Medicament for inhibiting nitric oxide formation

Abstract: The use is disclosed of methylguanidine or dimethylguanidine for the manufacture of a medicament particularly suitable for inhibiting nitric oxide formation in a warm blooded mammal. A pharmaceutical composition is disclosed containing methylguanidine or dimethylguanidine or a pharmaceutically acceptable salt thereof which is suitable in the treatment and/or prevention of diabetes-induced vascular dysfunction and complications in humans.

Description: Background of the Invention This invention relates to the use of methylguanidine or 1,1-dimethylguanidine for the manufacture of a medicament for inhibiting nitric oxide production in warm blooded mammals. Nitric oxide synthase catalyzes the mixed functional oxidation of L-arginine to L-citrulline and nitric oxide (NO · ), 1,2). NO · appears to function as either a signaling or an effector molecule depending on the isoform of the enzyme. The constitutive isoform of nitric oxide synthase produces small amounts of NO · which activate guanylate cyclase resulting in the formation of cGMP which mediates endothelium-dependent relaxation (2) and neural transmission (3). NO · is produced in much larger amounts by the cytokine and endotoxin inducible isoform of nitric oxide synthase, and in macrophages functions as an effector molecule which appears to mediate the cytotoxic actions of macrophages on target cells (4). Since NO · is a potent vasodilator and increases blood flow, and since vasoactive agents (such as histamine and bradykinin), which stimulate NO · production increase both blood flow and vascular permeability, NO · may be a candidate for mediating increases in blood flow and vascular permeability induced by diabetes and elevated glucose (5). Recently, Interleukin-1 (IL-1) has been shown to induce the expression of the cytokine inducible isoform of nitric oxide synthase in pancreatic islets. The production of NO · has been proposed to be the effector molecule which mediates IL-1's inhibitory affects on islet function (6,7). Generation of an IL-1-induced EPR detectable iron-nitrosyl complex, which is prevented by N G -monomethyl-L-arginine (NMMA), has been used to confirm the formation of nitric oxide by islets (8). Also, the protein synthesis inhibitor, cycloheximide, has been shown to block IL-1-induced nitrite formation, cGMP accumulation, and EPR detectable iron-nitrosyl complex formation by islets, thus establishing that IL-1 induces the cytokine inducible isoform of nitric oxide synthase in pancreatic islets (7). The pathogenesis of diabetic complications has been linked to imbalances in sorbitol, myo -inositol, and 1,2-diacyl- sn -glycerol metabolism, and to non-enzymatic glycation of cellular and extracellular constituents (5). The glycation link is supported by evidence that aminoguanidine, a nucleophilic hydrazine compound, interferes with the formation of these glycation products and also attenuates the development of several diabetes-induced vascular (5,9), neural (10), and collagen changes (11). Bucala et al. (12) recently reported that quenching of NO · in vitro by glycated albumin is attenuated by aminoguanidine (present during exposure of albumin to glycating agents) and suggested that glycation products may impair endothelium-dependent relaxation by attenuating NO · activity. K. Chang et al. (22) disclosed the use of aminoguanidine in the treatment of glucose- and sorbitol-induced vascular dysfunction. P.L. Balestri et al. (23) disclosed that methylguanidine has an effect on glucose metabolism and can decrease the blood glucose. The post-published article of N. Benjamin (24) (in as far as it concerns the subject matter of the references 1 to 3 cited in it, mentions the pressure action of guanidine compounds, including methylguanidine and dimethylguanidine on blood pressure and arterial hypertension. Brief Description of the Invention In accordance with the present invention the use is provided of methylguanidine or 1,1-dimethylguanidine for the manufacture of a medicament for inhibiting nitric oxide formation in warm blooded mammals. Furthermore, the invention provides for the use of methylguanidine or 1,1-dimethylguanidine for the manufacture of a pharmaceutical composition for inhibiting nitric oxide formation in a warm blooded mammal. This therapeutic application involves administering to a warm blooded mammal a small but effective amount of methylguanidine or 1,1-dimethylguanidine in the form of the said medicament or pharmaceutical composition to inhibit nitric oxide production. It will be understood that pharmaceutically acceptable salts of these compounds, e.g. the HCl and sulfate salts, also can be used for the manufacture of the said medicament. At the present time the pathogenesis of diabetic complications remains obscure and there is no known medication which has been shown to prevent them. Although diabetic complications are strongly linked to severity of diabetes as reflected by blood sugar levels and glycated hemoglobin, the efficacy of attempts to prevent and/or reverse diabetic complications by efforts to normalize blood sugar levels remains to be documented. In applicant's U.S. Patent 5,246,970, (copending application Ser. No. 07/807,912, filed December 16, 1991) a method is disclosed whereby aminoguanidine is administered to a warm blooded mammal at doses which inhibit nitric oxide production without producing a substantial elevation in arterial blood pressure. In accordance with the present invention, it has been found that methylguanidine is a potent inhibitor of constitutive and cytokine-induced NO production as manifested by increases in arterial blood pressure when injected intravenously in normal rats. However, in contrast to aminoguanidine, methylguanidine is relatively ineffective in preventing glucose-induced advanced glycation products manifested by the development of fluorescence products characteristic of advanced glycation end products. Thus, it is evident that the prevention by methylguanidine of diabetes-induced vascular dysfunction is attributable to its ability to block NO production rather than blocking advanced glycation end product formation. Detailed Description of the Invention FIG. 1 is a graphical representation which shows the changes in mean arterial blood pressure (MAP) induced by bolus intravenous injections of methylguanidine (MG), aminoguanidine (AG) (comparative) or N G -monomethyl-L-arginine (NMMA) (comparative) in which change in MAP is recorded in % increase in pressure above baseline and the dose of the bolus injection is recorded in µmol/kg. FIG. 2 is a graphical representation which shows the effects of methylguanidine (MG), aminoguanidine (AG) (comparative) or N G -monomethyl-L-arginine (NMMA) (comparative) on IL-1β-induced nitrite formation by Rin-m5F cells in which the effect on nitrite formation is recorded in % of IL-1βB-induced nitrite formation and the concentration of the test compounds is recorded in µM. FIG. 3 is a graphical representation which shows the effects of methylguanidine (MG), 1,1-dimethylguanidine (DMG) and aminoguanidine (AG) (comparative) on IL-1β-induced nitrite formation by Rin-m5F cells as in FIG. 2. FIG. 4 is a bar chart which shows the relative development of fluorescence products upon incubation of methylguanidine, aminoguanidine (comparative) or semicarbazide (comparative) in glucose-6-phosphate/lysine (G-6-P/Lysine) for six days. Effects of methylguanidine on constitutive (vascular) nitric oxide synthase activity were assessed by monitoring mean arterial blood pressure (MAP) changes following intravenous injection of methylguanidine in anesthetized, normal rats. The dose responses of methylguanidine, aminoguanidine (comparative) and NMMA (comparative) are shown in Figure 1. Since methylguanidine contains strong structural similarities to L-arginine and the competitive inhibitor of nitric oxide synthase, viz. NMMA, in that these compounds contain two chemically equivalent guanidino nitrogen groups, the effects of methylguanidine on IL-1β-induced formation of nitrite and cGMP by Rin m5F cells were examined and compared to the effects of NMMA (comparative) in similar tests (Figure 2). Also in similar tests, methylguanidine and its close analog, 1,1-dimethylguanidine, were each compared to the effects of aminoguanidine (AG) (comparative) (Figure 3). The Rin m5F cell line is an insulinoma cell line of the rodent β-cell that has been shown to contain the cytokine-inducibie isoform of nitric oxide synthase. Figures 2 and 3 demonstrate the dose response of methylguanidine, 1,1-dimethylguanidine, aminoguanidine (comparative) and NMMA (comparative) or AG (comparative) on IL-1β-induced formation of nitrite (an oxidation product of nitric oxide) from Rin m5F cells incubated for 18 hrs with 5 units/ml IL-1β ± the indicated concentrations of methylguanidine, 1,1-dimethylguanidine, aminoguanidine and NMMA or AG. The effects of methylguanidine on glycation were assessed by measuring the development of fluorescence products upon its incubation in glucose-6-phosphate/lysine and compared to the corresponding effects of aminoguanidine (comparative) and semicarbazide (comparative). The results are shown in Figure 4 and indicate that methylguanidine is relatively ineffective (compared to aminoguanidine and semicarbazide) in preventing glucose-induced glycation products as manifested by the devlopment of fluorescence products which are characteristic of glycation end products. In order to further illustrate the invention, the following detailed EXAMPLES were carried out although it should be understood that the invention is not limited to these specific EXAMPLES or the details described therein. The results obtained in these EXAMPLES are shown in Tables 1 to 5 and the accompanying Figures 1 to 4. EXAMPLE I This example illustrates a method to prevent diabetes-induced vascular dysfunction using methylguanidine to inhibit nitric oxide synthase. MATERIALS AND METHODS Animal Protocols All rats used in these tests were housed and cared for in accordance with the guidelines of the University Committee for the Humane Care of Laboratory Animals and in accordance with NIH guidelines on laboratory animal welfare. Rats were housed individually, allowed food (standard rat chow; Ralston Purina, Richmond, IN) and water ad libitum , and were on a 12 hour light/dark cycle. Male, Sprague-Dawley rats initially weighing 225-250 g were divided into four groups: group 1, untreated controls; group 2, methylguanidine (mg)-treated controls; group 3, untreated diabetics; and group 4, mg-treated diabetics. Diabetes was induced by intravenous injection of 45 mg/kg body weight streptozotocin (Sigma Chemical Co., St. Louis, MO) using ketamine anesthesia. Methylguanidine hydrochloride (Sigma Chemical Co.) was administered subcutaneously once daily at a dose of 25 mg/kg body weight. In addition, control rats were given water containing 2.5 g/L methylguanidine while diabetic rats were given water containing 1 g/L. Water consumption was monitored weekly for all rats. Body weights were measured weekly, nonfasting morning plasma glucose levels were assessed 3 days after injection of streptozotocin (to ensure induction of diabetes), then biweekly thereafter using the conventional glucose oxidase method of Lowry and Passoneau (14). After 4 weeks, all rats were placed into individual metabolic cages for 24 hours to determine food consumption (g/100 g body weight/24 hr) and urine output (ml/kidney/24 hr). A sample of urine was stored at -70°C for determination of urinary albumin excretion (see below). Five weeks after induction of diabetes, rats were used for the permeability and blood flow studies detailed below. Test Protocol Regional vascular albumin permeation was quantified by use of an isotope dilution technique based on the injection of bovine serum albumin (BSA) labeled with 2 different iodine isotopes, 131 I and 125 I (15-17). 125 I-BSA was used to quantify vascular albumin filtration after 10 min of tracer circulation, while 131 I-BSA served as a plasma volume marker for correction of 125 I-BSA tissue activity for tracer contained within vessels. Preparation of radiolabeled tracers . Purified monomer BSA (20 mg) was iodinated with 1 mCi of 131 I or 125 I (NEN Research Products, Boston, MA) by the iodogen method as previously described (15). 57 Co-EDTA was prepared as previously described (15, 16) and 46 Sc-microspheres (10 µm diameter) were used to assess regional blood flow as detailed below. Surgical Procedures . Rats were anesthetized with inactin (Byk Gulden, Konstanz, FRG) (∼100 mg/kg body weight injected i.p.), and core body temperature maintained at 37 ± 0.5°C using heat lamps, a 37°C surgical tray, and a rectal temperature probe. The left femoral vein, left iliac artery, right subclavian artery, and right carotid artery were cannulated with polyethylene tubing (0.58 mm i.d.) filled with heparinized saline (400 U heparin/ml). The femoral vein cannula was used for tracer injection and the subclavian artery cannula was connected to a pressure transducer for blood pressure monitoring. The left iliac artery was connected to a 1 ml syringe attached to a Harvard Model 940 constant withdrawal pump preset to withdraw at a constant rate of 0.055 ml/min. The tip of the cannula in the right carotid artery was placed in the left ventricle of the heart and was used for injection of radiolabeled microspheres. The trachea was intubated and connected to a small rodent respirator for continuous ventilatory support. Test Procedure . At time 0, 125 I-albumin (in 0.3 ml of saline) and 57 Co-EDTA (∼0.1 µCi in 0.1 ml of saline) were injected i.v. and the withdrawal pump was started simultaneously. Eight min after time 0, 0.2 ml of 131 I-BSA was injected and 1 min later, the 46Sc-microspheres were injected slowly over ∼30 sec. At the 10 min mark, the heart was excised to stop all blood flow, the withdrawal pump was stopped simultaneously, and various tissues were sampled for gamma spectrometry. Both kidneys, bladder, and connecting ureters were removed. Eyes were dissected as previously described (15, 16) and tissues from both eyes were pooled prior to gamma spectrometry. All tisue samples and arterial plasma samples were weighed, then counted in a gamma spectrometer interfaced with a Hewlett-Packard 1000A computer in which the data were corrected for background and stored for subsequent analysis. Data Analysis . A quantitative index of 125 I-BSA tissue clearance was calculated as previously described (15, 16, 17) and expressed as µg plasma/g tissue wet weight/min. Very briefly, 125 I-BSA tissue activity was corrected for tracer contained within the tissue by multiplying 125 I-BSA activity in the tissue by the ratio of 125 I-BSA/ 131 I-BSA activities in the arterial plasma sample obtained at the end of the test. The vascular-corrected 125 I-BSA tissue activity was divided by the time-averaged 125 I-BSA plasma activity (obtained from a well mixed sample of plasma taken from the withdrawal syringe) and by the tracer circulation time (10 min) and then normalized per g tissue wet weight. Glomerular filtration fate (GFR) was calculated as previously described (18). To calculate regional blood flows, the total activity of 46 Sc in the retina was divided by the total activity of 46 Sc in the reference blood sample obtained from the withdrawal syringe, then multiplied by the pump withdrawal rate, and expressed as ml/g tissue wet weight per minute (19). Preparation of advanced glycosylation end products . Lysine-derived advanced glycosylation products were prepared as described by Bucala et al. (12) by incubating 100 mM concentrations of glucose-6-phosphate and L-lysine in 0.2 M sodium phosphate buffer, pH 7.4. The incubations were maintained sterile, and were kept in the dark at room temperature for ∼6 days, at which time relative fluorescence was measured as an index of glycation using a Perkin-Elmer LS-5B luminescence spectrometer with excitation at 370 nm and emission at 440 nm. Samples were diluted 1:11 with saline prior to spectrometry. The ability of 10 and 100 mM concentrations of methylguanidine, aminoguanidine, or semicarbazide to inhibit the glycation process was compared in two separate tests (Figure 4). Blood Pressure Measurement. Systolic blood pressure was measured at weekly intervals in conscious rats using the tail-cuff method (20, 21). Animals were adapted to the procedure initially by placing them in a restrainer and inflating the sphygmomanometer several times. Blood pressure also was obtained from the iliac artery cannula in anesthetized rats during the permeability studies. Effects of bolus injections of methylguanidine on mean arterial blood pressure . Normal male, Sprague-Dawley rats weighing 250-300 g were anesthetized with 100 mg/kg body weight inactin, followed by 0.1 ml/kg body weight d -tubocurarine chloride, the left femoral vein (for tracer injection) and right iliac artery (for monitoring blood pressure) were cannulated with polyvinyl tubing (0.8 x 0.5 mm) filled with heparinized saline, and the trachea was cannulated and connected to a small rodent respirator for continuous ventilatory support. Following stabilization of arterial pressure, increasing amounts (3.1 and 50 µmol/kg body weight) of methylguanidine or N G -monomethyl-L-arginine (NMMA) were injected intravenously in a volume of 0.5 ml in separate animals and the peak pressure increase was recorded using a Gould RS 3200 recorder. Results were expressed as a percent increase in pressure above baseline. Statistical Analysis Data are expressed as mean ± 1SD. An analysis of variance was performed using the SAS general linear models procedure. To reduce potential type 1 errors related to multiple comparisons, overall differences among groups for each parameter were preliminarily assessed by the Van Der Waerden test. If statistically significant differences (at p < 0.05) were indicated among groups for a given parameter, pair-wise comparisons were assessed by least square means following a nonparametric (rank order) Blom transformation of all data. EXAMPLE II This example illustrates the effects of methylguanidine on IL-1β-induced nitrite formation by Rin m5F cells (Figure 2). Rin m5F cells, obtained from the Washington University Tissue Culture Support Center, were removed from growth flasks (55-80 million cells/flask) by trypsin/EDTA treatment, and aliquoted into 1 ml Petri dishes (1-2 million Rin m5F cells per condition). Cells were incubated for 18 hrs (under an atmosphere of 95% air and 5% CO 2 ) in 1 ml of complete CMRL-1066 tissue culture media (CMRL supplemented with 10% heat-inactivated, fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin), or complete CMRL-1066 supplemented with methylguanidine (MG), aminoguanidine (AG) (comparative) or NMMA (comparative). Following incubation, the supernatant was removed and nitrite was determined on 100 µl aliquots by conventional procedures as previously described (8,13). The results are expressed as the % of IL-1 induced nitrite formation, and are the mean ± SEM of 4 individual tests containing 3 replicates per test. The results demonstrate that both AG and NMMA are ∼10 fold more potent than MG at inhibiting nitric oxide formation by the cytokine inducible isoform of nitric oxide synthase. EXAMPLE III The effects of each of methylguanidine, 1,1-dimethylguanidine and aminoguanidine (comparative) on IL-1β-induced nitrite formation by Rin m5F cells were tested by the procedures described in EXAMPLE II and the results are shown in Figure 3. The results demonstrate the following order of potency, AG>DMG>MG, for inhibiting the cytokine inducible isoform of nitric oxide synthase. Tables 1 to 5, below, and the accompanying Figures 1 to 4 record the results obtained in the foregoing EXAMPLES. These results indicate that methylguanidine and 1,1-dimethylguanidine are potent inhibitors of constitutive and cytokine-induced NO production. This is manifested by increases in mean arterial blood pressure by methylguanidine when injected intravenously in normal rats as shown in Figure 1 and by inhibition of IL-1β-induced increases in nitrite in rodent insulinoma cells with methylguanidine and 1,1-dimethylguanidine as shown in Figures 2 and 3, respectively. Since methylguanidine is relatively ineffective (in contrast to aminoguanidine) in preventing glucose-induced glycation products (manifested by the development of fluorescence products characteristic of advanced glycation end products (Figure 4), the prevention by methyguanidine of diabetes-induced vascular dysfunction is believed to be attributable to its ability to block NO production. Methylguanidine and its close analog, 1,1-dimethylguanidine, thus should be useful for prevention of diabetic complications as well as inflammatory and immunological diseases in which increased NO production is involved. Effects of diabetes and methylguanidine (MG) on body weights, plasma glucose and water consumption. control control + MG diabetic diabetic + MG number of rats 10 11 14 18 body weights (g) initial 249 ± 20 256 ± 16 250 ± 19 248 ± 18 week 2 326 ± 25 300 ± 31 297 ± 23 271 ± 23 week 4 375 ± 41 351 ± 37 334 ± 45 294 ± 50 plasma glucose (mg/dL) 130 ± 15 131 ± 28 419 ± 120 420 ± 87 hematocrit (%) 43 ± 2 42 ± 1 42 ± 1 42 ± 2 blood pressure (mm Hg) conscious 125 ± 18 121 ± 7 123 ± 5 126 ± 5 anesthetized 118 ± 14 114 ± 14 120 ± 16 121 ± 14 water consumption (ml/day) 46 ± 6 32 ± 9 108 ± 42 93 ± 53 Effects of diabetes and methylguanidine (MG) on 125 I-albumin permeation control control + MG diabetic diabetic + MG number of rats 10 8 11 9 eye anterior uvea 266 ± 77 359 ± 146 623 ± 109 370 ± 60 posterior uvea 328 ± 106 312 ± 101 742 ± 134 358 ± 108 retina 47 ± 12 61 ± 11 116 ± 30 55 ± 18 sciatic nerve 47 ± 13 47 ± 10 121 ± 22 50 ± 10 aorta 62 ± 20 60 ± 18 155 ± 37 85 ± 41 kidney 727 ± 239 714 ± 214 1011 ± 265 738 ± 169 lung 1805 ± 532 1656 ± 454 1405 ± 324 1,498 ± 487 diaphram 201 ± 75 190 ± 27 210 ± 61 216 ± 46 heart 521 ± 135 731 ± 269 534 ± 62 599 ± 68 brain 5 ± 3 4 ± 3 5 ± 2 6 ± 4 Effects of diabetes and methylguanidine (MG) on regional blood flows ( n ) anterior uvea posterior uvea retina sciatic nerve kidney control (10) 2.0 ± 0.6 3.4 ± 0.6 0.43 ± 0.02 0.06 ± 0.02 6.5 ± 0.3 control + MG (8) 2.4 ± 0.5 3.3 ± 0.6 0.45 ± 0.07 0.07 ± 0.03 4.8 ± 0.3 diabetic (10) 2.7 ± 0.3 4.2 ± 0.5 0.57 ± 0.08 0.09 ± 0.01 6.0 ± 0.4 diabetic + MG (9) 2.3 ± 0.6 3.9 ± 0.6 0.45 ± 0.04 0.06 ± 0.02 5.8 ± 0.3 Effects of diabetes and methylguanidine (MG) on GFR ( n ) per whole kidney per g kidney control (10) 1.33 ± 0.19 0.85 ± 0.07 control + MG (8) 1.53 ± 0.29 0.87 ± 0.08 diabetic (10) 1.81 ± 0.25 0.92 ± 0.14 diabetic + MG (9) 1.59 ± 0.15 , 0.85 ± 0.09 Effects of diabetes and methylguanidine (MG) on tissue sorbitol and myo -inositol control control + MG diabetic diabetic + MG number of rats 7 8 11 9 retina sorbitol 102 ± 16 102 ± 31 933 ± 275 533 ± 265 myo -inositol 1613 ± 516 1529 ± 187 1564 ± 452 1513 ± 402 sciatic nerve sorbitol 183 ± 41 194 ± 75 1999 ± 334 1234 ± 710 myo -inositol 3943 ± 526 4263 ± 1587 3444 ± 639 3308 ± 792 erythrocytes sorbitol 6 ± 1 6 ± 2 44 ± 9 40 ± 8 myo -inositol 131 ± 47 104 ± 19 109 ± 20 103 ± 19 The methylguanidine and 1,1-dimethylguanidine inhibitors of nitric oxide formation described herein can be used for administration to warm blooded mammals by conventional means, preferably in formulations with pharmaceutically acceptable diluents and carriers. The amount of the active inhibitor to be administered must be an effective amount, that is, an amount which is medically beneficial but does not present toxic effects which overweigh the advantages which accompany its use. It would be expected that the adult human daily dosage would normally range upward from about one milligram per kilo of body weight of the drug. A suitable route of administration is orally in the form of capsules, tablets, syrups, elixirs and the like, although parenteral administration also can be used, e.g. intraveneously, intraperitoneally or subcutaneously. Intraveneous administration of the drug in aqueous solution such as physiologic saline is illustrative. Appropriate formulations of the drug in pharmaceutically acceptable diluents and carriers in therapeutic dosage form can be prepared by reference to general texts in the field such as, for example, Remington's Pharmaceutical Sciences , Ed. Arthur Osol. 16th ed., 1980, Mack Publishing Co., Easton, PA. The medicament according to the invention is suitable in the treatment and/or prevention of diabetes-induced vascular dysfunction and complications in warm-blooded mammals, particularly in humans. References cited in parenthesis in the disclosure are appended hereto as follows: 1. D. J. Stuehr, H. J. Cho, N. S. Kwon, M. F. Weise, C. F. Nathan, Proc. Natl. Acad. Sci. USA 88 , 7773 (1991). 2. S. Moncada, R. M. J. Palmer, E. A. Higgs, Pharmacol. Reviews 43 , 109 (1991). 3. J. Garthwaite, Trends Neurol. Sci. 14:60 (1991). 4. J. B. Hibbs, Jr., et al., in Nitric Oxide from L-Arginine: a Bioregulatory System , S. Moncada and E. Higgs, Eds. Elsevier, New York, (1990) pp 189-223. 5. G. Pugliese, R. G. Tilton, J. R. Williamson, Diabetes/Metabolism Reviews 7 , 35 (1991). 6. C. Southern, D. Schulster, 1. C. Green, Febs Lett. 276 , 42 (1990). 7. J. A. Corbett, J. L. Wang, M. A. Sweetland, J. R. Lancaster, Jr., M. L. McDaniel, Biochemical J. (submitted). 8. J. A. Corbett, J. R. Lancaster, Jr., M. A. Sweetland, M. L. McDaniel, J. Biol. Chem. 266 , 21351-21354 (1991). 9. J. R. Williamson et al., Diabete & Metab. 16 , 3369 (1990). T. Soulis-Liparota, M. Cooper, D. Papazoglou, B. Clarke, G. Jerums, Diabetes 40 , 1328 (1991). 10. M. Kihara et al., Proc. Natl. Acad. Sci. USA 88 , 6107 (1991). 11. M. Brownlee, A. Cerami, H. Vlassara, N. Engl. J. Med. 318 , 1315 (1988). M. Brownlee, H. Vlassara, A. Kooney, P. Ulrich, A. Cerami, Science 232 , 1629 (1986). 12. R. Bucala, K. J. Tracey, A. Cerami, J. Clin. Invest. 87 , 432 (1991). 13. L. C. Green et al., Anal. Biochem. 126 , 131 (1982). 14. O. H. Lowry, J. V. Passoneau (1972) A Flexible System of Enzymatic Analysis. Orlando: Academis Press. 15. R.G. Tilton, K. Chang, G. Pugliese, D. M. Eades, M.A. Province, W. R. Sherman, C. Kilo, J. R. Williamson, Diabetes 38 , 1258-1270 (1989). 16. G. Pugliese, R. G. Tilton, A. Speedy, K. Chang, M. A. Province, C. Kilo, J. R. Williamson, Metabolism 39 , 690-697 (1990). 17. G. Pugliese, R. G. Tilton, K. Chang, A. Speedy, M. Province, D. M. Eades, P. E. Lacy, C. Kilo, J. R. Williamson, Diabetes 39 , 323-332 (1990). 18. Y. Ido, R. G. Tilton, K. Chang, and J. R. Williamson, Kidney Int. , In press, 1992. 19. G. Pugliese, R. G. Tilton, A. Speedy, E. Santarelli, D. M. Eades, M. A. Province, C. Kilo, W. R. Sherman, J. R. Williamson, Diabetes 39 312-322 (1990). 20. M. J. Fregly, J. Lab. Clin. Med. 62 , 223-230 (1963). 21. J. M. Pfeffer, M. A. Pfeffer, E. D. Frohlich, J. Lab. Clin. Med. 78 , 957-962 (1971). 22. K. Chang et al., Diabetes , 40 , (suppl. 1), 210A, (1991). 23. P.L. Balestri et al., Clinical Science, 42 , 395-404, (1972). 24. N. Benjamin, The Lancet, 340/8811 , 116, (1992). Acknowledgement of Government Support This work was supported in part by National Institutes of Health grants DK06181, T32 DK07296, EY06600, HL39934, and DK20579.

Claims: Use of methylguanidine or 1,1-dimethylguanidine for the manufacture of a medicament for inhibiting nitric oxide formation in a warm blooded mammal. Use according to claim 1 of methylguanidine for the manufacture of a medicament for inhibiting nitric oxide formation in a warm blooded mammal diagnozed as being susceptible to diabetes-induced vascular dysfunction and complications. Use according to claim 1 of 1,1-dimethylguanidine for the manufacture of a medicament for inhibiting nitric oxide formation in a warm blooded mammal diagnozed as being susceptible to diabetes-induced vascular dysfunction and complications. Use according to any of claims 1 to 3 wherein the medicament is administered to the mammal intravenously or subcutaneously. Use according to any of claims 1 to 4 wherein the medicament is administered to the mammal at doses of methylguanidine or 1,1-dimethylguanidine which inhibit nitric oxide production without substantially preventing formation of glucose-induced, advanced glycation end products. Use according to any of claims 1 to 5 wherein the mammal is a human. Use of methylguanidine or 1,1-dimethylguanidine for the manufacture of a pharmaceutical composition for inhibiting nitric oxide formation in a warm blooded mammal, containing an effective amount of methylguanidine or 1,1-dimethylguanidine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or diluent. Use according to claim 7 wherein the composition is for use in the prevention and/or treatment of diabetes-induced vascular dysfunction and complications in humans.

Inventors: CORBETT JOHN ANDREW, MCDANIEL MICHAEL LYNN, TILTON RONALD GENE, WILLIAMSON JOSEPH ROBERT, CORBETT, JOHN ANDREW, MCDANIEL, MICHAEL LYNN, TILTON, RONALD GENE, WILLIAMSON, JOSEPH ROBERT
Applicants: UNIV WASHINGTON, WASHINGTON UNIVERSITY
Application year: 1993
Main classification: A61K31
IPC codes: A61P3, A61K31, A61P43