Drug Metabolism and Disposition最新文献

A human absorption, distribution, metabolism, and excretion (hADME) study is an essential clinical pharmacology study for small-molecule drugs. The study provides insights into circulating drug-related materials and the drug's elimination pathways in humans, which can guide future studies on safety and drug-drug interaction of metabolites as well as organ impairment and drug-drug interaction of the parent drug. The 2 hADME study types, namely conventional and microtracer, are comprehensively compared in this manuscript. A review of literature found that conventional hADME studies were approximately 7 times that of microtracer hADME studies for small molecule and peptide drugs based on publications in 3 peer-reviewed journals from 2010 to 2024. Each study type has advantages and disadvantages. The advantages of conventional hADME studies primarily include the ease, low cost, and flexibility of radiometric sample analysis. In contrast, the advantages of microtracer hADME studies primarily include exemption from prerequisite studies and use of non-good manufacturing practice ¹⁴C-labeled materials. The disadvantages of each study type are essentially the advantages of the other. The manuscript also discusses scenarios where a microtracer hADME study may be preferable. Finally, recommendations are provided on selecting the appropriate hADME study type for an investigational drug. SIGNIFICANCE STATEMENT: The manuscript discusses 2 primary human absorption, distribution, metabolism, and excretion study types: conventional and microtracer. It covers published literature studies, the pros and cons of each type, scenarios for conducting microtracer studies, and a recommended decision tree for selecting the appropriate human absorption, distribution, metabolism, and excretion study type.

Quantitative prediction of hepatic clearance is a key element in predicting the human pharmacokinetic profile in the nonclinical stages. In the present study, we focused on the major cytochrome P450 (P450) isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) and tested a relative activity factor (RAF) method to quantitatively predict in vivo hepatic intrinsic clearance (CL_h,int,vivo) and fraction metabolized (f_m) by the P450 isoforms directly from an in vitro recombinant P450 system. We selected multiple probe substrates for CYP1A2 (caffeine, tizanidine, phenacetin), CYP2C9 ((S)-acenocoumarol, glimepiride, lornoxicam, tolbutamide, (S)-warfarin), CYP2C19 ((S)-lansoprazole, omeprazole, pantoprazole), CYP2D6 (desipramine, metoprolol, nebivolol, tolterodine), and CYP3A4 (alprazolam, felodipine, midazolam, nisoldipine, sildenafil, triazolam) to calculate the representative RAF value for each P450 isoform based on the in vivo-to-in vitro clearance ratio of the multiple probe substrates. The most pronounced substrate dependency of the RAF values was noted for CYP3A4 (2698 [alprazolam] to 19073 [nisoldipine] pmol P450/kg). Using the geometric mean of the RAF values for each isoform, a within 3-fold prediction of the CL_h,int,vivo was obtained for all the 11 test drugs, except glibenclamide, which is a known substrate of hepatic uptake transporters. The f_m values of the responsible P450 isoform(s) could be well predicted for mexiletine, tamsulosin, risperidone, celecoxib, and glibenclamide. This simple, practical RAF method can be one of the useful nonclinical methods to estimate the CL_h,int,vivo and f_m mediated by the major P450 isoforms, which would promote earlier understanding of the impact of genetic polymorphisms and drug-drug interactions on the human pharmacokinetics of the substrate compounds. SIGNIFICANCE STATEMENT: The relative activity factor method has been used for extrapolating in vitro clearance from recombinant systems to liver microsomes, but this study utilized this method to predict in vivo hepatic clearance and fraction metabolized values. By applying relative activity factor values obtained from multiple probe substrates, this study was able to quantitatively predict the in vivo clearances mediated by CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. This simple, practical method will help optimize metabolic clearances via the major cytochrome P450 isoforms in the nonclinical stages.

Mefloquine is an antimalarial drug routinely used for prophylaxis and in the treatment of malaria. Approximately 50% of mefloquine metabolism, both in vitro and in vivo, is mediated by CYP3A4 with the remaining contributions by other CYP450 isoforms unaccounted for. This study aimed to determine the contribution of CYP450s to mefloquine metabolism and incorporate this knowledge into a physiologically-based pharmacokinetic model. The data in human liver microsomes demonstrated the involvement of CYP3A4/5 as well as the previously unreported contribution of CYP1A2 to mefloquine metabolism. The fraction metabolized by CYP1A2 (fm,CYP1A2) was estimated to be at least 50% using chemical inhibitors and pooled human liver microsomes and confirmed using recombinant human CYP450 enzymes. A physiologically-based pharmacokinetic model built in Simcyp using the fm,CYP values recaptured observed clinical pharmacokinetic data-71 % of the simulated area under the curve (AUC) values were within 1.25-fold of the observed clinical data, and all simulated AUC values were within 2-fold of observed data. Simulated mefloquine exposures increased by 88% when an interaction with fluvoxamine, a CYP1A2 inhibitor was modeled. Modeling showed that heavy smoking, and subsequent induction of CYP1A2, had a notable effect on mefloquine exposure. CYP1A2 genotype status also influenced mefloquine exposure with a predicted AUC ratio of 1.68 in a simulated population of CYP1A2 poor metabolizers. The involvement of CYP1A2 in mefloquine metabolism suggests a previously unreported drug-drug interaction risk. Looking forward, the analysis here suggests the clinical exploration of the interaction between mefloquine and CYP1A2. SIGNIFICANCE STATEMENT: Despite the widespread use of mefloquine in malaria prophylaxis and treatment, its metabolism is not completely characterized. This has implications for understanding and predicting drug-drug interactions involving mefloquine. Here, we identify CYP1A2 as a key enzyme involved in mefloquine metabolism and use physiologically-based pharmacokinetic modeling to demonstrate the contribution of this route to interactions with mefloquine. The in vitro data and revised physiologically-based pharmacokinetic model are important starting points for future exploration of this pathway using clinical data.

Phenol-containing drugs may exhibit limited oral bioavailability due to first-pass conjugation in the intestine and liver, and potentially unfavorable biopharmaceutical properties imparted by the hydrogen-bond donor. We present a novel prodrug strategy in which O-methylpyrimidine modification masks the phenolic moiety and employs aldehyde oxidase (AO) to release the parent drug. Prototypical prodrugs of 4-hydroxy-tamoxifen (4OH-TAM), raloxifene (RAL), rotigotine, 5-hydroxy-tolterodine, and phentolamine were all substrates for AO-mediated parent drug release in liver cytosol from humans and every preclinical species evaluated. Reaction phenotyping confirmed the role of AO; hydralazine inhibited production of 4OH-TAM and RAL from their respective prodrugs in the human liver cytosol, and recombinant human AO activated those same prodrugs. Based on the identified byproduct, 5-(hydroxymethyl)uracil, and characterized 4OH-TAM prodrug metabolite intermediates, a mechanism is proposed, involving oxidation of the pyrimidine 4-position, followed by rate-limiting oxidation at the 2-position and subsequent C-O bond cleavage via an imine-methide intermediate. To determine a preclinical animal for proof-of-concept prodrug activation in vivo, we measured both absolute AO protein concentration and parent release for 2 prodrugs in the liver cytosol of multiple species and found that hamster was a promising candidate to model humans. After confirming a similar balance of AO-mediated prodrug conversion versus nonproductive/subsequent biotransformation in human and hamster hepatocytes, the 4OH-TAM prodrug and RAL prodrug 1 were progressed to a pharmacokinetic study in hamsters. A 30 mg/kg oral dose of RAL prodrug 1 demonstrated a 2-fold increase in RAL exposure compared with dosing parent RAL, indicating that this novel prodrug strategy has the potential to improve bioavailability in humans. SIGNIFICANCE STATEMENT: An aldehyde oxidase-mediated biotransformation that cleaves O-linked methylpyrimidine-masked phenolic moieties was identified, and this system employed for a novel prodrug bioactivation strategy. The research herein expands existing knowledge surrounding the metabolism capabilities of this enzyme and provides medicinal chemists with a tool to enhance the oral bioavailability of phenolic compounds that otherwise would be limited due to extensive phase II metabolism and possibly low permeability.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays an important role in regulating drug metabolism and bile acid homeostasis in the liver. Recently, it was revealed that the switch/sucrose non-fermentable (SWI/SNF) complex, a chromatin remodeler, regulates transactivation by nuclear receptors, such as the pregnane X receptor and vitamin D receptor. However, studies on the involvement of the SWI/SNF complex in CAR-mediated transactivation are limited. Here, we demonstrated that the induction of cytochrome P450 CYP2B6 expression by CAR activators, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and phenobarbital, was enhanced by the inhibition of AT-rich interactive domain-containing protein (ARID) 1A, a canonical brahma-related gene 1-associated factor (cBAF) component, one of the SWI/SNF complexes, and was attenuated by inhibition of bromodomain-containing protein (BRD) 9, a noncanonical BAF (ncBAF) component, in primary hepatocytes from humanized mice. Coimmunoprecipitation assays revealed that ARID1A and BRD9 interacted with CAR. Chromatin immunoprecipitation assay revealed that the 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime-induced binding of CAR to the 5'-flanking region of CYP2B6 gene increased with ARID1A inhibition and reduced with BRD9 inhibition. These results suggest that cBAF negatively regulates CAR-mediated transactivation by attenuating CAR binding to its response element, whereas ncBAF positively regulates it by facilitating CAR binding. Furthermore, ARID1A inhibition enhanced phenobarbital-induced increases in UDP-glucuronosyltransferase 1A1 expression and multidrug resistance-associated protein 2 mRNA level and activity. Collectively, our findings indicate that cBAF and ncBAF play essential roles in xenobiotic metabolism by regulating CAR-mediated transactivation and that ARID1A inhibitors may offer therapeutic benefits for hyperbilirubinemia and cholestasis by inducing UDP-glucuronosyltransferase 1A1 and multidrug resistance-associated protein 2 expression. SIGNIFICANCE STATEMENT: This study revealed that canonical brahma-related gene 1-associated factor and noncanonical brahma-related gene 1-associated factor, members of the switch/sucrose non-fermentable family, negatively and positively regulate constitutive androstane receptor (CAR) transactivation, respectively, through changes in the chromatin structure around the CAR response element in the 5'-flanking regions of CAR target genes. The inhibition of AT-rich interactive domain-containing protein 1A may be beneficial for cholestasis treatment by enhancing CAR-mediated transactivation.

Cardiac cytochrome P450 2J2 (CYP2J2) plays a significant role in cardiovascular homeostasis due to its dual functions in drug metabolism and the epoxidation of polyunsaturated fatty acids. Additionally, the inhibition of CYP2J2 by xenobiotics has been linked to drug-induced cardiotoxicity, warranting further investigation of this critical enzyme in cardiac systems. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are physiologically relevant in vitro models that recapitulate relevant phenotypes important for cardiovascular research. However, no studies have so far characterized CYP2J2 expression and activities in these models. Here, we developed and validated H7 hESC-CMs as suitable in vitro models for investigating CYP2J2 in drug metabolism and cardiotoxicity. We first performed the genotyping and confirmed the presence of wild-type CYP2J2∗1/∗1 alleles in wild-type hESCs. Our optimized cardiomyocyte differentiation protocols yielded virtually pure (93.3% ± 6.8%) hESC-CMs, which exhibited P450 epoxygenase mRNA-expression profiles consistent with human cardiomyocytes, with CYP2J2 as the dominant isozyme and minor contributions from CYP2C8 and CYP2C9. By employing a CYP2J2-selective fluorescent substrate, ER-BnXPI, and astemizole as probe substrates, CYP2J2-mediated demethylation of both substrates exhibited typical Michaelis-Menten kinetics, which confirms functional CYP2J2 activities in vitro. Additionally, we demonstrated the capacity of CYP2J2 for arachidonic acid epoxidation, validating its ability to metabolize polyunsaturated fatty acid substrates. Finally, CYP2J2 activity in hESC-CMs was significantly inhibited by danazol and dronedarone, which are established CYP2J2 inhibitors known to cause cardiotoxicity. Ultimately, our study sheds novel insights on hESC-CMs as a suitable model for investigating CYP2J2-mediated metabolism and its inhibition in vitro. SIGNIFICANCE STATEMENT: H7 human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were developed and validated as an in vitro model for investigating CYP2J2-mediated drug metabolism and its inhibition. By characterizing CYP2J2 transcriptional expression, catalytic activity, and inhibition response to established CYP2J2 inhibitors, our study confirmed functional CYP2J2 in hESC-CMs and ascertained that the model recapitulates the physiology of primary cardiomyocytes. This pioneering research highlights the potential of hESC-CMs in advancing our understanding of CYP2J2-mediated metabolism, its inhibition, and implications in drug-induced cardiotoxicity.

6-Mercaptopurine (6-MP) is a nucleobase analog used in the therapy of acute lymphoblastic leukemia and inflammatory bowel disease. It is associated with numerous side effects including myelotoxicity, hepatotoxicity, and gastrointestinal complications, which can lead to patient adherence issues or discontinuation of treatment. This is further complicated by the wide variability in plasma levels of 6-MP and the therapeutic response to a standard dose. Although a number of enzyme polymorphisms have been linked to therapeutic response, it is unclear what factors underlie the variability in plasma levels. We have established that SLC43A3-encoded equilibrative nucleobase transporter 1 mediates the transport of 6-MP into cells in both mice and humans. To determine whether this transporter is critical for 6-MP absorption and biodistribution, we examined the effect of the genetic deletion of slc43a3 in mice on the absorption and tissue distribution of orally administered 6-MP. A high-performance liquid chromatography method was developed to measure tissue levels of 6-MP and its key metabolites, 6-methylmercaptoprine, 6-thiourate, and 6-thioguanine nucleotides. The results of this study show that loss of slc43a3 dramatically reduces the absorption of 6-MP from the gastrointestinal tract and attenuates the levels achieved in peripheral tissues. Furthermore, the loss of slc43a3 decreases the tissue:blood concentration ratios of 6-MP and its metabolites, particularly in those tissues that show high levels of expression of slc43a3, such as the heart and lungs. Therefore, it is possible that differences in SLC43A3 expression in humans may contribute to the variability seen in 6-MP plasma levels and therapeutic response. SIGNIFICANCE STATEMENT: The loss of slc43a3 in mice dramatically reduces the absorption and the biodistribution of the chemotherapeutic drug 6-mercaptopurine. These data suggest that variations in SLC43A3 expression in humans may contribute to the variability in plasma levels that have been reported when using this drug therapeutically.

The formation of antidrug antibodies (ADAs) against antibody-drug conjugates (ADCs) can trigger a humoral immune response and change drug exposure. Although the immunogenicity assessment of an ADC drug in nonclinical nonhuman primates may not directly translate to potential immunogenicity in humans, the nonclinical ADA assay facilitates understanding the pharmacokinetic profiles of biotherapeutics. The immune response against the human IgG4 monoclonal antibody-based ADC was suspected in cynomolgus monkey serum after intravenous administration at 1.5 mg/kg. However, the conventional bridging format ADA assay presented unique challenges for the ADC molecules due to the interaction of ADC-based capture and detection reagents, which generated high background noise. Solid-phase extraction with acid dissociation (SPEAD) sample treatment allowed the selective ADA transfer to a second plate for detection while avoiding the interaction between the capture and detection reagents. The signal-to-noise ratio in the ADA assay for ADCs was notably improved with SPEAD sample treatment compared with the results from the bridging assay. Importantly, the rapid drug clearance of the ADC molecules at the later time points was well correlated with the signal-to-noise ratio of the ADA assay in monkey serum, suggesting the validity of the results. Hence, we demonstrated the utility of the SPEAD sample treatment to mitigate the critical reagent interaction that triggered the unexpectedly high background in the ADA assay. SIGNIFICANCE STATEMENT: A fit-for-purpose antidrug antibody screening assay for the human IgG4 monoclonal antibody-based antibody-drug conjugate (ADC) molecule by solid-phase extraction with acid dissociation was developed to mitigate the high background noise due to the interaction of capture and detection ADCs. A positive antidrug antibody signal was observed in the monkey serum sample, which is in line with the significant decrease in the plasma concentration of ADCs at the later time points.

The human kidney is a critical organ for the elimination of numerous drugs and metabolites. The mechanisms of renal drug handling are manifold including unbound filtration, transporter-mediated active secretion, bidirectional passive diffusion, and occasionally active reabsorption and renal metabolism. These mechanisms collectively dictate the fate of drugs at various spatiotemporal points as drug molecules travel through the renal vasculature, tubules, and cells, posing a significant challenge in accurately describing and predicting renal drug disposition. Toward this end, a physiologically based kidney model serves as a promising tool to combine the anatomical and physiological features of the kidney (eg, tubular flow rate, pH, and transporter expression) with the unique properties of drugs (eg, protein binding, lipophilicity, ionization, and transporter substrate) to capture the dynamic system-drug interactions. Despite the exciting progress over the past several decades, physiologically based pharmacokinetic modeling has overall been predominantly used to predict intestinal absorption and hepatic drug-drug interaction. In comparison, pharmacokinetic modeling of renal drug handling has been underappreciated. In this review, we first provide an overview of kidney function and physiology, renal clearance mechanisms, and the evolutionary history of the physiologically based mechanistic kidney model. We then summarize the recent efforts spent in different areas of kidney model application, particularly: (1) renal transporter-mediated drug-drug interaction, (2) disease effect from both renal and hepatic impairment, and (3) model applications across the lifespan (eg, pediatrics and geriatrics). Finally, we identify remaining knowledge gaps, future directions, and potential model utilities. SIGNIFICANCE STATEMENT: This review summarizes pharmacokinetic model case studies that are related to renal drug disposition, illustrating the current framework of modeling renal drug handling, highlighting knowledge gaps in predicting renal transporter-mediated drug-drug interactions, and modeling the effects of disease and age on renal drug handling. A discussion on robust model validation and areas for future directions is also provided.