NO-prednisolone

Kinase experiment:

Peripheral blood mononuclear cells (PBMCs) are isolated from heparinized human whole venous blood using Ficoll-Hypaque (d=1.077?g). PBMCs are then washed three times with HEPES buffered RPMI 1640 containing 0.05% gentamycin and 10% donor serum. Cells (1×106, ~20% monocytes, the remainder having been lymphocytes) are transferred to a 48 well plate and pre-incubated for 1?h with Prednisolone, NO-prednisolone (10 μM-0.1 nM) or vehicle, followed by activation with LPS (1?μg/mL) for 24?h at 37°C and 5% CO2. Supernatants are removed and IL-1β production measured using a commercially available human IL-1β ELISA[2].

Animal experiment:

Mice[1]BALB/c and male Swiss Albino mice (6-8 weeks old) are used. Mice (12 per group) are injected intrarectally with 50% ethanol or 1, 1.5, 2, or 2.5 mg per mouse of TNBS. TNBS-treated mice then are treated s.c. with 5 mg/kg/day Prednisolone or NO-prednisolone for 7 days. Surviving mice are killed 8 days after TNBS administration, colonic inflammation is scored, and myeloperoxidase (MPO) activity is measured. After instillation of 1.5 mg per mouse of TNBS, animals are allocated randomly into one of three treatment groups [placebo, NO-prednisolone (5 or 0.5 mg/kg/day s.c.), or Prednisolone (10 or 5 mg/kg/day s.c.)] and followed for 7 days. Mice are monitored for the appearance of diarrhea, loss of body weight, and overall mortality. At the end of the experiment, surviving mice are killed, blood samples are collected by cardiac puncture, and a 7 cm segment of the colon is excised for macroscopic and microscopic damage evaluation. Colonic IL-2, IL-10, IFN-γ, and tumor necrosis factor-α (ELISA and RT-PCR) and MPO activity are assessed according to published methods. In duplicate experiments, the colons of surviving mice are excised for LPMC preparation.

References:

[1]. Fiorucci S, et al. NCX-1015, a nitric-oxide derivative of prednisolone, enhances regulatory T cells in the lamina propria and protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15770-5.
[2]. Paul-Clark M, et al. 21-NO-prednisolone is a novel nitric oxide-releasing derivative of prednisolone with enhanced anti-inflammatory properties. Br J Pharmacol. 2000 Dec;131(7):1345-54.