Pharmacokinetics-pharmacodynamics of micafungin in Japanese patients with deep-seated mycosis.

The objective of this study was to describe the pharmacokinetic profile and investigate the effective concentration of micafungin in Japanese male patients with deep-seated mycosis. 66 patients were treated with i.v. micafungin 12.5-150 mg intravenously for up to 56 days. At this dose range, micafungin showed linear pharmacokinetics, and the mean values of Cmax and Cmin amounted to 3.16-12.9 microg/mL and 0.70-3.68 microg/mL, respectively. The mean value for the elimination half-life was 13.5 h (95 samples from 65 patients), and it remained almost constant over the dose range. In addition, the elimination half-life was not influenced by age, gender or weight, and was similar to that found in healthy subjects. The active metabolites M1 and M2 were detectable, but their exposure was lower than that of the unchanged drug. The pharmacokinetic-pharmacodynamics ob micafungin were then investigated. The overall clinical response rate against aspergillosis and candidiasis showed good results at a dose of 50 mg and over. The Cmax and Cmin at the latter dose amounted to 5.16 and 1.41 microg/mL, respectively. In conclusion, micafungin showed linear pharmacokinetics at doses ranging from 12.5 to 150 mg, and the effective concentration was considered to be over 5 microg/mL as maximum level in Japanese patients with deep-seated mycosis such as candidiasis and aspergillosis.

Investigation of the electrochemical oxidation products of zotepine and their fragmentation using on-line electrochemistry/electrospray ionization mass spectrometry.

When zotepine, an antipsychotic drug, was electrochemically oxidized using electrospray ionization mass spectrometry (ESI-MS) coupled with a microflow electrolytic cell, [M + 16 + H]+ (m/z 348), [M-H]+ (m/z 330) and [M-14 + H]+ (m/z 318) were observed as electrochemical oxidation product ions (M represents the zotepine molecule). Although a major fragment ion that was derived from the dimethyl aminoethyl moiety was observed only at m/z 72 in the collision-induced dissociation (CID) spectrum of zotepine, new fragments such as m/z 315 and 286 ions could be generated in the CID spectrum by combining electrochemical oxidation and CID. Since these fragments were relatively specific with high ion strength, it was thought that they would be useful for developing a sensitive LC-MS/MS assay. The S-oxide and N-demethylated products were detected by electrolysis assuring that a portion of P450 metabolites of zotepine could be mimicked by the electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) system.

Contribution of human hepatic cytochrome p450 isoforms to the metabolism of psychotropic drugs.

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 Vmax/Km values in human liver microsomes were the highest for tofisopam, followed by tandospirone>clotiazepam>imipramine, 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 microM 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 microM 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.

Interindividual variability in 5-Fluorouracil metabolism and procainamide N-acetylation in human liver cytosol.

We investigated the enzymatic kinetics and interindividual variability of the metabolism of 5-fluorouracil and procainamide by human liver cytosol and/or microsomes. The Km values for the 5-fluorouracil dihydropyrimidine dehydrogenase (DPD) and procainamide N-acetyltransferase activities in pooled liver cytosol, and procainamide hydrolysis in pooled liver microsomes were 3.9, 1670, and 969 microM, respectively, and the intrinsic clearance (Vmax/Km) values for these reactions were 128, 0.192, and 0.0059 microl/min/mg protein, respectively. The cytosolic activities of 5-fluorouracil metabolism and procainamide N-acetylation ranged from 145 to 790 (469+/-156, mean+/-S.D., n=22) and <1 to 152 (52+/-48, n=12) pmol/min/mg protein, respectively, and the DPD activity of 5-fluorouracil was neither gender-related nor age-dependent. Procainamide N-acetylation activities among 12 human cytosol samples were highly correlated with sulfamethazine N-acetylation activities, suggesting that procainamide N-acetylation is catalyzed by N-acetyltransferase-2. These results suggest that the N-acetylation reaction is more important than the hydrolysis in the metabolic pathway of procainamide, and that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of 5-fluorouracil and procainamide in human liver.

Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031.

FR255031 (2-[(7S)-7-[5-(4-ethylphenyl)-2-thienyl]-1,1-dioxido-4-(2-pyridinylcarbonyl)hexahydro-1,4-thiazepin-7-yl]-N-hydroxyacetamide) 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. 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. FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. FR255031 at a dose of 100 mg kg(-1) also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 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.

Identification of cytochrome P450 enzymes involved in the metabolism of FK228, a potent histone deacetylase inhibitor, in human liver microsomes.

FK228 (FR901228, depsipeptide) is a potent histone deacetylase inhibitor currently in phase II clinical trials for cancer treatment. In the present study, the cytochrome P450 (P450) enzymes responsible for FK228 metabolism in human liver microsomes were investigated. Incubation with human liver microsomes in the presence of an NADPH-generating system revealed that FK228 is metabolized to at least 10 different metabolites. Km and Vmax values for FK228 disappearance were 20.3 microM and 561.9 pmol/min/mg protein, respectively. Further studies were performed at a substrate concentration of 10 microM (half the Km value for FK228 disappearance). FK228 disappearance activities in human liver microsomes from 12 individuals strongly correlated (r2=0.957) with testosterone 6beta-hydroxylase activities, a marker enzyme activity of CYP3A4/5, but not with other P450 enzyme-specific activities (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 4A). Among 14 recombinant heterologously expressed human P450s examined, CYP3A4 exhibited the highest activity of FK228 disappearance. CYP3A5, 1A1, 2B6, and 2C19 showed 16.8%, 5.2%, 1.6%, and 1.3% of the activity of CYP3A4, respectively. Other P450s showed no significant metabolic activity toward FK228. In addition, FK228 disappearance in human liver microsomes was markedly inhibited by ketoconazole, a potent CYP3A4 inhibitor, and an anti-CYP3A4 antibody. These results indicate that the metabolism of FK228 in human liver microsomes is catalyzed mainly by CYP3A enzymes, particularly CYP3A4.

Interindividual variability in 2-hydroxylation, 3-sulfation, and 3-glucuronidation of ethynylestradiol in human liver.

In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE2) using human liver microsomes and cytosol. Km values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 microM, respectively. Vmax/Km (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE2 in human liver. All further studies were performed at a substrate concentration of 0.1 microM. Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04+/-1.34, mean+/-S.D., n=35) and 0.20 to 4.84 (1.20+/-1.00, n=35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8+/-4.4, n=21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males (p<0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 microM ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6+/-16.0%, n=27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE2 and the relative contribution of CYP3A to the 2-hydroxylation of EE2 in human liver.

Utility of microtiter plate assays for human cytochrome P450 inhibition studies in drug discovery: application of simple method for detecting quasi-irreversible and irreversible inhibitors.

In this study, a simple in vitro method for detecting human P450 (CYP) quasi-irreversible and irreversible inhibitors was evaluated. For the method, cDNA-expressed CYPs were applied to microtiter plate assays, CYP inhibitors were co-incubated with fluorometric substrates, and IC(50) were continuously measured (without stopping enzyme reactions). The typical reversible inhibitors (sulfaphenazole, tranylcypromine, quinidine, ketoconazole) showed constant IC(50) throughout the reaction. In contrast, the typical quasi-irrversible inhibitors (isosafrole, erythromycin, troleandomycin, diltiazem) and the typical irreversible inhibitors (furafylline, propranolol, mifepristone) showed time-dependent decreases in IC(50). For CYP3A4 inhibition studies, two substrates, 7-benzyloxyresorufin (BzRes) and 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), were used. The IC(50) of the CYP3A4 inhibitors were dependent on the substrate. However, the quasi-irreversible and irreversible inhibitors could be detected by examining changes in the IC(50), regardless of the substrate. Further, the detection method was applied to josamycin and bergamottin. Josamycin did not show definite time-dependent decreases in IC(50) for CYP 3A4, suggesting that josamycin is neither a quasi-irrversible nor an irreversible inhibitor of CYP3A4. On the other hand, bergamottin showed time-dependent decreases in IC(50) for CYP1A2, CYP 2C9, CYP 2C19, CYP 2D6 and CYP 3A4, suggesting that bergamottin is a quasi-irrversible or an irreversible inhibitor of the 5 CYP isoforms. This method provides more rapid and reliable detection of quasi-irreversible and irreversible inhibitors and may be useful in drug discovery.

Tissue distribution after intravenous dosing of micafungin, an antifungal drug, to rats.

The tissue distribution after an intravenous dose of micafungin (1 mg/kg), a new echinocandin-like lipopeptide antifungal agent, to male rats was investigated. Micafungin in plasma disappeared biexponentially with a terminal half-life of 5.03 h. Micafungin concentrations in liver, kidney, and lung at the first sampling time (5 min) after dosing were 1.15, 1.64, and 2.58-fold higher than the plasma concentration, and the AUC(0- infinity ) were 1.61, 3.42, and 2.89-fold higher than that for plasma. The terminal half-lives for these tissues were 5.14, 4.87, and 5.31 h, respectively, which were comparable to those for plasma. These results suggest that micafungin distributes rapidly and moderately into tissues such as the liver, kidney, and lungs, and that the concentrations in tissues decreased in parallel with the unchanged drug in plasma.

Effect of nilvadipine, a dihydropyridine calcium antagonist, on cytochrome P450 activities in human hepatic microsomes.

The effects of nilvadipine, a dihydropyridine calcium antagonist, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Nilvadipine competitively inhibited CYP1A2-mediated 7-ethoxyresorufin O-deethylase, CYP2A6-mediated coumarin 7-hydroxylase, CYP2C8/9-mediated tolbutamide methylhydroxylase, CYP2C19-mediated S-mephenytoin 4'-hydroxylase, and CYP3A4-mediated nifedipine oxidase activities, and the inhibition constant (Ki) values were 13.0, 35.8, 5.02, 24.5 and 44.3 microM, respectively. On the other hand, no inhibition of CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2D6-mediated bufuralol 1'-hydroxylation, or CYP2E1-mediated chlorzoxazone 6-hydroxylation by nilvadipine at 40 microM concentration was observed. The free fractions of nilvadipine in the incubation mixture estimated by ultracentrifugation were 18.9-27.4%. These results suggest that nilvadipine would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.

Effect of cefixime and cefdinir, oral cephalosporins, on cytochrome P450 activities in human hepatic microsomes.

The effects of two kinds of oral cephalosporins, cefixime and cefdinir, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Both cefixime and cefdinir at 2 mM concentration neither inhibited nor stimulated CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, or CYP3A4-mediated testosterone 6beta-hydroxylation. The free fractions of cefixime and cefdinir in the incubation mixture, which were measured by ultracentrifugation, were 86.1-93.8% and 94.1-97.8%, respectively. These results suggest that both cefixime and cefdinir would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.

Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.

As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.

Strategy for structural elucidation of drugs and drug metabolites using (MS)n fragmentation in an electrospray ion trap.

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MS can 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.

Utility of hepatocytes in predicting drug metabolism: comparison of hepatic intrinsic clearance in rats and humans in vivo and in vitro.

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, UDP-glucuronosyltransferase, sulfotransferase, and/or esterase. CL(int,in vitro) was determined from substrate disappearance rate at 1 microM 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.

New SRM data dependent exclusion (MS)n measurement for structural determination of drug metabolites using LC/ESI/Ion trap MS.

New SRM (selected reaction monitoring) data dependent exclusion (MS)(n) measurement makes it possible to obtain MS(3) fragmentation data for all MS(2) fragments, useful for structural determination of drug metabolites using ESI ion trap. MS(2) fragments are produced by cleavage of all protonated molecules at the lone electron pairs of heteroatoms or the pi electrons of double and triple bonds, benzene rings and hetero-rings of drugs. Usually, data dependent MS(3) measurement cleaves only MS(2) fragment of highest intensity, that normally does not contain important metabolic sites. Fragmentation data from all parts of drug metabolites is required to determine structure. In addition to the usual basic measurement of protonated molecules and (MS)(n) fragmentation of drug metabolites, we demonstrate the use of SRM data dependent (MS)(n) measurement, plus new SRM data dependent exclusion (MS)(n) measurement for structural determination of metabolites.

Strategy for structure elucidation of drug metabolites derived from protonated molecules and (MS)n fragmentation of zotepine, tiaramide and their metabolites.

TSQ ESI MS/MS and ion trap ESI MS(2) cleave protonated molecules. MS(2) at m/z 332 of zotepine cleaved m/z 245 (10%), m/z 287 (5%) and m/z 315 (100%) fragment ions at protonated positions. MS(2) at m/z 356 of tiaramide cleaved m/z 338 (18%), 313 (10%), 226 (100%), 198 (78%) and 131 (60%) fragment ions at protonated positions. The ESI ion trap MS produced new internal protonated molecules in an ion trap, such as m/z 113 and m/z 88 from m/z 131 protonated piperazinonium, and m/z 245 protonated 8-chloro dibenzo[b,f]thiepin. ESI ion trap (MS)(n) (n>or=3) cleaved new internal protonated molecules. It also causes carbocation cleavage, alpha cleavage, onium cleavage and McLafferty cleavage. We can easily elucidate the structure of metabolites from the difference in m/z of corresponding fragments between unchanged compound and its metabolite. Reactive oxygen diradicals involved in cytochrome P-450 cycles react with electron rich groups and reactive C-H bonds of zotepine and tiaramide to produce metabolites of 2-hydroxyzotepine, 3-hydroxyzotepine, norzotepine, zotepine-N-oxide, zotepine-S-oxide, Tiaramide carboxylic acid, dehydroxyethyltiaramide and tiaramide-N-oxide. The strategy for structure elucidation of drug metabolites was established on the basis of the reactivity of unchanged drug with reactive oxygen diradicals involved in cytochrome P-450 cycles and theory associated with protonated molecules and (MS)(n) fragmentation of drug metabolites.

Evidence for singlet oxygen involvement in rat and human cytochrome P450-dependent substrate oxidations.

Recently, we proposed that singlet oxygen ((1)O(2)) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of (1)O(2) is general in the P450-dependent substrate oxidations. In the present study, we examined omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of beta-carotene and NaN(3) significantly suppressed these reactions in a concentration-dependent manner, and (1)O(2) during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a (1)O(2)-spin trapping reagent, where the addition of (1)O(2) quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-(1)O(2) adduct. Next, we examined the effect of (1)O(2) quenchers on P450-dependent reactions in the human liver microsomes, and (1)O(2) was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that (1)O(2) is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.