In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS.

This study explored the effects of cucurbitacin E (CuE), a bioactive compound from Cucurbitaceae, on the metabolism/pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chromatography-(tandem) mass spectrometry (LC-MS/MS) assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE (1.25-100 µmol L-1) competitively inhibited tolbutamide 4-hydroxylation (CYP2C11) activity only in concentration-dependent manner with a K i value of 55.5 µmol L-1 in vitro. In whole animal studies, no significant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, multiple pretreatments of CuE (200 µg kg-1 d-1, 3 d, i.p.) significantly decreased tolbutamide clearance (CL) by 25% and prolonged plasma half-time (T 1/2) by 37%. Moreover, CuE treatment (50-200 µg kg-1 d-1, i.p.) for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or natural products containing CuE to avoid unnecessary drug-drug interactions.

Dietary flavonoids modulate CYP2C to improve drug oral bioavailability and their qualitative/quantitative structure-activity relationship.

This study aims to improve the drug oral bioavailability by co-administration with flavonoid inhibitors of the CYP2C isozyme and to establish qualitative and quantitative (QSAR) structure-activity relationships (SAR) between flavonoids and CYP2C. A total of 40 naturally occurring flavonoids were screened in vitro for CYP2C inhibition. Enzyme activity was determined by measuring conversion of tolbutamide to 4-hydroxytolbutamide by rat liver microsomes. The percent inhibition and IC50 of each flavonoid were calculated and used to develop SAR and QSAR. The most effective flavonoid was orally co-administered in vivo with a cholesterol-reducing drug, fluvastatin, which is normally metabolized by CYP2C. The most potent CYP2C inhibitor identified in vitro was tamarixetin (IC50 = 1.4 µM). This flavonoid enhanced the oral bioavailability of fluvastatin in vivo, producing a >2-fold increase in the area under the concentration-time curve and in the peak plasma concentration. SAR analysis indicated that the presence of a 2,3-double bond in the C ring, hydroxylation at positions 5, 6, and 7, and glycosylation had important effects on flavonoid-CYP2C interactions. These findings should prove useful for predicting the inhibition of CYP2C activity by other untested flavonoid-like compounds. In the present study, tamarixetin significantly inhibited CYP2C activity in vitro and in vivo. Thus, the use of tamarixetin could improve the therapeutic efficacy of drugs with low bioavailability.

Evaluation of the pharmacokinetic interaction between ticagrelor and tolbutamide, a cytochrome P450 2C9 substrate, in healthy volunteers.

OBJECTIVES: To assess the effect of ticagrelor on the pharmacokinetics of tolbutamide (a CYP2C9 substrate), and the effect of tolbutamide on ticagrelor pharmacokinetics. METHODS: In this randomized, double-blind, two-period, crossover study, 23 healthy volunteers received either placebo or ticagrelor 180 mg twice daily (b.i.d.) for 9 days, with a single open-label oral dose of tolbutamide 500 mg on Day 5. After washout (14 days), volunteers received the alternate treatment. Plasma concentrations of tolbutamide, 4-hydroxytolbutamide, ticagrelor, and AR-C124910XX were determined for pharmacokinetic analyses. RESULTS: Ticagrelor had no effect on tolbutamide or 4-hydroxytolbutamide pharmacokinetic parameters. The geometric least square mean ratios for maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from Time 0 to infinity (AUC0-∞) were lose to unity, and the 90% confidence intervals (CI) were within the range 0.80 - 1.25 for both tolbutamide and 4-hydroxytolbutamide. The terminal elimination half-life (t1/2), and time to maximal plasma concentrations (tmax) for tolbutamide and its metabolite were unaffected by ticagrelor coadministration. Tolbutamide had no effect on the Cmax, area under the concentration curve over the 2-hour dosing interval (AUC0-τ), t1/2 or tmax of either ticagrelor or AR-C124910XX. Coadministration of ticagrelor and tolbutamide was well tolerated. CONCLUSIONS: These results suggest that ticagrelor does not affect tolbutamide metabolism and is therefore unlikely to affect CYP2C9-mediated metabolism of drugs.

Synthesis, antidiabetic and hypolipidemic activities of new diethylamine and triethoxysilyl derivatives of tolbutamide on rats.

Tolbutamide (N-[(butylamino)carbonyl]-4-methylbenzenesulfonamide, CAS 64-77-7, I) is the first generation of potassium channel blocker and sulfonylurea oral hypoglycemic drug that imparts marked blood glucose lowering effect in type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM) patients. In this study, I and its two new analogs with substituting butylamine side by 3-diethylamino-1-propylamine (3) and 3-triethoxysilyl-1-propylamine (4) were synthesized and their antidiabetic and hypolipidemic activities were evaluated applying known procedures, and compared with both I and glibenclamide (II, well known second-generation sulfonylurea antidiabetic drug). The results showed that these new drugs (III and IV) were more potent than I and II and it could be concluded that changing the amine side of I would produce more potentials in new drugs (III and IV) of the first generation, to be named as the third generation. Also investigations on hypoglycemic and lipid lowering effects of these drugs proved that IV could reduce glucose, triglyceride (TG) and a low density lipoprotein (VLDL) level in blood serum more than others (I-III), 16 days after STZ injection.

Synthesis and preliminary biological profile of new NO-donor tolbutamide analogues.

We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological characterization of these compounds, including stimulation of insulin release from cultured rat pancreatic ß-cells and in vitro vasodilator and anti-aggregatory activities, is reported.

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats.

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.

[The inhibition of CYP2C9 isoenzyme in Cunninghamella blakesleeana AS 3. 910].

AIM: To investigate the variation of CYP2C9 isoenzyme activity in the microbial model in response to inhibitors of CYP2C9. METHODS: Using C. blakesleeana AS 3. 910 as a model strain, the impact of CYP2C9 inhibitors on the metabolites yields of CYP2C9 substrates was determined and the drug-drug interactions among CYP2C9 substrates were evaluated. Liquid chromatography-mass spectrometry was used to analyze biotransformation products. RESULTS: Benzbromarone decreased the yield of 4'-hydroxytolbutamide from 100% to 14.5%; sulfaphenazole decreased the yield of O-demethylindomethacin from 75.2% to 9.9%; valproic acid decreased the yield of 4'-hydroxydiclofenac from 98.6% to 2.7%, separately. Tolbutamide, indomethacin and diclofenac interacted with each other, resulting in the decreased formation of metabolites catalyzed by CYP2C9. CONCLUSION: Three CYP2C9 inhibitors inhibit the activity of CYP2C9 isoenzyme in C. blakesleeana AS 3. 910 differently, and there are drug-drug interactions among CYP2C9 substrates.

Biotransformation of tolbutamide to 4'-hydroxytolbutamide by the fungus Cunninghamella blakesleeana.

The hypoglycemic drug tolbutamide is commonly used as a probe drug to evaluate CYP2C9 enzyme activity in terms of production of 4'-hydroxytolbutamide. In the present study, an initial screening of seven filamentous fungi was carried out to identify which was most competent to transform tolbutamide into 4'-hydroxytolbutamide. From this screening, the fungus Cunninghamella blakesleeana AS 3.910 was selected as a suitable bioconverter. At a concentration of 1.2 mg ml(-1), the growing fungus transformed 95.0% of tolbutamide into 4'-hydroxytolbutamide in 96 h. With resting culture, the yield could reach 91.7% and exceeded 91.0% even when the tolbutamide concentration was increased to 4.0 mg ml(-1). On scale-up to 3 l buffer containing 12.0 g tolbutamide, 90% of tolbutamide was transformed into 4'-hydroxytolbutamide in 96 h. Work-up of the broth by column chromatography and recrystallization yielded 6.5 g (53.9% recovered) of 4'-hydroxytolbutamide with a purity of more than 99%. These results suggest C. blakesleeana AS 3.910 is a useful biosynthetic tool in the preparation of 4'-hydroxytolbutamide.

Cytochrome P450 2C9 phenotyping using low-dose tolbutamide.

OBJECTIVES: The hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500 mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking. METHODS: We examined tolbutamide and its metabolites 4'-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24 h after intake of 125 mg tolbutamide using liquid chromatography-tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1-*3 combinations who received 500 mg tolbutamide. RESULTS: Plasma clearance and tolbutamide plasma concentrations 24 h after drug intake reflected the genotypes: 0.85 l/h and 1.70 microg/ml (95% confidence interval, CI, 0.80-0.89 l/h and 1.50-1.90 microg/ml) for CYP2C9*1 homozygotes (n=15), 0.77 l/h and 2.14 microg/ml (95%CI, 0.67-0.88 l/h and 1.64-2.63 microg/ml) for *1/*2 genotypes (n=7), 0.60 l/h and 3.13 microg/ml (95%CI, 0.58-0.62 l/h and 2.68-3.58 microg/ml) for *1/*3 genotypes (n=3), and 0.57 l/h and 3.27 microg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24 h after intake correlated to plasma clearance (r(2)=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r(2)=0.97, P<0.0000001). CONCLUSIONS: A low dose of 125 mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24 h after drug intake.

Effect of albumin on phenytoin and tolbutamide metabolism in human liver microsomes: an impact more than protein binding.

The cytochrome P450 (P450)-dependent conversion of phenytoin (PHT) to p-hydroxy phenytoin (pHPPH), and tolbutamide (TLB) to 4-hydroxy tolbutamide (hydroxy-TLB), in human liver microsomes was studied in the presence of increasing concentrations (0-4%) of bovine serum albumin (BSA). Therefore, the free fraction (f(u)) of PHT and TLB varied. Whereas the f(u) of PHT (5 microM) decreased, an increase (3-fold), rather than a decrease in the pHPPH formation rate was observed when BSA (<1%) was present. The stimulation was attributed to a significant decrease in apparent K(m). The change, however, was diminished as the BSA concentration reached 4% (PHT f(u) = 0.2), in which the reaction velocity remained the same as that measured in the absence of BSA. Therefore, unchanged K(m) (16.2 +/- 0.7 microM) and V(max) (9.4 +/- 0.2 pmol/min/mg of protein) values were determined based on total PHT concentrations, whereas correction for f(u) led to an unbound K(m) (K(mu)) of approximately 3.2 microM. Similarly, the metabolism of TLB (50 microM) was enhanced (approximately 2-fold) in the presence of 0.25% BSA but remained only 35% of the control activity (no BSA) at 1% BSA. However, the remaining activity was higher (3-fold) than that determined with an equivalent free concentration of TLB (4 microM) calculated according to its f(u) (0.08). The difference became less significant when BSA concentration was 4% (f(u) < 0.02). Collectively, the results suggest a 2-fold effect of BSA on PHT and TLB hydroxylation: first, facilitation of the reactions via a decrease in K(m); second, a decrease in f(u) leading to a drop in reaction rate. For a given P450 reaction, therefore, the effect of BSA may depend upon enzyme affinity, catalytic capacity, and the extent of protein binding.

Impact of CYP2C9 and CYP2C19 polymorphisms on tolbutamide kinetics and the insulin and glucose response in healthy volunteers.

Tolbutamide is known to be metabolized by cytochrome P450 2C9 (CYP2C9), and the effects of the CYP2C9 amino acid polymorphisms *2 (Arg144Cys) and *3 (Ile359Leu) could be important for drug treatment with tolbutamide and for use of tolbutamide as a CYP2C9 test drug. Tolbutamide pharmacokinetics and plasma insulin and glucose concentrations were studied in 23 healthy volunteers with all six combinations of the CYP2C9 alleles *1, *2 and *3, including two subjects with the combined CYP2C9*1/*1 and CYP2C19*2/*2 genotype. Volunteers received a single oral dose of 500 mg tolbutamide, followed by 75 g oral glucose at 1, 4.5 and 8 h after tolbutamide administration. Pharmacokinetic analysis was performed using a computer program for regression analysis of nonlinear mixed effects models. The mean oral clearances of tolbutamide were 0.97 (95% confidence interval [CI] 0.89-1.05), 0.86 (95% CI 0.79-0.93), 0.75 (95% CI 0.69-0.81), 0.56 (95% CI 0.51-0.61), 0.45 (95% CI 0.41-0.49) and 0.15 (95% CI 0.14-0.16) l/h in carriers of CYP2C9 genotypes 1/*1, *1/*2, *2/*2, *1/*3, *2/*3 and *3/*3, respectively. Tolbutamide pharmacokinetics in carriers of the functionally deficient CYP2C19*2/*2 genotype were not different from those in the CYP2C19 highly active genotype. Elimination in the six CYP2C9 genotype groups could be expressed as the linear combination of three constants (0.05, 0.04, 0.01 h(-1), which were specific to the respective CYP2C9 alleles *1, *2 and *3, thus indicating a co-dominant mode of inheritance. Insulin and glucose concentration-time curves did not change with differing CYP2C9 genotypes. Tolbutamide was confirmed as a substrate of the genetically polymorphic enzyme CYP2C9. The pronounced differences in pharmacokinetics due to the amino acid variants did not significantly affect plasma insulin and glucose concentrations in healthy volunteers.

Effects of CYP2C19 and CYP2C9 genetic polymorphisms on the disposition of and blood glucose lowering response to tolbutamide in humans.

Several recent in-vitro data have revealed that CYP2C19, in addition to CYP2C9, is also involved in the 4-methylhydroxylation of tolbutamide. We evaluated the relative contribution of CYP2C9 and CYP2C19 genetic polymorphisms on the disposition of blood glucose lowering response to tolbutamide in normal healthy Korean subjects in order to reappraise tolbutamide as a selective in-vivo probe substrate of CYP2C9 activity. A single oral dose of tolbutamide (500 mg) or placebo was administered to 18 subjects in a single-blind, randomized, crossover study with a 2-week washout period. Twelve subjects (of whom six were CYP2C19 extensive metabolizer (EM) and six were CYP2C19 poor metabolizer (PM) genotype) were of the homozygous wild-type CYP2C9*1 genotype; the other six subjects were of the CYP2C9*1/*3 and CYP2C19 EM genotype. Pharmacokinetic parameters were estimated from plasma and urine concentrations of tolbutamide and 4-hydroxytolbutamide. Serum glucose concentrations were measured before and after oral intake of 100 g dextrose. In subjects heterozygous for the CYP2C9*3 allele, C(max) and AUC of tolbutamide were significantly greater and the plasma half-life significantly longer than those in homozygous CYP2C9*1 subjects. No pharmacokinetic differences were found between CYP2C19 EM and PM genotype subjects. The estimated AUC of the increase in serum glucose after oral intake of 100 g dextrose was 2.7-fold higher in subjects with the wild-type CYP2C9 genotype than in those with CYP2C9*1/*3, but CYP2C19 genetic polymorphism did not alter the blood glucose lowering effect of tolbutamide. The plasma AUC of 4-hydroxytolbutamide and the ratio of 4-hydroxytolbutamide/tolbutamide did not differ significantly between CYP2C19 PM and EM genotype subjects, while these parameters were about twice as high in subjects with the wild-type CYP2C9 genotype than in heterozygous CYP2C9*3 subjects (P < 0.05). Our results strongly suggest that the disposition and hypoglycemic effect of tolbutamide are affected mainly by CYP2C9 genetic polymorphism, but not by CYP2C19 polymorphism. The in-vivo contribution of CYP2C19 to tolbutamide 4-methylhydroxylation appears to be minor in humans. This suggests that, at least in vivo, tolbutamide remains a selective probe for measuring CYP2C9 activity in humans.

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.

Quantitative determination of tolbutamide and its metabolites in human plasma and urine by high-performance liquid chromatography and UV detection.

An isocratic, high-performance liquid chromatography method has been developed for simultaneous determination of the oral antidiabetic tolbutamide and two of its metabolites, 4-hydroxytolbutamide and carboxytolbutamide, in human plasma and urine. The method was based on simple one-step liquid-liquid extraction with tertiary-butyl methyl ether as extraction solvent. The chromatographic eluent was 23:77 (v/v) methanol: 0.01 M aqueous sodium acetate buffer pH 3.0, and the UV detection was performed at a wavelength of 230 nm. The limit of detection was 0.1 microM for tolbutamide in plasma and 1.5 microM, 0.5 microM, and 0.75 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The limit of quantitation was 0.5 micro for tolbutamide in plasma and 2 microM, 0.75 microM, and 1.25 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The overall mean recoveries ranged from 91% to 109% for tolbutamide in plasma and from 80% to 98% in urine for all three compounds.

Identification of S-(n-butylcarbamoyl)glutathione, a reactive carbamoylating metabolite of tolbutamide in the rat, and evaluation of its inhibitory effects on glutathione reductase in vitro.

Tolbutamide (TOLB), a widely used hypoglycemic agent in the therapy of non-insulin-dependent diabetes mellitus, has been reported to be teratogenic and/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are linked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductase (GR), although the mechanism by which this inhibition occurs remains unknown. In the study presented here, rats were injected with TOLB (200 mg/kg ip), and bile was collected for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarbamoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl isocyanate, as a minor metabolite of TOLB in bile. Upon incubation of SBuG (0.25-1.0 mM) with GR from either yeast or bovine intestinal mucosa in the presence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentration-dependent manner. No inhibition was observed when NADPH was omitted from incubations, or when the natural substrate for the enzyme, glutathione disulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over the concentration range of 0.8-2.0 mM. It is concluded that metabolic activation of TOLB in vivo leads to the generation of reactive intermediates (n-butyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At critical periods of organogenesis, the resulting perturbation of GSH homeostasis in exposed tissues may play a key role in the teratogenic and/or embryotoxic effects of TOLB.

Pharmacokinetics of tolbutamide in ethnic Chinese.

AIMS: Ethnic differences in drug disposition have been described for many drugs. Despite the widespread use of tolbutamide in Asian populations, the pharmacokinetics of tolbutamide, a CYP2C9 substrate, have not been described in ethnic Chinese. METHODS: The pharmacokinetics of tolbutamide (500 mg orally) were studied in 10 young, healthy volunteers (seven male/three female; age 21-29 years), each of whom had four ethnic Chinese grandparents. Plasma concentrations of tolbutamide were measured for 32 h post-dose by high performance liquid chromatography. The concentrations of hydroxytolbutamide and carboxytolbutamide were also measured in urine for 32 h post-dose. Noncompartmental pharmacokinetic parameters were calculated using standard equations and compared with those previously reported in Caucasian subjects using the Mann-Whitney U test. RESULTS: Pharmacokinetic parameters in Chinese (mean+/-s.d.) including Cmax (63+/-11 microg ml(-1)), tmax (median 3.3 h; range 1.6-6.0 h), V/F (9.1+/-1.7 l) and t1/2, (9.1 h; harmonic mean) were similar to the values in Caucasians. CL/F (637+/-88 ml h(-1)) was higher in Chinese than Caucasians. The urinary recoveries of hydroxytolbutamide (13+/-1% of dose) and carboxytolbutamide (68+/-5% of dose) and the partial apparent metabolic clearance (0.15+/-0.02 ml min(-1) kg(-1)) in Chinese were comparable with Caucasians. CONCLUSIONS: The pharmacokinetics of tolbutamide have been described in ethnic Chinese and the disposition is similar to that reported in Caucasians. This study suggests that there is no substantial ethnic difference in the tolbutamide hydroxylase activity of CYP2C9.

Inhibition of CYP2C9 by selective serotonin reuptake inhibitors: in vitro studies with tolbutamide and (S)-warfarin using human liver microsomes.

OBJECTIVE: To investigate the in vitro potential of selective serotonin reuptake inhibitors (SSRIs) to inhibit two CYP2C9-catalysed reactions, tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. METHODS: The formation of 4-hydroxytolbutamide from tolbutamide and that of 7-hydroxywarfarin from (S)-warfarin as a function of different concentrations of SSRIs and some of their metabolites was studied in microsomes from three human livers. RESULTS: Both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation followed one enzyme Michaelis-Menten kinetics. Kinetic analysis of 4-hydroxytolbutamide formation yielded a mean apparent Michaelis-Menten constant (Km) of 133 microM and a mean apparent maximal velocity (Vmax) of 248 pmol x min(-1) x mg(-1); formation of 7-hydroxywarfarin yielded a mean Km of 3.7 microM and a mean Vmax of 10.5 pmol x min(-1) x mg(-1). Amongst the SSRIs and some of their metabolites tested, only fluvoxamine markedly inhibited both reactions. The average computed inhibition constant (Ki) values and ranges of fluvoxamine when tolbutamide and (S)-warfarin were used as substrate, were 13.3 (6.4-17.3) microM and 13.0 (8.4-18.7) microM, respectively. The average Ki value of fluoxetine for (S)-warfarin 7-hydroxylation was 87.0 (57.0-125) microM. CONCLUSION: Amongst the SSRIs tested, fluvoxamine was shown to be the most potent inhibitor of both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. Fluoxetine, norfluoxetine, paroxetine, sertraline, desmethylsertraline, citalopram, desmethylcitalopram had little or no effect on CYP2C9 activity in vitro. This is consistent with in vivo data indicating that amongst the SSRIs, fluvoxamine has the greatest potential for inhibiting CYP2C9-mediated drug metabolism.

Inhibitory effect of nicotine and its metabolites on tolbutamide hydroxylation in rat liver microsomes.

A simple HPLC/fluorescence method to detect hydroxytolbutamide (a major metabolite of the anti-diabetic drug tolbutamide) has been developed. The effects of nicotine and some of its metabolites on tolbutamide hydroxylation is described. An extraction procedure with diethyl ether was followed by isocratic HPLC analysis of tolbutamide hydroxylation with a binary mobile phase composed of 10 mM monobasic sodium phosphate in methanol (45:55, v/v, apparent pH 2.28). A detection limit of sub-nanogram amounts (0.353 ng) of hydroxytolbutamide was obtained with fluorescence detection at 226 nm for excitation and 318 nm for emission. Overall precision values for hydroxytolbutamide was determined with coefficients of variation of 1.4-4.6% when nanogram levels of the metabolite were analyzed. Differential inhibitory responses were demonstrated for tolbutamide hydroxylation to nicotine and its metabolites. Tolbutamide hydroxylation was apparently inhibited by cotinine and relatively less inhibited by nicotine. Nornicotine, however, caused very little inhibition of tolbutamide hydroxylation. The implication is that nornicotine may not share similar affinity for the substrate binding site for tolbutamide. The results also suggest that heavy smokers may experience reduction in tolbutamide metabolism. The assay system itself will be useful for future studies of tolbutamide, and possibly related sulfonylureas.

Characterization of acetohexamide reductases purified from rabbit liver, kidney, and heart: structural requirements for substrates and inhibitors.

The structural requirements of acetohexamide reductases purified from rabbit liver, kidney, and heart for substrates and inhibitors were examined. Acetohexamide, an oral antidiabetic drug with a ketone group, and analogs of it with various alkyl groups instead of the cyclohexyl group were used as substrates for these three enzymes. The results obtained as to substrate specificity suggested that the nature of the substrate-binding region of the heart enzyme is markedly different from those of the substrate-binding regions of the liver and kidney enzymes. Tolbutamide, which has no ketone group within its chemical structure, strongly inhibited the heart enzyme, whereas it had little ability to inhibit the liver or kidney enzyme. The inhibition of the heart enzyme by tolbutamide was competitive with respect to acetohexamide and uncompetitive with respect to NADPH. Furthermore, tolbutamide analogs with n-pentyl and n-hexyl groups instead of the n-butyl group exhibited very pronounced inhibition of only the heart enzyme. Therefore, it is reasonable to postulate that the heart enzyme, unlike the liver and kidney ones, has a cleft of a strongly hydrophobic nature near its substrate-binding region, and that this hydrophobic cleft plays a critical role in the interaction of the heart enzyme with the cyclohexyl group of acetohexamide.