The non-linear pharmacokinetics of the recombinant human granulocyte colony-stimulating factor derivative nartograstim (NTG) has been reported. To clear the role of G-CSF receptor in the non-linear elimination of NTG, in vivo total clearance of NTG at steady-state (CLss) in the rats was compared with in vitro NTG binding capacity in the bone marrow. The dosedependent decrease of CLss in the rats indicated that a part of NTG elimination process was saturated. The Km and Vmax values for this saturable process were 107 pM and 16 pmol/hr/kg, respectively. The saturable CLss in the cyclophosphamide-treated rat with a low NTG binding capacity in the bone marrow, was 17 % of that in control. Also, the saturable CLss in the NTG-repeated administered rats with a high NTG binding capacity was 2-fold greater than that in control. On the other hands, the initial uptake clearance (CLuptake) by the bone marrow and spleen were down-regulated within 8 hr after a subcutaneous administration of NTG at a saturable dose. These finding indicated that the saturable elimination of NTG from the circulation might be due to the receptormediated endocytosis (RME) of G-CSF receptor.
In vitro RME of NTG in the rat bone marrow cells was kinetically analyzed. The internalization rate constant of occupied receptor (kint) and degradation rate constant (kdeg), obatained by three experiments (pulsechase, initial binding and steady-state binding experiments), were ranged 0.033-0.108 min^-1 and 0.041-0.046 min^-1. Also, from the steady-state plot, the ratio of kint to internalization constant of free receptor could be calculated as 6.6. This indicated that the internalization of the receptor was accelerated by the NTG binding, resulting in the down-regulation of the receptor. The in vitro clearance at steady-state, 104 ml/hr/kg, was comparable with the in vivo saturable CLss. This suggests that the saturable elimination of NTG from the circulating plasma comes mainly from RME in the bone marrow.

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The effect of membrane surface potential on the permeation of ionic compounds through the intestinal brush-border membrane was investigated using uptake experiments by brush-border membrane vesicles (BBMV) and large unilamellar vesicles (LUV). The uptake of anionic compounds (ceftibuten, cefixime, benzylpenicillin etc.) was decreased with increase of membrane surface negativity. On the other hand, the uptake of a cationic compound, tryptamine, was increased with increase of surface negativity. The uptake of these ionic compounds was well correlated with membrane surface potential of BBMV and LUV monitored by ANS. These results suggest that the permeation of ionic compounds through the intestinal brush-border membrane is dependent on the membrane surface potential. The mechanism of inhibitory effect on the uptake among these Ionic compounds was also examined from the viewpoint of changes in the membrane surface potential. The inhibitory effect of tetracaine and imipramine on the uptake of tryptamine was well correlated with changes in the membrane surface potential induced by these organic cations. Similar relation was observed in the inhibitory effect of flufenamic acid on the uptake of cefixime. These results suggest that changes in the membrane surface potential contribute to the inhibitory effect on the uptake of these Ionic compounds.

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A pharmacokinetic analytical method is presented, which was developed in Sandoz during the preclinical evaluation of a cyclosporine derivative, SDZ IMM 125. A physiologically-based pharmacokinetic (PBPK) model established based on rat data was scaled-up to those for dog and human using 1) an allometric equation for estimating transmembrane drug exchange clearance for each organ in the larger mammals, and 2) relative activity ratios of a specific cyt.P-450 subtype among rat, dog, and human, which were measured in vitro. The resultant PBPK models for dog and human successfully predicted the blood kinetics of SDZ IMM 125 measured experimentally during preclinical and clinical pharmacokinetic studies. The technique seems to facilitate the safety assessment in humans at very early period of drug development.

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The purpose of the present study was to understand the permeability of tumor-derived endothelial cell monolayer using tumor-conditioned medium and primary cultured tumor-derived endothelial cells. B16 melanoma-conditioned medium increased permeabilities of bovine aortic, bovine vein and human umbilical vein-derived endothelial monolayers. This characteristic resemble that of tumor vascular endothelium in vivo. Ascorbic acid, which stimulated endothelial collagen synthesis, decreased endothelial permeability. 3, 4-Dihydroxybenzoic acid ethyl ester, a collagen synthesis inhibitor, increased the permeability. The conditioned medium decreased endothelial collagen content. These findings suggested that the hyperpermeability of the endothelial monolayer which were cultured with tumor-conditioned medium was due to low collagen content. The isolation of rat KMT-17 fibrosarcoma-derived endothelial cells by Percoll gradient centrifugation and an attaching speed separation technique is described, together with their properties in culture. Primary cultured tumor-derived endothelial monolayer was more permeable than normal tissue-derived endothelial monolayer. Leukocyte adhesion to tumor-derived endothelial cells was reduced compared to normal tissue-derived endothelial cells. These characteristics resemble those of tumor vascular endothelium. Our data indicate that tumor-conditioned medium or primary cultured tumor-derived endothelial cells are applicable for studies on physiological characteristics of tumor endothelial cells.

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The purpose of our study is to clarity the mechanism of cellular uptake of antisense DNA. Antisense DNA must enter cells before exerting its pharmacological effect. It is thus important to know the uptake mechanism of antisense DNA for designing antisense DNA that are efficiently taken up by target cells. We use HCT-15(human colorectal carcinoma cell line) and rat primary cultured hepatocytes.The difference of uptake of phosphodiester oligonucleotide(D-oligo) between the two cells was observed. The uptake of D-oligo by HCT-15 was highly concentrative and 20-fold larger than that by hepatocytes. The uptake of D-oligo by HCT-15 and hepatocytes was saturable. (Km0.12μM, 2.1μM, Vmax37.7pmol/min/mg, 0.94pmol/min/mg, respectively) and exhibited remarkable temperature dependency. The uptake of Doligo by hepatocytes was inhibited by metabolic inhibitors, anion charged compounds(ATP, dextran sulfate), while in HCT-15 those treatment didn't alter the uptake. Chloroquine and monensin, known to inhibit receptor-mediated endocytosis, didn't change the uptake of D-oligo by both cells. Our results suggest that Doligo uptake is mediated by a sort of transporter and that the uptake mechanism is different between both cells. Further studies are required to conclude whether the uptake occurs via receptor-mediated endocytosis or not.

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The purpose of the present study was to understand the skin permeation mechanism of amino acids. Skin permeation of amino acids through the excised rat skin was measured at various pH. The permeabilities varied with donor pH and amino acid. But, the permeabilities of cortisone and mannitol were almost constant in the same range of pH. It suggests that pH-dependent permeation of amino acids may be due to the different permeability of each ionic specie. The permeability coefficient of each ionic specie was estimated from the permeability-pH profiles using the dissociation constants. The estimated permeability coefficients of monocation and neutral zwitterion were not depend on the lipophilicity but on the size of the amino acids, suggesting porous mechanism of transport. The permeability coefficient was highest for dication, followed by monocation, positive zwitterion, neutral zwitterion and negative zwitterion. The electrical potential difference through the skin was too small to affect the permeability of ions. Then, the permselective property of skin seems to be caused by the difference of diffusion coefficient in pore pathway due to the hydration of ions.

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The maximum fluxes (Jmax) of six β-blockers across keratinized membranes were determined in vitro and compared with their melting points (mp). Rat abdominal skin and hamster cheek pouch mucosa were used as the model membranes. Linear relations of Jmax with mp were observed in both membrane. Permeation studies using the delipidized membranes and the isolated stratum corneum (SC) sheet of hamster cheek pouch clarified that the primary rate-limiting barrier function exists in the SC especially in the intercellular lipid. A lipid mixture system (ceramide : cholesterol : palmitic acid : cholesterol-3-sulfate = 40 : 25 : 25 : 10 by weight, a mimic of intercellular lipids of SC) was used as a lipid-coated membrane and liposomes to evaluate drug permeation through the SC. The Jmax values obtained in the lipid-coated membrane, which showed linear relations with those determined in rat skin and in hamster cheek pouch, correlated with the degree of drug partition to the liposomes prepared with the same model lipid mixture, suggesting the validity of this model lipid mixture system as an in vitro system substituted for animal skin in percutaneous drug permeation studies. The usefulness of the system in assessing the penetration enhancers which act on the lipids in the SC, such as oleic acid (OA), was also clarified.

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Novel biodegradable triglycerides having quaternary ammonium group (YKS) were synthesized as counter ions for ion-pair complexation with anionic drugs and evaluated for their transdermal penetration enhancing effects. Permeation of salicylate or cromoglicate, model anionic drugs, through the rat skin were measured in modified Franz-type diffusion cells. The steady-state flux, the permeability coefficient and the diffusion coefficient were calculated. The permeability of these anionic drugs were enhanced by an ion-pair mechanism in the presence of the YKS. Significant increases in the permeability parameters were observed after pretreatment with YKS. It was shown that YKS21 itself was an excellent penetration enhancer as well as an effective counter ion for ion-pair transport. The exact mechanisms of the increased permeability by pretreatment with YKS remain to be elucidated. When ion-pair complex of cromoglicate with YKS21 was applied in the donor compartment after pretreatment with 3% YKS21 for 24 hours, it was found that this ion-pair complex with the pretreatment was several times more effective than cromoglicate applied alone with the pretreatment or than ion-pair complex without the pretreatment. Judging from the primary irritation indices, YKS21 was 3 to 5 safer than Azone.

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It is well known that the oral delivery of peptide drugs is highly restricted by two barriers, degradation by enzymes and low membrane permeability. In this study, using the vascular perfusion technique of rat small intestine and the liver, we attempted the quantitative estimation of the contribution of two barriers to the low availability of orally administered peptide. As a model peptide, metkephamid (MKA), a stable analog of methionine enkephaline, was used. After intraintestinal administration, the most of MKA degraded in the intestinal tract, while only 0.5-1% of administered amount was absorbed to the vascular blood flow as an intact form. The calculation of the degradation and the permeation clearance (CLd and CLp) of MKA from the intestinal tract clearly showed that the rate of MKA degradation is much higher than that of the absorption. Also, MKA is degraded not only before the absorption but also during the absorption. Puromycin, an aminopeptidase inhibitor, increased the absorbed amount of MKA to 3.5%, by both decreasing CLd and increasing CLp. However, thiorphan, an endopeptidase inhibitor, showed no effect on both clearances. In addition, the CLp in the ileum was 2-times higher than that in the jejunum. From the liver perfusion experiment, it was demonstrated that about 30% of absorbed MKA was degraded at the liver. This firstpass metabolism increased to 60% when the albumin (BSA) was excluded from the perfusate, because only the free fraction of MKA (about 50 %) in the blood would be metabolized at the liver. Based on above results, the total bioavailability of MKA after intraintestinal administration can be predicted as 5 to 6 % when the appropriate enzyme inhibitor is utilized. In order to develop the convenient way to know the intestinal absorption of MKA, in vitro permeation study was carried out using Caco-2 monolayer. In this experiment, the degradation of MKA was not observed, suggeting thelow enzyme activity in Caco-2 monolayer. The permeability of MKA to Caco-2 monolayer was very low and was the same order with thatof dextran or PEG. Therefore, it is considered that not only the inhibition of the degradation but also theimprovement of the premeability was great importance to deliver MKA by oral administration.

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The effects of protease inhibitors on the intestinal absorption of insulin and human calcitonin were investigated in situ in closed small and large intestinal loops in rats. The intestinal absorption of insulin and human calcitonin was evaluated by its hypoglycemic and hypocalcemic effects, respectively. When insulin alone was administered into small or large intestinal loops, no marked hypoglycemic response was observed in either region. However, a significant hypoglycemic effect was obtained following large intestinal administration of insulin with various protease inhibitors, whereas we found little hypoglycemic effect following small intestinal co-administration of insulin with these protease inhibitors. In addition, when the luminal surface was washed with saline solution, we did not observe the regional difference in promoting insulin absorption by these inhibitors. Similar results were also noted in the effects of these inhibitors on the intestinal absorption of human calcitonin. In the stability experiments, insulin and human calcitonin were rapidly degraded in the small intestinal fluid and homogenates of the small or large intestinal mucosae. Further, these protease inhibitors were effective for improving the stability of these peptides in the intestinal homogenates. In conclusion, these findings indicate that the protease inhibitors increase the intestinal stability of these peptides, thereby improving their absorption from the gut. These results would give us basic information for developing the oral dosage form of peptide drugs.

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Major obstacles to the oral administration of peptide drugs are their low transmembrane permeability and enzymatic degradation in the gastrointestinal tracts. We glycosylated insulin (INS) to try to solve these problemes. When the derivative, B1-Phe monosubstituted p-(succinylamido)phenyl-α-D-glucopyranoside (SAPG)substituted insulin (SAPG-INS), was administered intraintestinally to rats, it showed a greater hypoglycemic effect than the native bovine insulin. One of the causes of this effect was considered to be the interaction of SAPG-INS with the D-glucose transporter. To investigate more clearly the possibility whether a glycosylated peptide is transported by the Na⁺, D-glucose cotransporter, we synthesized ¹²⁵I-labeled SAPG-Gly-Gly-Tyr-Arg (SAPG-GGYR) and examined its uptake to BBMV by a rapid filtration technique. In the presence of Na⁺-gradient inward to BBMV, the amount of SAPG-GGYR taken up to BBMV was significantly increased in conparison with that in the absence of Nat-gradient. These findings suggested that the glycosylated tetra-peptide is transported by the Na⁺ dependent D-glucose cotransporter. Consequently it is suggested that the glycosylation of peptide drugs increases the resistance to enzymatic degradation and is useful for the improvement of its intestinal absorption.

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The intestinal transport mechanism of 5-fluorouracil (5-FU) was investigated in rats using in vitro techniques, everted sacs and brush border membrane vesicles (BBMVs), and in situ perfusion technique. The gastrointestinal absorption of 5-FU was also evaluated in vivo by the analysis of gastrointestinal disposition and plasma concentration in order to examine the quantitative in vitroin situ-in vivo correlation in the absorption. In everted sacs, the uptake of 5-FU was Na⁺ and concentration-dependent, and inhibited by pyrimidines, but not by purines and nucleosides. These results are consistent with previous reports, ^3.4) and suggest the involvement of pyrimidines-specific carrier-mediated transport in the intestinal transport of 5-FU. It was also shown that the kinetic parameters were close to those in in situ perfusion. However, in BBMVs, Na⁺ dependency in the uptake of 5-FU was not confirmed. Although a slight concentration dependency and inhibition by pyrimidines were observed, the contribution of carrier-mediated transport appeared to be far smaller than that in everted sacs. Thus, it was suggested that, in addition to Na⁺, some other factor may also play an important role in the intestinal transport of 5-FU. On the other hand, oral absorption of 5-FU was shown to be gastric emptying-limited at a low dose, where the carriermediated transport is presumed to be in the linear phase (cocentration << Michaelis constant) and most efficient. The apparent membrane permeability clearance (in vivo) was in agreement with those in perfusion (in situ) and everted sacs (in vitro) at comparative concentrations. In conclusion, the carrier-mediated transport of 5-FU was confirmed to be Na⁺-dependent in everted sacs, but not in BBMVs. There may be some other factor than Na⁺ may also be required for the transport of 5-FU. It was also shown that there is a good quantitative in vitro (everted sacs)-in situ-in vivo correlation in the intestinal absorption of 5-FU.

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The transport characteristics of cefaclor, ceftibuten and S-1090, a new orally active antibiotic, in rat intestinal brush border membrane vesicles were compared. The uptake of ceftibuten showed a typical overshooting (up-hill) transport in the presence of W-gradient, while only a slightly stimulated uptake was observed in the case of cefaclor or S-1090. Although various types of dipeptides and tripeptides exhibited inhibitory and counter-transport effects on the uptake of ceftibuten, S-1090 uptake was inhibited by the dipeptides possessing cyclic ring in N-terminal amino acid but inhibitory effects were not observed when these amino acid was at C-terminal. Cefaclor, cephalexin and the dipeptides composed of amino acids with aliphatic chain did not show significant inhibitory effects. Countertransport effects on S-1090 uptake were observed in the vesicles preloaded only with histidyl-peptides. The uptake characterictics of these cephems into Caco-2 cells were also separately investigated. From the above results, these oral cephems are considered to be absorbed by dipeptides transport carrier, however, there seems to be the heterogeneity (multiplicity) in dipeptide transport systems which may depend on the structure of N-terminal amino acid.

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Transport mechanisms of intestinal absorption and renal excretion for new-quinolone antibactrial agents were investigated. The absorption of enoxacin (ENX) from rat jejunal loop exhibited a pH-dependent profile. The greater absorption at acidic pH of medium was observed, and ciprofloxacin (CPFX) reduced the ENX plasma concentration after simultaeously oral administration to rat. In the uptake experiments by the intestinal brush-border membrane vesicles (BBMV), initial rate and time-course of ENX and sparfioxacin (SPFX) were significantly dependent on the pH of medium (pH5.5 > pH7.5), and CPFX inhibited compleately the pH dependent uptake of ENX. The ion diffusion potential affected the uptake of these drugs by the intestinal BBMV. On the other hand, ENX uptake by the renal BBMV was contributed by not only the ionic diffusion potential but also the cation-H⁺ antiport system of organic cation secretion.

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In order to investigate the mechanism of the reabsorption of a protein in renal tubules of rats, we employed OCT-7000, which is a recombinant variant of natural human interleukin-1α with a molecular mass of approximately 18, 000. In this study, OCT-7000 uptake in renal tubules was examined using immunoelectron microscopic technique with immunogold staining. The effects of various proteins or synthetic polypeptides on the urinary excretion of OCT-7000 were also investigated. Immunoelectron microscopic observations showed that OCT-7000 was taken up into the endocytic vesicle close to the brush border membrane located in segment 2 of the proximal tubules, followed by accumulation of secondary lysosomes. Urinary excretion of OCT-7000 after systemic administration was extremely low, accounting for 0.014% of the dose. Human serum albumin had no effect on the excretion of OCT-7000, while increases in the urinary excretion of OCT-7000 were found in rats treated with a trypsin inhibitor, myoglobin and trypsinogen, in a dose-dependent manner. The order of potency for urinary excretion of OCT-7000 was trypsinogen > myoglobin > trypsin inhibitor. Poly-L-lysine, a synthetic polypeptide dose-dependently increased the urinary excretion of OCT-7000, whereas poly-L-glutamic acid had no effect on excretion. Specifically, the data reveal that reabsorption of OCT-7000 in the proximal tubules was inhibited by trypsinogen, myoglobin, trypsin inhibitor or poly-L-lysine, resulting in an increase of urinary excretion of OCT-7000. Furthermore, it was considered that negative charges on the brush border membrane in the proximal tubules were involved in the reabsorption of OCT-7000 because the inhibitory potency of proteins or synthetic polypeptides on the reabsorption of OCT-7000 was increased with a high isoelectric point. From the above, the mechanisms of reabsorption of protein in renal tubule are speculated as follows. (1) The reabsorption site of proteins is located in segment 1 and/or 2 of the proximal tubules in the kidneys. (2) Binders with negative charges likely exist on the surface of the brush border membrane, glomerular filtrated proteins are bound to the binder. The binder has an unusually broad binding specificity for filtered proteins, and the binding affinity between them is involved in only molecular charges. (3) After binding of filtered proteins to the binder on the membrane, the membrane invaginates into the cell, and then the endocytic vesicle including filtered proteins is incorporated into the cell. (4) Primary lysosome and endocytic vesicle are fused to constitute a secondary lysosome.

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In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man) -modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately 2-fold higher accumulation than non-modified MLV (PC-MLV). These hepatic uptake of liposomes were obviously inhibited by preheating the serum at 56 °C for 30 min or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.

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In vivo biodistribution of lipophilic drugs, injected with various lipid carrier systems such as micelles, liposomes, and emulsions, was systematically studied. It was clarified that drug lipophilicity is a determinant factor to control in vivo behavior of lipophilic drugs with lipid carriers. In situ sigle-pass rat liver perfusion experiment was also carried out to evaluate hepatic disposition of lipophilic drugs injected with lipid carriers, and to investigate retention ability of lipid carriers for lipophilic drugs. There was a large difference in release rates of lipophilic drug from carrier particles between lipid carriers. In addition, on the basis of these pharmacokinetic findings, potential control of pharmacological activity of highly lipophilic probucol, plasma cholesterol-lowering agent, was shown.

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We studied the metabolism of amines by mitochondrial monoamine oxidase (MAO) and substrate selectivity in mammalian brain. The following conclusions were obtained. 1) The regional distribution of MAO-B in human brain by autoradiographical use of ₁₁C-L-deprenyl was high in the caudate nucleus, putamen, thalamus, substantia nigra.The half-life for the turnover rate of MAO-B in pig brain by positron emission tomography using ¹¹C-L-deprenyl was calculated to be 6.5 days. 2) Relation between serotonin and dopamine uptake rates, monoamine concentrations and MAO activities were estimated in various regions of rat brain. Of main interest is the finding that MAO activities in general (conventional method) were positively correlated to seroton in uptake rates and to intra-5-HT-synaptosomal MAO, but not to dopamine uptake rates or intra-DA-synaptosomal MAO activities. 3) An inhibition of MAO-B of more than 40 % was required to reduce the MPTP toxicity and it was completely prevented at about 60 % MAO inhibition. Moreover, although MPTP toxicity in relation to age, dopamine up take and MAO activity was investigated in two rodent species, MAO-B activity was not the rate-limiting step for MPTP neurotoxicity. 4) Histamine H3-ligands inhibited the conversion of N-tele-methylhistamine to N-tele-methylimidazoleacetic acid by MAO-B in the rat brain.5) The oxidation of racemic chlorpheniramine was catalyzed by mitochondrial MAO of rat brain. Moreover, there was a stereoselective difference in oxidative deamination of chlorpheniramine by MAO-A and -B.

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Metabolism of (+)-S-145 was investigated within vitro studies to elucidate the metabolic pathway and responsible enzymes therein. Co-factor requirements and subcellular distribution indicated that a-side chain of (+)-S-145 was β-oxidized by peroxisomal enzymes, and that hydroxylation at C-5 or C-6 position of bicyclo-ring was catalyzed by cytochrome P-450s in microsomes. Results of these studies revealed that the most of (+)-S 145 incorporated into liver was activated to its acyl-CoA ester, and that β-oxidation was major pathway in metabolism of (+)-S-145. In peroxisome, there were two independent pathways in β-oxidation, thus (+)-S 145-CoA was generally β-oxidized to Bisnor-(+)S-145 and Tetranor-(+)-S 145, meanwhile its a-side chain was saturated by Δ5-reduction to form DH(+)-S-145 by NADPH dependent manner, then it was β-oxidized to DH-bisnor-(+)-S-145. As OH-(+)S-145 could never be n-oxidized, it was concluded that OH-Tetranor-(+)-S-145, one of major metabolites in vivo, was produced in the hydroxylation of Tetranor-(+)-S-145 catalyzed mainly by P-450 3A1/2.

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A cytochrome P450 isozyme (designated P450GPF-B) that catalyzes the oxidation of 7α-OH and 7β-OH-Δ⁸-THCs to 7-oxo-Δ⁸-THC has been purified from liver microsomes of untreated female guinea pigs. P450GPF-B had an apparent MW of 50kD, and specific content of 18.7 nmol/mg protein. Analysis of the N-terminal amino acid sequence suggests that the isozyme is a member of the P450 3A gene subfamily. Antibody against P450GPF-B significantly inhibited the microsomal formation of 7-oxo-Δ⁸-THC from 7β-OH-Δ⁸-THC, but not 7α-OH-Δ⁸-THC. The oxidation of 7α-OH and 7β-OH-Δ⁸-THCs to 7-oxo-Δ⁸THC in liver microsomes of male rats was significantly higher than that of female rats. The catalytic activity in rat was significantly induced by treatment of dexamethasone and phenobarbital, and was significantly inhibited by antibody against P450GPF-B. These results suggest that P450GPF-B is a major isozyme responsible for the formation of 7-oxo-Δ⁸-THC from 7β-OH-Δ⁸-THC in guinea pig liver microsomes and that the formation of 7-oxo-Δ⁸-THC in rat is also catalyzed by P450 isozyme(s) immunologically related to P450GPF-B.

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Rats are commonly used as experimental animals in an attempt to predict drug metabolism and toxicity in man and much data for drug metabolism with rats have been accumulated. Extrapolation of the data to humans can be improved by a comparative studies of characteristics and properties of drug metabolizing enzymes in experimental animals such as rats. In this study, we compared catalytic activities of purified human recombinant P450 with those of rats. Furthermore, we assayed the levels of P450 in human hepatic microsomes with specific antibody prepared with recombinant human P450. Like in rat hepatic microsomes, CYP2C and 3A forms were major P450s in human hepatic microsomes. Human and rat CYP3A forms had similar characteristics; they required phospholipid mixtures and cytochrome b5 for having efficient catalytic activity in a reconstituted system. Also, these forms had similar catalytic acitivity. However, rat CYP2C11 had different catalytic properties from human CYP2C8, 2C9, and 2C18. CYP2C11 had high activity toward testosterone and some drugs including lidocaine, but human CYP2C forms did not. Therefore, difference in drug metabolism between human and rat may occur when CYP2C11 contributes to metabolism. In drug metabolism, CYP2D6 is important, because it exhibits genetic polymorphism and metabolism of some drugs such as debrisoquine was delayed in poor metabolizer who has a mutated CYP2D6 gene. Rat CYP2D1 and human 2D6 seem to have similar catalytic activity. CYP2E1 is ethanol-inducible in both rat and human. Human and rat CYP2E1 had similar catalytic activities. Rat CYP2B1 and human CYP2B6 have high activity toward drugs including lidocaine. Rat CYP2B1 is strongly induced by phenobarbital and by many other drugs. In human, CYP2B6 may be induced by barbiturate as well as CYP3A4. In this case, drug metabolism maybe enhanced.

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Metabolism of mefenamic acid and diclofenac was studied using human liver microsomes and recombinant human P450 (CYP2C8, 9, 18 and 19) and compared with that of tolbutamide and S-mephenytoin. Anti-human CYP2C antibodies inhibited all the reactions mentioned above by upto 90% in human liver microsomes. However, the mean Vmax/Km values of diclofenac 4'-hydroxylation and mefenamic acid 3'-hydroxylation in the microsomes obtained from putative poor metabolizers of S-mephenytoin did not differ from those from extensive metabolizers. In addition, there were high correlations (r=0.91-96, P<0.01) among the activities of diclofenac 4'-hydroxylation, mefenamic acid 3'-hydroxylation and tolbutamide hydroxylation, whereas the correlation of these activities with S-mephenytoin 4'-hydroxylation capacity was insignificant or low. Among the CYP2C isoforms studied, CYP2C9 exhibited the highest activities in diclofenac 4'-hydroxylation and mefenamic acid 3'-hydroxylation, whereas CYP2C19 showed a comparable activity with CYP2C9 (70% of CYP2C9) for mefenamic acid. In conclusion, P450 isoform responsible for the metabolism of diclofenac, mefenamic acid and tolbutamide appears to be identical, whereas it seems to be different from that of S-mephenytoin. The study with recombinant P450 suggested that CYP2C9 is an isoform of P450 responsible for the 4'-hydroxylation of diclofenac and mefenamic acid, while CYP2C19 may also be involved in the metabolism of mefenamic acid in addition to CYP2C9.

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To identify cytochrome P450 isoform(s) involved in 8-hydroxylation, N-demethylation and N-oxidation of mianserin (US) enantiomers in humans, we studied the metabolism of S(+) and R(-)-MS using human liver microsomes and 6 different recombinant human P450 isoforms (i.e., CYP1A2, 2A6, 2B6, 2D6, 2E1 and 3A4). The rates of 8-hydroxylation and N-oxidation of MS were faster for S(+) than those of R(-)-enantiomer of MS, while that of N-demethylation of MS was faster for R(-) than S(+)-MS in human liver microsomes. When the correlation of the activities of individual forms of P450 with 8-hydroxylation. N-demethylation and N-oxidation of MS at low (5μM) and high (200μM) substrate concentration of MS were evaluated in 10 different human liver microsomes, there were significant correlations between 2-hydroxylation of desipramine and 8-hydroxylation of S(+)-MS (r=0.94, P<.01), between O-deethylation of phenacet i n and N-demethylation of S(+) (r=0.82. P<.05), R(-)-MS (r = 0.71. P<.05) or N-oxidation of S(+)-MS (r=0.95. P<.01) at low concentration of MS. and between 60-hydroxylation of teststerone and 8-hydroxylation, N-demethylation or N-oxidation of S(+)and R(-)-MS (r=0.68 ^ 0.93, P<.05) at high concentration of MS. Quinidine. a selective inhibitor of CYP2D6, inhibited 8-hydroxylation of S(+) and R(-)-MS by approximately 15 and 30%. respectively, while α-naphthoflavone, a selective inhibitor of CYP1A subfamily, inhibited N-demethylation and N-oxidation of S(+) and R(-)-MS by approximately 40 to 60% in human liver microsomes. In addition, troleandamycin, a inhibitor for CYP3A. inhibited 8-hydroxylation, N-demethylation and N-oxidation of S(+) and R(-)-MS by 30 to 80% in human liver microsomes. Among the 6 differnt recombinant human CYP isoforms, CYP2D6. CYP3A4 CYP1A2 and CYP286 catalyzed 8-hydroxylation of both MS enantiomers, while CYP1A2, CYP2B6, CYP2D6 and CYP3A4 showed catalytic activities for N-demethylation of both enantiomers of MS. Catalytic activities for N-oxidation of both enantiomers of MS were detected with CYP3A4 and CYP1A2. The results obtained from human liver microsomes and recombinant P450 isoforms suggested that 8-hydroxylation of MS is catalysed mainly by CYP2D6 and CYP3A4 and at least to some extent by CYP1A2 and CYP286, N-demethylation by CYP1A2, CYP286, CYP2D6 and CYP3A4.and N-oxidation by CYP1A2 and CYP3A4. Therfore, several P450 isoforms appear to be involved in major metabolic pathways of MS enantiomers in human. Since these isoforms have a different stereoselectivity for the metabolism of MS, stereoselective metabolism of MS observed in vivo in human appears to be determined by the sum of stereoselectivities of the different isoforms of P450 responsible for the metabolism of MS enantiomers.

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We have isolated 5 different CYP3A genes (P450/6βA, P450/6βB, P450/6βD, P450/6βE and P450/6βF) containing 5'-flanking region and the first exon. Several clones covering the whole structure of two genes (P450/6βA and P450/6βB) were isolated and analyzed nucleotide sequences around exon-intron boundary, resulting that these gene consisted in 13 exons. P450/6βA and P450/6βB may correspond to CYP3A2 and CYP3A1, respectively, based on their nucleotide sequences and expression profiles of the two mRNA. The 5'-flanking region of P450/6βA and P450/βB genes were further analyzed. A high conserved region was observed in proximal promoter region (-180bp to -1bp) of both genes. Using DNase I footprinting analysis and gel shift assay, three sites (6βA-A, 6βA-B and 6βA-C) interacting with nuclear proteins were found in the proximal promoter region of P450/6βA gene. Thus, we carried out to analyze the role of these sites on transcriptional activation of P450/6βA gene using CAT assay. A site (60A-A: -89bp to -106bp) was mainly responsible for the transcriptional activation of P450/6βA gene in HepG2 cells, but not in Y1 cells. The nuclear protein binding to 6βA-A site was competitively inhibited by the addition of APF 1 oligonucleotide which bound hepatocyte nuclear factor-4 (HNF-4) in gel shift assay. These results suggest that HNF-4 plays an essential role of the transcriptional activation of P450/6βA gene in rat liver.

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Most promutagens and procarcinogens exert their genotoxicity after undergoing metabolic activation. Metabolism of chemicals is one of important factors limiting the extents of the action of chemicals. In this study, we established cell lines which carried cDNAs coding for human CYP1A2 and N-acetyltransferase (NAT); the latter functions as O-acetyltransferase for N-hydroxyarylamines formed by CYP1A2. A cell line which expresses CYP1A2 together with P450 reductase activated aflatoxin B₁, but not heterocyclic amines. A cell line which carries CYP1A2 and polymorphic NAT(NAT2) in addition to P450 reductase activated 2-amino-3, 8-dimethylimidazo[4, 5 f]quinoline (IQ) and some other heterocyclic amines efficiently. However, a cell line which carries CYP1A2 and monomorphic NAT (NAT1) showed only low activity toward the same heterocyclic amines. In order to determine the presence and the frequency of genetic polymorphisms of CYP1A2 and NAT2 in humans, we performed caffeine phenotyping test on 205 Japanese volunteers. Analyses of metabolic ratios of urinaly metabolites showed a bimodal distribution, indicating that about 86% and 91% of Japanese were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively. The genotype of NAT2 determined by PCR-RFLP method agreed completely with the phenotype. To determine the mechanism of the differences in CYP1A2 activities, genomic DNA from peripheral lymphocytes of slow and rapid metabolizers was subjected to DNA sequencing. No differences in nucleotide sequence were observed between poor and extensive metabolizers in the exons, exon-intron junctions and 5'-flanking region of the CYP1A2 gene. Since CYP1A2 and NAT2 are enzymes activating heterocyclic amines efficiently, it is expected that extensive metabolizers of CYP1A2 and NAT2 are under high risk when exposed to these promutagens.

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The inducing activities of picrotoxin and strychnine were studied in rats. Inducibility was estimated by measuring P450 content, metabolizing activities toward benzphetamine, aniline, benzo[a]pyrene and testosterone, and by immunoblot analysis using anti-CYP2B1 antibody. Picrotoxin and the components, picrotoxinin and picrotin, equally induced hepatic CYP2B1 and 2B2. The magnitude of the induction was comparable to phenobarbital-induction. Strychnine and the analogue, brucine, with low toxicity also induced CYP2B1/2, although the extent was less than that of PB. Interestingly, these alkaloids induced CYP2B3 as well as CYP2B1/2. Based on these findings together with a recent concept, Animal-Plant Warfare, the evolution of CYP2B subfamily P450 is discussed.

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Hormonal effects on expression of constitutive and phenobarbitalinducible Cyp2b9/10 species were investigated in mouse hepatocytes in primary culture and in vivo. Although the expression decreased rapidly in monolayer-cultured cells, it maintained at equivalent levels to that in in vivo when the cells were cultured as spheroids(multiclellular aggregate). In control liver more Cyp2b9 than Cyp2b10 mRNA and protein were expressed, whereas their amounts in cultured cells showed the reverse order because of more rapid decline of Cyp2b9 in culture. Dexamethasone at 10^-6 or 10^-7 M induced the mRNA. Without dexamethasone addition of phenobarbital scarcely induced it. Although expression of rat C YP2B1/2, orthologous to mouse Cyp2b9/10, is higher in male, female mouse expresses them higher. Beta-estradiol at 10^-5 or 10^-6 M induced the Cyp2b9/10 and the induced mRNA amounts corresponded to those by 10^-3 M phenobarbital. Estrone also showed an inducing potency at 10^-6 M. These observations indicate that constitutive expression of mouse Cyp2b9/10 may be regulated by glucocorticoid and female hormones.

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Propranolol was converted by rat liver microsomes (Ms) into primary metabolites including 4-hydroxypropranolol (4-OH-PL). Preincubation of 4-OH-PL with Ms and NADPH caused inhibition of the ring hydroxylase activities of PL catalyzed by the CYP2D subfamily. 4-OH-PL eliminated quickly during incubation with Ms and NADPH. The 4-OH-PL elimination was changed not by inhibitors of CYP and FAD-monooxygenase or hydroxyl radical scavengers, but by ascorbic acid (1 mM). The microsomal 4-OH-PL elimination was markedly suppressed by cytosol, and the suppressive effect was blocked by KCN (5 mM). Purified Cu, Zn-SOD well mimicked the cytosolic effect, indicating that superoxide is resposible for microsomal 4-OH-PL elimination. 1, 4-Naphthoquinone (1, 4-NQ) was identified as a metabolite of 4-OH-PL in xanthine-xanthine oxidase and microsomal systems. Binding study using 3-³H-4-OH-PL and 4-OH-PL having ¹⁴C at the naphthalene ring showed that during incubation with Ms and NADPH, 3-5% of ³H and 20% of ¹⁴C-labeled substrates consumed covalently bound to microsomal protein. These results suggest that 4-OH-PL is converted into 1, 4-NQ by superoxide derived from CYP or fp₂, and the quinone metabolite binds to microsomal protein including CYP isozymes, indicating a possible mechanism in the inactivation of CYP2D subfamily.

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The purpose of the present study was to elucidate the mechanism of ALoxidation by cytochrome P-450 (P450). The N-dealkylation, ALoxygenation and reduction rates for dialkylarylamines and their NLoxide substrates, and kinetic hydrogen isotope effects for N-demethylation of N, N dimethylaniline (DMA) and N-oxidation of 1, 4-dihydropyridine were investigated. P450 was demonstrated to catalyze the formation of small amounts of N-oxide. The ratio of N-dealkylation:N-oxygenation varied from 1020 to 6. The slow decomposition of N-oxide was also observed by P450 and appears to occur via homolytic N-O bond scission. Low kinetic deuterium and tritium isotope effects were observed for DMA N-demethylation catalyzed by P450 and chloroperoxidase; high isotope effects were observed for peroxidases and hemoglobin. With all of these enzymes low isotope effects were observed for dehydrogenation of two 1, 4-dihydropyridines, where the aminium radicals have low pKas. It is concluded that aminium radicals are a branch point in Noxygenation and N-dealkylation reactions catalyzed by P450, and P450s use specific base catalysis (by the FeO²⁺ entity) to remove protons from aminium radicals, in contrast to the other hemeproteins such as horse radish peroxidase.

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Purification and characterization of the double bond reductases in mammalian species, bacteria and fish was performed. The NADPH- and NADH-linked double bond reductase activities towards 15-ketoprostaglandin F_1α, F_2α E₂, and E₂ were observed in rat liver cytosol. Five kinds of 15-ketoprostaglandin Δ¹³-reductases were isolated and purified from rat liver by chromatography on columns of DEAE-cellulose, hydroxyapatite, Blue-toyopearl and phenyl-toyopearl. These enzyme activities were not influence by oxygen, but inhibited by dicumarol and quercitrin. Some of these enzyme exhibited the double bond reductase activities towards benzylideneacetone and phenyl propenyl ketone. However, the double bonds of prostaglandin E₁, stilbene and testosterone were not reduced by these enzymes. Salmonella typhimurium exhibited a significant double bond reductase activity toward phenyl propenyl ketone. NAD(P)H-linked and NADPH-linked double bond reductase were isolated from cell-free extract of Salmonella typhimurium. NAD(P)H-linked enzyme was purified from the cell-free extract by chromatography on columns of DEAE-cellulose, hydroxyapatite and Phenyl-toyopearl. The enzyme activity was also oxygen-insensitive, but susceptible to inhibited by dicumarol. 15-Ketoprostaglandins were also reduced with the enzyme. Three double bond reductase activities toward phenyl propenyl ketone were isolated from sea-bream liver cytosol by DEAE-cellulose column chromatography. Thus, a variety of double bond reductases were found in mammalian species, fish and bacteria.

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Two new theta class glutathione S-transferases (GSTs), designated Yrs-Yrs' and Yrs'-Yrs', were isolated from rat liver cytosol and purified to homogeneity. Polyclonal antibody raised against the previously reported theta class GST Yrs-Yrs cross-reacted with GSTs Yrs-Yrs' and Yrs'-Yrs'. These three theta class GSTs had different pI values and were separated by chromatofocusing. The enzyme subunit Yrs' was identical to Yrs in the first 37 N-terminal amino acid sequence and electrophoretic mobility, but separable from Yrs on a hydrophobic column by reverse partition HPLC. Like GST Yrs-Yrs, both GSTs Yrs-Yrs' and Yrs'-Yrs' were not retained on an S-hexyl-GSH affinity column and had little activity toward 1-chloro-2, 4-dinitrobenzene. However, they showed potent activities toward the reactive sulfate ester of the carcinogen, 5-hydroxymethylchrysene. GST Yrs-Yrs showed much higher GSH peroxidase activities toward hydroperoxides of endogenous polyunsaturated fatty acids than did rat liver alpha class GSTs. GST Yrs'-Yrs' showed a much higher activity only toward the arachidonate hydroperoxide than did alpha class GSTs. The linolate and linolenate hydroperoxides were poorer substrates for GST Yrs'-Yrs' than for the alpha class GSTs. The above fact strongly suggests that the theta class GSTs may play an important role in retarding the oxidative stress induced by hydroperoxides of polyunsaturated fatty acids derived by pospholipase A2 from phospholipid hydroperoxides formed in hepatic intracellular membranes.

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Four forms of naloxone reductase (NR1, NR2, NR3 and NR4) have been purified to homogeneous proteins on SDS-PAGE from the cytosolic fraction of rabbit liver. All the isozymes are monomeric proteins with molecular weights of 36, 000 -38, 000. NR3 and NR4 exhibited highly restricted cofactor specificity for NADPH and other two isozymes utilyzed NADPH as prefered cofactor over NADH. All the isozymes catalyzed stereospecific reduction of naloxone; NRI formed 6α-naloxol and other three isozymes formed 6β-naloxol. In addition to naloxone, naloxone analogues which have a non-alkylated 3-hydroxyl group such as naltrexone, dihydromorphinone and morphinone were efficiently reduced by NR2, NR3 and NR4 but were poor or inactive substrates for NR1. Other than naloxone analogues, a variety of carbonyl compounds such as DL-glyceraldehyde, p-nitrobenzaldehyde, p-nitroacetophenone and phenanthrenequinone including oxosteroids were good substrates for all isozymes. Pyrazole and barbital had weak or no inhibitory effects on isozymes but quercetin acted as potent inhibitor. These findings indicate that the present enzymes satisfy most of the physicochemical and biochemical criteria expected as carbonyl reductase.

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Carbonyl reductases were purified from the cytosolic fraction of rabbit liver, kidney and heart by using acetohexamide as a substrate. The enzymes purified from the liver and kidney reduced many drugs with a ketone group. However, the enzyme purified from the heart had no ability to reduced drugs with a ketone group other than acetohexamide. A correlation was observed between the values of the specificity constant (kcat / Km) of the enzymes purified from the liver and kidney for 4-acetylpyridine analogs substituted by straight-chain alkyl groups and their partition coefficients. These results indicate that the hydrophobicity of 4-acetylpyridine analogs plays an inportant role in the catalytic activity and substrate-binding capacity of these enzymes. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibited the enzyme purified from the kidney noncompetitively with respect to acetohexamide and competitively with respect to NADPH. It is reasonable to postulate that NSAIDs cause the inhibition of acetohexamide reduction by competing with NADPH.

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Dihydrodiol dehydrogenase (DD) is involved in both the metabolic inactivation and intoxication of aromatic hydrocarbons. Human liver contains four DDs, DD1-DD4, of which DD3 was identified with aldehyde reductase. In order to further evaluate the nature of the other enzymes, we performed the molecular cloning of the two enzymes, DD2 and DD4, and examined the reactivity towards endogenous and xenobiotic compounds. cDNA cloning of the enzymes demonstrated significant sequence homology to members of the aldo-keto reductase family. The sequences of DD2 and DD4, having 82% identity, were virtually identical with those of human liver bile-acid binder and chlordecone reductase, respectively. The results of substrate specificity for endogenous compounds indicated that DD1, DD2 and DD4 act as 3(20)α or 3α-hydroxysteroid dehydrogenases, and suggested that DD1 and DD2 are also the major species of prostaglandin D2 11-ketoreductase in human liver cytosol. On the other hand, the DDs catalyzed the reduction of several drug ketones more efficiently than did aldehyde reductase and carbonyl reductase of human liver.

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