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We have isolated and characterized a novel human and rat organic anion transporter subtype, OATP-D. The isolated cDNA from human brain encodes a polypeptide of 710 amino acids ([M.sub.r] 76,534) with 12 predicted transmembrane domains. The rat clone encodes 710 amino acids ([M.sub.r] 76,821) with 97.6% amino acid sequence homology with human OATP-D. Human and rat OATP-D have moderate amino acid sequence homology with LST-1/rlst-1, the rat oatp family, the prostaglandin transporter, and moat1/ MOAT1/KIAA0880/OATP-B. Phylogenetic tree analysis revealed that OATP-D is branched in a different position from all known organic anion transporters. OATP-D transports prostaglandin [E.sub.1] ([K.sub.m] 48.5 nM), prostaglandin [E.sub.2] ([K.sub.m] 55.5 nM), and prostaglandin [F.sub.2[alpha]], suggesting that, functionally, OATP-D encodes a protein that has similar characteristics to those of the prostaglandin transporter. Rat OATP-D also transports prostaglandins. The expression pattern of OATP-D mRNA was abundant mainly in the heart, testis, brain, and some cancer cells. Immunohistochemical analysis further revealed that rat OATP-D is widely expressed in the vascular, renal, and reproductive system at the protein level. These results suggest that OATP-D plays an important role in translocating prostaglandins in specialized tissues and cells. prostaglandin transporter; eicosanoid

Vitamin C (L-ascorbic acid) is required for certain enzymatic reactions. Two L-ascorbic acid transporters, SVCT1 and SVCT2 have been isolated from rate complementary DNA libraries to study the importance of L-ascorbic acid transport in supply and metabolism regulation of vitamin C. Both transporters mediate high affinity L-ascorbic acid transport driven by the Na+ electrochemical gradient.

The selective inhibition of hepatic cholesterol synthesis by pravastatin, an inhibitor of the hydrophilic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was investigated. Pravastatin uptake by isolated rat hepatocytes was evaluated to test the possibility of the existence of a specific uptake mechanism for pravastatin in the liver. The results showed that a Na+ -independent, carrier-mediated active transport mechanism is involved in pravastatin uptake. This could account for the in vivo selective inhibition of hepatic cholesterol synthesis by pravastatin.

The antisense oligonucleotides (AONs) targeting the gene of each oatp subtype with subtype-specific sequences are selected, by using a gene alignment program (GAP) and antisense in vitro selection (AIVS), to compare both procedures to search for antisense sequences. The result shows the use of AONs to elucidate the mechanism of target genes related to diseases such as cancer, inflammation, and diabetes.

1. The biotransformation and disposition of tivantinib in humans, dogs and rats was examined after a single oral administration of [14C]tivantinib. Tivantinib constituted no more than one-third of the plasma radioactivity in all species, demonstrating significant contribution of the metabolites to plasma radioactivity. The major circulating metabolites in all species were M4 and M5, hydroxylated metabolites at the benzyl position of the tricyclic ring, accounting for 19.3 and 12.2% of the AUC of the total radioactivity, respectively, in humans. 2. The majority of radioactivity was excreted to the feces via bile. Tivantinib was detected at trace levels in urine, feces and bile, demonstrating extensive metabolism prior to biliary excretion and nearly complete tivantinib absorption under fed conditions. 3. Seven metabolic pathways were identified for tivantinib and included six oxidations (M4, M5, M7, M8, M9 and M11) and one glucuronidation (M23). The major metabolic and excretory pathways were found to be common among all species. Species differences in the metabolic pathways included lactam metabolite (M8) formation in humans and dehydrogenated metabolite (M11) formation in animals. 4. None of the metabolites identified in this work are believed to significantly impact the efficacy or toxicity of tivantinib in humans. [ABSTRACT FROM AUTHOR]

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High reactivity of [C]-methyl iodide ([C]CHI) with the thiol group was demonstrated with cysteamine and other compounds containing a thiol and another functional groups in each structure. The methylation of the thiol group in cysteamine with [C]CHI was very rapid at 0°C with no catalyst, and gave a high radiochemical yield and purity without any detectable by-product. Moreover, this reaction was not disturbed by the other functional groups, such as -NH, -OH and -COOH in the same structure. This S-methylation reaction is very useful for producing a new radiopharmaceutical labeled with the short lived positron emitting nuclide C-11. [ABSTRACT FROM AUTHOR]

Typical CYP2D6 substrates generally contain a basic nitrogen atom that interacts with Asp(301) and/or Glu(216) and an aromatic moiety adjacent to the site of metabolism. Recently, we found novel acidic substrates for CYP2D6, pactimibe, and its indole metabolite, R-125528, that are not protonated but are negatively charged at physiological pH. The K(m) value of R-125528 in CYP2D6-expressing microsomes was determined to be 1.74 microM, which was comparable with those of typical basic CYP2D6 substrates (1-10 microM). Pactimibe has lower affinity than R-125528; however, the K(m) value was comparable with that of metoprolol. Interestingly, their sites of metabolism, the omega-1 position of the N-octyl indoline/indole group, were relatively distant from the aromatic moiety. A pactimibe analog with an N-decyl chain was similarly labile against CYP2D6; however, analogs with N-hexyl or N-dodecyl chains were stable to CYP2D6. An induced fit docking of the ligands with an X-ray crystal structure of substrate-free CYP2D6 (Protein Data Bank code 2F9Q) indicated the involvement of an electrostatic interaction between the carboxyl group and Arg(221) and hydrophobic interaction between the aromatic moiety and Phe(483). The docking model correctly positioned the site of metabolism above the heme. The effect of the N-alkyl chain length of pactimibe analogs on their CYP2D6 metabolic stability was plausibly explained by the docking model. In conclusion, we report herein a novel CYP2D6 binding mode for the acidic substrates pactimibe and R-125528. Further investigation, such as a site-directed mutation, will be necessary to directly demonstrate the involvement of Arg(221) in CYP2D6 binding.

Pactimibe sulfate is a novel acyl coenzyme A:cholesterol acyltransferase inhibitor developed for the treatment of hypercholesterolemia and atherosclerotic diseases. Pactimibe has two equally dominant clearance pathways forming R-125528 by CYP3A4 and M-1 by CYP2D6 in vitro. R-125528 is a plasma metabolite and is cleared solely by CYP2D6 despite its acidity. To evaluate contributions of the cytochrome P450 enzymes on the pharmacokinetics of pactimibe and R-125528 in humans, drug-drug interaction studies using ketoconazole and quinidine were conducted. Eighteen healthy male subjects were given a single dose of pactimibe sulfate without and with 400 mg of ketoconazole (q.d.). With the concomitant treatment, the area under the plasma concentration-time curve (AUC(0-inf)) of pactimibe modestly increased 1.7-fold and AUC(0-tz) of R-125528 decreased by 55%. In addition, 17 healthy male subjects were given a single dose of pactimibe sulfate without and with 600 mg of quinidine (b.i.d.). With the concomitant treatment, the AUC(0-inf) for pactimibe modestly increased 1.7-fold. On the other hand, the AUC(0-tz) of R-125528 was markedly elevated 5.0-fold, although the AUC(0-inf) could not be adequately defined because the terminal elimination phase of R-125528 was not obtained in the study period up to 72 h. As the f(m CYP3A4) and f(m CYP2D6) values of pactimibe estimated from in vitro studies were 0.40 and 0.33, respectively, AUC increase ratios of pactimibe were estimated to be 1.7 with ketoconazole and 1.5 with quinidine. These values were well in accordance with the values observed in this study. Moreover, the f(m CYP2D6) of R-125528 estimated to be almost 1 would well explain the accumulation of R-125528 observed with the quinidine treatment.

Pactimibe sulfate is a novel acyl coenzyme A:cholesterol acyltransferase inhibitor. We conducted metabolic studies of pactimibe and its plasma metabolite, R-125528. Pactimibe had multiple metabolic pathways including indolin oxidation to form R-125528, omega-1 oxidation, N-dealkylation, and glucuronidation. Among them, the indolin oxidation and the omega-1 oxidation were dominant and were mainly catalyzed by CYP3A4 and CYP2D6, respectively. The intrinsic clearance (CL(int)) values for these pathways in human hepatic microsomes were 0.63 and 0.76 microl/min/mg-protein, respectively. On the other hand, the metabolic reaction for R-125528 was restricted. It was demonstrated that omega-1 oxidation was the only pathway that could eliminate R-125528 from the systemic circulation. To our surprise, only CYP2D6-expressing microsomes could catalyze the reaction, and omega-1 oxidation was strongly correlated with the CYP2D6 marker reaction, dextromethorphan O-demethylation (r(2) = 0.90), in human hepatic microsomes. Although R-125528 is an atypical substrate for CYP2D6 because of its acidity, the K(m) value was 1.8 microM for the reaction in human hepatic microsomes and the CL(int) value was as high as 75.0 microl/min/mg-protein. These results suggested that the systemic clearance of R-125528 was highly dependent on CYP2D6 activity and that several studies with CYP2D6 including drug-drug interaction and polymorphism sensitivity should be performed during development from the viewpoint of metabolite safety assessment. The finding that R-125528, an acidic compound devoid of basic nitrogen, was a good substrate for CYP2D6 raised a question about previously reported CYP2D6 models based on a critical electrostatic interaction with Asp(301) and/or Glu(216).

Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (K(m)) of 29.3 +/- 9.9 microM. Both Na(+)-dependent and Na(+)-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na(+)-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with K(m) values of 42.6 +/- 28.6 and 71.8 +/- 21.6 microM, respectively. The Na(+)-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.

On thyrotropin-releasing hormone (TRH) metabolism, pyroglutamyl aminopeptidase II (PAP-II), a zinc-dependent ectoenzyme primarily located in the central nervous system, is believed to play a predominant role. Recently we cloned pyroglutamyl aminopeptidase I (PAP-I) which is known for specifically removing a L-pyroglutamate (L-pGlu) residue from the amino terminus of proteins and peptides including TRH. To investigate possible contribution of PAP-I toward TRH metabolism, we conducted biochemical and immunohistochemical characterization using recombinant rat, mouse and human PAP-Is and an antibody raised against rat PAP-I. The Km values toward TRH by the recombinant PAP-Is were about 0.05 mM, being similar value to the reported value of recombinant PAP-II. The L-pGlu-cleaving activities toward TRH in rat brain homogenate were inhibited by a PAP-II specific inhibitor 1,10-phenanthroline, but not inhibited by the antibody against rat PAP-I. Immunohistochemical study in rats revealed heterogeneous distribution of PAP-I in the pituitary, the target tissue of TRH, but the distribution was cytosolic. Taken together, these results suggested that PAP-I might not be dominantly involved in the degradation of TRH in rats. Additionally, we found that PAP-I was localized in the renal proximal tubules. Further investigations are needed for elucidating the function of PAP-I in these restricted sites.

Pyroglutamyl aminopeptidase I (PAP-I) is known for specifically removing the L-pyroglutamate (L-pGlu) residue from the amino terminus of L-pGlu proteins and peptides. In general, substrate recognition of PAP-I as to L-pGlu moiety is tightly regulated. However, we recently identified PAP-I as a metabolic enzyme of an organic nitrate compound, RS-7897, which contains L-2-oxothiazolidine-4-carboxylic acid (L-OTCA). L-OTCA is a latent sulfhydryl group, which has moiety structurally related to L-pGlu. In this study, we investigated the substrate specificity of PAP-I toward modified L-pGlu-containing substrates using recombinant rat, mouse and human PAP-Is. PAP-I was tolerant of replacement of a carbon atom at the 4-position of the L-pGlu moiety by a sulfur atom (L-OTCA), an oxygen atom (L-2-oxooxazolidine-4-carboxylic acid, L-OOCA) and an NH group (L-2-oxoimidazolidine-4-carboxylic acid, L-OICA). The K(m) values for rat PAP-I in hydrolyzing L-pGlu-L-Ala, L-OTCA-L-Ala, L-OOCA-L-Ala and L-OICA-L-Ala were 0.057, 0.43, 0.71 and 0.42 mM, respectively. Similar results were observed in mouse and human PAP-Is as well. Moreover, the hydrolysis of RS-7897 in rat and mouse liver cytosols were both completely inhibited by an antibody against rat PAP-I, strongly suggesting that PAP-I is solely involved in the hydrolysis of L-OTCA-containing compounds in rat and mouse liver cytosols.

To gain a better understanding of the metabolic stability and transport of pitavastatin (CAS 147526-32-7), a new and potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, experiments were conducted using human hepatocytes and oocytes of Xenopus laevis expressing human organic anion transporting polypeptide-2 (OATP2), respectively. Almost the entire radioactivity was from the unchanged substance or lactone form in human hepatocytes, and the cytochrome P450 (CYP)-mediated metabolism of pitavastatin was negligible. The results suggested that CYPs are not critically involved in determining the metabolic fate of pitavastatin. The hepatic uptake of pitavastatin reached saturation with a Km of 2.99 +/- 0.79 micromol/L. Also, the uptake of pitavastatin was mediated by OATP2 expressed in oocytes with a Km of 5.53 +/- 1.70 micromol/L. These results indicated that OATP2 plays a major role in the distribution of pitavastatin in liver. Furthermore, to elucidate the increase in the plasma concentration of pitavastatin in a clinical setting, the inhibitory effect of ciclosporin (cyclosporin A, CAS 59865-13-3) on the uptake of pitavastatin was examined. The uptake of pitavastatin was inhibited in the presence of cyclosporin A and the apparent IC50 value was 2.91 +/- 0.78 micromol/L. This result may at least partly explain the drug-drug interaction between pitavastatin and cyclosporin A. In conclusion, the characterization of transporters needs to be taken into account to avoid transporter-mediated drug-drug interaction.

Pyroglutamyl aminopeptidase I (PAP-I) is a cytosolic cysteine peptidase, which hydrolytically removes the L-pyroglutamate residue from the amino terminus of endogenous proteins and peptides. L-Pyroglutamyl p-nitroanilide serves as the synthetic substrate of this enzyme, while there is a possibility of other hydrolases being involved in the hydrolysis of this xenobiotic substrate. We cloned a full-length cDNA encoding rat PAP-I from a rat liver cDNA library and expressed this cDNA in Escherichia coli to obtain a recombinant PAP-I as a single protein. The cDNA encoded a sequence of 209 amino acids with a calculated molecular weight of 22913 Da. The homology of the deduced amino acid sequence of rat PAP-I was 98.6 and 94.3% to mouse and human PAP-Is, respectively. The biochemical properties of the recombinant rat PAP-I were almost identical to those of the recombinant mouse and human PAP-Is and the purified rat liver cytosolic PAP-I in terms of the molecular weight, subunit structure, affinity to the substrate, inhibitor profile and pH optimum. Immunoblot analysis using an antibody raised against recombinant rat PAP-I showed that rat PAP-I is present almost exclusively in the cytosolic fraction of the rat liver. Moreover, the hydrolyzing activity for L-pyroglutamyl p-nitroanilide in rat liver cytosolic fraction was completely inhibited by the antibody, strongly suggesting that this xenobiotic substrate is hydrolyzed solely by PAP-I.

To select antisense oligonucleotides (AONs) that specifically target the genes of rat organic anion transporting polypeptide (oatp) subtypes, in vitro selection and an alignment program were used. When we incorporated a couple of our original 2'-O,4'-C-ethylene nucleoside (ENA) residues into the AONs at both the 3' and 5' ends, the inhibitory activity of these oatp subtype-specific AONs was enhanced. This strategy of combining in vitro selection with the use of an alignment program as well as incorporating ENA residues, was effective in synthesizing oatp subtype-specific AONs.