Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator,via phosphorylation and dephosphorylation in humans

1.?CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes.

2.?The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498?μM to 1060?μM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate.

3.?The Michaelis constants of four human ALPs and microsomes were in the range from 10.9?μM to 32.1?μM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes.

4.?FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.     

Placebo controlled evaluation of Xilei San,a herbal preparation in patients with intractable ulcerative proctitis

Background and Aim: Topical mesalamine or corticosteroid has shown efficacy in patients with ulcerative proctitis, but patients often become refractory to these interventions. Xilei San is a herbal preparation with evidence of anti‐inflammatory effects. We evaluated the efficacy of topical Xilei San in ulcerative proctitis patients. Methods: In a double blind setting, 30 patients with intractable ulcerative proctitis despite ≥ 4 weeks of topical mesalamine or corticosteroid were randomly assigned to True (n = 15) and placebo (n = 15). Patients in True received suppository Xilei San (0.1 g/dose per day of Xilei San), the other 15 received placebo suppository. The initial efficacy was evaluated on day 14. Primary endpoint of the trial was avoiding relapse during 180 days, relapse meant recurrence of active disease. Riley's index was applied for endoscopic and histological evaluations, while patients' quality of life was evaluated by an inflammatory bowel disease questionnaire. Results: On day 14, the number of patients who achieved remission, clinical activity index ≤ 4 in True was significantly higher versus placebo (P < 0.04). Likewise, at day 180, an 81.8% of patients in True were without relapse versus 16.7% in placebo (P < 0.001). Further, significant endoscopic (P < 0.01), histological (P < 0.02) and inflammatory bowel disease questionnaire (P < 0.04) improvements were observed in True, but not in placebo. Conclusions: This is the first controlled investigation showing significant clinical and endoscopic efficacy for Xilei San in patients with intractable ulcerative proctitis. Topical Xilei San was well tolerated, and was without safety concerns.     

Suppression of glucose-6-phosphate-isomerase induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of glucose-6-phosphate-isomerase

Objective: To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI_325–339) in mice model of GPI-induced arthritis (GIA).

Methods: We generated transgenic rice expressing T-cell epitope of hGPI_325–339 and APL12 contained in the seed endosperm. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined for IL-17 production in splenocytes and inguinal lymph node (iLN) cells, and analyzed the expression levels of functional molecules in splenocytes.

Results: Prophylactic treatment of GIA mice with APL12 transgenic (APL12-TG) rice seeds significantly reduced the severity of arthritis and titers of serum anti-GPI antibodies compared with non-transgenic (Non-TG) rice-treated mice. APL12-TG and hGPI_325–339 transgenic (hGPI_325–339-TG) rice seeds improved the histopathological arthritis scores and decreased IL-17 production compared with non-TG rice-treated mice. APL12-TG rice-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4?⁺?CD25?⁺?Foxp3^+?cells in the spleen compared with non-TG rice- and hGPI_325–339-TG rice-treated mice.

Conclusion: APL12-TG rice seeds improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on Treg cells in spleen.