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WO2011044230A2 - N-acétylcystéine amide (nac amide) destiné au traitement de maladies et de conditions - Google Patents

N-acétylcystéine amide (nac amide) destiné au traitement de maladies et de conditions Download PDF

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WO2011044230A2
WO2011044230A2 PCT/US2010/051623 US2010051623W WO2011044230A2 WO 2011044230 A2 WO2011044230 A2 WO 2011044230A2 US 2010051623 W US2010051623 W US 2010051623W WO 2011044230 A2 WO2011044230 A2 WO 2011044230A2
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amide
nac amide
nac
derivative
cells
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WO2011044230A9 (fr
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Glenn A. Goldstein
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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • N-ACETYLCYSTEINE AMIDE (NAC AMIDE) FOR THE TREATMENT
  • the present invention generally relates to the treatment of mammalian, including human, diseases with antioxidants. More particularly, the invention relates to treatments and therapies of a variety of diseases and conditions involving the administration of N- acetylcysteine amide (NAC amide) or a derivative thereof, alone or in combination with another agent, to a mammal in need thereof.
  • NAC amide N- acetylcysteine amide
  • Oxidative stress plays an important role in the progression of neurodegenerative and age-related diseases, causing damage to proteins, DNA, and lipids.
  • Low molecular weight, hydrophobic antioxidant compounds are useful in preventing conditions of peripheral tissues, such as acute respiratory distress syndrome, amyotrophic lateral sclerosis, atherosclerotic cardiovascular disease, multiple organ dysfunctions and central nervous system
  • Parkinson's disease e.g., Parkinson's disease, Alzheimer's disease and Creutzfeldt- Jakob's disease.
  • Oxidative stress has been causally linked to the pathogenesis of Parkinson's disease, Alzheimer's disease and Creutzfeldt- Jakob's disease, as well as other types of disorders.
  • U.S. Patent No. 6,420,429 to D. Atlas et al U.S. Patent No. 6,420,429 to D. Atlas et al.
  • GSH glutathione
  • GSH is synthesized by most cells and is one of the primary cellular antioxidants responsible for maintaining the proper oxidation state within the body. When oxidized, GSH forms a dimer, GSSG, which may be recycled in organs producing glutathione reductase. In human adults, reduced GSH is produced from GSSG, primarily in the liver, and to a smaller extent, by skeletal muscle and red and white blood cells, and is distributed through the blood stream to other tissues in the body.
  • NAC amide Glutathione N-acetylcysteine amide
  • NAC amide the amide form of N- acetylcysteine (NAC)
  • BuOOH tert.-butylhydroxyperoxide
  • RBCs red blood cells
  • BuOOH-induced thiol depletion and hemoglobin oxidation in the RBCs This restoration of thiol-depleted RBCs by externally applied NAC amide was significantly greater than that found using NAC.
  • NAC amide protected hemoglobin from oxidation.
  • NAC amide was shown to react with oxidized glutathione (GSSG) to generate reduced glutathione (GSH).
  • GSH oxidized glutathione
  • NAC amide readily permeates cell membranes, replenishes intracellular GSH, and, by incorporating into the cell's redox machinery, protects the cell from oxidation. Because of its neutral carboxyl group, NAC amide possesses enhanced properties of lipophilicity and cell permeability. (See, e.g., U.S. Patent No. 5,874,468 to D. Atlas et al). NAC amide is also superior to NAC and GSH in crossing the cell membrane, as well as the blood-brain barrier.
  • NAC amide may function directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells from free-radical mediated damage.
  • NAC amide is a potent cellular antioxidant responsible for maintaining the proper oxidation state within the body. NAC amide can recycle oxidized biomolecules back to their active reduced forms and may be as effective, if not more effective, than GSH as an antioxidant.
  • Glutamate an excitatory amino acid, is one of the major neurotransmitters in the central nervous system (CNS). Elevated levels of extracellular glutamate have been shown to be responsible for acute neuronal damage as well as many CNS disorders, including hyperglycemia, ischemia, hypoxia (Choi, D.W., Neuron, l(8):623-34, 1988), and chronic disorders such as Huntington's, Alzheimer's, and Parkinson's diseases (Meldrum B. and Garthwaite J., Trends Pharmacol Sci., 11(9):379-87, 1990; and Coyle J.T. and Puttfarcken P., Science, 262(5134):689-95, 1993). Two mechanisms have been proposed for glutamate toxicity.
  • the first mechanism explains the excitotoxicity of glutamate as being mediated through three types of excitatory amino acid receptors (Monaghan D.T. et al., Annu Rev Pharmacol Toxicol, 29:365-402, 1989).
  • receptor-mediated glutamate excitotoxicity it has also been proposed that elevated levels of extracellular glutamate inhibits cystine uptake, which leads to a marked decrease in cellular GSH levels, resulting in the induction of oxidative stress (Murphy T.H. et al., Neuron, 2(6): 1547-58, 1989).
  • Cysteine is a critical component for intracellular GSH synthesis. Because of redox instability, almost all of the extracellular cysteine is present primarily in its oxidized state, cystine, which is taken up by cells via a cystine/glutamate transporter, the X c— system. Studies indicate that glutamate and cystine share the same transporter; therefore, elevated levels of extracellular glutamate competitively inhibit cystine transport, which leads to depletion of intracellular GSH. (Bannai S. and itamura E., J Biol Chem. 255(6):2372-6, 1980; and Bannai S., Biochem Biophys Acta., 779(3):289-306, 1984).
  • antioxidants such as NAC, lipoic acid (LA), (Han D. et al., Am J Physiol, 273: 1771-8, 1997), tocopherol (Pereira CM. and Oliveira C.R., Free Radic Biol Med.,
  • ARDS acute respiratory distress syndrome
  • ALS amyotrophic lateral sclerosis
  • Lou Gerhig's disease atherosclerotic cardiovascular disease and multiple organ dysfunction
  • antioxidants such as GSH.
  • Depressed antioxidant levels either locally in particular organs or systemically, have been associated with a number of clinically defined diseases and disease states, including HIV/ AIDS, diabetes and macular degeneration, all of which progress because of excessive free radical reactions and insufficient antioxidants.
  • Other chronic conditions may also be associated with antioxidant deficiency, oxidative stress, and free radical formation, including heart failure and associated conditions and pathologies, coronary arterial restenosis following angioplasty, diabetes mellitus and macular
  • HIV is known to start pathologic free radical reactions, which lead to the destruction of antioxidant molecules, as well as their exhaustion and the destruction of cellular organelles and macromolecules.
  • oxidative stresses e.g. low intracellular levels of reduced antioxidants and relatively high levels of free radicals, activate certain cytokines, including NF- ⁇ and TNF-oc, which, in turn, activate cellular transcription of the DNA to mRNA, resulting in translation of the mRNA to a polypeptide sequence.
  • cytokines including NF- ⁇ and TNF-oc
  • the viral genome is transcribed, resulting in viral RNA production, generally necessary for viral replication of RNA viruses and retroviruses.
  • the amplification effect of oxidative intracellular conditions on viral replication is compounded by the actions of various viruses and viral products, which degrade antioxidants, such as GSH.
  • GSH antioxidants
  • gpl20 an HIV surface glycoprotein having a large number of disulfide bonds
  • gpl20 oxidizes GSH, resulting in reduced intracellular GSH levels.
  • GSH reduces the disulfide bonds of gpl20, thus reducing or eliminating its biological activity that is necessary for viral infectivity.
  • Antioxidants such as GSH therefore interfere with the production of such oxidized proteins and degrade them once formed.
  • a cascade of events may occur which can allow the cell to pass from a relatively quiescent stage with low viral activity to an active stage with massive viral replication and cell death. This is accompanied by a change in redox potential. By maintaining adequate levels of antioxidant, this cascade may be impeded.
  • HIV is transmitted through two predominant routes, namely, contaminated blood and/or sexual intercourse.
  • pediatric cases approximately one half of the newborn individuals are infected in utero and one half are infected at delivery. This circumstance permits a study of prevention of transmission since the time of spread is known.
  • there is an intense viral infection simulating a severe case of the flu, with massive replication of the virus.
  • this acute phase passes spontaneously as the body mounts a largely successful immune defense.
  • the individual has no outward manifestations of the infection.
  • the virus continues to replicate within immune system cells and tissues, e.g., lymph nodes, lymphoid nodules, macrophages and certain multidendritic cells that are found in various body cavities.
  • cytokines are normally occurring biochemical substances that signal numerous reactions and that typically exist in minuscule
  • AIDS Acquired Immune Deficiency Syndrome
  • HIV has a powerful ability to mutate. It is this capability that makes it difficult to create a vaccine or to develop long-term, antiviral pharmaceutical treatments. As more people fail to be successfully treated by the present complex regimens, the number of resistant viral strains is increasing. Resistant strains of HIV are a particularly dangerous population of the virus and pose a considerable health threat. These resistant HIV mutants also add to the difficulties in developing vaccines that will be able to inhibit the activity of highly virulent viral types. Further, the continuing production of free radicals and cytokines that may become largely independent of the virus perpetuate the dysfunctions of the immune system, the gastrointestinal tract, the nervous system, and many other organs in patients with AIDS.
  • New anti-HIV/AIDS therapies include additional drugs in the classes of Reverse Transcriptase inhibitors and protease inhibitors. Also, drugs are in development to block the integrase enzyme of the virus, which integrates the HIV DNA into the infected cell's DNA, analogous to splicing a small length of wire into a longer wire. Vaccine development also continues, although prospects seem poor because HIV appears to be a moving target and seems to change rapidly. Vaccine development is also impaired by the immune cell affinity of the virus.
  • Diabetes mellitus (“diabetes”) is found in two forms: childhood or autoimmune (Type I, IDDM) and late-onset or non-insulin dependent (Type II, NIDDM).
  • Type I constitutes about 30% of the cases of diabetes. The rest of the cases are represented by Type II.
  • Symptoms include excessive urination, hunger and thirst, with a slow and steady loss of weight associated with Type I.
  • Obesity is often associated with Type II and has been thought to be a causal factor in susceptible individuals. Blood sugar is often high and there is frequent spilling of sugar in the urine. If the condition goes untreated, the victim may develop ketoacidosis with a foul-smelling breath similar to some who has been drinking alcohol.
  • the immediate medical complications of untreated diabetes can include nervous system symptoms, and even diabetic coma.
  • glycation a non-enzymatic chemical reaction, called glycation, frequently occurs inside cells and causes a chronic inactivation of essential enzymes.
  • GSH is in high demand throughout the body for multiple, essential functions, for example, within all mitochondria, to produce chemical energy called ATP.
  • ATP chemical energy
  • brain cells, heart cells, nerve cells, blood cells and many other cell types are not able to function properly and can be destroyed through apoptosis associated with oxidative stress and free radical formation.
  • GSH is the major antioxidant in the human body and the only one that can be synthesized de novo. It is also the most common small molecular weight thiol in both plants and animals. Without GSH the immune system cannot function, and the central and peripheral nervous systems become aberrant and then cease to function.
  • GSH as the carrier of nitric oxide
  • a vasodilator responsible for control of vascular tone the cardiovascular system does not function well and eventually fails. Since all epithelial cells seem to require GSH, without GSH, intestinal lining cells also do not function properly and valuable micronutrients are lost, nutrition is compromised, and microbes are given portals of entry to cause infections.
  • GSH precursors In diabetes, the use of GSH precursors cannot help to control GSH deficiency due to the destruction of the rate-limiting enzyme by glycation. As GSH deficiency becomes more profound, the well-known sequelae of diabetes progress in severity. The complications that develop in diabetics are essentially due to runaway free radical damage since the available GSH supplies in diabetics are insufficient. For example, a diabetic individual becomes more susceptible to infections because the immune system approaches collapse when GSH levels fall, analogous to the situation in HIV/AIDS. In addition, peripheral vasculature becomes comprised and blood supply to the extremities is severely diminished because GSH is not available in sufficient amounts to stabilize nitric oxide to effectively exert its vascular dilation (relaxation) property. Gangrene is a common sequel and successive amputations often result in later years. Peripheral neuropathies, the loss of sensation commonly of the feet and lower extremities develop and are often followed by aberrant sensations like
  • Retinopathy and nephropathy are later events that are actually due to icroangiopathy, i.e., excessive budding and growth of new blood vessels and capillaries, which often will bleed due to weakness of the new vessel walls. This bleeding causes damage to the retina and kidneys with resulting blindness and renal shutdown, which requires dialysis treatment. Further, cataracts occur with increasing frequency as the GSH deficiency deepens. Large and medium sized arteries become sites of accelerated severe atherosclerosis, with myocardial infarcts at early ages, and of a more severe degree. If coronary angioplasty is used to treat the severe atherosclerosis, diabetics are much more likely to have re-narrowing of cardiac vessels, termed restenosis.
  • Macular degeneration as a cause of blindness is a looming problem as the population ages.
  • Age-related macular degeneration is characterized by either a slow (dry form) or rapid (wet form) onset of destruction and irrevocable loss of rods and cones in the macula of the eye.
  • the macula is the approximate center of the retina wherein the lens of the eye focuses its most intense light.
  • the visual cells known as the rods and cones, are an outgrowth and active part of the central nervous system. They are responsible and essential for the fine visual discrimination required to see clear details such as faces and facial expression, reading, driving, operation of machinery and electrical equipment and general recognition of surroundings. Ultimately, the destruction of the rods and cones leads to functional, legal blindness.
  • Drusen Since there is no overt pain associated with the condition, the first warnings of onset are usually noticeable loss of visual acuity. This may already signal late stage events. It is now thought that one of the very first events in this pathologic process is the formation of a material called "drusen", which first appears as either patches or diffuse drops of yellow material deposited upon the surface of the retina in the macula lutea or yellow spot. This is the area of the retina where sunlight is focused by the lens and which contains the highest density of rods for acuity. Although cones, which detect color, are lost as well in this disease, it is believed to be loss of rods, which causes the blindness. Drusen has been chemically analyzed and found to be composed of a mixture of lipids that are peroxidized by free radical reactions.
  • RPE retinal pigmented epithelial
  • UVB near ultraviolet
  • GSH glycosyl-semiconductor
  • T-cell proliferation T-cell proliferation
  • T- and B-cell differentiation cytotoxic T-cell activity
  • natural killer cell activity Adequate GSH levels have been shown to be necessary for microtubule polymerization in neutrophils.
  • Intraperitoneally administered GSH augments the activation of cytotoxic T-lymphocytes in mice, and dietary GSH was found to improve the splenic status of GSH in aging mice, and to enhance T-cell mediated immune responses.
  • the presence of macrophages can cause a substantial increase of the intracellular GSH levels of activated lymphocytes in their vicinity.
  • the cysteine supply function of the macrophages is an important part of the mechanism which enables T-cells to shift from a GSH-poor to a GSH-rich state.
  • Glutathione status is a major determinant of protection against oxidative injury.
  • GSH acts on the one hand by reducing hydrogen peroxide and organic hydroperoxides in reactions catalyzed by glutathione peroxidases, and on the other hand by conjugating with electrophilic xenobiotic intermediates capable of inducing oxidant stress.
  • the epithelial cells of the renal tubule have a high concentration of GSH, no doubt because the kidneys function in toxin and waste elimination, and the epithelium of the renal tubule is exposed to a variety of toxic compounds.
  • GSH transported into cells from the extracellular medium, substantially protects isolated cells from intestine and lung against t-butylhydroperoxide, menadione or paraquat-induced toxicity.
  • Isolated kidney cells also transport GSH, which can supplement endogenous synthesis of GSH to protect against oxidant injury.
  • Hepatic GSH content has also been reported to increase (i.e. to double) in the presence of exogenous GSH. This may be due either to direct transport, as has been reported for intestinal and alveolar cells, or via extracellular degradation, transport, and intracellular resynthesis.
  • GSH conjugation of a substrate generally requires both GSH and glutathiones-transferase activity.
  • glutathione-S-transferases with specific, but also overlapping, substrate specificities enables the enzyme system to handle a wide range of compounds.
  • Several classes of compounds are believed to be converted by glutathione conjugate formation to toxic metabolites.
  • halogenated alkenes, hydroquinones, and quinones have been shown to form toxic metabolites via the formation of S-conjugates with GSH.
  • the kidney is the main target organ for compounds metabolized by this pathway.
  • Selective toxicity to the kidney is the result of the kidney's ability to accumulate intermediates formed by the processing of S-conjugates in the proximal tubular cells, and to bioactivate these intermediates to toxic metabolites.
  • Morphine is known to be biotransformed into morphinone, a highly hepatotoxic compound, which is 9 times more toxic than morphine in mouse by subcutaneous injection, by morphine 6-dehydrogenase activity. Morphinone possesses an ⁇ , ⁇ -unsaturated ketone, which allows it to form a glutathione S-conjugate. The formation of this conjugate correlates with loss of cellular GSH. This pathway represents the main detoxification process for morphine. Pretreatment with GSH protects against morphine- induced lethality in the mouse.
  • GSH may complex with methylmercury, prevent its transport into the cell, and increase cellular antioxidant capabilities to prevent cell damage.
  • Methylmercury is believed to exert its deleterious effects on cellular microtubules via oxidation of tubulin sulfhydryls, and by alterations due to peroxidative injury.
  • GSH also protects against poisoning by other heavy metals such as nickel and cadmium.
  • nephrotoxic agents such as cisplatin, in order to reduce systemic toxicity.
  • GSH was administered intravenously to patients with advanced neoplastic disease, in two divided doses of 2,500 mg, shortly before and after doses of cyclophosphamide.
  • GSH was well tolerated and did not produce unexpected toxicity.
  • Other studies have shown that co-administration of GSH intravenously with cisplatin and/or cyclophosphamide combination therapy, reduces associated nephrotoxicity, while not unduly interfering with the desired cytotoxic effect of these drugs.
  • GSH has an extremely low toxicity, and oral LD50 measurements are difficult to perform due to the sheer mass of GSH, which has to be ingested by the animal in order to see any toxic effects.
  • GSH can be toxic, especially in cases of ascorbate deficiency, and these effects may be demonstrated in, for example, ascorbate deficient guinea pigs given 3.75 mmol/kg daily (1,152 mg/kg daily) in three divided doses, whereas in non- ascorbate deficient animals, toxicity was not seen at this dose, but were seen at double this dose.
  • antioxidant compounds other than GSH, that are safe and even more potent, to overcome high oxidative stress in the pathogenesis of diseases. Ideally, such compounds should readily cross the blood-brain barrier and easily permeate the cell membrane. Antioxidants such as vitamins E and C are not completely effective at decreasing oxidative stress, particularly because, in the case of vitamin E, they do not effectively cross through the cell membrane to reach the cytoplasm so as to provide antioxidant effects.
  • the present invention provides the use of a potent antioxidant N-acetylcysteine amide (NAC amide) or derivatives thereof, or a physiologically acceptable derivative, salt, or ester thereof, in new applications to treat disorders, conditions, pathologies and diseases that result from, or are associated with, the adverse effects of oxidative stress and/or the production of free radicals in cells, tissues and organs of the body.
  • NAC amide and its derivatives are provided for use in methods and compositions for improving, treating, and/or preventing such disorders, conditions, pathologies and diseases.
  • a "subject" within the context of the present invention encompasses, without limitation, mammals, e.g., humans, domestic animals and livestock including cats, dogs, cattle and horses.
  • a "subject in need thereof is a subject having one or more manifestations of disorders, conditions, pathologies, and diseases as disclosed herein in which administration or introduction of NAC amide or its derivatives would be considered beneficial by those of ordinary skill in the art.
  • methods and compositions comprising NAC amide provide an antioxidant to cells and tissues to reduce oxidative stress, and the adverse effects of cellular oxidation, in an organism.
  • the invention provides a method of reducing oxidative stress associated with the conditions, diseases, pathologies as described herein, by administering a pharmaceutically acceptable formulation of NAC amide or derivatives thereof to a human or non-human mammal in an amount effective to reduce oxidative stress.
  • NAC amide and its derivatives are provided to treat an organism having a disorder, condition, pathology, or disease that is associated with the overproduction of oxidants and/or oxygen free radical species.
  • NAC amide treatment can be prophylactic or therapeutic.
  • “Therapeutic treatment” or “therapeutic effect” means any improvement in the condition of a subject treated by the methods of the present invention, including obtaining a preventative or prophylactic effect, or any alleviation of the severity of signs or symptoms of a disorder, condition, pathology, or disease or its sequelae, including those caused by other treatment methods (e.g., chemotherapy and radiation therapy), which can be detected by means of physical examination, laboratory, or instrumental methods and considered statistically and/or clinically significant by those skilled in the art.
  • “Prophylactic treatment” or “prophylactic effect” means prevention of any worsening in the condition of a subject treated by the methods of the present invention, as well as prevention of any exacerbation of the severity of signs and symptoms of a disorder, condition, pathology, or disease or its sequelae, including those caused by other treatment methods (e.g., chemotherapy and radiation therapy), which can be detected by means of physical examination, laboratory, or instrumental methods and considered statistically and/or clinically significant by those skilled in the art.
  • other treatment methods e.g., chemotherapy and radiation therapy
  • NAC amide is used in the treatment and/or prevention of cosmetic conditions and dermatological disorders of the skin, hair, nails, and mucosal surfaces when applied topically.
  • compositions for topical administration include (a) NAC amide, or derivatives thereof, or a suitable salt or ester thereof, or a physiologically acceptable composition containing NAC amide or its derivatives; and (b) a topically acceptable vehicle or carrier.
  • the present invention also provides a method for the treatment and/or prevention of cosmetic conditions and/or dermatological disorders that entails topical administration of NAC amide- or NAC- amide derivative-containing compositions to an affected area of a patient.
  • the present invention provides methods and compositions useful for cancer and pre-cancer therapy utilizing NAC amide or a derivative thereof, or its pharmaceutically acceptable salts or esters.
  • the present invention particularly relates to methods and compositions comprising NAC amide or a derivative thereof in which apoptosis is selectively induced in cells of cancers or precancers.
  • the present invention provides compositions and methods comprising NAC amide or a derivative thereof for the suppression of allograft rejection in recipients of allografts.
  • the present invention provides a NAC amide or a derivative thereof in a method of supporting or nurturing the growth of stem cells for stem cell transplants, particularly stem cells cultured in vitro prior to introduction into a recipient animal, including humans.
  • the present invention provides methods of inhibiting, preventing, treating, or both preventing and treating, central nervous system (CNS) injury or disease, traumatic brain injury, neurotoxicity or memory deficit in a subject, involving the
  • CNS central nervous system
  • the present invention provides a method of killing or inhibiting the growth of microorganisms by providing NAC amide in an amount effective to increase cellular levels of HIF-1 or HIF- ⁇ to enhance the capacity of white blood cells to kill or inhibit the growth of the microorganisms.
  • NAC amide is used as a countermeasure for biodefensive purposes, e.g., in killing or growth inhibiting microorganisms, viruses, mycoplasma, etc., and in treating and/or preventing resulting diseases and conditions, as further described herein.
  • the present invention provides a method of preventing tissue destruction resulting from the effects of metalloproteinases, such as MMP-3, which has been found to cause normal cells to express the Raclb protein, an unusual form of Rho GTPase that has previously been found only in cancers.
  • Raclb stimulates the production of highly reactive oxygen species (ROS), which can promote cancer by activating major genes that elicits massive tissue disorganization.
  • ROS highly reactive oxygen species
  • NAC amide is used to block the effects of Rac lb-induced ROS production by administering or introducing NAC amide to cells, tissues, and/or the body of a subject in need thereof, to target molecules in the pathways leading to tissue damage and degradation.
  • NAC amide can be used to inhibit MMP-3 and its adverse functions, to target ROS indirectly or directly via the processes by which ROS activates genes to induce the EMT.
  • Another aspect of the present invention provides a method of stimulating endogenous production of cytokines and hematopoietic factors, comprising administering or introducing NAC amide to cells, tissues, and/or a subject in need thereof for a period of time to stimulate the endogenous production.
  • NAC amide can be used to stimulate production of cytokines and hematopoietic factors, such as but not limited to, TNF-cc, IFN-a, IFN- ⁇ , IFN- ⁇ , IL-1, IL- 2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF, and GM-CSF, which are factors that modulate the immune system and whose biological activities are involved in various human diseases, such as neoplastic and infectious diseases, as well as those involving hematopoiesis and immune depressions of various origin (such as, without limitation, erythroid, myeloid, or lymphoid suppression).
  • cytokines and hematopoietic factors such as but not limited to, TNF-cc, IFN-a, IFN- ⁇ , IFN- ⁇ , IL-1, IL- 2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF, and GM-
  • Stimulation of endogenous production of these cytokines and hematopoietic factors by NAC amide is particularly advantageous, since exogenous administration of these cytokines and hematopoietic factors have limitations associated with the lack of acceptable formulations, their exhorbitant cost, short half-life in biological media, difficulties in dose-determination, and numerous toxic and allergic effects.
  • the present invention encompasses methods and composition comprising NAC amide for detecting NAC-amide responsive changes in gene expression in a cell, tissue, and/or a subject, comprising administering or introducing NAC amide or derivative of NAC amide to the cell, tissue, and/or subject for a period of time to induce changes in gene expression and detecting the changes in gene expression.
  • NAC amide and derivatives thereof can induce changes in gene expression such as genes involved in apoptosis, angiogenesis, chemotaxis, among others.
  • the present invention provides directed delivery of NAC amide to cells, such as cancer cells that express high levels of receptors for folic acid (folate) or glutathione.
  • NAC amide (“NACA”) is coupled to a ligand for the receptor (e.g., folic acid or glutathione) to form a conjugate, and then this NACA-ligand conjugate is coated or adsorbed onto readily injectable nanoparticles using procedures known to those skilled in the art.
  • the nanoparticles containing NAC amide (“nano-NACA particles”) may be preferentially taken up by cancer or tumor cells where the NAC amide will exert its desired effects.
  • the present invention provides a method of directed delivery of NAC amide to host cells expressing high levels of surface receptor for a ligand, in which the method involves (a) coupling NAC amide to the surface receptor ligand to form a NAC amide-ligand conjugate; (b) adsorbing the NAC amide-ligand conjugate onto nanoparticles; and (c) introducing the nanoparticles of (b) into the host.
  • the invention further provides a method of directed delivery of NAC amide to host cells expressing high levels of surface receptor for a ligand, in which the method involves (a) conjugating acetylated dendritic nanopolymers to a ligand; (b) coupling the conjugated ligand of (a) to NAC amide to form NAC amide-ligand nanoparticles; and c) introducing the nanoparticles of (b) into the host.
  • Another aspect of the present invention provides a compound of the formula I: wherein: Rj is OH, SH, or S-S-Z;
  • X is C or N
  • R 3 is absent or wherein: R is NH or O;
  • R 5 is CF 3 , NH 2 , or CH 3 and wherein: Z is
  • R ⁇ is S-S-Z
  • X and X' are the same
  • Y and Y' are the same
  • R 2 and R 6 are the same
  • R 3 and R 7 are the same.
  • the present invention also provides a NAC amide compound and NAC amide derivatives comprising the compounds disclosed herein.
  • a process for preparing an L- or D- isomer of the compounds of the present invention comprising adding a base to L- or D-cystine diamide dihydrochloride to produce a first mixture, and subsequently heating the first mixture under vacuum; adding a methanolic solution to the heated first mixture; acidifying the mixture with alcoholic hydrogen chloride to obtain a first residue; dissolving the first residue in a first solution comprising methanol saturated with ammonia; adding a second solution to the dissolved first residue to produce a second mixture; precipitating and washing the second mixture; filtering and drying the second mixture to obtain a second residue; mixing the second residue with liquid ammonia and an ethanolic solution of ammonium chloride to produce a third mixture; and filtering and drying the third mixture, thereby preparing the L- or D-isomer compound.
  • the process further comprises dissolving the L- or D-isomer compound in ether; adding to the dissolved L- or D-isomer compound an ethereal solution of lithium aluminum hydride, ethyl acetate, and water to produce a fourth mixture; and filtering and drying the fourth mixture, thereby preparing the L- or D-isomer compound.
  • Another aspect of the invention provides a process for preparing an L- or D-isomer of the compounds disclosed herein, comprising mixing S-benzyl-L- or D-cysteine methyl ester hydrochloride or O-benzyl-L- or D-serine methyl ester hydrochloride with a base to produce a first mixture; adding ether to the first mixture; filtering and concentrating the first mixture; repeating steps (c) and (d), to obtain a first residue; adding ethyl acetate and a first solution to the first residue to produce a second mixture; filtering and drying the second mixture to produce a second residue; mixing the second residue with liquid ammonia, sodium metal, and an ethanolic solution of ammonium chloride to produce a third mixture; and filtering and drying the third mixture, thereby preparing the L- or D-isomer compound.
  • Yet another aspect of the invention provides a process for preparing a compound as disclosed herein, comprising mixing cystamine dihydrochloride with ammonia, water, sodium acetate, and acetic anhydride to produce a first mixture; allowing the first mixture to precipitate; filtering and drying the first mixture to produce a first residue; mixing the second residue with liquid ammonia, sodium metal, and an ethanolic solution of ammonium chloride to produce a second mixture; filtering and drying the second mixture, thereby preparing the compound.
  • a method is presented for preventing tissue damage in a human subject exposed to a high-energy impulse blasts.
  • the method comprises administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for preventing tissue damage due to exposure to high-energy impulse blasts.
  • the method may involve prophylactic treatment of lung injury due to exposure to a high-energy impulse blasts.
  • a method is presented for preventing pulmonary inflammation after exposure to blast overpressure or related conditions in a human subject in need thereof.
  • the method comprises administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing pulmonary inflammation or related conditions after exposure to blast overpressure.
  • the related conditions from exposure to blast are contusions or barotrauma-like injury to air-filled organs, wherein the air-filled organs are ears, lungs, and the gastrointestinal tract.
  • a method for preventing blunt chest trauma in a human subject in need thereof may comprise administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing blunt chest trauma.
  • NAC amide N- acetylcysteine amide
  • a method for preventing lung contusion in a human subject in need thereof may comprise administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing lung contusion.
  • NAC amide N- acetylcysteine amide
  • a method for preventing traumatic brain injury in a human subject in need thereof may comprise administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing traumatic brain injury.
  • NAC amide N-acetylcysteine amide
  • a method for preventing malaria in a human subject infected with a malarial parasite comprising administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for prevention of malaria.
  • the method may involve prophylactic treatment of malaria.
  • One aspect of the present invention provides for inhibiting replication of HIV virus in a human subject infected with the virus, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for inhibiting the viral replication.
  • NAC amide N-acetylcysteine amide
  • a method for preventing HIV/AIDS in a human subject infected with HIV virus, comprising administering N- acetyl cysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for prevention of HIV/ AIDS.
  • the method may involve prophylactic treatment of HIV/AIDS.
  • Another embodiment of the present invention involves a method for inhibiting replication of dengue virus in a human subject infected with the virus, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for inhibiting the viral replication.
  • NAC amide N-acetylcysteine amide
  • the present invention provides a method for preventing dengue fever in a human subject infected with dengue virus, comprising administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for prevention of dengue fever.
  • the method may involve prophylactic treatment of dengue fever.
  • a method for preventing tissue damage in a human subject exposed to radiological materials comprises administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for preventing tissue damage due to exposure to radiological materials.
  • the method may be for prophylactic treatment of radiological exposure.
  • the present invention provides a method for preventing tissue damage in a human subject exposed to airborne particulate matter, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for preventing tissue damage due to exposure to airborne particulate matter.
  • NAC amide N-acetylcysteine amide
  • the present invention provides a method for preventing tissue damage in a human subject exposed to toxic gas or fumes, comprising administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for preventing tissue damage due to exposure to toxic gas or fumes.
  • NAC amide N- acetylcysteine amide
  • the invention also provides a method for inhibiting replication of an influenza virus in a human subject infected with the virus, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for inhibiting the viral replication.
  • a method is also presented for preventing influenza in a human subject infected with an influenza virus, comprising administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for prevention of influenza.
  • the method may involve prophylactic treatment of influenza.
  • a method for inhibiting replication of a virus in a human subject infected with the virus comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for inhibiting the viral replication.
  • the method for preventing infection by a virus in a human subject comprises administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective.
  • the method involves prophylactic treatment of viral infection.
  • a method for improving survibility after exposure to a high energy impulse blast or blast overpressure comprising administering to a human subject in need thereof N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing pulmonary inflammation after exposure to to a high energy impulse blast or blast overpressure.
  • NAC amide N-acetylcysteine amide
  • a method for preventing pulmonary damage after exposure to blast overpressure in a human subject in need thereof comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing pulmonary inflammation after exposure to blast overpressure.
  • NAC amide N-acetylcysteine amide
  • Another embodiment provides a method for preventing multiple organ damage or related conditions after exposure to blast overpressure in a human subject in need thereof comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, to the subject in a dose effective for preventing organ damage or related conditions after exposure to blast overpressure.
  • NAC amide N-acetylcysteine amide
  • the related conditions of multiple organ damage are contusions or barotrauma-like injury to air-filled organs, where the air-filled organs are ears, lungs, and the gastrointestinal tract. Additional affected organs may be lung, heart, brain, liver, kidneys, or gastrointestinal tract.
  • the present invention provides a method for preventing or treating Parkinson's disease in a mammalian subject, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof, in a dose effective for preventing or treating neurodegenerative disease.
  • NAC amide N-acetylcysteine amide
  • the neurodegerative disease may be Parkinson's disease.
  • the mammalian subject according to the current invention may be a human.
  • the current invention also embodies prevention or inhibition of neurotoxin induced cell death, where the cell may be a neuronal cell in an animal, comprising administering N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof.
  • the neurotoxin may be N- acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof.
  • the neurotoxin may be N- acetylcysteine amide
  • the current invention also embodies prevention or inhibition of toxicity induced by an antibiotic in kidney cells, comprising administering N-acetylcysteine amide (NAC amide), or a pharmaceutically acceptable salt, ester, or derivative thereof.
  • NAC amide N-acetylcysteine amide
  • the antibiotic may be gentamycin.
  • N-acetylcysteine amide or a pharmaceutically acceptable salt, ester, or derivative thereof may be administed.
  • the dose may be 25-500 mg per dose, or in an equivalent amount.
  • NAC amide may be delivered orally via a capsule.
  • the present invention also provides a food additive comprising NAC amide or a NAC amide derivative as disclosed herein.
  • FIG. 1 A presents the structure of N acetyl cysteine.
  • FIG. IB presents the structure of N-acetylcysteine amide (NAC amide).
  • FIGS. 2A-2D show the cytotoxic response of PC 12 cells to glutamate and protection by NAC amide.
  • PC 12 cells were plated at a density 25 x 10 3 cells/well in a 24 well plate and grown for 24 h in culture medium. They were treated or not (control) with 10 mM Glu with or without NAC amide, as described in Example 1. Twenty-four hours later, cells were examined and photographed.
  • FIG. 2A Control
  • FIG. 2B NAC amide (NACA) only
  • FIG. 2C Glutamate only
  • FIG. 2D Glutamate and NACA.
  • FIG. 3 shows the protective effect of NAC amide against glutamate cytotoxicity.
  • Cells were plated and grown for 24 hours in a culture medium; then they were treated or not (control) with 10 mM Glu, with or without NAC amide. Twenty-four hours later, the % LDH release was determined using LDH analysis. Values represent means ⁇ SD. Statistically different values of * P ⁇ 0.0001 and ** P ⁇ 0.05 were determined, compared to control. *** P ⁇ 0.0001 compared to glutamate-treated group.
  • FIG. 4 shows the effect of NAC amide on glutamate- induced cytotoxicity.
  • Cells were exposed to 10 mM Glu, with or without NAC amide, for 24 hours; the effects were compared to the control.
  • Cell viability was quantified by the MTS assay. Values represent means ⁇ SD.
  • Statistically different values of *P ⁇ 0.0005 and ** P ⁇ 0.05 were determined, compared to control. *** P ⁇ 0.05 compared to glutamate-treated group.
  • FIG. 5 shows the effects of NAC amide [NAC amide] on cysteine levels in PC 12 cells.
  • Cells were plated and grown for 24 hours, and then they were exposed to glutamate (10 mM) in the presence or absence of NAC amide (750 ⁇ ). Twenty-four hours later, the cells were harvested and cysteine levels were measured. Values represent means ⁇ SD.
  • FIG.6 is a graph depicting a comparison of survival rates of Sprague-Dawley rats after X-ray irradiation treatment in combination with pre-treatment or post-treatment with NAC or NAC amide (TOVA).
  • FIG. 7 Gross pathological signs of lung injury after exposure to blast overpressure. Isolated areas of hemorrhage were observed in lungs 2 days (B) after exposure to 140-kPa level of blast. At 8 days (C), there were no detectable pathological signs of lung damage compared with control (A).
  • FIG. 8 Representative pictures of evolving lung injury in rats 2 days after exposure to 140-kPa blast. Hematoxylin and eosinYstained sections of lung tissue from control animal (A), placebo-treated animal (B), NACA-treated (C) animal. Adjacent to a large bronchus, there is a focally extensive area of hemorrhage and inflammation (B and C). Alveoli are filled with erythrocytes, fibrin, and cellular debris. Surrounding alveolar septa are expanded by an inflammatory infiltrate (open arrow, B). Similar, but less extensive, area of hemorrhage and inflammation in NACA-treated animal (C). Magnification: 40X.
  • FIG. 9 Area of hemorrhage (B) and inflammation (C) 2 days after blast at higher power. Arrows indicate alveolar septa expanded up to three times normal by erythrocytes, fibrin, inflammatory cells (mostly macrophages and lymphocytes), and fibroblasts (B).
  • Alveoli contain moderate numbers of inflammatory cells (macrophages, lymphocytes, fewer neutrophils), and low amounts of fibrin and edema (pink flocculent material), hemorrhage, and cellular debris (C). Magnification: 200X.
  • FIG. 10 Histopathology of lung injury in rats 8 days after exposure to 140-kPa blast. Arrows indicate small foci of inflammation in placebo- (A) and NACA-treated (B) animals. At higher magnification (C and D), the inflammation in both cases consists mostly of alveolar macrophages (large round cells in the alveolar space) and lymphocytes (mostly expanding the alveolar septa, C, D). Magnification: X 40 (A, B) or X 200 (C, D).
  • FIG. 1 Myeloperoxidase activity (A) and CD1 lb mRNA (B) level in rat lungs at 2 and 8 days after exposure to blast. Myeloperoxidase activity and CD1 lb mRNA was increased 2 days after exposure in animals injected with placebo. N- Acetylcysteine amide treatment significantly reduced MPO activity and CD1 lb mRNA induction compared with placebo-treated animals. No change in both groups compared with nonblasted controls was observed 8 days after blast. Data are mean ⁇ SD from five to eight animals in each group. *P ⁇ 0.05 compared with nonblasted controls; **P ⁇ 0.05 compared with controls and placebo- injected animals.
  • FIG. 12 Macrophage inflammatory protein 1 (A), MCP-1 (B), and CINC-1 mRNA (C) levels in rat lungs at 2 and 8 days after exposure to blast. mRNA levels were increased 2 days after exposure. N-Acetylcysteine amide treatment completely eliminated the mRNA increases in all chemokines. No change in both groups compared with controls was observed 8 days after blast. Data are mean ⁇ SD from five to eight animals in each group. *P ⁇ 0.05 compared with nonblasted controls.
  • FIG. 13 Heme oxygenase 1 mRNA (A), MnSOD mRNA (B), and GR mRNA (C) levels in rat lungs at 2 and 8 days after exposure to blast. Heme oxygenase 1 mRNA was increased in animals injected with placebo 2 days after exposure and returned to the control level 8 days after blast. N- Acetylcysteine amide treatment eliminated the HO-1 mRNA increase after blast. No change compared with nonblasted controls was observed at MnSOD and GR mRNA expression at 2 or 8 days after blast. Data are mean ⁇ SD from five to eight animals in each group. *P ⁇ 0.05 compared with nonblasted controls.
  • FIG. 14 NACA increased survival following blast injury by ⁇ 3-fold.
  • FIG. 15. Mean fold change in inflammatory gene mRNA in lung tissue measured 8 days after BOP injury. NACA-treated animals were protected from the up regulation in mRNA caused by BOP injury.
  • FIG. 16. Mean fold change in MIP niRNA in lung tissue measured 2 and 8 days after BOP injury. Chemokine levels caused by BOP injury in NACA-treated animals (blue bars) were decreased approximately 50% as compared the up regulation in mRNA in controls.
  • FIG. 17 Mean fold change in MCP mRNA in lung tissue measured 2 and 8 days after BOP injury. Chemokine levels in NACA-treated animals (blue bars) were significantly lower on day 2 as compared to controls.
  • FIG. 18 Mean fold change in IL- ⁇ mRNA in lung tissue measured 2 and 8 days after BOP injury. No significant differences were observed in chemokine levels caused by BOP injury in NACA-treated animals (blue bars) as compared to controls.
  • FIG. 19 Mean fold change in HO-1 mRNA in lung tissue measured 2 and 8 days after
  • FIG. 20 Histopathology of lung injury in control and NACA-treated animals on days 2 and 8 after exposure to BOP.
  • FIG. 21 TOVA is neuroprotective following TBI. Tissue Sparing assessment at 7 days post TBI; TOVA increases tissue sparing following TBI. Sections shown are
  • FIG. 23 TOVA reduces biomarkers for oxidative stress following TBI.
  • Tissue sections were stained for the lipid peroxidation marker, 4-F1NE, and for the protein nitrosylation maker, 3-NT. Sections were reacted with primary antibody (rabbit anti-FTNE polyclonal antibody, Calbiochem) (mouse anti-3-NT monoclonal antibody, Upstate).
  • FIG. 24 TOVA reduces oxidative stress following TBI.
  • TOVA reduced lipid peroxidation (FINE levels) following TBI.
  • TOVA did not reduce protein nitrosylation (3-NT levels); which was expected based on the upstream utilization of superoxide by peroxyni trite.
  • N 3/group, bars are group means, S.D. T test *p ⁇ 0.01.
  • FIG. 26 A and B Lack of HIV Induction and Cytotoxicity in Chronically Infected Ul Cells by TOVA. Day 4 post-stimulation.
  • FIG. 27 A and B Inhibitory Effects of TOVA on Cytokine-Induced HIV Expression in Chronically Infected Ul Cells. Day 5 post-stimulation.
  • Fig. 28 Schematic overview of study protocol for DEP-induced inflammation and oxidative stress in the lungs of C57BL/6 mice, and the role of N-acetylcysteineamide (ACA). Inflammation and oxidative stress were induced in male C57BL/6 mice following exposure to diesel exhaust particles (1.5 h and 3 h each day) for 9 days. All of the animals were pretreated with NACA or saline (on alternate days), 30 min before exposure to DEPs. The mice were sacrificed by urethane injection 24 h after the last exposure.
  • NACA N-acetylcysteineamide
  • Fig. 29 Representative photomicrographs of H&E stained lung section of male C57BL/6 mice exposed to DEPs or clean filtered air for 9 days and sacrificed 24 h thereafter. Large arrows indicate macrophages filled with DEPs. Small arrows indicate macrophages with little or no DEPs. Magnification 40*.
  • Fig. 30 Enumeration of macrophages in the lungs of mice that were pretreated with NACA or saline and then exposed to DEPs.
  • the number of macrophages per 5 high power field (hpf) was measured in H&E stained lung sections of C57BL/6 mice. * Values were significantly different from those of the control. #Values were significantly different from the DEPs 1.5 h exposed group. ##Values were significantly different from those of the DEPs 3 h exposed group. Data are expressed as mean ⁇ SD, p ⁇ 0.05.
  • Fig. 31 Representative photomicrograph of H&E stained lung sections of male C57BL/6 mice exposed to DEPs for 3 h every day for 9 days and then sacrificed 24 h thereafter. Large arrows indicate mucus. Small arrows indicate macrophages filled with DEPs. Magnification 40*.
  • Fig. 32 Effect of NACA on thiol levels in the lungs.
  • Fig. 33 Effect of NACA on lipid peroxidation in the lungs. MDA levels in the lungs of mice exposed to DEPs for 9 days, as mentioned in Section 2. * Values were significantly different from the control. #Values were significantly different from the DEPs 1.5 h exposed group. ##Values significantly different from those of the DEPs 3 h exposed group. Data are expressed as mean ⁇ SD, p ⁇ 0.05.
  • Fig. 34 Effect of NACA on the activity of the antioxidant enzyme catalase (CAT) in the lungs. Catalase activity in the lungs of mice exposed to DEPs for 9 days as mentioned in Section 2. *Values were significantly different from those of the control. Data are expressed as mean ⁇ SD,/? ⁇ 0.05.
  • CAT antioxidant enzyme catalase
  • Fig. 35 Effect of NACA on cytotoxicity levels in the lungs. Lactate dehydrogenase levels in the lungs of mice exposed to DEPs for 9 days. * Values were significantly different from those of the control. Data are expressed as mean ⁇ SD, /? ⁇ 0.05.
  • Fig. 36 Schematic representation of the role of N-acetylcysteineamide (NACA) in diesel particulate matter induced oxidative stress in the lungs.
  • NACA N-acetylcysteineamide
  • FIG. 37 provides a chromatogram of a plasma sample from an animal sacrificed 30 min after administration of 500 mg/kg body weight NACA. It shows NACA conversion to NAC, GSH, and cysteine.
  • FIG 38 shows exposure to Acetaminophen decreased the cell viability in dose dependent manner.
  • FIG 39 shows exposure to Acetaminophen (AAP) decreased the cell viability in dose dependent manner.
  • AAP Acetaminophen
  • FIG 40 shows the effect of NAC and NAC amide (NACA) on HEPG2 cell viability.
  • FIG 41 shows the protective effects of NAC and NACA against AAP toxicity.
  • HEPG2 cells were exposed to 20 mM of AAP with 5 mM of NAC or NACA for 24 hrs and viability was tested using MTS assay.
  • FIG 42 shows the protective effects of NAC and NACA against AAP toxicity.
  • HEPG2 cells were exposed to 30 mM of AAP with 5 mM of NAC or NACA for 24 hrs and viability was tested using MTS assay.
  • FIG 43 shows the effect of NAC and NAC amide (NACA) on cysteine levels in cells exposed to AAP (20 mM) for 24 hrs. Around 350,000 cells were seeded in 25 cm 2 and were allowed to attach overnight. The cells were pretreated with NAC and NACA for 2 hrs. and later exposed to acetaminophen. After the exposure the cells were analyzed for cysteine levels using HPLC- Flurometric method.
  • FIG 44 shows the ability of NACA to increase the viability by acting as thiol donor in the absence or depletion of GSH.
  • HEPG2 cells were seeded in 96 well plates and pretreated with NAC and NACA for 2 hrs and later exposed to BSO 5 mM, AAP 20 mM each for 2 days. MTS assay was done to determine the viability.
  • FIG 45 provides effects of various concentrations of gentamycin on kidney cells.
  • FIG 46 provides gentamycin induced apoptosis in kindney cells.
  • FIG 47 shows increased levels of iNOS, p38MAPK, NF- ⁇ , and Bcl-2 after gentamycin treatment.
  • FIG 48 shows inhibition of gentamycin induced apoptosis by NAC amide (NACA).
  • FIG 49 shows rescue of gentamycin mediated iNOS and p38MAPK induction by NAC amide (NACA).
  • FIG 50 shows rescue of gentamycin mediated NF- ⁇ and Bcl-2 induction by NAC amide (NACA).
  • FIG 51 shows rescue of gentamycin mediated Bax induction by NAC amide (NACA).
  • FIG 52 shows rescue of gentamycin mediated Bcl-2 and Bax induction by SB.
  • the present invention involves the use of an effective and potent antioxidant, glutathione N-acetylcysteine amide (NAC amide), (FIG. 1), or a physiologically or pharmaceutically acceptable derivative or salt or ester thereof, for use in a variety of disorders, conditions, pathologies and diseases in which oxidative stress and/or free radical formation cause damage, frequently systemic damage, to cells, tissues and organs of the body.
  • NAC amide glutathione N-acetylcysteine amide
  • FOG. 1 glutathione N-acetylcysteine amide
  • the invention encompasses a pharmaceutically acceptable composition comprising NAC amide, e.g., water-soluble NAC amide, or physiologically acceptable derivatives, salts, or esters thereof, which can be used in treatment and therapeutic methods in accordance with this invention.
  • NAC amide Glutathione N-acetylcysteine amide
  • NAC amide the amide form of N- acetylcysteine (NAC)
  • BOCs mammalian red blood cells
  • NAC amide has been shown to inhibit tert.- butylhydroxyperoxide (BuOOH)-induced intracellular oxidation and to retard BuOOH- induced thiol depletion and hemoglobin oxidation in the RBCs.
  • NAC amide This restoration of thiol- depleted RBCs by externally applied NAC amide was significantly greater than that found using NAC. Unlike NAC, NAC amide protected hemoglobin from oxidation. (L. Grinberg et al., Free Radic Biol Med., 2005 Jan 1, 38(1): 136-45). In a cell-free system, NAC amide was shown to react with oxidized glutathione (GSSG) to generate reduced glutathione (GSH). NAC amide readily permeates cell membranes, replenishes intracellular GSH, and, by incorporating into the cell's redox machinery, protects the cell from oxidation. Because of its neutral carboxyl group, NAC amide possesses enhanced properties of lipophilicity and cell permeability.
  • NAC amide is also superior to NAC and GSH in crossing the cell membrane, as well as the blood-brain barrier. NAC amide can be prepared as described in U.S. Patent No. 6,420,429 to D. Atlas et al., the contents of which are incorporated by reference herein.
  • NAC amide may function directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells from free-radical mediated damage.
  • NAC amide is a potent cellular antioxidant responsible for maintaining the proper oxidation state within cells. NAC amide is synthesized by most cells and can recycle oxidized biomolecules back to their active reduced forms. As an antioxidant, NAC amide may be as effective, if not more effective, than GSH.
  • the present invention encompasses methods and compositions comprising NAC amide for preventing, reducing, protecting, or alleviating glutamate-induced cytotoxicity in neurodegenerative diseases, particularly in neuronal cells and tissues (See, e.g., Example 1).
  • NAC amide can protect cells of the nervous system from the effects of oxidative toxicity induced by glutamate.
  • NAC amide treatment can function to supply GSH as a substrate for GSH peroxidase activity in affected cells.
  • NAC amide can inhibit lipid peroxidation, scavenge for reactive oxygen species (ROS) and enhance intracellular levels of GSH to combat and overcome oxidative stress.
  • ROS reactive oxygen species
  • NAC amide can chelate lead and protect against lead-induced oxidative stress.
  • NAC amide is particularly beneficial and advantageous for neurological disorders and diseases affecting the brain and associated parts thereof, because it more readily crosses the blood-brain barrier to enter the brain and provide its antioxidant effects.
  • NAC amide can be used in the reduction of brain damage during seizures; to provide resistance to induced epileptic seizures; for protection during traumatic brain injury through the effect on mitochondrial function, reduction of inflammation and attenuation of and improvement in re-profusion with decreased re-profusion injury; for reduction of traumatic brain injury; and for treating and/or preventing prion disease, such as Creutzfeldt- Jakob disease and mad cow disease, by acting as an NMDA receptor antagonist, by enhancing intracellular levels of the anti-apoptotic protein Bcl-2; and by increasing antioxidants to glutathione.
  • NAC amide can be used in neural protection, mitochondrial preservation and therapy potential after nerve injury, particularly to prevent primary sensory neuronal death.
  • the invention embraces methods and compositions comprising NAC amide for protecting cells and tissues from radiation-induced oxidative stress.
  • NAC amide is superior to NAC in protecting tissues from radiation-induced oxidative stress.
  • Example 2 The medical crisis following the Chernobyl incident and the threat of a terrorist nuclear attack have raised awareness that high-dose total body irradiation may occur and result in death due to the induction of three potentially lethal cerebrovascular, gastrointestinal and hematopoietic clinical syndromes, which result from high dose radiation exposure. The combination of the prodromal syndrome followed by the gastrointestinal syndrome and bone marrow death induces dehydration, anemia, and infection that lead to irreversible shock.
  • ROS reactive oxygen species
  • oxidation of the glucosamine synthetase active site sulfhydryl groups is a key factor in the toxicity of the gastrointestinal syndrome.
  • Polyunsaturated fatty acids, when exposed to ROS, can also be oxidized to hydroperoxides that decompose in the presence of metals to hydrocarbons and aldehydes such as malondialdehyde (MDA).
  • MDA malondialdehyde
  • This lipid peroxidation can cause severe impairment of membrane function through increased membrane permeability and membrane protein oxidation. DNA oxidation can lead to strand breakage and consequent mutation or cell death.
  • GSH is the principal intracellular thiol responsible for scavenging ROS and maintaining the oxidative balance in tissues, such as plasma, brain, kidney, liver and lung.
  • NAC amide significantly improves GSH levels in these tissues after radiation exposure. (Example 2).
  • the prevention of spinal cord damage resulting from radiation exposure is also encompassed by the use of NAC amide.
  • the present invention encompasses methods and compositions comprising NAC amide for stimulating endogenous production of cytokines and
  • NAC amide can be used to stimulate production of cytokines and hematopoietic factors, such as but not limited to, TNF-a, IFN-a, IFN- ⁇ , IFN- ⁇ , IL-1 , IL-2, IL-6, IL-10, erythropoietin, G- CSF, M-CSF, and GM-CSF, which are factors that modulate the immune system and whose biological activities are involved in various human diseases, such as neoplastic and infectious diseases, as well as those involving hematopoiesis and immune depressions of various origin (such as, without limitation, erythroid, myeloid, or lymphoid suppression).
  • cytokines and hematopoietic factors such as but not limited to, TNF-a, IFN-a, IFN- ⁇ , IFN- ⁇ , IL-1 , IL-2, IL-6, IL-10, erythropoietin, G- CSF, M-CSF, and GM-
  • endogenous means naturally occurring within a cell, tissue, or organism, or within a subject.
  • the present invention encompasses methods and composition comprising NAC amide for detecting NAC-amide responsive changes in gene expression in a cell, tissue, and/or a subject, comprising administering or introducing NAC amide or derivative of NAC amide to the cell, tissue, and/or subject for a period of time to induce changes in gene expression and detecting the changes in gene expression.
  • the cell can be an endothelial cell, smooth muscle cell, immune cell such as erythroid, lymphoid, or myeloid cell, progenitors of erythroid, lymphoid, or myeloid cells, epithelial cell, fibroblasts, neuronal cell and the like.
  • the tissue can be any tissue of the subject, such as hair, skin, or nail tissue, vascular tissue, brain tissue, among many others.
  • the changes in gene expression are detected by microarray analysis, but other detection means can encompass, without limitation, reverse-transcription polymerase chain reaction (RT-PCR), Northern Blotting, immunofluorescence, immunoblotting, or enzyme-linked immunosorbent assay, all of which are familiar techniques to those skilled in the art.
  • NAC amide and derivatives of NAC amide can induce changes in, for example, endothelial cells that are indicative of an anti-angiogenic effect.
  • NAC has been shown to inhibit chemotaxis of endothelial cells in culture, and produce anti-angiogenic effects, such as modulation of genes responsible for blood vessel growth and differentiation, through its antioxidant effects and upregulation of angiostatin (Pfeffer, U. et al, (2005) Mut. Res. 591 : 198-21 1).
  • NAC amide and NAC amide derivatives can be used to inhibit angiogenesis as an anti-cancer agent, for example, by preventing or inhibiting tumor growth and metastasis.
  • Cells, tissues, and/or a subject can be exposed to stimuli in the presence of NAC amide or derivatives of NAC amide.
  • Stimuli include, for example, cells cultured in the presence of chemotactic or chemoattractant agents, like chemokines CXCLl-16, CCLl-27, XCLl, XCL2, RANTES, MIP 1-5 (alpha, beta, and gamma isoforms), MCP-1 through 5, and the like.
  • Cells, tissues, and subjects can also be stimulated with pharmaceutical agents, drugs, or treatment modalities. After stimulation, DNA, RNA, or protein can be isolated from the cells, tissues, and/or subject, and changes in gene expression can be detected.
  • total RNA can be isolated from cells according to standard techniques known in the art and resultant cDNAs can be synthesized and subsequently hybridized to a solid support, such as a silicon chip for microarray analysis. Expression data and changes in the expression of genes in response to the stimuli can then be analyzed using computer software programs, such as GeneSpring (Silicon Genetics).
  • Non-limiting examples of such genes that exhibit changes in their expression include genes involved in or pertaining to cellular adhesion, apoptosis, chemokine and cytokine biosynthesis, synthesis of extracellular matrix components, endothelium, inflammation, MAP kinases, metalloproteinases, NF- ⁇ , nitric oxide, transforming growth factor (TGF) signaling, and blood vessels.
  • TGF transforming growth factor
  • NAC-responsive genes that are modulated (i.e., up- or downregulated) include HSP40 (heat shock protein 40; DnaJ homolog), SERCA2 (Ca2+ transporting ATPase in cardiac muscle), MKP2 (MAP kinase phosphatase), TIP30 (HIV-1 Tat interactive protein 2), BTG1 (B-cell translocation gene 1), TXL (thioredoxin-like), CRADD (Death receptor adaptor protein), WSX1 (Class I cytokine receptor), EMAP2 (endothelial monocyte-activating protein), Jagged 1 (ligand for Notch receptor), MEA5 (hyaluronoglucosaminidase), VRNA (Integrin V), COL4A1 (Type IV collagen ocl), uPA (urokinase plasminogen activator), CPE (carboxypeptidase E), TSPAN-6 (transmembrase E), TSPAN-6 (transmembra
  • the present invention encompasses methods and compositions comprising NAC amide for stimulating macrophages and neutrophils to phagocytize infectious agents and other foreign bodies and to eliminate microorganisms, mediated by reactive oxygen species and proteases.
  • NAC amide can be used to improve macrophage function by increasing glutathione availability, which, in turn, will improve alveolar function in fetal alcohol syndrome and to augment premature alveolar macrophage function.
  • the invention encompasses methods and compositions comprising NAC amide to increase levels of intracellular reduced glutathione levels, which blocks the formation of irreversibly sickled cell red blood cells.
  • Methods involving the administration of NAC amide to prevent and treat sickle cell anemia and thalassemia are provided.
  • the invention encompasses methods and compositions comprising NAC amide to treat leishmania through the mechanism of histopathological modulation, in which cytokine pattern is modified as demonstrated by a sustained higher frequency of interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor alpha producing cells.
  • IFN- ⁇ interferon- ⁇
  • NAC amide is used in the modulation of effector responses in animals, in conjunction with bi- glutathione.
  • NAC amide is used to down-regulate cytokine synthesis, activation and downstream processes and/or to exert an antagonistic effect on proinflammatory signals.
  • Such an effect is beneficial in the treatment of many diseases in which cytokines participate in the pathophysiology of the disease.
  • cytokines which are mediators of oxidative stress, can alter the redox equilibrium by affecting GSH/oxidized glutathione disulfide (GSSG) shuttling and recycling.
  • liver injury related to the administration of certain drugs can be initiated or intensified by inflammation states that stimulate unregulated production of proinflammatory cytokines or growth factors, such as interferon ⁇ , which leads to the down-regulation of enzymes and proteins involved in drug metabolism and elimination.
  • NAC amide, or derivative thereof as an agent that can decrease proinflammatory cytokine levels is thus useful for preventing and/or managing drug-induced hepatocytoxicity.
  • the invention encompasses methods and compositions comprising NAC amide or a derivative thereof for use as a chemoprotectant against bone marrow toxicity after or during chemotherapy, including alkylators with or without glutathione depletion.
  • the invention encompasses methods and compositions comprising NAC amide or a derivative thereof to treat various aspects of sepsis, particularly bacterial sepsis and septic shock, including gram-negative septic shock.
  • NAC amide and its derivatives can act as an inhibitor of the nuclear factor NF- ⁇ , which prevents staphylococcal enterotoxin A (SCC) fever by acting through the human peripheral blood mononuclear cells to block the stimulation and synthesis or release pyrogenic cytokines and to block
  • SCC staphylococcal enterotoxin A
  • NAC amide or a derivative thereof is used to block lipid peroxidation and to improve the disease status in children with acute purulent meningitis and encephalitis.
  • NAC amide and its derivatives can be used to block pertussis toxin secretion by Bordetella pertussis and for the treatment of lethal sepsis by limiting inflammation and potentiating host defense. Because decreased bacterial colonies improve survival, migration of neutrophils to the site of infection and to a distant site is upregulated and optimal GSH levels are important for an efficient response to sepsis.
  • ROS release by immune cells is important mediators in sepsis and septic shock. During a normal immune response antioxidant serves to down-regulate the ongoing immune response mostly through modulation of proinflammatory mediators.
  • compositions comprising NAC amide or a derivative thereof can be used in the treatment of infection and disease caused by
  • microorganisms and the like such as bacteria, parasites, nematodes, yeast, fungi, plasmodia, mycoplasma, spores, and the like, e.g., malarial infections and tuberculosis and rickettsia infection.
  • infection by a number of types of bacteria such as Streptococcus, Staphylococcus, Salmonella, Bacillus (Tubercule bacillus) etc., which cause diseases in humans, induce a direct response by leukocytes (i.e., white blood cells) in the body, to increase their levels of hypoxia inducible transcription factor- 1 , or HIF-1.
  • leukocytes i.e., white blood cells
  • the HIF-1 protein binds to cellular DNA and activates specific genes to help cells function in a low oxygen environment. HIF-1 , in turn, stimulates the white blood cells to produce and release antimicrobial compounds, e.g., small proteins, enzymes and nitric oxide, that work together to kill bacteria.
  • antimicrobial compounds e.g., small proteins, enzymes and nitric oxide
  • low oxygen levels which occur at the site of an infection, activate HIF-1 in macrophages and neutrophils, which typically ingest and destroy invading microorganisms.
  • the greater the increase in HIF-1 levels in the white blood cells the greater their anti-bacterial activity.
  • agents e.g., small molecules, that promote HIF-1 activity in white blood cells to boost their bacterial killing ability, thereby promoting a resolution to infection through the actions of the immune system's natural defense mechanisms.
  • One such agent is NAC amide, which can be used in a method of killing or inhibiting the growth of microorganisms by increasing cellular levels of HIF-1, i.e., HIF- ⁇ , thereby enhancing the capacity of white blood cells, such as macrophages, to kill the microorganisms.
  • NAC glutathione
  • ROS ROS scavenger
  • the present invention is further directed to the use of NAC amide or a derivative thereof as a bacteriostatic agent when used as a treatment for bacterial infection, particularly antibiotic resistant, or multi-antibiotic resistant bacteria such as tuberculosis- causing microorganisms.
  • the present invention is directed to the use of NAC amide or a derivative thereof as a biodefensive agent for inducing the killing of infecting or contaminating microorganisms.
  • NAC amide or a derivative thereof may pose a severe health threat if they should be disseminated to the public and/or genetically altered so as to be antibiotic resistant.
  • the following lists set forth categories of microorganisms, viruses, diseases and agents for which NAC amide or its derivative is provided as a suitable countermeasure, used alone, or in combination with other active compounds, agents and substances to treat affected organisms and/or cells thereof:
  • Infectious Diseases Aflatoxins, Alphavirus Eastern equine encephalitis virus, Alphavirus Venezuelan equine encephalitis virus, Antibiotic-resistant Mycobacterium tuberculosis, Arenavirus Junin Virus, Arenavirus Lassa Virus, Ascaris lumbricoides
  • Coccidioidomycosis immitis Coxiella burnetti (Q fever), Cryptosporidium parvum,
  • Blister agents including Lewisite, nitrogen and sulfur mustards; Blood agents, including hydrogen cyanide and cyanogens chloride; Exotic agents, including hybrid organisms, genetically modified organisms, antibiotic-induced toxins, autoimmune peptides, immune mimicry agents, binary bioweapons, stealth viruses and bioregulators and biomodulators; Heavy metals, including arsenic, lead and mercury; incapacitating agents, including BZ; nerve agents, including Tabun, Sarin, Soman, GF, VX, V-gas, third generation nerve agents, organophosphate pesticides and carbamate insecticides; nuclear and radiological materials, pulmonary agents, including phosgene and chorine vinyl chloride; volatile toxins, including benzene, chloroform and trihalomethanes.
  • NAC amide or derivatives thereof can serve as an innovative treatment for known and emerging natural infectious disease threats, as well as trauma, e.g., excessive bleeding and other events, associated with and/or
  • Rickettsia which causes the pathogenesis of typhus and spotted fever rickettsioses, results in serious adverse vascular and hemorrhagic conditions, (e.g., increased vascular permeability and edema) notably in the brain and lung, following its entry into vascular endothelial cells.
  • R. rickettsii-infected endothelial cells produce ROS causing peroxidative damage to cell membranes. (D.J. Silverman et al., 1990, Ann. N.Y. Acad. Sci., 590: 111-117; D.H. Walker et al., 2003, Ann. N.Y. Acad. Sci., 990:1-1 1).
  • oxidative-stress mediated damage to R. rickettsii-infected endothelial cells is associated with the depletion of host components such as GSH and levels of catalase that act as host defenses against ROS-induced damage, the concentration of hydrogen peroxide and ROS increase in the cells to cause ROS-induced cellular damage.
  • cells e.g., fibroblasts that are infected with Mycoplasma (e.g., Mycoplasma pneumoniae) also produce increased intracellular levels of hydrogen peroxide and decreased levels of catalase, resulting in oxidative stress that can lead to death of the infected cells.
  • Mycoplasma e.g., Mycoplasma pneumoniae
  • NAC amide or a derivative thereof is provided to an infected host as an antioxidant therapeutic.
  • NAC amide administration to cells and/or organisms (e.g., infected host mammals) in accordance with the present invention, alone or in combination with other agents and/or antioxidants, can limit the amount and/or extent of oxidative damage that is induced by microbial infection.
  • the invention encompasses methods and compositions comprising NAC amide or a derivative thereof for use in preventing periventricular leukomalacia (PVL).
  • NAC amide or a derivative thereof may provide neural protection and attenuate the degeneration of OPCs against LPS evoked inflammatory response in white matter injury in developing brain.
  • NAC amide or a derivative thereof may be used as a treatment for placental infection as a means of minimizing the risk of PVL and cerebral palsy (CP).
  • the invention encompasses methods and compositions comprising NAC amide or a derivative thereof for the treatment of osteoporosis.
  • the tumor necrosis factor member RANKL regulates the differentiation, activation and survival of osteoclasts through binding of its cognate receptor, RANK.
  • RANK can interact with several TNF-receptor-associated factors (TRAFs) and activate signaling molecules including Akt, NF-KB and MAPKs.
  • TNF-receptor-associated factors TNF-receptor-associated factors
  • Akt TNF-receptor-associated factors
  • MAPKs TNF-receptor-associated factors
  • NAC amide can be used to pretreat or treat osteoclasts so as to achieve a reduction in RANKL-induced Akt, NF- ⁇ , and ERK activation.
  • the reduced NF- ⁇ activity by NAC amide may be associated with decreased IKK activity and ⁇ phosphorylation.
  • Pretreatment with NAC amide or a derivative thereof can be used to reduce RANKL-induced actin ring formation required for bone resorbing activity and osteoclast survival.
  • the methods and compositions comprising NAC amide or a derivative thereof can be used for the improvement of osteoporosis through blockage and interference with osteoclasts, and to lower reactive oxidative stress levels so as to have beneficial effects on preventing bone loss by reducing RANKL-induced cellular function.
  • NAC amide or a derivative thereof is used in the treatment of osteoporosis by blockage of thiol thioredoxin-1 , which mediates osteoclast stimulation by reactive oxidation species (ROS), as well as blockage of TNF-a, which causes loss of bone, particularly in circumstances of estrogen deficiency.
  • ROS reactive oxidation species
  • the invention embraces methods and compositions comprising NAC amide or a derivative thereof are used for the treatment of polycystic ovary syndrome.
  • NAC amide or a derivative thereof may also be used as a therapeutic agent to ameliorate the homocysteine and lipid profiles in PCOS-polycystic ovary syndrome.
  • the invention encompasses the use of NAC amide or a derivative thereof in treatments and therapies for toxin exposure and conditions related thereto, e.g., sulfur mustard (HD-induced lung injury).
  • Treatment of individuals having been exposed to toxins or suffering from toxin exposure with NAC amide or a derivative thereof may reduce neutrophil counts to achieve a decreased inflammatory response.
  • NAC amide and its derivatives may be useful as a treatment compound for patients having sulfur mustard vapor exposure induced lung injury.
  • Administration of NAC amide or a derivative thereof can be either orally or as a bronchoalveolar lavage.
  • NAC amide and its derivatives are useful in methods and compositions for the blockage of brain and/or lung damage and cognitive dysfunction in mechanical warfare agents including CW, vesicants, sulfur mustard, nitrogen mustards, chloroethyl amine, lewisite, nerve agents O-ethyl S-(2-[di-isopropylamino] ethyl) methyl phosphorothioate (VX), tabun (GA) and sarin (GB) and soman DG and the blood agents cuianogenchloride, and in the prevention of organophosphate induced convulsions and neuropathological damage.
  • mechanical warfare agents including CW, vesicants, sulfur mustard, nitrogen mustards, chloroethyl amine, lewisite, nerve agents O-ethyl S-(2-[di-isopropylamino] ethyl) methyl phosphorothioate (VX), tabun (GA) and sarin (GB) and soman DG
  • the present invention encompasses methods and compositions comprising NAC amide for use in the treatment of burn trauma.
  • NAC amide or a derivative thereof can block NF- ⁇ , which has been shown to reduce burn and burn sepsis.
  • NAC amide or a derivative thereof can be used to protect microvascular circulation, reduce tissue lipid peroxidation, improve cardiac output and reduce volume of required fluid resuscitation.
  • NAC amide or a derivative thereof can be used in the prevention of burn related cardiac NF- KB nuclear migration, and improve cardiomyocyte secretion of TNF-a, IL- ⁇ ⁇ , and IL-6 and to improve cardiac malfunction.
  • NAC amide or a derivative thereof as an antioxidant that can inhibit free radical formation and/or scavenge free radicals to protect tissues and organs in patients with burn injury.
  • the present invention encompasses methods and compositions comprising NAC amide or a derivative of NAC amide for use in the prevention of lung injury due to the adverse effects of air pollution, smoke inhalation, poison gas or diesel exhaust particles.
  • the present invention encompasses methods and compositions comprising NAC amide or a derivative thereof for use in the treatment and therapy of cardiovascular disease and conditions.
  • NAC amide and its derivatives can be used as a blocker of angiotensin-converting enzyme.
  • NAC amide or a derivative thereof can be used to decrease oxidative stress, and to cause more rapid re- profusion, better left ventricular preservation, reduced infarct size, better preservation of global and regional left ventricular function and modification of QSR complex morphology and ECG.
  • NAC amide or a derivative thereof can also be used in the treatment of focal cerebral ischemia with protection of the brain and reduction of inflammation in experimental stroke.
  • NAC amide can be used in the treatment of reperfusion injuries, as well as apoptosis of myocardial endothelial cells and interstitial tissue.
  • NAC amide or a derivative thereof may assist in the elevation of nitric oxide levels, play an important role in the management of cardiovascular disease, reduce chronic inflammation in cardiovascular disease and prevent restenosis of cardiovascular stents placed in coronary arteries and carotid arteries.
  • NAC amide and its derivatives can be used in the prevention of cardiac failure following MI and cardiomyopathy due to prevention of oxidative stress and improvement of left ventricular remodeling.
  • NAC amide or a derivatives of NAC amide in this capacity supports the involvement of oxidative stress in myocardial vascular dysfunction and hypertension and provides a role for antioxidant strategies to preserve the myocardial micro vasculature.
  • NAC amide or a derivative thereof can also be used in the prevention of oxidized proteins in muscles.
  • NAC amide or a derivative thereof can be used to treat arterial sclerosis and to increase high density lipoprotein (HDL)-cholesterol serum levels in hyperlipidemic and normal lipidemic individuals with documented coronary stenosis.
  • HDL high density lipoprotein
  • NAC amide or a derivative thereof can also be used to decrease coronary and alpha-beta stress; to prevent further myocardial infarctions; and to cause a reduction in body fat thereby improving glucose tolerance, particularly in overweight or obese individuals.
  • NAC amide or a derivative thereof be used to improve muscular performance and decrease levels of tumor necrosis factor in old age.
  • the present invention is directed to the use of method and compositions comprising NAC amide or a derivative thereof in the treatment of thalassemic blood by ameliorating oxidative stress in platelets.
  • the activation of platelets causes thromboembolic consequences and produces a hypercoagulable state that is amenable to treatment by the antioxidant NAC amide or a derivative thereof.
  • NAC amide or a derivative thereof is useful as a wound dressing to permit enhancement of neutrophil function.
  • NAC amide or a derivative thereof is used to block the effects of leptin, which is a cardiovascular risk factor in diabetic patients.
  • NAC amide or a derivative thereof is used in the treatment of total plasma homocysteine and cysteine levels with increased urinary excretion, as well as in the treatment for hyperhomocysteinemic conditions, to improve oxidative stress. It has been found that elevated levels of homocysteine pose a significant risk in vascular disease, such as atherosclerosis, venous thrombosis, heart attack and stroke, as well as neural tube defects and neoplasia. Homocysteine promotes free radical reactions. In patients with defective homocysteine metabolism, relatively high levels of homocysteine are present in the blood.
  • NAC amide or a derivative thereof is administered to patients with elevated homocysteine levels.
  • NAC amide or a derivative thereof is used as a chemoprotectant against bone marrow toxicity after or during
  • NAC amide or a derivative thereof is used in the treatment of lithium induced renal failure.
  • NAC amide or a derivative thereof is used in the treatment of prostatic inflammation, which may contribute to prostatic carcinogenesis and
  • NAC amide or a derivative thereof is used in pulmonary disease medicine, particularly in oxygen-mediated lung disease.
  • NAC amide or a derivative thereof can improve oxygenation in cardiopulmonary bypass during coronary artery surgery and is useful in the treatment of chronic obstructive pulmonary disease and pulmonary hypertension.
  • NAC amide or a derivative thereof is used in the treatment of injury in the lung due to high-energy impulse noise-blasts, which can induce antioxidant depletion.
  • the administration of NAC amide or its derivatives provide an advantageous antioxidant source.
  • NAC amide or a derivative thereof is particularly useful if provided as a supplement prior to noise blast exposure.
  • NAC amide or a derivative thereof is useful in the treatment of asthma with increased oxidative stress.
  • NAC amide or a derivative thereof is useful for the treatment of adult respiratory distress syndrome; in the treatment of pulmonary fibrosis, in the treatment of idiopathic pulmonary fibrosis and asbestos exposure; and in the treatment of chronic lung rejection. Further, NAC amide or a derivative thereof is contemplated for use in occupational isocyanate exposure and the development of isocyanate allergy, which is believed to develop by two processes, namely, isocyanate-protein conjugation and airway epithelial cell toxicity. More specifically, NAC amide or a derivative thereof can serve to protect against hexamethylene diisocyanate (HDI) conjugation to cellular proteins and to reduce HDI toxicity to human airway epithelial cells following isocyanate exposure. Thus, NAC amide or a derivative thereof can help to prevent the development of allergic sensitization and asthma that are associated with this occupational hazard.
  • HDI hexamethylene diisocyanate
  • the present invention encompasses the use of NAC amide or a derivative thereof to inhibit HIV replication in chronically and acutely infected cells.
  • NAC amide can be used in GSH replacement therapy, as NAC amide and its derivatives may interfere with the expression of the integrated HIV genome, thus, attacking the virus in a manner that is different from that of the currently employed anti-retrovirals, e.g., AZT, ddl, ddC or D4T.
  • NAC amide or a derivative thereof can also be beneficial in countering the excess free radical reactions in HIV infection, which may be attributable to: 1) the hypersecretion of TNF-a by B-lymphocytes in HIV infection, and 2) the catalysis of arachidonic acid metabolism by the gp 120 protein of HIV.
  • NAC amide and its derivatives can serve as a suppressant of viral and bacterial species in vaginal tissues by the use of intravaginal placement of gel induced thiol.
  • NAC amide and its derivatives can be used to restore antioxidant levels in a mammal in need thereof, to arrest the replication of the virus at a unique point, and specifically prevent the production of toxic free radicals, prostaglandins, TNF-a, interleukins, and a spectrum of oxidized lipids and proteins that are
  • NAC amide or a derivative thereof to elevate or replace antioxidant levels could slow or stop the diseases progression safely and economically.
  • an aspect of this invention is to increase intracellular levels of antioxidant in infected cells, as well as to increase extracellular of antioxidant, by introducing or administering AD3 so as to interfere with the replication of HIV and to prevent, delay, reduce or alleviate the cascade of events that are associated with HIV infection.
  • AIDS may also be associated with reduced GSSG levels, providing an amount of NAC amide to cells and/or to an individual in need thereof, can overcome any interference with de novo synthesis of antioxidant such as GSH, as well as the oxidation of existing GSH, which may occur in HIV infected cells.
  • NAC amide or a derivative thereof is used to inhibit cytokine-stimulated HIV expression and replication in acutely infected cells, chronically infected cells, and in normal peripheral blood mononuclear cells.
  • NAC amide or derivatives thereof can be used to effect concentration-dependent inhibition of HIV expression induced by TNF-a or IL-6 in chronically infected cells. Due to NAC amide's superior ability to cross cellular membranes and enhanced lipophilic properties, NAC amide and derivatives thereof can be used at lower concentrations as compared to NAC or GSH, such as 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or lower,
  • the depletion of antioxidants by HIV in infected cells is also associated with a process known as apoptosis, or programmed cell death.
  • NAC amide or a derivative thereof to HIV infected individuals and/or cells, the intercellular processes, which artificially deplete GSH and which may lead to cell death can be prevented, interrupted, or reduced.
  • the NAC amide thiol can be used as a blocker of bio-replication from West Nile Virus and protection of cells from the cytopathic effect after infection of West Nile Virus, as well as other RNA and DNA virus infections.
  • NAC amide or a derivative thereof may be administered by several routes that are suited to the treatment or therapy method, as will be appreciated by the skilled practitioner.
  • routes and modes of administration for NAC amide and its derivatives include parenteral routes of injection, including subcutaneous, intravenous, intramuscular, and intrasternal.
  • Other modes of administration include, but are not limited to, oral, inhalation, topical, intranasal, intrathecal, intracutaneous, opthalmic, vaginal, rectal, percutaneous, enteral, injection cannula, timed release and sublingual routes.
  • Administration of NAC amide and its derivatives may also be achieved through continuous infusion.
  • NAC amide and its derivatives may be mediated by endoscopic surgery.
  • NAC amide or a derivative thereof can be introduced into the tissues lining the ventricles of the brain.
  • the ventricular system of nearly all brain regions permits easier access to different areas of the brain that are affected by the disease or disorder.
  • a device such as a cannula and osmotic pump, can be implanted so as to administer a therapeutic compound, such as NAC amide, or derivative thereof as a component of a pharmaceutically acceptable composition.
  • Direct injection of NAC amide and its derivatives are also encompassed.
  • the close proximity of the ventricles to many brain regions is conducive to the diffusion of a secreted or introduced neurological substance in and around the site of treatment by NAC amide.
  • a recipient for example, injectable administration, a
  • composition or preparation formulated to contain water-soluble NAC amide or a derivative thereof is typically in a sterile solution or suspension.
  • NAC amide or a derivative thereof can be resuspended in pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e. blood) of the recipient.
  • excipients suitable for use include water, phosphate buffered saline (pH 7.4), 0.15M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures.
  • Formulations comprising NAC amide or a derivative thereof for topical
  • NAC amide or a derivative thereof may be administered to mucous membranes in the form of a liquid, gel, cream, and jelly, absorbed into a pad or sponge.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions comprising NAC amide or a derivative thereof for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets.
  • Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
  • Formulations for parenteral administration may include, but are not limited to, sterile solutions, which may also contain buffers, diluents and other suitable additives.
  • the present invention also provides a food additive comprising NAC amide or a derivative thereof for mammalian, preferably human, consumption.
  • NAC amide and other cysteine derivatives have been detected in many different food products, including but not limited to, garlic, peppers, turmeric, asparagus, and onions. See, for example, Hsu, C.C., et al, (2004) J. Nutr. 134:149-152 and Demirkol, O. et al, (2004) J. Agric. Food Chem. 52.
  • the food additive can comprise NAC amide or its derivative in a liquid or solid material intended to be added to a foodstuff.
  • the food additives can be added to "food compositions" including any products—raw, prepared or processed— which are intended for human consumption in particular by eating or drinking and which may contain nutrients or stimulants in the form of minerals, carbohydrates (including sugars), proteins and/or fats, and which have been modified by the incorporation of a food additive comprising NAC amide or a derivative of NAC amide as provided herein.
  • the present modified food compositions can also be characterized as "functional foodstuffs or food compositions”.
  • Foodstuffs can also be understood to mean pure drinking water.
  • the term "food additive” is understood to mean any a liquid or solid material intended to be added to a foodstuff. This material can, for example, have a distinct taste and/or flavor, such as a salt or any other taste or flavor potentiator or modifier. It is to be noted, however, that the food additive comprising NAC amide or a NAC amide derivative does not necessarily have to be an agent having a distinct taste and/or flavor.
  • Other food additives that can be added in combination with NAC amide, or in food additive formulations of NAC amide include, but are not limited to, acids which are added to make flavours "sharper", and also act as preservatives and antioxidants, such as vinegar, citric acid, tartaric acid, malic acid, fumaric acid, lactic acid, acidity regulators, anti-caking agents, antifoaming agents, antioxidants such as vitamin C and tocopherols such as vitamin E, bulking agents, such as starch are additives, food coloring, color retention agents, emulsifiers flavors, flavor enhancers, humectants, preservatives, propellants, stabilizers, thickeners and gelling agents, like agar or pectin, and sweeteners .
  • acids which are added to make flavours "sharper” such as vinegar, citric acid, tartaric acid, malic acid, fumaric acid, lactic acid, acidity regulators, anti-caking agents, antifoaming
  • Doses, amounts or quantities of NAC amide, or derivative thereof as well as the routes of administration used, are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those having skill in the art. As is appreciated by the skilled practitioner in the art, dosing is dependent on the severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
  • a pharmaceutical formulation for orally administrable dosage form can comprise NAC amide, or a pharmaceutically acceptable salt, ester, or derivative thereof in an amount equivalent to at least 25-500 mg per dose, or in an amount equivalent to at least 50-350 mg per dose, or in an amount equivalent to at least 50-150 mg per dose, or in an amount equivalent to at least 25-250 mg per dose, or in an amount equivalent to at least 50 mg per dose.
  • NAC amide or a derivative thereof can be administered to both human and non-human mammals. It therefore has application in both human and veterinary medicine.
  • esters of NAC amide include alkyl and aryl esters, selected from the group consisting of methyl ester, ethyl ester, hydroxyethyl ester, t-butyl ester, cholesteryl ester, isopropyl ester and glyceryl ester.
  • AIDS AIDS
  • diabetes macular degeneration
  • congestive heart failure cardiovascular disease and coronary artery restenosis
  • lung disease asthma
  • virus infections e.g., toxic and infectious hepatitis, rabies, HIV
  • sepsis osteoporosis
  • toxin exposure radiation exposure
  • burn trauma prion disease
  • neurological diseases blood diseases, arterial disease, muscle disease, tumors and cancers.
  • exposure to toxins, radiation, medications, etc. may result in free radical reactions, including types of cancer chemotherapy.
  • the present invention provides NAC amide or a derivative thereof as an agent that can treat these diseases and conditions in a convenient and effective formulation, particularly for oral administration.
  • the administration of exogenous NAC amide or a derivative thereof can serve to supplement or replace the hepatic output of GSH and to assist in the maintenance of reduced conditions within the organism.
  • the failure to alleviate free radical reactions allows an undesirable cascade that can cause serious damage to macromolecules, as well as lipid peroxidation and the generation of toxic compounds. Maintaining adequate levels of GSH is necessary to block these free radical reactions.
  • NAC amide or a derivative thereof is able to provide efficient and effective remedial action.
  • NAC amide can form chelation complexes with copper and lead.
  • NAC amide may also form circulating complexes with copper in the plasma.
  • NAC amide or a derivative thereof can be administered to treat metal toxicity.
  • NAC amide-metal complexes will be excreted, thus reducing the metal load.
  • NAC amide or a derivative thereof may be administered for the treatment of toxicity associated with various metals, e.g., iron, copper, nickel, lead, cadmium, mercury, vanadium, manganese, cobalt, transuranic metals, such as plutonium, uranium, polonium, and the like.
  • the chelation properties of NAC amide are independent from its antioxidant properties. However, because some metal toxicities are free radical mediated, e.g., iron, NAC amide administration may be particularly advantageous for such conditions.
  • NAC amide or a derivative thereof can be provided in a relatively high concentration in proximity to the mucous membrane, e.g., the duodenum for oral administration.
  • NAC amide or a derivative thereof can be administered as a single bolus on an empty stomach.
  • the preferred dosage is between about 100-10,000 mg NAC amide or between about 250-3,000 mg NAC amide.
  • the NAC amide or NAC amide derivative formulation can be stabilized with a reducing agent, e.g., ascorbic acid, to reduce oxidation both during storage and in the digestive tract prior to absorption.
  • Capsules e.g., a two-part gelatin capsule
  • the capsule is preferably a standard two-part hard gelatin capsule of double-0 (OO) size, which may be obtained from a number of sources.
  • OO double-0
  • the capsules are preferably stored under nitrogen to reduce oxidation during storage.
  • the capsules are preferably filled according to the method of U.S. Patent No.
  • each capsule preferably contains 500 mg of NAC amide and 250 mg of crystalline ascorbic acid.
  • a preferred composition includes no other excipients or fillers; however, other compatible fillers or excipients may be added. While differing amounts and ratios of NAC amide and stabilizer may be used, these amounts are preferable because they fill a standard double-0 capsule, and provide an effective stabilization and high dose. Further, the addition of calcium carbonate is avoided as it may contain impurities and may accelerate the degradation of NAC amide in the small intestine due to its action as a base, which neutralizes stomach acid.
  • NAC amide or a derivative thereof is advantageously administered over extended periods. Therefore, useful combinations include NAC amide or NAC amide derivatives and drugs intended to treat chronic conditions. Such drugs are well absorbed on an empty stomach and do not have adverse interactions or reduced or variable combined absorption.
  • One particular class of drugs includes central or peripheral adrenergic or catecholenergic agonists, or reuptake blockers, which may produce a number of toxic effects, including neurotoxicity, cardiomyopathy and other organ damage. These drugs are used, for example, as cardiac, circulatory and pulmonary medications, anesthetics and psychotropic / antipsychotic agents. Some of these drugs also have abuse potential, as stimulants, hallucinogens, and other types of psychomimetics.
  • NAC amide or a derivative thereof can advantageously be provided in an oral pharmaceutical formulation in an amount of between about 50-10,000 mg, along with an effective amount of a pharmacological agent that is capable of initiating free radical reactions in a mammal.
  • the pharmacological agent is, for example, an adrenergic, dopaminergic, serotonergic, histaminergic, cholinergic, gabaergic, psychomimetic, quinone, quinolone, tricyclic, and/or steroid agent.
  • formulations of NAC amide or a derivative thereof provide an advantageous alternative to GSH administration.
  • NAC amide or a derivative thereof offers beneficial properties of lipophilicity and cell-permeability, allowing it to more readily enter cells and infiltrate the blood-brain barrier more readily than GSH, NAC or other compounds.
  • the properties of NAC amide or a derivative thereof may increase its bioavailability following administration to provide an improved treatment for the various diseases, disorders, pathologies and conditions as described herein.
  • Hepatic glutathione is consumed in the metabolism, catabolism and/or excretion of a number of agents, including aminoglycoside antibiotics, acetominophen, morphine and other opiates.
  • the depletion of hepatic glutathione may result in hepatic damage or a toxic hepatitis.
  • High dose niacin, used to treat hypercholesterolemia, has also been associated with a toxic hepatitis.
  • the present invention therefore encompasses an oral pharmaceutical formulation comprising NAC amide or a derivative thereof in an amount between about 50- 10,000 mg, administered in conjunction with an effective amount of a pharmacological agent that consumes hepatic glutathione reserves. A number of pathological conditions result in hepatic damage.
  • the present invention encompasses a pharmaceutical formulation comprising NAC amide or a derivative of NAC amide and an antiviral or antineoplastic agent.
  • the antiviral or antineoplastic agent is, for example, a nucleoside analog.
  • the formulations according to the present invention have an NAC amide or NAC amide derivative content greater than 50 mg, and are provided in one or more doses totaling up to about 10,000 mg per day.
  • NAC amide or a derivative thereof will treat the effects of HIV infection.
  • NAC amide, or derivative thereof administered according to the present method can be use in the treatment of congestive heart failure (CHF).
  • CHF congestive heart failure
  • the heart muscle is weakened, causing enlargement of the heart.
  • peripheral vasospasm is believed to be present, causing increased peripheral resistance.
  • NAC amide or a derivative thereof can be effective in enhancing the effects of nitric oxide, and therefore can be of benefit to these patients by decreasing vasoconstriction and peripheral vascular resistance, while increasing blood flow to the tissues.
  • the present invention thus encompasses the oral administration of NAC amide or a derivative thereof in conjunction with a congestive heart failure medication, for example, digitalis glycosides, dopamine, methyldopa, phenoxybenzamine, dobutamine, terbutaline, amrinone, isoproterenol, beta blockers, calcium channel blockers, such as verapamil, propranolol, nadolol, timolol, pindolol, alprenolol, oxprenolol, sotalol, metoprolol, atenolol, acebutolol, bevantolol, tolamolol, labetalol, diltiazem, dipyridamole, bretylium, phenytoin, quinidine, clondine, procainamide, acecainide, amiodarione, disopyramide, encainide,
  • the present invention embraces NAC amide or a derivative thereof to treat hepatitis of various types by oral administration.
  • NAC amide and its derivatives may also be effective in the treatment of toxicities to other types of cells or organs, which result in free radical damage to cells or reduced glutathione levels.
  • NAC amide or a derivative thereof can be used to supplement diabetic patients in order to prevent a major secondary pathology.
  • the present invention also encompasses an oral pharmaceutical formulation comprising NAC amide and an antihyperglycemic agent.
  • NAC amide or a derivative thereof may be of benefit for treating and/or preventing obesity and/or eating disorders, other addictive or compulsive disorders, including tobacco (nicotine) and opiate additions.
  • This invention also encompasses administering NAC amide or a derivative thereof in conjunction with nicotine.
  • the physiologic effects of nicotine are well known.
  • NAC amide or a derivative thereof may cause vasodilation and improve cerebral blood flow, thereby resulting in a synergistic cerebral function-enhancing effect.
  • the levels of glutathione in the plasma are relatively low, in the micromolar range, while intracellular levels are typically in the millimolar range. Therefore, intracellular cytosol proteins are subjected to vastly higher concentrations of glutathione than extracellular proteins.
  • the endoplasmic reticulum a cellular organelle, is involved in processing proteins for export from the cell. It has been found that the endoplasmic reticulum forms a separate cellular compartment from the cytosol, having a relatively oxidized state as compared to the cytosol, and thereby promoting the formation of disulfide links in proteins, which are often necessary for normal activity.
  • cells may be induced to produce proteins for export from the cells, and the progression of the pathology is interrupted by interference with the production and export of these proteins.
  • many viral infections rely on cellular production of viral proteins for infectivity. The interruption of the production of these proteins will interfere with infectivity.
  • certain conditions involve specific cell-surface receptors, which must be present and functional. In both cases, cells that are induced to produce these proteins will deplete reduced glutathione in the endoplasmic reticulum. It is noted that cells that consume glutathione will tend to absorb glutathione from the plasma, and may be limited by the amounts present.
  • the reducing conditions in the endoplasmic reticulum may be interfered with, and the protein production blocked.
  • Normal cells may also be subjected to some interference; however, in viral infected cells, or cells otherwise abnormally stimulated, the normal regulatory mechanisms may not be intact, and the redox conditions in the endoplasmic reticulum will not be controlled by the availability of extracellular glutathione.
  • the administration of NAC amide or its derivatives may serve to replenish GSH or the effects of GSH and provide significant effects for such conditions.
  • DNA virus and herpes virus infections may be treated by administering NAC amide or a derivative thereof.
  • infection by the rabies virus, an RNA virus may be treated by the administration of glutathione. While standard treatments are available, and indeed effective when timely administered, glutathione may be useful in certain circumstances.
  • rabies virus infection may be treated, at least in part, by administering NAC amide or a derivative thereof according to the present invention.
  • One available treatment for rabies is an immune serum.
  • the present invention encompasses the parenteral administration of NAC amide, or derivative thereof separately, or in combination with one or more immunoglobulins.
  • Coronary heart disease risk is increased by the consumption of a high-fat diet and is reduced by the intake of antioxidant vitamins, including vitamin E and vitamin C, as well as flavonoids.
  • High fat meals impair the endothelial function through oxidative stress, resulting in impaired nitric oxide availability. It has been found that vitamin C and vitamin E restore the vasoconstriction resulting from nitric oxide production by endothelium after a high fat meal.
  • NAC amide or a derivative thereof may be administered prophylactically to combat vascular disease.
  • one aspect of the invention provides synergistic therapies to patients by increasing antioxidant levels systemically or in specific organs as well as reducing oxidative, free radical generating and ionizing influences.
  • NAC amide therapy would be complemented with ultraviolet blocking sunglasses, and a tobacco smoking cessation plan, as necessary.
  • NAC amide or a derivative thereof can be used in combination with alpha tocopherol succinate, if necessary.
  • Free radicals occur in different parts or subparts of tissues and cells, with different inciting agents.
  • the injurious free radicals are in the fatty (lipid) coverings that insulate nerve fibers, i.e., the myelin sheaths.
  • MPSS methyl prednisolone sodium succinate
  • the present invention therefore provides a pharmaceutical composition comprising a combination of NAC amide or a NAC amide derivative and a glucocorticoid agent.
  • NAC amide can be used to curtail the virtually self-perpetuating, powerful biochemical cycles producing corrosive free radicals and toxic cytokines that are largely responsible for the signs and symptoms of AIDS. These biochemical cycles destroy considerable quantities of glutathione but they can eventually be brought under control, and normalized with sufficient, ongoing NAC amide therapy.
  • a typical example is the over production of a substance, 15 HPETE (15-hydroperoxy eicosatetraenoic acid), from activated macrophages.
  • HPETE is a destructive, immunosuppressing substance and requires glutathione for conversion into a non-destructive, benign molecule.
  • the problem is that once macrophages are activated, they are difficult to normalize. Once inside cells, GSH curtails the production of free radicals and cytokines, corrects the dysfunctions of lymphoctyes and of macrophages, reinforces defender cells in the lungs and other organs and halts HIV replication in all major infected cell types, by preventing the activation of the viral DNA by precluding the activation of NF- ⁇ , inhibiting the TAT gene product of HIV that drives viral replication and dismantling the g l20 proteins of the virus coat.
  • NAC amide can be provided to disrupt the gpl20 protein, thereby offering a potential mode of preventing transmission of virus not only to other cells in the patient, but perhaps to others.
  • NAC amide or a derivative thereof provides combinations of NAC amide or a derivative thereof with the following drugs: cycloporin A, thalidomide, pentoxifylline, selenium, desferroxamine, 2L- oxothiazolidine, 2L-oxothiazolidine-4-carboxylate, diethyldithiocarbamate (DDTC), BHA, nordihydroguairetic acid (NDGA), glucarate, EDTA, R-PIA, alpha-lipoic acid, quercetin, tannic acid, 2'-hydroxychalcone, 2-hydroxychalcone, flavones, alpha-angelicalactone, fraxetin, curcurmin, probucol, and arcanut (areca catechul).
  • drugs cycloporin A, thalidomide, pentoxifylline, selenium, desferroxamine, 2L- oxothiazolidine, 2L-oxothia
  • NAC amide and its derivatives may have application in the therapy for inflammatory diseases.
  • NAC amide or a derivative thereof may
  • NAC amide or a derivative thereof may be administered to patients suffering from an inflammatory disease, such as arthritis of various types, inflammatory bowel disease, etc.
  • the present invention also provides combination pharmaceutical therapy including NAC amide or NAC amide derivative and an analgesic or anti-inflammatory agent, for example, opiate agonists, glucocorticoids or non-steroidal antiinflammatory drugs (NSAIDS), including opium narcotics, meperidine, propoxyphene, nalbuphine, pentazocine, buprenorphine, aspirin, indomethacin, diflunisal, acetominophen, ibuprofen, naproxen, fenoprofen, piroxicam, sulindac, tolmetin, meclofenamate, zomepirac, penicillamine, phenylbutazone, oxyphenbutazone, chloroquine
  • an analgesic or anti-inflammatory agent for example, opiate agonists,
  • NAC amide and its derivatives may also be beneficial for the treatment of parotitis, cervical dysplasia, Alzheimer's disease, Parkinson's disease, aminoquinoline toxicity, gentamycin toxicity, puromycin toxicity, aminoglycoside nephrotoxicity, paracetamol, acetaminophen and phenacetin toxicity.
  • NAC amide or a derivative thereof may be added to a virus-contaminated fluid or potentially contaminated fluid to inactivate the virus. This occurs, for example, by reduction of critical viral proteins.
  • NAC amide or a derivative thereof is added to blood or blood components prior to transfusion.
  • the added NAC amide or derivative of NAC amide is added in a concentration of between about 100 micromolar to about 500 millimolar or to a solubility limit, whichever is lower, and more preferably in a concentration of about 10-50 millimolar.
  • the addition of NAC amide or a derivative thereof to whole blood, packed red blood cells, or other formed blood components may be used to increase the shelf like and/or quality of the cells or formed components.
  • the present invention encompasses the use of NAC amide, or derivative thereof or a pharmaceutically acceptable salt or ester thereof, in the treatment and/or prevention of cosmetic conditions and dermatological disorders of the skin, hair, nails, and mucosal surfaces when applied topically.
  • compositions for topical administration include (a) NAC amide, or derivative thereof or a suitable salt or ester thereof, or a physiologically acceptable composition containing NAC amide; and (b) a topically acceptable vehicle or carrier.
  • the present invention also provides a method for the treatment and/or prevention of cosmetic conditions and/or dermatological disorders that entails topical administration of NAC amide- or NAC amide-derivative containing compositions to an affected area of a patient.
  • Such compositions and methods are useful in anti-aging treatments and therapies, as well as for the treatment of wrinkles, facial lines and depressions, particularly around the eyes and mouth, creases in the skin, age spots and discolorations, and the like.
  • the present invention provides methods and compositions useful for cancer and pre-cancer therapy utilizing NAC amide, or derivative thereof or its pharmaceutically acceptable salts or esters.
  • the present invention particularly relates to methods and compositions comprising NAC amide or a derivative thereof in which apoptosis is selectively induced in cells of cancers or precancers.
  • the present invention relates to a method of selectively inducing apoptosis of precancer cells by administering an effective amount of NAC amide or a derivative thereof to a subject.
  • NAC amide or a derivative thereof can be topically administered to the subject.
  • the present invention relates to a method of selectively inducing apoptosis in cancer cells by administering an effective amount of NAC amide or a derivative thereof to a subject.
  • NAC amide or its derivative can be topically administered to the subject in this embodiment.
  • Selective apoptosis refers to a situation in which corresponding normal, non-transformed cells do not undergo NAC amide-induced cell death.
  • the present invention relates to a method comprising reducing the number of cancer cells present in a subject by administering NAC amide or a derivative thereof to the subject as an adjunct to chemotherapy or radiation therapies such that the susceptibility of the cancer cells to apoptosis is enhanced relative to the non-cancer cells of the subject.
  • the present invention relates to a method comprising administering an effective amount of NAC amide or a derivative thereof as an adjunct to p53 therapy, including p53 gene therapy.
  • the cancer or precancer cells in which apoptosis is induced are generally those which exhibit at least one functional p53 allele.
  • administration of NAC amide results in restoration of mutant p53 protein conformation and/or activity to a functional state. It is to be understood that an endogenous functional p53 allele is not necessary for methods comprising p53 therapy, including p53 gene therapy.
  • methods which comprise administering NAC amide or a derivative thereof to selectively induce cells which arise in hyperproliferative or benign dysproliferative disorders.
  • Another embodiment of the present invention encompasses the use of NAC amide or a derivative thereof in methods for selective cell cycle arrest comprising contacting the cell with an amount of NAC amide or a derivative thereof to selectively arrest cells at a particular stage of the cell cycle.
  • administration of NAC amide can lead to prolonged transition through Gl phase.
  • This cell cycle arrest may be influenced by an increase in p21 expression.
  • the methods of the present invention can also be utilized to reduce or inhibit tumor vascularization, or to induce differentiation in cancer cells.
  • the present invention is directed to the use of NAC amide or a derivative thereof to treat cancers and tumors that may be induced by faulty signals from the microenvironment that result in loss of tissue organization in cancerous organs and loss of genomic stability in individual cancer cells. Loss of tissue structure may lead to certain cancers. Involved in this process are matrix metalloproteinases (MMPs), which are enzymes that are important not only during an organism's development and during wound healing, but also in promoting tumorigenesis or carcinogenesis.
  • MMPs matrix metalloproteinases
  • MMPs contribute prominently to microenvironmental signals because these proteolytic enzymes degrade structural components of the basement membrane and extracellular matrix (ECM) and digest the contacts that bind epithelial cells into sheets, thereby permitting the invasion of tumor cells and metastasis. MMPs can also release cell-bound inactive precursor forms of growth factors; degrade cell-cell and cell-ECM adhesion molecules; activate precursor zymogen forms of other MMPs; and inactivate inhibitors of MMPs and other proteases. Further, these enzymes induce the epithelial-mesenchymal transition, or EMT, a transition of one cell state to another that causes epithelial cells to disassociate from their neighbors, break free and acquire the ability to move through the body. While this process is essential for normal development in the embryo, in cancers, such as breast cancer, EMT provides mobility for tumor cells and assists tumor cells in penetrating barriers, such as wall of lymph and blood vessels, thus facilitating metastasis.
  • EMT epithelial-mesenchymal transition
  • MMP-3 is a particular type of metalloproteinase that has been observed to induce transformation in mammary epithelial cells in culture and in transgenic mice. MMP-3 has been found to cause normal cells to express the Raclb protein, an unusual form of Rho GTPase that has previously been found only in cancers. Raclb dramatically alters the cell skeleton, which facilitates the separation and movement of epithelial cells from surrounding cells. (D.C. Radisky et al., 2005, Nature, 436: 123-127). Changes in the cell skeleton induced by Raclb stimulate the production of highly reactive oxygen molecules, called reactive oxygen species (ROS), which can promote cancer by leading to tissue disorganization and by damaging genomic DNA.
  • ROS reactive oxygen species
  • Rhosin The increased amounts of ROS induced by Raclb activate major genes that control the EMT, which then begins a cascade of massive tissue disorganization and stimulates the development of cancer by directly affecting genomic DNA, for example, causing deletion or duplication of large regions of the DNA.
  • MMPs can also activate oncogenes and comprising the integrity of the DNA in an organism's genome.
  • NAC amide in accordance with the present invention can be used to block the effects of ROS. This can be achieved, for example, by administering or introducing NAC amide or a derivative thereof to cells, tissues, and/or the body of a subject in need thereof, to affect or target molecules in the pathways leading to epithelial-mesenchymal transition.
  • NAC amide or a derivative thereof can be used to inhibit MMP-3 and its functions, such as MMP-3-induced downregulation of epithelial cytokeratins and
  • NAC amide or a derivative thereof can also be used to target ROS indirectly or directly, and/or the processes by which ROS activate genes that induce the EMT.
  • the present invention encompasses compositions and methods comprising NAC amide or a derivative thereof for the suppression of allograft rejection in recipients of allografts.
  • the present invention provides a NAC amide or derivative of NAC amide in a method of supporting or nurturing the growth of stem cells for stem cell transplants, particularly stem cells cultured in vitro prior to introduction into a recipient animal, including humans.
  • the present invention provides methods of inhibiting, preventing, treating, or both preventing and treating, central nervous system (CNS) injury or disease, neurotoxicity or memory deficit in a subject, involving the administration of a therapeutically effective amount of NAC amide, or derivative thereof or a pharmaceutically acceptable composition thereof
  • CNS injuries or disease include traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke, cerebral ischemia, neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease, Alzheimer's disease, brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS malaria or treatment with anti-malaria agents, and other CNS traumas.
  • TBI traumatic brain injury
  • PTE posttraumatic epilepsy
  • stroke cerebral ischemia
  • neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease, Alzheimer's
  • the present invention embraces a method of treating and/or preventing a subject suffering from a CNS injury or disease comprising administering to the subject a composition comprising a therapeutically effective amount of NAC amide or a derivative thereof.
  • the present invention relates to a method of preventing or inhibiting a CNS injury or disease in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of NAC amide or a derivative thereof.
  • the present invention embraces a method of preventing, inhibiting or treating neurotoxicity or memory deficit in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of NAC amide or a derivative thereof. Where the memory deficit may be induced by electroconvulsive shock therapy for treating and/or preventing diseases and disorders such as depression and schizophrenia, the composition may be administered before the
  • the CNS injury or disease may be traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke, cerebral ischemia, or a neurodegenerative disease.
  • CNS injury may be induced by fluid percussion, by trauma imparted by a blunt object, for example on the head of the subject, by trauma imparted by an object which penetrates the head of the subject, by exposure to radiation, ionizing or iron plasma, a nerve agent, cyanide, toxic concentrations of oxygen, CNS malaria, or an anti-malaria agent.
  • the therapeutically effective amount of NAC amide or a derivative thereof administered to the subject is the amount required to obtain the appropriate therapeutic effect, for example, about 0.001 mg to about 20 mg per kg of the subject, preferably about 1 mg to about 10 mg per kg of the subject, more preferably about 3 mg to about 10 mg per kg of the subject.
  • the total daily amount of NAC amide or a derivative thereof administered to the subject is about 50 mg to about 1200 mg, or about 100 mg to about 1000 mg, or about 200 mg to about 800 mg, or about 300 mg to about 600 mg.
  • the invention encompasses a method of treating a subject (e.g., an animal, including humans) before the subject is exposed or likely to be exposed to a risk of CNS injury or damage, or before the subject is exposed to conditions likely to cause neurotoxicity or memory deficit or both, by administering NAC amide or a derivative thereof to a subject in a period of time prior to the exposure of the subject to the risk of CNS injury or damage, etc.
  • a subject e.g., an animal, including humans
  • NAC amide or a derivative thereof to a subject in a period of time prior to the exposure of the subject to the risk of CNS injury or damage, etc.
  • neurotoxicity or memory deficit include electroconvulsive shock therapy, traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke, cerebral ischemia, neurodegenerative diseases, fluid percussion, a blunt object impacting the head of the subject, an object penetrating the head of the subject, radiation, ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, CNS malaria, and anti-malaria agents.
  • CNS injury or damage neurotoxicity or memory deficit
  • Other conditions that may cause CNS injury or damage, neurotoxicity or memory deficit include, without limitation, certain medical procedures or conditions associated with risk for CNS ischemia, hypoxia or embolism such as brain tumor, brain surgery, other brain-related disorders, open heart surgery, carotid endarterectomy, repair of aortic aneurysm, atrial fibrillation, cardiac arrest, cardiac or other catheterization, phlebitis, thrombosis, prolonged bed rest, prolonged stasis (such as during space travel or long trips via airplane, rail, car or other transportation), CNS injury secondary to air/gas embolism or decompression sickness.
  • certain medical procedures or conditions associated with risk for CNS ischemia such as brain tumor, brain surgery, other brain-related disorders, open heart surgery, carotid endarterectomy, repair of aortic aneurysm, atrial fibrillation, cardiac arrest, cardiac or other catheterization, phlebitis, thrombosis, prolonged bed rest, prolonged sta
  • the period of time may be about 72 hours prior to the time of expected exposure, or about 48 hours prior to the time of expected exposure, or about 12 hours prior to the time of expected exposure, or about 4 hours prior to the time of expected exposure, or about 30 minutes-2 hours prior to the time of expected exposure.
  • the administration of NAC amide may be continuous from the initial time of treatment to the end of treatment.
  • a transdermal patch or a slow-release formulation may be used to continually administer NAC amide or a derivative thereof to the subject for a given period of time.
  • NAC amide or a derivative thereof may be administered to the subject periodically.
  • NAC amide or a derivative thereof may first be administered at about 24 hours before the time of expected exposure and then administered at about every 2 hours thereafter.
  • the NAC amide- or NAC amide derivative-containing composition may further comprise a pharmaceutically acceptable excipient and the composition may be administered
  • the present invention encompasses a pharmaceutical composition for treating or preventing CNS injury, disease or neurotoxicity in a subject comprising a therapeutically effective amount of NAC amide or a derivative thereof and a pharmaceutically acceptable excipient.
  • the invention embraces a kit comprising a composition comprising a therapeutically effective amount of NAC amide or a derivative thereof.
  • the kit may further comprise a device for administering the composition to a subject such as an injection needle, an inhaler, a transdermal patch, as well as
  • anti-cancer treatments involving NAC amide or a derivative thereof are designed to specifically target cancer and tumor cells.
  • This embodiment is directed to the use of nano-sized particles for the in vivo and ex vivo administration of NAC amide or a derivative thereof to cancer and tumor cells.
  • cancer cells which display more receptors for the vitamin folic acid (or folate) and absorb more folic acid than do normal, healthy cells, are able to be preferentially targeted.
  • core or shell nanogels, or nanoparticles can be functionalized with folic acid or folate conjugated or linked to NAC amide or a derivative thereof without disrupting or destroying the folic acid binding site to its cell receptor.
  • Such functionalized nanoparticles can be introduced into a subject, particularly a folate-deprived subject, with a cancer, e.g., epithelial cancer, in whom the cancer cells have excess folic acid receptors which will preferentially bind the folic acid-NAC amide (or folic acid-NAC amide derivative) nanoparticles and endocytose them.
  • a cancer e.g., epithelial cancer
  • NAC amide or a derivative thereof exert its therapeutic effects, for example, by inhibiting ROS and/or other target molecules that play a role in initiating, fueling, and/or maintaining cancer cells, and/or ultimately killing the cancer cells.
  • PAMAM dendritic polymers ⁇ 5 nm in diameter can be used as carriers of NAC amide, as described in J.F. Kukowska-Latallo et al., 2005, Cancer Res., Jun
  • Acetylated dendrimers can be conjugated to folic acid as a targeting agent and then coupled to NAC amide or a derivative thereof and either fluorescein or 6- carboxytetramethylrhodamine.
  • NAC amide or a derivative thereof can be coupled to folic acid to form a conjugate and the conjugate can be coupled to the
  • nanoparticles These conjugates can be injected i.v. into a tumor-bearing patient or mammal, especially those tumors that overexpress the folic acid receptor.
  • the folate-conjugated nanoparticles can then concentrate in the tumor and tissue following administration, where the delivered NAC amide or NAC derivative can interact with ROS in the cells, and/or target other molecules to kill the cancer or tumor cells.
  • the tumor tissue localization of the folate- targeted polymer may be attenuated by prior i.v. injection of free folic acid.
  • polymers or nanoparticles can be functionalized to display glutathione-NAC amide or glutathione-NAC amide derivative conjugates, which can then be used to deliver NAC amide or a derivative thereof to cancer cells which display increased numbers of glutathione receptors on their cell surfaces.
  • the NAC amide-glutathione nanoparticles can then be targeted to those cancer cells having glutathione receptors and preferentially endocytosed by the cells.
  • the present invention provides directed delivery of NAC amide or a derivative thereof to cells, such as cancer cells that express high levels of receptors for folic acid (folate) or glutathione.
  • NAC amide (“NACA”) or a derivative thereof is coupled to a ligand for a cell surface receptor (e.g., folic acid or glutathione) to form a conjugate.
  • a cell surface receptor e.g., folic acid or glutathione
  • This NACA- ligand conjugate is coated or adsorbed onto readily injectable nanoparticles using procedures known to those skilled in the art. Accordingly, the nanoparticles containing NAC amide or a derivative thereof (“nano-NACA particles”) may be preferentially taken up by cancer or tumor cells where the NAC amide will exert its desired effects.
  • the present invention is drawn to a method of directed delivery of NAC amide or a derivative thereof to host cells expressing high levels of surface receptor for a ligand, comprising: a) conjugating acetylated dendritic nanopolymers to ligand; b) coupling the conjugated ligand of step (a) to NAC amide or a derivative thereof to form NAC amide-ligand nanoparticles; and c) injecting the nanoparticles of (b) into the host.
  • the present invention is drawn to a method of directed delivery of NAC amide or a derivative thereof to host cells expressing high levels of surface receptor for a ligand, comprising: a) coupling NAC amide or a derivative thereof to the surface receptor ligand to form a NAC amide-ligand conjugate; b) adsorbing the NAC amide-ligand conjugate onto nanoparticles; and c) injecting the nanoparticles of (b) into the host.
  • Another embodiment of the present invention provides a compound of the formula I: wherein: R] is OH, SH, or S-S-Z;
  • X is C or N
  • R 3 is absent or wherein: R 4 is NH or O;
  • R 5 is CF 3 , NH 2 , or CH 3
  • Ri is S-S-Z
  • X and X' are the same
  • Y and Y' are the same
  • R 2 and R 6 are the same
  • R 3 and R 7 are the same.
  • Ri is S
  • X is C
  • Y is NH-CH 3
  • R 2 is H
  • R 3 is
  • R4 is O, and R 5 is CH 3 .
  • the present invention also provides compounds of the formula I above, wherein Ri is S, X is
  • Compounds of the present invention also include compounds of formula I wherein R] is 0, X is C, Y is NH 2 , R 2
  • R4 is NH
  • R 5 is NH 2
  • Another embodiment of the present invention provides compounds of formula I wherein Ri is O, X is C, Y is OH, R 2 is absent, R 3 is
  • R 4 is O, and R5 is CH 3 .
  • the compounds disclosed herein can be chiral, i.e., enantiomers, such as L- and D- isomers, or can be racemic mixtures of D- and L-isomers.
  • Preferred compounds include, but are not limited to, the following:
  • Compounds I through XVIII comprise NAC amide or NAC amide derivatives.
  • a process for preparing an L- or D- isomer of the compounds of the present invention comprising adding a base to L- or D-cystine diamide dihydrochloride to produce a first mixture, and subsequently heating the first mixture under vacuum; adding a methanolic solution to the heated first mixture; acidifying the mixture with alcoholic hydrogen chloride to obtain a first residue; dissolving the first residue in a first solution comprising methanol saturated with ammonia; adding a second solution to the dissolved first residue to produce a second mixture; precipitating and washing the second mixture; filtering and drying the second mixture to obtain a second residue; mixing the second residue with liquid ammonia, and an ethanolic solution of ammonium chloride to produce a third mixture; and filtering and drying the third mixture, thereby preparing the L- or D-isomer compound.
  • the base can comprise liquid ammonia or methylamine.
  • the second solution comprises water, an acetate salt, and an anhydride, wherein the acetate salt can comprise sodium acetate or sodium trifluoroacetate, and the anhydride can comprise acetic anhydride or trifluoroacetic anhydride.
  • the second solution can comprise
  • dichloromethane triethylamine, and l,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
  • the second residue can be further mixed with sodium metal.
  • the process further comprises dissolving the L- or D-isomer compound in ether; adding to the dissolved L- or D-isomer compound an ethereal solution of lithium aluminum hydride, ethyl acetate, and water to produce a fourth mixture; and filtering and drying the fourth mixture, thereby preparing the L- or D-isomer compound.
  • the compounds of formula II and III are prepared by mixing L- or D-cystine diamide dihydrochloride with liquid ammonia; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution;
  • the compounds of formula IV and V are prepared by mixing L- or D-cystine diamide dihydrochloride with methylamine; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution;
  • the compounds of formula VII and VIII are prepared by mixing L- or D-cystine diamide dihydrochloride with ammonia; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution;
  • the compounds of formula XIII and XIV are prepared by mixing L- or D-cystine diamide dihydrochloride with ammonia; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution;
  • the compounds of formula XI and XII are prepared by mixing L- or D-cystine diamide dihydrochloride with liquid ammonia; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution; acidifying the filtrate with alcoholic hydrogen chloride for obtaining a first residue;
  • the compounds of formula XVII and XVIII are prepared by mixing L-or D-cystine diamide dihydrochloride with liquid ammonia; warming the mixture to remove volatiles; warming mixture in vacuo to 50°C; adding a warm methanolic solution; filtering the solution; acidifying the filtrate with alcoholic hydrogen chloride for obtaining a first residue;
  • Another embodiment of the invention provides a process for preparing an L- or D- isomer of the compounds disclosed herein, comprising mixing S-benzyl-L- or D-cysteine methyl ester hydrochloride or O-benzyl-L- or D-serine methyl ester hydrochloride with a base to produce a first mixture; adding ether to the first mixture; filtering and concentrating the first mixture; repeating steps (c) and (d), to obtain a first residue; adding ethyl acetate and a first solution to the first residue to produce a second mixture; filtering and drying the second mixture to produce a second residue; mixing the second residue with liquid ammonia, sodium metal, and an ethanolic solution of ammonium chloride to produce a third mixture; and filtering and drying the third mixture, thereby preparing the L- or D-isomer compound.
  • the base can comprise liquid ammonia or methylamine.
  • the second solution comprises water, an acetate salt, and an anhydride, wherein the acetate salt can comprise sodium acetate or sodium trifluoroacetate, and the anhydride can comprise acetic anhydride or trifluoroacetic anhydride.
  • the second solution can comprise
  • the process further comprises dissolving the L- or D-isomer compound in ether; adding to the dissolved L- or D-isomer compound an ethereal solution of lithium aluminum hydride, ethyl acetate, and water to produce a fourth mixture; and filtering and drying the fourth mixture, thereby preparing the L- or D-isomer compound.
  • the compounds of formula II and III are prepared by mixing S-benzyl-L- or D- cysteine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of ammonia over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding water, sodium acetate and acetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; and crystallizing the third residue from isopropanol
  • the compounds of formula IV and V are prepared by mixing S-benzyl-L- or D- cysteine methyl ester hydrochloride with a cold methanolic solution of methylamine; passing a stream of methylamine over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding water, sodium acetate and acetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt;
  • the compounds of formula VII and VIII are prepared by mixing S-benzyl-L- or D- cysteine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of methylamine over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding trifluoroacetic anhydride to this suspension; adding water, sodium trifluoroacetate and trifluoroacetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; and crystallizing the
  • the compounds of formula IX and X are prepared by mixing O-benzyl-L- or D-serine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of methylamine over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding water, sodium acetate and acetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; and crystallizing the third residue from isopropan
  • the compounds of formula XIII and XIV are prepared by mixing S-benzyl-L- or D- cysteine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of ammonia over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding dichloromethane, triethylamine, and l ,3-bis(benzyloxycarbonyl)-2- methyl-2-thiopseudourea; lowering the temperature to 0°C; precipitating the mixture;
  • the compounds of formula XI and XII are prepared by (a) mixing S-benzyl-L- or D- cysteine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of ammonia over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding of water, sodium acetate and acetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; dissolving the third
  • the compounds of formula XV and XVI are prepared by (a) mixing O-benzyl-L- or D-serine methyl ester hydrochloride with a cold methanolic solution of ammonia; passing a stream of ammonia over the mixture; sealing the flask securely; concentrating the mixture; adding ether; filtering the solution; concentrating the filtrate; adding ether and filtering again, to obtain a residue; suspending the residue with ethyl acetate; adding acetic anhydride to this suspension; adding of water, sodium acetate and acetic anhydride; raising the temperature to 65°C; cooling the mixture; filtering the crude solid; washing with ethyl acetate; drying the precipitate for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; dissolving the third residue
  • Yet another embodiment of the invention provides a process for preparing a compound as disclosed herein, comprising mixing cystamine dihydrochloride with ammonia, water, sodium acetate, and acetic anhydride to produce a first mixture; allowing the first mixture to precipitate; filtering and drying the first mixture to produce a first residue; mixing the second residue with liquid ammonia, sodium metal, and an ethanolic solution of ammonium chloride to produce a second mixture; filtering and drying the second mixture, thereby preparing the compound.
  • the compound of formula VI is prepared by mixing cystamine dihydrochloride with ammonia; adding water, sodium acetate and acetic anhydride; raising the temperature to 50°C; precipitating the mixture; washing the mixture with water; filtering the crude solid; drying the mixture for obtaining a second residue; mixing the second residue with liquid ammonia; slowly adding sodium metal; removal of the solvent; slowly adding an ethanolic solution of ammonium chloride; filtering and separating the inorganic salt; concentrating and cooling the filtrate to obtain a third residue; and crystallizing the third residue from
  • NAC Amide shows better protection that NAC in acetaminophen induced cell death in HEPG2 cells.
  • NAC amide was assessed for its protective effects against oxidative toxicity induced by glutamate in PC 12 cells.
  • N-(l-pyrenyl)-maleimide was purchased from N-(l-pyrenyl)-maleimide (NPM).
  • PC 12 cells were plated at a density of 25 x 10 cells/well in a 24-well, collagen-coated plate for morphological assessment.
  • the plate was divided into five groups in triplicate: 1) control: no glutamate, no NAC amide; 2) Nerve Growth Factor (NGF) control: NGF (100 ng/ml), no glutamate, no NAC amide; 3) NAC amide only: NGF (100 ng/ml), no glutamate, NAC amide (750 ⁇ ); 4) glutamate only: NGF (100 ng/ml), glutamate (10 mM), no NAC amide; and 5) Glu + NAC amide: NGF (lOOng/ml), glutamate (10 mM), NAC amide (750 ⁇ ).
  • LDH assay For the lactate dehydrogenase (LDH) assay, cells were plated at a density of 2.5 x 10 5 cells/well in a 24 well collagen-coated culture plate and, after 24 h; the medium was replaced with fresh D EM medium containing the desired concentration of glutamate and NAC amide.
  • the LDH activity released was determined using the kit as described below.
  • MTS assay cells were plated at a density of 10 5 cells/well in a 24 well collagen-coated plate. At the end of the experiments, cell viability was assayed using the kit as described.
  • the LDH activity assay was performed with the CytoTox96® Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA), which quantitatively measured the activity of LDH, a stable cytosolic enzyme that is released upon cell lysis [Technical Bulletin No. 163, Promega].
  • LDH in culture supernatants was measured with a 30-minute coupled enzymatic assay, which resulted in the conversion of a tetrazolium salt into a red formazan product.
  • the amount of color formed was proportional to the degree of damage to the cell membranes.
  • Absorbance data were collected using a BMG microplate reader (BMG Labtechnologies, Inc., Durham, NC, USA) at 490 nm. LDH leakage was expressed as the percentage (%) of the maximum LDH release in the cells treated with glutamate alone (100%), according to the formula:
  • MTS assay Cell Titer 96® Aqueous One solution cell proliferation Assay, Promega
  • MTS assay Cell Titer 96® Aqueous One solution cell proliferation Assay, Promega
  • GSH measurement Cellular levels of GSH were determined by using the method as described in Winters R.A. et al., Anal Biochem., 227(1): 14-21 , 1995. Cells were seeded at a density of 80,000 cells/cm 2 on poly-D-lysine coated (0.05 mg/ml) 75 cm 2 flasks (5 ml/flask) for GSH measurement. After 24 hours, the flasks were incubated with fresh medium containing glutamate (10 mM), or BSO (0.2 mM) or Glu + BSO + NAC amide (750 ⁇ ) at 370 C for another 24 h.
  • glutamate 10 mM
  • BSO 0.2 mM
  • Glu + BSO + NAC amide 750 ⁇
  • MDA measurement To prepare the solution, 350 ⁇ of straight cell homogenate, 100 ⁇ of 500 ppm BHT (butylated hydroxytoluene), and 550 ⁇ of 10% TCA (trichloroacetic acid) were combined and boiled for 30 min. The tubes were cooled on ice and centrifuged for 10 min at 2500 rpm. Five hundred (500) ⁇ of the supernatant were removed and 500 ⁇ of TBA (thiobarbituric acid) were added. The tubes were boiled again for 30 min, and then cooled on ice.
  • BHT butylated hydroxytoluene
  • TCA trichloroacetic acid
  • Protein determination and statistical analysis Protein levels were determined by the Bradford method with Coomassie Blue (Bio-Rad) (Bradford M.M., Anal Biochem., 72:248- 54, 1976). The data were given as the mean ⁇ SD. The one-way analysis of variance test was used to analyze the significance of the differences between the control and experimental groups.
  • Glutamate toxicity was evaluated by 1) morphological assessment of PC 12 cells in the presence of glutamate; 2) measuring the amount of LDH released in the media 24 h after glutamate exposure; and 3) measuring cell viability using the MTS assay. As shown in FIGS.
  • Example 1 demonstrate that NAC amide treatment significantly increased PC 12 cell GSH levels. When cells were exposed to 10 mM glutamate, a significant reduction in GSH levels was observed (Table 1).
  • PC 12 cells were seeded and grown for 24 hours, then they were treated with either GLU (10 mM); NAC amide (750 ⁇ ); GLU (10 mM) + NAC amide (750 ⁇ ); GLU (10 mM) + BSO (0.2 mM) + NAC amide (750 ⁇ ); or BSO (0.2 mM). Twenty hours later, cells were removed and analyzed for GSH levels, as described in the text. Values represent means ⁇ SD. Statistically different values of * P ⁇ 0.05 were determined, compared to control. ** P ⁇ 0.001 compared to glutamate-treated group. *** P ⁇ 0.05 compared to glutamate-treated group.
  • NAC amide increased the PC 12 cell GSH level two fold, compared to the control group.
  • similar results were obtained when Chinese hamster ovary (CHO) cells were incubated with NAC amide (data not shown).
  • NAC amide prevented the marked decline of cellular GSH levels that normally occurs after glutamate treatment (Table 1). Glutamate inhibits cystine uptake, resulting in the loss of cellular GSH, while buthionine-sulfoximine (BSO) inhibits ⁇ -GCS activity and thereby causes the depletion of intracellular GSH.
  • BSO buthionine-sulfoximine
  • NAC amide further protected cells against intracellular peroxide accumulation.
  • Malondialdehyde (MDA) is a by-product of a free radical attack on lipids. Marked increase in MDA levels was observed in glutamate- exposed cells, as compared with the corresponding control cells (Table 2). Treatment with NAC amide completely protected cells against glutamate toxicity by lowering MDA levels.
  • NAC amide itself may act as a sulfhydryl group donor for GSH synthesis.
  • Example 1 shows that NAC amide protects PC 12 cells against glutamate-induced cytotoxicity by preventing glutamate-induced loss of cellular GSH and inhibiting lipid peroxides.
  • NAC amide can play a role in the treatment of neurodegenerative disorders such as cerebral ischemia and Parkinson's disease in which GSH levels are depleted in certain regions of the brain.
  • This Example examines the radioprotective effects of NAC amide.
  • the radioprotective role of NAC amide was compared with that of NAC with respect to increasing the levels of GSH and returning oxidative stress parameters to their control values.
  • the radiation (XRT) control received whole body irradiation by 6 Gy of 16 MeV electrons.
  • the NAC amide+XRT group received 500 mg/kg/day NAC amide immediately before irradiation and for three days after until sacrifice.
  • the rats were anesthetized and heparinized blood was collected via cardiopuncture. Following sacrifice, liver, lung, brain and spleen were removed and stored at -70°C until homogenization.
  • Rats were shipped in paper crates (4 in each crate). Rats were delivered with a certificate including serological, bacteriological, pathological parasitological information. They were divided into 4 cages (3 rats in each cage) and kept in a temperature controlled (20°C) room equipped to maintain a 12h light-dark cycle. Standard rat chow (Purina rat chow) and tap water were supplied in individual glass bottle and given ad libitum. Water was changed daily. Weights of the animal were taken before giving the NAC amide treatment solution and amount of food eaten and water consumed was not measured because NAC amide was given orally but not in the drinking water or food.
  • Standard rat chow Purified Rat chow
  • tap water were supplied in individual glass bottle and given ad libitum. Water was changed daily. Weights of the animal were taken before giving the NAC amide treatment solution and amount of food eaten and water consumed was not measured because NAC amide was given orally but not in the drinking water or food.
  • NAC amide was provided by Novetide Ltd (Haifa Bay, Israel) including certificate of analysis and MSDS (lot# 40233-64).
  • NAC amide feeding solution was prepared freshly each day right before the administration by weighing 1.25g NAC amide solid sample (Type HR- 120 electronic balance, A&D Company limited, Japan. S/N: 12202464) and adding into 10ml PBS solution and put on ice. One ml of this solution was administrated (gavaged) per rat orally by using animal feeding biomedical needles and 3ml BD Luer-Lok Tip syringes.
  • the peak area for GSH in the sample is 90860.25.
  • the GSH concentration (nM) is calculated from the standard curve. After determining the protein content (mg/ml) of the sample, for example: 16.5mg/ml, the calculation is as follows:
  • tissue homogenate was used to react with 100 ⁇ of 500 ppm
  • the peak area for MDA in the sample as 65289.23, The MDA concentration (nM) is calculated from the standard curve. After determining the protein content (mg/ml) of the sample, for example: 16.5mg/ml, the resulting calculation is as follows:
  • k(enzyme activity) l/60*ln(A0/A60)*(Total Volume of reaction/volume of sample) AO- Absorbance at 0 second
  • K(specific activity) J protein concentration.
  • Oxidative Stress Parameters in Animals After the blood samples were drawn, the animals were perfused by a cold antioxidant buffer first and then liver, brain and kidney samples were collected aseptically, rinsed in ice-cold saline and placed in petri dishes maintained on ice. The tissue samples kept at -70°C for the GSH, GSSG, and MDA determinations were made.
  • Glutathione (GSH) and Glutathione Disulfide (GSSG) Determination Cells or tissue samples were homogenized on ice and derivatized with N-(l-pyrenyl)-maleimide ( PM). The derivatized samples were injected onto a 3 ⁇ CI 8 column (Column Engineering) in a reverse phase HPLC system with a mobile phase of 35% water, 65% acetonitrile containing 1 mL/L of acetic acid and ophosphoric acid (R. Winters, et al., Anal. Biochem., 227: 14-21 (1995) and H.H. Draper et al, Free Rad. Biol. Med., 15:353-363 (1993)). Malondialdehyde (MDA) determinations were made as described in J. Gutteridge, Anal. Biochem., 69: 518-526 (1975).
  • MDA Malondialdehyde
  • InStat® by GraphPad Software, San Diego, CA will use One-way Analysis of Variance (ANOVA) and the Student-Newman-Keuls Multiple Comparisons Test to analyze data from experimental and control groups. The p values ⁇ 0.05 is considered significant.
  • AD4 is synonymous with NAC amide.
  • AD4 or NAC administration (500mg/kg orally):
  • NAC amide functions as a strong thiol antioxidant in radiation-induced oxidative stress. NAC does not increase GSH levels in tissues, presumably because it does not cross the cell membranes. Although plasma Cys level increased significantly, this was not reflected in the liver. NAC generally provides GSH only during increased demand on the GSH pool.
  • GSH a tripeptide consisting of ⁇ -glutamyl-cysteinyl-glycine
  • GSH a tripeptide consisting of ⁇ -glutamyl-cysteinyl-glycine
  • Several distinct mechanisms of radioprotection by GSH can be identified and include radical scavenging, hydrogen donation to damaged molecules, reduction of peroxides, and protection of protein thiol oxidative status.
  • GSH has been shown to decrease in tissues following irradiation. Since GSH is an endogenous radioprotector, modification of GSH concentration may be useful as radiation protection.
  • Cysteine provides the rate-limiting step in GSH synthesis since its apparent Km value for ⁇ -glutamyl-cysteine synthetase is close to the intracellular concentration of the amino acid.
  • administration of cysteine is not the ideal way to increase intracellular GSH, since it auto-oxidizes rapidly and can lead to the production of hydroxy 1 and thiyl radicals.
  • NAC a cysteine analogue that is a mucolytic agent and a treatment for paracetamol intoxication, promotes hepatic GSH synthesis. It penetrates the cell membrane and is rapidly deacetylated to L-cysteine, while also stimulating GSSG reductase. NAC can rapidly increase the hepatic GSH levels and maintain these levels for at least 6 hours (B. Wong et al., J. Pharm. Sci., 75:878-880 (1986)). NAC has also been shown to protect Chinese hamster ovary cells from lead and ⁇ -aminolevulinic acid-induced toxicity through restoration of the oxidative status of the cells by GSH replenishment.
  • NAC protects liver and brain of C57BL/6 mice from GSH depletion as a result of lead poisoning. Radioprotective effects of select thiols such as indomethacin, WR-2721, cysteamine, and diethyldithiocarbamate have been reported, though at higher concentrations these induce cellular toxicity.
  • the radioprotective effect of NAC has been demonstrated in human granulocyte/macrophage-colony forming cells.
  • the more radioresistant SW-1573 human squamous lung carcinoma cell line was not protected from X-ray induced cell death by NAC.
  • NAC amide is more lipophilic and able to more easily cross cell membranes than NAC. In this Example, the radioprotective function of NAC amide was compared with that of NAC in terms of increasing GSH levels and returning oxidative stress parameters to their control values.
  • MDA is a degradation product of the highly unstable lipid peroxides.
  • irradiation of Sprague Dawley rats resulted in increased MDA levels in liver and lung.
  • lung MDA levels were significantly lowered, while treatment with NAC did not change the MDA levels significantly.
  • NAC amide may be considered for use as a thiol radioprotectant to protect against such a complication.
  • NAC amide significantly increases thiol levels in plasma and liver and performs better than NAC as a radioprotecting agent.
  • NAC amide is administered between 1 and three grams per day, in two divided doses, between meals (on an empty stomach).
  • Encapsulated NAC amide (a formulation of NAC amide comprising 500 mg NAC amide and optionally, 250 mg USP grade crystalline ascorbic acid, and not more than 0.9 mg magnesium stearate, NF grade in an OO-type gelatin capsule) is suitable for administration.
  • the administration of exogenous NAC amide is expected to provide a dose response effect in patients, despite the production of large quantities of glutathione in the human body.
  • This Example describes a combination pharmaceutical composition to ameliorate the detrimental effects of acetaminophen, a drug that consumes glutathione in the liver during metabolism and, in excess doses, causes liver damage due to oxidative damage.
  • the composition includes 500 mg NAC amide, 250 mg crystalline ascorbic acid and 350 mg acetaminophen.
  • NACA did not induce any cell death by itself even at higher concentrations (l OmM). Cysteine levels in cells were restored to control after exposed to AAP with NACA treatment.
  • HEPG2 cells showed a dose dependent decrease in viability change when exposed to acetaminophen, the cells were exposed to concentrations 4-5 times more than the concentrations observed in AAP poisoning.
  • HEPG2 cells modified liver cells
  • Stock solutions of AAP were prepared by dissolving the drug in water and later diluted with media for the required concentration for all the experiments. The solutions were prepared fresh every time. Around 8000 cells/well were seeded in a 96 well plate. After attachment of the cells overnight, the cells were exposed to different concentrations of AAP from 24 hrs and 48 hrs. The media was removed after required exposure time and cell viability was measure using MTS reagent.
  • NACA provides a better protection than NAC in acetaminophen induced cell death in HEPG2 cells.
  • NACA did not induce any cell death by itself even at higher concentrations (l OmM). Cysteine levels in cells were restored to control after exposed to AAP with NACA treatment.
  • HEPG2 cells showed a dose dependent decrease in viability change when exposed to acetaminophen, the cells were exposed to concentrations 4-5 times more than the concentrations observed in AAP poisoning.
  • Experiments have to be performed to determine the efficacy of NACA in increasing glutathione levels, decreasing ROS in cells when exposed to to acetaminophen
  • This Example describes a combination pharmaceutical composition to ameliorate the detrimental effects of chlorpromazine, a phenothiazine drug that causes side effects, including tardive dyskinesia, which may be associated with excess free radical reactions.
  • the composition includes 500 mg NAC amide, 250 mg crystalline ascorbic acid and 200 mg chlorpromazine.
  • This Example describes a combination pharmaceutical composition to ameliorate the detrimental effects of aminoglycoside drugs (antibiotics), nonlimiting examples of which include neomycin, kanamycin, amikacin, streptomycin, gentamycin, sisomicin, netilmicin and tobramycin, a drug class which may be associated with various toxicities.
  • This damage may be related to oxidative damage or consumption of glutathione during metabolism.
  • the composition according to the present invention is an intravenous formulation, including the aminoglycoside in an effective amount, and NAC amide in an amount of about 10-20 mg/kg. Ascorbic acid in an amount of 5-10 mg/kg may be added as a stabilizer, FIGs 45-52 show that NACA block kidney cell toxicity from the antibiotic gentamycin.
  • This Example describes a urethral insert comprising NAC amide.
  • a composition containing 200 mg NAC amide, 50 mg ascorbic acid per unit dosage is mixed with carageenan and/or agarose and water in a quick-gelling composition, and permitted to gel in a cylindrical form having a diameter of about 3 mm and a length of about 30 mm.
  • the composition is subjected to nitric oxide to cause between 0.1-10% of the NAC amide to be converted to nitroso-NAC amide.
  • the gelled agarose is then freeze dried under conditions that allow shrinkage.
  • the freeze-dried gel is than packaged in a gas barrier package, such as a foil pouch or foil "bubble-pack".
  • the freeze-dried gel may then be used as a source of nitroso-NAC amide for administration transmucosally.
  • the cylindrical freeze-dried gel may be inserted into the male urethra for treatment of impotence, or administered sublingually for systemic vasodilation.
  • This Example describes an oral formulation for prophylaxis of vascular disease, e.g., in men over 40.
  • the composition includes 500 mg NAC amide, 250 mg USP grade crystalline ascorbic acid and 50 mg USP acetyl salicylic acid (aspirin) in an OO-type gelatin capsule. Typical administration is twice per day.
  • the acetyl salicylic acid may be provided in enteric release pellets within the capsule to retard release.
  • This Example describes an oral formulation for prophylaxis of vascular disease.
  • the composition contains 500 mg NAC amide, 200 mg USP grade crystalline ascorbic acid, and 200 mg arginine in an OO-type gelatin capsule.
  • Arginine is the normal starting substrate for the production of nitric oxide. Because arginine is normally in limited supply, a relative deficiency of arginine may result in impaired vascular endothelial function.
  • This Example describes an oral formulation for prophylaxis of vascular disease.
  • the composition includes 500 mg NAC amide, 200 mg USP grade crystalline ascorbic acid, and 200 mg vitamin E succinate in an OO-type gelatin capsule. Vitamin E consumption reduces the risk of heart attack and other vascular disease. Vitamin E succinate (alpha-tocopherol succinate) is a dry powder.
  • Example 11
  • This Example describes an oral formulation for prophylaxis of vascular disease.
  • esters are formed between agents that are useful combination therapies in order to provide for efficient administration, high bioavailability, and
  • esters include alpha tocopherol-ascorbate, alpha tocopherol-salicylate, and ascorbyl-salicylate.
  • the tocopherol ester maintains the molecule in a reduced state, allowing full antioxidant potential after ester cleavage.
  • esters may be administered alone or in combination with other agents, for example NAC amide.
  • the esters are administered to deliver an effective dose of salicylate equivalent of 100 mg per day for prophylaxis, or 750-1000 mg per dose for treatment of inflammatory diseases.
  • Tocopherol is administered in an amount of 100-500 IU equivalent.
  • Ascorbate is
  • a non-specific esterase may be provided in the formulation to cleave the ester after dissolution of the capsule. Therefore, a non-specific esterase, such as a bacterial or saccharomyces
  • yeast enzyme or an enriched enzyme preparation, may be included in the formulation as a powder or as pellets in the capsule.
  • This Example describes an oral formulation for prophylaxis of vascular disease.
  • the composition includes 500 mg reduced NAC amide, 200 mg USP grade crystalline ascorbic acid, and 100 mg nordihydroguaretic acid, in an OO-type gelatin capsule. Typical administration is twice per day. Nordihydroguaretic acid is a known lipoxygenase inhibitor. Thus, this composition may be used to treat inflammatory processes or as prophylaxis against vascular disease.
  • This Example describes a study observing the survival of rats receiving whole body, single-dose irradiation by X-rays (XRT) in the presence or absence of NAC or NAC amide (TOVA).
  • XRT X-rays
  • TOVA NAC or NAC amide
  • the first treatment of NAC or TOVA was administered 30 minutes to 1 hour after the irradiation.
  • the same amount 500mg/kg NAC or TOVA daily was administered for 4 or 5 consecutive days.
  • Group 2 rats received NAC only (n 3) at an amount of 500mg/kg body weight NAC daily for 5 consecutive days without XRT.
  • Group 2 rats received NAC only (n 3) at an amount of 500mg/kg body weight NAC daily for 5 consecutive days without XRT.
  • Group 5 rats received one treatment of NAC at 500 mg/kg body weight before XRT
  • Group 7 rats received one treatment of NAC at 500 mg/kg body weight before XRT
  • the rats were observed twice a day, and the survival status of rats in each group will be recorded.
  • the mean survival days were calculated for each group and compared to the survival differences of the three groups of rats at the end of the experiment.
  • Table 15 shows the number of animals that survived under conditions where NAC or TOVA was administered pre- or post-XRT treatment.
  • Table 16 shows the survival rate percentage of rats receiving NAC or TOVA pre- or post-XRT treatment.
  • FIG. 6 is a graphical representation comparing the percentage survival rates as presented in Table 16. These results show that rats pre-treated with NAC or TOVA before XRT have a higher survival rate than those receiving XRT alone.
  • Lung contusion is a common problem from blunt chest trauma caused by mechanical forces and by exposure to blast overpressure, often with fatal consequences. Lung contusion is also a risk factor for the development of pneumonia, severe clinical acute lung injury
  • NACA antioxidant N-acetylcysteine amide
  • integrin CD1 lb mRNA and lung inflammatory chemokine mRNA expression corresponded with activation of integrin CD1 lb mRNA and lung inflammatory chemokine mRNA expression; macrophage inflammatory protein- 1 (MIP-1), monocyte chemotactic peptide- 1 (MCP-1), and cytokine-induced neutrophil chemoattractant-1 (CINC-1).
  • MIP-1 macrophage inflammatory protein- 1
  • MCP-1 monocyte chemotactic peptide- 1
  • CINC-1 cytokine-induced neutrophil chemoattractant-1
  • N- Acetylcysteine amide significantly reduced infiltration of neutrophils and CD1 lb mRNA activation in lungs, and completely blocked activation of MIP-1 , MCP-1 and CINC-1 mRNA.
  • the relatively higher inhibition of chemokine mRNAs compared with reduction in MPO activity and CD1 lb is in accordance with an antioxidant effect of NACA on reactive oxygen species (ROS) accumulation, rather than by an effect on neutrophil sequestration.
  • ROS reactive oxygen species
  • BOP blast overpressure
  • Pulmonary barotrauma is the most critical and immediately life-threatening injury after blast in nonprotected civilian population. Pressures within the lung parenchyma and air spaces can match or greatly exceed the BOP because the lung tissue and blood vessels are compressed more slowly than air in the respiratory tract, 0 051623 forcing air out through alveolar septa and blood through capillary walls, causing them to rupture (1). The resulting alveolar hemorrhage and edema greatly reduce the gas exchange capacity of the lung, contributing to bradycardia, hypotension, and apnea (5, 6). The extravasated blood initiates a cascade of reactions that involve activation and release of various vasoactive and proinflammatory factors.
  • Oxidative stress caused by excessive accumulation of reactive oxygen species (ROS) is a common mechanism of damage in many experimental models of acute lung injury (13, 14). Excessive ROS accumulation results in depletion of antioxidants and oxidative damage to major cellular components such as lipids, proteins, and DNA.
  • Evidence for oxidative stress in blast includes a decrease in endogenous antioxidant capacity and increase in lipid peroxidation and protein nitration in lungs after blast exposure (15, 16).
  • another ROS-mediated mechanism of damage after blast is activation of inflammation, with leucocytes, macrophages, and neutrophils being the most prodigious source of ROS. Decrease in antioxidant defense capacity is widely recognized as a central feature of many inflammatory lung diseases.
  • attempts to attenuate lung injury include the restoration of the oxidant/antioxidant balance and limiting the degree of oxidative cell damage by augmenting the intracellular pool of antioxidants.
  • Some antioxidants such as N-acetylcysteine (NAC) have been shown to protect pulmonary cells from oxidative injury and ameliorate inflammation (17, 18).
  • NAC N-acetylcysteine
  • treatment with intravenous NAC was associated with improved cardiorespiratory functions in patients with ARDS (19).
  • bioavailability of NAC is very low because of its low ability to cross biological membranes.
  • a newly designed amide form of NAC, N-acetylcysteine amide (NAC A) was shown to be more hydrophobic and membrane permeable (20).
  • NACA N-Acetylcysteine amide was also shown to ameliorate lung inflammatory injury in the mouse model of asthma (24).
  • phosphatebuffered saline 16; pH 7.4
  • the drug or placebo was injected 30 min, 60 min, and 24 h after exposure to blast.
  • a control group of animals (n 8), in which half received NACA and the other half received placebo, underwent the same treatment (anesthesia, suspension, time delays) except they were not exposed to blast.
  • the extent of injury was defined by the severity type elements score, which classifies pulmonary lesions in a 1 to 5 range: 1 indicates no damage; 2, trace; scattered surface petechiation, 3, slight; extensive petechiation to ecchymosis; 4, moderate; parenchymal contusions; and 5, extensive; confluent hepatized regions.
  • rats were euthanized with an intraperitoneal injection of pentobarbital, and the trachea was isolated and cannulated with an 18-gauge catheter.
  • the lungs were inflated and en block fixed with 4% paraformaldehyde at 20 cm H20 fixative pressure. After fixation and dehydration, lungs were embedded in paraffin and cut into 10-2m sections.
  • Sections were stained with hematoxylin and eosin and graded on a 1 to 5 scale as percent of tissue involved in the inflammation and hemorrhage (grade 1 G5%; grade 2, 5%Y15%; grade 3, 15%Y35%; grade 4, 36% to 70%; grade 5, 970%) by two blinded observers.
  • Myeloperoxidase (MPO) activity quantification in lung samples was carried out using colorimetric assay kit (CytoStore, Calgary, Canada). In brief, lung tissue was homogenized, sonicated, and centrifuged. The supernatant was mixed with 50 mmol/L potassium phosphate (pH 6) containing O-dianisidine and hydrogen peroxide and analyzed in duplicate on a spectrophotometric plate reader (1420 Multilabel Counter, Perkin-Elmer, Waltham, Mass) at 450 nm. Change in absorbance was measured over 1 min, and results are expressed as optical density per minute per mg lung wet weight.
  • a spectrophotometric plate reader (1420 Multilabel Counter, Perkin-Elmer, Waltham, Mass
  • RNAlater (Ambion, Austin, Tex) at 20-C and homogenized by a vortex. Total RNA from six to eight animals was extracted with Trizol according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif).
  • First-strand cDNA was prepared from 1 2g of total RNA by using iScript RTYpolymerase chain reaction kit (BioRad) and oligo(dT) primer in a 20 2L reaction.
  • 50 ng of first strand cDNA was used in a total volume 25 2L of the iQ SYBR Green Super Mix (BioRad) containing 200 nmol of each mRNA-specific primer (Sigma, St. Louis, Mo).
  • Polymerase chain reactions consisted of initial 10 min at 90-C and then 40 cycles of 15 s at 95-C and 60 s 60-C.
  • the ABI Prism 7700 Sequence Detection System was used to measure and quantify signatls by the comparative threshold cycle method with "-actin as a housekeeping gene reference. Statistics All results were expressed as mean T SD. For multiple group
  • Hemorrhage areas contained moderate numbers of macrophages that were often laden with erythrocytes and cell debris (Fig. 9B). Inflammation consisted of moderate numbers of lymphocytes, macrophages, and neutrophils (Fig. 9C). Affected areas were often lined by type II pneumocyte hyperplasia (indicative of epithelial cell damage in the alveolus) and the alveolar septa expanded by inflammation and mild fibrosis (Fig. 9B). Occasionally affected areas contained brightly eosinophilic, crystalline material located only in areas of
  • lymphocytes and macrophages Fig. 10, C and D. Occasionally, macrophages seemed to contain hemosiderin or erythrocytes as in animals 2 days postblast. However, there was no sign of hemorrhage (free erythrocytes) or any other changes such as necrosis, type II pneumocyte hyperplasia, or eosinophilic crystalline material.
  • Pulmonary neutrophil sequestration is a characteristic component of chest blunt injury and its progression/resolution.
  • Myeloperoxidase activity in lung samples was used to assess neutrophil infiltration into lung parenchyma. Results show a massive increase in MPO activity in lungs 2 days after exposure (Fig. 1 1 A). At 8 days, MPO activity returned to baseline, indicating resolution of inflammatory reaction.
  • CD1 lb is a member of the "2 integrin receptors and mediates neutrophil adhesion to the endothelial cells. As shown in Figure 1 IB, a significant increase in neutrophil CD1 lb was observed at 2 days after blast exposure.
  • CC chemokines macrophage inflammatory protein- 1 (MIP-1), monocyte chemotactic peptide- 1 (MCP-1) and CXC chemokine, cytokineinduced neutrophil chemoattractant-1 (CINC-1) were assayed in the lung 2 and 8 days after exposure to 140 kPa blast intensity.
  • MIP-1 macrophage inflammatory protein- 1
  • MCP-1 monocyte chemotactic peptide- 1
  • CXC chemoattractant-1 cytokineinduced neutrophil chemoattractant-1
  • inflammatory mediators in lungs returned to baseline levels (Fig. 12, AYC).
  • NACA decreased expression of CD1 lb in lungs after exposure to blast similar to that in MPO.
  • the increased mRNA levels of inflammatory chemokines MIP-1 , MCP-1, and CINC-1 in lungs after exposure to blast were completely eliminated 2 days later by treatment with antioxidant (Fig. 12, AYC).
  • HO-1 mRNA expression in lungs was induced more than 6-fold 2 days after exposure. By 8 days, the HO-1 mRNA level returned to controls (Fig. 13 A).
  • the MnSOD mRNA expression did not reach statistical significance compared with controls at 2 days, and there was no change in the mRNA expression 8 days after exposure to blast (Fig. 13B).
  • Alterations in glutathione metabolism are recognized as a central feature of many inflammatory lung diseases.
  • GSH-GSSG ratio is maintained in reaction catalyzed by glutathione reductase (GR).
  • GR glutathione reductase
  • Pulmonary contusion is a common injury seen after blunt chest trauma and blast exposure and is often associated with increased morbidity and mortality (10, 1 1). Blunt chest trauma is a common problem in the critical care of trauma patients and accounts for 10% to 30% of adult deaths caused by trauma (10). Lung contusions are an important risk for the development of other conditions such as pneumonia and ALI/ ARDS.
  • Mechanical factors relevant for lung contusion include the spalling effect where alveoli and small airways are disrupted by shearing forces, as well as an inertial effect where alveolar tissue is stripped from hilar structures. In addition to spalling, an implosion effect can occur as alveolar and airway tissues rebound from overexpansion of gas after passing of a pressure wave (25).
  • the CC chemokines MIP-1 and MCP-1 are chemotactic for and stimulate mostly monocytes and T lymphocytes (27).
  • Macrophage inflammatory protein- 1 is produced by a variety of immune cells such as macrophages, lymphocytes, and neutrophils and has been shown to stimulate secretion of cytokines from peritoneal macrophages (28).
  • Macrophage inflammatory protein- 1 in addition to its leukocyte chemoattractant properties, can also operate as autocrine activators of alveolar macrophages, facilitating the acute inflammatory process (29). It was shown that treatment with MIP-1 antibody significantly reduced both lung recruitments of neutrophils and the increase in vascular permeability in endotoxemia (29).
  • Monocyte chemotactic peptide 1 in addition to its monocyte chemotactic activity, has also been shown to induce a respiratory burst and upregulate expression of "2 integrin (30). Cytokine-induced neutrophil
  • chemoattractant is a rat CXC chemokine, an important neutrophil chemotactic factor produced by rat alveolar macrophages involved in neutrophilic lung inflammation (31).
  • Cytokine-induced neutrophil chemoattractant was activated in lungs at the same pattern observed in MPO, CD1 lb, MIP-1, and MCP-1.
  • the early temporal expression of CC and CXC chemokines together with MPO activity correlated with the neutrophil and macrophage influx to the lung and suggests a role for these mediators in coordinating the influx of immune cells to the site of injury in the blast model of lung trauma.
  • Heme oxygenase 1 is a stress protein induced by a variety of stimuli such as oxidative stress, endotoxins, cytokines, and its main substrate, hemin (32). It catalyzes the initial and rate-limiting step in the catabolism of heme to yield biliverdin, carbon monoxide, and iron. Whereas CO is supposed to be responsible for the antiinflammatory effect of HO-1 (inhibition of expression of cytokines and iNOS), biliverdin may contribute protection by its potent antioxidant effect.
  • HO-1 induction is a part of adaptive and protective mechanisms leading to the resolution of oxidative and inflammatory lung damage after exposure to moderate (nonlethal) levels of blast. Protection related to HO-1 may include multiple anti-inflammatory and antioxidant mechanisms such as downregulation of proinflammatory cytokines and adhesion factors, resulting in suppression of neutrophil infiltration in inflamed tissue or downregulation of iNOS expression by degradation of heme.
  • NAC N- acetylcysteine
  • NACA NACA
  • carboxyl group is neutralized is expected to be more hydrophobic and membrane permeable (20).
  • N-Acetylcysteine amide was shown to have more efficient membrane permeation (21, 22), high metal chelating activity, and better scavenging antioxidant properties (23) compared with NAC. Recently, it was shown that NACA administered before challenge significantly ameliorated
  • NACA N- Acetylcysteine amide was administered two times in 30-min intervals immediately after blast to quench ROS produced by release of hemoglobin and then 24 h later for the culmination of infiltration of neutrophils and activation of inflammatory response in lungs (16).
  • Our finding that NACA was effective when administered after exposure to blast is especially important because it is obviously impossible to anticipate the exact time and location of blast.
  • ROS have been implicated as second messengers in the activation of redox-sensitive signaling pathways and transcription of numbers of genes (38).
  • transcription factors involved in modulating ROS have been implicated as second messengers in the activation of redox-sensitive signaling pathways and transcription of numbers of genes (38).
  • nuclear factorY.B is the most important redox-sensitive transcription factor (38).
  • Nuclear factorY.B binding motif is present in the promoter regions of many genes that encode proinflammatory cytokines, especially chemokines that are important in the recruitments of neutrophils, macrophages, and lymphocytes (38).
  • the administration of both NAC and NACA resulted in significant reduction of nuclear factorY.B activation and attenuated lung inflammation caused by increased production of ROS (24, 31).
  • N- Acetyl cysteine amide protection against inflammation in our model was not related to the activation of antioxidant defense enzymes HO-1, MnSOD, or GR. Instead, NACA administration prevented the activation of HO-1 mRNA observed 2 days after blast and had no effect on MnSOD and GR mRNA transcription. In lung fibroblasts, HO-1 was induced during oxidative stress, and this induction paralleled a decrease in intracellular GSH, and treatment with NAC reduced expression of HO-1. This supports previous observations of the mechanism of NACA's antioxidant effect by restoration of GSH level in lung inflammation and our observation of inhibition of HO-1 mRNA activation.
  • Glutathione reductase is a crucially important cellular enzyme in the GSH redox cycle. Glutathione reductase in the presence of NADPH, a supplier of reducing equivalents, recycles GSSG back to GSH to increase the antioxidant pool required for detoxification. Our data indicate that NACA antioxidant effect was not related to the activation of GR expression.
  • Fink MP Role of reactive oxygen and nitrogen species in acute respiratory distress syndrome. Curr Opin Crit Care 8:6Y1 1, 2002.
  • NACA N- acetylcysteine amide
  • NAC Nacetyl cysteine
  • Crouch LD Lentsch AB, Sarma V, et al.: Role of CC chemokines (macrophage inflammatory proteinYl ", monocyte chemoattractant proteinYl, RANTES) in acute lung injury in rats. J Immunol 164:2650Y2659, 2000.
  • Baglole CJ, Sime PJ, Phipps RP Cigarette smoke-induced expression of heme oxygenaseYl in human lung fibroblasts is regulated by intracellular glutathione. Am J Physiol Lung Cell Mol Physiol 295:L624YL636, 2008.
  • Rotstein OD Modeling the two-hit hypothesis for evaluating strategies to prevent organ injury after shock/resuscitation. J Trauma 54:S203YS206, 2003.
  • NACA reactive oxygen species
  • hemeoxygenase an enzyme important in the catabolism of heme that is induced under conditions of oxidative stress, may indicate a relative decrease in the amount of hemorrhaging observed in NACA-treated lungs of animals, especially at two (2) days after exposure to blast (BOP).
  • BOP blast
  • the new additional data together with the previously submitted data further indicate that NACA may effectively prevent morbidity and mortality resulting from blast overpressure when given before or after injury.
  • Example 16 Post- Injury Administration, of a Cell-Permeant Glutathione Precursor, N- Acetylcysteine Amide, Increases Tissue Sparing and Reduces Oxidative Stress Following Traumatic Brain Injury
  • Traumatic brain injury is a silent epidemic, resulting in over one million new cases annually in the United States; unfortunately, treatment options have been limited and no approved pharmacological treatment has been effective thus far 1 .
  • TBI has been characterized as a biphasic injury, which includes a primary blunt force injury and a prolonged secondary injury cascade occurring in the penumbra surrounding the injury 2 . Associated with this secondary injury cascade is glutamate induced excitotoxicity resulting from increased intracellular Ca 2+ levels 3 4 .
  • Mitochondria act as calcium sinks during normal cellular functioning, however excessive calcium uptake during excitotoxic insult results in reduced mitochondrial membrane potential ⁇ 1 ?), increased reactive oxygen species (ROS) production, and decreased ATP production 5 .
  • ROS reactive oxygen species
  • Glutathione a thiol which acts as the primary intracellular antioxidant, plays a critical role in the attenuation of excessive ROS production. It has been shown that following injury both cellular and mitochondria] levels of glutathione are decreased, and that the loss of mitochondrial glutathione has been associated with increased tissue damage . Recently, several studies have evaluated the efficacy of using the novel antioxidant N-acetylcysteine amide (NACA), due to its permeability to both cellular and mitochondrial membranes .
  • NACA N-acetylcysteine amide
  • NACA a glutathione precursor
  • NACA the amide form of N-acetylcysteine
  • NACA has a neutralized carboxylic group making it more lipophilic, which enables it to be cellular and mitochondrial membrane permeable 8 . It has also been shown to chelate copper, attenuate MAPK activity, and decrease oxidative stress 9 ' 10 .
  • Previous studies have also shown that NACA crosses erythrocyte membranes and upon entering replenish intracellular glutathione levels 8 .
  • neuronal cell line studies have shown that NACA reduces the levels of ROS induced by glutamate and decreases lipid peroxidation".
  • NACA provides beneficial support to endogenous antioxidant systems after oxidative insult, it may be able to attenuate the rampant cascade of secondary injury after TBI. Therefore, in our current studies we have investigated the ability of NACA to spare tissue and decrease oxidative stress when administered after a controlled cortical impact model of TBI.
  • the miniosmotic pumps were assembled and implanted immediately after injury as previously described with few modifications and remained in the animals for 7 days 15 .
  • another set of rats received a moderate CCI injury.
  • Tissue sparing was expressed as a percentage of the contralateral cortex within each animal, by dividing the ipsilateral cortical volume by the contralateral cortical volume.
  • Sections were then blocked using 5 % goat serum, 0.25 % Triton X-100, and 1 % milk in a PBS solution for 2 hours at room temperature. Sections were then immunoreacted with primary antibody (rabbit anti-HNE polyclonal antibody, Calbiochem) (mouse anti-3-NT monoclonal antibody, Upstate). Sections were then rinsed with PBS and incubated with secondary antibody for 2 hours (800 secondary goat anti-rabbit antibody, Rockland) (IR Dye 700 D conjugated goat anti -mouse IgG antibody). Sections were rinsed with water and mounted. Imaging was preformed on Li-core Odyssey machine.
  • N-acetylcysteine amide is Neuroprotective following TBI
  • NACA treated rats with the NACA and CsA treated rats there was no significant difference in tissue sparring, therefore indicating a lack of synergistic effect with co-administration of the two compounds.
  • N-acetylcysteine amide Decreases Oxidative Stress Following TBI
  • NACA neuroprotective compound
  • TBI is classically associated with increased ROS production and oxidative damage; therefore we measured oxidative markers to assess the effect of NACA on tissue.
  • FINE levels were significantly reduced in injured animals treated with NACA when compared with vehicle treated animals.
  • 3-NT levels were not significantly reduced among vehicle treated and NACA treated rats.
  • NACA was not expected to reduce 3-NT levels, as observed, due to the absence of support for the reduction of peroxynitrite by glutathione. From these data it is shown that NACA significantly reduces oxidative stress following TBI.
  • Mitochondrial Uncouplers Increases Tissue Sparing and Improves Behavioral Outcome following Traumatic Brain Injury in Rodents. Journal of neurotrauma. 2007;24:798-81 1
  • NACA N-acetylcysteine amide
  • Sullivan PG Geiger JD, Mattson MP, Scheff SW. Dietary supplement creatine protects against traumatic brain injury. Annals of neurology. 2000;48:723-729 15. Sullivan PG, Thompson M, Scheff SW. Continuous infusion of cyclosporin A postinjury significantly ameliorates cortical damage following traumatic brain injury.
  • Mitochondrial uncoupling protein-2 protects the immature brain from excitotoxic neuronal death. Annals of neurology.
  • Example 17 NAC inhibits Dengue replication in human monocytic cells
  • NACA is metabolized to NAC in the body. See Fig. 36.
  • FIGs. 26-27 show that NACA (a.k.a. TOVA) blocked the cytokines and HIV replication and that strong blockage was seen at a dosage of NACA that was 1/1000 the dose previously reported for NAC (Poli 1992). 2010/051623
  • NACA N-acetylcysteineamide
  • Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are one of the major components of air borne particulate matter (PM) in the urban environment.
  • DEPs are composed of soot, polycyclic aromatic hydrocarbons (PAHs), redox active semi- quinones, and transition metals, which are known to produce pro-oxidative and proinflammatory effects, thereby leading to oxidative stress-induced damage in the lungs.
  • NACA N-acetylcysteineamide
  • Diesel engine exhaust is a complex mixture of organic and inorganic gases (NOx, SOx, CO), and particulate matters (PMs).
  • Diesel exhaust particles a by- product of DEE, are one of the major components of airborne particulate matter in the urban environment. Epidemiological studies have demonstrated a strong association between particulate matter and lung diseases like asthma and chronic obstructive pulmonary disease (Nel et al, 1998; Peterson and Saxon, 1996; Diaz-Sanchez, 1997; Li et al, 1996).
  • DEPs have mean diameters of 0.2_m or less, which render them easily respirable and capable of being deposited in the airways and the alveoli.
  • DEPs contain carbon, with large surface areas which readily adsorb chemicals like polyaromatic hydrocarbons (PAH),quinones, aldehydes, and heavymetals like iron, copper, chromium, and nickel (Schuetzle, 1983;
  • PAH polyaromatic hydrocarbons
  • Macrophages which are the first line of defense in the lungs and contain enzymes (like cytochrome P4501A1) which aid in the conversion of xenobiotics, have also been reported to generate ROS (Park et al., 1996; Kumagai et al., 1997; Ng et al., 1998).
  • SAPKs stress-activated protein kinases
  • Oxidative stress occurs when pro-oxidants outweigh antioxidant levels in the cells.
  • Glutathione GSH is one of the major intracellular thiol antioxidants that provide protection against oxidative stress in the body. Alteration in the GSH metabolism has been recognized as a key feature ofmany inflammatory diseases of the lung (Lee et al., 2007).
  • Thiol antioxidants like N-acetylcysteine (NAC), which increase GSH by affecting the cysteine levels in the body, have been shown to be effective in treating the oxidative and inflammatory effects of allergens in the respiratory tract (Astiet al., 1995; Whitekus et al., 2002; Lee et al., 2004).
  • NAC N-acetylcysteine
  • the antioxidant NAC has a negatively charged carboxyl group at physiological pH, which limits its ability to cross the cell membrane.
  • C57BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).
  • Nacetylcysteineamide was provided by Dr. Glenn Goldstein (David
  • N-(l-pyrenyl)-maleimide was purchased fromSigma (St.Louis, MO). High-performance liquid chromatography (HPLC) grade solvents were purchased fromFisher Scientific (Fair Lawn, NJ). All other chemicals were purchased from Sigma (St. Louis, MO), unless stated otherwise.
  • DEPs were generated from a diesel generator (Model No. 4039T, John Deere, IL).
  • the diesel exhausts from the generator were diluted with clean dry air, one part exhaust to six parts air, and fed to the exposure chamber.
  • concentration of the DEPs were monitored using a Cambustion DMS500 (Cambridge, UK), a state-of-the-art fast particulate spectrometer (Reavell et al, 2002).
  • the aerosol characteristics of the DEP, as calculated from the size distribution were:mean diameter 38 nm, geometric standard deviation 1.36, total number concentration D2.1 * 106 cm-3, and mass concentration 0.23mg/m3 using a soot density of 2 g/cm3 (Virtanen et al., 2002).
  • mice Male C57BL/6 mice (30-35 g, 7 weeks old) were housed in a controlled temperature (20-23 °C) and controlled-humidity (D55%) animal facility, with a 12 h light and dark cycle. The animals had unlimited access to rodent chowandwater, and were utilized after lweek of acclimatization. All animal procedureswere conducted under an animal protocol approved by the Institutional Animal Care and Use Committee of the Missouri University of Science and Technology. The mice were divided into two major groups: an experimental and a control group. The model was developed based on previous reports of diesel exposure in in vivo models (Dong et al.,2005).
  • the animals in the experimental group were exposed to DEPs and the control animals were exposed to clean filtered air (3 h) for nine consecutive days (Fig. 28).
  • mice were sacrificed 24 h after the last exposure by urethane injection. All mice were weighed at the beginning and at the end of the study. Following sacrifice, the lungs were harvested and divided into two parts, with two slices fixed in 10% buffered formalin and the remaining tissue being stored in an antioxidant buffer [8.6mM sodium phosphate dibasic (Na2HP04), 26.6mM sodium phosphate monobasic (NaH2P04), 50_M butylhydroxytoluene (BHT), 1 OmM aminotriazole, 0.1 mM
  • DTP A diethyltriamine pentaacetic acid
  • H&E hematoxylin and eosin
  • GSH and cysteine (CYS) levels The levels of GSH and CYS in the tissue were determined by RP-HPLC, according to the method developed in our laboratory (Winters et al., 1995).
  • the HPLC column used was a Reliasil ODS-1 CI 8 column (5-_m packing material) with
  • the mobile phase (70% acetonitrile and 30% water) was adjusted to a pH of 2 with acetic acid and o-phosphoric acid.
  • the NPM derivatives of CYS and GSH were eluted from the column isocratically at a flow rate of 1 ml/min.
  • the tissue samples were homogenized in a serine borate buffer, centrifuged, and 250_1 of the supernatant was added to 750_1 of ImM NPM.
  • the resulting solution was incubated at room temperature for 5min, and the reaction was stopped by adding 10 1 of 2N HC1.
  • the samples were then filtered through a 0.45-_m filter and injected into the HPLC system.
  • Lactate dehydrogenase levels were determined according to the method described by Uitto et al. (1972). Briefly, a cytosolic fraction of the tissue homogenate was added to a solution containing 50mM potassium phosphate buffer and lOmM sodium pyruvate. The reaction was initiated by adding the NADH (Nicotinamide adenine dinucleotide-reduced), and the absorbance was recorded at 340 nm. The activity of LDH was determined
  • the MDA levels were determined according to the method described by Draper et al. (1993). Briefly, 550J of 5% tricholoroacetic acid (TCA) and 100_1 of 500ppm butylated hydroxytoluene (BHT) in methanol were added to 350_1 of the tissue homogenates, and boiled for 30 min in a water bath. After cooling on ice, the mixtures were centrifuged, and the supernatant collectedwasmixed 1 : 1 with saturated thiobarbituric acid (TBA). The mixture was again heated in a water bath for 30 min, followed by cooling on ice. 500_1 of the mixture was extracted with 1ml of «-butanol and centrifuged to facilitate the separation of phases.
  • TCA tricholoroacetic acid
  • BHT butylated hydroxytoluene
  • the resulting organic layers were first filtered through 0.45_m filters and then injected into the HPLC system (Shimadzu, US), which consisted of a pump (model LC-6A), a Rheodyne injection valve and a fluorescence detector (model RF 535).
  • the column was a
  • the mobile phase used contained 69.4% sodium phosphate buffer, 30% acetonitrile, and 0.6%
  • Protein levels of the tissue samples were measured by the Bradford method (Bradford, 1976). Concentrated Coomassie Blue (Bio-Rad, Hercules, CA) was diluted 1 :5 (v/v) with distilledwater. 20 1 of the diluted tissue homogenatewas then added to 1.5ml of this diluted dye, and absorbance was measured at 595nm using a UV spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). Bovine serum albumin (BSA) was used as the protein standard.
  • BSA Bovine serum albumin
  • Lipid peroxidation is an important consequence of oxidative stress, and can be estimated by measuring the levels of malondialdehyde (MDA)— a stable by-product of lipid peroxidation.
  • MDA malondialdehyde
  • DEP-exposed animals had about a 1.5-fold increase in MDA levels, as compared to the levels in the controls (Fig. 33).
  • An increase in the dose-dependent MDA level was also observed in animals exposed to DEPs, although the difference was not significant.
  • the animals in the NACA-treated group experienced significantly less lipid peroxidation than the animals did in the DEP-only treated group.
  • the MDA levels in the NACA-treated group were similar to the levels in the controls.
  • Antioxidant enzymes like Catalase are involved in detoxification of peroxides in the body. Exposure of animals to DEPs decreased the levels of CAT activity in the lungs, as compared to the levels in the lungs of the control or NACA-alone treated group (Fig. 34). However, partial reversal in the loss of CAT activity was observed in DEP-exposed animals that had been pretreated with NACA.
  • CAT Catalase
  • Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are one of the major air pollutants worldwide. As a consequence of the small size of these particles, they can easily penetrate and settle deep inside the lungs.
  • DEPs are composed of soot, PAH, redox active semi-quinones, and traces of transition metals, which are also known to produce pro-oxidative and pro-inflammatory effects, thereby inducing oxidative stress and damage in the lungs.
  • Thiol antioxidants have been shown to be effective in treating oxidative effects in the respiratory tracts (Asti et al., 1995; Whitekus et al., 2002).
  • DEE is also composed of a variety of gases especially NOx ( ⁇ 90% NO and 10% N02), CO, and S02 (Reed et al., 2004).
  • NOx ⁇ 90% NO and 10% N02
  • CO carbon dioxide
  • S02 S02
  • NOx and CO have been reported to potentiate inflammation and oxidativedamage in lung
  • GSH glutathione
  • GSH GSH
  • pulmonary diseases like idiopathic pulmonary fibrosis and acute respiratory distress syndrome (Rahman et al., 1999; Rahman and MacNee, 2000).
  • GSH has also been reported to attenuate IL-13-induced asthma in mice (Lowry et al., 2008). This indicates that low levels of GSH may render individuals susceptible to the deleterious effects of exposure to inhaled toxicants and may also perpetuate inflammatory responses in their lungs.
  • the results from our study show that animals exposed to DEPs have significant decreases in their GSH levels, indicating that exposure to DEPs induces oxidative stress in these animals.
  • NACA sulfhydryl
  • Catalase is one such enzyme found in peroxisomes that aids in removal of hydrogen peroxide.
  • CAT is one such enzyme found in peroxisomes that aids in removal of hydrogen peroxide.
  • the results from our study indicate that exposure of animals to DEPs significantly decreases the CAT levels.
  • Pretreatment with NACA increases the CAT levels, thereby protecting the lungs by removing hydrogen peroxide and superoxide radicals.
  • Free radicals produced by oxidative stress, also attack lipids, especially polyunsaturated fatty acids in the cell membranes and lead to the formation of by-products like MDA (Karbownik and Reiter, 2000).
  • Apoptosis of the macrophages involves shedding of the apoptotic bodies after death, which spreads the toxic chemicals to the neighboring cells. This further induces cytotoxicity, as these apoptotic bodies contain active chemicals (PAHs), and their uptake by the surrounding inflammatory cellsmay further induce cytotoxicity and contribute to pathogenesis of respiratory diseases (Hiura et al, 1999b).
  • PAHs active chemicals
  • data from the present study indicates that, after exposure to DEE, DEPs enter into the lungs, where they are engulfed by the macrophages.
  • These particle-laden macrophages induce oxidative stress in the lungs, as indicated by decreases in GSH and catalase levels, and increases in MDA levels (Fig. 36).
  • Administration of NACA however, resulted in significant reductions in macrophages and oxidative stress-induced damage, thereby suggesting a therapeutic potential for this novel antioxidant.
  • Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling. J. Biol. Chem. 274, 26337-26343.
  • Nacetylcysteineamide attenuates allergic airway disease by regulating activation of NF- kappaB and hypoxia-inducible factor- 1 alpha. Exp. Mol. Med. 39, 756-768.

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Abstract

La présente invention concerne des procédés et des compositions comprenant un N-acétylcystéine amide (NAC amide) et des dérivés de celui-ci utilisés dans des traitements et des thérapies prophylactiques pour des maladies, des troubles, des conditions et des pathologies chez des mammifères humains et non humains associés à l'explosion d'une bombe ou autre bruit très énergétique ou à des explosions par impulsion. La présente invention concerne également des compositions pharmaceutiquement ou physiologiquement acceptables de NAC amide ou de dérivés de celui-ci, qui peuvent être administrées seules, ou en combinaison avec d'autres agents adaptés.
PCT/US2010/051623 2009-10-06 2010-10-06 N-acétylcystéine amide (nac amide) destiné au traitement de maladies et de conditions Ceased WO2011044230A2 (fr)

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CN103070294A (zh) * 2013-01-31 2013-05-01 武汉工业学院 N-乙酰半胱氨酰胺作为饲料添加剂的应用
WO2013175479A1 (fr) 2012-05-24 2013-11-28 Hadasit Medical Research Services And Development Ltd. Compositions comprenant de l'isosilybine b pour l'amélioration et la prévention de la toxicité induite par un médicament
WO2015112724A1 (fr) * 2014-01-24 2015-07-30 Brighton Biotech, Inc. Amide nac pour le traitement de dysfonctionnement cognitif aigu ou chronique
WO2015112715A1 (fr) * 2014-01-24 2015-07-30 Brighton Biotech, Inc. Naca pour le traitement d'un traumatisme cérébral à faible impact ou chronique
JP2016523975A (ja) * 2013-07-10 2016-08-12 グレン・エー・ゴールドスタイン 穿通性頭部外傷の処置におけるｎ−アセチルシステインアミドの使用
EP3302456A4 (fr) * 2015-06-05 2018-12-19 Glenn A. Goldstein Utilisation de n-acétylcystéine amide dans le traitement d'une surdose d'acétaminophène
WO2020146674A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. Traitement de la dégénérescence maculaire liée à l'âge, du glaucome et de la rétinopathie diabétique par l'amide de n-acétylcystéine (naca) ou le bis(2-acétamidopropanamide) de (2r,2r')-3,3'-disulfanediyle (dinaca)
WO2020146660A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. N-acétylcystéine amide (naca) et (2r, 2r')-3-3'-disulfanediyl bis (2-acétamidopropanamide) (dinaca) pour la prévention et le traitement de la pneumonie radio-induite et le traitement de la fonction pulmonaire dans la mucoviscidose
WO2020146666A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. N-acétylcystéine amide (naca) et (2r,2r')-3,3'-disulfanediyl bis(2-acétamidopropanamide) (dinaca) pour la prévention et le traitement de la radiodermite et de l'éclaircissement de la peau, le blanchiment de la peau et l'amélioration de la peau
WO2021217131A1 (fr) * 2020-04-24 2021-10-28 Ashvattha Therapeutics, Inc. Compositions de dendrimères et méthodes de traitement du syndrome de détresse respiratoire aiguë grave
WO2021217221A1 (fr) * 2020-05-01 2021-11-04 MUCPharm Pty Ltd Prévention et traitement d'infections virales
WO2021226235A1 (fr) * 2020-05-05 2021-11-11 Northwestern University Système et procédé pour détecter et traiter des régions arythmogènes dans une fibrillation auriculaire
WO2021239823A1 (fr) * 2020-05-26 2021-12-02 Daams Brechtje Johanna Prévention ou traitement d'états liés à une déficience en oxyde nitrique
US11753370B2 (en) 2017-11-09 2023-09-12 Nacuity Pharmaceuticals, Inc. Methods of making deuterium-enriched N-acetylcysteine amide (d-NACA) and (2R, 2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and using d-NACA and diNACA to treat diseases involving oxidative stress
WO2024160924A1 (fr) * 2023-01-31 2024-08-08 Queen Mary University Of London Utilisation de nitrate inorganique pour prévenir une néphropathie induite par le contraste chez un patient recevant un agent de contraste
US12472157B2 (en) 2014-11-07 2025-11-18 Nacuity Pharmaceuticals, Inc. Treatment of retinitis pigmentosa with n-acetylcysteine amide

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MA49906A (fr) 2017-08-14 2020-06-24 Axcella Health Inc Acides aminés à chaîne ramifiée pour le traitement d'une maladie du foie
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WO2013175479A1 (fr) 2012-05-24 2013-11-28 Hadasit Medical Research Services And Development Ltd. Compositions comprenant de l'isosilybine b pour l'amélioration et la prévention de la toxicité induite par un médicament
US9474763B2 (en) 2012-05-24 2016-10-25 Hadasit Medical Research Services And Development Ltd. Compositions and methods for amelioration and prevention of drug-induced toxicity
CN103070294A (zh) * 2013-01-31 2013-05-01 武汉工业学院 N-乙酰半胱氨酰胺作为饲料添加剂的应用
JP2016523975A (ja) * 2013-07-10 2016-08-12 グレン・エー・ゴールドスタイン 穿通性頭部外傷の処置におけるｎ−アセチルシステインアミドの使用
EP3019164A4 (fr) * 2013-07-10 2017-03-15 Glenn A. Goldstein Emploi de n-acétylcystéine amide dans le traitement de lésions profondes à la tête
WO2015112724A1 (fr) * 2014-01-24 2015-07-30 Brighton Biotech, Inc. Amide nac pour le traitement de dysfonctionnement cognitif aigu ou chronique
WO2015112715A1 (fr) * 2014-01-24 2015-07-30 Brighton Biotech, Inc. Naca pour le traitement d'un traumatisme cérébral à faible impact ou chronique
US12472157B2 (en) 2014-11-07 2025-11-18 Nacuity Pharmaceuticals, Inc. Treatment of retinitis pigmentosa with n-acetylcysteine amide
EP3302456A4 (fr) * 2015-06-05 2018-12-19 Glenn A. Goldstein Utilisation de n-acétylcystéine amide dans le traitement d'une surdose d'acétaminophène
US11753370B2 (en) 2017-11-09 2023-09-12 Nacuity Pharmaceuticals, Inc. Methods of making deuterium-enriched N-acetylcysteine amide (d-NACA) and (2R, 2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and using d-NACA and diNACA to treat diseases involving oxidative stress
JP2022518174A (ja) * 2019-01-11 2022-03-14 ナキュイティ ファーマシューティカルズ， インク． 放射線皮膚炎の予防及び治療、並びに皮膚ライトニング、皮膚ホワイトニング、並びに皮膚改善のためのｎ－アセチルシステインアミド（ｎａｃａ）及び（２ｒ，２ｒ’）－３，３’－ジスルファンジイルビス（２－アセトアミドプロパンアミド）（ｄｉｎａｃａ）
US12458608B2 (en) * 2019-01-11 2025-11-04 Nacuity Pharmaceuticals, Inc. N-Acetylcysteine Amide (NACA) and (2R,2R′)-3,3′ disulfanediyl BIS(2-acetamidopropanamide) (DINACA) for the prevention and treatment of radiation dermatitis and skin lightening, skin whitening and skin improvement
KR20210114460A (ko) * 2019-01-11 2021-09-23 나쿠이티 파마슈티컬스, 인코포레이티드 방사선 피부염, 피부 라이트닝 또는 피부 화이트닝을 방지 및 치료하고 피부를 개선하기 위한 n-아세틸시스테인 아미드(naca) 및 (2r,2r')-3,3'-디설판디일 비스(2-아세트아미도프로판아미드)(dinaca)
US20220105056A1 (en) * 2019-01-11 2022-04-07 Nacuity Pharmaceuticals, Inc. N-Acetylcysteine Amide (NACA) and (2R,2R')-3,3' disulfanediyl BIS(2-Acetamidopropanamide) (DINACA) for the Prevention and Treatment of Radiation Dermatitis and Skin Lightening, Skin Whitening and Skin Improvement
CN113573704A (zh) * 2019-01-11 2021-10-29 纳崔泰制药有限公司 用于预防和治疗辐射性皮炎和皮肤变亮、皮肤增白和皮肤改善的n-乙酰半胱氨酸酰胺（naca）和(2r,2r’)-3,3’-二硫烷二基双(2-乙酰氨基丙酰胺)（dinaca）
WO2020146660A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. N-acétylcystéine amide (naca) et (2r, 2r')-3-3'-disulfanediyl bis (2-acétamidopropanamide) (dinaca) pour la prévention et le traitement de la pneumonie radio-induite et le traitement de la fonction pulmonaire dans la mucoviscidose
WO2020146674A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. Traitement de la dégénérescence maculaire liée à l'âge, du glaucome et de la rétinopathie diabétique par l'amide de n-acétylcystéine (naca) ou le bis(2-acétamidopropanamide) de (2r,2r')-3,3'-disulfanediyle (dinaca)
US12245998B2 (en) 2019-01-11 2025-03-11 Nacuity Pharmaceuticals, Inc. N-acetylcysteine amide (NACA) and (2R,2R′)-3,3′ disulfanediyl bis(2-acetamidopropanamide) (diNACA) for the prevention and treatment of radiation pneumonitis and treatment of pulmonary function in Cystic Fibrosis
WO2020146666A1 (fr) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. N-acétylcystéine amide (naca) et (2r,2r')-3,3'-disulfanediyl bis(2-acétamidopropanamide) (dinaca) pour la prévention et le traitement de la radiodermite et de l'éclaircissement de la peau, le blanchiment de la peau et l'amélioration de la peau
US11766413B2 (en) 2019-01-11 2023-09-26 Nacuity Pharmaceuticals, Inc. Treatment of age-related macular degeneration, glaucoma, and diabetic retinopathy with n-acetylcysteine amide (NACA) or (2R,2R′)-3,3′-disulfanediyl BIS(2-acetamidopropanamide)(DiNACA)
US20200222344A1 (en) * 2019-01-11 2020-07-16 Nacuity Pharmaceuticals, Inc. Treatment of Age-Related Macular Degeneration, Glaucoma, and Diabetic Retinopathy with N-Acetylcysteine Amide (NACA) or (2R,2R')-3,3'-Disulfanediyl BIS(2-Acetamidopropanamide)(DiNACA)
WO2021217131A1 (fr) * 2020-04-24 2021-10-28 Ashvattha Therapeutics, Inc. Compositions de dendrimères et méthodes de traitement du syndrome de détresse respiratoire aiguë grave
CN116075322A (zh) * 2020-04-24 2023-05-05 阿什瓦塔治疗股份有限公司 用于治疗重度急性呼吸窘迫综合征的树枝状大分子组合物和方法
US11931418B2 (en) 2020-04-24 2024-03-19 Ashvattha Therapeutics, Inc. Methods of treating severe inflammation
WO2021217221A1 (fr) * 2020-05-01 2021-11-04 MUCPharm Pty Ltd Prévention et traitement d'infections virales
US11911178B2 (en) 2020-05-05 2024-02-27 Northwestern University System and method to detect and treat arrhythmogenic regions in atrial fibrillation
WO2021226235A1 (fr) * 2020-05-05 2021-11-11 Northwestern University Système et procédé pour détecter et traiter des régions arythmogènes dans une fibrillation auriculaire
WO2021239823A1 (fr) * 2020-05-26 2021-12-02 Daams Brechtje Johanna Prévention ou traitement d'états liés à une déficience en oxyde nitrique
WO2024160924A1 (fr) * 2023-01-31 2024-08-08 Queen Mary University Of London Utilisation de nitrate inorganique pour prévenir une néphropathie induite par le contraste chez un patient recevant un agent de contraste

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