CYP3A4 Activity Research Articles

Construction of humanized CYP1A2 rats using CRISPR/Cas9 to promote drug metabolism and pharmacokinetic research.

Cytochrome P450 family 1 subfamily A member 2 (CYP1A2), performs an indispensable role in metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not yet been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/Cas9 technology. Results showed that human CYP1A2 was successfully expressed in humanized rat liver and there were no statistically significant differences of physiological symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics. Significance Statement Human CYP1A2 takes active part both in the biotransformation of exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia, hypertension as well as various malignant tumors. This study successfully constructed humanized CYP1A2 rat model by CRISPR/Cas9 technology, providing a powerful model for promoting drug development and safety evaluation, as well as further exploring the role of CYP1A2 in human diseases.

The Pharmacokinetics of the Nonsteroidal Mineralocorticoid Receptor Antagonist Finerenone.

Finerenone, a selective and nonsteroidal antagonist of the mineralocorticoid receptor, has received regulatory approval with the indication of cardiorenal protection in patients with chronic kidney disease associated with type 2 diabetes. It is rapidly and completely absorbed and undergoes first-pass metabolism in the gut wall and liver resulting in a bioavailability of 43.5%. Finerenone can be taken with or without food. The pharmacokinetics of finerenone are linear and its half-life is 2 to 3 h in the dose range of up to 20 mg. Cytochrome P450 (CYP) 3A4 (90%) and CYP2C8 (10%) are involved in the extensive biotransformation of finerenone to pharmacologically inactive metabolites, which are excreted via both renal (80%) and biliary (20%) routes. Moderate or severe renal impairment, or moderate hepatic impairment result in area-under-the-curve increases of finerenone (< 40%), which do not require a dose adjustment per se, as the starting dose is based on estimated glomerular filtration rate (eGFR) and titrated according to serum potassium levels and eGFR decline. No relevant effects of age, sex, body size or ethnicity on systemic finerenone exposure were identified. Modulators of CYP3A4 activity were found to affect finerenone exposure, consistent with its classification as a sensitive CYP3A4 substrate. Serum potassium should be monitored during drug initiation or dosage adjustment of either a moderate or weak CYP3A4 inhibitor or finerenone, and the dose of finerenone should be adjusted as appropriate. Its use with strong inhibitors is contraindicated and strong or moderate inducers of CYP3A4 should be avoided. Finerenone has no potential to affect relevant CYP enzymes and drug transporters.

Pharmacokinetic interaction of saxagliptin with andrographolide and their hypoglycemic effect in type 2 diabetes rats

Controlling blood glucose is the primary therapeutic strategy for T2DM. Both saxagliptin and andrographolide possess hypoglycemic effects, which makes the combination easy. This study aimed to evaluate the co-administration of saxagliptin and andrographolide in rats and revealed their combined impact on type 2 diabetes (T2DM). T2DM rat models were established by high-fat diet and the injection of nicotinamide and streptozotocin. The blood glucose and insulin resistance index were monitored after modeling. The pharmacokinetics of saxagliptin was assessed by orally administrating 10 mg/kg saxagliptin. The co-administration was performed with the pre-treatment of 30 or 100 mg/kg andrographolide. In vitro, the metabolic stability of saxagliptin was assessed in rat liver microsomes. The co-administration of saxagliptin with andrographolide induced significant changes in the pharmacokinetics of saxagliptin, behaving as the increasing AUC(0-t), Cmax, t1/2, and the decreasing clearance rate. The effect of andrographolide was enhanced with the growing concentration. Combination with andrographolide enhanced the hypoglycemic effect and alleviated insulin resistance of saxagliptin in T2DM rats. Co-administration of saxagliptin with 100 mg/kg andrographolide induced hypoglycemia. In vitro, andrographolide significantly improved the metabolic stability of saxagliptin and showed a significant inhibitory effect on the activity of CYP3A4. Combining saxagliptin with andrographolide could increase the systemic exposure of saxagliptin via inhibiting CYP3A4 and improve the hypoglycemic effect, but the high concentration of andrographolide is the risk of inducing hypoglycemia.

Inhibitory Mechanisms of Lekethromycin in Dog Liver Cytochrome P450 Enzymes Based on UPLC-MS/MS Cocktail Method.

Lekethromycin (LKMS) is a synthetic macrolide compound derivative intended for use as a veterinary medicine. Since there have been no in vitro studies evaluating its potential for drug-drug interactions related to cytochrome P450 (CYP450) enzymes, the effect of the inhibitory mechanisms of LKMS on CYP450 enzymes is still unclear. Thus, this study aimed to evaluate the inhibitory effects of LKMS on dog CYP450 enzymes. A cocktail approach using ultra-performance liquid chromatography-tandem mass spectrometry was conducted to investigate the inhibitory effect of LKMS on canine CYP450 enzymes. Typical probe substrates of phenacetin, coumarin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and testosterone were used for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4, respectively. This study showed that LKMS might not be a time-dependent inhibitor. LKMS inhibited CYP2A6, CYP2B6, and CYP2D6 via mixed inhibition. LKMS exhibited mixed-type inhibition against the activity of CYP2A6 with an inhibition constant (Ki) value of 135.6 μΜ. LKMS inhibited CYP2B6 in a mixed way, with Ki values of 59.44 μM. A phenotyping study based on an inhibition assay indicated that CYP2D6 contributes to the biotransformation of LKMS. A mixed inhibition of CYP2D6 with Ki values of 64.87 μM was also observed. Given that this study was performed in vitro, further in vivo studies should be conducted to identify the interaction between LKMS and canine CYP450 enzymes to provide data support for the clinical application of LKMS and the avoidance of adverse interactions between other drugs.

St. John's wort formulations induce rat CYP3A23-3A1 independent of their hyperforin content.

The pregnane X receptor (PXR) is a ligand activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant® was used as it contains a high hyperforin content and Rebalance® because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant® and Rebalance® transactivated the CYP3A-reporter 3.8 fold and 2.8 fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared to control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant® we observed 1.8-times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount and a 1.6-fold increase in activity compared to control. For Rebalance® we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared to control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. Significance Statement Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the PXR-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro Importantly, CYP3A induction mimics findings in humans but our results suggest that another so far unknown constituent of SJW is responsible for the expression and function modifying effects in rat liver.

The effect of brain serotonin deficit (TPH2-KO) on the expression and activity of liver cytochrome P450 enzymes in aging male Dark Agouti rats

BackgroundLiver cytochrome P450 (CYP) greatly contributes to the metabolism of endogenous substances and drugs. Recent studies have demonstrated that CYP expression in the liver is controlled by the central nervous system via hormonal pathways. In particular, the expression of hepatic CYPs is negatively regulated by the brain serotoninergic system. The present study aimed to investigate changes in the function of the main liver drug-metabolizing CYP enzymes as a result of serotonin depletion in the brain of aging rats, caused by knockout of brain tryptophan hydroxylase gene (TPH2-KO).MethodsThe hepatic CYP mRNA (qRT-PCR), protein level (Western blotting) and activity (HPLC), and serum hormone levels (ELISA) were measured in Dark Agouti wild-type (WT) male rats (mature 3.5-month-old and senescent 21-month-old) and in TPH2-KO senescent animals.ResultsThe expression/activity of the studied CYPs decreased with age in the liver of wild-type rats. The deprivation of serotonin in the brain of aging males decreased the mRNA level of most of the studied CYPs (CYP1A/2A/2B/3A), and lowered the protein level of CYP2C11 and CYP3A. In contrast, the activities of CYP2C11, CYP3A and CYP2C6 were increased. The expression of cytochrome b5 decreased in aging rats, but increased in TPH2-deficient senescent animals. The serum concentration of growth hormone declined in the aged and further dropped down in TPH2-deficient senescent rats.ConclusionsRat liver cytochrome P450 functions deteriorate with age, which may impair drug metabolism. The TPH2 knockout, which deprives brain serotonin, affects cytochrome P450 expression and activity differently in mature and senescent male rats.

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations.

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model's functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12's robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10-14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development.

Cytochrome P450 activity in rheumatoid arthritis patients during continuous IL-6 receptor antagonist therapy

BackgroundInflammation suppresses cytochrome P450 (CYP) enzyme activity, and single-dose interleukin 6 receptor antagonists (anti-IL-6R) reverse this effect. Here, we assess the impact of continuous anti-IL-6R therapy in patients with rheumatoid arthritis.MethodsIn a clinical pharmacokinetic trial, the Basel cocktail was administered before and after 3 and 12 weeks of anti-IL-6R therapy to assess CYP enzyme activity (registered in the ClinicalTrials.gov database (identifier NCT04842981) on April 13th, 2021). In a retrospective study, the 4β-hydroxycholesterol/cholesterol ratio was measured as a biomarker for CYP3A4 activity before and after 3 and 6 months of anti-IL-6R therapy. The control group was patients initiating a tumor necrosis factor alfa (TNF-α) inhibitor.ResultsIn the clinical pharmacokinetic trial (n = 3), midazolam metabolic ratio (CYP3A4) was inconclusive due to the limited sample size. Midazolam AUC and Cmax indicate a weak impact on CYP3A4 activity after 3 weeks of anti-IL-6R therapy compared to baseline (AUC geometric mean ratio (GMR): 0.80, 95% CI: 0.64–0.99 and Cmax GMR: 0.58, 95% CI: 0.37–0.91), which returns to baseline levels after 12 weeks of therapy (AUC GMR 1.02, 95% CI: 0.72–1.46 and Cmax GMR 1.03, 95% CI 0.72–1.47). No effect on the 4β-hydroxycholesterol/cholesterol ratio was observed in the retrospective study.ConclusionBased on sparse data from three patients, continuous anti-IL-6R therapy seems to cause an acute but transient increase in CYP3A4 activity in rheumatoid arthritis patients, which may be due to a normalization of the inflammation-suppressed CYP activity. Further studies are warranted to understand the mechanism behind this putative transient effect.Trial registration Registered in the ClinicalTrials.gov database (identifier NCT04842981) on April 13th, 2021.

CYP2A6 associates with respiratory disease risk and younger age of diagnosis: a phenome-wide association Mendelian Randomization study.

CYP2A6, a genetically variable enzyme, inactivates nicotine, activates carcinogens, and metabolizes many pharmaceuticals. Variation in CYP2A6 influences smoking behaviors and tobacco-related disease risk. This phenome-wide association study examined associations between a reconstructed version of our weighted genetic risk score (wGRS) for CYP2A6 activity with diseases in the UK Biobank (N = 395 887). Causal effects of phenotypic CYP2A6 activity (measured as the nicotine metabolite ratio: 3'-hydroxycotinine/cotinine) on the phenome-wide significant (PWS) signals were then estimated in two-sample Mendelian Randomization using the wGRS as the instrument. Time-to-diagnosis age was compared between faster versus slower CYP2A6 metabolizers for the PWS signals in survival analyses. In the total sample, six PWS signals were identified: two lung cancers and four obstructive respiratory diseases PheCodes, where faster CYP2A6 activity was associated with greater disease risk (Ps < 1×10-6). A significant CYP2A6-by-smoking status interaction was found (Psinteraction < 0.05); in current smokers, the same six PWS signals were found as identified in the total group, whereas no PWS signals were found in former or never smokers. In the total sample and current smokers, CYP2A6 activity causal estimates on the six PWS signals were significant in Mendelian Randomization (Ps < 5×10-5). Additionally, faster CYP2A6 metabolizer status was associated with younger age of disease diagnosis for the six PWS signals (Ps < 5×10-4, in current smokers). These findings support a role for faster CYP2A6 activity as a causal risk factor for lung cancers and obstructive respiratory diseases among current smokers, and a younger onset of these diseases. This research utilized the UK Biobank Resource.

Glycyrrhizin protects against diosbulbin B-induced hepatotoxicity by inhibiting the metabolic activation of diosbulbin B.

Diosbulbin B (DIOB), isolated from herbal medicine Dioscorea bulbifera L. (DB), could induce severe liver injury, and its toxicology was closely associated with CYP3A4-mediated metabolic oxidation of furan moiety to the corresponding cis-enedial reactive metabolite. Glycyrrhizin (GL), the major bioactive ingredient in licorice, can inhibit the activity of CYP3A4. Thus, GL may ameliorate hepatotoxicity of DIOB when GL and DIOB are co-administrated. The study aimed to investigate the protective effect of GL on DIOB-induced hepatotoxicity and the underlying mechanism. Biochemical and histopathological analysis demonstrated that GL alleviated DIOB-induced hepatotoxicity in a dose-dependent manner. In vitro study with mouse liver microsomes (MLMs) demonstrated that GL reduced the formation of metabolic activation-derived pyrrole-glutathione (GSH) conjugates from DIOB. Toxicokinetic studies showed that the pretreatment with GL caused the increase of AUCs and Cmax of DIOB in blood of mice, resulting in accelerating the accumulation of DIOB in the circulation. In addition, the pretreatment with GL alleviated DIOB-induced hepatic GSH depletion. In summary, GL ameliorated DIOB-induced hepatotoxicity, possibly related to the inhibition of the metabolic activation of DIOB. Thus, development of a standardized combination of DIOB with GL may protect patients from DIOB-induced liver injury.

Biochemical and genetic biomarkers associated with nicotine dependence in Mexican smokers.

Cigarette smoking remains an important health concern and is still a leading cause of preventable mortality. Nicotine is the substance responsible for sustained tobacco use and dependence. Identification of biomarkers underlying nicotine dependence behavior is important to identify people at risk for this dependence. In the present study, we identified biochemical and genetic biomarkers of nicotine dependence detected by the Fagerström Test for Nicotine Dependence (FTDN) in Mexican smokers. The nicotine metabolites nicotine-N'-oxide, trans-3'-hydroxycotinine-glucuronide (3HC-O-Gluc), and nicotine-N-Gluc (Gluc) were useful to differentiate nicotine-dependent from non-dependent subjects (p < .0001) with an area under the curve (AUC) of 0.7818. Genetic variants in CYP2A6, FMO3, and UGT2B7 (rs2431413, rs28363545, and rs7439326, respectively) were associated with nicotine dependence (p = .03, p = .01, p = .01, respectively). Variations in the enzymatic activity of CYP2A6 were associated with altered nicotine-N'-oxide and 3HC-O-Gluc levels. Decreased urinary levels of 3HC-O-Gluc and increased nicotine-N'-oxide were associated with a decrease in the functional activity of CYP2A6. A strong positive correlation was observed between the ratio of urinary 3HC/cotinine, a measure of CYP2A6 activity, and the levels of 3HC-O-Gluc (p < .0001, r = .6835), while a strong negative correlation was observed with nicotine-N'-oxide (p < .0001, r = .6522) in nicotine-dependent subjects. No correlations were observed in non-nicotine-dependent subjects. These data suggest that particular urinary nicotine metabolites and genetic variants involved in nicotine metabolism are useful to identify subjects with nicotine dependence in the Mexican population.

The effect of genetic variation in estrogen transportation and metabolism on the severity of menopause symptoms: A study from the RIGHT 10K cohort.

The severity of menopause-related symptoms varies considerably among women. The determinants of this variation are incompletely understood. The aim of this study was to assess the association between genetic variation in estrogen metabolism and transport pathways and the severity of menopause symptoms. This was a cross-sectional study of 60 peri- and postmenopausal women in the Mayo Clinic RIGHT study (which involved sequencing of genes involved in drug metabolism and transport), who had also been evaluated in the Women's Health Clinic at Mayo Clinic in Rochester, MN. All participants completed the Menopause Rating Scale (MRS) for assessment of menopause symptoms, including hot flashes. The association between severity of menopause symptoms and the variation in genes encoding 8 enzymes and transporters involved in estrogen metabolism was evaluated. Lower CYP3A4 activity and higher COMT activity were associated with lower severity of somatic menopause symptoms (p=0.04 and 0.06, respectively). These associations did not persist after adjustment for hormone therapy use. No differences in MRS scores or hot flash severity were noted among other genetic variant groups. Age at natural menopause was not affected by variations in the genes studied. The current study did not show an association between genetic variation in estrogen metabolism and transport pathways and the severity of menopause symptoms. Further studies with larger sample sizes may be required to understand this potentially complex association.

Activation of CYP3A by ABC phenomenon potentiates the hepatocellular carcinoma-targeting therapeutic effects of PEGylated anticancer prodrug liposomes.

Reduced enzyme activity in hepatocellular carcinoma (HCC) and poor targeting limit the application of enzyme-activating prodrugs, which is also detrimental to the effective treatment of HCC. Here, we investigated whether accelerated blood clearance (ABC) phenomenon occurs in HCC models following repeated injections of PEGylated liposomes (PEG-L), thus inducing prodrugs accumulation and activation in the liver and exerting highly effective and low-toxicity therapeutic effects on HCC. Firstly, PEGylated liposomal cyclophosphamide (PEG-CP-L) was prepared by solvent injection and characterized. Importantly, pre-injection of PEG-L induced ABC phenomenon and activation of CYP3A in both HCC rats and HCC mice by investigating the effects of repeated injections of PEG-L on pharmacokinetics and tissue distribution. Next, the efficacy and toxicity of repeated injections of PEG-L in HCC mice were examined, and our data indicate that repeated injections are administered in a manner that significantly enhances the antitumor effect compared to controls with little or no toxicity to other organs. To further reveal the pharmacokinetic mechanism of PEG-L repeated administration for the treatment of HCC, the protein expression of hepatic CYP3A and the concentration of CP in the liver and spleen of HCC mice by inhibiting CYP3A were analyzed. These results revealed that inducing CYP3A to accelerate the rapid conversion of prodrugs that accumulate significantly in the liver is a key mechanism for the treatment of HCC with repeated injections of PEG-L. Collectively, this work taps into the application potential of the ABC phenomenon and provides new insights into the clinical application of PEGylated nanoformulations. Significance Statement This study revealed that repeated injections of PEG-L could induce the ABC phenomenon characterized by hepatic accumulation and CYP3A activation based on HCC rats and HCC mice. Furthermore, it was verified that induction of the ABC phenomenon dependent on hepatic accumulation and CYP3A activation could enhance the anti-hepatocellular carcinoma effects of PEGylated anticancer prodrugs in HCC mice. This elucidated the relevant pharmacokinetic mechanisms and unearths new clues for solving the clinical application of PEGylated nanoparticles.

Limonin mitigates cisplatin-induced acute kidney injury through metabolic reprogramming

BackgroundAcute kidney injury (AKI) is a known complication of cisplatin administration; currently, there are no effective ways to prevent it. Therefore, it largely limited the use of cisplatin in chemotherapy in the clinic. In this study, we reported that Limonin, a triterpenoid compound extracted from citrus, alleviated cisplatin-induced AKI through metabolic reprogramming in the diseased kidneys. MethodsCisplatin was employed to induce AKI in mice. Three groups were set up: Sham, cisplatin + vehicle, and cisplatin + Limonin. Using UHPLC-TOF/MS, we conducted metabolomics to profile the kidneys' endogenous metabolites and metabolic pathways. A network pharmacological method was performed to identify the targets of Limonin on AKI. The human proximal tubular epithelial cell line (HK-2) was applied for in vitro studies. ResultsLimonin preserved serum creatinine and blood urea nitrogen levels after cisplatin-induced AKI. Employing metabolomics, we identified 33 endogenous differentially expressed metabolites and 7 significantly disturbed metabolic pathways in the diseased kidneys within three groups. After AKI, Limonin significantly reduced linoleic acid and its downstream product, arachidonic acid, thus exerting a protective effect on the kidney. The network pharmacological method identified CYP3A4 as a key target of Limonin in treating AKI, while CYP3A4 also serve as a mediator of arachidonic acid metabolism. In vitro, Limonin markedly reduced the level of arachidonic acid and HK-2 cell apoptosis triggered by cisplatin, mainly related to the targeted inhibition of CYP3A4-mediated arachidonic acid metabolism. ConclusionLimonin ameliorates cisplatin-induced AKI by inhibiting CYP3A4 activity to regulate arachidonic acid metabolism, ultimately preserving kidney function.

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.

Unraveling the Effects of Acute Inflammation on Pharmacokinetics: A Model-Based Analysis Focusing on Renal Glomerular Filtration Rate and Cytochrome P450 3A4-Mediated Metabolism.

A physiologically based model incorporating renal and hepatic drug clearance was implemented. Functions correlating IL-6 levels with GFR and in vitro CYP3A4 activity were derived and incorporated into the modeling framework. We then simulated treatment scenarios for hypothetical drugs by varying the IL-6 levels, the contribution of renal and hepatic drug clearance, and protein binding. The relative change in observed area under the concentration-timecurve (AUC) was computed for these scenarios. Inflammation showed opposite effects on drug exposure for drugs eliminated via the liver and kidney, with the effect of inflammation being inversely proportional to the extraction ratio (ER). For renally cleared drugs, the relative decrease in AUC was close to 30% during severe inflammation. For CYP3A4 substrates, the relative increase in AUC could exceed 50% for low-ER drugs. Finally, the impact of inflammation-induced changes in drug clearance is smaller for drugs with a larger unbound fraction. This analysis demonstrates differences in the impact of inflammation on drug clearance for different drug types. The effects of inflammation status on pharmacokinetics may explain the inter-individual variability in pharmacokinetics in critically ill patients. The proposed model-based analysis may be used to further evaluate the effect of inflammation, i.e., by incorporating the effect of inflammation on other drug-metabolizing enzymes or physiological processes.

Quercetin inhibits the metabolism of arachidonic acid by inhibiting the activity of CYP3A4, thereby inhibiting the progression of breast cancer

BackgroundRecent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function.MethodsBioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo.ResultsQuercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice.ConclusionQuercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.

Will ChatGPT3 Substitute for us as Clozapine Experts?

ChatGPT3 is a new artificial intelligence program released on February 13, 2023. The authors tested ChatGPT3 on February 18, 2023, and repeated the test a week later. They used their expertise on the effects of ethnic ancestry in the stratification of clozapine dosing and the new idea that they published in March 2022 that African-Americans need higher clozapine doses because they have higher clozapine clearance. In the first interaction on February 18, ChatGPT3 provided reasonable and very up-to-date information, which included a comment that patients of African ancestry have higher clozapine metabolism. The other 4 interactions became progressively more concerning as we asked ChatGPT3 to provide references to justify the latter statement. ChatGPT3 provided non-existent "references" using articles from real journals, with real authors, false PubMed identifiers, and false titles. Moreover, ChatGPT3 said that the first author wrote in 2003 that African-Americans had higher CYP1A2 activity when that did not happen until 2022. One week later, the second author repeated the same set of questions. This time ChatGPT3 described the opposite, that African-Americans have "lower" CYP1A2 activity and "slower" metabolism. ChatGPT3 provided another set of articles to justify the information; some were real but did not comment on clozapine metabolism in African-Americans while others did not exist. ChatGPT3 provided a mixture of truth, twisted reality, and non-existent "facts." Within one week it defended opposite positions regarding a clinically relevant issue such as using higher or lower clozapine doses in African-Americans.

Guía internacional para una dosificación más segura de la clozapina en adultos mediante el uso de 6 titulaciones personalizadas de dosis basados en la etnicidad, la proteína C reactiva y los niveles de clozapina

This is the Spanish translation of an international guideline which proposes improving clozapine package inserts worldwide by using ancestry-based: 1) dosing and 2) titration. Adverse drug reaction (ADR) databases suggest clozapine: 1) is the third most toxic drug in the United States (US), and 2) produces worldwide pneumonia mortality four times greater than that of agranulocytosis or myocarditis. For trough steady-state clozapine serum concentrations, the therapeutic reference range is narrow, from 350 to 600 ng/mL with the potential for toxicity and ADRs as concentrations increase. Clozapine is mainly metabolized by CYP1A2 (female non-smokers require the lowest dose and male smokers the highest dose). Poor metabolizer (PM) status through phenotypic conversion is associated with co-prescription of inhibitors (including oral contraceptives and valproate), obesity or inflammation with C-reactive protein (CRP) elevations. People with ancestry from Asia (Pakistan to Japan) or the Americas’ original inhabitants have lower CYP1A2 activity and require lower clozapine doses to reach concentrations of 350 ng/ml. Daily doses of 300-600 mg/day are recommended in the US. Slow personalized titration may prevent early ADRs (including syncope, myocarditis and pneumonia). The core of this guideline consists of six personalized titration schedules for inpatients: 1) Asian/Amerindian ancestry with lower metabolism (in cases of obesity or valproate) needing minimum therapeutic dosages of 75-150 mg/day, 2) Asian/Amerindian ancestry with average metabolism needing 175-300 mg/day, 3) European/Western Asian ancestry with lower metabolism (in cases of obesity or valproate) needing 100-200 mg/day, 4) European/Western Asian ancestry with average metabolism needing 250-400 mg/day, 5) in the US of non-Asian/Amerindian ancestry with lower clozapine metabolism (in cases of obesity or valproate) needing 150-300 mg/day, and 6) in the US of non-Asian/Amerindian ancestry with average clozapine metabolism needing 300-600 mg/day. Baseline and weekly CRP monitoring for at least 4 weeks is required to identify any inflammation, including inflammation secondary to clozapine rapid titration.

GTP1 metabolic stability assessment: A study of the tau PET tracer [18F]GTP1

Tau PET imaging using the tau specific PET tracer [18F]GTP1 has been and is part of therapeutic trials in Alzheimer's disease to monitor the accumulation of tau aggregates in the brain. Herein, we examined the metabolic processes of GTP1 and assessed the influence of smoking on its metabolism through in vitro assays. The tracer metabolic profile was assessed by incubating GTP1 with human liver microsomes (HLM) and human hepatocytes. Since smoking strongly stimulates the CYP1A2 enzyme activity, we incubated GTP1 with recombinant CYP1A2 to evaluate the role of the enzyme in tracer metabolism. It was found that GTP1 could form up to eleven oxidative metabolites with higher polarity than the parent. Only a small amount (2.6 % at 60 min) of a defluorinated metabolite was detected in HLM and human hepatocytes incubations highlighting the stability of GTP1 with respect to enzymatic defluorination. Moreover, the major GTP1 metabolites were not the product of CYP1A2 activity suggesting that smoking may not impact in vivo tracer metabolism and subsequently GTP1 brain kinetics.