During the past ten years, the experiments based on the following three main propositions were carried out in our laboratory. (1) Drug receptor mechanisms. M3-Cholinoceptors and α1-adrenoceptors could be divided into two subtypes which were discriminated by β-chloroethylamines only in the presence of GTP. The full agonists interacted with both subtypes to induce responses. The partial agonists activated one of them to induce responses but behaved as competitive antagonists when they interacted with the other. The responses mediated through the receptors which were activated by the partial agonists were resistant to myosin light chain kinase inhibitors, while the responses by the activation of the other receptors were suppressed by the inhibitors. The possible mechanisms for responses mediated through α1-adrenoceptors and M3-cholinoceptors were discussed. β-Adrenoceptors had also two binding sites, high and low affinity sites, which could be discriminated by the partial agonists. (2) Effects of ageing on drug receptor mechanisms. Potencies of α- and β-adrenoceptor agonists increased from the yound stage to the adult stage and decreased slowly thereafter to the old stage. The affinities of adrenergic drugs for their receptors did not alter with ageing. The changes in the adrenoceptor mechanisms with ageing were mainly due to the changes in the amount of receptors. However, the decrease in the potency of β-agonists in the preparations from the older animals was due to the change in the post β-receptor processes in responsiveness. No age-related change was observed in serotonin, acetylcholine and tachykinin receptor mechanisms. However, the potencies of acetylcholine and tachykinin were modified by the change in the activity of related enzymes, which altered with ageing. (3) Drug design. Taking into account pharmacological studies on opioid receptors, N-cyclopropylmethyl normorphine derivatives were synthesized. They had more potent analgesic action than morphine through the activation of κ-opioid receptors. They might not possess dependence liability.
An efficient method for the disulfide bond formation in peptides by the silylchloride-sulfoxide system is described. Methyltrichlorosilane in trifluoroacetic acid, in the presence of diphenylsulfoxide, is found to cleave various S-protecting groups of cysteine to form cystine directly within 10 to 30 min. No side reactions were observed with nucleophilic amino acids such as Met, His, or Tyr, except for Trp, under the reaction conditions of the silylchloride-sulfoxide treatment. A chlorination of the indole moiety of unprotected Trp, rather than the sulfur-sulfur bond formation, is a dominant reaction when the peptide containing unprotected Trp is treated with the chlorosilane-sulfoxide. However, the disulfide bond can be formed efficiently with no modification at the indole ring by the treatment of the peptide having formyl-protected Trp residue with the silylchloride-sulfoxide system. The formyl group is removed by a brief treatment at basic pH without affecting the disulfide bond formed by the silylchloridesulfoxide treatment. Total synthesis of human insulin, a two chain peptide containing three disulfide bonds, was achieved unambiguously by sequential and selective formation of disulfide bonds in the protein for the first time. The key reaction in the synthesis is regioselective formation of three disulfide bonds separately using the silyl chloride method described above. Prior to the insulin synthesis, it was confirmed by the syntheses of double-disulfide peptides : b-hANP, unnatural parallel dimer of a-hANP, and human endothelin-1 that no disulfide exchange occurred during the silyl chloride treatment. Using three orthogonal thiol protecting groups, Trt, Acm, and But, three disulfide bonds of human insulin were efficiently constructed by the successive reactions using thiolysis, iodine oxidation, and the silyl chloride method. Each reaction for the stepwise disulfide formation proceeded within 15 to 60 min with no polymeric product and no solubility problem. The synthetic human insulin had the correct structure and was indistinguishable from natural human insulin.
We have performed nutritional and biochemical studies on vitamin D and its active derivatives and the following results are obtained. 1. Since recent studies have revealed that dietary supplement of vitamin D (D2 and D3) and calcium is effective for preventing osteoporosis, a simplified routine method for determination of vitamin D in foods is established and applied to the assay on the contents of vitamin D in various kinds of Japanese foods. 2. A simplified routine method for simultaneous determination of vitamin D and its metabolites in the plasma and milk is established and applied to nutritional and clinical studies. 3. Physiological activities of two kinds of novel vitamin D3 derivatives, 22-oxa-1α, 25-dihydroxyvitamin D3 (22-oxa-1, 25 (OH)2D3, OCT) and 2β-(3-hydroxypropoxy)-1, 25 (OH)2D3 (ED-71) have been studied. OCT, which has less calcemic and stronger cell differentiationon activities than 1, 25 (OH)2D3, is a candidate for curing leukemia and other cancers without hypercalcemia. We have clarified that the property is due to its weak binding affinity for vitamin D binding protein and rapid turn-over in the body and rapid excretion into bile. On the other hand, ED-71, which has stronger effects on intestinal calcium absorption and longer bone turn-over than 1, 25 (OH)2D3, is a candidate for curing osteoporosis. We have clarified that the properties are due to stronger binding affinity for DBP and longer half-life than 1, 25 (OH)2D3.
High-performance liquid chromatography (HPLC) has been used for the assays of pharmaceuticals and their impurities, and drugs and their metabolites in biological fluids. However, HPLC packing materials so far developed are not necessarily suitable for the above mentioned purposes. During the past decade, we developed of new HPLC packing materials for direct serum injection analysis of drugs, and for chiral resolution of drug enantiomers. A sample preparation step such as deproteinization and/or extraction is required for HPLC assays of drugs and their metabolites in biological fluids. Direct serum injection analysis of drugs can be achieved by the use of the packing materials having properties as follows : large molecules such as proteins are eluted in the void volume without destructive accumulation, but small molecules such as drugs and their metabolites can reach the hydrophobic sites and be separated. Such a material is called restricted access stationary phase (RASP). We developed two RASPs, an improved internal-surface reversed-phase (ISRP) material and a mixed functional phase (MFP) material. The preparation methods of the ISRP and MFP materials and their applications to the assays of drugs in the serum are described. HPLC chiral stationary phases based on chiral small molecules, and macromolecules such as polysaccharides and proteins have been used for separations of drug enantiomers. Disadvantages of protein-bonded stationary phases include low capacity, lack of column ruggedness and limited understanding of the chiral recognition mechanism. We tried to overcome the disadvantages of proteinbonded stationary phases by modification of side-chain amino acid and preparation of protein-domain or -fragment column. We isolated each ovomucoid domain and examined chiral recognition ability of each domain. The ovomucoid third domain exhibited chiral recognition for only a few benzodiazepines and 2-arylpropionic acid derivatives. The chiral binding site and mechanism on the ovomucoid third domain was investigated by proton NMR measurements and molecular modeling of the ligand-protein complex. Further, new protein, ovoglycoprotein, was isolated from crude ovomucoid preparations, characterized and bound to silica gels. It was found that the chiral recognition ability of the ovomucoid reported previously came from the ovoglycoprotein, which is present in crude ovomucoid as an impurity.
Novel quinoxaline 1, 4-dioxide derivatives were synthesized from benzofuroxans and the enolic form of 1, 3-diketones or 3-oxoalkanoic esters or 3-oxoalkanamides or butanedioic esters catalyzed by silica gel or molecular sieves and their antibacterial activities were evaluated. As the results of antibacterial screening tests in vitro, quinoxaline 1, 4-dioxides revealed strong activities against Bacteroides fragilis.
In this study, the transport of enoxacin (ENX) was investigated in a LLC-PK1 kidney epithelial cell line. The uptake of ENX by LLC-PK1 monolayers cultured in plastic dishes was shown to be temperature-dependent and concentration-dependent. Cimetidine and guanidine inhibited the uptake of ENX, whereas TEA and NMN did not. The basolateral to apical flux of ENX across LLC-PK1 monolayers cultured on permeable supports was about two times larger than the apical to basolateral flux. The basolateral to apical flux of ENX was remarkably inhibited by guanidine, whereas it was not inhibited by TEA, NMN and cimetidine. The apical to basolateral flux of ENX was inhibited by cimetidine and guanidine, whereas it was not inhibited by TEA and NMN.