Drug Metabolism and Disposition最新文献

The influence of transporters on the pharmacokinetics of drugs is being increasingly recognized, and drug-drug interactions (DDIs) via modulation of transporters could lead to clinical adverse events. Organic anion-transporting polypeptide 1B (OATP1B) is a liver-specific uptake transporter in humans that can transport a broad range of substrates, including statins. It is a challenge to predict OATP1B-mediated DDIs using preclinical animal models because of species differences in substrate specificity and abundance levels of transporters. PXB-mice are chimeric mice with humanized livers that are highly repopulated with human hepatocytes and have been widely used for drug metabolism and pharmacokinetics studies in drug discovery. In the present study, we measured the exposure increases [blood AUC (area under the blood/plasma concentration-time curve) and C_max] of 10 OATP1B substrates in PXB-mice upon coadministration with rifampin, a potent OATP1B specific inhibitor. These data in PXB-mice were then compared with the observed DDIs between OATP1B substrates and single-dose rifampin in humans. Our findings suggest that the DDIs between OATP1B substrates and rifampin in PXB-mouse are comparable with the observed DDIs in the clinic. Since most OATP1B substrates are metabolized by cytochromes P450 (CYPs) and/or are substrates of P-glycoprotein (P-gp), we further validated the utility of PXB-mice to predict complex DDIs involving inhibition of OATP1B, CYPs, and P-gp using cyclosporin A (CsA) and gemfibrozil as perpetrators. Overall, the data support that the chimeric mice with humanized livers could be a useful tool for the prediction of hepatic OATP1B-mediated DDIs in humans. SIGNIFICANCE STATEMENT: The ability of PXB-mouse with humanized liver to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs) in humans was evaluated. The blood exposure increases of 10 OATP1B substrates with rifampin, an OATP1B inhibitor, in PXB-mice have a good correlation with those observed in humans. More importantly, PXB-mice can predict complex DDIs, including inhibition of OATP1B, cytochromes P450 (CYPs), and P-glycoprotein (P-gp) in humans. PXB-mice are a promising useful tool to assess OATP1B-mediated clinical DDIs.

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [³H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [³H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [³H]2-chloroadenosine transport activity (V_max values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/μl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (K_i) for ENT2 (2.6 μM), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 μM). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (K_i) ranging from 8.6 μM for ticagrelor to 2,300 μM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Mobocertinib (formerly known as TAK-788) is a targeted covalent tyrosine kinase inhibitor of epidermal growth factor receptor with exon 20 insertion mutations. This article describes the metabolism and excretion of mobocertinib in healthy male subjects after a single oral administration of [¹⁴C]mobocertinib. Mobocertinib-related materials were highly covalently bound to plasma proteins such as human serum albumin. The mean extraction recovery of total radioactivity was only 3.9% for six individual Hamilton pooled plasma samples. After extraction, mobocertinib was the most abundant component accounting for 7.7% of total extracted circulating radioactivity (TECRA) in the supernatant. Each of identified metabolites accounted for <10% of TECRA. Mobocertinib underwent extensive first-pass metabolism with the fraction of the dose absorbed estimated to be approximately 91.7%. Fecal excretion of mobocertinib metabolites was the major elimination route. Mobocertinib was mainly eliminated via oxidative metabolism with a fraction of approximately 88% metabolized by CYP3A4/5. The other minor elimination pathways included cysteine conjugation, metabolism by other cytochrome P450s, and renal excretion of unchanged mobocertinib. SIGNIFICANCE STATEMENT: This article describes the metabolism and excretion of a targeted covalent inhibitor mobocertinib in humans after a single oral administration of [¹⁴C]mobocertinib. Mobocertinib was highly covalently bound to human plasma proteins. No metabolite accounted for >10% of total extracted circulating radioactivity in human plasma. Mobocertinib was mainly eliminated via CYP3A4/5 mediated oxidative metabolism followed by fecal excretion after approximately 91.7% of the dose was absorbed.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

UGT2B4 is a highly expressed drug-metabolizing enzyme in the liver contributing to the glucuronidation of several drugs. To enable quantitatively assessing UGT2B4 contribution toward metabolic clearance, a potent and selective UGT2B4 inhibitor that can be used for reaction phenotyping was sought. Initially, a canagliflozin-2'-O-glucuronyl transferase activity assay was developed in recombinant UGT2B4 and human liver microsomes (HLM) [±2% bovine serum albumin (BSA)]. Canagliflozin-2'-O-glucuronidation (C2OG) substrate concentration at half-maximal velocity value in recombinant UGT2B4 and HLM were similar. C2OG formation intrinsic clearance was five- to seven-fold higher in incubations containing 2% BSA, suggesting UGT2B4 susceptibility to the inhibitory unsaturated long-chain fatty acids released during the incubation. Monitoring for C2OG formation, 180 compounds were evaluated for UGT2B4 inhibition potency in the presence and absence of 2% BSA. Compounds that exhibited an apparent UGT2B4 IC₅₀ of < 1 μM in HLM with 2% BSA were evaluated for inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 catalytic activities to establish selectivity suitable for supporting UGT reaction phenotyping. In this study, clotrimazole was identified as a potent UGT2B4 inhibitor (HLM apparent IC₅₀ of 11 to 35 nM ± 2% BSA). Moreover, clotrimazole exhibited selectivity for UGT2B4 inhibition (>24-fold) over the other UGT enzymes evaluated. Additionally, during this study it was discovered that the previously described UGT2B7 inhibitors 16α- and 16β-phenyllongifolol also inhibit UGT2B4. Clotrimazole, a potent and selective UGT2B4 inhibitor, will prove essential during UGT reaction phenotyping. SIGNIFICANCE STATEMENT: To mechanistically evaluate drug interactions, it is essential to understand the contribution of individual enzymes to the metabolic clearance of a drug. The present study describes the development of a UGT2B4 activity assay that enabled the discovery of the highly selective and potent UGT2B4 inhibitor clotrimazole. Clotrimazole can be used in UGT reaction phenotyping studies to estimate fractional contribution of UGT2B4.

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long noncoding RNAs (lncRNAs) hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the downregulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the downregulation of some CYPs proteins, and upregulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. SIGNIFICANCE STATEMENT: Although the impact of long noncoding RNAs (lncRNAs) including hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and hepatocyte nuclear factor 4a antisense RNA 1 (HNF4A-AS1) on the downstream transcription factor (TF) and cytochrome P450 enzyme (CYP) expression is well studied, the upstream regulation of these two lncRNAs by methyltransferase-like 3 (METTL3) remains unexplored. This study reveals that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1 in HepG2 and Huh7 cells.

One-compartment (1C) and permeability-limited models were used to evaluate the ability of microsomal and hepatocyte intrinsic clearances to predict hepatic clearance. Well-stirred (WSM), parallel-tube (PTM), and dispersion (DM) models were evaluated within the liver as well as within whole-body physiologically based pharmacokinetic frameworks. It was shown that a linear combination of well-stirred and parallel-tube average liver blood concentrations accurately approximates dispersion model blood concentrations. Using a flow/permeability-limited model, a large systematic error was observed for acids and no systematic error for bases. A scaling factor that reduced interstitial fluid (ISF) plasma protein binding could greatly decrease the absolute average fold error (AAFE) for acids. Using a 1C model, a scalar to reduce plasma protein binding decreased the microsomal clearance AAFE for both acids and bases. With a permeability-limited model, only acids required this scalar. The mechanism of the apparent increased cytosolic concentrations for acids remains unknown. We also show that for hepatocyte intrinsic clearance in vitro-in vivo correlations (IVIVCs), a 1C model is mechanistically appropriate since hepatocyte clearance should represent the net clearance from ISF to elimination. A relationship was derived that uses microsomal and hepatocyte intrinsic clearance to solve for an active hepatic uptake clearance, but the results were inconclusive. Finally, the PTM model generally performed better than the WSM or DM models, with no clear advantage between microsomes and hepatocytes. SIGNIFICANCE STATEMENT: Prediction of drug clearance from microsomes or hepatocytes remains challenging. Various liver models (e.g., well-stirred, parallel-tube, and dispersion) have been mathematically incorporated into liver as well as whole-body physiologically based pharmacokinetic frameworks. Although the resulting models allow incorporation of pH partitioning, permeability, and active uptake for prediction of drug clearance, including these processes did not improve clearance predictions for both microsomes and hepatocytes.

Hepatic clearance (CL_H ) prediction is a critical parameter to estimate human dose. However, CL_H underpredictions are common, especially for slowly metabolized drugs, and may be attributable to drug properties that pose challenges for conventional in vitro absorption, distribution, metabolism, and elimination (ADME) assays, resulting in nonvalid data, which prevents in vitro to in vivo extrapolation and CL_H predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in CL_H Recent advances in understanding the interplay of metabolism and drug transport for clearance processes have aided in developing the extended clearance model. In this study, we demonstrate proof of concept of a novel two-step assay enabling the measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and cytochrome P450 inhibitors-the hepatocyte uptake and loss assay (HUpLA). HUpLA accurately predicted the CL_H of eight of the nine drugs (within twofold of the observed CL_H ). Distribution clearances were within threefold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the CL_H The CL_H of only two drugs was predicted within twofold of the observed CL_H Therefore, HUpLA is advantageous by enabling the measurement of enzymatic and transport processes concurrently within the same system, alleviating the need for applying scaling factors independently. The use of primary human hepatocytes enables physiologically relevant exploration of transporter-enzyme interplay. Most importantly, HUpLA shows promise as a sensitive measure for low-turnover drugs. Further evaluation across different drug characteristics is needed to demonstrate method robustness. SIGNIFICANCE STATEMENT: The hepatocyte uptake and loss assay involves measuring four commonly derived in vitro hepatic clearance endpoints. Since endpoints are generated within a single test system, it blunts experimental error originating from assays otherwise conducted independently. A key advantage is the concept of removing drug-containing media following intracellular drug loading, enabling the measurement of drug reappearance rate in media as well as the measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.

Since his graduate studies on alcohol induction of a novel cytochrome P450 (P450) enzyme, through his postdoctoral work on hormonal regulation of sexually differentiated cytochrome P450s (P450s), the author has maintained an interest in the regulation of drug metabolizing enzymes. This article is a recounting of his scientific career and focuses on his laboratory's work on inflammatory regulation of P450 enzymes that formed the basis for the Bernard B. Brodie Award. Key findings and publications are identified and discussed that contributed to the elucidation of some important principles: 1) inflammatory stimuli generally downregulate P450 enzymes, resulting in reduced metabolism of substrate drugs; 2) the main mechanism for this downregulation is transcriptional and involves both the activation of negatively acting transcription factors and the suppression of positive transcription factors; 3) inflammatory cytokines such as interleukin 1, interleukin 6, and tumor necrosis factor α act on hepatocytes to mediate this regulation; 4) these cytokines selectively regulate different P450 enzymes, and therefore different P450s are downregulated in different inflammatory diseases or disease models; 5) nitric oxide formed by inducible nitric oxide synthase 2 reacts with P450s in an enzyme-specific manner to stimulate their proteolytic degradation; and 6) both tyrosine nitration and heme nitrosylation are likely required for this NO-stimulated degradation. Finally, findings from clinical studies are discussed that shine a light on the importance of P450 regulation by inflammation for drug development, clinical practice, and personalized medicine. SIGNIFICANCE STATEMENT: This article discusses the key publications and findings in the author's laboratory that helped to identify inflammation as an important factor contributing to interindividual variation in drug metabolism.