Cytochrome P450 reaction phenotyping: State of the art.

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.

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

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

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

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

To develop physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and drug-drug interactions (DDI) of pravastatin, using the in vitro transport parameters. In vitro hepatic sinusoidal active uptake, passive diffusion and canalicular efflux intrinsic clearance values were determined using sandwich-culture human hepatocytes (SCHH) model. PBPK modeling and simulations were implemented in Simcyp (Sheffield, UK). DDI with OATP1B1 inhibitors, cyclosporine, gemfibrozil and rifampin, was also simulated using inhibition constant (Ki) values. SCHH studies suggested active uptake, passive diffusion and efflux intrinsic clearance values of 1.9, 0.5 and 1.2μL/min/10(6)cells, respectively, for pravastatin. PBPK model developed, using transport kinetics and scaling factors, adequately described pravastatin oral plasma concentration-time profiles at different doses (within 20% error). Model based prediction of DDIs with gemfibrozil and rifampin was similar to that observed. However, pravastatin-cyclosporine DDI was underpredicted (AUC ratio 4.4 Vs ~10). Static (R-value) model predicted higher magnitude of DDI compared to the AUC ratio predicted by the PBPK modeling. PBPK model of pravastatin, based on in vitro transport parameters and scaling factors, was developed. The approach described can be used to predict the pharmacokinetics and DDIs associated with hepatic uptake transporters.

Solid transplant recipients are at increased risk for invasive aspergillosis. Tacrolimus and Voriconazole is one of the most frequently utilized treatments for those recipients with invasive fungal infections. However, it is difficult to use them properly due to the interaction between them. This study aimed to investigate the potential drug-drug interaction between Tacrolimus and Voriconazole by multiple methods, including in vitro liver microsome method and the PBPK(Physiologically Based Pharmacokinetic) model. Midazolam and testosterone were used as probe substrates to evaluate individual differences in CYP3A4/5 metabolic activity. A comprehensive interaction analysis was also conducted based on the STITCH database and the DD-Inter system. Furthermore, a PBPK model was constructed by the data from the literature to simulate the real metabolic process in vivo. The research employed multiple methodologies to demonstrate that the co-administration of Voriconazole significantly enhances Tacrolimus concentrations, considering genotypes and the activity of CYP3A4/5 genotypes. The findings indicated a decrease in the relative percentages of midazolam and testosterone metabolites with increasing Voriconazole concentration. Moreover, the results for residual Tacrolimus in the 30-minute incubation group revealed that Voriconazole exerts a mild inhibitory effect on the in vitro metabolism of Tacrolimus. The STITCH database and DD-Inter system analysis also suggested that Tacrolimus and Voriconazole share a strong association in liver metabolism, most likely interacting with CYP3A4/5 and CYP2C19. Furthermore, the result of PBPK analysis indicated that Tacrolimus AUC increases with Voriconazole co-therapy. Moreover, the AUC of Tacrolimus in intermediate CYP2C19 metabolizers (IM) was the highest at 10.1 µmol·min/L, followed by poor metabolizers (PM) at 8.13 µmol·min/L, and extensive metabolizers (EM) at 2.18 µmol·min/L. And the genotype of CYP3A5 poor metabolizer (PM) had AUC of Tacrolimus at 3.13µmol·min/L and extensive metabolizer (EM) at 2.18µmol·min/L. Microsomal studies, PBPK models, and multiple other analyses have comprehensively elucidated the impact of Voriconazole on Tacrolimus concentrations. These findings can serve as a valuable point of reference for concurrently administering these two medications. These findings also indicate that the genotypes of CYP2C19 play an important role in the development of DDI during concurrent Tacrolimus and Voriconazole treatment, which may have some guidance for clinical medication.

Identifying the CYP450 enzymes underlying metabolism of investigational drugs (i.e., P450 reaction phenotyping; PRP) is critical to the development of effective therapeutics. Information gained from such studies can: a) provide insight into structural modifications of test drugs in order to improve metabolic stability and; b) specify an estimate of drug‐drug interaction potential as well as that of interindividual and species differences in oxidative metabolism. CYP450 chemical inhibitors (e.g., a‐naphthoflavone and sulfaphenazole) are often used in reaction phenotyping studies but the outcome is sometimes equivocal due to the non‐specific effects of these chemical agents plus their capacity to undergo P450‐dependent catabolism. In contrast, inhibitory P450 antibodies offer high specificity and irreversibility, and may thus represent a superior approach for characterizing individual P450 enzyme involvement in drug metabolism. We recently developed a compilation of highly‐specific polyclonal and monoclonal antibodies to the CYP450s comprising the major oxidative drug‐metabolizing enzymes in human liver, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (aka CYP ImmunoInhibit antibodies). As shown here, each of these antibodies proved capable of eliciting > 80% inhibition of probe drug metabolism by the cognate P450 antigens in human liver microsomes (HLMx), while giving insignificant effects on different CYP450s (Figure 1). The inhibition observed with each of the six CYP450 antibodies was not only highly‐specific but was also dose‐dependent, with maximal inhibition (80–90%) achieved at IgG:microsomal protein ratios ranging from 0.1 mg/mg to 3 mg/mg (Figure 2). Control (pre‐immune) antibodies had no effect on substrate metabolism. Such results show that inhibitory antibodies coupled with HLMx represent an alternative and powerful approach to chemical inhibitors for evaluating involvement of specific P450 enzymes in the hepatic metabolism of investigational drugs. Interestingly, studies ongoing with CYP ImmunoInhibit antibodies and MetMax™ cryopreserved human hepatocytes, which are liver cells that have been permeabilized and cryopreserved, have given analogous results to those achieved with HLMx. The utilization of inhibitory antibodies with HLMx represents an accurate model for PRP of new therapeutics due to the specificity of these immunoreagents, and the irreversible nature of the inhibition achieved, an important benefit when working with metabolically‐stable test agents.Specific Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 1Dose‐Response Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 2

Physiologically basedpharmacokinetic (PBPK) models are increasinglyused in drug development to simulate changes in both systemic andtissue exposures that arise as a result of changes in enzyme and/ortransporter activity. Verification of these model-based simulationsof tissue exposure is challenging in the case of transporter-mediateddrug–drug interactions (tDDI), in particular as these may leadto differential effects on substrate exposure in plasma and tissues/organsof interest. Gadoxetate, a promising magnetic resonance imaging (MRI)contrast agent, is a substrate of organic-anion-transporting polypeptide1B1 (OATP1B1) and multidrug resistance-associated protein 2 (MRP2).In this study, we developed a gadoxetate PBPK model and explored theuse of liver-imaging data to achieve and refine in vitro–invivo extrapolation (IVIVE) of gadoxetate hepatic transporter kineticdata. In addition, PBPK modeling was used to investigate gadoxetatehepatic tDDI with rifampicin i.v. 10 mg/kg. In vivo dynamic contrast-enhanced(DCE) MRI data of gadoxetate in rat blood, spleen, and liver wereused in this analysis. Gadoxetate in vitro uptake kinetic data weregenerated in plated rat hepatocytes. Mean (%CV) in vitro hepatocyteuptake unbound Michaelis–Menten constant (Km,u) of gadoxetate was 106 μM (17%) (n = 4 rats), and active saturable uptake accounted for 94% of totaluptake into hepatocytes. PBPK–IVIVE of these data (bottom-upapproach) captured reasonably systemic exposure, but underestimatedthe in vivo gadoxetate DCE–MRI profiles and elimination fromthe liver. Therefore, in vivo rat DCE–MRI liver data were subsequentlyused to refine gadoxetate transporter kinetic parameters in the PBPKmodel (top-down approach). Active uptake into the hepatocytes refinedby the liver-imaging data was one order of magnitude higher than theone predicted by the IVIVE approach. Finally, the PBPK model was fittedto the gadoxetate DCE–MRI data (blood, spleen, and liver) obtainedwith and without coadministered rifampicin. Rifampicin was estimatedto inhibit active uptake transport of gadoxetate into the liver by96%. The current analysis highlighted the importance of gadoxetateliver data for PBPK model refinement, which was not feasible whenusing the blood data alone, as is common in PBPK modeling applications.The results of our study demonstrate the utility of organ-imagingdata in evaluating and refining PBPK transporter IVIVE to supportthe subsequent model use for quantitative evaluation of hepatic tDDI.

Cytochrome P450 enzymes in the pediatric population: Connecting knowledge on P450 expression with pediatric pharmacokinetics.

A link between histone deacetylases (HDACs) and intestinal inflammation has been established. HDAC inhibitors that target gut-selective inflammatory pathways represent a potential new therapeutic strategy in patients with refractory inflammatory bowel diseases (IBD). To review the use of selective HDAC inhibitors to treat gut inflammation and to highlight potential improvements in selectivity/sensitivity by additional targeting of HDAC-regulating microRNAs (miRNAs). Original articles and reviews have been identified using PubMed search terms: 'histone deacetylase', 'HDAC inhibitor', 'inflammatory bowel disease', 'gut inflammation,' and 'microRNA and HDAC'. The use of butyrate in distal colitis provided the first evidence that inhibition of HDACs decreases intestinal inflammation in IBD. HDAC inhibitors, such as valproic acid, vorinostat and givinostat, reduce inflammation and tissue damage in experimental murine colitis. Potential mechanisms of action for HDAC inhibitors include increased apoptosis, reduction of pro-inflammatory cytokine release, regulation of transcription factors and modulation of HDAC-regulatory miRNAs. HDAC2, HDAC3, HDAC6, HDAC9 and HDAC10 isoforms seem to be specifically involved in chronic intestinal inflammation, justifying the use of selective inhibitors as new therapeutic strategies in IBD. Controlling miRNAs for these isoforms can be identified. The pro-inflammatory influence of HDACs in the gut has been confirmed, but mostly in murine studies. Considerably more human data are required to permit development of selective HDAC inhibitors for IBD treatment. Inhibition of key HDAC isoforms in combination with modulation of HDAC-regulatory miRNAs has potential as a novel therapeutic approach.

e13019 Background: MPC-3100 is an oral synthetic inhibitor of HSP90, in a phase I safety study in cancer patients. Major cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) isozymes were identified to define the inclusion and exclusion criteria necessary to ensure patient safety. Methods: CYP450 isozymes responsible for the metabolism of MPC-3100 were assessed by two methods. The first entailed the use of recombinant human Supersomes (1A2, 2C8, 2C9, 2C19, 2D6 and 3A4) suspended in phosphate buffer (pH 7.4) with 40 pmoles of CYP450. An NADPH regenerating system was used with a 5 minute pre-incubation prior to adding 10 µM MPC-3100. The second used human donor liver microsomes suspended in phosphate buffer (pH 7.4) with 0.5 mg/mL of protein. The microsomes were pre-incubated with CYP450 isozyme selective chemical inhibitors prior to addition of 10 µM MPC-3100 and NADPH. UGT isozymes responsible for glucuronidation of MPC-3100 and a major oxidative metabolite were identified using recombinant UGT Supersomes suspended in Tris-HCl buffer (pH 7.5) at a final protein concentration of 1 mg/mL. The UGT Supersomes were pre-incubated with alamethicin and the reaction was initiated with UDPGA. The disappearance of MPC-3100 and formation of metabolites was monitored by HPLC-ESI-MS/MS. Results: MPC-3100 was metabolized by CYP450 1A2, 2C19, 2D6 and 3A4, but not by 2C8 or 2C9. With the use of selective chemical inhibitors in pooled human liver microsomes, the percent of MPC-3100 metabolized at 40 minutes was inhibited greatest by azamulin (31.4%), ketoconazole (20.9%) and quinidine (5.5%). MPC-3100 was glucuronidated by UGT 1A3, 1A8 and 1A4, but not by 1A1, 1A6, 1A7, 1A9, 1A10, 2B4, 2B5, 2B15, and 2B17. The major oxidative metabolite of MPC-3100 was a substrate for UGT 1A3, 1A7, 1A9, 1A10, 2B4, 2B17 with 1A1, 1A8, and 2B7 predominating. Conclusions: CYP450 3A4 is the primary isozyme involved in the metabolism of MPC-3100. UGT 1A4 is the major isozyme responsible for direct glucuronidation of MPC-3100; whereas multiple UGTs (1A1, 1A8, and 2B7) are capable of glucuronidating the major oxidative metabolite. Therefore current clinical studies preclude co-administration of potent CYP3A4 inhibitors with MPC-3100.

Metapristone is the primary metabolite of the abortifacient mifepristone (RU486), and is being developed as a safe and effective cancer metastatic chemopreventive agent for both sexes. Here, we systematically investigated the sex-related pharmacokinetics of metapristone in both rats and dogs, and explored the related mechanisms of actions. Administration of metapristone to rats and dogs showed that plasma concentrations of metapristone (AUC, Cmax) were significantly higher in female dogs and rats than in males. The sex-related differences in pharmacokinetics become more significant after ten consecutive days of oral administration. Female liver microsomes metabolized metapristone significantly slower than the male ones. The results from P450 reaction phenotyping using recombinant cDNA-expressed human CYPs in conjunction with specific CYP inhibitors suggested that CYP1A2 and CYP3A4 are the predominant CYPs involved in the metapristone metabolism, which were further confirmed by the enhanced protein levels of CYP1A2 and CYP3A4 induced by 1-week oral administration of metapristone to rats. The highest tissue concentration of metapristone was found in the liver. The study demonstrates, for the first time, the sex-related pharmacokinetics of metapristone, and reveals that activities of liver microsomal CYP1A2 and CYP3A4 as well as the renal clearance are primarily responsible for the sex-related pharmacokinetics.

Piperaquine (PQ) and its pharmacologically active metabolite PQ N-oxide (PM1) can be metabolically interconverted via hepatic cytochrome P450 and FMO enzymes. The reductive metabolism of PM1 and its further N-oxidation metabolite (PM2) by intestinal microflora was evaluated, and its role in PQ elimination was also investigated. The hepatic and microbial reduction metabolism of PM1 and PM2 was studied in vitro. The reaction phenotyping experiments were performed using correlation analysis, selective chemical inhibition, and human recombinant CYP/FMO enzymes. The role of microbial reduction metabolism in PQ elimination was evaluated in mice pretreated with antibiotics. The effect of the reduction of metabolism on PQ exposures in humans was predicted using a physiologically-based pharmacokinetic (PBPK) model. Both hepatic P450/FMOs enzymes and microbial nitroreductases (NTRs) contributed to the reduction metabolism of two PQ N-oxide metabolites. In vitro physiologic and enzyme kinetic studies of both N-oxides showed a comparable intrinsic clearance by the liver and intestinal microflora. Pretreatment with antibiotics did not lead to a significant (P > 0.05) change in PQ pharmacokinetics in mice after an oral dose. The predicted pharmacokinetic profiles of PQ in humans did not show an effect of metabolic recycling. Microbial NTRs and hepatic P450/FMO enzymes contributed to the reduction metabolism of PQ N-oxide metabolites. The reduction in metabolism by intestinal microflora did not affect PQ clearance, and the medical warning in patients with NTRs-related disease (e.g., hyperlipidemia) will not be clinically meaningful.

Transporter‐mediated renal secretion plays a critical role in the systemic clearance of ~30% of the Food and Drug Administration (FDA)‐approved drugs and is attributed to clinically significant drug‐drug interactions (DDIs). For example, organic anion transporters (OATs) are inhibited by perpetrator drugs (e.g., probenecid), leading to higher circulating levels of OAT substrates (e.g., furosemide).1 Recently, OAT inhibition has been shown to be associated with increased plasma levels of endogenous substrates, e.g., pyridoxic acid (PDA) and homovanillic acid, which can serve as potential OAT biomarkers.2 The objective of our study was to develop and validate a physiologically‐based pharmacokinetic (PBPK) model of PDA to allow prospective evaluation of the magnitude of potential DDI of an OAT inhibitor‐substrate pair. Probenecid and furosemide were used as selective inhibitor and substrate of OATs, respectively. Physicochemical properties and in vitro data pertaining to the pharmacokinetics of probenecid and PDA were collated from literature. A PBPK model was first constructed to predict the plasma concentration‐time profiles of probenecid after oral administration using Simcyp (v20). Then, utilizing the fraction secreted versus filtered as well as the unbound fraction (fu = 0.092) data of PDA,2 the total elimination and synthesis rates of PDA at steady‐state were used to develop a baseline PBPK model. The effect of OAT inhibition by probenecid on the PDA levels was estimated and the predicted area under the plasma concentration‐time profile (AUC), change in clearance, and the highest plasma concentration (Cmax) values were compared with the observed data.2 The model was able to capture the changes within 2‐fold of the clinical data. Using the predicted AUC profile of PDA and assuming 100% inhibition at Cmax, the magnitude of inhibition was estimated across different time points. The biomarker‐informed magnitude of inhibition showed significant correlation (r2 = 0.9336) with the observed change in furosemide concentrations in the presence of probenecid (Fig. 1). PDA‐informed PBPK model developed here can be used to prospectively predict the magnitude of OAT inhibition, which is particularly applicable to assessing DDI potential of investigational drugs or prescribed drugs in special populations with sparse samples, without the requirement of an exogenous probe substrate.