Takashi Sohda

Reduction of Insulin Resistance in Obese and/or Diabetic Animals by 5-[4-(1-Methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione (ADD-3878, U-63,287, Ciglitazone), a New Antidiabetic Agent

Effects of 5-[4-(1-methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione (ADD-3878, U-63,287, Ciglitazone) on glucose and lipid metabolism were examined in various animal models. ADD-3878, administered as a dietary admixture (30–186 mg/kg/day) to obese-diabetic yellow KK (KK-Ay) mice, markedly suppressed the diabetic syndromes (hyperglycemie, hypertriglyceride-mia, and hyperinsulinemia), accompanied by the reduction of insulin resistance as manifested by improvement of overall insulin sensitivity in either the insulin tolerance test òr the steady-state blood glucose test. Chronic administration of ADD-3878 for as long as 12 wk to young yellow KK mice, which were in the early stage of diabetes and obesity, depressed age-dependent rises in blood glucose, plasma triglyceride, and insulin without exerting any effect on obesity. When orally administered to obese Zucker-fatty rats, ADD-3878 decreased plasma insulin and triglyceride in a dose-dependent manner (5–100 mg/kg/day). The treated rats showed increased tolerance and decreased insulin secretion in response to oral glucose. The glycemie response to insulin and the steady-state plasma glucose were also normalized in the treated rats. Chronic administration of ADD-3878 to young fatty rats for as long as 12 wk decreased the dose-dependent rises in blood glucose, plasma triglyceride, and insulin without exerting any effect on body weight. ADD-3878 had no effect on glucose and lipid metabolism of young Sprague-Dawley rats and mild strepto-zotocin-diabetic rats. However, in old Sprague-Dawley rats that were moderately insulin resistant and hyperli-pidemic compared with young ones, ADD-3878 decreased plasma triglyceride and insulin and improved insulin sensitivity. Five-day administration of ADD-3878 to beagle dogs with slightly impaired glucose tolerance increased glucose tolerance and suppressed postprandial rises in plasma glucose, insulin, and triglyceride. Based on these results, ADD-3878 is effective on abnormal glucose and lipid metabolism associated with insulin resistance or obesity through reduction of peripheral insulin resistance. Therefore, ADD-3878 is expected to be useful in the treatment of hyperglycemie, hyperinsulinemia, and hyperlipemia in obese type II diabetes and Obesity.

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.

TAK-603 SELECTIVELY SUPPRESSES TH1-TYPE CYTOKINE PRODUCTION AND INHIBITS THE PROGRESSION OF ADJUVANT ARTHRITIS

We have shown that TAK‐603, a new anti‐rheumatic drug, is more effective in animal models in which cellular immunity plays a central role. Here, we studied the effect of the drug on Th1 cytokines, which are dominantly produced in this type of immune reaction, in an in vitro system and an in vivo model. We established Th1‐ and Th2‐dominant T‐cell lines, and studied the effect of TAK‐603 on their cytokine production. Th1 cell lines were BALB/c mouse allo‐reactive T cells and C57BL mouse mite antigen‐reactive T cells, and the Th2 cell line was BALB/c mouse ovalbumin‐reactive T cells. TAK‐603 suppressed the production of Th1 cytokines [ interferon‐&ggr; (IFN‐&ggr;) and interleukin‐2 (IL‐2)] and not that of Th2 cytokines (IL‐4, IL‐5) in these cell lines. Furthermore, selective suppression of Th1 cytokine production was also observed in the T‐cell clones obtained from the ovalbumin‐reactive T‐cell line. To investigate the effect on cytokine production in animal models of arthritis, we analysed the expression of cytokine messenger RNA using reverse transcription–polymerase chain reaction. In adjuvant arthritis rats, Th1‐dominant cytokine production was observed both in the arthritic joint and the spleen, and the time–course paralleled the progression of arthritis. On the other hand, in type‐II collagen‐induced arthritis, in which TAK‐603 has little effect, Th1‐dominant cytokine production was not observed and Th2 cytokines were shown to be more important. The adjuvant arthritis rats treated with TAK‐603 (6·25 mg/kg/day, per os) showed significantly lower cytokine mRNA expression both locally and systemically. These data suggest that TAK‐603 selectively suppresses Th1 cytokine production, which is consistent with its effect on cellular immunity in animal models.

Automated high-performance liquid chromatographic determination of hydroxylysylpyridinoline and lysylpyridinoline in urine using a column-switching method

An on-line urine clean-up system was developed for the simultaneous determination of free and total pyridinoline, hydroxylysyl-pyridinoline (HP) and lysylpyridinoline (LP) by high-performance liquid chromatography (HPLC) using a column-switching technique. The method is based on a combination of gel permeation chromatography (GPC) and ion-pair reversed-phase HPLC. In the GPC column, pyridinoline is preseparated from endogenous urinary substances with 0.03 M heptafluorobutyric acid (HFBA) as the mobile phase. After column switching, the eluate fraction containing pyridinoline is further separated by ion-pair chromatography using an octadecylsilica (ODS) column with 0.03 M HFBA-acetonitrile (81:19) as the mobile phase. The detection limits were 36 and 44 pmol/ml for free and total HP, respectively, and 44 pmol/ml for both free and total LP at a signal-to-noise ratio of 3. The coefficients of variation for free and total pyridinoline were 1.5 and 3.5%, respectively. The determination of one sample including the clean-up is completed within 25 min. This system is precise and is useful for the determination of pyridinoline in large amounts of urine. The usefulness of pyridinoline as a biomedical marker for bone resorption was also examined.

Immunomodulating and articular protecting activities of a new anti-rheumatic drug, TAK-603

We investigated the pharmacological activities of a newly synthesized anti-rheumatic drug, TAK-603. (1) In vivo: In adjuvant arthritic (AA) rats, TAK-603 inhibited the hind paw swelling and the body weight loss. The minimum effective dose was 3.13 mg/kg/day (p.o.). Histological and radiographic studies showed that TAK-603 suppressed the development of synovial lesions and joint and bone destruction. TAK-603 was also effective in AA rats when administered for the first 7 days after the adjuvant injection. It suppressed type IV allergy (25 mg/kg/day, p.o.) but had no effect on type III allergy. It had little effect in acute inflammation, analgesic and antipyretic models. These data suggest that TAK-603 acts on the immune system, especially on cellular immunity. (2) In vitro: TAK-603 suppressed the mitogen-induced proliferation of mouse lymphocytes and the ConA-induced IFN-gamma and IL-2 production by rat lymphocytes at 10(-7) to 10(-5) M. It also significantly inhibited the IL-1 induced extracellular matrix reduction in rabbit chondrocytes. It had no effects on prostaglandin E2 (PGE2) production in rat peritoneal cells. These data show that TAK-603 has the ability to suppress the immune system and protect cartilage from destruction. TAK-603 is expected to be a promising drug for rheumatoid arthritis.