Underlying genetic structure impacts the association between CYP2B6 polymorphisms and response to efavirenz and nevirapine.

OBJECTIVE: CYP2B6 variation predicts pharmacokinetic characteristics of its substrates. Consideration for underlying genetic structure is critical to protect against spurious associations with the highly polymorphic CYP2B6 gene. DESIGN: The effect of CYP2B6 variation on response to its substrates, nonnucleoside reverse transcriptase inhibitors (NNRTIs), was explored in the Women's Interagency HIV Study. METHODS: Five putative functional polymorphisms were tested for associations with virologic suppression within 1 year after NNRTI initiation in women naive to antiretroviral agents (n = 91). Principal components were generated to control for population substructure. Logistic regression was used to test the joint effect of rs3745274 and rs28399499, which together indicate slow, intermediate, and extensive metabolizers. RESULTS: Rs3745274 was significantly associated with virologic suppression [odds ratio = 3.61, 95% confidence interval (CI) 1.16-11.22, P trend = 0.03]; the remaining polymorphisms tested were not significantly associated with response. Women classified as intermediate and slow metabolizers were 2.90 (95% CI 0.79-12.28) and 13.44 (95% CI 1.66 to infinity) times as likely to achieve virologic suppression compared to extensive metabolizers after adjustment for principal components (P trend = 0.005). Failure to control for genetic ancestry resulted in substantial confounding of the relationship between the metabolizer phenotype and treatment response. CONCLUSION: The CYP2B6 metabolizer phenotype was significantly associated with virologic response to NNRTIs; this relationship would have been masked by simple adjustment for self-reported ethnicity. Given the appreciable genetic heterogeneity that exists within self-reported ethnicity, these results exemplify the importance of characterizing underlying genetic structure in pharmacogenetic studies. Further follow-up of the CYP2B6 metabolizer phenotype is warranted, given the potential clinical importance of this finding.

Induction of cytochrome P450 2B6 activity by the herbal medicine baicalin as measured by bupropion hydroxylation.

PURPOSE: This study investigated the effect of the herbal medicine baicalin on bupropion hydroxylation, a probe reaction for CYP2B6 activity related to different CYP2B6 genotype groups. METHOD: Seventeen healthy male volunteers (6 CYP2B6*1/*1, 6 CYP2B6*1/*6, and 5 CYP2B6*6/*6) received orally administered bupropion alone and during daily treatment with baicalin. Blood samples were taken up to 72 h after each bupropion dose, and pharmacokinetics profiles were determined on days 1 and 25 for bupropion and hydroxybupropion. RESULT: Baicalin administration increased hydroxybupropion maximum plasma concentration (C(max)) by 73% [90% confidence interval (CI), 44-108%; P < 0.01] and the area under the concentration time curve extrapolated to infinity (AUC(0-infinity)) of hydroxybupropion by 87% (90% CI, 48-137%; P < 0.01), with no change in the elimination half-life of hydroxybupropion. Baicalin increased the AUC(0-infinity) ratio of hydroxybupropion to bupropion by 63% (90% CI, 38-92%; P < 0.01). CONCLUSION: Baicalin significantly induced CYP2B6-catalyzed bupropion hydroxylation, and the effects of baicalin on other CYP2B6 substrate drugs deserve further investigation.

Efavirenz induces CYP2B6-mediated hydroxylation of bupropion in healthy subjects.

OBJECTIVE: To characterize the effect of efavirenz on bupropion hydroxylation as a marker of cytochrome P450 (CYP) 2B6 activity in healthy subjects. METHODS: Thirteen subjects received a single oral dose of bupropion SR 150 mg before and after 2 weeks of efavirenz administration for comparison of bupropion and hydroxybupropion pharmacokinetics. Efavirenz plasma concentrations were also assessed. Subjects were genotyped for CYP2B6 (G516T, C1459T, and A785G), CYP3A4 (A-392G), CYP3A5 (A6986G), and multidrug resistance protein 1 (C3435T). RESULTS: The area under the concentration vs. time curve ratio of hydroxybupropion:bupropion increased 2.3-fold after efavirenz administration (P=0.0001). Bupropion area under the concentration vs. time curve and Cmax decreased by 55% and 34%, respectively (P<0.002). None of the CYP2B6 or CYP3A genotypes evaluated were associated with a difference in bupropion or efavirenz clearance. The 2 individuals homozygous for multidrug resistance protein 1 3435-T/T had 2.5- and 1.8-fold greater bupropion and efavirenz clearance, respectively, relative to C/C and C/T individuals (P<0.05). CONCLUSIONS: Our results confirm that efavirenz induces CYP2B6 enzyme activity in vivo, as demonstrated by an increase in bupropion hydroxylation after 2 weeks of efavirenz administration.

Roles of nitric oxide in inflammatory downregulation of human cytochromes P450.

The purpose of this study was to determine the role of nitric oxide (NO) in the downregulation of human cytochrome P450 (CYP) enzymes and mRNAs by an inflammatory stimulus in cultured human hepatocytes. We focused on CYP2B6 because previous studies showed that rat CYP2B proteins undergo an NO-dependent degradation in response to inflammatory stimuli. To ensure high-level expression of CYP2B6, the inducer phenytoin was present at all times. Stimulation of cells with a mixture of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma (ILmix) downregulated CYP2B6 mRNA and protein to 9 and 19% of control levels. The NO donor NOC-18 downregulated CYP2B6 protein to 30% of control, with only a small effect on CYP2B6 mRNA. Nitric oxide synthase inhibitors attenuated the downregulation of CYP2B6 protein but not mRNA by ILmix. These findings demonstrate that the posttranscriptional NO-dependent downregulation of CYP2B enzymes, observed previously in rat hepatocytes, is conserved in human CYP2B6. This mechanism is specific for CYP2B6 among the enzymes tested. No evidence was found for regulation of CYP2E1 mRNA or protein by NO. NOC-18 treatment downregulated CYP3A4 mRNA to 50% of control. However, NOS inhibitors failed to block the effects of ILmix on CYP3A4 expression.

Increase of R-/S-methadone enantiomer concentration ratio in serum of patients treated with either nevirapine or efavirenz.

An increased methadone enantiomer ratio (R/S) was associated to both nevirapine (179%, n=5) and efavirenz (36%, n=9) treatments when compared with that of controls (n=52). Additionally, in four follow-up patients, both R- and S-methadone normalized concentrations decreased (19%-93%) while R/S increased (22%-314%) following nevirapine/efavirenz treatment. R/S decreased (42%) after non-compliance with efavirenz treatment. Therefore, the methadone-maintenance-treatment outcome should be evaluated when patients are treated with drugs which are supposed to induce CYP3A4 and CYP2B6 isoforms.

CYP2B6 genotype alters abstinence rates in a bupropion smoking cessation trial.

BACKGROUND: CYP2B6 is the primary enzyme involved in bupropion (Zyban; GlaxoSmithKline, Research Triangle Park, North Carolina) metabolism. Genetic polymorphisms in CYP2B6, such as CYP2B6*6, can alter bupropion metabolism and may affect bupropion treatment outcome. METHODS: Subjects participated in a smoking cessation clinical trial of bupropion versus placebo. The main outcome was a 7-day point prevalence abstinence rate measured 10 weeks after the start of treatment (i.e., end of treatment) and at the 6-month follow-up; secondary outcomes were severity of adverse effects, withdrawal, and urge to smoke. Subjects were haplotyped for the CYP2B6*6 variants. RESULTS: Among smokers in the CYP2B6*6 group (CYP2B6*1/*6 or CYP2B6*6/*6 genotype, n = 147, 45% of the population), bupropion produced significantly higher abstinence rates than placebo at the end of treatment (32.5% vs. 14.3%, p = .01) and at the 6-month follow-up (31.2% vs. 12.9%, p = .008). In contrast, bupropion was no more effective than placebo for smokers in the CYP2B6*1 group (CYP2B6*1/*1, n = 179) at the end of treatment (31.0% vs. 31.6%, p = .93) or at the 6-month follow-up (22.0% vs. 21.5%, p = .94). There was a significant genotype by treatment interaction at the end of treatment (odds ratio [OR] = 2.97, confidence interval [CI] = 1.05-8.40, p = .04), which was similar at 6-month follow-up (OR = 2.98, CI = .98-9.06, p = .05). CONCLUSIONS: These data suggest that smokers with the CYP2B6*6 genotype have a higher liability to relapse on placebo and that they may be good candidates for bupropion treatment for smoking cessation.

[Sex-related differences in the antinociceptive effect of some non-narcotic analgetics in rats: the role of biotransformation].

Non-narcotic analgetics sodium diclofenac, indomethacin, naproxen, nimesulid, ketorolac, and celebrex (cytochrome P-450(2)c substrates) produce more pronounced and prolonged analgesic effect in pubertate female rats than in males. This can be related to the slower elimination of drugs from the female organism. The liver of females is characterized by a lower content of cytochrome P-450 and by less pronounced activity of amidopyrine-N-, indomethacin-O-, and naproxen-O-demethylase activity. No sex-related differences in pharmacodynamics were observed for meloxicam, and ethoricoxib, benzofurocaine, and amison, and acetylsalicylic acid, which are the substrates predominantly for CYP3A.

Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices.

Recent studies have suggested that both constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B and 3A through a mechanism called cross-talk. In this study we intend to determine if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen structurally diverse compounds was evaluated by using rat precision-cut liver slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs in rat liver slices was quantified by real-time polymerase chain reaction. Rat PXR activation was measured by induction of luciferase activity in rat PXR reporter-gene system. Linear regression analysis of the fold of induction of mRNA in liver slices and the fold of luciferase activity in rat PXR reporter-gene system shows that a reasonable correlation (r2 = 0.6) exists between the CYP3A induction and the rat PXR activation. A much lower correlation was observed between CYP2B induction and the rat PXR activation (r2 = 0.1). The results from this study suggest that the PXR may play a major role in the induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay may be useful in a high-throughput screening to predict CYP3A induction in rats.

Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva.

In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription.

Diltiazem inhibits human intestinal cytochrome P450 3A (CYP3A) activity in vivo without altering the expression of intestinal mRNA or protein.

AIMS: To determine the effect of diltiazem on intestinal CYP3A activity and protein and mRNA expression in vivo in healthy subjects. METHODS: Intestinal biopsies were obtained from ten healthy controls and from ten healthy subjects after receiving diltiazem 120 mg bid for 7 days. Intestinal CYP3A activity, CYP3A4 protein and mRNA concentrations were quantified in both groups. Intestinal CYP3A activity was determined by incubation of small bowel homogenate with midazolam (25 microM) and NADPH for 5 min and the rate of formation of 1'-hydroxymidazolam was quantified. RESULTS: All subjects in the treatment group had detectable diltiazem concentration in the serum. While there was no significant difference in CYP3A4 protein and mRNA expression between the control and treatment groups, the formation of 1'-hydroxymidazolam (446 pmol min(-1) mg(-1) 6 (control) vs. 170 (CI 112, 228) pmol min(-1) mg(-1) 95% confidence interval (CI 269, 623) (diltiazem group)) was significantly reduced (P < 0.05). CONCLUSION: Diltiazem decreased small bowel CYP3A activity by 62% as a result of irreversible inhibition with no corresponding change in intestinal CYP3A4 mRNA or protein concentrations.

Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P450 3A metabolism.

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (P450)-activated anticancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, a panel of human liver microsomes activated MMDX with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6beta-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC(50) values in growth-inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells), and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that rat CYP3A1 and human CYP3A4 activated MMDX more efficiently than rat CYP3A2 and that human P450s 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in Chinese hamster ovary cells after stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that overexpresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t(1/2) of approximately 5.5 h at 37 degrees C. MMDX was rapidly activated by CYP3A at low ( approximately 1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2-h exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived, and cell-permeable cytotoxic metabolite and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.

Role of cytochrome P4503A in cysteine S-conjugates sulfoxidation and the nephrotoxicity of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A) in rats.

The volatile anesthetic sevoflurane is degraded to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) in anesthesia machines. FDVE is nephrotoxic in rats. FDVE undergoes glutathione conjugation, subsequent conversion to cysteine and mercapturic acid conjugates, and cysteine conjugate metabolism by renal beta-lyase, which is a bioactivation pathway mediating nephrotoxicity in rats. Recent in vitro studies revealed cytochrome P4503A-catalyzed formation of novel sulfoxide metabolites of FDVE cysteine-S and mercapturic acid conjugates in rat liver and kidney microsomes. FDVE-mercapturic acid sulfoxides were more toxic than other FDVE conjugates to renal proximal tubular cells in culture. Nevertheless, the occurrence and toxicological significance of FDVE sulfoxides formation in vivo remain unknown. This investigation determined, in rats in vivo, the existence, role of P4503A, and nephrotoxic consequence of FDVE conjugates sulfoxidation. Rats were pretreated with dexamethasone, phenobarbital, troleandomycin, or nothing (controls) before FDVE, and then, nephrotoxicity, FDVE-mercapturate sulfoxide urinary excretion, and FDVE-mercapturate sulfoxidation by liver microsomes were assessed. The formation of FDVE-mercapturic acid sulfoxide metabolites in vivo and their urinary excretion were unambiguously established by mass spectrometry. Dexamethasone and phenobarbital increased, and troleandomycin decreased (i) liver microsomal FDVE-mercapturic acid sulfoxidation in vitro, (ii) FDVE-mercapturic acid sulfoxide urinary excretion in vivo, and (iii) FDVE nephrotoxicity in vivo assessed by renal histology, blood urea nitrogen concentrations, and urine volume and protein excretion. Urine 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid, reflecting beta-lyase-dependent FDVE-cysteine S-conjugates metabolism, was minimally affected by the pretreatments. These results demonstrate that FDVE S-conjugates undergo P4503A-catalyzed sulfoxidation in rats in vivo, and this sulfoxidation pathway contributes to nephrotoxicity. FDVE S-conjugates sulfoxidation constitutes a newly discovered mechanism of FDVE bioactivation and toxicification in rats, in addition to beta-lyase-catalyzed metabolism of FDVE-cysteine S-conjugates.

Effect of prototypical inducing agents on P-glycoprotein and CYP3A expression in mouse tissues.

P-glycoprotein (P-gp) and CYP3A have considerable overlap in inducers in vitro. Characterizing P-gp induction in vivo and potential coregulation with CYP3A are important goals for predicting drug interactions. This study examined P-gp expression in mouse tissues and potential coinduction with CYP3A following oral treatment with 1 of 7 prototypical inducing agents for 5 days. P-gp expression in brain or liver was not induced by any treatment as determined by Western blot, whereas dexamethasone, pregnenolone-16alpha-carbonitrile (PCN), St. John's wort (SJW), and rifampin induced hepatic CYP3A expression. In intestine, rifampin and SJW induced P-gp expression 3.7- and 1.6-fold and CYP3A 3.5- and 2.4-fold, respectively, whereas dexamethasone and PCN induced CYP3A only. These observations suggest that P-gp in mouse small intestine is inducible by some, but not all, CYP3A inducers, whereas P-gp expression in liver or brain is not readily induced. Intriguingly, rifampin and SJW, both activators of the human pregnane X receptor (PXR), induced CYP3A in both liver and intestine but induced P-gp only in intestine, whereas PCN, an activator of murine PXR, did not induce P-gp in any tissue. Rifampin disposition was evaluated, and hepatic exposure to rifampin was comparable to intestine; in contrast, brain concentrations were low. Overall, these observations demonstrate that P-gp induction in vivo is tissue-specific; furthermore, there is a disconnect between P-gp induction and CYP3A induction that is tissue- and inducer-dependent, suggesting that PXR activation alone is insufficient for P-gp induction in vivo. Tissue-specific factors and inducer pharmacokinetic/pharmacodynamic properties may underlie these observations.

Ethanol inhibits in-vitro metabolism of nifedipine, triazolam and testosterone in human liver microsomes.

Although extended exposure to ethanol induces CYP3A metabolism in-vivo, the acute effects of ethanol on CYP3A metabolism have not been fully evaluated in-vitro. We assessed the effect of ethanol on CYP3A-mediated biotransformation using human liver microsomes in-vitro with three prototypic CYP3A-mediated reactions: nifedipine to oxidized nifedipine, triazolam to its 1-hydroxy (1-OH TRZ) and 4-hydroxy (4-OH TRZ) metabolites, and testosterone to 6beta-hydroxytestosterone (6beta-OH TST). Ethanol inhibited metabolism of nifedipine (oxidized nifedipine IC50 3 mg dL(-1), where the IC50 value is the inhibitor concentration corresponding to a 50% reduction in metabolite formation velocity), triazolam (1-OH TRZ IC50 1.1 mg dL(-1), 4-OH TRZ IC50 2.7 mg dL(-1)) and testosterone (6beta-OH TST IC50 2.4 mg dL(-1)). The inhibitory potency of ethanol was similar for the three substrates representing the three hypothetical CYP3A substrate categories. The IC50 values obtained were lower than clinically relevant blood alcohol concentrations. In conclusion, ethanol is an inhibitor of human CYP3A metabolism and may contribute to clinically important interactions.

Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane x receptor and its target gene CYP3a in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. The in vivo effects of lipopolysaccharide (LPS) on expression of PXR and its target gene cytochrome P450 3A (CYP3A) in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using reverse transcription polymerase chain reaction. Results indicate that LPS significantly inhibits the expression of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also represses the upregulation of CYP3A11 mRNA levels and erythromycin N-demethylase (ERND) catalytic activity in mice pretreated with PXR ligands dexamethasone, rifampicin, mifepristone, and phenobarbital. LPS-induced downregulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with gadolinium chloride, a selective Kupffer cell toxicant. Pretreatment with a single dose of gadolinium chloride (10 mg/kg) also significantly attenuated LPS-induced downregulation of dexamethasone-, rifampicim-, mifepristone-, and phenobarbital-inducible, CYP3A11 mRNA expression and ERND activity in mouse liver. Furthermore, LPS-induced downregulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated with allopurinol, an inhibitor of xanthine oxidase, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. Allopurinol and diphenyleneiodonium chloride pretreatment also attenuated the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, has no effect on LPS-induced downregulation of PXR and CYP3A11 mRNA. Finally, LPS-induced downregulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. These results indicate that Kupffer cells contribute to LPS-induced downregulation of PXR and CYP3A in mouse liver. Reactive oxygen species, produced possibly by NADPH oxidase and perhaps by xanthine oxidase, are involved in LPS-induced downregulation of nuclear receptor PXR and its target gene CYP3A in mouse liver.

Induction of hepatic cyp2b and cyp3a subfamily enzymes by nicardipine and nifedipine in mice.

1. Nicardipine (Nic) or nifedipine (Nif) was given to male and female C57BL/6J mice by a single gavage at doses of 100, 200 and 400 micromolkg(-1), and changes in the levels of mRNA and apoprotein of hepatic cytochrome P450 (P450) enzymes, including Cyp2b9, Cyp2b10, Cyp3a11 and Cyp3a41, were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, hepatic microsomal activities for pentoxyresorufin O-dealkylation (PROD) and nifedipine oxidation, which are mainly mediated by Cyp2b and Cyp3a subfamily enzymes, respectively, were measured. 2. Results from RT-PCR analysis revealed that Nic, but not Nif, showed a capacity for activating the Cyp3a11 gene in either sex of mice and that both chemicals could induce a male-selective activation of Cyp2b10 gene, although they had no capacity for activating the Cyp2b9 and Cyp3a41 genes in either sex. 3. Increased levels of the mRNAs of Cyp2b10 and Cyp3a11 were closely correlated with those of apoprotein and activity of the corresponding P450 subfamily enzymes. 4. The study demonstrated for the first time that Nic, but not Nif, showed the ability to induce Cyp3a11 in both sexes of mice, although both Nif and Nic led to a male-selective induction of Cyp2b10, and that Nic and Nif had no ability to induce Cyp2b9 and Cyp3a41 in either sex.

Relative receptor expression is a determinant in xenobiotic-mediated CYP3A induction in rat and human cells.

1. Species differences in xenobiotic-mediated transcriptional activation of CYP3A genes are known to exist. These differences are proposed to be due, in part, to host cell differences. 2. Host cell effects were investigated by trans-species transient transfection of reporter genes containing either the rat CYP3A23 or human CYP3A4 proximal promoters into human HepG2 and rat FaO and H4IIEC3 hepatoma cells. HepG2 and FaO cells supported activation of both CYP3A constructs by xenobiotics in a species-specific manner, whereas H4IIEC3 cells were non-permissive. 3. The mRNA complement of the cell lines was then quantified by semiquantitative RT-PCR for adult CYP3As (CYP3A23, CYP3A4/5), steroid hormone receptors (constitutive androstane receptor, glucocorticoid receptor-alpha, pregnane X receptor) and transcription factors (Hepatic nuclear factor 4alpha, retinoid X receptor). 4. Principal component analysis of absolute receptor levels demonstrated a wide scattering, with no coherent pattern. In contrast, PCA of relative receptor ratios segregated H4IIEC3 cells from all other samples. 5. The observation is confirmed that species differences in response to xenobiotics are a result of host cell environment. In addition, new evidence is provided to support the hypothesis that in addition to individual receptor activation profiles, the relative abundance of steroid hormone receptors that control CYP3A gene expression play an important role in this observed species difference.

CYP3A induction by N-hydroxyformamide tumor necrosis factor-alpha converting enzyme/matrix metalloproteinase inhibitors use of a pregname X receptor activation assay and primary hepatocyte culture for assessing induction potential in humans.

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.

Cytochrome P450 3A4 activity after surgical stress.

OBJECTIVE: To evaluate the relationship between the acute inflammatory response after surgical trauma and changes in hepatic cytochrome P450 3A4 activity, compare changes in cytochrome P450 3A4 activity after procedures with varying degrees of surgical stress, and to explore the time course of any potential drug-cytokine interaction after surgery. DESIGN: Prospective, open-label study with each patient serving as his or her own control. SETTING: University-affiliated, acute care, general hospital. PATIENTS: A total of 16 patients scheduled for elective repair of an abdominal aortic aneurysm (n = 5), complete or partial colectomy (n = 6), or peripheral vascular surgery with graft (n = 5). INTERVENTIONS: Cytochrome P450 3A4 activity was estimated using the carbon-14 [14C]erythromycin breath test (ERMBT) before surgery and 24, 48, and 72 hrs after surgery. Abdominal aortic aneurysm and colectomy patients also had an ERMBT performed at discharge. Blood samples were obtained before surgery, immediately after surgery, and 6, 24, 32, 48, and 72 hrs after surgery for determination of plasma concentrations of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha. Clinical markers of surgical stress that were collected included duration of surgery, estimated blood loss, and volume of fluids administered in the operating room. MEASUREMENTS AND MAIN RESULTS: ERMBT results significantly declined in all three surgical groups, with the lowest value at the time of the 72-hr study in all three groups. There was a trend toward differences in ERMBT results among groups that did not reach statistical significance (p =.06). The nadir ERMBT result was significantly and negatively correlated with both peak interleukin-6 concentration (r(s) = -.541, p =.03) and log interleukin-6 area under the curve from 0 to 72 hrs (r(s) = -.597, p =.014). Subjects with a peak interleukin-6 of >100 pg/mL had a significantly lower nadir ERMBT compared with subjects with a peak interleukin-6 of <100 pg/mL (35.5% +/- 5.2% vs. 74.7% +/- 5.1%, p <.001). CONCLUSIONS: Acute inflammation after elective surgery was associated with a significant decline in cytochrome P450 3A4 activity, which is predictive of clinically important changes in the metabolism of commonly used drugs that are substrates for this enzyme.