Human Liver Subcellular Fractions Research Articles

Electrochemical Probing of Human Liver Subcellular S9 Fractions for Drug Metabolite Synthesis.

Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O2-saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s-1, with a formal potential of -0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography-mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms.

Electrochemical Activation of Metabolic Enzymes in Human Liver S9 Fractions

Human liver subcellular fractions such as liver microsomes, liver cytosol fractions, or S9 fractions are widely used in in-vitro assays to predict liver metabolism. The S9 fractions are the supernatants of human liver homogenates consist of both human liver microsomes and the cytosol. Therefore, these fractions contain majority of cytochrome P450 (CYP) enzymes and soluble phase II enzymes like glucuronosyltransferases, N-acetyl transferases, sulfotransferases, and glutathione-S-transferases. Here we report the direct electrochemistry and biocatalytic features of redox active enzymes impregnated in S9 fractions. We investigated the electrochemical properties of S9 films by immobilizing them onto a high purity graphite electrode and performing cyclic voltammetry in anaerobic (Ar saturated) and aerobic (O2 saturated) conditions. The electron transfer between the S9 film and the high purity graphite electrode was observed at the formal potential of -0.48 V, with respect to Ag/AgCl reference electrode, which confirmed the activation of CYP450 reductase enzyme in the film. These films also demonstrated a catalytic oxygen reduction with a maximum current density of ~0.5 mA cm-2 at the scan rate of 0.050V/s. We investigated the CYP activity in the S9 biofilm for phase 1 metabolism using diclofenac hydroxylation as a probe reaction and could able to identify metabolic products using liquid chromatography-mass spectrometry (LC-MS) technique. Eventually, we envision to develop a platform to perform both phase I and II metabolic reactions in a single electrochemical assay.

In Vitro Metabolism of Isopropylated and tert-Butylated Triarylphosphate Esters Using Human Liver Subcellular Fractions.

Isopropylated and tert-butylated triarylphosphate esters (ITPs and TBPPs, respectively) are plasticizers and flame retardants that are ubiquitous in indoor environments; however, no studies to date have characterized their metabolism. Using human liver subcellular S9 fractions, phase I and II in vitro metabolism of triphenyl phosphate (TPHP), 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) was investigated at 1 and 10 μM doses. Parent depletion and the formation of known or suspected metabolites (e.g., likely hydrolysis or hydroxylated products), including diphenyl phosphate (DPHP), hydroxyl-triphenyl phosphate (OH-TPHP), isopropylphenyl phenyl phosphate (ip-PPP), and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored and quantified via GC/MS or LC-MS/MS. tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP and accounted for 71% and 49%, respectively, of the parent molecule that was metabolized during the incubation. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying suspected metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry and screened for in recently collected human urine where mono-isopropenylphenyl diphenyl phosphate was detected in one of nine samples analyzed. This study provides insight into the biological fate of ITP and TBPP isomers in human tissues and is useful in identifying appropriate biomarkers of exposure to monitor, particularly in support of epidemiological studies.

Drug-gut microbiota metabolic interactions: the case of UniPR1331, selective antagonist of the Eph-ephrin system, in mice

The interest on the role of gut microbiota in the biotransformation of drugs and xenobiotics has grown over the last decades and a deeper understanding of the mutual interactions is expected to help future improvements in the fields of drug development, toxicological risk assessment and precision medicine. In this paper, a microbiome drug metabolism case is presented, involving a lipophilic small molecule, N-(3β-hydroxy-Δ5-cholen-24-oyl)-l-tryptophan, UniPR1331, active as antagonist of the Eph–ephrin system and effective in vivo in a murine orthotopic model of glioblastoma multiforme (GBM). Following the administration of a single 30 mg/kg dose (p.o.) to mice, maximal plasma levels were reached 30 min after dosing and rapidly declined thereafter.To explain the observed in vivo behaviour, in vitro phase I and II metabolism assays were conducted employing mouse and human liver subcellular fractions and profiling main metabolites by means of tandem (HPLC-ESI-MS/MS) and high resolution mass spectrometry (HPLC-ESI-HR-MS).In the presence of in vitro mouse liver fractions, UniPR1331 showed a low phase I metabolic clearance, despite the identification of a 3-oxo and several hydroxylated metabolites. Conversely, after oral administration of UniPR1331 to mice, a novel isobaric metabolite was detected that (i) was subjected, as parent UniPR1331, to enterohepatic circulation (ii) had not been previously identified in vitro in mouse liver microsomes and (iii) was not observed forming after intraperitoneal (i.p.) administration of UniPR1331. An in vitro faecal fermentation assay produced the same chemical entity supporting a major role of gut microbiota in the in vivo clearance of UniPR1331.

Metabolism of Strained Rings: Glutathione S-transferase-Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation.

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione-related metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs, including GSTA1, A2-2, M1a, M2-2, T1-1, and GST from human placenta, were incubated with AZD1979. All GSTs tested catalyzed the formation of M12, with GSTA2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

Impacts of Unregulated Novel Brominated Flame Retardants on Human Liver Thyroid Deiodination and Sulfotransferation.

The inhibitory effects of five novel brominated flame retardants, 1,2-bis(2,4,5-tribromophenoxy)ethane (BTBPE), decabromodiphenylethane (DBDPE), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), bis(2-ethylhexyl)tetrabromophthalate (BEH-TEBP), and β-tetrabromoethylcyclohexane (β-TBECH), on thyroid hormone deiodinase (DIO) and sulfotransferase (SULT) activity were investigated using human in vitro liver microsomal and cytosolic bioassays. Enzymatic activity was measured by incubating active human liver subcellular fractions with thyroid hormones (T4 and rT3 separately) and measuring changes in thyroid hormone (T4, T3, rT3, and 3,3'-T2) concentrations. Only DBDPE showed inhibition of both outer and inner ring deiodination (O and IRD) of T3 and 3,3'-T2 formation from T4, respectively, with an estimated IC50 of 160 nM; no statistically significant inhibition of SULT activity was observed. ORD inhibition of 3,3'-T2 formation from rT3 was also observed (IC50 ∼ 100 nM). The kinetics of T4 O and IRD were also investigated, although a definitive mechanism could not be identified as the Michaelis-Menten parameters and maximal rate constants were not significantly different. Concentrations tested were intentionally above expected environmental levels, and this study suggests that these NBFRs are not potent human liver DIO and SULT inhibitors. To our knowledge, DBDPE is the first example of a nonhydroxylated contaminant inhibiting DIO activity, and further study of the mechanism of action is warranted.

Database of Optimized Proteomic Quantitative Methods for Human Drug Disposition-Related Proteins for Applications in Physiologically Based Pharmacokinetic Modeling.

The purpose of this study was to create an open access repository of validated liquid chromatography tandem mass spectrometry (LC‐MS/MS) multiple reaction monitoring (MRM) methods for quantifying 284 important proteins associated with drug absorption, distribution, metabolism, and excretion (ADME). Various in silico and experimental approaches were used to select surrogate peptides and optimize instrument parameters for LC‐MS/MS quantification of the selected proteins. The final methods were uploaded to an online public database (QPrOmics; www.qpromics.uw.edu/qpromics/assay/), which provides essential information for facile method development in triple quadrupole mass spectrometry (MS) instruments. To validate the utility of the methods, the differential tissue expression of 107 key ADME proteins was characterized in the tryptic digests of the pooled subcellular fractions of human liver, kidneys, intestines, and lungs. These methods and the data are critical for development of physiologically based pharmacokinetic (PBPK) models to predict xenobiotic disposition.

Oral pharmacokinetics and in-vitro metabolism of metyrapone in male rats.

The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11β-HSD1 in the male rats and humans.

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

Applying human and pig hepatic in vitro experiments for sulfur mustard study: screening and identification of metabolites by liquid chromatography/tandem mass spectrometry.

Sulfur mustard is a chemical warfare agent (CWA) with high toxicity and complex metabolism. This study aimed at identification of new metabolic biomarkers for sulfur mustard using in in vitro exposures and various mass spectrometric techniques. Human and pig liver subcellular fractions were used as biocatalysts. Metabolites were screened by liquid chromatography and tandem mass spectrometry (LC/MS/MS) using positive electrospray ionization (ESI). For structural identification, product ion scans (MS/MS, MS(3) ) and accurate mass measurements using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) were acquired. Sulfur mustard is metabolized in vitro by S-oxidation and glutathione (GSH) conjugations. One S-oxidized metabolite, bis(2-chloroethyl) sulfoxide (m/z 175), was formed in both species only when liver microsomes were present in incubations, and it was the main metabolite if GSH was not added into the reaction mixture. However, conjugation with GSH was found to be a spontaneous reaction in physiological pH and buffered solution. Three GSH conjugates of sulfur mustard were detected and identified, among which two were novel; 2-((2-(S-glutathionyl)ethyl)thio)ethanol (m/z 412) and 2-((2-(S-glutathionyl)ethyl)thio)ethyl phosphate (m/z 492). To our knowledge, this was the first time that S-oxidized metabolites and GSH conjugates of sulfur mustard have been detected and identified from human samples in vitro by LC/MS/MS. The usefulness of the GSH conjugates to serve as biomarkers for sulfur mustard exposure in human samples requires further studies.

The role of carbonyl reducing enzymes in oxcarbazepine in vitro metabolism in man

Oxcarbazepine, a second generation antiepileptic drug belonging to the family of dibenz[b,f]azepines, is subjected to a rapid and extensive biotransformation. Oxcarbazepine demonstrates a low potential for drug interactions because its biotransformation is mainly mediated by the reduction pathway instead of oxidative pathways, which are very susceptible to drug interactions. The reductive metabolism of oxcarbazepine yields a 10-monohydroxy derivative (10,11-dihydro-10-hydroxy-carbazepine), which is responsible for the pharmacological activity. The identity and localization of enzymes participating in the reduction of oxcarbazepine in response to this active metabolite have remained unknown until now. Thus, we investigated the reductive metabolism of oxcarbazepine in human liver subcellular fractions and using recombinant carbonyl reducing enzymes. The reduction of oxcarbazepine was shown to occur largely in the liver cytosol rather than liver microsomes. Furthermore, the activity and stereospecificity of cytosolic carbonyl reducing enzymes toward oxcarbazepine were assessed. Of the eight tested enzymes, six reductases were identified to contribute to the reduction of oxcarbazepine. The highest activities were demonstrated by AKR1C1, AKR1C2, AKR1C3, and AKR1C4. The contribution of CBR1 and CBR3 to the reduction of oxcarbazepine was also significant, although their role in oxcarbazepine metabolism in vivo is unclear.

Regioselective glucuronidation of the isoflavone calycosin by human liver microsomes and recombinant human UDP-glucuronosyltransferases

Hepatic conjugation plays important roles in systemic exposure and drug interactions of flavonoids. In the present study, the hepatic metabolism of calycosin, a major isoflavone from Astragali Radix, was characterized and the regioselectivity in the predominant glucuronidation pathway was first delineated in human liver microsomes (HLMs) and a panel of recombinant human UDP-glucuronosyltransferases (UGTs). Calycosin underwent major glucuronidation and minor oxidation and sulfation in human liver subcellular fractions. The major glucuronide (G2) of calycosin was isolated and identified as calycosin 3′-glucuronide by NMR analysis, and thus, the minor glucuronide (G1) was tentatively assigned as calycosin 7-glucuronide. The formations of both glucuronides in HLMs fit typical Michaelis–Menten kinetics. HLMs exhibited higher affinity (Km, G2 12.37±1.20μM vs G1 40.90±5.51μM) and velocity (Vmax, G2 5.39±0.13nmol/min/mgprotein vs G1 2.80±0.13nmol/min/mgprotein) on G2 formation, leading to the intrinsic clearance of calycosin via 3′-glucuronidation 6times that through 7-glucuronidation. UGT1A1, 1A3 and 1A10 showed activities on both 3′-OH and 7-OH, whereas UGT1A7, 1A8, 1A9, and 2B7 were only capable of catalyzing 3′-OH glucuronidation of calycosin. Among them, UGT1A9 exhibited the highest activity (Clint, 2169.50μL/min/mgprotein) for 3′-glucuronide formation followed by UGT1A7 (Clint, 396.38μL/min/mgprotein). UGT1A1 showed the highest activity towards 7-OH glucuronidation (Clint, 224.34μL/min/mgprotein), which was comparable to its activity on 3′-OH glucuronidation (Clint, 203.82μL/min/mgprotein). Propofol (UGT1A9 inhibitor) produced a complete inhibition of 3′-glucuronide formation accompanied by an increase of 7-glucuronide in HLMs, while bilirubin (UGT1A1 inhibitor) only partially (∼60%) inhibited the 7-OH glucuronidation. These findings demonstrated the regioselective glucuronidation at the 3′-OH of the isoflavone calycosin in HLMs and shed light on potential drug interactions of calycosin with other UGT1A9 substrates.

Effect of commonly used organic solvents on Aldehyde oxidase-mediated Vanillin, Phthalazine and Methotrexate oxidation in human, rat and mouse liver subcellular fractions

1. Aldehyde oxidase (AOX) is a cytosolic molybdoflavoprotein enzyme widely distributed across many tissues. In this study, we report the effect of commonly used organic solvents such as dimethyl sulfoxide (DMSO), acetonitrile (ACN), methanol and ethanol on AOX activity in human, rat and mouse liver S9 fractions using vanillin, phthalazine and methotrexate as probe substrates.2. Methanol was found to be the most potent solvent in inhibiting vanillic acid and 1-phthalazinone formation in comparison to DMSO, ACN and ethanol across the species tested, except 7-hydroxy methotrexate.3. Treatment with these solvents at approximate IC50 (% v/v) concentrations showed significant reduction in Clint and Vmax of the probe substrates and also resulted in different effects on Km across the species.4. Marked differences in the activity and affinity towards AOX were observed with different probe substrates with methotrexate showing least activity and affinity as compared to vanillin and phthalazine.5. Overall, AOX activity seemed to be more resilient to the presence of organic solvents at higher concentrations in human and rodent species. These results suggest that low concentrations of organic solvents are acceptable for in vitro incubations involving AOX-mediated metabolism.

Deeper Insight into the Reducing Biotransformation of Bupropion in the Human Liver

Bupropion is widely used as an antidepressant drug and also as a smoking cessation aid. In humans, this drug is extensively metabolized to form several metabolites. Oxidised hydroxybupropion and two reduced metabolites, threohydrobupropion and erythrohydrobupropion, are major metabolites. All of these metabolites are considered to be active. Although the oxidative metabolic pathway and the central role of CYP2B6 are known, the enzymes that participate in the reduction have not been identified to date. The aim of this study was to confirm the role of human liver subcellular fractions in the metabolism of bupropion and elucidate the contribution of particular carbonyl-reducing enzymes. An HPLC method for the determination of bupropion metabolites was utilised. Bupropion is reduced to threohydrobupropion and less to erythrohydrobupropion in human liver cytosol, microsomes and also mitochondria. Surprisingly, intrinsic clearance for formation of both metabolites is the highest in mitochondrial fraction. Moreover this study provides the first direct evidence that 11β-hydroxysteroid dehydrogenase 1, AKR1C1, AKR1C2, AKR1C3 and CBR1 participate in the reducing biotransformation of bupropion in vitro. The enzyme kinetics of all of these reductases was investigated and kinetic parameters were calculated.

Identification of domperidone metabolites in plasma and urine of gastroparesis patients with LC–ESI-MS/MS

1. Domperidone is a prokinetic agent used to treat gastroparesis. Previous studies reported oxidative metabolites of domperidone, detected by radiometric high-performance liquid chromatography or single quadrupole mass spectrometric techniques. Our aim was to identify domperidone Phase I and Phase II metabolites using liquid chromatography combined with electrospray ionization-enabled tandem mass spectrometry.2. Domperidone metabolites were identified in the plasma and urine of 11 gastroparesis patients currently being treated with domperidone. In addition, oxidative and conjugative metabolites of domperidone were characterized in human liver subcellular fractions.3. Seven metabolites were detected in vivo. Domperidone was metabolized to two mono-hydroxylated metabolites (M1 and M2), a de-alkylated metabolite (M5) and a di-hydroxylated metabolite (M7). The mono-hydroxylated metabolites were further glucuronidated to M8, M9 and sulfated to M11. To the best of our knowledge, M7, M8, M9 and M11 have not been reported previously. Five additional metabolites were identified in vitro in human subcellular fractions which comprise two additional mono-hydroxylated metabolites (M3 and M4), an alcohol metabolite (M6) possibly formed from an aldehyde intermediate, and other conjugative metabolites (M10 and M12). M6, M10 and M12 have not been characterized previously.4. In total, 12 domperidone metabolites including 7 new metabolites were identified in the present study. These results allow a better understanding of domperidone disposition in humans.

P.5.010 The behavioural and neurochemical effects of NGF dipeptide fragment GK-2 in MPTP-lesioned C57BL mice

Trabectedin (YONDELIS®) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [14C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 × g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [14C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.

Repaglinide‐gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism

To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.

Characterization of Metabolism and in Vitro Permeability Study of Notoginsenoside R1 from Radix Notoginseng

As a main and characteristic constituent in Radix notoginseng, the fate of notoginsenoside R1 (NGR1) in human is largely unknown. The present study investigated, for the first time, NGR1 metabolism by human intestinal bacteria and liver subcellular fractions, and permeability properties of NGR1 and resultant metabolites on a Caco-2 model. Samples were qualitatively analyzed using HPLC-MS/MS and quantitatively determined using HPLC-UV. When incubated with pooled human intestinal bacteria anaerobically, NGR1 showed biphasic elimination: an insignificant decrease in the first 8 h followed by a rapid elimination during 8-48 h. Four metabolites, three unambiguously identified as ginsenosides Rg1, F1 and 20(S)-protopanaxatriol formed via stepwise deglycosylation, and one tentatively assigned as a dehydrogenated protopanaxatriol with transformation occurring at the tetracyclic triterpenoid skeleton, were produced sequentially. Rg1 and F1 were formed transiently at low apparent velocities, while 20(S)-protopanaxatriol was the major metabolite with a formation rate close to the rate of NGR1 elimination and a low elimination rate. NGR1 remained intact in human liver S9 or microsomes over 1 h. Transport study of NGR1 and its metabolites revealed an ascending permeability order with stepwise deglycosylation. Taken together, the results revealed a determinant role of intestinal bacteria in the overall disposition and potential bioactivity of NGR1 in human.

Stereospecific reduction of a potent kinesin spindle protein (KSP) inhibitor in human tissues

Compound A, 1-{(3 R,3a R)-3-[3-(4-acetylpiperazin-1-yl)propyl]-7-fluoro-3-phenyl-3a,4-dihydro-3 H-pyrazolo[5,1- c][1,4]benzoxazin-2-yl}ethanone, is a novel and potent inhibitor of the mitotic kinesin spindle protein. Metabolism studies with human hepatocytes showed that Compound A underwent significant ketone reduction to its biologically active metabolite M1. Here, we describe the studies that characterized the metabolic interconversion between Compound A and M1 in vitro in human tissues. LC–MS/MS analysis showed that the ketone reduction was stereospecific for M1 with no diastereomer of M1 detected in incubations with human hepatocytes. Interestingly, such stereospecific ketone reduction was not observed with Compound B, the enantiomer of Compound A. No reductive products were observed when Compound B was incubated with human hepatocytes. Studies with human liver subcellular fractions showed that Compound A was reduced to M1 primarily by human liver cytosol with little contribution from human liver microsomes and mitochondria. NADPH was the preferred cofactor for the reduction reaction. Reverse oxidation of M1 back to Compound A was also observed, preferentially in human liver cytosol with NADP + as the cofactor. The interconversion between Compound A and M1 in human liver cytosol was inhibited significantly by flufenamic acid and phenolphthalein (potent inhibitors for aldo–keto reductase 1Cs, p < 0.05), but not by menadione, a selective inhibitor for carbonyl reductase. In addition to the liver, S9 from human lung and kidney was also capable of catalyzing this interconversion. Collectively, the results implicated the aldo–keto reductase 1Cs as the most likely enzymes responsible for the metabolic interconversion of Compound A and its active metabolite M1.

In vitro studies on the metabolism of trabectedin (YONDELIS ®) in monkey and man, including human CYP reaction phenotyping

Trabectedin (YONDELIS ®) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [ 14C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 × g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without ( N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [ 14C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.