The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Pharmaceutical development and specification of stereoisomers.

The pharmaceutical development of chiral drugs requires the activities of many different research and development groups. Guidelines which help to coordinate the activities of these groups and assist in the successful development of compounds with either single or multiple chiral centers are outlined and discussed.

Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays.

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15,000 micrograms/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 micrograms/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 micrograms/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 micrograms/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.

Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities.

Benomyl (methyl [1-[(butylamino)carbonyl]-1H-benzimidazol-2- yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate (samples tested up to 500 and 1200 micrograms/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (> or = 5000 micrograms/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20,000 micrograms/plate) or without (up to 16,000 micrograms/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 micrograms/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 microgram/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10 ppm, respectively, would be positive in the Salmonella/Ames test with activation when tested at 5000 micrograms/plate. Purified carbendazim is not mutagenic.

Liver homogenate-mediated mutagenesis in chinese hamster V79 cells by polycyclic aromatic hydrocarbons and aflatoxins.

Several chemical carcinogens that require metabolic activation have been examined for their cytotoxic and mutagenic activity in Chinese hamster V79 cells. Mutagenic activity was measured as the induced frequency of 6-thioguanine-resistant colonies. Metabolic activation was provided by the 9000 g supernatant fraction of rat liver plus cofactors. The cytotoxicity and mutagenicity of aflatoxin B1 and B2, benzo(a)pyrene, 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, dibenz(a,h)-anthracene, dibenz(a,c)anthracene, and benz(a)anthracene were examined as functions of concentration. Except for the two isomers of dibenzanthracene, the mutagenic activity in general paralleled the carcinogenic activity. An assay of this type may be useful as a prescreen for environmental chemicals that require metabolic activation.

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine.

Low concentrations (less than or equal 20 mug/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 mug of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 mug/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; non-mutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH or US and low concentrations of AG were not mutants, but those from medium containing high concentrations (greater than or equal 30 mug/ml) of AG were mutants. Spontaneous and N-methyl-N-nitro-N-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (greater than or equal 30 mug/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH. HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.