Drug Metabolism and Disposition最新文献

Small interfering RNA (siRNA) therapeutics represent an emerging class of pharmacotherapy with the potential to address previously hard-to-treat diseases. Currently approved siRNA therapeutics include lipid nanoparticle-encapsulated siRNA and tri-N-acetylated galactosamine-conjugated siRNA. These siRNA therapeutics exhibit distinct pharmacokinetic characteristics and unique absorption, distribution, metabolism, and elimination (ADME) properties. As a new drug modality, limited clinical data are available for siRNA therapeutics in specific populations, including pediatrics, geriatrics, individuals with renal or hepatic impairment, and pregnant women, making dosing challenging. In this Minireview, a mechanistic overview of the ADME properties of the 5 currently approved siRNA therapeutics is presented. A concise overview of the clinical data available for therapeutic siRNAs in special populations, focusing on the potential impact of physiologic changes during pregnancy on siRNA disposition, is provided. The utility of physiologically based pharmacokinetic (PBPK) modeling as a tool to elucidate the characteristics and disposition of siRNA therapeutics in pregnant women is explored. Additionally, opportunities to integrate known physiologic alterations induced by pregnancy into PBPK models that incorporate siRNA ADME mechanisms to predict the effects of pregnancy on siRNA disposition are discussed. Clinical data regarding the use of therapeutic siRNA in special populations remain limited. Data for precise parameterization of maternal-fetal siRNA PBPK models are lacking presently and underscore the need for further research in this area. Addressing this gap in knowledge will not only enhance our understanding of siRNA pharmacokinetics during pregnancy but also advance the possible development of siRNA therapeutics to treat pregnancy-related conditions. SIGNIFICANCE STATEMENT: This Minireview proposes a framework on how small interfering RNA (siRNA) disposition can be predicted in pregnancy based on mechanistic absorption, distribution, metabolism, and elimination (ADME) information using physiologically-based pharmacokinetic (PBPK) modeling. The mechanistic ADME information and available clinical data in special populations of currently Food and Drug Administration-approved siRNA therapeutics are summarized. Additionally, how physiological changes during pregnancy may affect siRNA disposition is reviewed, and the opportunities to use PBPK modeling to predict siRNA disposition in pregnant women is explored.

Physiologically based pharmacokinetic (PBPK) modeling is a mechanistic dynamic modeling approach that can be used to predict or retrospectively describe changes in drug exposure due to drug-drug interactions (DDIs). With advancements in commercially available PBPK software, PBPK DDI modeling has become a mainstream approach from early drug discovery through to late-stage drug development and is often used to support regulatory packages for new drug applications. This Minireview will briefly describe the approaches to predicting DDI using PBPK and static modeling approaches, the basic model structures and features inherent to PBPK DDI models, and key examples where PBPK DDI models have been used to describe complex DDI mechanisms. Future directions aimed at using PBPK models to characterize transporter-mediated DDI, predict DDI in special populations, and assess the DDI potential of protein therapeutics will be discussed. A summary of the 209 PBPK DDI examples published to date in 2023 will be provided. Overall, current data and trends suggest a continued role for PBPK models in the characterization and prediction of DDI for therapeutic molecules. SIGNIFICANCE STATEMENT: Physiologically based pharmacokinetic (PBPK) models have been a key tool in the characterization of various pharmacokinetic phenomena, including drug-drug interactions. This Minireview will highlight recent advancements and publications around physiologically based pharmacokinetic drug-drug interaction modeling, an important area of drug discovery and development research in light of the increasing prevalence of polypharmacology in clinical settings.

Environmentally persistent free radicals (EPFRs) are a recently recognized component of particulate matter that cause respiratory and cardiovascular toxicity. The mechanism of EPFR toxicity appears to be related to their ability to generate reactive oxygen species (ROS), causing oxidative damage. EPFRs were shown to affect cytochrome P450 (P450) function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs is their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230, the laboratory-generated EPFR made by heating silica 5% copper oxide, and silica [<0.2 μm in diameter] and 2-monochlorophenol at ≥230 °C) exposure. Because CYP2E1 is also known to generate ROS, it is important to understand the ability of EPFRs to influence the function of this enzyme and to identify the mechanisms involved. CYP2E1 was shown to be inhibited by EPFRs and to a lesser extent by non-EPFR particles. Because EPFR-mediated inhibition was more robust at subsaturating NADPH-P450 reductase (POR) concentrations, disruption of POR•CYP2E1 complex formation and electron transfer were examined. Surprisingly, neither complex formation nor electron transfer between POR and CYP2E1 was inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR control particle (CuO-Si). When a POR/CYP2E1-containing reconstituted system was added to the pollutant-particle systems, there was a synergistic stimulation of ROS production. The results indicate that EPFRs cause inhibition of CYP2E1-mediated substrate metabolism, yet do not alter electron transfer and actually stimulate ROS generation. Taken together, the results are consistent with EPFRs affecting CYP2E1 function by inhibiting substrate metabolism and increasing the generation of ROS. SIGNIFICANCE STATEMENT: Environmentally persistent free radicals affect CYP2E1 function by inhibition of monooxygenase activity. This inhibition is not due to disruption of the POR•CYP2E1 complex or inhibition of electron transfer but due to the uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of reactive oxygen species. These results show that environmentally persistent free radicals block the metabolism of foreign compounds and synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.

Silybin, a milk thistle extract, is a flavonolignan compound with hepatoprotective effect. It is commonly used in dietary supplements, functional foods, and nutraceuticals. However, the metabolism of silybin has not been systematically characterized in organisms to date. Therefore, we established a novel high-performance liquid chromatography quadrupole time-of-flight mass spectrometry method to analyze and identify the prototype and metabolites of silybin in rats. In total, 29 (of 32) new metabolic pathways and 56 (of 59) unreported metabolite products were detected. Moreover, we found that the liver had a high first-pass effect of 63.30% ± 13.01% for silybin, and only 1 metabolite was detected. Moreover, the metabolites identified in gastrointestinal tract possessed 88% of all unreported metabolite products (52 of 59). At the same time, the high concentration of silybin in the liver also indicated that large amounts of silybin may be accumulated in the liver instead of being metabolized. These results indicated that the primary metabolizing organ of silybin in rats was intestine rather than liver, which offers a solid chemical foundation for exploring more pharmacological effects of silybin. SIGNIFICANCE STATEMENT: This study confirmed that the primary location of metabolism of silybin in rats after intragastric administration was the gastrointestinal tract instead of the liver and that intestinal microbes were closely involved. In total, 29 (of 32) metabolism pathways and 56 (of 59) metabolites were identified for the first time in rats, to the authors' knowledge. To further study the liver disposition of silybin, its hepatic first-pass effect was determined for the first time. This work is capable of furnishing a robust material foundation for the forthcoming pharmacological investigations regarding silybin.

Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. An increasing number of therapeutic drugs on the market underscores the necessity to accurately assess new drug combinations during preclinical evaluation for DDIs. In vitro primary human hepatocytes (PHH) models are only applicable for short-term induction studies because of their rapid loss of metabolic function. Though coculturing nonhuman stromal cells with PHH has been shown to stabilize metabolic activity long-term, there are concerns about human specificity for accurate clinical assessment. In this study, we demonstrated a PHH-only liver microphysiological system in the Liver Tissue Chip is capable of maintaining long-term functional and metabolic activity of PHH from 3 individual donors and thus a suitable platform for long-term DDI induction studies. The responses to rifampicin induction of 3 PHH donors were assessed using cytochrome P450 activity and mRNA changes. Additionally, victim pharmacokinetic studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following perpetrator drug treatment, rifampicin-mediated induction, which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values, respectively, compared with the untreated liver microphysiological system. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on cytochrome P450 activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study illustrates the utility of the Liver Tissue Chip for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs. SIGNIFICANCE STATEMENT: This study demonstrated the utility of the Liver Tissue Chip with a primary human hepatocyte-only liver microphysiological system for drug-drug interaction induction studies. This unique in vitro system with continuous recirculation maintains long-term functionality and metabolic activity for up to 4 weeks, enabling the study of perpetrator and victim drug pharmacokinetics, quantification of drug-induced cytochrome P450 mRNA and activity levels, investigation of patient variability, and ultimately clinical predictions.

Pirfenidone (PIR) is used in the treatment of idiopathic pulmonary fibrosis. After oral administration, it is metabolized by cytochrome P450 1A2 to 5-hydroxylpirfenidone (5-OH PIR) and further oxidized to 5-carboxylpirfenidone (5-COOH PIR), a major metabolite excreted in the urine (90% of the dose). This study aimed to identify enzymes that catalyze the formation of 5-COOH PIR from 5-OH PIR in the human liver. 5-COOH PIR was formed from 5-OH PIR in the presence of NAD⁺ by human liver microsomes (HLMs) more than by human liver cytosol (HLC), with the concomitant formation of the aldehyde form (5-CHO PIR) as an intermediate metabolite. By purifying enzymes from HLMs, alcohol dehydrogenases (ADHs) were identified as candidate enzymes catalyzing 5-CHO PIR formation, although ADHs are localized in the cytoplasm. Among constructed recombinant ADH1-5 expressed in HEK293T cells, only ADH4 efficiently catalyzed 5-CHO PIR formation from 5-OH PIR with a K_m value (29.0 ± 4.9 μM), which was close to that by HLMs (59.1 ± 4.6 μM). In contrast to commercially available HLC, HLC prepared in-house clearly showed substantial 5-CHO PIR formation, and ADH4 protein levels were significantly (rs = 0.772, P < .0001) correlated with 5-CHO PIR formation in 25 HLC samples prepared in-house. Some components of the commercially available HLC may inhibit ADH4 activity. Disulfiram, an inhibitor of aldehyde dehydrogenases (ALDH), decreased 5-COOH PIR formation and increased 5-CHO PIR formation from 5-OH PIR in HLMs. ALDH2 knockdown in HepG2 cells by siRNA decreased 5-COOH PIR formation by 61%. SIGNIFICANCE STATEMENT: This study clarified that 5-carboxylpirfenidone formation from 5-hydroxylpirfenidone proceeds via a 2-step oxidation reaction catalyzed by ADH4 and disulfiram-sensitive enzymes, including ALDH2. Interindividual differences in the expression levels or functions of these enzymes could cause variations in the pharmacokinetics of pirfenidone.

To further the development of an in vitro model that faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from 3 donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell inserts, and confirmed transformation into a largely enterocyte population via RNA sequencing analysis and immunocytochemistry (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH)₂ vitamin D₃-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, because, to our knowledge, this is the first study to assess drug metabolizing enzymes and transporters expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. SIGNIFICANCE STATEMENT: This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers that maintain barrier function and metabolic functionality for up to 42 days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.

Many factors cause interperson variability in the activity and expression of the cytochrome P450 (CYP) drug-metabolizing enzymes in the liver, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors are associated with CYP expression, with estrogen receptor α (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the 2 top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the 3 most abundant ESR1 isoforms in 260 liver samples derived from African Americans (n = 125) and European Americans (n = 135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that compared with gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and 3 UDP-glucuronosyltransferases (UGTs), whereas ESR1 isoforms improved predictive models for the UGTs and CYP2D6 but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, noncanonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) was only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level transcription factor expression may help to explain variation in CYP or UGT expression between individuals. SIGNIFICANCE STATEMENT: We quantified 18 NR1I3 splice isoforms and the 3 most abundant ESR1 splice isoforms in 260 liver samples derived from African American and European American donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared with gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some cytochrome P450s and UDP-glucuronosyltransferases, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

Physiologically based pharmacokinetic (PBPK) modeling is a physiologically relevant approach that integrates drug-specific and system parameters to generate pharmacokinetic predictions for target populations. It has gained immense popularity for drug-drug interaction, organ impairment, and special population studies over the past 2 decades. However, an application of PBPK modeling with great potential remains rather overlooked-prediction of diarrheal disease impact on oral drug pharmacokinetics. Oral drug absorption is a complex process involving the interplay between physicochemical characteristics of the drug and physiological conditions in the gastrointestinal tract. Diarrhea, a condition common to numerous diseases impacting many worldwide, is associated with physiological changes in many processes critical to oral drug absorption. In this Minireview, we outline key processes governing oral drug absorption, provide a high-level overview of key parameters for modeling oral drug absorption in PBPK models, examine how diarrheal diseases may impact these processes based on literature findings, illustrate the clinical relevance of diarrheal disease impact on oral drug absorption, and discuss the potential and challenges of applying PBPK modeling in predicting disease impacts. SIGNIFICANCE STATEMENT: Pathophysiological changes resulting from diarrheal diseases can alter important factors governing oral drug absorption, contributing to suboptimal drug exposure and treatment failure. Physiologically based pharmacokinetic (PBPK) modeling is an in silico approach that has been increasingly adopted for drug-drug interaction potential, organ impairment, and special population assessment. This Minireview highlights the potential and challenges of using physiologically based pharmacokinetic modeling as a tool to improve our understanding of how diarrheal diseases impact oral drug pharmacokinetics.

The placenta acts as a barrier, excluding noxious substances while actively transferring nutrients to the fetus, mediated by various transporters. This study quantified the expression of key placental transporters in term human placenta (n = 5) and BeWo, BeWo b30, and JEG-3 placenta cell lines. Combining these results with pregnancy physiologically based pharmacokinetic (PBPK) modeling, we demonstrate the utility of proteomic analysis for predicting placental drug disposition and fetal exposure. Using targeted proteomics with quantification concatemer standards, we found significant expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein (MRP) 2, MRP4, and MRP6 in the human placenta (0.05-0.25 pmol/mg membrane protein) with only regional differences observed for P-gp. Unexpectedly, both P-gp and BCRP were below the limit of quantification in the regularly used BeWo cells, indicating that this cell line may not be suitable for the study of placental P-gp and BCRP-mediated transport. In cellular and vesicular overexpression systems, P-gp and BCRP were detectable as expected. Vesicle batches showed consistent P-gp expression correlating with functional activity (N-methyl-quinidine transport). However, BCRP activity (estrone 3-sulfate transport) did not consistently align with expression levels. Incorporating in vitro transporter kinetic data, along with placental transporter abundance, into a PBPK model enabled the evaluation of fetal exposure. Simulation with a hypothetical drug indicated that estimating fetal exposure relies on the intrinsic clearances of relevant transporters. To minimize interlaboratory discrepancies, expression data was generated using consistent proteomic methodologies in the same lab. Integration of this data in pregnancy PBPK modeling offers a promising tool to investigate maternal, placental, and fetal drug exposure. SIGNIFICANCE STATEMENT: This study quantified the expression of key placental transporters in human placenta and various placental cell lines, revealing significant expression variations. By integrating these data with physiologically based pharmacokinetic modeling, the study highlights the importance of transporter abundance data in understanding and predicting placental drug disposition, essential for maternal and fetal health during pregnancy.