Liver CYP3A4 Research Articles

Drug-induced enzyme activity inhibition and CYP3A4 genetic polymorphism significantly shape the metabolic characteristics of furmonertinib

This study aimed to elucidate the impact of variations in liver enzyme activity, particularly CYP3A4, on the metabolism of furmonertinib. An in vitro enzyme incubation system was established for furmonertinib using liver microsomes and recombinant CYP3A4 baculosomes, with analytes detected by LC-MS/MS. The pharmacokinetic characteristics of furmonertinib were studied in vivo using Sprague-Dawley rats. It was found that telmisartan significantly inhibited the metabolism of furmonertinib, as demonstrated by a significant increase in the AUC of furmonertinib when co-administered with telmisartan, compared to the furmonertinib-alone group. Mechanistically, it was noncompetitive in rat liver microsomes, while it was mixed competitive and noncompetitive in human liver microsomes and CYP3A4. Considering the genetic polymorphism of CYP3A4, the study further investigated its effect on the kinetics of furmonertinib. The results showed that compared to CYP3A4.1, CYP3A4.29 had significantly increased activity in catalyzing furmonertinib, whereas CYP3A4.7, 9, 10, 12, 13, 14, 18, 23, 33, and 34 showed markedly decreased activity. The inhibitory activity of telmisartan varied in CYP3A4.1 and CYP3A4.18, with IC50 values of 8.56 ± 0.90 μM and 27.48 ± 3.52 μM, respectively. The key loci affecting the inhibitory effect were identified as ARG105, ILE301, ALA370, and LEU373. Collectively, these data would provide a reference for the quantitative application of furmonertinib.

Ionic Liquid 1-Octyl-3-Methylimidazolium (M8OI) Is Mono-Oxygenated by CYP3A4 and CYP3A5 in Adult Human Liver.

Environmental sampling around a landfill site in the UK previously identified the methylimidazolium ionic liquid, 1-octyl-3-methylimidazolium (M8OI), in the soil. More recently, M8OI was shown to be detectable in sera from 5/20 PBC patients and 1/10 controls and to be oxidised on the alkyl chain in the human liver. The objective of this study was to examine the metabolism of M8OI in humans in more detail. In human hepatocytes, M8OI was mono-oxygenated to 1-(8-Hydroxyoctyl)-3-methyl-imidazolium (HO8IM) then further oxidised to 1-(7-carboxyheptyl)-3-methyl-1H-imidazol-3-ium (COOH7IM). The addition of ketoconazole-in contrast to a range of other cytochrome P450 inhibitors-blocked M8OI metabolism, suggesting primarily CYP3A-dependent mono-oxygenation of M8OI. Hepatocytes from one donor produced negligible and low levels of HO8IM and COOH7IM, respectively, on incubation with M8OI, when compared to hepatocytes from other donors. This donor had undetectable levels of CYP3A4 protein and low CYP3A enzyme activity. Transcript expression levels for other adult CYP3A isoforms-CYP3A5 and CYP3A43-suggest that a lack of CYP3A4 accounted primarily for this donor's low rate of M8OI oxidation. Insect cell (supersome) expression of various human CYPs identified CYP3A4 as the most active CYP mediating M8OI mono-oxygenation, followed by CYP3A5. HO8IM and COOH7IM were not toxic to human hepatocytes, in contrast to M8OI, and using a pooled preparation of human hepatocytes from five donors, ketoconazole potentiated M8OI toxicity. These data demonstrate that CYP3A initiates the mono-oxygenation and detoxification of M8OI in adult human livers and that CYP3A4 likely plays a major role in this process.

Examining dietary supplements containing Euterpe oleracea Mart. (açaí) for potential botanical-drug interactions involving CYP3A4 inhibition

BackgroundBotanical dietary supplements (BDS) are consumed more by cancer patients than otherwise healthier patients to alleviate side effects of chemotherapy drugs or increase quality of life. The enzyme responsible for the metabolism of most anticancer drugs is CYP3A4, an enzyme predominantly located in the liver. HypothesisOur research team examined the potential risk between CYP3A4-interactive cancer drugs and Euterpe oleracea Mart. (açaí) berry and found that the compounds that passively and non-passively diffused from methanol açaí extract displayed significant inhibition of liver CYP3A4 – suggesting the potential for interactions between açaí and drugs metabolized by CYP3A4 if used synchronously. This study aims to identify the compounds responsible for the CYP3A4 inhibition using the newly automated Bioactivity – Global Natural Product Social Molecular Networking (GNPS) tool. MethodsDietary supplement capsules from two manufacturers (F1 and F2) containing aqueous extracts of açaí fruits were extracted separately by methanol (ME) and acidic methanol (AC) and standardized to cyanidin-3-O-glucoside (CG) concentration at a human-relevant dose. After solvent removal, a parallel artificial membrane permeability assay (PAMPA) was utilized to filter intestinal passive diffusion of the four extract constituents so that compounds could be tested for inhibition of hepatic CYP3A4. Passively and non-passively diffused constituents of extracts from PAMPA assays were injected into the liquid chromatography-mass spectrometry (LC–MS) and characterized for visualization and comparison of chemical entities. To elucidate the non-passively diffused compounds responsible for significant CYP3A4 inhibition, the Bioactivity – GNPS tool was used to hypothesize which compounds within the formulation extract were potentially active to accelerate their identification. ResultsThere was no significant inhibition of CYP3A4 by passively diffused constituents for any extract. There was, however, significant inhibition by non-passively diffused constituents of both F1ME and F1AC extracts with 50 % CYP3A4 inhibition at the donor compartment concentrations 5.764 and 15.58 ng/ml CG, respectively. Six compounds were predicted to be inhibitors of CYP3A4 from methanol F1 extract, including betaine and 5 unknown compounds. ConclusionThis indicates that BDS containing açaí may not inhibit CYP3A4 through passively diffused compounds. Compounds from açaí BDS that inhibit CYP3A4 may be absorbed through other mechanisms, such as transporters.

The acute toxic effect of Chinese medicine Fuzi is exacerbated in kidney yang deficiency mice due to metabolic difference

Ethnopharmacological relevanceThe proper application of toxic medicines is one of the characteristics of traditional Chinese medicines, and the use of traditional Chinese medicines follows the principle of dialectical treatment. It is necessary to combine different “syndrome” or “disease” states with the toxicity of traditional Chinese medicines to form a reliable toxicity evaluation system. Fuzi, the lateral root of Aconitum carmichaelii Debx, is recognized as a panacea for kidney yang deficiency syndrome, however, its toxic effects significantly limit its clinical application. Aim of the studyHerein, our research aimed to explore the toxic effects of Fuzi on syndrome models, and tried to reveal the underlying mechanisms. Materials and methodsFirstly, the mouse model of kidney yang deficiency syndrome was established through intramuscular injection of 25 mg/kg hydrocortisone per day for 10 consecutive days. Then, the acute toxicity of Fuzi in normal mice and kidney yang deficiency model mice was explored. Finally, the plasma metabolite concentrations and liver CYP3A4 enzyme activity were analyzed to reveal the possible mechanisms of the different pharmacological and toxicological effects of Fuzi in individuals with different physical constitutions. ResultsIt was found that the treatment with Fuzi (138 g/kg) had serious toxic effects on kidney yang deficiency mice, leading to the death of 80% of the mice, whereas it showed no lethal toxicity in normal mice. This indicates that Fuzi induced greater toxicity in kidney yang deficiency mice than in normal ones. The liver CYP3A4 enzyme activity in kidney yang deficiency mice was decreased by 20% compared to the controls, resulting in slower metabolism of the toxic diester diterpenoid alkaloids in Fuzi. ConclusionIn conclusion, our study showed that changes of the metabolic enzyme activity in individuals with different syndromes led to different toxic effects of Chinese medicines, emphasizing the crucial importance of considering individual physical syndromes in the clinical application of traditional Chinese medicine, and the significance of conducting safety evaluations and dose predictions on animal models with specific syndromes for traditional Chinese medicines.

Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs

Skatole, a strong fecal odor substance, is generated through microbial degradation of tryptophan in the animal hindgut. It easily accumulates in adipose tissue and affects meat quality. In this study, the effect of mulberry leaf supplementation on skatole in finishing pigs was studied. In a 35-day trial, 20 finishing pigs (barrows and gilts) were fed with a basal diet or basal diet with 6% mulberry leaves. Growth performance of the pigs (n = 10) was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests. Skatole and short-chain fatty acids were detected using HPLC and gas chromatography, respectively. Fecal microbiota were analyzed using 16S rRNA gene sequencing. The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS. The major cytochrome P450 (CYP) enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot. Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line HepG2 in an in vitro test (n = 3). In finishing pigs, mulberry leaf supplementation had no significant effect on the average daily gain, average daily feed intake, and feed conversion ratio (P > 0.05), but reduced skatole levels in feces, serum, and backfat (P < 0.05), and increased acetic acid levels in feces (P = 0.027). Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella (P < 0.05). Indole-3-acetic acid, the intermediate that is essential for skatole production, was significantly reduced in feces by mulberry leaf supplementation (P < 0.05) and was positively correlated with skatole content in feces (P = 0.004). In pigs treated with mulberry leaves, liver CYP1A1 expression was increased (P < 0.05) and was negatively correlated with skatole content in backfat (P = 0.045). The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells. These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.

Serum Phosphorus, Serum Bicarbonate, and Renal Function in Relation to Liver CYP1A2 Activity.

The liver plays an important role in normal metabolism and physiological functions such as acid-base balance; however, limited epidemiologic studies have investigated how the liver contributes toward acid-base balance using non-invasive biomarkers. We determined associations between serum biomarkers related to acid-base balance and renal function with liver CYP1A2 activity. We used data from 1381 participants of the 2009-2010 National Health and Nutrition Examination Survey (NHANES) with measurements of serum phosphorus, serum bicarbonate, caffeine intake, caffeine metabolites, and estimated glomerular filtration rate (eGFR). Liver CYP1A2 activity was estimated using urine caffeine metabolite indices, which were calculated as the ratio of one of the urine caffeine metabolites (i.e., paraxanthine and 1-methyluric acid) to caffeine intake. We analyzed associations in the whole data set and in different strata of hepatic steatosis index (HSI) based on different cut-points. We found that serum bicarbonate was positively associated with CYP1A2 activity in the whole data set when comparing persons with bicarbonate at Q4 to Q1 (β = 0.18, p = 0.10 for paraxanthine; β = 0.20, p = 0.02 for 1-methyluric acid). Furthermore, serum phosphorus was positively associated with CYP1A2 activity only in the stratum of 30 ≤ HSI < 36. Lastly, low eGFR was significantly associated with lower CYP1A2 activity measured with paraxanthine in the whole dataset and in all the strata with HSI < 42; when comparing eGFR < 60 to eGFR > 90, β estimates ranged from -0.41 to -1.38, p-values ranged from 0.0018 to 0.004. We observed an opposite trend in the highest stratum (HSI ≥ 42). Non-invasive measurements of serum bicarbonate, serum phosphorus, and eGFR have dynamic associations with CYP1A2 activity. These associations depend on the extent of liver damage and the caffeine metabolite used to assess CYP1A2 activity.

Application of fused-grid-based CYP-Template systems for genotoxic substances to understand the metabolisms

Understanding of metabolic processes is a key factor to evaluate biological effects of carcinogen and mutagens. Applicability of fused-grid Template* systems of CYP enzymes (Drug Metab Pharmacokinet 2019, 2020, 2021, and 2022) was tested for three phenomena. (1) Possible causal relationships between CYP-mediated metabolisms of β-naphthoflavone and 3-methylcholanthrene and the high inducibility of CYP enzymes were examined. Selective involvement of non-constitutive CYP1A1, but not constitutive CYP1A2, was suggested on the oxidative metabolisms of efficient inducers, β-naphthoflavone and 3-methylcholanthrene. These results supported the view of the causal link of their high inducibility with their inefficient metabolisms due to the lack of CYP1A1 in livers at early periods after the administration of both inducers. (2) Clear differences exist between human and rodent CYP1A1 enzymes on their catalyses with heterocyclic amines, dioxins and polyaromatic hydrocarbons (PAHs). Reciprocal comparison of simulation results with experimental data suggested the rodent specific site and distinct sitting-preferences of ligands on Template for human and rodent CYP1A1 enzymes. (3) Enhancement of metabolic activation and co-mutagenicity have been known as phenomena associated with Salmonella mutagenesis assay. Both the phenomena were examined on CYP-Templates in ways of simultaneous bi-molecule bindings of distinct ligands as trigger and pro-metabolized molecules. α-Naphthoflavone and norharman served consistently as trigger-molecules to support the oxidations of PAHs and arylamines sitting simultaneously as pro-metabolized molecules on Templates of CYP1A1, CYP1A2 and CYP3A4. These CYP-Template simulation systems with deciphering capabilities are promising tools to understand the mechanism basis of metabolic activations and to support confident judgements in safety assessments.

Effects of aqueous extract from Baiyedancong-Oolong tea on cytochrome P450 enzymes activities, P-gp and OATs transport abilities and transcription levels in mice.

Recent studies have been conducted on its influence on drug metabolism and its potential mechanisms, among which the most studies have been focused on CYP3A enzymes. In this study, Baiyedancong Oolong tea (BOT) was processed by freeze- and hot air-drying techniques separately to obtain the aqueous extracts of freeze-and hot-dried BOT (FBOT and HBOT, respectively). High and low doses of FBOT (1463.7 and 292.74 mg/kg/d, respectively) and HBOT (1454.46 mg/kg/d, 290.89, respectively) were administered to mice for 7 days. Aqueous extracts from BOT simultaneously improved liver CYP3A, CYP2E1, and CYP2C37 activities and weakened the transport ability of P-gp and OATs in a dose-dependent manner, thus affecting multiple links of oral drug metabolism in liver, intestinal absorption and metabolism, and renal excretion. Moreover, aqueous extracts from BOT significantly increased the mRNA expressions of liver CYP3A11 and CYP2C37 as well as intestinal CYP3A11. Decreased transcription levels of MDR1 encoding P-gp in small intestine and renal OAT1 and OAT3, which was in the same direction as the regulation of the above enzyme activities and transport capacities. Besides, the transcription level of liver CYP2E1 was weakened, which was inconsistent with its corresponding enzyme activity, suggesting that the increased CYP2E1 activity may be caused by other mechanisms. Daily consumption or high dose administration of BOT and its related products may affect drug absorption, metabolisms, and excretion.

Enhancement of the Oral Availability of Cabazitaxel Using the Cytochrome P450 3A (CYP3A) Inhibitor Ritonavir in Mice.

There is currently great interest in developing oral taxanes due to their lower costs and greater patient friendliness. We here wanted to test whether oral ritonavir, a cytochrome P450 3A (CYP3A) inhibitor, could boost the pharmacokinetics and tissue distribution of orally administered cabazitaxel (10 mg/kg) in male wild-type, Cyp3a-/-, and Cyp3aXAV (transgenic overexpression of human CYP3A4 in liver and intestine) mice. Ritonavir was initially administered at a dose of 25 mg/kg, but lower dosages of 10 and 1 mg/kg were also studied to assess the remaining amount of boosting, aiming to minimize possible side effects. Compared to the respective vehicle groups, plasma exposure of cabazitaxel (AUC0-24h) was enhanced 2.9-, 10.9-, and 13.9-fold in wild-type mice and 1.4-, 10.1-, and 34.3-fold in Cyp3aXAV mice by treatment with 1, 10, and 25 mg/kg ritonavir, respectively. Upon treatment with 1, 10, and 25 mg/kg of ritonavir, the peak plasma concentration (Cmax) was increased by 1.4-, 2.3-, and 2.8-fold in wild-type mice, while it increased by 1.7-, 4.2-, and 8.0-fold in Cyp3aXAV mice, respectively. AUC0-24h and Cmax remained unchanged in Cyp3a-/-. Biotransformation of cabazitaxel to its active metabolites still took place when coadministered with ritonavir, but this process was delayed due to the Cyp3a/CYP3A4 inhibition. These data indicate that CYP3A is the primary limiting factor in the plasma exposure to cabazitaxel and that cabazitaxel oral bioavailability could be dramatically enhanced by coadministration of an effective CYP3A inhibitor such as ritonavir. These findings could be a starting point for the setup of a clinical study, which would be needed to verify the boosting of cabazitaxel by ritonavir in humans.

A Study on Pharmacokinetic Functionalities and Safety Margins of an Optimized Simvastatin Nanoformulation.

A pharmaceutical formulation with favorable pharmacokinetic parameters is more likely to be efficacious and safe to overcome the failures of the drug resulting from lack of efficacy, poor bioavailability, and toxicity. In this view, we aimed to evaluate the pharmacokinetic functionalities and safety margin of an optimized CS-SS nanoformulation (F40) by in vitro/in vivo methods. The everted sac technique was used to evaluate the improved absorption of a simvastatin formulation. In vitro protein binding in bovine serum and mice plasma was performed. The formulation's liver and intestinal CYP3A4 activity and metabolic pathways were investigated by the qRT-PCR technique. The excretion of cholesterol and bile acids was measured to demonstrate the formulation's cholesterol depletion effect. Safety margins were determined by histopathology as well as fiber typing studies. In vitro protein binding results revealed the existence of a high percentage of free drugs (22.31 ± 3.1%, 18.20 ± 1.9%, and 16.9 ± 2.2%, respectively) compared to the standard formulation. The controlled metabolism in the liver was demonstrated from CYP3A4 activity. The formulation showed enhanced PK parameters in rabbits such as a lower Cmax, clearance, and a higher Tmax, AUC, Vd, and t1/2. qRT-PCR screening further proved the different metabolic pathways followed by simvastatin (SREBP-2) and chitosan (PPAR-γ pathway) in the formulation. The results from qRT-PCR and histopathology confirmed the toxicity level. Hence, this pharmacokinetic profile of the nanoformulation proved it has a unique synergistic hypolipidemic modality.

A simple LC-MS/MS method for the simultaneous quantification of drug metabolic enzymes

ObjectiveThe aim of this study is to develop a LC-MS/MS method for the quantitation of seven cytochrome P450 (CYP450) enzymes. MethodsA high-performance liquid chromatography-tandem mass spectrometry method was developed using multiple reaction monitoring mode with positive electrospray ionization. The method was validated with selectivity, linearity, stability, accuracy and precious. In addition, the abundance of seven CYP450 enzymes in human liver microsomes and CYP3A4 in placenta were determined using the current method. ResultsThe linear range for CYP1A2, CYP2B6 and CYP2C8 was 0.036–3.6 nM and for CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was 0.090–9.0 nM. No interference was found between the blank matrix and each specific peptides. The accuracy and precious results were in accord with the requirement of analytical methods for biological samples in Chinese Pharmacopoeia. In addition, the peptides were stable under current stability conditions. The content of CYP3A4 in placenta and the seven CYP450 enzymes in human liver microsomes were accurately quantified. ConclusionThe developed method is sensitive and specific and can be applied to the quantification of enzymes abundance in different human derived samples like placenta and liver microsomes.

Evidence for exposure biomarkers in dictamnine-induced liver injury resulting from metabolic activation in vitro and in mice.

Dictamnine (DTN), a furoquinoline alkaloid isolated from Dictamni Cortex, is responsible for the liver injury caused by Dictamni Cortex and the preparations. Discovering new biomarkers with high specificity and sensitivity for diagnosis and tracing the source of DTN-induced liver injury is urgently needed. Considering that metabolic activation of DTN has been suggested as a primary trigger initiating hepatotoxicity, the present study aimed to investigate the bio-activation process of DTN in vitro and in mice and to explore whether the adducts could be developed as exposure biomarkers. When trapping with N-acetyl-cysteine (NAC) and glutathione (GSH) in mouse liver microsomes and CYP3A4 overexpressed L02 cells, two isomers of DTN-NAC adducts were detected in both systems and one DTN-GSH adduct was found in mouse liver microsomes. As expected, one DTN-NAC adduct was also found in plasma and bile of mice with liver injury after DTN exposure. Moreover, mouse liver microsomes were used to simulate the conjugation of serum albumin with metabolically activated DTN. The sole modified peptide 25 DAHKSEVAHR34 was found, and the oxidative metabolites of DTN might bind to the side chain amino of albumin at Arg34. The above findings not only provided confirmative evidence that DTN was metabolically activated to induce liver injury but also suggested that the adducts had the potential to be developed as exposure biomarkers of DTN-induced liver injury.

Circadian oscillator NPAS2 regulates diurnal expression and activity of CYP1A2 in mouse liver

We aimed to investigate the potential role of NPAS2 in controlling diurnal expression and activity of hepatic CYP1A2 and to determine the underlying mechanisms. Regulatory effects of NPAS2 on CYP1A2 were determined using Npas2 knockout (Npas2-/-) mice as well as AML-12, Hepa1-6 and HepG2 cells. mRNA and protein levels were detected by reverse transcription-quantitative real-time PCR and western blotting, respectively. In vitro and in vivo CYP1A2 activities were respectively evaluated using the probe substrates phenacetin and theophylline. Transcriptional regulation was investigated using luciferase reporter assays and ChIP-Seq analysis. Loss of Npas2 in mice decreased CYP1A2 expression (at both mRNA and protein levels) and blunted its rhythmicity in the liver. Likewise, Npas2 ablation down-regulated the enzymatic activity of CYP1A2 (probed by metabolism of phenacetin and theophylline) and abrogated its time-dependency. Cell-based assays confirmed that NPAS2 positively regulated CYP1A2 expression. Mechanistic study indicated that NPAS2 trans-activated Cyp1a2 through its specific binding to the −416 bp E-box-like element within the gene promoter. In conclusion, NPAS2 was identified as a key transcriptional regulator of diurnal expression of hepatic CYP1A2 in mice. Our findings have implications for improved understanding of circadian metabolism and chronopharmacokinetics.

Effects of Gukang Capsules on activity and protein expression of hepatic cytochrome P450 enzymes in rats

Gukang Capsules are often used in combination with drugs to treat fractures, osteoarthritis, and osteoporosis. Cytochrome P450(CYP450) mainly exists in the liver and participates in the oxidative metabolism of a variety of endogenous and exogenous substances and serves as an important cause of drug-metabolic interactions and adverse reactions. Therefore, it is of great significance to study the effect of Gukang Capsules on the activity and expression of CYP450 for increasing its clinical rational medication and improving the safety of drug combination. In this study, the Cocktail probe method was used to detect the changes in the activities of CYP1A2, CYP3A2, CYP2C11, CYP2C19, CYP2D4, and CYP2E1 in rat liver after treatment with high-, medium-and low-dose Gukang Capsules. The rat liver microsomes were extracted by the calcium chloride method, and protein expression of the above six CYP isoform enzymes was detected by Western blot. The results showed that the low-dose Gukang Capsules could induce CYP3A2 and CYP2D4 in rats, medium-dose Gukang Capsules had no effect on them, and high-dose Gukang Capsules could inhibit them in rats. The high-dose Gukang Capsules did not affect CYP2C11 in rats, but low-and medium-dose Gukang Capsules could induce CYP2C11 in rats. Gukang Capsules could inhibit CYP2C19 in rats and induce CYP1A2 in a dose-independent manner, but did not affect CYP2E1. If Gukang Capsules were co-administered with CYP1A2, CYP2C19, CYP3A2, CYP2C11, and CYP2D4 substrates, the dose should be adjusted to avoid drug interactions.

Distinct effects of form selective cytochrome P450 inhibitors on cytochrome P450-mediated monooxygenase and hydrogen peroxide generating NADPH oxidase

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as “NADPH oxidase” activity. H2O2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched β-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 μM) for CYP1A2 and 4-methylpyrazole (32 μM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 μM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.

The Chinese herb Styrax triggers pharmacokinetic herb-drug interactions via inhibiting intestinal CYP3A.

Human cytochrome P450 3A4 (hCYP3A4) is a predominant enzyme to trigger clinically relevant drug/herb-drug interactions (DDIs or HDIs). Although a number of herbal medicines have been found with strong anti-hCYP3A4 effects in vitro, the in vivo modulatory effects of herbal medicines on hCYP3A4 and their potential risks to trigger HDIs are rarely investigated. Herein, we demonstrate a case study to efficiently find the herbal medicine(s) with potent hCYP3A4 inhibition in vitro and to accurately assess the potential HDIs risk in vivo. Following screening over 100 herbal medicines, the Chinese herb Styrax was found with the most potent hCYP3A4 inhibition in HLMs. In vitro assays demonstrated that Styrax could potently inhibit mammalian CYP3A in liver and intestinal microsomes from both humans and rats. In vivo pharmacokinetic assays showed that Styrax (i.g., 100 mg/kg) significantly elevated the plasma exposure of two CYP3A-substrate drugs (midazolam and felodipine) when midazolam or felodipine was administered orally. By contrast, the plasma exposure of either midazolam or felodipine was hardly affected by Styrax (i.g.) when the victim drug was administered intravenously. Further investigations demonstrated that seven pentacyclic triterpenoid acids (PTAs) in Styrax were key substances responsible for CYP3A inhibition, while these PTAs could be exposed to intestinal tract at relatively high exposure levels but their exposure levels in rat plasma and liver were extremely low. These findings well explained why Styrax (i.g.) could elevate the plasma exposure of victim drugs only when these agents were orally administrated. Collectively, our findings demonstrate that Styrax can modulate the pharmacokinetic behavior of CYP3A-substrate drugs via inhibiting intestinal CYP3A, which is very helpful for the clinical pharmacologists to better assess the HDIs triggered by Styrax or Styrax-related herbal products.

ABCB1 restricts brain accumulation of the novel RORγ agonist cintirorgon, while OATP1A/1B and CYP3A limit its oral availability

Cintirorgon (LYC-55716), a first-in-class, small-molecule, oral selective RORγ agonist, has been developed as a new immuno-oncology drug for solid tumors. We here studied the functions of the ABCB1 and ABCG2 multidrug efflux transporters, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A enzyme complex in cintirorgon pharmacokinetics using genetically modified mouse models. Cintirorgon was modestly transported by human ABCB1 and mouse Abcg2 in vitro. Upon oral administration at 40 mg/kg, net cintirorgon brain penetration was enhanced in Abcb1a/1b−/− (2.1-fold) and Abcb1a/1b;Abcg2−/− (2.7-fold) relative to wild-type mice. Deficiency of Oatp1a/1b led to a substantial (2.4-fold) increase in cintirorgon systemic exposure, with a corresponding (2.3-fold) decrease in hepatic distribution. However, these changes were not rescued in mice overexpressing human OATP1B1 or human OATP1B3 in liver, although this did partially reverse the altered cintirorgon glucuronide pharmacokinetics in Oatp1a/1b−/− mice. In Cyp3a−/− mice, the cintirorgon plasma AUC0-8h was 1.4-fold increased, and then decreased by 1.5-fold upon overexpression of transgenic human CYP3A4 in intestine and liver. Cintirorgon brain accumulation was thus markedly restricted by ABCB1. Mouse Oatp1a/1b mediated cintirorgon uptake into the liver, thus limiting its plasma exposure. Moreover, oral availability of cintirorgon was limited by CYP3A. These insights could help optimizing cintirorgon’s clinical application.

Alterations in circadian rhythms aggravate Acetaminophen-induced liver injury in mice by influencing Acetaminophen metabolization and increasing intestinal permeability

ABSTRACT Acetaminophen (APAP) is the most common antipyretic and analgesic drug causing drug-induced liver injury (DILI). Alterations in circadian rhythms can adversely affect liver health, especially metabolic and detoxification functions. However, the effect of circadian rhythm alterations induced by environmental factors on APAP-induced liver injury and the underlying mechanisms are not well known. In this study, a mouse model of circadian rhythm alterations was established by light/dark cycle shift and then treated with excessive APAP. The liver injury indexes, APAP-related metabolic enzymes, and intestinal permeability in mice were evaluated by biochemical analysis, quantitative real-time PCR, enzyme-linked immunosorbent assays, and histopathology. Results showed that circadian rhythm alterations resulted in increased reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased liver superoxide dismutase (SOD), glutathione, and CYP1A2 and CYP3A11 mRNA expression, and increased serum diamine oxidase, lipopolysaccharide, and D-lactate in the mice. Compared with control mice, APAP induced higher serum alanine aminotransferase and aspartate aminotransferase, liver interleukin-1β and tumor necrosis factor-α mRNA, ROS and MDA, lower SOD, glutathione, and UDP-glucuronosyltransferases /sulfotransferases mRNA and more severe liver necrosis and intestinal damage in mice with alterations in circadian rhythms. In conclusion, circadian rhythm alterations by light/dark cycle shift resulted in increased oxidative stress and intestinal permeability in the mice and exacerbated APAP-induced liver injury by influencing APAP metabolization and increasing intestinal permeability.

Pharmacokinetic Herb-Drug Interactions of Xiang-Sha-Liu-Jun-Zi-Tang and Paclitaxel in Male Sprague Dawley Rats and Its Influence on Enzyme Kinetics in Human Liver Microsomes.

Paclitaxel is a prescribed anticancer drug used to treat various cancers. It is a substrate of cytochrome P-450 (CYP-450) enzymes. Despite its efficacy, paclitaxel has severe side effects. Herbal medicines are commonly used to treat the side effects of chemotherapy. They can be administered before, during, and after chemotherapy. Xiang-Sha-Liu-Jun-Zi Tang (XSLJZT) is a herbal formula commonly used in breast cancer patients. The main purpose of this study was to assess the pharmacokinetic (PK) influence of XSLJZT on paclitaxel PK parameters, determine its effect on CYP-450 enzyme expression, and evaluate its effect on enzyme activity. Sprague Dawley rats were classified into pretreatment and co-treatment groups, where XSLJZT was pre-administered for 3, 5, and 7 days and co-administered 2 h before paclitaxel administration. The rat liver tissues and Hep-G2 cells were used to determine the effects of XSLJZT on CYP3A1/2 and CYP3A4 enzymes respectively. Western blot analysis was used to detect changes in the CYP3A1/2 and CYP3A4 enzymes expression. The influence of XSLJZT on enzyme activity was evaluated using human liver microsomes, and a liquid chromatography-tandem mass spectrometric system was developed to monitor paclitaxel levels in rat plasma. Results demonstrated that XSLJZT increased the area under the concentration versus time curve (AUC) for paclitaxel in pretreatment groups by 2-, 3-, and 4-fold after 3, 5, and 7 days, respectively. In contrast, no significant change in the AUC was observed in the co-treatment group. However, the half-life was prolonged in all groups from 17.11 min to a maximum of 37.56 min. XSLJZT inhibited CYP3A1/2 expression in the rat liver tissues and CYP3A4 enzymes in Hep-G2 cells in a time-dependent manner, with the highest inhibition observed after 7 days of pretreatment in rat liver tissues. In the enzyme kinetics study, XSLJZT inhibited enzyme activity in a competitive dose-dependent manner. In conclusion, there is a potential interaction between XSLJZT and paclitaxel at different co-treatment and pretreatment time points.

ABCB1 limits brain exposure of the KRASG12C inhibitor sotorasib, whereas ABCB1, CYP3A, and possibly OATP1a/1b restrict its oral availability

Sotorasib (Lumakras™) is the first FDA-approved KRASG12C inhibitor for treatment of patients with non-small cell lung cancer (NSCLC) carrying this mutation. Using genetically modified mouse models, we studied the influence of the efflux transporters ABCB1 and ABCG2, the OATP1a/1b uptake transporters, and the CYP3A drug-metabolizing enzyme complex on the plasma pharmacokinetics and tissue distribution of oral sotorasib. In vitro, sotorasib was a potent substrate for human ABCB1 and a modest substrate for mouse Abcg2, but not for human ABCG2. In vivo, the brain-to-plasma ratio of sotorasib (40 mg/kg) was highly increased in Abcb1a/1b-/- (5.9-fold) and Abcb1a/1b;Abcg2-/- (7.6-fold) compared to wild-type mice, but not in single Abcg2-/- mice. Upon coadministering elacridar, an ABCB1/ABCG2 inhibitor, sotorasib brain accumulation increased 7.5-fold, approaching the levels observed in Abcb1a/1b-deficient mice. No acute CNS toxicity emerged upon boosting of the sotorasib exposure. In Oatp1a/1b-deficient mice, we observed a 2-fold reduction in liver disposition compared to wild-type mice, although these uptake transporters had no noticeable impact on sotorasib plasma exposure. However, plasma exposure was limited by mouse Cyp3a and human CYP3A4, as the AUC0–4 h in Cyp3a-/- mice was increased by 2.5-fold compared to wild-type mice, and subsequently strongly decreased (by 3.9-fold) in Cyp3aXAV mice transgenically overexpressing human CYP3A4 in liver and intestine. Collectively, the oral availability of sotorasib was markedly limited by CYP3A and possibly also by ABCB1 and OATP1a/b, whereas its brain accumulation was strongly restricted by ABCB1. The obtained results may help to further optimize the safety and efficacy of sotorasib in clinical use.