Treatment with the antidepressant nefazodone has been associated with clinical idiosyncratic hepatotoxicty. Using membranes expressing human bile salt export pump (BSEP), human sandwich hepatocytes, and intact rats, we compared nefazodone and its marketed analogs, buspirone and trazodone. We found that nefazodone caused a strong inhibition of BSEP (IC(50) = 9 microM), inhibition of taurocholate efflux in human hepatocytes (IC(50) = 14 microM), and a transient increase in rat serum bile acids 1 h after oral drug administration. Buspirone or trazodone had no effect on biliary transport system. Nefazodone produced time- and concentration-dependent toxicity in human hepatocytes with IC(50) = 18 microM and 30 microM measured by inhibition of protein synthesis after 6 h and 24 h incubation, respectively. Toxicity was correlated with the amount of unmetabolized nefazodone. Partial recovery in toxicity by 24 h has been associated with metabolism of nefazodone to sulfate and glucuronide conjugates. The saturation of nefazodone metabolism resulted in sustained decrease in protein synthesis and cell death at 50 microM. The toxicity was not observed with buspirone or trazodone. Addition of 1-aminobenzotriazole (ABT), an inhibitor of CYP450, resulted in enhancement of nefazodone toxicity at 10 microM and was associated with accumulation of unmetabolized nefazodone. In human liver microsomes, ABT also prevented metabolism of nefazodone and formation of glutathione conjugates. We suggest that inhibition of bile acid transport by nefazodone is an indicator of potential hepatotoxicity. Our findings are consistent with the clinical experience and suggest that described methodology can be applied in the selection of nonhepatotoxic drug candidates.

The importance of uridine 5'-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway.

Here we present a preclinical model to assess drug-drug interactions due to inhibition of glucuronidation. Treatment with the antiepileptics phenobarbital (PB) or phenytoin (PH) has been associated with increased incidence of acetaminophen (APAP) hepatotoxicity in patients. In human hepatocytes, we found that the toxicity of APAP (5 mM) was increased by simultaneous treatment with phenobarbital (2 mM) or phenytoin (0.2 mM). In contrast, pretreatment with PB for 48 h prior to APAP treatment did not increase APAP toxicity unless both drugs were present simultaneously. Cells treated with APAP in combination with PB or PH experienced decreases in protein synthesis as early as 1 h, ultrastructural changes by 24 h, and release of liver enzymes by 48 h. Toxicity correlated with inhibition of APAP glucuronidation. PB or PH also inhibited APAP glucuronidation in rat and human liver microsomes and expressed human UGT1A6, 1A9, and 2B15. As with intact hepatocytes, PB and PH were neither hydroxylated nor glucuronidated, suggesting the direct inhibition of UGTs. Our findings suggest that, in multiple drug therapy, an inhibitory complex between UGT and one of the drugs can lead to decreased glucuronidation and increased systemic exposure and toxicity of a coadministered drug.

ABSTRACT:We investigated the effect of bergamottin, a major furanocoumarin in grapefruit juice, on phase I and phase II drug-metabolizing enzymes using cultured human and monkey hepatocytes. Both cultured systems were compared and evaluated for the direct effects of bergamottin as well as control treatments on liver enzymes. Treatment of hepatocytes with 0.1, 1, 5, and 10 M bergamottin resulted in a concentration-dependent reduction in CYP3A4 activity (40-100%) in both human and monkey cells, as measured by testosterone 6␤-hydroxylase activity. Bergamottin was potent at eliciting these inhibitory effects at both basal and induced states of CYP3A. Bergamottin (5 M) completely inhibited ␣-naphthoflavone-induced ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in human hepatocytes and caused a 100% decrease in EROD activity in monkey hepatocytes. A 48-h exposure of cultured human hepatocytes to bergamottin resulted in increased levels of immunoreactive CYP3A4, CYP1A1, and CYP1A2 proteins, and CYP3A4, CYP1A1, CYP1A2, CYP2B6, and UDP-glucuronosyl transferase mRNAs. There was only a 20 to 30% reduction in glucuronidation and sulfation of 4-methylumbelliferone in human hepatocytes by 10 M bergamottin and no effect on conjugation in the monkey hepatocytes. These results suggest that bergamottin causes both inhibition of CYP3A and CYP1A1/2 enzymatic activities and induction of correspondent proteins and mRNAs.

Four species of Gangamopteris and some species of Glossopteris have been described from the shales about 70 feet above Argada 'S' Seam. The dominance of Gangamopteris indicates the Karharbari age. Twelve species of Glossopteris, out of which three are new, have been described from the shales of Argada Seam, Lower Nakkari seam and Upper Nakkari Seam. Glossopteris fusa sp. nov. is distinguished by the evanescent nature of midrib and closely spaced elongate meshes of equal size. Glossopteris barakarensis sp. nov. is distinguished by a petiolate leaf and closely spaced, more or less straight secondary veins which "form narrow polygonal meshes. Glossopteris karanpurensis sp. nov. is distinguished by its large size, arched secondary veins, which run straight to the margin and form open, hexagonal meshes of equal size.