Yoshihiko Itokazu

Effects of ZNC-2381, a New Oral Compound, on Several Hepatic Injury Models and on Hepatocellular Apoptosis in Mice and Rats

The hepatoprotective effect of ZNC‐2381 (1‐(4‐aminophenyl) methyl‐3‐(3‐nitrophenyl)‐1,3‐dihydroimidazo[4,5‐b]pyridine‐2‐one), a novel 2‐one dihydroimidazopyridine derivative, has been evaluated in several experimental models of hepatic injury.

Effects of a novel hepatoprotective drug, ZNC-2381, on fas-induced hepatocellular caspase-3 activity and apoptosis in mice.

We examined the effects of ZNC-2381 (1-(4-aminophenyl)methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b] pyridine-2-one), a new oral hepatoprotective agent, on hepatocellular caspase-3 activity and apoptosis induced by anti-mouse Fas antibody (anti-Fas ab) in mice. Oral ZNC-2381, administered at doses of 10, 30 and 100 mg/kg 1 h before inducing hepatic injury with anti-Fas ab, dose-dependently inhibited the increase in serum alanine aminotransferase (s-ALT) activity 8 h after injection of anti-Fas ab. Increases in DNA fragmentation (nucleosome assay) and caspase-3 activity in the liver 2 h after injection of anti-Fas ab were also inhibited by ZNC-2381 in a dose-dependent manner. As shown by histopathological examination, ZNC-2381 dose-dependently inhibited the appearance of TUNEL-positive apoptotic cells in the liver. Moreover, in studies in vitro, ZNC-2381 (1– 100 µmol/l) concentration-dependently inhibited increases in DNA fragmentation and caspase-3 activity caused by anti-Fas ab in isolated mouse hepatocytes. N- Acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-cho), a caspase-3-specific inhibitor, inhibited hepatocellular apoptosis caused by anti-Fas ab both in vivo and in vitro, as well as the increase in s-ALT activity in vivo. These results demonstrate that orally administered ZNC-2381 inhibits hepatocellular apoptosis induced by anti-Fas ab and presents the progression of hepatic injury. We propose that the mechanism of action of ZNC-2381 may involve blockade of the signal transduction pathway (caspase-3) of apoptosis mediated by anti-Fas ab.

Effects of anti-rheumatic drugs on rhIL-1.BETA.-induced nitric oxide production in rat synovial fibroblast-like cells.

Effects of anti-rheumatic drugs, gold sodium thiomalate (GST), methotrexate (MTX), D-penicillamine (D-P) and bucillamine (BU), on rhIL-1β-induced nitric oxide (NO) production in rat synovial fibroblast-like cells (SFC) cultured in vitro were investigated. The SFC (passage 4) were cultured for 48hrs in Dulbeccos modified Eagles medium containing an NO inducer, LPS or rhIL-1β. NO production by SFC was assessed by using Griess reagent. RhIL-1β but not LPS, induced a significant increase in NO production in SFC. The rhIL-1β-induced NO production was inhibited by the presence of GST in a dose dependent manner, whereas MTX (100 ng/ml) had no effect. In contrast, both D-P (10 μg/ml) and BU (3 μg/ml) significantly stimulated rhIL-1β-induced NO production in SFC. This effect of SH-containing antirheumatic drugs was completely abolished by the presence of NO inhibitors, aminoguanidine (AG : 1 mM) and cycloheximide (CH : 2 μM) . AG but not CH, added at a later stage of culture (24-48hrs) abolished rhiL 1β induced NO production, indicating that these SH-compounds stimulate NO synthase activity in SFC.The present study suggests that SH-anti-rheumatic drugs stimulate rhIL-1β-induced NO production in SFC, and that the increase in NO may partly inhibit the bone destruction in rheumatoid arthritis.

The effect of methotrexate on the urinary pyridinium crosslink level in adjuvant induced arthritis in rats.

The effect of methotrexate on bone metabolism was investigated in adjuvant arthritis in rats (AA rats) . Methotrexate was orally administered to rats for 28 days from the next day after adjuvant inoculation. Twenty four-hour urine samples were obtained before sacrifice, pyridinoline (PY) and deoxypyridinoline (DPY), and creatinine concentrations in urine were measured by HPLC and the alkaline picronate method, respectively. Urinary PY and DPY in AA rats were increased 3.8 and 2.7 times that of age-matched controls, respectively. Doses of 0.05, 0.1 and 0.2 mg/kg caused a dose-dependent inhibition of the inflammatory parameters (fibrinogen, A/G) and the excretion of urinary PY and DPY in AA rats. Indomethacin (1 mg/kg, p. o.) had a significant inhibitory effect on both inflammatory parameters and urinary PY, but not on DPY. Moreover, the fourth lumbar of vertebra was used for determination of morphological changes. The trabecular number in the vertebra in the AA rats significantly decreased in comparison with that of age-matched normal. Methotrexate (0.05, 0.1 and 0.2 mg/kg) dose- dependently and completely inhibited severe changes observed in the vertebra of AA rats, while indomethacin only partly inhibited these changes. The present study indicates that MTX can prevent bone destruction in AA rats and measurement of urinary DPY is a useful marker for examination the effect of drugs on bone destruction.

The effects of anti-inflammatory and anti-rheumatic drugs, immunomodulators on bone resorption.

Rheumatoid arthritis (RA) is a systemic and chronic disorder involving joint damage. Recent studies have demonstrated changes in bone metabolism in patients with RA. Therefore, the aim of this study was to investigate the effects of some therapeutic drugs for RA on bone resorptionin vitro. The bone resorption was studied by measuring of calcium contents in medium released from mouse calvaria in tissue culture. Parathyroid hormone (PTH) and recombinant human interleukin 1β (rhIL-1β), bone resorting factor, was used in this experiment. The calcium contents in the medium with mouse calvaria was significantly increased by the presence of PTH (10-100nM) and rhIL-1β (3 -30U/ml) in a dose dependent manner. The increase in the medium calcium contents induced by PTH was inhibited by the coexistence of cyclosporin (l0μM) and methotrexate (1-10μM) whereas was not blocked by the presence of 10μM sulfasalazine, aurothioglucose, indomethacin, diclofenac Na, ketoprofen and bucillamine. However, rhIL-1β stimulating bone resorption was inhibited completely by indomethacin, diclofenac Na, ketoprofen and cyclosporin at 10μM. However sulfasalazine, aurothioglucose and methotrexate had not effect on rhIL-1β stimulating calcium release. At 10μM, bucillamine tend to enhance the calcium release from mouse calvaria by rh1L-1β. Furthermore, the calcium release by PTH and rhIL-1β was strongly prevented by bafilomycin A1 in doses between 1 nM and 10 nM.The effect of therapeutic drugs for RA was investigated in cloned osteoblast-like osteosarcoma cell (Saos-2) . Cells were cultured for 1 day at 37°C in a CO2incubator in plastic dishes containing McCoys 5 A medium supplemented with 10% fetal bovine serum (FCS) . After the cultures, the medium exchanged for FCS-free medium containing upper drugs and the cells were cultured further for 72h. Alkaline phosphatase (ALP) activity and DNA content in Saos-2 cells was significantly inhibited by cyclosporin (10μM) . Also, only DNA content in Saos-2 cells was significantly inhibited by methotrexate (10μM) . However, diclofenac Na, indomethacin, ketoprofen, bucillamine, sulfasalazine and aurothioglucose at the concentration of 10μM did not effect on ALP activity and DNA contents in the cells.These results indicated that NSAIDs had an inhibitory effects of bone resorption by rhIL-1β rather than PTH, but DMARDs had not directly inhibition by them, and, immunosuppresser, cyclosporine and methotrexate, had an inhibitory effects of bone resorption by PTH and/or rhIL-1β, but these two drugs also prevented osteoblastic function. From a clinical standpoint, the development of inhibitory drugs for bone resorption blocked or reduced the rate of progression of bone damage in RA without the inhibitory effect of osteoblast may be important.