Drug Metabolism and Disposition最新文献

Rheumatoid arthritis (RA) patients often develop comorbid RA-associated interstitial lung disease, which is a severe extra-articular manifestation associated with high incidence and mortality rates. Nintedanib, an oral small-molecule tyrosine kinase inhibitor, has shown promise in clinical studies by mitigating disease progression and enhancing lung function among RA-associated interstitial lung disease patients. Adverse reactions to nintedanib are closely linked to its blood concentration, potentially leading to discontinuation due to gastrointestinal side effects and elevated liver enzymes. However, existing research has yet to delve into the pharmacokinetic impact of RA on nintedanib. Therefore, we established a collagen-induced arthritis rat model to examine how RA influences the pharmacokinetics of nintedanib. Additionally, we conducted Caco-2 transport experiments to explore potential factors contributing to these pharmacokinetic alterations. Our findings reveal that nintedanib's pharmacokinetics change in collagen-induced arthritic rats at different disease stages compared to the control group. This alteration results in a notable increase in blood drug concentration and significant changes in pharmacokinetic parameters such as maximum serum concentration (C_max), AUC_0-t, AUC_0→∞, and time to reach peak concentration (T_max). The outcomes from the Caco-2 transport experiments indicate that interleukin 6 stimulation impedes nintedanib efflux, aligning with the observed pharmacokinetic alterations. Further Western blot experiments suggest that the changes in nintedanib's pharmacokinetics are associated with reduced expression of P-glycoprotein. Moreover, our findings suggest that RA may reduce intestinal P-glycoprotein expression by activating the C-Jun N-terminal kinase signaling pathway. SIGNIFICANCE STATEMENT: Rheumatoid arthritis significantly increases the drug concentration of nintedanib. These data suggest that rheumatoid arthritis may be caused by reducing the expression of P-glycoprotein by affecting the C-Jun N-terminal kinase signaling pathway.

Although exenatide is approved for patients with type 2 diabetes mellitus (T2DM) with mild to moderate renal impairment, specific dosing guidelines for this population remain undefined. To address this gap, we developed a physiologically based pharmacokinetic model using PK-Sim & MoBi software, integrating target-mediated drug disposition to simulate exenatide's nonlinear pharmacokinetics in normal renal function. The model was extrapolated to renal impairment populations by adjusting physiological parameters and validated against clinical data. The plasma concentrations of exenatide predicted by the established physiologically based pharmacokinetic models for populations with normal renal function and those with renal impairment were in high concordance with the observed values, with fold errors of major pharmacokinetic parameters falling within the 0.5- to 2-fold range. After reducing simulated doses for the renal impairment population to 75%, 50%, and 25% of the 10 μg standard dose, area under the concentration-time curve and C_max were re-predicted to identify optimal doses that bring this population's pharmacokinetic parameters within the normal ranges. On the basis of our findings, we recommend a model-guided dosing strategy for patients with T2DM with renal impairment, consisting of an initial dose of 2.5 μg twice daily, followed by 5-7.5 μg (mild impairment) or 5 μg (moderate impairment) twice daily for maintenance dose. This study suggests that, compared with patients with T2DM with normal renal function, patients with T2DM with renal impairment should begin at half the initial dose and also receive a reduced maintenance dose. SIGNIFICANCE STATEMENT: Exenatide is approved by the US Food and Drug Administration for patients with type 2 diabetes mellitus with mild to moderate renal impairment, but dosing guidelines are still lacking. This study developed and validated physiologically based pharmacokinetic models of exenatide in renal impairment. These new models close the evidence gap for optimal dosing in this population.

Organic anion transporting polypeptide 1B1 (OATP1B1), the hepatic-specific uptake transporter, plays key roles in the absorption, distribution, and excretion of a broad range of endogenous and exogenous compounds. Altered expression and function of OATP1B1 affect the bioavailability and pharmacokinetics of various clinically important drugs. In this study, OATP1B1 uptake function was found to be significantly suppressed by SRC proto-oncogene, non-receptor tyrosine kinase family kinase inhibitors, with SU6656 demonstrating the most potent inhibitory effect. Knockdown and overexpression experiments revealed that YES1 is the specific SRC proto-oncogene, non-receptor tyrosine kinase family kinase responsible for regulating OATP1B1. Further, YES1 was found to interact with OATP1B1, and the tyrosine phosphorylation status of the transporter was suppressed by both the SU6656 treatment and the knockdown of the tyrosine kinase. Moreover, Caveolin 1 (CAV-1), the oligomeric scaffolding protein, was found to interact with OATP1B1. CAV-1 knockdown significantly suppressed the uptake function of OATP1B1. Although the reduction of CAV-1 did not affect the interaction between YES-1 and the transporter, it affected the phosphorylation level of OATP1B1. Immunofluorescence analysis indicated that CAV-1 colocalized with OATP1B1, and the disruption of lipid rafts significantly reduced the association of CAV-1 with OATP1B1, suggesting that the integrity of lipid rafts is essential for the effect of CAV-1 on OATP1B1. In conclusion, YES1 was identified as a regulator for OATP1B1. The tyrosine kinase physically interacts with OATP1B1, and the conformation that facilitates the phosphorylation of OATP1B1 by YES1 is likely maintained by CAV-1. SIGNIFICANCE STATEMENT: The present study found that YES-1 regulates the function of organic anion transporting polypeptide 1B1 (OATP1B1) by interacting with the transporter and influencing its tyrosine phosphorylation status. Caveolin-1 was shown to interact with OATP1B1 as well. Abrogation of Caveolin-1 exhibited no effect on the interaction between YES-1 and OATP1B1 but reduced the phosphorylation level of the transporter. Taken together, inhibitors of YES-1 may alter the uptake function of OATP1B1, potentially leading to drug-drug interactions related to post-translational modification.

Drug clearance and drug-drug interactions (DDIs) are important in the pharmacokinetic assessment of investigational drugs, yet predicting in vivo fraction metabolized (f_m) and DDI intensity remains challenging, particularly for low-clearance compounds. This study demonstrates how human liver chimeric mice (hu-PXB mice) can predict CYP2C9-mediated drug disposition for low-clearance compounds in humans. To estimate human in vitro CYP2C9 fraction metabolized (f_{m,CYP2C9,in vitro}), 3 CYP2C9 substrates (phenytoin, tolbutamide, and warfarin) were incubated in human hepatocytes with or without sulfaphenazole (CYP2C9 inhibitor). The f_{m,CYP2C9,in vitro} was calculated based on hepatic intrinsic clearance. For in vivo estimation (f_{m,CYP2C9,in vivo}), clinical DDI data obtained using CYP2C9 inhibitors were analyzed to calculate f_{m,CYP2C9,in vivo} based on observed clearance changes. To evaluate human DDI predictability, the 3 drugs were administered intravenously to hu-PXB and SCID mice with or without CYP2C9 inhibitors (sulfaphenazole or tienilic acid). Clearance changes were calculated and compared among humans, hu-PXB mice, and SCID mice. Results showed that f_{m,CYP2C9,in vitro} values for phenytoin and tolbutamide were overestimated compared to f_{m,CYP2C9,in vivo}, whereas warfarin could not be evaluated under current conditions. Hu-PXB mice demonstrated a better correlation with humans in both clearance changes and absolute values compared to SCID mice. Notably, hu-PXB mice predicted CYP2C9-mediated DDI magnitude within 15% of clinical values and predicted clearance for CYP2C9 substrates within 2-fold of clinical values. These findings establish hu-PXB mice as a reliable preclinical model for predicting human CYP2C9-mediated drug disposition. SIGNIFICANCE STATEMENT: Human liver chimeric mice can accurately predict the clearance and magnitude of drug-drug interaction for CYP2C9 substrate drugs. Findings from humanized mice enable the selection of better candidates in drug discovery and facilitate the design of efficient clinical trials for investigational drugs.

Although tyrosine kinase inhibitor and immune checkpoint inhibitor (TKI-ICI) combination therapy has emerged as a promising treatment for hepatocellular carcinoma (HCC), reliable biomarkers for predicting long-term survival remain underexplored. Here, we conducted metabolomics and lipidomics profiling in baseline plasma samples from 58 patients with HCC who received TKI-ICI therapy in a prospective phase II trial. Prognostic features were identified using an integrated machine learning framework combining random forest survival analysis, LASSO regression, and Cox modeling. Untargeted metabolomics identified 2 lipids, phosphatidylinositol lyso 18:1 and O-phosphorylethanolamine, that were associated with progression-free survival. Lipidomics further revealed 9 prognostic lipids, including cholesteryl ester (18:1), triacylglycerol (TG) (15:0/15:0/21:6), TG (18:1/20:5/20:5), TG (41:3), phosphatidylserine (42:3), phosphatidylethanolamine (38:5), sphingosine (d18:1), phosphatidylcholine (43:5), and ceramide (d18:2/22:0), as independent predictors of progression-free survival. Multivariate Cox modeling integrating metabolomic and lipidomic markers reinforced their prognostic relevance. Meanwhile, 6 lipids, including phosphatidylcholine (39:8p), phosphatidylserine (18:0/20:4), TG (18:1/20:4/22:5), TG (15:0/15:0/21:6), ph sphingomyelin (d38:5), and sphingomyelin (d41:5), were found to be associated with overall survival. Functional enrichment analysis revealed that these prognostic lipids were involved in sphingolipid-related metabolism and immune-related signaling, highlighting the importance of lipid-immune crosstalk in reshaping responses to TKI-ICI therapy in patients with HCC. As sphingolipids are also known to modulate drug metabolism enzymes and transporters, they may thereby affect interindividual variability in TKI pharmacokinetics. In conclusion, our findings demonstrated that circulating lipidomic features, particularly sphingolipid-related species, were predictive of long-term survival in patients with HCC receiving TKI-ICI combination therapy. These lipids may serve as noninvasive biomarkers for survival prediction, patient stratification, and informed therapeutic decision making, while offering insights into lipid-immune interplay in immunotherapy-based cancer treatment. SIGNIFICANCE STATEMENT: This study integrates metabolomic and lipidomic profiling of baseline plasma from patients with hepatocellular carcinoma receiving tyrosine kinase inhibitor and immune checkpoint inhibitor combination therapy, identifying sphingolipid-related lipid species as strong predictors of long-term survival. Unlike prior work focused on short-term response or monotherapy, these findings highlight lipidomic markers as noninvasive tools for survival prediction and treatment stratification, providing new insights into lipid-immune interactions and supporting the clinical utility of lipidomic signatures in guiding therapeutic decisions.

Microphysiological systems (MPSs) are emerging in vitro technologies designed to recapitulate human physiology for applications in drug development and safety assessment. Compared with conventional in vitro systems, MPSs may contain multiple types of cells and display dynamic and mechanical features of organ microenvironments. As part of new approach methodologies, MPSs are expected to contribute to reducing reliance on animal testing by providing human-relevant models that align with the principles of replacement, reduction, and refinement. This review discusses the advantages of MPSs over conventional in vitro systems for drug absorption, distribution, metabolism, and excretion and toxicity evaluation. We then systematically examines organ-specific MPS platforms used in absorption, distribution, metabolism, and excretion and toxicity studies. Next, we briefly evaluated the reproducibility of MPSs across different systems. Finally, we provide our perspectives and considerations on employing MPSs in regulatory applications. SIGNIFICANCE STATEMENT: This minireview provides an overview of the current trends in the field of microphysiological systems within the framework of new approach methodologies. This review will give readers insights into key differences between microphysiological systems and other in vitro methods in drug absorption, distribution, metabolism, and excretion, followed by recent advances in several organ chips for drug evaluation.

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.

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 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.

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.