CYP2C19 Activity Research Articles

Effects of multi-walled carbon nanotubes in soil on earthworm growth and reproduction, enzymatic activities, and metabolomics

Increased production and environmental release of multi-walled carbon nanotubes (MWCNTs) increase soil exposure and potential risk to earthworms. However, MWCNT toxicity to earthworms remains unclear, with some studies identifying negative effects and others negligible effects. In this study, to determine whether exposure to MWCNTs negatively affects earthworms and to elucidate possible mechanisms of toxicity, earthworms were exposed to sublethal soil concentrations of MWCNTs (10, 50, and 100 mg/kg) for 28 days. Earthworm growth and reproduction, activities of cytochrome P450 (CYP) isoforms (1A2, 2C9, and 3A4) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione-s-transferase (GST)), and metabolomics were determined. Effects of MWCNTs on earthworms depended on exposure concentration. Exposure to MWCNTs did not significantly affect growth and reproduction of individual earthworms. Exposure to 50 mg/kg MWCNTs significantly increased activities of CYP2C9, CYP3A4, SOD, CAT, and GST but clearly reduced levels of L-aspartate, L-asparagine, and glutamine. With exposure to 100 mg/kg MWCNTs, toxic effects on earthworms were observed, with significant inhibition in activities of CYP isoenzymes and SOD, significant reductions in L-aspartate, L-asparagine, glutamine, and tryptophan, and simultaneous accumulations of citrate, isocitrate, fumarate, 2-oxoglutarate, pyruvate, D-galactose, carbamoyl phosphate, formyl anthranilate, hypoxanthine, and xanthine. Results suggest that toxicity of MWCNTs to earthworms is associated with reduced detoxification capacity, excessive oxidative stress, and disturbance of multiple metabolic pathways, including amino acids metabolism, the tricarboxylic acid cycle, pyruvate metabolism, D-galactose metabolism, and purine metabolism. The study provides new insights to better understand and predict the toxicity of MWCNTs in soil.

Effect of chronic unpredicted mild stress-induced depression on clopidogrel pharmacokinetics in rats.

BackgroundClopidogrel is widely used to prevent and treat cardiovascular atherosclerosis and thrombosis. However, disturbance in the expression and activity of liver cytochrome metabolic enzymes significantly changes clopidogrel efficacy. Therefore, the effect of chronic unpredictable mild stress (CUMS)-induced depression on the expression of liver cytochrome metabolic enzymes and clopidogrel pharmacokinetics in rats were explored.MethodsNine different CUMSs were selected to establish a rat model of depression. Open field experiment and sucrose preference test were applied to explore the depressive behaviors. The concentration of serotonin in the cortex of depressed rats was determined using enzyme linked immunosorbent assay (ELISA). All rats were given 10 mg/kg clopidogrel orally after 12 weeks, and blood samples were collected at different time points. The clopidogrel concentration and CYP2C19/ CYP2C9 activity in rat liver microsomes were assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The rat liver drug enzymes expression was determined by Real-Time Quantitative Reverse Transcription PCR (RT-qPCR).ResultsOpen field experiment and sucrose preference test indicated the successful construction of the CUMS-induced depression model. The concentration of serotonin in the cortex of depressed rats decreased by 42.56% (∗∗p < 0.01). The area under the curve of clopidogrel pharmacokinetics decreased by 33.13% (∗p < 0.05) in the depression rats, while distribution volume and clearance increased significantly (∗∗p < 0.01). The half-time and distribution volume did not significantly differ. The CYP2C19 and CYP2C9 activity of liver microsomes in the CUMS-induced depression group were significantly higher than that in the control group (∗∗p < 0.01). CYP2C11 and CYP1A2 mRNA expression up-regulated approximately 1.3 - fold in the depressed rat livers compared with that in the control, whereas that of CYP2C13 was down-regulated by 27.43% (∗∗p < 0.01). CYP3A1 and CYP2C12 expression were slightly up-regulated, and that of CES1 did not change.ConclusionsThese results indicated that CUMS-induced depression altered clopidogrel pharmacokinetics, and the change in CYP450 activity and expression in depressed rat livers might contribute to the disturbance of clopidogrel pharmacokinetics.

Impact of type 2 diabetes on in vivo activities and protein expressions of cytochrome P450 in patients with obesity.

Previous studies have not accounted for the close link between type 2 diabetes mellitus (T2DM) and obesity when investigating the impact of T2DM on cytochrome P450 (CYP) activities. The aim was to investigate the effect of T2DM on in vivo activities and protein expressions of CYP2C19, CYP3A, CYP1A2, and CYP2C9 in patients with obesity. A total of 99 patients from the COCKTAIL study (NCT02386917) were included in this cross-sectional analysis; 29 with T2DM and obesity (T2DM-obesity), 53 with obesity without T2DM (obesity), and 17 controls without T2DM and obesity (controls). CYP activities were assessed after the administration of a cocktail of probe drugs including omeprazole (CYP2C19), midazolam (CYP3A), caffeine (CYP1A2), and losartan (CYP2C9). Jejunal and liver biopsies were also obtained to determine protein concentrations of the respective CYPs. CYP2C19 activity and jejunal CYP2C19 concentration were 63% (-0.39 [95% CI: -0.82, -0.09]) and 40% (-0.09 fmol/μg protein [95% CI: -0.18, -0.003]) lower in T2DM-obesity compared with the obesity group, respectively. By contrast, there were no differences in the in vivo activities and protein concentrations of CYP3A, CYP1A2, and CYP2C9. Multivariable regression analyses also indicated that T2DM was associated with interindividual variability in CYP2C19 activity, but not CYP3A, CYP1A2, and CYP2C9 activities. The findings indicate that T2DM has a significant downregulating impact on CYP2C19 activity, but not on CYP3A, CYP1A2, and CYP2C9 activities and protein concentrations in patients with obesity. Hence, the effect of T2DM seems to be isoform-specific.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Drug metabolic enzyme genotype-phenotype discrepancy: High phenoconversion rate in patients treated with antidepressants.

Cytochromes from the P450 family (CYP) play a central role in the primary metabolism of frequently prescribed antidepressants, potentially affecting their efficacy and tolerance. There are however important differences in the drug metabolic capacities of each individual resulting from a combination of intrinsic and environmental factors. This variability can present an important risk for patients and increases the difficulty of drug prescription in clinical practice. Pharmacogenetic studies have uncovered a number of alleles defining the intrinsic metabolizer status, however, additional factors affecting cytochrome activity can modify this activity and result in a phenoconversion. The present study investigates the discrepancy between the genetically predicted and actually measured activities for the six most important liver cytochromes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) in a cohort of patients under antidepressant treatment, previously shown to have a high proportion of patients with low metabolizing activities. We now performed the genetic characterization of this cohort to determine the extent of the genetic versus environmental contribution in these decreased activities. For all enzyme tested, we observed an important rate of phenoconversion, affecting between 33 % and 65 % of the patients, as well as a significant (p<1E-06) global reduction in the effective but not predicted activities of CYP2D6, CYP2C9 and CYP2C19 compared to the general population. Our results highlight the advantages of phenotyping versus genotyping as well as the increased risk of treatment failure or adverse effect occurrence in a polymedicated population.

Inhibition of imrecoxib on mRNA and protein expression of CYP2C11 enzyme in rats.

To evaluate the effect of imrecoxib on CYP2C11 enzyme activity, mRNA, and protein expression, a UPLC method was established. Tolbutamide was selected as the CYP2C11 enzyme-specific probe drug and incubated with imrecoxib in rat liver microsomes. The yield of 4-hydroxytolbutamide was measured using UPLC to investigate the effect of imrecoxib on CYP2C11 enzyme activity. Imrecoxib (10mg/kg) was administered intragastrically twice daily. After 1, 7, and 14 days of administration, the liver tissues were analyzed. The expression of CYP2C11 enzyme mRNA was determined using reverse transcription-polymerase chain reaction, and its protein expression was determined using Western blot analysis. Imrecoxib concentration was inversely proportional to the production of 4-hydroxytolbutamide in liver microsomes. Imrecoxib demonstrated a dose-dependent inhibitory effect on CYP2C11 activity with IC50 = 74.77 μM. After administration, reverse transcription-polymerase chain reaction showed CYP2C11 enzyme mRNA expressions were 65% (P < 0.05), 35%, and 34% of the control group, respectively (P < 0.01). Western blot analysis showed CYP2C11 enzyme protein expressions were 80, 37, and 34% of the control group, respectively (P < 0.01). Imrecoxib can reduce mRNA and protein expression of CYP2C11 enzyme in rat liver and inhibit the activity of CYP2C11 enzyme in a dose-dependent manner. However, it does not produce clinically significant drug interactions.

Effects of ginkgo leaf tablet on the pharmacokinetics of rosiglitazone in rats and its potential mechanism

Context Ginkgo leaf tablet (GLT), a traditional Chinese herbal formula, is often combined with rosiglitazone (ROS) for type 2 diabetes mellitus treatment. However, the drug-drug interaction between GLT and ROS remains unknown. Objective To investigate the effects of GLT on the pharmacokinetics of ROS and its potential mechanism. Materials and methods The pharmacokinetics of 10 mg/kg ROS with 100/200 mg/kg GLT as single-dose and 10-day multiple-dose administration were investigated in Sprague-Dawley rats. In vitro, the effects of GLT on the activity of CYP2C8 and CYP2C9 were determined in recombinant human yeast microsomes and rat liver microsomes with probe substrates. Results The t 1/2 of ROS increased from 2.14 ± 0.38 (control) to 2.79 ± 0.37 (100 mg/kg) and 3.26 ± 1.08 h (200 mg/kg) in the single-dose GLT administration. The AUC0-t (139.69 ± 45.46 vs. 84.58 ± 39.87 vs. 66.60 ± 15.90 h·μg/mL) and t 1/2 (2.75 ± 0.70 vs. 1.99 ± 0.44 vs. 1.68 ± 0.35 h) decreased significantly after multiple-dose GLT treatment. The IC50 values of quercetin, kaempferol, and isorhamnetin, GLT main constituents, were 9.32, 7.67, and 11.90 μmol/L for CYP2C8, and 27.31, 7.57, and 4.59 μmol/L for CYP2C9. The multiple-dose GLT increased rat CYP2C8 activity by 44% and 88%, respectively. Discussion and conclusions The metabolism of ROS is attenuated in the single dose of GLT by inhibiting CYP2C8 and CYP2C9 activity, and accelerated after the multiple-dose GLT treatment via inducing CYP2C8 activity in rats, indicating that the clinical dose of ROS should be adjusted when co-administrated with GLT.

Optimisation of the HepaRG cell line model for drug toxicity studies using two different cultivation conditions: advantages and limitations.

The HepaRG cell line represents a successful model for hepatotoxicity studies. These cells are of human origin and are differentiated in vitro into mature and functional hepatocyte-like cells. The objective of this research was to compare two different culture protocols, Sison-Young et al. 2017 (hereinafter referred as Sison) and Gripon et al. 2002 (hereinafter referred as Biopredic) for HepaRG cells in order to optimise this model for drug metabolism and toxicity testing studies. HepaRG cells obtained from the same batch were cultured according to the described protocols. Using both protocols, differentiated HepaRG cells retained their drug metabolic capacity (major phase I/II enzymes) and transporters, as well as their morphological characteristics. Morphologically, HepaRG cells cultured after the Biopredic protocol formed more apical membranes and small ductular-like structures, than those cultivated using the Sison protocol. Also, the efflux activity of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) as well as the activity of uridine-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) were significantly reduced in HepaRG cultured using the Sison protocol. Applying well-established drug cocktails to measure cytochrome P450 (CYPs) activity, we found that production of the corresponding metabolites was hampered in Sison-cultured HepaRG cells, indicating that the activity of CYP1A2, CYP2C9, CYP3A4, CYP2B6 and CYP2C19 was significantly reduced. Moreover, HepaRG sensitivity to well-known drugs, namely diclofenac, amiodarone, imipramine and paracetamol, revealed some differences between the two culture protocols. Furthermore, the HepaRG cells can be maintained with higher viability and sufficient CYPs activity and expression (i.e. CYP3A4, CYP1A2 and CYP2B6) as well as liver-specific functions, using Biopredic compared with the Sison culture protocol. These maintained liver-specific functions might be dependent on the prolongation of the culture conditions in the case of the Biopredic protocol. In conclusion, based on the metabolic activity of HepaRG cells using the standard protocol from Biopredic, we believe that this protocol is optimal for investigating drug metabolism and pharmacokinetic screening studies.

Phenytoin Metabolic Ratio, a Marker of CYP2C9 Activity, is Superior to the CYP2C9 Genotype as a Predictor of (S)-Warfarin Clearance.

CYP2C9 is a member of the cytochrome P450 (CYP) superfamily responsible for the metabolism of 16% of drugs that undergo oxidative metabolism. The activity of CYP2C9 exhibits marked inter-individual variability, which translates into prominent differences in the pharmacokinetics of CYP2C9 substrates, some of which are characterized by a narrow therapeutic window. Genetic polymorphisms in the gene encoding for CYP2C9 account for a fraction of the variability in CYP2C9 activity. The phenytoin metabolic ratio (PMR) is a marker of CYP2C9 activity in vivo, which correlates with CYP2C9 genetic polymorphisms. The purpose of the current study was to evaluate the ability of the PMR to predict the oral clearance of (S)-warfarin (SWOCL) and its formation clearance towards its CYP2C9-mediated metabolites (SWCLf) [i.e., 6- and 7-hydroxy-(S)-warfarin]. The study was conducted in 150 healthy non-smoker subjects (segment 1) and 60 patients treated with warfarin (segment 2). In the first segment, the participants received on two separate occasions a single 300-mg dose of phenytoin and at least 7 days later a single dose of warfarin (5 or 10 mg). The same PMR procedure was performed in the second segment, except that it was performed either before warfarin initiation or after the patients had reached stable anticoagulation. The PMR was derived from the ratio of 5-(4-hydroxyphenyl)-5-phenyl-hydantoin content in a 24-hour urine collection to plasma phenytoin concentration 12- (PMR24/12) or 24- (PMR24/24) post-dosing. In segment 1, SWOCL was calculated from the ratio of (S)-warfarin dose to the warfarin area under the plasma concentration-time curve extrapolated to infinity and the SWCLf from the ratio of urine content of 6- and 7-hydroxy-(S)-warfarin to (S)-warfarin area under the (S)-warfarin plasma concentration-time curve until the last measured timepoint. In segment 2, estimated SWOCL was derived from the ratio of (S)-warfarin dose to the mid-interval plasma concentration of (S)-warfarin. The PMR, SWOCL, and SWCLf varied significantly between carriers of different CYP2C9 genotypes in both healthy subjects (p < 0.001) and patients (p < 0.005). However, PMR and SWOCL values exhibited substantial intra-genotypic variability. PMR24/12 and PMR24/24 were significantly correlated with SWOCL both in healthy subjects (r = 0.62 and r = 0.67, respectively, p < 0.001) and in patients (r = 0.57 and r = 0.61, respectively, p < 0.001). In a multiple regression model that included all variables that correlated with SWOCL, PMR was the strongest predictor, explaining 44% and 38% of the variability in SWOCL among healthy subjects and patients, respectively, and accounting for 95.7% (44%/46%) and 90.5% (38%/42%) of the total explained variability in SWOCL among healthy subjects and patients, respectively. The PMR is the strongest predictor of SWOCL, and as such, it exhibits a significant advantage over the CYP2C9 genotype. The inclusion of PMR in future dosing algorithms of CYP2C9 substrates characterized by a narrow therapeutic window should be encouraged and further investigated.

Analyses of Pharmacokinetic, Pharmacogenetic and Psychometrics Correlates of Antidepressants Use during Pregnancy and the Post-Partum Period

IntroductionAbout 15% of women experience a depressive episode during pregnancy, and 19% during the postpartum. Studies on safety of Antidepressants use during pregnancy have given controversial results. Obstetric-gynecological changes of pregnancy determine modifications in the pharmacokinetics of medications, through an altered metabolism of the CYP enzymes. Patient’s therapeutic response might also be influenced by polymorphisms of the genes encoding CYP enzymes.ObjectivesIn this perspective, we evaluated the correlation between pharmacokinetics, pharmacogenetics and psychopathological measures, analyzing SSRIs or SNRIs concentrations during the three trimesters of pregnancy, at birth and at postpartum, in order to define efficacy, tolerability and safety of Antidepressants (ADs) in the treatment of affective and anxiety disorders during pregnancy.Methods87 patients were enrolled at the Depressive Disorders Treatment Centre (CTDD) of the Department of Psychiatry of Sacco University Hospital (Milano, Italy). Plasma concentrations of ADs were measured during first (T1), second (T2), third (T3) trimester, at birth (T4) and at postpartum (T5). Psychometric assessments were carried out. The genotype of hepatic CYP isoforms were also analysed.ResultsADs mainly metabolized by CYP2C19 (es. Sertraline) are less frequently below therapeutic range than ADs metabolized by CYP2D6. In fact, the metabolic activity of CYP2C19 is slowed down during pregnancy. The majority of ADs concentrations below therapeutic range were found in women with an accelerated metaboslism, carrier of a CYP polymorphism.ConclusionsOur results underline that the systematic use of pharmacokinetic and pharmacogenetic analyses during pregnancy could constitute a valid support in the management of therapy in the last phases of pregnancy.DisclosureNo significant relationships.

Analysis of Genetic Polymorphism for CYP2D6 &amp; CYP2C19 on a Forensic Autopsy Case Involving Venlafaxine Poisoning

Venlafaxine (VEN) is a type of serotonin-norepinephrine reuptake inhibitor. It is known that the pharmacogenetics of VEN is mainly affected by metabolizer status of the Cytochrome P450 (CYP) 2D6 and CYP2C19 enzymes. We investigate a fatal overdose case of VEN in relation to CYP2D6 and CYP2C19 genetic polymorphisms. Qualitative and quantitative analyses for VEN were performed using gas chromatography-mass spectrometry and it is subsequently confirmed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Genetic analysis for CYP2D6 and CYP2C19 polymorphism was performed using polymerase chain reaction (PCR) followed by DNA sequencing. The result was determined to be a fatal overdose of VEN in a Korean man with CYP2D6 heterozygous genotype *1/*2 that did not affect enzyme activity and wild allele of CYP2C19. The use of genotyping of CYP2D6 and CYP2C19 may assist interpretation for a considerable fatal dose of VEN in individuals with different CYP2D6 genotypes.

Methysticin Acts as a Mechanism-Based Inactivator of Cytochrome P450 2C9.

Methysticin is one of the naturally occurring bioactive constituents extracted from Piper methysticum Forst. In the present study, we intended to investigate the inhibitory effect of methysticin on cytochrome P450 (P450) enzymes. Methysticin exhibited time-, concentration-, and NADPH-dependent inhibition on CYP2C9 using diclofenac as a probe substrate. Approximately 85% of CYP2C9 activity was inhibited by methysticin at 50 μM after a 30 min preincubation with human liver microsomes in the presence of NADPH. The kinetic parameters KI, kinact, and t1/2,inact were 13.32 ± 1.35 μM, 0.054 ± 0.005 min-1, and 12.83 ± 3.23 min, respectively. Sulfaphenazole (competitive inhibitor of CYP2C9) displayed a significant protective effect on methysticin-induced CYP2C9 inactivation. However, the inclusion of catalase/superoxide dismutase or glutathione (GSH) showed no such protection. A carbene intermediate was postulated to be involved in methysticin-induced CYP2C9 inactivation as K3Fe(CN)6 recovered 14.96% of CYP2C9 activity. A methysticin-derived ortho-quinone intermediate dependent on NADPH was trapped by GSH, and this intermediate was believed to be involved in CYP2C9 inactivation. CYP1A2, 2C9, and 3A4 were the major enzymes responsible for methysticin bioactivation. Taken together, the present work demonstrated that methysticin was a mechanism-based inactivator of CYP2C9. Both ortho-quinone and carbene intermediates appeared to be involved in the inactivation of CYP2C9 induced by methysticin.

Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism.We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed.The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT.A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.

Effects of CYP2C19 variants on the metabolism of tapentadol in vitro.

Objective(s):This study aims to evaluate the catalytic activities of 31 CYP2C19 alleles and their effects on the metabolism of tapentadol in vitro. Materials and Methods:Insect microsomes expressing the CYP2C19 alleles were incubated with 50–1250 μM tapentadol for 40 min at 37 °C and terminated by cooling to -80 °C, immediately. Tapentadol and N-desmethyl tapentadol were analyzed by a UPLC-MS/MS system. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl tapentadol were determined. Results:As a result, the intrinsic clearance (Vmax/Km) values of most variants were significantly altered, while CYP2C19.3 and 35FS had no detectable enzyme activity. Only one variant, N277K, showed no significant difference from CYP2C19.1B. Two variants CYP2C19.29 and L16F displayed markedly increased intrinsic clearance values of 302.22% and 199.97%, respectively; whereas 24 variants exhibited significantly decreased relative clearance ranging from 0.32% to 79.15% of CYP2C19.1B. Especially, CYP2C19.2G, 2H, R124Q, and R261W exhibited a drastic decrease in clearance (>80%) compared with wild-type CYP2C19.1B. Conclusion:As the first study of all aforementioned alleles for tapentadol metabolism, the comprehensive data in vitro may provide novel insights into the allele-specific and substrate-specific activity of CYP2C19.

Effects of the total flavonoid extracts and the monomers of Daphne genkwa on CYP2C8 activity

This study aimed to assess the effects of total flavonoid extracts (TFDG) and the monomers of Daphne genkwa on the CYP2C8 activity in vitro and in vivo. The 50% inhibitory concentration (IC50) values were used to determine the inhibitory effect of TFDG and its four monomers for the CYP2C8 activity by recombinant human CYP2C8 (RHCYP2C8) yeast microsome system in vitro, and the volume per dose index (VDI) was predicted the potential inhibition in vivo. The effects of multiple-dose administration of TFDG on the pharmacokinetic parameters of rosiglitazone in rats were evaluated. The IC50 values of apigenin, luteolin, hydroxy-genkwanin, genkwanin, and TFDG were 7.27 μmol/L, 11.9 μmol/L, 28.1 μmol/L, 127 μmol/L, and 13.4 μg/mL, respectively. The VDI values of apigenin and TFDG were 2.15 L and 6.60 L. In vivo study, compared with the control group, the elimination phase half-life and mean residence time in the TFDG treatment group were significantly increased by 96.9% and 106.8% (p <.05), respectively. Apigenin showed a moderate inhibitory effect on the CYP2C8 activity in vitro, while the other three monomers were weak inhibitors. TFDG had a strong inhibitory effect on CYP2C8 in vitro and in vivo, and also inhibited the metabolism of rosiglitazone in rats.

Hepatic drug metabolism in older people with body composition changes.

Dosage adjustment is essential in older individuals because they are prone to experience a decline in liver function and changes in body composition. However, quantitative tests or equations for evaluating the activity of hepatic drug metabolism have not yet been clearly established. We examined hepatic drug metabolism activities in older individuals, focusing on changes in body composition parameters. Lansoprazole and nifedipine, substrates of the metabolic enzymes cytochrome P450 (CYP) 2C19 and 3A4, respectively, were selected to study hepatic drug metabolism. Residual samples from blood test for older patients were evaluated to determine drug metabolism. The body composition of relevant patients was determined by analyzing characteristic parameters of skeletal muscle mass index (SMI), handgrip strength (HGS) and hepatic steatosis index (HSI). The differences in hepatic drug metabolism were studied statistically among categories in terms of the cut-off value of these parameters. Older male patients receiving lansoprazole and nifedipine in the low SMI (<7.0 kg/m2 ) category showed an 85-90% reduction in respective CYP2C19 and CYP3A4 metabolic activities compared with the normal SMI category. For the female patients, CYP2C19 and CYP3A4 metabolic activities showed no significant correlation with SMI and HGS. Fatty liver disease (HSI ≥36) was found to reduce CYP2C19 metabolic activity particularly in older female patients. Low CYP2C19 metabolic activity was statistically correlated with low SMI in male patients and high HSI in female patients, whereas low CYP3A4 metabolic activity was statistically correlated with low HGS in male patients. Geriatr Gerontol Int 2022; 22: 449-454.

A physiologically based pharmacokinetic model of clopidogrel in populations of European and Japanese ancestry: An evaluation of CYP2C19 activity.

ABSTRACTTreatment response to clopidogrel is associated with CYP2C19 activity through the formation of the active H4 metabolite. The aims of this study were to develop a physiologically based pharmacokinetic (PBPK) model of clopidogrel and its metabolites for populations of European ancestry, to predict the pharmacokinetics in the Japanese population by CYP2C19 phenotype, and to investigate the effect of clinical and demographic factors. A PBPK model was developed and verified to describe the two metabolic pathways of clopidogrel (H4 metabolite, acyl glucuronide metabolite) for a population of European ancestry using plasma data from published studies. Subsequently, model predictions in the Japanese population were evaluated. The effects of CYP2C19 activity, fluvoxamine coadministration (CYP2C19 inhibitor), and population‐specific factors (age, sex, BMI, body weight, cancer, hepatic, and renal dysfunction) on the pharmacokinetics of clopidogrel and its metabolites were then characterized. The predicted/observed ratios for clopidogrel and metabolite exposure parameters were acceptable (twofold acceptance criteria). For all CYP2C19 phenotypes, steady‐state AUC0‐τ of the H4 metabolite was lower for the Japanese (e.g., EM, 7.69 [6.26–9.45] ng·h/ml; geometric mean [95% CI]) than European (EM, 24.8 [20.4–30.1] ng·h/ml, p < .001) population. In addition to CYP2C19‐poor metabolizer phenotype, fluvoxamine coadministration, hepatic, and renal dysfunction were found to reduce H4 metabolite but not acyl glucuronide metabolite concentrations. This is the first PBPK model describing the two major metabolic pathways of clopidogrel, which can be applied to populations of European and Japanese ancestry by CYP2C19 phenotype. The differences between the two populations appear to be determined primarily by the effect of varying CYP2C19 liver activity.

Evaluation of CYP2C19 activity using microdosed oral omeprazole in humans

PurposeTo investigate the suitability of microdosed oral omeprazole for predicting CYP2C19 activity in vivo in combination with simultaneous assessment of CYP3A and CYP2D6 activity using both microdosed midazolam and yohimbine.MethodsAn open, fixed-sequence study was carried out in 20 healthy participants. Single microdosed (100 µg) and therapeutic (20 mg) doses of omeprazole were evaluated without comedication and after administration of established CYP2C19 perpetrators fluconazole (inhibition) and rifampicin (induction). To prevent degradation of the uncoated omeprazole microdose, sodium bicarbonate buffer was administered. The pharmacokinetics of omeprazole and its 5-hydroxy-metabolite were assessed as well as the pharmacokinetics of midazolam and yohimbine to estimate CYP3A4 and CYP2D6 activity.ResultsCalculated pharmacokinetic parameters after administration of 100 µg and 20 mg omeprazole in healthy subjects suggest dose proportionality. Omeprazole clearance was significantly decreased by fluconazole from 388 [95% CI: 266–565] to 47.2 [42.8–52.0] mL/min after 20 mg omeprazole and even further after 100 µg omeprazole (29.4 [24.5–35.1] mL/min). Rifampicin increased CYP2C19-mediated omeprazole metabolism. The omeprazole hydroxylation index was significantly related to omeprazole clearance for both doses. Both fluconazole and rifampicin altered CYP3A4 activity whereas no change of CYP2D6 activity was observed at all.ConclusionsMicrodosed oral omeprazole is suitable to determine CYP2C19 activity, also during enzyme inhibition and induction. However, the administration of sodium bicarbonate buffer also had a small influence on all victim drugs used.Trial registrationEudraCT: 2017–004270-34.

Selective Serotonin Reuptake Inhibitor Pharmacokinetics During Pregnancy: Clinical and Research Implications.

Pregnancy and associated physiologic changes affect the pharmacokinetics of many medications, including selective serotonin reuptake inhibitors—the first-line pharmacologic interventions for depressive and anxiety disorders. During pregnancy, SSRIs exhibit extensive pharmacokinetic variability that may influence their tolerability and efficacy. Specifically, compared to non-pregnant women, the activity of cytochrome P450 (CYP) enzymes that metabolize SSRIs drastically changes (e.g., decreased CYP2C19 activity and increased CYP2D6 activity). This perspective examines the impact of pharmacokinetic genes—related to CYP activity on SSRI pharmacokinetics during pregnancy. Through a simulation-based approach, plasma concentrations for SSRIs metabolized primarily by CYP2C19 (e.g., escitalopram) and CYP2D6 (e.g., fluoxetine) are examined and the implications for dosing and future research are discussed.

In vitro evaluation of the inhibition potential of echinacoside on human cytochrome P450 isozymes

BackgroundEchinacoside (ECH) possesses a wide range of biological activity. This present study analyzes the effect of ECH on cytochrome P450 isozymes (CYPs) activities of human liver microsomes.MethodsThe effect of ECH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), chlorzoxazone (2E1), S-mephenytoin (2C19), testosterone (3A4), coumarin (2A6), diclofenac (2C9), paclitaxel (2C8), and dextromethorphan (2D6). The IC50 values for CYP1A2, CYP2E1, CYP2C19, and CYP3A4 isoforms were examined to express the strength of inhibition. Further, the inhibition of CYPs was checked for time-dependent or not, and then fitted with competitive or non-competitive inhibition models. The corresponding parameters were also obtained.ResultsECH caused inhibitions on CYP1A2, CYP2E1, CYP2C19 and CYP3A4 enzyme activities in HLMs with IC50 of 21.23, 19.15, 8.70 and 55.42 μM, respectively. The obtained results showed that the inhibition of ECH on CYP3A4 was time-dependent with the KI/Kinact value of 6.63/0.066 min− 1·μM− 1. Moreover, ECH inhibited the activity of CYP1A2 and CYP2E1 via non-competitive manners (Ki = 10.90 μM and Ki = 14.40 μM, respectively), while ECH attenuated the CYP2C19 activity via a competitive manner (Ki = 4.41 μM).ConclusionsThe results of this study indicate that ECH inhibits CYP1A2, CYP2E1, CYP2C19 and CYP3A4 activities in vitro. In vivo and clinical studies are warranted to verify the relevance of these interactions.