Eiichiro Suzuki

Phase I study of sorafenib in Japanese patients with hepatocellular carcinoma

Sorafenib is an orally active multikinase inhibitor that targets serine and threonine, and tyrosine kinases that are involved in tumor‐cell signal transduction and tumor angiogenesis. This phase I trial was conducted to evaluate the pharmacokinetics (PK), safety, and preliminary efficacy of sorafenib in Japanese patients with hepatocellular carcinoma (HCC) with underlying liver dysfunction. Patients with unresectable HCC, Child–Pugh status A or B, and adequate organ functions were treated. A single dose of sorafenib was administered, followed by a 7‐day wash‐out period, after which patients received either sorafenib 200 mg (cohort 1) or 400 mg (cohort 2) twice daily. The PK were investigated after a single dose and during steady state. The efficacy was evaluated using the Response Evaluation Criteria in Solid Tumors. A total of 27 patients were evaluated for PK, safety, and efficacy. Although both area under the concentration–time curve for 0–12 h and maximal concentration at steady state were slightly lower in Child–Pugh B patients than in Child–Pugh A patients, the difference was not considered to be clinically relevant. Common adverse drug events included elevated lipase, amylase, rash or desquamation, diarrhea, and hand–foot skin reaction. A dose‐limiting toxicity of hand–foot skin reaction was observed in one patient (cohort 2). Among the 24 patients evaluable for tumor response, one patient (4%) achieved a partial response, 20 (83%) had stable disease, and three (13%) had progressive disease. Sorafenib demonstrated a favorable tolerability and safety profile in Japanese HCC patients. Moreover, promising preliminary antitumor activity has been observed. Finally, there were no clinically relevant differences in PK between Child–Pugh A and B patients. (Cancer Sci 2008; 99: 159–165)

Identification and characterization of oxidized human serum albumin

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases. However, little is known regarding the pathophysiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both the structural and functional differences between reduced and oxidized HSA. Using LC‐ESI‐TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide‐bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand‐binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions.

X-ray structures of a novel acid phosphatase from Escherichia blattae and its complex with the transition-state analog molybdate.

The structure of Escherichia blattae non‐specific acid phosphatase (EB‐NSAP) has been determined at 1.9 Å resolution with a bound sulfate marking the phosphate‐binding site. The enzyme is a 150 kDa homohexamer. EB‐NSAP shares a conserved sequence motif not only with several lipid phosphatases and the mammalian glucose‐6‐phosphatases, but also with the vanadium‐containing chloroperoxidase (CPO) of Curvularia inaequalis. Comparison of the crystal structures of EB‐NSAP and CPO reveals striking similarity in the active site structures. In addition, the topology of the EB‐NSAP core shows considerable similarity to the fold of the active site containing part of the monomeric 67 kDa CPO, despite the lack of further sequence identity. These two enzymes are apparently related by divergent evolution. We have also determined the crystal structure of EB‐NSAP complexed with the transition‐state analog molybdate. Structural comparison of the native enzyme and the enzyme–molybdate complex reveals that the side‐chain of His150, a putative catalytic residue, moves toward the molybdate so that it forms a hydrogen bond with the metal oxyanion when the molybdenum forms a covalent bond with NE2 of His189.

Crystal Structure of Red Sea Bream Transglutaminase

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-Å resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.

Recombinant curculin heterodimer exhibits taste-modifying and sweet-tasting activities.

Curculin from Curculigo latifolia is a unique sweet protein that exhibits both sweet‐tasting and taste‐modifying activities. We isolated a gene that encodes a novel protein highly homologous to curculin. Using cDNAs of the previously known curculin (designated as curculin1) and the novel curculin isoform (curculin2), we produced a panel of homodimeric and heterodimeric recombinant curculins by Escherichia coli expression systems. It was revealed that sweet‐tasting and taste‐modifying activities were exhibited solely by the heterodimer of curculin1 and curculin2.

Efficacy of Sorafenib in Intermediate-Stage Hepatocellular Carcinoma Patients Refractory to Transarterial Chemoembolization

Objective: We compared the benefits of sorafenib therapy with continued transarterial chemoembolization (TACE) in TACE-refractory patients with intermediate-stage hepatocellular carcinoma (HCC). Methods: This retrospective study reviewed intermediate-stage HCC patients who underwent the first TACE. Patients were defined as TACE-refractory and divided into two cohorts: (1) patients who switched from TACE to sorafenib and (2) those who continued TACE. We evaluated the patient overall survival (OS) and time to disease progression (TTDP; the time patients reached Child-Pugh C or developed advanced-stage HCC). Results: A total of 509 patients with HCC underwent TACE. Of 249 intermediate-stage HCC patients undergoing the first TACE, 122 were deemed refractory. At the time they were identified as refractory, 20 patients converted to sorafenib, whereas 36 patients continued TACE. We excluded patients with Child-Pugh scores of ≥8, those with advanced-stage HCC, those who had undergone hepatic arterial infusion chemotherapy or other systemic therapy, and those treated with best supportive care alone. The median TTDP and OS were 22.3 and 25.4 months, respectively, in the conversion group, and 7.7 and 11.5 months, respectively, in the continued group (p = 0.001 and p = 0.003, respectively). Conclusions: It is possible that sorafenib conversion might prolong OS and TTDP in TACE-refractory patients with intermediate-stage HCC.

Structure determination of metabolites isolated from urine and bile after administration of AY4166, a novel d-phenylalanine-derivative hypoglycemic agent

Molecular structures of 10 metabolites, which were isolated from urine (M1-M8) or bile (M9 and M10) after administration of AY4166 (N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine), a novel amino acid derivative with hypoglycemic activity, have been elucidated by mass spectrometry and nuclear magnetic resonance. Four of these (M1, M2, M3 and M8) were determined to be hydroxyl derivatives of AY4166, two (M9 and M10) were carboxylate derivatives via oxidization of M2 and M3, three (M4, M5 and M6) were glucronic acid conjugates and the other (M7) was a dehydro derivative. The estimated structures for M1, M2, M3, M7, M8, M9 and M10 were confirmed by the coincidence of the retention time of HPLC, MS and 1H NMR spectra between the isolated metabolites and authentic synthesized substances. For three glucronic acid conjugates, M4, M5 and M6, structural confirmation was performed by a selective enzymatic digestion with beta-glucronidase. M1 and M2/3 were about 5-6 and 3 times less potent than AY4166, respectively, and M7 was almost as potent as AY4166.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis.

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca2+-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

Structural studies of the Maillard reaction products of a protein using ion trap mass spectrometry.

The early stage products of the Maillard reaction of egg white lysozyme with D-glucose were studied. Incubation with D-glucose at 50 degrees C for 20 days caused reaction on the Lys and Arg residues of lysozyme as follows: all of the six Lys residues and 10 of the 11 Arg residues in lysozyme reacted with D-glucose; Arg 61 did not react with D-glucose. The Lys residues reacted with D-glucose with 1 mol of dehydration per mole of residue, and the Arg residues reacted with 2 mol of dehydration per mole of residue. The major constituent of the Amadori product with the epsilon-amino group of the Lys residue and the D-glucose was found to be the beta-pyranose form. The structure of the early stage product of the Maillard reaction of a protein with a sugar is the same as that of an amino acid with a sugar.

Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 Å space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX®. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.