Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding.

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

Efficacy of ivermectin and its metabolites against Plasmodium falciparum liver stages in primary human hepatocytes.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.

Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

The impact of CYP3A4 genetic polymorphism on crizotinib metabolism and drug-drug interactions.

To elucidate the impact of CYP3A4 activity inhibition and genetic polymorphism on the metabolism of crizotinib. Enzymatic incubation systems for crizotinib were established, and Sprague-Dawley rats were utilized for in vivo experiments. Analytes were quantified using LC-MS/MS. Upon screening 122 drugs and natural compounds, proanthocyanidins emerged as inhibitor of crizotinib metabolism, exhibiting a relative inhibition rate of 93.7%. The IC50 values were 24.53 ± 0.32 µM in rat liver microsomes and 18.24 ± 0.12 µM in human liver microsomes. In vivo studies revealed that proanthocyanidins markedly affected the pharmacokinetic parameters of crizotinib. Co-administration led to a significant reduction in the AUC(0-t), Cmax of PF-06260182 (the primary metabolite of crizotinib), and the urinary metabolic ratio. This interaction is attributed to the mixed-type inhibition of liver microsome activity by proanthocyanidins. CYP3A4, being the principal metabolic enzyme for crizotinib, has its genetic polymorphisms significantly influencing crizotinib's pharmacokinetics. Kinetic data showed that the relative metabolic rates of crizotinib across 26 CYP3A4 variants ranged from 13.14% (CYP3A4.12, 13) to 188.57% (CYP3A4.33) when compared to the wild-type CYP3A4.1. Additionally, the inhibitory effects of proanthocyanidins varied between CYP3A4.12 and CYP3A4.33, when compared to the wild type. Our findings indicate that proanthocyanidins coadministration and CYP3A4 genetic polymorphism can significantly influence crizotinib metabolism.

Differential inhibition of sildenafil and macitentan on saxagliptin metabolism.

The development of diabetes mellitus (DM) is generally accompanied by erectile dysfunction (ED) and pulmonary arterial hypertension (PAH), which increases the use of combination drug therapy and the risk of drug-drug interactions. Saxagliptin for the treatment of DM, sildenafil for the treatment of ED and PAH, and macitentan for the treatment of PAH are all substrates of CYP3A4, which indicates their potential involvement in drug-drug interactions. Therefore, we investigated potential pharmacokinetic interactions between saxagliptin and sildenafil/macitentan. We investigated this speculation both in vitro and in vivo, and explored the underlying mechanism using in vitro hepatic metabolic models and molecular docking assays. The results showed that sildenafil substantially inhibited the metabolism of saxagliptin by occupying the catalytic site of CYP3A4 in a competitive manner, leading to the alterations in the pharmacokinetic properties of saxagliptin in terms of increased maximum plasma concentration (Cmax), area under the plasma concentration-time curve from time 0 to 24 h (AUC(0-t)), area under the plasma concentration-time curve from time 0 extrapolated to infinite time (AUC(0-∞)), decreased clearance rate (CLz/F), and prolonged terminal half-life (t1/2). In contrast, a slight inhibition was observed in saxagliptin metabolism when concomitantly used with macitentan, as no pharmacokinetic parameters were altered, except for CLz/F. Thus, dosage adjustment of saxagliptin may be required in combination with sildenafil to achieve safe therapeutic plasma concentrations and reduce the risk of potential toxicity, but it is not necessary for co-administration with macitentan.

CYP3A4 inhibitors may influence the quantification of [123I]I-FP-CIT SPECT scans.

PURPOSE: [123I]I-FP-CIT SPECT is an imaging tool to support the diagnosis of parkinsonian syndromes characterized by nigrostriatal dopaminergic degeneration. After intravenous injection, [123I]I-FP-CIT is metabolized for a small part by the enzyme CYP3A4, leading to the formation of [123I]I-nor-ß-CIT. [123I]I-nor-ß-CIT passes the blood-brain barrier and has a very high affinity for the serotonin transporter (SERT). The SERT is expressed in the striatum and cortical areas. So, at least theoretical, the use of frequently used CYP3A4 inhibitors (like amiodarone) may influence the specific to non-specific striatal [123I]I-FP-CIT ratio. Here we tested this novel hypothesis. METHODS: Using a retrospective design, we determined the specific to non-specific striatal [123I]I-FP-CIT ratio (using BRASS software) in 6 subjects that were using an CYP3A4 inhibitor and 18 matched controls. Only subjects were included with a normal rated [123I]I-FP-CIT SPECT scan, and all participants were scanned on the same brain-dedicated SPECT system. RESULTS: The specific to non-specific (assessed in the occipital cortex) striatal [123I]I-FP-CIT binding ratio was significantly higher in CYP3A4 users than in the control group (3.52 ± 0.33 vs. 2.90 ± 0.78, p < 0.001). CONCLUSION: Our preliminary data suggest that the use of CYP3A4 inhibitors may influence striatal [123I]I-FP-CIT binding ratios. This information, when reproduced in larger studies, may be relevant for studies in which quantification of [123I]I-FP-CIT SPECT imaging is used for diagnostic or research purposes.

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.

The extent of cytochrome P450 3A induction by antiseizure medications: A systematic review and network meta-analysis.

OBJECTIVE: Antiseizure medications (ASMs) are commonly categorized as enzyme-inducers and non-enzyme-inducers based on their propensity to enhance the metabolism of concomitantly administered drugs. This systematic review and network meta-analysis aimed to rank ASMs as cytochrome P450 3A (CYP3A)-inducers based on a comparative assessment of ASM-induced reduction in the concentrations of sensitive substrate drugs. METHODS: The protocol was registered with PROSPERO (International Prospective Register of Systematic Reviews; CRD42022335846), and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) standards were followed. We searched MEDLINE, Embase, and Cochrane until March 14, 2023 without an initial date restriction. Data were additionally obtained via the US Food and Drug Administration database. Studies had to be prospective, with ASM monotherapy for ≥5 days. The primary parameter was the magnitude of change in the area under the concentration-time curve of CYP3A substrates following treatment with the ASM. The standardized mean difference (SMD) was used as the point estimate for the indirect comparisons between ASMs using the pairwise method. Bias risk was assessed using the PKclin tool. RESULTS: We identified 14 open-label, fixed-sequence studies with 370 participants. The effect size of 600 mg/day carbamazepine did not differ from those of 300 mg/day phenytoin (SMD = -.06, 95% confidence interval [CI] = -.18 to .07) and 200 mg/day cenobamate (SMD = -.11, 95% CI = -.26 to .04). Carbamazepine at 600 mg/day was the strongest CYP3A-inducer (P-score = .88), followed by carbamazepine 400 mg/day (.83), phenytoin 300 mg/day (.79), and cenobamate 200 mg/day (.73). Eslicarbazepine (800 mg/day) ranked higher than cenobamate 100 mg/day and oxcarbazepine 900 mg/day (.60, .39, and .37, respectively). SIGNIFICANCE: Despite the limited number of studies, our network meta-analysis emphasizes that the magnitude of ASM effects on CYP3A substrate metabolism is a dose-dependent continuum. When possible, ASM classification as inducers should apply cutoff values tailored to the outcome. Prescribers should monitor plasma concentrations or clinical effects of CYP3A substrates and consider selecting concomitant medications accordingly.

Frailty as a comprehensive health measure beyond seizure control in patients with epilepsy: A cross-sectional study.

OBJECTIVE: Due to the high clinical heterogeneity of epilepsy, there is a critical need for novel metrics aimed at capturing its biological and phenotypic complexity. Frailty is increasingly recognized in various medical disciplines as a useful construct to understand differences in susceptibility to adverse outcomes. Here, we develop a frailty index (FI) for patients with epilepsy (PwE) and explore its association with demographic and clinical features. METHODS: In this cross-sectional study, we consecutively enrolled 153 PwE from an outpatient epilepsy clinic. Participants were assessed for various health deficits to calculate the FI. Associations between FI and demographic/clinical features, antiseizure medications (ASMs), and patient-reported outcomes were analyzed using general linear models and Spearman correlation. RESULTS: The median age at the time of study visit was 47 years (interquartile range = 33-60), and 89 (58.2%) patients were females. Multiple linear regression revealed that the developed 33-item FI showed an independent association with age, female sex, higher body mass index, family history of epilepsy, intellectual disability, and the number of ASMs used. A robust analysis of covariance showed higher FI levels in patients using cytochrome P450 3A4-inducer ASMs. We found a moderate positive correlation between FI and psychological distress, lower quality of life, and physical frailty, measured by the Hospital Anxiety and Depression Scale, Quality of Life in Epilepsy Inventory-10, and handgrip strength, respectively. Finally, a weak association was observed between higher FI scores and an increased number of epileptic falls. SIGNIFICANCE: This study highlights the significance of frailty as a comprehensive health measure in epilepsy. It suggests that frailty in this specific population is not only a manifestation of aging but is inherently linked to epilepsy and treatment-related factors. Future research is warranted to validate and refine the FI in diverse epilepsy populations and investigate its impact on specific adverse outcomes in longitudinal studies.

Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.

Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

OBJECTIVES: Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. METHODS: A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. RESULTS: Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. CONCLUSION: In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

Evaluation of the potential drug-drug interactions of carotegrast methyl with midazolam, prednisolone or atorvastatin in healthy adults.

AIMS: This study evaluated drug-drug interactions between the CYP3A4 inhibitor carotegrast methyl and the other CYP3A4 substrates, midazolam, atorvastatin and prednisolone. METHODS: A total of 88 healthy volunteers orally received carotegrast methyl 960 mg 3 times daily for 14 days. A single oral (5 mg) or intravenous (0.017 mg kg-1 ) midazolam, oral (5 mg) prednisolone or oral (10 mg) atorvastatin was administered before, with and after carotegrast methyl treatment. When the 90% confidence interval (CI) for the geometric mean ratios of the pharmacokinetic (PK) parameters with coadministration with carotegrast methyl (Day 14) to those before carotegrast methyl administration was between 0.80 and 1.25, no PK interaction were deemed. RESULTS: The Cmax and AUC0-t of oral midazolam before administration of carotegrast methyl were 30.9 ± 9.8 ng mL-1 and 74.5 ± 21.9 ng h mL-1 , respectively. The geometric mean ratio of the Cmax and AUC0-t of midazolam on Day 14 to those on Day -1 was 1.86 (90% CI, 1.64-2.11) and 3.07 (90% CI, 2.81-3.35), which did not fall within the range of 0.80-1.25, suggesting that carotegrast methyl had a PK interaction with midazolam. Similar PK interactions were found for intravenous midazolam and atorvastatin, but not for prednisolone. The inhibitory effect of carotegrast methyl on CYP3A4-mediated metabolism of midazolam and atorvastatin had almost disappeared by 14 days after the end of administration. CONCLUSION: Carotegrast methyl was classified as a moderate CYP3A4 inhibitor in humans. Carotegrast methyl might enhance the action of drugs that are metabolized by CYP3A4.

Posaconazole-ibrutinib interaction cannot be avoided by staggered dosing: How to optimize ibrutinib dose during posaconazole treatment.

AIMS: Ibrutinib is used in the treatment of certain B-cell malignancies. Due to its CYP3A4-mediated metabolism and highly variable pharmacokinetics, it is prone to potentially harmful drug-drug interactions. METHODS: In a randomized, placebo-controlled, three-phase crossover study, we examined the effect of the CYP3A4-inhibiting antifungal posaconazole on ibrutinib pharmacokinetics. Eleven healthy participants ingested repeated doses of 300 mg of posaconazole either in the morning or in the evening, or placebo. A single dose of ibrutinib (30, 70 or 140 mg, respectively) was administered at 9 AM, 1 or 12 h after the preceding posaconazole/placebo dose. RESULTS: On average, morning posaconazole increased the dose-adjusted geometric mean area under the plasma concentration-time curve from zero to infinity (AUC0-∞ ) and peak plasma concentration (Cmax ) of ibrutinib 9.5-fold (90% confidence interval [CI] 6.3-14.3, P < 0.001) and 8.5-fold (90% CI 5.7-12.8, P < 0.001), respectively, while evening posaconazole increased those 10.3-fold (90% CI 6.7-16.0, P < 0.001) and 8.2-fold (90% CI 5.2-13.2, P < 0.001), respectively. Posaconazole had no significant effect on the half-life of ibrutinib, but substantially reduced the metabolite PCI-45227 to ibrutinib AUC0-∞ ratio. There were no significant differences in ibrutinib pharmacokinetics between morning and evening posaconazole phases. CONCLUSIONS: Posaconazole increases ibrutinib exposure substantially, by about 10-fold. This interaction cannot be avoided by dosing the drugs 12 h apart. In general, a 70-mg daily dose of ibrutinib should not be exceeded during posaconazole treatment to avoid potentially toxic systemic ibrutinib concentrations.

Effect of lemborexant on pharmacokinetics of clozapine: A potential drug-drug interaction mediated by time-dependent inhibition of CYP3A4.

Clozapine (CLZ) is extensively used for treatment-resistant schizophrenia (TRS) with caution to avoid serious adverse events such as agranulocytosis and drug-drug interactions (DDIs). In the current report, we present a case of a 35-year-old male non-smoking TRS patient whose steady-state plasma trough concentrations (Ctrough ) of CLZ and its active metabolite, N-desmethylclozapine (NDMC), were significantly increased after initiating oral administration of lemborexant (LEM), a dual orexin receptor antagonist, for the treatment of insomnia. The patient experienced oversedation with sleepiness and fatigue while maintaining high levels of Ctrough of CLZ. The increased concentrations of CLZ returned to normal ranges after the discontinuation of LEM dosing, implying a pharmacokinetic DDI between CLZ and LEM. To gain insight into possible mechanisms, we performed in vitro assays of CYP1A2- and CYP3A4-mediated CLZ metabolism by measuring the formations of NDMC and clozapine N-oxide (CNO). In accordance with previous studies, the incubation of CLZ with each enzyme resulted in the production of both metabolites. LEM had only a weak inhibitory effect on CYP1A2- and CYP3A4-mediated CLZ metabolism. However, the preincubation of LEM with CYP3A4 in the presence of NADPH showed a significant enhancement of inhibitory effects on CLZ metabolism with IC50 values for the formations of CNO and NDMC of 2.8 µM and 4.1 µM, respectively, suggesting that LEM exerts as a potent time-dependent inhibitor for CYP3A4. Taken together, the results of the current study indicate that co-medication of CLZ with LEM may lead to increase in exposure to CLZ and risks of CLZ-related adverse events.

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.

Effect of letermovir initiation on tacrolimus concentrations among lung transplant recipients receiving concomitant azole antifungal prophylaxis.

BACKGROUND: The antiviral letermovir has been increasingly used as off-label cytomegalovirus prophylaxis in solid organ transplant recipients. Observational studies have reported notable increases in tacrolimus (FK) exposure following letermovir; however, whether a significant interaction occurs in the setting of existing moderate-to-strong CYP3A4 inhibition is unknown. Therefore, the purpose of this study was to evaluate FK trough changes before and after letermovir among lung transplant recipients receiving azole antifungal prophylaxis. METHODS: This retrospective cohort study included lung transplant recipients newly initiated on letermovir between 2019-2022 following valganciclovir intolerance. Tacrolimus doses and concentrations were collected up to 30 days before and after the letermovir start date. No pre-emptive FK dose adjustments occurred prior to letermovir initiation. Patients admitted to the hospital or lacking an appropriately timed trough in the pre- or post-period were excluded. RESULTS: A total of 78 lung transplant recipients receiving FK (1.5 mg median total daily dose) and itraconazole (56.4%), isavuconazole (25.6%) or posaconazole (17.9%) prophylaxis were included. Letermovir was started at a median of 8.4 months post-transplant. The pre-/post-letermovir median FK trough was 9.6/9.0 ng/mL (p = .151), median dose-corrected trough was 4.2/4.7 ng/mL/mg (+11.9%, p = .032), and median weight-based dose-corrected trough was 362/326 [ng/mL]/[mg/kg/day] (-9.9%, p = .036). There was no significant difference in the proportion of patients within their goal trough range before and after letermovir initiation (62% vs. 72%, p = .229). CONCLUSION: Empiric FK dose adjustments do not appear warranted before letermovir initiation in lung transplant recipients receiving antifungal prophylaxis with moderate-to-strong CYP3A4 inhibitors.

Inhibition Effect of Okanin Toward Human Cytochrome P450 3A4 and 2D6 with Multi-spectroscopic Studies and Molecular Docking.

Okanin, a major flavonoid of a popular herb tea, Coreopsis tinctoria Nutt., showed strong inhibition on CYP3A4 and CYP2D6. The strong interaction between okanin and CYPs were determined by enzyme kinetics, multispectral technique and molecular docking. The inhibition type of two enzymes, CYP3A4 and CYP2D6, by okanin are mixed and non-competitive inhibition type, respectively. The IC50 values and the binding constant of okanin to CYP3A4 can be deduced that the interaction was stronger than that of CYP2D6. The Conformations of CYP3A4 and CYP2D6 were changed by okanin. The evidence from fluorescence measurement along with molecular docking verified that these two CYPs were bound with okanin by hydrogen bonds and hydrophobic forces. Our investigation suggested that okanin may lead to interactions between herb and drug by inhibiting CYP3A4 and CYP2D6 activities, thus its consumption should be taken with caution.

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.

Drug-drug interaction perpetrators of oxycodone in patients with cancer: frequency and clinical relevance.

AIM: Oxycodone is known to have numerous drug-drug interactions (DDIs) that can potentially decrease efficacy or lead to adverse drug reactions (ADRs). However, there is limited research on the frequency of DDIs associated with oxycodone, which is important in optimising pharmacovigilance and the need for additional research on certain DDIs. In this study, the frequency of pharmacologically and clinically relevant DDI perpetrators was studied in patients with cancer. METHODS: This was a cross-sectional study using hospital pharmacy records of patients with cancer who were prescribed oxycodone between September 2021 and September 2022. Medication records of patients prescribed oxycodone during a period of ≥ 5 consecutive days (= oxycodone treatment episodes) were reviewed to identify the concomitant use of pharmacologically relevant perpetrators, based on reference sources (Lexicomp®, Micromedex®, the Dutch Kennisbank and the Dutch Commentaren Medicatiebewaking). The clinical relevance was examined by a clinical pharmacologist and a medical oncologist. Additionally, the frequency of double interactions-concomitant oxycodone use with two CYP3A4 and / or CYP2D6 perpetrators-was studied. RESULTS: Overall, 254 oxycodone treatment episodes were included, of which 227 (89.4%) were found to contain at least one pharmacologically relevant DDI perpetrator. Of these, 210 (82.7%) were considered to be clinically relevant. A total of 80 different pharmacologically relevant perpetrators were identified, with 65 (81.3%) being considered clinically relevant. Double interactions were observed in 21 (8.3%) oxycodone treatment episodes. CONCLUSION: A high frequency of pharmacologically and clinically relevant perpetrators of oxycodone was observed in our cohort. Moreover, a high number of double interactions involving oxycodone was registered. More intense monitoring of DDIs may be needed to improve medication safety of patients with cancer taking oxycodone.

Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.