Drug Metabolism Letters - Volume 12, Issue 2, 2018

Background: Acyl glucuronides of xenobiotics have been a subject of wide interest from the pharmaceutical industry with respect to biochemical reactivity, hepatic disposition, and enterohepatic circulation. The reactivity and lack of stability of an acyl glucuronide for a clinical candidate could pose major developability concerns. To date, multiple in vitro assays have been published to assess the risk associated with acyl glucuronides. Despite this fact, the translation of these findings to predicting clinical safety remains poor. Methods: In the present investigation, we aimed to provide simplified in vitro strategy to understand the bioactivation potential of acyl glucuronides of 10 commercial, carboxylic acid containing drugs that have been categorized as “safe,” “warning,” or “withdrawn” with respect to their marketed use. Acyl migration was measured as a function of the number of peaks observed in LC-MSn analysis. In addition, we carried out reactive intermediate trapping studies with glutathione and methoxylamine to identify the key intermediates in the transacylation bioactivation and glycation pathways, respectively. We also conducted reaction phenotyping with recombinant UDP-glucuronosyltransferase (UGT) Supersomes® to investigate if the formation of acyl glucuronides could be linked to specific UGT isoform(s). Results: Our results were in line with reported values in the literature. Our assay could be used in discovery research where half-life calculation completely eliminated the need to chemically synthesize the acyl glucuronide standard for risk assessment. We captured our results for risk assessment in a flow chart to simplify the various complex in vitro techniques historically presented. Conclusion: While the compounds tested from “withdrawn” and “warning category” all formed the glutathione adduct in buffer, none from “safe” category formed the glutathione adduct. In contrast, none of the compounds tested from any category formed methoxylamine conjugate, a reaction with putative aldehyde moiety formed via acyl migration. These results, highly favor the nucleophilic displacement as a cause of the reactivity rather than the acyl migration via aldehyde formation. The workflow presented could also be applied in the discovery setting to triage new chemical entities of interest.

Background: There is a continued need for improvements in the efficiency of metabolite structure elucidation. Objective: We propose to take LC Retention Time (RT) into consideration during the process of structure determination. Methods: Herein, we develop a simple methodology that employs a Chromatographic Hydrophobicity Index (CHI) framework for standardizing LC conditions and introduce and utilize the concept of a predictable CHI change upon Phase 1 biotransformation (CHIbt). Through the analysis of literature examples, we offer a Quantitative Structure-Retention Relationship (QSRR) for several types of biotransformation (especially hydroxylation) using physicochemical properties (clogP, hydrogen bonding). Results: The CHI system for retention indexing is shown to be practical and simple to implement. A database of CHIbt values has been created from re-incubation of 3 compounds and from analysis of an additional 17 datasets from the literature. Application of this database is illustrated. Conclusion: In our experience, this simple methodology allows complementing the discovery efforts that saves resources for in-depth characterization using NMR.

Background: The use of polypharmacy in the present day clinical therapy has made the identification of clinical drug-drug interaction risk an important aspect of drug development process. Although many drugs can be metabolized to sulfoxide and/or sulfone metabolites, seldom is known on the CYP inhibition potential and/or the metabolic fate for such metabolites. Objective: The key objectives were: a) to evaluate the in vitro CYP inhibition potential of selected parent drugs with sulfoxide/sulfone metabolites; b) to assess the in vitro metabolic fate of the same panel of parent drugs and metabolites. Methods: In vitro drug-drug interaction potential of test compounds was investigated in two stages; 1) assessment of CYP450 inhibition potential of test compounds using human liver microsomes (HLM); and 2) assessment of test compounds as substrate of Phase I enzymes; including CYP450, FMO, AO and MAO using HLM, recombinant human CYP enzymes (rhCYP), Human Liver Cytosol (HLC) and Human Liver Mitochondrial (HLMit). All samples were analysed by LC-MS-MS method. Results: CYP1A2 was inhibited by methiocarb, triclabendazole, triclabendazole sulfoxide, and ziprasidone sulfone with IC50 of 0.71 μM, 1.07 μM, 4.19 μM, and 17.14 μM, respectively. CYP2C8 was inhibited by montelukast, montelukast sulfoxide, montelukast sulfone, tribendazole, triclabendazole sulfoxide, and triclabendazole sulfone with IC50 of 0.08 μM, 0.05 μM, 0.02 μM, 3.31 μM, 8.95 μM, and 1.05 μM, respectively. CYP2C9 was inhibited by triclabendazole, triclabendazole sulfoxide, triclabendazole sulfone, montelukast, montelukast sulfoxide and montelukast sulfone with IC50 of 1.17 μM, 1.95 μM, 0.69 μM, 1.34 μM, 3.61 μM and 2.15 μM, respectively. CYP2C19 was inhibited by triclabendazole and triclabendazole sulfoxide with IC50 of 0.25 and 0.22, respectively. CYP3A4 was inhibited by montelukast sulfoxide and triclabendazole with IC50 of 9.33 and 15.11, respectively. Amongst the studied sulfoxide/sulfone substrates, the propensity of involvement of CY2C9 and CYP3A4 enzyme was high (approximately 56% of total) in the metabolic fate experiments. Conclusion: Based on the findings, a proper risk assessment strategy needs to be factored (i.e., perpetrator and/or victim drug) to overcome any imminent risk of potential clinical drug-drug interaction when sulfoxide/sulfone metabolite(s) generating drugs are coadministered in therapy.

Background: The high levels of blood alcohol achieved with chronic plus binge alcohol exposures are somewhat reduced by co-administration of tobacco-specific Nicotine-Derived Nitrosamine Ketone (NNK) suggesting that NNK may alter alcohol metabolism. Objective: We examined ethanol and acetaldehyde-metabolizing enzyme activities and malondialdehyde adduct formation in rats exposed to ethanol (chronic + binge), NNK, or both. Methods: 4-week old Long Evans rats were fed liquid diets containing 0% or 26% caloric ethanol for 8 weeks. Ethanol-fed rats were binge-administered ethanol (2 g/kg; on Mondays, Wednesdays, and Fridays) by intraperitoneal (i.p.) injection, while control group administered saline in weeks 7 and 8 (n=12/group). Six rats from each group were administered i.p. injections of NNK (2 mg/kg) or saline on Tuesdays, Thursdays, and Saturdays of weeks 3 through 8. Alcohol dehydrogenase, catalase, and aldehyde dehydrogenase activities were measured using commercial assays. Cytochrome P450 mRNA levels (17 isoforms) were measured by quantitative reverse transcription-polymerase chain reaction. Malondialdehyde immunoreactivity was measured by enzyme-linked immunosorbent assay. Results: Dual exposures to ethanol and NNK significantly increased frontal lobe ADH activity relative to control (P=0.01) and ethanol only (P=0.04) treatments, and ALDH relative to control (P=0.02). In contrast, malondialdehyde-protein expression was not significantly altered by ethanol+NNK. Ethanol decreased CYP1A1 mRNA expression relative to control (P=0.02), and combined ethanol+NNK exposures decreased the expression of CYP1A1 (P=0.01) and CYP2C6 (P=0.03). Conclusion: Dual exposures to ethanol and NNK increase brain ethanol metabolism and inhibit the expression of CYP450s that regulate xenobiotic metabolism.

Background: Age has a significant impact on activities of hepatic metabolizing enzymes in humans and animals. Flavin-containing Monooxygenase (FMO) and Aldehyde Oxidase (AO) are two important hepatic enzymes. Understanding of the impact of age on these two enzymes is still limited in pigs. Objective: The aim of this work was to assess hepatic FMO and AO activities of male domestic pigs at five different ages of 1 day, 2, 5, 10 and 20 weeks. Methods: Porcine liver microsomes and cytosol were prepared from the livers of male domestic pigs at ages of 1 day, 2, 5, 10 and 20 weeks. FMO activity was assessed using N-oxidation of benzydamine in porcine liver microsomes and AO activity was evaluated using oxidation of O6-benzylguanine in the porcine liver cytosol. Results: Porcine hepatic FMO activity was substantial at the age of 1 day, rapidly increased in 2 weeks, and remained high afterwards. Porcine hepatic AO activity was minimal at the age of 1 day and gradually increased to the maximum in 5 weeks and remained relatively constant to the age of 20 weeks. Porcine hepatic FMO activity is higher than other species, including humans. Age-dependent FMO developmental pattern in porcine liver is different from porcine hepatic CYP450 and human hepatic FMO. Porcine hepatic AO activity is much lower than humans although their developmental patterns are similar. Conclusion: Age impact on hepatic activities of both FMO and AO is obvious in domestic male pigs although age patterns of both enzymes are different.

Background: We report here an evaluation of a novel experimental system- cofactorsupplemented permeabilized cryopreserved human enterocytes (MetMax™ cryopreserved human enterocytes (MMHE), patent pending) for applications in the evaluation of enteric drug metabolism. A major advantage of MMHE over Conventional Cryopreserved Human Enterocytes (CCHE) is the simplification of the use procedures including storage at -80°C instead of in liquid nitrogen, and use of the cells immediately after thawing without a need for centrifugation and microscopic evaluation of cell density and viability and cell density adjustment. Methods: In this study, we compared MMHE and CCHE in key phase 1 oxidation and phase 2 conjugation Drug Metabolism Enzyme (DME) activities that we recently reported for cryopreserved human enterocytes: CYP2C9 (diclofenac 4'- hydroxylation), CYP2C19 (s-mephenytoin hydroxylation), CYP3A4 (midazolam 1'-hydroxylation), CYP2J2 (astemizole O-demethylation), uridine 5'-diphosphoglucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7- hydroxycoumarin sulfation), N-acetyl transferase-1 (NAT-1; p-benzoic acid N-acetylation), and carboxyesterase- 2 (CES-2; hydrolysis of irinotecan to SN38). Both CCHE and MMHE were active in all the DME pathways evaluated, with specific activities of MMHE ranged from 142% (CYP2C9) to 1713% (UGT) of that for CCHE. β-hydroxylation and testosterone 6. Result and Conclusion: Our results suggest that the MMHE system represents a convenient and robust in vitro experimental system for the evaluation of enteric drug metabolism.

Background: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavonoids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. Objective: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. Methods: Real-time RT–PCR was used to examine the expression of genes families involved in xenobiotic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). Results: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 μM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. Conclusion: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing enzymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other pathologies.

Background: Few studies have systematically investigated pregnancy-induced changes in protein abundance of drug transporters in organs important for drug/xenobiotic disposition. Objective: The goal of this study was to compare protein abundance of important drug/xenobiotic transporters including Abcb1a, Abcg2, Abcc2, and Slco1b2 in the liver, kidney and brain of pregnant mice on gestation day 15 to that of non-pregnant mice. Methods: The mass spectrometry-based proteomics was used to quantify changes in protein abundance of transporters in tissues from pregnant and non-pregnant mice. Results: The protein levels of hepatic Abcc2, Abcc3, and Slco1a4 per μg of total membrane proteins were significantly decreased by pregnancy by 24%, 72%, and 70%, respectively. The protein levels of Abcg2, Abcc2, and Slco2b1 per μg of total membrane proteins in the kidney were significantly decreased by pregnancy by 43%, 50%, and 46%, respectively. After scaling to the whole liver with consideration of increase in liver weight in pregnant mice, the protein abundance of Abcb1a, Abcg2, Abcc2, Abcb11, Abcc4, Slco1a1, and Slco1b2 in the liver was ~50-100% higher in pregnant mice, while those of Abcc3 and Slco1a4 were ~40% lower. After scaling to the whole kidney, none of the transporters examined were significantly changed by pregnancy. Only Abcg2 and Abcb1a were quantifiable in the brain and their abundance in the brain was not influenced by pregnancy. Conclusion: Protein abundance of drug transporters can be significantly changed particularly in the liver by pregnancy. These results will be helpful to understand pregnancy-induced changes in drug/xenobiotic disposition in the mouse model.