Investigating the Contribution of Drug-Metabolizing Enzymes in Drug-Drug Interactions of Dapivirine and Miconazole

Dapivirine (DPV) is a potent NNRTI used to prevent the sexual transmission of HIV. In a phase 1 trial (IPM 028), the concomitant use of a DPV vaginal ring and an antifungal miconazole (MIC) vaginal capsule was found to increase the systemic exposure to DPV in women, suggesting a potential for drug-drug interactions. This study’s objective was to investigate the mechanism of DPV-MIC interactions using drug-metabolizing enzymes (DMEs; CYPs and UGTs) that are locally expressed in the female reproductive tract (FRT). In vitro studies were performed to evaluate the metabolism of DPV and its inhibition and induction potential with DMEs. In addition, the impact of MIC on DPV metabolism and the inhibitory potential of DPV with DMEs were studied. Our findings suggest that DPV is a substrate of CYP1A1 and CYP3A4 enzymes and that MIC significantly decreased the DPV metabolism by inhibiting these two enzymes. DPV demonstrated potent inhibition of CYP1A1 and moderate/weak inhibition of the six CYP and eight UGT enzymes evaluated. MIC showed potent/moderate inhibition of seven CYP enzymes and weak/no inhibition of eight UGT enzymes. The combination of DPV and MIC showed potent inhibition of seven CYP enzymes (1A1, 1A2, 1B1, 2B6, 2C8, 2C19, and 3A4) and four UGT enzymes (1A3, 1A6, 1A9, and 2B7). DPV was not an inducer of CYP1A2, CYP2B6, and CYP3A4 enzymes in primary human hepatocytes. Therefore, the increased systemic concentrations of DPV observed in IPM 028 were likely due to the reduced metabolism of DPV because of CYP1A1 and CYP3A4 enzymes inhibition by MIC in the FRT.

Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

A Novel System for Evaluating the Inhibition Effect of Drugs on Cytochrome P450 Enzymes in vitro Based on Human-Induced Hepatocytes (hiHeps)

Cytochrome P450 (CYP) is the most important phase I drug-metabolizing enzyme, and the effect of drugs on CYP enzymes can lead to decreased pharmacological efficacy or enhanced toxicity of drugs, but there are many deficiencies in the evaluation models of CYP enzymes in vitro. Human-induced hepatocytes (hiHeps) derived from human fibroblasts by transdifferentiation have mature hepatocyte characteristics. The aim was to establish a novel evaluation system for the effect of drugs on CYP3A4, 1A2, 2B6, 2C9, and 2C19 in vitro based on hiHeps. Curcumin can inhibit many CYP enzymes in vitro, and so the inhibition of curcumin on CYP enzymes was compared by human liver microsomes, human hepatocytes, and hiHeps using UPLC-MS and the cocktail method. The results showed that the IC50 values of CYP enzymes in the hiHeps group were similar to those in the hepatocytes group, which proved the effectiveness and stability of the novel evaluation system in vitro. Subsequently, the evaluation system was applied to study the inhibitory activity of notoginseng total saponins (NS), safflower total flavonoids (SF), and the herb pair of NS–SF on five CYP enzymes. The mechanism of improving efficacy after NS and SF combined based on CYP enzymes was elucidated in vitro. The established evaluation system will become a powerful tool for the research of the effect of drugs on the activity of CYP enzymes in vitro, which has broad application prospects in drug research.

Abstract P283: CCL5 Acts As A Vasoconstrictor On Penetrating Arterioles In The Cerebral Circulation By Enhancing 20-HETE Activity

20-HETE is synthesized from arachidonic acid by cytochrome P450 (CYP) enzymes 4A and 4F. Inactivating mutations in the CYP enzymes that produce 20-HETE are associated with hypertension and stroke in man. We previously revealed that inactivating variants of CYP4A/F enzymes are associated with dementia in the Atherosclerosis Risk in Communities Neurocognitive Study (ARIC-NS) population. 20-HETE is involved with sodium regulation in the kidney and is a powerful vasoconstrictor. It was recently discovered that CCL5 and 20-HETE share the same receptor, GPR75. We previously found that 20-HETE constricts and augments the myogenic response (MR) of the middle cerebral artery (MCA) and renal afferent arteriole. However, whether CCL5 has any effect on penetrating arterioles (PAs) and interacts with 20-HETE is unknown. We found that GPR75 is expressed in PAs and pericytes in the brain. CYP4A is also expressed in pericytes and is inversely proportional to levels of GPR75 in the brain. In the present study, we found that 20-HETE contributes to the basal myogenic tone of PAs in SD rats. Administration of HET0016, a 20-HETE synthesis inhibitor, dilated the PA by 34 ± 3% (n = 6) under 10 mmHg perfusion pressure. Administration of WIT003, a 20-HETE agonist, constricted the vessel by 23 ± 4% (n = 6) under the same perfusion pressure. We found that CCL5 also reduced PA diameter by 20 ± 4% (n = 7) in SD rats under 10 mmHg perfusion pressure. Moreover, we compared the response to CCL5 in SS rats that are 20-HETE deficient and SS.CYP4A1 transgenic rats in which 20-HETE production is restored. PAs isolated from SS rats treated with 0.1 nM CCL5 constricted by 9 ± 5% (n = 6) while those treated with 10 nM constricted by 12 ± 3% (n = 6). CCL5 had a greater response in PAs from the SS.CYP4A1 strain, and the diameter of the PAs constricted by 14 ± 2% (n = 5) and 24 ± 5% (n = 5) in response to 0.1 and 10 nM CCL5, respectively. These results demonstrate that CCL5 has a direct effect on PAs similar to 20-HETE that acts via the GPR75 receptor. However, further study is needed to determine how CCL5 and 20-HETE interact to promote vasoconstriction. These studies would help further understand the involvement of 20-HETE in disease and potentially identify novel drug targets.

Cytochrome P450 enzymes: a review on drug metabolizing enzyme inhibition studies in drug discovery and development

Assessment of drug candidate's potential to inhibit cytochrome P450 (CYP) enzymes remains crucial in pharmaceutical drug discovery and development. Both direct and time-dependent inhibition of drug metabolizing CYP enzymes by the concomitant administered drug is the leading cause of drug–drug interactions (DDIs), resulting in the increased toxicity of the victim drug. In this context, pharmaceutical companies have grown increasingly diligent in limiting CYP inhibition liabilities of drug candidates in the early stages and examining risk assessments throughout the drug development process. This review discusses different strategies and decision-making processes for assessing the drug–drug interaction risks by enzyme inhibition and lays particular emphasis on in vitro study designs and interpretation of CYP inhibition data in a stage-appropriate context.

The Effect of Chronic Iloperidone Treatment on Cytochrome P450 Expression and Activity in the Rat Liver: Involvement of Neuroendocrine Mechanisms

In order to achieve a desired therapeutic effect in schizophrenia patients and to maintain their mental wellbeing, pharmacological therapy needs to be continued for a long time, usually from the onset of symptoms and for the rest of the patients’ lives. The aim of our present research is to find out the in vivo effect of chronic treatment with atypical neuroleptic iloperidone on the expression and activity of cytochrome P450 (CYP) in rat liver. Male Wistar rats received a once-daily intraperitoneal injection of iloperidone (1 mg/kg) for a period of two weeks. Twenty-four hours after the last dose, livers were excised to study cytochrome P450 expression (mRNA and protein) and activity, pituitaries were isolated to determine growth hormone-releasing hormone (GHRH), and blood was collected for measuring serum concentrations of hormones and interleukin. The results showed a broad spectrum of changes in the expression and activity of liver CYP enzymes, which are important for drug metabolism (CYP1A, CYP2B, CYP2C, and CYP3A) and xenobiotic toxicity (CYP2E1). Iloperidone decreased the expression and activity of CYP1A2, CP2B1/2, CYP2C11, and CYP3A1/2 enzymes but increased that of CYP2E1. The CYP2C6 enzyme remained unchanged. At the same time, the level of GHRH, GH, and corticosterone decreased while that of T3 increased, with no changes in IL-2 and IL-6. The presented results indicate neuroendocrine regulation of the investigated CYP enzymes during chronic iloperidone treatment and suggest a possibility of pharmacokinetic/metabolic interactions produced by the neuroleptic during prolonged combined treatment with drugs that are substrates of iloperidone-affected CYP enzymes.

Cytochrome P450 in GtoPdb v.2021.2

The cytochrome P450 enzyme superfamily (CYP), E.C. 1.14.-.-, are haem-containing monooxygenases with a vast range of both endogenous and exogenous substrates. These include sterols, fatty acids, eicosanoids, fat-soluble vitamins, hormones, pesticides and carcinogens as well as drugs. Listed below are the human enzymes, their relationship with rodent CYP enzyme activities is obscure in that the species orthologue may not metabolise the same substrates. Some of the CYP enzymes located in the liver are particularly important for drug metabolism, both hepatic and extrahepatic CYP enzymes also contribute to patho/physiological processes. Genetic variation of CYP isoforms is widespread and likely underlies a proportion of individual variation in drug disposition. The superfamily has the root symbol CYP, followed by a number to indicate the family, a capital letter for the subfamily with a numeral for the individual enzyme. Some CYP are able to metabolise multiple substrates, others are oligo- or mono- specific.

Pharmacologic characterization of TACH101, a first-in-class KDM4 inhibitor for development as a cancer therapeutic.

e15067 Background: The histone Lysine (K) Demethylase 4 family, KDM4 family, is involved in nuclear functions such as programming development, activation or repression of transcription, timing and control of the cell cycle, and initiating DNA replication and repair through chromatin remodeling. Overexpression of KDM4 leading to mistakes in post-translational histone modification has been associated with many cancer types and is being investigated as a potential therapeutic drug target. TACH101 is a novel potent and small molecule inhibitor of the KDM4 family. It demonstrated strong efficacy in multiple cancer types in vitro and in vivo and favorable pharmacokinetics (PK) and safety profiles in preclinical models. Methods: The target interaction properties of TACH101 were evaluated using purified enzymes for biochemical, biophysical, and inhibition mechanism studies. For PK parameter measurements, Sprague-Dawley rats and Beagle dogs were used for intravenous or oral administration of TACH101. Hepatocytes or liver microsomes were used to measure cytochrome P450 (CYP) inhibition and induction studies. Cell lines expressing cardiac channels were used for radioligand displacement assays and patch clamping. Results: Biochemical, biophysical, and inhibition mechanism studies using purified enzymes showed TACH101 is a reversible, alpha-KG (co-factor) competitive, and slow-binding inhibitor with IC50 values less than 100 nM. Pharmacologic studies showed plasma protein binding of TACH101 to be ≥99% bound in mouse, rat, dog, and human. TACH101 appeared to be selective in off-target panel assays of receptors, ion channels and transporters. TACH101 did not show any significant binding displacement activity against cardiac ion channels or any effect on hERG in CHO cells using manual patch clamp techniques. In rats and dogs, TACH101 exhibited low systemic clearance, a moderate volume of distribution in rats and dogs, linear exposure profiles with a terminal half-life (̃ 6-hour) and high bioavailability (46-100%). In repeated dose studies in rats and dogs, mean Cmax and AUC values increased in a dose-related manner over the dose range evaluated. There was no significant impact of food on systemic exposure in dogs. TACH101 did not show inhibition or induction of CYP enzymes tested. Conclusions: TACH101 is a potent KDM4 specific inhibitor without significant off-target activity in in vitro and in vivo studies. It did not show inhibition or induction of CYP enzymes suggesting low probability of CYP mediated drug-drug interaction. The exposure from oral administration in rats and dogs was dose proportional and was not significantly affected by food intake in dogs. Given the favorable target activity, PK properties and safety profile, a Phase 1 study is being planned to advance TACH101 for patients with advanced cancer.