Lysosomes play a key role in the accumulation, catabolism, and transport of endogenous and exogenous metabolites and proteins and are involved in drug metabolism and prodrug activation. However, the protein abundance and interindividual variability of lysosomal drug-metabolizing enzymes and transporters (DMETs) remain underexplored. In this study, we performed a global proteomics analysis of the enriched human liver lysosomal fraction to characterize and annotate lysosomal proteins and compared these results with the proteomics data of hepatocyte homogenates and the liver microsomal fraction. We annotated and quantified 66 hydrolases and 41 membrane transporters in the lysosomal fractions. These included proteins involved in prodrug activation, transport, and metabolism or functioning as drug targets. After confirming the identity of lysosomal proteins, we investigated age-dependent changes in the abundance of these proteins in human hepatocytes (n = 58) across various age groups, ranging from neonatal (0-12 days) to adulthood (>18 years). We observed age-specific variations in the expression of key hydrolases (CTSA, CTSL, NAGLU, PLD3, and GALNS) and transporters (ATP6V1B2, ATP6V1C1, TMEM63A, and SLC39A14). Together, these findings highlight the lysosomal localization of proteins involved in drug disposition and their dynamic developmental changes, providing critical insights for refining physiologically based pharmacokinetic (PBPK) models to support precision dosing and improve therapeutic outcomes in pediatric populations.

The International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group analyzed survey results submitted by 17 member companies describing in vitro and in vivo data for drug-drug interactions (DDI) based on drug transporter inhibition. Trends for in vitro-in vivo correlation and impact of physicochemical properties on potential for clinical inhibition were explored for intestinal (P-glycoprotein/breast cancer resistance protein), hepatic (organic anion transporting polypeptide [OATP]1Bs), and renal (organic cation transporter 2/organic anion transporter/multidrug and toxin extrusion proteins) drug transporters. The dataset comprised 58 clinical inhibition studies involving 42 compounds as DDI perpetrators, balanced across Biopharmaceutics Classification System/Biopharmaceutical Drug Disposition Classification System classes and therapeutic areas. Studies were often triggered by in vitro data indicating potential clinical DDI risk or based on anticipated comedications. Overall findings suggest that the magnitude of transporter-mediated drug interactions was relatively low for the majority of the studies (<2-fold increase in exposures). Larger area under the curve, Cmax, or renal clearance changes in the presence of inhibitors were often seen with compounds that inhibited more than 1 pathways. Interactions >2-fold were only reported for statin probe substrates with OATP1B, breast cancer resistance protein, and/or CYP3A4 inhibitors. Consistent with previous reports, low false negative and high false positive rates were observed when applying static cutoff criteria suggested by regulatory agencies for both P-glycoprotein and OATPs. Lastly, the physicochemical analyses demonstrated that clinical inhibitors of P-glycoprotein and breast cancer resistance protein tended to be more lipophilic than noninhibitors (median log D, 2.9 vs 1.7), and OATP1B1/1B3 inhibitors also tended to have higher molecular weights (median, 700 vs 530 Da). This work highlights current strategies for identifying transporter-mediated DDI risks and the need to incorporate additional approaches, such as biomarker profiling and predictive modeling, for nuanced insights. SIGNIFICANCE STATEMENT: A diverse dataset comprising 58 clinical studies evaluating transporter inhibition showed that inhibitors of P-glycoprotein, breast cancer resistance protein, and organic anion transporting polypeptide-1B transporters tend to be more lipophilic and larger than noninhibitors. Greater than 2-fold interactions were generally observed with substrates involving multipathway inhibitors of organic anion transporting polypeptide-1B, breast cancer resistance protein, and/or CYP3A. Low false negative and high false positive rates were observed when applying the static cutoff values in the regulatory guidance, indicating adequacy of static approach with a role for additional approaches such as modeling or biomarkers for nuanced insights.

Cytochrome P450 (CYP) enzymes play a central role in drug metabolism. Understanding the contribution of individual CYP isoforms, known as CYP reaction phenotyping, is essential for elucidating metabolic pathways and predicting drug-drug interactions. Reversible inhibitors are commonly used in human liver microsome (HLM)-based CYP reaction phenotyping. However, selecting appropriate inhibitor concentrations is challenging as experimental results will vary depending on the HLM concentration, substrate concentration, and incubation duration selected. These factors collectively influence the observed inhibitory potency, often complicating data interpretation. The use of mechanism-based inactivators (MBIs) of P450 enzyme can potentially overcome these issues. However, the lack of standardized rigorously evaluated protocols and concerns about off-target inhibition have limited the broader application of MBIs for reaction phenotyping. This study systematically evaluated assay conditions for the use of P450 isoform-selective MBIs across the 7 major hepatic P450 isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) to establish an HLM concentration-independent, universal protocol for CYP reaction phenotyping. Inactivation kinetics were determined, and potential off-target P450 inhibition of each MBI across all 7 P450 isoforms was assessed. Optimized assay conditions were established for each P450 isoform-MBI pair, including HLM concentration, MBI concentration, and incubation time. Under these optimized assay conditions, all selected MBI demonstrated potent and selective inhibition of their target P450 isoforms, demonstrating ≥90% activity loss and minimal off-target inhibition for most MBIs (none >16%). Overall, the established protocol provides a robust and reproducible framework for HLM-based P450 reaction phenotyping using MBIs. This approach enhances the specificity of P450 isoform contribution assessments and supports improved drug-drug interaction prediction in early drug development. SIGNIFICANT STATEMENT: Mechanism-based inactivators offer a powerful approach for defining individual P450 isoform contributions. This study systematically optimized mechanism-based inactivator assays across all major hepatic P450 isoforms in human liver microsomes (HLMs), establishing a unified protocol that balances inactivator concentration, HLM level, and incubation duration to achieve ≥90% target inhibition with minimal off-target effects. The resulting framework improves assay specificity, enhances reliability of P450 reaction phenotyping in HLM, and supports more accurate prediction of metabolic pathways and drug-drug interaction risk.

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 (Km = 39 μM) and OAT3 (Km = 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.

Over the last several decades, efforts in medicinal chemistry have aimed to reduce the extent of CYP metabolism of new chemical entities. This approach, however, has led to increased susceptibility to metabolism by non-CYP-mediated pathways, particularly involving other phase I enzymes such as aldehyde oxidase (AO). Commonly used in vitro models, such as suspended or cocultured primary human hepatocytes, have limitations in evaluating the disposition of compounds metabolized by AO due to low or variable levels of enzyme activity. Thus, an in vitro model that exhibits high to moderate levels of AO activity that can better predict the contribution of AO to drug metabolism and its impact on drug clearance is needed. A novel, 2D+ primary human hepatocyte model, TruVivo, was evaluated for its potential utility to improve hepatic clearance (CLh) predictions and determine the contribution of AO to drug metabolism in humans. TruVivo demonstrated stable levels of AO activity for at least 2 weeks that were higher than levels in other hepatocyte models. CLh predictions generated using TruVivo for the reference compounds carbazeran, zoniporide, zaleplon, and O6-benzylguanine were within 2-fold of reported in vivo CL values. Furthermore, the estimated fraction metabolized by AO for zaleplon and zoniporide was within 25% of reported in vivo values, whereas that for carbazeran and O6-benzylguanine was similar to those generated in other systems.These findings suggest TruVivo may offer a novel means to assess CLh of AO substrates more accurately, even over extended incubation times for low clearance compounds. Significance Statement The TruVivo in vitro primary human hepatocyte model maintains high levels of aldehyde oxidase (AO) activity for at least 14 days in culture, making the system suitable for evaluating slowly metabolized compounds, particularly those metabolized by AO. This novel system may therefore be useful for improving human clearance predictions for AO substrates.

Iclepertin was a promising drug candidate being developed for the treatment of cognitive impairment associated with schizophrenia. Interestingly, the predominant major metabolite in vivo in humans, BI 761036 (M232), was not observed after incubations in hepatocytes or hepatic subcellular fractions. Subsequent studies revealed that the metabolic pathway leading to the formation of M232 involves 2 sequential oxidation steps. First, iclepertin undergoes CYP3A-mediated oxidation to form the unstable carbinolimide (M526), followed by plasma-mediated imide hydrolysis to release M232 and a minor metabolite, M312. The putative plasma imidase responsible for the hydrolysis of M526 was identified by incubating M526 in plasma with selective inhibitors of butyrylcholinesterase, paraoxonase, and human serum albumin (HSA) and by monitoring the hydrolysis of M526 in the presence of purified or recombinant esterases. M526 was exclusively hydrolyzed by HSA in plasma to form M232, demonstrating for the first time that imide hydrolysis by HSA contributes to the formation of a major human drug metabolite. The potential impact of HSA-mediated hydrolysis of M526 on the stability of M232 plasma concentrations in plasma samples collected from individuals administered a single dose of iclepertin (25 mg) was also investigated in the context of incurred sample reanalysis. These findings highlight the unexpected metabolic role of HSA in the generation of a major metabolite of iclepertin. Significance Statement Human serum albumin exclusively hydrolyzed the intermediate metabolite of iclepertin, M526, leading to the formation of M232, a major metabolite of iclepertin. This discovery marks the first evidence of human serum albumin’s direct involvement in the formation of a major human drug metabolite, underscoring the significant and previously unrecognized metabolic role of human serum albumin.

Uridine 5'-diphospho-glucuronosyltransferase (UGT) reaction phenotyping studies have posed significant challenges due to the limited availability of isoform-selective inhibitors. This recognized gap in reagent availability impedes the accurate determination of the contribution of specific UGT isoforms to the metabolism of UGT substrates. To address this challenge, 9 antibiotics were evaluated for their potential inhibitory effects on UGT isoforms. We identified 2 macrolide antibiotics, troleandomycin and erythromycin, as potent and selective inhibitors of UGT1A3 and UGT1A8, respectively. The mechanism of UGT inhibition by troleandomycin and erythromycin was investigated using recombinant UGT1A3 (mefenamic acid as probe substrate) and UGT1A8 (7-hydroxy-4-(trifluoromethyl)coumarin and apigenin as probe substrates). The results revealed a mixed-type inhibition mechanism, where troleandomycin and erythromycin allosterically inhibit UGT1A3 and UGT1A8, respectively. A slight positive cooperativity between erythromycin and substrate binding to UGT1A8 and a slight negative cooperativity between troleandomycin and substrate binding to UGT1A3 was observed. At saturating inhibitor concentrations, greater than 90% inhibition of glucuronidation catalyzed by UGT1A3 and UGT1A8 was observed. To validate these findings in human liver microsomes and human intestinal microsomes, telmisartan, a selective substrate of UGT1A3 and UGT1A8, was utilized. Similar to the results in expressed UGT isoforms, troleandomycin selectively inhibited UGT1A3 in human liver microsomes and erythromycin selectively inhibited UGT1A8 in human intestinal microsomes. The identification of these UGT isoform-selective inhibitors provides researchers with important new tools expanding the utility of in vitro UGT reaction phenotyping studies. SIGNIFICANCE STATEMENT: Identification of uridine 5'-diphospho-glucuronosyltransferase (UGT)1A3 (troleandomycin) and UGT1A8 (erythromycin) selective inhibitors addresses an important and heretofore unmet need for in vitro reaction phenotyping studies by facilitating the determination of the contribution of these enzymes to drug glucuronidation in humans.

Human α-1-acid glycoprotein (hAGP) immobilized directly on NHS ester (N-hydroxysuccinimide) activated magnetic beads (hAGP-beads) was developed as a novel tool for studying small molecule hAGP binding. This method offers a straightforward, one-step immobilization process compared to traditional biotin-streptavidin immobilization technique. The hAGP-beads system provides a rapid and convenient alternative to conventional methods like equilibrium dialysis (ED) or ultracentrifugation (UF) for assessing hAGP small molecule interactions. Characterization and evaluation of the hAGP-beads system revealed that equilibrium dissociation constant (Kd) values of various small molecules obtained by hAGP-beads method correlated well with those determined by ED. This result suggests that the conformation of hAGP binding site is not altered after hAGP covalently binds to magnetic beads through NHS ester conjugation. Key advantages of the hAGP-beads method include shorter assay incubation times compared to ED ( 3 min versus 4–6 h) and the ability to quantify both free and hAGP-bound small molecule species, facilitated by simple magnetic separation. Furthermore, long-term storage tests demonstrated that hAGP-beads remain stable and retain its binding functionality at -80°C, significantly reducing the need for frequent preparations. These properties make the hAGP-beads system not only well-suited for general hAGP-related studies, such as small molecule binding, but especially advantageous for high-throughput screening applications.

Uridine 5'-diphospho-glucuronosyltransferase (UGT) reaction phenotyping studies have posed significant challenges due to the limited availability of isoform-selective inhibitors. This recognized gap in reagent availability impedes the accurate determination of the contribution of specific UGT isoforms to the metabolism of UGT substrates. To address this challenge, 9 antibiotics were evaluated for their potential inhibitory effects on UGT isoforms. We identified 2 macrolide antibiotics, troleandomycin and erythromycin, as potent and selective inhibitors of UGT1A3 and UGT1A8, respectively. The mechanism of UGT inhibition by troleandomycin and erythromycin was investigated using recombinant UGT1A3 (mefenamic acid as probe substrate) and UGT1A8 (7-hydroxy-4-(trifluoromethyl)coumarin and apigenin as probe substrates). The results revealed a mixed-type inhibition mechanism, where troleandomycin and erythromycin allosterically inhibit UGT1A3 and UGT1A8, respectively. A slight positive cooperativity between erythromycin and substrate binding to UGT1A8 and a slight negative cooperativity between troleandomycin and substrate binding to UGT1A3 was observed. At saturating inhibitor concentrations, greater than 90% inhibition of glucuronidation catalyzed by UGT1A3 and UGT1A8 was observed. To validate these findings in human liver microsomes and human intestinal microsomes, telmisartan, a selective substrate of UGT1A3 and UGT1A8, was utilized. Similar to the results in expressed UGT isoforms, troleandomycin selectively inhibited UGT1A3 in human liver microsomes and erythromycin selectively inhibited UGT1A8 in human intestinal microsomes. The identification of these UGT isoform-selective inhibitors provides researchers with important new tools expanding the utility of in vitro UGT reaction phenotyping studies. Significance Statement Identification of uridine 5'-diphospho-glucuronosyltransferase (UGT)1A3 (troleandomycin) and UGT1A8 (erythromycin) selective inhibitors addresses an important and heretofore unmet need for in vitro reaction phenotyping studies by facilitating the determination of the contribution of these enzymes to drug glucuronidation in humans.

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGTbeads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/ Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmaxis the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (Km), reversible inhibition (Ki), or inactivation (KI) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (fu,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high fu,mic value) and significant (low fu,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The fu,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high fu,mic values, all methods generated similar fu,mic values. However, fu,mic values varied markedly across assay formats when high binding to HLM occurred, where fu,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed.

In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (Km). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (kcat or Vmax). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of Km, kcat or Vmax without the need for authentic metabolite standards. This approach, using the carbon-14 [14C] labeled metabolite to calibrate the unlabeled metabolite (14C calibration method), combines radiometric with LC-MS/MS detection to generate both [14C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the 14C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using 14C calibration method and evaluated the enzyme kinetics of metabolite formation. The Km values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the 14C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites. In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC–MS/MS. Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.

OBJECTIVE:An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair.METHODS:In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations.RESULTS:The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR.CONCLUSIONS:This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.

Inhibition of sodium-glucose cotransporter-2 (SGLT2) has been shown to be a safe and efficacious approach to support managing Type 2 diabetes. In the 2-year carcinogenicity study with the SGLT2 inhibitor empagliflozin in CD-1 mice, an increased incidence of renal tubular adenomas and carcinomas was identified in the male high-dose group but was not observed in female mice. An integrated review of available nonclinical data was conducted to establish a mode-of-action hypothesis for male mouse-specific tumorigenesis. Five key events were identified through systematic analysis to form the proposed mode-of-action: (1) Background kidney pathology in CD-1 mice sensitizes the strain to (2) pharmacology-related diuretic effects associated with SGLT2 inhib ition. (3) In male mice, metabolic demand increases with the formation of a sex- and species-specific empagliflozin metabolite. These features converge to (4) deplete oxidative stress handling reserve, driving (5) constitutive cellular proliferation in male CD-1 mice. The proposed mode of action requires all five key events for empagliflozin to present a carcinogenicity risk in the CD-1 mouse. Considering that empagliflozin is not genotoxic in the standard battery of genotoxicity tests, and not all five key events are present in the context of female mice, rats, or humans, nor for other osmotic diuretics or other SGLT2 inhibitors, the observed male mouse renal tumors are not considered relevant to humans.