Role of Cytochrome P450 3A4 in Cancer Drug Resistance: Challenges and Opportunities.

One of the biggest obstacles to the treatment of diseases, particularly serious conditions like cancer, is therapeutic resistance. The process of drug resistance is influenced by a number of important variables, including MDR genes, drug efflux, low-quality medications, inadequate dosage, etc. Drug resistance must be addressed, and new combinations based on the pharmacokinetics/pharmacodynamics (PK-PD) characteristics of the partner pharmaceuticals must be developed in order to extend the half-lives of already available medications. The primary mechanism of drug elimination is hepatic biotransformation of medicines by cytochrome P450 (CYP) enzymes; of these CYPs, CYP3A4 makes up 30-40% of all known cytochromes that metabolize medications. Induction or inhibition of CYP3A4-mediated metabolism affects the pharmacokinetics of most anticancer drugs, but these details are not fully understood and highlighted because of the complexity of tumor microenvironments and various influencing patient related factors. The involvement of CYPs, particularly CYP3A4 and other drug-metabolizing enzymes, in cancer medication resistance will be covered in the current review.

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.

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 influence of the model pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases in HepaRG cells.

INTRODUCTION: Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the cytochrome P450 system to their corresponding oxon analogues to act as potent inhibitors of acetylcholinesterase. It is known that interactions between cytochrome P450 and pesticides include the inhibition of major xenobiotic metabolizing cytochrome P450 enzymes and changes on the genetic level. METHODS: In this in vitro study, the influence of the pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases was investigated with a metabolically competent cell line (HepaRG cells). First, the viability of the cells after exposure to parathion and paraoxon was evaluated. The inhibitory effect of both pesticides on cytochrome P450 3A4, which is a pivotal enzyme in the metabolism of xenobiotics, was examined by determining the dose-response curve. Changes on the transcription level of 92 oxygenase associated genes, including those for important cytochrome P450 enzymes, were evaluated. RESULTS: The exposure of HepaRG cells to parathion and paraoxon at concentrations up to 100 µM resulted in a viability of 100 per cent. After exposure for 24 hours, pronounced inhibition of cytochrome P450 3A4 enzyme activity was shown, indicating 50 per cent effective concentrations of 1.2 µM (parathion) and 2.1 µM (paraoxon). The results revealed that cytochrome P450 involved in parathion metabolism were significantly upregulated. DISCUSSION: Relevant changes of the cytochrome P450 3A4 enzyme activity and significant alteration of genes associated with cytochrome P450 suggest an interference of pesticide exposure with numerous metabolic processes. The major limitations of the work involve the use of a single pesticide and the in vitro model as surrogate to human hepatocytes. CONCLUSION: The data of this study might be of relevance after survival of acute, life-threatening intoxications with organophosphorus compounds, particularly for the co-administration of drugs, which are metabolized by the affected cytochrome P450.

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.

Pharmacogenetics of Carbamazepine: A Systematic Review on CYP3A4 and CYP3A5 Polymorphisms.

BACKGROUND AND OBJECTIVE: The association between carbamazepine (CBZ) metabolism and resistance in epilepsy and the genetic polymorphisms of CYP3A5 (rs776746 and rs15524) and CYP3A4 (rs2242480, rs2740574, rs35599367, rs12721627, and rs28371759) has been the subject of previous investigations with controversial results. We conducted a systematic review to assess the potential link between these polymorphisms and CBZ metabolism and resistance. METHODS: Identifying relevant studies, was carried out bay searching PubMed, Scopus, PharmGKB, EPIGAD, and PHARMAADME databases up until June 2023. The studies included in our analysis investigated the connection between CYP3A5 (rs776746 and rs15524) and CYP3A4 (rs2242480, rs2740574, rs35599367, rs12721627, and rs28371759) polymorphisms and CBZ metabolism and resistance. RESULTS: This review included a total of 23 studies and more than 2177 epilepsy patients. As a result the CYP3A4 (rs12721627 and rs28371759) polymorphisms are associated with reduced catalytic activity, where the CYP3A4 (rs2740574) polymorphism is linked to lower levels of CBZ-diol and decreased activity. It's been found also that the CYP3A5 (rs776746) polymorphism influences the dose-adjusted plasma levels of CBZ. CONCLUSION: Although these findings highlight the impact of genetic variations in the CYP3A4 and CYP3A5 genes on CBZ pharmacokinetics and pharmacodynamics, further studies across diverse populations are essential to enhance personalized epilepsy therapy in clinical settings.

The influence of drug-induced metabolic enzyme activity inhibition and CYP3A4 gene polymorphism on aumolertinib metabolism.

The purpose of this study is to clarify the drug interaction profile of aumolertinib, and the influence of CYP3A4 genetic polymorphism on aumolertinib metabolic characteristics. Through microsomal enzyme reactions, we screened 153 drugs and identified 15 that significantly inhibited the metabolism of aumolertinib. Among them, telmisartan and carvedilol exhibited potent inhibitory activities in rat liver microsomes (RLM) and human liver microsomes (HLM). In vivo, the pharmacokinetic parameters of aumolertinib, including AUC and Cmax, were significantly altered when co-administered with carvedilol, with a notable decrease in the clearance rate CLz/F. Interestingly, the pharmacokinetic parameters of the metabolite HAS-719 exhibited a similar trend as aumolertinib when co-administered. Mechanistically, both telmisartan and carvedilol exhibited a mixed-type inhibition on the metabolism of aumolertinib. Additionally, we used a baculovirus-insect cell expression system to prepare 24 recombinant CYP3A4 microsomes and obtained enzymatic kinetic parameters using aumolertinib as a substrate. Enzyme kinetic studies obtained the kinetic parameters of various CYP3A4 variant-mediated metabolism of aumolertinib. Based on the relative clearance rates, CYP3A4.4, 5, 7, 8, 9, 12, 13, 14, 17, 18, 19, 23, 24, 33, and 34 showed significantly lower clearance rates compared to the wild-type. Among the different CYP3A4 variants, the inhibitory potency of telmisartan and carvedilol on the metabolism of aumolertinib also varied. The IC50 values of telmisartan and carvedilol in CYP3A4.1 were 6.68 ± 1.76 µM and 0.60 ± 0.25 µM, respectively, whereas in CYP3A4.12, the IC50 exceeded 100 µM. Finally, we utilized adeno-associated virus to achieve liver-specific high expression of CYP3A4*1 and CYP3A4*12. In the group with high expression of the less active CYP3A4*12, the magnitude of the drug-drug interaction was significantly attenuated. In conclusion, CYP3A4 genetic polymorphism not only influences the pharmacokinetic characteristics of aumolertinib, but also the inhibitory potency of telmisartan and carvedilol on it.

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.

Physiologically-Based Pharmacokinetic Modelling to Investigate the Effect of CYP3A4/3A5 Maturation on Tacrolimus Pharmacokinetics in Paediatric HSCT Patients.

Tacrolimus (FK506) is a cornerstone of GVHD-prophylaxis treatment in paediatrics undergoing haematopoietic stem cell transplantation (HSCT). However, due to concerns about highly inter/intra-individual variability, precision dosing of FK506 is crucial. Cytochrome P450(CYP) 3A4 and 3A5 are considered important sources of FK506 pharmacokinetic variability. Nevertheless, the impact of age-related maturation in hepatic and intestinal CYP3A4/3A5 enzymes remains unknown in paediatric HSCT patients. Physiologically-based pharmacokinetic (PBPK) models were developed and verified in adult volunteers and adult HSCT patients using GastroPlusTM (version 9.0), and then extrapolated to paediatric HSCT patients, taking into account the maturation of CYP3A4 and CYP3A5. Default CYP3A4 and CYP3A5 ontogeny profiles were updated based on the latest reports. The paediatric PBPK model was evaluated with independent data collected from Sun Yat-sen Memorial Hospital (86 paediatric HSCT patients, 1 to 16 -year-old). Simulations were performed to evaluate a reported FK506 dosing regimen in infants and children with different CYP3A5 genotypes. Extensive PBPK model validation indicated good predictability, with the predicted/observed (P/O) ratios within the range of 0.80-fold to 1.25-fold. Blood tacrolimus concentration-time curves were comparable between the real and virtual patients. Simulations showed that the higher levels of tacrolimus in 9-month-old to 3-year-old infants were mainly attributed to the CYP3A4/3A5 ontogeny profiles, which resulted in lower clearance and higher exposure relative to dose. The oral dosage of 0.1 mg/kg/day (q12 h) is considered appropriate for paediatric HSCT patients 9 months to 15 years of age with CYP3A5 *1/*1 genotypes. Lower doses were required for paediatric HSCT patients with CYP3A5 *1/*3 (0.08 mg/kg/day, q12h) or CYP3A5 *3/*3 genotypes (0.07 mg/kg/day, q12h), and analyses demonstrated 12.5%-20% decreases in ≤3-year-old patients. The study highlights the feasibility of PBPK modelling to explore age-related enzyme maturation in infants and children(≤3-year-old) undergoing HSCT and emphasizes the need to include hepatic and gut CYP3A4/3A5 maturation parameters.

Applying Physiologically Based Pharmacokinetic Modeling to Interpret Carbamazepine's Nonlinear Pharmacokinetics and Its Induction Potential on Cytochrome P450 3A4 and Cytochrome P450 2C9 Enzymes.

Carbamazepine (CBZ) is commonly prescribed for epilepsy and frequently used in polypharmacy. However, concerns arise regarding its ability to induce the metabolism of other drugs, including itself, potentially leading to the undertreatment of co-administered drugs. Additionally, CBZ exhibits nonlinear pharmacokinetics (PK), but the root causes have not been fully studied. This study aims to investigate the mechanisms behind CBZ's nonlinear PK and its induction potential on CYP3A4 and CYP2C9 enzymes. To achieve this, we developed and validated a physiologically based pharmacokinetic (PBPK) parent-metabolite model of CBZ and its active metabolite Carbamazepine-10,11-epoxide in GastroPlus®. The model was utilized for Drug-Drug Interaction (DDI) prediction with CYP3A4 and CYP2C9 victim drugs and to further explore the underlying mechanisms behind CBZ's nonlinear PK. The model accurately recapitulated CBZ plasma PK. Good DDI performance was demonstrated by the prediction of CBZ DDIs with quinidine, dolutegravir, phenytoin, and tolbutamide; however, with midazolam, the predicted/observed DDI AUClast ratio was 0.49 (slightly outside of the two-fold range). CBZ's nonlinear PK can be attributed to its nonlinear metabolism caused by autoinduction, as well as nonlinear absorption due to poor solubility. In further applications, the model can help understand DDI potential when CBZ serves as a CYP3A4 and CYP2C9 inducer.

Evaluation and optimization of a microcavity plate-based human hepatocyte spheroid model for predicting clearance of slowly metabolized drug candidates.

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning® Elplasia® microcavity 96-well microplates enable culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared to microsomes, hepatocyte suspensions, 2D plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted CLint and CLb within 3-fold for 53% (9/17; AAFE=3.9) and 82% (14/17; AAFE=2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE=4.0) and CLb (14/17; AAFE=2.1) without need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 µM itraconazole was within 25% of observed values for 6/8 compounds with 5/8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance, as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. Significance Statement Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance which is improved with the consideration of mass transport within the spheroid. Incubations with 3 µM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.

Exploring Acute Liver Damage: Slimming Health Foods and CYP3A4 Induction.

Background: Patients taking multiple drugs and various health foods often develop acute hepatitis. We hypothesized that the interaction between health foods and drug metabolism was the cause of severe liver injury in these patients. Therefore, we studied changes in the activity of the drug-metabolizing enzyme, cytochrome P450 (CYP), using slimming health food extracts and elucidated the molecular mechanism of liver injury onset through hepatotoxicity evaluation. Methods: For cytotoxicity testing, health food extract samples were added to HepG2 cells derived from hepatic parenchymal cells and culture medium, and cell viability was calculated 48 h after culture. To evaluate CYP3A4 induction, 3-1-10 cells constructed with a reporter linked to CYP3A4 gene were used, and reporter activity was measured 48 h after culture. Results: In the chronological order of the slimming health food intake history of the patient, niacinamide and Gymnema sylvestre extracts strongly inhibited HepG2 cell viability. In contrast, dietary supplements A and Coleus forskohlii extract strongly induced CYP3A4 reporter activity.To confirm CYP3A4 induction in humans, humanized CYP3A/pregnane X receptor (PXR) mice were treated with forskolin. CYP3A4 mRNA expression levels were elevated 3.9 times compared to that of the control group (P < 0.05). Conclusion: Coleus forskohlii extract showed the strongest transcriptional activation of CYP3A4 gene. In a mouse model of human-type drug metabolism, forskolin induced CYP3A4 transcription. Thus, we concluded that CYP3A4 induction by Coleus forskohlii is one of the causes of crucial hepatocellular injury, which is a type of liver injury caused by the active metabolite of acetaminophen produced by CYP3A4.

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.

Pharmacokinetics and pharmacodynamics of drug‒drug interactions in hospitalized older adults treated with direct oral anticoagulants.

PURPOSE: Polypharmacy is a frequent situation in older adults that increases the risk of drug-drug interactions (DDIs), both pharmacokinetic (PK) and pharmacodynamic (PD). Direct oral anticoagulants (DOACs) are frequently prescribed in older adults, mainly because of the high prevalence of atrial fibrillation (AF). DOACs are subject to cytochrome P450 3A4 (CYP3A4)- and/or P-glycoprotein (P-gp)-mediated PK DDIs and PD DDIs when co-administered with drugs that interfere with platelet function. The aim of our study was to assess the prevalence of DDIs involving DOACs in older adults and the associated risk factors at admission and discharge. METHODS: This was a cross-sectional study conducted in an acute geriatric unit between January 1, 2018 and December 31, 2022, including patients over 75 years of age treated with DOACs at admission and/or discharge, for whom a comprehensive collection of co-medications was performed. RESULTS: From 909 hospitalizations collected, the prevalence of PK DDIs involving DOACs was 16.9% at admission and 20.7% at discharge, and the prevalence of PD DDIs was 20.7% at admission and 20.2% at discharge. Factors associated with DDIs were bleeding history [adjusted odds ratio (ORa) 1.74, 95% confidence interval (CI) 1.13-2.68], number of drugs > 6 (ORa 2.54, 95% CI 1.88-3.46) and reduced dose of DOACs (ORa 0.39, 95% CI 0.28-0.54) at admission and age > 87 years (ORa 0.74, 95% CI 0.55-0.99), number of drugs > 6 (ORa 2.01, 95% CI 1.48-2.72) and reduced dose of DOACs (ORa 0.41, 95% CI 0.30-0.57) at discharge. CONCLUSION: This study provides an indication of the prevalence of DDIs as well as the profile of DDIs and patients treated with DOACs.

Prediction of Pediatric Pharmacokinetics for CYP3A4 Metabolized Drugs: Comparison of the Performance of Two Hepatic Ontogeny Within a Physiologically Based Pharmacokinetic Model.

The rapid growth in the use of pediatric physiologically based pharmacokinetic (PBPK) models, particularly for regulatory applications, has focused emphasis on model verification and ensuring system parameters are robust, including how these change with age. Uncertainty remains regarding the ontogeny of some enzymes and transporters, in this study 2 published ontogeny profiles for hepatic CYP3A4 were compared. Clinical pharmacokinetic data on 4 intravenously administered CYP3A4 substrates (alfentanil, fentanyl, midazolam, and sildenafil) used across the pediatric age range was collected from the literature. The PBPK models were verified in the adult population and then used to compare the Salem and a modified Upreti ontogeny profiles for CYP3A4 in terms of parent drug clearance and area under the curve from birth onward. Overall, the modified Upreti ontogeny profile resulted in 15 out of 17 age-related predictions within 2-fold and 12 out of 17 predictions within 1.5-fold ranges of observed values, for the Salem ontogeny these values were 12 out of 17 and 8 out of 17, respectively. The Upreti ontogeny profile performed better than Salem, average fold error and absolute average fold error were 1.14 and 1.35 compared to 1.56 and 1.90, respectively. Identifying the optimal CYP3A4 ontogeny is important for regulatory use of PBPK especially given the number of drugs cleared by this enzyme. This study broadens the evidence from previous studies that Upreti is more favorable than Salem, but further work is needed especially in the neonatal and early infant age range.

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.

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.

Design, synthesis, molecular modeling, in vitro and in vivo biological evaluation of potent anthranilamide derivatives as dual P-glycoprotein and CYP3A4 inhibitors.

Paclitaxel (PTX) is considered the blockbuster chemotherapy treatment for cancer. Paclitaxel's (PTX) oral administration has proven to be extremely difficult, mostly because of its susceptibility to intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP3A4). The concurrent local inhibition of intestinal P-gp and CYP3A4 is a promising approach to improve the oral bioavailability of paclitaxel while avoiding potential unfavorable side effects of their systemic inhibition. Herein, we report the rational design and evaluation of novel dual potent inhibitors of P-gp and CYP3A4 using an anthranilamide derivative tariquidar as a starting point for their structural optimizations. Compound 14f, bearing N-imidazolylbenzyl side chain, was found to have potent and selective P-gp (EC50 = 28 nM) and CYP3A4 (IC50 = 223 nM) inhibitory activities with low absorption potential (Papp (A-to-B) <0.06). In vivo, inhibitor 14f improved the oral absorption of paclitaxel by 6 times in mice and by 30 times in rats as compared to vehicle, while 14f itself remained poorly absorbed. Compound 14f, possessing dual P-gp and CYP3A4 inhibitory activities, offered additional enhancement in paclitaxel oral absorption compared to tariquidar in mice. Evaluating the CYP effect of 14f on oral absorption of paclitaxel requires considering the variations in CYP expression between animal species. This study provides further medicinal chemistry advice on strategies for resolving concerns with the oral administration of chemotherapeutic agents.

Identification of non-adherence to adjuvant letrozole using a population pharmacokinetics approach in hormone receptor-positive breast cancer patients.

BACKGROUND: Letrozole, an aromatase inhibitor metabolised via CYP2A6 and CYP3A4/5 enzymes, is used as adjuvant therapy for women with hormone receptor (HR)-positive early breast cancer. The objective of this study was to quantify the impact of CYP2A6 genotype on letrozole pharmacokinetics (PK), to identify non-adherent patients using a population approach and explore the possibility of a relationship between non-adherence and early relapse. METHODS: Breast cancer patients enrolled in the prospective PHACS study (ClinicalTrials.gov NCT01127295) and treated with adjuvant letrozole 2.5 mg/day were included. Trough letrozole concentrations (Css,trough) were measured every 6 months for 3 years by a validated LC-MS/MS method. Concentration-time data were analysed using non-linear mixed effects modelling. Three methods were evaluated for identification of non-adherent subjects using the base PK model. RESULTS: 617 patients contributing 2534 plasma concentrations were included and led to a one-compartment PK model with linear absorption and elimination. Model-based methods identified 28 % of patients as non-adherent based on high fluctuations of their Css,trough compared to 3 % based on patient declarations. The covariate analysis performed in adherent subjects revealed that CYP2A6 intermediate (IM) and slow metabolisers (SM) had 21 % (CI95 % = 12 - 30 %) and 46 % (CI95 % = 41 - 51 %) lower apparent clearance, respectively, compared to normal and ultrarapid metabolisers (NM+UM). Early relapse (19 patients) was not associated with model-estimated, concentration-based or declared adherence in the total population (p = 0.41, p = 0.37 and p = 0.45, respectively). CONCLUSIONS: These findings will help future investigations focusing on the exposure-efficacy relationship for letrozole in adjuvant setting.

Gene polymorphism as a cause of hemorrhagic complications in patients with non-valvular atrial fibrillation treated with oral vitamin K-independent anticoagulants.

Atrial fibrillation (AF) accounts for 40% of all cardiac arrhythmias and is associated with a high risk of stroke and systemic thromboembolic complications. Dabigatran, rivaroxaban, apixaban, and edoxaban are direct oral anticoagulants (DOACs) that have been proven to prevent stroke in patients with non-valvular AF. This review summarizes the pharmacokinetics, pharmacodynamics, and drug interactions of DOACs, as well as new data from pharmacogenetic studies of these drugs. This review is aimed at analyzing the scientific literature on the gene polymorphisms involved in the metabolism of DOACs. We searched PubMed, Cochrane, Google Scholar, and CyberLeninka (Russian version) databases with keywords: 'dabigatran', 'apixaban', 'rivaroxaban', 'edoxaban', 'gene polymorphism', 'pharmacogenetics', 'ABCB1', 'CES1', 'SULT1A', 'ABCG2', and 'CYP3A4'. The articles referred for this review include (1) full-text articles; (2) study design with meta-analysis, an observational study in patients taking DOAC; and (3) data on the single-nucleotide polymorphisms and kinetic parameters of DOACs (plasma concentration), or a particular clinical outcome, published in English and Russian languages during the last 10 years. The ages of the patients ranged from 18 to 75 years. Out of 114 reviewed works, 24 were found eligible. As per the available pharmacogenomic data, polymorphisms affecting DOACs are different. This may aid in developing individual approaches to optimize DOAC pharmacotherapy to reduce the risk of hemorrhagic complications. However, large-scale population studies are required to determine the dosage of the new oral anticoagulants based on genotyping. Information on the genetic effects is limited owing to the lack of large-scale studies. Uncovering the mechanisms of the genetic basis of sensitivity to DOACs helps in developing personalized therapy based on patient-specific genetic variants and improves the efficacy and safety of DOACs in the general population.

Gene polymorphism as a cause of hemorrhagic complications in patients with non-valvular atrial fibrillation treated with oral vitamin K-independent anticoagulantsAtrial fibrillation (AF) accounts for 40% of all cardiac arrhythmias and is associated with a high risk of stroke and systemic thromboembolic complications. Dabigatran, rivaroxaban, apixaban, and edoxaban are direct oral anticoagulants (DOACs) that have been proven to prevent stroke in patients with non-valvular AF. This review summarizes the pharmacokinetics, pharmacodynamics, and drug interactions of DOACs, as well as new data from pharmacogenetic studies of these drugs.