磷酸二酯酶7:一个新的抗炎免疫药物靶点研究进展

磷酸二酯酶(phosphod iesterases,PDEs)作为体内特异性水解第二信使cAMP和cGMP的唯一蛋白酶家族,参与多种生理病理过程。研究表明PDE7是cAMP特异性的,分为PDE7A、PDE7B两个亚型,主要分布在免疫和炎症细胞中。PDE7作为一个新的潜在抗炎免疫药物靶点,其选择性抑制剂有可能用于治疗慢性阻塞性肺疾病(COPD)、类风湿性关节炎、阿尔采末病(AD)等与免疫炎症密切相关的疾病。     

磷酸二酯酶及其抑制剂的研究进展

磷酸二酯酶(PDEs)是一类可水解细胞内第二信使环磷酸腺苷(cyclic adenosine monophosphate,cAMP)和环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)的酶类,可调节细胞内的多种信号传递和生理活动。PDEs由11种不同的家族组成,且各家族包含不同的亚型,各个亚型在细胞内分布、表达、调节方式以及对抑制剂的敏感性均不同,参与了炎症、哮喘、抑郁、勃起功能障碍等多种病理过程的发生发展,这些特点使得PDE作为新的药物靶点得到了越来越多的关注。该文将从PDE各家族生物学特点、生理病理学意义及其抑制剂的应用作一简单综述。     

磷酸二酯酶-4的中枢功能研究进展

磷酸二酯酶-4(phosphodiesterase-4,PDE4)对细胞内环腺苷酸(cyclic adenosine monophosphate,cAMP)浓度及其下行信号传导具有重要调控作用,是近年来备受关注的新型药物治疗靶点。PDE4在中枢神经系统具有广泛表达,其4个亚型的分布和功能各具特征。国内外近期研究发现,PDE4在抑郁、认知记忆障碍、药物依赖、神经损伤等疾病进程中发挥重要作用。特异性PDE4抑制剂的研发,可为上述疾病的治疗提供新的路径。该文就近年来PDE4中枢功能的研究进展作一简要综述,并探讨其作为神经精神疾病新型治疗靶点的应用前景。     

磷酸二酯酶4抑制剂临床及安全性研究进展

磷酸二酯酶4 (phosphodiesterase 4, PDE4)是磷酸二酯酶家族中重要成员之一,可特异性水解环磷酸腺苷(cyclic adenosine monophosphate, cAMP),通过改变cAMP浓度引发下游磷酸化级联反应,与多种疾病密切相关。靶向PDE4的抑制剂临床研究包括呼吸系统疾病、自身免疫性疾病、中枢神经系统疾病及皮肤病等多种疾病领域。但是在已上市的PDE4抑制剂临床研究中发现,大多数药物的恶心、呕吐等胃肠道不良反应发生率较高,限制了其临床应用。因此本综述总结了已上市PDE4抑制剂的临床进展及安全性问题,结合PDE4蛋白结构分析、PDE4抑制剂的作用机制及相关不良反应机制研究,阐述了PDE4抑制剂产生不良反应的主要原因,旨在为开发安全有效的PDE4抑制剂提供参考。     

磷酸二酯酶-4抑制剂的研究进展

目的综述磷酸二酯酶-4(phosphodiesterase-4,PDE-4)的生理活性及其抑制剂的研究进展。方法查阅近年来国内外相关文献31篇,对其进行归纳、总结和分析。结果 PDE-4抑制剂通过对PDE-4的抑制作用,阻止环磷酸腺苷的水解,在哮喘和慢性阻塞性肺炎等炎症疾病方面作用显著。目前,罗氟司特已经通过美国FDA批准上市,许多活性较强、不良反应较少的PDE-4抑制剂相继进入了临床阶段。结论 PDE-4作为重要的炎症靶点之一得到了深入的研究,其抑制剂发展迅速,具有良好的开发前景。     

磷酸二酯酶4抑制剂罗氟司特改善认知功能研究进展

罗氟司特(roflumilast)是2011年美国FDA批准用于治疗慢性阻塞性肺疾病的第二代磷酸二酯酶4(PDE4)抑制剂,具有抗炎、抗肿瘤、抗酒精成瘾和抗糖尿病等多种作用。近年发现,与其他PDE4抑制剂一样,其也具有神经保护作用,尤其对认知功能具有一定改善作用。在多种动物模型上,可改善诱导的认知功能障碍,并能有效改善患者的认知功能。因此,罗氟司特可能作为潜在的阿尔茨海默病和帕金森病等中枢神经退行性疾病治疗药物。其作用机制主要与调控cAMP信号转导通路及相关信号分子有关。本文对其非临床和临床研究的最新进展进行了综述。     

选择性磷酸二酯酶Ⅳ抑制剂的研究

磷酸二酯酶Ⅳ（PDE－4）主要分布于各类炎性细胞，能专一性水解环腺苷酸（cAMP),促进炎症的发展。PDE－4抑制剂通过选择性抑制PDE－4、使胞内cAMP水平升高，在抗炎和抗哮喘方面显示了广阔的应用前景。将PDE－4抑制剂分为儿茶酚醚类、苯甲酰胺类、喹唑啉二酮类、黄嘌呤类、苯并呋喃类等，分别综述其构效关系研究和临床开发应用。     

GEBR-7b, a novel PDE4D selective inhibitor that improves memory in rodents at non-emetic doses

BACKGROUND AND PURPOSE

Strategies designed to enhance cerebral cAMP have been proposed as symptomatic treatments to counteract cognitive deficits. However, pharmacological therapies aimed at reducing PDE4, the main class of cAMP catabolizing enzymes in the brain, produce severe emetic side effects. We have recently synthesized a 3-cyclopentyloxy-4-methoxybenzaldehyde derivative, structurally related to rolipram, and endowed with selective PDE4D inhibitory activity. The aim of the present study was to investigate the effect of the new drug, namely GEBR-7b, on memory performance, nausea, hippocampal cAMP and amyloid-β (Aβ) levels.

EXPERIMENTAL APPROACH

To measure memory performance, we performed object recognition tests on rats and mice treated with GEBR-7b or rolipram. The emetic potential of the drug, again compared with rolipram, was evaluated in rats using the taste reactivity test and in mice using the xylazine/ketamine anaesthesia test. Extracellular hippocampal cAMP was evaluated by intracerebral microdialysis in freely moving rats. Levels of soluble Aβ peptides were measured in hippocampal tissues and cultured N2a cells by elisa.

KEY RESULTS

GEBR-7b increased hippocampal cAMP, did not influence Aβ levels and improved spatial, as well as object memory performance in the object recognition tests. The effect of GEBR-7b on memory was 3 to 10 times more potent than that of rolipram, and its effective doses had no effect on surrogate measures of emesis in rodents.

CONCLUSION AND IMPLICATIONS

Our results demonstrate that GEBR-7b enhances memory functions at doses that do not cause emesis-like behaviour in rodents, thus offering a promising pharmacological perspective for the treatment of memory impairment.     

Characterization of phosphodiesterase 4 in guinea-pig macrophages: multiple activities,association states and sensitivity to selective inhibitors

1. The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated.
2. Differential centrifugation of macrophage lysates revealed that ∼90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC₅₀s of 240 and 540 nM, respectively.
3. Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 μM), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 μM), but were markedly suppressed by RS-rolipram (10 μM). The two peaks of PDE activity were arbitrarily designated CPPDE4α and CPPDE4β with respect to the order from which they were eluted from the column where the prefix, CP, refers to the species, Cavia porcellus.
4. The hydrolysis of cyclic AMP catalyzed by CPPDE4α and CPPDE4β conformed to Michaelis-Menten kinetic behaviour with similar K_ms (13.4 and 6.4 μM, respectively).
5. Thermal denaturation of membrane-bound PDE4 at 50°C followed bi-exponential kinetics with t_1/2 values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4α and CPPDE4β each decayed mono-exponentially with significantly different thermostabilities (t_1/2=2.77 and 1.15 min, respectively).
6. Gel filtration of CPPDE4β separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a ∼180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4α resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa.
7. Of ten PDE inhibitors examined, none distinguished CPPDE4α from CPPDE4β with respect to their IC₅₀ values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K_is of 2 μM and 1.5 μM for CPPDE4α and CPPDE4β, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4α and CPPDE4β.
8. In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive.
9. These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.

     

Phosphodiesterase-4 as a potential drug target

Phosphodiesterase-4 (PDE4) is the predominant enzyme in some specific cell types that is responsible for the degradation of the second messenger, cAMP. Consequently, PDE4 plays a crucial role in cell signalling and, as such, it has been the target of clinical drug development of various indications, ranging from anti-inflammation to memory enhancement. In this review, the fundamental biological role of PDE4 in intracellular signalling, its tissue distribution and regulation are described. The historical development of various chemical classes of PDE4 inhibitors and the challenges that face these inhibitors as therapeutics are also discussed. Finally, recent advances in the structural biology of PDE4 and their complexes with various inhibitors, as well as its potential impact on the rational design of potent and selective PDE4 inhibitors, are presented.     

Type 4 cAMP phosphodiesterase (PDE4) inhibitors augment glucocorticoid-mediated apoptosis in B cell chronic lymphocytic leukemia (B-CLL) in the absence of exogenous adenylyl cyclase stimulation

cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The primary objective of the current study was to determine whether PDE4 inhibitors, a class of compounds in late clinical development that raise intracellular cAMP levels by inhibiting type 4 cyclic nucleotide phosphodiesterases (PDE4), increase the efficacy of glucocorticoid-mediated apoptosis in leukemic cells from patients with B cell chronic lymphocytic leukemia (B-CLL). Rolipram, a prototypic PDE4 inhibitor, synergized with glucocorticoids in inducing B-CLL but not T cell apoptosis. Rolipram also augmented glucocorticoid receptor element (GRE) transactivation in B-CLL cells. In contrast, inhibition of protein kinase A (PKA) with the cAMP antagonist Rp-8Br-cAMPS reversed both glucocorticoid-induced apoptosis and GRE transactivation. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. Consistent with these results, inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram treatment up-regulated PDE4B in B-CLL, forskolin treatment up-regulated PDE4D in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating glucocorticoid receptor signal transduction. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL are indicated.     

磷酸二酯酶4及其抑制剂在学习记忆中的作用

环核苷酸磷酸二酯酶(phosphodiesterase,PDE)是环核苷酸的唯一细胞内水解酶,因而PDE抑制剂可以通过阻碍环核苷酸的分解,参与生物功能的调节。磷酸二酯酶4(PDE4)是PDE大家族的成员之一,PDE4抑制剂在临床上主要用于治疗慢性阻塞性肺病(COPD)、哮喘、自身免疫性疾病(如银屑病)、过敏性疾病及抑郁症。近年来,越来越多的研究表明PDE4可以通过多种途径影响学习记忆的过程,相应的抑制剂能够通过调控细胞内cAMP水平来改善中枢神经系统退行性疾病所致的学习记忆障碍。本文综述了PDE4在学习记忆调节中的作用及其抑制剂在治疗学习记忆障碍疾病中的应用。     

磷酸二酯酶-4抑制剂罗氟司特的药理与临床研究新进展

罗氟司特为磷酸二酯酶-4(PDE4)抑制剂,对肺部系统具有高效的抗炎活性。动物和临床试验结果显示,其对慢性阻塞性肺病(COPD)具有很好的疗效,且不良反应较少。罗氟司特可减轻伴慢性支气管炎和有加重史患者严重COPD恶化的风险,更多的研究有待进一步评价。现对罗氟司特治疗COPD的作用机制、药效学、药动学、临床评价和安全性做一综述。     

Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: Differential subcellular localization and regulated expression by cyclic AMP

1. A combination of pharmacological, molecular biological and biochemical approaches were used to investigate the differential expression of two cyclic GMP-inhibited cyclic nucleotide phosphodiesterase genes (PDE3A and PDE3B) in the rat.
2. RT–PCR using PDE3A- or PDE3B-specific oligonucleotide primers allowed amplification of products encoding PDE3A (508 bp) or PDE3B (499 bp) sequences from several rat tissues (heart, aorta, liver, kidney and epididymal fat), from primary cultures of aortic vascular smooth muscle cells (VSMC) as well as from an SV40 large T-antigen immortalized aortic VSMC line.
3. Immunoblotting experiments with PDE3-selective antisera allowed the detection of both PDE3A and PDE3B immunoreactive proteins in several rat tissues, including tissues of the cardiovascular system, in primary cultures of aortic VSMC and in an SV40 large T-antigen immortalized aortic VSMC line. In all cases, PDE3A was expressed as a 120 kDa protein which was only detected in the cytosolic fraction. PDE3B was expressed as a 135 kDa protein and its expression was limited to the particulate fraction of all tissues and cells studied.
4. Prolonged incubation of cultured aortic VSMC with agents that increase VSMC cyclic AMP (forskolin or 8-bromo-cyclic AMP) produced marked time-dependent increases in PDE3 activity which correlated with increases in PDE3A and PDE3B RT–PCR signals and a marked increase in particulate PDE3 activity and PDE3B protein.
5. The physiological, pharmacological and biochemical implications of these findings are discussed based on previous reports of the effects of PDE3 inhibitors in the cardiovascular system and the relevance of our findings are presented in the context of the development of PDE3A and/or PDE3B-selective pharmacological agents.

     

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram

1. The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated.
2. Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity>99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L-selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay.
3. Our results indicate that rolipram inhibited eotaxin-induced CD11b up-regulation up to 60.6±7.6% at the highest tested dose (10 μM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 μM.
4. We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen-induced eosinophil infiltration in different animal models of allergic inflammation.

     

磷酸二酯酶5抑制剂的进展与评价

目的:评价磷酸二酯酶5抑制剂的进展与临床合理应用.方法:采用近期国内、外相关文献进行综述.结果与结论:伴随社会的发展,性健康逐渐引起人们的认识.20世纪90年代一项药物研究的突破,促使药物的性质由治疗转向提高生活质量.磷酸二酯酶抑制剂可使环磷酸鸟苷酸的水平增高,促使男性阴茎勃起,受得了临床和患者的青睐.西地那非的上市为男性勃起功能障碍的治疗提供了全新的效果,标志着一个新的里程的开始.