Celecoxib is a CYP1A2 inhibitor in vitro but not in vivo

Background and objective We recently discovered that rofecoxib is a potent mechanism-based inhibitor of CYP1A2. The effect of the widely used cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drug celecoxib on CYP1A2 activity has not been reported. Methods The effect of celecoxib on CYP1A2 activity (phenacetin O-deethylation) was first studied in vitro using human liver microsomes. This was followed by a randomized, placebo-controlled, cross-over study in which 12 healthy volunteers were given celecoxib (200 mg twice daily) or placebo for 4 days. On day 3, a caffeine test was performed. On day 4, the subjects ingested 2 mg tizanidine. Plasma samples for the measurement of the concentrations of tizanidine, its metabolites and celecoxib were collected up to 24 h post-administration. Pharmacodynamic variables (e.g. blood pressure, subjective drowsiness and drug effect) were recorded up to 12 h post-adm. Results Celecoxib was found to be a moderately potent competitive inhibitor of CYP1A2 in vitro with a K_i (inhibitor constant) of 25.4 μM. However, in vivo, celecoxib did not affect the caffeine test, or the peak concentration, time to peak concentration, area under the concentration-time curve or half-life of tizanidine. The pharmacodynamic variables of tizanidine also remained unchanged. Conclusions Unlike rofecoxib, celecoxib does not clinically to significantly inhibit CYP1A2. The lack of significant in vivo inhibition of CYP1A2 can be correctly predicted on the basis of in vitro K_i data and the free peripheral or portal plasma concentration of celecoxib.     

Induction of suppressor cell activity in vivo and suppression of lymphoproliferative responses in vitro by SK&F 105.685 in the dog.

SK&F 105.685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in rat models of adjuvant-induced arthritis and experimental autoimmune encephalomyelitis as well as in the murine lupus model. The mechanism of action in these models appears to be the induction of non-specific suppressor cell activity which is measured by the ability of cells from treated animals to partially inhibit the proliferative response of lymphocytes from control animals to concanavalin A (ConA) in a co-culture assay. In this study we have shown that oral administration of 0.1-3 mg/kg/day of SK&F 105.685 to purebred beagle dogs induced suppressor cell activity in the spleen and bone marrow but not in the peripheral blood. In vitro, SK&F 105.685 partially suppressed the proliferative response of dog splenocytes to the mitogens phytohemagglutinin (PHA), ConA and, to a lesser extent, pokeweed mitogen (PWM). Peripheral blood lymphocytes (PBLs) differed from spleen cells in their susceptibility to suppression by SK&F 105.685. While the PWM and ConA responses of PBLs and spleen cells showed similar levels of inhibition, the PHA response of PBLs, in marked contrast to spleen cells, was resistant to suppression by the compound. Our results show that the immunoregulatory effects of SK&F 105,685 are not limited to rodents and that suppressor cell activity in dogs is induced quickly and by relatively low doses of the compound.     

Improvement of bioavailability of the HIV protease inhibitor SC-52151 in the beagle dog by coadministration of the CYP3A4 inhibitor,ketoconazole

1. SC-52151. an HIV protease inhibitor, is mainly metabolized by CYP3A4 and is poorly bioavailable after oral administration. After i.v. administration of SC-52151 to the female beagle dog (2 5?mg kg), SC-52151 was rapidly eliminated in plasma with an elimination half-life of about 1 h, a plasma clearance of 44 ml?min kg and an apparent steady-state volume distribution of 2 2 litre kg. The high value of plasma clearance of SC-52151 suggests an extensive hepatic first-pass metabolism since SC-52151 is highly protein bound and does not partition itself into red blood cells. 2. The extensive hepatic first-pass metabolism was reduced by coadministration of a CYP3A4 inhibitor, ketoconazole. 3. Dogs were dosed daily with ketoconazole at dose of 100?mg ketoconazole per dog (10?mg kg) for 5days prior to theinitiationof coadministration of SC-52151 for 15 days. The doses used for SC-52151 was 0, 60 and 120?mg SC-52151 kg day (divided t.i.d., 8-h dosing interval). Coadministration of ketoconazole improved the bioavailability of SC52151 from 4 1 to 9 6% and also improved the C of SC-52151 from 0 41 to 0 83 mu g ml. max 4. Although the absolute bioavailability of SC-52151 was still low (10%), the C max and AUC achieved in this study were satisfactory for conducting chronic toxicology studies. No toxicity associated with the coadministration of ketoconazole was evident. Results from this study suggest that coadministration of ketoconazole might be a practical approach to increase the exposure of SC-52151 in both preclinical and clinical studies.     

Cloning, tissue expression, and regulation of beagle dog CYP4A genes.

In addition to its function as a fatty acid hydroxylase, the peroxisome proliferator-activated receptor alpha (PPARalpha) target gene, CYP4A, has been shown to be important in the conversion of arachidonic acid to the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, suggesting a role for this enzyme in mediating vascular tone. In the present study, the cDNA sequence of beagle dog CYP4A37, CYP4A38, and CYP4A39 from the liver was determined. Open reading frame analysis predicted that CYP4A37, CYP4A38, and CYP4A39 each comprised 510 amino acids with approximately 90% sequence identity to one another, and approximately 71 and 78% sequence identity to rat CYP4A1 and human CYP4A11, respectively. PCR analysis revealed that the three dog CYP4A isoforms are expressed in kidney > liver > lung > intestine > skeletal muscle > heart. Treatment of primary dog hepatocytes with the PPARalpha agonists GW7647X and clofibric acid resulted in an increase in CYP4A37, CYP4A38, and CYP4A39 mRNA expression (up to fourfold), whereas HMG-CoA synthase mRNA expression was increased to a greater extent (up to 10-fold). These results suggest that dog CYP4A37, CYP4A38, and CYP4A39 are expressed in a tissue-dependent manner and that beagle dog CYP4A is not highly inducible by PPARalpha agonists, similar to the human CYP4A11 gene.     

Bufalin inhibits CYP3A4 activity in vitro and in vivo

Aim： To investigate the inhibitory interactions of bufalin and CYP3A4. Methods： Recombinant human CYP3A4 was incubated with bufalin in vitro. Bufalin was administered ig and iv to Wistar rats to further estimate its impact on CYP3A4, and midazolam was given to index the activity of CYP3A4. Results： The IC50 of bufalin was 14.52 μmol/L. Bufalin affected CYP3A4 activity with increases in AUC0-t and t1/2, and decreases in CL and the formation of 1-hydroxy-midazolam after ig or iv administration of midazolam （P〈0.05）. An increase in Cmax after ig bufalin administration （P〈0.05） was observed. Conclusion： Bufalin showed a modest but significant inhibition of CYP3A4 both in vitro and in vivo. The likelihood of an interaction between bufalin and the CYP3A4-metabolized drugs in human might not be negated.     

Modulation of UDP-glucuronosyltransferase 2B7 function by cytochrome P450s in vitro: differential effects of CYP1A2, CYP2C9 and CYP3A4

Effects of cytochrome P450 isoforms, CYP1A2, CYP2C9 and CYP3A4, on the catalytic activity of UDP-glucuronosyltransferase 2B7 (UGT2B7) expressed in COS cell microsomal membranes were investigated using morphine as a substrate. When detergent-untreated COS cell microsomes were used as the enzyme source, the activity of morphine-3-glucuronide formation by UGT2B7 was reduced by addition of purified CYP1A2 and CYP2C9 in a concentration-dependent manner. The effect of CYP1A2 was greater than that of CYP2C9. In contrast, exogenous CYP3A4 had little effect on morphine glucuronidation activity. These results suggest that CYP1A2 and CYP2C9 have ability to modify UGT2B7 function. However, the mechanism(s) underlying the modulation of UGT2B7 function by these P450s seems to differ from that by CYP3A4.     

In vivo and in vitro inhibition of CYP1A-dependent activity in Fundulus heteroclitus by the polynuclear aromatic hydrocarbon fluoranthene.

Certainpolynuclear aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) induce CYP1A-dependent enzyme activities. Because PAHs are ubiquitous environmental contaminants, and some are aryl hydrocarbon agonists, CYP1A has been used as a biomarker for PAH exposure. However, PAHs exist in the environment in complex mixtures that may confound biomarker results. In in vitro studies, the PAH fluoranthene (FL) failed to increase or enhance CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity in cells, but rather inhibited activities induced by AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(k)fluoranthene. In order to determine the in vivo effects of FL on CYP1A and DNA adduct levels, Fundulus heteroclitus were given single ip injections of either BaP (5 mg/kg), BaP + FL (5 mg/kg each), BaP + FL (5 and 50 mg/kg, respectively), FL (5 mg/kg), FL (50 mg/kg), or corn oil control. BaP-treated fish had liver microsome EROD activities significantly higher than controls, whereas FL-treated fish were not different from controls. EROD activities in BaP + FL cotreatments were significantly lower compared to fish treated with BaP alone. When FL was incubated with BaP-induced microsomes, the IC50 for inhibition of EROD activity was 1.4 x 10(-5) M FL. Kinetic studies indicated a significant noncompetitive component to the FL inhibition. When fish were treated with [(14)C]FL, the concentration of radiolabel associated with microsomal preparations was four orders of magnitude lower than the IC50. Therefore, the presence of FL or a FL metabolite was insufficient to account for the inhibition by a kinetic mechanism. In contrast to cell studies, CYP1A immunoreactive protein was significantly decreased in vivo by FL cotreatment, indicating that FL may inhibit EROD activity by down-regulating the CYP1A protein. A covalent interaction of [(14)C]FL with CYP1A was not detected. Total (32)P-postlabeled DNA adducts were not significantly changed by cotreatment of FL and BaP; however, cotreatment with 50 mg/kg FL decreased one adduct and increased another significantly. Because FL and perhaps other inhibitory PAHs, co-occur in the environment with CYP1A inducers, CYP1A-dependent bioassays may cause an underestimation of PAH exposures.     

A clinical study to assess CYP1A2 and CYP3A4 induction by AZD7325, a selective GABA(A) receptor modulator - an in vitro and in vivo comparison

AIM(S)

To investigate the potential of AZD7325 to induce CYP1A2 and CYP3A4 enzyme activities.

METHODS

Induction of CYP1A2 and CYP3A4 by AZD7325 was first evaluated using cultured human hepatocytes. The effect of multiple doses of 10 mg AZD7325 on the pharmacokinetics of midazolam and caffeine was then examined in healthy subjects.

RESULTS

The highest CYP1A2 and CYP3A4 induction responses were observed in human hepatocytes treated with 1 or 10 µm of AZD7325, in the range of 17.9%–54.9% and 76.9%–85.7% of the positive control responses, respectively. The results triggered the further clinical evaluation of AZD7325 induction potential. AZD7325 reached a plasma C_max of 0.2 µm after 10 mg daily dosing to steady-state. AZD7325 decreased midazolam geometric mean AUC by 19% (0.81-fold, 90% CI 0.77, 0.87), but had no effect on midazolam C_max (90% CI 0.82, 0.97). The mean CL/F of midazolam increased from 62 l h⁻¹ (midazolam alone) to 76 l h⁻¹ when co-administered with AZD7325. The AUC and C_max of caffeine were not changed after co-administration of AZD7325, with geometric mean ratios (90% CI) of 1.17 (1.12, 1.23) and 0.99 (0.95, 1.03), respectively.

CONCLUSIONS

While AZD7325 appeared to be a potent CYP3A4 inducer and a moderate CYP1A2 inducer from in vitro studies, the expected efficacious dose of AZD7325 had no effect on CYP1A2 activity and only a weak inducing effect on CYP3A4 activity. This comparison of in vitro and in vivo results demonstrates the critical role that clinical exposure plays in evaluating the CYP induction risk of a drug candidate.     

Pharmacokinetics of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat and dog

The pharmacokinetics of gefitinib and its metabolites in rat and dog were investigated in preclinical studies conducted to support the safety evaluation and clinical development of gefitinib, the first EGFR tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer. Following intravenous dosing (5 mg kg(-1), gefitinib plasma half-life was 3-6h in rats and dogs, although studies using a more sensitive HPLC-MS assay produced longer estimates of half-life (7-14h). In these studies, plasma clearance was high (male rat: 25 ml min(-1) kg(-1); female rat: 16 ml min(-1) kg(-1); male dog: 16 ml min(-1) kg(-1)), as was the volume of distribution (8.0-10.41 kg(-1) in rat; 6.31 kg(-1) in dog), and exposure in female rats was double that in males. Following administration of [14C]-gefitinib, concentrations of radioactivity in plasma exceeded gefitinib throughout the profile, indicating the presence of circulating metabolites in both rat and dog. An HPLC-MS assay was developed to measure concentrations of gefitinib and five potential metabolites in plasma. All five metabolites were detected in the rat, but at levels much lower than gefitinib. In the dog, exposure to gefitinib and M523595 was similar, with much lower concentrations of M537194 and only trace levels of the other metabolites. This profile of metabolites is similar to that observed in man.     

Pharmacokinetics of gefitinib,an epidermal growth factor receptor tyrosine kinase inhibitor,in rat and dog

1.?The pharmacokinetics of gefitinib and its metabolites in rat and dog were investigated in preclinical studies conducted to support the safety evaluation and clinical development of gefitinib, the first EGFR tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer.

2.?Following intravenous dosing (5?mg?kg^?1), gefitinib plasma half-life was 3–6?h in rats and dogs, although studies using a more sensitive HPLC-MS assay produced longer estimates of half-life (7–14?h).

3.?In these studies, plasma clearance was high (male rat: 25?ml?min^?1?kg^?1; female rat: 16?ml?min^?1?kg^?1; male dog: 16?ml?min^?1?kg^?1), as was the volume of distribution (8.0–10.4?l?kg^?1 in rat; 6.3?l?kg^?1 in dog), and exposure in female rats was double that in males.

4.?Following administration of [¹⁴C]-gefitinib, concentrations of radioactivity in plasma exceeded gefitinib throughout the profile, indicating the presence of circulating metabolites in both rat and dog.

5.?An HPLC-MS assay was developed to measure concentrations of gefitinib and five potential metabolites in plasma. All five metabolites were detected in the rat, but at levels much lower than gefitinib. In the dog, exposure to gefitinib and M523595 was similar, with much lower concentrations of M537194 and only trace levels of the other metabolites. This profile of metabolites is similar to that observed in man.     

Induction of CYP3A expression by dehydroepiandrosterone: involvement of the pregnane X receptor.

Dehydroepiandrosterone (DHEA) is a steroid produced by the human adrenal gland. Administration of pharmacological doses of DHEA to rats changes expression of many genes, including the cytochrome P450 family members CYP4A1 and CYP3A23. It is known that induction of CYP4A expression by DHEA requires the peroxisome proliferator-activated receptor alpha (PPAR(alpha)). In the current study, PPAR(alpha)-null mice were used to examine the role of PPAR(alpha) in expression of CYP3A. In wild-type mice, 150 mg/kg DHEA-sulfate induced Cyp4a and Cyp3a11 mRNAs by 5- and 2-fold, respectively. Induction of Cyp4a expression by DHEA-sulfate was not observed in PPAR(alpha)-null mice, whereas induction of Cyp3a11 expression by DHEA-sulfate was similar between genotypes. This suggests that PPAR(alpha) is not involved in induction of Cyp3a11 expression by DHEA. Because expression of CYP3A family members can be induced by activation of another member of the nuclear receptor superfamily, the pregnane X receptor (PXR), we examined the ability of DHEA to activate PXR. In transient transfection assays, DHEA and its metabolites androst-5-ene-3beta,17beta-diol (ADIOL), androst-5-ene-3,17-dione, and androst-4-ene-3,17-dione were activators of PXR. Maximal induction of a PXR-responsive reporter gene of approximately 3-fold was observed at concentrations of 50 to 100 microM, indicating that these steroids are relatively weak activators of PXR. Human and murine PXR exhibited different specificities for DHEA and its metabolites. ADIOL activated reporter gene expression in the presence of murine but not human PXR. Results of these studies suggest that the induction of rodent CYP3A expression upon treatment with high doses of DHEA occurs through activation of PXR.     

Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies

Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresorufin indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a significant NADPH oxidation catalyzed by CYP2E1 supersomes, strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present findings may contribute to understand better the physiological function of CYP450 in relation with inflammation and other physiological processes in which HA may have a relevant role.     

Preclinical safety evaluation of avasimibe in beagle dogs: an ACAT inhibitor with minimal adrenal effects.

Avasimibe, a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), is currently being developed as an antiatherosclerotic agent. The preclinical safety and toxicokinetics of the compound were assessed in beagle dogs in an escalating-dose study and in repeated-dose studies of 2-, 13-, and 52-week duration. Oral (capsule) doses up to 1000 mg/kg b.i.d. were assessed in the escalating dose study and once-a-day doses up to 300 mg/kg, 1000 mg/kg, and 1000 mg/kg were assessed in the 2-, 13-, and 52-week studies, respectively. Avasimibe was found to be a substrate and inducer of hepatic CYP 3A, producing pronounced decreases in plasma drug concentrations subsequent to Day 1. Plasma drug concentrations plateaued markedly at doses above 100 mg/kg. Significant toxicologic findings were restricted to the higher doses (> or =300 mg/kg) and included emesis, fecal consistency changes, salivation, body weight loss, microscopic and clinical pathologic evidence of hepatic toxicity, and red blood cell (RBC) morphology changes. Mortality occurred at 1000 mg/kg due to hepatic toxicity. Toxicity was more closely associated with the exaggerated pharmacodynamic effects of the compound (e.g., marked serum cholesterol decreases) seen at the high doses of avasimibe used in these studies rather than with measures of systemic exposure (Cmax or AUC). Adrenal effects were noted only in the 52-week study and consisted of minimal to mild cortical cytoplasmic vacuolization and fibrosis at doses > or =300 mg/kg, with no change in adrenal weight. In conclusion, avasimibe is an ACAT inhibitor that has minimal adrenal effects in dogs, with dose-limiting toxicity defined by readily monitored and reversible changes in hepatic function.     

In vitro/in vivo scaling of alprazolam metabolism by CYP3A4 and CYP3A5 in humans.

We attempted to predict the in vivo metabolic clearance of alprazolam from in vitro metabolic studies using human liver microsomes and human CYP recombinants. Good correlations were observed between the intrinsic clearance (CL(int)) for 4-hydroxylation and CYP3A4 content and between the CL(int) for alpha-hydroxylation and CYP3A5 content in ten human liver microsomal samples. Using the recombinant CYP isoforms expressed in insect cells, the CL(int) for CYP3A4 was about 2-fold higher than the CL(int) for CYP3A5 in the case of 4-hydroxylation. However, the CL(int) for CYP3A5 was about 3-fold higher than the CL(int) for CYP3A4 in the case of alpha-hydroxylation. The metabolic rates for 4- and alpha-hydroxylation increased as the added amount of cytochrome b(5) increased, and their maximum values were 3- to 4-fold higher than those without cytochrome b(5). The values of CL(int), in vivo predicted from in vitro studies using human liver microsomes and CYP3A4 and CYP3A5 recombinants were within 2.5 times of the observed value calculated from literature data. The average CL(int) value (sum of 4- and alpha-hydroxylation) obtained using three human liver microsomal samples was 4-fold higher than that obtained using three small intestinal microsomal samples from the same donors, indicating the minor contribution of intestinal metabolism to alprazolam disposition. The area under the plasma concentration-time curve (AUC) of alprazolam is reported to increase following co-administration of ketoconazole and the magnitude of the increase predicted from the in vitro K(i) values and reported pharmacokinetic parameters of ketoconazole was 2.30-2.45, which is close to the value observed in vivo (3.19). A quantitative prediction of the AUC increase by cimetidine was also successful (1.73-1.79 vs 1.58-1.64), considering the active transport of cimetidine into the liver. In conclusion, we have succeeded in carrying out an in vitro/in vivo scaling of alprazolam metabolism using human liver microsomes and human CYP3A4 and CYP3A5 recombinants.     

Simultaneous assessment of CYP3A4 and CYP1A2 activity in vivo with alprazolam and caffeine

Alprazolam (ALP) and caffeine (CAF) were suggested as probe drugs for the activities of CYP3A4 and CYP1A2, respectively. We investigated the disposition of oral ALP (1 mg) and CAF (100 mg) in 17 normal volunteers to establish and validate a procedure for the simultaneous assessment of CYP3A4 and CYP1A2 enzyme activity. Nine received ALP alone, ALP and CAF and CAF alone in an open three-way crossover study to test for pharmacokinetic interaction. Four received ALP after a 2-day pretreatment with ketoconazole, an inhibitor of CYP3A4, and four normal volunteers received ALP after 4 days of rifampin, an inducer of CYP3A4. AUC values of ALP and CAF administered alone were not different from AUC values when both drugs were administered combined, indicating that there is no metabolic interaction. The ratio formed of paraxanthine and CAF correlated significantly with systemic CAF clearance at 3, 4, 6, 8, 10 and 24 h. There was a strong correlation between AUC values of ALP and CAF and the plasma concentration obtained 6, 8, 10, or 24 h after ingestion of the drug. Ketoconazole and rifampin pretreatment significantly changed AUC values of ALP (mean AUC values in microg/l h: ALP = 242.2, ALP + ketoconazole = 426.2, ALP + rifampin = 28.4, ANOVA F = 17.7, P < 0.001). We conclude that ALP and CAF can be administered simultaneously for the assessment of CYP activity. Plasma concentrations 6, 8, 10, and 24 h after drug ingestion reflect AUC of ALP and CAF and therefore in-vivo CYP3A4 and CYP1A2 activity, respectively.     

Induction of CYP1A2 activity by carbamazepine in children using the caffeine breath test

Aims Carbamazepine is a known enzyme inducer. The aim of this study was to determine whether carbamazepine induces the metabolism of caffeine in children.
Methods Children due to receive carbamazepine for epilepsy were recruited into the study. The caffeine breath test was carried out prior to the administration of carbamazepine and after a minimum of 2–3 weeks therapy. Five children were studied and they received 200–600  mg carbamazepine daily.
Results The mean values of the 2  h cumulative labelled carbon dioxide were 3.47% before and 7.65% during carbamazepine. There was a significant increase in the percentage labelled caffeine exhaled as carbon dioxide during the administration of carbamazepine (Student's paired t -test, P <0.05).
Conclusions The results suggest that carbamazepine induces the metabolism of caffeine by the CYP1A2 pathway in the children studied.