Metabolites In Human Hepatocytes Research Articles

Hepatoprotective effect of tiliroside and characterization of its metabolites in human hepatocytes by ultra-high performance liquid chromatography-high resolution mass spectrometry

Hepatoprotective effect of tiliroside and characterization of its metabolites in human hepatocytes by ultra-high performance liquid chromatography-high resolution mass spectrometry

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

Metabolism of the active carfentanil metabolite, 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester in vitro

Carfentanil, a µ-opioid receptor (MOR) agonist with an analgesic potency 10,000 times that of morphine, is extensively metabolized to norcarfentanil (M1), 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester (M0 in this article), and other low abundant metabolites in human hepatocytes and liver/lung microsomes. M0 possessed comparable MOR activity to carfentanil, and accounted for approximately 12 % of the total carfentanil metabolite formation in human liver microsomes (HLMs). Little is known about the subsequent elimination of M0. This study investigated its metabolic pathway in HLMs, separation and preliminary identification of metabolites by liquid chromatography-tandem mass spectrometry, and possible involvement of cytochrome P450 enzymes in M0 metabolism with kinetic analysis. M0 produced 9 metabolites via N-dealkylation (M1), oxidation (M3, M6–9), N-dealkylation followed by oxidation (M2 and M4), and glucuronidation (M5). Formation of the major metabolite M1 fitted typical Michaelis-Menten kinetics. Recombinant human CYP3A5 showed the highest activity toward M1 formation followed by CYP3A4 and CYP2C8, while M8 was primarily formed by CYP3A4 followed by CYP2C19 and CYP2C8. These findings reveal the main involvement of CYP3A5 and 3A4 in human hepatic elimination of M0 with a kinetic profile similar to carfentanil which may inform development of treatment protocols for carfentanil exposure.

In Vivo Metabolites of AB-PINACA in Solid Tissues Obtained from Its Abuser: Comparison with In Vitro Experiment.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with β-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)-high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC-quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

Identification of metabolites of evobrutinib in rat and human hepatocytes by using ultra-high performance liquid chromatography coupled with diode array detector and Q Exactive Orbitrap tandem mass spectrometry.

Evobrutinib is a highly selective inhibitor of Bruton's tyrosine kinase (BTK) which may be clinically effective in treating certain autoimmune diseases. The purpose of the present study was to investigate the metabolism of evobrutinib in rat and human hepatocytes. Evobrutinib was incubated with rat and human hepatocytes at 37°C for 2hours after which the samples were analyzed by ultra-high performance liquid chromatography with diode array detection and Q Exactive Orbitrap tandem mass spectrometry (UPLC-DAD-Q Exactive Orbitrap-MS). The acquired data were processed by MetWorks™ software using mass effect filter and background subtraction functions. Under these conditions, 23 metabolites were detected and their identities proposed. Among these metabolites, M13 and M15 were identified by comparison of their retention times, accurate masses, and fragment ions with those of authentic reference standards. The metabolic pathways of evobrutinib were proposed accordingly. Our results demonstrated that evobrutinib was metabolized via hydroxylation, hydrolysis, O-dealkylation, glucuronidation, and GSH conjugation. Species-related metabolic differences between rat and human hepatocytes were observed. M1-M4 were rat-specific metabolites. M13 (hydroxyl-evobrutinib) was the major metabolite whereas M15 (evobrutinib-diol) was a minor metabolite in rat hepatocytes. On the other hand, M6, M11, M16, M17, and M19 were human-specific metabolites. M15 was the most abundant metabolite whereas M13 was the minor metabolite in human hepatocytes. This study provides preliminary information regarding the metabolism of evobrutinib that may be helpful in understanding the pharmacology of evobrutinib.

Pharmacokinetics and pharmacodynamics of thiopurines in an in vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay

AimTo apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. MethodsImmortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10−4 M azathioprine and 1.1 × 10−3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). ResultsAzathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). ConclusionDetailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites.

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion-transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.

Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations.

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and Cytochrome P450s.

Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-drug interactions (DDIs) could alter the exposure of this potent therapeutic; therefore, a thorough investigation of pathways responsible for metabolism is required. In the present study, the major drug-metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver microsomes, microsomal EH (mEH) but not soluble EH (sEH) was found to be responsible for oprozomib diol formation. Coincubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor, resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect oprozomib metabolism. Pretreatment of hepatocytes with the pan-cytochrome P450 (P450) inhibitor 1-aminobenzotriazole resulted in a modest reduction (∼20%) of oprozomib metabolism. These findings indicated that mEH plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other hand, pharmacokinetics of oprozomib is unlikely to be affected by coadministered P450 and sEH inhibitors and/or inducers.

Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry

AB-FUBINACA, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide, is an indazole synthetic cannabinoid identified in drug seizures around the world. Few metabolism data are available, despite the need for human urinary markers to detect AB-FUBINACA intake. Our main objective was to identify suitable analytical targets by analyzing human hepatocyte incubation samples with high-resolution mass spectrometry (HRMS) and to confirm the results in authentic urine specimens. We also determined AB-FUBINACA’s metabolic stability in human liver microsomes (HLMs) and compared hepatocyte and urine results with in silico predictions. The metabolic stability of AB-FUBINACA was determined in pooled HLMs (1 µmol/l, up to 1 h). The metabolite profile of human hepatocytes (10 µmol/l, 1 and 3 h) and urine samples from two subjects were determined by HRMS using information-dependent tandem-mass spectrometry (MS-MS) acquisition. Data were analyzed with MetabolitePilot™ software utilizing different processing algorithms, including generic peak finding, mass defect filtering, neutral loss, and product ion filtering. In silico metabolite prediction was performed with MetaSite™ software. AB-FUBINACA’s half-life in HLMs was 62.6 ± 4.0 min. AB-FUBINACA produced 11 metabolites (2 glucuronides) in human hepatocytes and 10 were identified in authentic human urine. Major metabolic pathways were terminal amide hydrolysis, acyl glucuronidation and hydroxylation at the aminooxobutane moiety. Epoxidation followed by hydrolysis, hydroxylation at the indazole moiety and dehydrogenation were minor pathways. Defluorination did not occur. Seventeen first-generation metabolites were predicted in silico, of which seven were observed in vitro and eight in vivo. We recommend AB-FUBINACA carboxylic acid, hydroxy AB-FUBINACA carboxylic acid, dihydrodiol AB-FUBINACA and dihydrodiol AB-FUBINACA carboxylic acid as suitable urinary markers.

Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-13C, 15N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography–high resolution accurate mass spectrometry (LC–HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.

Sorafenib Hepatobiliary Disposition: Mechanisms of Hepatic Uptake and Disposition of Generated Metabolites

Sorafenib is an orally active tyrosine kinase inhibitor used in the treatment of renal and hepatocellular carcinoma. This study was designed to establish whether transport proteins are involved in the hepatic uptake of sorafenib and to determine the extent of biliary excretion of sorafenib and its metabolites in human hepatocytes. Initial uptake was assessed in freshly isolated, suspended human hepatocytes in the presence of inhibitors and modulators. [(14)C]Sorafenib (1 µM) uptake at 4°C was reduced by about 61-63% of the uptake at 37°C, suggesting a high degree of passive diffusion. Hepatocyte uptake of [(14)C]sorafenib was not Na(+) dependent or influenced by the organic anion transporter 2 inhibitor ketoprofen. However, initial [(14)C]sorafenib hepatocyte uptake was reduced by 46 and 30% compared with control values in the presence of the organic anion transporting polypeptide inhibitor rifamycin SV and the organic cation transporter (OCT) inhibitor decynium 22, respectively. [(14)C]Sorafenib (0.5-5 µM) uptake was significantly higher in hOCT1-transfected Chinese hamster ovary cells compared with mock cells, and inhibited by the general OCT inhibitor, 1-methyl-4-phenylpryidinium. OCT1-mediated uptake was saturable with a Michaelis-Menten constant of 3.80 ± 2.53 µM and a V(max) of 116 ± 42 pmol/mg/min. The biliary excretion index and in vitro biliary clearance of sorafenib (1 µM) in sandwich-cultured human hepatocytes were low (∼11% and 11 ml/min/kg, respectively). Results suggest that sorafenib uptake in human hepatocytes occurs via passive diffusion, by OCT1, and by organic anion transporting polypeptide(s). Sorafenib undergoes modest biliary excretion, predominantly as a glucuronide conjugate(s).

In Vitro Drug Metabolism of Green Tea Catechins in Human, Monkey, Dog, Rat and Mouse Hepatocytes

The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and M11 were major metabolites in rat hepatocytes, and M3 was a major metabolite in mouse hepatocytes. The experimental results have demonstrated that fate of catechins in in vitro hepatocytes depends on metabolism and chemical stability. In certain experimental conditions, the chemical reaction may become a dominant pathway. Keywords: Catechins, metabolism, humans, monkeys, dogs, rats, mice, hepatocytes

In Vitro Drug Metabolism of Green Tea Catechins in Human, Monkey, Dog, Rat and Mouse Hepatocytes

The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and M11 were major metabolites in rat hepatocytes, and M3 was a major metabolite in mouse hepatocytes. The experimental results have demonstrated that fate of catechins in in vitro hepatocytes depends on metabolism and chemical stability. In certain experimental conditions, the chemical reaction may become a dominant pathway.

Prediction of Relative In Vivo Metabolite Exposure from In Vitro Data Using Two Model Drugs: Dextromethorphan and Omeprazole

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUC(m)/AUC(p)) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUC(m)/AUC(p) ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data.

Identification of benzo[ c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry

The metabolism of the benzo[ c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/ O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[ c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/ z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.

Preferential glutathione conjugation of a reverse diol epoxide compared with a bay region diol epoxide of benzo[a]pyrene in human hepatocytes.

Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 --> m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.

Abstract 4698: Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of benzo[a]pyrene in human hepatocytes: Relevance to glutathione-S-transferase genotyping in epidemiology studies

Abstract Many studies have examined the relationship between polymorphisms in glutathione-S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We previously addressed this question using the three ring bay region PAH phenanthrene as a model compound and found that, in human urine, the only detectable mercapturic acids which result from metabolic degradation of glutathione conjugates were formed from a reverse diol epoxide of phenanthrene (phenanthrene-3,4-diol-1,2-epoxide), not from the bay region diol epoxide (phenanthrene-1,2-diol-3,4-epoxide) which would be typical of those associated with PAH carcinogenicity. We obtained similar results when we incubated human hepatocytes with these diol epoxides of phenanthrene. In the study reported here, we extended our investigation to BaP, a prototypic PAH carcinogen. We incubated human hepatocytes from ten donors with 10 µM racemic BaP-7,8-diol-9,10-epoxide, believed to be a major ultimate carcinogen of BaP, or with the non-carcinogenic reverse diol epoxide, racemic BaP-9,10-diol-7,8-epoxide. Incubations were carried out for 12 or 24h. We used high performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464→317 to analyze the incubation mixtures for the mercapturic acid products which would result from glutathione conjugation followed by normal metabolic processing of the conjugates. The standard mercapturic acids were synthesized by reaction of racemic BaP-diol epoxides with N-acetylcysteine. Major products resulting from cis- and trans- addition to each diol epoxide were characterized by proton NMR and MS. A mercapturic acid derived from phenanthrene-3,4-diol-1,2-epoxide was used as an internal standard in the hepatocyte analyses. We obtained convincing evidence for time dependent mercapturic acid formation from the non-carcinogenic reverse diol epoxide BaP-9,10-diol-7,8-epoxide in all 10 samples. Levels ranged from 0.12 to 17 pmol/ml. However, we could detect mercapturic acids from the bay region diol epoxide BaP-7,8-diol-9,10-epoxide in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BaP-7,8-diol-9,10-epoxide with glutathione is a minor pathway in humans, suggesting that glutathione-S-transferase genotyping is not an effective method of assessing risk of PAH induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4698.

Markers of Electrophilic Stress Caused by Chemically Reactive Metabolites in Human Hepatocytes

The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites.

Syntheses of potential metabolites of a potent kappa-opioid receptor agonist, TRK-820.

Chemical syntheses of three kinds of potential metabolites of TRK-820, a potent kappa-opioid receptor agonist, were described. One of the potential metabolites 2, 17-N-dealkylated TRK-820, was synthesized starting from noroxycodone through 8 steps in 21% total yield. Glucuronidation of intermediate 10 and compound 1, the free base of TRK-820, was carried out stereoselectively to give 3-O-beta-D-glucuronides 15 and 16 in good yields, respectively. Syntheses of potential conjugated metabolites 3 and 4 were accomplished through 10 steps and 2 steps in 11% and 43% total yields, respectively. Among the potential metabolites of TRK-820, compounds 2 and 4 were identified as metabolites in human hepatocytes. The results of pharmacological studies of compounds 2, 3, and 4 are described.