Drug Metabolism and Disposition最新文献

The pharmacokinetic characterization of drug candidates is an essential step in drug development. To date, primary suspension hepatocytes have been widely used for this purpose; however, their poor stability has limited the application of in vitro systems for compounds with low metabolic turnover rates. Highly functional HepaSH cells, prepared from chimeric mice with humanized livers, maintain a cobblestone-like morphology and cytochrome P450-dependent drug-metabolizing activity for up to 168 hours in monolayer culture without medium change using a commercially available long-term hepatocyte culture medium. In this study, we attempted to investigate the utility of long-term culture systems and predict the hepatic clearance of 12 drugs with 9 low and 3 moderate-to-high CL_int in humans using multiple HepaSH monolayers. This culture system successfully monitored the depletion of low (such as diazepam and quinidine) and moderate-to-high CL_int drugs (midazolam). Two low-clearance drugs, disopyramide and warfarin, showed no depletion over 168 hours, indicating limitations in the application of this method for clearance evaluation. Hepatic clearance values obtained from incubation with HepaSH monolayers were predicted for 6-8 of 12 compounds tested with deviations within 3-fold, with an average fold error of 1.14- to 1.19-fold and an absolute average fold error of 1.52- to 1.97-fold, roughly correlating with the clinical reference data. In conclusion, a functionally stable culture method for HepaSH monolayers is highly effective for evaluating low-clearance compounds by greatly extending the metabolic reaction time and will be a valuable tool for determining the pharmacokinetic properties of new drug candidates. SIGNIFICANCE STATEMENT: This study demonstrated that combining highly functional HepaSH monolayers with extended drug incubation enables accurate monitoring of low-turnover compound clearance, an outcome that has been difficult to achieve with traditional assays.

The drug discovery and development process faces significant challenges, including high attrition rates and substantial financial investment, in part due to the limitations of traditional 2-dimensional (2D) cell culture systems and animal models to predict human drug metabolism, efficacy, and toxicity. This review highlights the emergence of novel in vitro human cell culture and organoid systems, such as 3-dimensional (3D) cultures, self-assembling organoids, induced pluripotent stem cell-derived models, and microphysiological system or organ-on-a-chip systems, as transformative solutions to the issues raised when extrapolating from 2D cell culture. These advanced platforms offer enhanced physiological relevance by better recapitulating complex in vivo microenvironments, thus improving the predictability and accuracy of preclinical drug assessment. In this study, we systematically cover the utility of these advanced systems in studying drug metabolism and toxicology across key organs like the liver, intestine, and kidney, emphasizing their advantages over conventional models in terms of cellular diversity, architectural complexity, and long-term functional maintenance. We also discuss the potential of integrating these novel systems into the drug development pipeline, particularly their compatibility with high-throughput screening and their alignment with the 3Rs principle (replacement, reduction, and refinement) for ethical research. Despite their immense promise, challenges remain; including the lack of standardized protocols, the complexity of data analysis, and the need for further advancements in vascularization, innervation, and immune component integration. We conclude by exploring future directions, including the crucial role of artificial intelligence and machine learning in analyzing complex datasets and the potential for personalized medicine through patient-derived organoids. Overcoming these challenges will be vital for these innovative platforms to revolutionize pharmaceutical development, leading to safer, more effective, and more efficiently produced pharmaceuticals. SIGNIFICANCE STATEMENT: This article reviews the design, construction, and implementation of novel in vitro cell culture and organoid systems for preclinical drug metabolism and pharmacokinetics and toxicology studies. As such, it serves as a resource for interested parties who would like to learn about, and implement, these cutting-edge technologies into their drug discovery and development workflow.

Cytochrome P450 (P450) enzymes in the small intestine play a critical role in determining the systemic availability of orally administered drugs. In dogs, the major intestinal drug-metabolizing P450 enzymes are CYP3A98, an intestine-specific isoform, and CYP2B11, which are expressed in both the liver and intestines. This study aimed to establish differentiated canine duodenal organoids and evaluate the expression, inducibility, and enzymatic activity of these key intestinal P450 enzymes. Duodenal organoids were generated from healthy canine intestinal biopsies and cultured under expansion and differentiation conditions. CYP3A98 and CYP2B11 gene expression was assessed by quantitative reverse transcription polymerase chain reaction, while enzyme function was evaluated using midazolam (CYP3A98) and bupropion (CYP2B11) hydroxylation assays. To assess P450 induction, organoids were treated with rifampicin (a pregnane X receptor [PXR] selective inducer) and phenobarbital (a constitutive androstane receptor [CAR] inducer). Organoid differentiation significantly upregulated CYP3A98 and CYP2B11 mRNA expression and enzyme activity. Rifampicin (50 μM) strongly induced CYP3A98 gene expression (7.1-fold) and enzyme activity (2.5-fold) without affecting CYP2B11 expression. CYP3A98 and CYP2B11 expression were unaffected by phenobarbital treatment at a CAR-selective concentration (250 μM). However, treatment with phenobarbital at a high concentration (2 mM), known to directly bind and activate PXR, resulted in a significant increase in CYP3A98 expression (3.6-fold) and activity (1.4-fold) without substantially affecting CYP2B11 expression. Differentiated canine duodenal organoids expressed functional CYP3A98 and CYP2B11. CYP3A98 was inducible through PXR, while CYP2B11 was not regulated by CAR or PXR. This platform may provide a valuable tool for evaluating drug absorption, metabolism, and drug-drug interactions in veterinary drug development. SIGNIFICANCE STATEMENT: A physiologic canine intestinal in vitro model for drug development is lacking in veterinary medicine. The canine differentiated duodenal organoids used in this study expressed CYP3A98 and CYP2B11 enzymes and may provide a physiological platform for studying drug metabolism and drug-drug interactions during the development of veterinary pharmaceuticals.

The significance of the gut microbiome on drug metabolism has been demonstrated, yet much less is known about the pathobiome's potential impact on systemic drug metabolism outside of the β-lactam antibiotics, especially for bacterial species prone to multidrug resistance, which often leads to acute or chronic infections. CYP107S1, a cytochrome P450 (P450) from the opportunistic pathogen Pseudomonas aeruginosa, which exhibits substrate promiscuity and allosteric features, was able to tightly bind (K_{d, app} of 0.755 μM) and rapidly metabolize with high affinity (K_m of 1.63 μM) the nonsteroidal anti-inflammatory drug celecoxib. It formed the same hydroxy metabolite as human CYP2C9, the primary enzyme responsible for the metabolism of this selective cyclooxygenase-2 inhibitor. In liquid cultures of the P. aeruginosa PAO1 strain expressing a relatively high level CYP107S1 during the initial bacterial growth phase, dosing of celecoxib resulted in an increase in the hydroxyl product formation over time, attesting to translation from the P450 in vitro recombinant drug-metabolizing activity to live bacterial cultures. Furthermore, the celecoxib metabolite formation by the CYP107S1 recombinant enzyme or in PAO1 culture was partially inhibited by the pan-CYP inhibitor 1-aminobenzotriazole and exhibited preincubation time-dependency characteristics. Thus, P. aeruginosa CYP107S1 capability to metabolize drugs continues to expand, driving new knowledge and potential for new useful substrate probes to study P450 function and regulation in P. aeruginosa. SIGNIFICANCE STATEMENT: This study provides further insights into the metabolic ability of CYP107S1, a cytochrome P450 enzyme belonging to the azetidine biosynthetic gene cluster of Pseudomonas aeruginosa, which is capable of metabolizing the nonsteroidal anti-inflammatory drug celecoxib, further widening the promiscuity feature of the enzyme and offering a novel probe to study its regulation in the PAO1 strain of P. aeruginosa.

Esophageal squamous cell carcinoma (ESCC) is a major global health threat characterized by high incidence and mortality rates. The aberrant suppression of CYP3A5 is frequently observed in ESCC. However, its precise function and the epigenetic mechanism mediating its transcriptional repression remain poorly elucidated. Herein, we found that CYP3A5 expression is significantly reduced in ESCC tumor tissues compared to normal tissues. Crucially, high CYP3A5 expression was associated with a favorable prognosis and reduced tumor metastasis in ESCC. Intriguingly, administration of the histone deacetylase inhibitor trichostatin A resulted in the upregulation of CYP3A5 expression. Further mechanistic experiments revealed that histone deacetylase 4 is the key deacetylase responsible for reducing H3K18/K27 acetylation levels at the CYP3A5 promoter, mediated by P300/CREB binding protein. Functionally, CYP3A5 overexpression effectively inhibited ESCC cell migration and invasion both in vitro and in vivo. In conclusion, CYP3A5 was crucial in ESCC and may serve as a promising therapeutic target for the prevention of tumor metastasis in ESCC. SIGNIFICANCE STATEMENT: CYP3A5 expression was downregulated in esophageal squamous cell carcinoma (ESCC) due to histone hypoacetylation at CYP3A5 promoter region. Because ESCC develops, CYP3A5 suppression promotes tumor metastasis and invasion. CYP3A5 is a potential biomarker and therapeutic target for ESCC.

Sulfamethoxazole (SMX) is associated with idiosyncratic drug-induced liver injury, which remains difficult to predict. SMX is metabolized by N-acetyltransferases (NAT1/NAT2) to form N₄-acetyl sulfamethoxazole (NA-SMX), and by cytochrome P450-mediated oxidation to form SMX-hydroxylamine. This study aimed to characterize SMX metabolism in vitro and investigate how NAT1 and NAT2 variation influences NA-SMX formation, including the relationship between NAT2 protein levels and metabolite formation. Human liver microsomes, S9 fractions, and primary human hepatocytes were used to generate SMX metabolites. NA-SMX was the most abundant metabolite in primary human hepatocytes, showing 4.2-fold variability across n = 26 donors. Interestingly, NAT2 genotype-inferred acetylator phenotype did not reliably predict NA-SMX formation in 6 of 9 slow acetylators, whose formation exceeded the mean of intermediate acetylators. However, N-acetyl sulfamethazine (NA-SMZ) formation was accurately predicted using the NAT2 probe substrate, SMZ, revealing significant differences between NAT2 phenotype groups (P < .05). Activities of NAT1 and NAT2, as measured by p-aminobenzoic acid and SMZ N-acetylation, respectively, significantly correlated with NA-SMX formation (r = 0.576, P = .006; r = 0.459, P = .036). The stronger correlation with NAT1 activity supports the relationship of NAT1 to SMX metabolism. Novel targeted proteomic quantification of NAT2 showed significant correlations between NAT2 protein concentration and NAT2 activity (r = 0.823; P < .0001 and r = 0.734, P = .0002; for 2 peptides). This work demonstrates interindividual variability in SMX metabolism and highlights the importance of considering genetic and nongenetic factors in SMX-induced drug-induced liver injury risk. SIGNIFICANCE STATEMENT: This study provides new insights into sulfamethoxazole (SMX) metabolism using in vitro hepatic systems and quantifies interindividual variability in N₄-acetyl SMX formation. Although NAT2 genotype did not predict SMX slow acetylator metabolism in all individuals, N₄-acetyl SMX formation was significantly correlated with NAT1 and NAT2 enzyme activity. These findings show the importance of considering both genetic and phenotypic data to better understand SMX metabolism and individual risk for drug-induced liver injury.

This study investigated the impact of genetic variations in organic anion transporting polypeptides (OATPs) 1A2 and 2B1 on their transport activity at pH 6.3 and 7.4 by using HEK293 cells expressing OATP variants, focusing on stoichiometric transport kinetic parameters corrected for the number of transporters on the plasma membrane. In the OATP2B1 Asp215Val, the maximal velocity per OATP molecule and intrinsic clearance at pH 6.3 were drastically reduced to 0.0648- and 0.0178-fold, respectively, compared with the wild type. All tested OATP1A2 variants exhibited increased transport activity at pH 6.3, suggesting that OATP1A2 is more sensitive to extracellular pH. Furthermore, we used the AlphaFold model to explain the observed differences in transport activity among genetic variants. In OATP1A2, the Glu172Asp mutation replaces a longer glutamate side chain with a shorter aspartate, which may enhance substrate interactions while weakening the salt-bridge interactions with neighboring residues, potentially compromising structural integrity. In OATP2B1, the Asp215Val variant was found to disrupt a key salt-bridge interaction with Lys595, which destabilizes the outward-open conformation. Moreover, the Val201Met mutation appears to lock the transporter in a single conformational state. Our findings underscore the importance of transmembrane helix 4 in maintaining functional conformational dynamics and suggest that mutations in this region can significantly alter substrate binding and transport efficiency in OATP1A2 and 2B1. SIGNIFICANCE STATEMENT: This study combined uptake assays using transporter-expressing cell lines, liquid chromatography-tandem mass spectrometry transporter quantification, and computer modeling to clarify the changes in transport activity per molecule, and these mechanisms caused by amino acid substitutions in organic anion transporting polypeptides 1A2 and 2B1.

NADPH cytochrome P450 reductase is the required redox partner for the majority of human cytochrome P450 enzymes, which are critically important for phase I drug metabolism of a wide variety of substrates. It is well understood that cytochrome P450 reductase supports P450 catalysis when its flavin mononucleotide (FMN)-containing domain (FMND) binds to the proximal side of P450 enzymes to deliver electrons to the P450 heme. Herein, we describe mass spectrometry-based footprinting approaches to compare the surface labeling of CYP2A6 and that of an artificial fusion protein composed of the reductase FMND linked to the N-terminus of CYP2A6 (FMND/CYP2A6). Three complementary footprinting approaches were used: hydrogen-deuterium exchange, benzoyl fluoride labeling, and fast photochemical oxidation of proteins (FPOP). Although the different labeling approaches target different amino acids and occur over varying reaction timescales, their outcomes generally agree. These experiments did not detect differential protection on the proximal P450 face where FMND is expected to bind. Instead, they consistently demonstrated increased exposure of CYP2A6 surface residues, indicative of structural changes in CYP2A6 in the presence of the FMND. Overall, the reduced protection is consistent with the FMN domain causing long-range allosteric modulation of the CYP2A6 structure. This structural evidence is consistent with increasing functional evidence that the reductase is an allosteric modulator of P450 enzymes in addition to its role in electron transfer. SIGNIFICANCE STATEMENT: Both established and new mass-spectrometry footprinting methods support structural changes in the CYP2A6 structure upon interaction with the FMN-containing domain of its reductase. This evidence supports the idea that the reductase is an allosteric modulator of P450 enzymes, in addition to its established role in electron transfer.

A dual prodrug linking clopidogrel and indobufen-an established dual antiplatelet therapy combination-was designed to enhance the bioactivation of clopidogrel while enabling coordinated inhibition of the ADP and thromboxane A₂ pathways of platelet activation. Because these 2 agents differ markedly in mechanism and duration of action, conventional combination therapy necessitates asymmetrical dosing. The fixed 1:1 molar ratio imposed by covalent conjugation introduces an inherent constraint on achieving balanced dual-pathway inhibition, a key consideration for defining the conjugate's therapeutic positioning. Three conjugates-deuterated clopidogrel-indobufen (1a), clopidogrel-indobufen (1b), and clopidogrel-(S)-indobufen (1c)-were synthesized and evaluated in rats. A single dose of these conjugates produced a delayed time to maximum plasma concentration and a sustained-release profile for both active metabolites. Covalent conjugation enhanced systemic exposure to the clopidogrel active metabolite while reducing exposure to released indobufen. Because conjugates 1b and 1c exhibited pharmacokinetic profiles more comparable to equimolar coadministration, they were selected for pharmacodynamic assessment. ADP receptor P2Y₁₂ occupancy and plasma thromboxane B₂ served as pathway-specific biomarkers, each bridging the pharmacokinetics and pharmacodynamics of the irreversible inhibition by clopidogrel and the reversible inhibition by indobufen, respectively. Both biomarkers showed strong correlations with inhibition of the corresponding platelet activation pathways. A single dose of 1b or 1c yielded synchronized maximal inhibition of both pathways at 8 hours-4 hours later than conventional coadministration-while retaining comparable peak efficacy. In the repeated dosing study, assessments aligned with the maximal-effect time point of the coadministration reference demonstrated that both conjugates-when supplemented with an interdose of indobufen-achieved pathway inhibition equivalent to the clinical regimen. These findings support conjugates 1b and 1c as promising alternatives to standard clopidogrel therapy and as potential tools for controlled de-escalation of antiplatelet therapy. SIGNIFICANCE STATEMENT: The clopidogrel-indobufen dual prodrugs enable synchronous, sustained release of both antiplatelet species in rats. P2Y₁₂ receptor occupancy and plasma thromboxane B₂ effectively capture the pharmacokinetic-pharmacodynamic relationships of this irreversible/reversible dual-antagonist combination.

Plasma levels of Coproporphyrin I (CP-I), an endogenous biomarker used to gauge hepatic organic anion transporting polypeptide (OATP)1B1 and OATP1B3 activities, are linked to covariates, namely ethnicity, sex, and hemoglobin level. We developed and verified a mechanistic physiologically based pharmacokinetic model for CP-I considering these covariates in basal conditions and with a range of OATP1B perpetrators in virtual healthy subjects of various ethnicities. Simulations recovered the observed steady-state baseline levels and concentrations with interaction (C_max and area under the curve ratios, n = 12 studies) within 2-fold. Published CP-I plasma data in hepatic impairment (HI) indicated a progressive reduction in OATP1B activity in vivo. We applied our verified CP-I model to simulate CP-I plasma levels reported in individuals with increasing severity of HI as classified based on the Child-Pugh classes (A, B, and C) to assess the hepatic OATP1B transporter activity in cirrhotic virtual populations. A biomarker-informed physiologically based pharmacokinetic (BI-PBPK) approach was applied to close the gap between known expression differences for human hepatic OATP1B and multidrug resistance-associated protein 2 (MRP2) in HI and observed activity differences in HI relative to healthy individuals. HI-associated relative activity factor scalars derived from BI-PBPK simulations were developed and verified using 9 OATP1B substrates (n = 7 studies), with an average fold error and absolute average fold error of 0.93 and 1.74 for C_max, and 1.29 and 1.47 for area under the plasma concentration-time curve ratios between HI and healthy. The BI-PBPK approach offers a powerful means to establish model system parameters to improve predictive performance, particularly in disease populations and to explore the mechanisms behind the changes in plasma level. SIGNIFICANCE STATEMENT: Biomarker-informed physiologically based pharmacokinetic approach was used to bridge abundance differences in transporter expression and observed activity differences between healthy volunteer and hepatically impaired patients. A PBPK model for the endogenous biomarker, Coproporphyrin I, was developed where its synthesis rate is linked to body weight, sex, ethnicity, and hemoglobin levels. The model was verified with an extensive set of weak-to-strong OATP1B perpetrator drugs and applied to recover plasma concentrations for multiple OATP1B substrates using associated drug-drug interactions.