US5545625.Pdf

||||||||| US005545625A United States Patent 19 11 Patent Number: 5,545,625 Gross et al. (45. Date of Patent: Aug. 13, 1996

54). PREVENTING CONVERSION OF Schmidt, H. H. H. W., et al, Eur. J. Pharmacol. 154, CITRULLINE TO ARGINNOSUCCNATE TO 213–216 (1988). LIMIT PATHOLOGICAL NITRIC OXDE Sessa, W. C., et al. Proc. Natl. Acad. Sci. USA, OVERPRODUCTION 87.8607-8611 (Nov. 1990). Corbett, J. A., et al, Biochemistry 32, 13767-13770 (1993). 75 Inventors: Steven S. Gross, New York, N.Y.: Cross, A. H., et al., J. Clin. Invest. 93, 2684-2690 (1994). Owen W. Griffith, Milwaukee, Wis. McCartney-Francis, N., et al, J. Exp. Med. 178, 749–754 (1993). (73) Assignee: The Medical College of Wisconsin Misko, T. P., et al, Eur. J. Pharmacol. 233, 119-125 (1993). Research Foundation, Inc., Suarez-Pinzon, W. L., et al, Endocrinology, 134, 1006-1010 Milwaukee, Wis. (1994). Takada, S., et al., J. Biochem. 85, 1309-1314 (1979). 21 Appl. No.: 354,585 Freytag, S. O., Medline Abstract of J. Biol. Chem..., 259, 22 Filed: Dec. 12, 1994 3160-6 (1984). Grisolia, S., et al, The Urea Cycle, John Wiley & Sons, p. (51 Int. Cl." ...... A61K 31/70; A61 K 31/195 191 (1976). 52 U.S. Cl...... 514/33; 514/561; 514/564; Mitchell, J. A., et al, Eur, J. Pharmacol., 182, 573-576 514/930 (1990). 58 Field of Search ...... 514/33, 561,564, Mulligan, M. S., et al. Proc. Natl. Acad. Sci. USA, 88, 514/930 6338-6342 (Jul 1991). Weinberg, J. B., et al., J. Exp. Med. 148, 651–660 (Feb. 56 References Cited 1994). U.S. PATENT DOCUMENTS Wu, G., et al. Biochem. J., 281, 45-48 (1992). 4,775,623 10/1988 Katsumata et al...... 435/114 Hattori, Y., et al., J. Biol. Chem. 269,9405-9408 (1994). 5,028,627 7/1991 Kilbourn et al...... 514/565 Ialenti, A., ct al., Eur, J. Pharmacol. 211, 177-182 (1992). 5,059,712 10/1991 Griffith ...... 562/560 Mulligan, M. S., et al, Br. J. Pharmacol., 107, 1159–1162 5, 196,195 3/1993 Griffith et al...... 424/94.6 (1992). 5,216,025 6/1993 Gross et al...... 514/565 Nathan, C. F., et al, Current Opinion in Immunology, 3, 5,364,881 11/1994 Griffith et al...... 54/508 65–70 (1991). 5,424,447 6/1995 Griffith et al...... 594/74. 5,464,858 11/1995 Griffith et al...... 514/399 Yang, X., et al, J. Clin. Invest., 94, 714-721 (1994). FOREIGN PATENT DOCUMENTS Primary Examiner-Kimberly Jordan WO95/O1972 1/1995 WIPO 57 ABSTRACT OTHER PUBLICATIONS Administration of argininosuccinate synthetase activity Carignan, J. A., et al, Hosp. Formul. 21, 1025-1030 and reducing agents, e.g., argininosuccinate synthetase induction 1033 (1986). blocking agents (e.g., antibiotics that bind to DNA Corbett, J. A., et al., J. Clin. Invest. 90, 2384-2391 (1992). Sequences present in the upstream regulatory region of the Jinno, Y., et al, EMBL Database, Sequence from J. Biochem. argininosuccinate synthetase gene, such as mithramycin) 98, 1395-1403 (1985). and argininosuccinate synthetase inhibitors (e.g., L-citrul Kleeman, R., et al, FEBS, 328, 9–12 (Aug. 1993). line antagonists such as methyl citrulline and L-aspartatic Nussler, A. K. etal, J. Biol. Chem., 269, 1257–1261 (1994). antagonists such as D-aspartate) is useful to prevent or treat Schmidt, H. H. H. W., et al, Eur, J. Pharmacol., 148, sepsis or cytokine-induced systemic hypotension, is useful 293-295 (1988). in the treatment of sepsis or cytokine-induced systemic Corbett, J. A., et al, Proc. Natl. Acad. Sci. USA, 90, hypotension to restore vascular sensitivity to the effects of 1731-1735 (Mar. 1993). O-adrenergic agonists, and is useful to suppress an immune Corbett, J. A., et al., Proc. Natl. Acad. Sci. USA, 90, response, e.g., in treating inflammation. In one embodiment, 8992-8995 (Oct. 1993). certain argininosuccinate synthetase activity reducing agents Kroncke, K-D, et al, Biochem. Biophys. Res. Commun. are used together with arginine antagonists to treat sepsis or 175, 752–758 (1991). cytokine induced hypotension. Lukic, M. L., et al, Biochem. Biophys. Res. Commun. 178, 913–920 (1991). 35 Claims, 5 Drawing Sheets U.S. Patent Aug. 13, 1996 Sheet 1 of 5 5,545,625 O-O (uyeo.Id fu/ugu/OUId) KLIAILOW SON a 3 3 c

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07 9| Z! (Wil) NOLLOnCIOld ELIA LIN 5,545,625 1. 2 PREVENTING CONVERSION OF To date, pathological overproduction of nitric oxide has CITRULLINE TO ARGINNOSUCCINATE TO been controlled by administration of nitric oxide synthase LIMIT PATHOLOGICAL, NTRIC OXDE inhibiting arginine antagonists, plasma arginine depleting OVERPRODUCTION enzymes and tetrahydrobiopterin induction or utilization blocking agents. This invention was made at least in part with Govern ment support under National Institutes of Health Grant HL46403 and under National Institutes of Health Grant SUMMARY OF THE INVENTION DK371 16. It has been discovered herein that administration of O argininosuccinate synthetase activity reducing agents is use TECHNICAL FIELD ful to prevent or treat sepsis or cytokine-induced systemic hypotension, is useful in the treatment of sepsis or cytokine This invention is directed to a novel approach for inhib induced systemic hypotension to restore vascular sensitivity iting biological nitric oxide production. to the effects of O-adrenergic agonists, is useful to suppress 15 an immune response, e.g., in treating inflammation, and is BACKGROUND OF THE INVENTION useful to prevent or treat a subject for a stroke. One embodiment herein is directed to a method of pro For several decades nitroglycerin has been administered phylaxis or treatment of a subject for systemic hypotension to humans as a vasodilating agent in the treatment of caused by pathological overproduction of nitric oxide from cardiovascular disease. It has been shown that nitroglycerin 20 arginine in vascular cells in said subject induced by therapy so administered is converted in the body to nitric oxide with a cytokine or by exposure to a bacterial endotoxin, said which is the pharmacologically active metabolite. Recently, method comprising administering to a subject expected to nitric oxide has been shown to be formed enzymatically as develop or having such systemic hypotension a therapeuti a normal metabolite from arginine in vascular endothelium cally effective amount of an argininosuccinate synthetase to provide an important component of endothelium-derived 25 activity reducing agent. The agent can be either an argini relaxing factors (EDRFs) which are currently being inten nosuccinate synthetase induction blocking agent or an sively studied as participating in regulation of blood flow argininosuccinate synthetase inhibitor. A therapeutically and vascular resistance. Macrophages have also been shown effective amount of an argininosuccinate synthetase activity to produce nitric oxide in the body as a component of their reducing agent in this method is one that causes reduction in cell killing and/or cytostatic function. 30 induced nitric oxide production to the extent of causing an More recently it has been established that the enzyme increase in blood pressure. forming nitric oxide from arginine, i.e., nitric oxide syn Another embodiment herein is directed to a method for thase, occurs in at least two distinct types, namely the treatment of a subject for systemic hypotension caused by constitutive forms and an inducible form. Constitutive forms 35 pathological overproduction of nitric oxide from arginine in are present in normal endothelial cells, neurons and some vascular cells in said subject induced by therapy with a other tissues. Formation of nitric oxide by a constitutive cytokine or by exposure to a bacterial endotoxin, said form in endothelial cells is thought to play a role in normal method comprising administering to a subject having such blood pressure regulation. The inducible form of nitric oxide systemic hypotension a therapeutically effective amount of synthase has been found to be present in activated macroph 40 an O-adrenergic agonist, and an amount of argininosucci ages and is induced in endothelial cells and vascular smooth nate Synthetase activity reducing agent to restore vascular muscle cells, for example, by various cytokines and/or contractile sensitivity to the effects of said O-adrenergic microbial products. It is thought that in sepsis or cytokine agonist. The argininosuccinate synthetase activity reducing induced shock, overproduction of nitric oxide by the induc agent can be either an argininosuccinate synthetase induc ible form of nitric oxide synthase plays an important role in 45 tion blocking agent or an argininosuccinate synthetase the observed life-threatening hypotension. Furthermore, it is inhibitor. A therapeutically effective amount of O-adrener thought that overproduction of nitric oxide by the inducible gic agonist is one that causes increase in blood pressure. form of nitric oxide synthase is a basis for insensitivity to Still another embodiment herein is directed to a method clinically used pressor agents such as O-adrenergic agonists for treating a subject for systemic hypotension caused by in the treatment of septic or cytokine-induced shock 50 pathological overproduction of nitric oxide from arginine in patients. Moreover, it is thought that overproduction of nitric vascular cells in said subject induced by therapy with a oxide by inducible form of nitric oxide synthase is involved cytokine or by exposure to a bacterial endotoxin, said in inflammation incident to an immune response. method comprising administering to said subject a thera Overproduction of nitric oxide is due either to overstimu peutically effective amount of an arginine antagonist of lation of a constitutive nitric oxide synthase (cNOS) or to 55 nitric oxide synthesis by nitric oxide synthase and a thera overexpression of inducible nitric oxide synthase (iNOS). In peutically effective amount of an argininosuccinate syn either case, overproduction of nitric oxide is also dependent thetase activity reducing agent selected from the group on adequate availability of arginine, the substrate of nitric consisting of argininosuccinate synthetase induction block oxide synthase. Arginine supply is maintained in three ways: ing agents and argininosuccinate synthetase inhibitors which (i) protein degradation, (ii) uptake of arginine from plasma, 60 do not block the activity of inducible nitric oxide synthase (iii) conversion of citrulline to arginine by pathways involv (by inhibiting inducible nitric oxide synthase by binding to ing conversion of citrulline and aspartate to argininosucci it or its heme cofactor). The therapeutically effective amount nate (ASA) by argininosuccinate synthetase and conversion of arginine antagonist of nitric oxide synthesis by inducible of ASA to arginine and fumarate by argininosuccinate lyase. nitric oxide synthase is an induced nitric oxide production In the case of iNOS, enzyme activity and overproduction of 65 limiting amount, i.e., a blood pressure raising amount. The nitric oxide is also dependent on availability of required therapeutically effective amount of the argininosuccinate cofactors including tetrahydrobiopterin. synthetase activity reducing agent in this method is that 5,545,625 3 4 which when administered to a subject sufficiently limits the versus nitric oxide synthase activity (open circles) and induction, or further induction, of argininosuccinate syn depicts results of Example I. thetase or inhibits the activity of argininosuccinate Syn FIG. 2 shows the time course of lipopolysaccharide/ thetase such that the induced argininosuccinate synthetase interferon-gamma-induced changes in mRNA levels for dependent citrulline to arginine metabolic pathway does not argininosuccinate synthetase (AS), argininosuccinate lyase contribute significantly to the availability of arginine needed (AL), nitric oxide synthase (NOS) and glyceraldehyde-3- to sustain overproduction of nitric oxide by inducible nitric phosphate dehydrogenase (GAPDH) and depicts results of oxide synthase, thereby to potentiate duration or magnitude Example II. of the blood pressure increase caused by the administration FIG.3 effect of the change concentration of mithramycin of the arginine antagonist of nitric oxide synthesis by nitric 10 on levels of argininosuccinate synthetase mRNA and nitric oxide synthase. oxide synthase mRNA and on GTP cyclohydrolase I mRNA Therapy with a cytokine includes therapy with interferons in induced rat aortic smooth muscle cells as determined by including interferon-gamma, tumor necrosis factor, interleu reverse transcriptase-polymerase chain reaction analysis and kin-1 or interleukin-2, e.g., chemotherapeutic treatment with depicts results of Example III. tumor necrosis factor or interleukin-2. Treatment with nitric 15 FIG. 4 depicts graphs of mithramycin concentration ver oxide synthase inducing cytokines will cause systemic sus nitrite production with mithramycin administered post hypotension by pathological overproduction of nitric oxide LPS/interferon gamma administration (open circles) and if continued for a long enough period. Thus, the expectation administered concurrently with LPS/interferon gamma of systemic hypotension caused by pathological overpro (closed circles), and depicts results of Example IV. duction of nitric oxide is currently a limitation on the extent 20 and duration of cytokine therapy. FIG. 5 depicts graphs showing the effect on nitrite pro Bacterial endotoxin induced system hypotension, also duction in induced intact cells of varying the concentration known as sepsis or septic shock, may arise, e.g., from of citrulline at fixed concentrations of arginine and depicts bacterial infection or immunosuppression therapy. There are results of Example V. 25 subjects that are expected to develop this condition, i.e., who DETAILED DESCRIPTION are at risk for developing this condition, e.g., those suffering from burn or hemorrhage or undergoing immunosuppres The argininosuccinate synthetase activity reducing agents sion therapy. herein consist of argininosuccinate synthetase induction In another embodiment, the invention herein is directed to blocking agents and argininosuccinate synthetase inhibitors. a method of prophylaxis or treatment of a subject for 30 They do not include agents which globally inhibit gene inflammation caused by induced nitric oxide production transcription such as actinomycin D, or agents which glo from arginine in cells, said method comprising administer bally inhibit protein synthesis such as cycloheximide, or ing to a subject at risk for or having such inflammation, a glucocorticoids. therapeutically effective amount of an argininosuccinate The argininosuccinate synthetase induction blocking synthetase activity reducing agent. The inflammation may 35 agents are preferably antibiotics that bind to DNA sequences arise, e.g., from non-acute allergic reactions including con present in the upstream regulatory region of the arginino tact dermatitis, from autoimmune conditions including rheu succinate synthetase gene (described in Freytag, S.O., et al, matoid arthritis, myasthenia gravis, multiple schlerosis, J. Biol. Chem..., 259(5), 3160-3166, 3/84 and Jinno, Y, et al, lupus erythematosus and Parkinson's disease, and host J. Biochem. 98, 1395–1403, 1985 and is archived in The defense immune mechanisms, e.g., allograft rejection reac 40 European Molecular Biology Database under EMBL acces tions, mediated by immunologically induced nitric oxide sion number 03258), such as the sequences for the binding production. Those at risk for such condition, e.g., those of of the transcriptional activating factor SP1. Antibiotics high probability to become afflicted with this condition known to do this include mithramycin (also known as include those undergoing allograft treatment. The agent can plicamycin), chromomycins (e.g., chromomycin A) and be either an argininosuccinate synthetase induction blocking 45 olivomycins (e.g., olivomycins A, B, C and D). Some of agent or an argininosuccinate synthetase inhibitor. A thera these antibiotics also have been found to block the induction peutically effective amount of an argininosuccinate syn of nitric oxide synthase gene expression. Such antibiotics thetase activity reducing agent in this method is an amount limit induced production of nitric oxide in two ways, namely which causes reduction in induced nitric oxide synthesis to by limiting induction of argininosuccinate synthetase result the extent of suppressing the immune response that is part of 50 ing in limiting of production of substrate arginine from the inflammatory response thereby attenuating the inflam citrulline and also by limiting induction of nitric oxide mation. synthase catalyst so that the conversion of arginine into In another embodiment, the invention herein is directed to nitric oxide and citrulline is limited. Administration of these a method of prophylaxis or treatment of a subject for a 55 antibiotics also has the effect of depleting calcium levels. stroke, said method comprising administering to said subject However, this is not a problem when these antibiotics are a therapeutically effective amount, e.g., a neuronal cell administered to treat acute conditions (e.g., when used alone protecting amount, of an argininosuccinate synthetase activ or together with O-adrenergic agonists in treatment of ity reducing agent. sepsis or cytokine-induced hypotension) or for short term In another embodiment, the invention is directed at 60 treatment for prophylaxis of expected induced hypotension N'-C-alkyl-L-citrullines as novel compounds. or for short term treatment of inflammation. The argininosuccinate synthetase inhibitors bind to argini The term "subject' is used herein to mean a mammal. nosuccinate synthetase to block the activity thereof and thereby prevent the argininosuccinate synthetase from con BRIEF DESCRIPTION OF THE DRAWINGS 65 verting citrulline to argininosuccinate. FIG. 1 is a graph of concentration of mithramycin versus The argininosuccinate synthetase inhibitors can be, for argininosuccinate synthetase activity (filled in circles) and example, L-citrulline antagonists, i.e., compounds which 5,545,625 S 6 interfere with the action of argininosuccinate synthetase on 125 g/kg for prophylaxis and may be administered intra L-citrulline, or L-aspartate antagonists, i.e., compounds venously. As these agents are known to lower plasma levels which interfere with the action of argininosuccinate syn of calcium, calcium supplementation may be provided to thetase on L-aspartate. patients in need thereof. For prophylaxis, the argininosuc The L-citrulline antagonists include, for example, L-thio cinate synthctase induction blocking agents should be citrulline and L-homothiocitrulline which also block the administered at the dosages recited for this purpose daily for activity of inducible nitric oxide synthase by binding to no more than about 4 days. We turn now to where argini heme cofactor of inducible nitric oxide synthase (i.e., the nosuccinate synthetase inhibitors are used as the arginino catalytically important heme cofactor enfolded in a nitric succinate synthetase activity reducing agents. Where L-cit oxide synthase molecule) and canavanine which also inhib O rulline antagonists are used as the argininosuccinate its inducible nitric oxide synthase as well as L-citrulline synthetase inhibitors, for treatment of systemic hypotension antagonists different from L-thiocitrulline and L-homothio already occurring, these are administered in a blood pressure citrulline and canavanine and which do not also block the raising amount, generally 1 mg/kg to 100 mg/kg for L-enan activity of nitric oxide synthase by binding to inducible tiomer (preferably 2 mg/kg to 20 mg/kg for L-thiocitrulline nitric oxide synthase or its heme cofactor, e.g., N'-C- 5 and 2 mg/kg to 40 mg/kg for L-homothiocitrulline and 20 alkyl-L-citrullines, such as N'-methyl-L-citrulline, and mg/kg to 100 mg/kg for N'-methyl-L-citrulline) by a route amino acids which naturally occur in mammalian species, of administration obtaining a fast response, normally except for arginine, preferably L-norvaline, L-isoleucine, parenteral, preferably intravenous, and for treatment in cases L-valine and L-serine. where systemic hypotension is expected (i.e., for prophy 20 laxis, i.e., prevention or delay of the condition occurring or The L-aspartate antagonists include, for example, O-me to provide reduced severity of the condition which occurs), thyl-D-L-aspartate, D-aspartate, erythro-3-hydroxyl-DL-as these are administered to provide a plasma level of L-cit partate, threo-3-hydroxy-3-methyl-DL-aspartate and ruline antagonist sufficient to eliminate or delay the occur erythro-3-hydroxy-3-methyl-DL-aspartate. ring of the hypotension or reduce the severity of the For the embodiment where O-adrenergic agonists are 25 hypotension which occurs, generally a plasma concentration utilized, C.-adrenergic agonists are used for the same pur of L-citruline antagonist ranging from 1 LM to 1000 uM for pose now (i.e., to increase blood pressure in a hypotensive L-enantiomer (preferably 10 um to 50 uM for L-thiocitrul subject) but eventually stop working because of loss of line and L-homothiocitrulline and 200 to 1000 uM for vascular contractile sensitivity. The O-adrenergic agonists N'-methyl-L-citrulline) by a route of administration which include epinephrine, norepinephrine, dopamine, phenyleph 30 can be parenteral (e.g., intravenous) but also can be oral rine, metaraminol, methoxamine, ephedrine and nephenter (doses to provide this concentration may be determined by mine. considering the half-life of the compounds in the body). For the embodiment where arginine antagonists of nitric L-Citrulline antagonists can be administered for the same oxide synthesis by nitric oxide synthase are used, i.e., purposes by continuous infusion, in which case the doses compounds which interfere with the action of iNOS on 35 would range from 1 mg/kg/hr to 100 mg/kg/hr generally, L-arginine, these bind to iNOS to block the activity thereof (preferably 2 mg/kg/hr to 20 mg/kg/hr for L-thiocitrulline and thereby prevent the iNOS from converting arginine to and 2 mg/kg/hr to 40 mg/kg/hr for L-homothiocitrulline and nitric oxide and citrulline. The arginine antagonists contem 20 mg/kg/hr to 100 mg/kg/hr for N'-methyl-L-citrulline). plated are those which are not also argininosuccinate syn Where L-aspartate antagonists are used as the argininosuc thetase inhibitors. These include N'-methyl-L-arginine, N 40 cinate synthetase inhibitors, for treatment of systemic amino-L-arginine, N-nitro-L-arginine, N-nitro-L- hypotension already occurring, these are administered in a arginine methyl ester, N-iminomethyl-L-ornithine, blood pressure raising amount, generally 10 mg/kg to 1000 N'-(hydrazinoiminomethyl)lysine, and guanidino substi mg/kg (preferably 10 mg/kg to 100 mg/kg for D-aspartate) tuted arginines or homoarginines based on monoalkyl car by a route of administration obtaining a fast response, bon-substituted ornithines or lysines (as described in U.S. 45 normally parenteral, preferably intravenous, and for treat Pat. No. 5,281627). ment in cases where systemic hypotension is expected (i.e., We turn now to the method herein for the prophylaxis or for prophylaxis), these are administered to provide a plasma treatment of a subject for systemic hypotension caused by level of L-aspartate antagonist sufficient to eliminate or pathological overproduction of nitric oxide from arginine in delay the occurring of the hypotension or reduce the severity vascular cells in said subject induced by therapy with a 50 of the hypotension which occurs, generally a plasma con cytokine or by exposure to a bacterial endotoxin, said centration of L-aspartate antagonist ranging from 50 uM to method comprising administering to a subject expected to 1000 uM (preferably 50 uM to 200 uM for D-aspartate) by develop or having such systemic hypotension a therapeuti a route of administration which can be parenteral (e.g., cally effective amount of argininosuccinate synthetase activ intravenous) but also can be oral (doses to provide the ity reducing agent. We turn firstly to where argininosucci 55 concentration may be determined by considering the half nate synthetase induction blocking agents are used as the life of the compounds in the body). L-Aspartate antagonists argininosuccinate synthetase activity reducing agents. Mith can be administered for the same purposes by continuous ramycin is utilized preferably at a dosage ranging from 15 to infusion, in which case the doses would range from 10 25ug/kg for acute treatments (e.g., treatment of hypotension mg/kg/hr to 1000 mg/kg/hr (preferably 10 mg/kg/hr to 100 resulting from Septic shock) and 12 to 15 ug/kg for prophy 60 mg/kg/hr for D-aspartate). laxis and may be administered intravenously as a bolus We turn now to the method herein for the treatment of a injection or as an infusion over 1 to 3 hours. Chronomycins subject for systemic hypotension caused by pathological are utilized preferably at a dosage ranging from about 15 to overproduction of nitric oxide from arginine in vascular 26 ug/kg for acute treatments and 12 to 16 ug/kg for cells in said subject induced by therapy with a cytokine or prophylactic treatments and may be administered intrave 65 by exposure to a bacterial endotoxin, said method compris nously. Olivomycins are utilized at a dosage preferably ing administering to a subject having such systemic ranging from 15 to 200 ug/kg for acute treatments and 12 to hypotension atherapeutically effective amount of an O-adr 5,545,625 7 8 energic agonist and an amount of argininosuccinate syn oxide synthase by binding to it or its heme cofactor), thetase activity reducing agent to restore vascular contractile hereinafter referred to as the third method herein. sensitivity to the effects of the O-adrenergic agonist, here The arginine antagonist competes with arginine for the inafter referred to as the second method herein. active site of nitric oxide synthase. Therefore, lowering The O-adrenergic agonists for the second method herein intracellular arginine level by administering an argininosuc are used in the same dosages as they are now used for the cinate synthetase activity reducing agent enables lowering same purpose (i.e., to increase blood pressure in a hypoten the dosage of arginine antagonist necessary to limit induced sive patient), i.e., in conventional therapeutically effective nitric oxide production and raise blood pressure, e.g., to less amounts. Doses for the O-adrenergic agonist dopamine than 50% of the dosage of arginine antagonist otherwise typically range from 2 ug/kg/min to 50 lug/kg/min. Doses for 0 necessary. This diminishes the potential toxicity of the the O-adrenergic agonist norepinephrine typically range arginine antagonist. from 2 ug/min to 4 lug/min. Doses for the O-adrenergic agonist phenylephrine can range from 0.1 to 10 ug/kg. The The arginine antagonists are used in the third method route of administration of the most popular O-adrenergic herein in a blood pressure raising amount. The dosages agonists (norepinephrine and dopamine) is intravenous and generally range from 0.1 to 100 mg/kg and/or from 0.1 to for the others the route of administration is intravenous or in 15 100 mg/kg/hr, with the actual dosage depending on the Some cases subcutaneous. arginine antagonist selected. Doses for N-methyl-L-argin We turn now to where argininosuccinate synthetase ine range from 1 to 40 mg/kg and/or 1 to 40 mg/kg/hr. The induction blocking agents are used as the argininosuccinate route of administration is preferably intravenous or other synthetase activity reducing agents in the second method route providing a fast response. herein. Mithramycin is utilized at a dosage preferably rang 20 The argininosuccinate synthetase induction blocking ing from 15 to 25 ug/kg and may be administered intrave agents for the third method herein are those generally nously as a bolus injection or as an infusion over 1 to 3 described above. The argininosuccinate synthetase inhibi hours. Chromomycins are utilized preferably at a dosage tors which do not block the activity of inducible nitric oxide ranging from about 15 to 26 g/kg and may be administered synthase (by inhibiting inducible nitric oxide synthase by intravenously. Olivomycins are utilized at a dosage prefer 25 binding to it or its heme cofactor) for the third method herein ably ranging from 15 to 200 ug/kg and may be administered can be (1) L-citrulline antagonists which do not block the intravenously. activity of iNOS by binding to iNOS or its heme cofactor, or We turn now to where argininosuccinate synthetase (2) L-aspartate antagonists. The L-citrulline antagonists for inhibitors are used as the argininosuccinate synthetase activ 30 the third method herein can be, for example, N'-Cs ity reducing agents in the second method herein. Where alkyl-L-citrullines such as N'-methyl-L-citrulline and L-citrulline antagonists are used as the argininosuccinate amino acids which naturally occur in mammalian-species, synthetase inhibitors, these are administered in a vascular except for arginine, preferably L-norvaline, L-isoleucine, contractile sensitivity restoring amount, generally 1 mg/kg L-valine and L-serine. L-Aspartate antagonists are generally described above. to 100 mg/kg for L-enantiomer (preferably 2 mg/kg to 20 35 mg/kg for L-thiocitrulline and L-homothiocitrulline and 20 Argininosuccinate synthetase activity reducing agents are mg/kg to 100 mg/kg for N'-methyl-L-citrulline) by a route used in the third method herein in an amount such that the of administration obtaining a fast response, normally induced argininosuccinate synthetase dependent citrulline to parenteral, preferably intravenous. L-Citruline antagonists arginine metabolic pathway does not contribute significantly can be administered for the same purpose by continuous 40 to the availability of arginine needed to sustain overproduc infusion, in which case the doses would range from 1 tion of nitric oxide by nitric oxide synthase, as may be mg/kg/hr to 100 mg/kg/hr generally (preferably 2 mg/kg/hr determined by the ability to administer less arginine antago to 20 mg/kg/hr for L-thiocitrulline and L-homothiocitrulline nist to achieve the same result as administering more argi and 20 mg/kg/hr to 100 mg/kg/hr for N'-methyl-L-citrul nine antagonist without the argininosuccinate synthetase line). Where L-aspartate antagonists are used as the argini 45 activity reducing agent being administered. nosuccinate synthetase inhibitors, these are administered in We turn now to when argininosuccinate synthetase induc a vascular contractile sensitivity restoring amount, generally tion blocking agents are used as the argininosuccinate syn 10 mg/kg to 1000 mg/kg (preferably 10 mg/kg to 100 mg/kg thetase activity reducing agents in the third method herein. for D-aspartate) by a route of administration obtaining a fast Mithramycin is utilized preferably at a dosage ranging from response, normally parenteral, preferably intravenous. 50 about 12 to 15ug/kg and may be administered intravenously L-Aspartate antagonists can be administered for the same as a bolus injection or as an infusion over 1 to 3 hours. purposes by continuous infusion in which case the doses Chronomycins are utilized preferably at a dosage ranging would range from 10 mg/kg/hr to 1000 mg/kg/hr (preferably from about 15 to 26 g/kg and may be administered intra 10 mg/kg/hr to 100 mg/kg/hr for D-aspartate). venously. Olivomycins are utilized preferably at a dosage We turn now to the method herein for the treatment of a 55 ranging from about 12 to 125 ug/kg and may be adminis subject for systemic hypotension caused by pathological tered intravenously. overproduction of nitric oxide from arginine in vascular We turn now to where argininosuccinate synthetase cells in said subject induced by therapy with a cytokine or inhibitors which do not block the activity of inducible nitric by exposure to a bacterial endotoxin, said method compris oxide synthase are used in the third method herein. The ing administering to said subject a therapeutically effective 60 L-citrulline antagonists are generally administered in a dos amount of an arginine antagonist of nitric oxide synthesis by age of 1 mg/kg to 100 mg/kg (preferably 20 mg/kg to 100 nitric oxide synthase and a therapeutically effective amount mg/kg for N'-methyl-L-citruline) by a route of administra of an argininosuccinate synthetase activity reducing agent tion obtaining a fast response, normally parenteral, prefer selected from the group consisting of argininosuccinate ably intravenous. The L-citrulline antagonists can be admin synthetase induction blocking agents and argininosuccinate 65 istered for the same purpose by continuous infusion, in synthetase inhibitors which do not block the activity of which case the doses would range from 1 mg/kg/hr to 100 inducible nitric oxide synthase (by inhibiting inducible nitric mg/kg/hr generally (preferably, 20 mg/kg/hr to 100 mg/kg/ 5,545,625 10 hr for N'-methyl-L-citrulline. The L-aspartate antagonists of the inflammation which occurs, generally a plasma con are generally administered in a dosage of 10 mg/kg to 1000 centration of L-aspartate antagonist ranging from 50 to 1000 mg/kg (preferably 10 mg/kg to 100 mg/kg for D-aspartate) uM (preferably 50 to 200 uM for D-aspartate). Methods of by a route of administration obtaining a fast response, administration include oral, intramuscular, Subcutaneous, normally parenteral, preferably intravenous. The L-aspartate intrasynovial and intravenous. The dosages recited, which antagonists can be administered for the same purposes by may be repeated, are administered for a period of time to continuous infusion, in which case the doses would range cause suppression of immune response and attenuation of from 10 mg/kg/hr to 1000 mg/kg/hr (preferably 10 mg/kg/hr inflammation, i.e., treating for two days or more, e.g., for to 100 mg/kg/hr for D-aspartate. two to three weeks. For prophylaxis, doses to provide the We turn now to the method herein of prophylaxis or 10 treatment of a subject for inflammation caused by induced aforestated plasma concentrations may be determined by nitric oxide production from arginine in immune cells, said considering the half-life of the compounds in the body. method comprising administering to a subject at risk for or The invention is illustrated in the following examples. having such inflammation, a therapeutically effective amount of an argininosuccinate synthetase activity reducing EXAMPLE I agent, hereinafter referred to as the fourth method herein. 5 Aortic smooth muscle cells were cultured by explanting We turn now to where argininosuccinate synthetase segments of the medial layer of aortae from adult male induction blocking agents are utilized as the argininosucci Fischer 344 rats. Aortae were removed aseptically and freed nate synthetase activity reducing agents in the fourth method of adventitial and endothelial cells by scraping both the herein. Mithramycin is utilized at a dosage ranging from 20 lumenal and abluminal surfaces. Medial fragments (1-2 about 0.1 to 50 ug/kg, for intravenous administration pref mm) were allowed to attach to dry Primaria 25 cm' tissue erably at a dosage ranging from about 12 to 15 ug/kg. culture flasks (Falcon; Oxnard, Calif.) which were kept Chromomycins are utilized at a dosage ranging from about moist with growth medium until cells emerged. Cultures 0.1 to 50 ug/kg, for intravenous administration preferably at were fed twice weekly with medium 199 containing 10% a dosage ranging from about 12 to 16 ug/kg. Olivomycins 25 fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 40 are utilized at a dosage ranging from about 0.1 to 125 ug/kg, ug/ml endothelial cell growth supplement (Biomedical for intravenous administration preferably ranging from Technologies; Stoughton, Mass.) and 10 g/ml gentamycin about 12 to 125 ug/kg. Methods of administration include (GIBCO; Grand Island, N.Y.). When primary cultures oral, intramuscular, subcutaneous, intrasynovial and intra became confluent, they were passaged by trypsinization and venous. The dosages set forth are daily dosages and are 30 administered for prophylaxis purposes or for a period of the explants were discarded. Cells in passage 10-15 were time to cause suppression of immune response and attenu seeded at 20,000/well in 96-well plates. ation of inflammation, normally 2 days or more but for no When the cells became confluent (density of 60-80x10 more than 7 days. cells in a well), the medium was removed by suction and We turn now to where argininosuccinate synthetase 35 fresh medium consisting of 200 pil of RPMI 1640 (Whittaker inhibitors are utilized as the argininosuccinate synthetase Laboratories) containing 10% bovine calf serum, 2.5 mM activity reducing agents in the fourth method herein. Where glutamine and penicillin (80U/ml), streptomycin (80 pg/ml) L-citrulline antagonists are used as the argininosuccinate and fugizone (2 ug/ml) was added to each well via a pipette. synthetase inhibitors, for treatment of inflammation, these Groups of 4 wells were administered fixed concentrations are administered in an inflammation attenuating amount, 40 of mithramycin (0.001 to 10 g/ml); control wells received generally 0.01 mg/kg to 100 mg/kg and/or 0.01 mg/kg/hr to no mithramycin. To each was also added bacterial 100 mg/kg/hr for L-enantiomer (preferably 0.1 mg/kg to 20 lipopolysaccharide (endotoxin); Serotype: E. Coli. 01.11:B4, mg/kg and/or 0.1 mg/kg/hr to 20 mg/kg/hr for L-thiocitrul 30 g/ml) plus rat interferon-gamma (50 ng/ml). The wells line and L-homothiocitrulline and 0.2 mg/kg to 100 mg/kg were then incubated at 37° C. in a humidified incubator for and/or 0.2 mg/kg/hr to 100 mg/kg/hr for N'-methyl-L- 45 24 hours. Cells were then harvested and prepared for assay citrulline). Where L-citrulline antagonists are used as the of argininosuccinate synthetase (AS activity) and nitric argininosuccinate synthetase inhibitors, for prophylaxis of oxide synthase (NOS activity). inflammation from induced production of nitric oxide, these Nitric oxide formation by smooth muscle cell cytosol was are administered to provide a plasma level of L-citrulline measured using a spectrophotometric kinetic microplate antagonist sufficient to eliminate or delay the occurring of 50 assay based on the increase in Aaos accompanying the inflammation from induced nitric oxide production or reduce oxidation of Fe”-myoglobin by nitric oxide. Cytosol the severity of the inflammation which occurs, generally a (50-100 g of protein) was incubated with 40 um dithionite plasma concentration of L-citrulline antagonist ranging from reduced myoglobin (prepared as described in Gross, S. S., et 1 M to 1000 puM for L-enantiomer (preferably 10 uM to 50 al, Biochem. Biophys. Res. Commun. 160,881-886, 1989) uM for L-thiocitrulline and L-homothiocitrulline and 200 to 55 in assay buffer containing 80 mM Tris, 0.5 mM L-arginine, 1000 IM for N'-methyl-L-citrulline). Where L-aspartate 0.5 mM NADPH, and 10 um tetrahydrobiopterin (pH 7.6). antagonists are used as the argininosuccinate synthetase Assays were performed in triplicate with As recorded for inhibitors, these are administered in an inflammation attenu each sample every 18 seconds for a period of 15 minutes. ating amount, generally 0.1 mg/kg to 1000 mg/kg and/or 0.1 The rate of nitric oxide synthesis was calculated from the mg/kg/hr to 1000 mg/kg/hr (preferably 0.1 mg/kg to 100 60 rate of increase in Aaos by linear regression analysis. The mg/kg and/or 0.1 mg/kg/hr to 100 mg/kg/hr for D-aspartate). conversion factor relating Aaos to the nitric oxide synthesis Where L-aspartate antagonists are used as the argininosuc rate under the conditions employed was 1.05 milli optical cinate synthetase inhibitors, for prophylaxis of inflammation density units (also referred to as m0D) min' pmol of from induced production of nitric oxide, these are adminis nitric oxide formed. tered to provide a plasma level of L-aspartate antagonist 65 Argininosuccinate synthetase activity was assayed based sufficient to eliminate or delay the occurring of inflammation on the conversion of 'Caspartate to "Cargininosucci from induced nitric oxide production or reduce the severity nate, as described in O'Brien, W. E., Biochemistry 18, 5,545,625 11 12 5353–5356, 1979, with the exception that aspartate was described in Surh, L. C., et al, Nucleic Acids Res. 16, 9352 present at a concentration of 30 um (0.021 uCi/nmol). (1988). Subcloning and dideoxynucleotide sequencing of Reaction mixtures also contained L-citrulline (5 mM), Tris the polymerase chain reaction product for nitric oxide syn (10 mM, pH 7.5), ATP 0.1mM), phosphoenolpyruvate (1.5 thase revealed a sequence that was identical to the cloned mM), pyruvate kinase (4.5 units), myokinase (4 units), and cDNA from cytokine-activated rat aortic smooth muscle as pyrophosphatase (0.2 unit) in a total volume of 0.3 ml. described in Nunokawa, Y., et al, Biochem. Biophys. Res. Reactions were allowed to proceed for 90 minutes at 37°C Commun. 191, 89-94 (1993). before termination and quantification of the ''Carginino The results are shown in FIG. 2 wherein "LPS/IFN' succinate formed, using ion exchange chromatography on stands for bacterial lipopolysaccharide/interferon-gamma; Dowex 1-X8 (200-400 mesh, Bio-Rad). 10 “AS” stands for argininosuccinate synthetase; "AL' stands The results are shown in FIG. 1 where the filled in circles for argininosuccinate lyase; "NOS' stands for nitric oxide denote argininosuccinate synthetase (denoted AS in FIG. 1) synthase; and "GAPDH' stands for glyceraldehyde-3-phos activity and the open circles denote nitric oxide synthase phate dehydrogenase. (denoted NOS in FIG. 1) activity. FIG. 2 shows that argininosuccinate synthetase mRNA is FIG. 1 shows that mithramycin, as a function of its 15 not normally present at detectable levels in rat aortic smooth concentration blocks argininosuccinate synthetase activity muscle cells but appears on treatment with immunostimu as well as nitric oxide synthase activity. The concentrations lants when nitric oxide synthase mRNA appears. It further of mithramycin that are effective in this regard are the same shows that argininosuccinate lyase mRNA is initially present concentrations obtained in patients administered doses of at detectable levels. Thus, the induced argininosuccinate mithramycin for hypercalcemia as described in The Merck 20 synthetase endows the cells with the capacity to recycle Manual, 16th edition, page 1013. citrulline into arginine. Similar results of blocking argininosuccinate synthetase activity as a function of concentration are obtained using the EXAMPLE III same concentrations of chromomycins and with up to 5 times greater concentrations of olivomycins. 25 Rat aortic smooth muscle cells were grown as described in Example I and were untreated or treated with 30 ug/ml bacterial lipopolysaccharide in combination with 50 ng/ml EXAMPLE II interferon-gamma alone and with the concentrations of mithramycin indicated in FIG. 3. After 6 hours, cells were Rat aortic smooth muscle cells were grown as described 30 harvested and RNA was prepared and assayed by reverse in Example I and were treated with 30 ug/ml bacterial transcriptase-polymerase chain reaction as described in lipopolysaccharide in combination with 50 ng/ml interferon Example II. Results using primers specific for glyceralde gamma. At the indicated times as shown in FIG. 2, cells were hyde-3-phosphate dehydrogenase as described in Example harvested and RNA was prepared and assayed by reverse II and other primers specific for GTP cyclohydrolase I as transcriptase-polymerase chain reaction using gene specific 35 primers. RNA was extracted by a modified guanidinium described in Hattori, Y., et al, Biochem. Biophys. Res. isothiocyanate method (RNAZol; Cinna/Biotecz, Houston, Comm. 195, 435-441 (1993) are shown for comparison. Tex.). Reverse transcriptase-polymerase chain reaction was The results are depicted in FIG. 3 wherein “LPS/IFN' performed by standard methods using 1 ug of total RNA. stands for bacterial lipopolysaccharide/interferon gamma; This was followed by polymerase chain reaction amplifica 40 “AS” stands for argininosuccinate synthetase; "AL' stands tion using synthetic gene-specific primers for rat arginino for argininosuccinate lyase, "NOS' stands for nitric oxide succinate synthetase as described in Amaya, Y., et al., J. synthase; “GTPCHi' stands for GTP cyclohydrolase I; and Biochem. (Tokyo) 103, 177-181 (1988), for argininosucci "GAPDH' stands for glyceraldehyde-3-phosphate dehydro nate lyase as described in Surh, L. C., et al, Nucleic Acids genase. Research, 16, 9352 (1988) and murine-inducible nitric oxide 45 FIG. 3 shows that mithramycin inhibits gene transcription synthase as described in Lyons, C. R., et al., J. Biol. Chem. of argininosuccinate synthetase and nitric oxide synthase but 267, 6370–6374 (1992). The primers are specifically not of GTP cyclohydralase I, i.e., that mithramycin does not described in Hattori, Y., et al., J. Biol. Chem. 269,9405-9408 block all gene transcription but selectively inhibits transcrip (1994). Polymerase chain reaction amplification was per tion of argininosuccinate synthetase and nitric oxide syn formed according to the following schedule: denaturation, 50 thase. annealing, and elongation at 95, 55 and 72 C. for 30 sec, 30 sec and 1 min, respectively, for 30 cycles. Parallel amplifi EXAMPLE IV cation of glyceraldehyde-3-phosphate dehydrogenase was performed for reference using primers as described in Rat aortic smooth muscle cells were grown as described Terada, Y, et al., J. Clin. Invest. 90, 659-665 (1992). 55 in Example I and were continuously treated with 30 ug/ml Polymerase chain reaction products were electrophoresed on of bacterial lipopolysaccharide in combination with 50 a 1.5% agarose gel containing ethidium bromide and visu ng/ml of interferongamma. Concentrations of mithramycin alized by UV-induced fluorescence. All polymerase chain as indicated in FIG. 4 were added either simultaneously with reactions resulted in the amplification of a single product of immunostimulant (filled in circles in FIG. 4) or 4 hours later the predicted size for argininosuccinate synthetase (612 bp), 60 (open circles in FIG. 4). argininosuccinate lyase (600 bp) and nitric oxide synthase Nitrite was used as an indicator of cellular NO synthesis (807 bp). To confirm the identity of the polymerase chain and assay was carried out as described in Gross, S. S., et al, reaction products, the products were cut with EcoRI and J. Biol. Chem. 267,25722–25729 (1992). In the assay, cells found to each give two fragments of the expected sizes based were treated with serum- and arginine-free RPMI 1640 on reported sequences for rat argininosuccinate synthetase 65 medium containing the desired test agents for 24 hours. The as described in Amaya, Y., et al., J. Biochem. (Tokyo) 103, accumulation of nitrite in the cell culture medium was 177-181 (1988) and for rat argininosuccinate lyase as quantified colorimetrically after adding 100 pil of Griess 5,545,625 13 14 reagent (1% sulfanilamide and 0.1% naphthalene diamine in When L-canawanine was included at a concentration of 1 5% o-phosphoric acid) to an equal volume of sample. mM, counts per minute in the argininosuccinate synthetase The results are shown in FIG. 4 where points are mean assay in duplicate runs were 355.00 (10.61% error) and values + standard error of mean (n= 4) for nitrite accumu 393.00 (10.09% error) counts per minute. lation in the cell culture medium after 24 hours. The results When N'-methyl-L-arginine was included at a concen show that mithramycin inhibits induction of NO synthetic tration of 1 mM, counts per minute in the argininosuccinate activity but is not a direct inhibitor of argininosuccinate synthetase assay in duplicate runs were 591.00 (8.23% error) synthetase or nitric oxide synthase. This is indicated because and 520.00 (8.77% error) counts per minute. the results show that if induction is allowed to proceed for The background count to be subtracted from the above 4 hours so that argininosuccinate synthetase and nitric oxide 10 count results was obtained by omitting citrulline from the synthase are present, mithramycin effectiveness to block NO argininosuccinate synthetase assay and in three runs was production is markedly diminished. determined to be 217, 237 and 252 counts per minute. Similar results indicating blocking of induction but failure The experiment shows that L-thiocitrulline and L-cana to inhibit enzyme activities is shown when chromomycins in 15 vanine are inhibitors of immunostimulant induced arginino the same concentrations or olivomycins in concentrations up succinate synthetase in rat aortic smooth muscle cells but to five times higher replace mithramycin. that N-methyl-L arginine is not. When N'-methyl-L-citrulline at a concentration of 1 mM is included, inhibition of immunostimulant induced argini EXAMPLE V 20 nosuccinate synthetase is shown which is more potent than Rat aortic smooth muscle cells were grown as described obtained with L-canavanine but less potent than obtained in Example I and were treated with 30 ug/ml of bacterial with L-thiocitrulline is obtained. lipopolysaccharide in combination with 50 ng/ml of inter When L-norvaline at a concentration of 1 mM is included, feron-gamma. Concentrations of L-citrulline and L-arginine inhibition of immunostimulant induced argininosuccinate as indicated in FIG. 5 were added simultaneously with 25 synthetase is shown. immunostimulant. Nitrite was used as an indicator of cel When D-aspartate at a concentration of 1 mM is included, lular NO synthesis and assay therefor was carried out as inhibition of immunostimulant induced argininosuccinate described in Example IV. synthetase is shown. The results are shown in FIG. 5 where points are mean 30 values it standard error of mean (n=4) of nitrite concentra EXAMPLE VII tion measured in the cell culture medium after 24 hours. The results show that, due to induction of argininosucci A patient with Gram negative septicemia and blood nate synthetase activity, cells can utilize L-citrulline just as pressure of 80/55 mm Hg is administered mithramycin at a efficiently as L-arginine to provide a substrate for nitric dose of 25 ug/kg intravenously in 50 ml of saline over a 3 oxide production. When there is 0 or low L-arginine, maxi 35 to 6 hour interval. The blood pressure returns to normal mal nitrite is obtained (i.e., the same nitrite production as range within 12 to 48 hours. with unlimited arginine) if the concentration of L-citrulline Similar results are obtained when chromamycin antibiotic is sufficiently high. Thus blocking L-citrulline synthesis complex at a dose of 25 g/kg or olivomycin antibiotic diminishes cells' capacity to make nitric oxide since L-argi– complex at a dose of 50 g/kg is substituted for the mith nine is being consumed by nitric oxide synthase. 40 ramycin. Cells normally obtain L-arginine from diet or via circu lation from synthesis in the kidney. While some L-arginine EXAMPLE VIII is supplied by the kidney, it is not the maximally usable amount. The experiment shows that cells can make L-argi 45 A human is treated for renal cell cancer with interleukin-2 nine from L-citrulline to supply the maximally usable (1x10' units) for 5 days. By the fifth day, the patient's blood aOunt. pressure falls to 80/55 mm Hg and the patient encounters nausea and extreme discomfort. Three weeks later, the patient returns for a second course of interleukin-2 treatment EXAMPLE VI 50 but is pretreated (before commencing interleukin-2 infusion) with mithramycin (25 ug/kg) and is treated at 24 hour Immunostimulant treated rat aortic smooth muscle cells intervals thereafter with 25 g/kg of mithramycin. The prepared as in Example I were homogenized in 10 volumes mithramycin is administered intravenously in 50 ml saline (10 ml/gm rat aortic smooth muscle cells) of 100 mM tris over a 3 to 6 hour interval. When mithramycin is adminis buffer containing 1 mM phenylmethylsulfonyl fluoride (a 55 tered, hypotension does not develop. protein synthesis inhibitor) and 1 mM dithiothreitol (to Similar results to what are obtained with mithramycin are maintain enzyme in reduced form). obtained when chromomycin antibiotic complex at a dose of For the control, the crude homogenate was assayed for 25 g/kg or olivomycin antibiotic complex at a dose of 50 argininosuccinate synthetase by the procedure as described ug/kg is substituted for the mithramycin. in Example I except that L-citrulline was present at a 60 concentration of 150 uM instead of 5 mM. In duplicate runs, counts per minute were 534.00 (8.65% error) and 443.00 EXAMPLE IX (9.50% error) counts per minute. A patient with Gram negative septicemia and blood When L-thiocitrulline was included at a concentration of pressure of 80/55 mm Hg is administered dopamine at 10 1 mM, counts per minute in the argininosuccinate synthetase 65 ug/kg/min to acutely increase blood pressure and concur assay in duplicate runs were 272.00 (12.13% error) and rently is administered mithramycin (25ug/kg) intravenously 227.00 (13.27% error) counts per minute. in 50 ml saline over 3-6 hours. Blood pressure increases 5,545,625 15 16 acutely to the mild hypotensive range and the patient citrulline or L-norvaline or L-isoleucine is used at quadruple becomes normotensive by 12 to 48 hours. the dose for L-thiocitrulline or D-aspartate is used at double Similar results to what are obtained with mithramycin are the dose for L-thiocitrulline, blood pressure is raised within obtained when chromomycin antibiotic complex at a dose of about 1 to 4 hours of administration of the bolus dose. 25 ug/kg or olivomycin antibiotic complex at a dose of 50 5 Lig/kg is Substituted for the mithramycin. EXAMPLE XIV EXAMPLE X A human is treated for renal cell cancer with interleukin-2 A patient with Gram negative septicemia and blood (1x10') for 5 days. Simultaneously with commencing inter pressure of 80/55 mm Hg is administered N-methyl-L- leukin-2 infusion, L-thiocitrulline (10 mg/kg/day) is admin arginine in a bolus dose of 20 mg/kg intravenously in saline istered. Hypotension does not develop. and thereafter is given a sustained infusion of N-methyl Similar results to what are obtained with L-thiocitrulline L-arginine at a dosage of 10 mg/kg/hr. Mithramycin (25 are obtained with the double dose for L-thiocitrulline of ug/kg) is given intravenously in 50 ml saline over 3–6 hours is D-aspartate or quadruple the dose for L-thiocitrulline of starting at the time of the bolus dose of N-methyl-L- N'-methyl-L-citrulline or N'-ethyl-L-citrulline or L-norva arginine. Blood pressure becomes normotensive within min utes of administration of the bolus dose of N-methyl-L- line or L-isoleucine. arginine and the normotensive state is sustained. The patient is released after 2 days. 20 EXAMPLE XV Similar results to what are obtained with mithramycin are obtained when chromomycin antibiotic complex at a dose of A patient with Gram negative septicemia and blood 25 g/kg or olivomycin antibiotic complex at a dose of 50 pressure of 80/55 mm Hg is administered dopamine at 10 ug/kg is substituted for the mithramycin. ug/kg/min to acutely increase blood pressure and concur 25 rently is intravenously administered an initial bolus dose of EXAMPLE X L-thiocitrulline of 20 mg/kg and thereafter L-thiocitrulline A patient with rheumatoid arthritis with swelling localized as a sustained infusion at a dose of 10 mg/kg/hr. Blood to two painful joints is administered mithramycin at a pressure becomes normal within minutes and this state is dosage of 0.25 g/kg intra-articularly into each joint. Local sustained. The patient is released within 2 days. synovitis is reduced in 12 to 24 hours. 30 When the double the dose for L-thiocitrulline of D-as partate or quadruple the dose for L-thiocitrulline of N'-me Similar results to what are obtained with mithramycin are thyl-L-citrulline or L-norvaline or L-isoleucine are substi obtained when chromomycin antibiotic complex at a dose of tuted for the L-thiocitrulline, blood pressure increases 0.25 g/kg or olivomycin antibiotic complex at a dose of acutely to the mild hypotensive range and the patient 0.50 g/kg is substituted for the mithramycin. 35 becomes normotensive by 24 to 72 hours. EXAMPLE XII Sprague-Dawley rats are injected with 25 cc air subder EXAMPLE XVI mally in the dorsal area in accordance with the air pouch inflammatory model (Selye, H., Proc. Soc. Exper. Biol. and 40 A patient with a Gram negative septicemia and blood Med., 82,328-333 (1953). Into the air pouch formed, an pressure of 80/55 mm Hg is administered N-methyl-L- inflammatory stimulus, croton oil (0.5% in 0.5 ml corn oil), arginine in a bolus dose of 20 mg/kg intravenously in saline is injected. The rats in one group do not receive drug. The and thereafter is given a sustained infusion of N-methyl rats in the second group receive mithramycin at 25 g/kg L-arginine at a dose of 10 mg/kg/hr. N'-Methyl-L-citrulline intraperitoneally with repeat doses at 24 hour intervals. At 45 (40 mg/kg) is given intravenously in 50 ml saline over 3-6 the end of 5 days, the group of rats given mithramycin have hours starting at the time of the bolus dose of N-methyl significantly less nitrite in the fluid exudate contained in the L-arginine. Blood pressure becomes mildly hypotensive or granulomycous lesion and less inflammation than the rats in normal within minutes of administration of the bolus dose of the other group. N-methyl-L-arginine and this state is sustained. Similar results to what are obtained with mithramycin are 50 Similar results are obtained as are obtained with N'-me obtained when chromomycin antibiotic complex at a dose of thyl-L-citrulline when N'-ethyl-L-citrulline at 40 mg/kg or 25 g/kg or olivomycin antibiotic complex at a dose of 50 D-aspartate at 20 mg/kg or L-norvaline at 20 mg/kg or ug/kg is substituted for the mithramycin. L-isoleucine at 40 mg/kg is given in place of the N'-methyl L-citrulline. EXAMPLE XIII 55 A patient with Gram negative septicemia and blood pressure of 80/55 mm Hg is administered L-thiocitrulline in EXAMPLE XVI a bolus dose of 20 mg/kg intravenously in saline and Apatient with rheumatoid arthritis with swelling localized thereafter is given a sustained infusion of L-thiocitrulline at 60 to two painful joints is administered L-thiocitrulline at a a dose of 10 mg/kg/hr. Blood pressure becomes normoten dose of 0.1 mg/kg intra-articularly into each joint. Local sive within 5 to 10 minutes of administration of the bolus synovitis is reduced within 12 to 72 hours. dose of L-thiocitrulline and the normotensive state is sus When N'-methyl-L-citrulline or L-norvaline or L-isoleu tained. The patient is released after 2 days. cine is given intra-articularly into each joint at a dose of 0.2 Similar results are obtained to what are obtained with 65 mg/kg or D-aspartate is given at a dose of 0.1 mg/kg L-thiocitrulline, when L-homothiocitrulline is used at intra-articularly into each joint, local synovitis is reduced double the dose for L-thiocitrulline. When N-methyl-L- within 12 to 72 hours. 5,545,625 17 18 EXAMPLE XVIII 4. The method of claim 3 wherein the antibiotic is selected from the group consisting of mithramycin, chromomycins Sprague-Dawley rats are injected with 25 cc air subder and olivomycins. mally in the dorsal area in accordance with the air pouch 5. The method of claim 4 wherein the antibiotic is inflammatory model (Selye, H., Proc. Soc. Exper. Biol. and mithramycin. Med., 82,328-333 (1953). Into the air pouch formed, an 6. The method of claim 1 wherein the argininosuccinate inflammatory simulus, croton oil (0.5% in 0.5 ml corn oil), synthetase activity reducing agent is an argininosuccinate is injected. The rats in one group do not receive drug. The synthetase inhibitor. rats in the second group receive L-thiocitrulline at 20 mg/kg 7. The method of claim 6 wherein the argininosuccinate intraperitoneally with repeat doses at 6-24 hour intervals. At O synthetase inhibitor is an L-citrulline antagonist. the end of 5 days, the group of rats given L-thiocitrulline 8. The method of claim 7 wherein the L-citrulline antago have significantly less nitrite in the fluid exudate contained nist is not L-thiocitruline or L-homothiocitrulline. in the granulomycous lesion and less inflammation than the 9. The method of claim 8 wherein the L-citrulline antago rats in the other group. nist is N'-alkyl-L-citrulline wherein the alkyl contains 1 to Similar results as to what are obtained with L-thiocitrul 15 6 carbon atoms. line are obtained when 20 mg/kg of D-aspartate or 40 mg/kg 10. The method of claim 9 wherein the L-citrulline of N'-methyl-L-citrulline or N'-propyl-L-citrulline or antagonist is N'-methyl-L-citrulline. L-norvalinc or L-isoleucine is substituted for the L-thio 11. The method of claim 6 wherein the argininosuccinate citruline. synthetase inhibitor is an L-aspartate antagonist. 20 12. The method of claim 11 wherein the L-aspartate EXAMPLE XIX antagonist is D-aspartate. 16.86 gm (0.1 mol) of L-ornithine hydrochloride is dis 13. A method for treatment of a subject for systemic Solved in 200 ml of M. NaOH. To the clear solution is hypotension caused by pathological overproduction of nitric added 5.71 gm of methyl isocyanate (0.1 mol) dropwise at oxide from arginine in vascular cells in said subject induced room temperature. The pH is maintained at 9.5-10.5 by 25 by therapy with a cytokine or by exposure to a bacterial occasional cautious addition of 1M NaOH. After stirring endotoxin, said method comprising administering to a sub overnight at room temperature, the reaction mixture is ject having such systemic hypotension a therapeutically reduced to dryness by rotary evaporation at reduced pres effective amount of an O-adrenergic agonist and an amount sure. The residue is suspended in concentrated HCl, and the of argininosuccinate synthetase activity reducing agent to precipitate of NaCl is filtered off. The filtrate, which contains 30 restore vascular contractile sensitivity to the effects of the N'-methyl-L-citrulline is evaporated to dryness under O-adrenergic agonist. reduced pressure and redissolved in a small volume of water. 14. The method of claim 13 wherein the argininosuccinate That solution is applied to a column 2.0 by 50 cm of Dowex synthetase activity reducing agent is an argininosuccinate 50 (Na" form). The column is washed with 2 L of distilled synthetase induction blocking agent. 35 15. The method of claim 14 wherein the argininosuccinate water and fractions of approximately 25 ml are collected synthetase induction blocking agent is also a nitric oxide sequentially. Under these conditions of chromatography, synthase induction blocking agent and is an antibiotic that N'-methyl-L-citrulline is eluted and residual unreacted binds to DNA sequences present in the upstream regulatory L-ornithine remains bound to the Dowex resin. Fractions region of the argininosuccinate synthetase gene. containing N'-methyl-L-citrulline (detected using a conven 40 16. The method of claim 15 wherein the antibiotic is tional ninhydrin spot assay) are pooled, rotary evaporated to selected from the group consisting of mithramycin, chro dryness under reduced pressure, and the residual oil is momycins and olivomycins. crystallized from ethanol/water. The overall yield is approxi 17. The method of clairn 16 wherein the antibiotic is mately 75% based on the amount of ornithine used. mithramycin. N'-Alkyl-L-citrullines with different N'-alkyl groups 45 18. The method of claim 13 wherein the argininosuccinate from methyl are prepared by substituting an equivalent synthetase activity reducing agent is an argininosuccinate molar amount of the corresponding alkyl isocyanate for the synthetase inhibitor. methyl isocyanate. 19. The method of claim 18 wherein the argininosuccinate Many variations will be obvious to those skilled in the art. synthetase inhibitor is an L-citrulline antagonist. Thus, the invention is defined by the claims. 50 20. The method of claim 19 wherein the L-citrulline What is claimed is: antagonist is not L-thiocitrulline or L-homothiocitrulline. 1. A method of prophylaxis or treatment of a subject for 21. The method of claim 20 wherein the L-citrulline systemic hypotension caused by pathological overproduc antagonist is N'-alkyl-L-citrulline wherein the alkyl con tion of nitric oxide from arginine in vascular cells in said tains 1 to 6 carbon atoms. subject induced by therapy with a cytokine or by exposure 55 22. The method of claim 21 wherein the L-citrulline to a bacterial endotoxin, said method comprising adminis antagonist is N'-methyl-L-citrulline. tering to a subject expected to develop or having such 23. The method of claim 18 wherein the argininosuccinate systemic hypotension a therapeutically effective amount of synthetase inhibitor is an L-aspartate antagonist. an argininosuccinate synthetase activity reducing agent. 24. The method of claim 23 wherein the L-aspartate 2. The method of claim 1 wherein the argininosuccinate 60 antagonist is D-aspartate. synthetase activity reducing agent is an argininosuccinate 25. A method for treating a subject for systemic hypoten synthetase induction blocking agent. sion caused by pathological overproduction of nitric oxide 3. The method of claim 2 wherein the argininosuccinate from arginine in vascular cells in said subject induced by synthetase induction blocking agent is also a nitric oxide therapy with a cytokine or by exposure to a bacterial synthase induction blocking agent and is an antibiotic that 65 endotoxin, said method comprising administering to said binds to DNA sequences present in the upstream regulatory subject a therapeutically effective amount of an arginine region of the argininosuccinate synthetase gene. antagonist of nitric oxide synthesis by nitric oxide synthase 5,545,625 19 and a therapeutically effective amount of an argininosucci 30. The method of claim 25 wherein the argininosuccinate nate synthetase activity reducing agent selected from the synthetase activity reducing agent is an argininosuccinate group consisting of orgininosuccinate synthetase induction synthetase inhibitor which does not block the activity of blocking agents and argininosuccinate synthetase inhibitors inducible nitric oxide synthase. which do not block the activity of inducible nitric oxide 5 31. The method of claim30 wherein the argininosuccinate synthase. synthetase activity reducing agent is an L-citrulline antago 26. The method of claim 25 wherein the argininosuccinate nist. synthetase activity reducing agent is an arginiosuccinate 32. The method of claim 31 wherein the L-citrulline synthetase induction blocking agent. anagonist is N'-alkyl-L-citrulline wherein the alkyl contains 27. The method of claim 26 wherein the argininosuccinate 10 1 to 6 carbon atoms. synthetase induction blocking agent is also a nitric oxide 33. The method of claim 32 wherein the L-citrulline synthase induction blocking agent and is an antibiotic that antagonist is N'-methyl-L-citrulline. binds to DNA sequences present in the upstream regulatory 34. The method of claim 30 wherein the argininosuccinate region of the argininosuccinate synthetase gene. synthetase activity reducing agent is an L-aspartate antago 28. The method of claim 27 wherein the antibiotic is 15 nist. selected from the group consisting of mithramycin, chro 35. The method of claim 34 wherein the L-aspartate momycins and olivomycins. antagonist is D-aspartate. 29. The method of claim 28 wherein the antibiotic is mithramycin. ck :k k k :

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,545,625 DATED : August 13, 1996 INVENTOR(S) : STEVEN S. GROSS, et al.

it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby Corrected as shown below:

Claim 25 (column 19, line 3), change "orgininosuccinate" to --argininosuccinate--.

Claim 26 (column 19, line 8), change "arginiosuccinate' to --argininosuccinate--.

Signed and Sealed this

Eleventh Day of March, 1997 (a teen

BRUCE LEHMAN

Attesting Officer Commissioner of Patents and Trademarks