Blood levels and excretion of ³H-TTC-909, an oil-in-water (o/w) emulsion of isocarbacyclin methyl ester (³H-TEI-9090), after intravenous administration to rats were investigated in comparison with those of ³H-TEI-9090.
1. Blood levels of radioactivity in male rats after rapid intravenous injection of ³H-TTC-909 declined tri-phasically, with parameters (T_1/2, AUC) which were almost similar to those for ³H-TEI-9090. The AUC increased in dose-dependent manner at the dose range of 0.5 to 8 μg/kg. In female rats, the AUC of blood level was about 1.8 times higher than that in male rats.
2. Isocarbacyclin (TEI-7165) levels in blood accounted for 11-15% of the total radioactivity by AUC, irrespective of dosage forms and administration mode (rapid injection/infusion).
3. Within 120 hr, 49.7% and 47.0% of the administered radioactivity was recovered in the urine and feces, respectively, after administration of ³H-TTC-909 to male rats. A similar excretion pattern was observed for ³H-TEI-9090. In female rats, urinary excretion after ³H-TTC-909 administration was about 10% higher than that in male rats.
4. In bile duct-cannulated male and female rats, 43.2% and 48.2% of the dose was excreted in the bile, respectively. Of the radioactivity excreted in the bile, 34.3% and 45.6% were reabsorbed from the intestinal tracts in male and female rats, respectively. This difference of reabsorption may be a cause of the higher blood levels found in female rats.

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Biotransformation of isocarbacyclin methyl ester (TEI-9090) in the rat was studied both in vivo and in vitro.
1. The chemical structures of the metabolites present in blood, urine and bile samples were elucidated by gas chromatography-mass spectrometry analysis using an ion cluster technique and high performance liquid chromatography. Twenty-three metabolites of TEI-9090 were identified, reflecting various combinations of metabolism by hydrolysis, β- or ω-oxidation, 13, 14- and 6, 9α-reduction, and 15-hydrogenation.
2. Blood levels of the metabolites and the composition of metabolites in 24 hr urine and feces after intravenous administration of ³H-TEI-9090 in the form of an oil-in-water (o/w) emulsion were similar to those after administration of a saline solution form of the drug. Some differences in the composition of biliary metabolites were observed between male and female rats.
3. The hydrolysis rate of TEI-9090 in plasma was extremely high compared with that in a red blood cells suspension and the homogenates of several other tissues.
4. The transformation rate of isocarbacyclin to its 13, 14-dihydro-15-oxoform (M-4), which is first generated in the degradation process, in rat kidney and lung cytosol was higher than that in liver.
On the basis of these observations, the metabolic activation and inactivation dynamics of TEI-9090 in the body are discussed.

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Tissue distribution and plasma protein binding of ³H-TTC-909, an oil-in-water (o/w) emulsion of isocarbacyclin methyl ester (³H-TEI-9090), after rapid intravenous injection to rats were investigated, in comparison with ³H-TEI-9090 and isocarbacyclin (³H-TEI-7165).
1. Radioactivity was distributed rapidly in most tissues after intravenous administration of ³H-TTC-909 to male rats. At 5 min, the level in the kidney and liver was 18 times and 14 times higher than that in the plasma, respectively, while the levels in other tissues were similar to or lower than the plasma level. Tissue levels decreased with half-lives similar to the plasma level. Distribution profiles of ³H-TEI-9090 did not differ from those of ³H-TTC-909, except that the spleen levels were lower than those of ³H-TTC-909.
2. A similar distribution pattern of ³H-TTC-909 was observed in female rats, but most of the tissue levels were about 2 times higher than those in male rats.
3. Plasma protein binding 5 min after administration of ³H-TTC-909 and ³H-TEI-9090 was 74.3% and 83.7%, respectively. Thereafter, both binding rates decreased to almost equal values 2 hr and 8 hr after the administration.
4. Five min after administration of ³H-TTC-909, ³H-TEI-9090 and ³H-TEI-7165, TEI-7165 was detected as the principal metabolite in the plasma, heart, spleen and brain, whereas most of the radioactivity in the liver, kidney and lung consisted of various inactive metabolites. In the brain, 58.1% and 69.9% of the radioactivity corresponded to TEI-7165 after the administration of ³H-TTC-909 and ³H-TEI-9090, respectively, while only 7.2% for the administration of ³H-TEI-7165.

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Blood levels, excretion and metabolism of ³H labeled isocarbacyclin methyl ester given in the form of an oil-in-water emulsion (³H-TTC-909) were compared with those after administration of the saline solution form (³H-TEI-9090) in the dog.
1. After rapid intravenous injection of ³H-TTC-909, the blood level of radioactivity decreased tri-phasically, while a plateau level was established between 7 min and 1 hr after ³H-TEI-9090 dosing. The ratio of AUC values of unchanged drug and isocarbacyclin (TEI-7165) to those of total redioactivity after administration of ³H-TTC-909 were slightly higher than the values obtained after ³H-TEI-9090 dosing. Similar results were observed in the case of constant rate infusion for 1 hr of ³H-TTC-909 and ³H-TEI-9090.
2. Radioactivity was excreted predominantly in urine and accounted for 78.3% and 70.4% within 7 days after dosing of ³H-TTC-909 and ³H-TEI-9090, respectively.
3. The 13, 14-dihydro-15-oxo form of the unchanged drug (M-2), which was not previously detected in rat blood, existed in the blood samples immediately after intravenous rapid injection of ³H-TTC-909 and ³H-TEI-9090 in dogs. After hydrolysis of TEI-9090 and M-2, TEI-7165 and its 13, 14-dihydro-15-oxo form (M-4) were widely metabolized in a variety of pathways in dogs, as in the case of rats.
4. Furthermote, two types of undefined conjugates (the ester-form conjugate and an amino acid conjugate) were postulated in the urine samples after administration of ³H-TTC-909. No significant differences in the composition of urinary metabolites was observed between ³H-TTC-909 and ³H-TEI-9090 dosing.

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The effects of FK037 on the aldehyde dehydrogenase and the drug metabolizing enzyme system in male rat liver were studied.
1. When male rats were given ethanol intraperitoneally at 24 hours after a single administration of FK037 (100 or 1000 mg/kg), the blood acetaldehyde concentration was not affected by the administration of FK037. On the other hand, a marked increase of blood acetaldehyde level was observed after by administration of cefoperazone (1000 mg/kg) and disulfiram (500 mg/kg), which were reported to have disulfiram-like reaction. In addition, the administration of cefoperazone and disulfiram caused a depression of hepatic mitochondrial lowKm aldehyde dehydrogenase (ALDH) activity. However, mitochondrial low-Km ALDH activity was not changed by the administration of FK037. Further, disulfiram, cefoperazone and 1-methyl-tetrazol-5-thiol (MTT) which is derived from 3-substituent of cefoperazone caused a inhibition of low Km-ALDH activity, while FK037 and 5-amino-1'-(2-hydroxyethyl)-pyrazole (AHP) which is derived from 3-substituent of FK037 did not inhibit this enzyme activity in vitro. From these results, we conclude that FK037 does not affect the aldehyde dehydrogenase in male rats.
2. There were no significant effects on cytochrome P-450 and b₅ contents, NADPH-cytochrome c reductase, aminopyrine demethylase, aniline hydroxylase and ethoxyresorufin deethylase on three days after intravenous administration of FK037 with 20 and 100 mg/kg/day to male rats. These results suggest that FK037 does not affect the hepatic drug metabolizing enzyme system in male rats.

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Research efforts made in past decades accumulated a large amount of knowledges on interspecies differences in the properties of drug metabolizing enzymes, which might be responsible for the occurrence of species differences of drug actions. However, some of such informations on the “differences” cannot be applied to a practical research for new drug development. Changing the angle to look at the enzymes involved in drug metabolism, we have proposed to analyze the interspecies homology rather than difference to find out an appropriate experimental animal to predict human drug metabolism. Thus, interspecies homology in the sequences of nucleic acids as well as amino acids were searched focusing on some major enzymes for drug metabolism. Results shown suggest that the homology values varied among enzymes, probably resulting from the endogenous roles of the enzyme and the extents of conserved regions.

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By the recent advance in the study of human cytochrome P450, the information on the role of individual isoform of human cytochrome P450 in the metabolism of therapeutic agents, has been accumulated. Consequently, even for a drug under development, it becomes possible to predict drug-drug interactions and whether genetics, aging, gender, and environmental factors may influence the metabolism of the drug or not, by determining which isoform of human cytochrome P450 is mainly responsible for the metabolism of the drug. This article reviews the characters of individual form of human cytochrome P450s, in vitro and in vivo methods to determine human P450 isoforms, and the significance of the human cytochrome P450 identification in the area of clinical pharmacology and new drug development.

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A group of Japanese subjects was phenotyped for CYP2D6 activity by determination of urinary metabolic ratios of sparteine. The relationship between genotypes of CYP2D6 gene and the metabolic phenotypes was investigated in one poor metabolizer (a proband). The genotype of the proband was not consistent with any of other previously described mutations in the CYP2D6 gene. To identify a new allele responsible for PM phenotype, we analyzed the CYP2D6 gene from the proband. The results revealed a nine base insertion in exon 9, designated as 2D6(9-bp). The proband also carried the deletional 2D6 (D) allele. The 2D6 (9-bp) /2D6 (D) proband thus showed reduced CYP2D6 activity. The 2D6 (9-bp) and 2D6 (D) alleles of the proband were demonstrated to inherit from the mother [2D6 (W)/2D6 (9-bp)] and the father [2D6 (W)/2D6 (D)], respectively. This 9-base insertion caused loss of catalytic activity of CYP2D6 by an experiment using the enzyme expressed in yeast. Four of 308 Japanese inherited 2D6 (9-bp) allele with heterologous type (0.7%, 4/616 chromosomes) examined by PCR analysis.

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Current studies on human hepatocyte cultures are surveyed. Human hepatocytes are generally isolated according to collagenase perfusion method by introducing the isolation solution into the vascular orifice on the cut surface of a human liver tissue fragment. The major problem in isolating viable hepatocytes is obtaining fresh specimens; usually several hours elapse after dissection of the fragment from surgical procedures. Inter-indivisual difference, premedication, period of coma and the time lag between dissection and the start of perfusion can produce large variety of the preparation. In spite of such technical limits, studies on primary cultures of human hepatocytes offered valuable information. Though the hepatocytes gradually lose drug-metabolizing activity during culture, they respond to typical enzyme inducers such as phenobarbital, β-naphthoflavone, omeprazole or rifampicin, and induced specific cytochrome P-450 subfamilies. Species differences in the effect of peroxisome proliferator or cytotoxicity of certain chemicals have also been reported.
Cultured dog hepatocytes resemble human hepatocytes in terms of enzyme induction suggesting a possibility to serve as an experimental model for predicting drug interaction in human. As an example, induction of hepatic P450 Ill A subfamily by a non-steroidal antiinflammatory drug in humans, dogs and primary cultured dog hepatocytes is presented.

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