Human CYP3A4 Research Articles

Novel Synthetic Analogues of 19(S/R)-Hydroxyeicosatetraenoic Acid Exhibit Noncompetitive Inhibitory Effect on the Activity of Cytochrome P450 1A1 and 1B1.

Cytochrome P450 (CYP) 1A1 and CYP1B1 enzymes play a significant role in the pathogenesis of cancer and cardiovascular diseases (CVD) such as cardiac hypertrophy and heart failure. Previously, we have demonstrated that R- and S-enantiomers of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid endogenous metabolite, enantioselectively inhibit CYP1B1. The current study was conducted to test the possible inhibitory effect of novel synthetic analogues of R- and S-enantiomers of 19-HETE on the activity of CYP1A1, CYP1A2, and CYP1B1. The O-dealkylation rate of 7-ethoxyresorufin (EROD) by recombinant human CYP1A1 and CYP1B1, in addition to the O-dealkylation rate of 7-methoxyresorufin (MROD) by recombinant human CYP1A2, were measured in the absence and presence of varying concentrations (0-40 nM) of the synthetic analogues of 19(R)- and 19(S)-HETE. Also, the possible inhibitory effect of both analogues on the catalytic activity of EROD and MROD, using RL-14 cells and human liver microsomes, was assessed. The results showed that both synthetic analogues of 19(R)- and 19(S)-HETE exhibited direct inhibitory effects on the activity of CYP1A1 and CYP1B1, while they had no significant effect on CYP1A2 activity. Nonlinear regression analysis and comparisons showed that the mode of inhibition for both analogues is noncompetitive inhibition of CYP1A1 and CYP1B1 enzymes. Also, nonlinear regression analysis and Dixon plots showed that the R- and S-analogues have KI values of 15.7 ±4.4 and 6.1 ±1.5 nM for CYP1A1 and 26.1 ±2.9 and 9.1 ±1.8 nM for CYP1B1, respectively. Moreover, both analogues were able to inhibit EROD and MROD activities in a cell-based assay and human liver microsomes. Therefore, the synthetic analogues of 19-HETE could be considered as a novel therapeutic approach in the treatment of cancer and CVD.

Abstract 1250: ODM-208, a novel, small-molecule CYP11A1 inhibitor, demonstrates strong inhibition of steroid biosynthesis and antitumor activity in castration-resistant prostate cancer (CRPC) model

Abstract Several new treatments have been developed to target the androgen signaling axis in CRPC over the last decade. Despite the new treatment options, the challenge for managing CRPC is the limited duration of clinical benefit from treatments due to primary and acquired resistance. Altered steroid biosynthesis and adaptive signaling by endogenous steroids especially in AR mutated tumors have been suggested to be one of the resistance mechanisms. Inhibition of CYP11A1 enzyme (cytochrome P450scc) that catalyzes the first step in the steroidogenic pathway and leads to suppression of the synthesis of all steroid hormones and their precursors is expected to be a target with therapeutic potential in CRPC patients. Here we show, using multiple in vitro and in vivo models, selectivity and anti-tumor activity of a potent, orally bioavailable, inhibitor of CYP11A1 enzyme, ODM-208. The potency and specificity of ODM-208 was tested by using targeted screening assay and human recombinant CYP11A1 enzyme. The inhibition potential of ODM-208 in vitro was studied in H295R cells. The selectivity of ODM-208 to wide panel of different target classes including GPCRs, nuclear receptors, ion channels and enzymes was evaluated. Inhibition of steroidogenesis in vivo was studied in intact male dogs after repeated 4-week dosing of ODM-208, with and without corticosteroid replacement therapy. Steroid hormone concentrations in plasma and cell culture media were analyzed by LC-MS/MS. In vivo antitumor effects of ODM-208 were assessed in murine subcutaneous CRPC VCaP xenograft model. Strong reduction of androgen and other steroid hormones in H295R cells was shown with ODM-208, whereas with abiraterone clear increases in concentrations of pregnenolone, progesterone and corticosterone were observed. ODM-208 showed high selectivity to CYP11A1 and no significant off-target binding was detected in the broad panel. ODM-208 exposure-dependent decreases in plasma testosterone and cortisol concentrations were detected. ODM-208 inhibited substantially tumor growth in murine CRPC VCaP xenograft model. A phase 1/2 study of ODM-208 administered concomitantly with corticosteroid replacement and androgen deprivation therapy in metastatic CRPC subjects is ongoing (NCT03436485). In subjects with ODM-208 treatment all studied serum steroid hormones were decreased to under lower limit of quantification in the LC-MS/MS assays. These preclinical and clinical data suggest that ODM-208 has potential to provide clinical benefit to patients with treatment-resistant CRPC bearing altered steroid synthesis and/or mutated AR. Citation Format: Mari Karimaa, Henna Kettunen, Meri Ramela, Suvi Mansikka-Savolainen, Marcin Chrusciel, Outi Simola, Päivi Taavitsainen, Gerd Wohlfahrt, Petteri Rummakko, Annamari Vuorela, Karim Fizazi, Riikka Oksala. ODM-208, a novel, small-molecule CYP11A1 inhibitor, demonstrates strong inhibition of steroid biosynthesis and antitumor activity in castration-resistant prostate cancer (CRPC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1250.

Inhibition of drug-metabolizing enzymes by Jingyin granules: implications of herb-drug interactions in antiviral therapy.

Jingyin granules, a marketed antiviral herbal medicine, have been recommended for treating H1N1 influenza A virus infection and Coronavirus disease 2019 (COVID-19) in China. To fight viral diseases in a more efficient way, Jingyin granules are frequently co-administered in clinical settings with a variety of therapeutic agents, including antiviral drugs, anti-inflammatory drugs, and other Western medicines. However, it is unclear whether Jingyin granules modulate the pharmacokinetics of Western drugs or trigger clinically significant herb–drug interactions. This study aims to assess the inhibitory potency of the herbal extract of Jingyin granules (HEJG) against human drug-metabolizing enzymes and to clarify whether HEJG can modulate the pharmacokinetic profiles of Western drug(s) in vivo. The results clearly demonstrated that HEJG dose-dependently inhibited human CES1A, CES2A, CYPs1A, 2A6, 2C8, 2C9, 2D6, and 2E1; this herbal medicine also time- and NADPH-dependently inhibited human CYP2C19 and CYP3A. In vivo tests showed that HEJG significantly increased the plasma exposure of lopinavir (a CYP3A-substrate drug) by 2.43-fold and strongly prolonged its half-life by 1.91-fold when HEJG (3 g/kg) was co-administered with lopinavir to rats. Further investigation revealed licochalcone A, licochalcone B, licochalcone C and echinatin in Radix Glycyrrhizae, as well as quercetin and kaempferol in Folium Llicis Purpureae, to be time-dependent CYP3A inhibitors. Collectively, our findings reveal that HEJG modulates the pharmacokinetics of CYP substrate-drug(s) by inactivating CYP3A, providing key information for both clinicians and patients to use herb–drug combinations for antiviral therapy in a scientific and reasonable way.

An insight on the nature of biochemical interactions between glycyrrhizin, myricetin and CYP3A4 isoform.

Drug interaction studies are imperative to gain insights into the beneficial or harmful effects of therapeutic and dietary agents. This study investigated the mechanism of modulatory roles of glycyrrhizin (GLH) and myricetin (MYC) on the human CYP3A4 isoform using in silico and in vitro methods. While MYC had concentration-dependent inhibitory effect on CYP3A4 (IC50 : 10.5±0.55μM) with characteristic Km and Vmax values of 1.13μM and 1.54nM/min, respectively, GLH exhibited no inhibitory effect on CYP3A4 activity in vitro. These observations are consistent with the results of in silico evaluations where the effect of MYC compared well with that of ketoconazole (a known CYP3A4 inhibitor) against CYP3A4. Overall, the established interactions between the study compounds and CYP3A4 could potentiate clinically vital drug-drug interactions and has lent credence to the mechanism of modulatory effect of MYC and GLH on CYP3A4 that could guide their safe use as therapeutic agents. PRACTICAL IMPLICATIONS: Myricetin (MYR) and glycyrrhizin (GLH) occur freely in commonly ingested foods and their supplements are recommended for the treatment of several debilitating diseases such as diabetes, cancer, and cardiovascular complications. This study provided an insight on the possible interactions that could be established between these compounds (MYR and GLH) and CYP3A4 when ingested and metabolized by the liver. The results suggested possibilities of potential clinical drug-drug interactions and advocates for their cautious use within the therapeutic dose in food supplements or medications to avoid probable liver damage.

Solar-Powered Whole-Cell P450 Catalytic Platform for C-Hydroxylation Reactions.

Photobiocatalysis is a green platform for driving redox enzymatic reactions using solar energy, not needing high-cost cofactors and redox partners. Here, a visible light-driven whole-cell platform for human cytochrome P450 (CYP) photobiocatalysis was developed using natural flavins as a photosensitizer. Photoexcited flavins mediate NADPH/reductase-free, light-driven biocatalysis by human CYP2E1 both in vitro and in the whole-cell systems. In vitro tests demonstrated that the photobiocatalytic activity of CYP2E1 is dependent on the substrate type, the presence of catalase, and the acid type used as a sacificial electron donor. A protective effect of catalase was found against the inactivation of CYP2E1 heme by H2 O2 and the direct transfer of photo-induced electrons to the heme iron not by peroxide shunt. Furthermore, the P450 photobiocatalysis in whole cells containing human CYPs 1A1, 1A2, 1B1, and 3A4 demonstrated the general applicability of the solar-powered, flavin-mediated P450 photobiocatalytic system.

Human CYP enzyme-activated clastogenicity of 2-ethylhexyl diphenyl phosphate (a flame retardant) in mammalian cells

2-Ethylhexyl diphenyl phosphate (EHDPP) is a common flame retardant and environmental pollutant, exposing humans with endocrinal disrupting potentials. Its mutagenicity, especially following metabolism, remains unclear. In this study, molecular docking analysis indicated that EHDPP was a potential substrate for several human CYP enzymes except for CYP1A1. Among V79-derived cell lines genetically engineered for the expression of each CYP, EHDPP (6 h exposure/18 h recovery) did not induce micronuclei in the V79 or V79-derived cells expressing human CYP1A1, however, it was positive in V79-derived cell lines expressing human CYP2E1, 3A4, and 2B6. In a human hepatoma (HepG2) cell line, EHDPP (48 h exposure) moderately induced micronuclei, which was blocked by 1-aminobenzotriazole (ABT, 60 μM, inhibitor of CYPs); pretreating HepG2 cells with bisphenol AF, another organic pollutant as inducer of CYPs (0.1 μM for 16 h), significantly potentiated micronuclei formation by EHDPP, threshold being decreased from 10 to 1.25 μM. This effect was blocked by ABT, drastically reduced by ketoconazole (inhibiting CYP3A expression/activity), and moderately inhibited by trans-1,2-dichloroethylene (selective CYP2E1 inhibitor). Immunofluorescent centromere protein B staining indicated that EHDPP-induced micronuclei in V79-derived cell lines expressing human CYP2E1 and 3A4 were predominantly centromere-negative, and that in HepG2 cells pretreated with bisphenol AF (for inducing multiple CYPs) were purely centromere-negative. In bisphenol AF-pretreated HepG2 cells EHDPP potently induced DNA breaks, as indicated by the comet assay and Western blot analysis of γ-H2AX. In conclusion, our study suggests that EHDPP is potently clastogenic, following activation by several human CYP enzymes, CYP3A4 being a major one.

Characterization of a Novel CYP1A2 Knockout Rat Model Constructed by CRISPR/Cas9.

CYP1A2, as one of the most important cytochrome P450 isoforms, is involved in the biotransformation of many important endogenous and exogenous substances. CYP1A2 also plays an important role in the development of many diseases because it is involved in the biotransformation of precancerous substances and poisons. Although the generation of Cyp1a2 knockout (KO) mouse model has been reported, there are still no relevant rat models for the study of CYP1A2-mediated pharmacokinetics and diseases. In this report, CYP1A2 KO rat model was established successfully by CRISPR/Cas9 without any detectable off-target effect. Compared with wild-type rats, this model showed a loss of CYP1A2 protein expression in the liver. The results of pharmacokinetics in vivo and incubation in vitro of specific substrates of CYP1A2 confirmed the lack of function of CYP1A2 in KO rats. In further studies of potential compensatory effects, we found that CYP1A1 was significantly upregulated, and CYP2E1, CYP3A2, and liver X receptor β were downregulated in KO rats. In addition, CYP1A2 KO rats exhibited a significant increase in serum cholesterol and free testosterone accompanied by mild liver damage and lipid deposition, suggesting that CYP1A2 deficiency affects lipid metabolism and liver function to a certain extent. In summary, we successfully constructed the CYP1A2 KO rat model, which provides a useful tool for studying the metabolic function and physiologic function of CYP1A2. SIGNIFICANCE STATEMENT: Human CYP1A2 not only metabolizes clinical drugs and pollutants but also mediates the biotransformation of endogenous substances and plays an important role in the development of many diseases. However, there are no relevant CYP1A2 rat models for the research of pharmacokinetics and diseases. This study successfully established CYP1A2 knockout rat model by using CRISPR/Cas9. This rat model provides a powerful tool to study the function of CYP1A2 in drug metabolism and diseases.

Recombinant expression and characterization of novel P450s from Actinosynnema mirum

Cytochrome P450 monooxygenases (P450s) are the major contributor in the metabolism of xenobiotics, including therapeutic agents. Thus, P450s find broad application in the pharmaceutical industry to synthesize metabolites of new active pharmaceutical ingredients in order to evaluate toxicity and pharmacokinetics. As an alternative to human hepatic P450s, microbial P450s offer several advantages, such as an easier and more efficient heterologous expression as well as higher stability under process conditions. Recently, the wild-type strain Actinosynnema mirum has been reported to catalyze hydroxylation reactions with high activity on a broad range of substrates. In this study, one of these substrates, ritonavir, was used to analyze the transcriptional response of the wild-type strain. Analysis of the differential gene expression pattern allowed the assignment of genes potentially responsible for ritonavir conversion. Heterologous expression of these candidates and activity testing led to the identification of a novel P450 that efficiently converts ritonavir resembling the activity of the human CYP3A4.

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Pharmacokinetic Drug Interaction between Tofacitinib and Voriconazole in Rats.

Fungal infections are prevalent in patients with immune diseases. Voriconazole, a triazole antifungal drug, inhibits the cytochromes CYP3A4 and CYP2C, and tofacitinib, a Janus kinase inhibitor for the treatment of rheumatoid arthritis, is metabolized by CYP3A4 and CYP2C19 in humans. Here, we investigated their interaction during simultaneous administration of both drugs to rats, either intravenously or orally. The area under the plasma concentration–time curve from time zero to time infinity (AUC) of tofacitinib was significantly greater, by 166% and 171%, respectively, and the time-averaged non-renal clearance (CLNR) of tofacitinib was significantly slower (59.5%) than that for tofacitinib alone. An in vitro metabolism study showed non-competitive inhibition of tofacitinib metabolism in the liver and intestine by voriconazole. The concentration/apparent inhibition constant (Ki) ratios of voriconazole were greater than two, indicating that the inhibition of tofacitinib metabolism could be due to the inhibition of the CYP3A1/2 and CYP2C11 enzymes by voriconazole. The pharmacokinetics of voriconazole were not affected by the co-administration of tofacitinib. In conclusion, the significantly greater AUC and slower CLNR of tofacitinib after intravenous and oral administration of both drugs were attributable to the non-competitive inhibition of tofacitinib metabolism via CYP3A1/2 and CYP2C11 by voriconazole in rats.

Design of a Phase 1/2 Open-Label, Dose-Escalation Study of the Safety and Efficacy of Gene Therapy in Adults With Classic Congenital Adrenal Hyperplasia (CAH) Due to 21-hydroxylase Deficiency Through Administration of an Adeno-Associated Virus (AAV) Serotype 5-Based Recombinant Vector Encoding the Human CYP21A2 Gene

Abstract The CYP21A2 gene, which encodes the 21-hydroxylase enzyme, plays a critical role in glucocorticoid (GC) and mineralocorticoid synthesis by the adrenal cortex. CYP21A2 pathogenic variants cause 21-hydroxylase deficiency (21OHD), the most common type of CAH, characterized by variable degrees of adrenal insufficiency and androgen excess. Standard treatment of classic 21OHD consists of daily doses of GC and mineralocorticoid. However, suppressive GC doses are often required to reduce androgen excess, and it is often not possible to dose exogenous GC in a manner that provides adequate disease control while avoiding overtreatment. Disease-related and treatment-related comorbidities are common and include life-threatening adrenal crises, impaired growth and development during childhood, adult short stature, virilization in females, subfertility in both sexes, obesity and cardiovascular risk factors, and decreased bone mineral density. Novel treatment approaches are needed to address these challenges and a treatment that restores the ability of the adrenals to produce cortisol and aldosterone in a physiologically-regulated manner would be particularly helpful. Here we present the design and rationale of a clinical trial using BBP-631, an AAV5 gene replacement therapy for adults with classic CAH due to 21-OHD. This treatment approach is based on the demonstration that a single intravenous administration of BBP-631 corrects the enzyme deficiency in the H2-aw18 CYP21-/- CAH mouse model of 21OHD, including response to stress. This correction was robust, dose-dependent and durable. BBP-631 treatment also resulted in robust and durable expression of the human CYP21A2 transgene in the non-human primate adrenal cortex. BBP-631 appears to be safe and well-tolerated in mice with 21-OHD, healthy mice and non-human primates. Taken together, these data support initiating clinical trials in adults with classic CAH due to 21-OHD. The trial will sequentially enroll individuals in up to 3 successive dose-escalation cohorts. Each subject will receive a single dose of BBP-631 and safety will be assessed prior to dose escalation. Endogenous production rates of adrenal steroids (cortisol, 17-hydroxyprogesterone, androstenedione) will be determined pre- and post- dose, and their concentrations assessed over a 1-year period after which subjects will roll over into an extension study for at least 4 years. The Phase 1 study will determine the tolerability of a single dose of BBP-631. The magnitude and durability of BBP-631 and effects on adrenal steroids, ACTH and, where relevant, aldosterone levels will be monitored. The ability of BBP-631 to allow tapering of GC doses will also be explored. This first in human study of gene therapy for CAH represents a milestone in the development of novel and improved treatment approaches for patients with classic CAH.

Adrenodoxin and Substrate Binding Modulate the Vitamin‐D Metabolizing Human Cytochrome P450 24A1

Vitamin-D3 insufficiency is associated with rickets, cardiovascular disease, and chronic kidney disease, and therefore impacts nearly 50% of the U.S. population. In humans, vitamin-D3 is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans first hydroxylates either the carbon-23 or carbon-24 position of 1,25-(OH)2D3. The human isoform of CYP24A1 is not easily expressed and purified recombinantly. Therefore, most current structural studies are based on the rat isoform, which does not display carbon-23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a cleavable fusion construct of human CYP24A1 with its reduction-oxidation protein partner adrenodoxin. Cleaved and isolated human CYP24A1 provides a platform for interrogating the structural effects of an Adx-CYP24A1 complex on substrate, analog, and inhibitor binding. Notably, we observed that the presence Adx weakens the interaction between human CYP24A1 1.5-fold, while also increasing the population of the high spin shift that results from displacement of a water ligand. This effect is not seen with 1-(OH)D3, a vitamin-D3 analog, suggesting that specific substrate binding relates allosterically to Adx recognition. The path of this allostery is investigated by specific point mutations. These findings provide mechanistic insight into the interrelated nature of step I (substrate binding) and step II (redox partner binding) of a multi-functional mitochondrial cytochrome P450 enzyme.

Calcitonin gene-related peptide (CGRP) receptor antagonists: Heterocyclic modification of a novel azepinone lead

In our efforts to identify orally bioavailable CGRP receptor antagonists, we previously discovered a novel series of orally available azepinone derivatives that unfortunately also exhibited the unwanted property of potent time-dependent human CYP3A4 inhibition. Through heterocyclic replacement of the indazole ring, we discovered a series of heterocycle derivatives as high-affinity CGRP receptor antagonists. Some of them showed reasonable oral exposures, and the imidazolone derivatives that showed good oral exposure also exhibited substantially reduced time-dependent CYP3A4 inhibition. Several compounds showed strong in vivo efficacy in our marmoset facial blood flow assay with up to 87% inhibition of CGRP-induced activity. However, oral bioavailability generally remained low, emphasizing the challenges we and others encountered in discovering clinical development candidates for this difficult Class B GPCR target.

The dietary carcinogen PhIP activates p53-dependent DNA damage response in the colon of CYP1A-humanized mice.

Species differences in the metabolism of xenobiotics by cytochrome P450 are critical in evaluating the use of experimental animals in studying toxic compounds relevant to human diseases. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced by high-temperature cooking of fish and meat, is activated to become a carcinogen by cytochrome P4501A2 (CYP1A2) through N2 -hydroxylation in humans, but is detoxified by Cyp1a2 through 4'-hydroxylation in mice. CYP1A-humanized (hCYP1A) mice, in which mouse Cyp1a is replaced with human CYP1A, show constitutive human xenobiotic metabolism by hCYP1A, thereby serving as a suitable model for studying PhIP. Previous studies have demonstrated that oral administration of PhIP induces colon tumors in hCYP1A mice; however, these studies used a super-high dose, raising concerns regarding the relevance of the mechanism to human cancer. Herein, we systematically investigated PhIP-induced colon carcinogenesis in hCYP1A mice treated with lower doses. We found that a dose 2000 times lower than that used previously, which is comparable to human daily intake levels, could induce colon tumors, albeit at a lower incidence rate. We further investigated the transcriptome changes in the colon of hCYP1A mice treated with PhIP and identified that PhIP treatment increased the expression of Bax, Btg2, Ccng1, Cdkn1a, and Trp53inp1 and decreased the expression of Igf1 and Ccnd1. Since these genes are key components of the p53-dependent DNA damage response, the altered expression patterns indicated PhIP-induced DNA damage in hCYP1A mice. Together, these results prove that hCYP1A mice are suitable for studying PhIP-induced carcinogenesis and show that PhIP is an important colorectal cancer carcinogen in human diet.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6β-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6β-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6β-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6β-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6β-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6β-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

ABCB1 and ABCG2 Restrict Brain and Testis Accumulation and, Alongside CYP3A, Limit Oral Availability of the Novel TRK Inhibitor Selitrectinib.

Selitrectinib (BAY2731954; LOXO-195) is a promising oral tropomyosin receptor kinase (TRK) inhibitor currently in phase I/II clinical trials for the treatment of histology-agnostic cancers positive for TRK fusions. With therapeutic resistance eventually developing with first-generation TRK inhibitors, selitrectinib was designed to overcome resistance mediated by acquired kinase domain mutations. Using genetically modified mouse models and pharmacological inhibitors, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing CYP3A enzyme complex in selitrectinib pharmacokinetics. In vitro, selitrectinib was markedly transported by mouse Abcg2 and human ABCB1, and modestly by human ABCG2. Following oral administration at 10 mg/kg, selitrectinib brain-to-plasma ratios were increased in Abcb1a/1b-/- (twofold) and Abcb1a/1b;Abcg2-/- (5.8-fold) compared with wild-type mice, but not in single Abcg2-/- mice. Testis distribution showed similar results. mAbcb1a/1b and mAbcg2 each restricted the plasma exposure of selitrectinib: With both systems absent oral availability increased by 1.7-fold. Oral administration of the ABCB1/ABCG2 inhibitor elacridar boosted plasma exposure and brain accumulation in wild-type mice to the same levels as seen in Abcb1a/1b;Abcg2-/- mice. In Cyp3a-/- mice, plasma exposure of selitrectinib over 4 hours was increased by 1.4-fold and subsequently reduced by 2.3-fold upon transgenic overexpression of human CYP3A4 in liver and intestine. The relative tissue distribution of selitrectinib remained unaltered. Thus, selitrectinib brain accumulation and oral availability are substantially restricted by ABCB1 and ABCG2, and this can be reversed by pharmacological inhibition. Moreover, oral availability of selitrectinib is limited by CYP3A activity. These insights may be useful to optimize the clinical application of selitrectinib.

Dibenzyl trisulfide binds to and competitively inhibits the cytochrome P450 1A1 active site without impacting the expression of the aryl hydrocarbon receptor

The toxicological manifestation of many pollutants relies upon their binding to the aryl hydrocarbon receptor (AHR), and it follows a cascade of reactions culminating in an elevated expression of cytochrome P450 (CYP) 1 enzymes. CYP1A1 and CYP1B1 are associated with enhanced carcinogenesis when chronically exposed to certain polyaromatic hydrocarbons, and their inhibition may lead to chemoprevention. We evaluated dibenzyl trisulfide (DTS), expressed in the ethnomedical plant, Petiveria alliacea, for such potential chemoprevention. Using recombinant human CYP1A1 and CYP1B1 bactosomes on a fluorogenic assay, we first demonstrated that DTS moderately inhibited both enzymes with half maximal inhibitory concentration (IC50) values of 1.3 ± 0.3 and 1.7 ± 0.3 μM, respectively. Against CYP1A1, DTS was a reversible, competitive inhibitor with an apparent inhibitory constant (Ki) of 4.55 ± 0.37 μM. In silico molecular modeling showed that DTS binds with an affinity of −39.8 kJ·mol−1, situated inside the binding pocket, approximately 4.3 Å away from the heme group, exhibiting interactions with phenylalanine residue 123 (Phe-123), Phe-224, and Phe-258. Lastly, zebrafish (Danio rerio) embryos were exposed to 0.08–0.8 μM DTS from 24 to 96 h post fertilization (hpf) with the in vivo ethoxyresorufin-O-deethylase (EROD) assay, and, at 96 hpf, DTS significantly suppressed EROD CYP1A activity in a dose-dependent manner, with up to 60% suppression in the highest 0.8 μM exposure group. DTS had no impact on gene transcription levels for cyp1a and aryl hydrocarbon receptor 2 (ahr2). In co-exposure experiments, DTS suppressed CYP1A activity induced by both B[a]P and PCB-126, although these reductions were not significant. Taken together, these results demonstrate that DTS is a direct, reversible, competitive inhibitor of the carcinogen-activating CYP1A enzyme, binding in the active site pocket close to the heme site, and shows potential in chemoprevention.

Binimetinib Is a Potent Reversible and Time-Dependent Inhibitor of Cytochrome P450 1A2

Binimetinib is a selective MEK1/2 inhibitor, which is indicative of melanoma. We aimed to investigate the inhibitory effect of binimetinib on cytochrome P450 using human liver microsomes. Binimetinib was demonstrated to display reversible and time-dependent inhibitory effects on human CYP1A2. Binimetinib can inhibit the activity of phenacetin deethylation with IC50 of 5.6 μM. A 30 min preincubation of binimetinib with NADPH-supplemented human liver microsomes raised a significant left IC50 shift (6.5-fold), from 5.69-0.88 μM. The inactivation parameters Kinact and KI were 0.063 min-1 and 15.47 μM, and the half-life of inactivation was 11 min. Glutathione (GSH) and catalase/superoxide exhibited minor or no protective effect on binimetinib-induced enzyme inactivation. Trapping experiment by GSH induced a detection of GSH adduct, of which the formation was believed to be through the oxidation of electron-rich 1,4-benzenediamine to reactive 1,4-diiminoquinone species. Cytochrome P450 3A4, 2C9, and 2D6 were involved in the bioactivation of binimetinib. In conclusion, binimetinib was proven to display reversible and time-dependent inhibitory effect on CYP1A2, which may have implications for the toxicity of binimetinib.

Inhibitory Effects of Schisandra Lignans on Cytochrome P450s and Uridine 5'-Diphospho-Glucuronosyl Transferases in Human Liver Microsomes.

Schisandra chinensis has been widely used as a traditional herbal medicine to treat chronic coughs, fatigue, night sweats, and insomnia. Numerous bioactive components including lignans have been identified in this plant. Lignans with a dibenzocyclooctadiene moiety have been known to possess anti-cancer, anti-inflammatory, and hepatoprotective activity. Fragmentary studies have reported the ability of some lignans to modulate some cytochrome P450 (P450) enzymes. Herein, we investigated the drug interaction potential of six dibenzocyclooctadiene lignans (schisandrin, gomisin A, B, C, and N, and wuweizisu C) on nine P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A) and six uridine 5′-diphosphoglucuronosyl transferase (UGT) enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) using human liver microsomes. We found that lignans with one or two methylenedioxyphenyl groups inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1 activities in a time- and concentration-dependent like their CYP3A inhibition. In comparison, these lignans do not induce time-dependent inhibition of CYP1A2, CYP2A6, and CYP2D6. The time-dependent inhibition of gomisin A against CYP2C8, CYP2C19, and CYP3A4 was also elucidated using glutathione as a trapping reagent of reactive carbene metabolites given that gomisin A strongly inhibits these P450 enzymes in a time-dependent manner. A glutathione conjugate of gomisin A was generated in reactions with human recombinant CYP2C8, CYP2C19, and CYP3A4. This suggests that the time-dependent inhibition of gomisin A against CYP2C8, CYP2C9, and CYP3A4 is due to the production of carbene reactive metabolite. Six of the lignans we tested inhibited the activities of six UGT to a limited extent (IC50 > 15 μM). This information may aid the prediction of possible drug interactions between Schisandra lignans and any co-administered drugs which are mainly metabolized by P450s.

Effects of 1,25 and 24,25 Vitamin D on Corneal Fibroblast VDR and Vitamin D Metabolizing and Catabolizing Enzymes

ABSTRACT Purpose: To investigate the effects of 1,25-Vit D3 and 24,25-Vit D3 on corneal fibroblast expression of the vitamin D-associated enzymes CYP27B1 and CYP24A1 and the roles of the vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3) in these cells. Methods: CYP24A1, CYP27B1, VDR, and Pdia3 expression in corneas was detected using immunohistochemistry. Western blotting was used to measure protein expression in human and mouse fibroblasts, including VDR KO mouse cells, treated with 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM). The Pdia3 inhibitor LOC14 was used to explore the role of Pdia3 as a Vit D3 receptor in these cells. Results: CYP24A1, CYP27B1, VDR, and Pdia3 were all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 significantly increased VDR expression in human and mouse fibroblasts. 1,25-Vit D3 and 24,25-VitD3 significantly increased CYP24A1 and CYP27B1 expression level in human, VDR WT mouse, and VDR KO mouse corneal fibroblasts. CYP24A1 and CYP27B1 expression was unchanged in VDR KO mouse fibroblasts treated with 1,25-Vit D3 or 24,25-Vit D3 plus LOC14. Human fibroblast VDR, CYP24A1, and CYP27B1 expression were unaffected by LOC14. Conclusions: Vitamin D metabolic enzymes, VDR, and Pdia3 are all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 modulates fibroblast vitamin D enzymes through both the VDR and Pdia3 pathways in a species-dependent manner. 24,25-Vit D3 can increase expression of fibroblast CYP24A1 and CYP27B1 in the absence of VDR and is likely involved in fibroblast regulation independent of 1,25-Vit D3 or VDR.