Drug metabolism and disposition: the biological fate of chemicals (Drug Metabol Dispos)

Publications in this journal

• Article: Milk Thistle's Active Components Silybin and Isosilybin: Novel Inhibitors of PXR-mediated CYP3A4 Induction.

  Kim D Mooiman, Roel F Maas-Bakker, Ed E Moret, Jos H Beijnen, Jan H M Schellens, Irma Meijerman
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  ABSTRACT: As cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause up or down regulation of drug transporters or cytochrome P450 (CYP) enzymes, in particular CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the Pregnane X Receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists is available. Some of these antagonists belong to the so-called Complementary and Alternative Medicines (CAM), such as sulphoraphane and coumestrol. Therefore, the aim was to determine the potential of selected CAM (β-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, vitamin B6, B12 and C) to inhibit of PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene and RT-PCR assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen® TR-FRET PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists in order to prevent drug-drug interactions via CYP3A4 in cancer patients.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Comparison of Endogenous 4β-hydroxycholesterol with Midazolam as Markers for CYP3A4 Induction by Rifampicin.

  Linda Bjorkhem-Bergman, Tobias Backstrom, Hanna Nylen, Yuko Ronquist-Nii, Eva Bredberg, Tommy B Andersson, Leif Bertilsson, Ulf Diczfalusy
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  ABSTRACT: Cytochrome P450 3A4 (CYP3A4) is considered the most important enzyme in drug metabolism and is often involved in drug-drug interactions. When developing new drugs appropriate markers for detecting CYP3A4-induction are needed. The aim of the present study was to compare the endogenously formed 4β-hydroxycholesterol with midazolam clearance in plasma and with 6β-hydroxycortisol / cortisol ratio in urine as markers for CYP3A4 induction. To do this we performed a clinical trial in which 24 healthy subjects were randomized to 10, 20 or 100 mg daily doses of rifampicin for 14 days (n=8 in each group) to achieve a low and moderate CYP3A4 induction. The CYP3A4-induction could be detected, even at the lowest dose of rifampicin (10 mg), by estimated midazolam clearance and 4β-hydroxycholesterol ratio (both p<0.01) and by 6β-hydroxycortisol ratio (p<0.05). The median fold-induction from baseline was 2.0, 2.6 and 4.0 for estimated midazolam clearance, 1.3, 1,6 and 2.5 for 4β-hydroxycholesterol / cholesterol ratio and 1.7, 2.9 and 3.1 for 6β-hydroxycortisol / cortisol ratio for the three dosing groups (10, 20 and 100 mg). In conclusion, 4β-hydroxycholesterol ratio was comparable to midazolam clearance as a marker of CYP3A4 induction and each may be used to evaluate CYP3A4-induction in clinical trials evaluating drug-drug interactions for new drugs.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Regulation of Murine Hepatic Hydroxysteroid Sulfotransferase Expression in Hyposulfatemic Mice and in a Cell Model of 3'-Phosphoadenosine-5'-Phosphosulfate Deficiency.

  Kathleen G Barrett, Hailin Fang, Mary D Gargano, Daniel Markovich, Thomas A Kocarek, Melissa Runge-Morris
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  ABSTRACT: The cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of nucleophilic substrates, and the cofactor for sulfonation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is biosynthesized from sulfate and ATP. The phenotype of male knock-out mice for the NaS1 sodium sulfate transporter includes hyposulfatemia and increased hepatic expression of Sult2a and Sult3a1. Here we report that in 8 week-old female NaS1-null mice, hepatic Sult2a1 mRNA levels were ~51-fold higher than they were in wild-type liver but expression of no other Sult was affected. To address whether hyposulfatemia-inducible Sult2a1 expression might be due to reduced PAPS levels, we stably knocked down PAPS synthases 1 and 2 in HepG2 cells (shPAPSS1/2 cells). When a reporter plasmid containing at least 233 nt of Sult2a1 5'-flanking sequence was transfected into shPAPSS1/2 cells, reporter activity was significantly increased relative to the activity that was seen for reporters containing 179 or fewer nt. Mutation of an IR0 nuclear receptor motif at nt -191 to 180 significantly attenuated the PAPSS1/2 knockdown-mediated increase. PAPSS1/2 knockdown significantly activated farnesoid X receptor (FXR)-, retinoid-related orphan receptor-, and pregnane X receptor-responsive reporters, and treatment with the FXR agonist GW4064 increased Sult2a1 promoter activity when the IR0 was intact. Transfection of shPAPSS1/2 cells with FXR siRNA significantly reduced Sult2a1 promoter activity. The impact of PAPSS1/2 knockdown on Sult2a1 promoter activity was recapitulated by knocking down endogenous SULT2A1 expression in HepG2 cells. We propose that hyposulfatemia leads to hepatic PAPS depletion, which causes loss of SULT2A1 activity, which results in accumulation of non-sulfated bile acids and FXR activation.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Tissue Distribution, Ontogeny, and Chemical Induction of Aldo-Keto Reductases in Mice.

  Matthew Pratt-Hyatt, Andrew J Lickteig, Curtis D Klaassen
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  ABSTRACT: Aldo-keto reductases (Akrs) are a conserved group of NAD(P)H-dependent oxido-reductase enzymes. This study provides a comprehensive examination of the tissue distribution of the 16 substrate metabolizing Akrs in mice, their expression during development and whether they are altered by chemicals that activate distinct transcriptional factor pathways. Akr1c6, 1c14, 1c20, and 1c22 are primarily present in liver; Akr1a4, 1c18, 1c21, and 7a5 in kidney; Akr1d1 in liver and kidney; Akr1b7 in small intestine; Akr1b3 and Akr1e1 in brain; Akr1b8 in testes; Akr1c14 is in ovaries; whereas Akrs1c12, 1c13, and 1c19 are expressed in numerous tissues. Liver expression of Akr1d1 and Akr1c genes are lowest during pre-natal and post-natal development. However, by 20 days of age, liver Akr1d1 increases 120-fold, and Akr1c mRNAs increase as much as 5-fold (Akr1c19) to 1000-fold (Akr1c6). Treatment of mice with chemical activators of transcription factors CAR, PXR, and Nrf2 alters liver mRNAs of Akrs. Specifically, CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases mRNAs of Akr1b7, Akr1c6, Akr1c19, and Akr1d1; whereas PXR activation by 5-pregnennenolone-16α-carbonitrile (PCN) increase the mRNA of Akrqb7 and suppresses mRNAs of Akr1c13 and Akr1c20. The Nrf2 activator 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) induces mRNAs of Akr1c6 and Akr1c19. Moreover, Nrf2-null and Nrf2 over-expressing mice demonstrate that this induction is Nrf2-dependent. This study also examined the ontogeny of Akr mRNAs and the mRNAs of Akr1c6, 1c14, 1c19, 1c20 and 1d1 increase after birth and continue to increase to adulthood.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 (GLP-1) Receptor.

  Heather Eng, Raman Sharma, Thomas S McDonald, David J Edmonds, Jean-Phillippe Fortin, Xianping Li, Benjamin D Stevens, David A Griffith, Chris Limberakis, Whitney M Nolte, David A Price, Margaret Jackson, Amit S Kalgutkar
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  ABSTRACT: 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following intravenous (but not oral) administration. In order to explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after intravenous and oral dosing. Failure to detect BETP in circulation after oral administration of a 10 mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon intravenous administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma and liver microsomes from rodents and human indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of nicotinamide adenine dinucleotide phosphate was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes, and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Aspirin Hydrolysis in Human and Experimental Animal Plasmas and the Effect of Metal Cations on Their Hydrolase Activities.

  Fatma Goksin Bahar, Teruko Imai
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  ABSTRACT: The hydrolyzing properties of plasma esterases for aspirin were investigated in human plasma and plasma from experimental animals. The observed rates of aspirin hydrolysis were in the order rabbit > human > monkey > rat > mouse > dog > minipig. In human, monkey and dog plasma, aspirin was hydrolyzed by their major hydrolases, paraoxonase (PON), butyrylcholinesterase (BChE) and albumin. In rabbit, mouse and rat plasma, carboxylesterase (CES) was determined to be the enzyme responsible for aspirin hydrolysis, and in mouse and rat plasma, especially the latter, hydrolase activity was increased by the addition of ethopropazine, a specific inhibitor of BChE. Interestingly, divalent cations affected the plasma activity by enhancing or inhibiting the hydrolase activity of plasma BChE. The addition of 2 mM calcium increased the hydrolysis of aspirin in human, monkey and dog plasma by 2.7-, 1.9- and 2.3-fold, respectively. Magnesium showed a similar but lesser effect. Increasing concentrations of calcium and magnesium resulted in a two-phase stimulatory effect on aspirin hydrolysis in human plasma. In contrast, the addition of zinc had an inhibitory effect on plasma BChE activity. It is postulated that calcium and magnesium bind to BChE and thereby change the conformation of the enzyme to a more appropriate position for aspirin hydrolysis.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Combinatorial Metabolism Notably Affects Human Systemic Exposure to Ginsenosides from Orally Administered Extract of Panax notoginseng Roots (Sanqi).

  Zheyi Hu, Junling Yang, Chen Cheng, Yuhong Huang, Feifei Du, Fengqing Wang, Wei Niu, Fang Xu, Rongrong Jiang, Xiumei Gao, Chuan Li
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  ABSTRACT: Ginsenosides are medicinal ingredients of the cardiovascular herb Panax notoginseng roots (Sanqi). Here, we implemented a human study (ChiCTR-ONC-09000603; www.chictr.org) to characterize pharmacokinetics and metabolism of ginsenosides from an orally ingested Sanqi extract (a 1:10 water extract of Sanqi) and the human plasma and urine samples were analyzed by liquid chromatography-mass spectrometry. Plasma and urinary compounds derived from ginsenosides included (1) intestinally absorbed ginsenosides Ra3, Rb1, Rd, F2, Rg1, and notoginsenoside R1 and (2) the deglycosylated products compound-K, 20(S)-protopanaxadiol, 20(S)-protopanaxatriol, and their oxidized metabolites. The systemic exposure levels of the first group compounds increased as the Sanqi extract dose increased, but those of the second group compounds were dose-independent. The oxidized metabolites of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol represented the major circulating forms of ginsenosides in the bloodstream, despite their large interindividual differences in exposure level. The metabolites were formed via combinatorial metabolism that consisted of a rate-limiting step of ginsenoside deglycosylation by the colonic microflora and a subsequent step of sapogenin oxidation by the enterohepatic cytochrome P450 enzymes. Significant accumulation of plasma ginsenosides and the metabolites occurred in the human subjects receiving three-week subchronic treatment with the Sanqi extract. Plasma 20(S)-protopanaxadiol and 20(S)-protopanaxatriol could be used as pharmacokinetic markers to reflect the subject's microbial activities, as well as the timely-changes and interindividual differences in plasma levels of their respective oxidized metabolites. The information gained from the current study is relevant to pharmacology and therapeutics of Sanqi.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Dynamic Modeling of Cytochrome P450 Inhibition In Vitro: Impact of Inhibitor Depletion on IC50-shift.

  Loren M Berry, Zhiyang Zhao, Min-Hwa Jasmine Lin
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  ABSTRACT: The impact of inhibitor depletion on the determination of shifted IC50 (IC50 determined after 30 minute preincubation with inhibitor) is examined. In addition, IC50 shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as, reversible and time-dependent inhibition. Anomalies such as smaller than expected shift in IC50 and even increases in IC50 with pre-incubation were explained by depletion of inhibitor during the pre-incubation. The IC50 shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methodologies, it is recommended that IC50-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC50-shift methodology, shifted IC50 should be calculated using observed inhibitor concentrations at the end of the incubation, rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes such as competitive inhibition, enzyme inactivation and inhibitor depletion can be used to accurately describe observed IC50 and shifted IC50 curves. For those compounds showing an IC50 fold shift of >1.5 based on observed inhibitor concentrations, reanalyzing the IC50 shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI and kinact directly from the IC50 shift experiments.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Integrated Approach of In Vivo and In Vitro for Evaluation of the Involvement of Hepatic Uptake Organic Anion Transporters in the Drug Disposition in Rats Using Rifampicin as an Inhibitor.

  Tomoki Imaoka, Tsuyoshi Mikkaichi, Koji Abe, Masakazu Hirouchi, Noriko Okudaira, Takashi Izumi
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  ABSTRACT: Cumulative studies describe the importance of drug transporters as one of the key determinants of pharmacokinetics that necessitate investigation and assessment of the involvement of drug transporters in drug discovery and development. The present study investigated an integrated in vivo and in vitro approach to determine the involvement of organic anion transporting polypeptides (Oatps) in the disposition of drugs in rats using rifampicin as an inhibitor. When bromosulfophthalein (BSP) and HMG-CoA reductase inhibitors (statins), which were used as model substrates for Oatps, were administered intravenously (3 and 1 mg/kg, respectively) to rats pretreated with rifampicin orally (30 mg/kg), the total plasma clearance of BSP and statins was attenuated compared to that in control rats, suggesting the involvement of Oatps in the disposition of these drugs in vivo. On the other hand, the pharmacokinetics of midazolam, used as a model substrate of Cyp3a, was unchanged between control rats and rifampicin-pretreated rats. The involvement of Oatps in the disposition of statins observed in vivo was further clarified by employing an in vitro hepatic uptake study and media-loss assay in the presence or absence of 100 μM rifampicin. Hepatic intrinsic clearance was reduced in the presence of rifampicin in both the media-loss assay and hepatocyte uptake study. The present study suggests in vivo investigations in rats using rifampicin together with in vitro investigations with a media-loss assay and/or uptake assay using rat hepatocytes can help determine whether a clinical drug-drug interaction study is necessary in drug development.
  Drug metabolism and disposition: the biological fate of chemicals 05/2013;
• Article: Characterization of Recombinantly Expressed Rat and Monkey Intestinal Alkaline Phosphatases: In Vitro Studies and In Vivo Correlations.

  Murali Subramanian, Sundeep Paruchury, Shashyendra Singh, Sheelendra P Singh, Rambabu Arla, Sonia Pahwa, Snehasis Jana, Prasannakumar Katnapally, Vadari Yoganand, Basanth Lakshmaiah, Debarati Mazumder, Kaushik Ghosh, Punit H Marathe, Sandhya Mandlekar
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  ABSTRACT: Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiological role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from pre-clinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, rIALP-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in-vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and minimal parent exposure in vivo. Finally, the apparent Km values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Acrolein, an α, β-Unsaturated Aldehyde, Irreversibly Inhibits the Acetylation of Aromatic Amine Xenobiotics by Human Nat1.

  Linh C Bui, Amine Manaa, Ximing Xu, Romain Duval, Florent Busi, Jean-Marie Dupret, Fernando Rodrigues Lima, Julien Dairou
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  ABSTRACT: Acrolein is an electrophilic α, β-unsaturated aldehyde of industrial, pharmaceutical and toxicological importance to which we are exposed in environmental, occupational and therapeutic situations. Acrolein is known to exert different biological effects through reactions with cellular macromolecules such as DNA, certain proteins or glutathione. In many situations (such as in tobacco smoke or other fumes), exposure to acrolein occurs concomitantly with other compounds such as aromatic amine chemicals. Interestingly, it has been shown that acrolein could impact the cellular metabolism of aromatic xenobiotics through an indirect mechanism based on the transcriptional induction of phase II xenobiotic-metabolizing enzymes. Here we report a novel mechanism by which acrolein acts on the metabolism of aromatic foreign chemicals. We provide molecular, kinetic and cellular evidence that acrolein can react directly and irreversibly with arylamine N-acetyltransferases, a major family of xenobiotic-metabolizing enzymes involved in the metabolization of aromatic amine chemicals. Formation of an acrolein adduct with a catalytic cysteine residue in the active site is responsible for the impairment of aromatic amine acetylation by the enzyme. This biochemical process may represent an additional mechanism by which acrolein impacts the metabolism and fate of aromatic amine drugs and pollutants.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Effects of Traditional Chinese Medicine Wuzhi Capsule on Pharmacokinetics of Tacrolimus in Rats.

  Hua Wei, Xia Tao, Peng Di, Yingbo Yang, Jingxian Li, Xiaofeng Qian, Jin Feng, Wansheng Chen
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  ABSTRACT: Wuzhi capsule (WZC) is a preparation of an ethanol herbal extract of Schisandra sphenanthera(Nan-Wuweizi), with its main active ingredients including schisandrin, schisandrol B, schisantherin A, schisanhenol and deoxyshisandrin. WZC and tacrolimus are often co-administered for the treatment of drug-induced hepatitis in organ transplant recipients in China. Recently, it was reported that WZC could significantly increase the blood concentration of tacrolimus. The purpose of this study was to investigate whether and how WZC affects the pharmacokinetics of tacrolimus in rats. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the plasma concentration of tacrolimus. The results showed that WZC increased the mean plasma concentration of tacrolimus. Compared with administration of tacrolimus alone (Cmax, 18.87±10.29 ng/mL; AUC0&→t, 40.98±37.07), a single intragastric administered dose of WZC increased, the pharmacokinetic parameters of tacrolimus (Cmax, 59.42±30.32 ng/mL; AUC0→t, 239.71±28.86) by five folds in rat plasma. After pretreatment with WZC for 12 days, there were still significant increases in AUC0→t (from 40.98±37.07 to 89.21±26.39 h ng/mL; p < 0.05) and Cmax (from 18.87±10.29 to 43.16±10.61 ng/mL; p < 0.05) of tacrolimus compared with oral of tacrolimus alone, suggesting that WZC increased the exposure of tacrolimus by one or more mechanisms. The increase in tacolimus Cmax by WZC was dose dependent. The effect of WZC on Tacrolimus AUC0→t also increased with dose with a maximal effect observed at 450 mg/kg (825.34 ng h/mL). No further increases in tacolimus AUC0→t were observed at WZC dose above 450 mg/kg. It is suggested that due to the effect of WZC on the pharmacokinetics of tacrolimus, the herb-drug interaction between WZC and tacrolimus should be taken into considered in clinical practice.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Inhibition of CYP2C19 and CYP3A4 by Omeprazole Metabolites and their Contribution to Drug-Drug Interactions.

  Yoshiyuki Shirasaka, Jennifer E Sager, Justin D Lutz, Connie Davis, Nina Isoherranen
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  ABSTRACT: The aim of this study was to evaluate the contribution of metabolites to drug-drug interactions (DDI) using the inhibition of CYP2C19 and CYP3A4 by omeprazole and its metabolites as a model. Of the metabolites identified in vivo, 5-hydroxyomeprazole, 5'-O-desmethylomeprazole, omeprazole sulfone and carboxyomeprazole had an AUCm/AUCp ≥ 0.25 when either total or unbound concentrations were measured following a single 20 mg dose of omeprazole in a cocktail. All of the metabolites inhibited CYP2C19 and CYP3A4 reversibly. In addition omeprazole, omeprazole sulfone and 5'-O-desmethylomeprazole were mechanism-based inhibitors (MBI) of CYP2C19 while omeprazole and 5'-O-desmethylomeprazole were found to be MBIs of CYP3A4. Reversible [I]/Ki ratios and irreversible λ/kdeg ratios were used to evaluate whether characterization of the metabolites affected DDI risk assessment. Identifying omeprazole as an MBI of both CYP2C19 and CYP3A4 was the most important factor in DDI risk assessment. Consideration of reversible inhibition by omeprazole and its metabolites would not identify DDI risk with CYP3A4, and with CYP2C19 reversible inhibition values would only identify DDI risk if the metabolites are included in the assessment. Based on inactivation data, CYP2C19 and CYP3A4 inactivation by omeprazole would be sufficient to identify risk, but metabolites were predicted to contribute 30-63% to the in vivo hepatic interactions. Hence, consideration of metabolites may be important in quantitative predictions of in vivo DDIs. The results of this study show that while metabolites contribute to in vivo DDIs their relative abundance in circulation or logP values do not predict their contribution to in vivo DDI risk.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: The Effect of BCRP, MDR1 and OATP1B3 on the Antitumor Efficacy of the Lipophilic Camptothecin AR-67 In Vitro.

  Eleftheria Tsakalozou, Eyob Debebe Adane, Kuei-Ling Kuo, Abigail Daily, Jeffrey A Moscow, Markos Leggas
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  ABSTRACT: AR-67 is a lipophilic camptothecin analogue, currently under early stage clinical trials. Transporters are known to have an impact on the disposition of camptothecins and on the response to chemotherapeutics in general due to their expression in tumor tissues. Therefore, the interplay between the BCRP, MDR1 and OATP1B1/OATP1B3 transporters and AR-67 and their impact on the toxicity profile of AR-67 was investigated. Using cell lines expressing the aforementioned transporters, we showed that the lipophilic AR-67 lactone form is a substrate for efflux transporters BCRP and MDR1. Additionally, OATP1B1 and OATP1B3 facilitated the uptake of AR-67 carboxylate in SLCO1B1 and SLCO1B3-transfected cell systems compared to the mock-transfected ones. Notably, both BCRPand MDR1, conferred resistance to AR-67 lactone. Prompted by recent studies showing increased OATP1B3 expression in certain cancer types, we investigated the effect of OATP1B3 expression on cell viability after exposure to AR-67 carboxylate. OATP1B3 expressing cells had increased carboxylate uptake as compared to mock transfected cells, but were not sensitized because the intracellular amount of lactone was fifty-fold higher than that of carboxylate and comparable between OATP1B3 expressing and non-expressing cells. In conclusion, BCRP- and MDR1-mediated efflux of AR-67 lactone confers resistance to AR-67, but OATP1B3-mediated uptake of the AR-67 carboxylate does not sensitize OATP1B3 expressing tumor cells.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Application of Receiver Operating Characteristic (ROC) Analysis to Refine the Prediction of Potential Digoxin Drug Interactions.

  Harma Ellens, Shibing Deng, Joann Coleman, Joe Bentz, Mitchell E Taub, Isabelle Ragueneau-Majlessi, Sophie Chung, Krisztina Heredi-Szabo, Sibylle Neuhof, Johan E Palm, [......], Elke Perloff, Ganesh Rajaraman, Laurent Salphati, Jesse Taur, Dietmar Weitz, Heleen Wortelboer, Cindy Xia, Guangqing Xiao, Tetsuo Yamagata, Caroline A Lee
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  ABSTRACT: In the 2012 FDA draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when [I1]/IC50 is ≥ 0.1 or [I2]/IC50 is ≥10. In this manuscript, refined criteria are presented, determined by receiver operating characteristic (ROC) analysis, utilizing IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for MDR1 over-expressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this manuscript take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of ([I1]/IC50 > 0.03 and [I2]/IC50 > 45) and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, while the number of false positives (DDI predicted but not observed) substantially decreased, from 51% to 40%, relative to the FDA criteria. Based on estimated overall variability in IC50 values a theoretical 95% confidence interval calculation was developed for single laboratory IC50's, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, due to variability in IC50 values.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various In Vitro Experimental Systems: Implications for Universal Digoxin DDI Risk Assessment Decision Criteria.

  Joe E Bentz, Mike O'Connor, Dallas Bednarczyk, Joann Coleman, Caroline A Lee, Johan E Palm, Anne Pak, Elke S Perloff, Eric L Reyner, Praveen Balimane, [......], Mitchell E Taub, Jesse Taur, Dietmar Weitz, Heleen Wortelboer, Cindy Xia, Guangqing Xiao, Jocelyn Yabut, Tetsuo Yamagata, Lei K Zhang, Harma Ellens
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  ABSTRACT: A P-glycoprotein (P-gp) IC50 working group was established with twenty-three participating pharmaceutical and contract research laboratories and one academic institution to assess inter-laboratory variability in P-gp IC50 determinations. Each lab followed their in-house protocol to determine in vitro IC50 values for sixteen inhibitors using four different test systems: Caco-2 (11 labs), MDCKII-MDR1 (6 labs) and LLC-PK1-MDR1 (4 labs) cells, and membrane vesicles containing human P-gp (5 labs). For cell models, various equations to calculate remaining transport activity (e.g. efflux ratio, unidirectional flux, net secretory flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio based-equation generally resulted in several fold lower IC50 values compared to unidirectional or net-secretory flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to inter-laboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion paper and recommendations are provided.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Human UGT2B10 in Drug N-glucuronidations: Substrate Screening and Comparison with UGT1A3 and UGT1A4.

  Yukiko Kato, Takeshi Izukawa, Shingo Oda, Tatsuki Fukami, Moshe Finel, Tsuyoshi Yokoi, Miki Nakajima
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  ABSTRACT: Recent observations revealed that human UDP-glucuronosyltransferase (UGT) 2B10 catalyzes N-glucuronidation of amine-containing compounds. Knowledge of the substrate specificity and clinical significance of UGT2B10 is still limited. The purpose of this study was to expand the knowledge of UGT2B10 substrates and to evaluate its significance in drug clearance. Using recombinant UGT2B10, we found that it catalyzes the N-glucuronidation of amitriptyline, imipramine, ketotifen, pizotifen, olanzapine, diphenhydramine, tamoxifen, ketoconazole and midazolam. These are drugs that were previously reported to be substrates for UGT1A4 or UGT1A3 and that contain in their structure either tertiary aliphatic amines, cyclic amines, or an imidazole group. UGT2B10 was inactive in the glucuronidation of desipramine, nortriptyline, carbamazepine and afloqualone. This group of drugs contains secondary or primary amines, and these results suggest that UGT2B10 preferably conjugates tertiary amines. This preference is partial because UGT2B10 did not conjugate the tertiary cyclic amine in trifluoperazine. Kinetic analyses revealed that the affinity and clearance of UGT2B10 for amitriptyline, imipramine, and diphenhydramine are significantly higher than the corresponding values of UGT1A4 and UGT1A3, although the Vmax values of UGT1A4 toward these drugs are considerably higher. These findings suggest that UGT2B10 plays a major role in the N-glucuronidation of these drugs at therapeutic concentrations. These results are also supported by inhibition studies with nicotine and hecogenin. In conclusion, this study expands the understanding of the substrate specificity of UGT2B10, highlighting its preference for tertiary amines with higher affinities and clearance values than those of UGT1A4 and UGT1A3.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Reactive Metabolite Trapping Studies on Imidazo- and 2-Methylimidazo[2,1-b]thiazole-based Inverse Agonists of the Ghrelin Receptor.

  Amit S Kalgutkar, Tim Ryder, Gregory S Walker, Suvi T M Orr, Shawn Cabral, Theunis C Goosen, Kimberly Lapham, Heather Eng
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  ABSTRACT: The current study examined the bioactivation potential of ghrelin receptor inverse agonists, 1-(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(imidazo[2,1-b]thiazol-6-yl)ethanone (1) and 1-(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethanone (2), containing a fused imidazo[2,1-b]thiazole motif in the core structure. Both compounds underwent oxidative metabolism in NADPH- and glutathione-supplemented human liver microsomes to yield glutathione conjugates, which was consistent with their bioactivation to reactive species. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in the thiol conjugates was on the thiazole ring within the bicycle. Two glutathione conjugates were discerned with the imidazo[2,1-b]thiazole derivative 1. One adduct was derived from the Michael addition of glutathione to a putative S-oxide metabolite of 1, whereas, the second adduct was formed via the reaction of a second glutathione molecule with the initial glutathione-S-oxide adduct. In the case of the 2-methylimidazo[2,1-b]thiazole analog 2, glutathione conjugation occurred via an oxidative desulfation mechanism, possibly involving thiazole ring epoxidation as the rate-limiting step. Additional insights into the mechanism were obtained via 18O exchange and trapping studies with potassium cyanide. The mechanistic insights into the bioactivation pathways of 1 and 2 allowed the deployment of a rational chemical intervention strategy that involved replacement of the thiazole ring with a 1,2,4-thiadiazole group to yield -(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)ethanone (3). These structural changes not only abrogated the bioactivation liability but also retained the attractive pharmacological attributes of the prototype agents.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: In vitro and in vivo human metabolism of degarelix, a GnRH receptor blocker.

  Anders Sonesson, Birgitte Buur Rasmussen
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  ABSTRACT: Degarelix is a decapeptide that shows high affinity/selectivity to human gonadotropin-releasing hormone receptors and have been approved for the treatment of advanced prostate cancer in US, EU and Japan. To investigate the metabolism of degarelix in humans, in vitro metabolism was addressed in liver tissue and in vivo metabolism was studied in plasma and excreta samples collected in clinical studies. Also, drug transporter interaction potential of degarelix with selected efflux transporters and uptake transporters was studied using in vitro membrane vesicles based assays and whole cell based assays. In vitro degradation was observed in fresh hepatocytes, less than 25 % of the initial concentration of degarelix remained after incubation at 37°C for 2 hours. One metabolite was detected representing a truncated nonapeptide of degarelix. The same metabolite was also detected at low concentrations in plasma. The in vivo investigations also showed that degarelix is excreted unchanged via the urine, but is undergoing extensive sequential peptidic degradation during its elimination via the hepato-biliary pathway. No unique human metabolites of degarelix were detected in the circulation or in the excreta. Degarelix did not show any interaction with selected efflux transporters and uptake transporters up to concentrations representing 200 times the clinical concentration. Since degarelix does not seem to interact with the CYP450 enzyme system as substrate, inhibitor or inducer, and does not show any interaction with hepatic and renal uptake and efflux transporters the risk for pharmacokinetic drug-drug interactions with this compound is highly unlikely.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;
• Article: Mechanistic Pharmacokinetic-Pharmacodynamic Modeling of BACE1 Inhibition in Monkeys: Development of a Predictive Model for Amyloid Precursor Protein Processing.

  Xingrong Liu, Harvey Wong, Kimberly Scearce-Levie, Ryan Watts, Melis Coraggio, Young Shin, Kun Peng, Kristin Wildsmith, Jasvinder Atwal, Jason Mango, Stephen Schauer, Kelly Regal, Kevin Hunt, Allen Thomas, Mike Siu, Joseph Lyssikatos, Gauri Deshmukh, Cornelis Hop
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  ABSTRACT: This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1), GNE-629 and GNE-892, and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid β (Aβ) in the plasma and cerebrospinal fluid (CSF), and secreted APPβ (sAPPβ) and secreted APPα (sAPPα) in the CSF were measured following a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aβ and CSF Aβ, sAPPα and sAPPβ using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 following adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC50 of 0.0033 μM and 0.065 μM, respectively. These differences in CSF and free plasma IC50 suggest different mechanisms are involved in Aβ formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aβ, sAPPβ, and sAPPα in the CSF, and Aβ in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.
  Drug metabolism and disposition: the biological fate of chemicals 04/2013;