CYP3A4 Research Articles

NANONIZED CURCUMIN IMPROVED ORAL BIOAVAILABILITY OF BROMOCRIPTINE THROUGH CYP3A ENZYME INHIBITION IN RATS

Curcumin (CUR) a water insoluble dietary flavonoid and is reported to inhibit CYP3A4 enzyme. The objective of present study was to assess the effect of CUR and nanonized curcumin (NC) on bromocriptine (BRO) metabolism in rats. NC was prepared by antisolvent precipitation method and was evaluated for particle size and solubility. CYP3A inhibitory activity of CUR was assessed in vitro using erythromycin-N-demethylase (EMD) assay. CYP3A inhibitory effect of CUR and NC confirmed in vivo using pharmacokinetic study of midazolam. These findings were further confirmed by an in vivo pharmacokinetic study in which BRO was administered (10 mg/kg, p.o) to CUR and NC (60 mg/kg, p.o.) pretreated (10 days) rats and its plasma concentrations were determined by HPLC analysis. CUR significantly (p less than 0.05) inhibited CYP 3A activity in EMD assay. CUR and NC significantly (p less than 0.05) increased plasma concentrations of midazolam in dexamethasone pretreated rats. In addition, in vivo studies revealed that CUR and NC pretreatments significantly (p less than 0.05) increased Cmax and AUC and decreased CL/F of BRO compared with BRO group.NC pretreatment may increase the bioavailability of BRO which could be mediated through the inhibition of CYP 3A enzymes.

Biochemical and molecular study on the beneficial effect of Solubilized coenzyme Q10 on thioacetamide-induced liver fibrosis in adult male rats

The current work aims to evaluate the therapeutic effect of both solubilized Coenzyme Q10 and Silymarin on liver injury. Thirty adult male albino rats, weigh 200±10 g, were allocated into five groups. Hepatic injury was induced by intraperitoneal injection of TAA (20 mg/ kg body weight) twice a week. Solubilized CoQ10 (20 mg/kg) and Silymarin (50 mg/kg) were taken orally for 28 days. The present results showed that the values for aspartate/platelet ratio (APRI) as well as aspartate aminotransferase/alanine aminotransferase ratio (AAR) were increased by TAA treatment. While treatments using coenzyme Q10 and Silymarin decreased these values near the control levels. The solubilized CoQ10 could attenuate TAA liver injury throughout controlling the oxidative stress markers (GSH, GSSG), NO level. Also, down-regulation gene expression of fibrotic markers TGF-β, collagen-1α, and TIMP1 and enhancing the expression for MMP2 and cytochrome P 450 (CYP 2E1& CYP 3A2). Collectively, solubilized CoQ10 singly or combined with Silymarin showed higher therapeutic impacts more than or equivalents to Silymarin treatment for liver fibrosis induced by TAA.

Discovery of first-in-class imidazothiazole-based potent and selective ErbB4 (HER4) kinase inhibitors.

This article reports on novel imidazothiazole derivatives as first-in-class potent and selective ErbB4 (HER4) inhibitors. There are no other reported selective inhibitors of this kinase in the literature, that's why they are considered as first-in-class. In addition, none of the reported non-selective ErbB4 inhibitors possesses imidazothiazole nucleus in its structure. Therefore, there is novelty in this work in both kinase selectivity and chemical structure. Compounds Ik and IIa are the most potent ErbB4 kinase inhibitor (IC50=15.24 and 17.70nM, respectively). Compound Ik showed promising antiproliferative activity. It is selective towards cancer cell lines than normal cells. Its ability to penetrate T-47D cell membrane and inhibit ErbB4 kinase inside the cells has been confirmed. Moreover, both compound Ik and IIa have additional merits such as weak potency against hERG ion channels and against CYP 3A4 and 2D6. Molecular docking and dynamic simulation studies were carried out to explain binding interactions.

Amino-Heterocycle Tetrahydroisoquinoline CXCR4 Antagonists with Improved ADME Profiles via Late-Stage Buchwald Couplings.

This work surveys a variety of diamino-heterocycles as an isosteric replacement for the piperazine substructure of our previously disclosed piperarinyl-tetrahydroisoquinoline containing CXCR4 antagonists. A late-stage Buchwald coupling route was developed for rapid access to final compounds from commercial building blocks. Among 13 analogs in this study, compound 31 embodying an aza-piperazine linkage was found to have the best overall profile with potent CXCR4 inhibitory activity and favorable in vitro absorption, distribution, metabolism, and excretion (ADME) properties. An analysis of the calculated physiochemical parameters (ROF, cLogD) and the experimental ADME attributes of the analogs lead to the selection of 31 for pharmacokinetic studies in mice. Compared with the clinical compound AMD11070, compound 31 has no CYP450 3A4 or 2D6 inhibition, higher metabolic stability and PAMPA permeability, greatly improved physiochemical parameters, and superior oral bioavailability (%F = 24). A binding rationale for 31 within CXCR4 was elucidated from docking and molecular simulation studies.

Metabolic Toxification of 1,2-Unsaturated Pyrrolizidine Alkaloids Causes Human Hepatic Sinusoidal Obstruction Syndrome: The Update

Saturated and unsaturated pyrrolizidine alkaloids (PAs) are present in more than 6000 plant species growing in countries all over the world. They have a typical heterocyclic structure in common, but differ in their potential toxicity, depending on the presence or absence of a double bond between C1 and C2. Fortunately, most plants contain saturated PAs without this double bond and are therefore not toxic for consumption by humans or animals. In a minority of plants, however, PAs with this double bond between C1 and C2 exhibit strong hepatotoxic, genotoxic, cytotoxic, neurotoxic, and tumorigenic potentials. If consumed in error and in large emouns, plants with 1,2-unsaturated PAs induce metabolic breaking-off of the double bonds of the unsaturated PAs, generating PA radicals that may trigger severe liver injury through a process involving microsomal P450 (CYP), with preference of its isoforms CYP 2A6, CYP 3A4, and CYP 3A5. This toxifying CYP-dependent conversion occurs primarily in the endoplasmic reticulum of the hepatocytes equivalent to the microsomal fraction. Toxified PAs injure the protein membranes of hepatocytes, and after passing their plasma membranes, more so the liver sinusoidal endothelial cells (LSECs), leading to life-threatening hepatic sinusoidal obstruction syndrome (HSOS). This injury is easily diagnosed by blood pyrrolizidine protein adducts, which are perfect diagnostic biomarkers, supporting causality evaluation using the updated RUCAM (Roussel Uclaf Causality Assessment Method). HSOS is clinically characterized by weight gain due to fluid accumulation (ascites, pleural effusion, and edema), and may lead to acute liver failure, liver transplantation, or death. In conclusion, plant-derived PAs with a double bond between C1 and C2 are potentially hepatotoxic after metabolic removal of the double bond, and may cause PA-HSOS with a potential lethal outcome, even if PA consumption is stopped.

Characterization of the metabolites of dichotomitin in rat, monkey and human by ultra-high-performance liquid chromatography/high-resolution mass spectrometry.

Dichotomitin is one of the bioactive constituents isolated from Belamcanda chinensis. The goal of this study was to identify the metabolites of dichotomitin produced by liver microsomes and hepatocytes. Using ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UPLC/HRMS), the metabolites were profiled and identified. The exact masses, elemental compositions and product ions of the metabolites were used to characterize their structures. A total of ten metabolites were found and identified. The main metabolites identified in the incubation samples were M6 (3',5,6,7-tetrahydroxy-4',5'-dimethoxyisoflavone) and M8 (8-hydroxydichotomitin). Opening of 1,3-benzodioxole, demethylation, hydroxylation, glucuronidation and sulfation were among the metabolic modifications for dichotomitin. A human recombinant cytochrome P450 enzyme study revealed that CYP 1A2, 2C19, and 2D6 facilitated the formation of M6, whereas CYP 1A2 catalyzed the formation of M8 exclusively. For the first time, data on the in vitro metabolic fates of dichotomitin were revealed in this work which would be helpful for us to understand the disposition of this bioactive constituent.

Effect of Aspirin on Mitochondrial Dysfunction and Stress in the Pancreas and Heart of Goto-Kakizaki Diabetic Rats.

Our previous study in Goto-Kakizaki (GK) type 2 diabetic rats provided significant evidence that aspirin treatment improves pancreatic β-cell function by reducing inflammatory responses and improving glucose tolerance. In the present study, we aimed to elucidate the mechanism of action of aspirin on the pathophysiology and progression of type 2 diabetic complications in the heart and pancreas of insulin-resistant GK rats. Aspirin treatment demonstrated a reduction in mitochondrial reactive oxygen species (ROS) production and lipid peroxidation, accompanied by improved redox homeostasis. Furthermore, the recovery of metabolic and mitochondrial functions, as well as cytochrome P450 enzyme activities, which were altered in the pancreas and heart of GK rats, were observed. Aspirin treatment brought the activity of CYP 2E1 to the control level in both tissues, whereas the CYP 3A4 level decreased only in the pancreas. This suggests the tissue-specific differential metabolism of substrates in these rats. The recovery of redox homeostasis could be the key target in the improvement of oxidative-stress-dependent alterations in mitochondrial functions which, in turn, facilitated improved energy metabolism in these tissues in the aspirin-treated GK rats. These results may have implications in determining the therapeutic use of aspirin, either alone or in combination with other clinically approved therapies, in insulin-resistant type 2 diabetes.

CYPstrate: A Set of Machine Learning Models for the Accurate Classification of Cytochrome P450 Enzyme Substrates and Non-Substrates.

The interaction of small organic molecules such as drugs, agrochemicals, and cosmetics with cytochrome P450 enzymes (CYPs) can lead to substantial changes in the bioavailability of active substances and hence consequences with respect to pharmacological efficacy and toxicity. Therefore, efficient means of predicting the interactions of small organic molecules with CYPs are of high importance to a host of different industries. In this work, we present a new set of machine learning models for the classification of xenobiotics into substrates and non-substrates of nine human CYP isozymes: CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. The models are trained on an extended, high-quality collection of known substrates and non-substrates and have been subjected to thorough validation. Our results show that the models yield competitive performance and are favorable for the detection of CYP substrates. In particular, a new consensus model reached high performance, with Matthews correlation coefficients (MCCs) between 0.45 (CYP2C8) and 0.85 (CYP3A4), although at the cost of coverage. The best models presented in this work are accessible free of charge via the “CYPstrate” module of the New E-Resource for Drug Discovery (NERDD).

Abstract 1370: ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201

Abstract Imipridone ONC201 is a first-in-class DRD2 antagonist and ClpP agonist that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients with once weekly dosing. We evaluated the toxicology, absorption, distribution, metabolism and excretion of ONC201 in animals. Repeat-dose 28-day studies with weekly oral ONC201 in rats and dogs revealed NOAELs (75 and 60 mg/kg, respectively) that represent higher dose levels compared to the 625 mg clinical dose. Genotoxicity (AMES, in vitro and in vivo micronucleus assays) assays demonstrated that ONC201 is not mutagenic at exposures above the RP2D and 2µM average Cmax in patients. The 3T3 neural red uptake assay showed ONC201 is not a phototoxin (photo-irritant factor 1.1). In embryofetal development studies, ONC201 did not lead to maternal effects in rats or rabbits and caused fetal malformations in 3/61 rabbit fetuses with daily dosing (from gestation day 7-17 or 19) at 62.5 and 25 mg/kg, respectively. A quantitative whole-body autoradiography study conducted in rats with one dose of [14C]-ONC201 showed rapidly tissue distribution peaking at 1h in most tissues and exhibiting a plasma terminal half-life of 5.6h. The endocrine, metabolic/excretory, ocular and GI tract tissues contained the highest distribution of [14C]ONC201. [14C]-ONC201 was distributed evenly across CNS substructures, including the midline region. Consistent with the uniform distribution, ONC201 displayed high permeability (23-31×10-6 cm/s, 7-700 μM) and was not a substrate of efflux transporters in Caco-2 cells (efflux ratio 0.46-0.79). ONC201 exerted inhibitory potential on MDR1- and BCRP-mediated transport at 200μM. Clinical trials with ONC201 restrict use of concomitant medications that induce or inhibit CYP enzymes, but several supportive medications can affect these enzymes. In vitro studies revealed that ONC201 is not an inducer of 7 major human CYP enzymes but does inhibit and is a substrate of CYP 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 (IC50 20-90µM). Analysis of dexamethasone, a known inducer of CYP3A4, did not show a correlation between baseline dose level and ONC201 plasma concentrations in H3 K27M-mutant glioma patients (n=22). Metabolic profiling in human hepatocytes revealed only one metabolite, ONC207, that was >10% in abundance. ONC207 did not achieve an IC50 in cancer cell viability and DRD2 antagonism assays. Assaying by LC-MS/MS of plasma samples obtained from ONC201-treated H3 K27M-mutant glioma patients (n=20) confirmed the presence of ONC207 as the major metabolite. A mass balance study conducted in rats with one dose of [14C]-ONC201 showed that the main route of excretion was biliary (61.18%) via the feces (69.78%), with urine (24.99%) also contributing to overall recovery. Together, these data inform the administration of ONC201 to advanced cancer patients who often require a range of comedications and comorbidities. Citation Format: Sara Morrow, Rohinton Tarapore, Ailan Guo, Magdalena Nej, Yunlan Fang, Gina Theerman, Leah Lake, Martin Stogniew, Varun V. Prabhu, Joshua E. Allen. ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201 [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 1370.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Tolvaptan in Hyponatremia – A Pharmacologic approach

Tolvaptan is the first orally active vasopressin receptor 2 antagonist approved by FDA in 2009 for the treatment of clinically significant hypervolemic and euvolemic hyponatremia associated with heart failure, syndrome of inappropriate antidiuretic hormone secretion, liver cirrhosis. Binding of vasopressin to V2 receptors which are located on renal collecting duct cells were competitively inhibited by Tolvaptan thus prevents vasopressin-mediated activation of these receptors and leads to an increase in the water permeability of the collecting duct causing an increase in the concentration of urine, results in an increase in free water clearance, a decrease in urine osmolality and a fall in serum sodium concentration. Tolvaptan is different from other conventional diuretics because it does not stimulate the sodium channel and increases free water excretion without affecting urinary sodium and potassium excretion. It is metabolized principally via the cytochrome P 450 (CYP 3A) isoenzymes and is eliminated by routes other than renal. Concurrent administration of tolvaptan with ketoconazole or other strong CYP 3A inhibitor decrease the total clearance of tolvaptan and increases tolvaptan concentrations. Urine sodium potassium ratio, weight loss, soluble CD14, urinary aquaporin 2 levels are known predictors of tolvaptan response. Tolvaptan is associated with decreased cost and high effectiveness.

Peak Plasma Rivaroxaban Levels in Individuals Weighing over 120 Kilograms

BackgroundRivaroxaban, a direct oral anticoagulant (DOAC), has been shown to be an efficacious and safe agent for treatment and secondary prevention of thromboembolism (TE). Currently, there is limited evidence available regarding its use in individuals weighing > 120 kg. Given the increasing prevalence of obesity in the general population, and that elevated weight itself is a risk factor for TE, this lack of data is a substantial issue. Theoretically, the greater volume of distribution in obesity may decrease DOAC peak drug concentration and half-life, potentially resulting in sub therapeutic anticoagulation. In fact, current ISTH guidelines recommend against empiric use of DOACs in patients over 120 kg, with the caveat that their use may be considered if drug specific peak and trough levels fall within the expected range (Martin et al. J Thromb Haemost. 2016). Additional data is required to guide the clinician regarding DOAC use for the management of TE in this growing demographic.ObjectivesOur primary objective was to assess peak rivaroxaban plasma levels (PRPL) in patients weighing > 120 kg on the standard 20 mg daily dose for treatment or secondary prevention of VTE and to compare these values to the expected range derived from pharmacokinetic studies in non-obese patients. The secondary objective was to assess for TE complications in this cohort.MethodsElectronic patient records were retrospectively reviewed at a thrombosis clinic at a single center from January 1, 2014 to May 31, 2017. Patients of age > 18, on rivaroxaban for treatment or secondary prevention of VTE, of weight > 120kg, and with at least one PRPL, measured 2-4 hours post drug ingestion at steady state, were included in the study. Patients with decreased renal function (creatinine clearance < 30mL/min), liver dysfunction (Child-Pugh score B or C), on concomitant strong P-glycoprotein or CYP 3A4 inducers or inhibitors, or with an additional indication for anticoagulation were excluded. Reference ranges for PRPL were extracted from pharmacokinetic studies (Buller et al. Blood. 2008). PRPL were measured using an anti-Xa assay calibrated to rivaroxaban (BIOPHENTM DiXal, HYPHEN BioMed).ResultsThere were 17 unique patients, each with a PRPL, weighing > 120kg, on rivaroxaban for treatment or secondary prevention of TE, over our study period. Six were female. The mean weight was 146.75 (SD +/- 20.19) kg. The mean calculated creatinine clearance (Cockcroft-Gault) was 208.53 (SD +/- 57.36) mL/min. The mean length of follow up from measurement of PRPL was 312.29 (SD +/- 265.47) days.The mean PRPL was 248.51 (5th to 95th percentile 174.6-380.4) ng/mL compared to the reference of 5th to 95th PRPL percentile of 175-360 ng/mL in non obese patients. Only 1 PRPL was found to be below the expected range at 161ng/mL (see figure 1). There were no recurrent TE events in any of the patients during the study.ConclusionsWe found that the majority of individuals weighing > 120 kg on standard dose rivaroxaban for treatment and secondary prevention of TE had a PRPL within the expected range, with only one value out of the seventeen falling below that range. There were no recurrent TE events in any of the patients. It is possible that the increased volume of distribution in obesity played a lesser role in decreasing PRPL given the lower lipophilicity of rivaroxaban compared to apixaban and dabigatran, although similar to edoxaban (Remko. Journal of Molecular Structure: THEOCHEM. 2009, Remko et al. Molecules. 2016). Despite the small sample size and lack of other pharmacokinetic values, this preliminary data is clinically reassuring that the use of rivaroxaban in patients weighing > 120kg seems to be safe. Further studies are required to fully elucidate the efficacy and safety of DOACs in the treatment of TE in morbidly obese patients. [Display omitted] DisclosuresSholzberg:Shire: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; NovoNordisk: Honoraria. Yeo:Bayer: Membership on an entity's Board of Directors or advisory committees.

Diabetes mellitus aggravates ranolazine-induced ECG changes in rats.

Diabetes mellitus (DM) is known to affect the pharmacokinetics of drugs. In this study, we evaluated the effect of DM on the liver content of CYP 3A2 enzyme. We also explored the ECG changes after administration of ranolazine in non-DM and DM rats. First phase: 24 male Wistar rats were separated into 4 groups. The control group (n = 6) received normal saline and the DM groups (n = 18) were treated with a single dose (55mg/kg) of streptozocin (STZ; i.p. injection), then were held for 10, 20, and 30days, respectively. After study duration for each group, the liver CYP 3A2 protein content was determined using western blotting. Second phase: 48 male Wistar rats were classified into two groups of non-DM and DM; and each group was divided into 4 subgroups (n: 6). Experimental groups received oral doses of 20, 40, and 80mg/kg ranolazine. DM and non-DM control groups received normal saline. Treatment lasted for 28days, and then the ECG was recorded. Experimental DM induced by STZ caused a significant decrement in liver CYP3A2 protein content of rats on days 10 and 20 (P < 0.01), and 30 (P < 0.05) compared to the control animals. Significant increases in QT and corrected QT (QTc) intervals (P < 0.01), and bradycardia (P < 0.01) without any significant effect on PR and QRS intervals were observed in DM in comparison with non-DM groups after ranolazine treatment. In summary, DM induction in animals resulted in CYP 3A2 inhibition and the prolongation of QT and QTc interval as well as bradycardia after ranolazine treatment.

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.

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) activates Aryl hydrocarbon receptor (AhR) mediated ROS and NLRP3 inflammasome/p38 MAPK pathway inducing necrosis in cochlear hair cells

Tetrabromodiphenyl ether (BDE-47) is widely used as commercial flame retardants that can be released into the environment and finally enter human body through the food chain. It has been identified to generate neurotoxicity, but little is known about auditory damage and the underlying mechanism following BDE-47 exposure. This study aimed to assess the cell viability with BDE-47 concentration ranging from 0 to 150 μM in mouse organ of Corti-derived cell lines (HEI-OC1). Aryl hydrocarbon receptor (AhR) as an environmental sensor, reactive oxygen species (ROS), NLRP3 inflammasome and p38 MAPK pathways were detected. Results: (1) BDE-47 inhibited the viability in a time- and dose-dependent way in HEI-OC1 cells. Cell cycle was arrested in G1 phase by BDE-47; (2) Elevated intracellular ROS, LDH levels and necrosis were found, which was alleviated by pretreatment with ROS scavenger N-acetylcysteine (NAC); (3) AhR plays an essential role in ligand-regulated transcription factor activation by exogenous environmental compounds. We found increased expression of AhR and decreased downstream targets of CYP 1A1 and CYP 1B1 in BDE-47-treated HEI-OC1 cells, which was reversed by the AhR antagonist CH-223191 for 2 h before BDE-47 exposure. No significant change was detected in CYP 2B; (4) Enhanced expressions of NLRP3 and caspase-1 were induced by BDE-47, with up-regulations of both pro-inflammatory factors for IL-1β, IL-6 and TNF-α, and anti-inflammatory factors for IL-4, IL-10 and IL-13, but down-regulation for IL-1α; (5) Additionally, the p38 MAPK signaling pathway was activated with increased phosphorylation levels of MKK/3/6, p38 MAPK and NF-kB. Overall, our findings illustrate a role of AhR in ROS-induced necrosis of cochlear hair cells by BDE-47 exposure, in which NLRP3 inflammasome and p38 MAPK signaling pathways are activated. The current study first elucidates the sense of hearing damage induced by BDE-47, and cell-specific or mixture exposures in vivo or human studies are needed to confirm this association.

Effects of 27 CYP3A4 protein variants on saxagliptin metabolism in vitro.

Saxagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor widely used in patients with type 2 diabetes. It can increase the amount of insulin after meals and lower blood sugar. CYP450 3A4 (CYP3A4) can metabolize about 30%-40% of therapeutic drugs. Individual differences caused by CYP3A4 genetic polymorphisms can lead to treatment failure, unpredictable side effects, or severe drug toxicity. The aim of this study was to evaluate the catalytic activities of 27 CYP3A4 variants on saxagliptin metabolism in vitro, which were identified in human CYP alleles. We successfully constructed27 kinds of wild-type and variant vectors of pFast-dual-OR-3A4 by overlap extension PCR and prepared 27 kinds of CYP3A4 highly expressed cell microsomes by baculovirus insect cell expression system. The ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to detect the concentrations of the metabolite of saxagliptin (5-hydroxysaxagliptin) and the internal standard. Compared with the wild-type CYP3A4.1, the intrinsic clearance values of most varieties decreased to 1.91%-77.08%. Most of these varieties showed a decrease in Vmax and an increase in Km values compared with wild type. We are the first to report the vitro metabolic data of 27 CYP3A4 variants of the metabolism of saxagliptin which candeepen our understanding of individualized drug use by combining previous studies about the effects of CYP3A4 variants of drug metabolism. With further in vivo studies, we hope it can guide individualized drug use in the clinic when the variants with low metabolic activity to saxagliptin were sequenced in the human body.

P546 The safety and efficacy of the new-generation budesonide-MMX in the aspect of the cytochrome P-450 enzyme genotype

Abstract Background Unlike previous forms of budesonide absorbing from the ileal and ascending colon region, the new-generation budesonide-MMX contains a formula, that allows absorption throughout the whole colon. Budesonide is degraded in the liver by cytochrome P450 3A enzyme, but so far, there is no study examining the relationship between the budesonide’s effect and the enzyme activity. CYP3A5 is absent in 90% of the European/caucasian population due to a functional loss mutation (CYP3A5*3), whereas patients with the wild-type CYP3A5*1 allele may be expected to have increased metabolism. The most common genetic polymorphisms in CYP3A4 (CYP3A4*1B and CYP3A4*22) result in increased and decreased expression, respectively. Methods We enrolled 33 patients with UC in this prospective study until January of 2021. Patients received 9 mg oral budesonide-MMX once daily until 8 weeks. Laboratory parameters (cholesterol, triglyceride, CRP) and serum hormone levels (parathormone, dehydroepiandrosterone and cortisol) were monitored before and after the 8-week therapy to follow metabolic and hormonal changes. During these visits, body composition analysis was also performed with InBody 770 machine to observe the adverse effects of budesonide-MMX in respect of body fat mass, body mass index, protein content of the body and bone mineral content. We examined the CYP450 3A (CYP3A5 and CYP3A4) enzyme genotype of the patients, to see, whether the different alleles of this drug-degrading enzyme affect the efficacy and safety. Results 33 patients had received the 2-month therapy. By the end of follow-up, based on partial Mayo score, 26 (78.8%) patients experienced remission and 6 patients (18.2%) were primary non-responders. Mean pMayo score decreased from 4.18 to 1.63 (p&amp;lt;0.001). No significant changes were observable regarding body composition. Serum cholesterol level showed significant increase (p&amp;lt;0.001), while triglyceride and CRP did not show significant changes. Serum cortisol levels were decreased (p&amp;lt;0.001), while PTH and DHEA showed no significant decrease. Only two patients experienced side effects: one of them hypertonia, headache and acnes, while the other mild diarrhoea. 3 patients have CYP3A5*1/*3 genotype, and 16 have CYP3A5*3/*3. There was no significant difference between the two groups, regarding safety and efficacy. Only 1 patient have CYP3A4*1B genotype, while the rest have CYP3A4*1, hence, no statistics could be performed. Conclusion In our study, budesonide-MMX proved to be safe and effective in the therapy of UC, however, cholesterol was elevated in the serum. Based on our cohort, different genotypes of CYP3A don’t have an impact on the effect of the drug, however, CYP3A allele variants are rare, therefore, further examinations should be performed.

Versicolorin A enhances the genotoxicity of aflatoxin B1 in human liver cells by inducing the transactivation of the Ah-receptor

Aflatoxins are a group of mycotoxins that have major adverse effects on human health. Aflatoxin B1 (AFB1) is the most important aflatoxin and a potent carcinogen once converted into a DNA-reactive form by cytochrome P450 enzymes (CYP450). AFB1 biosynthesis involves the formation of Versicolorin A (VerA) which shares structural similarities with AFB1 and can be found in contaminated commodities, often co-occurring with AFB1. This study investigated and compared the toxicity of VerA and AFB1, alone or in combination, in HepG2 human liver cells. Our results show that both toxins have similar cytotoxic effects and are genotoxic although, unlike AFB1, the main genotoxic mechanism of VerA does not involve the formation of DNA double-strand breaks. Additionally, we show that VerA activates the aryl hydrocarbon receptor (AhR) and significantly induce the expression of the CYP450-1A1 (CYP1A1) while AFB1 did not induce AhR-dependent CYP1A1 activation. Combination of VerA with AFB1 resulted in enhanced genotoxic effects, suggesting that AhR-activation by VerA influences AFB1 genotoxicity by promoting its bioactivation by CYP450s to a highly DNA-reactive metabolite. Our results emphasize the need for expanding the toxicological knowledge regarding mycotoxin biosynthetic precursors to identify those who may pose, directly or indirectly, a threat to human health.

Investigating Intestinal Transporter Involvement in Rivaroxaban Disposition through Examination of Changes in Absorption

The involvement of the intestinally expressed xenobiotic transporters P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) have been implicated in rivaroxaban disposition based on in vitro studies, similar to what had previously been proposed for apixaban. We recently showed that these efflux transporters were not clinically relevant for apixaban disposition and examine here their relevance for this second Factor Xa inhibitor. Using recently published methodologies to discern metabolic- from transporter- mediated drug interactions, a critical evaluation was undertaken of 9 rivaroxaban studies reporting 12 DDIs, one study of food effects and one study of hepatic function. Rationale examination of these clinical studies using basic pharmacokinetic theory finds little support for the clinical significance of intestinal efflux transporters in rivaroxaban disposition. Drug-drug interactions are most likely adequately predicted based on the level of CYP 3A metabolism. These analyses indicate that inhibition of efflux transporters appears to have negligible, clinically insignificant effects on the rivaroxaban absorption process, which is consistent with the concern that predictions based on in vitro measures may not translate to a clinically relevant interaction in vivo. We emphasize the need to evaluate gastric emptying, dissolution and other processes related to absorption when using MAT changes to indicate efflux transporter inhibition.