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Effects of Phosphodiesterase Inhibitors on Human Phosphodiesterase Activity and Airway Inflammation in Rats

Author: ChenJunChun
Tutor: ChenJiQiang
School: Zhejiang University
Course: Pharmacology
Keywords: Phosphodiesterase inhibitors 3’ 5’-cyclic-nucleotide-phosphodiesterase Ciclamilast Piclamilast Saccharomyces cerevisiae Eosinophils Rats
CLC: R96
Type: Master's thesis
Year: 2004
Downloads: 103
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Abstract

Introduction: The cyclic nucleotides cAMP and cGMP are key regulators of diverse cellular processes. The levels of the cyclic nucleotides are regulated by the rates of the related synthesis and degradation: the synthesis is generated by adenylate and guanylate cyclases and the degradation is linked to phosphodiesterase activity. cAMP specific PDE4, which is composed of four subtypes (PDE4A to PDE4D), has emerged as the predominant isoenzyme in inflammatory cells, pulmonary smooth muscle cells, heart cells and brain cells. Numerous studies have demonstrated the modulation of inflammatory and immunocompetent cell activation by selective PDE4 inhibitors.Piclamilast and ciclamilast both are the second generation selective PDE4 inhibitors. Ciclamilast is a newly developed "me-too" drug of piclamilast by our country. It has been reported that piclamilast can inhibit the accumulation of eosinophil in many animal models.AIM: In the first part of experiment, to improve the specific activity of human phosphodiesterase 4A (PDE4A) expressed in yeast cell GL62 and investigate the effects of selective phosphodiesterase 4 (PDE4) inhibitors ciclamilast, piclamilast, rolipram, selective phosphodiesterase 5 (PDE5) inhibitor zaprinast andcyclooxygenase (COX) inhibitors aspirin, indomethacin on human PDE4A activity expressed in yeast cell GL62; In the second part of experiment, to investigate the effects of piclamilast, ciclamilast, dexamethasone, and indomethacin on rat lung inflammation induced by Sephadex beads.METHODS:1. Human PDE4A was expressed in yeast cell GL62 after CuSO4 induction and the specific activity of human PDE4A was improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. The activity of PDE4A was measured by high performance liquid chromatography (HPLC).2. To establish the rat model of lung eosinophilia airway inflammation after Sephadex administration and investigate the effects of dexamethasone, indomethacin, piclamilast, ciclamilast on lung inflammation induced by Sephadex beads.RESULTS:1. Induced PDE4A activity expressed in crude yeast cell GL62 supernatant and pellet was (340 21) nmol g-1 min"1 and (250 25) nmol g-1 min"1 respectively.2. The specific activity of recombinant PDE4A in supernatant was improved 6.4 fold.3. Ciclamilast, piclamilast, and rolipram could inhibit PDE4A activity in a concentration-dependent manner. The IC50 values (95 % confidence limits) of ciclamilast, piclamilast, and rolipram were 1.27 (0.84-1.91), 66.4 (33.3-132.2), and 3.73 (2.51-5.53) mol L"1 respectively. Zaprinast, aspirin, and indomethacin had no obvious inhibitory effect on PDE4A activity.4. 72 h after a single i.v. injection of Sephadex beads, mononuclear cells, especially macrophages, were packed tightly around the beads, and eosinophils and neutrophils were conspicuous in the periphery of the lesions. The score of histological examination in Sephadex group was higher than that in control group,and dexamethasone, indomethacin, piclamilast, ciclamilast all reduced the filtration of eosinophils and improved mucus edema and epithelial lesion of bronchi and bronchioles.5. Exposure of rats to Sephadex produced a substantial increase in numbers of total and mononuclear cells in BAL fluid. Total and mononuclear cell numbers peaked after 72 h and declined to baseline levels by 7 days. A significant infiltration of neutrophils into BALF was observed 24 to 72 h after exposure to Sephadex but by 7 days neutrophils were almost undetectable. Eosinophils appeared in BALF at significant levels 24 h after exposure and continued to rise to peak at 72 h, thereafter declining substantially.6. Dexamethasone 2 mg kg"1 markedly reduced total leukocyte numbers in BALF, and almost completely inhibited eosinophil accumulation. Piclamilast 2 mg- kg"1, ciclamilast 2 mg kg-1 and indomethacin 2 mg kg-1 also inhibited eosinophil accumul

Effects of Phosphodiesterase Inhibitors on Human Phosphodiesterase Activity and Airway Inflammation in Rats

Abstract

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