Induction Of Cytochrome P450 Enzymes Research Articles

Relationship Between Preoperative Alcohol Consumption and Postoperative Nausea and Vomiting in Liver Resection: A Propensity Score Matched Analysis in a Single Institute.

Patients with alcohol drinking habits have less nausea and vomiting during chemotherapy because of cytochrome P450 enzyme induction. However, few studies have examined the effect of alcohol consumption on postoperative nausea and vomiting (PONV). We conducted a study to clarify the relationship between alcohol drinking habits and PONV. Data of patients undergoing hepatectomy under general anesthesia between 2016 and 2020 were retrospectively collected. Since alcohol drinking habits vary by gender, age, and comorbidities, propensity score matching was performed to adjust patient background before multivariate logistic regression analysis. Seventy-eight patients in the alcohol consumption and non-consumption groups were matched by propensity matching. Univariate analysis showed that alcohol consumption (P = 0.04) and male (P ＜ 0.001) were the factors that significantly reduced PONV. Multiple logistic regression analysis including intraoperative factors showed that alcohol consumption (odds ratio, 0.36; 95% confidence interval [CI], 0.15-0.90) and female (odds ratio, 5.34; 95% CI, 2.0-14.2) were associated with PONV as factors affecting PONV. Patients with no alcohol drinking habits may be at higher risk of PONV.

Relationship Between Preoperative Alcohol Consumption and Postoperative Nausea and Vomiting in Liver Resection: A Propensity Score Matched Analysis in a Single Institute.

Patients with alcohol drinking habits have less nausea and vomiting during chemotherapy because of cytochrome P450 enzyme induction. However, few studies have examined the effect of alcohol consumption on postoperative nausea and vomiting (PONV). We conducted a study to clarify the relationship between alcohol drinking habits and PONV.Data of patients undergoing hepatectomy under general anesthesia between 2016 and 2020 were retrospectively collected. Since alcohol drinking habits vary by gender, age, and comorbidities, propensity score matching was performed to adjust patient background before multivariate logistic regression analysis.Seventy-eight patients in the alcohol consumption and non-consumption groups were matched by propensity matching. Univariate analysis showed that alcohol consumption (P = 0.04) and male (P ＜ 0.001) were the factors that significantly reduced PONV. Multiple logistic regression analysis including intraoperative factors showed that alcohol consumption (odds ratio, 0.36; 95% confidence interval [CI], 0.15-0.90) and female (odds ratio, 5.34; 95% CI, 2.0-14.2) were associated with PONV as factors affecting PONV.Patients with no alcohol drinking habits may be at higher risk of PONV.

MO420: Effects of Indoxyl Sulfate and Apixaban in Liver Gene Expression

Abstract BACKGROUND AND AIMS Uraemic toxins accumulate in the blood and tissues of patients with chronic kidney disease (CKD). Previous animal studies have shown that chronic kidney disease (CKD) not only alters the elimination of drugs excreted by the kidneys, but it also impacts the metabolism of drugs subject to non-renal clearance, which involves mainly the liver and the gut. The use of apixaban, an oral anticoagulant, has been approved in the USA for dialysis patients. However, the coexistence of thrombotic and haemorrhagic risk in patients with CKD makes dose adjustment difficult. The liver has a major role in drug metabolization. Hepatocytes express high levels of AhR, a ligand-inducible transcription factor that mediates the induction of various liver cytochrome P450 enzymes by xeno and endobiotic. Tryptophan-derived uraemic toxins (TDUT) are AhR agonists. Indoxyl sulfate (IS) is the main TDUT involved in uraemic syndrome. IS increases the expression and activity of P-glycoprotein (P-gp) in the liver. Apixaban metabolism is mediated by P-gp and Cyp3a4 (Cyp3a11 in mice). We aim to study the effect of apixaban and IS in the expression of drug metabolism genes. Our hypothesis is that AhR activation by IS could severely modify drug metabolism during CKD. METHOD C57BL/6J Wild-type mice purchased from The Jackson Laboratory were fed ad libitum with a standard diet. At 10 weeks of age, mice drinking water was substituted with a 5% sucrose water solution with either 0.1% indoxyl sulphate or KCl (control) added at equivalent concentrations. 48-h before sacrifice some mice were gavaged with an apixaban solution (0.6 mg/mL) twice a day with the last dose given 4 h prior to sacrifice. All mice were sacrificed at 11 weeks of age. Liver samples were stored in an RNA-later solution at −20°C. Samples were thawed and lysed in Trizol using the Tissue-Ruptor system (Qiagen). RNA was extracted and purified with chloroform and precipitated with isopropanol. RNA concentration was estimated by spectrophotometry. Gene expression was analysed by q-RT-PCR using Gusb as a housekeeping gene. Apixaban levels were quantified by LC-MS. Kruskal–Wallis followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (q < 0.05) was performed using GraphPad Prism 9.2.1. RESULTS Four groups of mice per sex were thus created: WT-KCl, WT-IS, WT-KCl Apix, WT-IS Apix. In the IS/KCl model we observe higher mRNA basal expression of Abcb1a (q = 0.008) and Sult1a1 (q = 0.006) and lower basal expression of Abcg2 (q = 0.0005) in females compared to males when treated with KCl. IS treatment increases Cyp1a2 expression in females (q = 0.0139) and Cyp1a1 in both males (q = 0.0145) and females (q = 0.026). In the IS/KCl-Apix model the same sex-related differences are maintained for Abcb1a (q = 0.0031), Sult1a1 (q = 0.0316) and Abcg2 (q < 0.0001). In males, CYP2e1 expression is increased by apixaban in KCl (q = 0.067) and IS (q = 0.0278) treated mice. Moreover, apixaban counteracts the increased expression of Cyp1a1 induced by IS (q = 0.0177). The expression of Cyp3a11 is augmented in males treated with KCl-apixaban (q = 0.0143), an effect that seems to be reversed by IS (q = 0.0066). In females, apixaban has no effect in gene expression. Apixaban serum concentration is higher in KCl (q = 0.0350) and IS (q = 0.0019) treated females when compared to males. CONCLUSION The effect of IS as an agonist of AhR in the liver is confirmed by the increased expression of Cyp1a1. Apixaban provokes a remarkable increase of Cyp3a11 in male mice, which could lead to higher degradation rates decreasing its activity. IS seems to reverse this effect. This could lead to increased activity of apixaban during CKD which could derive in an increased risk of bleeding. Higher BCRP rates could explain lower rates of apixaban in male serum. Apixaban did not alter gene expression in females. Clinical trials in CKD are generally biased regarding sex (fewer women represented) possibly masking important sex-dependent drug adverse effects. Our results show great differences in basal expression of various genes, confirming the importance to study men and women separately with respect to drug metabolism.

The role of heat-not-burn, snus and other nicotine-containing products as interventions for epileptic patients who take phenytoin and smoke cigarettes

BackgroundCigarette smoke increases the metabolism of phenytoin, a widely used anti-epileptic agent, by inducing cytochrome P450 enzymes in the liver. Therefore, cigarette smoke may reduce the clinical effect of phenytoin. Switching from cigarettes to smoke-free products (e.g., heat-not-burn, snus and e-cigarettes) is likely to have an impact on phenytoin metabolism. The absence of tobacco combustion in these smoke-free products reduces the production of the chemicals that induce the metabolism of phenytoin. ObjectivesThe primary objective was to determine whether smoke-free products have a role to play in epileptic patients who take phenytoin and continue to smoke cigarettes. The secondary objectives were to assess: (1) the influence of cigarette smoke on phenytoin metabolism including the metabolic pathways involved, (2) the influence of nicotine on phenytoin metabolism including the metabolic pathways involved, if any, and (3) the influence of nicotine on epilepsy in humans, if any. MethodsThe literature review was conducted in 2019 and 2020 using a structured search to identify relevant articles. The potential mechanisms underlying the effects of cigarette smoke and nicotine on phenytoin metabolism and the pathways for phenytoin metabolism were evaluated to determine overlapping mechanisms/pathways. The key methodological deficiencies in the studies were recorded, where applicable. ResultsThirty-five studies were reviewed. The literature showed that cigarette smoke influenced the metabolism of phenytoin by increasing the maximum metabolism rate of phenytoin by an average of 16% in humans. Cigarette smoke is known to contain several polycyclic aromatic hydrocarbons (PAHs), which can lead to faster elimination of numerous medicines, including phenytoin. There is no literature assessing the direct influence of nicotine on phenytoin metabolism in humans and animals. The metabolic pathways of phenytoin and nicotine do not overlap indicating that nicotine does not influence the metabolism of phenytoin. Hence, complete switching to smoke-free products with similar nicotine concentrations to cigarettes, may reduce the influence smoking has on phenytoin metabolism in epileptic patients who take phenytoin and smoke. There was also no evidence to demonstrate that nicotine has the potential to trigger epilepsy in humans. ConclusionThe literature showed that the increase in metabolic rate of phenytoin due to tobacco smoke is probably attributable to PAHs and not nicotine. The similar nicotine content between smoke-free products and cigarettes and the reduced levels of PAHs in smoke-free products indicate that there is a role for smoke-free products in epileptic patients who take phenytoin and smoke.

Toxicological and Pharmacokinetic Analysis by Insilico Bioinformatics Tools of an Ancient Medicine Compound Cantharidin Derived from Spanish fly

Background: Cantharidin, a vesicant released by spanish fly belongs to species beetles of zoological order Coleoptera. It has a long history in both folk and traditional medicine use for topical cantharidin to treat warts and molluscum and as an aphrodisiac. They are also known to cause poisoning by the presence of toxic principle.Aims and Objectives: The aims of the study were to study possible drug likeness, pharmacokinetic and toxicological profiling of known toxicological active principle cantharidin by insilico analysis and prediction tools.Materials & Methods: This study was investigated on web-based tools PubChem to extract the chemical structure, followed by authentication and validation with the chemical formula. The two-dimensional structures are further converted to three-dimensional (3D) structure with ChemSketch software; The structures are then screened for molecular properties with Simplified Molecular Input Line Entry System, followed by absorption, distribution, metabolism, elimination, and toxicity through SWISS ADME software web based tool. The reports are analyzed and predicted for drug likeness, pharmacokinetic and toxicity characters of cantharidin.Results: The compounds screened cantharidin for drug likeness had a Log P score of >4.15, Bioavailability Score of 0.55, +ve blood brain barrier +ve Intestinal penetration and -ve to Cytochrome P450 enzyme induction and inhibition. The toxic hazard classification by Cramer resulted as HIGH –CLASS III with Skin Sensitization score of 0.861Conclusion: The insilico analysis predicts cantharidin belongs to class III Toxic hazard classification but potential as drug like candidate for topical administration due to its high skin sensitivity and irritation ability as a vesicant in treatment of warts and molluscum.

In vitro drug-drug interactions of decitabine and tetrahydrouridine involving drug transporters and drug metabolising enzymes

1. NDec is a novel, oral, fixed dose formulation of decitabine and tetrahydrouridine that is currently being developed for the treatment of patients with sickle cell disease. Here, we examine the potential for both components of NDec to interact with key drug metabolising enzymes (tetrahydrouridine only) and drug transporters (decitabine and tetrahydrouridine). 2. This study assessed the inhibition and induction of cytochrome P450 (CYP) enzymes by tetrahydrouridine, as well as the involvement of specific drug metabolising enzymes in tetrahydrouridine metabolism. Inhibition of efflux and uptake transporters by both decitabine and tetrahydrouridine was also studied. 3. Tetrahydrouridine did not inhibit or induce relevant CYP enzymes at concentrations ranging from 0.1–100 μM. Metabolism of tetrahydrouridine did not occur in the presence of the human drug metabolising enzymes tested. Tetrahydrouridine showed weak inhibition towards the MATE2-K transporter (∼30% inhibition at 5 and 50 μM), which was not deemed clinically relevant. Tetrahydrouridine did not inhibit any of the remaining uptake or efflux transporters. Decitabine (0.5 and 5 μM) did not inhibit any of the evaluated uptake or efflux drug transporters. 4. Data presented confirm that tetrahydrouridine and decitabine are unlikely to be involved in metabolism- or transporter-based drug-drug interactions.

793. Rifabutin Use in Staphylococcal Prosthetic Material Infections: A Case Series

BackgroundBacterial biofilm formation is of clinical concern among patients with staphylococcal infections involving prosthetic material. While rifampin has in-vitro, animal and clinical data to support its adjunctive role in these types of infections, its potent induction of multiple cytochrome P450 enzymes and P-glycoprotein transport system proteins can pose significant drug-drug interactions. Rifabutin has comparable in-vitro anti-staphylococcal activity but less drug-drug interaction potential than rifampin. However, minimal clinical data exists to support rifabutin use as adjunctive treatment of prosthetic infections.MethodsThis case series describes 7 patients who received adjunctive rifabutin for staphylococcal prosthetic material infections between February 2018 and January 2021 at Massachusetts General Hospital. The primary outcome of infection recurrence was defined as need for surgical intervention for suspected or proven recurrence infection within 6 months after starting rifabutin therapy. Incidence of adverse effects was the main secondary outcome.ResultsMost patients (6/7) had methicillin-sensitive S. aureus (MSSA) and one patient had S. epidermidis infection. Three patients had spinal fusion hardware infection, one patient had hardware-associated spinal osteomyelitis/ diskitis with epidural abscess, two patients had prosthetic joint infection, and one patient had MSSA bacteremia with left ventricular assistance device involvement. All patients except one underwent surgical management prior to starting rifabutin. Infection recurrence was noted in one of seven patients who required surgical washout. Adverse events were uncommon (n=1 treatment-related nausea, n=1 leukopenia).ConclusionThis small case series suggests favorable outcomes with use of rifabutin instead of rifampin for staphylococcal infections with prosthetic material involvement.Disclosures Sandra B. Nelson, MD, UpToDate (Other Financial or Material Support, author)

Evaluation of Nigerian Medicinal Plants Extract on Human P-glycoprotein and Cytochrome P450 Enzyme Induction: Implications for Herb-drug Interaction.

Herbal medicine represents a significant component of disease prevention and therapy in most African countries. Herb-drug interactions (HDI) can arise from the co-administration of herbal and orthodox medicines. This study assessed the potential for HDI of V. amygdalina, O. gratissimum, M. oleifera, A. indica, and P. nitida extracts using in vitro assays. Little is known about these medicinal plants' potential for drug interaction despite their extensive use in Nigeria for several disease conditions. The medicinal plant crude extracts were evaluated for Cytochrome P450 (CYP) enzyme induction using cryopreserved human hepatocytes. Enzyme activity was determined by quantifying probe substrate metabolism and metabolite formation using liquid chromatography-mass spectrometry/mass spectrometry. The extracts were evaluated for the potential to inhibit P-glycoprotein (P-gp) activity using human embryonic kidney membrane vesicles over-expressing human P-gp. The herbal extracts in vivo drug interaction potential was predicted based on the USFDA drug interaction guidance. O. gratissimum and P. nitida methanol extracts induced CYP1A2 enzyme activity by greater than 3-fold. P. nitida methanol extracts showed over 2-fold induction of CYP1A2 mRNA expression. O. gratissimum methanol extract induced CYP2B6 mRNA expression over 2-fold. P. nitida and A. indica methanol extracts showed potent inhibition of P-gp activity (IC50: 3.8 and 5.4 μg/mL), respectively, while V. amygdalina and M. oleifera methanol extracts showed moderate P-gp inhibition (IC50: 12.1 and 37.2 μg/mL, respectively). Our studies suggested that the medicinal plants' extracts can modulate CYP enzymes and P-gp activity with the potential to cause herb-drug interaction in vivo.

Protective effect of Bridelia tomentosa due to its phenolic acids and flavonoids against oxidative stress-mediated hepatic toxicity induced by carbofuran

The aim of this study was to analyze the protective role of Bridelia tomentosa Blume fruit against oxidative stress-mediated hepatic damage induced by carbofuran in rats and anti-radical effects of phenolic acids and flavonoids. High-performance liquid chromatography analysis was used to determine the presence of benzoic acid, gallic acid, salicylic acid, quercetin, and rutin which have in-vitro anti-radical effects. In-vivo results suggested that carbofuran produced toxic reactive oxygen species and malondialdehyde which significantly affected serum biochemical parameters, endogenous hepatic antioxidants, and histological architecture of hepatocytes. Carbofuran also induced cytochrome P450 enzymes that increased reactive chemical species mediated cellular damage. However, oral treatment with B. tomentosa fruit methanol extract significantly attenuated the toxicant-induced hepatic toxicity on serum biochemical markers such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase, total bilirubin, total protein and lipid profile. Co-administration of fruit extract also significantly increased the hepatic endogenous antioxidants superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione. Cytochrome P450 inhibitory activity and histopathological findings further confirmed the protective role against carbofuran induced damage. It could be concluded that phenolic acids and flavonoids of B. tomentosa fruit might neutralize the toxic free radical mediated oxidative hepatic injury.

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

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

In vitro inhibition and induction of human liver cytochrome P450 enzymes by a novel anti-fibrotic drug fluorofenidone

Fluorofenidone (AKF-PD) is an analog of pirfenidone and shows stronger antifibrotic effect and lower toxicity compared to pirfenidone in preclinical studies. However, the inhibitory and inducible effects of AKF-PD on human CYP450s are unclear. The aim of this study was to evaluate the ability of AKF-PD to inhibit and induce CYP450s in vitro. In inhibition study, the inhibitory effects of CYP1A2, CYP3A4, CYP2C9, CYP2E1, CYP2C19 and CYP2D6 by AKF-PD were evaluated with the metabolic rate of probe drug of each enzyme in pooled human liver microsomes. The enzyme inducible potential of AKF-PD was evaluated by the mRNA expression and enzyme activity of CYP1A2, CYP2B6 and CYP3A4 in human hepatocytes. The results suggested that AKF-PD produced weak inhibition on CYP1A2 and CYP2C19, while no inhibitory effects were found on the other enzymes. Since the plasma concentration of AKF-PD is much lower than the IC50 values of both CYP1A2 and CYP2C19, the inhibitory effects can be reasonably ignored. On the other hand, AKF-PD showed no inducible effects on CYP1A2 while showed potential inducible ability on CYP2B6 and CYP3A4 in some test groups. Further study of this novel anti-fibrotic drug should take into account in clinical therapies.

Genotoxicity and subchronic toxicity studies of supercritical carbon dioxide and acetone extracts of rosemary

Toxicology studies conducted with oil-soluble rosemary extracts to support authorization as a food additive (antioxidant) in the EU include an Ames test using a supercritical carbon dioxide extract (D74), a full 90-day study using D74 and an acetone extract (F62), and an investigative 90-day study with a 28-day recovery period (using D74 only). D74 was non-mutagenic in the Ames test. In the full 90-day study, where rats (20/sex/group) were either provided control diet or diets containing D74 (300, 600, or 2400 mg/kg) or F62 (3800 mg/kg), liver enlargement and hepatocellular hypertrophy were observed. To determine a mode of action and assess the reversibility of the hepatic effects, an investigative 90-day study was conducted using female rats (10/group receiving control diet or diet containing 2400 mg/kg D74). Liver enlargement was fully reversible after 28 days and microsomal enzyme analysis revealed reversible induction of cytochrome P450 enzymes (CYP2A1, CYP2A2, CYP2C11, CYP2E1, and CYP4A), demonstrating that the hepatic effects were adaptive and of no toxicological concern. Therefore, the highest dietary concentrations were established as the NOAELs. The investigative 90-day study NOAEL (providing 64 mg/kg bw/day carnosol and carnosic acid [the primary antioxidant components]) was used to establish a temporary ADI for rosemary extracts.

Involvement of cytochrome P450 enzymes in inflammation and cancer: a review.

Cytochrome P450 (CYP) enzymes are responsible for the biotransformation of drugs, xenobiotics, and endogenous substances. This enzymatic activity can be modulated by intrinsic and extrinsic factors, modifying the organism's response to medications. Among the factors that are responsible for enzyme inhibition or induction is the release of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ), from macrophages, lymphocytes, and neutrophils. These cells are also present in the tumor microenvironment, participating in the development of cancer, a disease that is characterized by cellular mutations that favor cell survival and proliferation. Mutations also occur in CYP enzymes, resulting in enzymatic polymorphisms and modulation of their activity. Therefore, the inhibition or induction of CYP enzymes by proinflammatory cytokines in the tumor microenvironment can promote carcinogenesis and affect chemotherapy, resulting in adverse effects, toxicity, or therapeutic failure. This review discusses the relevance of CYPs in hepatocarcinoma, breast cancer, lung cancer, and chemotherapy by reviewing in vitro, in vivo, and clinical studies. We also discuss the importance of elucidating the relationships between inflammation, CYPs, and cancer to predict drug interactions and therapeutic efficacy.

Ensuring Safe Use of Isotretinoin

For individuals with severe acne, isotretinoin is a first-line therapy. As this agent is both a cytochrome P-450 enzyme inducer and a potent teratogen,

Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 μM sodium phenobarbital (NaPB), 3–100 μM fluquinconazole (FQZ), or 3–300 μM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 μM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.

AB005. Vitamin D in children—role beyond bones

: Vitamin D is a fat-soluble steroid hormone that contributes to the maintenance of normal calcium homeostasis and skeletal mineralization. The prevalence of subclinical vitamin D deficiency is not only restricted to rural population, but it is highly prevalent across the whole globe. It affects the individuals irrespective of their gender, race and geography. It’s deficiency is common in all age groups including the neonates. Although, it is commonly known as vitamin, but is actually not an essential dietary vitamin in the strict sense, as it can be easily synthesized by all mammals from sunlight in adequate amount. Vitamin D not only plays an essential role in calcium homeostasis and bone mineral metabolism, but it also helps in performing a wide range of fundamental biological functions such as cell differentiation, inhibition of cell growth and immunomodulation. By inhibiting uncontrolled proliferation and stimulation of cell differentiation, Vitamin D helps to prevent development of autoimmune disorders and even common cancers. Prolonged usage of certain drugs (Glucocorticoids, cytochrome P450 enzyme inducers, anticonvulsants, antiretro viral agents etc.) and other health problems including chronic kidney disease, asthma, pneumonia, coeliac disease, liver disease, tuberculosis, cancers (prostate, breast and colon), diabetes etc. are associated with Vitamin D deficiency. The concept of vitamin D possessing important pleiotropic actions and owing to its multiple implications on health, the epidemic of vitamin D deficiency significantly contributes to the burden on the healthcare system and society. Thus, it is desirable to update our knowledge about current research related to role of vitamin D beyond bone metabolism.

Clinical relevance of St. John's wort drug interactions revisited

The first clinically relevant reports of preparations of St. John's wort (SJW), a herbal medicine with anti‐depressant effects, interacting with other drugs, altering their bioavailability and efficacy, were published about 20 years ago. In 2000, a pharmacokinetic interaction between SJW and cyclosporine caused acute rejection in two heart transplant patients. Since then, subsequent research has shown that SJW altered the pharmacokinetics of drugs such as digoxin, tacrolimus, indinavir, warfarin, alprazolam, simvastatin, or oral contraceptives. These interactions were caused by pregnane‐X‐receptor (PXR) activation. Preparations of SJW are potent activators of PXR and hence inducers of cytochrome P450 enzymes (most importantly CYP3A4) and P‐glycoprotein. The degree of CYP3A4 induction correlates significantly with the hyperforin content in the preparation. Twenty years after the first occurrence of clinically relevant pharmacokinetic drug interactions with SJW, this review revisits the current knowledge of the mechanisms of action and on how pharmacokinetic drug interactions with SJW could be avoided.Linked ArticlesThis article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND:Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes.OBJECTIVE:For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes.METHODS:POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method.RESULTS:The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models.CONCLUSION:In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Ischemic and Thrombotic Events Associated with Concomitant Xa-inhibiting Direct Oral Anticoagulants and Antiepileptic Drugs: Analysis of the FDA Adverse Event Reporting System (FAERS)

Factor Xa-inhibiting direct oral anticoagulants (FXa-DOACs) undergo hepatic metabolism via cytochrome P-450 (CYP450). Concomitant use of rifampicin, an inducer of these enzymes, with FXa-DOACs, has been shown to decrease FXa-DOAC concentrations in healthy subjects. Several common antiepileptic drugs (AEDs) are known to induce CYP450 enzymes as well. However, little is known regarding the impact of this potential interaction on treatment outcomes with FXa-DOACs. We analyzed adverse event cases submitted to the Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) from January 2013 to December 2018. We compared the proportion of cases reporting thromboembolic and ischemic adverse events (TAIAEs) with the concomitant use of FXa-DOACs and enzyme-inducing AEDs to the proportion of cases with FXa-DOACs and other AEDs. During this period, 9693 adverse event cases reported concomitant use of FXa-DOACs and AEDs. Almost all reports (> 99%) involved the use of rivaroxaban or apixaban. Compared with other AEDs, enzyme-inducing AEDs were associated with an 86% increase in the odds of reporting TAIAEs [reporting odds ratio (ROR) 1.86, 95% confidence interval (CI) 1.61-2.15; p < 0.0001]. In secondary separate analyses of rivaroxaban and apixaban, enzyme-inducing AEDs were similarly associated with increased reporting of a TAIAE (ROR 1.79, 95% CI 1.50-2.12, and ROR 1.88, 95% CI 1.41-2.48, respectively). Using real world data, we observed an increase in the odds of reporting anticoagulation treatment failure among patients treated with FXa-DOACs and concomitant enzyme-inducing AEDs compared to those treated with other AEDs.

The Nonclinical Pharmacokinetics and Prediction of Human Pharmacokinetics of SPH3127, a Novel Direct Renin Inhibitor.

SPH3127 is a novel direct renin inhibitor designed as an oral drug for the regulation of blood pressure and body fluid homeostasis via the renin-angiotensin-aldosterone system (RAAS). This candidate is now being evaluated in a phase I clinical trial in China. The purpose of this study is to investigate detailed nonclinical pharmacokinetic data, and to predict human pharmacokinetic parameters. In vivo pharmacokinetic studies of SPH3127 were performed to investigate the exposure, absorption, clearance, distribution and metabolism after intravenous and oral administration in rats, beagle dogs and cynomolgus monkeys. The cynomolgus monkey pharmacokinetics/pharmacodynamics study was conducted to investigate the effect-concentration relationship of SPH3127. Its human pharmacokinetic properties were predicted employing an allometric scaling approach based on non-clinical species data. In vitro studies were also employed in a cytochrome P450(CYP) enzyme phenotyping study, an inhibition and induction study, and a Caco-2 cell permeation and metabolites profile analysis. After a single intravenous administration of SPH3127 in rats and monkeys, high clearance and volume of distribution and a short terminal elimination half-life were seen for both species. The oral bioavailability of SPH3127 to rats and monkeys was about 11.5-24.5% and 3.3-11.3%, respectively, with the short peak time, Tmax, ranging from 0.25 to 1.3h. SPH3127 shows low permeability across Caco-2 cell membranes, and as the substrate of p-gp with apparent efflux characteristics. SPH3127 is mainly distributed in the gastrointestine, liver, kidney, pancreas and lung after oral dose in rats, and which decreased quickly to a 1% peak concentration during 12h. The plasma protein binding ratio of SPH3127 is low as 11.7-14.8% for all species. Excretion studies in rats suggested that fecal, urine and bile excretion represented about 15% of the intake dose, indicating that SPH3127 undergoes extensive metabolism after oral dosing. Phenotyping data revealed that CYP3A4 was the most active enzyme catalyzing the metabolism of SPH3127. The key metabolites were likely N-hydroxylation (M8-7), mono-oxidation-dehydrogenation (M7-4) and mono-oxidation (M8-1, M8-2), both for in vitro liver microsome incubation of all species and in vivo results in rats. The in vitro CYP inhibition study only found very weak action for CYP3A4 (midazolam 1'-hydroxylation) and CYP3A4 (midazolam 6β-hydroxylation) with IC50 of 56.8µM and 41.1µM, respectively. Monkey pharmacokinetic/pharmacodynamic data showed favorable safety margins when compared with the exposure of the effect dose and that of the monkey NOAEL level. Simple four-species allometric scaling led to predicted human plasma clearance and volume of distribution, and then simulated the oral human plasma concentration-time profile, which are both in good consistency with phase I clinical trial pharmacokinetic data. SPH 3127 has appropriate pharmacokinetic properties for further clinical exploration.