Effects of intestinal flora on the expression of cytochrome P450 3A in the liver

Living organisms eliminate foreign low-antigenic substances, such as drugs and environmental pollutants, by detoxification mediated by metabolizing cytochrome P450 (CYP). We have examined the possible regulation of CYP expression by enteric bacteria. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in hepatic microsomal fractions were compared in germ-free (GF) and specific pathogen-free (SPF) mice. We evaluated hepatic Cyp3a11 mRNA expression levels and Cyp3a metabolic activity in GF and SPF mice after five days of antibiotic administration. The fecal levels of lithocholic acid (LCA)-producing bacteria and hepatic taurolithocholic acid (TLCA) were also measured. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in SPF mice were higher than those in GF mice, indicating that enteric bacteria increases hepatic Cyp3a expression. The effects of enteric bacteria-reducing antibiotics on Cyp3a expression were examined. We observed that decreasing enteric bacteria with antibiotics in SPF mice caused a significant decrease in the hepatic Cyp3a11 mRNA expression, TLCA, and fecal LCA-producing bacteria compared to the group that did not receive antibiotics. No change in Cyp3a11 expression was observed in GF mice that were treated with antibiotics. Administration of LCA to GF mice showed an increase in Cyp3a11 expression similar to that of SPF mice. The enzymes of the enteric bacteria are believed to metabolize and detoxify drugs by either reduction or hydrolysis. The results of this study indicate that changes in enteric bacteria may alter the expression and activity of hepatic drug metabolizing enzymes and pharmacokinetics. Therefore, enteric bacteria should be closely monitored to ensure the safe use of drugs.     

Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.     

Characterization of Intestinal and Hepatic CYP3A-Mediated Metabolism of Midazolam in Children Using a Physiological Population Pharmacokinetic Modelling Approach

Purpose

Changes in drug absorption and first-pass metabolism have been reported throughout the pediatric age range. Our aim is to characterize both intestinal and hepatic CYP3A-mediated metabolism of midazolam in children in order to predict first-pass and systemic metabolism of CYP3A substrates.

Methods

Pharmacokinetic (PK) data of midazolam and 1-OH-midazolam from 264 post-operative children 1–18 years of age after oral administration were analyzed using a physiological population PK modelling approach. In the model, consisting of physiological compartments representing the gastro-intestinal tract and liver,intrinsic intestinal and hepatic clearances were estimated to derive values for bioavailability and plasma clearance.

Results

The whole-organ intrinsic clearance in the gut wall and liver were found to increase with body weight, with a 105 (95% confidence interval (CI): 5–405) times lower intrinsic gut wall clearance than the intrinsic hepatic clearance (i.e. 5.08 L/h (relative standard error (RSE) 10%) versus 527 L/h (RSE 7%) for a 16 kg individual, respectively). When expressed per gram of organ, intrinsic clearance increases with increasing body weight in the gut wall, but decreases in the liver, indicating that CYP3A-mediated intrinsic clearance and local bioavailability in the gut wall and liver do not change with age in parallel. The resulting total bioavailability was found to be age-independent with a median of 20.8% in children (95%CI: 3.8–50.0%).

Conclusion

In conclusion, the intrinsic CYP3A-mediated gut wall clearance is substantially lower than the intrinsic hepatic CYP3A-mediated clearance in children from 1 to 18 years of age, and contributes less to the overall first-pass metabolism compared to adults.

     

Host microbiota dictates the proinflammatory impact of LPS in the murine liver

Gut microbiota can impact liver disease development via the gut-liver axis. Liver inflammation is a shared pathological event in various liver diseases and gut microbiota might influence this pathological process. In this study, we studied the influence of gut microbiota on the inflammatory response of the liver to lipopolysaccharide (LPS). The inflammatory response to LPS (1–10 μg/ml) of livers of specific-pathogen-free (SPF) or germ-free (GF) mice was evaluated ex vivo, using precision-cut liver slices (PCLS). LPS induced a more pronounced inflammatory response in GF PCLS than in SPF PCLS. Baseline TNF-α gene expression was significantly higher in GF slices as compared to SPF slices. LPS treatment induced TNF-α, IL-1β, IL-6 and iNOS expression in both SPF and GF PCLS, but the increase was more intense in GF slices. The anti-inflammatory markers SOCS3 and IRAK-M gene expression was significantly higher in GF PCLS than SPF PCLS at 24h with 1 µg/ml LPS treatment, and IL-10 was not differently expressed in GF PCLS than SPF PCLS. In addition, TLR-4 mRNA, but not protein, at basal level was higher in GF slices than in SPF slices. Taken together, this study shows that, in mice, the host microbiota attenuates the pro-inflammatory impact of LPS in the liver, indicating a positive role of the gut microbiota on the immune homeostasis of the liver.     

CYP3A-dependent drug metabolism is reduced in bacterial inflammation in mice

BACKGROUND AND PURPOSE

Gene expression of Cyp3a11 is reduced by activation of Toll-like receptors (TLRs) by Gram-negative or Gram-positive bacterial components, LPS or lipoteichoic acid (LTA) respectively. The primary adaptor protein in the TLR signalling pathway, TIRAP, plays differential roles in LPS- and LTA-mediated down-regulations of Cyp3a11 mRNA. Here, we have determined the functional relevance of these findings by pharmacokinetic/pharmacodynamic (PK/PD) analysis of the Cyp3a substrate midazolam in mice. Midazolam is also metabolized by Cyp2c in mice.

EXPERIMENTAL APPROACH

Adult male C57BL/6, TIRAP+/+ and TIRAP−/− mice were pretreated with saline, LPS (2 mg·kg−1) or LTA (6 mg·kg−1). Cyp3a11 protein expression, activity and PK/PD studies using midazolam were performed.

KEY RESULTS

Cyp3a11 protein expression in LPS- or LTA-treated mice was reduced by 95% and 60% compared with saline-treated mice. Cyp3a11 activity was reduced by 70% in LPS- or LTA-treated mice. Plasma AUC of midazolam was increased two- to threefold in LPS- and LTA-treated mice. Plasma levels of 1′-OHMDZ decreased significantly only in LTA-treated mice. Both LPS and LTA decreased AUC of 1′-OHMDZ-glucuronide. In the PD study, sleep time was increased by ∼2-fold in LPS- and LTA-treated mice. LTA-mediated decrease in Cyp3a11 protein expression and activity was dependent on TIRAP. In PK/PD correlation, AUC of midazolam was increased only in LPS-treated mice compared with saline-treated mice.

CONCLUSIONS AND IMPLICATIONS

LPS or LTA altered PK/PD of midazolam. This is the first study to demonstrate mechanistic differences in regulation of metabolite formation of a clinically relevant drug by Gram-negative or Gram-positive bacterial endotoxins.     

Midazolam and cyclosporin a metabolism in transgenic mice with liver-specific expression of human CYP3A4.

Cytochrome P450 3A4 (CYP3A4) is a major determinant of the metabolism of many drugs, including important anticancer drugs, with sometimes profound impact on therapeutic efficacy and toxic side effects. To study in vivo CYP3A(4) functions, we have generated and characterized transgenic mice with functional expression of human CYP3A4 cDNA in the liver. Two transgenic lines displayed substantial, physiologically relevant and stable CYP3A4 levels in liver and moderate levels in kidney, but not in small intestine. The mice did not display obvious physiological abnormalities. The CYP3A4 substrate drugs midazolam and cyclosporin A were used to test functional activity of CYP3A4 in liver. The area under the plasma concentration versus time curve (AUC) of intravenously administered midazolam (30 mg/kg) was 2.2-fold decreased in the transgenic mice compared with wild-type (5.45 +/- 0.21 versus 11.7 +/- 0.46 microg . h ml(-1); P < 0.01), and early formation of the primary metabolite 1-hydroxymidazolam was about 2-fold increased, demonstrating the functionality of CYP3A4 in the liver. Similarly, following intravenous administration of cyclosporin A (20 mg/kg), CYP3A4 transgenic mice displayed a reduced plasma AUC compared with wild-type (24.3 +/- 0.66 versus 35.8 +/- 0.53 microg . h ml(-) (1); P < 0.01). Thus, midazolam and cyclosporin A, compounds with markedly different clearance rates and half-lives, both demonstrated clearly accelerated kinetics in the CYP3A4 transgenic mice. We expect that this CYP3A4 transgenic model will provide a useful tool to study the impact of CYP3A4 on drug levels, especially when combined with other transgenic and knockout strains.     

Role of CYP1A2 and CYP2E1 in the pentoxifylline ciprofloxacin drug interaction

In this study the drug interaction between ciprofloxacin (CIPRO) and pentoxifylline (PTX) was investigated and the role of CYP1A2 in the drug interaction was determined with the aid of a selective CYP1A2 inhibitor, furafylline (FURA), and the Cyp1A2 knockout mouse. Serum concentrations of PTX (83.4+/-1 micromol/l) and metabolite-1 (M-1) (13.7+/-2.8 micromol/l) following a single injection of PTX (100 mg/kg i.p.) were significantly higher (P<0.05) in mice treated with CIPRO (25 mg/kg i.p. 9 days) compared to serum concentrations of PTX (46.3+/-0.5 micromol/l) and M-1 (6.4+/-1.1 micromol/l) in mice administered saline. Murine hepatic microsomes were incubated with PTX alone or the combination of PTX and CIPRO. The metabolism of PTX in the murine hepatic microsomes containing both CIPRO and PTX was significantly decreased compared to microsomes incubated with PTX alone, suggesting that CIPRO may inhibit the metabolism of PTX. To further clarify the role of CYP1A2 in the metabolism of PTX in mice, the effect of a selective CYP1A2 mechanism based inhibitor, FURA, on the metabolism of PTX was investigated and our results indicate that FURA inhibited metabolism of PTX. We then investigated PTX elimination in the Cyp1A2 knockout mouse. Blood levels of PTX were assessed at 2 and 20 min following a single injection of PTX (32 mg/kg i.v). Serum concentration of PTX was determined in Cyp1A2 knockout mice compared to Cyp1A2 wild type control mice. The serum concentration of PTX in Cyp1A2 wild type mice (n=9) was 22.2+/-3.2 micromol/l at 20 min following injection of PTX. The serum concentration of PTX in Cyp1A2 knockout mice (n=11) was significantly elevated at 20 min following injection of PTX compared to Cyp1A2 wild type mice. These results clearly indicate that inhibition of CYP1A2 catalytic activity that occurs in the Cyp1A2 knockout mice is sufficient to alter metabolism of PTX and result in markedly elevated levels in serum of Cyp1A2 knockout mice. The results of Western analysis in murine microsomes suggest that CYP1A2 protein levels were not altered by CIPRO indicating that CIPRO did not downregulate Cyp1A2. The results of Western analysis also indicated that CIPRO treatment increased CYP2E1 in mouse microsomes and the implications of these will be discussed.     

Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.     

Midazolam and cortisol metabolism before and after CYP3A induction in humans

INTRODUCTION: CYP3A is responsible for the metabolism of numerous endogenous and exogenous compounds. Several substrates of CYP3A have been investigated to assess the CYP3A-metabolizing capacity of an individual in an attempt to predict the rate of metabolism of other CYP3A substrates. Two such tests of CYP3A activity are the midazolam plasma clearance after its intravenous administration and the 6beta-OH cortisol urinary ratio. Possible correlations between these 2 tests were investigated before and after treatment with rifampin in a group of healthy volunteers. METHODS: Pharmacokinetic parameters of midazolam and 6beta-OH cortisol urinary ratio were evaluated in 8 volunteers before and after 6 days treatment with rifampin, a potent inducer of CYP3A, and after cessation of rifampin treatment. RESULTS: Midazolam systemic clearance and the 6beta-OH cortisol urinary ratio were significantly higher at Days 7 and 10 than at Day 0. There was a strong positive correlation between these 2 parameters (r = 0.70, p < 0.001). In contrast, no correlation was observed between the ratio of the AUCs of 1'-OH midazolam vs. midazolam (AUC0-1(1'-OH)/AUC0-t(MDZ)) or the ratio of plasma concentration of 1'-OH midazolam vs. midazolam (C30 min(1'-OH)/C30 min(MDZ)) and the 6beta-OH cortisol urinary ratio (r = 0.05, p = 0.82; r = 0.04, p = 0.88, respectively). Considering only data obtained before or after treatment with rifampin, however, no correlation was observed between midazolam systemic clearance and the 6beta-OH cortisol urinary ratio. CONCLUSIONS: These data demonstrate that there is a strong positive correlation between systemic midazolam clearance and 6beta-OH cortisol urinary ratio before and after induction. This suggests that the 6beta-OH cortisol urinary ratio test is a non-invasive alternative to the use of systemic midazolam clearance for monitoring the time-course of CYP3A induction.     

Enzyme-catalyzed processes of first-pass hepatic and intestinal drug extraction

Oral bioavailability of pharmacologically effective drugs is often limited by first-pass biotransformation. In humans, both hepatic and intestinal enzymes can catalyze the metabolism of a drug as it transits between the gastrointestinal lumen and systemic blood for the first time. Although a spectrum of drug biotransformations can occur during first-pass, the most common are oxidations catalyzed by cytochromes P450. It is the isozymes CYP2D6, CYP3A4, CYP1A2, CYP2C9 and CYP2C19 that are most often implicated in first-pass drug elimination. For any given substrate, enzyme specificity, enzyme content, substrate binding affinity and sensitivity to irreversible catalytic events all play a role in determining the overall efficiency, or intrinsic clearance, of elimination. Several models have been proposed over the past twenty-five years that mathematically describe the process of drug extraction across the liver. The most widely used, the well-stirred model, has also been considered for depiction of first-pass drug elimination across the intestinal wall. With these models it has been possible to examine sources of interindividual variability in drug bioavailability including, variable constitutive enzyme expression (both genetic and environmentally determined), enzyme induction by drugs, disease and diet, and intrinsic or acquired differences in plasma protein binding and organ blood flow (food and drug effects). In recent years, the most common application of hepatic clearance models has been the determination of maximum organ availability of a drug from in vitro derived estimates of intrinsic metabolic clearance. The relative success of the in vitro-in vivo approach for both low and highly extracted drugs has led to a broader use by the drug industry for a priori predictions as part of the drug selection process. A considerable degree of effort has also been focused on gut wall first-pass metabolism. Important pathways of intestinal Phase II first-pass metabolism include the sulfation of terbutaline and isoproterenol and glucuronidation of morphine and labetalol. It is also clear that some of the substrates for CYP3A4 (e.g., cyclosporine, midazolam, nifedipine, verapamil and saquinavir) undergo significant metabolic extraction by the gut wall. For example, the first-pass extraction of midazolam by the intestinal mucosa appears, on average, to be comparable to extraction by the liver. However, many other CYP3A substrates do not appear susceptible to a gut wall first-pass, possibly because of enzyme saturation during first-pass or a limited intrinsic metabolic clearance. Both direct biochemical and indirect in vivo clearance data suggest significant inter-individual variability in gut wall CYP3A-dependent metabolism. The source of this constitutive variability is largely unknown. Because of their unique anatomical location, enzymes of the gut wall may represent an important and highly sensitive site of metabolically-based interactions for orally administered drugs. Again, interindividual variability may make it impossible to predict the likelihood of an interaction in any given patient. Hopefully, though, newer models for studying human gut wall metabolic extraction will provide the means to predict the average extraction ratio and maximum first-pass availability of a putative substrate, or the range of possible inhibitory or inductive changes for a putative inhibitor/inducer.     

Intraindividual variability in male hepatic CYP3A4 activity assessed by alfentanil and midazolam clearance.

Clinical investigations using isoform-selective probes to phenotype cytochrome P450 activity and interaction studies using isoform-selective inhibitors to determine P450 involvement in drug metabolism assume minimal interday variability in P450 activity. CYP3A4 is the most abundant human P450 isoform and metabolizes approximately half of all therapeutic agents. This investigation evaluated interday variability in hepatic CYP3A4 activity in males, using the clearances of midazolam and alfentanil as metabolic probes. Midazolam (1 mg) followed 1 hour later by alfentanil (20 micrograms/kg) were administered by intravenous bolus to 9 nonsmoking male volunteers (ages 30 +/- 8 years). Drug administration was repeated 12 and 20 days later. Venous plasma midazolam and alfentanil concentrations were determined by gas chromatography/mass spectrometery. Drug clearances were determined by noncompartmental and multiexponential analysis. There were no significant interday differences in plasma drug concentrations or clearances (3.9 +/- 1.4, 3.9 +/- 1.7, and 4.2 +/- 1.7 ml/kg/min for alfentanil, respectively, and 6.6 +/- 2.0, 7.9 +/- 2.4, and 7.9 +/- 2.5 ml/kg/min for midazolam, respectively, on days 1, 13, and 21 [mean +/- SD]). Interday variability in clearance was 13% +/- 6% and 19% +/- 12% for alfentanil and midazolam, respectively. Interday variability in the clearance of these probes, and presumably hepatic CYP3A4 activity, was small compared with interindividual variability. Consideration of interday variability in the hepatic metabolism of CYP3A4 substrates does not appear significant in the design of clinical trials.     

CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH-cytochrome P450 reductase activity

Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ. Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1'-OH-MDZ. Significantly higher K(m) and lower V(max) for the formation of 1'-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P?=?0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1'-OH-MDZ displayed Michaelis-Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity. The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.     

Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate

CYP1A1 and CYP1B1 metabolically activate many polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene, to reactive intermediates associated with toxicity, mutagenesis, and carcinogenesis. Paradoxically, however, Cyp1a1-/- knockout mice are more sensitive to oral benzo[a]pyrene exposure, compared with wild-type Cyp1a1+/+ mice (Mol Pharmacol 65:1225, 2004). To further investigate the mechanism for this enhanced sensitivity, Cyp1a1-/-, Cyp1a2-/-, and Cyp1b1-/- single-knockout, Cyp1a1/1b1-/- and Cyp1a2/1b1-/- double-knockout, and Cyp1+/+ wild-type mice were analyzed. After administration of oral benzo[a]pyrene (125 mg/kg/day) for 18 days, Cyp1a1-/- mice showed marked wasting, immunosuppression, and bone marrow hypocellularity, whereas the other five genotypes did not. After 5 days of feeding, steady-state blood levels of benzo[a]pyrene were approximately 25 and approximately 75 times higher in Cyp1a1-/- and Cyp1a1/1b1-/- mice, respectively, than in wild-type mice. Benzo[a]pyrene-DNA adduct levels were highest in liver, spleen, and marrow of Cyp1a1-/- and Cyp1a1/1b1-/- mice. Many lines of convergent data obtained with oral benzo[a]pyrene dosing suggest that: 1) inducible CYP1A1, probably in both intestine and liver, is most important in detoxication; 2) CYP1B1 in spleen and marrow is responsible for metabolic activation of benzo[a]pyrene, which results in immune damage in the absence of CYP1A1; 3) both thymus atrophy and hepatocyte hypertrophy are independent of CYP1B1 metabolism but rather may reflect long-term activation of the aryl hydrocarbon receptor; and 4) the magnitude of immune damage in Cyp1a1-/- and Cyp1a1/1b1-/- mice is independent of plasma benzo[a]pyrene and total-body burden and clearance. Thus, a balance between tissue-specific expression of the CYP1A1 and CYP1B1 enzymes governs sensitivity of benzo[a]pyrene toxicity and, possibly, carcinogenicity.     

Lack of correlation between in vitro inhibition of CYP3A-mediated metabolism by a PPAR-gamma agonist and its effect on the clinical pharmacokinetics of midazolam, an in vivo probe of CYP3A activity

RG 12525 (2-[[4-[[2-(1H-tetrazole-5-ylmethyl)phenyl]methoxy]phenoxy]methyl] quinolone) is a novel peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist. In vitro microsomal inhibition assays indicated that RG 12525 is a potent inhibitor of CYP3A4, with a Ki value of 0.5 microM. With the conservative assumption that the total plasma concentration of drug was available to metabolic enzymes following RG 12525 oral administration, marked inhibition of CYP3A4 was expected to substantially reduce the systemic clearance of compounds metabolized by this enzyme. The possibility also existed for inhibition of intestinal and hepatic CYP3A4 by RG 12525 to reduce "first-pass" metabolism and increase absolute bioavailability of CYP3A4 substrates orally coadministered. Consequently, an in vivo drug-drug interaction study was performed to evaluate the effects of orally administered RG 12525 on in vivo CYP3A4 activity in healthy male subjects. The pharmacokinetics of oral midazolam, a probe for intestinal and hepatic CYP3A activity, was not influenced by either the low (100 mg qd for 4 days) or high (600 mg qd for4 days) RG 12525 dosing regimen despite the resulting total plasma concentrations of inhibitor that were well above in vitro Ki values. The point estimates and 90% confidence intervals for the ratios of mean midazolam AUC for subjects administered 100 mg RG 12525 (110.6; 98.7-124.1) and 600 mg RG 12525 (98.4; 84.4-114.7) versus midazolam alone were within 80% to 125%. To explain these results, factors that could limit the accuracy of in vitro models in predicting metabolic drug interactions, mainly the high degree of RG 12525 protein binding (> 99.9%), were considered. The lack of correlation between the in vitro inhibition of CYP3A4 by RG 12525 and the inconsequential effects of this compound on midazolam pharmacokinetics accentuate the need to recognize factors other than plasma drug concentrations and potency of in vitro enzyme inhibition when extrapolating in vitro data to predict in vivo drug-drug interactions.     

A critical analysis of the interplay between cytochrome P450 3A and P-glycoprotein: recent insights from knockout and transgenic mice

CYP3A is one of the most important drug-metabolizing enzymes, determining the first-pass metabolism, oral bioavailability, and elimination of many drugs. It is also an important determinant of variable drug exposure and is involved in many drug-drug interactions. Recent studies with CYP3A knockout and transgenic mice have yielded a number of key insights that are important to consider during drug discovery and development. For instance, studies with tissue-specific CYP3A-transgenic mice have highlighted the importance of intestinal CYP3A-dependent metabolism. They also revealed that intestinal CYP3A plays an important role in the regulation of various drug-handling systems in the liver. Intestinal CYP3A activity can thus have far-reaching pharmacological effects. Besides CYP3A, the active drug efflux transporter P-glycoprotein also has a strong effect on the pharmacokinetics of numerous drugs. CYP3A and P-glycoprotein have an extensive overlap in their substrate spectrum. It has been hypothesized that for many drugs, the combined activity of CYP3A and P-glycoprotein makes for efficient intestinal first-pass metabolism of orally administered drugs as a result of a potentially synergistic collaboration. However, there is only limited in vitro and in vivo evidence for this hypothesis. There has also been some confusion in the field about what synergy actually means in this case. Our recent studies with Cyp3a/P-glycoprotein combination knockout mice have provided further insights into the CYP3A-P-glycoprotein interplay. We here present our view of the status of the synergy hypothesis and an attempt to clarify the existing confusion about synergy. We hope that this will facilitate further critical testing of the hypothesis and improve communication among researchers. Above all, the recent findings and insights into the interplay between CYP3A and P-glycoprotein may have implications for improving oral drug bioavailability and reducing adverse side effects.     

PXR-mediated induction of human CYP3A4 and mouse Cyp3a11 by the glucocorticoid budesonide

Budesonide, a glucocorticoid with a high first-pass metabolism, is used for the oral treatment of inflammatory bowel disease. Cytochrome P450 3A4 (CYP3A4) is an enzyme involved in the metabolism of numerous drugs, including budesonide. Since inhibition or induction of CYP3A4 is often the cause of drug–drug interactions we analyzed how budesonide affects the activity and expression of this enzyme. CYP3A4 activity was assessed by the metabolism of a luminogenic substrate (luciferin-benzylether) using recombinant human CYP3A4 protein. We observed no inhibition of the metabolism in the presence of budesonide at concentrations up to 25 μM. Induction experiments in human LS180 colon carcinoma cells showed an increased expression of CYP3A4 mRNA after budesonide treatment. Transactivation assays revealed that budesonide activates the CYP3A4 promoter via the pregnane X receptor (PXR). In mice, oral budesonide administration (25 mg/kg) for 4 days induced the murine homolog Cyp3a11 in the intestine 3-fold, whereas liver expression was notably less influenced. In knockout mice devoid of PXR, budesonide-mediated inductions were reduced compared to wild-type mice. In conclusion, we could demonstrate that budesonide is not an efficient inhibitor but rather an inducer of CYP3A via a PXR-mediated mechanism. In vivo, however, oral budesonide administration to mice showed only modest gene induction, which occurred mainly in the intestine. Therefore, the risk for budesonide-mediated drug interactions seems to be low but cannot be ruled out entirely.     

Inhibitory effect of docosahexaenoic acid (DHA) on the intestinal metabolism of midazolam: in vitro and in vivo studies in rats

The aim of this study was to evaluate the effects of docosahexaenoic acid (DHA) on the intestinal cytochrome P450 isoenzyme (CYP3A) and P-glycoprotein (P-gp) functions using midazolam and rhodamine-123 as specific substrates of CYP3A and P-gp, respectively. Perfused everted intestinal segments from rats were employed to determine the effects of DHA on midazolam metabolism and rhodamine-123 transport. In addition, the effects of DHA on in vitro midazolam metabolism in rat intestinal microsomes and on midazolam bioavailability in rats were examined. The intestinal extraction ratio (ER G) of midazolam was determined to be 0.43 and decreased significantly to 0.12, 0.07, and 0.06 in the presence of 50, 100, and 200 microM DHA, respectively, in a concentration-dependent manner. The results from an in vitro study using rat intestinal microsomes demonstrated that DHA competitively inhibited the intestinal CYP3A activity with Ki of 15.7 and 27.1 microM for the formations of 1'-OH midazolam and 4-OH midazolam, respectively. Moreover, the oral administration of DHA (100mg/kg) increased the AUC infinity, Cmax, and oral bioavailability (F) of midazolam by about 50% in rats, without affecting the T 1/2, V dss/F, or CL tot/F. In contrast, DHA did not change the serosal-to-mucosal transport of rhodamine-123 in the perfused everted intestine and oral administration of DHA (100mg/kg) had no influence on the pharmacokinetics of intravenously administered midazolam in rats, thus suggesting that DHA has little effect on the intestinal P-gp activity and hepatic clearance of midazolam. This study provided the first direct evidence to show that DHA has an inhibitory effect on the intestinal pre-systemic metabolism of a CYP3A substrate and that DHA has little, if any, effect on the P-gp activity in the gut.     

Synthesis of carbon‐14 labelled midazolam

Cytochrome P450 (CYP) enzymes are responsible for much of the phase I oxidative metabolism observed in vivo. Many important pharmaceutical compounds are metabolized by CYP. Co‐administration of a drug with another agent can alter the efficacy or the toxicity, especially in cases where drug clearance depends primarily on the CYP metabolism. Compounds that induce or inhibit the CYP activity are often used in drug–drug interaction studies. Midazolam is one such compound that is routinely used in drug–drug interaction studies because it is a known substrate for CYP3A enzymes. The synthesis of this important tool molecule has been documented, but unfortunately a detailed preparation of carbon‐14‐labelled midazolam has not been reported in the literature. This paper describes a two‐step synthesis leading to [¹⁴C]midazolam. A total of 4.5 mCi of [¹⁴C]midazolam was obtained having a specific activity of 120.1 µCi/mg (39.12 mCi/mmol). The radiochemical purity as determined by HPLC was 99.8% and the overall radiochemical yield was 9%. Copyright © 2009 John Wiley & Sons, Ltd.     

Menstrual cycle variability in midazolam pharmacokinetics

Activity of cytochrome P450 3A4 (CYP3A4), the most abundant human P450 isoform and responsible for metabolizing approximately half of all therapeutic agents, has been speculated to vary during the menstrual cycle. This investigation evaluated CYP3A4 activity during the menstrual cycle, using midazolam clearance as a metabolic probe. Midazolam (1 mg i.v.) was administered to nonsmoking, nonpregnant female volunteers (N = 11, age 26 +/- 5 years) with normal menstrual cycles on three separate occasions during the same cycle: days 2 (menstrual phase), 13 (estradiol peak), and 21 (progesterone peak). Venous plasma midazolam concentrations were determined by gas chromatography-mass spectrometry. Midazolam clearance was determined by noncompartmental and compartmental analysis. Midazolam plasma disposition did not differ between phases of the menstrual cycle. There was no significant difference in any measure of midazolam clearance. Noncompartmental clearances (mean +/- SD) were 7.36 +/- 2.73, 6.34 +/- 3.59, and 6.23 +/- 2.04 ml/kg/min, respectively, on days 2, 13, and 21 of the menstrual cycle. These results suggest no difference in hepatic CYP3A4 activity on menstrual cycle days 2, 13, and 21. Consideration of menstrual cycle variability in the metabolism of CYP3A4 substrates does not appear indicated in the dosing or design of clinical trials.