Cytochrome P450 Reaction Phenotyping Research Articles

Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes.

Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role.

Defining the Selectivity of Chemical Inhibitors Used for Cytochrome P450 Reaction Phenotyping: Overcoming Selectivity Limitations with a Six-Parameter Inhibition Curve-Fitting Approach.

The utility of chemical inhibitors in cytochrome P450 (CYP) reaction phenotyping is highly dependent on their selectivity and potency for their target CYP isoforms. In the present study, seventeen inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 commonly used in reaction phenotyping were evaluated for their cross-enzyme selectivity in pooled human liver microsomes. The data were evaluated using a statistical desirability analysis to identify (1) inhibitors of superior selectivity for reaction phenotyping and (2) optimal concentrations for each. Among the inhibitors evaluated, α-naphthoflavone, furafylline, sulfaphenazole, tienilic acid, N-benzylnirvanol, and quinidine were most selective, such that their respective target enzymes were inhibited by ~95% without inhibiting any other CYP enzyme by more than 10%. Other commonly employed inhibitors, such as ketoconazole and montelukast, among others, were of insufficient selectivity to yield a concentration that could adequately inhibit their target enzymes without affecting other CYP enzymes. To overcome these shortcomings, an experimental design was developed wherein dose response data from a densely sampled multi-concentration inhibition curve are analyzed by a six-parameter inhibition curve function, allowing accounting of the inhibition of off-target CYP isoforms inhibition and more reliable determination of maximum targeted enzyme inhibition. The approach was exemplified using rosiglitazone N-demethylation, catalyzed by both CYP2C8 and 3A4, and was able to discern the off-target inhibition by ketoconazole and montelukast from the inhibition of the targeted enzyme. This methodology yields more accurate estimates of CYP contributions in reaction phenotyping. Significance Statement Isoform-selective chemical inhibitors are important tools for identifying and quantifying enzyme contributions as part of a CYP reaction phenotyping assessment for projecting drug-drug interactions. However, currently employed practices fail to adequately compensate for shortcomings in inhibitor selectivity and the resulting confounding impact on estimates of the CYP enzyme contribution to drug clearance. In this report, we describe a detailed IC50 study design with 6-parameter modeling approach that yields more accurate estimates of enzyme contribution.

An improved method for cytochrome p450 reaction phenotyping using a sequential qualitative-then-quantitative approach.

Cytochrome P450 reaction phenotyping to determine the fraction of metabolism values (fm) for individual enzymes is a standard study in the evaluation of a new drug. However, there are technical challenges in these studies caused by shortcomings in the selectivity of P450 inhibitors and unreliable scaling procedures for recombinant P450 (rCYP) data. In this investigation, a two-step "qualitative-then-quantitative" approach to P450 reaction phenotyping is described. In the first step, each rCYP is tested qualitatively for potential to generate metabolites. In the second step, selective inhibitors for the P450s identified in step1 are tested for their effects on metabolism using full inhibition curves. Forty-eight drugs were evaluated in step 1 and there were no examples of missing an enzyme important to in vivo clearance. Five drugs (escitalopram, fluvastatin, pioglitazone, propranolol, and risperidone) were selected for full phenotyping in step2 to determine fm values, with findings compared to fm values estimated from single inhibitor concentration data and rCYP with intersystem-extrapolation-factor corrections. The two-step approach yielded fm values for major drug clearing enzymes that are close to those estimated from clinical data: escitalopram and CYP2C19 (0.42 vs 0.36-0.82), fluvastatin and CYP2C9 (0.76 vs 0.76), pioglitazone and CYP2C8 (0.72 vs 0.73), propranolol and CYP2D6 (0.68 vs 0.37-0.56) and risperidone and CYP2D6 (0.60 vs 0.66-0.88). Reaction phenotyping data generated in this fashion should offer better input to physiologically-based pharmacokinetic models for prediction of DDI and impact of genetic polymorphisms on drug clearance. The qualitative-then-quantitative approach is proposed as a replacement to standard reaction phenotyping strategies. Significance Statement P450 reaction phenotyping is important for projecting drug-drug interactions and interpatient variability in drug exposure. However, currently recommended practices can frequently fail to provide reliable estimates of the fractional contributions of specific P450 enzymes (fm) to drug clearance. In this report, we describe a two-step qualitative-then-quantitative reaction phenotyping approach that yields more accurate estimates of fm.

Reaction Phenotyping of Low-Turnover Compounds in Long-Term Hepatocyte Cultures Through Persistent Selective Inhibition of Cytochromes P450.

Recognizing the challenges of determining the relative contribution of different drug metabolizing enzymes to the metabolism of slowly metabolized compounds, a cytochrome P450 reaction phenotyping (CRP) method using cocultured human hepatocytes (HEPATOPAC) has been established. In this study, the emphasis on the relative contribution of different cytochrome P450 (P450) isoforms was assessed by persistently inhibiting P450 isoforms over 7 days with human HEPATOPAC. P450 isoform-selective inhibition was achieved with the chemical inhibitors furafylline (CYP1A2), tienilic acid (CYP2C9), (+)-N-3-benzylnirvanol (CYP2C19), paroxetine (CYP2D6), azamulin (CYP3A), and a combination of 1-aminobenzotriazole and tienilic acid (broad spectrum inhibition of P450s). We executed this CRP method using HEPATOPAC by optimizing for the choice of P450 inhibitors, their selectivity, and the temporal effect of inhibitor concentrations on maintaining selectivity of inhibition. In general, the established CRP method using potent and selective chemical inhibitors allows to measure the relative contribution of P450s and to calculate the fraction of metabolism (f m) of low-turnover compounds. Several low-turnover compounds were used to validate this CRP method by determining their hepatic intrinsic clearance and f m, with comparison with literature values. We established the foundation of a robust CRP for low-turnover compound test system which can be expanded to include inhibition of other drug metabolizing enzymes. This generic CRP assay, using human long-term hepatocyte cultures, will be an essential tool in drug development for new chemical entities in the quantitative assessment of the risk as a victim of drug-drug interactions. SIGNIFICANCE STATEMENT: An ongoing trend is to develop drug candidates which have limited metabolic clearance. The current studies report a generic approach to conducting reaction phenotyping studies with human HEPATOPAC, focusing on P450 metabolism of low-turnover compounds. Potent and selective chemical inhibitors were used to assess the relative contribution of the major human P450s. Validation was achieved by confirming hepatic intrinsic clearance and fraction of metabolism for previously reported low-turnover compounds. This approach is adaptable for assessment of all drug metabolizing enzymes.

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes.

Pinostrobin (PI, 5-hydroxy-7-methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450-selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro. In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC50 >100 μm). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.

REDX08608, a Novel, Potent and Selective, Reversible BTK Inhibitor with Efficacy and Equivalent Potency Against Wild-Type and Mutant C481S BTK

Here we report the preclinical profile of REDX08608 our novel, potent and selective, reversible BTK inhibitor that is equipotent against wild-type and mutant C481S BTK.Bruton's tyrosine kinase (BTK) is a member of the src-related Tec family of cytoplasmic tyrosine kinases and plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK is a clinically validated target to treat B-cell malignancies that are dependent on BCR signaling i.e.CLL and NHL with ibrutinib approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors such as ibrutinib, acalabrutinib and GS-4059 specifically target a cysteine residue C481 within BTK and mutations at this site clearly interfere with covalent drug binding. C481S, C481Y, C481R, C481F mutations have been reported and linked to cases of resistance that have emerged in patients with CLL progression following treatment with ibrutinib (Byrd2016, Inhye2016, Maddocks2015, Woaych2014). Redx reversible BTK inhibitor, REDX08608, aims to overcome this resistance mechanism by targeting both wild type and C481-mutated BTK. Redx have recently presented REDX06961 our BTK probe (Guisot2016, AACR#4795) and, following lead optimization, we are now disclosing REDX08608, our lead compound, a potent, reversible and selective BTK inhibitor, which displays an improved profile including superior pharmacokinetics.REDX08608, reversibly, inhibits WT and C481S BTK and displays nanomolar binding affinity and potency in biochemical and cellular-based assays.REDX08608 inhibits BTK signaling and growth in cell lines dependent on the BTK pathway such the OCI-LY10 ABC-DLBCL cell line. Importantly, REDX08608 also inhibits BTK signaling in primary CLL cells. In human whole blood and isolated human PBMCs, REDX08608 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 in CD19+cells.REDX08608 is highly selective when tested against a panel of 468 kinases and exhibits improved target specificity with >100-fold selectivity against other Tec and Src kinase family members (ITK, TXK, BMX, TEC, BLK, CSK, FYN, HCK, LCK, SRC) and >400-fold selectivity against EGFR.REDX08608 was fully profiled through DMPK in vitro assays including metabolic stability, plasma stability, cytochrome P450 inhibition, PXR activation/cytochrome P450 activity, time dependent inhibition and cytochrome P450 reaction phenotyping. REDX08608 was shown to have an acceptable metabolic and plasma stability profile across species (mouse, rat, dog, monkey and human). REDX08608 displayed no evidence of PXR activation or time dependent inhibition. IC50s were determined for human cytochrome P450s (1A2, 2D6, 2C9, 2C19 and 3A4) and were all >10 µM. Good exposure, oral bioavailability and half-life were demonstrated for REDX08608 in mouse, rat and dog, with dose linearity assessment performed in mouse (F = 73-100%, CL= 11% liver blood flow in mice; F = 55-84%, CL = 28% liver blood flow in rat; F = 85%, CL= 10% liver blood flow in dog). In vivo efficacy studies in preclinical models will also be disclosed.In conclusion, REDX08608 is a potent and selective, reversible BTK inhibitor with efficacy in lymphoma cell lines that offers the potential to target both wild-type BTK and an important emerging resistance mechanism in patients with CLL progression following ibrutinib-treatment. DisclosuresGuisot:Redx Oncology Ltd - Redx Pharma Plc: Employment. Best:Redx Oncology Ltd - Redx Pharma Plc: Employment. Wright:Redx Oncology Ltd - Redx Pharma Plc: Employment. Thomason:Redx Oncology Ltd - Redx Pharma Plc: Employment. Woyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding. Abet:Redx Oncology Ltd - Redx Pharma Plc: Employment. Castagna:Redx Oncology Ltd - Redx Pharma Plc: Employment. Cousin:Redx Oncology Ltd - Redx Pharma Plc: Employment. Emmerich:Redx Oncology Ltd - Redx Pharma Plc: Employment. Ho:Redx Oncology Ltd - Redx Pharma Plc: Employment. Kelly:Redx Oncology Ltd - Redx Pharma Plc: Employment. King-Tours:Redx Oncology Ltd - Redx Pharma Plc: Employment. Lyons:Redx Oncology Ltd - Redx Pharma Plc: Employment. Muller:Redx Oncology Ltd - Redx Pharma Plc: Employment. Refuerzo:Redx Oncology Ltd - Redx Pharma Plc: Employment. Sargent:Redx Oncology Ltd - Redx Pharma Plc: Employment. Talab:Redx Oncology Ltd - Redx Pharma Plc: Employment. Bingham:Redx Oncology Ltd - Redx Pharma Plc: Employment. Phillips:Redx Oncology Ltd - Redx Pharma Plc: Employment. Armer:Redx Oncology Ltd - Redx Pharma Plc: Employment.

Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method.

A novel cytochrome P450 (P450) reaction phenotyping method for low-clearance compounds has been developed for eight P450 enzymes (CYP1A2, 2B6, 2D6, 2C8, 2C9, 2C19, 3A, and 3A4) and pan-cytochrome using the hepatocyte relay approach. Selective mechanism-based inhibitors were used to inactivate the individual P450 enzymes during preincubation, and inactivators were removed from the incubation before adding substrates to minimize reversible inhibition and maximize inhibitor specificity. The inhibitors were quite selective for specific P450 isoforms using the following inhibitor concentrations and preincubation times: furafylline (1 µM, 15 minutes) for CYP1A2, phencyclidine (20 µM, 15 minutes) for 2B6, paroxetine (1.8 µM, 15 minutes) for CYP2D6, gemfibrozil glucuronide (100 µM, 30 minutes) for 2C8, tienilic acid (15 µM, 30 minutes) for 2C9, esomeprazole (8 µM, 15 minutes) for 2C19, troleandomycin (25 µM, 15 minutes) for 3A4/5, CYP3cide (2 µM, 15 minutes) for 3A4, and 1-aminobenzotriazole (1 mM, 30 minutes) supplemented with tienilic acid (15 µM, 30 minutes) for pan-cytochrome. The inhibitors were successfully applied to the hepatocyte relay method in a 48-well format for P450 reaction phenotyping of low-clearance compounds. This novel method provides a new approach for determining the fraction metabolized of low-turnover compounds that are otherwise challenging with the traditional methods, such as chemical inhibitors with human liver microsomes and hepatocytes or human recombinant P450 enzymes.

Software-aided cytochrome P450 reaction phenotyping and kinetic analysis in early drug discovery.

Cytochrome P450 (CYP450) reaction phenotyping (CRP) and kinetic studies are essential in early drug discovery to determine which metabolic enzymes react with new drug entities. A new semi-automated computer-assisted workflow for CRP is introduced in this work. This workflow provides not only information regarding parent disappearance, but also metabolite identification and relative metabolite formation rates for kinetic analysis. Time-course experiments based on incubating six probe substrates (dextromethorphan, imipramine, buspirone, midazolam, ethoxyresorufin and diclofenac) with recombinant human enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and human liver microsomes (HLM) were performed. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) analysis was conducted with an internal standard to obtain high-resolution full-scan and MS/MS data. Data were analyzed using Mass-MetaSite software. A server application (WebMetabase) was used for data visualization and review. CRP experiments were performed, and the data were analyzed using a software-aided approach. This automated-evaluation approach led to (1) the detection of the CYP450 enzymes responsible for both substrate depletion and metabolite formation, (2) the identification of specific biotransformations, (3) the elucidation of metabolite structures based on MS/MS fragment analysis, and (4) the determination of the initial relative formation rates of major metabolites by CYP450 enzymes. This largely automated workflow enabled the efficient analysis of HRMS data, allowing rapid evaluation of the involvement of the main CYP450 enzymes in the metabolism of new molecules during drug discovery.

In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA.

Disposition and metabolic profiling of [(14)C]cerlapirdine using accelerator mass spectrometry.

Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist of the 5-hydroxytryptamine 6 (5-HT6) receptor. Cerlapirdine and other 5-HT6 receptor antagonists have been in clinical development for the symptomatic treatment of Alzheimer's disease. A human absorption, distribution, metabolism, and excretion study was conducted to gain further understanding of the metabolism and disposition of cerlapirdine. Because of the low amount of radioactivity administered, total (14)C content and metabolic profiles in plasma, urine, and feces were determined using accelerator mass spectrometry (AMS). After a single, oral 5-mg dose of [(14)C]cerlapirdine (177 nCi), recovery of total (14)C was almost complete, with feces being the major route of elimination of the administered dose, whereas urinary excretion played a lesser role. The extent of absorption was estimated to be at least 70%. Metabolite profiling in pooled plasma samples showed that unchanged cerlapirdine was the major drug-related component in circulation, representing 51% of total (14)C exposure in plasma. One metabolite (M1, desmethylcerlapirdine) was detected in plasma, and represented 9% of the total (14)C exposure. In vitro cytochrome P450 reaction phenotyping studies showed that M1 was formed primarily by CYP2C8 and CYP3A4. In pooled urine samples, three major drug-related peaks were detected, corresponding to cerlapirdine-N-oxide (M3), cerlapirdine, and desmethylcerlapirdine. In feces, cerlapirdine was the major (14)C component excreted, followed by desmethylcerlapirdine. The results of this study demonstrate that the use of the AMS technique enables comprehensive quantitative elucidation of the disposition and metabolic profiles of compounds administered at a low radioactive dose.

Metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes

Aildenafil, 1-{[3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d] primidin-5-yl)-4-ethoxyphenyl] sulfonyl}-cis-3, 5-dimethylpiperazine, a phosphodiesterase type V enzyme inhibitor (PDE5I), is under development for treatment of erectile dysfunction (ED). The purpose of this study was to elucidate metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes. Thirty-one phase I metabolites have been found by LTQ/Orbitrap hybrid mass spectrometry in rat urine, faeces, and bile after oral administration. Major biotransformation pathways of aildenafil included N-dealkylation of the piperazine ring, hydroxylation and dehydrogenation, aliphatic hydroxylation and loss of alkyl group of piperazine ring. Minor pathways involved hydroxylation on the phenyl ring, pyrazole N-demethylation, O-deethylation, loss of piperazine ring (cleavage of N-S bond) and dehydrogenation on the piperazine ring. Similar metabolic pathways of aildenafil were observed in the incubations of liver microsomes from mouse, rat, and dog as well as from human. The depletion rate of parent drug in mouse and rat liver microsomes was significantly different from that in human liver microsomes. The cytochrome P450 reaction phenotyping analysis was conducted using isozyme-specific inhibitors. The results indicated that CYP3A was the main isoenzyme involved in oxidative metabolism of aildenafil. Overall, these in vitro and in vivo findings should provide valuable information on possible metabolic behaviours of aildenafil in humans.

Strategies to Address Drug Interaction Potential for Antibody–Drug Conjugates in Clinical Development

Antibody-drug conjugates (ADCs) are a unique class of therapeutic proteins with both small and large molecular components. In vivo, ADCs are processed to multiple clinically relevant analytes, each with distinct PK properties. This increases the complexity for ADC drug interaction (DI) assessment. Furthermore, given the usually narrow therapeutic range for ADCs, a thorough risk assessment is essential to establish benefit/risk for patients. Therefore, an early understanding of the ADC catabolism and elimination pathways and cytochrome P450 reaction phenotyping, cytochrome P450 inhibition and induction potential, transporter interaction and inhibition potential for the cytotoxic drug catabolites assessed by in vitro and preclinical studies is essential. This information would be integrated with the clinical PK and PD properties of the ADC-related analytes for a theoretical risk assessment of ADC DI in combination therapy. ADC DI assessment in clinical studies will further support the theoretical risk assessment and the conclusions for the labeling statement.

In Vitro Metabolism of the Mycotoxin Enniatin B in Different Species and Cytochrome P450 Enzyme Phenotyping by Chemical Inhibitors

Enniatins are cyclic hexapeptidic mycotoxins produced by fungi growing on field grains, especially in wet climates. They show considerable resistance to food and feed processing technologies and might cause intoxication of humans and animals. Enniatins are also under exploration as anticancer drugs. The observed difference of in vitro and in vivo toxicities suggests low absorption or fast elimination of the enniatins after oral uptake. In the study presented here, in vitro metabolism studies of enniatin B were performed using rat, dog, and human liver microsomes under conditions of linear kinetics to estimate the respective elimination rates. Furthermore, cytochrome P450 reaction phenotyping with chemical inhibitors selective for human enzymes was carried out. Twelve metabolites were separated and characterized by multiple high-performance liquid chromatographic/mass spectrometric analyses as products of oxidation and demethylation reactions. Biotransformation rates and metabolite patterns varied considerably in the three species. The intrinsic clearances determined in assays with rat, dog, and human liver microsomes were 1.16, 8.23, and 1.13 l/(h · kg), respectively. The predicted enniatin B in vivo blood clearances were 1.57 l/(h · kg) in rats, 1.67 l/(h · kg) in dogs, and 0.63 l/(h · kg) in humans. CYP3A4 was important for enniatin B metabolism in human microsomes as shown by 80% inhibition and impaired metabolite formation in the presence of troleandomycin. CYP1A2 and CYP2C19 were additionally involved. Preliminary results showed that CYP3A and CYP1A might also be relevant in rats and dogs. The extensive hepatic metabolism could explain the reduced in vivo potential of enniatin B.

In Vitro Evaluation of Potential Drug-Drug Interactions with Ticagrelor: Cytochrome P450 Reaction Phenotyping, Inhibition, Induction, and Differential Kinetics

Ticagrelor is an orally administered, antiplatelet agent that inhibits the prothrombotic effects of ADP on the platelet by antagonizing the P2Y(12) receptor. Ticagrelor is a reversibly binding direct-acting P2Y(12) antagonist and does not require metabolic activation to achieve its antiplatelet effect. CYP3A4 and CYP3A5 appear to be the enzymes predominantly responsible for the formation of the ticagrelor active and inactive metabolites, AR-C124910XX and AR-C133913XX. The apparent K(m) values in human liver microsomes are 27.0 and 38.8 μM, with V(max) values of 730 and 417 pmol/min/mg for AR-C124910XX and AR-C133913XX, respectively. Ticagrelor moderately inhibited CYP2C9 activity in human liver microsomes with an IC(50) of 10.5 μM, while exhibiting little or no inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, and CYP2E1. In human liver microsomes, ticagrelor inhibited midazolam 4-hydroxylation with an IC(50) of 8.2 μM, while activating 1'-hydroxylation of midazolam. Studies with recombinant enzymes suggested that cytochrome b(5) and CYP3A4 interactions play a significant role in this differential kinetic behavior. Evaluated in fresh human hepatocytes at concentration up to 20 μM, ticagrelor was not an inducer of CYP1A2 or CYP3A4. Although ticagrelor exhibited a tendency for CYP2B6 and CYP2C9 induction, its potential to cause drug interactions via the induction of these enzymes is low when its exposure at a therapeutic dose is considered.

Metabolic Profiling and Cytochrome P450 Reaction Phenotyping of Medroxyprogesterone Acetate

Medroxyprogesterone acetate (MPA) is one of the most frequently prescribed progestins for conception, hormone replacement therapy, and adjuvant endocrine therapy. MPA has a low oral bioavailability because of extensive metabolism; however, its metabolism was poorly documented. This study was intended to profile the phase I metabolites of MPA and the cytochrome P450 (P450) isoforms involved. After MPA was incubated with human liver microsomes and the NADPH-generating system, five main metabolites (namely M-1, M-2, M-3, M-4, and M-5) were isolated by high-performance liquid chromatography. Three major metabolites (M-2, M-4, and M-3) were tentatively identified to be 6beta-, 2beta-, and 1beta-hydroxy MPA by liquid chromatography/mass spectrometry and (1)H nuclear magnetic resonance. By consecutive metabolism of purified M-2, M-3, and M-4, M-1 and M-5 were proposed to be 2beta-, 6beta-dihydroxy MPA, and 1,2-dehydro MPA, respectively. CYP3A4 was identified to be the isoform primarily involved in the formation of M-2, M-3, and M-4 in studies with specific P450 inhibitors, recombinant P450s, and correlation analysis. Rat and minipig liver microsomes were included evaluating species differences, and the results showed little difference among the species. In human liver microsomes, the K(m) values ranged from 10.0 to 11.2 muM, and the V(m) values ranged from 194 to 437 pmol/min/mg for M-2, M-3, and M-4. In conclusion, CYP3A4 was the major P450 isoform involved in MPA hydroxylation, with 6beta, 2beta, and 1beta being the possible hydroxylation sites. Minipig and rat could be the surrogate models for man in MPA pharmacokinetic studies.

In vitro Cytochrome P450 Reaction Phenotyping for Metabolism‐linked hemotoxicity of NPC1161B, a novel 8‐aminoquinoline antimalarial agent

8‐Aminoquinolines are important class of drugs, holding promise in many infectious diseases. Primaquine is a prototype of this class, while its 5‐dichlorophenoxy derivative, NPC1161B has been identified as a promising candidate with enantioselective toxicity and efficacy profiles. Use of 8‐AQs is restricted due to their hematological toxicities, for which biotransformation seems to be a prerequisite. Earlier metabolic studies have been hindered due to the unstable nature of hematotoxic intermediates. An in vitro metabolism‐linked hematoxicity assay has been employed to evaluate generation of methemoglobin and other hematotoxic responses by 8‐AQs. Direct incubation of the drug with microsomes or recombinant human CYP isoforms afforded in situ generation of toxic metabolites that mediate a robust hemoglobin oxidation. Three main approaches of reaction phenotyping i.e., correlation analysis, chemical inhibition and recombinant CYPs were employed to investigate hematoxicity of NPC1161B. CYP3A4 selectively metabolized NPC1161B leading to methemoglobin toxicity. It also correlated with CYP3A4 content of different batches of human liver microsomes. Inhibitors of CYP3A4 dramatically reduced methemoglobin formation in in vitro and in vivo studies. Data provide the basis for a mechanism based approach to abrogate hematotoxicity of NPC1161B, currently under evaluation for clinical advancement.

UDP-GLUCURONOSYLTRANSFERASE 2B7 IS THE MAJOR ENZYME RESPONSIBLE FOR GEMCABENE GLUCURONIDATION IN HUMAN LIVER MICROSOMES

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.