Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria-rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo.

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria-rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN3 ), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN3 -treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN3 inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN3 will lead to a molecular understanding of cytoplasmic and cortical reorganization.

Nateglinide controlled release tablet containing compressionable enteric coated granules.

We designed a single unit type controlled release tablet containing nateglinide to decrease both postprandial blood glucose level (PBG) and fasting blood glucose level (FBG) in normal beagle dogs. The tablet contains 60 mg of nateglinide in an immediate release portion, and 90 mg of nateglinide in a controlled release portion. Compressionable enteric coated granules were selected as the controlled release portion to primarily decrease FBG, and they were prepared by an aqueous coating with Eudragit. Three types of nateglinide controlled release tablets were obtained, and their weights were 418.1-425.1 mg/tablet containing the above compressionable enteric coated granules. Even after tableting, the dissolution behavior of enteric coated granules was maintained approximately. In vivo single oral administration studies using normal male beagle dogs demonstrated that these tablets were able to decrease both PBG and FBG. The relative bioavailability values of the obtained tablets containing enteric coated granules having a dissolution pH of 6.0 and 6.8 were estimated at about 57.2 and 60.8% respectively against nateglinide immediate release tablets. In an in vivo repeated administration study with the tablets containing enteric coated granules having a dissolution pH of 6.8 (an interval: 8 h), decreases in both PBG and FBG were observed continuously twice. On the basis of the above results, it is expected to enable control of both PBG and FBG for moderate and severe diabetes patients with a controlled release formulation containing a short-acting type oral blood glucose regulator, not only nateglinide but also meglitinides (repaglinide, mitiglinide, etc.).

Design of nateglinide controlled release tablet containing erosion matrix tablet and multiple administration study in normal beagle dogs.

We designed a single unit type tablet formulation containing nateglinide to decrease both postprandial blood glucose level (PBG) and fasting blood glucose level (FBG) in normal beagle dogs. The tablet was a dry coated tablet comprising both a core tablet (an erosion matrix tablet: a controlled release portion(nateglinide: 90 mg)) and an outer shell (an immediate release portion (nateglinide: 60 mg)). The weight, the diameter and the hardness of the obtained tablet were 416.1 mg, 10 mmpsi, about 60 N, respectively. The dissolution study of the obtained tablet in pH 1.2 or 6.8 showed that the nateglinide in the immediate release portion dissolved in almost 30 min., and that 30 min after the dissolution test started, the nateglinide in the controlled release portion had dissolved slowly. An in vivo single oral administration study using normal beagle dogs showed the bioavailability value of the obtained nateglinide dry coated tablets against nateglinide immediate release tablets was 73.6%, although the value of nateglinide controlled release tablets containing enteric coated granules was 57.2-60.8%. An in vivo multiple oral administration study (b.i.d. (interval: 12 h), 8 d) using normal beagle dogs showed the reproducibility of nateglinide absorption. In addition, decreases in both PBG and FBG were observed. The ability to decrease the blood glucose level did not weaken during a multiple administration. On the basis of the above results, a controlled release formulation containing a short-acting type oral blood glucose regulator, not only nateglinide but meglitinides (repaglinide, mitiglinide, etc.) was suggested to enable control of both PBG and FBG for moderate and severe diabetes patients.

Effect of decrease in both postprandial blood glucose (PBG) and fasting blood glucose (FBG) levels in normal beagle dogs with nateglinide enteric coated granules and immediate release tablets.

Nateglinide is a new quick action/short duration (QRSD) type of oral blood glucose regulator, and nateglinide immediate release tablets are used for patients with mild diabetes under the trade name of Fastic((R)) tablets. In this study, we attempted to determine if it was possible to control both post-prandial blood glucose level (PBG) and fasting blood glucose level (FBG) for moderate or severe diabetes through controlled release of nateglinide. Enteric coated granules were selected for the administration form for controlled release of nateglinide, and three types of enteric coated granules were prepared having dissolution pH values of 5.5, 6.5 and 7.2. The three types of enteric coated granules were each administered separately or the enteric coated granules having an dissolution pH of 6.5 were administered simultaneous to administration of nateglinide immediate release tablets to normal beagle dogs just before feeding followed by measurement of plasma nateglinide concentration, plasma insulin concentration and blood glucose level. In the case of administering enteric coated granules alone (nateglinide: 9 mg/kg), the absorption of nateglinide was confirmed to tend to be delayed as the dissolution pH increased. In the case of an dissolution pH of 5.5, decreases in both PBG and FBG were observed. In the case of dissolution pH values of 6.5 and 7.2, only decrease in FBG was observed. In case of nateglinide immediate release tablets (nateglinide: 9 mg/kg), only decrease in PBG was observed. Decreases in both PBG and FBG were observed in the case of simultaneous administration of dissolution pH 6.5 enteric coated granules and nateglinide immediate release tablets just before feeding (nateglinide: 90 mg/head+60 mg/head). A correlation was observed between plasma nateglinide concentrations and blood glucose levels. On the other hand, there were no correlations observed between changes in plasma insulin concentrations and blood glucose levels. In case of nateglinide immediate release tablets (nateglinide: 150 mg/head), Decreases in both PBG and FBG were observed. However, the nateglinide controlled release formulation is more useful than the nateglinide immediate release tablets from the view point of avoidance of side effect, or of easy control of both PBG and FBG. On the basis of these results, the design of a controlled release formulation that contains nateglinide was suggested to enable control of both PBG and FBG for moderate and severe diabetes patients.