Levels of the oxidative stress marker γ-glutamyltranspeptidase at different stages of nonalcoholic fatty liver disease.

OBJECTIVES: This study investigated oxidative stress in the liver, by determining hepatic expression and serum levels of γ-glutamyltranspeptidase (GGT) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in different stages of nonalcoholic fatty liver disease (NAFLD), and assessed whether GGT can differentiate between the various stages of NAFLD. METHODS: Expression of GGT and 8-OHdG was examined in biopsy specimens by immunohistochemistry, and serum GGT and 8-OHdG levels were measured by enzyme-linked immuno sorbent assays in patients with simple fatty liver (n = 10), nonalcoholic steatohepatitis (NASH; n = 10) and, as a control, in alcoholic liver disease (ALD; n = 10). RESULTS: Hepatic tissue expression of GGT and 8-OHdG was seen in ALD, NASH and fatty liver patients. The percentage of hepatocytes positive for 8-OHdG expression and serum 8-OHdG levels was significantly higher in patients with NASH than simple fatty liver. Serum GGT levels were increased in all cases with ALD, NASH and fatty liver, and correlated significantly with serum levels of 8-OHdG in ALD and NASH, but not in simple fatty liver. CONCLUSIONS: Levels of GGT in fatty liver patients may compensate for mild oxidative stress by repressing 8-OHdG levels and preventing progression to NASH; however further oxidative stress leads to increased levels of 8-OHdG and the development of NASH.

Characteristic genotypes of vascular endothelial growth factor are susceptible to ascites in patients with cirrhosis.

This study was designed to investigate whether different vascular endothelial growth factor (VEGF) genotypes are associated with ascites formation in cirrhotic patients. Seventy cirrhotic patients were included in the study: 25 cirrhotic patients with ascites and 45 cirrhotic patients without ascites. Patient characteristics were investigated and compared between the two groups. With regard to VEGF genotype, 42 patients were C/C and 28 patients were T/T or C/T. The genotypes T/T or C/T were observed in 23 cases (51%) among the non-ascites group, but in only five cases (20%) among the ascites group. Serum levels of albumin and creatinine, and the VEGF genotypes were significantly different between the two groups. Multiple regression analysis showed that serum levels of creatinine and the VEGF genotypes were significantly correlated with ascites formation. Thus, it can be concluded that VEGF genotyping might be a valuable susceptibility marker for ascites formation in cirrhotic patients.

Sustained-release microcapsules of a bone formation stimulant, TAK-778, for local injection into a fracture site.

The benzothiepin derivative (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) is a potent bone formation stimulant. A sustained release formulation was prepared by encapsulating the drug into biodegradable microcapsules for local application to fracture repair in rats. The microcapsules consisted of TAK-778 (10% w/w) and a biodegradable polymer, copoly (d,l-lactic/glycolic acid), with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 15 k. The TAK-778 amount at the injection site progressively diminished for 4 weeks after administration, and the serum level of TAK-778 was sustained over the same period. The local concentration of TAK-778 after administration of the microcapsules was simulated by a two-compartment open model. In the model, a first-order release rate constant and a transfer rate constant were obtained from the release profile of the microcapsules and the serum level of TAK-778 after administration of the TAK-778 solution, respectively. Localization at the injection site was examined by radiography using microcapsules in which iodoform was encapsulated as a contrast agent. The microcapsules formed a clot at the injection site, and their spread was narrowly restricted. The local concentration was calculated to be maintained within the range 10(-3)-10(-6) M over 4 weeks on the assumption that the dose and spread volume were 5 mg of TAK-778/site and 3 mL, respectively.

Hemochromatosis with HFE gene mutation in a Japanese patient.

A case of hemochromatosis associated with HFE gene mutation has never been previously reported in a Japanese patient. A 65-yr-old Japanese woman presenting with primary hemochromatosis underwent HFE mutation analyses, which demonstrated a C282Y mutation, this being the definitive gene mutation of Caucasian hemochromatosis.

Mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) variants in American and Japanese populations.

M6P/IGF2R encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and cytotoxic T cell-induced apoptosis. M6P/IGF2R is imprinted and expressed only from the maternally inherited allele in marsupials and rodents. In contrast, humans were initially reported to differ from the imprinted mammalian orders by not having an imprinted M6P/IGF2R; however, some studies now suggest M6P/IGF2R imprinting may be a human polymorphic trait. Mutational and functional evidence are consistent with M6P/IGF2R also being a tumor suppressor in human colon, liver, lung, breast, and ovarian cancers. M6P/IGF2R expression is also pathologically downregulated following mammalian in vitro embryo culture, resulting in fetal overgrowth and "large offspring syndrome." Therefore, the M6P/IGF2R imprint status in humans is an unresolved question that critically impacts upon biological issues ranging from human cancer predisposition to evolution. Attempts to further characterize the imprint status of human M6P/IGF2R and loss of heterozygosity at this locus in cancer have been hindered by a lack of readily usable polymorphisms. To facilitate these genetic analyses, we have screened American and Japanese populations for M6P/IGF2R single nucleotide polymorphisms (SNPs). We have identified nine novel SNPs intragenic to human M6P/IGF2R, and have described experimental conditions for their optimal use. Three identified amino-acid variants in the M6P/IGF2R ligand-binding domains may be under selection in humans.

Increased expression of transforming growth factor-alpha in a patient with recurrent hepatocellular carcinoma following partial hepatectomy.

A 45-year-old woman with chronic hepatitis B underwent partial hepatectomy for hepatocellular carcinoma (HCC). However, the HCC recurred 2 months after surgery and rapid progression of the disease resulted in her death. Immunohistochemistry showed that transforming growth factor-alpha (TGFalpha) was barely expressed in the liver specimens obtained at hepatic resection, whereas autopsy specimens were strongly stained with anti-TGFalpha antibody in the cytoplasm of both non-tumourous and tumourous liver cells. A higher level of Ki67 expression, a proliferating marker, was observed in the recurrent HCC, similar to that of TGFalpha. Thus, we speculate that the partial hepatectomy increased the level of TGFalpha leading to recurrence and progression of HCC through an autocrine/paracrine mechanism.

Enhancement of fracture repair in rats with streptozotocin-induced diabetes by a single injection of biodegradable microcapsules containing a bone formation stimulant, TAK-778.

The feasibility of using microcapsules containing a bone formation stimulant, (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) to enhance fracture repair was assessed in rats with streptozotocin-induced diabetes. The release profile of the microcapsules was designed to mimic a dosing regimen of multiple injections of TAK-778 solution. The solution was injected locally every third day from day 0 (the day of operation) to day 27 according to several dosing regimens, and fracture repair was assessed at day 28. The production of callus was most prominent when TAK-778 solution was injected so that 50-75% of the total dose (5 mg TAK-778/site) was administered during the first half of the treatment period. Thus, injectable microcapsules of 30 micrometer in mean diameter were prepared in order to release TAK-778 over 4 weeks using a biodegradable polymer, poly(d,l-lactic/glycolic) acid, with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 14,000. A single local injection of the microcapsules markedly enhanced fracture repair, which resulted in recovery of destructive bending strength of the bone at day 28. Histologically, the injection of TAK-778 microcapsules stimulated both fibrous and cartilaginous proliferation and periosteal ossification in the callus at day 7; bony bridge formation was observed at day 28. At day 56, the callus was remodeled and cortical bony union was evidenced in the microcapsule-treated fractures compared with the controls, which showed only fibrous union.

Allele-specific polymerase chain reaction for genotyping human cytochrome P450 2E1.

Allele-specific polymerase chain reaction (AS-PCR) was applied to investigate the cytochrome P450 2E1 (CYP2E1) genotype. AS-PCR is a competitive multiplex PCR method in which PCR amplification is successfully performed only by using the sequence of 3' oligonucleotide ends as a DNA template in order to obtain an absolutely complementary product. I was able to produce allele-specific primers whose 3' ends had the base specific to Pst I polymorphism located within the 5'-flanking region of the CYP2E1 gene. Electrophoresis of the products showed that bands derived from common PCR products, allele C1 and C2, were clearly separate from each other due to the difference in the size of the products. I tested 102 unrelated Japanese individuals, and the results of both restriction fragment length polymorphism (RFLP) by Pst I or Rsa I and direct sequencing were in complete agreement with those of AS-PCR. These results lead me to conclude that AS-PCR is a simple and useful technique for investigating CYP2E1 genotype.

Studies on disease-modifying antirheumatic drugs. IV. Synthesis of novel thieno[2,3-b:5,4-c']dipyridine derivatives and their anti-inflammatory effect.

The syntheses and anti-inflammatory activities of novel thieno[2,3-b]pyridine and thieno[2,3-b:5,4-c']-dipyridine derivatives are described. These compounds were designed by modification of the quinoline template of a new type of disease-modifying antirheumatic drug (DMARD), TAK-603, and prepared by the Friedländer reaction as a key reaction. Their anti-inflammatory effects were evaluated using an adjuvant arthritis rat model. Most of the compounds which included a diethylamino moiety in the side chain had potent anti-inflammatory effect. In particular, ethyl 2-(diethylaminomethyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b:5,4-c'] dipyridine-3-carboxylate (21) exhibited more potent activity than TAK-603.

Enhancement of osteogenesis in vitro and in vivo by a novel osteoblast differentiation promoting compound, TAK-778.

TAK-778 [(2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxyamide; mw 505.53], a novel osteoblast differentiation promoting compound, was characterized in vitro and in vivo models. TAK-778 at doses of 10(-6) M and higher promoted potently bone-like nodule formation in the presence of dexamethasone in rat bone marrow stromal cell culture. This was accompanied by increases in cellular alkaline phosphatase activity, soluble collagen release, and osteocalcin secretion. Under the culture conditions, TAK-778 also stimulated the secretion of transforming growth factor-beta and insulin-like growth factor-I, indicating that TAK-778 may exert regulatory effects on osteoblast differentiation via autocrine/paracrine mechanisms. Furthermore, the in vivo osteogenic potential of TAK-778 was studied in bony defect and osteotomy animal models, using sustained release microcapsules consisted of a biodegradable polymer, poly (dl-lactic/glycolic) acid (PLGA). Single local injection of TAK-778/PLGA-microcapsules (PLGA-MC) (0.2-5 mg/site) to rat skull defects resulted in a dose-dependent increase in new bone area within the defects after 4 weeks. When the pellet containing TAK-778/PLGA-MC (4 mg/pellet) was packed into place to fill the tibial segmental defect in rabbit, this pellet induced osseous union within 2 months, whereas the placebo pellet did not. In addition, single local application of TAK-778/PLGA-MC (10 mg/site) to rabbit tibial osteotomy site enhanced callus formation accompanied by an increase in breaking force after 30 days. These results reveal for the first time that a nonendogenous chemical compound promotes potently osteogenesis in vitro and enhances new bone formation during skeletal regeneration and bone repair in vivo and should be useful for the stimulation of fracture healing.

Frequencies in the Japanese population of HFE gene mutations.

We studied the frequencies of C282Y and H63D mutations in the HFE gene, thought to be responsible for hereditary hemochromatosis (HH), in 504 chromosomes obtained from 252 unrelated Japanese. Allele-specific PCR and PCR-restriction fragment length polymorphism methods revealed that the C282Y mutation was not found and the H63D mutation was low in frequency (at only 0.99%) compared with data from European people. Since most HH is thought to be associated with the HFE gene mutation, the low incidence of these mutations is a likely reason for the rarity of this disease in the Japanese population.

Putative hemochromatosis gene mutations and alcoholic liver disease with iron overload in Japan.

It is well known that alcoholic liver disease is associated with iron overload. To study the role of hemochromatosis gene mutations on the pathogenesis of alcoholic liver disease (ALD), we have analyzed C282Y and H63D mutations on the chromosomes obtained from 95 Japanese alcoholics. Patients were divided in two groups [i.e., 64 alcoholic patients with liver damage (group I) and 31 alcoholics without liver damage (group II)]. In group I, biochemical examinations showed that serum levels of iron and ferritin were significantly high, and unsaturated iron binding capacity levels were low, compared with those of group II. An analysis by means of allele-specific polymerase chain reaction demonstrated that C282Y mutation was not observed in both groups I and II. H63D mutation was observed in only two heterozygotes of group I and in one heterozygote of group II. Results could not indicate the relationship between ALD and these mutations. We speculate that other causes of iron overload may exist in ALD with iron overload.

Studies on disease-modifying antirheumatic drugs. III. Bone resorption inhibitory effects of ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmethyl) quinoline-3-carboxylate (TAK-603) and related compounds.

In the course of our studies aimed at obtaining new drugs for treatment of bone and joint diseases, chemical modification of the potent bone resorption inhibitors justicidins, was performed and various naphthalene lactones, quinoline lactones and quinoline derivatives bearing an azole moiety at the side chain were prepared. Their inhibitory effects on bone resorption were evaluated by Raisz's method, and several compounds, including ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmeth yl)quinoline -3-carboxylate (6c, TAK-603), were found to have activities comparable with or superior to the justicidins. The 4-(3-isopropoxy-4-methoxy)-phenyl derivative (6d), in particular, displayed a marked increase in potency. TAK-603 and compound 6d were very effective in preventing osteoclast formation and bone resorption by mature osteoclasts. Further, TAK-603 was shown to be effective in preventing bone loss in ovariectomized mice.

Synthesis of novel 2-benzothiopyran and 3-benzothiepin derivatives and their stimulatory effect on bone formation.

In a search for therapeutic agents for the treatment of osteoporosis and bone fracture, we found that 2-benzothiopyran-1-carboxamide derivatives 1, derived from ipriflavone as a lead compound, increase cellular alkaline phosphatase activity in cultures of rat bone marrow stromal cells. Further modification of 1 has led to the discovery of more potent 3-benzothiepin-2-carboxamide derivatives 2. Of these, 3-benzothiepin derivatives bearing a 4-(dialkoxyphosphorylmethyl)phenyl group on the 2-carboxamide moiety such as 2h and 2q exhibited significant improvement of activity compared to ipriflavone. Asymmetric synthesis of 2h and 2q revealed that the (-)-isomers possessed activities superior to those of the (+)-isomers. Further evaluation of these compounds using the mouse osteoblastic cell line MC3T3-E1 revealed that (-)-2q enhanced the effect of bone morphogenetic protein. In addition, application of a sustained-release agent containing 2q increased the area of newly formed bone in a rat skull defect model. Based on these findings, (-)-2q was selected for further investigation as a new drug stimulating bone formation. Synthesis and structure-activity relationships for this novel series of 2-benzothiopyran and 3-benzothiepin derivatives are detailed.

Synthesis of peptide aldehyde derivatives as selective inhibitors of human cathepsin L and their inhibitory effect on bone resorption.

Cathepsin L, a lysosomal cysteine protease, is secreted by osteoclasts and participates in bone collagen degradation. In a search for cathepsin L inhibitors as antiosteoporotic agents, a series of peptide aldehyde derivatives were prepared by two synthetic approaches, DMSO oxidation of the corresponding alcohol derivatives and DIBAL-H reduction of the corresponding N, O-dimethylhydroxylamide derivatives, and evaluated for inhibitory activity against human cathepsin L and for inhibitory effects on bone resorption. Some of the peptide aldehyde derivatives including alpha-acylamino aldehyde derivatives showed potent activities. Among these compounds, N-(1-naphthalenylsulfonyl-L-isoleucyl-L-tryptophanal (12) was selected as a candidate for further investigation. Compound 12, a potent, selective, and reversible inhibitor of human cathepsin L with an IC50 of 1.9 nM, inhibited the release of Ca2+ and hydroxyproline from bone in in vitro bone culture system and also prevented bone loss in ovariectomized mice at an oral dose of 50 mg/kg.

Case report: Insulin-like growth factor II expression in hepatocellular carcinoma with alcoholic liver fibrosis accompanied by hypoglycaemia.

This case of hepatocellular carcinoma (HCC) with alcoholic liver fibrosis, which was not associated with hepatitis viruses, was accompanied by hypoglycaemia. The immunoreactive insulin level was low and other hormonal examinations were almost normal. Immunohistochemical studies showed a high level of insulin-like growth factor II (IGF2) peptide in the HCC section and the size heterogeneity of serum IGF2 investigated by western blot revealed a large form at approximately 15 kDa. These results suggest that the HCC with alcoholic liver fibrosis produced IGF2 and that the hypoglycaemia was caused by tumour-associated IGF2.

Studies on disease-modifying antirheumatic drugs. II. Synthesis and activity of the metabolites of ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmethyl)quino line-3-carboxylate (TAK-603).

The metabolites of ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmeth yl)quinoline -3-carboxylate (1, TAK-603), which is under clinical evaluation as a new type of disease-modifying antirheumatic drug (DMARD), were prepared to confirm their structures and to study their pharmacological properties. Of the metabolites identified, the 4-(4-hydroxy-3-methoxyphenyl) derivative (2c, M-I) was found to have an anti-inflammatory effect in an adjuvant arthritic rat model, although its potency in this model was slightly lower than that of the parent compound.

Promoter-specific insulin-like growth factor 2 gene imprinting in human fetal liver and hepatoblastoma.

Insulin-like growth factor 2 (IGF2) gene imprinting has been demonstrated to be promoter-specific, in that expression from the P1 promoter is biallelic whereas that from the P2-P4 promoters is monoallelic. In the present study, in order to investigate IGF2 gene imprinting status at the cellular level, allelic analysis was performed of IGF2 gene expression transcribed from the P1 and P3 promoters, using reverse transcription polymerase chain reaction (RT-PCR) on human fetal liver and hepatoblastoma. In situ hybridization was also undertaken, to obtain information about the cellular localization of transcripts expressed from the P1 and P3 promoters. The results indicated that transcripts expressed from the P1 and P3 promoters co-localized in the same fetal or neoplastic hepatocytes. These data should provide information regarding the molecular basis of genomic imprinting, suggesting that an imprint recognized for the differential expression may be strictly local and localized downstream of the IGF2 P1 promoter.