Measurement of DNA Length Changes Upon CpG Hypermethylation by Microfluidic Molecular Stretching.

Abnormal DNA methylation in CpG-rich promoters is recognized as a distinct molecular feature of precursor lesions to cancer. Such unintended methylation can occur during in vitro differentiation of stem cells. It takes place in a subset of genes during the differentiation or expansion of stem cell derivatives under general culture conditions, which may need to be monitored in future cell transplantation studies. Here we demonstrate a microfluidic device for investigating morphological length changes in DNA methylation. Arrayed polymer chains of single DNA molecules were fluorescently observed by parallel trapping and stretching in the microfluidic channel. This observational study revealed that the shortened DNA length is due to the increased rigidity of the methylated DNA molecule. The trapping rate of the device for DNA molecules was substantially unaffected by changes in the CpG methylation.

In vitro metabolism of dexamethasone cipecilate, a novel synthetic corticosteroid, in human liver and nasal mucosa.

Dexamethasone cipecilate (DX-CP, 9-fluoro-11ß,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel synthetic corticosteroid used to treat allergic rhinitis. The pharmacological effect of DX-CP is considered to be mainly due to its active de-esterified metabolite (DX-17-CPC). To investigate the in vitro metabolism of DX-CP in human liver, DX-CP was incubated with human liver microsomes and S9. In addition, a metabolism study of DX-CP with human nasal mucosa was carried out in order to elucidate whether DX-17-CPC is formed in nasal mucosa, the site of action of DX-CP. DX-17-CPC was the major metabolite in both liver microsomes and S9. Two new epoxide metabolites, UK1 and UK2, were detected in liver S9, while only UK1 was detected in liver microsomes. This suggests that cytosol enzymes are responsible for the formation of UK2. In human nasal mucosa, DX-CP was mainly transformed into DX-17-CPC. By using recombinant human carboxylesterases (CESs), the reaction was shown to be catalyzed by CES2. These results provide the evidence that the active metabolite DX-17-CPC is the main contributor to the pharmacological action after the intranasal administration of DX-CP to humans.

Pharmacological profile of the novel anti-inflammatory corticosteroid NS-126, a therapeutic agent for allergic rhinitis.

NS-126 (9-fluoro-11beta,17,21-trihydroxy-16alpha-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel, highly lipophilic anti-inflammatory corticosteroid. We compared NS-126 and the widely used intranasal corticosteroid fluticasone propionate (FP) in a guinea-pig model of allergic rhinitis and a rat model of airway eosinophilia. In the allergic rhinitis model, NS-126 and FP reduced sneezing and nasal obstruction to similar extents. In the airway eosinophilia model, both compounds inhibited the infiltration of eosinophils into the bronchoalveolar lavage fluid, but the effect of NS-126 was longer-lasting than that of FP. In vitro, NS-126 showed lower affinity than FP for the glucocorticoid receptor and was a weaker inhibitor of Th(2) cytokine and chemokine production and mast-cell secretory responses. We also investigated DX-17-CPC, a metabolite of NS-126 generated in nasal tissue by carboxylesterase-catalyzed hydrolysis at the 17-position. DX-17-CPC showed greater affinity than NS-126 for the glucocorticoid receptor and was a stronger inhibitor of Th(2) cytokine and chemokine production and mast-cell secretory responses. The long duration of the anti-allergic effects of NS-126 may be explained by its high lipophilicity, while the strength of its anti-allergic effects may be explained by the generation of the active metabolite DX-17-CPC. NS-126 is a long-acting intranasal corticosteroid and a promising therapeutic agent for allergic rhinitis.