Effects of the selective AMPA modulator NBI-1065845 on the pharmacokinetics of midazolam or ethinyl estradiol-levonorgestrel in healthy adults.

This parallel-arm, phase I study investigated the potential cytochrome P450 (CYP)3A induction effect of NBI-1065845 (TAK-653), an investigational α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator in phase II development for major depressive disorder. The midazolam treatment arm received the sensitive CYP3A substrate midazolam on Day 1, followed by NBI-1065845 alone on Days 5-13; on Day 14, NBI-1065845 was administered with midazolam, then NBI-1065845 alone on Day 15. The oral contraceptive treatment arm received ethinyl estradiol-levonorgestrel on Day 1, then NBI-1065845 alone on Days 5-13; on Day 14, NBI-1065845 was administered with ethinyl estradiol-levonorgestrel, then NBI-1065845 alone on Days 15-17. Blood samples were collected for pharmacokinetic analyses. The midazolam treatment arm comprised 14 men and 4 women, of whom 16 completed the study. Sixteen of the 17 healthy women completed the oral contraceptive treatment arm. After multiple daily doses of NBI-1065845, the geometric mean ratios (GMRs) (90% confidence interval) for maximum observed concentration were: midazolam, 0.94 (0.79-1.13); ethinyl estradiol, 1.00 (0.87-1.15); and levonorgestrel, 0.99 (0.87-1.13). For area under the plasma concentration-time curve (AUC) from time 0 to infinity, the GMRs were as follows: midazolam, 0.88 (0.78-0.98); and ethinyl estradiol, 1.01 (0.88-1.15). For levonorgestrel, the GMR for AUC from time 0 to the last quantifiable concentration was 0.87 (0.78-0.96). These findings indicate that NBI-1065845 is not a CYP3A inducer and support its administration with CYP3A substrates. NBI-1065845 was generally well tolerated, with no new safety signals observed after coadministration of midazolam, ethinyl estradiol, or levonorgestrel.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research.

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Unraveling the Ligand-Binding Sites of CYP3A4 by Molecular Dynamics Simulations with Solvent Probes.

Cytochrome P450 3A4 (CYP3A4) is one of the most important drug-metabolizing enzymes in the human body and is well known for its complicated, atypical kinetic characteristics. The existence of multiple ligand-binding sites in CYP3A4 has been widely recognized as being capable of interfering with the active pocket through allosteric effects. The identification of ligand-binding sites other than the canonical active site above the heme is especially important for understanding the atypical kinetic characteristics of CYP3A4 and the intriguing association between the ligand and the receptor. In this study, we first employed mixed-solvent molecular dynamics (MixMD) simulations coupled with the online computational predictive tools to explore potential ligand-binding sites in CYP3A4. The MixMD approach demonstrates better performance in dealing with the receptor flexibility compared with other computational tools. From the sites identified by MixMD, we then picked out multiple sites for further exploration using ensemble docking and conventional molecular dynamics (cMD) simulations. Our results indicate that three extra sites are suitable for ligand binding in CYP3A4, including one experimentally confirmed site and two novel sites.

Induction of hepatic CYP3A4 expression by cholesterol and cholic acid: Alterations of gene expression, microsomal activity, and pharmacokinetics.

Human cytochrome P450 3A4 (CYP3A4) is a drug-metabolizing enzyme that is abundantly expressed in the liver and intestine. It is an important issue whether compounds of interest affect the expression of CYP3A4 because more than 30% of commercially available drugs are metabolized by CYP3A4. In this study, we examined the effects of cholesterol and cholic acid on the expression level and activity of CYP3A4 in hCYP3A mice that have a human CYP3A gene cluster and show human-like regulation of the coding genes. A normal diet (ND, CE-2), CE-2 with 1% cholesterol and 0.5% cholic acid (HCD) or CE-2 with 0.5% cholic acid was given to the mice. The plasma concentrations of cholesterol, cholic acid and its metabolites in HCD mice were higher than those in ND mice. In this condition, the expression levels of hepatic CYP3A4 and the hydroxylation activities of triazolam, a typical CYP3A4 substrate, in liver microsomes of HCD mice were higher than those in liver microsomes of ND mice. Furthermore, plasma concentrations of triazolam in HCD mice were lower than those in ND mice. In conclusion, our study suggested that hepatic CYP3A4 expression and activity are influenced by the combination of cholesterol and cholic acid in vivo.

Clinical assessment of momelotinib drug-drug interactions via CYP3A metabolism and transporters.

Momelotinib-approved for treatment of myelofibrosis in adults with anemia-and its major active metabolite, M21, were assessed as drug-drug interaction (DDI) victims with a strong cytochrome P450 (CYP) 3A4 inhibitor (multiple-dose ritonavir), an organic anion transporting polypeptide (OATP) 1B1/1B3 inhibitor (single-dose rifampin), and a strong CYP3A4 inducer (multiple-dose rifampin). Momelotinib DDI perpetrator potential (multiple-dose) was evaluated with CYP3A4 and breast cancer resistance protein (BCRP) substrates (midazolam and rosuvastatin, respectively). DDI was assessed from changes in maximum plasma concentration (Cmax), area under the concentration-time curve (AUC), time to reach Cmax, and half-life. The increase in momelotinib (23% Cmax, 14% AUC) or M21 (30% Cmax, 24% AUC) exposure with ritonavir coadministration was not clinically relevant. A moderate increase in momelotinib (40% Cmax, 57% AUC) and minimal change in M21 was observed with single-dose rifampin. A moderate decrease in momelotinib (29% Cmax, 46% AUC) and increase in M21 (31% Cmax, 15% AUC) were observed with multiple-dose rifampin compared with single-dose rifampin. Due to potentially counteracting effects of OATP1B1/1B3 inhibition and CYP3A4 induction, multiple-dose rifampin did not significantly change momelotinib pharmacokinetics compared with momelotinib alone (Cmax no change, 15% AUC decrease). Momelotinib did not alter the pharmacokinetics of midazolam (8% Cmax, 16% AUC decreases) or 1'-hydroxymidazolam (14% Cmax, 16% AUC decreases) but increased rosuvastatin Cmax by 220% and AUC by 170%. Safety findings were mild in this short-term study in healthy volunteers. This analysis suggests that momelotinib interactions with OATP1B1/1B3 inhibitors and BCRP substrates may warrant monitoring for adverse reactions or dose adjustments.

The Impact of Baohuoside I on the Metabolism of Tofacitinib in Rats.

Purpose: To study the potential drug-drug interactions between tofacitinib and baohuoside I and to provide the scientific basis for rational use of them in clinical practice. Methods: A total of eighteen Sprague-Dawley rats were randomly divided into three groups: control group, single-dose group (receiving a single dose of 20 mg/kg of baohuoside I), and multi-dose group (receiving multiple doses of baohuoside I for 7 days). On the seventh day, each rat was orally administered with 10 mg/kg of tofacitinib 30 minutes after giving baohuoside I or vehicle. Blood samples were collected and determined using UPLC-MS/MS. In vitro effects of baohuoside I on tofacitinib was investigated in rat liver microsomes (RLMs), as well as the underlying mechanism of inhibition. The semi-inhibitory concentration value (IC50) of baohuoside I was subsequently determined and its inhibitory mechanism against tofacitinib was analyzed. Furthermore, the interactions between baohuoside I, tofacitinib and CYP3A4 were explored using Pymol molecular docking simulation. Results: The administration of baohuoside I orally has been observed to enhance the area under the concentration-time curve (AUC) of tofacitinib and decrease the clearance (CL). The observed disparity between the single-dose and multi-dose groups was statistically significant. Furthermore, our findings suggest that the impact of baohuoside I on tofacitinib metabolism may be a mixture of non-competitive and competitive inhibition. Baohuoside I exhibit an interaction with arginine (ARG) at position 106 of the CYP3A4 enzyme through hydrogen bonding, positioning itself closer to the site of action compared to tofacitinib. Conclusion: Our study has demonstrated the presence of drug-drug interactions between baohuoside I and tofacitinib, which may arise upon pre-administration of tofacitinib. Altogether, our data indicated that an interaction existed between tofacitinib and baohuoside I and additional cares might be taken when they were co-administrated in clinic.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Undifferentiated HepaRG cells show reduced sensitivity to the toxic effects of M8OI through a combination of CYP3A7-mediated oxidation and a reduced reliance on mitochondrial function.

The methylimidazolium ionic liquid M8OI (1-octyl-3-methylimidazolium chloride, also known as [C8mim]Cl) has been detected in the environment and may represent a hazard trigger for the autoimmune liver disease primary biliary cholangitis, based in part on studies using a rat liver progenitor cell. The effect of M8OI on an equivalent human liver progenitor (undifferentiated HepaRG cells; u-HepaRG) was therefore examined. u-HepaRG cells were less sensitive (>20-fold) to the toxic effects of M8OI. The relative insensitivity of u-HepaRG cells to M8OI was in part, associated with a detoxification by monooxygenation via CYP3A7 followed by further oxidation to a carboxylic acid. Expression of CYP3A7 - in contrast to the related adult hepatic CYP3A4 and CYP3A5 forms - was confirmed in u-HepaRG cells. However, blocking M8OI metabolism with ketoconazole only partly sensitized u-HepaRG cells. Despite similar proliferation rates, u-HepaRG cells consumed around 75% less oxygen than B-13 cells, reflective of reduced dependence on mitochondrial activity (Crabtree effect). Replacing glucose with galactose, resulted in an increase in u-HepaRG cell sensitivity to M8OI, near similar to that seen in B-13 cells. u-HepaRG cells therefore show reduced sensitivity to the toxic effects of M8OI through a combination of metabolic detoxification and their reduced reliance on mitochondrial function.

Genome-Wide Association Study of Atorvastatin Pharmacokinetics: Associations With SLCO1B1, UGT1A3, and LPP.

In a genome-wide association study of atorvastatin pharmacokinetics in 158 healthy volunteers, the SLCO1B1 c.521T>C (rs4149056) variant associated with increased area under the plasma concentration-time curve from time zero to infinity (AUC0-∞) of atorvastatin (P = 1.2 × 10-10), 2-hydroxy atorvastatin (P = 4.0 × 10-8), and 4-hydroxy atorvastatin (P = 2.9 × 10-8). An intronic LPP variant, rs1975991, associated with reduced atorvastatin lactone AUC0-∞ (P = 3.8 × 10-8). Three UGT1A variants linked with UGT1A3*2 associated with increased 2-hydroxy atorvastatin lactone AUC0-∞ (P = 3.9 × 10-8). Furthermore, a candidate gene analysis including 243 participants suggested that increased function SLCO1B1 variants and decreased activity CYP3A4 variants affect atorvastatin pharmacokinetics. Compared with individuals with normal function SLCO1B1 genotype, atorvastatin AUC0-∞ was 145% (90% confidence interval: 98-203%; P = 5.6 × 10-11) larger in individuals with poor function, 24% (9-41%; P = 0.0053) larger in those with decreased function, and 41% (16-59%; P = 0.016) smaller in those with highly increased function SLCO1B1 genotype. Individuals with intermediate metabolizer CYP3A4 genotype (CYP3A4*2 or CYP3A4*22 heterozygotes) had 33% (14-55%; P = 0.022) larger atorvastatin AUC0-∞ than those with normal metabolizer genotype. UGT1A3*2 heterozygotes had 16% (5-25%; P = 0.017) smaller and LPP rs1975991 homozygotes had 34% (22-44%; P = 4.8 × 10-5) smaller atorvastatin AUC0-∞ than noncarriers. These data demonstrate that genetic variation in SLCO1B1, UGT1A3, LPP, and CYP3A4 affects atorvastatin pharmacokinetics. This is the first study to suggest that LPP rs1975991 may reduce atorvastatin exposure. [Correction added on 6 April, after first online publication: An incomplete sentence ("= 0.017) smaller in heterozygotes for UGT1A3*2 and 34% (22%, 44%; P × 10-5) smaller in homozygotes for LPP noncarriers.") has been corrected in this version.].

Understanding CYP3A4 and P-gp mediated drug-drug interactions through PBPK modeling - Case example of pralsetinib.

Pralsetinib, a potent and selective inhibitor of oncogenic RET fusion and RET mutant proteins, is a substrate of the drug metabolizing enzyme CYP3A4 and a substrate of the efflux transporter P-gp based on in vitro data. Therefore, its pharmacokinetics (PKs) may be affected by co-administration of potent CYP3A4 inhibitors and inducers, P-gp inhibitors, and combined CYP3A4 and P-gp inhibitors. With the frequent overlap between CYP3A4 and P-gp substrates/inhibitors, pralsetinib is a challenging and representative example of the need to more quantitatively characterize transporter-enzyme interplay. A physiologically-based PK (PBPK) model for pralsetinib was developed to understand the victim drug-drug interaction (DDI) risk for pralsetinib. The key parameters driving the magnitude of pralsetinib DDIs, the P-gp intrinsic clearance and the fraction metabolized by CYP3A4, were determined from PBPK simulations that best captured observed DDIs from three clinical studies. Sensitivity analyses and scenario simulations were also conducted to ensure these key parameters were determined with sound mechanistic rationale based on current knowledge, including the worst-case scenarios. The verified pralsetinib PBPK model was then applied to predict the effect of other inhibitors and inducers on the PKs of pralsetinib. This work highlights the challenges in understanding DDIs when enzyme-transporter interplay occurs, and demonstrates an important strategy for differentiating enzyme/transporter contributions to enable PBPK predictions for untested scenarios and to inform labeling.

Equilibrium landscape of ingress/egress channels and gating residues of the Cytochrome P450 3A4.

The Cytochrome P450 (CYP) enzymes metabolize a variety of drugs, which may potentially lead to toxicity or reduced efficacy when drugs are co-administered. These drug-drug interactions are often manifested by CYP3A4, the most prevalent of all CYP isozymes. We carried out multiple MD simulations employing CAVER to quantify the channels, and Hidden Markov Models (HMM) to characterize the behavior of the gating residues. We discuss channel properties, bottleneck residues with respect to their likelihood to deem the respective channel ingress or egress, gating residues regarding their open or closed states, and channel location relative to the membrane. Channels do not display coordinated motion and randomly transition between different conformations. Gateway residues also behave in a random fashion. Our findings shed light on the equilibrium behavior of the gating residues and channels in the apo state.

Humanization of SLCO2B1 in Rats Increases rCYP3A1 Protein Expression but Not the Metabolism of Erlotinib to OSI-420.

The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 (SLCO2B1)] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of SLCO2B1+/+ knockin and rSlco2b1-/- knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used SLCO2B1+/+ and rSlco2b1-/ - rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing SLCO2B1+/+ and rSlco2b1-/- rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.

Predicting Drug-Drug Interactions Involving Rifampicin Using a Semi-mechanistic Hepatic Compartmental Model.

AIMS: To develop a semi-mechanistic hepatic compartmental model to predict the effects of rifampicin, a known inducer of CYP3A4 enzyme, on the metabolism of five drugs, in the hope of informing dose adjustments to avoid potential drug-drug interactions. METHODS: A search was conducted for DDI studies on the interactions between rifampicin and CYP substrates that met specific criteria, including the availability of plasma concentration-time profiles, physical and absorption parameters, pharmacokinetic parameters, and the use of healthy subjects at therapeutic doses. The semi-mechanistic model utilized in this study was improved from its predecessors, incorporating additional parameters such as population data (specifically for Chinese and Caucasians), virtual individuals, gender distribution, age range, dosing time points, and coefficients of variation. RESULTS: Optimal parameters were identified for our semi-mechanistic model by validating it with clinical data, resulting in a maximum difference of approximately 2-fold between simulated and observed values. PK data of healthy subjects were used for most CYP3A4 substrates, except for gilteritinib, which showed no significant difference between patients and healthy subjects. Dose adjustment of gilteritinib co-administered with rifampicin required a 3-fold increase of the initial dose, while other substrates were further tuned to achieve the desired drug exposure. CONCLUSIONS: The pharmacokinetic parameters AUCR and CmaxR of drugs metabolized by CYP3A4, when influenced by Rifampicin, were predicted by the semi-mechanistic model to be approximately twice the empirically observed values, which suggests that the semi-mechanistic model was able to reasonably simulate the effect. The doses of four drugs adjusted via simulation to reduce rifampicin interaction.

Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 µM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

Deep Learning Models Compared to Experimental Variability for the Prediction of CYP3A4 Time-Dependent Inhibition.

Most drugs are mainly metabolized by cytochrome P450 (CYP450), which can lead to drug-drug interactions (DDI). Specifically, time-dependent inhibition (TDI) of CYP3A4 isoenzyme has been associated with clinically relevant DDI. To overcome potential DDI issues, high-throughput in vitro assays were established to assess the TDI of CYP3A4 during the discovery and lead optimization phases. However, in silico machine learning models would enable an earlier and larger-scale assessment of TDI potential liabilities. For CYP inhibition, most modeling efforts have focused on highly imbalanced and small data sets. Moreover, assay variability is rarely considered, which is key to understand the model's quality and suitability for decision-making. In this work, machine learning models were built for the prediction of TDI of CYP3A4, evaluated prospectively, and compared to the variability of the experimental assay. Different modeling strategies were investigated to assess their influence on the model's performance. Through multitask learning, additional data sets were leveraged for model building, coming from public databases, in-house CYP-related assays, or other pharmaceutical companies (federated learning). Apart from the numerical prediction of inactivation rates of CYP3A4 TDI, three-class predictions were carried out, giving a negative (inactivation rate kobs < 0.01 min-1), weak positive (0.01 ≤ kobs ≤ 0.025 min-1), or positive (kobs > 0.025 min-1) output. The final multitask graph neural network model achieved misclassification rates of 8 and 7% for positive and negative TDI, respectively. Importantly, the presented deep learning-based predictions had a similar precision to the reproducibility of in vitro experiments and thus offered great opportunities for drug design, early derisk of DDI potential, and selection of experiments. To facilitate CYP inhibition modeling efforts in the public domain, the developed model was used to annotate ∼16 000 publicly available structures, and a surrogate data set is shared as Supporting Information.

Pharmacogenomic implications of the differential distribution of CYP3A5 metabolic phenotypes among Latin American populations.

This study shows that the distribution of CYP3A5 alleles (*1, *3, *6 and *7) and genotype-predicted CYP3A5 phenotypes vary significantly across Latin American cohorts (Brazilians and the One Thousand Genomes Admixed American superpopulation), as well as among subcohorts comprising individuals with the highest proportions of Native, European or sub-Saharan African ancestry. Differences in biogeographical ancestry across the study groups are the likely explanation for these results. The differential distribution of CYP3A5 phenotypes has major pharmacogenomic implications, affecting the proportion of individuals carrying high risk CYP3A5 phenotypes for the immunosuppressant tacrolimus and the number of patients that would need to be genotyped to prevent acute rejection in kidney transplant recipients under tacrolimus treatment.

Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform.

OBJECTIVES: Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. METHODS: We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. RESULTS: Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. CONCLUSIONS: Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

Time Course of the Interaction Between Oral Short-Term Ritonavir Therapy with Three Factor Xa Inhibitors and the Activity of CYP2D6, CYP2C19, and CYP3A4 in Healthy Volunteers.

BACKGROUND: We investigated the effect of a 5-day low-dose ritonavir therapy, as it is used in the treatment of COVID-19 with nirmatrelvir/ritonavir, on the pharmacokinetics of three factor Xa inhibitors (FXaI). Concurrently, the time course of the activities of the cytochromes P450 (CYP) 3A4, 2C19, and 2D6 was assessed. METHODS: In an open-label, fixed sequence clinical trial, the effect and duration of a 5-day oral ritonavir (100 mg twice daily) treatment on the pharmacokinetics of three oral microdosed FXaI (rivaroxaban 25 µg, apixaban 25 µg, and edoxaban 50 µg) and microdosed probe drugs (midazolam 25 µg, yohimbine 50 µg, and omeprazole 100 µg) was evaluated in eight healthy volunteers. The plasma concentrations of all drugs were quantified using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and pharmacokinetics were analysed using non-compartmental analyses. RESULTS: Ritonavir increased the exposure of apixaban, edoxaban, and rivaroxaban, but to a different extent the observed area under the plasma concentration-time curve (geometric mean ratio 1.29, 1.46, and 1.87, respectively). A strong CYP3A4 inhibition (geometric mean ratio > 10), a moderate CYP2C19 induction 2 days after ritonavir (0.64), and no alteration of CYP2D6 were observed. A CYP3A4 recovery half-life of 2.3 days was determined. CONCLUSION: This trial with three microdosed FXaI suggests that at most the rivaroxaban dose should be reduced during short-term ritonavir, and only in patients receiving high maintenance doses. Thorough time series analyses demonstrated differential effects on three different drug-metabolising enzymes over time with immediate profound inhibition of CYP3A4 and only slow recovery after discontinuation. CLINICAL TRIAL REGISTRATION: EudraCT number: 2021-006643-39.

Mechanistic Framework to Predict Maternal-Placental-Fetal Pharmacokinetics of Nifedipine Employing Physiologically Based Pharmacokinetic Modeling Approach.

Nifedipine is used for treating mild to severe hypertension and preventing preterm labor in pregnant women. Nevertheless, concerns about nifedipine fetal exposure and safety are always raised. The aim of this study was to develop and validate a maternal-placental-fetal nifedipine physiologically based pharmacokinetic (PBPK) model and apply the model to predict maternal, placental, and fetal exposure to nifedipine at different pregnancy stages. A nifedipine PBPK model was verified with nonpregnant data and extended to the pregnant population after the inclusion of the fetoplacental multicompartment model that accounts for the placental tissue and different fetal organs within the Simcyp Simulator version 22. Model parametrization involved scaling nifedipine transplacental clearance based on Caco-2 permeability, and fetal hepatic clearance was obtained from in vitro to in vivo extrapolation encompassing cytochrome P450 3A7 and 3A4 activities. Predicted concentration profiles were compared with in vivo observations and the transplacental transfer results were evaluated using 2-fold criteria. The PBPK model predicted a mean cord-to-maternal plasma ratio of 0.98 (range, 0.86-1.06) at term, which agrees with experimental observations of 0.78 (range, 0.59-0.93). Predicted nifedipine exposure was 1.4-, 2.0-, and 3.0-fold lower at 15, 27, and 39 weeks of gestation when compared with nonpregnant exposure, respectively. This innovative PBPK model can be applied to support maternal and fetal safety assessment for nifedipine at various stages of pregnancy.