Yoshiro Kohno

Comparative pharmacokinetics of clarithromycin (TE-031), a new macrolide antibiotic, and erythromycin in rats.

Clarithromycin (TE-031) is a newly synthesized macrolide with high stability in acidic conditions. In the present study, the pharmacokinetics of [N-methyl-14C]clarithromycin were compared with those of [N-methyl-14C]erythromycin in rats by radioassay and bioassay. Both radioactivity and bioactivity of [14C]clarithromycin in plasma and tissues were found to be significantly higher than those of [14C]erythromycin at the same oral dose of 20 mg/kg (body weight). Among the tissues, the peak level of [14C]clarithromycin in the lung was especially high. Levels of radioactivity and bioactivity amounted to 15 and 73 times the corresponding levels of [14C]erythromycin. In the urine, bioactivity recovered after administration of [14C]clarithromycin was sevenfold higher than that for [14C]erythromycin. This accounted for about 60 and 20% of the total radioactivity in the urine for [14C]clarithromycin and [14C]erythromycin, respectively. An examination of metabolites in the urine for [14C]clarithromycin revealed appreciable amounts of bioactive unchanged clarithromycin. These results indicate that clarithromycin has pharmacokinetic properties superior to those of erythromycin. The desirable properties of clarithromycin include high levels in plasma resulting from its high stability in gastric acid; a high tissue affinity, especially to the lung; and favorable urinary excretion. Images

High-throughput screening for the assessment of time-dependent inhibitions of new drug candidates on recombinant CYP2D6 and CYP3A4 using a single concentration method

The inhibitory effects of various test compounds on recombinant human CYP3A4 activity assayed by fluorescent metabolite formation from 7-benzyloxyquinoline (7-BQ) and the effect of pre-incubation on inhibition were evaluated using the microtitre plate assay with multiple concentrations of test compounds (multiple concentration method). Among the test compounds studied, ketoconazole inhibited CYP3A4 activity most extensively, followed by miconazole, troleandomycin, terfenazine and midazolam. The IC50 values of other compounds exceeded 10 (iM, but those of many compounds decreased after pre-incubation. The inhibitory effects of verapamil, amiodarone and diltiazem after pre-incubation were 205, 154 and 833 times greater than those in the case of co-incubation, respectively. To assess the inhibitory effects more readily, the validity of the microtitre plate assay with a single concentration of the test compound (single concentration method) was studied. The accuracy of the automated dispensation and the coefficient of variation on enzyme activity were approximately 3%. The IC50 values estimated using the per cent of residual activity from the single concentration method matched closely those from the multiple concentration method. When the ICSO value as inhibitor concentration was used for a single concentration method, the method enabled easy estimation of inhibitory patterns (such as competitive or time-dependent inhibition) on cytochromes P450. Therefore, from the ease of the technique, automation of the microtitre plate assay and application of the single concentration method might be useful for inhibitory assessment of cytochromes P450 more than that of current conventional methods.

Comparative studies on the gastrointestinal lesions caused by several nonsteroidal anti-inflammatory agents in rats

The gastrointestinal toxicity of a single oral administration of five nonsteroidal anti-inflammatory agents (NSAIDs) to rats was compared, by a method using51Cr-labeled red blood cells (RBC), and by macroscopic and microscopic examination. From the profile of gastrointestinal bleeding, the NSAIDs could be divided into a group consisting of aspirin (ASA), oxaprozin (OXP) and 2-[4-(3-methyl-2-butenyl)phenyl]propionic acid (TA), which caused only a transient increase in fecal blood loss based on a gastric lesion, and another group including indomethacin (IM) and ibuprofen (IP), which produced a biphasic increase in the blood loss. The initial phase was caused not only by a gastric lesion but also an intestinal lesion, and the secondary phase originated only in the intestinal lesion. The order of potency causing blood loss was IM≫IP>ASA≫TA>OXP. The safety ratio of OXP and TA was shown to be more favorable than that of the other three drugs.

High-throughput screening to estimate single or multiple enzymes involved in drug metabolism: microtitre plate assay using a combination of recombinant CYP2D6 and human liver microsomes

1. The purpose of this study was to estimate readily involvement of single or multiple enzymes in the metabolism of a drug through inhibitory assessment. Inhibitory effects of various compounds on CYP2D6 activity assayed by formation of fluorescent metabolite from 3-[2- (N, N -diethyl-N-methyl-ammonium)ethyl]-7-methoxy-4-methyl-coumarin (AMMC) were assessed using microtitre plate (MTP) assays with a combination of recombinant CYP2D6 and human liver microsomes (HLM). 2. Among various compounds studied, antipsychotic drugs extensively inhibited recombinant CYP2D6 activity and the IC 50 values were generally lower than those of antidepressants and antiarrhythmic drugs. 3. After pre-incubation, the IC 50 values of mianserin, chlorpromadine, risperidone, thioridazine, alprenolol, propafenone and dextromethorphan increased but the values of timolol, S- metoprolol and propranolol substantially decreased compared with those in case of co-incubation 4. The IC 50 values of typical substrates of CYP2D6 (bufuralol and dextromethorphan at lower substrate concentration) in inhibition studies using HLM, were similar to those in the case of recombinant CYP2D6, but the values of the compounds that are metabolized by multiple CYP forms (perphenazine and chlorpromazine) in HLM were much larger. 5. If the ratio (HLM/rCYP ratio) of IC 50 values between HLM and recombinant CYP2D6 exceeds approximately 2, it suggests that other CYP forms in addition to CYP2D6 might be involved in the metabolism of the test compounds. From the advantage such as speed, high throughput and ease of the technique, the MTP assay using a combination of the recombinant CYP2D6 and HLM is useful to estimate the involvement of single or multiple enzymes in the metabolism of drugs at the stage of drug discovery.

Prediction of oral clearance from in vitro metabolic data using recombinant CYPs: Comparison among well-stirred, parallel-tube, distributed and dispersion models

Intrinsic clearances (CLint-HLM-total) for the metabolism of NE-100, metoprolol, clarithromycin (CAM), lornoxicam and tenoxicam were predicted from in vitro data with recombinant cytochorme P450s (CYPs) using relative activity factor (RAF) and then compared with CLint-HLM observed in human liver microsomes (HLM). The predicted CLint-HLM-total correlated well with the observed CLint-HLM in HLM. When oral clearances (CLoral) of low-clearance drugs such as metoprolol, CAM, lornoxicam and tenoxicam were predicted from the in vitro data using four physiological models (well-stirred, parallel tube, distributed and dispersion models), the predicted CLoral corresponded well with the observed CLoral in vivo and were similar among the four models. For a high-clearance drug, the predicted CLoral of NE-100 in extensive CYP2D6 metabolizers (EMs) was substantially different between individual models, although the predicted CLoral in a poor metabolizer of CYP2D6 (PMs) was similar. The CLoral ratio of NE-100 between the EMs and the PMs predicted from the dispersion model, which leads to a reliable prediction for the high-clearance drug, was 48.4, but the ratio decreased depending on the increase of the NE-100 plasma concentration. The results suggest that the CLoral decrease in the EMs is caused by saturation of NE-100 metabolism mediated by CYP2D6 and is based on increases in plasma NE-100 concentrations dependent on the dose of NE-100. The study suggests that the RAF and the in vitro–in vivo scaling approaches are useful for predicting CLoral from in vitro data with recombinant CYPs without using HLM and hepatocytes.

An epoxysuccinic acid derivative (Loxistatin)-induced hepatic injury in rats and hamsters.

Loxistatin is a possible therapeutic agent of muscular dystrophy. A single oral administration of loxistatin to male rats caused focal necrosis of the liver with inflammatory cell infiltration. The severity of the lesions was dose-dependent up to 200 mg/kg and also manifest by an increase in serum alanine aminotransferase and aspartate aminotransferase activities. Hepatic glutathione (GSH) levels decreased with a maximum 20% depletion within 5 hr after the oral administration of loxistatin. Pretreatment with diethyl maleate did not potentiate the loxistatin-induced hepatic injury. On the other hand, the hepatoprotective effect of cysteamine was observed when cysteamine was administered 24 hr before loxistatin dosing, but the effect was not observed when the antidote was administered concomitantly with loxistatin. Pretreatment of rats with phenobarbital or trans-stilbene oxide provided partial protection against the hepatotoxic effect of loxistatin. Pretreatment with SKF-525A resulted in increased hepatic injury, while pretreatment with piperonyl butoxide, cimetidine, or 3-methylcholanthrene had no effect on hepatic damage by loxistatin. Five hours after [14C]loxistatin administration to rats, the covalent binding of the radioactivity to proteins was greatest in the liver, followed by the kidney, then muscle and blood to a lesser extent. [14C]Loxistatin acid, the pharmacologically active form of loxistatin, irreversibly bound to rat liver microsomal proteins; more binding occurred when the NADPH-generating system was omitted and when the microsomes were boiled first. GSH did not alter the extent of irreversible binding, whereas N-ethylmaleimide decreased the binding of [14C]loxistatin acid to rat liver microsomal proteins by 75%. Unlike the rat, administration of loxistatin to hamsters caused neither hepatic injury nor hepatic GSH depletion even at a high dose (500 mg/kg). Both the distribution and covalent binding of radioactivity in the hamster liver were one-third of those in rats following [14C]loxistatin dosing. These results suggest that loxistatin causes species-specific hepatotoxicity and that, at least in part, some of the toxic effects of loxistatin are mediated by the nonenzymatic covalent binding of loxistatin acid to thiol residues on cellular macromolecules.

Thein vitro metabolism of desglymidodrine, an active metabolite of prodrug midodrine by human liver microsomes

SummaryThe human cytochrome P450 (CYP) isoforms catalyzing the oxidation metabolism of desglymidodrine (DMAE), an active metabolite of midodrine, were studied. Recombinant human CYP2D6, 1A2 and 2C19 exhibited appreciable catalytic activity with respect to the 5′-O-demethylation of DMAE. TheO-demethylase activity by the recombinant CYP2D6 was much higher than that of other CYP isoforms. Quinidine (a selective inhibitor of CYP2D6) inhibited the O-demethylation of DMAE in pooled human microsomes by 86%, while selective inhibitors for other forms of CYP did not show any appreciable effect. Although the activity of CYP2D6 was almost negligible in the PM microsomes, the O-demethylase activity of DMAE was found to be maintained by about 25% of the pooled microsomes. Furafylline (a selective inhibitor of CYP1A2) inhibited the M-2 formation in the PM microsomes by 57%. The treatment of pooled microsomes with an antibody against CYP2D6 inhibited the formation of M-2 by about 75%, whereas that of the PM microsomes did not show drastic inhibition. In contrast, the antibody against CYP1A2 suppressed the activity by 40 to 50% in the PM microsomes. These findings suggest that CYP2D6 have the highest catalytic activity of DMAE 5′-O-demethylation in human liver microsomes, followed by CYP1A2 to a small extent.

Entrapping Efficiency and Drug Release Profile of an Oil-in-Water (o/w) Emulsion Formulation Using a Polydimethylsiloxane-Coated Glass Bead Assay

Evaluation of entrapping efficiency is difficult for an o/w emulsion formulation containing a lipophilic oily drug, isocarbacyclin methyl ester (TEI-9090), by commonly employed techniques (dialysis, ultrafiltration, or gel filtration), because of its adsorption to the system materials. Employing this characteristic of TEI-9090, we developed an adsorption technique with polydimethylsiloxane-coated glass beads (PDMS-GB). The assay is based on the quantitative adsorption of unentrapped TEI-9090 to the PDMS-GB. The entrapping efficiency of a 10% soybean oil emulsion containing [3H]TEI-9090 (1 µg/mL) assayed by this method approached 100%. The PDMS-GB assay was performed for the emulsion diluted 100 times with physiological saline at different time intervals after dilution over a period of 24 hr. A plot of [3H]TEI-9090 in the emulsion particles versus time showed rapid release within 1 hr, followed by very slow release, reaching equilibrium. Applying first-order kinetics, the data were found to fit to a biexponential equation over the first hour of release. The terminal release resembled the first-order release of the drug from the phospholipid-rich infranatant, which was separated from the creamy layer by ultracentrifugation of the emulsion and contained 35% [3H]TEI-9090. These results suggest that the drug is released from two components in the emulsion.

Autobacteriographic studies of clarithromycin and erythromycin in mice.

The antimicrobial activity of clarithromycin was compared with that of erythromycin in experimentally infected mice by whole-body autobacteriography. In mice with systemic staphylococcal infections, the number of vital microbes in the body was relatively low in the early period after oral administration of erythromycin, but increased thereafter to the levels found in nonmedicated control mice. On the other hand, with clarithromycin treatment, a significantly smaller number of microbes was evident throughout the body. The microbes were scarcely seen in the parenchyma of any organs during the examination period. This potent antimicrobial activity of clarithromycin compared with that of erythromycin was further demonstrated in mice with respiratory infections. On the other hand, to examine the distribution properties of both antibiotics in the whole body, an autoradiographic study was carried out with [N-methyl-14C]clarithromycin and [N-methyl-14C]erythromycin. Both labeled antibiotics were distributed widely throughout the body after oral administration in both uninfected control mice and mice with systemic infections. However, the radioactivity was more marked and persistent for [14C]clarithromycin than it was for [14C]erythromycin, particularly in the lungs. The observations described above indicate the superior in vivo antimicrobial activity of clarithromycin compared with that of erythromycin and suggest that the superiority of clarithromycin is largely attributed to its favorable distribution properties. The advantages of whole-body autobacteriography, coupled with whole-body autoradiography, are discussed. Images

Mechanistic studies on metabolic chiral inversion of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of the new anti-rheumatic agent 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), in rats.

The chiral inversion properties of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), were compared with those of ibuprofen in rats. After administration of R(-)-[2 alpha-2H]KE-748, S(+)-KE-748 was present in the rat plasma, and the deuterium atoms of the S(+)-enantiomer were almost all replaced by hydrogen atoms. After administration of S(+)-[2 alpha-2H]KE-748, the deuterium content of S(+)-KE-748 in the plasma remained intact. In the in vitro study, using a cell-free system and rat liver homogenates, the chiral inversion of ibuprofen was apparent when both CoA and ATP were present; however, KE-748 was not inverted. In the study on isolated rat hepatocytes, the unidirectional chiral inversion from R(-)-to S(+)-enantiomer was observed for both ibuprofen and KE-748. When R(-)-ibuprofen was incubated with medium and long chain fatty acids (carbon chain length C6 to C16), using isolated hepatocytes, the chiral inversion decreased significantly. On the other hand, when R(-)-KE-748 was incubated with short and medium chain fatty acids (carbon chain length C3 to C8), chiral inversion was inhibited markedly. To induce hepatic microsomal long chain fatty acid CoA ligase, rats were treated with clofibric acid (CF rats). In both in vitro and in vivo experiments on CF rats, chiral inversion from R(-)-to S(+)-ibuprofen was enhanced significantly compared with that in controls, whereas the enhancement was not observed in the case of R(-)-KE-748. There was no influence of benzoic acid, a typical substrate on medium chain fatty acid CoA ligase in the mitochondrial matrix, on chiral inversion of R(-)-ibuprofen, using, isolated hepatocytes. In contrast, the chiral inversion from R(-)-to S(+)-KE-748 was strongly inhibited in the presence of benzoic acid. These results indicate that chiral inversion of R(-)-KE-748 may proceed via formation of the CoA-thioester intermediate with loss of the 2 alpha-methine proton, in a manner similar to that seem with R(-)-ibuprofen. However, the enzymes needed to form CoA-thioester of R(-)-KE-748 differ from those for R(-)-ibuprofen.