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WO1998013037A1 - Procedes d'utilisation de l'agmatine pour reduire le niveau de polyamine a l'interieur des cellules et inhiber le monoxyde d'azote synthetase inductible - Google Patents

Procedes d'utilisation de l'agmatine pour reduire le niveau de polyamine a l'interieur des cellules et inhiber le monoxyde d'azote synthetase inductible Download PDF

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Publication number
WO1998013037A1
WO1998013037A1 PCT/US1997/017424 US9717424W WO9813037A1 WO 1998013037 A1 WO1998013037 A1 WO 1998013037A1 US 9717424 W US9717424 W US 9717424W WO 9813037 A1 WO9813037 A1 WO 9813037A1
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agmatine
cells
derivative
composition
odc
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PCT/US1997/017424
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Joseph Satriano
Roland C. Blantz
Carolyn J. Kelly
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The Regents Of The University Of California
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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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)

Definitions

  • This invention relates generally to the fields of biochemistry and medicine, and more specifically to controlling polyamine levels and inhibiting inducible nitric oxide synthase in cells and tissues.
  • ODC ornithine decarboxylase
  • ODC enzyme constitutively activated in cells transformed by oncogenes, carcinogens or viruses. Preventing polyamine synthesis in mammalian cells through the use of inhibitors can result in complete cessation of growth.
  • the search for inhibitors that block polyamine biosynthesis for use as therapeutic agents is an ongoing endeavor. Some compounds have been effective in blocking polyamine biosynthesis, but toxic effects of these compounds on cells or organisms occur.
  • ODC nitric oxide synthase enzyme
  • NOS nitric oxide synthase enzyme
  • Toxic cellular effects are observed when nitric oxide (NO) is generated by cells. NO is produced by the conversion of the ammo acid L-argimne to L-citrullme by NOS.
  • NO can confer cellular antimicrobial activity
  • this protection can also result in inflammatory damage to host cells and tissues by the potential injurious nature of high NO levels.
  • Septic shock for example, is the leading cause of death in intensive care units and occurs when microbial products trigger systemic inflammatory responses.
  • the resultant induction of inflammatory cytokmes causes a dramatic, irrepressible fall in central blood pressure and, eventually, organ failure.
  • NO is the major contributor to this non- responsive vasodilation, as high NO levels are known to increase during infection, and NOS inhibitors can reverse hypotension and increase survival
  • NOS inhibitors can reverse hypotension and increase survival
  • m autoimmune disease such as glomerulonephritis and arthritis
  • NO production may be important in the pathogenesis of autoimmune disease.
  • Abnormalities in tumor vasculature may also be attributed to increased NO.
  • NO production has also been associated with increased vascularization in nude mice, resulting in rapid progression of tumor growth. It is advantageous, therefore, to selectively modify NOS activity without altering NO levels required for normal homeostasis
  • compositions useful for reducing intracellular polyamine levels and inhibiting deleterious effects of NO accumulation in cells in order to ameliorate or prevent various cellular pathologies.
  • the present invention satisfies this need and provides additional benefits as well.
  • the present invention provides methods of using an arginine derivative to reduce intracellular polyamine levels and to inhibit inducible NOS activity.
  • the invention provides methods of reducing polyamine levels intracellularly by administering a composition, comprising an arginine derivative such as the compound agmatine.
  • agmatine inhibits the enzyme ODC and represses polyamine uptake into cells.
  • the invention also provides a pharmacological composition, comprising agmatine in a physiologically acceptable buffer, that can be administered to a subject in order to reduce intracellular polyamines.
  • the invention further provides methods of inhibiting hyperplasias such as kidney hypertrophy, liver and smooth muscle hyperplasia and the growth of tumor cells by administering agmatine to the affected cells.
  • the invention provides methods of selectively inhibiting inducible nitric oxide synthase (iNOS), while maintaining constitutive nitric oxide synthase (cNOS) , by administering an arginine derivative.
  • An arginine derivative can be, for example, agmatine or an agmatine metabolite, agmatine-aldehyde (guanidinobutyraldehyde) .
  • the invention also provides methods of treating endotoxic shock in a mammal by administering a composition, comprising agmatine to the mammal.
  • the invention further provides methods of treating conditions resulting from excessive NO generation, including arthritis, glomerulonephritis, angiogenesis in tumors, transplantation and tissue graft rejection, neurodegeneration, stroke, ischemic injury, chronic inflammation and diabetes, by administering an arginine derivative to an individual suffering from the condition .
  • Figure 1 shows the increase in ODC activity in tubules at 24 hours post-nephrectomy, compared with the suppression of arginine decarboxylase (ADC) activity in tubules at 24 hours post-nephrectomy.
  • ADC arginine decarboxylase
  • Figure 2 shows ODC activity of tubules post-nephrectomy when incubated for 1 hour with buffer, with
  • Figure 3 shows ODC activity of immortalized proximal tubule cells (MCT) .
  • Figure 3A shows ODC activity when the cells were incubated for 16 hours in the presence of varying concentrations of agmatine.
  • Figure 3B shows ODC activity of MCT cells incubated for 16 hours without inhibitors, or in the presence of 10 mM eflornithine (DFMO) , 1 mM agmatine, or 1 M putrescine.
  • DFMO mM eflornithine
  • Figure 4 shows the change in ODC activity in MCT cells, comparing control cells with cells exposed to 1 mM agmatine for the times indicated.
  • Figure 5 shows ODC activity in MCT cells in the presence of various inhibitors.
  • Figure 5A shows ODC activity of control cells compared to cells incubated with agmatine in the presence or absence of cycloheximide (CHX) .
  • Figure 5B compares ODC activity of control cells to cells incubated with agmatine in the presence or absence of actinomycin-D .
  • CHX cycloheximide
  • Figure 6 shows the effect of inhibitors and agmatine on 3 H-agmat ⁇ ne uptake into MCT cells
  • Figure 6A shows inhibition by the polyamines putrescine, spermidme, and spermme, the polyamine transport inhibitor paraquat, guanidmobutyric acid (GBA) , ornithine, lysine, arginine, and the arginine catiomc transporter inhibitor N J -monomethyl-L-arginme (L-NMMA)
  • Figure 6B shows the effect of premcubation of agmatine (1 mM) on ⁇ -agmatme uptake m MCT cells over time
  • Figure 7 shows the effect of DNA synthesis in MCT cells as indicated by ⁇ -thymidme incorporation at 48 hours after addition of 1 mM agmatine or 1 mM agmatine plus 50 ⁇ M putrescine.
  • Figure 8 shows the effect of increasing concentrations of agmatine (10 ⁇ M to 1 mM) , putrescine, spermidine, paraquat, ornithine, lysine and arginine on ⁇ -putrescme uptake in MCT cells
  • Figure 9 shows the effect of premcubation of agmatine (1 mM) on " ⁇ -putrescine uptake in MCT cells over time (0 to 24 -hours) .
  • Figure 10 shows the effect of agmatine m the presence of actinomycin-D or cycloheximide (CHX) on H-putrescme uptake in MCT cells
  • Figure 11 shows agmatine inhibition of both polyamine transport (TSP) and ODC activity in the presence of actinomycin-D or CHX.
  • Figure 12 shows ODC inhibition using extracts from MCT cells treated with increasing amounts of agmatine
  • Figure 13 shows ODC inhibition m the presence or absence of anti-antizyme IgG (anti-AZ) or an antizyme inhibitor (Ain) using extracts from MCT cells treated with 10 mM agmatine.
  • anti-AZ anti-antizyme IgG
  • Ain antizyme inhibitor
  • Figure 14 shows the effect of agmatine, spermme, spermidine, putrescine, GBA or ornithine on the generation of cytokine-induced nitric oxide end products in MCT cells.
  • Figure 15 shows the effect of agmatine (1 mM) on the generation of cytokine -induced NO end products in MCT cells over time.
  • Figure 16 shows the effect of diamine oxidase
  • DAO cytokine-mduced NO end products
  • Figure 17 shows the effect of the DAO inhibitor, pentamidine, and agmatine (1 mM) on the cytokine- induced NO end products in MCT cells.
  • Figure 18 shows the effect of pentamidine with increasing concentrations of agmatine on cytokine-mduced NO end products in MCT cells.
  • Figure 19 shows the effect of aldehyde dehydrogenase (AldDH) on the generation of cytokine- mduced NO end products in MCT cells.
  • AldDH aldehyde dehydrogenase
  • Figure 19A shows the effect of increasing amounts of AldDH.
  • Figure 19B shows the effect of increasing amounts of AldDH and agmatine (1 mM)
  • the effect of the AldDH cofactor, nicotinamide aden e dinucleotide (NAD) is also shown.
  • Figure 20 shows the effect of AldDH and increasing amounts of agmatine on the generation of cytokine- induced NO end products in MCT cells.
  • Figure 21 shows the inhibition of NO end product accumulation as a result of increasing amounts of agmatine in various cell lines.
  • Figure 22 shows the effect of administering lipopolysaccharide (LPS) and agmatine plus LPS in Wistar Fromter rats.
  • Figure 22A shows the change in blood pressure.
  • Figure 22B shows the change in glomerular filtration rate (GFR) .
  • the present invention provides a method of reducing polyamine levels intracellularly by administering the compound agmatine to cells or tissues of a mammal.
  • agmatine which is an arginine derivative, can inhibit the enzyme ornithine decarboxylase (ODC), reduce polyamine uptake into cells, and inhibit the inducible nitric oxide synthase (iNOS) enzyme while maintaining constitutive nitric oxide synthase (cNOS) enzyme levels in cells.
  • ODC enzyme ornithine decarboxylase
  • iNOS inducible nitric oxide synthase
  • cNOS constitutive nitric oxide synthase
  • polyamine refers to the naturally occurring polyamines spermidine, spermine, as well as the diamine precursor putrescine.
  • Putrescine is derived from ornithine through the action of ODC.
  • spermidine is formed from putrescine via the enzyme spermidine synthase in conjunction with a decarboxylated-adenosyl ethionine
  • spermine is formed from spermidine and decarboxylated-adenosylmethionine using the enzyme spermine synthase.
  • the diamine putrescine (HN(CH StammNH ) and the polyamines spermidine (H,N(CH )-,NH(CH ),,NH 7 ) and spermine (HnN(CH ) -,NH(CH 2 ) ,NH(CH) 3 NH 2 ) are present in all mammalian cells and are involved in the initiation and progression of the cell cycle, as well as cell hypertrophy and differentiation
  • the biosynthesis of polyamines precedes both protein and nucleic acid synthesis in the cell cycle
  • ODC catalyzes the decarboxylation of ornithine to produce putrescine, from which spermidine and spermine are produced
  • Agmatine is a polyamine, specifically, a decarboxylated arginine derivative having the chemical name guanidmobutylamine or 4 - (a inobutyl) guanid e (C-H H N .
  • Agmatine has a molecular weight of 130 19
  • Arginine is converted to agmatine by arginine decarboxylase (ADC)
  • ADC arginine decarboxylase
  • Arginine is critical to normal cellular growth and multiple physiological processes Arginine concentration in extracellular fluid is maintained at 100 to 200 ⁇ M and is regulated by gastrointestinal absorption, conversion to ornithine by the urea cycle, and synthesis from citrullme in the kidney (Lortie et al . , J. Clin.
  • the metabolites of arginine include NO, which is generated from arginine by NOS, and agmatine, which is a decarboxylated arginine derivative produced from arginine by ADC
  • Derivatives that increase the half-life of the agmatine molecule by decreasing susceptibility to diamme oxidase are one type of derivative Since the agmatine molecule is a four carbon chain separating two positively charged groups, agmatine derivatives useful in the invention maintain the four carbon chain with two positively charged groups at either end.
  • DAO diamme oxidase
  • amme group at one end can be modified, for example, by methylation of the amine or by substitution of a second guanidmium group in place of the amine, or by other types of modification that would prevent oxidation of the amine to the aldehyde by DAO, thus producing an agmatine derivative Agmatine can be used as a pharmaceutical composition, for example, a salt formulation, or a zwitterionic form of the molecule, or can be formulated in a composition or conjugated to a carrier molecule.
  • Agmatine-aldehyde (guanidinobutyraldehyde) is a particular agmatine derivative m which the am e group of agmatine is oxidized to an aldehyde Agmatine is converted by DAO to agmatine-aldehyde, which is unstable and is further metabolized to GBA by aldehyde dehydrogenase (AldDH) .
  • AldDH aldehyde dehydrogenase
  • arginme derivatives in addition to agmatine, are useful in practicing the methods of the invention
  • Arginine derivatives are metabolites of argin e that have a demonstrated ability to reduce NO end product accumulation or biological activity as determined using the assays described in the Examples, or other similar assays known to those in the art
  • Arginme derivatives generally are products of the arginine decarboxylase pathway, including agmatine and agmatine-aldehyde, but also can be synthetic arginine derivatives .
  • agmatine is described by Kosel (Physiol. Chem 68:170 (1910)), which is incorporated herein by reference Agmatine is available from commercial vendors such as Sigma Chemical Company (St. Louis, MO) . Agmatine is a naturally occurring endogenous molecule that concentrates in some organs and is also a component of plasma, which allows distribution of agmatine to all tissues. Agmatine enters cells by the polyamine transport system and its uptake can be competitively inhibited using polyamines or polyamine transport inhibitors (see Figure 6 and Example I) .
  • ODC is the rate limiting enzyme of polyamine biosynthesis and is one of the most highly regulated eukaryotic enzymes.
  • ODC which is the convergence point of many oncogenic signaling pathways, is a proto- oncogene, whose over-expression leads to transformation of certain cells. Therefore, polyamines are also thought to play a proximate role in the transformation process.
  • ODC exhibits the shortest half-life of any described enzyme and is transiently induced m cells in response to various conditions, including following growth factor addition, activation of tyrosine kinase receptors, in hypoxia, following cellular free radical formation, through prostaglandin activity and by immediate early gene activity. In contrast, ODC is constitutively active in cells transformed by oncogenes, carcinogens or viruses .
  • ODC is related to the process of cellular transformation
  • modulating this enzyme with inhibitors such as eflormthine (DFMO) does not prevent proliferation or hypertrophy of cells in experimental models reported to date.
  • the levels of spermidine and spermine inside cells were largely unaffected by inhibition of ODC and these levels were sufficient to allow the normal progression of events models of kidney hypertrophy, liver hyperplasia and smooth muscle hyperplasia.
  • Cells that are unable to synthesize polyamines, for example, due to the presence of the inhibitor DFMO are still capable of taking up polyamines from the environment.
  • Polyamines regulate their biosynthesis by feedback inhibition, indirectly inducing the translational expression of the protein ODC antizyme (AZ) .
  • AZ has a dual function, inhibiting both ODC activity and polyamine transport into the cell
  • Abnormal polyamine biosynthesis is associated with abnormal cell growth such as cancer, cell enlargement and hypertrophy. Preventing polyamine synthesis mammalian cells through the use of inhibitors results in complete cessation of growth unless exogenous polyamines are provided (Pegg, Cancer Res. 48:759-774 (1988)) .
  • the use of inhibitors that block polyamine biosynthesis as therapeutic agents is directed towards a variety of diseases involving pathological cell proliferation or cell enlargement.
  • Synthetic ODC inhibitors have been tested for therapeutic impact on abnormal cell proliferation and cell enlargement
  • Presently known inhibitors of ODC can be classified as reversible inhibitors, such as direct competitors, and enzyme-activated irreversible inhibitors.
  • the latter often referred to as "suicide” inhibitors, are chemically inert substrates for the enzyme, that inactivate the enzyme upon binding.
  • the most commonly used inhibitor is DFMO (Pegg, supra . 1988) .
  • ODC inhibitors does not always produce the intended results.
  • models of smooth muscle hyperplasia in the rat (Luck et al., Am. J. Physiol. 267 :G1021-G1027 (1994)), as well as in a model of liver regeneration (Beyer et al . , Am. J Physiol . 262 :G677-G684 (1992))
  • the administration of the ODC inhibitor DFMO did not prevent hyperplasia or the regeneration of liver cells.
  • Putrescine content in the tissues was reduced after the administration of DFMO, but the level of spermine and spermidine was unchanged. Reducing the biosynthesis of polyamines intracellularly increased uptake of polyamines from the surrounding tissue .
  • Polyamines in normal cell types, are the products of a highly regulated intracellular biosynthetic pathway Polyamines are transported into and out of cells through temperature sensitive, energy dependent transporters (Humphries et al , Am J Physiol . 255 : F270-F277 (1988)). Polyamine uptake can substitute for de novo synthesis. Extracellular polyamine uptake can be enhanced by many of the same factors that induce ODC activity, for example, growth factors and hormones.
  • Polyamine transport is inhibited by the induction of intracellular biosynthesis and, conversely, biosynthesis is inhibited by the induction of transport, thereby demonstrating a system highly sensitive to intracellular polyamine levels
  • extracellular polyamine levels generally are far lower than intracellular concentrations, plasma polyamine levels, as well as cellular uptake, are often markedly elevated in malignancy. Therefore, inhibitors targeting only polyamine biosynthesis have had very limited success as cancer therapeutics.
  • AZ inhibits polyamine uptake and inhibits and destabilizes ODC.
  • the degradation of ODC is catalyzed by 26S protease (Murakami et al . , Nature 360:597-599 (1992)) .
  • ODC is destabilized by AZ, where the C-terminal half of AZ binds to ODC, inducing a conformational change (Li et al., Mol . Cell. Biol. 13:2377-2383 (1993)) allowing an additional internal sequence in AZ to promote destabilization of ODC (Li et al . , Mol. Cell. Biol. 14:87-92 (1994); Ichiba et al., Biochem. Biophvs. Res.
  • ODC is not only short-lived like other key proteins, but its turnover is regulated.
  • the induced destabilization by AZ is analogous to the human papilloma virus oncoprotem E6 action on the tumor suppressor p53 (Scheffner et al . , Cell 53:1129-1136 (1990); Matsufuji et al , Cell 80:51-60 (1995) ) .
  • AZ has a second function, repressing polyamine uptake, thereby effectively preventing polyamine accumulation m cells.
  • researchers using ODC-overproducmg cells transfected with an AZ cDNA found a decrease in polyamine transport into the cells and subsequent cellular toxicity when an ODC inhibitor was administered, compared with controls where polyamine transport increased in response to the use of the ODC inhibitor DFMO (Suzuki et al . , Proc. Natl. Acad. Sci., USA 91.8930-8934 (1994)). Therefore, induction of AZ inhibits polyamine biosynthesis as well as transport. However, until the present invention, only the natural polyamines, putrescine, spermidine and spermine were known to induce AZ in a feedback regulatory manner.
  • AZ induction by polyamines occurs at the translational level. AZ synthesis was blocked by cycloheximide JCHX) , but not by actmomycm D (Fong et al., Biochim. Biophys. Acta 428.456-465 (1976); Matsufuji et al . , J. Biochem. 107:87-91 (1990)). While very low levels of AZ are present m mammalian tissues, mRNA levels are relatively high and not further elevated by polyamines (Matsufu i et al . , supra . 1990) . The mechanism of polyamine feedback inhibition through AZ involves modulation of frameshifting due to the cell concentration of polyamines (Matsufuji et al . , supra . 1995) .
  • the present invention provides methods of decreasing intracellular polyamine levels by administering an arginine derivative, such as agmatine or a derivative thereof, to cells or tissues of a mammal.
  • an arginine derivative such as agmatine or a derivative thereof
  • administering agmatine to a cell culture or tissue inhibits ODC activity and uptake of polyamines through the induction of AZ .
  • the present invention also provides methods of inhibiting the ODC enzyme by administering a composition, containing an arginine derivative, such as agmatine or a derivative thereof, to the cells or tissues of a mammal.
  • Example I The inhibitory effect of agmatine on ODC activity is demonstrated in Example I.
  • MCT cells, SV-40 transformed proximal tubule cells like other transformed cell lines, demonstrate high constitutive ODC levels (Olanrewaju et al., Am. J. Physiol. 63 (2Ptl) : E282-E286 (1992) which is incorporated herein by reference) .
  • the transformed cells mimic the cellular hypertrophy (Luck et al . , supra. 1994) or the profile of ODC expression in diabetes (Levine et al., Diabetes 29:532-535 (1980)).
  • Tubule cells from nephrectomized animals have marked inverse changes in the ODC and ADC activities from control tubule * cells .
  • ODC activity is elevated 24 hours post-nephrectomy, while ADC activity is decreased.
  • ADC catalyzes the conversion of arginine to agmatine, and its activity is considered indicative of agmatine production in various tissues (Lortie et al . , supra. 1996) .
  • Agmatine effectively suppresses ODC activity in the tubules. At 1 mM agmatine concentration, ODC activity was almost completely suppressed (Figure 3) .
  • Agmatine inhibits polyamine uptake into cells (see Example III) . Concentrations of agmatine (10 ⁇ M or higher) effectively reduced putrescine uptake into cells. CHX, but not actinomycin-D, affected the inhibition demonstrating that only translation is required for agmatine inhibition of both ODC and polyamine transport. These results are consistent with the induction of AZ by agmatine. The role of AZ was demonstrated directly in Example IV. Agmatine was administered to MCT cells, then extracts of these agmatine- treated cells were used to inhibit ODC. Extracts of agmatine- treated cells decreased ODC activity in a dose-dependent manner. Extracts of MCT cells treated with 10 M agmatine were added to an ODC assay mixture.
  • the present invention further provides a pharmacological composition containing an arginine derivative, such as agmatine or a derivative thereof, and a physiologically acceptable carrier.
  • an arginine derivative such as agmatine or a derivative thereof
  • a physiologically acceptable carrier includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, or emulsions such as an oil/water or water/oil emulsion and various types of wetting agents
  • dosages of between approximately 5 ⁇ g/kg body weight to 80 g/kg body weight are preferred for the pharmacological compositions of the invention
  • the preferred dosage will vary with the mode of administration Multiple or intravenous administrations, for example, allow lower dosages than intramuscular or other routes of administration Very h gh dosages of agmatine administered to test animals (up to 80 mg/kg body weight) every three hours were well tolerated
  • a broad range of dosages is available for the treatment of various pathological conditions
  • the dosage will vary with the condition being treated as well as the method of administration .
  • a pharmacological composition of the invention can also include other components to enhance the effectiveness or stability of the argmme derivative.
  • DAO inhibitors such as aminoguanidine or pentamidine can be included to inhibit agmatine metabolism and increase agmatine half -life. DAO converts agmatine to guamdmobutyraldehyde
  • a pharmacological solution can be administered using a number of methods known in the art, for example, intravenously, intraperitoneally, intramuscularly, intranasally , or subcutaneously. In some cases, the pharmacological composition can be infused directly into the tissue that is targeted.
  • the present invention also provides methods of treating pathological conditions by administering a composition, comprising an arginine derivative such as agmatine or a derivative thereof.
  • a composition of the invention comprising an arginine derivative such as agmatine or a derivative thereof.
  • the pathological conditions most suited for treatment using a composition of the invention are characterized, in part, by abnormal cellular proliferation or hypertrophy, for example, tumor development.
  • Compositions containing agmatine for example, can be administered directly to cells to prevent proliferation of the cells, or can be administered to an individual to prevent angiogenesis associated with tumor development.
  • a composition of the invention can also be administered to prevent clonal cell expansion in the immune response in order to reduce inflammation or to increase tolerance for transplanted organs.
  • arginine derivative such as agmatine is capable of inducing the same biochemical responses to hypertrophy as it does with respect to proliferative conditions.
  • Methods of treating conditions such as cardiac hypertrophy, renal disease progression and the hypertrophy characteristic of diabetic renal disease by administering a composition comprising an arginine derivative such as agmatine or a derivative thereof are provided by the invention.
  • Polyamines have multiple other functions including acting as antioxidants , modulating differentiation and apoptosis, and regulating transport through channels. Therefore, influencing the levels of deleterious polyamines in the cells can potentially affect all of these conditions.
  • AZ was believed to be induced only by ODC m a feedback dependent manner.
  • the present invention provides methods of inducing AZ in a non- feedback dependent manner through the administration of an arginine derivative such as agmatine or a derivative thereof
  • other naturally occurring or synthetic molecules can be screened for modulation of AZ expression, thereby manipulating both polyamine biosynthesis and transport
  • Such a screening method would involve, for example, the use of an anti-AZ antibody, which can be a monoclonal or polyclonal antibody specific for the AZ and made as described, for example, in Harlow and Lane, Antibodies. A laboratory manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference AZ has been isolated and characterized (Hayashi et al . , Ornithine Decarboxylase Biology. Enzymology. and Molecular
  • Agmatine and other arginme derivatives are polyamines that function as inhibitors of the enzyme ODC.
  • Other enzymes, including iNOS, can also be inhibited by agmatine.
  • the present invention also provides methods of selectively inhibiting inducible nitric oxide synthase (iNOS) , while maintaining or enhancing constitutive nitric oxide synthase (cNOS) production
  • NO is an inorganic free radical NO is produced in many cell types by the conversion of
  • L-argmme to L-citrullme and NO through NOS NOS converts L-argmine to N G -hydroxy-L-argmme , which is then further converted to citrullme and NO
  • the biological activity of NO is the result of the activation of various enzymes, for example, guanylyl cyclase, and the inhibition of others, for example, acomtase or ribonucleotide reductase, or activation by alternative mechanisms such as damaging nucleic acids NO is produced by either cNOS or iNOS enzymes
  • NO synthases occur as a family of isoenzymes Two of the cNOS isozymes are constitutively produced (NOS I and NOS III) and iNOS (NOS II) is induced by immunological stimuli such as endotoxin or inflammatory cytokines .
  • cNOS first described in brain and endothelial cells, is activated by acetylcholine , bradykinm, and other substances, resulting in shortlived production of NO in picomolar amounts.
  • the NO released by constitutive enzymes acts as an important signaling molecule in cardiovascular and nervous systems
  • the NO released by iNOS n response to cytokines or endotoxin is generated for long periods and in nanomolar amounts (Nathan et al . , Cell 76:915-918 (1994); Ketteler et al . , Am J Physiol 36:F197-F207 (1994))
  • the iNOS is made by macrophages, hepatocytes, vascular smooth muscle cells, mesangial cells, renal tubular cells, and other cell types and has been shown to be cytostatic and cytotoxic for tumor cells and a variety of organisms. Each isoform contains a reductase as well as a heme domain and requires a number of cofactors
  • the enzymes are produced by at least three different genes and range in molecular weight from about 130 kDa to 160 kDa.
  • LPS lipopolysaccharides
  • interferon y and other cytokines induce the synthesis of NOS in macrophages and related cells (Granger et al . , J. Immunol. 146:1294-1302 (1991)) .
  • the NO produced arrests the growth of microbes and tumor cells by several mechanisms.
  • iNOS protection against microbes also cause inflammatory damage to host cells and tissues by the potential injurious nature of high NO levels. Deleterious effects include the combination with 0 : , or superoxide ion, where NO can damage DNA and induce mutations.
  • the genotoxic potential may be responsible for initiating various genetic disorders including some cancers.
  • iNOS induction of iNOS has been implicated in numerous pathological conditions, including sepsis- related hypotension, disturbances of the hemostatic- thrombotic balance, and local vascular lesions such as atherosclerosis and post angioplasty arterial injury.
  • NO-induced hypotension leads to cardiovascular complications in septic shock patients as well as during cytokine-based immunotherapy .
  • Studies in iNOS deficient mice have elaborated on the role of NO in septic shock. When iNOS deficient mice were challenged with bacterial endotoxic LPS, they did not suffer from the fall in central arterial blood pressure and subsequent death caused by septic shock when compared with the control wild type mice (MacMicking et al . , Cell 81:641-650 (1995) ) .
  • Nonspecific NOS inhibitors can alter various autoimmune diseases including glomerulonephritis and arthritis, indicating that elevated NO production is important in the pathogenesis of autoimmune disease.
  • Studies with the mouse model of spontaneous mu ⁇ ne autoimmune disease indicate that increased NO production corresponds with the onset of autoimmune disease and the manifestations of the disease can be reduced by administering a NO inhibitor (Wemberg et al . , J . Exp . Med. 179:651-660 (1994))
  • Abnormalities described in tumor vasculature are attributed to increased NO production m the tumor.
  • NO production is also associated with increased vascularization in nude mice resulting in rapid progression of tumor growth (Andrade et al., Br J Pharmacol.
  • NO affects immune suppression in transplantation and graft rejection
  • Increased NO levels correlate to the degree of graft rejection
  • Use of the immunosuppressive drugs cyclosporin A or FK506 result in the inhibition of NO production in vivo (Langrehr et al . , J. Chn Invest 90:679-683 (1992)).
  • ammoguanidine administration selectively inhibits iNOS and was beneficial to survival (Devlin et al . , supra .
  • ADC coverts arginme to agmatine.
  • the ADC activity is indicative of agmatine production various tissues, such as in membrane-enriched fractions of the brain, liver, and kidney cortex and medulla (Lortie et al., supra. 1996).
  • Constitutive ADC activity in mammalian systems is highest in the kidney (glomeruli and tubules) and the liver (Lortie et al , supra. 1996) .
  • Arginine is the only physiological nitrogen donor for the NOS catalyzed NO synthesis.
  • Proximal tubules are a major site of arginine synthesis in the kidney (Levillian et al . , Am . J .
  • a mouse kidney proximal tubule cell line MCT (Olanrewaju et al . , supra, 1992, which is incorporated herein by reference), was used as a model to determine if metabolites of the ADC pathway could modulate NO production.
  • Agmatine is structurally similar to the polyamine putrescine, being composed of two catiomc regions separated by a four carbon chain backbone It differs from the polyamines in having a guanidmium moiety as one catiomc moiety.
  • arginine metabolites cells include the argmme metabolite agmatine and the agmatine metabolite agmatine-aldehyde
  • Cytokine-stimulated MCT cells produced NO end products as determined by the Greiss reaction (see below) .
  • the NO end products of the stimulated cells correspond to NO produced by iNOS .
  • agmatine addition to the cells inhibited NO end product accumulation (Example V)
  • DAO resulted in the reduction of NO end products
  • DAO with agmatine farther reduced NO endproduct accumulation.
  • AldDH increased the amount of NO end products.
  • polyamines spermme andspermidine also were potent inhibitors of iNOS (Example V) However, these polyamines were toxic to cells at dosages greater than 100 ⁇ M, and their aldehyde derivatives were even more toxic (Example V) . In contrast, agmatine is not toxic to cells or animals, even at multiple dosages of 80 mg/kg.
  • Several cell lines were screened for the efficacy of agmatine derivatives in reducing cytokine-stimulated iNOS production. iNOS production was reduced in all cell lines tested by exogenous agmatine addition (Example V) . Variation in the transport of exogenous agmatine into the cells is responsible for the difference in efficacy.
  • Bacterial LPS was administered to Wistar Fromter rats as an in vivo model of septic shock. Multiple agmatine doses as high as 80 mg/kg administered intraperitoneally were well tolerated by the animals. Blood pressure and GFR, an indicator of kidney function, were both normalized by administration of agmatine. Thus, the extreme hypotension encountered in septic shock was normalized by agmatine. Furthermore, the animals had no side effects in response to agmatine administration and were alert and active afterwards. Schwartz et al . ,
  • the present invention provides a method of specifically inhibiting iNOS without inhibiting cNOS by administering an arginine derivative such as agmatine or a derivative thereof to a mammal.
  • the present invention further provides a pharmacological composition containing an arginine derivative in a physiologically acceptable carrier.
  • Argiiine derivatives preferred in the present invention include agmatine and its metabolite agmatine-aldehyde.
  • a pharmacological composition of the invention can include other components enhancing the effectiveness or stability of the active agent.
  • the DAO enzyme co-administered with agmatine increases intracellular agmatine-aldehyde concentration derived from the agmatine administered and enhances the inhibitory effect on NO end product accumulation (see Example V) .
  • DAO alone decreases NO end product accumulation due to its action of producing agmatine-aldehyde.
  • Inhibitors of AldDH also serve to increase the cellular pool of agmatine-aldehyde by preventing breakdown of this metabolite to the acid.
  • a pharmacological composition of the invention advantageously can include components stabilizing the pool of agmatine-aldehyde, such as DAO, an agonist of DAO, or an inhibitor of AldDH.
  • the present invention further provides a method of treating a pathological condition by administering a composition comprising an arginme derivative such as agmatine or a derivative of agmatine
  • a condition can be any pathological condition resulting, at least in part, from an excess of NO generation due to the stimulation of iNOS .
  • pathological conditions include, for example, septic shock, arthritis, glomerulonephritis, angiogenesis in tumors, transplantation and graft rejection, neurodegeneration, stroke, ischemic injury, chronic inflammation and diabetes.
  • a method of the invention is particularly useful for treating septic shock.
  • septic shock refers to a condition of acute circulatory failure in a subject secondary to infection or the presence of toxic microbial products, for example, bacterial LPS, which trigger systemic inflammatory responses in the subject Septic shock can be associated, for example, with hypotension, coldness of the skin, tachycardia and anxiety Septic shock also can be associated, for example, with the induction of inflammatory cytokines, which trigger a dramatic fall in blood pressure, organ failure and death.
  • the present invention provides a method of treating septic shock by administering a pharmaceutical composition comprising an argmme derivative such as agmatine or a derivative thereof.
  • Preferred dosages for the treatment of septic shock range from 5 ⁇ g/kg body weight to 80 mg/kg body weight.
  • the present invention provides methods of reducing polyamine levels and inhibiting iNOS activity by administering agmatine or a derivative thereof, thereby inducing AZ
  • Administering agmatine both inhibits ODC and reduces polyamine uptake into cells
  • the methods of the invention inhibit iNOS activity while maintaining or enhancing cNOS activity Therefore, the method is useful therapeutically for controlling certain pathological conditions involving excessive cell proliferation or enlargement
  • Argin e is converted to agmatine by ADC
  • ADC activity is considered indicative of agmatine production in various tissues (Lortie et al , supra . 1996), which is incorporated herein by reference
  • ADC activity has been demonstrated in the membrane -enriched fraction of the brain, liver, and kidney cortex and medulla by a radiochemical assay (Lortie et al . , supra , 1996)
  • Compensatory renal hypertrophy in response to unilateral nephrectomy is associated with early induction of ODC activity and increased polyamine levels, as described, for example, in Humphries et al . , Am. J. Physiol 255 (24) :F270-F277 (1988), which is incorporated herein by reference.
  • a single kidney (unilateral nephrectomy) is removed from the experimental animal, and the remaining kidney experiences compensatory renal hypertrophy (increase in the size and weight of the kidney) in response
  • a unilateral nephrectomy isolated tubule cells prepared as described below
  • the iidney cortex was dissected and separated into glomerular and tubule preparations by sequential sieving (Tucker et al . , J. Cl . Invest. 78:680-688 (1986)) .
  • the preparations were then homogenized m ice-cold ODC reaction buffer (10 mM Tris, pH 7.4 , 2.5 mM DTT, 0.3 mM pyridoxal- 5 -phosphate, and 0.1 mM EDTA) .
  • MCT cells (Olanrewaju et al . , supra, 1992) were plated on 10 cm culture dishes and allowed to grow to confluence in DMEM with 5% FCS .
  • tissue preparation 250 ⁇ l was added to large bare glass reaction tubes .
  • Ex vivo tubular or glomerular preparations were incubated with agmatine (1 mM) for 1 hour at 37°C prior to the start of the enzyme reactions.
  • MCT cells were incubated with experimental agents in DMEM plus 5% FCS for 16 hours unless otherwise noted, ODC and ADC reactions were started by the addition of 0.1 ⁇ Ci C-carboxyl labeled L-ornithme or L-argmine, respectively, to the tissue preparations.
  • Tubes were capped with rubber stoppers fitted with metabolic wells (KONTES) containing 250 ⁇ l of trapping agent (Solvable, Dupont Corporation, Boston, MA) .
  • Incubations were for 1 hour at 37°C. Reactions were stopped by injection of 200 ⁇ l of 50% TCA and allowed to equilibrate for an additional hour prior to counting trapped 1 C-CO, .
  • MCT cells were plated at 5000 cells/well in
  • Tubular cells demonstrated marked inverse changes in the ODC and ADC activities in control versus nephrectomized tubule preparations (Figure 1) .
  • ODC activity increased in tubules 24 hours post-nephrectomy, while ADC activity was suppressed post-nephrectomy
  • Nephrectomy- prised ODC activity m tubules was blunted by the addition of agmatine ex v vo ( Figure 2) .
  • Tubules were incubated for one hour with 1 mM agmatine or 1 mM putrescine .
  • FIG. 4 A time course (30 minutes to 24 hours) for agmatine inhibition of ODC activity is shown ( Figure 4)
  • the effect of CHX or actinomycin-D on agmatine inhibition of ODC activity is presented (Figure 5A) MCT cells were incubated with or without 100 ⁇ g/ml CHX in the presence or absence of agmatine. Agmatine effectively inhibited ODC activity by about 74.5% in the absence of CHX In the presence of CHX the level of inhibition is reduced to about 19.4% of the control.
  • Agmatine inhibition requires new protein synthesis
  • Agmatine effectively inhibits ODC activity in the absence (by 88.7%) or presence (by 97.0%) of actinomycm-D, indicating new transcription was not required for inhibition of ODC by agmatine ( Figure 5B) .
  • Agmatine inhibits ODC activity in a number of additional cell lines. All cell lines were grown as described above in the presence or absence of 1 mM agmatine. At 48 hours the cells were harvested and assayed for ODC activity. The inhibition was compared to the control untreated cells.
  • the various cell lines include MDCK canine kidney epithelial -like cell line (ATCC, Rockville, MD) , ENDO endothelial cell line (Hothofer et al , Lab Invest 69 (2 ) • 183 - 192 (1993)), MCT cells (Haverty et al , supra, 1988), JS-1 cancer transformed Schwann cell line, MC mouse kidney mesangial cell line, NKD-49-fibroblast cell line, LLCPK pig kidney cell line, Hep-G2 cancer transformed liver hepatocyte cell line, J774 and RAW309 monocyte/macrophage cell lines (ATCC, Rockville, MD) .
  • the ODC activity m all cell lines was inhibited to between 75% and 100% of control activity.
  • ⁇ -agmatine uptake by polyamines (putrescine, spermidine and spermine) ; and by a polyamine transport inhibitor paraquat.
  • the results demonstrate that competition for agmatine transport was specific for polyamines Conversely, agmatine uptake was not competitively inhibited by the arginine catiomc (system y+) transporter agents L-NMMA or ornithine, lysine or arginine, or by agmatine 's stable acid metabolite guanidmobutyric acid (GBA) .
  • putrescine 50 ⁇ M
  • MCT are transformed proximal tubule cells and demonstrate high constitutive ODC levels
  • a lag period of 30 minutes prior to agma ine -dependent inhibition of ODC activity correlates with observed polyamine induction of AZ (Hayashi et al , Biochem J. 306 1-10 (1995))
  • experiments with CHX and actmoraycin-D demonstrate that ODC inhibition is dependent upon new protein synthesis, but not new transcription ( Figures 5A and 5B)
  • Polyamine activation of AZ displays these characteristics due to translational frame-shifting (Matsufuji et al , supra. 1995) indicating that agmatine acts by inducing AZ
  • the uptake of 3 H-putrescine into MCT cells was measured in the presence of increasing concentrations of agmatine (10 ⁇ M to 1000 mM) , putrescine, spermidine, paraquat, ornithine, lysine, and arginine ( Figure 8) .
  • Agmatine (10 ⁇ M) reduced uptake to about 90% of the control, while a higher concentration (1 mM) completely inhibited J H-putrescine uptake.
  • Polyamines also inhibited putrescine uptake, while the amino acids did not.
  • agmatine is capable of down-regulating polyamine transport, even at low concentrations.
  • agmatine is capable of increasing the inhibition of ⁇ -putrescine uptake in a time-dependent manner, comparable to the regulation of polyamine transport.
  • Agmatine decreased ODC activity in a dose -dependent manner m MCT cells ( Figure 12) .
  • Cells were grown to approximately 70% confluence 10 cm dishes and medium changed approximately 24 hours prior to the addition of increasing concentrations of agmatine or the DAO inhibitor am oguanidine Twenty- four hours later, cells were harvested, extracts of the cells prepared by three cycles of freezing and thawing, the cell extract suspended in 0.3 ml of 0.155 M KC1 containing 1 mM DTT, and the extract was centrifuged at 3000 x g for 20 minutes The supernatant was assayed for ODC inhibitory activity by adding 50 ⁇ l of the supernatant to the ODC reaction mixture, described in Example I above, to a final volume of 0.140 ml.
  • Agmatine induced the ODC inhibitory activity AZ ( Figure 13) .
  • the activity was precipitated with anti-AZ antibody and neutralized with an AZ inhibitor, indicating that the inhibitor is AZ
  • the same procedure was used as described in Figure 12 except that the MCT extract was treated with 10 mM agmatine.
  • part of the extract was treated with control IgG or anti-AZ antibody bound to an immunoadsorbent (Kana ota et al., J. Biol. Chem. 268 ( 1 ) : 3 3 - 9399 (1993), which is incorporated herein by reference) .
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • IFN- ⁇ interferon- ⁇
  • the Greiss reaction measures only NO ; (Granger et al . , J. Immunol. 146:1294-1302 (1991); Southan et al ... Biochem. Biophys. Res. Commun. 203:1638-1644 (1944), each is incorporated herein by reference) .
  • Nitrate is reduced to nitrite with bacterial nitrate reductase, and then nitrite is measured using the Greiss reagent (1% sulfanilamine and 0.1% naphthylethylenediamide in 5% phosphoric acid, as described in Report of the American Institute of Nutrition Ad hoc Committee on
  • Pentamidine (Sigma Chemical Company) is an effective inhibitor of DAO, but has no inhibitory effect on NO end product accumulation. Agmatine and increasing concentrations of pentamidine were administered to MCT cell cultures ( Figure 17) . Agmatine, pentamidine and cytokines were added and the cells incubated for 48 hours, samples of media were taken, and the samples analyzed for total nitrite by the Greiss reaction. Protein content of the cells was determined by the Lowry reaction. Pentamidine repressed the inhibitory effects of agmatine on iNOS ( Figure 17) . Pentamidine blocks the conversion of agmatine to agmatine-aldehyde by DAO. Increasing concentrations of agmatine can overcome the repression of pentamidine ( Figure 18) .
  • Cell lines were stimulated to induce iNOS activity for 48 hours and nitrite end products measured by the Greiss reaction described above.
  • Cell lines include NRK-49 fibroblast cells, J774 monocyte cell cultures (ATCC, Rockville, MD) , esangial cell lines, endothelial cell lines (Hothofer et al . , supra. 1993) and MCT cells (Haverty, et al., supra. , 1988).
  • Cells were grown as described above, and increasing concentrations of agmatine were added to the cell lines. All of the cell lines showed a decrease in NO end product accumulation compared to the control with increasing concentrations of agmatine ( Figure 21) .
  • the response to agmatine however, varies substantially between cell types, due to variations in agmatine uptake in the different cells.

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Abstract

La présente invention concerne un procédé pour réduire le niveau de polyamine à l'intérieur des cellules en administrant un dérivé d'arginine à un mammifère. La présente invention concerne également une composition pharmacologique comprenant de l'agmatine dans un tampon physiologiquement acceptable. En conséquence, la présente invention concerne également un procédé pour traiter les états pathologiques qui résultent d'un niveau anormalement élevé de polyamine à l'intérieur des cellules, en administrant un dérivé d'arginine ou de l'agmatine aux cellules dans un état cancéreux ou d'hypertrophie. La présente invention concerne également un procédé pour réguler le monoxyde d'azote synthétase inductible tout en conservant le monoxyde d'azote synthetase constitutif, en administrant de l'agmatine ou un dérivé d'arginine à un mammifère. La présente invention concerne également un procédé pour traiter les chocs septiques chez un mammifère, en lui administrant une composition comprenant de l'agmatine ou un dérivé d'arginine. En outre, la présente invention concerne un procédé pour traiter les états pathologiques résultant d'une production excessive de monoxyde d'azote inductible, y compris le traitement des chocs septiques, de l'arthrite, de la néphrite glomérulaire, de l'angiogénèse dans les tumeurs, de la transplantation et du rejet de greffe tissulaire, de la dégénérescence neuronale, des accidents cérébrovasculaires, des lésions d'origine ischémique, des inflammations chroniques et du diabète.
PCT/US1997/017424 1996-09-25 1997-09-25 Procedes d'utilisation de l'agmatine pour reduire le niveau de polyamine a l'interieur des cellules et inhiber le monoxyde d'azote synthetase inductible WO1998013037A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035431A2 (fr) * 1998-12-11 2000-06-22 University College London Traitement de troubles de l'erection chez des patients diabetiques
WO2000074701A2 (fr) * 1999-06-05 2000-12-14 The Board Of Trustees Of The Leland Stanford Junior University Methode et composition permettant d'inhiber la proliferation de cellules du systeme cardio-vasculaire
WO2001097794A2 (fr) * 2000-06-21 2001-12-27 Georgetown University Decouverte a l'aide d'une structure d'inhibiteurs de matriptase destines au traitement du cancer et d'autres pathologies
WO2003017994A1 (fr) * 2001-08-31 2003-03-06 Neurochem (International) Limited Derives d'amidine destines au traitement de l'amylose
US7262223B2 (en) 2004-01-23 2007-08-28 Neurochem (International) Limited Amidine derivatives for treating amyloidosis
US7557087B2 (en) 2004-03-01 2009-07-07 Lumen Therapeutics, Llc Compositions and methods for treating diseases
FR2985427A1 (fr) * 2012-01-10 2013-07-12 Nutrialys Medical Nutrition Sa Compositions contenant de l'agmatine et leurs utilisations dans la preparation de medicaments ou de substances nutraceutiques
US11311565B2 (en) 2020-06-25 2022-04-26 Celagenex Research (India) Pvt. Ltd. Synergistic nutritional compositions for promoting axonal regeneration

Citations (2)

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US5574059A (en) * 1995-10-27 1996-11-12 Cornell Research Foundation, Inc. Treating disorders mediated by vascular smooth muscle cell proliferation
US5677349A (en) * 1995-04-27 1997-10-14 Gilad; Gad M. Agmatine for the treatment of neurotrauma and neurodegenerative diseases

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5677349A (en) * 1995-04-27 1997-10-14 Gilad; Gad M. Agmatine for the treatment of neurotrauma and neurodegenerative diseases
US5574059A (en) * 1995-10-27 1996-11-12 Cornell Research Foundation, Inc. Treating disorders mediated by vascular smooth muscle cell proliferation

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035431A2 (fr) * 1998-12-11 2000-06-22 University College London Traitement de troubles de l'erection chez des patients diabetiques
WO2000035431A3 (fr) * 1998-12-11 2000-11-09 Univ London Traitement de troubles de l'erection chez des patients diabetiques
WO2000074701A2 (fr) * 1999-06-05 2000-12-14 The Board Of Trustees Of The Leland Stanford Junior University Methode et composition permettant d'inhiber la proliferation de cellules du systeme cardio-vasculaire
WO2000074701A3 (fr) * 1999-06-05 2001-08-30 Univ Leland Stanford Junior Methode et composition permettant d'inhiber la proliferation de cellules du systeme cardio-vasculaire
WO2001097794A2 (fr) * 2000-06-21 2001-12-27 Georgetown University Decouverte a l'aide d'une structure d'inhibiteurs de matriptase destines au traitement du cancer et d'autres pathologies
WO2001097794A3 (fr) * 2000-06-21 2003-08-21 Univ Georgetown Decouverte a l'aide d'une structure d'inhibiteurs de matriptase destines au traitement du cancer et d'autres pathologies
WO2003017994A1 (fr) * 2001-08-31 2003-03-06 Neurochem (International) Limited Derives d'amidine destines au traitement de l'amylose
US7262223B2 (en) 2004-01-23 2007-08-28 Neurochem (International) Limited Amidine derivatives for treating amyloidosis
US7557087B2 (en) 2004-03-01 2009-07-07 Lumen Therapeutics, Llc Compositions and methods for treating diseases
FR2985427A1 (fr) * 2012-01-10 2013-07-12 Nutrialys Medical Nutrition Sa Compositions contenant de l'agmatine et leurs utilisations dans la preparation de medicaments ou de substances nutraceutiques
WO2013104871A1 (fr) 2012-01-10 2013-07-18 Nutrialys Medical Nutrition Sa Compositions contenant de l'agmatine et leurs utilisations dans la preparation de medicaments ou de substances nutraceutiques
US11311565B2 (en) 2020-06-25 2022-04-26 Celagenex Research (India) Pvt. Ltd. Synergistic nutritional compositions for promoting axonal regeneration

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