Hepatic Cytochrome P450 3A Research Articles

Clinical Pharmacokinetics and Summary of Efficacy and Tolerability of Atazanavir

The efficacy of HIV-1 protease inhibitors (PIs) as part of highly active antiretroviral therapy is now well established and has provided benefits to many patients with HIV infection. Atazanavir is a new azapeptide PI compound that was recently approved in the US and Europe. Atazanavir is recommended in combination with other antiretroviral agents for the treatment of HIV-1 infection. Atazanavir is rapidly absorbed and administration of a single dose of atazanavir with a light meal resulted in a 70% increase in area under the plasma concentration-time curve (AUC); therefore atazanavir should be taken with food. Atazanavir is 86% bound to human serum protein independently of concentration. Concentration in body fluids appeared to be lower than plasma concentration. Like other PIs, atazanavir is extensively metabolised by hepatic cytochrome P450 (CYP) 3A isoenzymes. The mean terminal elimination half-life in healthy volunteers was approximately 7 hours at steady state following administration of atazanavir 400 mg daily with a light meal. When atazanavir 300 mg was coadministered with ritonavir 100 mg on a once-daily dosage regimen, atazanavir AUC from 0 to 24 hours and minimum plasma concentration were increased by 3- to 4-fold and approximately 10-fold, respectively, compared with atazanavir 300 mg alone. Therefore, ritonavir boosted atazanavir regimen (ritonavir 100 mg and atazanavir 300 mg once daily) is increasingly favoured in some patients. Efavirenz, a potent CYP3A inducer, decreased atazanavir concentrations by 75% and, unexpectedly, tenofovir, a nucleotide reverse transcriptase inhibitor, decreased atazanavir concentrations by 25%. Average predose concentrations in HIV-infected patients who received atazanavir 400mg once daily were 273 ng/mL, which was believed to be several-fold higher than protein-binding corrected 50% inhibitory concentration of wild-type viruses. In HIV-infected patients who received once-daily ritonavir (100mg) boosted atazanavir (300 mg), mean (+/-SD) trough concentration was 862 (+/-838) ng/mL. Several clinical trials showed the efficacy of atazanavir 400 mg once daily with a nucleoside analogue backbone in antiretroviral-naive patients. The atazanavir 300/ritonavir 100 mg once-daily combination coadministered with other antiretrovirals showed the efficacy of this strategy in patients receiving efavirenz or in moderately antiretroviral-experienced HIV-infected patients. Recommended once-daily doses of atazanavir taken with food are either 400 mg or 300 mg in combination with low dose ritonavir (100 mg) in moderately antiretroviral-experienced patients. Major advantages of atazanavir to date are its simplicity of administration (once-daily administration) and its less undesirable effect on the lipid profiles in patients.

Effects of naringin on the pharmacokinetics of verapamil and one of its metabolites, norverapamil, in rabbits

It was reported that verapamil is metabolized via hepatic microsomal cytochrome P450 (CYP) 3A4 and that naringin (a component of grapefruit juice) inhibits CYP3A4 in humans. Hence, after oral administration of verapamil, the total area under the plasma concentration-time curve from time zero to time infinity (AUC) of verapamil and the AUC(verapamil)/AUC(D-617 (a metabolite of verapamil)) ratio were significantly greater after oral grapefruit juice in humans. The aim of this study was to determine whether similar results could be obtained from rabbits. The pharmacokinetics of verapamil and one of its metabolites, norverapamil, were investigated after oral administration of verapamil at a dose of 9 mg/kg without or with oral naringin at a dose of 7.5 mg/kg in rabbits. With naringin, the AUC of verapamil was significantly greater (28.4 versus 18.4 microg min/ml). Although, the AUC values of norverapamil were not significantly different between groups without and with naringin, the AUC(verapamil)/AUC(norverapamil) ratio was considerably greater (1.49 versus 1.11) with naringin. The above data suggested that the metabolism of verapamil and the formation of norverapamil was inhibited by naringin possibly by inhibition of CYP3A in rabbits.

The pharmacokinetics of nelfinavir and M8 during pregnancy and post partum

The objective of this study was to explore the pharmacokinetics of nelfinavir and its active metabolite hydroxy-t-butylamidenelfinavir (M8) during pregnancy and post partum. Eleven human immunodeficiency virus type 1-infected pregnant women receiving 1250 mg nelfinavir twice daily were enrolled. Pharmacokinetics of nelfinavir and M8 were assessed over a 12-hour period during pregnancy (median, 32 weeks' gestation; range, 31-36 weeks) and post partum (median, 8 weeks post partum; range, 6-15 weeks). Drug concentrations were analyzed by HPLC coupled to tandem mass spectroscopy, and pharmacokinetic parameters were calculated by use of noncompartmental methods. The median area under the plasma concentration-time curve from 0 to 12 hours (AUC 0-12), the maximal plasma concentration (C max), and the concentration at the end of the dosing interval (C 12) for nelfinavir post partum were 33.5 h . microg/mL, 5.80 microg/mL, and 1.40 microg/mL, respectively. The values for the geometric mean ratio (GMR) (third trimester/post partum) for the nelfinavir AUC 0-12 , C max , and C 12 were 0.76 (90% confidence interval [CI], 0.54-1.06), 0.81 (90% CI, 0.57-1.15), and 0.43 (90% CI, 0.25-0.76), respectively. The GMR values for the M8 AUC 0-12 , C max , and C 12 were 0.32 (90% CI, 0.18-0.55), 0.31 (90% CI, 0.19-0.51), and 0.30 (90% CI, 0.14-0.64), respectively. The median ratio values of the AUC 0-12 of M8 and nelfinavir (M8/nelfinavir) during the third trimester and post partum were 11% and 27%, respectively (GMR, 0.42 [90% CI, 0.33-0.53]). Nelfinavir exposure was reduced during pregnancy, and the reduction was statistically significant for C 12 . M8 concentrations were about 70% lower during pregnancy compared with post partum, suggesting either induction of hepatic cytochrome P450 (CYP) 3A4 or inhibition of CYP2C19, or both, during pregnancy. Because 8 of 11 women had subtherapeutic nelfinavir trough concentrations during pregnancy, the safety and efficacy of therapeutic drug monitoring should be investigated.

Genetically decreased CYP2A6 and the risk of tobacco dependence: a prospective study of novice smokers

Objective: Case control studies in adults suggest that defective alleles in the gene that codes for the hepatic cytochrome P450 2A6 (CYP2A6) protect against nicotine dependence (ND) and higher levels...

Assessment of cytochrome P450 1A in harbour seals (Phoca vitulina) using a minimally-invasive biopsy approach

Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.

Intravenous and oral alfentanil as in vivo probes for hepatic and first-pass cytochrome P450 3A activity: Noninvasive assessment by use of pupillary miosis

Systemic clearance of intravenous (IV) alfentanil (ALF) is an in vivo probe for hepatic cytochrome P450 (CYP) 3A activity, miosis is a surrogate for plasma ALF concentrations, and IV ALF miosis is a noninvasive probe for hepatic CYP3A. This investigation characterized the bioavailability and first-pass metabolism of oral ALF and tested the hypotheses that (1) first-pass ALF clearance reflects first-pass CYP3A activity, (2) miosis after oral ALF will reflect intestinal and hepatic CYP3A activity, and (3) miosis can approximate plasma concentration-based pharmacokinetic measures for IV and oral ALF as a noninvasive in vivo probe for hepatic and first-pass CYP3A activity and drug interactions. Results were compared with those for midazolam (MDZ), an alternative CYP3A probe. Ten volunteers were studied by use of a randomized, 9-way, crossover design after administration of rifampin (INN, rifampicin) (hepatic and intestinal CYP3A induction), troleandomycin (TAO) (hepatic and intestinal CYP3A inhibition), grapefruit juice (selective intestine CYP3A inhibition), or nothing (control). For each condition, they received 1 mg IV MDZ and then 15 microg/kg IV ALF, as well as 3 mg oral MDZ and then oral ALF (23 or 60 microg/kg) on another day. Plasma concentrations were determined by liquid chromatography-mass spectrometry. Dark-adapted pupil diameters were measured coincident with blood sampling. ALF effect was analyzed similarly to concentration to yield an effect "clearance" (Dose/Area under the pupil diameter change versus time curve). Bioavailability (Foral), hepatic extraction (EH), and intestinal availability (FG) were 0.26 +/- 0.08, 0.52 +/- 0.09, and 0.56 +/- 0.20, respectively, for MDZ and 0.42 +/- 0.15, 0.28 +/- 0.09, and 0.56 +/- 0.18, respectively, for ALF. Oral clearance (CL/F) was 34.7 +/- 12.8 and 10.9 +/- 3.5 mL.kg -1.min -1 , respectively, for MDZ and ALF. After rifampin, TAO, and grapefruit juice, ALF F oral was 0.04 +/- 0.02 (P <.05, versus control), 0.99 +/- 0.18 (P <.05, versus control), and 0.62 +/- 0.18 (P <.05, versus control), respectively; E H was 0.69 +/- 0.14 (P < .05, versus control), 0.04 +/- 0.01 (P <.05, versus control), and 0.26 +/- 0.08, respectively; F G was 0.16 +/- 0.10 (P <.05, versus control), 1.0 +/- 0.2 (P <.05, versus control), and 0.85 +/- 0.30 (P <.05, versus control), respectively; CL/F was 339 +/- 233 (P <.05, versus control), 0.62 +/- 0.26 (P <.05, versus control), and 6.7 +/- 2.5 (P <.05, versus control), respectively, and effect clearance was 2.1 +/- 1.1 (P <.05, versus control), 0.087 +/- 0.056 (P <.05, versus control), and 0.54 +/- 0.30 (0.73 +/- 0.43 mg.mm -1.h -1 in controls), respectively. There were significant correlations between ALF and MDZ systemic clearances (r2= 0.92), EH (r2=0.93), and CL/F (r2= 0.97), as well as between oral ALF effect (miosis) clearance and oral clearance (r2=0.59). ALF and MDZ have similar intestinal extraction but low and intermediate hepatic extraction, respectively. Systemic and oral clearances of ALF are excellent in vivo probes for hepatic and first-pass CYP3A activities and drug interactions. Miosis was an acceptable surrogate for plasma ALF. ALF miosis may be a suitable noninvasive in vivo probe for both hepatic and first-pass CYP3A.

Allelic expression imbalance of the human CYP3A4 gene and individual phenotypic status.

The human cytochrome P450 3A4 (CYP3A4) plays a dominant role in the metabolism of numerous clinically useful drugs. Alterations in the activity or expression of this enzyme may account for a major part of the variation in drug responsiveness and toxicity. However, it is generally accepted that most of the known single nucleotide polymorphisms in the coding and 5'-flanking regions are not the main determinants for the large inter-individual variability of CYP3A4 expression and activity. We show that the allelic variation is critically involved in determining the individual total hepatic CYP3A4 mRNA level and metabolic capability. There exists a definite correlation between the total CYP3A4 mRNA level and allelic expression ratio, the relative transcript level ratio derived from the two alleles. Individuals with a low expression ratio, exhibiting a large difference of transcript level between the two alleles, revealed extremely low levels of total hepatic CYP3A4 mRNA, and thus low metabolic capability as assessed by testosterone 6beta-hydroxylation. These results present a new insight into the individualized CYP3A4-dependent pharmacotherapy and the importance of expression imbalance to human phenotypic diversity.

CYP3A4 and P-Glycoprotein Activity in Healthy Controls and Transplant Patients on Cyclosporin vs. Tacrolimus vs. Sirolimus

This study aimed to determine the impact of maintenance immunosuppressive therapy with cyclosporin A (CsA), tacrolimus (FK506) and sirolimus (Rapa) on the in vivo activity of both intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (PGP) in renal transplant patients. The activity of these four elimination pathways was measured by the recently validated intravenous (iv) and per oral (po)14C erythromycin breath and urine test. In addition, overall hepatic P450 activity was measured by the 13C aminopyrin breath test. Three groups of stable renal transplant patients on maintenance therapy with corticosteroids (CS) and mycophenolate mofetil (MMF) plus either CsA or FK506 or Rapa were examined. A significant increase in intestinal CYP3A4 activity and a significant decrease in hepatic and intestinal PGP activity was seen in patients on CsA in comparison with those on FK506 or Rapa (p < 0.01). A similar analysis in six healthy volunteers at baseline and after intake of CsA, FK506 and Rapa confirmed the results seen in the patients. There was no difference in CYP3A4 and PGP activity in the patients taking either FK506 or Rapa and healthy controls. These data suggest that a different pattern of drug interactions might be expected in patients treated with CsA vs. FK506/Rapa.

Evidence for concurrent effects of exposure to environmental cadmium and lead on hepatic CYP2A6 phenotype and renal function biomarkers in nonsmokers.

We examined the interrelationships between phenotype of hepatic cytochrome P450 2A6 (CYP2A6), nephropathy, and exposure to cadmium and lead in a group of 118 healthy Thai men and women who had never smoked. Their urinary Cd excretion ranged from 0.05 to 2.36 μg/g creatinine, whereas their urinary Pb excretion ranged from 0.1 to 12 μg/g creatinine. Average age and Cd burden of women and men did not differ. Women, however, on average showed a 46% higher urinary Pb excretion (p < 0.001) and lower zinc status, suggested by lower average serum Zn and urinary Zn excretion compared with those in men. Cd-linked nephropathy was detected in both men and women. However, Pb-linked nephropathy was seen only in women, possibly because of higher Pb burden coupled with lower protective factors, notably of Zn (p < 0.001), in women compared with men. In men, Pb burden showed a negative association with CYP2A6 activity (adjusted β= −0.29, p = 0.003), whereas Cd burden showed a positive association with CYP2A6 activity (adjusted β= 0.38, p = 0.001), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. The weaker correlation between Cd burden CYP2A6 activity in women despite similarity in Cd burden between men and women is consistent with opposing effects of Pb and Cd on hepatic CYP2A6 phenotypic expression. A positive correlation between Cd-linked nephropathy (urinary N-acetyl-β-d-glucosaminidase excretion) and CYP2A6 activity in men (r = 0.39, p = 0.002) and women (r = 0.37, p = 0.001) suggests that Cd induction of hepatic CYP2A6 expression and Cd-linked nephropathy occurred simultaneously.

Effects of phenobarbital on metabolism and toxicity of diclofenac sodium in rat hepatocytes in vitro

Diclofenac sodium (DF-Na) was a nonsteroidal anti-inflammatory drug used in various aspects of inflammatory disease. The purpose of this study was to examine the effects of phenobarbital (PB) on metabolism and toxicity of DF-Na in vitro and explore the potential mechanism of DF-Na induced hepatotoxicity. Rat hepatocytes were isolated by a modification of the two-step in situ collagenase perfusion technique and the harvested rat hepatocytes were cultured with sandwich method. Control or PB (2 mM) pre-treated hepatocytes were incubated with DF-Na (0.1, 0.05 or 0.01 mM) in vitro and cytosolic enzyme leakage levels, cytochrome P450 (CYP) 3A activity, and metabolite content of DF-Na in cell culture medium were measured. The results showed that without any treatment hepatocyte CYP 3A activity gradually decreased with culture time. On day four, CYP 3A activity was 53% of the initial value. The decline of CYP 3A was partially reversed by CYP inducer PB, and the maximum induction of CYP 3A was 2.2-fold over control after continuous exposure of hepatocytes to 2 mM PB for 48 h. Lactic dehydrogenase (LDH), aspartate transaminase (AST), and alanine transamine (ALT) activity and the contents of the DF-Na metabolites 4′-hydroxydiclofenac (4′-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) in media appeared to increase with increasing DF-Na concentrations, though there were no significant differences between DF-Na exposed and control hepatocytes. However, if the hepatocytes first were pre-treated with 2 mM PB for 2 days and then exposed to DF-Na, the concentrations of DF-Na metabolites and the activity of LDH in the media were significantly higher than that of control group. These findings suggest that the hepatotoxicity and metabolism of DF-Na in rat hepatocytes are increased when hepatic CYP 3A activity is increased.

Alterations of CYP3A4 and P-glycoprotein activity in vivo with time in renal graft recipients

Catabolism by intestinal and hepatic cytochrome P450 3A4 (CYP3A4), and excretion by P-glycoprotein (PGP), has a major influence on oral bioavailability of calcineurin inhibitors. In this study, the activity of intestinal and hepatic CYP3A4 and PGP in vivo was assessed in renal transplant recipients during the first year after transplantation (Tx). Stable Caucasian renal transplant patients were tested at 1 week, 3 months, and 1 year after Tx, and compared with the results obtained in drug-free healthy volunteers. Intestinal and hepatic CYP3A4 and PGP activity were determined by measurement of (14)C-excretion dynamics in breath and urine after oral and intravenous administration of [N-methyl-(14)C]-erythromycin. Compared with 1 week after Tx, intestinal and hepatic CYP3A4 activity significantly decreased at 3 months and 1 year after Tx (-33% and -45%; -7% and -33%, respectively). Compared with the healthy volunteers, intestinal and hepatic CYP3A4 activity of the patients was significantly increased at 1 week after Tx, but normalized at 1 year after Tx. A similar pattern, though not significant, was seen for intestinal PGP activity. Phenotypic expression of hepatic and intestinal CYP3A4 was increased immediately after Tx, but gradually decreased to basal levels toward the end of the first year after Tx. The most plausible explanation for this evolution was the tapering of corticosteroid (CS) doses. These findings may also explain the increasing bioavailability of tacrolimus with time after Tx.

Pharmacokinetic changes in drugs during protein-calorie malnutrition: correlation between drug metabolism and hepatic microsomal cytochrome P450 isozymes.

The rats with protein-calorie malnutrition (PCM, 5% casein diet for a period of 4-week) were reported to exhibit 60 and 80% suppression in the hepatic microsomal cytochrome P450 (CYP) 1A2 and CYP2C11 levels, respectively, and 40-50% decreases in CYP2E1 and CYP3A1/2 levels compared to control (23% casein diet for a period of 4-week) based on Western blot analysis. In addition, Northern blot analysis showed that CYP1A2, CYP2E1, CYP2C11, and CYP3A1/2 mRNAs decreased in the state of PCM as well. Hence, pharmacokinetic changes of the drugs in rats with PCM [especially the area under the plasma concentration-time curve from time zero to time infinity (AUC) changes of metabolite(s)] reported from literatures were tried to explain in terms of CYP isozyme changes in the rats. Otherwise, the time-averaged nonrenal clearance (CL NR) of parent drug was compared. Pharmacokinetic changes of the drugs in other types of malnutritional state, such as kwashiorkor and marasmus, in both human and animal models were also compared. The drugs reviewed are as follows: diuretics, antibiotics, anticancer agents, antiepileptics, antiarrythmics, analgesics, xanthines, antimalarials, and miscellaneous.

Expression of Pregnane-X-Receptor Transcript in Peripheral Blood Mononuclear Cells and Correlation with Mdr1 Mrna

P-glycoprotein (P-gp) limits bioavailability and accumulation of HIV protease inhibitors (PIs). PIs are ligands for the pregnane-X-receptor (PXR), which regulates P-gp expression. This occurs when ligands activate the receptor, initiating binding to response elements in the MDR1 promoter. PXR also activates cytochrome P4503A4 (CYP3A4) and a correlation between hepatic PXR and CYP3A4 mRNA has been reported. We have examined the relationship between MDR1 and PXR mRNA in peripheral blood cells and demonstrate a significant correlation in 18 volunteers (R2=0.4; P<0.005). PXR was approximately 250-fold lower in peripheral blood mononuclear cells than in liver (1.6+/-1.2 vs 450+/-298; n=6; P<0.01).

Chronic fuel oil toxicity in American mink ( Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil

Petroleum oil enters the coastal marine environment through various sources; marine mammals such as sea otters that inhabit this environment may be exposed to low concentrations of petroleum hydrocarbons through ingestion of contaminated prey. The inability to perform controlled studies in free-ranging animals hinders investigations of the effects of chronic petroleum oil exposure on sea otter morbidity and mortality, necessitating the development of a reliable laboratory model. We examined the effects of oral exposure to 500 ppm bunker C fuel oil over 113–118 days on American mink, a species phylogenetically related to the sea otter. Hematological parameters and organs were examined for fuel oil-associated changes. Hepatic cytochrome P4501A1 mRNA expression and fecal cortisol concentrations were also measured. Ingestion of fuel oil was associated with a decrease in erythrocyte count, hemoglobin concentration (Hgb), hematocrit (HCT), and an increase in mean corpuscular volume (MCV). Total leukocytes were elevated in the fuel oil group from increases in neutrophils, lymphocytes, and monocytes. Significant interactions between fuel oil and antigen challenge were found for erythrocyte parameters, monocyte and lymphocyte counts. Liver and adrenal weights were increased although mesenteric lymph node weights were decreased in the fuel oil group. Hepatic cytochrome P4501A1 mRNA was elevated in the fuel oil group. Fecal cortisol concentration did not vary between the two groups. Our findings show that fuel oil exposure alters circulating leukocyte numbers, erythrocyte homeostasis, hepatic metabolism and adrenal physiology and establish a framework to use mink as a model for sea otters in studying the systemic effects of marine contaminants.

Involvement of cytochrome P450 1A in sanguinarine detoxication

Sanguinarine (SA), a member of the benzo[c]phenanthridine alkaloids, is a potent anti-microbial agent with anti-inflammatory and anti-neoplastic properties. However, toxicity of the alkaloid severely limits its medical applications. Recent report by Williams et al. [Vet. Hum. Toxicol. 42 (2000) 196] implicated rat hepatic cytochrome P450 (CYP) 1A2 as a likely modulator of SA toxicity. Indeed, the in vitro toxicity of SA in primary culture of rat hepatocytes and human hepatic cell line HepG2, demonstrated as lactate dehydrogenase leakage and metabolic capability (MTT assay), was diminished following induction of CYP1A by 2,3,7,8-tetrachlorodibenzo- p-dioxin, 3-methylcholanthrene, and β-naphtoflavone. Using microsomes containing recombinant CYP1A1 or CYP1A2 we show that SA causes non-competitive inhibition of the former and competitive inhibition of the latter as assessed by ethoxyresorufin de-ethylation (EROD). In human hepatic microsomes SA exhibits competitive inhibition of EROD activity with apparent K i of 2 μM, a value identical to that observed for CYP1A2 inhibition in recombinant system. Pre-incubation of SA with human liver microsomes resulted in time-dependent, but not dose-dependent decline in EROD activity suggesting CYP1A2 inhibition is not mechanism based. SA also inhibits activity of NADPH:CYP reductase, an enzyme required for CYP activity, with IC 50 very similar to that observed for EROD inhibition. Tentative mechanism for CYP1A involvement in decreased in vitro SA toxicity is discussed.

St. John's Wort ( Hypericum perforatum) induces overexpression of multidrug resistance protein 2 (MRP2) in rats: a 30-day ingestion study

St. John's Wort ( Hypericum perforatum, SJW) has been used as a herbal medicine for the treatment of depression in oral doses of 900–1050 mg/day in humans. However, the ingestion of SJW was reported to cause interactions with drugs. In the present study, we examined the effects of SJW treatment on the induction of drug transporters and enzymes in rats. An immunoblot analysis was performed to quantify the expression of the transporters and enzymes. SJW was given at a dose of 400 mg/kg/day, since it was reported that 400 mg/kg/day is antidepressant effective dose in rats. When SJW was administered for 10 days, the amounts of multidrug resistance protein 2 (MRP2), glutathione S-transferase-P (GST-P) and cytochrome P450 1A2 (CYP1A2) in the liver were increased to 304%, 252% and 357% of controls, respectively, although the amounts of P-glycoprotein and multidrug resistance protein 1 were not changed. Under the same conditions, an increase of MRP2 in the kidney was not observed. The increase in the levels of each protein was maximal at 10 days after SJW treatment and lasted for at least 30 consecutive days. These results suggest that SJW induces hepatic MRP2, GST-P and CYP1A2 overexpressions, and thus, it could affect drug metabolism, conjugation and disposition.

The variability of liver graft function and urinary 6beta-hydroxycortisol to cortisol ratio during liver regeneration in liver transplant recipients.

The urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) is considered to be the simplest and most practical method for estimation of hepatic cytochrome P450 3A4 (CYP3A4) activity as a non-invasive marker of human in vivo CYP3A4 activity. However, the inter- and intra-individual variability of the urinary 6beta-OHF/F ratio during liver regeneration and the effect of variability on optimal dose of tacrolimus have not yet been clarified. The objective of this study was to clarify the change in the urinary 6beta-OHF/F ratio during liver regeneration and to determine the effect of the liver graft function on the optimal tacrolimus dose in liver transplant recipients. Two liver transplant recipients (one male and one female) and eight healthy volunteers (five males and three females) were enrolled in this study. Urine samples were collected from the recipients from 08.00 hours for 24 h on post-transplant period, 1-10 and 21-30 days postoperatively. In the healthy volunteers, morning spot urine samples were collected at 08.00 hours. The mean urinary 6beta-OHF/F ratio in the immediate postoperative period was significantly low (p < 0.05). However, a marked difference in the regulation of CYP3A4 activity during liver regeneration was found in the two recipients. A significant correlation was found between the urinary 6beta-OHF/F ratio and the C/D ratio of tacrolimus (R = 0.658, p < 0.05). The urinary 6beta-OHF/F ratio is a useful probe for estimating the variability of CYP3A4 activity in liver transplant recipients in early postoperative phase. Future studies should evaluate the clinical usefulness of the urinary 6beta-OHF/F ratio as a predictor of tacrolimus pharmacokinetics in liver transplantation.

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[ p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC 50 of ∼5 × 10 −6 M (indirubin 3 ′-oxime EC 50 ∼5 × 10 −7 M, indirubin EC 50 ∼1 × 10 −7 M). A comparison of these EC 50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) (∼3 × 10 −9 M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo- p-dioxins. Competitive binding assays for AhR occupancy showed similar IC 50 values for indirubin and TCDBF (∼2 × 10 −9 and 5 × 10 −9 M), with the IC 50 values of indigo and indirubin 3 ′-oxime being ∼10-fold higher. When rats were treated with these indigoids in the range of 1.5–50 mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.

Should serum voriconazole (Vori) levels be monitored in allogeneic hematopoietic stem cell transplant (HSCT) recipients?

Vori has significant activity against Candida and Aspergillus. It is metabolized by the hepatic cytochrome P450 enzymes 2C19, 2C9 and 3A4. CYP2C19, which plays a significant role in the metabolism, exhibits significant genetic polymorphism. As a result, some patients metabolize Vori poorly. The clinical significance of this and resultant possible increases in Vori levels is unknown, and the utility of monitoring Vori levels is unclear. We monitored steady-state trough serum Vori levels in 22 allogeneic HSCT recipients using a new HPLC assay (Pennick et al. Antimicrob Agents Chemother 2003;47:2348–50). All had received non-myeloablative conditioning with 100 mg/m2 melphalan (+50 mg/kg cyclophosphamide if not prior autograft). GVHD prophylaxis comprised cyclosporine/tacrolimus with mycophenolate mofetil. Vori was started at 200 mg BID PO in 20 patients, and 400 mg BID for a day followed by 200 mg BID in 2. One had aspergillosis, and the rest were receiving Vori prophylactically or empirically. Patients had drug levels checked once (n = 12), twice (n = 8), or ≥3 times (n = 2); 5 days to 9 months (median 10 days) after starting Vori or dose modification. The intention was to achieve a level greater than 0.5–1.0 μg/mL corresponding to the in vitro MIC90 of Vori for Aspergillus spp. The 22 baseline levels ranged from 0.2 to 6.8 μg/mL (median 1.1). Overall, the 36 levels were 0.2–6.8 (median 1.7); with 5 levels <0.5. An increase in the dose to 300 mg twice daily in 3 patients (2 with levels <0.5 and 1 with a level of 1.0 in the presence of aspergillosis) increased the level 6- to 8-fold. There was no significant correlation between Vori levels and serum creatinine, bilirubin, and ALT. Vori levels correlated with alkaline phosphatase (r = 0.44; P = 0.008) and AST (r = 0.52; P = 0.001). Since liver dysfunction is relatively common after allogeneic HSCT, it was not possible to determine if elevated AST and alkaline phosphatase levels were the cause or the consequence of higher Vori levels. Based on these data, we conclude that trough Vori levels vary considerably between patients with a small proportion having potentially subtherapeutic levels on standard doses. We suggest checking serum Vori levels in patients receiving the drug for confirmed fungal infections. Additionally, patients developing elevation of AST or alkaline phosphatase on therapy should also have Vori levels monitored. Vori levels should also be checked if the drug dose is changed.

Severe reduction in tacrolimus levels with rifampin despite multiple cytochrome P450 inhibitors: a case report

Although the rifampin-cyclosporine interaction is well described, information on the extent, duration, and potency of the rifampin-tacrolimus interaction is limited. We describe a renal transplant recipient who demonstrated an increase in tacrolimus metabolism as a result of rifampin administration. A 40-year-old Asian woman received a cadaveric renal transplant for end-stage renal disease due to IgA nephropathy and was administered tacrolimus, thymoglobulin, mycophenolate mofetil, and prednisone, along with diltiazem for hypertension. On postoperative day (POD) 5, donor bronchioalveolar lavage revealed active tuberculosis. The recipient received rifampin 600 mg/d, and the diltiazem dose was increased. Over the next 12 days, the tacrolimus dose was increased to 32 mg/d to achieve a target trough level of 10 to 15 ng/mL, finally reached on POD34, when the serum creatinine was 145 μmol/L. The patient also received a course of fluconazole 100 mg/d and clarithromycin 1000 mg/d starting on POD38 and POD41, respectively. Despite this, there was no increase in tacrolimus levels. Rifampin was discontinued on POD76, after which therapeutic tacrolimus levels were finally attained with usual doses by POD132. Rifampin had potent and prolonged effects on tacrolimus metabolism. Induction of the hepatic cytochrome P4503A4 system by rifampin was sufficient to overcome the inhibitory effects of diltiazem; fluconazole, and clarithromycin, necessitating the use of large doses of tacrolimus. Close monitoring of tacrolimus levels and frequent dose adjustments are required whenever rifampin is administered posttransplant, regardless of P450 inhibitors used, to optimize allograft function.