Cytochrome P450 3A4 Activity Research Articles

Effects of green light-emitting diode irradiation on hepatic differentiation of hepatocyte-like cells generated from human adipose-derived mesenchymal cells

Light-emitting diode (LED) irradiation has been used in the differentiation of mesenchymal stem cells into a variety of cell types. This study investigated the effect of green LED (GLED) irradiation on the differentiation of adipocyte-derived mesenchymal cells into hepatocyte-like cells (HLCs) and the mechanism of its action. HLCs in the hepatocyte maturation phase were irradiated with GLED (520 nm, 21 W/m2, 5 min/day for 10 days). The cells were then assessed for expression of hepatocyte maturity genes and opsin 3 (OPN3), hepatocyte function, viability, apoptosis, and levels of reactive oxygen species (ROS), intracellular adenosine triphosphate (ATP) and calcium ions (Ca2+). GLED irradiation increased Alpha-1 antitrypsin and Ornithine transcarbamylase gene expression, promoted Cytochrome P450 3A4 activity and urea synthesis, and elevated intracellular ROS, ATP and Ca2+ levels. OPN3 expression was significantly more upregulated in GLED-irradiated HLCs than in the non-irradiated HLCs. No significant difference in cell viability or apoptosis was observed between GLED-irradiated and non-irradiated HLCs. GLED irradiation can promote hepatocyte maturation and functions through OPN3. GLED irradiation also stimulated mitochondrial function via Ca2+/ATP/ROS activation. GLED irradiation has potential to support cell-based transplantation in patients.

The effects of vitamins C and E on the redox parameters of cytochrome P450 3A4

Abstract The research on the influence of antioxidant vitamins C and E on the catalytic activity of cytochrome P450 3A4 in electrochemical experiments is relevant, as they are widely used as additional biologically active substances in human diets and can affect biochemical processes in the body. Understanding these effects is of significant importance for pharmacology and medicine. The purpose of this study is to investigate how antioxidant vitamins C and E impact the catalytic activity of cytochrome P450 3A4 within the framework of electrochemical experiments. The voltammetry was performed in the presence of potassium phosphate buffer of neutral pH value (0.2 M) and 0.5 M erythromycin solution (Er). The ratio between the measures of current obtained in the presence of O2 and Er was 2.10 ± 0.09 (100 %, P < 0.05). With vitamin C and Er, it corresponded to 2.80 ± 0.12 (133 %, P < 0.05). The voltammograms of Cyt with Dic show a higher current stimulated by ascorbic acid. The effect of vitamin C on the H-dimethylation process of Er was investigated with a spectrophotometer (value 408 nm). The catalytic current of cytochrome P450 3A4 in the presence of diclofenac was stimulated by ascorbic acid, the effect of which on the N-demethylation of erythromycin was studied spectrophotometrically at 408 nm based on the formation of formaldehyde using the acetylacetone reagent. The obtained results suggest the potential impact of antioxidant vitamins not only on the stimulation of electrochemical reduction of cytochrome P450 3A4 but also on the clinical metabolism of substrates in patients. These findings provide valuable insights for further clinical investigations aimed at predicting the risk of drug interactions with antioxidants.

Is Higher Docetaxel Clearance in Prostate Cancer Patients Explained by Higher CYP3A? An In Vivo Phenotyping Study with Midazolam.

Patients with prostate cancer (PCa) have a lower docetaxel exposure for both intravenous (1.8-fold) and oral administration (2.4-fold) than patients with other solid cancers, which could influence efficacy and toxicity. An altered metabolism by cytochrome P450 3A (CYP3A) due to castration status might explain the observed difference in docetaxel pharmacokinetics. In this in vivo phenotyping, pharmacokinetic study, CYP3A activity defined by midazolam clearance (CL) was compared between patients with PCa and male patients with other solid tumors. All patients with solid tumors who did not use CYP3A-modulating drugs were eligible for participation. Patients received 2 mg midazolam orally and 1 mg midazolam intravenously on 2 consecutive days. Plasma concentrations were measured with a validated liquid chromatography-tandem mass spectrometry method. Genotyping was performed for CYP3A4 and CYP3A5. Nine patients were included in each group. Oral midazolam CL was 1.26-fold higher in patients with PCa compared to patients with other solid tumors (geometric mean [coefficient of variation], 94.1 [33.5%] L/h vs 74.4 [39.1%] L/h, respectively; P= .08). Intravenous midazolam CL did not significantly differ between the 2 groups (P = .93). Moreover, the metabolic ratio of midazolam to 1'-hydroxy midazolam did not differ between the 2 groups for both oral administration (P = .67) and intravenous administration (P = .26). CYP3A4 and CYP3A5 genotypes did not influence midazolam pharmacokinetics. The observed difference in docetaxel pharmacokinetics between both patient groups therefore appears to be explained neither by a difference in midazolam CL nor by a difference in metabolic conversion rate of midazolam.

Effect of Omeprazole Administration on the Pharmacokinetics of Oral Extended-Release Nifedipine in Healthy Subjects.

Oral extended-release (ER) dosage forms have been used to sustain blood drug levels, reduce adverse events, and improve patient compliance. We investigated potential effects of comedication on pharmacokinetic exposure of nifedipine ER products with different formulation designs and manufacturing processes. A clinical study compared a generic version of nifedipine ER tablet with pH-dependent dissolution behavior with an osmotic pump product with pH independent drug release under fasting condition. In this study, two nifedipine tablet products were tested with or without short-term omeprazole comedication in healthy subjects. Seven-day administration of omeprazole before nifedipine dosing significantly increased the gastric pH, and subsequently increased the geometric least square (LS) means of area under the concentration-time curve from time zero to the last measurable timepoint (AUC0-t ) and maximum plasma concentration (Cmax ) of nifedipine to 132.6% (90% confidence interval (CI): 120.6-145.7%) and 112.8% (90% CI: 100.8-126.3%) for pH-dependent ER tablets, and 120.6% (90% CI: 109.7-132.5%) and 122.5% (90% CI: 113.7-131.9%) for the pH-independent ER tablets, respectively. Similar extent of increase in AUC0-t and Cmax was confirmed in the subpopulations whose gastric pH was ≥ 4 or ≤ 3 in subjects with or without omeprazole administration. Given that similar increases in drug exposures were observed for both pH-dependent and pH-independent nifedipine formulations and the geometric LS mean ratios were between 112% and 133% with and without short-term omeprazole comedication, the gastric pH may have limited effects on omeprazole-induced nifedipine PK changes on the tested formulations. The inhibition of cytochrome P450 3A4 activity may play a significant role causing nifedipine exposure changes for both formulations, which would warrant additional assessment.

Synthesis of 6β-hydroxy androgens from a 3,5-diene steroid precursor to test for cytochrome P450 3A4-catalyzed hydroxylation of androstenedione

6β-Hydroxytestosterone is a biomarker for the activity of human cytochrome P450 3A4 (P450 3A4), the major drug metabolizing cytochrome P450 enzyme. Despite its significance, efficient routes for the chemical synthesis of 6β-hydroxytestosterone are rare. In this study, 6β-hydroxytestosterone was synthesized through the oxidation of a 3,5-diene precursor under the Uemura-Doyle reaction conditions using a dirhodium catalyst in the presence of tert-butylhydroperoxide. Mechanistic studies showed that some oxygen is incorporated from molecular oxygen and CH abstraction is partially rate-limiting. This reaction was used to synthesize 6β-hydroxyandrostenedione, which was used as a standard to test the hypothesis of whether P450 3A4 catalyzes the hydroxylation of androstenedione. Upon incubation of P450 3A4 with androstenedione, a hydroxylated product was formed, which matched the retention time of synthetic 6β-hydroxyandrostenedione. This reaction can be exploited to study other biochemical processes involving compounds with a 6 β -hydroxy-3-keto-Δ4 steroid backbone.

Evaluation of effects of indoxyl sulfate and parathyroid hormone on CYP3A activity considering the influence of CYP3A5 gene polymorphisms.

Indoxyl sulfate and parathyroid hormone (PTH), which accumulate in chronic kidney disease (CKD), have been reported to reduce cytochrome P450(CYP)3A activity. Homozygotes of the CYP3A5*3 allele have reduced CYP3A5 activity compared to carriers of at least one CYP3A5*1 allele. 4β-Hydroxycholesterol (4β-OHC) has been established as an endogenous substrate reflecting CYP3A activity. 4β-OHC is produced through hydroxylation by CYP3A4 and CYP3A5 and by autoxidation of cholesterol, whereas 4α-hydroxycholesterol (4α-OHC) is produced solely by autoxidation of cholesterol. This study focused on CKD patients and evaluated the effects of plasma indoxyl sulfate and intact-PTH concentrations on plasma 4β-OHC concentration, 4β-OHC/total cholesterol ratio and 4β-OHC-4α-OHC, with consideration of the influence of CYP3A5 polymorphism. Sixty-three CKD patients were analysed and divided into CYP3A5 carrier group (n = 26) and non-carrier group (n = 37). Plasma indoxyl sulfate significantly correlated inversely with 4β-OHC concentration and with 4β-OHC-4α-OHC in both the CYP3A5*1 carrier group (r = -0.42, P = .034; r = -0.39, P = .050, respectively) and the non-carrier group (r = -0.45, P = .0054; r = -0.39, P = .019, respectively). However, multiple regression analysis did not identify plasma indoxyl sulfate concentration as a significant independent factor associated with any of the CYP3A activity indices. There was no significant correlation between plasma intact-PTH concentration and any of the CYP3A activity indices. The present results suggest that plasma indoxyl sulfate and intact-PTH concentrations do not have clinically significant effects on CYP3A activity in patients with CKD.

Cytochrome P450 3A4 activity and genetic variants as predictors of liver failure in patients with obstructive jaundice

Liver failure in patients with obstructive jaundice is a significant contributor to mortality within this patient cohort. The exact mechanism and triggers of this occurrence are yet to be fully understood. With this in mind, our study aimed to assess the correlation between the urinary 6 β-OHC/C ratio and various biochemical parameters of liver function. Furthermore, we conducted genotyping of CYP3A4*22 (rs35599367), CYP3A5*3 (rs776746) polymorphic markers to investigate the potential effects of their variants on the probability of liver failure in obstructive jaundice. Our study included 75 patients diagnosed with severe obstructive jaundice. All test subjects underwent functional liver tests, and control blood tests were administered on the seventh day following biliary decompression. Patients were categorized into two groups: group 1 - patients without liver failure (n = 60) and group 2 - patients with liver failure (n = 15). Laboratory indexes such as 6 β –OHC concentration and 6 β- OHC/cortisol ratio can serve as significant predictors of liver failure in patients with moderate and severe degree obstructive jaundice after biliary decompression. Based on the study of “wild” and polymorphic variants of CYP3A4*22 (CC and CT) and polymorphism of CYP3A5*3A6986G (GG, GA, AA), it was discovered that liver failure in the CYP3A4*22 variant may be associated with the CC genotype, and in the CYP3A5*3 variant - with the GA genotype. Hence, the determination of 6β- OHC concentration and 6β- OHC/C ratio, as well as the analysis of polymorphic and “wild” variants of CYP3A4*22 (CC and CT) and CYP3A5*3 polymorphism A6986G (GG, GA, AA), may play a crucial role in predicting liver failure in patients with obstructive jaundice.

Artificial cells eavesdropping on HepG2 cells.

Cellular communication is a fundamental feature to ensure the survival of cellular assemblies, such as multicellular tissue, via coordinated adaption to changes in their surroundings. Consequently, the development of integrated semi-synthetic systems consisting of artificial cells (ACs) and mammalian cells requires feedback-based interactions. Here, we illustrate that ACs can eavesdrop on HepG2 cells focusing on the activity of cytochrome P450 1A2 (CYP1A2), an enzyme from the cytochrome P450 enzyme family. Specifically, d-cysteine is sent as a signal from the ACs via the triggered reduction of disulfide bonds. Simultaneously, HepG2 cells enzymatically convert 2-cyano-6-methoxybenzothiazole into 2-cyano-6-hydroxybenzothiazole that is released in the extracellular space. d-Cysteine and 2-cyano-6-hydroxybenzothiazole react to form d-luciferin. The ACs respond to this signal by converting d-luciferin into luminescence due to the presence of encapsulated luciferase in the ACs. As a result, the ACs can eavesdrop on the mammalian cells to evaluate the level of hepatic CYP1A2 function.

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions invitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-β), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-β1 stimulation. Therefore, this hierarchical invitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Air-ventilated normothermic machine perfusion alleviates hepatic injury from DCD rat through CYP1A2

Background.Normothermic machine perfusion (NMP) could provide protection to organs from donation after circulatory death (DCD) before transplantation, and its molecular mechanism remains unclear. Our previous study discovered that the air-ventilated NMP confers a better DCD liver recovery than oxygen-ventilated NMP. The purpose in the current study was to investigate the protective mechanism of air-ventilated NMP in a rat model of DCD liver by metabolomics, and to select biomarker to predict liver function recovery.Materials and Methods.Peroxisome proliferator activator receptor-α (PPARα) agonist or antagonist was administered via the perfusion circuit in the air-ventilated NMP. Perfusate samples were taken for measurements of aminotransferases using standard biochemical methods, tumor necrosis factor-alpha and interleukin-6. Liver biopsies were allocated for detection of metabolomics, PPARα and cytochrome P450 1A2 (CYP1A2).Results.Metabolomics analysis revealed the significant increased γ-linolenic acid and decreased adrenic acid during the air-ventilated NMP, indicating linoleic acid metabolism pathway was associated with a better DCD liver recovery; as a major enzyme involved in linolenic acid metabolism, CYP1A2 was found correlated with a less inflammation and better liver function with the air-ventilated NMP; PPARα agonist could increase CYP1A2 expression and activity, decrease inflammation response, and improve liver function with the air-ventilated NMP, while PPARα antagonist played the opposite.Conclusion.Air-ventilated NMP confers a better liver recovery from DCD rats through the activated linoleic acid metabolism and CYP1A2 upregulation; CYP1A2 expression and activity might function as biomarker to predict DCD liver function recovery with NMP.

Physiologically Based Pharmacokinetic Modeling to Simulate CYP3A4-Mediated Drug-Drug Interactions for Pyrotinib.

Pyrotinib is a newly developed tyrosine kinase inhibitor whose in vivo clearance relies heavily on cytochrome P450 3A4 (CYP3A4) activity. Clinical trials are ongoing to explore the effects of coadministration with CYP3A4 perpetrators on pyrotinib exposure. The present study aims to utilize physiologically based pharmacokinetic (PBPK) modeling to predict CYP3A4-based drug interactions of pyrotinib. Pyrotinib PBPK model was developed in the PK-Sim® multicompartmental physiology structure. Physiochemical parameters were obtained from the literature, and clearance-related parameters were optimized by fitting clinical single-dose pharmacokinetic data. Pharmacokinetic parameters from the model output were compared with the observed data to validate the model predictive performance. Using validated CYP3A4 perpetrator models, we conducted PBPK simulations for drug interactions in a virtual population to explore the impacts of comedication with these perpetrators. The PBPK model accurately describes pyrotinib single- and multi-dose pharmacokinetics. The model also predicts dramatic exposure change of pyrotinib in the presence of itraconazole and rifampicin, though the impact of rifampicin is somewhat underestimated. According to model predictions, coadministration with typical potent or moderate CYP3A4 perpetrators increases pyrotinib concentration by over sixfold, extinguishing the possibility of dose adjustment for pyrotinib. A weak CYP3A4 inhibitor has minimal influence on pyrotinib pharmacokinetics. PBPK modeling provides valuable information to avoid irrational medication when receiving pyrotinib chemotherapy.

Comprehensive in vitro analysis evaluating the variable drug–drug interaction risk of rifampicin compared to rifabutin

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Evaluation of Drug-Drug Interaction Potential of Enarodustat (JTZ-951) Using a Cytochrome P450 Probe Cocktail.

The drug interaction potential of enarodustat (doses: 25, 50mg) on the activity of cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 was evaluated after once-daily administration for 15 days in a phase 1 multiple-ascending-dose study in healthy subjects. Probe substrates specific for the enzymes, i.e.,caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4), were administered orally as a cocktail with (day15) and without (day -3) enarodustat. Drug interaction was based on geometric mean maximum plasma concentration (Cmax ) and area under the plasma concentration-time curve from the time of dosing to infinity (AUCinf ) ratios (day 15/day -3) for CYP1A2, 2C9, 2C19, 2D6, 3A4, and urinary excretion of dextromethorphan metabolite dextrorphan for CYP2D6. At the 2 enarodustat doses, for caffeine, the geometric mean ratios (range) for Cmax and AUCinf were 0.99-1.06 and 1.61-1.63, respectively. The ratios for peak concentrations and total exposures were 0.98-1.07 and 0.71-1.78 for tolbutamide and omeprazole, respectively. For dextrorphan the Cmax and AUCinf ratios were 0.83-0.90 and 1.02-1.04, respectively. The mean dextrorphan cumulative amount excreted into the urine from the time of dosing to 24 hours values on day -3 and day 15 were 8.25 mg and 8.20 mg at the lower dose, and 9.40 mg and 9.51 mg at the higher dose. The ratios for midazolam Cmax and AUCinf were 1.42-1.63. Overall, there was a lack of enarodustat dose dependency regarding the geometric mean ratios and 90% confidence intervals and urinary excretion of dextrorphan. There were some cases where the 90% confidence intervals at the 2 enarodustat doses were outside the 0.80-1.25 range, but changes in the geometric mean ratios were all <2-fold.

Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions.

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.

Rationally Engineered CYP3A4 Fluorogenic Substrates for Functional Imaging Analysis and Drug-Drug Interaction Studies.

Cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme-mediated drug metabolism and drug-drug interaction (DDI). Herein, an effective strategy was used to rationally construct a practical two-photon fluorogenic substrate for hCYP3A4. Following two-round structure-based substrate discovery and optimization, we have successfully constructed a hCYP3A4 fluorogenic substrate (F8) with desirable features, including high binding affinity, rapid response, excellent isoform specificity, and low cytotoxicity. Under physiological conditions, F8 is readily metabolized by hCYP3A4 to form a brightly fluorescent product (4-OH F8) that can be easily detected by various fluorescence devices. The practicality of F8 for real-time sensing and functional imaging of hCYP3A4 has been examined in tissue preparations, living cells, and organ slices. F8 also demonstrates good performance for high-throughput screening of hCYP3A4 inhibitors and assessing DDI potentials in vivo. Collectively, this study develops an advanced molecular tool for sensing CYP3A4 activities in biological systems, which strongly facilitates CYP3A4-associated fundamental and applied research studies.

The antidiabetic effect of methanolic extract of Holarrhena pubescens seeds is mediated through multiple mechanisms of action

Holarrhena pubescens is widely used in Indian and Chinese medicine in the treatment of diabetes. The current work determined the oral hypoglycemic and antidiabetic effects of seed extract in rats. The probable mechanism of action was evaluated in-vitro by α - glucosidase inhibition, glucose metabolism in insulinoma (INS-1) cells to reflect secretion of insulin, and protein glycation inhibition. Its potential for herb-drug interaction was evaluated in the cytochrome P450 3A4 (CYP3A4) inhibition assay. The seed extract increased serum insulin levels and reduced serum blood glucose levels in the oral glucose tolerance test. It also reduced the serum glucose levels in streptozocin-induced diabetes. The extract also inhibited α –glucosidase enzyme activity and demonstrated that it can increase the secretion of insulin from INS to 1-rat insulinoma cell line cells in-vitro in a concentration-dependent manner. However, it had a very weak inhibitory effect on protein glycation and it did not affect the activity of CYP3A4. The results of the study showed that H. pubescens seed extract increases insulin secretion and inhibits glucose absorption both in-vivo and in-vitro with a weak protein glycation inhibitory effect. The herb is devoid of CYP3A4 inhibitory effect indicating that it may not have pharmacokinetic interaction with the drug metabolized by this enzyme.

Abstract 2683: Investigation to explore how duocarmycin prodrugs bioactivated by cytochrome P450 enzymes can be used in combination therapy to treat breast cancer cells

Abstract Introduction: Breast cancer is the most diagnosed cancer worldwide1 and, among other factors, hormones1 and pollutants2 have been suggested to contribute to disease progression. Due to its role in hormone environmental pollutant metabolism and involvement in breast cancer proliferation and survival, the cytochrome P450 1A1 (CYP1A1) isoform represents an interesting therapeutic target in breast cancer3.The present study builds on our understanding of reengineering the duocarmycin class of compounds4, but with focus on comparing the duocarmycin prodrug ICT2700 as single agent or in combination with the PARP inhibitor Olaparib, to uncover the potential of CYP1A1 and DNA minor groove alkylating agents in treating breast cancer. Methods: Functional CYP1A1 activity was evaluated using the EROD assay in a panel of breast cancer cell lines (MCF-7, T47D, MDA-MB-468, MDA-MB-231, MDA-MB-436), using as a control the isogenic cell line pair CHO and CHO1A1. CYP1A1 activity was also measured after serum starving the cells (0% FBS) for 24 and 48 hours. MTT assay was performed to measure the potency of the active duocarmycin CI-MI, ICT2700 and Olaparib in breast cancer cells, alone and in combination. DNA damage was measured using γH2AX as a marker, while cell cycle analysis was performed using FACS after exposing the cells to ICT2700 and Olaparib, alone and in combination. Results: The ICT2700 prodrug is activated, in part, by CYP1A1 which activity was shown to increase as a response to acute nutrient stress. Moreover, both the cytotoxic CI-MI and ICT2700 compounds were shown to enhance cell kill when combined with Olaparib in MDA-MB-468 and T47D breast cancer cells. Conclusion: The current investigation represents a promising starting point to study the activity of duocarmycin prodrugs such as ICT2700 in combination with other drugs such as Olaparib or modalities such as radiation therapy; the latter research is ongoing and will be reported at the meeting.

Time course of CYP3A activity during and after metamizole (dipyrone) in healthy volunteers.

In patients of all ages, metamizole is a frequently used analgesic. Recently, metamizole has been identified as an inducer of, among others, cytochrome P450 (CYP) 3A activity, but the time course of this interaction has not been evaluated. Using repeated oral microdoses (30 μg) of the CYP3A index substrate midazolam, we assessed changes in midazolam pharmacokinetics (area under the concentration-time curve from 2-4h: AUC2-4 and estimated partial metabolic clearance: eClmet ) before, at steady-state, and after discontinuation of 3× 1000 mg metamizole/day orally for 8days. Significant changes in pharmacokinetic parameters were detected already 3 days after start of metamizole treatment. At the steady-state of enzyme induction, the geometric mean ratio of midazolam AUC2-4 was substantially reduced to 0.18 (90% confidence interval: 0.14-0.24) with a corresponding 5.43-fold (4.15-7.10) increase of eClmet . After discontinuation of metamizole, the changes slowly recovered, but were still significant at the end of the observation period on the fifth day after discontinuation of metamizole therapy (AUC2-4 reduced to 0.50 [0.41-0.63] and eClmet 1.99-fold increased [1.60-2.47, P< 0.05]). Metamizole acts as a strong inducer of CYP3A already few days after start of metamizole administration and, thus, should be avoided in patients using drugs with narrow therapeutic index and major clearance via CYP3A. If their administration is essential, close monitoring and dose adjustment of comedication should be performed as early as the first week after the initiation and after discontinuation of metamizole therapy.

TET3-mediated DNA demethylation and chromatin remodeling regulate T-2 toxin-induced human CYP1A1 expression and cytotoxicity in HepG2 cells

T-2 toxin is a hazardous environmental pollutant that poses a risk to both farm animals and humans. Our previous research has reported that T-2 toxin highly induced the expression of human cytochrome P450 1A1 (CYP1A1), which may be a representative inducible marker of T-2 toxin and mediate the toxicity of T-2 toxin. In this study, we found that T-2 toxin decreased the DNA methylation levels of the CpG islands on the CYP1A1 promoter by inducing the expression of eleven translocation family protein 3 (TET3) and facilitating its binding to the promoter. These DNA methylation changes then generated an activated chromatin structure on the CYP1A1 promoter by releasing the repressor complex methyl-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2), increasing the active histone modification markers, including H3K4ac, H3K9ac and H3K14ac, and facilitating RNA pol II and NRF1/Sp1 recruitment, which ultimately led to the transcriptional activation of CYP1A1. Interestingly, TET3-mediated CYP1A1 induction enhanced the cytotoxicity of T-2 toxin through inhibiting cell proliferation. Our results demonstrate that T-2 toxin-induced CYP1A1 expression is detrimental to cells and clearly show how T-2 toxin inhibits cell proliferation through regulating CYP1A1 expression from an epigenetic perspective. The findings broaden our current knowledge of the epigenetic mechanisms regulating environmental factors-induced CYP1A1 expression and cytotoxicity. TET3 may serve as a potential new target for toxicogenic detoxification.

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity.

CYP3A4, which contributes to the metabolism of more than 30% of clinically used drugs, exhibits high variation in its activity; therefore, predicting CYP3A4 activity before drug treatment is vital for determining the optimal dosage for each patient. We aimed to develop and validate an LC-tandem mass spectrometry (LC-MS/MS) method that simultaneously measures the levels of CYP3A4 activity-related predictive biomarkers (6β-hydroxycortisol (6β-OHC), cortisol (C), 1β-hydroxydeoxycholic acid (1β-OHDCA), and deoxycholic acid (DCA)). Chromatographic separation was achieved using a YMC-Triart C18 column and a gradient flow of the mobile phase comprising deionized water/25% ammonia solution (100 : 0.1, v/v) and methanol/acetonitrile/25% ammonia solution (50 : 50 : 0.1, v/v/v). Selective reaction monitoring in the negative-ion mode was used for MS/MS, and run times of 33 min were used. All analytes showed high linearity in the range of 3-3000 ng/mL. Additionally, their concentrations in urine samples derived from volunteers were analyzed via treatment with deconjugation enzymes, ignoring inter-individual differences in the variation of other enzymatic activities. Our method satisfied the analytical validation criteria under clinical conditions. Moreover, the concentrations of each analyte were quantified within the range of calibration curves for all urine samples. The conjugated forms of each analyte were hydrolyzed to accurately examine CYP3A4 activity. Non-invasive urine sampling employed herein is an effective alternative to invasive plasma sampling. The analytically validated simultaneous quantification method developed in this study can be used to predict CYP3A4 activity in precision medicine and investigate the potential clinical applications of CYP3A4 biomarkers (6β-OHC/C and 1β-OHDCA/DCA ratios).