Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates.

Cytochrome P450 inhibition studies are performed in the pharmaceutical industry in the discovery stage to screen candidates that may have the potential for clinical drug-drug interactions. A 96-well microtiter plate assay using recombinant cytochrome P450 (Supersomes) has been used to increase the overall throughput. The IC(50) values for the inhibition of CYP3A4 by 52 new chemical entities (NCEs) were determined using the Supersomes assay with resorufin benzyl ether as a substrate, and the data were compared with those obtained in human liver microsomes (HLM) using midazolam as a substrate. Among the 52 compounds tested, 25 showed IC(50) values within a 5-fold difference in the two assays. For all compounds that showed a >5-fold difference, the IC(50) values in the Supersomes assay were lower than those obtained in HLM, except for one compound. Further studies suggested that this discrepancy was not related to difference in protein concentrations between the two assays. In addition, the IC(50) values for 16 compounds with a wide range of inhibition potency were determined in HLM using testosterone and dextromethorphan as substrates. The results showed an 80 to 93% match within a 5-fold difference between the three probe substrates. However, for certain compounds including ketoconazole, there were substrate-dependent differences in the inhibition. The results suggest that the difference between the Supersomes and HLM could be partially attributed to differences in the substrate used, and to metabolism by other cytochrome P450s present in the HLM but not in the Supersomes. Furthermore, multiple CYP3A4 substrates should be used to improve the reliability of estimating potential drug-drug interaction of NCEs.

Influence of surgically induced gastric and gastroduodenal content reflux on esophageal carcinogenesis--experimental model in Wistar female rats.

Studies in human beings and animals have shown that esophageal exposure to duodenal and gastric contents may be important for the development of Barrett's esophagus and its complications, including adenocarcinoma and epidermoid carcinoma. Diethylnitrosamine (DEN) is a carcinogen that stimulates the development of epidermoid carcinoma in the esophagus of mice. The aim of this study was to evaluate the effect of gastroduodenal and gastric content reflux on induction of esophageal carcinogenesis. Gastroesophageal reflux (GER) and gastroduodenoesophageal reflux (GDER) were produced by cardioplasty and esophagoduodenostomy. The chosen carcinogen was DEN, diluted in drinking water, given 3 days a week for 20 consecutive weeks. One hundred Wistar female rats were divided into six groups, as follows: group 1 (18 rats), cardioplasty without DEN; group 2 (18 rats), cardioplasty with DEN; group 3 (10 rats), only water; group 4 (17 rats), cardioplasty with DEN; group 5 (17 rats), esophagoduodenostomy with DEN; group 6 (20 rats), only DEN. GER in isolation induced papillomatosis or ulceration in 22.2% of rats and, when associated with DEN, induced papillomatosis in 61.1% of rats. GDER in isolation induced marked esophagitis in 61.1% of rats, Barrett's esophagus in 16.7% and esophageal adenocarcinoma in 16.7%; when associated with DEN, 23.5% of rats presented marked esophagitis, papillomatosis or ulceration, whereas 76.5% had esophageal carcinoma, with 70.6% epidermoid carcinoma and 5.9% adenocarcinoma. Rats treated with water alone did not show histologic abnormalities of the esophageal mucosa. Rats treated with DEN alone developed papillomas in 50.0% of the cases and remained histologically unchanged in 50.0%. There was no development of low- or high-grade dysplasia in any group. The conclusions are that (1) GDER is significantly more deleterious to esophageal mucosa than GER; (2) in this study, GER did not present carcinogenic potential in relation to the esophagus; (3) GDER in isolation is an esophageal carcinogen, producing Barrett's esophagus and esophageal adenocarcinoma; (4) esophageal oncogenesis caused by GDER is potentiated by DEN, inducing esophageal epidermoid carcinoma; (5) in this study, DEN in isolation did not generate tumors in the esophagus of rats.

Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.

Status of in vitro ocular irritation testing.

This paper reviews advances in the validation of alternative methods for eye irritation testing since the 1987 publication, A Critical Evaluation of Alternatives to Acute Ocular Irritation Testing (1). We have highlighted details of methods that appear promising and identified the minimum needs and endpoints necessary to develop a battery or batteries of in vitro tests to evaluate eye irritancy. We have recommended a series of workshops to provide identified batteries for specific classes of chemicals or for specific uses of eye irritancy testing. We have also identified the need for consensus meetings and peer-reviewed publication to ensure that the most predictive batteries become parts of validation studies. Finally, we note that academic scientists, industry, government and the animal protection community must work together in order to replace in vivo eye irritancy testing with appropriately validated in vitro methods.

Adaptation of a gamma-glutamyl transpeptidase assay to microtiter plates.

A kinetic assay for measuring gamma-glutamyl transpeptidase (GGT) activity has been adapted to microtiter plates and an automated microtiter plate reader. This method permits the simultaneous analysis of enzyme activity in a large number of samples incubated with the chromogenic GGT substrate gamma-glutamyl-p-nitroanilide. A major advantage of this assay over previously reported methods is the substantial reduction in the time needed for measuring sample enzyme activity. In addition, reduction of the total assay volume to 0.28 ml conserves both sample and reagents. This method has been calibrated at 23 degrees C using purified GGT, and used to analyze GGT activity in human sera. The assay is sensitive over a range of 3-200 U/liter.

Early biological indicators of S-(1,2-dichlorovinyl)-L-cysteine nephrotoxicity in the rabbit.

The progression of changes in rabbit kidney function following dosing with the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC, 20-50 mg/kg) was determined. Proteinuria was observed 0.5-1 hr after administration of DCVC at doses of 20-50 mg/kg. Blood urea nitrogen levels, glomerular filtration rates, urinary glucose excretion, and urine volume were also altered following DCVC dosing; however, these parameters were less sensitive than proteinuria as markers of early renal dysfunction. None of these latter four indicators were affected by low DCVC doses, nor were they altered by high DCVC doses until 1.5-2.5 hr after dosing. Dose-dependent morphological changes to kidney structure were also observed 5 hr after DCVC administration. Low doses caused damage restricted to brush border membranes in the pars recta, while higher doses produced a necrotic lesion encompassing all regions of the proximal tubule. This study indicates that DCVC can cause rapid renal dysfunctional changes which are first detected by elevated urinary protein excretion.

Binding of porcine von Willebrand factor to human platelets in the presence of ristocetin.

The interaction of porcine von Willebrand factor (vWF) with human platelets in the presence of ristocetin was examined. Binding was rapid, specific, saturable and dependent on ristocetin concentration with half-maximal stimulation occurring at a ristocetin concentration of 0.5 mM. Assuming vWF to be a tetramer with a MW of 9.5 x 10(5), approximately 94,000 vWF binding sites per platelet were found with an average binding constant of 2.1 x 10(-8)M. Human vWF competed with the porcine protein for this site only in the presence of ristocetin. Binding of porcine vWF to platelets was found to be less sensitive to reduction with dithiothreitol than platelet agglutinating activity.

Phospholipid biosynthesis in some anaerobic bacteria.

We have identified and characterized enzymes of phospholipid synthesis in two plasmalogen-rich anaerobes. Megasphaera elsdenii and Veillonella parvula, and one anaerobe lacking plasmalogens. Desulfovibrio vulgaris. All three species contained phosphatidate cytidylyltransferase and phosphatidylserine synthase. Phosphatidylglycerophosphate synthesis was detected only D. vulgaris extracts. Phosphatidylserine (diacyl form) was the major product of the phosphatidylserine synthase assay with particles from M. elsdenii or V. parvula. The amounts of phosphatidylethanolamine formed were very low. Only D. vulgaris particles had an active phosphatidylserine decarboxylase.

Interaction of porcine von Willebrand factor (platelet aggregating factor) with human platelets.

The binding of 125I-labeled porcine von Willebrand factor to washed human platelets was examined. In the absence of ristocetin, binding was found to be rapid, specific and saturable and was decreased in the presence of urea or at high ionic strength. Platelets were found to contain approx. 4760 binding sites for porcine von Willebrand factor with an average binding constant of 2.92 x 10(-7) M assuming the von Willebrand factor to be a tetramer with a molecular weight of 9 x 10(5). Although ristocetin was not absolutely required for binding, in its presence binding was increased.

The enzymes of phospholipid synthesis in Clostridium butyricum.

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.

Purification and properties of a diacetyl reductase from Escherichia coli.

A reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with the ability to reduce diacetyl has been isolated from Escherichia coli and has been purified 800-fold to near homogeneity. The product of the reduction of diacetyl was shown to be acetoin. The enzyme proved to catalyze the oxidation of NADPH in the presence of both uncharged alpha- and beta-dicarbonyl compounds. Even monocarbonyl compounds showed slight activity with the enzyme. On the basis of its substrate specificity, it is suggested that the enzyme functions as a diacetyl reductase. In contrast to other diacetyl reductases, the one reported here is specific for NADPH and does not possess acetoin reductase activity. The pH optimum of this enzyme was found to be between 6 and 7. The maximal velocity for the NADPH-dependent reduction of diacetyl was determined to be 9.5 mumol per min per mg of protein and the K(m) values for diacetyl and NADPH were found to be 4.44 mM and 0.02 mM, respectively. The molecular weight was estimated by gel filtration on Sephadex G-100 to be approximately 10,000.