Recent findings that D-amino acids, especially D-aspartic acid and D-serine, exist in vivo in the mammalian tissues (brain and peripheries), prompted us now to investigate their biological and pathological roles in mammals. In this review, the overview of the progress of analytical chemistry and biochemistry of D-amino acids is described.

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Derivatization reactions to enhance the sensitivity and selectivity of gas and high-performance liquid chromatographic detection of biologically important substances are reviewed, with emphasis on the papers reported in the last decade. The achiral and chiral reagents for various functional groups, such as amino, carboxy, thiol, carbonyl and hydroxy groups, include phthalimidyl, quinolyl, quinoxalyl, benzoxazolyl and benzoxadiazolyl analogous having a reacting group to make a covalent bond with analytes. The derivatization methods for increasing sensitivity in liquid chromatography combined with mass spectrometry are also discussed.

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In tryptophan metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been reported to show a carcinogenic action to mice bladder and the relation of the metabolites to human bladder cancer has been discussed. We developed methods for the fluorometric assay of these compounds and showed that the excretion of 3-hydroxyanthranilic acid increased in the patients with bladder cancer. We also devised methods for the fluorometric assay of glucuronide and sulfate of 3-hydroxyanthranilic acid and showed that the minor excretion of these conjugated forms was shown in humans. The distribution of these compounds was also studied and the obtained data suggests that 3-hydroxykynurenine has affinity for the pancreas. We then developed methods for the determination of other metabolites of tryptophan. A fluorescence reaction with UV irradiation was found and applied to the determination. This method is the most sensitive to kynurenic acid but can be applied to kynurenine, nicotinamide and quinolinic acid. Furthermore, this methods also applied to the determination of some medicines, e.g. indomethacin, isoniazid, nalidixic acid, nicorandil and disodium cromoglicate in the serum or urme. We further devised other methods for the determination of xanthurenic acid and 5-hydroxyindoles.

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Selenium is an essential trace element and a toxicant for animals. Seleno-cystine, a selenium-containing amino acid, is one of the chemical forms in which selenium exists in food. This review summarized recent studies on the toxicity mechanism of seleno-cystine in experimental animals. Hepatotoxicity is caused by repeated oral administration of selenocystine. Selenocystine is metabolized by reduced glutathione and/or glutathione reductase to hydrogen selenide via seleno-cysteine-glutathione selenenyl sulfide. The hydrogen selenide is a key intermediate in the selenium methylation metabolism of inorganic and organic selenium compounds. Accumulation of the hydrogen selenide resulting from inhibition of the selenium methylation metabolism, detoxification metabolic pathway of selenium, is found in animals following repeated administration of a toxic dose of selenocystine. The excess of the hydrogen selenide produced by inhibition of the selenium methylation metabolism contributes to the hepatotoxicity caused by selenocystine.

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Guanidino compounds are known as uremic toxins which increase in the blood of patients with renal failure. Guanidino succinic acid (GSA) and methyl guanidine (MG) have been intensively studied since they are toxic and are candidate markers which reflect the pathological stage of nephritis. GSA correlates well with blood urea nitrogen and therefore indicates the reduction of renal function. MG does not appear in the early stage of renal failure and abruptly increases at the stage of serious uremia. MG is produced by the oxidation of creatinine (CTN) with active oxygen. The MG/CTN ratio in the serum therefore reflects the degree of the generation of active oxygen. Accordingly, active oxygen scavengers may be useful for the treatment of uremia.

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The term neurosteroids applies to those steroids that are both synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors, and that accumulate in the nervous system to levels that are at least in part independent of steroidogenic gland secretion rates. Neurosteroids consist of 17-or 20-oxosteroids and accumulate in the brain as unconjugated form and their sulfates, fatty acid esters and sulfolipids. The characterization and determination of neurosteroids including conjugates in the brain are summarized in this review. For example, the separation and characterization of 3-fatty acid esters (stearate, palmitate) of pregnenolone and dehydroepiandrosterone in the rat brain are carried out using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) operating in the positive-ion mode. The fatty acid esters obtained from the rat brain were derivatized with O-methylhydroxylamine to give the respective methyloximes, which were identified in comparison with their chromatographic behavior with authentic samples during LC/APCI-MS. The function of the steroids are also briefly described.

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This brief article is dedicated to the late Professor Morizo Ishidate, and concerned in a methodology developed by the author and collaborators, aiming at the regulation of polyamine, especially, the regulation of spermidine synthase. The content is separated in 3 sections. The first section on the development of analytical methods, contains seven items, e.g. fluorometric determination of polyamines by fluorescamine, analysis of naturally occurring polyamines by GC and GC-MS, etc. The second section on the syntheses of needed compounds, contains three items, e.g. syntheses of decarboxylated S-adenosylmethionine which is a substrate for spermidine synthase and its related compounds, syntheses of ¹5N-enriched polyamines applicable to the preparation of various polyamines, etc. The last section on the studies of aminopropyl transferases, contains three items, e.g. purification of spermidine synthase from mammalian tissues using ATPA-Sepharose, a novel affinity carrier, newly developed inhibitors for the enzymes, etc.

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In a study to solve a mystery of venom toxicity of the joro spider, Nephila clavata, we purified and identified novel spider toxins such as clavamine, spidamine and joramine. Chemical analyses, bioassays and physical analyses were specifically elaborated in these procedures. The structure-activity relationship of the spider toxins was discussed biologically and chemically in comparison with the other spider toxins. We considered that the diversity of the joro spider toxins by reserving 2, 4-dihydroxyphenylacetyl-L-asparaginylcadaverine as a common moiety gave rise to an important insight into not only the toxic reaction, but also the ontology.

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Spiders belonging to the genera, Nephila, Nephilengys, Araneus, etc., posses various polyamine toxins in their venom glands which paralyze insects by blocking the nerve-muscle signal transduction of glutaminergic synapses. More than 50 kinds of polyamine toxin analogs have been characterized which consist, in general, of the aromatic or heteroaromatic moiety connected with the combination of various types of polyamine chains. We are developing a new analytical system by means of a modern mass spectrometric technique which satisfies more rapid, highly sensitive and simple clean-up procedure without complicated chromatographic treatment. The trial of such approach has been performed by employing crude spider venom as the model material. This review article concerns with such proceeding of mass spectrometric analysis by microcolumn HPLC hyphenated FAB-MS system, continuous flow FAB-MS/MS system with high energy collision charge-remote fragmentation, solid and liquid matrix assisted laser desorption ionization (MALDI-and liquid MALDI-) technique connected with tandem mass spectrometer performing in the institute and in addition, femto-molar characterization of new spider venom spider polyamine toxins as a result of the application of such new techniques is addressed.

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Sulfation is an important conjugation reaction in the metabolism of a diversity of xenobiotics and endogenous compounds such as drugs, food additives, carcinogens, steroids and neurotransmitters. Sulfate conjugation is catalyzed by various kinds of sulfotransferase (ST) such as hydroxysteroid ST (HS-ST), phenol ST (P-ST) and estrogen ST (E-ST) present in cytosols. Our laboratory has been studying the multiplicity of rat and mouse STs. We found that tertiary amines such as triethylamine selectively inhibited rat hepatic HS-ST. Developmental changes and zonal distributions of rat liver HS-ST and P-ST isoenzymes provided evidence for their complex regulatory mechanisms. Studies on site-directed mutagenesis and chimeras of rat HS-ST cDNAs, ST-40 and ST-20, revealed the importance of the C-terminal region for the substrate specificity and involvement of multiple regions for the enzyme activities. These two cDNAs have been mapped to the same chromosomal region 1q21.3→q22.1 by fluorescence in situ hybridization. Mouse olfactory P-ST was present in the cytoplasm of olfactory sustentacular cells and its cloning study revealed that it is 94% identical with rat ST1C1 in amino acid sequences.

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Research on the kallikrein-kinin system started from the discovery of urinary hypotensive substance in Germany around 1940-1950. Since then, numbers of researchers have explored this field including related inhibitors, enzymes and autacoids, from all over the world. Components of the kallikrein-kinin system have been analysed extensively, especially since the epoch of the discovery of the deficient patients in these components. Recent progress of gene techniques also enhanced the progress of the study in the kallikrein-kinin field. In this review I discuss about 1) Components and the difference of plasma kallikrein system and glandular kallikrein system ; 2) Impact of the discovery of the deficient plasma in Factor XII, prekallikrein, kininogens as well as in C1INH, and consequent knowledge from the studies of these deficients ; 3) Biological roles of the kallikrein-kinin system ; and recent topics in this field.

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Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA and have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of TΨC-loop in P-site tRNA base pair exist along with the conventional base pairs of adjacent codon-anticodon interactions. This A-P tRNA pair model can be translocated to the P-E tRNA model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain. On the other hand, it is noteworthy that C1378 of E. coli 16S rRNA cross-links to the 32 position on the anticodon loop of A-site tRNA in the pre-translocational state, and also to the same position of E-site tRNA in the post-translocational state, instead of the corresponding position of P-site tRNA. It resulted in a relationship between the A-P and P-E tRNA docking pair models in the pre- and post-translocational states, respectively, caused by a rotation with the angle of 25° around the axis of rotation symmetry. Furthermore, nucleotide sequence analysis showed that CGAGC1107 of 16S rRNA is complementary with the conserved GTΨCG57 of tRNA. When it is combined with the P-E tRNA pair model, the crystallograp-hically obtained L-shaped tRNA model fits both A-site codon with base pairings and the free TΨC-loop of P-site tRNA without base pairings. The base pairings between the GTΨCG57 of tRNA and the CGAGC1107 of 16S rRNA destabilize the bound aminoacyl-tRNA and result in a flow of discarding noncognate and near-cognate ternary complexes until cognate one arrives at the A-site codon. Recognition of a cognate ternary complex could occur, starting from breaking a hydrogen bond between N3 atom of U33 and O5' atom of A36 in the aminoacyl-tRNA, with base pairings of the codon-anticodon interactions, the conserved A 1394 in the 16S rRNA to the conserved U33 in the anticodon loop of the tRNA. The exposed U33 of the aminoacyl-tRNA is paired with A1394 in the recognitionmode of A site, and finally passed to A1398 of A-site tRNA in the A-P tRNA pair model of the pre-translocational state. The exposure of U33 base at the A site is a key event in the mechanism of codon recognition.

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Differences in color and molecular structure between phthalein-pigments and sulfonphthalein-pigments were investigated using X-ray crystallography and absorption spectrophotometry of their aqueous solutions. The molecular structure of sulfonefluorescein (H₂⁺SF^- ) was determined as a zwitter ion, 2-(3-hydroxonio-6-hydroxy-3H-xanthen-9-yl) benzenesulfonate. The absorption spectra of H₂⁺SF^- demonstrated the dissociation profile of a dibasic acid, while those of fluorescein (H₂FL) indicated a tribasic acid and further, at pH>10, SF^2- and FL^2-, while at pH<0, H₂⁺SF^- and H₃⁺FL to be dominant. The spectra of H₂⁺SF^- were analyzed to obtain the values of pK₁ and pK₂ together with the spectrum of HSF^- . Similarly, from the spectra of H₃⁺FL the values of pK₁, pK₂ and pK₃ together with the spectra of HFL^- and H₂FL were obtained. Further by adding 1/5 of the H₃⁺FL spectrum to 2/15 of HFL^- spectrum, an indicated spectrum as that of H₂FL was obtained. From these results, the features of dissociation of H₂SF^- and H₃⁺FL were estimated. The molecular structure of phenolsulfonphtalein (H₂PS^-) was determined as a zwitter-ion, α-(4-hydroxonio-2, 5-cyclohexadiene-1-ylidene)-α-(4-hydroxy-phenyl)-2-toluenesulfonate. The absorption spectra of H₂PS^- demonstrated that H₂PS^-, HPS^- and PS^2- became dominant at pH«0, pH=4 and pH>11, respectively. On the contrary, phenolphthalein (H₂PP) displayed only one type of absorption spectrum in the visual region, the shape of which was similar to that of PS^2- while the molar extinction coefficient was smaller. The spectra were analyzed to obtain the valus of pK₁=9.05 and pK₂=9.5O. The spectra also demonstrated a slow addition reaction of OH^- to PP^2-= and pK₃= 12 was obtained by measuring the absorbance after equilibration. From these results, the features of dissociation and coloration of H₂⁺PS^- and H₂PP were estimated.

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In 1939 the author reported the results of measured refractive indices of about a hundred crystalline drugs listed in [JP V] at the Takeda Reseach Laboratory using a Leitz PM polarizing microscope and newly developed immersion oils. When the author had reopened the study of crystalline drugs using a polarizing microscope at the Kobe-Gakuin University starting from 1975 one of the main purposes was to clarify the relation between crystal habits and refractive indices. It had been found that in most cases of crystal habits refractive indices were uniquely measured from a predominant pair of faces forming superior the habit, and they were called as "key refractive indices". The author and his co-workers tried to investigate the possibility of measuring the key refractive indices widely from all the obtainable crystalline drugs listed in the [JP X] or [JP XI], co-operating with the Pharmacy of Kobe University Hospital. Thus, more than 170 kinds of crystalline drugs were tested for their key refractive indices and found that they were measured from about 60-70% of tested drugs. It was also clarified that the difference of 2 key refractive indices, (n₂-n₁), the birefringence of the section, was also an unique invariable number for the habit, and it played an important role not only for the graphic representation of log(n₂-n₁), abscissa, against (n₁, n₂), ordinate, for the sake of an analytical purpose but also to measure a thickness of a section (habit) using a retardation color. Then, it had been cleared that the similarity of crystal habits in the microscopic field was based on the facts of measuring the same key refractive indices, and the author had developed a chart for measuring key refractive indices as well as producing a 3 dimensional orthographic projection of a crystal habit simultaneously applying a thickness measuring method using a birefringence. Finally 3 dimensional parameter a, b, c of a crystal habit and "habit coefficients" T : √(ab)/c and L : b/c were determined from the orthographic projection. In conclusion using the similarity in crystal habits the distributions of particle sizes and specific surface areas of all the crystals in the microscopic field had been calculated by a personal computer putting in necessary habit coefficients and obtained data of parameter b. The relation between 2 dispersions of perticle sizes in log (V) and specific surface areas in log (SSA) were shown under the rectangular coordinates log (V) on the abscissa and log (SSA) on the ordinate, where the loci of log (SSA) formed simple striped pattern composed of parallel straight lines depending on habit coefficients. It would be possible to estimate the value of a specific surface area of any crystalline substance by plotting the value of log (V) on the straight line of a locus of log (SSA) having the same habit coefficients.

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The Mitsunobu reaction of commercial 4-hydroxycoumarin with both (R)-(-)- and (S)-(+)-tert-butyl mandelates derived from commercial enantiopure mandelic acids furnished (S)-(+)- and (R)-(-)-tert-butyl O-coumarinylman-delates which were, then, treated with trifluoroacetic acid to give novel crystal-line optically pure (S)-(+)- and (R)-(-)-O-coumarinylmandelic acids [SCMOH and RCMOH], respectively in good overall yields. Diastereotopic nonequivalence ¹H-NMR examination of the resultant esters and amides without any racemization or kinetic resolution by way of a Steglich's procedure (a DCC-DMAP method) has proved each acid to be a useful, efficient and reliable chiral derivatizing agent for the enantiomeric excess determination of chiral alcohols and amines.

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Studies have been made to design chiral bidentate lithium amides and chiral tetradentate amines, and to explore the use of these chiral bases for enantioselective formation and reactions of lithium enolates. Chiral bidentate lithium amides having a chiral amide nitrogen made by virtue of chelation were successfully applied to the enantioselective deprotonation reaction of prochiral cyclic ketones, the kinetic resolution of racemic cyclohexanone derivatives by deprotonation, and the regioselective deprotonation of optically active 3-keto steroids. Structures of some of these chiral bidentate lithium amides in the solid state and in solution were elucidated by X-ray and NMR spectroscopic analyses. By the use of chiral tetradentate amines, enantioselective reactions of lithium enolates with electrophiles, such as alkylation, protonation, and Michael addition, proceeded successfully. Examples of catalytic enantioselective deprotonation, alkylation, and protonation by the present strategy are also presented and discussed.

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We developed a new method for high performance liquid chromatography (HPLC) using packing materials modified with a temperature responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). A homogeneous PNIPAAm polymer and its copolymer with butyl methacrylate (BMA) were synthesized and grafted to aminopropyl silica by activated ester-amine coupling and they were used as packing materials. These grafted silica surfaces show hydrophilic properties at lower temperatures and, as temperature increases, transform to hydrophobic surface properties. These features are due to rapid changes in the polymer conformation that is attributed to the mobility of grafted polymers. Using the column packed with PIPAAm-modified silica, separation of steroids was carried out by changing temperature. With increasing temperature, the interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases increased. The temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The separation selectivity and retention are controlled by a small change in column temperatures without any change in the eluent. The separation of the mixture of three peptides, insulin chains A and B and a β-endorphin fragment 1-27, was achieved by changing column temperature with a NaCl aqueous solution as a sole eluent. A gradient elution-like effect in HPLC is demonstrated with a single mobile phase by controlling external temperature. The retention times of peptides and proteins were controlled both by column temperatures and by NaCl concentration in the aqueous mobile phases in this chromatographic system.

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It has been known that the stimulation of skin cells by physiologically active substances is accompanied by the quantitative and qualitative alterations of the glycosaminoglycans (GAGs) in skin. This phenomenon make it possible to evaluate the activity of test substances for the stimulation of skin cells. The purpose of our study is to elucidate the effectiveness of inorganic compounds for the stimulation of skin cells. (1) The cultured skin cells including keratinocytes and fibroblasts, (2) the cultured model skin constituted of collagen gel, fibroblasts and keratinocytes, and (3) the mouse damaged skin were used in the present study. The results obtained from these studies demonstrate that inorganic substances commonly have a possibility to activate cyclic AMP-dependent protein kinase (A-kinase), and the A-kinase activation results in an increase in GAGs. These findings brought the scientific basis for the effectiveness of the Japanese traditional balneotherapy for damaged skin.

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General aspects of the deterioration and typical examples in some hot springs (Atami, Shirahama, Isawa·Kasugai, and Syuzenji Hot Springs) are outlined in this report in connection with the change of chemical constituents. (1) Deterioration of thermal spring resources generally occurs accompanied with the following phenomena : a) drawdown of the water level in thermal water wells. b) lowering of temperature of thermal springs. c) change of the water quality (salt water encroachment in the coastal region and decrease in concentration of chemical components resulting from the intrusion of underground water in the inland area).(2) Typical examples of the deterioration are summarized as follows ; [Table] (3) It has been revealed from the analyses of the statistical data of Environment Agency that the deterioration of hot spring resources in Japan is gradually progressimg.

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Survey of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofu-rans has been carried out for four years starting from 1992 to 1995, which showed that about 25% or more of marine products are contaminated by trace amount of dioxins and related compounds. But intake of dioxins and related compounds through fishes and shellfishes was below 5 pg/kg/d. Further monitoring is indispensable.

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The chemiluminescent reaction of lucigenin with various biological substances has been studied. Chemiluminescence of lucigenin is produced by the addition of either hydrogen peroxide or organic reducing compounds to lucigenin in an alkaline solution. On the basis of these reaction, we have developed highly sensitive chemiluminescent methods for the detection of enzyme immunoassay, especially using alkaline phosphatase as a label enzyme, and also for HPLC of corticosteroids or p-nitrophenacyl esters of carboxylic acids. The detection limits of enzymes were 10^-19-10^-20 mol per assay, corticosteroids and p-nitrophenacyl esters were 500 fmol per injection.

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Historically the developmant of new anticancer drugs in Japan was initiated in 1950s by M. Ishidate et al. for alkylating agents and by H. Umezawa, T. Hata et al. for antitumor antibiotics. In current cancer chemotherapy, appreciable clinical responses have been reported against some types of hematological and childhood malignancies and solid cancers such as chorionic, ovarian, testicular cancers. However, solid cancers such as many GI-tract, lung, breast and other cancers are still drug-refractory. Since many of these cancers have been discovered at their advanced forms, the drug treatment has been a very important modality. From these reasons, efforts have been placed on the search of new anticancer drugs. Recently, new anticancer drugs relating to taxanes, camptothecins, platinum compounds have exhibited excellent clinical activities against some solid cancers. From these experiences, searches of novel anticancer drugs including those against molecular targets have been continued. The author is greatly indebted to the late Professor Morizo Ishidate for the initiation of his career in cancer research.

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The metabolic fates of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and N, N-dibutylnitrosamine (DBN) were investigated in the rat and other animal species, to elucidate a possible relationship between metabolism and organotropic carcinogenicity to the urinary bladder of these N-nitrosamines. The principalurinary metabolite of BBN as well as of DBN in the rat was N-butyl-N-(3-carboxypropyl) nitrosamine (BCPN), which was demonstrated to be the active form of these compounds as bladder carcinogen. The species difference in response to BBN or DBN is discussed on the basis of the urinary excretion rate of BCPN. Metabolism in vivo and carcinogenicity of a number of BBN analogues were investigated in the rat and a general scheme for biotransformation of N-alkyl-N-(ω-hydroxyalkyl) nitrosamines is given. A possible correlation of structure and metabolism with organotropic carcinogenicity of BBN analogues is discussed, with special reference to selective induction of urinary bladder tumors.

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Xenobiotic metabolizing enzymes are known to play a role in the metabolic activation of environmental mutagens and carcinogens to exert their carcinogenic effects as well as detoxification by increasing their hydrophilicity. These enzymes include cytochrome P450s, glutathione S-transferases (GSTs), acetyltransferases (NATs) and sulfotransferases. Genetic polymorphisms in many of these enzymes, such as CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP2E1, NAT1, NAT2, GSTM1, GSTP1 and GSTT1, have been shown to occur, which result in the altered expression of enzymatic activities. This suggests that the genetic polymorphisms may affect the individual susceptibility to environmental carcinogens and thus play a role in human carcinogenesis. Recently, the mutations that confer those polymorphisms of xenobiotic metabolizing enzymes have been identified and genotyping methods for the genetic polymorphisms have been developed. Specific phenotypes and genotypes for CYP1A1, CYP2D6, CYP2E1, NAT1, NAT2, GSTM1 and GSTP1 have been associated with susceptibility to malignant diseases including lung, bladder and colon cancers, although the association was not confirmed in some studies. A number of factors such as degree of exposure to environmental carcinogens and the role of xenobiotic metabolizing enzymes in human carcinogenesis should carefully be evaluated in understanding genetic susceptibility.

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In 1993 eighteen Japanese patients with cancer and herpes zoster, a viral disease, died from interactions of the new oral antiviral drug, sorivudine (SRV : 1-β-D-arabinofuranosyl-(E)-5-(2-bromovinyl) uracil), with oral anticancer prodrugs of 5-fluorouracil (5-FU) within 40 d after SRV was approved by the Japanese government and began to be used clinically. Before the death, most of these patients had severe symptoms of toxicity, including diarrhea with bloody flux and marked decreases in white blood cell and platelet counts. All of these patients received SRV daily for several days while being administered long-term anticancer chemotherapy with one of the oral 5-FU prodrugs. There was no acute toxic symptom in patients who received SRV alone or SRV and the other types of anticancer drugs. A toxicokinetic study was carried out using rats to investigate the mechanism of the acute death in the patients caused by drug interactions between SRV and 5-FU prodrugs. Rats were orally coadministered SRV with tegafur (FT : 1-(2-tetrahydrofuryl)-5-fluorouracil), a 5-FU prodrug that most of the patients were considered to receive before the death. All the rats receiving SRV and FT once daily showed extremely elevated levels of 5-FU in the plasma and tissues, including bone marrow and small intestines, and died within 10d, while the animals given the same repeated dose of SRV or FT alone were still alive over 20 d without any appreciable toxic symptom. Before their death, there were a marked damage of bone marrow, a marked atrophy of intestinal membrane mucosa, marked decreases in white blood cells and platelets, diarrhea with bloody flux, and severe anorexia as reported for the patients. Data obtained by in vivo and in vitro studies indicated that (E)-5-(2-bromovinyl) uracil (BVU), generated from SRV by the gut flora and absorbed through the intestinal membrane, was reduced in the presence of NADPH to a reactive form by hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the tissue 5- FU levels from FT, bound covalently to DPD as a suicide inhibitor, and markedly retarded the catabolism of 5-FU. An irreversible inactivation by BVU of rat and human DPDs, expressed in E. coli for the latter, was observed in the

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The doping test method used in a horserace requires the accurate detection of a wide variety of drugs and metabolites as well as the rapidity in order to examine a large number of samples within a limited time. For this purpose, the routine method consists of a preliminary screening and a confirmatory test. In this paper, a historical review for the development of the doping test method in Japan is described. The metabolism and pharrnacology of drugs in horses are also discussed.

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The experimental reproduction of SMON using several kinds of animals given a prolonged administration of chinoform has been carried out by many investigators because of the importance to solve the problem of etiology in the SMON. In these experiments, it is demonstrated that a marked species difference was observed in the relationship between the doses given to animals and the frequency of the onset of neurologic symptoms. Although dogs were accepted as the most suitable animal model for SMON, pathological changes in the peripheral nerve of the dog were not observed. The blood level or tissue distribution of chinoform after oral, intravenous or intraperitoneal administration of the drug differed in the animal species. Thus, it is considered that the species difference in the onset of neurologic symptoms is principally caused by the difference in pharmacokinetics of chinoform in each animal. Moreover, for the onset of neurologic symptoms in animals, perhaps it is necessary to maintain the level of unconjugated chinoform in the nerve tissues around several to over ten μg/ml for three or four weeks as well as that in SMON patients while the neurologic symptoms or pathological changes do not appear in some kinds of animals at these levels. In a study on the cellular toxicity of chinoform, many other problems remain to be solved although degeneration or uncoupling on oxidative phosphorylation in mitochondria of the axons by chinoform and lipid peroxidation of the membrane by chinoform-ferric chelate have been already shown.

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Prof. Morizo Ishidate synthesized "Promin" for the treatment of leprosy/Hansen's disease which had been considered "incurable" until the discovery of antileprosy effect of that drug by Dr. Faget of U.S.A. in 1941. Prof. Ishidate was the first to synthesize the drug in Japan in 1946 based on a brief news item on a Swiss journal smuggled in during the War. For this achievement, he is known as "father of chemotherapy for leprosy in Japan." Prof. Ishidate also contributed to the global fight against leprosy as the Chairman of the Board of Directors of Sasakawa Memorial Health Foundation, which he helped to establish in May 1974, with a full financial backing of Mr. Ryoichi Sasakawa, President of Japan Shipbuilding Industry Foundation. Prof. Ishidate, with his scientific knowledge as well as christianity based humanitarian concern, advised Mr. Sasakawa how to spend JSIF money wisely for eliminating leprosy and nearly US$200 million was channeled through WHO and SMHF. The successful outcome of global multidrug therapy (MDT) programme in the' 80s resulted in the adoptation of resolution by the World Health Assembly, "Elimination of Leprosy, as a public health problem" by the Year 2000. The synthesis of "Promin" in Japan and promoting the global implementation of MDT, both achievement can be attributed to Prof. Ishidate.

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New drug development can be made by providing products of higher "selectivity for the drug" for medical treatment. There are two ways for the approach to get higher "selectivity of drug" : 1) discovery of new compounds with high selectivity of drug ; 2) innovation of new drug administration, that is new formulation and/or method with high selectivity of drug by integration and harmonization of various hard/soft technologies. An extensive increase of biological information and advancement of surrounding science and technology may modify the situation as the latter overcomes the former in the 21 century. As the science and technology in the 21 century is said to be formed on "3H", that is, 1. hybrid ; 2. hi-quality ; 3. husbandry, the new drug development by innovative drug administration is exactly based on the science and technology of 3H. Its characteristic points are interdisciplinary/interfusion, international, of philosophy/ethics, and systems of hard/hard/heart. From these points of view, not only the advance of unit technology but also a revolution in thinking way should be "must" subjects. To organize this type of research well, a total research activity such as ROR (research on research) might take an important and efficient role. Here the key words are the "Optimization technology" and "Change in Pharmaceutical Fields." As some examples of new drug innovation, our trials on several topical mucosal adhesive dosage forms and parenteral administration of peptide drugs such as insulin and erythropoietin will be described.

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