¹⁴C-Troglitazone was administered to male cynomolgus monkeys (monkey) orally or intraduodenally, and the time course of the plasma concentrations of the radioactivity and of the biliary excretions of the metabolites were investigated. The same was investigated for male marmosets after oral administration of ¹⁴C-troglitazone.
1. The main metabolite in the plasma after oral administration to monkeys was the sulfoconjugate of troglitazone (M1). The quinone form metabolite (M3) and glucuronide (M2) of troglitazone were also detected in the plasma.
2. The ratio of the biliary excretion of the radioactivity over a 24-hr period after intraduodenal administration to a bile-fistula cynomolgus monkey was 54.4%, which was as high as that observed in rats and dogs. The main metabolites in the bile were M1 and M2, each accounting for about 40% of the biliary radioactivity. An unknown metabolite, U3, was also detected at a ratio of about 6%. U3 was also detected in the bile after administration of M3, and was show to contain the quinone-like structure.
3. The plasma metabolites after oral administration to marmosets were almost the same as those in the plasma of monkeys. U3 was also observed in the plasma of marmosets.
4. U3 was isolated from the bile and urine of monkey and marmoset after administration of ¹⁴C-M3 or M3. According to the MS and NMR spectra, the chemical structure of U3 was determined as the glucuronide of the hydroquinone form of troglitazone, and its was conjugated at position 8"a.

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塩酸ドネペジルのヒトにおける代謝的特徴を予測することを目的として，代謝に関与するヒトP450分子種を明らかにするとともに，典型的なP450阻害剤との薬物相互作用について検討した．なお，P450分子種の特定にはヒトCYPIA1，CYPIA2，CYP2A6，CYP2B6，CYP2C9，CYP2D6，CYP2E1およびCYP3A4のcDNAをAHH-1TK+/一cellline導入し，それぞれの分子種が特異的に発現している細胞から調製したミクロソームを用いた．その結果，(1)M4は主としてCYP3A4によって生成し，CYP2C9もわずかに関与していること，(2)M1とM2の生成には主としてCYP2D6が関与していること，(3)発現系ミクロソームにおけるV_max/Kmを肝臓中における各P450分子種の存在比で補正したCL_intは，ヒト肝臓ミクロソームを用いた結果と一致し，発現系ミクロソームで得られた結果の妥当性が立証された．また，ヒト肝ミクロソームを用いて塩酸ドネペジルの主代謝物であるM1，M2およびM4の生成に対するシメチジン，テオフィリソ，エリスロマイシン，キニジンおよびケトコナゾールの影響について検討した結果，(1)シメチジン，テオフィリソおよびエリスロマ・イシンは塩酸ドネペジルと併用しても，その代謝に影響を及ぼさないこと，(2)キニジンはM1およびM2の生成を阻害する可能性があること，(3)ケトコナゾールはM1，M2およびM4の生成を阻害する可能性があることが明らかとなった．臨床用量におけるキニジンおよびケトコナゾールの血漿中遊離型薬物濃度を考慮すると，キニジンは塩酸ドネペジルの肝固有クリアランスを82.0%に，ケトコナゾールは57.9%に低下させるが，塩酸ドネペジルの血漿中濃度の上昇はそれぞれ，最大で1.2倍および1.7倍の上昇にとどまることが示唆された。

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We succeeded the quantitative prediction of plasma and tissue concentrations of β-lactam antibitotics, insulin, pentazocine, quinolone antibacterial agents, inaperizone and digoxin by utilyzing physiologically-based pharmacokinetic models incorporated their plasma membrane transport mechanisms. Although passive diffusion, which depends on the lipid solubility, is a fundamental mechanism for the membrane transport of many of compounds, water-soluble compounds also cross cell membranes by the specialized carriermediated transport mechanisms. We have demonstrated that several drugs are transported by the tissuespecific transporters in intestinal and renal epithelial cells, hepatocytes and brain capillary endothelial cells which form the blood-brain barrier. They include oligopeptide transporter (PepT1), monocarboxylic acid transporter (MCT1), anion antiporter (AE2), organic anion transporter (Npt1), cation transporter (OCTN1 and OCTN2) and P-glycoprotein (MDR1). Most of them function for the uptakes of drugs into the cells leading to the increased permeability, others exclude drugs out of cells, thereby decreasing apparent permeability into cells. This finding demonstrates that it would be possible to expect tissue selective delivery of drugs by utilizing transporters which have different characteristics among tissues. Further mechanistic clarification and quantitative analysis of pharmacokinetic importance of such transporters will help the development of effective strategies for the site specific drug delivery.

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Here, I briefly review studies on an active metabolite of morphine and characterization of enzymes responsible for the formation of the glucuronide in our laboratory. Morphine has been used extensively in pain clinic and was found biotransformed mainly by glucuronidation to a major inactive glucuronide; morphine-3-glucuronide (M-3-G) and also to a minor active metabolite; morphine-6-glucuronide (M-6-G). The very potent metabolite has been attracted much interest in a role of morphine analgesia in humans. The pharmacokinetic results are now interpreted as demonstrating that the active glucuronide had a positive effect on the analgesia. Recently, researchers have disclosed the presence of an unique opioid receptor for the glucuronide. Our recent experimental evidence from expression of two cDNA clones of glucuronidating enzymes from guinea pig liver supported the view that the enzymes form hetero-oligomers by accessing a broader range of compounds as in the active metabolite than homo-oligomers. A natural follow-up to the present evidence would be to carry out for the genomic information in regulatory molymorphism in drug metabolism by glucuronidation.

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GC/MS revealed that α-ecdysone is biosynthsized by the prothoratic glands. The characteristic reactions of derivatization for GC/MS was utilized to elucidate the structures of the rat urinary metabolites of Mydocalm, and the unexpected chain elongation metaolites of 5-(chlo-n-butyl)picolinic acid in a novel metabolic pathway. The stable isotope techniques were applied to measure exactly the pharmacokinetics (PK) of d-and l-chlorpheniramines (CPA) in human using a pseudoracemate of d-CPA-d₆ and l-CPA d₀, and to measure both PK and pharmacodynamics of glycerol trinitrate and it's two kinds of dinitrates in dog and human. New silylating agents were synthesized and the resulted silyl ether derivatives of hydroxylated compounds provided some advantages for GC/MS. Furthermore, a new retention index, Δ [Um]_E was defined by differences in the methylene unit values of the DMES and TMS ether derivatives, and was used for estimating the number of the hydroxyl groups. Alcoholic and phenolic hydroxyl groups in a compound was discrimated easily by the use of TMS-DMES exchange reaction. The profile analyses of biologically important substances were carried out by GC/MS for clarifing their physiological roles. The presence of PGD₂ in human brain was identified and quantified by GC/HR/SIM using the Me-MO-DMiPS ether derivatives. The simultaneous determination of the activities of HMG-CoA reductase and cholesterol 7α-hydroxylase in human cauld be performed by quantifing the amounts of mevalonate and 7α-hydroxylcholesterol in plasma.

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It has been reported that some transporters are located on the blood-brain and blood-cerebrospinal fluid barriers to exclude their substrates from the central nervous system. In this article, the molecular mechanism for the vectorial transport of xenobiotics is summarized particularly focusing on our own studies. In addition to MDR1 P-glycoprotein, we could suggest the significant role of multidrug resistance associated proteins in the drug disposition in the central nervous system. Moreover, the role of organic anion transporting polypeptides and organic anion transporters is discussed in relation to the transport studies in the blood-cerebrospinal fluid barrier.

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Recently, pharmacokinetic analysis in clinical study has indicated that small intestinal metabolisms of certain drugs give a great impact on their exposures due to extensive first-pass effects particularly by CYP3A4. However, the in vitro metabolic activities in the small intestinal microsomes are, in general, lower than the relevant hepatic activities, and the intestinal extraction ratio predicted from the activities appeared to be inconsistent with the in vivo data. The reason for this may lie in the fact that both methodologies for the estimations from in vitro and in vivo have some issues (involvement of protein binding and effect of inducers/inhibitors on Q, respectively) to be clarified, or P-glycoprotein with the similar substrate specificity to CYP3A might be involved in the first pass metabolism. The detailed studies in an animal with a comparable CYP3A activity to human will be required to elucidate the contribution of small intestinal first-pass to overall metabolism.

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The CYP2D subfamily has received much attention during the past decade due largely to the involvement of CYP2D6 in human, particularly as the enzyme metabolizes over 50 clinically important drugs. More than 40 genetic polymorphisms exist in human CYP2D6. The frequency of the poor metabolizer (PM) with deficient CYP2D6 activity is reported to be 5% to 10% in Caucasians and less than 1% in Japanese. The CYP2D6^*10 allele, which includes both the CYP2D6^*10A and CYP2D6^*10B variants, is widely observed in Japanese (38%; our data) and Chinese (50%), and has two amino acid changes Pro34→Ser and Ser486→Thr.
We have found a significant influence on the pharmacokinetics of venlafaxine by CYP2D6^*10 allele in a Japanese population, and have emphasized a clinical significance of CYP2D6^*10 allele in addition to PM-related alleles in a Japanese population. Recently, several studies on the relationship between CYP2D6^*10 and pharmacokinetic of CYP2D6 substrates have reported. We have also expressed CYP2D6.10 using yeast expression systems to determine its metabolic specificity.
In this review, we introduce our findings in vivo and in vitro on CYP2D6^*10 and prospects of its clinical relevance in the future.

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Three related forms of phenol sulfotransferase, thermostable ST1A2 and ST1A3 and a thermolabile ST1A5 are known to exist in human livers. Thermostable forms, whose activities are polymorphically distributed in human tissues, have been shown to mediate the bioactivation of carcinogenic N-hydroxy aromatic amines as well as phenolic substrates. Variant forms of ST1A3 mRNAs (i.e. Arg213His and Met223Val) have been found in human livers. In a Japanese population, allele frequencies of ²¹³Arg and ²¹³His were 0.83 and 0.17, respectively. No remarkable difference in [³⁵S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol was observed between recombinant ²¹³Arg- and ²¹³His-type ST1A3, although it was reported that ²¹³His homozygosity was associated with both lower (less than one-sevenths) p-nitrophenol sulfation and thermolability in human platelets. The recombinant ST1A3 (²¹³His) did exhibit unstability at 45° and 37°C as compared with ST1A3 (²¹³Arg). Liver cytosols from ²¹³His homozygotes did not always show low p-nitrophenol-sulfating activities. Different molecular mechanisms in sulfation polymorphism are suggested in livers and platelets of humans.

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We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transport taurocholate (Km; 13.6μM) in a sodium- independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17β-glucuronide and estrone-3-sulfate), eicosanoids (prostaglandin E₂, thromboxane B₂, leukotriene C₄, leukotriene E₄), and thyroid hormones, reflecting hepatic multispecificity. LST-1 is probably the most important transporter in the human liver for clearance of bile acids and organic anions since hepatic levels of another organic anion transporter, OATP is very low. We have also isolated rat counter part, rlst-1. rlst-1 is excusively expressed in the liver and in both the bile duct ligation model and the cecum ligation and puncture model, the mRNA expression levels of rlst-1 were downregulate, suggesting rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

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We identified and characterized novel Na⁺-dependent carnitine/organic cation transporter family, OCTNs from human and mouse. We isolated two and three members of OCTN family from human and mouse, respectively. OCTNs are present in various tissues, including kidney, heart, skeletal muscle and placenta strongly, and in several human-derived cancer cell lines. By immunohistochemical analysis in kidney, mouse OCTNs localized commonly in luminal membrane of tubular epithelial cells. Most of human and mouse OCTNs exhibited multifunctionality by transporting both of carnitine and organic cation, tetraethylammonium (TEA). Furthermore, sodium ions were essential for carnitine transport by human and mouse OCTN1 and 2. In systemic carnitine deficiency (SCD) phenotype mouse model, juvenile visceral steotosis (jvs) mouse, mutation in OCTN2 gene was found. Furthermore, several kinds of mutation in human SCD patients were found, demonstrating that OCTN2 is a physiologically important carnitine transporter. Interestingly, TEA transport was sodium independent. In addition, OCTNs transporterd various cationic drugs such as quinidine, verapamil, and actinomycin D. Furthermore, since one mutation of human OCTN2 lost carnitine transport activity but retained TEA transport activity, it was suggested that OCTN2 have differential functional sites for carnitine and organic cations. So, OCTNs are thought to be multifunctional transporters by transporting carnitine and organic cations by sodium ion dependent and independent manner, respectively, and would be important for disposition of organic cationic drugs as well as carnitine.

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It has been recognized that the pharmacologic effect and the adverse reaction of a drug are closely related to the concentration of its free form at the site of action. The measurement of this concentration is very important to manage the optimal therapeutic response with the minimum adverse effects but it is practically impossible. The cerebrospinal fluid (CSF) concentration of a drug which acts on the central nervous systems is believe to be instantaneously equilibrated with its concentration at the site of action. The tear concentration of a drug reflects not only its plasma free concentration but also its CSF concentration. The individualization of the dosage regimen become to be very important for the drugs with narrow therapeutic windows such as anticonvulsants and antiasthmatics. The purpose of the present study is to develop a pharmacokinetic model for theophylline and valproic acid in tear and CSF, and to examine whether their CSF concentrations can be predicted by their tear concentrations in patients. The time courses of theophylline and valproic acid concentrations in guinea pig CSF and tear were similar to those of their free concentrations in plasma, and could be described by a basic physiological model in which their CSF and tear compartments are independently connected with their plasma compartments (free drugs) based on the apparent diffusion constants. The CSF concentrations of theophylline at the steady states of both guinea pigs and patients could be predicted by its tear concentrations using the resulting pharmacokinetic parameters. The measurement of the drug concentrations in tear which can be collected non-invasively might be a very useful method for individual dosage regiment of these drugs in patients.

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Orally active cephalosporin antibiotics are known to be absorbed from the small intestine via the oligopeptide transporter (PepT1). Although several methods have been proposed for the prediction of oral absorption in humans, no in vitro method has been established to predict oral absorption of the drugs that are absorbed by a carrier-mediated process. In this mini review, we propose a method to predict oral absorption of cephalosporins in humans from in vitro studies using rat intestinal brush border membrane vesicles (BBMV) or Caco-2 cells. Uptake into BBMV or Caco-2 cells via PepT1 was estimated by subtracting the uptake at 4°C from that at 25°C (BBMV) or 37°C (Caco-2 cells), which was well correlated with the extent of oral absorption according to the complete radial mixing (CRM) model reported by Amidon et al. The present method gives fairly good prediction of oral absorption of cephalosporins and may be used for the screening of well absorbed PepT1 substrates.

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A novel method of assessing the extent of bioavailability (EBA) of drugs from pharmacological data was presented. Disopyramide, a class Ia antiarrhythmic agent, arginine-vasopressin (AVP), a nona-peptide hormone, and human insulin were used as the model drugs. The prolongation of QT interval, the anti-diuretic response and the hypoglycemic response were used as the pharmacological indexes of disopyramide, AVP and human insulin, respectively. After intra-vascular administration (I.V. bolus, short-term infusion or long-term infusion) of drugs to the rats, the relationship between blood (plasma or serum) concentrations and their pharmacological responses were described on the basis of an integrated pharmacokinetic-pharmacodynamic (PK-PD) model. A sigmoid Emax model or a linear model was applied to describe the drugreceptor interaction. Pharmacological responses after intra-vascular administration of these 3 drugs were reasonably described by the PK-PD model. Since PK-PD relationship after oral administration was assumed to be identical with that after intra-vascular administration, the PK-PD model obtained under I.V. bolus study was used to assess the extent of oral bioavailability (EBAp.o.). The EBAp.o. values, estimated from pharmacological effects after oral administration of disopyramide (25 mg/kg to 100 mg/kg) was 96.6% and this value were almost identical with the actual EBAp.o. values (57.6 to 99.4%). However, the EBAp.o. values, estimated from pharmacological effects after oral administration of AVP were significantly underestimated the actual EBAp.o. values. While, the EBA values, estimated from pharmacological effects after subcutaneous administration of human insulin using an osmotic mini-pump were significantly overestimated the actual EBA values. These results indicated that PK-PD relationship was significantly influenced by intra-vascular input rate of drugs, especially the peptide drugs. Then we re-estimated the EBA values using the PK-PD model constructed under the long-term infusion study, and obtained reasonable results. From these results, we concluded that the EBA was reasonably predicted by a PK-PD model, provided that appropriate pharmacological effects and appropriate intra-vascular dosing rate as a reference formulation are available. The method may be the alternative to methods based on plasma concentrations, when drug concentration cannot be measured and when appropriate pharmacological data are available.

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