Kimiyo Takeshita

Effects of a new anti-rheumatic drug KE-298 and its active metabolite: KE-758 on secretion of thioredoxin and on the level of intracellular glutathione in human monocytes and T cells.

Thioredoxin (TRX) and glutathione (GSH) are key regulators of the cellular balance of reduction/oxidation (redox). The impaired redox balance in joint cellular circumstances participates in immune dysfunctions seen in patients with rheumatoid arthritis (RA). We analyzed effects of a newly developed anti-rheumatic drug, KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid) and it is active metabolite; KE-758 (2-mercaptomethyl-4-(4-methylphenyl)-4-oxobutanoic acid) on the secretion of TRX and the level of intracellular GSH in THP-1 cells, a human monocytic cell line and in Jurkat cells, a human T cell leukemia cell line, then we compared their effects with N-acetyl-L-cysteine (NAC). KE-298 (10-100 microg/ml) and KE-758 (10-100 microg/ml) as well as a high concentration of NAC (10mM) dose-dependently inhibited the secretion of TRX by THP-1 and Jurkat cells. RT-PCR analysis indicated that the suppressive effects of KE-298 and KE-758 on TRX secretion could be partly explained by the inhibition of TRX mRNA expression. On the other hand, KE-758 as well as a high concentration of NAC significantly increased the level of intracellular GSH. Thus, KE-298 is a novel sulphydryl drug which regulates the redox state of cellular circumstances. The potential of KE-298 to suppress the secretion of TRX and to increase the level of intracellular GSH may partly explain the efficacy in cases of RA.

Studies ofd-penicillamine on strain variability and lymph node cellularity in adjuvant arthritis

Experiments were performed in order to ascertain the action ofd-penicillamine (PA) on adjuvant arthritis (AA) in rats and to develop a quantitative evaluation method for PA-like drugs, using Lewis, SD, and Wistar rats. Rats were inoculated in the tail with 0.6 mg ofMycobacterium butyricum suspended in 0.1 ml of liquid paraffin. PA apparently induced enhancement of arthritis only in Lewis rats with a good reproducibility. The enhancing effect of PA was seen when it was administered during the period from day −7 to day −1, from day 0 to day 6, or from day 14 to day 20. In control group of Lewis and Wistar rats, adjuvant caused a rapid increase in the cell number of lymph nodes just after the inoculation, and also a marked increase in spleen cells coinciding with the development of arthritis. In PA-treated Lewis rats, the cell numbers of lymph nodes and spleen significantly surpassed those of control rats. However, PA induced no difference from control in Wistar rats, which were not sensitive to PA treatment during the course of arthritis. These results indicate that AA in Lewis rats is a good model for evaluating the activities of PA-like drugs and that PA may affect lymphocytes in lymph nodes and spleen and induce severer arthritis in Lewis rats.

Synthesis and antirheumatic activity of the metabolites of esonarimod.

We have developed esonarimod, (+/-)-2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid, as a new antirheumatic drug. Now we describe herein the preparation of the enantiomers of (+/-)-deacetylesonarimod, the pharmaceutically active metabolites of esonarimod, and comparison of their antirheumatic activities. No significant difference has been observed between the two enantiomers. In a pre-clinical study of esonarimod, other metabolites were detected in rat blood or urine. We also synthesized these compounds as authentic samples to analyze the human metabolites in clinical studies of esonarimod.

Microbial Oxidation of KE-298 Metabolites by Rhizopus sp. and Rhodococcus sp. Strains

The metabolites of the antirheumatic agent KE-298 in humans, (-)-(2R)-M-4 [(-)-(2R)-4-(4-hydroxymethylphenyl)-2-methylthiomethyl-4-oxobutanoic acid], (-)-(2R)-M-5 [diastereomers of (-)-(2R)-4-(4-hydroxymethyl-phenyl)-2-methylsulfinyl-methyl-4-oxobutanoic acid], (-)-(2R)-M-6 [(-)-(2R)-4-(4-carboxyphenyl)-2-methylthio-methyl-4-oxobutanoic acid], and (-)-(2R)-M-7 [di- astereomers of (-)-(2R)-4-(4-carboxyphenyl)-2-methyl-sulfinylmethyl-4-oxobutanoic acid] were synthesized based on microbial transformation. The substrate KE-748 (racemic form of (-)-(2R)- and (+)-(2S)-4-(4-methyl-phenyl)-2-methylthiomethyl-4-oxobutanoic acid: 7.5 g) was converted to (-)-(2R)-M-4 (1.84 g) using Rhizopus sp. TF0040 in a 50-l jar fermentor. Specific cytochrome P-450 inhibitors, SKF-525-A and metyrapone strongly inhibited the hydroxylation reaction. It was suggested that cytochrome P-450 is responsible for the microbial reaction. Furthermore, (-)-(2R)-M-4 (200 mg) was transformed to (-)-(2R)-M-6 (144 mg) by co-oxidation with n-hexadecane as a carbon source using Rhodococcus sp. TA0250 in a 1.4-l jar fermentor. Starting from (-)-(2R)-M-4 and (-)-(2R)-M-6 obtained as above, (-)-(2R)-M-5 and (-)-(2R)-M-7, respectively were chemically synthesized by m-chloroperoxybenzoic acid oxidation.

Effect of KE-298 on production of immunoglobulins and IgG- and IgM-rheumatoid factors by rheumatoid arthritis mononuclear cells.

The effects of new immunomodulators, KE-298, on the production of immunoglobulins and IgG- and IgM-rheumatoid factors (IgGRF, IgMRF) by mono-nuclear cells from patients with rheumatoid arthritis (RA) were studied.KE-298 at the concentrations ranging from 3.13 to 200 μg/ml inhibited IgG, IgM and IgA production and IgGRF and IgMRF production by RA mononuclear cells. Mean % inhibition for IgG, IgM, IgA, IgGRF and IgMRF by 100 μg/ml KE-298 was 38.3%, 43.8%, 40.4%, 73.8% and 50.3%, respectively. Since enhanced RF production by RA mononuclear cells has an immunopathogenetic role in RA, the inhibitory action of KE-298 on RF production by RA mononuclear cells may be one of the mechanisms by which KE-298 exerts clinical efficacy in RA patients.