研究業績2003

薬品部第一室研究業績　2003.5-2004.4

誌上発表

Effects of sodium tetraborate and boric acid on nonisothermal mannitol crystallization in frozen solutions and freeze-dried solids. Izutsu, K., Ocheda, S.O.*, Aoyagi, N., Kojima, S. Int. J. Pharm., 273: 85-93 (2004): The purpose of the present study was to elucidate the effects of sodium tetraborate (borax) and boric acid on the crystallization of mannitol in frozen aqueous solutions and freeze-dried solids. Thermal analysis of frozen solutions showed that sodium tetraborate inhibits mannitol crystallization at sodium tetraborate/mannitol molar concentration ratios of approximately 0.05, which is much lower than the other co-solutes studied (boric acid, sucrose, sodium phosphate buffer). Inhibition of the mannitol crystallization in frozen solutions resulted in highly amorphous mannitol in the freeze-dried solids. Mannitol remained in an amorphous state in some of the combination freeze-dried solids, even at elevated temperatures. Changes in the thermal transition temperatures (glass transition temperature of maximally freeze-concentrated solute (Tg') and glass transition temperature of freeze-dried solid (Tg)) suggested reduced mannitol molecular mobility with increases in the sodium tetraborate ratio. Fourier transform infrared spectroscopy (FT-IR) analysis of the bovine serum albumin secondary structure showed apparent protein structure-stabilizing effects of the amorphous mannitol and sodium tetraborate combination during the freeze-drying process. The mannitol and sodium tetraborate combination also protected lactate dehydrogenase from inactivation during freeze-drying. We conclude that the complex formation and the accompanying reduction in molecular mobility make sodium tetraborate an effective mannitol crystallization inhibitor in frozen solutions and freeze-dried solids.
Protection of protein secondary structure by saccharides of different molecular weights during freeze-drying. Izutsu, K, Aoyagi, N., Kojima S. Chem. Pharm. Bull., 52: 199-203 (2004): The protective effects of saccharides with various molecular weights (glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltoheptaose, dextran 1,060, dextran 4,900, and dextran 10,200) against lyophilization-induced structural perturbation of model proteins (BSA, ovalbumin) were studied. FT-IR analysis of the proteins in initial solutions and freeze-dried solids indicated that maltose conferred the greatest protection against secondary structural change. The structure-stabilizing effect of maltooligosaccharides decreased in increasing the number of saccharide units. Larger molecules of dextran also showed a smaller structure-stabilizing effect. Increasing the effective saccharide molecular size by a borate-saccharide complexation reduced the protein structure-stabilizing effect of all of the saccharides except glucose. The results indicate that the larger saccharide molecules, and/or the complex formation with borate ion, reduce the free and accessible hydroxyl groups to interact with and stabilize the protein structure by a water-substitution mechanism.
Abnormal dissolutions of chlorpromazine hydrochloride tablets in water by paddle method under a high agitation condition Aoyagi. N., Rimando. A. P., Izutsu. K., Katori. N., Kojima. S.: Chem.Pharm.Bull., 51, 1021-1024 (2003).: All sugar-coated tablets of chlorpromazine hydrochloride except for those produced by one manufacture showed concave dissolution profiles in water by paddle method at 100 rpm but not at 50 rpm. The study was undertaken to clarify the agitation-dependent abnormal dissolutions. The strange dissolutions were also observed in water at different ionic strengths but not in buffer solutions of pH1.2, 4.0 and 6.8. When monitored, the pH’s of water in dissolution vessels for the abnormal tablets increased with time at 100 rpm and some of them exceeded pH 8 but did not at 50 rpm. The solubility of chlorpromazine hydrochloride decreased with the increase of pH which was too low to dissolve the whole amount of drug contained in a tablet at pH 8. The elevation of pH seemed to be mainly brought about by dissolution of calcium carbonate popularly used for sugar-coated tablets, because larger amount of calcium ion was dissolved out from the abnormal tablets at 100 rpm than from a normal tablet and from them at 50 rpm. These findings indicate that the concave dissolution profiles should be caused by the decrease of drug solubility with increase in pH of water, probably because of dissolution of calcium carbonate. We should pay attention to the change in pH of water which may differ depending on the agitation speed of dissolution tests.
Effect of sodium tetraborate (borax) on the thermal properties of frozen aqueous sugar and polyol solutions. Izutsu, K., Rimando, A.*, Aoyagi, N., Kojima, S.: Chem. Pharm. Bull.，51, 63-66 (2003): The effect of sodium tetraborate (Na2B4O7, borax) on the thermal property of frozen aqueous sugar and polyol solutions was studied through thermal analysis. Addition of borax raised the thermal transition temperature (glass transition temperature of maximally freeze-concentrated solutes; Tg´) of frozen sucrose solutions depending on the borax/sucrose concentration ratios. Changes in the Tg´ of frozen mono- and disaccharide solutions suggested various forms of complexes, including those of a borate ion and two saccharide molecules. Borax exerted the maximum effect to raise the oligosaccharide and dextran Tg´s at borax/saccharide molar ratios of approximately 1-2 (maltose and maltooligosaccharides), 2 (dextran 1,060), 5 (dextran 4,900), and 10 (dextran 10,200). Further addition of borax lowered Tg´s of the saccharide solutions. Borax also raised Tg and Tg´ temperatures of frozen aqueous glycerol solutions. The decreased solute mobility in frozen solutions by the borate-polyol complexes suggested higher collapse temperature in the freeze-drying process and improved stability of biological systems in frozen solutions.
UGT1A1 Haplotypes associated with reduced glucuronidation and increased serum bilirubin in irinotecan-administered Japanese cancer patients. K. Sai, M. Saeki, Y. Saito, S. Ozawa, N. Katori, H. Jinno, R. Hasegawa, N. Kaniwa, J. Sawada, K. Komamura*4, K. Ueno*4, S. Kamakura*4, M. Kitakaze*4, Y. Kitamura*2, N. Kamatani*2, H. Minami*3, A. Ohtsu*3, K. Shirao*3, T. Yoshida*3, and N. Saijo *3: Clin. Pharmacol. Ther. ,75, 501-515 (2004): Purpose: A comprehensive haplotype analysis of UGT1A1 in the Japanese population was conducted, and the effects of these haplotypes were investigated with respect to UGT1A1-related phenotypic parameters in patients with cancer who received irinotecan.Methods: The UGT1A1 gene, including the enhancer, the promoter, and all 5 exons and their flanking regions, was sequenced from 195 Japanese subjects. The gene was divided into 2 blocks, and the haplotypes of each block were assigned. The association of these haplotypes with area under the concentration-time curve (AUC) ratios (7-ethyl-10-hydroxycamptothecin glucuronide [SN-38G]/7-ethyl-10-hydroxycamptothecin [SN-38]) and pretreatment levels of serum total bilirubin was investigated in 85 cancer patients who received irinotecan.
Results: Four haplotype groups (*1, *60, *28, and *6) were assigned in block 1, and 2 haplotype groups (*IA and *IB) were in block 2. The majority of the *IB haplotypes in block 2 were linked to either the *1 or the *60 haplotype but not to *28 in block 1. Highly significant associations were obtained between the *28 haplotypes and both a reduced AUC ratio (P=0.0014, Jonckheere-Terpstra [JT] test) and an increased total bilirubin level (P=0.0007, JT test). Increased total bilirubin levels in the *60 (P=0.0048, JT test) and *IB groups (P=0.0224, JT test) were also observed. The reduction in the AUC ratio by the *6 group was moderate (P=0.0372, JT test) but was remarkable in combination with *60 (*6/*60) or *28 (*6/*28) as compared with the *1 group (*1/*1) (P=0.049 and P=0.0071, respectively; nonparametric Dunnett test). Conclusion: This study identified several UGT1A1 haplotypes significantly associated with the reduced AUC ratio (*28 and *6) and with the increased total bilirubin level (*28, *60, and *IB) and suggested that the novel haplotype *IB might be functionally important. These findings will be useful for further pharmacogenetic studies on adverse reactions to irinotecan. (Clin Pharmacol Ther 2004;75:501-15.)
ホウ素-ポリオール錯体凍結水溶液の物性と凍結乾燥時のタンパク質安定化作用 伊豆津健一　青柳伸男　小嶋茂雄 低温生物工学会誌 49: 115-118 (2003).: タンパク質医薬品も製剤モデルを用い、物性とタンパク質二次構造に対するホウ砂（４ホウ酸ナトリウム）の影響を検討した。二糖類とホウ砂の錯体形成は凍結溶液の凍結乾燥品ガラス転移温度の上昇など分子運動抑制作用を示す一方で、糖によるタンパク質二次構造安定化作用を抑制する事が明らかとなった。