Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.     

In vitro evidence for auto-induction of artemisinin metabolism in the rat.

Artemisinin disappearance rate was more rapid in incubations with liver microsomes from rats pre-treated with oral artemisinin (60 mg/kg/day for 5 days) compared with microsomes from control animals. A single pathway Michaelis-Menten saturable elimination model was fitted to the concentration-time data of artemisinin incubations by non-linear regression. Model parameters were obtained after fitting results for each animal separately and by pooling data for pre-treated and control animals. Parameter estimates (% coefficient of variation) from fitting the pooled data was maximum velocities (Vmax) = 1.8 (12) mmole/min/mg protein and Michaelis constants (Km) = 20(22) microM for artemisinin pre-treated and Vmax = 0.85 (35) mmole/min/mg protein and Km = 67(52) microM for control animals indicating a 2-fold increase in Vmax and a 3-fold decrease in Km with microsomes from artemisinin pre-treated animals. Estimates of intrinsic clearance in microsomes from the pre-treated animals were 8-fold higher compared with controls. Thus, artemisinin appears to be a potent auto-inducer of drug metabolism in rats as has also been observed in humans. The present findings suggest caution in the interpretation of repeat-dose rat toxicity studies with artemisinin unless its pharmacokinetics are simultaneously monitored, since during multiple administration, the exposure of the drug will not be constant over time.     

Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer

1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARalpha/gamma activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARgamma-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3-5, 5 and 5-11 in male rat, monkey, female rat, and dog, respectively). 2. M1 (%) formed in vitro (5, 53, 57-65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vivo (24-45, 53-57, 78, 75-85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1). 3. The plasma clearance of M1 was higher in male rats (32 ml min(-1) kg(-1) compared with 6, 7 and 2 ml min(-1) kg(-1) in female rat, male monkey and male dogs, respectively). 4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1.     

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin

AIMS: The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. METHODS: Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. RESULTS: The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. CONCLUSIONS: Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.     

Model for the drug-drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1. Cytochrome P450 (CYP) 3A catalysis of testosterone 6beta-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80 mg kg(-1) day(-1) dexamethasone (DEX) for 3 days. 2. Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent K(m) was 1.8 microM, similar to that in human microsomes. 3. Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent K(i) values were 0.088 and 91.2 microM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans. 4. After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively. 5. Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (C(max)) increased when ketoconazole was co-administered with midazolam. 6. It was shown that the drug-drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug-drug interactions based on CYP3A enzyme inhibition.     

Nilutamide inhibits mephenytoin 4-hydroxylation in untreated male rats and in human liver microsomes

1. The effects of nilutamide (an anti-androgen with a hydantoin moiety) on the 4-hydroxylation of mephenytoin were studied in rat liver microsomes. Nilutamide, at a concentration expected in human liver (100uM) during prolonged administration of nilutamide, inhibited by 40% mephenytoin (0-3 mM) 4-hydroxylase activity in liver microsomes from untreated male rats, but not in microsomes from untreated female rats, or in microsomes from dexamethasone-treated male or female rats.

2. Administration to male rats of nilutamide, in doses (20mg/kg i.p. twice daily) known to reproduce plasma concentrations observed in human therapeutics, decreased by 60% the 24?h urinary excretion of 4-hydroxymephenytoin after administration of mephenytoin (15mg/kg oral).

3. Nilutamide (100uM) markedly inhibited mephenytoin 4-hydroxylase activity in human liver microsomes. Inhibition kinetics were consistent with mixed inhibition. It is concluded that nilutamide inhibits mephenytoin 4-hydroxylase activity in untreated male rats and in human liver microsomes. It is suggested that inhibition is likely to occur in vivo in humans receiving therapeutic doses of nilutamide.     

Artemisinin pharmacokinetics in healthy adults after 250, 500 and 1000 mg single oral doses

Eight healthy male, Vietnamese subjects were administered 1×250, 2×250 and 4×250 mg artemisinin capsules in a cross-over design with randomized sequence with a 7-day washout period between administrations. The inter-individual variability in artemisinin pharmacokinetics was large with parameter coefficients of variation (CV) typically between 50–70%. The parameter with the smallest variability was the elimination half-life (CV≈30–40%). Analysis of variance indicated also a large intra-subject variability (CV≤24%) for the dose-normalized area under the plasma concentration–time curve (AUC/dose). The pharmacokinetic results suggested artemisinin to be subject to high pre-systemic extraction. Artemisinin half-life could not predict the extent ofin vivo exposure to the drug, there being no correlation between half-life and oral clearance. Artemisinin oral plasma clearance was about 400 L h⁻¹ exhibiting a slight decrease with dose, although the effect was weak. Thus results from studies using different artemisinin doses may, within the studied dose range, be compared without the complication of disproportionate changes in drug exposure with varying dose levels. Half-lives appeared to increase with dose. An observed period effect in the analysis of variance was tentatively associated with time-dependency in artemisinin pharmacokinetics. There was a high correlation between artemisinin plasma concentrations determined at various time-points after drug administration and the AUCs after the 500 and 1000 mg doses, but less so after the 250 mg dose. This may show a tentative approach to assess the systemic exposure of the patients to artemisinin from the determination of artemisinin plasma concentrations in one or two plasma samples only. Artemisinin was well tolerated with no apparent dose or time dependent effects on blood pressure, heart rate or body temperature. © 1998 John Wiley & Sons, Ltd.     

Sex differences in pharmacokinetics of cilostazol in rats

The pharmacokinetics of cilostazol was investigated after oral and intravenous administration in both male and female rats. After oral administration, area under serum concentration-time curve (AUC) was about 35-fold higher in female rats than in male rats, and absolute bioavailability was about 5.8-fold higher in female rats than in male rats. Total body clearance (CL(total)) for female rats was around one-sixth of that for male rats. In vivo hepatic clearance (CL(h)) calculated based on isolated liver perfusion studies was even higher than or around 90% of the in vivo CL(total) of cilostazol for female and male rats, respectively, indicating that cilostazol is mainly eliminated by the liver in both male and female rats. In vitro metabolism studies utilizing hepatic microsomes and recombinant cytochrome (CYP) isoforms clearly indicated that major metabolites of cilostazol were generated extensively with hepatic microsomes of male rats and that male-predominant CYP3A2 and male-specific CYP2C11 were mainly responsible for the hepatic metabolism of cilostazol. Therefore, the great sex differences in the pharmacokinetics of cilostazol were mainly attributed to the large difference in hepatic metabolism. Our experimental results also suggested that the substantial metabolism of cilostazol in the small intestine and its possible saturation would be responsible for dose-dependent bioavailability in both male and female rats.     

Preclinical pharmacokinetics and metabolism of 6-(4-(2,5-difluorophenyl)oxazol-5-yl)-3-isopropyl-[1,2,4]-triazolo[4,3-a]pyridine, a novel and selective p38alpha inhibitor: identification of an active metabolite in preclinical species and human liver microsomes

The disposition of 6-(4-(2,5-difluorophenyl)oxazol-5-yl)-3-isopropyl-[1,2,4]-triazolo[4,3-a]pyridine (1), a potent and selective inhibitor of mitogen activated protein (MAP) kinase p38alpha, was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 1 demonstrated generally favorable pharmacokinetic properties in all species examined. Following intravenous (i.v.) administration, 1 exhibited low volumes of distribution at steady state (Vd(ss)) ranging from 0.4-1.3 l/kg (2.4-26 l/m(2)) in the rat, dog and monkey. Systemic plasma clearance was low in cynomolgus monkeys (6.00 ml/min/kg, 72.0 ml/min/m(2)) and Sprague-Dawley rats (7.65+/-1.08 ml/min/kg, 45.9+/-6.48 ml/min/m(2) in male rats and 3.15+/-0.27 ml/min/kg, 18.9+/-1.62 ml/min/m(2) in female rats) and moderate in beagle dogs (12.3+/-5.1 ml/min/kg, 246+/-102 ml/min/m(2)) resulting in plasma half-lives ranging from 1 to 5 h in preclinical species. Moderate to high bioavailability of 1 was observed in rats (30-65%), dogs (87%) and monkeys (40%) after oral (p.o.) dosing consistent with the in vitro absorption profile of 1 in the Caco-2 permeability assay. In rats, the oral pharmacokinetics were dose dependent over the dose range studied (5, 50 and 100 mg/kg). The principal route of clearance of 1 in rat, dog, monkey and human liver microsomes and in vivo in preclinical species involved oxidative metabolism mediated by cytochrome P450 enzymes. The major metabolic fate of 1 in preclinical species and humans involved hydroxylation on the isopropyl group to yield the tertiary alcohol metabolite 2. In human liver microsomes, this transformation was catalysed by CYP3A4 as judged from reaction phenotyping analysis using isozyme-specific inhibitors and recombinant CYP enzymes. Metabolite 2 was also shown to possess inhibitory potency against p38alpha in a variety of in vitro assays. 1 as well as the active metabolite 2 were moderately to highly bound to plasma proteins (f(u) approximately 0.1-0.33) in rat, mouse, dog, monkey and human. 1 as well as the active metabolite 2 did not exhibit competitive inhibition of the five major cytochrome P450 enzymes namely CYP1A2, 2C9, 2C19, 2D6 and 3A4 (IC(50)>50 microM). Overall, these results indicate that the absorption, distribution, metabolism and excretion (ADME) profile of 1 is relatively consistent across preclinical species and predict potentially favorable pharmacokinetic properties in humans, supporting its selection for toxicity/safety assessment studies and possible investigations in humans as an anti-inflammatory agent.     

Correlation of the intrinsic clearance of donepezil (Aricept) between in vivo and in vitro studies in rat, dog and human

1. Donepezil hydrochloride (Aricept) is used for the treatment of Alzheimer's disease. Here the correlation of the intrinsic clearance (Cl(int)) of donepezil between the in vivo and in vitro states was studied in rat, dog and human. 2. In an experiment with 14C-donepezil and human microsomes the routes of metabolism were identified as N-dealkylation and O-demethylation, and no unknown metabolites were detected. 3. The Cl(int) of donepezil in the male rat, female rat, dog and human liver microsomes were 33.7, 13.4, 37.0 and 6.35 microl/min/mg microsomal protein respectively, and sex difference in rat and interspecies difference in the estimated Cl(int) were found. 4. After a single intravenous administration to the male rat, female rat and dog, total plasma clearance (ClP(total)) was 78.6, 29.5 and 88.3 ml/min/kg respectively, and a sex difference was observed in rat. 5. After a single oral administration to the male rat, dog and healthy volunteer, ClP(total) was 140, 105 and 2.35 ml/min/kg respectively, and remarkable differences were observed between animals and man. 6. The contribution of renal clearance to blood clearance (Cl(r)) was low in all species. The predicted in vitro hepatic clearance (Cl(h-pre)) was in the rank order: male rat (15.91 ml/min/kg) > dog (7.96) > female rat (7.67) > human (1.04). Although Cl(h-pre) was underestimated, Cl(h-pre) was significantly correlated with that of ClB(total) in the different animal species and in man, indicating that the in vitro-in vivo ranking order was conserved.     

Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer

1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARα/γ activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARγ-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3–5, 5 and 5–11 in male rat, monkey, female rat, and dog, respectively).

2. M1 (%) formed in vitro (5, 53, 57–65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vitro (24–45, 53–57, 78 and 75–85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1).

3. The plasma clearance of M1 was higher in male rats (32 ml min^-1 kg^-1 compared with 6, 7 and 2 ml min^-1 kg^-1 in female rat, male monkey and male dogs, respectively).

4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1.     

染料木黄酮在雌雄大鼠肝微粒体中的代谢差异

目的研究染料木黄酮在♀、♂大鼠肝微粒体中的代谢差异。方法制备♀、♂大鼠肝微粒体,确定染料木黄酮代谢的酶动力学条件,分别用CYP1A2抗体和选择性CYP1A2抑制剂呋喃茶碱与大鼠肝微粒体和染料木黄酮共同温孵,测定染料木黄酮在♀、♂大鼠肝微粒体中的代谢速率,评价♀、♂大鼠CYP1A2的相对百分比活性。结果在CYP1A2抗体浓度为1∶400,孵育时间为30 m in条件下,♂大鼠肝微粒体代谢染料木黄酮的相对代谢率为(20.95±2.13)%,♀动物为(13.73±1.26)%。在选择性CYP1A2抑制剂呋喃茶碱浓度为3.125μmol.L-1,孵育时间为30 m in条件下,♂动物为(58.02±3.35)%,而♀大鼠肝微粒体代谢染料木黄酮的相对代谢率为(43.82±2.65)%,两者之间差异有显著性(P<0.01)。结论染料木黄酮在♂大鼠肝微粒体中代谢较♀大鼠快,提示♂大鼠肝微粒体CYP1A2酶活性高于♀大鼠。     

Pharmacokinetics of omeprazole in rats with water deprivation for 72 hours

Dehydration can occur by excessive sweating, polyuria, severe diarrhea and hyperthermia. Previous studies reported that the expressions of CYP1A1/2 and 3A1(23)/2 were not changed in male Sprague-Dawley rats with 72 h water deprivation (dehydrated rats), and that the metabolism of omeprazole is mainly catalysed via CYP1A1/2, 2D1 and 3A23/2 in rats. Hence, it could be expected that the hepatic metabolism of omeprazole would not be changed considerably in dehydrated rats, if the contribution of CYP2D1 to the metabolism of omeprazole in dehydrated rats is not considerable. Therefore, the pharmacokinetics of omeprazole were compared after intravenous (20 mg/kg) and oral (40 mg/kg) administration in control rats and in dehydrated rats. After intravenous administration, the time-averaged nonrenal clearance (Cl(nr)) values of omeprazole were comparable between the two groups of rats. This could be supported by comparable in vitro intrinsic clearance (Cl(int)) values for the disappearance of omeprazole in rat hepatic microsomes and the comparable free (unbound to plasma proteins) fractions of omeprazole in plasma in the two groups of rats. After oral administration, the AUC values of omeprazole were also comparable in the two groups of rats. The above data suggest that the dehydration state did not affect considerably the pharmacokinetics of omeprazole in rats.     

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.     

Gender differences in ondansetron pharmacokinetics in rats

It has been reported that ondansetron is primarily metabolized via hepatic CYP2D and 3A1/2 in male Sprague-Dawley rats, and CYP2D1 and 3A2 are male dominant and male specific isozymes, respectively, in rats. Thus, it could be expected that the pharmacokinetics of ondansetron would be changed in male rats compared with those in female rats. Thus, gender-different ondansetron pharmacokinetics were evaluated after its intravenous or oral administration at a dose of 8 mg/kg to male and female Sprague-Dawley rats. After intravenous administration of ondansetron to male rats, the AUC and time-averaged non-renal clearance (Clnr) of the drug were significantly smaller (22.6% decrease) and faster (27.3% increase), respectively, than those in female rats. This probably could be due to faster hepatic blood flow rate in male rats. After oral administration of ondansetron to male rats, the AUC of the drug was also significantly smaller (58.8% decrease) than that in female rats, and this could have been due mainly to increased intestinal metabolism of ondansetron in addition to increased hepatic metabolism of the drug in male rats.     

Evidence for polymorphism in the canine metabolism of the cyclooxygenase 2 inhibitor, celecoxib.

The pharmacokinetics of celecoxib, a cyclooxygenase-2 inhibitor, was characterized in beagle dogs. Celecoxib is extensively metabolized by dogs to a hydroxymethyl metabolite with subsequent oxidization to the carboxylic acid analog. There are at least two populations of dogs, distinguished by their capacity to eliminate celecoxib from plasma at either a fast or a slow rate after i.v. administration. Within a population of 242 animals, 45.0% were of the EM phenotype, 53.5% were of the PM phenotype, and 1.65% could not be adequately characterized. The mean (+/-S.D.) plasma elimination half-life and clearance of celecoxib were 1.72 +/- 0.79 h and 18.2 +/- 6.4 ml/min/kg for EM dogs and 5.18 +/- 1.29 h and 7.15 +/- 1.41 ml/min/kg for PM dogs. Hepatic microsomes from EM dogs metabolized celecoxib at a higher rate than microsomes from PM dogs. The cDNA for canine cytochrome P-450 (CYP) enzymes, CYP2B11, CYP2C21, CYP2D15, and CYP3A12 were cloned and expressed in sf 9 insect cells. Three new variants of CYP2D15 as well as a novel variant of CYP3A12 were identified. Canine rCYP2D15 and its variants, but not CYP2B11, CYP2C21, and CYP3A12, readily metabolized celecoxib. Quinidine (a specific CYP2D inhibitor) prevented celecoxib metabolism in dog hepatic microsomes, providing evidence of a predominant role for the CYP2D subfamily in canine celecoxib metabolism. However, the lack of a correlation between celecoxib and bufuralol metabolism in hepatic EM or PM microsomes indicates that other CYP subfamilies besides CYP2D may contribute to the polymorphism in canine celecoxib metabolism.     

The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs.

Bergamottin, a furanocoumarin isolated from grapefruit juice, was investigated for the ability to increase diazepam bioavailability and for its effect on cytochrome P450 (P450) enzymes in the beagle dog liver and intestine. To study the effect of bergamottin on diazepam pharmacokinetics, male beagle dogs were dosed with bergamottin (1 mg/kg) p.o. 0 or 2 h before p.o. diazepam (10 mg). In a second experiment, bergamottin (0.1 mg/kg) was dosed i.v. or p.o. 1 h before p.o. diazepam (10 mg). Plasma samples were collected over 24 h postdose, analyzed by liquid chromatography/mass tandem spectrometry, and diazepam pharmacokinetic parameters were determined. To study the effect of bergamottin on P450 enzymes, beagle dog liver and jejunum was harvested after a 10-day dosing regimen of bergamottin (1 mg/kg) p.o. per day; microsomes were prepared and analyzed for CYP3A12, CYP2B11, CYP1A1/2, and tolbutamide hydroxylase activity. Bergamottin predosing increased the plasma levels of diazepam as observed by C(max) (278.75 ng/ml versus 5.49 ng/ml) and the area under the curve [AUC((0-TLDC))] (247.69 versus 2.79 ng x hr/ml) in bergamottin versus placebo groups, respectively, indicating P450 enzyme inhibition. Diazepam plasma concentrations were increased to a similar level in the presence of i.v. and p.o. administered bergamottin. In hepatic microsomes, bergamottin treatment for 10 days reduced the activity of CYP3A12 by 50% and CYP1A1/2 by 75%. Tolbutamide hydroxylase activity did not change, and CYP2B11 activity was moderately induced. In jejunal microsomes, CYP3A12 activity doubled with bergamottin treatment. CYP2B11, CYP1A1/2 activity and tolbutamide hydroxylation was not detected. In conclusion, bergamottin is both an inhibitor and an inducer of P450 enzymes.     

Multiple dose study of interactions between artesunate and artemisinin in healthy volunteers

AIMS: To investigate whether coadministration of the antimalarials artesunate and artemisinin alters the clearance of either drug. METHODS: Ten healthy Vietnamese males (Group AS) were randomized to receive a single dose of 100 mg oral artesunate (pro-drug of dihydroartemisinin) on day -5 and then once daily for 5 consecutive days (days 1-5). Oral artemisinin (500 mg) was coadministered on days 1 and 5. Another 10 subjects (Group AM) were given 500 mg oral artemisinin on day -5 and then further doses on days 1-5. Artesunate 100 mg was given on days 1 and 5. Artemisinin and dihydroartemisinin plasma concentrations on days -5, 1 and 5 were quantified by h.p.l.c. with on-line postcolumn derivatization and u.v. detection. RESULTS: In Group AS, dihydroartemisinin oral clearance values (mean (95% CI)) were similar on day 1 (32 (22, 47)) l h(-1) and day 5 (38 (28, 51)) l h(-1) of daily artesunate administration but these mean values were approximately three fold higher compared with day -5 after a single dose (95 (56, 159)). In this group, artemisinin oral clearance increased from 196 (165, 232) l h(-1) on day 1-315 (241, 410) l h(-1) on day 5. In Group AM, dihydroartemisinin oral clearance on day 1 was 39 (34, 46) l h(-1) and increased 1.6 fold to 64 (48, 85) l h(-1) on day 5. In this group, artemisinin oral clearance increased sequentially (1.5 and 4.7 fold, respectively) from 207 (151, 285) l h(-1) on day -5-308 (257, 368) l h(-1) on day 1 and to 981 (678, 1420) l h(-1) on day 5. The increase in artemisinin oral clearance between days -5 and 1 (in the absence of artesunate) was similar to that between days 1 and 5 in Group AS subjects who took daily artesunate. Dihydroartemisinin was not a significant metabolite of artemisinin. CONCLUSIONS: Artesunate (dihydroartemisinin) did not alter the elimination of artemisinin. However, dihydroartemisinin elimination was inhibited by artemisinin. Artemisinin induced its own elimination even 5 days after a single oral dose. There was no evidence for the formation of dihydroartemisinin from artemisinin.     

Pharmacokinetics and tissue distribution of galantamine and galantamine-related radioactivity after single intravenous and oral administration in the rat

The plasma kinetics and tissue distribution of galantamine hydrobromide [4aS-(4a alpha,6beta,8aR*)]-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro-[3a,3,2-ef] [2benzazepin-6-ol hydrobromide, CAS-1953-04-4], a reversible acetylcholinesterase inhibitor, were studied in male and female non-pregnant and pregnant SPF Wistar rats and in male Fisher x Copenhagen pigmented rats. Most studies were performed using 3H-labelled galantamine hydrobromide, measuring unchanged drug (UD) and non-volatile radioactivity (NVR) in plasma and tissues by high-performance liquid chromatography (HPLC), liquid scintillation counting and quantitative whole-body autoradiography (QWBA). Plasma levels after single intravenous administration of UD (1.25-2.5 mg/kg) declined bi- or triphasically, with an elimination half-life of 3.5 h in male, and 5.1 h in female rats. The plasma clearance (Cl) averaged 1.9 l/kg/h (male rats) and 0.9 l/kg/h (female rats), and the volume of distribution (VdSS) was about 5 l/kg for both male and female rats. Following oral administration (2.5-10 mg/kg), galantamine was rapidly absorbed in both sexes, with an absolute oral bioavailability of 77%. Distribution studies after oral administration of 3H-galantamine showed an almost immediate equilibrium between plasma and tissues, with highest tissue levels of NVR and UD in liver, kidney, salivary glands, adrenal glands and, for the female rat, spleen, and lowest in white fat. To most tissues and especially to brain, the distribution of UD was more pronounced than that of its metabolites. Tissue concentrations of UD and NVR declined at a similar rate as plasma, showing no undue retention. QWBA in the pigmented rat showed the same distribution and elimination pattern of NVR. Only in hair follicles and choroid some retention of NVR was seen, but the calculated half-life was less than one day. In the female pregnant SPF Wistar rat, maternal tissue distribution of NVR was similar to that of the non-pregnant rat. NVR tissue levels in the foetus were similar to those found in maternal blood during the whole experiment, indicating a rapid equilibrium without accumulation.