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Publications (10)10.13 Total impact

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    ABSTRACT: Atazanavir (marketed as Reyataz®) is an important member of the human immunodeficiency virus protease inhibitor class. LC-UV-MS(n) experiments were designed to identify metabolites of atazanavir after incubations in human hepatocytes. Five major (M1-M5) and seven minor (M7-M12) metabolites were identified. The most abundant metabolite, M1, was formed by a mono-oxidation on the t-butyl group at the non-prime side. The second most abundant metabolite, M2, was also a mono-oxidation product, which has not yet been definitively identified. Metabolites, M3 and M4, were structural isomers, which were apparently formed by oxidative carbamate hydrolysis. The structure of M5 comprises the non-prime side of atazanavir which contains a pyridinyl-benzyl group. Metabolite M6a was formed by the cleavage of the pyridinyl-benzyl side chain, as evidenced by the formation of the corresponding metabolic product, the pyridinyl-benzoic acid (M6b). Mono-oxidation also occurred on the pyridinyl-benzyl group to produce the low abundance metabolite M8. Oxidation of the terminal methyl groups produced M9 and M10, respectively, which have low chemical stability. Trace-level metabolites of di-oxidations, M11 and M12, were also detected, but the complexity of the molecule precluded identification of the second oxidation site. To our knowledge, metabolites M6b and M8 have not been reported. Copyright © 2013 John Wiley & Sons, Ltd.
    Biological Mass Spectrometry 06/2013; 48(6):640-50. · 3.41 Impact Factor
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    ABSTRACT: Apparent intrinsic clearance (CL(int,app)) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL(int,app) values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL(int,app). This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (k(up)) and metabolism (k(met)). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [k(up)/(k(met) + k(up))]/[1 + (C(hep)/C(buffer)) x (V(hep)/V(buffer))], where the C(hep), C(buffer), V(hep), and V(buffer) represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.
    Drug Metabolism and Disposition 10/2006; 34(9):1600-5. · 3.36 Impact Factor
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    ABSTRACT: Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A beta-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC(50) approximately 18.0, 10.0, and 13.2 microM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC(50) approximately 11.5 microM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC(50) values > 30 microM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.
    Drug Metabolism and Disposition 04/2006; 34(4):702-8. · 3.36 Impact Factor
  • Richard Gallegos
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    ABSTRACT: Paroxetine, a frequently-prescribed serotonin reuptake inhibitor, is a potent CYP2D6 mechanism-based inactivator (1). As such, paroxetine is associated with significant drug-drug interactions due to the role of CYP2D6 in drug metabolism. CTP-347 is a new chemical entity that differs from paroxetine by specific deuterium incorporation at key positions of the molecule. CTP-347 possesses an in vitro pharmacology profile similar to paroxetine, but shows an increased rate of clearance in vitro and in vivo. This difference in clearance is due to a significant decrease in the mechanism-based inactivation of CYP2D6 relative to paroxetine. To our knowledge, CTP-347 is the first compound to have deuterium isotope effects upon mechanism-based inhibition. These results suggest that the potential for drug-drug interactions for CTP-347 may be substantially reduced compared to paroxetine.
    16th North American Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Antioxidants such as tocopherol succinate and oxo-thiazolidine have been reported to increase several markers of the hepatocyte differentiation including ureagenesis, cytochrome P450 (CYP) expression, and the inducibility of glucuronidation and sulfation activities when added in plating medium to the hepatocyte cultures. In the present studies, we evaluated the effects of antioxidants (AOX), tocopherol succinate and oxo-thiazolidine, in the plating medium on the morphology and CYP inductions in rat hepatocytes sandwich-cultured under different matrices. Freshly isolated Sprague-Dawley (SD) rat hepatocytes were plated in the DMEM medium supplemented with or without FBS or AOX, and maintained in either gelled-rigid or rigid-rigid collagen matrices. Dexmethasone and pregnenolone-16-carbonitrile were used as CYP3A inducers, phenobarbital was used as CYP2B inducer, and -naphthoflavone was used as CYP1A inducer. The hepatocyte cultures were treated with inducers for two days after the hepatocytes were overlaid with different matrices. The enzyme activities were measured via the production of metabolites from testosterone (for CYP3A and CYP2B) and phenacetin (for CYP1A). AOX in FBS free plating medium helped to preserve a high CYP1A activity, but not CYP2B and 3A activities, in hepatocytes. AOX did not affect the CYP induction. FBS containing plating medium helped the attachment of hepatocytes based on morphological appearance and the increased protein amount. Our studies has demonstrated that out of eight different culture conditions, AOX free and FBS containing plating medium with gelled matrix overlaid on the hepatocyte monolayers is the best system for CYP induction assays according to morphology and CYP activities.
    8th International International society for the study of xenobiotics Meeting;
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    ABSTRACT: Lapatinib (Tykerb) is a potent, orally available small-molecule reversible dual EGFR and HER-2 tyrosine kinase inhibitor. It is indicated, in combination with capecitabine, for the treatment of advanced or metastatic breast cancer. In humans, lapatinib undergoes extensive metabolism and is eliminated primarily in the feces. The predominant metabolite observed in human feces is the potentially reactive and hence undesired phenol M1 resulting from CYP3A4/5 mediated O-debenzylation of lapatinib. CoNCERT prepared several precision deuterium-modified isotopologs of lapatinib to examine the effects of deuteration on the metabolic fate of the analogs. While deuterium isotope effects on CYP450-mediated metabolism are well documented, the consequences of deuteration on the overall metabolite profile of a drug are unpredictable. Hence, in vitro and in vivo testing is required to determine the effects of deuterium substitution on the metabolism and pharmacokinetics of a drug. We investigated the formation of the O-debenzylated metabolite M1 from nine deuterated lapatinib isotopologs in human liver microsomes (HLM). Only a select subset of the isotopologs showed a substantial reduction in the formation of the phenolic metabolite compared to lapatinib. Interestingly, several of the more heavily deuterated lapatinib analogs showed a less marked effect on the formation of the phenolic metabolite. Kinetic isotope effects, DV and DV/K, for a d2-isotoplog were also observed. In addition, metabolite profiling in hepatocytes was performed. The results of these experiments supported the observations from the HLM studies and are indicative of metabolic shunting.
    17th North American Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Sandwich-cultured hepatocytes have been used for characterizing the function of hepatic transporters and cytochrome P450 (CYP450) enzymes. The objective of the present studies was to evaluate the effect of fetal bovine serum (FBS, a common nutrition supply for cell cultures) in the cell culture maintenance medium on the expressions and activities of hepatic transporters and P450 enzymes in sandwich-cultured Wistar rat hepatocytes. Compared to the cultured hepatocytes without FBS or with ITS, canalicular biliary network was more pronounced in the hepatocytes with FBS from phase contrast microscopy studies. The mRNA levels and functions of transporters and CYP450 enzymes in the cultured hepatocyte with ITS, or 0%, 2%, 5%, or 10% FBS was compared with the fresh isolated hepatocytes. FBS at 5% or above significantly increased Mrp2 mRNA expression but did not change the mRNA level of P-gp, Bcrp and Bsep after 5-days culture. The expression levels of uptake transporters such as Ntcp and Oatp in hepatocytes were reduced after 5-day culture while FBS could help to maintain the Ntcp expression at a concentration depended manner. The taurocholate uptake and efflux studies also demonstrated that the Ntcp function could be maintained by the addition of 5% or 10% FBS in the culture medium, which is correlated with the mRNA expression of Ntcp. The activities and mRNA expressions of CYP2B1 were significantly decreased in the presence of 2% FBS or above while the activities and mRNA levels of other CYPs such as 1A1, 2C11 and 3A1 were not significantly affected by FBS. Thus, culture medium containing FBS can be used to maintain Ntcp expressions and activities in the sandwich-cultured rat hepatocytes although the regulation pathways of FBS on the transporters and CYP450 enzyme are still unknown.
    8th International International society for the study of xenobiotics Meeting;
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    ABSTRACT: Hepatic transporters may have a great impact on drug disposition and drug-drug interactions in the clinic. Sandwich-cultured rat hepatocytes (B-CLEAR-RT) maintain the hepatocyte architecture, including tight junctions, canalicular biliary network, and the functional transporters. In this presentation, we employed B-CLEAR-RT to define the hepatobiliary transporter-mediated drug-drug interaction (DDI). Accumulation of compounds in hepatocytes and bile ducts, or in hepatocytes only is determined in Ca2+ or Ca2+ free buffer, respectively. The compound's elimination in bile is then calculated as the difference of the two measurements. The biliary excretion index (BEI), a ratio of the compound in the bile over the compound in hepatocytes and bile ducts, is also calculated. Several transporter substrates such as digoxin, taurocholate, methotrexate, DPDPE and estron sulfate, as well as inhibitors such as LY335979, verapamil, estradiol-17-glucuronide, ritonavir, cyclosporine, Ko143, omeprazole, and probenecid were chosen to evaluate their DDI potential. An inhibitor of uptake transporters could inhibit the substrate uptake and reduce the hepatocyte accumulation but might not change the BEI. An inhibitor of efflux pumps could inhibit the substrate excretion into the bile and reduce its BEI while causing accumulation of substrate in hepatocytes. An inhibitor for both efflux and uptake transporters could decrease the substrate accumulation in bile and BEI but might not change accumulation of the substrate in hepatocytes. Our studies clearly demonstrate the predictability of hepatobiliary transporter-mediated DDI by B-CLEAR.
    14th North American Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Indiplon (Fig. 1), a high affinity GABAA receptor agonist, is under development for the treatment of insomnia. The metabolism and pharmacokinetics (PK) of indiplon have been extensively investigated in rats and humans. In both species, indiplon is rapidly absorbed with tmaxvalues ≤ 2h, and rapidly eliminated from plasma with tvalues of < 2h. In vitro studies with human liver microsomes indicate that N-demethylation and N-deacteylation are the major metabolic pathways leading to the formation of pharmacologically inactive metabolites. Indiplon is also extensively metabolized in vivo with <1% dose excreted unchanged in human excreta. N-desmethyl-indiplon, the major circulating metabolite in humans, is formed primarily by CYP3A4 with minor contributions from CYP1A2. Thus, the PK profile of indiplon makes it a good candidate for providing rapid sleep onset with low risk for next day sedation. However, the short t1/2 of indiplon has required the development of a modified-release (MR) formulation for sleep maintenance. The MR formulation shows variability in PK and is undergoing further clinical evaluation. CoNCERT Pharmaceuticals has developed C-20045 (Fig. 2), a precision-deuterated analog of indiplon, with the goal of reducing the metabolic clearance and thereby providing longer sleep maintenance. C-20045 differs from indiplon by having deuterium atoms in place of hydrogen atoms on the N-methyl group. The C-D bonds are known to be approximately eight times stronger than the corresponding C-H bonds. The rate of CYP450 mediated N-demethylation of C-20045 was investigated in rat and human liver microsomes. These studies showed increased t1/2 values for disappearance of C-20045 compared to indiplon. The in vitro t1/2 of C-20045 increased by approximately 30% and 20% in human and rat liver microsomal incubations, respectively. Oral administration of 20 mg/kg C-20045 to rats resulted in about 1.7-fold increase in Cmax, 2.6-fold increase in exposure, and an approximately 2-fold increase in t1/2 compared to indiplon. The binding of C-20045 to the central benzodiazepine receptor was similar to indiplon indicating that deuteration of the N-methyl substituent did not alter the desirable pharmacodynamic attributes of indiplon. These initial investigations suggest that C-20045 has an improved PK profile with the potential of eliminating the need for an MR formulation and reduced PK variability due to blunting of CYP450 metabolism.
    15th North American Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Bilirubin, the end product of heme catabolism, is taken up from blood into hepatocytes by the sinusoidal membrane transporter, OATP2, conjugated with UGT in the hepatocyte and then excreted into the bile through the bile canallicular membrane transporter, MRP2, mainly as bilirubin glucuronides. MLN-X when dosed via oral gavage to Sprague-Dawley rats resulted in increased serum levels of both bilirubin and bilirubin glucuronides. The purpose of this study was to investigate the potential mechanism underlying MLN-X-induced hyperbilirubinemia in rats. MRP2 and MRP3 expressing membrane vesicles, hepatocyte suspension and Caco-2 cells were used to assess the inhibitory effect of MLN-X on MRP2, MRP3, OATP2/8 and P-gp, respectively. MLN-X inhibited P-gp-mediated taxol efflux in Caco-2 with an IC50 of 18 M as well as inhibited MRP2 and MRP3 mediated Leukotriene C4 uptake in MRP2 and MRP3 expressing membrane vesicles with IC50 values of 4.3 and 1.9 M, respectively. MLN-X also inhibited OATP-mediated E17βG uptake in cryopreserved human and rat hepatocytes with IC50 values of 9.5 and 16 M, respectively. The effect of MLN-X on UGT1A1 was also evaluated in rat liver microsomes. The IC50 and IC90 of MLN-X on the bilirubin monoglucuronide formation in rat liver microsomes were 177 and 394 M, respectively. Thus the hyperbilirubinemia in rats dosed with MLN-X may have been caused by a combination of mechanisms including inhibition of bilirubin uptake transporters, OATP, bilirubin efflux transporters P-gp, MRP2 and MRP3, and inhibition of UGT1A1 catalyzed bilirubin glucuronidation. This is the first report to demonstrate that the drug-induced hyperbilirubinemia can be caused by the inhibitions of all bilirubin disposition steps.
    14th North American Regional International society for the study of xenobiotics Meeting;