Preclinical pharmacokinetics of SB-203580, a potent inhibitor of p38 mitogen-activated protein kinase.

1. SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)imidazole) is a potent, selective inhibitor of p38 MAP kinase used extensively as a tool inhibitor in various pharmacological and toxicological models. This study was designed to evaluate the pharmacokinetics of SB-203580 in several preclinical species, both to assist with the interpretation of existing studies and to aid in the design of future studies with this inhibitor. 2. In vitro, SB-203580 was stable in mouse, rat, dog, monkey and human plasma over 24 h. However, species differences in plasma protein binding were observed; SB-203580 was 96-97% bound in human plasma and 78-92% bound in other species. These data suggest that protein binding may influence the results of in vitro studies using SB-203580, particularly when comparing results from different in vitro systems that incorporate plasma components. In vivo, SB-203580) demonstrated moderate to high clearance in all species tested, with non-linear elimination observed in the rat at plasma concentrations > 1,000 ngml(-1). Although good solution bioavailability was observed in non-rodents (78% in dog, 32% in monkey), lower and more variable bioavailability was observed in the rat and mouse (3 -48%). 3. These interspecies differences in bioavailability, and the non-linear pharmacokinetics observed in rat, highlight the importance of monitoring SB-203580 systemic exposure in parallel witb the pharmacological endpoint during in vivo pharmacology

Preclinical pharmacokinetics and interspecies scaling of a novel vitronectin receptor antagonist.

Allometric scaling may be used in drug development to predict the pharmacokinetics of xenobiotics in humans from animal data. Although allometry may be successful for compounds that are excreted unchanged or that are oxidatively metabolized (with corrections for metabolic capacity), it has been more challenging for compounds excreted primarily as conjugates in bile. (S)-10, 11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[ a, d]cycloheptene-10-acetic acid (SB-265123) is a novel alphavbeta3 ("vitronectin receptor") antagonist. In this study, the in vivo pharmacokinetics and in vitro plasma protein binding of SB-265123 were examined in four species: mice, rats, dogs, and monkeys. In monkeys and dogs, SB-265123 exhibited moderate clearance, whereas low clearance (<20% hepatic blood flow) was observed in the rat, and high clearance (>70% hepatic blood flow) was seen in the mouse. The concentration-time profiles indicated the possibility of enterohepatic recirculation; subsequent studies in bile duct-cannulated rats demonstrated extensive biliary excretion of an acyl-glucuronide of SB-265123. In allometric scaling to predict the disposition of SB-265123 in humans, various standard correction factors were applied, including protein binding, maximum lifespan potential, and brain weight; each failed to produce adequate interspecies scaling of clearance (r(2) < 0.72). Consequently, a novel correction factor incorporating bile flow and microsomal UDP-glucuronosyltransferase activity in each species was applied, demonstrating substantial improvement in the correlation of the allometric plot (r(2) = 0.96). This study demonstrates a novel allometric correction that may be applicable to compounds that undergo conjugation and biliary excretion.

Microsomally-mediated denitrosation of nitrosoguanidinium compounds.

A major metabolic fate of 1-methyl-2-nitro-1-nitrosoguanidine (MNNG) and nitrosocimetidine (NC) in rodents is denitrosation to generate the unmodified, parent guanidinium compound. MNNG is a potent, locally-acting carcinogen. NC is the nitrosated derivative of cimetidine, an important clinical drug administered orally for the treatment of stomach ulcers. Contrary to expectations based on the results of various short-term in vitro tests for carcinogenic potential, NC is not a carcinogen when administered to rats or mice. Rat liver microsomal enzymes have been found to be capable of catalyzing the denitrosation of MNNG, NC and an NC analog, 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) in an NADPH-dependent reaction. The denitrosated guanidinium compound generated accounts for 50-70% of the nitroso compound metabolized in a microsomal incubate; nitrite is generated with a yield which represents 40-60% of the guanidinium compound produced. The cytochrome P450 inhibitors metyrapone, n-octylamine, 1-n-hexylimidazole and ellipticine inhibit the conversion of CyanoDMNG to 1,3-dimethyl-2-cyanoguanidine (Cyano-DMG) and nitrite. Microsomal NADPH-cytochrome c reductase activity is not perturbed by this series of organic compound inhibitors. Diethyl maleate at high concentrations weakly stimulates the reaction. The rates of production of the CyanoDMNG degradation products CyanoDMG, nitrite and nitrate are markedly diminished in nitrogen-saturated and in carbon dioxide-saturated microsomal incubates. Preincubating microsomes for 1 h at 37 degrees C prior to substrate and NADPH addition has no effect on the denitrosation activity. Kinetic analysis of the conversion of CyanoDMNG to CyanoDMG indicates a Km of 1.0 mM and a Vmax of 2.7 nmol/min/mg protein. Microsomes isolated from rats pretreated with the cytochrome P450 inducers pyrazole or phenobarbital show enhanced denitrosation activity. The denitrosation capacity of hamster liver microsomes is similar to that observed for rat microsomes.

Evidence for cytosolic glutathione transferase-mediated denitrosation of nitrosocimetidine and 1-methyl-2-nitro-1-nitrosoguanidine.

Nitrosocimetidine (NC) and 1-methyl-2-nitro-1-nitrosoguanidine (MNNG) are closely related N-nitrosamidines. NC is the nitrosated derivative of cimetidine (Tagamet), an orally administered compound used extensively in the treatment of gastric ulcers. MNNG is a potent carcinogen capable of initiating tumors close to the site of administration and used experimentally to produce stomach cancer. It has become evident that the primary metabolic fate of both of these agents is denitrosation. We have discovered an activity in the cytosol fraction of hamster liver which is capable of denitrosating these nitrosamidines with an efficiency approaching 100%. The activity is heat sensitive and requires reduced glutathione as a cofactor. Inhibition of the denitrosating activity with compounds which inhibit in parallel the conjugation of glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) provides evidence that the activity is glutathione transferase. One molecule of reduced glutathione is consumed in each denitrosation event. Nitrite is formed as denitrosation proceeds with a yield equivalent to 25-50% of the denitrosated product produced. Glutathione disulfide is a minor reaction product, representing 3% of the denitrosation product yield in the MNNG case and 12% in the NC case. Thus far in our survey of N-nitrosamines, N-nitrosamides and N-nitrosamidines, only the nitrosamidines appear to be vulnerable to the cytosolic denitrosating activity. In an attempt to evaluate the importance of the glutathione-dependent reaction in the intact hamster, we have depleted glutathione by pretreatment with the commonly used agents diethyl maleate (DEM) and L-buthionine-S,R-sulfoximine (L-BSO). Nitroso compound was administered i.v. and the circulating blood levels of intact and denitrosated compound 5 min after dosing quantified. NC- and MNNG-derived methylation of organ DNA was also monitored. Pretreatment had no effect on the cytosolic denitrosating or CDNB-conjugating activities. L-BSO pretreatment had no apparent effect on the denitrosative metabolism of NC or MNNG. With DEM pretreatment we obtained clear indications of a decreased rate of denitrosation and observed a 10-fold increase in MNNG-derived liver DNA methylation. The differential effects of these pretreatments are taken as an indication that DEM-sensitive processes other than those requiring glutathione dominate N-nitrosamidine denitrosation in the hamster.

Species differences in blood-mediated nitrosocimetidine denitrosation.

Nitrosocimetidine (NC) is the nitrosated derivative of cimetidine (Tagamet), a p.o. administered drug used widely in the treatment of stomach ulcers. NC is capable of methylating DNA in vitro and in cultured cells in a manner similar to that of the laboratory carcinogens 1-methyl-2-nitro-1-nitrosoguanidine and methylnitrosourea (MNU) and gives positive indications in short-term in vitro tests for genotoxicity, generally held to be prognostic of compound carcinogenic potential. Nevertheless NC has been found to be a weak or non-carcinogen in the rat and mouse model systems and to produce minimal levels of tissue DNA alkylation when dosed p.o. or i.v. to rats. The results from our earlier experiments (D. E. Jensen, Cancer Res., 43: 5258-5267, 1983) indicated that compound denitrosation is the primary fate of NC in the rat and suggested that denitrosation is the blood, mediated by hemoglobin sulfhydryl residues, is perhaps the major detoxification mechanism. We now report that whole blood and hemoglobin isolated from various mammalian species differ in their capacity for NC degradation rate enhancement and for compound denitrosation. The observed whole blood activity in the degradation reaction (rat greater than mouse/guinea pig greater than human/hamster) paralleled the hemoglobin activity. The NC half-life in isolated rat blood, 37 degrees C, was found to be about 2 min and in hamster or human blood 27 min. For reference, the MNU half-life in isolated blood is 8 min. Compound denitrosation accounted for at least 75% of the degradation in rat blood and 40 to 55% in human and hamster blood. Parallel NC denitrosation activity was found in the various hemoglobin preparations. The NC degradation rates in the presence of the several hemoglobin species were roughly proportional to the number of sulfhydryls on the hemoglobin tetramers available for reaction with p-chloromercuribenzoate and approximated the rates observed in solutions containing equivalent concentrations of L-cysteine. The percentage of total decomposition due to compound denitrosation in the presence of rat hemoglobin, 95%, was found to be unique relative to the L-cysteine-mediated reactions (about 35%) and the reactions studied over the pH range 6 through 10, the denitrosation process never accounted for more than 50% of the total degradation. Chemically blocking the sulfhydryls on human hemoglobin using iodoacetamide deleted the NC degradation rate enhancement. We found no evidence for nitrosylhemoglobin formation.(ABSTRACT TRUNCATED AT 400 WORDS)