Clinical significance of CADM1/TSLC1/IgSF4 expression in adult T-cell leukemia/lymphoma.

Cell adhesion molecule 1 (CADM1/TSLC1) was recently identified as a novel cell surface marker for adult T-cell leukemia/lymphoma (ATLL). In this study, we developed various antibodies as diagnostic tools to identify CADM1-positive ATLL leukemia cells. In flow cytometric analysis, the percentages of CD4(+)CADM1(+) double-positive cells correlated well with both the percentages of CD4(+)CD25(+) cells and with abnormal lymphocytes in the peripheral blood of patients with various types of ATLL. Moreover, the degree of CD4(+)CADM1(+) cells over 1% significantly correlated with the copy number of the human T-lymphotropic virus type 1 (HTLV-1) provirus in the peripheral blood of HTLV-1 carriers and ATLL patients. We also identified a soluble form of CADM1 in the peripheral blood of ATLL patients, and the expression levels of this form were correlated with the levels of soluble interleukin 2 receptor alpha. Moreover, lymphomas derived from ATLL were strongly and specifically stained with a CADM1 antibody. Thus, detection of CD4(+)CADM1(+) cells in the peripheral blood, measurement of serum levels of soluble CADM1 and immunohistochemical detection of CADM1 in lymphomas would be a useful set of markers for disease progression in ATLL and may aid in both the early diagnosis and measurement of treatment efficacy for ATLL.

In vitro activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation in human liver microsomes.

1. The effects of substrate concentration and enzyme source (human liver microsomes and recombinant cytochrome P450s, CYP) on the activation of 7-benzyloxyresorufin O-debenzylation and nifedipine oxidation were investigated. 2. 7-Benzyloxyresorufin O-debenzylase activity in human liver microsomes was inhibited by a monoclonal antibody against CYP2B6 and a polyclonal antibody against CYP3A2 by 53-69 and 19-44%, respectively, suggesting that CYP2B6 and CYP3A4 mainly catalyse 7-benzyloxyresorufin O-debenzylation in human liver microsomes. 3. 7-Benzyloxyresorufin O-debenzylase activity at 0.2-5 micro M substrate concentrations in human liver microsomes was increased by the addition of alpha-naphthoflavone, quinidine, testosterone and progesterone, and the V(max) of 7-benzyloxyresorufin O-debenzylation increased with increasing alpha-naphthoflavone concentrations, whereas the K(m) remained constant. Additionally, 7-benzyloxyresorufin O-debenzylation by recombinant CYP3A4 was increased by the addition of alpha-naphthoflavone, testosterone and progesterone but not by quinidine, whereas no chemicals tested could activate the O-debenzylation of 7-benzyloxyresorufin by CYP2B6. 4. The K(m) for nifedipine oxidation activity by CYP3A4 decreased by the addition of progesterone, whereas the V(max) remained constant. Quinidine and testosterone increased 7-benzyloxyresorufin O-debenzylase and nifedipine oxidase activities, respectively, in human liver microsomes, whereas activation was not observed in CYP3A4. 5. The results suggest that in vitro activation patterns are substrate dependent and that selection of the enzyme source can influence the activation phenomenon.

Stereoselective metabolism of cibenzoline, an antiarrhythmic drug, by human and rat liver microsomes: possible involvement of CYP2D and CYP3A.

Stereoselective metabolism of cibenzoline succinate, an oral antiarrhythmic drug, was investigated on hepatic microsomes from humans and rats and microsomes from cells expressing human cytochrome P450s (CYPs). Four main metabolites, M1 (p-hydroxycibenzoline), M2 (4,5-dehydrocibenzoline), and unknown metabolites M3 and M4, were formed by human and rat liver microsomes. The intrinsic clearance (CL(int)) of the M1 formation from R(+)-cibenzoline was 23-fold greater than that of S(-)-cibenzoline in human liver microsomes, whereas the R(+)/S(-)-enantiomer ratio of CL(int) for M2, M3, and M4 formation was 0.39 to 0.83. The total CL(int) for the formation of the four main metabolites from S(-)- and R(+)-cibenzoline was 1.47 and 1.64 microl/min/mg, respectively, suggesting that the total CL(int) in R(+)-enantiomer was slightly greater than that in S(-)-enantiomer in human liver microsomes. The M1 formation from R(+)-cibenzoline was highly correlated with bufuralol 1'-hydroxylation and CYP2D6 content and was inhibited by quinidine, a potent inhibitor of CYP2D6. Additionally, only microsomes containing recombinant CYP2D6 were capable of M1 formation. These results suggest that the M1 formation from R(+)-cibenzoline was catalyzed by CYP2D6. The formation of M2, M3, and M4 from S(-)- and R(+)-cibenzoline was highly correlated with testosterone 6beta-hydroxylation and CYP3A4 content. Ketoconazole, which is a potent inhibitor of CYP3A4/5, had a strong inhibitory effect on their formation, and the M4 formation from R(+)-cibenzoline was inhibited by quinidine by 45%. The formation of M2 was also inhibited by quinidine by 46 to 52% at lower cibenzoline enantiomers (5 microM), whereas the inhibition by quinidine was not observed at a higher substrate concentration (100 microM). In male rat liver microsomes, ketoconazole and quinidine inhibited the formation of the main metabolites, M1 and M3, >74% and 44 to 59%, respectively. These results provide evidence that CYP3A and CYP2D play a major role in the stereoselective metabolism of cibenzoline in humans and male rats.

Sulfotransferase catalyzing sulfation of heterocyclic amines.

Cytosolic sulfation of arylamines to form sulfamates is found to be mediated by sulfotransferases of three gene families (SULT1 to 3). Among them, a SULT3 form (ST3A1) showed a high selectivity for N-sulfation of N-substituted aryl and alicyclic compounds. SULT1 (phenol) and SULT2 (hydroxysteroid) sulfotransferases showed N-sulfating activities of carcinogenic heterocyclic amines. For N-hydroxyarylamine O-sulfation, SULT1 forms showed high activity. In rats, ST1C1 mediated the metabolic activation of N-hydroxyarylamines. However, the related form (ST1C2) in humans showed the negligible activity. Instead, ST1A3 showed high metabolic activating abilities among human sulfotransferases.

N-sulphoconjugation of amines by human cytosolic hydroxysteroid sulphotransferase.

1. N- and O-sulphoconjugation of various substrates were studied with human liver cytosol and purified cytosolic sulphotransferase in the presence of 3-phosphoadenosine 5-phosphosulphate. 2. Human liver cytosol catalysed N-sulphoconjugation of alicyclic and aryl-amines, and O-sulphoconjugation of hydroxysteroid and phenol. Activities of amine sulphoconjugation in the cytosol correlated well with those of hydroxysteroid but not with phenol. 3. Alicyclic amine sulphotransferase in human liver cytosol was purified to homogeneity by anion exchange, affinity and hydroxyapatite chromatography. Sulphoconjugating activities of alicyclic amine co-purified with those for hydroxysteroid conjugation. Subunit molecular weight of the purified sulphotransferase was 34 kDa. Contents of the purified enzyme correlated with the sulphoconjugating activities of hydroxysteroid and alicyclic amine. From these results, we concluded that the alicyclic amine sulphotransferase purified in this study was identical to hydroxysteroid sulphotransferse in human liver cytosol. 4. The results of this study indicate that hydroxysteroid sulphotransferase in human liver cytosol catalyses N-sulphoconjugation of alicyclic and aryl-amines. Hydroxysteroid sulphotransferase in the cytosol is reported to catalyse O-sulphoconjugations of various compounds including hydroxysteroids, bile acids, cholesterol, and aliphatic and benzylic alcohols. The present and previously reported results indicate that hydroxysteroid sulphotransferase in the cytosol catalyses both N- and O-sulphoconjugations of several substrates.

The disposition of 14C-labeled tacrolimus after intravenous and oral administration in healthy human subjects.

Tacrolimus is a macrolide lactone with potent immunosuppressive properties. It has been shown in clinical studies to prevent allograft rejection. The pharmacokinetics of tacrolimus in healthy subjects and transplant patients has been described in earlier studies using immunoassay methods; however, detailed information on the absorption, distribution, metabolism, and excretion of tacrolimus using a radiolabeled drug is lacking. The objective of the present study was to characterize the disposition of tacrolimus after single i.v. (0.01 mg/kg) and oral (0.05 mg/kg) administration of 14C-labeled drug in six healthy subjects. Tacrolimus was absorbed rapidly after oral dosing with a mean Cmax and Tmax of 42 ng/ml and 1 h, respectively. The oral bioavailability was about 20%. After i.v. and oral dosing, most of the administered dose was recovered in feces, suggesting that bile is the principal route of elimination. Urinary excretion accounted for less than 3% of total administered dose. In systemic circulation, unchanged parent compound accounted for nearly all the radioactivity; however, less than 0.5% of unchanged drug was detectable in feces and urine. The excretion of the metabolites was formation-rate-limited. The mean total body clearance at 37.5 ml/min was equivalent to about 3% of the liver blood flow. Renal clearance was less than 1% of the total body clearance. The mean elimination half-life was 44 h.

Identification of cytochrome P450 enzymes involved in the metabolism of zotepine, an antipsychotic drug, in human liver microsomes.

1. Studies using human liver microsomes and recombinant human cytochrome P450 (P450) enzymes and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the formation of zotepine metabolites in man. 2. Human liver microsomes produced four metabolites and a tentative order of importance was: norzotepine, 3-hydroxyzotepine, zotepine S-oxide and 2-hydroxyzotepine. Zotepine N-oxide was also detected, but it could not be quantified. 3. The rates of formation of the major metabolite, norzotepine, and zotepine S-oxide (at a substrate concentration of 20 microM) were significantly correlated with the testosterone 6beta-hydroxylase activities and CYP3A4 contents of the 12 different human liver microsomal samples. Inhibition studies with P450 enzyme selective inhibitors and anti-rat CYP3A2 antibodies also indicated a predominant role of CYP3A4 in the formation of norzotepine and zotepine S-oxide. Furafylline and sulphaphenazole inhibited the N-demethylation of zotepine by up to approximately 30%. 4. Correlation and inhibition data for the 2- and 3-hydroxylation of zotepine were consistent with the predominant role of CYP1A2 and 2D6 in the formation of these metabolites, respectively. 5. Recombinant CYP1A1, 1A2, 2B6, 2C19, 3A4 and 3A5 efficiently catalysed N-demethylation of zotepine. CYP1A1, 1A2, 2B6 and 3A4 were also active for S-oxidation. CYP1A2 and 2D6*1-Val374 efficiently produced 2-hydroxyzotepine and 3-hydroxyzotepine, respectively. Recombinant human FMO3 did not catalyse zotepine S-oxidation. 6. These results suggest that both the N-demethylation and S-oxidation of zotepine are mediated mainly by CYP3A4, and that CYP1A2 and 2D6 play an important role in the 2- and 3-hydroxylation of zotepine, respectively.

Purification and characterization of two amine N-sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from liver cytosols of male rabbits.

Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS-PAGE. AST-RB1 efficiently catalyzed N-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3, 6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.

Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/Peptide transporter PepT1.

BACKGROUND & AIMS: Dietary regulation is one of the most important factors of intestinal peptide transport. However, the cellular and molecular mechanisms of dietary regulation of the intestinal peptide transport system remain unknown. This study investigated the molecular mechanism of transcriptional activation of intestinal peptide transporter (PepT1) gene by the dietary protein. The promoter region of the rat PepT1 gene was isolated and characterized. METHODS: PepT1 messenger RNA levels were determined by Northern blot analysis. In transient transfection experiments, effects of amino acid and dipeptide on luciferase activity were investigated. RESULTS: The proximal promoter region of the rat PepT1 gene has a TATA-like box and a GC box sequence. The luciferase activities of the clone -351 RPT-LUC responded to particular amino acids (phenylalanine, arginine, and lysine) and dipeptides (Gly-Sar, Gly-Phe, Lys-Phe, and Asp-Lys). An AP-1 binding site and an amino acid-responsible element were present at -295 and -277 nucleotides relative to the transcription start site in this region. CONCLUSIONS: These results suggest that the up-regulation of dipeptide transport activity by dietary protein is caused by transcriptional activation of the PepT1 gene by selective amino acids and dipeptides in the diet.

Molecular cloning and expression of an amine sulfotransferase cDNA: a new gene family of cytosolic sulfotransferases in mammals.

A cDNA of amine sulfotransferase-RB1 (AST-RB1), which efficiently catalyzes 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) sulfation, has been isolated by immunoscreening of a rabbit liver cDNA library. The cDNA consisted of 1,117 base pairs and encoded a protein of 301 amino acids with a molecular weight of 35,876. The deduced amino acid sequence matched at six positions those of peptide fragments obtained from purified AST-RB1 protein. The sequence had less than 38% identity at the amino acid level with cytosolic sulfotransferases in mammals, although high degrees of similarity were observed with regions conserved throughout mammalian sulfotransferases. These results indicate that AST-RB1, arbitrarily named sulfotransferase 3A1 (ST3A1), constitutes a new and third gene family of cytosolic sulfotransferases in mammals. ST3A1 expressed in Escherichia coli as a fused protein catalyzed sulfation of amines such as PTHP, aniline, 4-chloroaniline, 2-naphthylamine, and desipramine, but barely O-sulfation of typical aryl and hydroxysteroid sulfotransferase substrates. These data unequivocally demonstrate the existence of a cytosolic sulfotransferase showing a high selectivity for amine substrates, and indicate that multiple forms of sulfotransferase mediate sulfation of xenobiotics in mammalian livers.

Molecular cloning, expression, and enzymatic characterization of rabbit hydroxysteroid sulfotransferase AST-RB2.

Cytosolic sulfotransferases, which consist of at least three gene families, play a major role in activation and detoxification of both endogenous and exogenous chemicals. We recently purified a rabbit sulfotransferase, AST-RB2, showing high activities to both hydroxysteroids and amines. To characterize this enzyme, a rabbit cDNA library was screened using anti-AST-RB2 antibodies. The isolated cDNA was judged to encode AST-RB2 (ST2A8) based on the amino acid sequences of peptide fragments obtained from purified AST-RB2. The cDNA showed high similarity to other mammalian hydroxysteroid sulfotransferases (ST2) at the amino acid level (58-68%), but low similarity to aryl sulfotransferases (ST1) (less than 37%). The protein expressed in Escherichia coli catalyzed sulfation of typical ST2 substrates. Therefore, ST2A8 was judged to belong to the ST2 family from both its primary structure and substrate specificity. The ST2A8 protein expressed in E. coli clearly differed from rat ST2A1 and ST2A2 on its localization (cytosol/insoluble fraction ratio). ST2A8 had no activity to lithocholate, but showed the highest catalysis on dehydroepiandrosterone and testosterone among the four forms (ST2A1, ST2A2, ST2A3, and ST2A8), indicating a clear difference between ST2A forms in substrate specificity to endogenous chemicals.

Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury.

BACKGROUND & AIMS: The oligopeptide transport system of the small intestine is resistant to mucosal injury. The mechanism of this resistance was investigated by examining the activity level and expression of the peptide transporter PepT1 in the intestine of rats treated with 5-fluorouracil. METHODS: The expression and localization of PepT1 were examined by immunoblot analysis of brush border membrane vesicles and immunohistochemical analysis of intestinal sections with PepT1-specific rabbit polyclonal antibodies. Also, Northern blot analysis was used for the expression of PepT1 messenger RNA (mRNA). RESULTS: Although the amounts of sucrase and an Na+-dependent glucose transporter protein in intestinal vesicles decreased markedly after 5-fluorouracil treatment, the amount of PepT1 protein remained largely unaffected. Immunohistochemical analysis also showed that the PepT1 immunoreactivity level was preserved in the brush border membrane of the remaining villi of 5-fluorouracil-treated rats. Levels of amino acid, glucose, and phosphate transporter mRNAs were profoundly depressed in 5-fluorouracil-treated animals, whereas the level of PepT1 mRNA conversely increased. CONCLUSIONS: The resistance of intestinal peptide transport to tissue injury may be attributable to increased synthesis of PepT1 rather than to a change in the kinetic properties of the residual absorbing cells.

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter.

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

Interactions of FK506 (tacrolimus) with clinically important drugs.

The effect of eight drugs on the metabolism of FK506 (tacrolimus) by human liver microsomes was studied at a substrate concentration of 10 microM. NADPH-dependent oxidative metabolism of FK506 was inhibited 20, 15, and 10% by quinidine, omeprazole, and sulindac, respectively, at 100 microM. Theophylline, diclofenac, indomethacin, phenylbutazone, and cimetidine (at ten times molar excess of FK506) and all eight drugs (at equal molar concentration) had slight effects on metabolism. The effects of these drugs were much weaker than that of cyclosporin A. The effect of FK506 on NADPH-dependent oxidation of prednisolone and theophylline by human liver microsomes were also studied. FK506 inhibited prednisolone metabolism in a concentration-dependent manner but exhibited a negligible effect on theophylline metabolism. The results suggest potential for interactions between FK506 and drugs metabolized by the cytochrome P450 3A subfamily.

Species and sex differences of testosterone and nifedipine oxidation in liver microsomes of rat, dog and monkey.

1. Species and sex differences in testosterone hydroxylation and nifedipine oxidation in liver microsomes from rat, dog and monkey have been investigated. 2. The formation of 2 alpha-, 2 beta-, 6 beta-, and 16 alpha-hydroxytestosterone and androstenedione in the male rat was higher than that in the female rat. Microsomes prepared from the male rat oxidized nifedipine about eight times faster than did those from the female rat. In contrast, marked sex-related differences were not seen in the dog and monkey. 3. Nifedipine oxidase activity in rat, dog and monkey correlated significantly with the activities for both testosterone 2 beta-hydroxylation and 6 beta-hydroxylation, suggesting the involvement of P4503A isozymes in these reactions. The ratios of formation of the 2 beta- to 6 beta-hydroxytestosterone in male rat and monkey were 0.17 and 0.18 respectively, whereas that in dog was 0.46. The corresponding activity ratios catalysed by P450DPB-1, a P4503A isoform purified from dog liver microsomes, was 0.36. 4. The formation of 16 beta-hydroxytestosterone was higher than that of the 16 alpha-hydrolated metabolite in liver microsomes from monkey, whereas 16 alpha-hydroxytestosterone was the predominant metabolite in the rat and dog, indicating species differences in stereoselectivity at the 16-position.

Species- and gender-related differences in amine, alcohol and phenol sulphoconjugations.

1. Species-, gender- and strain-related differences in amine sulphoconjugations were studied in 105,000 g supernatants of liver samples isolated from mouse, rat, guinea pig, rabbit, dog, monkey and man and were compared with those of alcoholic and phenolic compounds. Substrates examined were desipramine (an alkylamine), piperazine and piperidine derivatives (alicyclic amines), aniline (an arylamine), tiaramide and dehydroepiandrosterone (alcoholic compounds) and 2-naphthol (a phenolic compound). 2. Sulphoconjugating activities of alicyclic and aryl-amines and tiaramide varied depending on the animal species, sex and strain used. In all animal species examined, the activity for desipramine was low or negligible but for 2-naphthol was consistently detected and high. Amine sulphoconjugations were higher in rabbit than in other animal species. Dog hepatic 105,000g supernatants exhibited low or neglible activities for amines and tiaramide. Females showed higher sulphoconjugating activities for all substrates in mouse and for amines and tiaramide in rat; males exhibited higher activities for 2-naphthol in rat and monkey and for amines in rabbit; there were no clear sex-related differences in other sulphoconjugations. 3. Among BALB/c, C57BL/6, DBA/2, and AKR mouse strains, the AKR strain showed higher activities towards amines and tiaramide than others. 4. In human liver 105,000g supernatants, sulphoconjugating activities for alicyclic amines, dehydroepiandrosterone, and 2-naphthol were detected. Among them, higher activities were observed in piperazine and phenol sulphoconjugations. There were no sex-related differences in the activities of all substrates examined. Good correlations were observed in activities between alicyclic amine and dehydroepiandrosterone sulphoconjugations. 5. These results indicate that activities of amine and alcohol sulphoconjugations vary considerably depending on the substrate, species, sex and strain but phenol sulphoconjugation is consistently detected in all species examined.

Further metabolism of FK506 (tacrolimus). Identification and biological activities of the metabolites oxidized at multiple sites of FK506.

To characterize the metabolic pathway of FK506 (tacrolimus), FK506 or its 31-O-desmethyl metabolite was incubated with liver microsomes prepared from dexamethasone-treated rats in the presence of a NADPH-generating system under aerobic conditions. Besides the four oxidized metabolites already reported, four new metabolites were isolated and identified by HPLC, mass spectrometry, and NMR spectroscopy, and their biological activities were examined. The di-demethylated metabolites at the 15- and 31-, 13- and 31-, and 13- and 15-methoxy groups of FK506, were designated respectively as M-V, M-VI, and M-VII. The fourth, M-VIII, was the metabolite produced after O-demethylation at the 31-methoxy group and formation of a fused 10-membered ring structure through the 19- to 22-carbon of the macrolide ring after oxidation of the 19-methyl group, and of the 36- and 37-vinyl group of FK506. The immunosuppressive activity of the isolated metabolites was estimated in a mouse mixed lymphocyte reaction system and the IC50 values for M-V, M-VI, M-VII, M-VIII, and FK506 were > 1000, 8.78, > 1000, 15.27, and 0.11 ng/ml, respectively. Reactivity of the metabolites with mouse anti-FK506 monoclonal antibody was studied and immunocrossreactivity of M-V was 92.3% of FK506, but no reactivity was observed for M-VI, M-VII, and M-VIII. FK506 thus was metabolized at multiple sites by rat hepatic microsomes and the metabolites formed (M-V) - (M-VIII) exhibited weak or negligible immunosuppressive activity.

Isolation and characterization of an alicyclic amine N-sulfotransferase from female rat liver.

An alicyclic amine N-sulfotransferase sulfonating 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) was purified from female rat liver cytosol and showed a homogenous band with a molecular weight of 30500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme, designated NST-1, catalyzed sulfonation not only of the alicyclic amine but also dehydroepiandrosterone, a typical substrate of hydroxysteroid sulfotransferase (STs), but had little sulfonating activity towards 2-naphthol, a typical substrate of aryl STs. The N-terminal amino acid sequence for the first 24 residues had a high homology with those of rat liver hydroxysteroid STs. Therefore, it is suggested that NST-1 is classified as a member of the hydroxysteroid ST. Immunoblot analysis of male and female rat liver cytosol, carried out by using rabbit antisera raised against NST-1, indicated that the female cytosol contained a higher level of the enzyme than that of male. The marked sex difference in the expression level of NST-1 was in good accordance with the previous demonstration that female rat liver cytosol catalyzed sulfonation of PTHP to a greater extent than that of male.

Effect of repeated injection of somatomedin C on drug metabolizing enzyme activities in rats.

The effect of somatomedin C on the hepatic drug-metabolizing enzyme system in rat liver after sc injection, a clinically applicable dosing route, was studied. Although liver weight was slightly increased after somatomedin C (1 mg/kg) was injected for 7 days, no significant changes were observed in other dosing group (0.1 and 10 mg/kg). There were no significant effects of somatomedin C on cytochrome P-450 and b5 contents, and NADPH-cytochrome c reductase, aminopyrine demethylase, aniline hydroxylase, and ethoxyresorufin deethylase. Additionally, somatomedin C treatment did not affect the activities of 2 alpha-, 2 beta-, 6 beta- and 16 alpha-hydroxytestosterone and androstenedione formation from testosterone. Changes relation to dosing period were also examined with 1 mg/kg of somatomedin C. In this case, hepatic levels of cytochrome P-450 and b5, NADPH-cytochrome c reductase and drug oxidations did not differ significantly among groups treated for 3, 7, or 14 days. These results suggest that somatomedin C has no effect on the hepatic mixed-function oxidase system.