Cytochrome P450 3A4 Inhibitors Research Articles

Interaction of CYP3A4 with the inhibitor cobicistat: Structural and mechanistic insights and comparison with ritonavir

Cobicistat is a derivative of ritonavir marketed as a pharmacoenhancer for anti-HIV therapy. This study investigated the interaction of cobicistat with the target protein, drug-metabolizing cytochrome P450 3A4 (CYP3A4), at the molecular level using spectral, kinetic, functional, and structural approaches. It was found that, similar to ritonavir, cobicistat directly coordinates to the heme via the thiazole nitrogen but its affinity and the binding rate are 2-fold lower: 0.030 μM and 0.72 s−1, respectively. The newly determined 2.5 Å crystal structure of cobicistat-bound CYP3A4 suggests that these changes arise from the inability of cobicistat to H-bond to the active site S119 and establish multiple stabilizing contacts with the F–F’ connecting fragment, which becomes disordered upon steric clashing with the bulky morpholine moiety. Nonetheless, cobicistat inhibits recombinant CYP3A4 as potently as ritonavir (IC50 of 0.24 μM vs 0.22 μM, respectively) due to strong ligation to the heme and formation of extensive hydrophobic/aromatic interactions via the phenyl side-groups. To get insights into the inhibitory mechanism, the K257 residue, known to be solely and irreversibly modified by the reactive ritonavir metabolite, was substituted with alanine. Neither this nor control K266A mutation changed the extent of time-dependent inhibition of CYP3A4 by cobicistat and ritonavir, suggesting the existence of alternative inactivation mechanism(s). More importantly, K257 was found to be functionally important and contributed to CYP3A4 allosterism, possibly by modulating protein-ligand interactions through conformational dynamics.

Iminium ion metabolites are formed from nintedanib by human CYP3A4

Nintedanib is used to treat idiopathic pulmonary fibrosis, systemic sclerosis, interstitial lung disease, and progressive fibrotic interstitial lung disease. It is primarily cleared via hepatic metabolism, hydrolysis, and glucuronidation. In addition, formation of the iminium ion, a possible reactive metabolite, was predicted based on the chemical structure of nintedanib. To obtain a hint which may help to clarify the cause of nintedanib-induced liver injury, we investigated whether iminium ions were formed in the human liver. To detect unstable iminium ions using liquid chromatography-tandem mass spectrometry (LC-MS/MS), potassium cyanide was added to the reaction mixture as a trapping agent. Human liver and intestinal microsomes were incubated with nintedanib in the presence of NADPH to form two iminium ion metabolites on the piperazine ring. Their formation is strongly inhibited by ketoconazole, a potent cytochrome P450 (CYP) 3A4 inhibitor. Among the recombinant P450s, only CYP3A4 formed cyanide adducts. The role of CYP3A4 was supported by the positive correlation between CYP3A4 protein abundance, as determined by LC-MS-based proteomics, and the formation of cyanide adducts in 25 individual human liver microsomes. In conclusion, we have demonstrated that iminium ion metabolites are formed from nintedanib by CYP3A4 as potential reactive metabolites.

3D spheroid HepaRG and fluorescent biphasic tracer for CYP3A4-mediated antibiotic interaction monitoring in sepsis.

Cytochrome P450 3A4 (CYP3A4) is a crucial enzyme in the metabolism of xenobiotics, particularly in drug metabolism interactions (DDIs), making it a significant factor in clinical drug use. However, current assay techniques are both laborious and costly, making it difficult to construct a high-throughput monitoring method that can be used in conjunction with the clinic. This poses certain safety hazards for drug combination. Therefore, it is crucial to develop a synchronized monitoring method for the inhibition and induction of CYP3A4. In this study, we utilized 3D culture technology to develop a HepaRG cells spheroid model. The CYP450 and transporter expression, the albumin secretion, and urea synthesis capacity characteristics were analyzed. The NEN probe was utilized as a tracer molecule for CYP3A4. The fluorescence intensity of metabolites was characterized by laser confocal technique to determine the inhibition and expression of CYP3A4 in the HepaRG cell spheroid model by the antibiotics for sepsis. The results indicate that the HepaRG sphere model successfully possessed the physiological phenotype of the liver, which could be used for drug interaction monitoring. Through positive drug testing, NEN probe was able to achieve bidirectional characterization of CYP3A4 induction and inhibition. The monitoring method described in this paper was successfully applied to drug interaction monitoring of commonly used antibiotics in sepsis patients, which is a convenient and rapid monitoring method. The proposed method offers a new strategy for monitoring CYP3A4-mediated drug-drug interactions with a high-throughput assay, which will help to improve the safety of clinical drug combination.

Effect of cytochrome P450 3A4 on tissue distribution of humantenmine, koumine, and gelsemine, three alkaloids from the toxic plant Gelsemium

Humantenmine, koumine, and gelsemine are three indole alkaloids found in the highly toxic plant Gelsemium. Humantenmine was the most toxic, followed by gelsemine and koumine. The aim of this study was to investigate and analyze the effects of these three substances on tissue distribution and toxicity in mice pretreated with the Cytochrome P450 3A4 (CYP3A4) inducer ketoconazole and the inhibitor rifampicin. The in vivo test results showed that the three alkaloids were absorbed rapidly and had the ability to penetrate the blood-brain barrier. At 5 min after intraperitoneal injection, the three alkaloids were widely distributed in various tissues and organs, the spleen and pancreas were the most distributed, and the content of all tissues decreased significantly at 20 min. Induction or inhibition of CYP3A4 in vivo can regulate the distribution and elimination effects of the three alkaloids in various tissues and organs. Additionally, induction of CYP3A4 can reduce the toxicity of humantenmine, and vice versa. Changes in CYP3A4 levels may account for the difference in toxicity of humantenmine. These findings provide a reliable and detailed dataset for drug interactions, tissue distribution, and toxicity studies of Gelsemium alkaloids.

Nirmatrelvir/ritonavir treatment of patients with COVID-19 taking tacrolimus: case series describing the results of drug-drug interactions.

Nirmatrelvir/ritonavir is a novel drug combination that is authorized by the Food and Drug Administration for the treatment of coronavirus disease 2019 (COVID-19). Ritonavir is a cytochrome P450 3A inhibitor and a P-glycoprotein inhibitor that increases the plasma concentration of tacrolimus and other medications. We describe the cases of two patients treated with nirmatrelvir/ritonavir: a patient who had undergone kidney transplantation and another with a history of hematopoietic stem cell transplantation. Toxic concentrations of tacrolimus were induced in both. This case series highlights the risk associated with the concomitant administration of tacrolimus and nirmatrelvir/ritonavir.

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.

Clinical Pharmacology of the Antibody-Drug Conjugate Enfortumab Vedotin in Advanced Urothelial Carcinoma and Other Malignant Solid Tumors.

Enfortumab vedotin is an antibody-drug conjugate comprised of a human monoclonal antibody directed to Nectin-4 and monomethyl auristatin E (MMAE), a microtubule-disrupting agent. The objectives of this review are to summarize the clinical pharmacology of enfortumab vedotin monotherapy and demonstrate that the appropriate dose has been selected for clinical use. Pharmacokinetics (PK) of enfortumab vedotin (antibody-drug conjugate and total antibody) and free MMAE were evaluated in five clinical trials of patients with locally advanced or metastatic urothelial carcinoma (n=748). Intravenous enfortumab vedotin 0.5-1.25 mg/kg on days 1, 8, and 15 of a 28-day cycle showed linear, dose-proportional PK. No significant differences in exposure or safety of enfortumab vedotin and free MMAE were observed in mild, moderate, or severe renal impairment versus normal renal function. Patients with mildly impaired versus normal hepatic function had a 37% increase in area under the concentration-time curve (0-28 days), a 31% increase in maximum concentration of free MMAE, and a similar adverse event profile. No clinically significant PK differences were observed based on race/ethnicity with weight-based dosing, and no clinically meaningful QT prolongation was observed. Concomitant use with dual P-glycoprotein and strong cytochrome P450 3A4 inhibitors may increase MMAE exposure and the risk of adverse events. Approximately 3% of patients developed antitherapeutic antibodies against enfortumab vedotin 1.25 mg/kg. These findings support enfortumab vedotin 1.25 mg/kg monotherapy on days 1, 8, and 15 of a 28-day cycle. No dose adjustments are required for patients with renal impairment or mild hepatic impairment, or by race/ethnicity.

Docetaxel, cyclophosphamide, and epirubicin: application of PBPK modeling to gain new insights for drug-drug interactions.

The new adjuvant chemotherapy of docetaxel, epirubicin, and cyclophosphamide has been recommended for treating breast cancer. It is necessary to investigate the potential drug-drug Interactions (DDIs) since they have a narrow therapeutic window in which slight differences in exposure might result in significant differences in treatment efficacy and tolerability. To guide clinical rational drug use, this study aimed to evaluate the DDI potentials of docetaxel, cyclophosphamide, and epirubicin in cancer patients using physiologically based pharmacokinetic (PBPK) models. The GastroPlus™ was used to develop the PBPK models, which were refined and validated with observed data. The established PBPK models accurately described the pharmacokinetics (PKs) of three drugs in cancer patients, and the predicted-to-observed ratios of all the PK parameters met the acceptance criterion. The PBPK model predicted no significant changes in plasma concentrations of these drugs during co-administration, which was consistent with the observed clinical phenomenon. Besides, the verified PBPK models were then used to predict the effect of other Cytochrome P450 3A4 (CYP3A4) inhibitors/inducers on these drug exposures. In the DDI simulation, strong CYP3A4 modulators changed the exposure of three drugs by 0.71-1.61 fold. Therefore, patients receiving these drugs in combination with strong CYP3A4 inhibitors should be monitored regularly to prevent adverse reactions. Furthermore, co-administration of docetaxel, cyclophosphamide, or epirubicin with strong CYP3A4 inducers should be avoided. In conclusion, the PBPK models can be used to further investigate the DDI potential of each drug and to develop dosage recommendations for concurrent usage by additional perpetrators or victims.

Evaluation of machine learning models for cytochrome P450 3A4, 2D6, and 2C9 inhibition.

Cytochrome P450 (CYP) enzymes are involved in the metabolism of approximately 75% of marketed drugs. Inhibition of the major drug-metabolizing P450s could alter drug metabolism and lead to undesirable drug-drug interactions. Therefore, it is of great significance to explore the inhibition of P450s in drug discovery. Currently, machine learning including deep learning algorithms has been widely used for constructing in silico models for the prediction of P450 inhibition. These models exhibited varying predictive performance depending on the use of machine learning algorithms and molecular representations. This leads to the difficulty in the selection of appropriate models for practical use. In this study, we systematically evaluated the conventional machine learning and deep learning models for three major P450 enzymes, CYP3A4, CYP2D6, and CYP2C9 from several perspectives, such as algorithms, molecular representation, and data partitioning strategies. Our results showed that the XGBoost and CatBoost algorithms coupled with the combined fingerprint/physicochemical descriptor features exhibited the best performance with Area Under Curve (AUC) of 0.92, while the deep learning models were generally inferior to the conventional machine learning models (average AUC reached 0.89) on the same test sets. We also found that data volume and sampling strategy had a minor effect on model performance. We anticipate that these results are helpful for the selection of molecular representations and machine learning/deep learning algorithms in the P450 model construction and the future model development of P450 inhibition.

Impact of loperamide on the pharmacokinetics and tissue disposition of ritonavir-boosted oral docetaxel therapy; a preclinical assessment.

An oral docetaxel formulation boosted by the Cytochrome P450 (CYP) 3A inhibitor ritonavir, ModraDoc006/r, is currently under clinical investigation. Based on clinical data, the incidence of grade 1-2 diarrhea is increased with this oral docetaxel formulation compared to the conventional intravenous administration. Loperamide, a frequently used diarrhea inhibitor, could be added to the regimen as symptomatic treatment. However, loperamide is also a substrate of the CYP3A enzyme, which could result in competition between ritonavir and loperamide for this protein. Therefore, we were interested in the impact of coadministered loperamide on the pharmacokinetics of ritonavir-boosted oral docetaxel. We administered loperamide simultaneously or with an 8-hour delay to humanized CYP3A4 mice (with expression in liver and intestine) receiving oral ritonavir and docetaxel. Concentrations of docetaxel, ritonavir, loperamide and two of its active metabolites were measured. The plasma exposure (AUC and Cmax) of docetaxel was not altered during loperamide treatment, nor were the ritonavir plasma pharmacokinetics. However, the hepatic and intestinal dispositions of ritonavir were somewhat changed in the simultaneous, but not 8-hour loperamide treatment groups, possibly due to loperamide-induced delayed drug absorption. The pharmacokinetics of loperamide itself did not seem to be influenced by ritonavir. These results suggest that delayed loperamide administration can be added to ritonavir-boosted oral docetaxel treatment, without affecting the overall systemic exposure of docetaxel.

Effect of Strong CYP3A4 Inhibition, CYP3A4 Induction, and OATP1B1/3 Inhibition on the Pharmacokinetics of a Single Oral Dose of Sotorasib.

Sotorasib is a small molecule that irreversibly inhibits the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein with a G12C amino acid substitution mutant protein. The impact of cytochrome P450 (CYP) 3A4 inhibition and induction on sotorasib pharmacokinetics (PKs) was evaluated in 2 separate studies in healthy volunteers (N = 14/study). The impact of CYP3A4 inhibition was interrogated utilizing repeat doses of 200mg of itraconazole, a strong CYP3A4 inhibitor, on 360mg of sotorasib PKs. The impact of CYP3A4 induction was interrogated utilizing multiple doses of 600mg of rifampin, a strong CYP3A4 inducer. Additionally, the impact of organic anion transporting polypeptide (OATP) 1B1/3 inhibition on 960mg of sotorasib PKs was interrogated after a single dose of 600mg of rifampin. CYP3A4 inhibition did not significantly impact sotorasib Cmax but did lead to a 26% increase in sotorasib AUCinf. CYP3A4 induction decreased sotorasib Cmax by 35% and AUCinf by 51%. OATP1B1/3 inhibition decreased sotorasib Cmax and AUCinf by 16% and 23%, respectively. These results support that sotorasib can be given together with strong CYP3A4 and OATP1B1/3 inhibitors but the co-administration of sotorasib and strong CYP3A4 inducers should be avoided.

Effect of drug interactions with non-vitamin-K oral anticoagulants on thromboembolic events in patients with nonvalvular atrial fibrillation.

Few real-world studies have investigated drug-drug interactions (DDIs) involving non-vitamin-K antagonist oral anticoagulants (NOACs) in patients with nonvalvular atrial fibrillation (NVAF). The interactions encompass drugs inducing or inhibiting cytochrome P450 3A4 and permeability glycoprotein. These agents potentially modulate the breakdown and elimination of NOACs. This study investigated the impact of DDIs on thromboembolism in this clinical scenario. Patients who had NVAF and were treated with NOACs were selected as the study cohort from the National Health Insurance Research Database of Taiwan. Cases were defined as patients hospitalised for a thromboembolic event and who underwent a relevant imaging study within 7 days before hospitalisa-tion or during hospitalisation. Each case was matched with up to 4 controls by using the incidence density sampling method. The concurrent use of a cytochrome P450 3A4/permeability glycoprotein inducer or inhibitor or both with NOACs was identified. The effects of these interactions on the risk of thromboembolic events were examined with univariate and multivariate conditional logistic regressions. The study cohort comprised 60,726 eligible patients. Among them, 1288 patients with a thromboembolic event and 5144 matched control patients were selected for analysis. The concurrent use of a cytochrome P450 3A4/permeability glycoprotein inducer resulted in a higher risk of thromboembolic events (adjusted odds ratio [AOR] 1.23, 95% confidence interval [CI] 1.004-1.51). For patients with NVAF receiving NOACs, the concurrent use of cytochrome P450 3A4/ permeability glycoprotein inducers increases the risk of thromboembolic events.

Projections of Drug-Drug Interactions Caused by Time-Dependent Inhibitors of Cytochrome P450 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 Using In Vitro Data in Static and Dynamic Models.

In vitro time-dependent inhibition (TDI) kinetic parameters for cytochrome P450 (CYP) 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6, were determined in pooled human liver microsomes for 19 drugs (and 2 metabolites) for which clinical drug-drug interactions (DDI) are known. In vitro TDI data were incorporated into the projection of the magnitude of DDIs using mechanistic static models and Simcyp®. Results suggest that for the mechanistic static model, use of estimated average unbound exit concentration of the inhibitor from the liver resulted in a successful prediction of observed magnitude of clinical DDIs and was similar to Simcyp®. Overall, predictions of DDI magnitude (i.e., fold increase in AUC of a CYP-specific marker substrate) were within 2-fold of actual values. Geometric mean-fold errors were 1.7 and 1.6 for static and dynamic models, respectively. Projections of DDI from both models were also highly correlated to each other (r2 = 0.92). This investigation demonstrates that DDI can be reliably predicted from in vitro TDI data generated in HLM for several CYP enzymes. Simple mechanistic static model equations as well as more complex dynamic PBPK models can be employed in this process. Significance Statement Cytochrome P450 time-dependent inhibitors (TDI) can cause drug-drug interactions (DDI). An ability to reliably assess the potential for a new drug candidate to cause DDI is essential during drug development. In this report, TDI data for 19 drugs (and 2 metabolites) were measured and used in static and dynamic models to reliably project the magnitude of DDI resulting from inhibition of CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6.

Physiologically based pharmacokinetic modeling to predict the clinical effect of azole antifungal agents as CYP3A inhibitors on azelnidipine pharmacokinetics

In this study, a physiologically based pharmacokinetic (PBPK) model of the cytochrome P450 3A (CYP3A) substrate azelnidipine was developed using in vitro and clinical data to predict the effects of azole antifungals on azelnidipine pharmacokinetics. Modeling and simulations were conducted using the Simcyp™ PBPK simulator. The azelnidipine model consisted of a full PBPK model and a first-order absorption model. CYP3A was assumed as the only azelnidipine elimination route, and CYP3A clearance was optimized using the pharmacokinetic profile of single-dose 5-mg azelnidipine in healthy participants. The model reproduced the results of a clinical drug–drug interaction study and met validation criteria. PBPK model simulations using azole antifungals (itraconazole, voriconazole, posaconazole, fluconazole, fosfluconazole) and azelnidipine or midazolam (CYP3A index substrate) were performed. Increases in the simulated area under the plasma concentration–time curve from time zero extrapolated to infinity with inhibitors were comparable between azelnidipine (range, 2.11–6.47) and midazolam (range, 2.26–9.22), demonstrating that azelnidipine is a sensitive CYP3A substrate. Increased azelnidipine plasma concentrations are expected when co-administered with azole antifungals, potentially affecting azelnidipine safety. These findings support the avoidance of azole antifungals in patients taking azelnidipine and demonstrate the utility of PBPK modeling to inform appropriate drug use.

A comprehensive review of the use of lemborexant in treating insomnia

Abstract Lemborexant (LEM), a dual orexin receptor antagonist, represents a significant advancement in treating insomnia, a prevalent sleep disorder affecting a considerable portion of the population, especially older adults in India. LEM has several benefits over conventional sedatives in the treatment of insomnia, such as the selective mechanism of action, lower risk of tolerance and dependence, better sleep architecture, reduced possibility of cognitive impairment, various dosing options, the risk of rebound insomnia, and the risk of drug–drug interactions are also decreased. This thorough examination explores the pharmacological mechanisms, pharmacokinetics, clinical effectiveness, and safety profile of LEM, providing valuable insights into its distinct mode of action compared with traditional medications targeting gamma-aminobutyric acid receptors. By blocking orexin receptors (orexin 1 receptor and orexin 2 receptor), LEM regulates neuropeptides orexin A and orexin B, crucial for controlling wakefulness and arousal. Clinical trials demonstrate its predictable pharmacokinetics, dose-dependent effects, and minimal adverse reactions, confirming its efficacy in improving sleep initiation and maintenance. Long-term studies on LEM’s sustained therapeutic benefits show no signs of rebound insomnia or withdrawal. Its favorable safety profile, including minimal impact on next-day functioning, cognitive abilities, and postural stability, sets it apart from other treatments. The exploration of potential drug interactions underscores the importance of cautious prescribing, especially concerning cytochrome P450 3A inhibitors and inducers. As LEM becomes integrated into healthcare protocols, it promises to be a well-tolerated and effective solution, reshaping standard treatment approaches for insomnia and overcoming the limitations of older medications. In summary, LEM is a valuable addition to the pharmacotherapeutic toolbox, offering a novel strategy for managing sleep disturbances with sustained efficacy and favorable safety features.

Alkylphenols disrupt estrogen homeostasis via diradical cross-coupling reactions: A novel pathway of endocrine disruption

Estrogen, being an essential class of sex hormone, is an important target of endocrine disruption chemicals. It is well known that environmental disruptors could activate or inhibit estrogen receptors, acting as agonists or antagonists, and thus affect the circulating estrogen concentrations. Here, we report enzyme-mediated diradical cross-coupling reactions between alkylphenols (e.g., 2,4-di-tert-butylphenol [DBP], 4-nonylphenol [4-NP], and 4-tert-octylphenol [4-t-OP]) and estrogens (e.g., estradiol [E2]) that generate coupling metabolites and disrupt estrogen homeostasis. Among the phenolic xenobiotics, the screening of metabolic products revealed that alkylphenols had the highest reaction activities and generated coupling metabolites with high abundances (DBP-O-E2, 4-t-OP-O-E2, and 4-NP-O-E2). The coupling reactions were catalyzed by cytochrome P450 3A4 (CYP3A4) and verified by the detection of the coupling products in general populations. In vitro and in vivo exposures together with CYP3A4 inhibition demonstrated that cross-coupling reactions of phenols and E2 significantly reduced the normal levels of E2. We further established a unique spin-trapping-based high-throughput screening method to show the existence of diradicals in the coupling reaction. Density functional theory calculations revealed that spin aromatic delocalization was the fundamental cause of the high rebound barrier and sufficient lifetime of phenoxy radicals that enabled phenolic cross-coupling triggered by cytochrome P450. The identified mechanistic details for diradical cross-coupling reactions provide a novel pathway for phenolic chemicals to disrupt estrogen homeostasis.

Delayed clearance of the pro-carcinogen benzo[a]pyrene in PLHC-1 cells when co-exposed to the antifungal drug clotrimazole and effects on the CYP1A biomarker

Cytochrome P450 1 A (CYP1A) is a key enzyme in the metabolism of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in animals, and a biomarker for environmental PAH exposure. The common antimycotic imidazole drug clotrimazole (CLO) has been detected in the aquatic environment and likely co-exists with BaP. Like BaP, CLO can bind to CYP1A enzymes and can act as a CYP1A inhibitor. Co-exposure of BaP with CLO significantly delayed BaP elimination in a fish liver cell line (PLHC-1). Intracellular BaP concentration was 2.4 times higher after 6 h in co-exposed cells, compared to cells exposed to BaP alone. Higher BaP concentrations in cells co-exposed to CLO positively correlated with CLO dose, indicating CLO-mediated delays in BaP clearance. After 24 h, BaP was undetectable irrespective of CLO co-exposure. In contrast, intracellular CLO concentrations remained constant over the 72 h experimental period. Co-exposure of BaP with CLO caused synergistic and time-dependent increases on the CYP1A biomarker both on CYP1A mRNA levels and on CYP1A enzyme activity, in accordance with an apparent delayed BaP elimination in the presence of CLO. These results indicate a toxicokinetic interaction between BaP and CLO on the CYP1A enzyme that delays metabolic clearance of BaP.

An Uncertainty-Guided Deep Learning Method Facilitates Rapid Screening of CYP3A4 Inhibitors.

Cytochrome P450 3A4 (CYP3A4), a prominent member of the P450 enzyme superfamily, plays a crucial role in metabolizing various xenobiotics, including over 50% of clinically significant drugs. Evaluating CYP3A4 inhibition before drug approval is essential to avoiding potentially harmful pharmacokinetic drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Despite the development of several CYP inhibitor prediction models, the primary approach for screening CYP inhibitors still relies on experimental methods. This might stem from the limitations of existing models, which only provide deterministic classification outcomes instead of precise inhibition intensity (e.g., IC50) and often suffer from inadequate prediction reliability. To address this challenge, we propose an uncertainty-guided regression model to accurately predict the IC50 values of anti-CYP3A4 activities. First, a comprehensive data set of CYP3A4 inhibitors was compiled, consisting of 27,045 compounds with classification labels, including 4395 compounds with explicit IC50 values. Second, by integrating the predictions of the classification model trained on a larger data set and introducing an evidential uncertainty method to rank prediction confidence, we obtained a high-precision and reliable regression model. Finally, we use the evidential uncertainty values as a trustworthy indicator to perform a virtual screening of an in-house compound set. The in vitro experiment results revealed that this new indicator significantly improved the hit ratio and reduced false positives among the top-ranked compounds. Specifically, among the top 20 compounds ranked with uncertainty, 15 compounds were identified as novel CYP3A4 inhibitors, and three of them exhibited activities less than 1 μM. In summary, our findings highlight the effectiveness of incorporating uncertainty in compound screening, providing a promising strategy for drug discovery and development.

Broad-spectrum antiviral activity of two structurally analogous CYP3A inhibitors against pathogenic human coronaviruses in vitro

Coronaviruses pose a permanent risk of outbreaks, with three highly pathogenic species and strains (SARS-CoV, MERS-CoV, SARS-CoV-2) having emerged in the last twenty years. Limited antiviral therapies are currently available and their efficacy in randomized clinical trials enrolling SARS-CoV-2 patients has not been consistent, highlighting the need for more potent treatments. We previously showed that cobicistat, a clinically approved inhibitor of Cytochrome P450-3A (CYP3A), has direct antiviral activity against early circulating SARS-CoV-2 strains in vitro and in Syrian hamsters. Cobicistat is a derivative of ritonavir, which is co-administered as pharmacoenhancer with the SARS-CoV-2 protease inhibitor nirmatrelvir, to inhibit its metabolization by CPY3A and preserve its antiviral efficacy. Here, we used automated image analysis for a screening and parallel comparison of the anti-coronavirus effects of cobicistat and ritonavir. Our data show that both drugs display antiviral activity at low micromolar concentrations against multiple SARS-CoV-2 variants in vitro, including epidemiologically relevant Omicron subvariants. Despite their close structural similarity, we found that cobicistat is more potent than ritonavir, as shown by significantly lower EC50 values in monotherapy and higher levels of viral suppression when used in combination with nirmatrelvir. Finally, we show that the antiviral activity of both cobicistat and ritonavir is maintained against other human coronaviruses, including HCoV-229E and the highly pathogenic MERS-CoV. Overall, our results demonstrate that cobicistat has more potent anti-coronavirus activity than ritonavir and suggest that dose adjustments could pave the way to the use of both drugs as broad-spectrum antivirals against highly pathogenic human coronaviruses.

Revumenib Monotherapy in Patients with Relapsed/Refractory KMT2Ar Acute Leukemia: Topline Efficacy and Safety Results from the Pivotal Augment-101 Phase 2 Study

Background: Most patients (pts) with histone-lysine N-methyltransferase 2A (KMT2A)-rearranged (KMT2Ar) acute leukemia, relapse after conventional chemotherapy and hematopoietic stem cell transplant (HSCT). Remission rates after relapse (complete remission [CR], 5%) and median overall survival (2.4 mo) in ≥2nd salvage therapies in adults remain low (Blood Cancer J. 2021;11[9]:162). In KMT2Ar leukemia, interaction of menin with KMT2A fusion proteins is a key driver of leukemogenesis. However, no therapies targeting the menin-KMT2A interaction have been approved. Revumenib (SNDX-5613; rev), a small-molecule inhibitor of menin-KMT2A interactions, demonstrated preliminary efficacy and safety in a phase 1 study of R/R KMT2Ar and nucleophosmin 1-mutated (NPM1m) acute leukemias. We report topline efficacy and safety for pts with R/R KMT2Ar acute leukemia treated with rev in a pivotal phase 2 study (AUGMENT-101; NCT04065399). Methods: Pts aged ≥30 days with R/R KMT2Ar acute leukemia were enrolled in cohort A (acute lymphoblastic leukemia [ALL]/mixed phenotype acute leukemia [MPAL]) and B (acute myeloid leukemia [AML]); cohort C continues to enroll pts with NPM1m and is not included in this analysis. Pts received rev (163 mg or 95 mg/m2 if body weight <40 kg) q12h with a strong cytochrome P450 3A4 inhibitor orally in 28-day cycles. Treatment continued until unacceptable toxicity or lack of at least morphological leukemia-free state (MLFS) after 4 cycles of treatment. Phase 2 primary objectives were safety and tolerability of rev and CR+CR with partial hematologic recovery (CRh) rate. Key secondary endpoints included composite CR rate (CRc, CR+CRh+CR with incomplete platelet recovery [CRp]+CR with incomplete count recovery [CRi]) and overall response rate (ORR, CRc+MLFS+partial remission). A planned interim analysis (IA) of pooled adult and pediatric pts with KMT2Ar acute leukemia was conducted. Results:As of July 24, 2023, 94 pts with R/R KMT2Ar acute leukemia received ≥1 dose of study drug and were included in the safety analysis (Table 1). Median age was 37.0 y (range, 1.3-75.0). Of the 94 pts, 23 (24.5%) were aged <18 y and 13 (13.8%) were aged ≥65 y. Seventy-eight (83%) had AML and 16 (17.0%) had ALL or MPAL. Over half of pts were female; 17.5% of pts were non-White. Pts were heavily pretreated (median 2 [range, 1-11] prior lines of therapy), with 41 pts (43.6%) having received ≥3 prior lines. Fifty-four pts (57.4%) had refractory relapse disease (unresponsive to most recent salvage treatment), and 47 pts (50%) had prior HSCT. Treatment-related adverse events (TRAEs) were reported in 81.9% of the safety population. Most common TRAEs (≥20%) were nausea (27.7%), differentiation syndrome (26.6%), and QTc prolongation (23.4%). Grade ≥3 TRAEs were observed in 51 pts (54.3%), the most common being differentiation syndrome (16.0%), febrile neutropenia (13.8%), and QTc prolongation (13.8%) (Table 2). Overall, 6.4% of pts discontinued therapy due to TRAEs; none discontinued rev due to differentiation syndrome or QTc prolongation. The pooled efficacy population for the IA (n=57) included all phase 2 pts with centrally confirmed KMT2Ar and ≥5% blasts in bone marrow at baseline who had received ≥1 dose of study drug and started treatment on or before the 38th adult AML efficacy evaluable patient. The IA was specified to occur when the 57 patients have had the opportunity to be followed for 6 months. After median follow-up of 6.1 mo, 13 pts (22.8% [95% confidence interval (CI), 12.7-35.8]) achieved CR+CRh, surpassing the predefined IA efficacy boundary for the pooled KMT2Ar population; CR+CRh rate was similar in adult and pediatric pts. Median duration of CR+CRh was 6.4 mo (95% CI, 3.4-not reached). CRc was 43.9% (95% CI, 30.7-57.6); ORR was 63.2% (95% CI, 49.3-75.6). Most pts with a CR or CRh response, and for whom measurable residual disease (MRD) status was reported, achieved MRD negativity (7/10, 70.0%); most pts with CRc and MRD reported also achieved MRD negativity (15/22, 68.2%). Fourteen of 36 responders (38.9%) proceeded to HSCT, with half resuming rev post HSCT. Conclusions: Rev demonstrated clinically meaningful results in a heavily pretreated KMT2Ar population, including high ORR and rates of MRD negativity and subsequent HSCT. At IA, this pivotal study met its primary endpoint and the KMT2Ar cohorts were stopped early for efficacy.