Drug Metabolism and Disposition最新文献

Vicadrostat, an aldosterone synthase inhibitor in development in combination with empagliflozin for chronic kidney disease, heart failure, and cardiovascular risk reduction, undergoes extensive hepatic glucuronidation primarily by UDP-glucuronosyltransferase (UGT)2B7 to form BI 689875, an ether glucuronide metabolite. Despite its hepatic formation, BI 689875 is predominantly excreted in urine, as determined in a human ADME study of vicadrostat. This study elucidated mechanisms underlying BI 689875 disposition in humans. BI 689875 was evaluated as a substrate of various drug transporters using transporter-expressing membrane vesicles and HEK293 cells. BI 689875 was identified as a substrate of MRP2, MRP3, MRP4, BCRP, OAT3, OATP1B1, and OATP1B3, but not of P-gp, OAT1, OAT2, OAT4, MATE1, or MATE2-K. The affinity of BI 689875 for MRP3 (K_m = 39 μM) and OAT3 (K_m = 46 μM) was substantially greater than that for other uptake/efflux transporters (not saturable up to 300 μM). In vitro-in vivo extrapolation using a proteomics-informed approach correcting for in vitro versus in vivo transporter expressions revealed that MRP3- and OAT3-mediated intrinsic clearance values for BI 689875 were substantially higher than those of other transporters. These findings suggest that basolateral efflux via MRP3 is the dominant hepatic elimination pathway for BI 689875, explaining its minimal fecal excretion observed in the human ADME study. They also indicate that OAT3-mediated uptake is the primary renal elimination route, with renal basolateral uptake substantially higher than hepatic uptake, consistent with the preferential urinary elimination of BI 689875. Transporter interplay between hepatic MRP3 and renal OAT3 determines the primary route of BI 689875 disposition. SIGNIFICANCE STATEMENT: BI 689875, a glucuronide metabolite, is formed in the liver but eliminated in urine. Through proteomics-informed in vitro-in vivo extrapolation, hepatic MRP3 and renal OAT3 were identified as key contributors to its predominant urinary elimination, highlighting interorgan transporter interplay.

Proteolysis targeting chimeras (PROTACs), a class of targeted protein degraders, are advancing in clinical development, necessitating the accurate prediction of human pharmacokinetics (PK). This study developed a physiologically based pharmacokinetic (PBPK) modeling approach informed by in vitro to in vivo extrapolation to predict the human PK of 2 PROTACs: vepdegestrant (ARV-471) and bavdegalutamide (ARV-110). Bottom-up PBPK models were built in mouse (ARV-471), and in mouse, rat, and dog (ARV-110) using physicochemical and in vitro absorption, distribution, metabolism, and excretion data, including solubility, permeability from a modified Genentech Madin-Darby canine kidney cells assay with 4% bovine serum albumin, and liver microsomal intrinsic clearance (CL). In vitro to in vivo extrapolation gaps were identified and addressed using empirical scalars, including additional systemic CL and tissue partition coefficient scalars, to capture observed intravenous PK. Oral absorption and exposure in preclinical species were predicted using a mechanistic absorption model, assuming passive diffusion driven by total drug concentration. Based on the preclinical PBPK strategy, predicted human apparent CL after oral administration and apparent volume of distribution after oral dosing values for ARV-110 at 35 mg aligned within 2-fold of clinical observations. For ARV-471 at 30 mg oral dose, apparent volume of distribution after oral dosing predictions were within range, but apparent CL after oral administration was overpredicted. To improve alignment with the observed clinical PK, model refinement was limited to adjusting the additional systemic CL scalar, whereas absorption and distribution parameters remained unchanged. The refined PBPK models successfully simulated human oral PK within 2-fold of observed values across multiple doses (60-360 mg for ARV-471 and 70-140 mg for ARV-110). This PBPK modeling framework may support human PK prediction of PROTACs during late-stage drug discovery and development. SIGNIFICANCE STATEMENT: This study highlights that a physiologically based pharmacokinetic (PK)-in vitro to in vivo extrapolation strategy can reliably predict the human PK of proteolysis targeting chimeras, an emerging therapeutic class with complex absorption, distribution, metabolism, and excretion properties. Incorporating mechanistic absorption modeling and permeability data from modified in vitro assays (Genentech Madin-Darby canine kidney cells with 4% bovine serum albumin) improved oral absorption predictions, whereas the integration of multispecies preclinical PK data enhanced the translational accuracy of human PK predictions. Together, these findings establish a translational physiologically based PK framework for estimating oral exposure in first-in-human studies and supporting model-informed development of proteolysis targeting chimeras drug candidates.

The severe acute respiratory syndrome coronavirus-2 main protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), has been approved by the US Food and Drug Administration as an oral treatment option for coronavirus disease 2019 patients. In this perspective, we share the expediated absorption, distribution, metabolism, and excretion strategies, which were incorporated as part of discovery efforts, to design orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitors. PF-07321332 (nirmatrelvir) emerged as a potential oral clinical candidate within ∼ 6 months from the time discovery efforts were first initiated. The review also delves into a discussion around the successful use of quantitative fluorine-19 nuclear magnetic resonance spectroscopy in the characterization of the human mass balance and excretion pathways of nirmatrelvir. Human absorption, distribution, metabolism, and excretion data that emerged from the fluorine-19 nuclear magnetic resonance study were used to support the Emergency Use Authorization and new drug application filing, which was accepted by regulatory agencies worldwide. Efficient operational and technical strategies, incorporating the elements of speed without sacrificing data quality, which were crucial to the success of the program, are highlighted. SIGNIFICANCE STATEMENT: This perspective discusses the expedited absorption, distribution, metabolism, and excretion efforts utilized in the discovery and development of the orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitor nirmatrelvir, which in combination with the cytochrome P450 3A inhibitor ritonavir (Paxlovid), is used in the oral treatment of COVID-19. Paxlovid was granted an Emergency Use Authorization by global regulatory agencies in less than 2 years from the initiation of the discovery program and has since been fully approved by the US Food and Drug Administration.

The pregnane X receptor (PXR), a ligand-activated transcription factor, regulates the expression of genes involved in endobiotic and xenobiotic metabolism, inflammation, and fibrosis. Disruption of PXR functions can affect processes critical to metabolic dysfunction-associated steatohepatitis (MASH) progression. Although ligand-dependent PXR functions are well studied, its regulation by post-translational modification, particularly phosphorylation, remains unclear. PXR has a conserved phosphorylation motif within its ligand binding domain (Ser347 in mice; Ser350 in humans). In vitro studies showed that this site mutation impairs human PXR transcriptional activity; however, the mechanism remains elusive. To investigate this phosphorylation site role in MASH development, wild-type and PXR Ser347Ala knock-in mutation (PXR-KI) mice were fed either a high-fat diet or a control chow diet for 16 weeks. On control chow diet, PXR-KI mice exhibited decreased expression of alternative bile acid (BA) synthesis genes compared with wild-type mice. On a high-fat diet, PXR-KI mice manifested more severe hepatic steatosis, revealed by elevated serum total cholesterol, and increased expression of genes involved in lipid metabolism. In addition, changes in BA metabolism and transporter genes suggested a cholestatic pattern in this group of mice. BA profiling showed higher levels of conjugated, hydrophilic, primary BA in the serum and liver, and increased unconjugated BA in the intestine. The data suggest that PXR Ser347 phosphorylation motif is essential for regulating PXR functions to maintain endobiotic metabolism and alleviate hepatotoxicity during MASH progression. SIGNIFICANT STATEMENT: The ligand-independent role of pregnane X receptor (PXR) is unclear. In phosphodeficient PXR knock-in mice, loss of Ser347 phosphorylation worsened hepatic steatosis and altered bile acid homeostasis under high-fat diet feeding, uncovering a novel role and therapeutic potential of PXR phosphorylation in fatty liver diseases.

In our previous study, long-term cocultured hepatocytes were used to estimate the fraction of a drug metabolized by CYP3A4 (f_m,CYP3A4). Metabolic turnover was measured with and without a CYP3A4 selective inhibitor, and the results were verified against in vivo reference data. The current study followed a similar approach using direct or time-dependent inhibitors to evaluate f_m,CYP1A2, f_m,CYP2C8, f_m,CYP2C9, f_m,CYP2C19, and f_m,CYP2D6 for a set of marketed drugs. The used inhibitors were for CYP1A2 (20 μM furafylline), CYP2C8 (40 μM montelukast), CYP2C9 (40 μM sulfaphenazole), CYP2C19 (3 μM (-)N-3-benzyl-phenobarbital), and CYP2D6 (5 μM quinidine). We found that in vitro f_m values above 0.5 were comparable to in vivo values, falling within a 0.5 to 2-fold error in 9 of 11 CYP1A2 substrates, 5 of 8 CYP2C8 substrates, 5 of 8 CYP2C9 substrates, 2 of 3 CYP2C19 substrates, and 11 of 20 CYP2D6 substrates. The study also showed how uncertainty in measured metabolic turnover affects the estimated f_m,CYPs, revealing that when estimated f_m errors are <25%, 89% of predictions are within 2-fold of in vivo f_m, but this drops to 40% when there is higher uncertainty in measured turnover. Although some f_m values were poorly predicted and clinical studies revealed off-target inhibition by certain inhibitors, the chemical inhibition approach using human long-term cocultured hepatocytes showed useful prediction performance for early drug discovery enabling moderate-to-sensitive drug-drug interaction risk assessments, when metabolic turnover is adequate, and inhibitor selectivity is well defined. SIGNIFICANCE STATEMENT: Calculating in vitro fraction metabolized by cytochrome P450 enzymes in liver is vital in drug discovery for assessing the object drug-drug interaction risk of new chemical entities metabolized by cytochrome P450 enzymes before clinical data are available. Despite some limitations, the current study demonstrated that using long-term cocultured hepatocytes with chemical inhibitors is a reliable method for estimating fraction metabolized by cytochrome P450 enzymes in liver, complementing the drug interaction risk assessment.

Organic cation transporter 1 (OCT1, SLC22A1) is a key determinant in the hepatic disposition of cationic drugs, primarily supported by pharmacogenomic studies. However, evidence for OCT1-mediated drug-drug interactions (DDIs) remains limited. This study aimed to elucidate the role of OCT1 in DDIs using cynomolgus monkeys through comprehensive in vitro and in vivo experiments. Cynomolgus monkey OCT1 (cOCT1) shares 94.2% amino acid identity with human OCT1 (hOCT1). Transport assays in transfected human embryonic kidney 293 cells showed that sumatriptan, fenoterol, metformin, quinidine, and 1-methyl-4-phenylpyridinium were transported by cOCT1 at rates comparable to hOCT1 (less than 2-fold difference). The K_m and V_max values for cOCT1-mediated transport of sumatriptan and fenoterol were similar or within 2-fold to those of hOCT1 (K_m: 188 ± 56 vs 178 ± 25 and 1.6 ± 0.48 vs 0.73 ± 0.47 μM, respectively, V_max: 49.4 ± 8.3 vs 83.9 ± 5.2 and 124 ± 8.9 vs 158 ± 22 pmol/min per mg, respectively). Inhibition studies demonstrated that quinidine, rifamycin SV, and ketoprofen inhibited sumatriptan uptake in monkey hepatocytes to a similar extent as in human hepatocytes, with IC₅₀ values within a 2- to 3-fold range. In addition, axitinib, nintedanib, and erlotinib were identified as inhibitors of both cOCT1 and hOCT1. In vivo, coadministration of axitinib (15 mg/kg), nintedanib (40 mg/kg), and erlotinib (15 mg/kg) increased sumatriptan area under the plasma concentration-time curve from zero to 24 hours by 1.3, 2.0, and 1.9-fold, respectively, compared with sumatriptan alone (2 mg/kg). These findings underscore the crucial role of OCT1 in the hepatic disposition and DDIs of cationic drugs, and indicate that cynomolgus monkeys may serve as a valuable model for studying OCT1-mediated drug disposition and interactions. SIGNIFICANCE STATEMENT: This study provides the first evidence that cynomolgus monkey organic cation transporter 1 (OCT1) transport and inhibition characteristics closely align with its human ortholog. Consistent with our in vitro findings, coadministration of OCT1 inhibitors (axitinib, nintedanib, and erlotinib) significantly increased the systemic exposure of sumatriptan in monkeys. These findings offer valuable insights into the role of OCT1 in drug-drug interactions and highlight the potential of cynomolgus monkeys as a useful and potentially translational model for OCT1-mediated disposition and interactions.

This study examined the absorption, distribution, metabolism, and excretion of branaplam in healthy adult male volunteers and, additionally, compared the adult metabolite profiles obtained in plasma and urine to those obtained in infants with type 1 spinal muscular atrophy. Six volunteers received a single oral dose of 140 mg ¹⁴C-branaplam. Blood, plasma, urine, and fecal samples were analyzed using liquid scintillation counting, accelerator mass spectrometry, and liquid chromatography coupled with high-resolution or tandem mass spectrometry to assess radioactivity, generate metabolite profiles, and structurally characterize branaplam metabolites. Pediatric samples from various age groups were also evaluated. Mechanistic in vitro experiments enabled direct comparison between adult and pediatric results. The ¹⁴C-branaplam dose was well tolerated. Pharmacokinetic analysis showed that branaplam and metabolite UFB112 were the main circulating species, exhibiting delayed T_max (10 and 28 hours, respectively) and prolonged half-lives (218 and 199 hours, respectively). UFB112 formation was exclusively catalyzed by CYP3A4, and its plasma levels increased with age, reflecting hepatic enzyme maturation. Branaplam was primarily eliminated through metabolism. Renally excreted metabolites were formed via oxygenation, glucuronidation, glucosidation, or ribose conjugation, whereas metabolites in feces included glucosidation and oxidative products. Mass balance was almost complete, with 86.6% of the administered radioactivity recovered in urine and feces over 47 days. These findings highlight the pharmacokinetic behavior of branaplam and UFB112, including the role of the 2,2,6,6,-tetramethylpiperidine-1-oxyl moiety, in how metabolism of branaplam changes during physiological development. Mechanistic insights confirm that CYP enzyme ontogeny significantly influences metabolic profiles. SIGNIFICANCE STATEMENT: This study provides a comprehensive overview of the metabolism of the tetramethyl piperidine moiety, contextualizing enzyme maturation by comparing metabolic fates in infants and adults. It also clearly explains human metabolism of branaplam and summarizes a rare Adenosine Triphosphate pathway observed in these studies.

Mycobacterium abscessus (Mab, MAB) poses a rising health threat worldwide. Infections occur in hospital settings, affecting immunocompromised and immunocompetent patients alike. Individuals with underlying lung diseases, such as cystic fibrosis and bronchiectasis, are particularly at risk. Mab is intrinsically multidrug resistant to most antibiotics, has high treatment failure to current treatment regimens, and lacks a vaccine. Targeting bacterial metabolism has historically resulted in successful therapies. We discovered that the cytochrome P450 isoform CYP123 encoded by MAB_1216c is required for host infection. To determine the role of CYP123 during host infection, we generated highly active recombinant CYP123 and found CYP123 interactions with steroid hormones, which are key players in host immune response. All tested steroids induced a reverse type I shift when titrated to the enzyme. Their binding affinity was dictated by the presence of hydroxyl groups at certain positions in the steroid scaffold. Metabolism assays with a surrogate redox system revealed that CYP123 is a steroid hydroxylase and can convert 11-deoxycorticosterone and progesterone to a single monohydroxylated product, respectively. Mab infection has been associated with fungal coinfection, and cytochrome P450 enzymes have been shown to interact with azoles. We found that CYP123 binds to various triazole and azole drugs in the low micromolar range. Our results indicate that Mab CYP123 can interfere with host endobiotics with a potential implication in host cell reprogramming and can bind antifungal therapeutics possibly leading to worse polymicrobial infections. CYP123 could emerge as a potential drug target for an orthogonal approach to treating Mab infections. SIGNIFICANCE STATEMENT: Infections with the pathogen Mycobacterium abscessus are on the rise with limited treatment options. The M. abscessus cytochrome P450 CYP123 was identified to play an essential role for host infection. Steroids do not only bind to CYP123 but are also metabolized to monohydroxylated products implicating the potential to interfere with steroidogenesis and immune antagonism by this bacterium.

Arachidonic acid (AA) is a polyunsaturated essential fatty acid and a precursor for eicosanoids. It is metabolized by cyclooxygenases, lipoxygenases, and cytochrome P450 (P450) enzymes, which convert AA into hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), chiral eicosanoids with distinct biological activities. Although racemic HETEs and EETs have been studied in cardiovascular diseases, the enantiospecific roles of their enantiomers and the enantioselectivity of P450 enzymes remain largely unexplored. This study aimed to investigate the enantioselective metabolism of AA by human recombinant P450 enzymes, focusing on the formation of R/S-HETEs and (R, S)/(S,R)-EETs. Metabolites were analyzed using liquid chromatography electrospray ionization mass spectrometry. CYP1A2 exhibited the highest activity in forming R-midchain HETEs, followed by CYP3A4. CYP2C19 was the most active enzyme in producing R-subterminal HETEs, with CYP1A2 and CYP1A1, CYP4F3B, and CYP2E1 ranking second. Similarly, CYP2C19 showed the highest activity in generating S-midchain and S-subterminal HETEs, with CYP3A4, CYP2C8, CYP1A1, and CYP1A2 contributing to varying degrees. For EETs, CYP2C19 and CYP1A2 primarily catalyzed the formation of both (R, S)/(S, R)-EETs. These findings emphasize the significant roles of CYP2C19 and CYP1A2 in the regio- and stereoselective metabolism of HETEs and EETs, highlighting their contributions to lipid signaling and potential physiological implications. SIGNIFICANT STATEMENT: This work highlights the importance of profiling P450 with respect to their enantioselectivity in arachidonic acid metabolism. The findings indicate that major P450 differ in the magnitude of their hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acid formation rates, which is a significant for studying diseases that is known to be influenced by alterations in these pathways. Altered enantioselectivity could have implications in diseases such as hypertension, cancer, inflammation, and cardiovascular disorders.

Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.