Drug Metabolism and Disposition最新文献

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.

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.

3-dimensional (3D) primary hepatic spheroid cultures better mimic in vivo liver architecture and maintain a more stable cellular phenotype than the current gold standard of sandwich-cultured primary human hepatocytes. Therefore, they are a promising in vitro model for CYP enzyme induction and metabolism. This study aimed to evaluate long-term stability and suitability of primary human hepatic spheroids for regulatory-relevant in vitro applications, focusing on CYP enzyme metabolism and induction. Assay-ready spheroids were purchased from InSphero AG in monocultured, cocultured, single-, and multidonor formats. These were compared with 2-dimensional (2D) sandwich-cultured hepatocytes of the same donor. Basal activity of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2B6, CYP2D6, CYP3A4, CYP2J2, UDP-glucuronosyltransferases, UGT1A1, and sulfotransferases was successfully measured in spheroids. Further, CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2B6, and CYP3A4 were inducible by prototypical inducers at mRNA levels. The 6-fold induction threshold recommended by regulatory guidelines was exceeded in 4 of 5 donors. CYP1A2 (9- to 16-fold), CYP2B6 (6- to 15-fold), and CYP3A4 (7- to 35-fold) were robustly induced, qualifying the spheroid model for in vitro induction studies, despite lower induction levels compared with 2D cultures. No significant differences were observed between monocultured and cocultured spheroids. Spheroids maintained stable morphology and CYP enzyme activity for up to 4 weeks. Overall, primary human hepatic spheroids demonstrated suitability for CYP induction studies and for assessing compound metabolism in short- and long-term in vitro applications. A comprehensive assessment of how culture dimensionality and cellular composition affect CYP enzyme metabolism and induction is provided, supporting that spheroid cultures can be suitable for in vitro drug metabolism and pharmacokinetics applications. SIGNIFICANCE STATEMENT: This study explores the impact of culture dimensionality and cellular composition on CYP enzyme metabolism and induction. Primary human hepatocytes were characterized as monocultured, cocultured, single-, and multidonor 3-dimensional (3D) spheroids and compared with 2-dimensional primary human hepatocyte culture. The study provides a first thorough characterization of InSphero 3D InSight Human Liver Microtissues for suitability in in vitro drug metabolism and pharmacokinetics studies. By addressing interdonor variability and long-term functionality, the findings support the enhanced relevance and applicability of 3D liver models in drug development.

Organic anion transporting polypeptide (OATP) 1B3 plays a clinically significant role in hepatic drug disposition. Lysine acetylation, a key post-translational modification, has not been investigated for OATP1B3. This study determined the lysine acetylation status of OATP1B3 by proteomics and assessed the impact of inhibition of lysine deacetylase (KDAC) 6, a major cytosolic KDAC, on OATP1B3 acetylation and transport function. Proteomics revealed 7 acetylation sites, including 5 with additional ubiquitin-like modifications, and 4 phosphorylation sites (T10, S293, S295, S683). In human embryonic kidney 293 (HEK293)-Myc-FLAG-OATP1B3 cells, preincubation with the selective KDAC6 inhibitor tubacin (TBC) (5 μM, 24 hours), markedly reduced OATP1B3-mediated transport of [³H]cholecystokinin-8 (CCK-8), a specific substrate, and [³H]estradiol-17β-D-glucuronide to 0.15 ± 0.03-fold and 0.19 ± 0.01-fold of the control, respectively, without affecting OATP1B3 mRNA, protein levels, or membrane localization determined by real-time reverse transcription polymerase chain reaction, immunoblotting, and confocal microscopy. TBC treatment increased K664 acetylation to 2.12 ± 1.03-fold of the control (P < .05). Consistently, the acetylation-mimetic K664Q variant exhibited reduced transport compared with the acetylation-null K664R variant (P < .05). Treatment with a second KDAC6 selective inhibitor, WT-161 (3 μM, 5 hours), similarly reduced OATP1B3-mediated [³H]CCK-8 transport. In cultured primary human hepatocytes, TBC treatment for 4, 8, and 24 hours decreased [³H]CCK-8 transport to 0.34 ± 0.02-fold, 0.27 ± 0.03-fold, and 0.37 ± 0.03-fold of the control, respectively (all P < .05). The study reveals a novel post-translational modification of OATP1B3 by lysine acetylation and demonstrates impaired transporter function following KDAC6 inhibition, likely involving increased acetylation at K664, thereby providing new insight into OATP1B3-mediated drug-drug interactions driven by KDAC6 activity. SIGNIFICANCE STATEMENT: This study identifies lysine acetylation as a novel post-translational modification of organic anion transporting polypeptide (OATP)1B3 and demonstrates that altered lysine acetylation following inhibition of lysine deacetylase 6 reduces OATP1B3 transport function. These findings provide a mechanistic basis for altered hepatic drug disposition and highlight a new pathway through which drug-drug interactions involving OATP1B3 may occur.

Recent clinical success for RNA therapeutics supports the promise of these modalities to unlock additional drug targets with pharmacodynamic responses that are considerably more durable than other drug classes. Metabolic clearance in tissue is a key determinant of dosing requirements and therapeutic durability. To build our understanding of the mechanisms of RNA metabolic clearance, we quantified the catalytic efficiency of 3'- and 5'-exonucleases, and endonucleases in S9 fractions from various rat tissues using novel fluorescent RNA probes. We validated the specificity of these probes using recombinant nucleases and rat liver S9 fractions, demonstrating their ability to accurately report enzyme activity without cross-reactivity between nuclease classes. Key experimental parameters, such as solution pH and enzyme-substrate ratios, were optimized to maximize dynamic range. Profiling nuclease activity across various rat tissues revealed tissue-specific variations, with kidney, muscle, and plasma showing the highest catalytic efficiency for 3' exonuclease, 5' exonuclease, and endonuclease, respectively. Using a model siRNA targeting hypoxanthine phosphoribosyltransferase, comparative degradation studies in rat liver homogenate, liver S9 fractions, and liver tritosomes revealed divergent metabolic profiles; S9 fractions and homogenate processed both double-stranded siRNA and single-stranded antisense RNA to a similar extent, whereas in tritosomes single-stranded RNA was observed to be degraded more rapidly than a double-stranded form. These differences are consistent with distinct nuclease activities in each compartment, reflecting both enzyme identity and relative abundance as revealed by the probe analyses. Collectively, these findings offer critical mechanistic insights into RNA metabolism and establish a robust platform to improve the development and predictability of siRNA therapeutics. SIGNIFICANT STATEMENT: Metabolic clearance of RNA therapeutics is a critical determinant of their pharmacological durability and potency. This work quantitatively describes the activity of 3 major classes of ribonucleases within various tissues and subcellular compartments, revealing specific patterns of RNA metabolism and enable discovery of novel RNA therapeutics with improved pharmacokinetic properties.

A comprehensive understanding of the in vivo fate of low-molecular-weight polyethylene glycol (PEG) oligomers is essential for optimizing PEGylated therapeutics. This study delineates the polymerization-dependent absorption, distribution, metabolism, and excretion of discrete PEG600 oligomers (n = 9-18) in rats using a validated ultrahigh-performance liquid chromatography-tandem mass spectrometry method. Systemic exposure, assessed by dose-normalized area under the plasma concentration-time curve, was highest for shorter oligomers (n = 9-10) and declined for longer chains (n ≥ 17), whereas all oligomers exhibited similarly short terminal half-lives (12.7-15.2 minutes), indicating rapid elimination irrespective of chain length. Tissue distribution revealed pronounced renal accumulation, peaking at midchain lengths (n = 11-13), consistent with size-dependent glomerular filtration and tubular reabsorption. Oxidative metabolism yielded both monocarboxylated and dicarboxylated derivatives, confirming active enzymatic processing. Excretion studies demonstrated chain length-dependent elimination, with reduced recovery of longer oligomers, suggesting substantial in vivo biodegradation. These findings provide the first monomer-resolved absorption, distribution, metabolism, and excretion profile of PEG600 oligomers, highlighting the critical role of polymerization degree in governing their pharmacokinetics and informing the rational design of PEGylated drug formulations. SIGNIFICANCE STATEMENT: This study provides the monomer-resolved insight into the in vivo fate of polyethylene glycol 600 oligomers, revealing that chain length critically governs their pharmacokinetics, tissue distribution, and elimination. The identification of dual oxidative metabolic pathways and substantial biodegradation of longer oligomers offers guidance for the rational design and safety evaluation of PEGylated therapeutics.

Organic anion transporting polypeptide 1B1 (OATP1B1) is an important member of the solute carrier organic anion transporter family and is exclusively expressed on the basolateral membrane of human hepatocytes. As a membrane protein, its extracellular cysteine residues are very likely forming disulfide bonds. The cryo-electron microscopy structure of OATP1B1 reveals that 16 of its 20 cysteine residues are located on the extracellular side. Our present study showed that OATP1B1 has no free cysteines to react with the sulfhydryl-reactive biotinylation reagent maleimide-PEG₂-biotin. However, mutation of 1 of the 2 cysteines in each disulfide bond pair would release a free cysteine that can be labeled by maleimide-PEG₂-biotin. These results indicate that all 16 extracellular cysteine residues in OATP1B1 are involved in the formation of disulfide bonds. Among the 8 extracellular disulfide bonds, C430-C530 and C599-C613 are essential for the surface expression of OATP1B1, because their breakage makes OATP1B1 almost entirely retained intracellularly; disulfide bonds C142-C463, C465-C485, C474-C524, C489-C504, and C506-C459 have a significant effect on both surface expression and transport activity of OATP1B1 per se, whereas C162-C607 is dispensable for both surface expression and function of OATP1B1. In addition, the N-glycosylation status of OATP1B1 would be changed on disulfide bond breakage, which may play a role in rescuing the surface expression of disulfide bond-disrupted OATP1B1. Taken together, most extracellular disulfide bonds are crucial for normal surface expression and function of OATP1B1. SIGNIFICANCE STATEMENT: This study revealed that all extracellular cysteine residues in organic anion transporting polypeptide 1B1 (OATP1B1) form disulfide bonds and play important roles in the expression and function of OATP1B1. Nonsynonymous single nucleotide variations for some key cysteine residues of OATP1B1 such as C430, C530, C599, and C613 have been observed. Our current findings may provide a good basis to predict the in vivo function of OATP1B1 cysteine variants and potential OATP1B1-mediated adverse drug reactions in those individuals who carry these genetic variants.

The development of artificial intelligence (AI) tools and technology has made AI-driven drug discovery a more prominent field. We are firmly in the AI era, with hybrid designs that eventually comprise deep learning (DL) and conventional machine learning (ML). Although traditional models can predict ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties, they remain relatively unsuccessful, and improving the accuracy of predictions remains challenging. Recently, several researchers have developed a hybrid learning model that successfully addresses these problems and improves prediction accuracy. The systematic tendencies facing AI-powered transformation from conventional DL and ML to hybrid learning AI models are examined in this review. Compared with traditional ML and DL, hybrid AI models have increased efficiency by reducing drug development time and costs, and improved success rates. In this context, the ongoing development of new ADMET software based on hybrid AI and multimodeling techniques can enhance the accuracy of pharmacokinetic-pharmacodynamic predictions, improve ADMET endpoint predictions, and expedite the drug discovery of new chemical entities. Moreover, this review covers the future of AI in pharmaceutical sciences and ADMET predictions, including AI-driven prediction models that range from basic ML/DL to newly developed hybrid models, evaluation parameters, and their applications in ADMET property prediction. SIGNIFICANCE STATEMENT: The article covers the compilation of ongoing research in the development of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) software based on hybrid artificial intelligence and multimodeling techniques, which may increase the accuracy of pharmacokinetic-pharmacodynamic predictions, improve ADMET endpoint predictions, and accelerate drug discovery.

Pharmacokinetics (PKs) changes in the aging state are relatively common in clinical practice, but their underlying mechanisms remain unclear. This study aims to explore the potential effects of gut microbiota on PK of P450 probe drugs in old and young mice. A cocktail of probe drugs including phenacetin (PHE), midazolam (MID), dextromethorphan tartrate (DEX), and chlorzoxazone was gavage administered to control and pseudo-sterile old and young mice, and the PK parameters were compared. Subsequently, fecal microbiota transplantation (FMT) from young to old mice was performed to assess the impact of FMT on PK of the probe drugs. We observed that gut microbiota significantly affected the systemic exposure of PHE and MID, whereas age-related increase in DEX exposure in the old mice could be reversed by clearance of microbiota. No changes in PK parameters of the probe drugs were observed in old mice with FMT from young mice, suggesting that the alterations in PHE, MID, and DEX metabolism in the old mice could not be explained by unique microbiota from young mice. Our findings provide valuable guidance on how to improve the individualized medication for the elderly population. SIGNIFICANCE STATEMENT: This article offers new insights into the role of gut microbiota in the pharmacokinetic changes with aging, which is conducive to individualized medication for elderly patients and provides new insight for the research and development of drugs for elderly population.