out of plane, typically perpendicular to the indicated planar region.
- Figures 24A through 241 show exemplified species of salen metal complexes.
- FIG. 25 shows example generic structures of salicyladehydes (panel A) and diamines (panel B) suitable for making salen-metal complexes of the invention via condensation reaction as described herein and by reference to incorporated literature and patent publications.
- X 1 , X 2 , X 3 , and/or X 4 are methoxy, ethoxy, chlorine, bromine, fluorine, hydroxyl, nitro, or hydrogen.
- R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of hydrogen, hydroxy, nitrate, halides, alkyls, aryls, arylalkyls, silyl groups, aminos, alkyls or aryls bearing heteroatoms; aryloxys, alkoxys, and halide; preferably, R 1 , R 2 , R 3 , and/or R 4 are hydrogen; Z 1 , Z 2 , Z 3 , and Z 4 are independently selected from the group consisting of hydrogen, hydroxy, nitrate, halides, alkyls, aryls, arylalkyls, silyl groups, aminos, alkyls or aryls bearing heteroatoms; aryloxys, alkoxys, and halide;
- R 1 , R 2 , R 3 , and/or R 4 are hydrogen; Q is a
- n 0, 1, 2, 3, 4, 5, 6, 7 or 8, and the group (CQ 2 )n may comprise a benzene ring.
- Figure 26A through 26E show structural formulae of preferred genuses of salen-metal complexes.
- M is a transition metal selected from Mn, Cu, V, Zn, Fe, Pd, Cr, Co;
- X 1 , X 2 , X 3 , and X 4 are independently halide, hydrogen, alkoxy, aryloxy, hydroxy, amine, -NHCOR where R is an optionally substituted hydrocarbyl, C 6 H 5 , or lower alkyl;
- Y 1 , Y 2 , Y 3 , and Y 4 are independently halide, hydrogen, alkoxy, aryloxy, hydroxy, amine, -NHCOR where R is an optionally substituted hydrocarbyl, C 6 H 5 , or lower alkyl;
- A is an axial ligand composed of a halide, acetate, formate, PF 6 , triflate,
- R 1 through R 4 are independently H, optionally substituted hydrocarbyl, CH 3 , C 2 H 5 , C 6 H 5 , O-benzyl, primary alkyls, fatty acid esters, substituted alkoxyaryls, heteroatom-bearing aromatic groups, arylalkyls, secondary alkyls, or tertiary alkyls.
- Z 1 , Z 2 , Z 3 , and Z 4 are independently selected from hydrogen, halide, lower alkoxy, and lower alkyl.
- the bridge structure is an optionally substituted hydrocarbyl, typically -(CH 2 )n-, where n is generally 1, 2, 3, 4, 5, 6, 7 or 8, often 2 or 6 , and when 6 , often C(n) is a benzene ring.
- FIG. 27 shows catalytic SOD activity of C7
- Figure 28 shows inhibition of lipid peroxidation by C7, C53, and Vitamin E. Lipid peroxidation was induced in brain microsomes by iron and ascorbate, and was analyzed based on malonyldialdehyde content as described infra for Example 2.
- Figure 29 shows protection by C40 and C7 in a rat model for myocardial infarct. Rats were subjected to
- Figure 30 shows C40 delays rejection in a mouse skin transplantation model.
- donor and recipient mice were immunologically mismatched (Classl/Class II MHC
- a piece of skin ( ⁇ 1 cm 2 ) from the tail of a donor mouse was transplanted onto the back of a recipient mouse.
- the graft was bandaged and observed daily for
- FIG 32 shows C40 protects dopaminergic neurons in the mouse MPTP model for Parkinson's Disease. Neuronal damage was induced in mice by injection with MPTP as described in Example 1. Where indicated, mice were also treated with intraperitoneal injections of C40 at 0.02 or 0.2 mg/kg. The integrity of the nigrostriatal dopaminergic neurons was assessed based upon 3 H-Mazindol binding to striatal membranes harvested from the brains of these mice about 1 week after MPTP administration.
- Figure 33 shows C40 is protective in a rat model for stroke. Rats were subjected to a Middle Cerebral Artery (MCA) Occlusion model involving permanent occlusion of the parietal branch of the left middle cerebral artery and MCA Occlusion model involving permanent occlusion of the parietal branch of the left middle cerebral artery and MCA Occlusion model involving permanent occlusion of the parietal branch of the left middle cerebral artery and
- mice (“Presensitized” and "Presensitized + C7” groups) were presensitized with oxazolone on the abdomen.
- One group (“Not presensitized”) received only vehicle on the abdomen at this time.
- each mouse was challenged with the oxazolone hapten on one ear and given vehicle only on the opposite ear.
- mice also received a topical administration of C7 in 90% acetone (2.5 micrograms C7 per ear) on both ears immediately prior to hapten challenge.
- the other two groups received an equivalent volume of 90% acetone.
- Twenty-four hr after challenge mice were sacrificed and ear edema was assessed by determining the wet weight/dry weight ratio. (Wet weight was determined by weighing the freshly dissected ear and dry weight was determined after lyophilization to a constant weight.)
- FIG. 35 shows chronic treatment with C7 prolongs the life of an autoimmune strain of mice.
- MRL/lpr mice develop autoantibodies and numerous autoimmune associated pathologies and die prematurely (mean lifespan ⁇ 150 days) . They are considered a mouse model for autoimmune disorders such as lupus.
- MRL/lpr mice were treated intraperitoneally three times per week with C7 (1 mg/mouse) from the age of about 8 weeks until their death. Control mice received vehicle injections only or were left untreated.
- Figure 36 shows C7 protects neuronal tissue from beta-amyloid peptide-induced cytotoxicity. Rat hippocampal slices in culture were incubated with the beta-amyloid peptide (1-42) at the indicated concentrations. Cell viability was assessed by two criteria: release of lactate dehydrogenase (L*H) into the culture medium and staining with propidium (LDM) iodide (PI) which binds to exposed DNA.
- L*H lactate dehydrogen
- an "antioxidant” is a substance that, when present in a mixture or structure containing an oxidizable substrate biological molecule, significantly delays or prevents oxidation of the substrate biological molecule. Antioxidants can act by scavenging biologically important reactive free radicals or other reactive oxygen species (•O 2 -, H 2 O 2 , •OH, HOCl, ferryl, peroxyl, peroxynitrite, and alkoxy1), or by preventing their formation, or by catalytically
- An antioxidant salen-transition metal complex of the invention generally has detectable SOD activity.
- a salen-transition metal complex of the invention has antioxidant activity if the complex, when added to a cell culture or assay reaction, produces a detectable decrease in the amount of a free radical, such as superoxide, or a
- nonradical reactive oxygen species such as hydrogen peroxide
- the relative amount of free radiacal species is often determined by detection of a
- Suitable concentrations can be determined by various methods, including generating an empirical dose-response curve,
- Antioxidant salen metal complexes of the invention may have glutathione peroxidase activity or peroxidase activity in general.
- a "salen-transition metal complex” refers to a compound having a structure according to Structure I, Structure II, Structure III, or Structure IV, Structure V, Structure VI, Structure VII, Structure VIII, Structure IX, Structure X, Structure XI, Structure XII, Structure XIII, Structure XIV, Structure XV, Structure XVI, Structure XVII, Structure XVIII, Structure XIX, Structure XX, Structure XXI, Structure XXIII, Structure XXIV, (see.
- the axial ligand (A) is typically halide, acetate, propionate, butyrate, or
- the transition metal (M) is typically selected from the group consisting of: Mn, Mg, Co, Fe, Cu, Zn, V, Cr, and Ni; and is most
- the axial ligand (A) is often anionic, such as halide, acetate, propionate, butyrate, formate, PF 6 , triflate,
- tosylate or is an oxygen atom.
- free radical-associated disease refers to a pathological condition of an individual that results at least in part from the production of or exposure to free radicals, particularly oxyradicals, and other reactive oxygen species in vivo. It is evident to those of skill in the art that most pathological conditions are multifactorial, in that multiple factors contributing to the disease state are present, and that assigning or identifying the predominant causal factor (s) for any individual pathological condition is frequently extremely difficult.
- free radical associated disease encompasses pathological states that are recognized in the art as being conditions wherein damage from free radicals or reactive oxygen species is believed to contribute to the pathology of the disease state, or wherein administration of a free radical inhibitor (e.g., desferrioxamine), scavenger (e.g., tocopherol,
- a free radical inhibitor e.g., desferrioxamine
- scavenger e.g., tocopherol
- glutathione glutathione
- catalyst e.g., SOD, catalase
- the disease states discussed herein are considered free radical-associated diseases (e.g., ischemic reperfusion injury, inflammatory diseases, systemic lupus erythematosus, myocardial infarction, stroke, traumatic hemorrhage, spinal cord trauma, Crohn's disease, autoimmune diseases (e.g., rheumatoid arthritis, diabetes), cataract formation, uveitis, emphysema, gastric ulcers, oxygen
- free radical-associated diseases e.g., ischemic reperfusion injury, inflammatory diseases, systemic lupus erythematosus, myocardial infarction, stroke, traumatic hemorrhage, spinal cord trauma, Crohn's disease, autoimmune diseases (e.g., rheumatoid arthritis, diabetes), cataract formation, uveitis, emphysema, gastric ulcers, oxygen
- Such diseases can include "apoptosis-related ROS" which refers to reactive oxygen species (e.g., O 2 -, HOOH) which damage critical cellular components (e.g., lipid peroxidation) in cells stimulated to undergo apoptosis, such apoptosis- related ROS may be formed in a cell in response to an apoptosis-related ROS.
- reactive oxygen species e.g., O 2 -, HOOH
- critical cellular components e.g., lipid peroxidation
- apoptotic stimulus and/or produced by non-respiratory electron transport chains i.e., other than ROS produced by oxidative phosphorylation
- the present invention provides methods for therapy and prophylaxis of free radical-associated disease comprising administering to a patient a therapeutically-effective dose of an antioxidant salen-metal complex pharmaceutical composition.
- the method is used for preventing, arresting, or treating (1) neurological damage such as
- Parkinson's disease or Alzheimer's disease (2) cardiac tissue necrosis resulting from cardiac ischemia, (3) autoimmune neurodegeneration (e.g., encephalomyelitis), (4) acute lung injury such as in sepsis and endotoxemia, and (5) neuronal damage resulting from ischemia (e.g., stroke, drowning, brain surgery) or trauma (e.g., concussion or cord shock).
- ischemia e.g., stroke, drowning, brain surgery
- trauma e.g., concussion or cord shock
- SOD mimetic As used herein the terms “SOD mimetic”, “SOD mimic”, “superoxide dismutase mimetic”, and “superoxide catalyst” refer to compounds which have detectable catalytic activity for the dismutation of superoxide as determined by assay.
- an SOD mimetic possesses at least about 0.001 percent of the SOD activity of human Mn-SOD or Zn,Cu-S0D, on a weight basis, as determined by standard assay methods such as for example the SOD assay used herein below.
- pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
- alkyl refers to a cyclic, branched, or straight chain alkyl group containing only carbon
- Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
- substituents e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
- lower alkyl refers to a cyclic, branched or straight chain monovalent alkyl radical of one to six carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, n-amyl, and hexyl.
- aryl refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio,
- substituted alkoxy refers to a group having the structure -O-R, where R is alkyl which is
- arylalkoxy refers to a group having the structure -O-R-Ar, where R is alkyl and Ar is an aromatic substituent.
- Arylalkoxys are a subset of substituted alkoxys. Examples of preferred substituted alkoxy groups are: benzyloxy,
- aryloxy refers to a group having the structure -O-Ar, where Ar is an aromatic group.
- a preferred aryloxy group is phenoxy.
- heterocycle refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzo[b]thienyl) and having at least one heteroatom, defined as N, O, P, or S, within the ring, which can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
- heteroaryl or “HetAr” refers to
- Arylalkyl refers to the groups -R-Ar and
- Ar is an aryl group
- HetAr is a heteroaryl group
- R is straight-chain or branched-chain aliphatic group.
- arylalkyl groups include benzyl and furfuryl.
- Arylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
- halo or halide refers to fluoro, bromo, chloro and iodo substituents.
- OBn means benzyloxy
- amino refers to a
- quaternary amine refers to the positively charged group -N + R'R"R'", where R', R", and R'" are independently selected and are alkyl or aryl.
- a preferred amino group is -NH 2 .
- silyl refers to organometallic substituents, wherein at least one silicon atom is linked to at least one carbon atom; an example of a silyl substituent is the trimethylsilyl substituent, (CH 3 ) 3 Si-.
- hydrocarbyl shall refer to an organic radical comprised of carbon chains to which hydrogen and other elements are
- alkyl alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and
- heteroaryl refers to aromatic monovalent mono- or poly-cyclic radical having at least one heteroatom within the ring, e.g., nitrogen, oxygen or sulfur.
- heteroalkyl refers to a branched or straight chain acyclic, monovalent saturated radical of two to twenty atoms in which at least one of the atoms in the chain is a heteroatom, such as, for example, nitrogen, oxygen or sulfur.
- heterocycloalkyl refers to a monovalent saturated cyclic radical of one to twelve atoms, having at least one heteroatom (such as nitrogen, oxygen or sulfur) within the ring.
- hydrocarbyl refers to a hydrocarbyl group which can optionally be mono-, di-, or tri-substituted, independently, with hydroxylower-alky1, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro,
- pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly
- a basis of the present invention is the unexpected finding that members of a class of compounds described
- compositions can be prepared with an effective amount or concentration of at least one antioxidant salen-transition metal complex species.
- the catalytic activity of salen-metal complexes to interconvert epoxides may also be used to advantage to scavenge or prevent formation in vivo of cytotoxic and/or carcinogenic epoxide species, such as may be formed by the cytochrome P-450 monooxygenation system (e.g., benzo-[a]- pyrene diol epoxide).
- Catalytic salen-metal complexes may be advantageously included into foodstuffs or dietary supplements (or administered in other forms) to individuals who are at risk of exposure to polycyclic hydrocarbon chemical
- catalytically active salen- metal complexes may be formulated for administration to smokers (including passive smokers) to enhance detoxification of reactive epoxides formed from cigarette smoke.
- ALS amyotrophic lateral sclerosis
- Antioxidant salen metal complexes of the present invention can be used for treatment and prophylaxis of such neurodegenerative diseases (e.g., ALS, MS, Parkinson's disease, Alzheimer's disease).
- neurodegenerative diseases e.g., ALS, MS, Parkinson's disease, Alzheimer's disease.
- the salen-transition metal complex has the following
- M is a transition metal ion, preferably Mn
- A is an axial ligand (anion) composed of a halide, acetate, acetyl, acetoxy, ethoxy, formate, formyl, methoxy, PF 6 , triflate, tosylate, or is an oxygen atom typically bound via a double bond to the transition metal (M)
- A is typically Cl, Br, F, MeO or OAc
- n is either 0, 1, 2, or 6.
- X 1 , X 2 , X 3 and X 4 are independently selected from the group consisting of
- X 2 and X 3 are from the same functional group, usually hydrogen, ethoxy, methoxy, quaternary amine, or tertiary butyl, and X 2 and X 4 are
- X 1 and X 3 are each F, Cl, Br, OAc, OMe, OH, or H and X 2 and X 4 are each F, Cl, Br, OAc, OMe, OH, or H, typically when X 1 and X 3 are other than H, X 2 and X 4 are both H, and vice versa.
- Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of hydrogen, halides, alkyls, aryls, arylalkyls, silyl groups, aminos, alkyls or aryls bearing heteroatoms; aryloxys,
- R 1 , R 2 , R 3 and R 4 are independently selected from the group consisting of H, CH 3 , C 2 H 5 , C 6 H 5 , O-benzyl, primary alkyls, fatty acid esters, substituted alkoxyaryls, heteroatom-bearing aromatic groups, arylalkyls, secondary alkyls, and tertiary alkyls.
- one of R 1 and R 2 is covalently linked to one of R 3 or R 4 forming a cyclic structure; preferred cyclic structures include a six-membered ring, such as a benzene ring.
- At least one of the X 1 and X 3 sites, and preferably both X 1 and X 3 include a substituent selected from the group of blocking substituents consisting of
- X 1 and X 3 sites bear the same substituent, which substituent is most preferably a tertiary alkyl group, such as tertiary butyl.
- X 1 and X 3 bear a blocking substituent
- X 2 and X 4 are selected from a group of non-blocking substituents such as H, CH 3 , C 2 H 5 , and primary alkyls, most preferably, H.
- either three or four of X 1 , X 2 , X 3 , and X 4 can be selected from the group of blocking substituents.
- R 1 , R 2 , R 3 and R 4 are selected from a group consisting of H, CH 3 , C 2 H 5 , and primary alkyls.
- this group will be referred to as the non-blocking group.
- R 1 is selected from the non-blocking group
- R 2 and R 3 are preferably selected from the blocking group
- typically R 2 and R 3 are identical and are phenyl or benzyloxy.
- R 1 and R 4 are preferably selected from the blocking group.
- R 3 is selected from the non- blocking group
- R 1 and R 4 are preferably selected from the blocking group.
- R 4 is selected from the non- blocking group
- R 2 and R 3 are preferably selected from the blocking group.
- Phenyl and benzyloxy are particularly
- blocking groups for substitution at any of R 1 , R 2 , R 3 and R 4 .
- the blocking groups selected are
- a preferred class of embodiments have R 2 and R 4 as benzyloxy or phenyl and R 2 and R 3 as hydrogen.
- one class of embodiments of the first aspect of the invention requires that, of the four sites available for substitution on the two carbon atoms adjacent . to nitrogen, at least one or two of these preferably will include a substituent from the non-blocking group.
- the non-blocking substituent is either hydrogen or methyl, but most preferably, hydrogen.
- the blocking substituent is either a phenyl group, a benzyloxy, or a tertiary butyl group, more preferably a phenyl group or a benzyloxy group, most usually a phenyl group.
- Y 3 and Y 6 are hydrogen, methyl, alkyl, or aryl. More preferably, they are hydrogen or methyl. Most preferably, they are hydrogen.
- the Y 1 , Y 2 , Y 4 , and Y 5 sites are selected
- Y 1 and Y 4 are examples of substituents independently selected from the group consisting of hydrogen, halides, alkyls, aryls, alkoxy groups, substituted alkoxy groups, nitro groups, and amino groups.
- substituents independently selected from the group consisting of hydrogen, halides, alkyls, aryls, alkoxy groups, substituted alkoxy groups, nitro groups, and amino groups.
- Y 1 and Y 4 are examples of substituents independently selected from the group consisting of hydrogen, halides, alkyls, aryls, alkoxy groups, substituted alkoxy groups, nitro groups, and amino groups.
- tertiary butyl preferably occupied by methoxy, ethoxy, chloro, bromo, iodo, primary alkyl, tertiary butyl, primary amine, secondary amine, or tertiary amine substituents, most preferably methoxy, chloro, tertiary butyl, or methyl.
- the salen-transition metal complex has the structure:
- M is a transition metal ion, preferably Mn
- A is an axial ligand (anion) composed a halide, acetate, acetyl, acetoxy, ethoxy, formate, formyl, methoxy, PF 6 , triflate, tosylate, or is an oxygen atom typically bound via a double bond to the transition metal (M) ;
- A is typically Cl, Br, F, MeO or OAc, typically Cl;
- at least one of X 1 or X 2 is selected from the group consisting of aryls, primary alkyls, secondary alkyls, tertiary alkyls, and heteroatoms or H;
- at least one of X 1 or X 3 is selected from the group consisting of aryls, primary alkyls, secondary alkyls, tertiary alkyls, arylalkyls, heteroatoms, and hydrogen, preferably tertiary butyl or hydrogen;
- Y 1 and Y 4 are selected from the group consisting of lower alkyls, alkoxy, halide, and amino groups, more preferably from the group consisting of methoxy, chloro, and primary amine.
- Y 1 and Y 4 are methoxy: X 1 and X 3 are
- the salen-transition metal has the following structure:
- M is a transition metal ion such as Mn, Mg, Co, Fe, Zn, Cu, V, Cr, and Ni
- A is an axial ligand composed of a halide, acetate, formate, PF 6 , triflate, tosylate, or is an oxygen atom typically bound via a double bond to the metal (M) ; and A is typically Cl and M is typically Mn; where n is either 4, 5, or 6;
- X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of aryls, arylalkyls, aryloxys, primary alkyls, secondary alkyls, tertiary alkyls, alkoxy, substituted alkoxy, heteroatoms, aminos, quaternary amines, and hydrogen; preferably, at least one of X 1 or X 3 are selected from the group consisting of aryls, primary alkyls, secondary alkyls, ter
- Y 1 or Y 4 are selected from the group consisting of aryls, primary alkyls, secondary alkyls,
- tertiary alkyls substituted alkoxy, heteroatoms, amines, and halides; more preferably Y 1 and Y 4 are identical and are either methoxy, chloro, bromo, iodo, tertiary butyl, or amine.
- R 1 and R 4 are independently selected from the group consisting of hydrogen, halides, primary alkyls, secondary alkyls, tertiary alkyls, fatty acid esters, alkoxys, or aryls.
- Y 1 and Y 2 are independently selected from the group consisting of methoxy, ethoxy, methyl, ethyl, formyl, acetyl, t-butyl, chloro, bromo, iodo, fluoro, amino, quaternary amine, alkylamino, dialkylamino, and hydrogen; R 1 and R 2 are independently selected from the group consisting of: phenyl, benzyloxy, chlorobenzyloxy, hydrogen, amino, quaternary amine, or fatty acid ester.
- Y 1 and Y 2 are identical.
- R 1 and R 2 are selected independently from the group consisting of: phenyl, benzyloxy, chlorobenzyloxy, methoxy, ethoxy, hydrogen, amino, quaternary amine, methoxy, ethoxy, or fatty acid ester.
- R 1 and R 2 are identical.
- Y 1 and Y 2 are independently selected from the group consisting of methoxy, ethoxy, methyl, ethyl, t-butyl, chloro, bromo, iodo, amino, quaternary amine, alkylamino,
- R 1 and R 2 are selected
- Y 1 and Y 2 are identical, and R 1 and R 2 are identical.
- X is selected from the group consisting of methoxy, ethoxy, methyl, ethyl, formyl, acetyl, t-butyl, chloro, bromo, iodo, fluoro, amino, quaternary amine, alkylamino,
- Y is selected from the group consisting of t-butyl, methoxy, ethoxy, formyl, acetyl, Cl, Br, F, quaternary amine, amino, and hydrogen.
- R 1 and R 2 are independently selected from the group consisting of aryloxys, alkoxys, aryls, and hydrogen; R' and R" are independently selected from the group consisting of alkyls, aryls, and hydrogen.
- at least one of the amino groups is protonated at physiological pH (i.e., pH 7.3- 7.8).
- Preferred R' or R'' alkyls include but are not limited to: methyl, ethyl, and propyl.
- Preferred R 1 and R 2 aryloxys include but are not limited to benzyloxy and chlorobenzyloxy.
- Preferred R 1 and R 2 alkoxys include but are not limited to ethoxy and methoxy.
- a preferred subgenus of Structure VIII includes, but is not limited to: where R is selected from the group consisting of alkyls and hydrogen. Preferably, at least one of the amino groups are protonated at physiological pH (i.e., pH 7.3-7.8).
- Additional preferred structural genuses include, but are not limited to Structures X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, and XXI, XXII, XXIII, XXIV as shown in Fig. 11 and Fig. 26A through 26E. Additional preferred exemplified species are shown in Figs. 24A-24I.
- Cyclic structures linking the 3 and 3' positions often enhance catalase properties (i.e., catalytic rate, endpoint, turnover rate, and peroxidase activity) in a manner comparable to the enhancement seen with separate substituents (e.g., C82 and C48>C47.
- aromatic ring structures are preferred bridge modifications which enhance planarity (e.g., compare C31 to C43, C47 and C7 to C44).
- Substituents added to the imine tend to reduce catalase activity.
- Table I shows activity determinations for a variety of disclosed salen-metal species relative to C7
- the following species are preferred antioxidant salen-transition metal complexes for formulation in
- compositions e.g., dietary supplements, foodstuff preservatives, cosmetics, sunburn preventatives, and other compositions of the invention, and are referenced by structure number (e.g., C1 through C30) for clarity throughout.
- compositions of the present invention comprise a therapeutically or
- salicylaldehyde derivative a derivative-based complex of a transition metal ion.
- solden is used herein to refer to those ligands typically formed through a condensation reaction of two molecules of a salicylaldehyde derivative with one molecule of a diamine derivative. While salen ligands are formed from
- ethylenediamine derivatives may also be used to give analogous salen and salen
- Salen derivatives are preferred and their general structure is shown in Figs. 1, 12, and 26A-26E.
- a salen derivative where n is 0 is shown in Fig. 2.
- the two nitrogens and the two oxygens are oriented toward the center of the salen ligand and thus provide a complexing site for the transition metal ion M.
- this metal ion is selected from the group
- the transition metal ion is selected from the group consisting of Mn, Mg, Cr, Fe, Ni, and Co.
- the metal ion is Mn.
- the anion is selected from the group consisting of PF 5 , (aryl) 4 , BF 4 , B(aryl) 4 , halide, acetate, acetyl, formyl, formate, triflate, tosylate, with halide, acetate, or PF 6 being more preferred, and chloride and acetate being most preferred.
- Fig. 1 also shows the many sites available for substitution on the salen ligand. Of these sites, it is believed that R 1 , R 2 , R 3 , R 4 , and X 1 , X 2 , X 3 , X 4 , Y 3 and Y 6 are the most important in this first salen-transition metal complex. Structures I, III, IV, VI, VII, VIII, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, and XXI, XXII,
- XXIII, XXIV may have independently selected fatty acid ester substituents at the R 1 , R 2 , R 3 , and R 4 (or the Z 1-4 poisitions for structures in Figs. 24A-24I having Z 1-4 substituent positions).
- the fatty acid esters typically occupy no more than two substituent positions and are usually identical.
- the unsaturated acids occur in isomeric forms due to the presence of the one or more unsaturated positions.
- the compounds of the present invention are intended to include the individual double bond isomers, as well as mixtures thereof.
- the fatty acid esters of the present invention can be obtained by known acylation techniques. See, e.g., March, Advanced Organic Chemistry. 3rd Ed., John Wiley & Sons, New York (1985), pp. 299, 348-351, and 353-354,
- Figures 3 and 24A-24H show structures of preferred antioxidant salen-transition metal complexes of the invention.
- Example antioxidant salen-transition metal complexes are shown in Figs. 3, 19A, and 24A-24H.
- C84, C85, C86, C87, C88, C89, C90, C91, C92, C93, and C94 are particularly preferred for formulation in pharmaceuticals and other antioxidant compositions of the invention. It is believed that C7, C31, C32, and C40 is particularly preferred because of their facile preparation and relatively hydrophilic nature which is well-suited to pharmaceutical usage.
- a preferred salen-transition metal complex having high superoxide dismutase activity is the C12 compound having the structure: additional preferred congeners of C12 are:
- a particularly preferred antioxidant salen-metal complex of the invention is C7:
- Antioxidant salen-transition metal complexes generally have detectable superoxide dismutase activity and preferably also have catalase activity.
- C7, C31, C32, and C40 are both simple to prepare and relatively hydrophilic
- the preferred route to prepare the antioxidant salen-transition metal complexes of the present invention is a condensation reaction with the substituted salicylaldehyde and the substituted diamine.
- quantities of these compounds are reacted in a 2 to 1 molar ration in absolute ethanol.
- the solutions are refluxed typically for 1 hour, and the salen ligand is either
- the metal complex is generated directly by addition of the metal as its acetate, halide, or triflate salt.
- the starting diamine is R,R- or S,S-1,2-diamino- 1,2-diphenylethane and the starting salicylaldehyde is 3-tert- butylsalicylaldehyde.
- a solution of 2.0 mmol of 3-tert- butylsalicylaldehyde in 3 ml of absolute ethanol is added dropwise to a solution of 1.0 mmol of (R,R)-1,2-diamino-1,2- diphenylethane in 5 ml of ethanol.
- reaction mixture is heated to reflux for 1 h and then 1.0 mmol of Mn(Oac) 2 ⁇ 4H 2 O is added in one portion to the hot (60°C) solution.
- the color of the solution immediately turns from yellow to brown upon addition. It is refluxed for an additional 30 min and then cooled to room temperature.
- a solution of 10% NaCl (5ml) is then added dropwise and the mixture stirred for 0.5h. The solvents are then removed in vacuo and the residue is
- antioxidant salen-transition metal complexes of the invention may be routinely accomplished by those of ordinary skill in the art according to the cited publications.
- the SOD activity of the prepared salen-Mn complexes is determined according to standard assay methods for SOD activity known in the art and exemplified infra .
- Salen-metal complexes having at least 0.001 percent of human SOD activity on a weight basis in aqueous solution are antioxidant salen- metal complexes; preferably antioxidant salen-metal complexes have at least about 0.01 percent of SOD activity per unit weight; and more preferably have at least about 0.1 percent of SOD activity per unit weight.
- catalase activity is preferably supplemented, it is
- the SOD mimetic salen-metal complex also possesses detectable catalase activity (e.g., C4 , C7, C9, CIO, C11, C12, C32, C40, C41, C67, C68 , and others; see Table I).
- detectable catalase activity e.g., C4 , C7, C9, CIO, C11, C12, C32, C40, C41, C67, C68 , and others; see Table I).
- compositions comprising an antioxidant salen-transition metal complex of the present invention are useful for topical and parenteral
- compositions for parenteral administration will commonly comprise a solution of an antioxidant salen- transition metal complex or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier or organic solvent (e.g., DMSO, solvated PEG, etc.). Since many of the salen-Mn complexes of the invention are lipophilic, it is preferable to include in the carrier a hydrophobic base (e.g., polyethylene glycol, Tween 20).
- aqueous carrier e.g., DMSO, solvated PEG, etc.
- a hydrophobic base e.g., polyethylene glycol, Tween 20.
- compositions can be sterilized by conventional, well known sterilization techniques.
- the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium
- concentration of the antioxidant salen-transition metal complex (es) in these formulations can vary widely, i.e., from less than about 1 nM, usually at least about O.lmM to as much as 100 mM and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Most usually, the antioxidant salen-transition metal complex (es) in these formulations can vary widely, i.e., from less than about 1 nM, usually at least about O.lmM to as much as 100 mM and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Most usually, the antioxidant salen-transition metal complex (es) in these formulations can vary widely, i.e., from less than about 1 nM, usually at least about O.lmM to as much as 100 mM and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the
- antioxidant salen-metal complex is present at a concentration of 0.1 mM to 10 mM.
- a typical formulation for intravenous injection comprises a sterile solution of an antioxidant salen-metal complex (e.g., C7, C32, C40) at a concentration of 1 mM in physiological saline or Ringer's solution.
- the generally hydrophobic nature of some of the preferred antioxidant salen-metal complexes indicates that a hydrophobic vehicle may be used, or that an aqueous vehicle comprising a detergent or other lipophilic agent (e.g., Tween, NP-40, PEG); alternatively, the antioxidant salen complexes may be administered as a suspension in an aqueous carrier, or as an emulsion.
- a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile water, and about 0.1-100 mg of antioxidant salen- transition metal complex (es).
- a typical composition for intravenous infusion can be made up to contain 250 ml of sterile saline or Ringer's solution, and about 10-1000 mg of antioxidant salen-transition metal complex (es).
- Lipophilic agents may be included in formulations of lipophilic salen- metal complexes.
- Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example. Remington's Pharmaceutical Science. 15th Ed., Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference.
- a typical pharmaceutical composition for topical application can be made with suitable dermal ointments, creams, lotions, ophthalmic ointments and solutions, respiratory aerosols, and other excipients.
- antioxidant salen-transition metal complex that are the active ingredient(s) of the preparation, and generally should not increase decomposition, denaturation, or aggregation of active ingredient (s). Frequently, excipients will have lipophilic components such as oils and lipid emulsions.
- the antioxidant salen-transition metal complex (es) of this invention can be lyophilized for storage and
- compositions containing the present antioxidant salen-transition metal complex (es) or cocktails thereof can be administered for prophylactic and/or therapeutic treatments.
- compositions are administered to a patient already affected by the particular free radical- associated disease, in an amount sufficient to cure or at least partially arrest the condition and its complications. An amount adequate to accomplish this is defined as a
- Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration, but generally range from about 1 mg to about 10g of antioxidant salen-transition metal complex (es) per dose, with dosages of from 10 mg to 2000 mg per patient being more commonly used.
- a antioxidant salen metal complex e.g., C7, C32, C40
- a antioxidant salen metal complex may be administered systemically by intravenous infusion; at least about lmg to 500 mg of antioxidant salen-metal
- complex(es) may be administered by intrapericardial injection to provide elevated local concentrations of SOD activity in the myocardium.
- compositions containing the antioxidant salen-transition metal complex (es) or cocktails thereof are administered to a patient not already in a disease state to enhance the patient's resistance or to retard the progression of disease.
- Such an amount is defined to be a "prophylactically effective dose.”
- the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 1 mg to 10 g per dose, especially 10 to 1000 mg per patient.
- a typical formulation of an antioxidant salen-metal complex such as C7, C31, C32, or C40 will contain between about 2.5 and 250 mg of the salen-metal complex in a unit dosage form.
- compositions can be carried out with dose levels and dosing pattern being selected by the treating physician.
- pharmaceutical formulations should provide a quantity of the antioxidant salen-transition metal complex (es) of this invention sufficient to effectively treat the patient.
- Kits can also be supplied for use with the subject antioxidant salen-transition metal complex (es) for use in the protection against or therapy for a free radical-associated disease.
- the subject composition of the present invention can also be supplied for use with the subject antioxidant salen-transition metal complex (es) for use in the protection against or therapy for a free radical-associated disease.
- inventions may be provided, usually in a lyophilized form or aqueous solution in a container, either alone or in
- antioxidant salen-transition metal complex e.g., sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium phosphate
- Salen-Mn complexes preferably compounds C12, C7, C32, C40, or the like can be incorporated into a hypothermic cardioplegia solution at a concentration of at least about 1 mM into a solution formulation according to Amano et al.
- C7 is included in the cardioplegia solution.
- the dosage of SOD-mimetic salen-metal complex will vary with each particular application.
- the composition is administered either systemically or topically.
- Systemic administration includes per os and parenteral routes; topical administration includes in situ applications.
- the in situ means includes, for example, administering an SOD-mimetic salen-metal complex by endoscopic bolus wash and/or paravenous injection, or in the case of lower GI treatments, by enema.
- Parenteral routes may include, for example, subcutaneous, intradermal, intramuscular, and intravenous routes.
- the amount of SOD-mimetic salen-metal complex will range from about 0.02 to 5,000 mg or more, typically 1 to 1000 mg, depending on the administration interval and route, which can range from a single oral dose, parenteral dose and/or topical dose to multiple oral doses, parenteral doses, and/or topical doses over a few days or greater than 5 weeks.
- the dosage may also vary with the severity of the disease.
- antioxidant salen-transition metal complexes of the invention are employed to modulate the expression of naturally-occurring genes or other polynucleotide sequences under the transcriptional control of an oxidative stress response element (e.g., an antioxidant responsive element, ARE) , such as an antioxidant response element of a glutathione S-transferase gene or a NAD(P)H:quinone reductase gene (Rozen et al. (1992) Arch.
- an oxidative stress response element e.g., an antioxidant responsive element, ARE
- an antioxidant response element of a glutathione S-transferase gene or a NAD(P)H:quinone reductase gene Roszen et al. (1992) Arch.
- Transgenes, homologous recombination constructs, and episomal expression systems comprising a polynucleotide sequence under the transcriptional control of one or more ARE linked to a promoter will be made by those of skill in the art according to methods and guidance available in the art, as will
- the antioxidant salen-metal complexes may be used to modulate the transcription of ARE- regulated polynucleotide sequences in cell cultures (e.g., ES cells) and in intact animals, particularly in transgenic animals wherein a transgene comprises one or more AREs as transcriptional regulatory sequences.
- a dose-response curve is generated by titrating transcription rate of the ARE-controlled
- polynucleotide sequence against increasing concentrations of antioxidant salen-metal complex es
- antioxidant salen-metal complex es
- Similar dose-response titration can be performed in transgenic animals, such as transgenic mice, harboring an ARE-controlled transgene sequence.
- a therapeutically or pharmaceutically effective amount of an antioxidant salen- transition metal complex is administered to a patient to treat or prevent a free radical-associated disease.
- the required dosage will depend upon the nature of the free radical- associated disease, the severity and course of the disease, previous therapy, the patient's health status and response to the antioxidant salen-transition metal complex, and the judgment of the treating physician.
- antioxidant salen-Mn complex is administered as the sole active ingredient, or in combination with one or more other active ingredients, typically selected from the group consisting of: N-2-mercaptopropionylglycine, N-acetylcysteine, glutathione, dimethyl thiourea, desferrioxamine, mannitol, ⁇ - tocopherol, ascorbate, allopurinol, 21-aminosteroids, calpain inhibitors, glutamate receptor antagonists, tissue plasminogen activator, streptokinase, urokinase, nonsteroidal anti- inflammatory agent, cortisone, and carotenoids.
- Antioxidant salen-Mn complexes may also be administered in conjunction with polypeptides having SOD and/or catalase activity, particularly in view of the capacity of the salen-Mn
- the present invention includes a method of treating patients, such as humans, who have a free radical-associated disease with a prophylactically effective or therapeutically effective amount of a antioxidant salen-transition metal complex, typically a salen-Mn complex, preferably C7, C31, C32, or C40.
- a prophylactically effective or therapeutically effective amount of a antioxidant salen-transition metal complex typically a salen-Mn complex, preferably C7, C31, C32, or C40.
- This method can be used to treat patients at various stages of their diseases or to prevent development of free radical-associated diseases in patients.
- the treatment can be administered to prevent or reduce, as a prophylactic, the age-adjusted probability of developing a neoplasm and/or the age-adjusted mortality rate and/or the rate of senescence.
- the antioxidant salen-metal complexes of the invention can also be administered to patients who are infected with a human immunodeficiency virus (e.g
- TNF tumor necrosis factor
- TNF or other inflammatory mediators both in AIDS and in other conditions (e.g., septic shock).
- a dosage of about 50 to 5000 mg will be administered to a patient with HIV and/or with excessive or inappropriate levels of TNF, either in single or multiple doses, to reduce or retard the development of pathology and clinical symptoms.
- Antioxidant salen-metal complexes may be administered therapeutically to treat viral diseases other than HIV.
- antioxidant salen-transition metal complexes Since oxidative damage occurs proportionately to the abundance of free radicals and reactive oxygen species, it is expected that administration of antioxidant salen-transition metal complexes at even low levels will confer a protective effect against oxidative damage; thus it is expected that there is no threshold level below which antioxidant salen-Mn complexes are ineffective.
- a suitable effective dose of the antioxidant salen- Mn complex will be in the range of 0.001 to 1000 milligram (mg) per kilogram (kg) of body weight of recipient per day, preferably in the range of 0.1 to 100 mg per kg of body weight per day.
- the desired dosage is preferably presented in one, two, three, four or more subdoses administered at appropriate intervals throughout the day. These subdoses can be
- unit dosage forms for example, containing 0.01 to 10,000 mg, preferably 10 to 1000 mg of active ingredient per unit dosage form.
- composition used in these therapies can be in a variety of forms. These include, for example, solid, semi- solid and liquid dosage forms, such as tablets, pills,
- compositions also preferably include conventional
- administration will be by oral or parenteral (including subcutaneous, intramuscular,
- the pharmaceutically acceptable formulations of the present invention comprise at least one compound of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically or
- Preferred carriers include inert, non-toxic solids (e.g., mannitol, talc) and buffered saline.
- mannitol, talc a solids
- buffered saline a buffered saline.
- Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, and others.
- Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, NJ, incorporated herein by reference.
- pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers such as sterile solutions, tablets, coated tablets, and capsules. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acids or salts
- magensium or calcium sterate talc, vegetable fats or oils, gums, glycols, or other known excipients.
- Such carriers may also include flavor and color additives or other
- compositions comprising such carriers are
- compositions may be in the form of solid, semi-solid, or liquid dosage forms, such, for example, as powders, granules, crystals, liquids, suspensions, liposomes, pastes, cremes, salves, etc., and may be in unit-dosage forms suitable for administration of relatively precise dosages.
- semi-solid compositions as would be appropriate for pastes and creams intended for topical administration, the salen-metal complexes can be provided separately or may be compounded with
- compositions may contain about 0.005-100% active
- compositions include lotions containing water and/or alcohols and emollients such as hydrocarbon oils and waxes, silicone oils, vegetable, animal or marine fats or oils, glyceride derivatives, fatty acids or fatty acid esters or alcohols or alcohol ethers, lecithin, lanolin and
- emulsifiers nonionic, cationic or anionic, although some of the emollients inherently possess emulsifying properties.
- compositions are referred to herein as dermatologically acceptable carriers.
- the pharmaceutical compositions will be administered by parenteral or oral administration for prophylactic and/or therapeutic treatment.
- the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
- unit dosage forms suitable for oral administration include powder, tablets, pills, capsules, and dragees.
- compositions for intravenous administration which comprise a solution of the compound dissolved or suspended in an
- antioxidant salen-metal complex(es) such as C7, C12, C32, or C40 and others may be dissolved in an organic solvent (e.g., dimethylsulfoxide) and either applied directly or diluted into an aqueous solvent.
- organic solvent e.g., dimethylsulfoxide
- compositions will sometimes be sterilized by conventional, well known sterilization techniques, or can preferably be sterile filtered.
- aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
- the compositions can contain
- auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium
- conventional nontoxic solid carriers can be used which include, for example,
- composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 0.001-95% of active ingredient, preferably about 20%.
- compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.
- compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as
- terapéuticaally effective amount or dose Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
- compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease.
- a patient susceptible to or otherwise at risk of a particular disease is defined to be a "prophylactically effective amount or dose.”
- the precise amounts again depend on the patient's state of health and weight.
- conventional non-toxic solid excipients include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum,
- celluloses glucose, sucrose, magnesium carbonate, and the like may be used.
- the active compound as defined above may be formulated as suppositories using, for example, triglycerides, for example, the Witepsols, as the carrier.
- compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
- a excipient such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like.
- the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or
- emulsifying agents for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
- pH buffering agents for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
- Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th Edition, 1985.
- formulation to be administered will, in any event, contain an effective amount of the active compound (s).
- a pharmaceutically acceptable non-toxic composition is formed by the
- any of the normally employed excipients such as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, celluloses, glucose, sucrose, magnesium, carbonate, and the like.
- compositions take the form of solutions, suspensions, tablets, capsules, powders, sustained release formulations and the like. Such compositions may contain 0.01-95% active
- Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously.
- injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to
- Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
- the pharmaceutically acceptable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
- the pharmaceutically acceptable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
- compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or
- emulsifying agents such as for example, sodium acetate, sorbitan monolaurate,
- a more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated herein by reference.
- Antioxidant salen-metal complexes may be administered by transdermal patch (e.g., iontophoretic transfer) for local or systemic application.
- a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of the disease symptoms or as a prophylactic measure to prevent disease symptom recurrence.
- Antioxidant salen-metal complex can also be added to extravasated blood for transfusion to inhibit
- antioxidant salen-metal complexes can also reduce oxyradical damage to blood cells in vivo.
- Antioxidant salen-metal complex can also be added to perfusion, rinse or storage solutions for organs and tissues, such as for organ transplantation or for surgical rinses.
- organs and tissues such as for organ transplantation or for surgical rinses.
- excised organs are often placed in a preservation solution prior to transplant into a recipient.
- the antioxidant salen-metal complex is present in the rinse or storage solution at a concentration of about 1 ⁇ M to about 1 mM, and most usually is present at 10- 100 ⁇ M.
- a concentration of about 1 ⁇ M to about 1 mM and most usually is present at 10- 100 ⁇ M.
- a concentration of about 1 ⁇ M to about 1 mM and most usually is present at 10- 100 ⁇ M.
- suitable rinse solution comprises Ringer's solution (102 mM NaCl, 4 mM KCl , 3 mM CaCl 2 , 28 mM sodium lactate, pH 7.0) or Ringer's solution with 0.1 mM adenosine, and the antioxidant salen-Mn complex C7 at a final concentration of 50 ⁇ M.
- the rinse solution can further comprise additional antioxidants (e.g., glutathione, allopurinol).
- Preservation, perfusion, or rinse solutions containing an antioxidant salen-metal complex can be used to provide enhanced storage or irrigation of organs (e.g., kidney, liver, pancreas, lung, fetal neural tissue, heart, vascular grafts, bone, ligament, tendon, skin) which is believed to enhance the viability of the tissue and increase resistance to oxidative damage (e.g., as a
- the capacity of the antioxidant salen-metal complexes to catalyze the decomposition of reactive oxygen species can be used to advantage to inhibit or slow damage to biological tissues and cells.
- benzoyl peroxide is a widely used treatment for acne lesions; excessive or inappropriate application of benzoyl peroxide (e.g., accidental application to the eyes) may be treated by local (or if desired, systemic) administration of an antioxidant salen-metal complex (e.g., C7, C32, C40).
- oxyradical-induced damage to connective tissues e.g., collagen
- connective tissues e.g., collagen
- senescence may be reduced by administration of an antioxidant salen-metal complex approximately concomitant with the exposure to UV light, cigarette smoking, or other
- oxyradical-generating process e.g., cellular senescence
- Antioxidant salen-transition metal complexes typically antioxidant salen-Mn complexes, such as compound C7, C32, C40 are used to protect cells and tissues from free radical-producing agents, such as ionizing radiation and chemotherapeutic agents (e.g., bleomycin).
- free radical-producing agents such as ionizing radiation and chemotherapeutic agents (e.g., bleomycin).
- a protective dosage comprising at least about l ⁇ g of salen-Mn complex/kg bodyweight is administered by one or more of several routes (e.g., oral, intraveneous, intraperitoneal, intragastric lavage, enema, portal vein infusion, topical, or inhalation of mist), preferably by injection of liposomes or immunoliposomes for targeted delivery of the antioxidant salen-Mn complexes to protect normal cells, for example, against free radical toxicity associated with chemotherapy or radiotherapy of a neoplasm.
- the antioxidant salen-transition metal complexes are preferably preadministered to the patient prior to the commencement of the chemotherapy and/ or
- Antioxidant salen-Mn may be continually administered to the patient during the course of therapy.
- a solution of an antioxidant salen- metal complex can be encapsulated in micelles to form
- the immunoliposomes containing the antioxidant salen-metal species will comprise a targeting moiety (e.g., monoclonal antibody) that targets the immunoliposomes to non- neoplastic cells that are otherwise sensitive to radiotherapy or chemotherapy.
- a targeting moiety e.g., monoclonal antibody
- immunoliposomes having a monoclonal antibody that binds specifically to a hematopoietic stem cell antigen not present on the cancer cells of the individual may be used to target antioxidant salen-metal complexes to hematopoietic stem cells and thereby protect said stem cells against radiotherapy or chemotherapy used to treat the cancer.
- chemotherapeutic agent forms free radicals in vivo (e.g., bleomycin).
- Antioxidant salen-Mn complexes are also administered to individuals to prevent radiation injury or chemical injury by free radical generating agents. Military personnel and persons working in the nuclear, nuclear medicine, and/or chemical industries may be administered salen-Mn complexes prophylactically. Antioxidant salen-metal complexes may also be used as chemoprotective agents to prevent chemical
- reactive epoxide intermediates e.g., benzo-[a]-pyrene, benzanthracene
- carcinogens or promoting agents which form free radicals directly or indirectly (e.g.,
- proliferators ciprofibrate, clofibrate. Persons exposed to such chemical carcinogens are pretreated with an antioxidant salen-metal complex to reduce the incidence or risk of
- Antioxidant salen-metal complexes can also be formulated into a lipophilic base (or, if desired, an aqueous carrier) for topical application in cosmetics or sunburn- prevention creams and lotions.
- a typical cosmetic or sunburn- prevention cream or lotion will comprise about between 1 ⁇ g to 50 mg of antioxidant salen-metal complex per gram of cosmetic or sunburn-prevention cream or lotion.
- Antioxidant salen-metal complexes may also be administered to deep-divers or individuals exposed to
- an efficacious dose of an antioxidant salen-metal complex to an individual may permit the breathing or hyberbaric and/or oxygen-enriched gases with a reduced risk of oxygen toxicity. It is also believed that administration of an efficacious dosage of an antioxidant salen-metal complex can reduced toxicity and biological damage associated with exposure to ozone. Prophylactic administration of an
- antioxidant salen-metal complex to humans who are or will be exposed to ozone is expected to confer an enhanced resistance to ozone toxicity, such as the ozone-induced lung damage noted in geographical areas with high ozone levels (e.g., Los
- antioxidant salen-metal complexes of the invention can be formulated into a cosmetic base for topical application and/or for reducing oxidation of the cosmetic by molecular oxygen and oxyradicals.
- antioxidant salen-metal agents of the invention can be formulated with an anti-inflammatory agent in a cosmetic base or dental linament (periodontal disease) for topical application for local prevention of inflammation and/or tissue damage consequent to inflammation.
- a cosmetic base or dental linament peripheral disease
- a variety of steroidal and non-steroidal anti-inflammatory agents can be combined with an antioxidant salen-metal compound.
- Steroidal anti-inflammatory agents including but not limited to, corticosteroids such as hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone,
- fluadrenolone fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene
- fluprednylidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate,
- methylprednisolone triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, flupreclnisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof may be used.
- non-steroidal anti-inflammatory agents useful in the composition of the present invention include, but are not limited to: piroxicam, isoxicam, tenoxicam, sudoxicam, CP-14,304, aspirin, disalcid, benorylate,
- diclofenac diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, a ⁇ emetacin, fentiazac, zomepirac, clidanac, oxepinac, felbinac, mefenamic, meclofenamic, flufenamic, niflumic, tolfenamic acids,
- ibuprofen ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic, phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone, among others.
- non-steroidal anti-inflammatory agents may also be employed, as well as the pharmaceutically-acceptable salts and esters of these agents.
- etofenamate a flufenamic acid derivative
- nonsteroidal anti-inflammatory agents ibuprofen, naproxen, flufenamic acid, mefenamic acid,
- meclofenamic acid piroxicam and felbinac are preferred and ibuprofen, naproxen, and flufenamic acid are most preferred.
- compositions of the present invention formulated as solutions typically include a pharmaceutically- or cosmetically-acceptable organic solvent.
- pharmaceutically-acceptable organic solvent and “cosmetically-acceptable organic solvent” refer to an organic solvent which, in addition to being capable of having
- the salen-metal compound and optionally also an anti-inflammatory agent, also possesses acceptable safety (e.g. irritation and sensitization
- a solvent is isopropanol.
- suitable organic solvents include: propylene glycol, polyethylene glycol (200-600), polypropylene glycol (425-2025), glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol,
- solutions contain from about 0.0001% to about 20%, preferably from about 0.01% to about 1%, antioxidant salen-metal complex, from about 0.01% to about 5%, preferably from about 0.5% to about 2% of an anti-inflammatory agent, and from about 80% to about 99%, preferably from about 90% to about 98%, of an acceptable organic solvent.
- emollients refer to materials used for the prevention or relief of dryness, as well as for the protection of the skin.
- suitable emollients are known and may be used herein. Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972), incorporated herein by reference, contains numerous examples of suitable materials. Examples of classes of useful emollients.
- emollients include the following: 1. Hydrocarbon oils and waxes. Examples include mineral oil, petrolatum, paraffin, ceresin, ozokerite,
- microcrystalline wax polyethylene, and perhydrosqualene.
- Silicone oils such as dimethyl polysiloxanes
- methylphenyl polysiloxanes water-soluble and alcohol-soluble silicone glycol copolymers.
- Triglyceride esters for example vegetable and animal fats and oils. Examples include castor oil, safflower oil, cottonseed oil, corn oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, and soybean oil.
- Ethoxylated glycerides such as ethoxylated glyceryl monostearate.
- Alkenyl esters of fatty acids having 10 to 20 carbon atoms examples include oleyl myristate, oleyl stearate, and oleyl oleate.
- Fatty acids having 10 to 20 carbon atoms include pelargonic, lauric, myristic, palmitic, stearic, isostearic, hydroxystearic, oleic, linoleic,
- ricinoleic arachidic, behenic, and erucic acids.
- Fatty alcohols having 10 to 20 carbon atoms Lauryl, myristyl, cetyl, hexadecyl, stearyl, isostearyl,
- erucyl alcohols as well as 2-octyl dodecanol, are examples of satisfactory fatty alcohols.
- Ethoxylated fatty alcohols of 10 to 20 carbon atoms include the lauryl, cetyl, stearyl, isostearyl, oelyl, and cholesterol alcohols having attached thereto from 1 to 50 ethylene oxide groups or 1 to 50
- Ether-esters such as fatty acid esters of ethoxylated fatty alcohols.
- Lanolin and derivatives Lanolin, lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids, isopropyl
- lanolate ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxylated cholesterol, propoxylated lanolin alcohols, acetylated lanolin, acetylated lanolin alcohols, lanolin alcohols linoleate, lanolin alcohols ricinoleate, acetate of lanolin alcohols ricinoleate, acetate of ethoxylated
- alcohols-esters hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and semisolid lanolin absorption bases are illustrative of emollients derived from lanolin.
- Ethylene glycol mono- and di-fatty acid esters diethylene glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglycerol
- poly-fatty acid esters ethoxylated glyceryl monostearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol
- polyoxyethylene polyol fatty acid ester polyoxyethylene polyol fatty acid ester
- sorbitan fatty acid esters polyoxyethylene sorbitan fatty acid esters
- Wax esters such as beeswax, spermaceti, myristyl myristate, stearyl stearate.
- Beeswax derivatives e.g. polyoxyethylene sorbitol beeswax. These are reaction products of beeswax with
- ethoxylated sorbitol of varying ethylene oxide content, forming a mixture of ether-esters.
- Vegetable waxes including carnauba and candelilla waxes.
- Phospholipids such as lecithin and derivatives.
- Sterols Cholesterol and cholesterol fatty acid esters are examples thereof.
- Amides such as fatty acid amides, ethoxylated fatty acid amides, solid fatty acid alkanolamides.
- Preferred skin conditioning agents are the propoxylated glycerol derivatives.
- antioxidant salen- transition metal complexes preferably salen-Mn complexes
- myocardial infections preferably myocardial infections
- the compounds of the invention inhibit the deleterious effects of ischaemia
- the compounds are effective in animal models for cardiovascular and CNS diseases, and will be useful for the treatment of myocardial infarction, stroke, brain injury, and transplant surgery, particularly with reperfusion of infarcted areas, arrhythmias, variant and exercise-induced angina, congestive heart failure, stroke and other circulatory disorders, in mammals, particularly in human beings.
- the salen-Mn complexes are also included in preservation solutions used to bathe excised organs (e.g., heart, kidney, pancreas, liver, lung) during transport and storage of the excised organ prior to transplantion surgery, including skin grafting and corneal grafting.
- the preservation solutions will typically comprise at least about 0.1 ⁇ M of an antioxidant salen-metal complex, preferably at least about 10 ⁇ M of an antioxidant salen-metal complex.
- Administration of the active compound and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include oral,
- composition parenteral, transdermal, subcutaneous and other systemic modes.
- the preferred method of administration is oral, except in those cases where the subject is unable to ingest, by himself, any medication. In those instances it may be necessary to administer the composition parenterally . If the composition comprises an antioxidant salen-metal species having an amino substituent that can be protonated at
- the antioxidant salen-metal complex is dissolved or suspended in a solution having a pH at which the amino substituent is protonated.
- the amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing
- an effective dosage is in the range of 0.001-50 mg/kg/day, preferably 0.01-25 mg/kg/day. For an average 70 kg human, this would amount to 0.07-3500 mg per day, or preferably about 0.7-1750 mg/day.
- the SOD activity of the compounds was determined by evaluating the inhibition of the reduction of cytochrome C produced by the oxygen free radical generating system,
- Cytochrome C reduction is monitored spectrophotometrically at 550 nm according to the method described in Darr et al. (1987) Arch. Biochem. Biophys. 258: 351, incorporated herein by reference.
- concentration of xanthine oxidase is adjusted such that it produces a rate of reduction of cytochrome C at 550 nm of 0.025 absorbance unit per minute. Under these conditions, the amount of SOD activity required to inhibit the rate of cytochrome C
- Salen- metal complexes are identified as antioxidants if they have at least 0.1 unit of activity at a concentration of 1 mM under these standard assay conditions.
- Catalase activity was measured using a
- One unit of catalase activity is defined as the amount of enzyme (or salen-metal complex) required to decompose 1 ⁇ mole of hydrogen peroxide in one minute.
- each of the compounds was formulated in saline and was stable with no loss of activity observed after several weeks of storage at room temperature. Frequently, it is desirable to first dissolve the salen-metal complex in an organic solvent (e.g., DMSO) and then dilute the solution into a more polar solvent such as water. This is particularly preferred for salen-metal species that are relatively
- hydrophobic e.g., C12
- Fig. 11 shows a generic structure of salen-metal complexes of the invention which can have antioxidant
- M is selected from the group consisting of Mn, Co, Cu, Fe, V, Cr, and Ni;
- A is an axial ligand selected from the group Cl, F, O, Br, or acetyl
- X 1, X 2 , X 3 and X 4 are independently selected from the group consisting of hydrogen, lower alkoxys, halides, and aryloxys;
- Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are independently selected from the group consisting of hydrogen, lower alkoxys, aryloxys, and halide;
- R is selected from the group consisting of: 1,2-ethane diyl; 1,2-benzenediyl; 2,3-pyridine diyl; (2-hydroxy)-2,3- propane diyl; 1,2-ethene diyl; 1,2-epoxy ethane diyl; alkylene diyl; and cyclohexane diyl.
- a preferred subgenus of salen metal complex are those where R is 1,2-benzene diyl, which is a hydrophobic moiety.
- Fig. 11 shows a generic salen-metal complex structure in (A), and shows the preferred R substituents of the generic formula in (B).
- Table IV shows the in vitro SOD and catalase activities of the various salen-Mn complexes tested. SOD and catalase activities are expressed as units/mM.
- a widely used assay to determine the therapeutic potential of molecules in brain ischemia consists of evaluating their ability to prevent irreversible damage induced by an anoxic episode in brain slices maintained under physiological conditions.
- Rat brain slices were maintained at 35°C in an interface chamber in an artificial cerebrospinal fluid containing: 124 mM NaCl, 3 mM Cl , 1.25 mM KH 2 PO 4 , 3 mM CaCl, 1 mM MgCl 2 , 26 mM NaHCO 3 , 10 mM D-glucose, and 2 mM L- ascorbate, continuously gassed with a mixture of O 2 :CO 2 (95:5).
- the atmosphere of the chamber was also continuously gased with the mixture of O 2 :CO 2 (95:5), except during the anoxic episode when it was replaced by N 2 .
- Axons were electrically
- EBPs excitatory post-synaptic potentials
- Fig. 4 shows the schematic of an EPSP recorded under normal conditions (A), five minutes following replacement of O 2 with N 2 (ischemic episode, B), and 30 to 40 minutes
- the extent of permanent damage can be quantified by measuring both the amplitude (in mV) and the initial slope (in mV/msec) of the EPSP.
- Figs. 5 and 6 show the protective effect of the antioxidant salen-Mn complex designated C7 in the rat brain slice ischemia EPSP system.
- Brain slices were incubated in the absence or presence of 50 ⁇ M C7 and subjected to an episode of ischemia/reoxygenation. After 5 minutes of baseline recording, O 2 was replaced by N 2 for an average of 5 minutes. O 2 was then reintroduced and recording was continued for another 50 minutes. Samples with 50 ⁇ M C7 showed that both the amplitude and slopes of the EPSPs recovered to pre- ischemia levels. In contrast, recovery in untreated brain slices was only about 40% of pre-ischemia levels.
- Fig. 7 demonstrates that, while without any treatment this percentage is very low (6%) , it was as high as 70% in slices treated with 50 ⁇ M C7. A slice was considered viable if an EPSP of 3 mV amplitude could be elicited by increasing stimulation intensity.
- Tritiated mazindol was used for binding studies on samples of the globus pallidus, caudate nucleus, and striatum of mouse brain according to conventional methods; specific binding of tritiated mazindol was determined
- the perfusion fluid was a modified Krebs-Henseleit buffer containing (in mmol/1) : NaCl 118, KCl 5.9, NaHC0 3 25, MgCl 2 1.2, NaH 2 PO 4 0.6, CaCl 2 2.4, Glucose 11. pH was maintained at 7.4 ⁇ 0.05 when the perfusion medium was saturated with O 2 -CO 2 (95%-5%) at 37°C.
- the perfusion apparatus was fully
- LVDP Left Ventricular Developed Pressure
- DMTU dimethylthiourea
- Table V shows heart rates (HR), systolic pressures (SP), diastolic pressures (DP), and the products HR x LVDP, in the three experimental groups, after 15 minutes of perfusion, before ischemia (Before), 1 minute after reperfusion (1 After) and 15 minutes after reperfusion
- VF ventricular fibrillation 1 minute after reperfusion
- Table VI summarizes the results from the electron microscopy evaluation of the hearts. Mitochondria were classified into Type A (normal), Type B (swollen, unbroken), and Type C (ruptured membranes). Sarcomeres were classified into Type A (normal) and Type B (contacted and/or necrosis). The results are expressed as percentages. The numbers of mitochondria analyzed were 1293, 1632 and 1595 for controls, DMTU and C7 groups, respectively. The numbers of sarcomeres analyzed were 1046, 1173, and 1143 for controls, DMTU and C7 groups, respectively.
- EAE is an animal model of multiple sclerosis.
- mice 20 mice (control) and 10 mice (C7 treated) .
- mice in both groups were immunized with an encephalitogenic PLP peptide in complete Freund's adjuvant subcutaneously, followed by Petrussis Toxin (IV) .
- Petrussis toxin was repeated on day 3 post immunization.
- mice in the C7 group were treated daily (1 mg/mouse, approximately 40 mg/kg) by IP injection, starting from 2 days prior to immunization through day 14 after immunization.
- mice in the control group developed symptomatic EAE: 2 Stage I, 4 Stage II/III, 2 Stage IV.
- mice in the C7 treated group developed symptomatic EAE (Stage II) .
- mice in the C7 group developed symptomatic EAE, 4 Stage II and 2 Stage IV.
- ROM's Reactive oxygen metabolites
- ALI acute lung injury
- lipopolysaccharide LPS;endotoxin
- Hippocampal slices 400 ⁇ m thick were obtained from Sprague-Dawley rats (150-200g) and collected in preoxygenated (95% O 2 / 5% CO 2 ) Krebs-Ringer phosphate medium (pH 7.4) containing NaCl 120 mM, KCl 5 mM, CaCl 2 1.3 mM, MgCl 2 1.2 mM, NaPhosphate 16 mM (pH 7.4) and glucose 10 mM.
- the buffer was replaced with the same buffer (control) or a modified buffer (lactate buffer) containing NaCl 90 mM, KCl 5 mM, CaCl 2 1.3 mM, MgCl 2 1.2 mM, NaPhosphate 16 mM and lactic acid 30 mM (pH 5.0).
- C7 50 ⁇ M was added during the preincubation and the incubation periods.
- slices were collected and homogenized in 0.9 ml of TCA 5%, whereas 0.35 ml of TCA 5% was added to 0.5 ml of the incubation medium.
- the Fig. 9 shows lipid peroxidation at time 0
- 6-OHDA 6-OHDA in mice.
- Adult male CFW mice were anesthetized with ketamine and rumpun, and immobilized in a stereotaxic device.
- 6-OHDA as the hydrobromide salt, was dissolved in normal saline with 1% ascorbate, and 50 ⁇ g was administered in lateral ventricle by means of a 10 ⁇ l Hamilton syringe.
- C7 (66 mg/kg, i.p.) was administered daily for 4 days. Animals were sacrificed 7 days later, and neuronal pathology was assessed by measuring 3 H-mazindol binding in striatal homogenates.
- Fig. 10 shows I.e.v. injection of 6-OHDA (50 ⁇ g) resulted in a 60-70% decrease in mazindol binding in
- Synthetic Catalytic Scavenger (SCS), C7 in various models of tissue damage.
- C7 was able to protect neurons from acute early manifestations of neuronal damage, such as lipid peroxidation and loss of synaptic viability, as well as long- term manifestations of neuronal injury, such as neuronal loss 7 days after toxin injection.
- Synthetic catalytic scavengers of reactive oxygen species may have clinical value in alleviating tissue damage associated with numerous acute and chronic diseases.
- ROS reactive oxygen species
- SOD superoxide dismutase
- C7 has been found to be protective in several models for ROS-associated tissue injury.
- the catalytic properties of C7 are further characterized demonstrating that it also utilizes hydrogen peroxide as a substrate, exhibiting both catalase and peroxidase activities.
- the synthesis of a new series of salen manganese complexes that are analogs of C7 are described and their multiple catalytic activities summarized. All of these compounds showed SOD activities comparable or identical to that of C7.
- Many of the compounds, like C7 also function as catalases and peroxidases. In contrast to their similar SOD activities, the salen manganese complexes
- SOD/catalase/peroxidase mimics with clinical utility and applicability, as well as finding use in other applications (e.g., as antioxidative reagents, stabilizers, and the like).
- C7 In cultured hippocampal slices, C7 protects against functional synaptic damage induced by anoxia-reperfusion and blocks acidosis-induced lipid peroxidation.
- C7 has also been found to reduce the degeneration of dopaminergic neurons in vivo, in two mouse models for Parkinson's disease (Example 1, supra) and to protect neurons against amyloid peptide toxicity in vitro. In addition, C7 is protective in an in vitro model for
- Certain of these salen manganese complexes in addition to having SOD activity, also function as catalases, converting hydrogen peroxide to oxygen. Furthermore, the compounds exhibit peroxidase activity in the presence of an oxidizable substrate. This is consistent with their ability to mimic the proteinaceous catalases.
- the XTT reagent was obtained from
- SOD Superoxide dismutase
- NBT reduction was also monitored at 550 nm.
- An estimated extinction coefficient for NBT reduction, 20,800 M -1 cm -1 determined empirically by exhaustive reduction of NBT in the reaction mixture described above, was employed where indicated. This value agreed well with reported extinction coefficients for reduced tetrazolium dyes.
- Control reactions to ensure that the compounds did not directly inhibit xanthine exidase were performed by monitoring urate production at 290 nm in reaction mixtures lacking cytochrome C of NBT. Conversion of xanthine to urate was calculated using e290's of 12,200 M 1 cm -1 for urate and 4050 M -1 cm -1 for xanthine.
- Catalase activity was assayed by monitoring the conversion of H 2 O 2 to oxygen using a Clark-type polarographic oxygen electrode.
- the apparatus consisted of a Mini Clark Style electrode, a 600 ⁇ l Oxygen Uptake Chamber, and a
- Catalase reaction mixtures consisted of 50 mM sodium
- initial rates were calculated by determining the slope of the linear portion of time dependent plots of oxygen generation, usually comprising the first five seconds of the reaction. Unless otherwise indicated, these were corrected by subtracting the rate obtained with H 2 O 2 alone.
- endpoint oxygen generated was calculated from time-dependent plots, such as those shown in Fig. 14, as the difference between the baseline oxygen concentration immediately prior to substrate addition and the maximal oxygen concentration achieved during the course of the reaction. All reactions subjected to these calculations were run for a sufficient time to ensure that oxygen generation had ceased.
- Peroxidase activity was assayed by monitoring the hydrogen peroxide-dependent oxidation of 2,2'-azino-bis(3- ethylbenzthiazoline-6) sulfonic acid (ABTS)
- Standard assay mixtures consisted of 50 mM sodium phosphate, pH 8.1, 0.9% sodium chloride, 0.5 mM ABTS, and H 2 O 2 and salen manganese complex as indicated. Where indicated, 50 mM sodium phosphate buffers of pH 6.0 or pH 7.1 were substituted. Assays were conducted at 27 ⁇ 0.2°C. ABTS oxidation was monitored at 740 or 500 nm to eliminate
- the amount of oxidized ABTS was estimated using an ⁇ 740 of 20,300 M -1 cm -1 or an ⁇ 500 of 3400 M -1 cm -1 calculated based upon the published molar extinction
- rat brains minus the pons and cerebellum, were each homogenized in 7 volumes of an artificial cerebral spinal fluid (ASCF) containing 124 mM NaCl, 3 mM KCl, 1.25 mM KH 2 PO 4 , 3 mM CaCl 2 , 1 mM MgCl 2 , 26 mM NaHC0 3 , 10 mM D-glucose, and 2 mM L-ascorbate, equilibrated with O 2 :CO 2 (95:5). Lipid peroxidation was induced by ASCF
- ASCF artificial cerebral spinal fluid
- HF cells Human dermal fibroblasts
- a medium consisting of Dulbecco's Modified Eagle's Medium with 4.5 g/1 D-glucose, 10% calf serum 4 mM glutamine, 50 units/ml penicillin, and 50 ⁇ g/ml streptomycin in a 37°C humidified incubator
- HF cells were seeded at a density of about 15,000 cells per cm2 onto 96-well culture plates and allowed to grow to confluence.
- t-BHP tert-butylhydroperoxide
- test compounds concentrations of test compounds dissolved in HF medium for 18 hr. The medium was then replaced with fresh medium
- t-BHP tert-butylhydroperoxide
- Fig. 12 shows the structures of salen manganese complexes evaluated in this Example.
- the Schiff base ligands used to complex manganese (III) are derivatives of the
- chloride axial ligand and the other having an acetate axial ligand were synthesized. All compounds have a mirror plane or symmetry. In general, those compounds with an axial acetate ligand were found to be more water soluble than the corresponding chlorides. In addition, the acetate axial ligand can be rapidly converted to the chloride in the
- the other complexes contain salen ligands with substituents, either methoxy or fluorine, on the aromatic rings as shown in Table I.
- the chloride and acetate pairs are, respectively, C7 and C31, C37 and C36, C41 and C38, C40, C32, C39, and C35, and C34, and C33.
- the two members of each pair showed similar, if not identical, activity in the various assay systems, as discussed further below.
- Salen-metal complexes having antioxidant activity in aqueous environments are suitable for use a pharmaceutical agents.
- An antioxidant composition comprising a salen metal complex of Fig. 11, Fig. 12, Figs. 26A-E, or Figs. 24A-24I can be formulated, typically with an excipient, vehicle, or inert compound, into a tablet, capsule, ampule, suppository, inhaler, hypodermic syringe, or other pharmaceutical form.
- the salen-metal complexes can be co-formulated with other pharmaceutical agents.
- One variation is the co- formulation of an antioxidant salen-metal complex with a pharmaceutical which is susceptible to undesired oxidation or free radical degradation; for example and no limitation, L- dopa (Levadopa) can be co-formulated with an antioxidant salen-metal complex to stabilize L-dopa, and can provide additional therapeutic or prophylactic pharmacological benefit to the patient.
- Other pharmaceutical agents susceptible to oxygen radical-mediated degradation can be co-formulated with an antioxidant salen-metal complex (e.g., C7, C31, C32, C40, C81).
- Table I and Table VII show catalytic activities of various salen-metal complexes.
- Example 1 demonstrates that certain salen manganese compounds have superoxide dismutase (SOD) activity, based upon their ability to inhibit the reduction of nitroblue
- tetrazolium NBT
- C7 inhibited the rate of NBT reduction in a concentration-dependent manner, with no effect on xanthine oxidase activity (Fig. 13B).
- the stoichiometries observed in these experiments support a catalytic mechanism for C7, since large molar excesses of superoxide were apparently scavenged by the salen manganese complex.
- Fig. 13A tetrazolium
- Catalase activity was detected by monitoring the generation of oxygen, as described in Example 2, in the presence of H 2 O 2 .
- the addition of H 2 O 2 to a solution of C7 resulted in a phase of rapid oxygen
- H 2 O 2 -dependent C7 degradation was investigated further using HPLC as described below. As Fig. 14 also indicates, both the initial rate of oxygen generation and the total amount of oxygen produced increased with the concentration of H 2 O 2 .
- C7 completed more catalytic cycles before ceasing to react.
- the catalase activity of C7 did not appear saturable within the range of H 2 O 2 concentrations examined.
- kinetic analyses of mammalian catalases indicate that the enzymes lack a K m for H 2 O 2 and therefore exhibit increased activity as the intracellular H 2 O 2 concentration increases.
- C7 also exhibited peroxidase activity, which is consistent with its function as a catalase.
- the catalase reaction involves conversion of two moles H 2 O 2 to one mole oxygen and two moles of water.
- C7 catalyzed a peroxidative reaction between H 2 O 2 and the oxidizable substrate ABTS.
- the peroxidase activity of C7 was dependent on H 2 O 2 concentration, with no apparent saturation reached at any concentration tested.
- catalase activity of C7 showed a similar pH dependence and both activities were even faster at pH 8.9. Under these assay conditions, bovine liver catalase (19 units/ml) showed no peroxidase activity toward ABTS at pH 6.0, 7.1, 8.1. In comparison, in our catalase assays, the same concentration of bovine liver catalase produced oxygen at the rate of
- manganese compounds were assayed in a similar system as that described above, except that cytochrome C was used as
- cytochrome C a better choice for quantitative comparisons among analogs.
- the half-maximally active concentration was determined as described in Experimental Procedures. As summarized in Table VII, most of the compounds exhibited similar SOD activities, with IC 50 s ranging from 0.9 to 1.7 ⁇ M. The only markedly different SOD activities were exhibited by C7 and C39, the analogs with two methoxy groups on each salen ring, which had IC 50 s of 3.2 and 3.7 ⁇ M,
- Table VII also summarizes the catalase activities of the various salen manganese complexes when assayed under equivalent reaction conditions, that is, 10 ⁇ M salen manganese complex and 10 mM H 2 O 2 . All analogs displayed a time course analogous to that exhibited by C7, with the reaction ceasing prior to consumption of all substrate. Table VII presents initial rates as well as the maximal amount of oxygen
- ROS promote tissue destruction in part through oxidative damage to cellular macromolecules, in particular, by inducing lipid peroxidation.
- manganese compounds were tested for the ability to protect brain tissue from lipid peroxidation induced by incubating brain homogenates with iron in an oxygen-rich atmosphere.
- Lipid peroxidation was induced in brain homogenates and assessed based upon malonyldialdehyde (MDA) content as described in Example 2,.
- MDA malonyldialdehyde
- the effects of salen manganese complexes, included in the incubation mixtures at the indicated concentrations, are expressed as percent of MDA levels in control (i.e. without salen manganese complex) incubations. Each value represents the mean of 2 to 4 experiments.
- Salen manganese compounds were also evaluated for the ability to protect human fibroblasts against
- t-BHP tert-butylhydroperoxide
- T-BHP is believed to cause oxidative damage to cells due to its intracellular decomposition to alkoxyl and methoxyl free radicals. It has been reported that SOD, particularly when encapsulated into liposomes, protects hepatocytes from t-BHP toxicity, implying that intracellular superoxide may play a role in the cytotoxicity of this organic hydroperoxide. The ability of several salen manganese
- Cell protection assays were performed as described in Example 2 with salen- manganese complexes administered at the indicated concentrations and t-BHP at 0.5 mM. Cell viability was assessed using the XTT reagent as described and is expressed as the absorbance at 490 nm uncorrected for blank. The value represent mean +/- s.d. of triplicate samples.
- t-BHP was fully toxic against the human fibroblasts. (Based on a lack of spectrophotometric change, t-BHP, unlike H 2 O 2 , has no apparent ability to oxidatively destroy C7). All the salen manganese complexes exhibited full protection, although their minimally effective concentrations differed. For C7 and C31,
- IC 50 's ranging from 0.9 to 3.7 ⁇ M. This is in contrast to the more structurally diverse series examined in Example 1, whose IC 50 's ranged over two orders of magnitude, with C7 being among the most active.
- the present series of compounds compare favorably to a manganese-porphyrin complex, which has an IC 50 of -0.7 ⁇ M when assayed under similar
- C41 which had a catalase rate only slightly higher than C7 and comparable to that of the fluorinated analogs, produced over twice as much oxygen as either compound before ceasing to react. This may indicate that the presence of methoxy substituents at the R3 position confers more resistance to H 2 O 2 -dependent inactivation.
- the stability of the entire series of compounds to H 2 O 2 likely affects the apparent catalase and peroxidase activities exhibited in our assay systems.
- the present example demonstrates that salen manganese complexes can display SOD as well as
- catalase/peroxidase activities and that the ratios of these activities can be structurally manipulated. Furthermore, many of these compounds are protective biologically.
- FIG. 19A shows structural formulae of the salen-metal compounds C42-C52, which were synthesized and evaluated.
- Fig. 19B shows the catalase rate, catalase endpoint, peroxidase rate, and SOD activity of these compounds relative to C7.
- Fig. 23 shows structural features important in antioxidant activity of salen-metal species.
- Fig. 24A-24H show structural formulae of further salen-metal compounds which were synthesized and evaluated.
- Fig. 25 shows exemplified types of salicylaldehyde and diamine species for synthesis of active salen metal species.
- Figs. 26A-26E show generic structural formulae of active salen-metal complexes.
- Fig. 28 shows inhibition of lipid peroxidation by C7, C53, and Vitamin E.
- Fig. 29 shows protection by C40 and C7 in a rat model for myocardial infarct. Rats were subjected to
- Fig. 30 shows C40 delays rejection in a mouse skin transplantation model.
- donor and recipient mice were immunologically mismatched (Classl/Class II MHC
- a piece of skin ( ⁇ 1 cm 2 ) from the tail of a donor mouse was transplanted onto the back of a recipient mouse.
- the graft was bandaged and observed daily for
- Fig. 31 shows C40 protects against ischemia- reperfusion induced kidney damage in the rat.
- Fig. 32 shows C40 protects dopaminergic neurons in the mouse MPTP model for Parkinson's Disease. Neuronal damage was induced in mice by injection with MPTP as described in Example 1. Where indicated, mice were also treated with intraperitoneal injections of C40 at 0.02 or 0.2 mg/kg. The integrity of the nigrostriatal dopaminergic neurons was assessed based upon 3 H-Mazindol binding to striatal membranes harvested from the brains of these mice about 1 week after MPTP administration.
- Fig. 33 shows C40 is protective in a rat model for stroke.
- Rats were subjected to a Middle Cerebral Artery (MCA) Occlusion model involving permanent occlusion of the parietal branch of the left middle cerebral artery and temporary (60 min) occlusion of the common carotid arteries.
- MCA Middle Cerebral Artery
- the figure shows mean infarct volumes ( ⁇ sd) for each group.
- Total brain volumes ( ⁇ 1200 cm 3 ) did not differ significantly between groups.
- Fig. 34 shows topically administered C7 is protective in a mouse model for delayed hypersensitivity.
- mice (“Presensitized” and "Presensitized + C7” groups) were presensitized with oxazolone on the abdomen.
- One group (“Not presensitized”) received only vehicle on the abdomen at this time.
- each mouse were challenged with the oxazolone hapten on one ear and given vehicle only on the opposite ear.
- mice also received a topical administration of C7 in 90% acetone (2.5 micrograms C7 per ear) on both ears immediately prior to hapten challenge.
- the other two groups received an equivalent volume of 90% acetone.
- Twenty-four hr after challenge mice were sacrificed and ear edema was assessed by determining the wet weight/dry weight ratio. (Wet weight was determined by weighing the freshly dissected ear and dry weight was determined after lyophilization to a constant weight.)
- Fig. 35 shows chronic treatment with C7 prolongs the life of an autoimmune strain of mice.
- MRL/lpr mice develop autoantibodies and numerous autoimmune associated pathologies and die prematurely (mean lifespan ⁇ 150 days). They are considered a mouse model for autoimmune disorders such as lupus. For this study, MRL/lpr mice were treated
- mice intraperitoneally three times per week with C7 (1 mg/mouse) from the age of about 8 weeks until their death. Control mice received vehicle injections only or were left untreated.
- Fig. 36 shows C7 protects neuronal tissue from beta- amyloid peptide-induced cytotoxicity.
- Rat hippocampal slice cultures were incubated with the beta-amyloid peptide (1-42) at the indicated concentrations.
- Cell viability was assessed by two criteria: release of lactate dehydrogenase (L*H) into the culture medium and staining with propidium iod*de (PI) which binds to exposed DNA. Where indicated, C7 (25 ⁇ M) was present in the medium throughout the experiment.
- O-Vanillin 2-hydroxy-4-methoxybenzaldehyde, 4,6- dimethoxysalicylaldehyde, 2,4-dihydroxybenzaldehyde, 2,5- dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde,
- the ligands were prepared by the addition of 1 equivalent of the diamine in absolute ethanol to a solution of 2 equivalents of the substituted aldehyde in absolute ethanol (0.05 to 0.2 M solution). After stirring at ambient (2 to 48 hrs), the precipitate was filtered, washed with ethanol, and air dried to give the desired product in 79 to 96% yield. Effect of bridge modifications on catalytic activity of salen-manganese compounds:
- Modification of the ethylenediamine bridge of salen- manganese compounds can affect the catalase activity (i.e., initial rate) as well as the number of turnovers completed (i.e., the catalase endpoint).
- the latter parameter is influenced by the stability of the compound in the presence of hydrogen peroxide.
- the presence of an aromatic ring at the bridge results in compounds that are faster catalases than C7 or C31 and that complete more turnovers. For example, compare C43 to C31 (Fig. 20, Fig. 21, Table X) or C45 to C32 (Table X).
- Such compounds are not necessarily faster as peroxidases (Table X, Fig. 21), indicating that peroxidase and catalase activities can be manipulated independently by such bridge modifications.
- Peroxidase assays contained 10 ⁇ M salen-manganese complex and 0.2 mM hydrogen peroxide. Glucose-glucose oxidase toxicity assays were performed using human dermal fibroblasts as described in Example 2.
- Antioxidant salen-metal complexes are formulated according to the following protocols:
- a moisturizing lotion is prepared by combining the following components utilizing conventional mixing techniques.
- This lotion may be topically applied to inhibit damage caused by acute or chronic UV exposure.
- Use of an amount of lotion sufficient to deposit about 0.1 to 100 ⁇ g/cm 2 of C7 to the skin immediately prior to UV exposure is
- a skin lotion is prepared by combining the following components utilizing conventional mixing techniqu ⁇ ees-.
- This lotion is useful for topical application to inhibit damage caused by acute or chronic UV exposure or exposure to an oxyradical environment. Use of an amount of lotion
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU63328/96A AU719450B2 (en) | 1995-06-07 | 1996-06-06 | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
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JP50227297A JP4160115B2 (en) | 1995-06-07 | 1996-06-06 | Synthetic catalytic free radical scavengers useful as antioxidants for disease prevention and treatment |
EP96922461A EP0831836A4 (en) | 1995-06-07 | 1996-06-06 | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
US11/362,454 US7582786B2 (en) | 1992-12-07 | 2006-02-23 | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
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US08/485,489 US5696109A (en) | 1992-12-07 | 1995-06-07 | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
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PCT/US1996/010267 WO1996040149A1 (en) | 1992-12-07 | 1996-06-06 | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
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JP (1) | JP4160115B2 (en) |
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CA (1) | CA2223510C (en) |
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US5696109A (en) * | 1992-12-07 | 1997-12-09 | Eukarion, Inc. | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
-
1995
- 1995-06-07 US US08/485,489 patent/US5696109A/en not_active Expired - Lifetime
-
1996
- 1996-06-06 AU AU63328/96A patent/AU719450B2/en not_active Ceased
- 1996-06-06 US US08/973,577 patent/US6046188A/en not_active Expired - Fee Related
- 1996-06-06 AU AU62725/96A patent/AU6272596A/en not_active Abandoned
- 1996-06-06 CA CA002223510A patent/CA2223510C/en not_active Expired - Fee Related
- 1996-06-06 EP EP96922461A patent/EP0831836A4/en not_active Withdrawn
- 1996-06-06 WO PCT/US1996/010037 patent/WO1996040148A1/en active Application Filing
- 1996-06-06 WO PCT/US1996/010267 patent/WO1996040149A1/en active Application Filing
- 1996-06-06 JP JP50227297A patent/JP4160115B2/en not_active Expired - Fee Related
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2000
- 2000-04-04 US US09/542,182 patent/US6573257B2/en not_active Expired - Fee Related
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2003
- 2003-05-30 US US10/452,695 patent/US6900198B2/en not_active Expired - Fee Related
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2005
- 2005-05-27 US US11/139,141 patent/US20050249796A1/en not_active Abandoned
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US5403834A (en) * | 1992-12-07 | 1995-04-04 | Eukarion, Inc. | Synthetic catalytic free radical scavengers useful as antioxidants for prevention and therapy of disease |
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US6589948B1 (en) | 2000-11-28 | 2003-07-08 | Eukarion, Inc. | Cyclic salen-metal compounds: reactive oxygen species scavengers useful as antioxidants in the treatment and prevention of diseases |
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US9573108B2 (en) | 2011-10-28 | 2017-02-21 | The Chemours Company Tt, Llc | Treated inorganic core particles having improved dispersability |
US20140338852A1 (en) * | 2011-10-28 | 2014-11-20 | E I Du Pont De Nemours And Company | Treated inorganic pigments having improved dispersability and use thereof in paper products |
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Also Published As
Publication number | Publication date |
---|---|
JPH11507646A (en) | 1999-07-06 |
US20020002157A1 (en) | 2002-01-03 |
US5696109A (en) | 1997-12-09 |
CA2223510A1 (en) | 1996-12-19 |
CA2223510C (en) | 2008-05-20 |
EP0831836A1 (en) | 1998-04-01 |
US6046188A (en) | 2000-04-04 |
US6900198B2 (en) | 2005-05-31 |
US20030216371A1 (en) | 2003-11-20 |
AU719450B2 (en) | 2000-05-11 |
US20050249796A1 (en) | 2005-11-10 |
US6573257B2 (en) | 2003-06-03 |
US20090311230A1 (en) | 2009-12-17 |
EP0831836A4 (en) | 2000-09-27 |
AU6332896A (en) | 1996-12-30 |
AU6272596A (en) | 1996-12-30 |
JP4160115B2 (en) | 2008-10-01 |
WO1996040148A1 (en) | 1996-12-19 |
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