Hepatic Cytochrome P450 3A Activity Research Articles

Intrasubject Variability in Intravenous and Oral Probes for Hepatic and First-Pass CYP3A Activity.

Clearances and the area under the plasma concentration-time curve extrapolated to infinity (AUC0-∞) of intravenous (IV) and oral midazolam and alfentanil are probes for hepatic and first-pass cytochrome P450 3A (CYP3A) activity, drug interactions, and phenotyping. Single-time plasma concentrations are also used as a proxy for clearance and AUC0-∞. Pupil diameter change is a noninvasive surrogate for plasma alfentanil. An ideal probe should have minimal intrasubject (interday) variability. Despite their widespread use, the intrasubject variability of CYP3A probes is not well characterized. This investigation determined the intrasubject (interday) variability of midazolam and alfentanil metrics of hepatic and first-pass CYP3A. Twelve volunteers were studied in a four-period protocol, with each period identical and separated by approximately 2 weeks. In each period, participants received 1 mg IV midazolam then 15 μg/kg IV alfentanil 1 h later. The next day, they received 3 mg oral midazolam then 60 μg/kg oral alfentanil. Plasma drug concentrations were determined by liquid chromatography-mass spectrometry (LCMS). Dark-adapted pupil diameters were measured coincident with blood sampling. Plasma concentrations and pupil effects (miosis) were analyzed using noncompartmental methods. The results were the coefficient of variation (%CV, mean ± SD) across four sessions in 12 participants. For IV midazolam: AUC0-∞, clearance, and 5 h concentration, the %CVs were 12 ± 3, 12 ± 3, and 18 ± 8. For IV alfentanil AUC0-∞, clearance, 2 h concentration, and area under the effect curve from time zero to infinity (AUEC0-∞), the %CVs were 16 ± 5, 15 ± 4, 22 ± 7, and 50 ± 28. For oral midazolam AUC0-∞, clearance, and 5 h concentration, %CVs were 19 ± 5, 18 ± 4, and 28 ± 11. For oral alfentanil: AUC0-∞, clearance, 4 h concentration, and AUEC0-∞, %CVs were 20 ± 4, 21 ± 4, 42 ± 26, and 37 ± 14. Midazolam and alfentanil had comparable intrasubject variabilities of clearance and AUC0-∞. Single-time point metrics had greater intrasubject variability than AUC0-∞ and clearance. Miosis was a surrogate for alfentanil concentrations and provided real-time results, but intrasubject variability was greater than that of clearances and AUC0-∞.

Induction of hepatic CYP3A4 expression by cholesterol and cholic acid: Alterations of gene expression, microsomal activity, and pharmacokinetics.

Human cytochrome P450 3A4 (CYP3A4) is a drug-metabolizing enzyme that is abundantly expressed in the liver and intestine. It is an important issue whether compounds of interest affect the expression of CYP3A4 because more than 30% of commercially available drugs are metabolized by CYP3A4. In this study, we examined the effects of cholesterol and cholic acid on the expression level and activity of CYP3A4 in hCYP3A mice that have a human CYP3A gene cluster and show human-like regulation of the coding genes. A normal diet (ND, CE-2), CE-2 with 1% cholesterol and 0.5% cholic acid (HCD) or CE-2 with 0.5% cholic acid was given to the mice. The plasma concentrations of cholesterol, cholic acid and its metabolites in HCD mice were higher than those in ND mice. In this condition, the expression levels of hepatic CYP3A4 and the hydroxylation activities of triazolam, a typical CYP3A4 substrate, in liver microsomes of HCD mice were higher than those in liver microsomes of ND mice. Furthermore, plasma concentrations of triazolam in HCD mice were lower than those in ND mice. In conclusion, our study suggested that hepatic CYP3A4 expression and activity are influenced by the combination of cholesterol and cholic acid invivo.

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform

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

Correlations between 4β-hydroxycholesterol and hepatic and intestinal CYP3A4: protein expression, microsomal ex vivo activity, and in vivo activity in patients with a wide body weight range

PurposeVariability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4β-hydroxycholesterol (4βOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4βOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range.MethodsThe patients (n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4βOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam.Results4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (ρ = 0.53, p < 0.001), and hepatic CYP3A4 concentrations (ρ = 0.30, p = 0.027), but not with intestinal CYP3A4 concentrations (ρ = 0.18, p = 0.28) or intestinal microsomal CYP3A4 activity (ρ = 0.15, p = 0.53). 4βOHC concentrations correlated weakly with midazolam absolute bioavailability (ρ = − 0.23, p = 0.027) and apparent oral clearance (ρ = 0.28, p = 0.008), but not with systemic clearance (ρ = − 0.03, p = 0.81).ConclusionThese findings suggest that 4βOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4βOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs.Trial registrationClinical.Trials.gov identifier: NCT02386917.

Urinary metabolic markers reflect on hepatic, not intestinal, CYP3A activity in healthy subjects

Intestinal cytochrome P450 3A (CYP3A) plays an important role in oral drug metabolism, but only endogenous metabolic markers for measuring hepatic CYP3A activity were identified. Our study evaluated whether hepatic CYP3A markers reflected intestinal CYP3A activity. An open-label, three-period, six-treatment, one-sequence clinical trial was performed in 16 healthy Korean males. In the control phase, all subjects received a single dose of intravenous (IV) and oral midazolam (1 mg and 5 mg, respectively). Clarithromycin (500 mg) was administered twice daily for 4 days to inhibit hepatic and intestinal CYP3A, and 500 mL of grapefruit juice was given to inhibit intestinal CYP3A. Clarithromycin significantly inhibited total CYP3A activity, and the clearance of IV and apparent clearance of oral midazolam decreased by 0.15- and 0.32-fold, respectively. Grapefruit juice only reduced the apparent clearance of oral midazolam by 0.84-fold, which indicates a slight inhibition of intestinal CYP3A activity. Urinary markers, including 6β–OH–cortisol/cortisol and 6β–OH–cortisone/cortisone, were significantly decreased 0.5-fold after clarithromycin administration but not after grapefruit juice. The fold changes in 6β–OH–cortisol/cortisol and 6β–OH–cortisone/cortisone did not correlate to changes in intestinal availability but did correlate to hepatic availability. In conclusion, endogenous metabolic markers are only useful to measure hepatic, but not intestinal, CYP3A activity.

Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo

1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice.2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice.3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant.4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.

Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice.

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.

Assessment of Hepatic Cytochrome P450 3A Activity Using Metabolic Markers in Patients with Renal Impairment.

BackgroundThe renal function of individuals is one of the reasons for the variations in therapeutic response to various drugs. Patients with renal impairment are often exposed to drug toxicity, even with drugs that are usually eliminated by hepatic metabolism. Previous study has reported an increased plasma concentration of indoxyl sulfate and decreased plasma concentration of 4β-hydroxy (OH)-cholesterol in stable kidney transplant recipients, implicating indoxyl sulfate as a cytochrome P450 (CYP) inhibiting factor. In this study, we aimed to evaluate the impact of renal impairment severity-dependent accumulation of indoxyl sulfate on hepatic CYP3A activity using metabolic markers.MethodsSixty-six subjects were enrolled in this study; based on estimated glomerular filtration rate (eGFR), they were classified as having mild, moderate, or severe renal impairment. The plasma concentration of indoxyl sulfate was quantified using liquid chromatography-mass spectrometry (LC-MS). Urinary and plasma markers (6β-OH-cortisol/cortisol, 6β-OH-cortisone/cortisone, 4β-OH-cholesterol) for hepatic CYP3A activity were quantified using gas chromatography-mass spectrometry (GC-MS). The total plasma concentration of cholesterol was measured using the enzymatic colorimetric assay to calculate the 4β-OH-cholesterol/cholesterol ratio. The correlation between variables was assessed using Pearson's correlation test.ResultsThere was a significant negative correlation between MDRD eGFR and indoxyl sulfate levels. The levels of urinary 6β-OH-cortisol/cortisol and 6β-OH-cortisone/cortisone as well as plasma 4β-OH-cholesterol and 4β-OH-cholesterol/cholesterol were not correlated with MDRD eGFR and the plasma concentration of indoxyl sulfate.ConclusionHepatic CYP3A activity may not be affected by renal impairment-induced accumulation of plasma indoxyl sulfate.

Assessment of hepatic cytochrome P450 3A activity using steroid metabolic markers in patients with renal impairment

Assessment of hepatic cytochrome P450 3A activity using steroid metabolic markers in patients with renal impairment

Impact of nanomedicine on hepatic cytochrome P450 3A4 activity: things to consider during pre-clinical and clinical studies

Hepatic cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme as it is responsible for metabolizing over 30% of currently marketed drugs across most therapeutic classes. CYP3A4 is regulated at various stages including transcriptional, post-transcriptional, translational and post-translational expression. Changes made at any of these stages of CYP3A4 regulation could result in fluctuations in CYP3A4 levels and/or activity. These alterations are important to understand as they could lead to drug–drug interactions, sub-therapeutic levels or toxic levels of pharmaceutical drugs. Nanomedicine, the use of nanotechnology for treatment, diagnosis and monitoring of biological systems, is a growing market. This includes use of viral vectors for gene transfer and vaccine applications and nanoparticles based on gold and silver for various applications. Both have shown to alter CYP3A4 activity and function. Hence, during pre-clinical and clinical studies it is important to elucidate any effect of nanomedicines on CYP3A4 to avoid unintended changes in maintenance of medications due to changes in drug metabolism.

Relationship between Differential Hepatic microRNA Expression and Decreased Hepatic Cytochrome P450 3A Activity in Cirrhosis

Background and AimLiver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis.MethodsHepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity.ResultsHepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08).ConclusionThe strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.

Hepatic CYP3A expression and activity in low birth weight developing female rats

We aimed to investigate the effects of low birth weight (LBW) on the hepatic expression of cytochrome P-450 3A (CYP3A) in developing female rats. Pregnant rats were divided into two groups, a nourished group and an under-nourished group. The offspring of the nourished rats were defined as a normal weight, normal diet group (NN group). The offspring of the under-nourished rats were designated as a LBW, normal diet group (LN group). CYP3A mRNA expression, protein expression, protein localization and activity were determined. The CYP3A1 mRNA expression levels of the LN group on days 3, 21, and 56 were significantly higher than those of the same age in the NN group (P≤0.01). The mRNA expression of CYP3A2 in the LN group on day 21 was higher than in rats of the same age in the NN group (P<0.01). The staining intensity and frequency of CYP3A1-positive hepatocytes were significantly lower on days 7 and 21 in the LN group than those of rats of the same age in the NN group (P<0.05). The staining intensity and frequency of CYP3A2-positive hepatocytes on days 14 and 21 were higher in the LN group than those on the same days in the NN group (P<0.05). The mean CYP3A activity of the LN group on day 3 was significantly higher than that of the NN group (P<0.001). We found the effect of LBW on CYP3A activity was most prominent during the early days of life in rats. Investigators and clinicians should consider the effect of LBW on CYP3A in both pharmacokinetic study design and data interpretation, when prescribing drugs to LBW infants.

The mycotoxin T-2 inhibits hepatic cytochrome P4503A activity in pigs

Mycotoxins are toxic metabolites produced by fungi that readily colonize crops. After ingestion, these mycotoxins can compromise intestinal health, and once entering the blood stream, even affect the liver and its metabolizing enzymes. It was therefore the aim of the present study to investigate the effect of T-2 toxin, an emerging and potent Fusarium mycotoxin, on the enzymatic activity of cytochrome P4503A (CYP3A) metabolizing enzymes in the liver of pigs. In addition, a yeast-derived feed additive that claims to bind T-2 toxin was included in the study to evaluate its efficacy. Our results demonstrated that a 14-days intake of T-2 toxin contaminated feed at a dose of 903 μg/kg feed, whether or not combined with the mycotoxin binder, results in a substantial inhibition of the CYP3A activity in the liver of pigs. This result may be of importance for animal health, the pharmacokinetics and the withdrawal time of drugs that are substrate of CYP3A enzymes, and consequently can be a threat for public health with respect to tissue residues of these drugs.

In vivo effect of Schisandrin B on cytochrome P450 enzyme activity

To investigate the possible drug interaction, this study is designed to evaluate the ability of Schisandrin B (Sch B) to modulate cytochrome P450 3A activity (CYP3A) in vivo and to alter the pharmacokinetic profiles of CYP3A substrate (midazolam) in treated rats. Rats were repeated administered with physiological saline (negative control group), ketoconazole (75mg/kg, positive control group) or varied doses of Sch B (experimental groups) for three consecutive days. Subsequently, changes in hepatic microsomal CYP3A activity and the pharmacokinetic profiles of midazolam and 1′-hydroxy midazolam in plasma were studied to evaluate CYP3A activity. The results indicated that Sch B significantly dose-dependently inhibited rat hepatic microsomal CYP3A activity with Ki value of 16.64mg/kg and showed the characteristic of a noncompetitive inhibitor. Oral administration of Sch B for 3 days in rats produced significant effect on the pharmacokinetics of oral midazolam. Sch B resulted in a significant, dose-dependent increase in midazolam AUC0−∞ except at the dose of 2mg/kg, while AUC0−∞ increased by 26.1% (8mg/kg) and 60.6% (16mg/kg), respectively. In the pharmacokinetic profiles of 1′-hydroxy midazolam, the significant, dose-dependent decrease in AUC0−∞ was observed except at the dose of 2mg/kg, while AUC0−∞ reduced by 44.5% (8mg/kg) and 49.2% (16mg/kg), respectively. These results suggested that 3-day treatment of Sch B could increase concentration and oral bioavailability of drug metabolized by CYP3A. When the drug, consisting of Sch B, is used in the clinic for more than 3 days, the possible drug–drug interactions should be taken into consideration.

Rate of onset of inhibition of gut-wall and hepatic CYP3A by clarithromycin

To determine the extent and time-course of hepatic and intestinal cytochrome P450 3A (CYP3A) inactivation due to the mechanism-based inhibitor clarithromycin. Intestinal and hepatic CYP3A inhibition was examined in 12 healthy volunteers following the administration of single and multiple doses of oral clarithromycin (500 mg). Intestinal biopsies were obtained under intravenous midazolam sedation at baseline and after the first dose, on days 2-4, and on days 6-8 of the clarithromycin treatment. The formation of 1'-hydroxymidazolam in biopsy tissue and the serum 1'-hydroxymidazolam:midazolam ratio were indicators of intestinal and hepatic CYP3A activity, respectively. Intestinal CYP3A activity decreased by 64 % (p = 0.0029) following the first dose of clarithromycin, but hepatic CYP3A activity did not significantly decrease. Repeated dosing of clarithromycin caused a significant decrease in hepatic CYP3A activity (p = 0.005), while intestinal activity showed little further decline. The CYP3A5 or CYP3A4*1B genotype were unable to account for inter-individual variability in CYP3A activity. Following the administration of clarithromycin, the onset of hepatic CYP3A inactivation is delayed compared to that of intestinal CYP3A. The time-course of drug-drug interactions due to clarithromycin will vary with the relative contribution of intestinal and hepatic CYP3A to the clearance and bioavailability of a victim substrate.

Does Roux-en-Y Gastric Bypass Affect the Metabolism of the Orally Administered Medications?: 2010 Presidential Poster

Purpose: The effect of Roux-en-Y gastric bypass (RYGB) on the bioavailability of orally administered drugs is unknown. Cytochrome P450 3A (CYP3A) is the major human drug metabolizing enzyme and it metabolizes many important compounds (e.g., macrolides, tacrolimus, diltiazem). CYP3A enzyme is present in both small intestine and liver. We conducted a study to systematically examine the effect of RYGB on CYP3A in humans. Methods: Sixteen subjects who were at least one year post RYGB and 16 age, race, gender and BMI-matched healthy volunteers without RYGB participated in this study. CYP3A activity was measured using oral and IV midazolam as in vivo probe drugs. Participants received midazolam 1mg orally followed by midazolam 0.01ml/kg intravenously 6 hours later and timed peripheral venous blood sampling was performed per protocol. CYP3A enzyme activity was estimated using the plasma ratios of area under the serum concentration-time curves (AUC 0-t) of oral midazolam and its primary metabolite, 1 ‘OH midazolam. Absorption pharmacokinetics (Cmax, Tmax, and AUC) were also compared between the two groups. Results: There were no statistically significant differences in the demographics between the two groups (Table 1). Difference in ratios of AUC 0-6 values of oral midazolam and 1 ‘OH midazolam between the RYGB and control groups were not statistically significant (5.5±1.1 uM vs. 33.4±24.7 uM, p=0.26). Compared to controls, patients with RYGB did not have significantly different Cmax (2.33±0.48 ng/mL vs. 6.44±2.85 ng/mL, p=0.19) or AUC 0-6 (3.88±0.68 ng*hr*mL-1 vs. 14.07±5.98 ng*hr*mL-1, p=0.11), but their Tmax significantly shorter (0.53±0.05 vs. 0.82±0.09 hrs, p=0.04). There were no statistically significant differences in the Cmax, Tmax, AUC or hepatic CYP3A activity following IV midazolam (Table 1).Table 1: Demographics and Pharmacokinetic data on RYGB and age, race, gender, BMI-matched controlsConclusion: Shorter Tmax of oral midazolam in patients with RYGB suggests that they absorb oral CYP3A substrates more rapidly than controls, but similar Cmax and AUC values between two groups indicate that absorption kinetics of oral CYP3A substrates are not significantly affected by RYGB. Small bowel and liver CYP3Aactivity in patients with RYGB is not different when compared to controls. Our results suggest that patients with RYGB do not need dosage adjustment of orally administered CYP3A substrates.

Effects of cytochrome P450 3A (CYP3A) and the drug transporters P‐glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) on the pharmacokinetics of lopinavir

Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2. Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/- ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine. Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration - time curve (AUC)(oral) was increased in Abcb1a/b(-/-) mice (approximately ninefold vs. wild-type) but not in Abcc2(-/-) mice. Increased lopinavir AUC(oral) (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a(-/-)) mice compared with wild-type mice. No difference in AUC(oral) between Cyp3a(-/-) and Cyp3a/Abcb1a/b/Abcc2(-/-) mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC(oral) (>100-fold), compared with Cyp3a(-/-) mice. Ritonavir markedly increased lopinavir AUC(oral) in all CYP3A-containing mouse strains. CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity.

Methadone Pharmacokinetics Are Independent of Cytochrome P4503A (CYP3A) Activity and Gastrointestinal Drug Transport

Methadone clearance is highly variable, and drug interactions are problematic. Both have been attributed to CYP3A, but actual mechanisms are unknown. Drug interactions can provide such mechanistic information. Ritonavir/indinavir, one of the earliest protease inhibitor combinations, may inhibit CYP3A. We assessed ritonavir/indinavir effects on methadone pharmacokinetics and pharmacodynamics, intestinal and hepatic CYP3A activity, and intestinal transporters (P-glycoprotein) activity. CYP3A and transporters were assessed with alfentanil and fexofenadine, respectively. Twelve healthy human immunodeficiency virus-negative volunteers underwent a sequential three-part crossover. On three consecutive days, they received oral alfentanil/fexofenadine, intravenous alfentanil, and intravenous plus oral (deuterium-labeled) methadone, repeated after acute (3 days) and steady-state (2 weeks) ritonavir/indinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were assessed by miosis. Alfentanil apparent oral clearance was inhibited more than 97% by both acute and steady-state ritonavir/indinavir, and systemic clearance was inhibited more than 90% due to diminished hepatic and intestinal extraction. Ritonavir/indinavir increased fexofenadine area under the plasma concentration-time curve four- to five-fold, suggesting significant inhibition of gastrointestinal P-glycoprotein. Ritonavir/indinavir slightly increased methadone N-demethylation, but it had no significant effects on methadone plasma concentrations or on systemic or apparent oral clearance, renal clearance, hepatic extraction or clearance, or bioavailability. Ritonavir/indinavir had no significant effects on methadone plasma concentration-effect relationships. Inhibition of both hepatic and intestinal CYP3A activity is responsible for ritonavir/indinavir drug interactions. Methadone disposition was unchanged, despite profound inhibition of CYP3A activity, suggesting little or no role for CYP3A in clinical methadone metabolism and clearance. Methadone bioavailability was unchanged, despite inhibition of gastrointestinal P-glycoprotein activity, suggesting that this transporter does not limit methadone intestinal absorption.

Short Communication: Insulin Alters Hepatic Progesterone Catabolic Enzymes Cytochrome P450 2C and 3A in Dairy Cows

High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500mL of water (n=10) or propylene glycol (a gluconeogenic substrate; n=7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0μg of insulin/kg of BW per h; n=6 each) or remained as controls (saline infused; n=7) for 96h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.

Effects of ursodeoxycholic acid on the pharmacokinetics and pharmacodynamics of intravenous and oral midazolam in healthy volunteers

Animal and in vitro studies suggest that ursodeoxycholic acid (UDCA) can induce cytochrome P450 3A (CYP3A) expression and enhance its activities. On the other hand, Becquemont et al. demonstrated that UDCA had no influence on intestinal CYP3A activities. The aim of this study was to investigate the effects of UDCA on the intestinal and hepatic CYP3A activities by administration of midazolam (MDZ), as a specific probe for CYP3A activity, in humans. This was a randomized, open-label, crossover study with two phases in 14 healthy volunteers. The volunteers received UDCA (300 mg/day) or placebo orally for 9 days. The pharmacokinetics and pharmacodynamics of intravenous MDZ (5 microg/kg) and oral MDZ (15 microg/kg) were assessed on days 8 and 9, respectively. The pharmacodynamics of MDZ was estimated by measuring peak saccadic velocity, postural away length, critical fusion flicker frequency, and visual analogue scale. UDCA did not affect the pharmacokinetic and pharmacodynamic parameters of intravenous and oral MDZ administrations. Our study suggests that the clinical dosage of UDCA could not affect both hepatic and intestinal CYP3A activities and that the drug interaction between UDCA and substrates for CYP3A is unlikely in humans.