Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate.

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL(int)) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CL(int) of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL(int) of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL(int) of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

In vitro activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation in human liver microsomes.

1. The effects of substrate concentration and enzyme source (human liver microsomes and recombinant cytochrome P450s, CYP) on the activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation were investigated. 2. 7-Benzyloxyresorufin O-debenzylase activity in human liver microsomes was inhibited by a monoclonal antibody against CYP2B6 and a polyclonal antibody against CYP3A2 by 53-69 and 19-44%, respectively, suggesting that CYP2B6 and CYP3A4 mainly catalyse 7-benzyloxyresorufin O-debenzylation in human liver microsomes. 3. 7-Benzyloxyresorufin O-debenzylase activity at 0.2-5 micro M substrate concentrations in human liver microsomes was increased by the addition of alpha-naphthoflavone, quinidine, testosterone and progesterone, and the V(max) of 7-benzyloxyresorufin O-debenzylation increased with increasing alpha-naphthoflavone concentrations, whereas the K(m) remained constant. Additionally, 7-benzyloxyresorufin O-debenzylation by recombinant CYP3A4 was increased by the addition of alpha-naphthoflavone, testosterone and progesterone but not by quinidine, whereas no chemicals tested could activate the O-debenzylation of 7-benzyloxyresorufin by CYP2B6. 4. The K(m) for nifedipine oxidation activity by CYP3A4 decreased by the addition of progesterone, whereas the V(max) remained constant. Quinidine and testosterone increased 7-benzyloxyresorufin O-debenzylase and nifedipine oxidase activities, respectively, in human liver microsomes, whereas activation was not observed in CYP3A4. 5. The results suggest that in vitro activation patterns are substrate dependent and that selection of the enzyme source can influence the activation phenomenon.

Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans.

We investigated the quantitative prediction of human hepatic metabolic clearance from in vitro experiments focusing on cytochrome P450 metabolism with eight model compounds, FK1052, FK480, zolpidem, omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem. For the compounds, in vivo human hepatic extraction ratios ranged widely from 0.03 to 0.87. In vitro and in vivo hepatic intrinsic clearance (CL(int)) values for each compound were measured and calculated in rats and/or dogs and humans. CL(int,in vitro) was determined from a substrate disappearance rate at 1 microM in hepatic microsomes, which was a useful method. CL(int,in vivo) was calculated from in vivo pharmacokinetic data using three frequent mathematical models (the well stirred, parallel-tube, and dispersion models). The human scaling factor values (CL(int,in vivo)/CL(int,in vitro)) showed marked difference among the model compounds (0.3-26.6-fold). On the other hand, most of the animal scaling factors were within 2-fold of the values in humans, suggesting that scaling factor values were similar in the different animal species. When human CL(int,in vitro) values were compared with the actual CL(int,in vivo), correlation was not necessarily good. By contrast, using human CL(int,in vitro) corrected with the rat and/or dog scaling factors yielded better predictions of CL(int,in vivo) that were mostly within 2-fold of the actual values. Furthermore, successful predictions of human CL(oral) and hepatic extraction ratio (E(H)) were obtained by use of the human CL(int,in vitro) corrected with animal scaling factors. The new variant method is a simple one, incorporating additional information from animal studies and providing a more reliable prediction of human hepatic clearance.

Stereoselective metabolism of cibenzoline, an antiarrhythmic drug, by human and rat liver microsomes: possible involvement of CYP2D and CYP3A.

Stereoselective metabolism of cibenzoline succinate, an oral antiarrhythmic drug, was investigated on hepatic microsomes from humans and rats and microsomes from cells expressing human cytochrome P450s (CYPs). Four main metabolites, M1 (p-hydroxycibenzoline), M2 (4,5-dehydrocibenzoline), and unknown metabolites M3 and M4, were formed by human and rat liver microsomes. The intrinsic clearance (CL(int)) of the M1 formation from R(+)-cibenzoline was 23-fold greater than that of S(-)-cibenzoline in human liver microsomes, whereas the R(+)/S(-)-enantiomer ratio of CL(int) for M2, M3, and M4 formation was 0.39 to 0.83. The total CL(int) for the formation of the four main metabolites from S(-)- and R(+)-cibenzoline was 1.47 and 1.64 microl/min/mg, respectively, suggesting that the total CL(int) in R(+)-enantiomer was slightly greater than that in S(-)-enantiomer in human liver microsomes. The M1 formation from R(+)-cibenzoline was highly correlated with bufuralol 1'-hydroxylation and CYP2D6 content and was inhibited by quinidine, a potent inhibitor of CYP2D6. Additionally, only microsomes containing recombinant CYP2D6 were capable of M1 formation. These results suggest that the M1 formation from R(+)-cibenzoline was catalyzed by CYP2D6. The formation of M2, M3, and M4 from S(-)- and R(+)-cibenzoline was highly correlated with testosterone 6beta-hydroxylation and CYP3A4 content. Ketoconazole, which is a potent inhibitor of CYP3A4/5, had a strong inhibitory effect on their formation, and the M4 formation from R(+)-cibenzoline was inhibited by quinidine by 45%. The formation of M2 was also inhibited by quinidine by 46 to 52% at lower cibenzoline enantiomers (5 microM), whereas the inhibition by quinidine was not observed at a higher substrate concentration (100 microM). In male rat liver microsomes, ketoconazole and quinidine inhibited the formation of the main metabolites, M1 and M3, >74% and 44 to 59%, respectively. These results provide evidence that CYP3A and CYP2D play a major role in the stereoselective metabolism of cibenzoline in humans and male rats.

Residual short-range order in the heavy fermion compound CeInCu2.

By using the X-ray diffraction technique, residual short-range order was detected in CeInCu(2), cerium dicopper indium, which is known to be a heavy fermion compound. In spite of the long-range order of this substance, diffuse scattering exhibiting short-range order was observed at room temperature. The correlation parameters obtained showed that an incorrectly occupied lattice site has a tendency to gather atoms of different species at the neighboring sites along the <111> directions. Thus, the disordered region would form a cluster composed of several disordered atoms. Furthermore, a chain-type correlation which has a period of 20-23 Å along the same directions was indicated. The superstructure-like feature of the disordered atoms hardly increases the residual resistivity. It is consistent with the behavior of the residual resistivity under high pressure.

Osteotropic drug delivery system (ODDS) based on bisphosphonic prodrug. I: synthesis and in vivo characterization of osteotropic carboxyfluorescein.

An osteotropic drug delivery system (ODDS) based on a bisphosphonic prodrug was designed as a novel method for site-specific and controlled delivery of drugs to the bone. Due to the chemical adsorption of bisphosphonic promoiety to the mineral component, hydroxyapatite, a bisphosphonic prodrug is predominantly taken up into the bone. To verify the concept, bisphosphonic promoiety was chemically introduced into 6-carboxyfluorescein (CF) as a model compound and the disposition after intravenous injection was studied in rats. The bisphosphonic prodrug of CF, disodium (fluorescein-6-carbonyloxy) acetoaminomethylene bisphosphonate (CF-BP) was highly taken up to the skeleton (62.1% of dose) and the remainder was excreted into the urine (35.9% of dose). Subsequently, regeneration of CF by hydrolysis of CF-BP in the bone was observed. The microscopic observation showed that CF-BP was buried into the bone with a calcification of the bone. According to the remodeling of the bone, bisphosphonic prodrug buried was supposed to be released in the vicinity of the osteoclast or resorption surface of the bone. Thus, it is suggested that ODDS has a potential to achieve osteoclast-specific or resorption surface-specific targeting of the drugs.

Oral absorption of FK506 in rats.

The oral absorption of FK506 in solid dispersion formulation was studied in rats. The obtained area under the concentration versus time curve (AUC) increased in a nonlinear fashion with a small dose-dependent increase in the peak blood concentrations (Cmax). The peak concentration time (Tmax) was observed within 30 min after administration in all dosing groups (1-10 mg/kg) with or without feeding, whereas the oral absorption of FK506 was reduced to about 50% by gavage at a dose of 1 mg/kg. Participation of first-pass elimination was suggested by comparing the blood levels after infusion via the portal vein with those after infusion via the femoral vein. Further, in an in vitro stability study and an in situ loop absorption study, FK506 was fairly stable in the gastrointestinal juice and was absorbed predominantly from the upper part of the small intestine.