Robert Michael Wallis

Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro

Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5), which has been shown to be a clinically effective treatment for erectile dysfunction. Its action results from increased levels of cyclic guanosine monophosphate (cGMP), which is normally degraded by PDE5. This cyclic nucleotide is a second messenger for nitric oxide, which is involved in the regulation of numerous functions, including vascular smooth muscle tone. In an attempt to better predict the effects of sildenafil on cardiovascular function, the distribution of PDE activity was determined with anti-PDE1 and anti-PDE5 antibodies in the human cardiac ventricle and saphenous vein, and in vitro studies were performed on the isolated human cardiac ventricle, corpus cavernosum, saphenous vein, and mesenteric artery as well as on rabbit aorta, dog coronary artery, dog trabecular tissue, and rabbit and human platelets. The major PDE activity in the human cardiac ventricle was shown to be calcium/calmodulin-dependent PDE1, but there was no detectable level of PDE5. In contrast, the human saphenous vein contained PDEs 1, 4, and 5, and the human mesenteric artery contained PDEs 1, 2, 3, 4, and 5. The distribution of PDE5 in the cardiovascular system is consistent with the observed pharmacodynamic and clinical effects of sildenafil. Sildenafil, unlike milrinone, a selective PDE3 inhibitor, had no effect on the isolated trabeculae carneae; this is consistent with the lack of PDE5 expression in cardiac myocytes. Sildenafil selectively increased cGMP levels in coronary vascular smooth muscle tissue but produced no change in cyclic adenosine monophosphate (cAMP) levels, which is consistent with the drugs selectivity for PDE5. In phenylephrine-contracted isolated rabbit aortic rings, sildenafil enhanced the relaxation induced by the nitric oxide donor glyceryl trinitrate, suggesting that sildenafil may potentiate the hypotensive effects of nitric oxide donor agents on the vasculature, an effect that has been observed clinically. Human platelets were found to contain PDE5, which was inhibited by 50% (IC50) by sildenafil at a concentration of 6.3 nM, consistent with the IC50 value in the corpus cavernosum. Sildenafil alone had no direct effect on platelet function, but it potentiated the in vitro antiaggregatory activity of sodium nitroprusside on rabbit and human platelets. The pharmacodynamic and adverse event profiles observed in clinical trials with sildenafil are consistent with the in vitro profile of the tissue distribution of PDE5 and its known mechanism of action as a selective inhibitor of PDE5.

Muscarinic antagonists in development for disorders of smooth muscle function

Compounds with high affinity for muscarinic M3 receptors have been used for many years to treat conditions associated with altered smooth muscle tone or contractility such as urinary urge incontinence, irritable bowel syndrome or chronic obstructive airways disease. M3 selective antagonists have the potential for improved toleration when compared with non-selective compounds. Darifenacin has high affinity (pKi 9.12) and selectivity (9 to 74-fold) for the human cloned muscarinic M3 receptor. Consistent with this profile, the compound potently inhibited M3 receptor mediated responses of smooth muscle preparations (guinea pig ileum, trachea and bladder, pA2 8.66 to 9.4) with selectivity over responses mediated through the M1 (pA2 7.9) and M2 receptors (pA2 7.48). Interestingly, darifenacin also exhibited functional tissue selectivity for intestinal smooth muscle over the salivary gland. The M3 over M1 and M2 selectivity of darifenacin was confirmed in a range of animal models. In particular, in the conscious dog darifenacin inhibited intestinal motility at doses lower than those which inhibit gastric acid secretion (M1 response), increase heart rate (M2 response) or inhibit salivary secretion. Clinical studies are ongoing to determine if darifenacin has improved efficacy and or toleration when compared with non-selective agents.

Integrated risk assessment and predictive value to humans of non-clinical repolarization assays

The potential for drugs to be associated with the life‐threatening arrhythmia, Torsades de Pointes (TdeP), continues to be a topic of regulatory, academic and industrial concern. Despite being an imperfect biomarker, prolongation of the QT interval of the surface ECG is used to assess the risk of a drug being associated with TdeP such that a thorough examination of drug effects on the QT interval is required for all new chemical entities. Numerous studies have investigated the relationship between non‐clinical findings and the risk of TdeP and QT prolongation in the general population. There are many literature references supporting the strong correlation between the clinical safety margin over human ether‐a‐go‐go (hERG) inhibitory potency and the risk of drug‐induced arrhythmia and sudden death. A quantitative analysis of the relationship between non‐clinical studies and the outcome of a human Thorough QT study has also been reported. In the current manuscript, based on the outcome of the non‐clinical assays the sensitivity and specificity of each assay and an integrated risk assessment for predicting the outcome of the human Thorough QT study has been conducted. The data suggest that for QT prolongation mediated through inhibition of the hERG current the non‐clinical assays are highly predictive of drug effects on the QT interval. Based on the literature review and specific quantitative analysis reported above it is concluded that non‐clinical assays predict the risk of compounds to prolong the QT interval and cause TdeP in humans if the mechanism is through inhibition of the hERG current.

The effect of sildenafil on corpus cavernosal smooth muscle relaxation and cyclic GMP formation in the diabetic rabbit

Sildenafil, a type V phosphodiesterase inhibitor, enhances smooth muscle relaxation in normal human and rabbit corpus cavernosum. We investigated the in vitro effects of sildenafil on non-adrenergic, non-cholinergic and nitric oxide (NO)-mediated cavernosal smooth muscle relaxation in diabetic rabbits, since alterations in this pathway are recognised in diabetic erectile dysfunction. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan. Cavernosal strips from age-matched control, 3- and 6-month diabetic animals were mounted in organ baths. Relaxation responses to electrical field stimulation (1-20 Hz) or sodium nitroprusside (10(-8)-10(-4) M) were assessed in the absence and presence of sildenafil (10(-8) and 10(-7) M). The effect of sildenafil on cGMP formation by the corpus cavernosum was also assessed following stimulation with sodium nitroprusside, A23187 and acetylcholine. Sodium nitroprusside-stimulated relaxations were significantly (P<0.03) impaired in the corpus cavernosum from both diabetic groups, (IC(50)=4.6 x 10(-6) M following 3 months of diabetes mellitus and 4.0 x 10(-6) M following 6 months of diabetes mellitus; compared to 7.5 x 10(-7) M for pooled age-matched controls). Sildenafil (10(-7) M) significantly enhanced sodium nitroprusside-stimulated relaxation in control (P<0.05) and diabetic groups (P<0.03). Electrical field stimulation-mediated relaxations of the corpus cavernosum were significantly impaired after 6-month diabetes mellitus and enhanced by sildenafil (10(-8) M). cGMP formation by the diabetic corpus cavernosum was impaired significantly, but restored towards normal by sildenafil. We suggest that the impairment of NO-mediated relaxation of the corpus cavernosum reflect, at least in part, a defect in guanylyl cyclase activity. These findings support the use of sildenafil as an effective, orally administered, treatment for diabetic erectile dysfunction.

Pre-clinical and clinical pharmacology of selective muscarinic M3 receptor antagonists

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.