This article is available online at http://dmd.aspetjournals.org ABSTRACT:We investigated hepatic in vitro intrinsic clearance (CL int,in vitro ) in freshly isolated or cryopreserved hepatocytes and compared with CL int,in vivo by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDPglucuronosyltransferase, sulfotransferase, and/or esterase. CL int,in vitro was determined from substrate disappearance rate at 1 M in hepatocytes. CL int,in vivo was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL int,in vitro in freshly isolated hepatocytes, the rat scaling factor values (CL int,in vivo /CL int,in vitro ) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL int,in vitro values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL int,in vitro were determined by use of cryopreserved hepatocytes. When human CL int,in vitro was regarded as the predicted CL int,in vivo , the observed and predicted CL int,in vivo for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL int,in vitro corrected with the rat scaling factors yielded better predictions of CL int,in vivo that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.Recently, pharmacokinetic investigation has played an increasingly important role in drug discovery. In particular, it is very important to predict human hepatic metabolic clearance because most drugs are eliminated from the body predominantly by hepatic metabolism. For predicting hepatic clearance, theoretical aspects of in vitro/in vivo scaling based on a physiological model and clearance concepts have been developed (Roberts and Rowland, 1986). Application of this method has been successful in predicting in vivo hepatic clearance in rats for many drugs metabolized by P450 1 from in vitro metabolism data using rat liver microsomes and isolated hepatocytes (Sugiyama et al., 1988;Houston and Carlile, 1997). Because human liver samples have become more readily available, it would also be very useful to predict in vivo outcomes from in vitro data in humans. However, there has been relatively limited application of this approach (Hoener, 1994), and there have been some failed attempts at the prediction of human hepatic clearance. For example, Iwatsubo et al. (1997) and Houston and Carlile (1997) reported that CL int,in vitro generally exhibited a positive correlation with CL int,in vivo , but in some cases animal or human clearance values were not well predicted from in vitro studies. To improve the predict...

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.

Micafungin, a new echinocandin-like lipopeptide antifungal agent, has potent in vitro and in vivo activity against a variety of pathogenic Candida species and Aspergillus species by inhibiting the biosynthesis of 1,3-b-D-glucan, a major and specific component of the fungal cell wall, and its minimal inhibitory concentrations at which 90% of the isolates were inhibited (MIC 90 ) against Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Aspergillus flavus are less than 0.125 mg/ml.1-4) Micafungin has a significant therapeutic effect against deepseated mycoses caused by Candida or Aspergillus, the major pathogenic fungi. The clinical responses in trials that examined the safety and efficacy of micafungin monotherapy (micafungin dosage: 12.5-150 mg) in Japan were 60% in invasive pulmonary aspergillosis, 67% in clonic necrotizing pulmonary aspergillosis, 55% in pulmonary aspergilloma, 100% in candidiasis, and 71% in esophageal candidiasis. 2,5) It has been reported that micafungin exhibits linear pharmacokinetics after intravenous dosing to rats, mice, dogs, and rabbits, as well as humans. [6][7][8][9] The radioactivity after intravenous dosing of 14 C-labeled micafungin to male rats is widely distributed immediately and the radioactivity in tissues decreases almost in parallel with the radioactivity in plasma.10) Unchanged micafungin concentrations in rabbit tissues, including the liver, kidney, lungs, and spleen, at near peak plasma concentrations 30 min after the last of multiple doses over eight days are several-fold in excess of the MIC 90 against the clinical isolates of Candida spp. and Aspergillus spp. 7) However, there are few detailed studies on the tissue distribution kinetics of unchanged drug after an intravenous dose of micafungin in animals and humans.In the present study, we investigated the distribution kinetics in tissues, such as liver, kidney, and lungs, after an intravenous dose of micafungin to male rats. MATERIALS AND METHODS MaterialsMicafungin and internal standard (FR195743) 11) were synthesized and supplied by Fujisawa Pharmaceutical Co., Ltd. All other reagents were of the highest purity commercially available.Animal Studies Male Sprague-Dawley rats of 7 weeks of age and weighing between 287 and 318 g, obtained from Charles River Japan (Kanagawa, Japan), were used. During the experiments, the rats were housed at a temperature of 23Ϯ2°C and relative humidity of 55Ϯ5% with a 12-h night/day cycle. Micafungin sodium was dissolved in saline (1 mg/ml), and administered as a single intravenous bolus at a dose of 1 mg/kg. Blood and tissue (liver, kidney, and lung) samples were collected predose and at the following times after the dose: 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h. All blood samples were collected from the abdominal aorta under ether anesthesia and immediately centrifuged at 4°C to obtain the plasma. The plasma and tissue samples were frozen at Ϫ20°C until analysis. Determination of Micafungin in Plasma and TissuesThe plasma concentrat...

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.

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MScan be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.

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) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type 1 MMP (MT1-MMP/MMP-14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP-14, on a rat collagen-induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti-IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg À1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT-MMP, gelatinases are also involved in joint destruction in arthritis.

The metabolic activities of six psychotropic drugs, diazepam, clotiazepam, tofisopam, etizolam, tandospirone, and imipramine, were determined for 14 isoforms of recombinant human hepatic cytochrome P450s (CYPs) and human liver microsomes by measuring the disappearance rate of parent compounds. In vitro kinetic studies revealed that V max /K m values in human liver microsomes were the highest for tofisopam, followed by tandospironeϾclotiazepamϾimipramine, diazepam, and etizolam. Among the recombinant CYPs, CYP3A4 exhibited the highest metabolic activities of all compounds except for clotiazepam and imipramine. The metabolism of clotiazepam was catalyzed by CYP2B6, CYP3A4, CYP2C18, and CYP2C19, and imipramine was metabolized by CYP2D6 most efficiently. In addition, the metabolic activities of diazepam, clotiazepam, and etizolam in human liver microsomes were inhibited by 2.5 m mM ketoconazole, a CYP3A4 inhibitor, by 97.5%, 65.1%, and 83.5%, respectively, and the imipramine metabolism was not detected after the addition of 1 or 10 m mM quinidine, a CYP2D6 inhibitor. These results suggest that the psychotropic drugs investigated are metabolized predominantly by CYP3A4, except that CYP2D6 catalyzes the metabolism of imipramine. In addition, this approach based on the disappearance rate appears to be useful for the identification of the responsible CYP isoform(s) of older drugs, for which metabolic profiles have not been reported.