NK-104 is a very potent competitive inhibitor of HMG-CoA reductase. The pharmacokinetic properties of NK-104 were evaluated after intravenous and/or oral administration to rats, rabbits, dogs, monkeys and humans. The plasma concentration of NK 104 showed triexponential elimination after intravenous administration, with a half-life of 4.0-5.3 h in all four animal species. The absorption was relatively rapid after the oral administration. The relationship between the dose and the AUC was linear at a relatively high dose range in the animal species. In humans, the correlation line was located between those of dogs and rats. High bioavailability (about 80%) at a dose of 1 mg/kg was observed in all animal species except monkeys. These results indicated that the pharmacokinetic properties of NK-104 after oral administration in humans resembled those in dogs, suggesting that the BA of NK-104 is large in humans as well as dogs.
NK-104 was excreted mainly into feces via biliary route, and the renal handling was negligible in rats, dogs and humans. On the other hand NK-104 was excreted mainly into urine in rabbits, and was excreted poorly into urine and feces in monkeys.
The unchanged drug was excreted mainly in rat bile. Although several β-oxidation products as metabolites of NK-104 were detected, the levels of these metabolites were fairly low. In dogs, the major component in plasma, urine and feces after oral administration was the unchanged NK-104. The β-oxidation products (M-5 and M-8) were also slightly detected only in dogs. In humans, the major components in plasma after repeated oral administration (2 mg/day for 5 days) were NK-104 and its lactone form.
These results suggest that NK-104 may be hardly metabolized in humans than in animal species.

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The absorption, distribution, metabolism and excretion of radioactivity were investigated in rats after oral administration of ¹⁴C-azasetron hydrochloride.
1. Azasetron hydrochloride was mainly absorbed from the small intestine. The extent of absorption was estimated to be more than 91% by the sum of radioactivity excretion in the urine and bile after dosing. The plasma levels of radioactivity showed a maximum (C_max) within 0.6 hour, and then was eliminated with the terminal half-lives (t_1/2Z) of 6.7-8.0 hours after administration of 0.4, 2 and 10 mg/kg doses. No dose-related difference in t_1/2Z was observed. The C_max and the areas under the plasma concentration-time curve increased greater than dose proportionally.
2. Radioactivity distributed rapidly into various tissues, showing a peak level at 1 hour after dosing in most tissues. Radioactivity levels were high in the gut and urinary bladder, followed by the liver, pituitary gland, kidney, submaxillary gland, pancreas and lung. Radioactivities in most tissues at 24 hours after dosing were not detected.
3. Within 48 hours after administration to rats at the doses of 0.4, 2 and 10 mg/kg, total excretion of radioactivity was more than 96%. Radioactivity disappeared rapidly from the body. At 96 hours after administration of 2 mg/kg, radioactivity was not detected in the body. Following administration to bile-duct cannulated rats, urinary excretion increased significantly, and biliary excretion decreased significantly with elevation of the dose. About 24% of radioactivity excreted in the bile was reabsorbed from the intestine via the enterohepatic circulation.
4. No qualitative difference was observed in metabolite pattern in the urine, feces and bile between the dose levels, but a quantitative difference was observed. Following administration to bile-duct cannulated rats, azasetron was excreted primarily as unchanged drug and metabolite M1 in the urine, while primarily as M1 and M3 in the bile. At 10 mg/kg dose, as compared with 0.4 and 2 mg/kg doses, urinary excretion of azasetron increased significantly, and biliary excretion of main metabolites M1 and M3 decreased significantly. The extent of absorption decreased by feeding, but alteration of metabolism was not observed.
5. These findings suggest that the cause of the non-linear pharmacokinetic behavior of ¹⁴C-azasetron hydrochloride is due mainly to saturation of a metabolic capacity of azasetron in the liver.

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The in vitro metabolism of azasetron was investigated in the presence of liver microsomes each from male and female rats, and male dogs, in order to elucidate the cause of sex or species difference in the excretion of metabolites of azasetron hydrochloride observed in vivo. The NADPH generating system was required for each reaction process in azasetron metabolism. The N-oxidation of the azabicyclooctane ring was increased markedly with increasing pH from 7.4 to 8.5 in dog liver microsomal incubation mixture, while decreased markedly by exposure of microsomes to mild heat (50°C for 60 sec). Moreover, this N-oxidation was inhibited by methimazole (MTZ), but was hardly inhibited by metyrapone (MP). These results indicate that the N-oxidation of the azabicyclooctane ring is mainly catalyzed by the flavin-containing monooxygenases (FMO) in dog liver microsomes. The N-oxidation of the azabicyclooctane ring in rat liver microsomes were also increased markedly with increasing pH from 7.4 to 8.5 in the specimen, while still remained more than 40% of control activity when mildly heated microsomes were used. Moreover, this oxidation was partially inhibited by MTZ, MP and SKF-525A. These results indicate that both FMO and the cytochrome P-450 (P-450) take part equally in N-oxidation of the azabicyclooctane ring in rats. The activities of N-demethylation and hydroxylation of M1 were hardly decreased, when mildly heated microsomes were used, while strongly inhibited by SKF-525A and MP. Accordingly, it seems that N-demethylation and hydroxylation of Ml are catalyzed by P-450. As no difference was observed in the intrinsic clearance by liver (CL_int) for azasetron N-demethylation between male rats and dogs, this reaction is apparently not related to species difference. The CL_int of N-oxidation of the azabicyclooctane ring and hydroxylation of the benzene ring in male dogs were large in comparison with the values in male and female rats. Hence, the results strongly suggest that species difference in the excretion is caused by the difference of these enzyme activities. The CL_int of N-demethylation of azasetron in male rats was large in comparison with the values in female rats. Therefore, sex difference in the excretion would attribute to the difference of this enzymatic activity between male and female rats.

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Cytochrome P450s (P450s) in the CYP4 family metabolize both drugs and endogenous substances such as arachidonic acid and prostaglandins. To clarify the biological roles of these P450s, they were purified by HPLC with an ion-exchange column and a hydroxylapatite column. All purified P450s metabolized lauric acid at the co-position. CYP4A and 4F metabolized arachidonic acid to ω-hydroxyarachidonic acid (20-HETE) which is reported to be a potent vasoconstrictor. On the contrary, CYP4B1 activated aromatic amines such as 3, 3-dichlorobenzidine and 2-naphthylamine to mutagenic substances. Antibodies against these P450s were prepared and localization of these P450s was investigated by Western blotting. These P450s revealed tissue-specific expression. CYP4A was present in the liver and kidney of human and experimental animals and CYP4B1 in the lung and bladder. CYP4F was present in the liver. Regulation of rat CYP4A2 was investigated. CYP4A2 was a male-dominant form in rat kidney and induced by diabetes. Castration decreased the CYP4A2 level in the kidney of male rats and testosterone increased CYP4A2 in the kidneys of both castrated male and untreated female rats, demonstrating that CYP4A2 is regulated directly by androgen. Human renal P450 was purified from renal microsomes and its cDNA was cloned with CYP4A2 cDNA as a probe. Human renal P450 was designated to CYP4A11 which had an identical N-terminal amino acid sequence with purified human P450 and similar characteristics as rat CYP4A2.

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In order to provide mechanistic insights into the pharmacokinetics of peptide drugs (including cytokines and growth factors), I investigated the mechanisms underlying the clearance and distribution of opioid peptides (β-endorphin, dynorphin, and dynorphin-like analgesic peptide), human insulin, and synthetic cyclopeptides (cyclosporine and PSC 833), by use of in vivo animals, perfused organs, and in vitro experimental systems. For opioid peptides, their tissue distribution was suggested to be governed by specific binding with K-type opioid receptors present in peripheral tissues including lung and liver, whereas for insulin the distribution and clearance were suggested to be governed by receptor binding and receptor-mediated endocytosis (RME), respectively, at the physiological concentration range in target organs. The “receptorrecycling” model, in which the internalized receptors are recycled back to the surface to be reutilized for subsequent binding, was developed to predict the hepatic handling of insulin in mice at low and high doses, and successfully incorporated in a physiologically-based pharmacokinetic model, together with transcapillary permeability and static receptor binding in extrahepatic tissues. For cyclopeptides, moreover, their brain penetration was shown to be modulated by P-glycoprotein-mediated efflux functioning at the blood-brain barrier. The kinetic RME analysis enables the prediction of not only the nonlinear target-mediated clearance and distribution of peptides, but also the down-regulation and subsequent recovery of surface receptors, which is useful for assessing the time-dependent changes of in vivo efficacy of peptide drugs. In conclusion, the therapeutic efficacy and protocols of peptide drugs should be assessed from its microscopic pharmacology based on the RME mechanisms, in conjunction with macroscopic pharmacokinetic modeling.

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It has long been believed that most of drugs are absorbed by passive diffusion mechanism. However, some drugs have been suggested to be absorbed by specialized transporters, although no clear evidence for the involvement of such membrane transport mechanisms has been obtained. In the present study, carrier-mediated transport mechanisms involved in the drug absorption was clarified by the approach of molecular cloning and functional expression of the transporters as well as membrane physiological analysis of drug transport. pH-Dependent intestinal absorption of monocarboxylic acids was demonstrated to be partially ascribed to the monocarboxylic acid-proton cotransporter, MCT1 and pH-sensitive anion antiporter AE2. An involvement of such pH-dependent transporters in the intestinal absorption of weak organic acids is important, because they can be alternative mechanisms against passive diffusion according to pH-partition hypothesis. PepT1 cloned from intestinal epithelial cells as the peptide transporter was clarified to be utilized for the enhanced intestinal absorption of peptide-mimetics such as β-lactam antibiotics and others by a proton-gradient dependent mechanism. In contrast, P-glycoprotein decreases apparent intestinal absorption of various drugs by its secretory transport activity into intestinal lumen, thereby causing nonlinear phenomena in intestinal absorption kinetics. These lines of studies of the clarification of carrier-mediated drug absorption mechanisms will provide a new knowledge for the targets and strategies in controlling intestinal absorption of drugs.

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There are remarkable differences in oral bioavailabilities among β-lactam antibiotics, ranging from poorly absorbed to completely absorbed, despite that most of these drugs posses similar degrees of hydrophilicity. Many of the well-absorbed β-lactam antibiotics have been shown to be substrates for the transport system by which peptides are absorbed (proton-coulpled transporter, Pep Tl), whereas the poorly absorbed analogs are not. Although the proton-coupled transport system has the greatest influence on intestinal absorption of β-lactam antibiotics, passive diffusion contributes significantly to the total transport. Considerling that passive diffusion of some β-lactam antibiotics is fairly rapid, the low oral bioavailability of other related compound, like cefazolin, could seem inconsistent.
Recently, it has been found that the mechanisms which induce secretory-oriented permeation of orally inactive β-lactam antibiotics are factors limiting intestinal absorption of such antibiotics. This energy-demanding effiux system is distinct from P-glycoprotein-mediated transport. It should be noted that as long as a β-lactam antibiotic is the substrate with a high affinity for this intestiinal effiux system, the efflux system can pump it out efficiently from cytosol, even if an absorptive peptide transport system on the brush-border membrane mediates their translocation from the lumen into cytosol. Previously, we reported that the well-absorbed β-lactam antibiotics bind to the cytosolic component, fraction b, extensively, whereas the orally inactive analogs do not bind to this fraction. Therefore, it may be possible that the well-absorbed β-lactam antibiotics have a way to escape the intestinal effiux system; for example, binding to the fraction b could prevent the well-absorbed β-lactam antibiotics from interacting with the energy-dependent efflux system, stimulating absorption of these antibiotics. The role of the binding factor, fraction b, for intestinal absorption mechanisms should be evaluated in more detail.

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