Potency, Selectivity, and Consequences of Nonselectivity of PDE Inhibition

Home , IC50, PDE11, PDE5 inhibitor, Potency (pharmacology), Sildenafil, Tadalafil

Potency, selectivity, and consequences of nonselectivity of PDE inhibition

E Bischoff1*

1Bayer Pharma Research, Bayer Healthcare Wuppertal, PH-R-EU-CV-ll, Wuppertal, Germany

Phosphodiesterases (PDEs) play a decisive role in cyclic nucleotide-mediated intracellular signaling. As PDEs are expressed in a variety of tissues, selectivity is a prerequisite for a therapeutically applicable PDE inhibitor. Sildenafil, vardenafil, and tadalafil are selective for PDE5, with vardenafil exhibiting the highest potency and minimal inhibition of other PDEs, with the exception of PDE6. Tadalafil is extremely selective for PDE5, but also potently inhibits PDE11, an enzyme with unknown physiological function. As PDE1 is expressed in the brain, myocardium, and vascular smooth muscle cells, nonselectivity with respect to this enzyme (selectivity: tadalafil4 vardenafil4sildenafil) may result in vasodilation and tachycardia. Inhibition of PDE6 (selectivity: tadalafil4vardenafilDsildenafil), which is expressed only in retina and functions in visual transduction, can transiently disturb vision. PDE5 inhibitors may also indirectly inhibit PDE3 by increasing cyclic guanosine monophospate levels, thereby elevating heart rate and vasodilation while inhibiting platelet aggregation. International Journal of Impotence Research (2004) 16, S11–S14. doi:10.1038/sj.ijir.3901208

Keywords: phosphodiesterase inhibitors; 30,50-cyclic-nucleotide phosphodiesterase; sildenafil; vardenafil; tadalafil

Introduction nafil, vardenafil, and tadalafil—sildenafil and vardenafil are typical substrate analogues as their structures mimic that of cGMP, which is the natural The 11 known phosphodiesterase (PDE) families substrate of PDE5. Both sildenafil and vardenafil comprise at least 60 distinct PDE species. They bind at the catalytic active center of PDE5, but differ in their substrate specificity, their kinetic because they are not hydrolyzed, sildenafil and properties, and in their tissue distribution (Table 1). vardenafil can inhibit the catalytic activity of the PDE enzymes participate in the regulation of all enzyme in a competitive manner. cellular functions, depending on the cyclic nucleo- The structures of sildenafil and vardenafil differ tides that serve as second messengers. PDE enzymes in particular by the molecular configuration of their hydrolyze the phosphodiester bond of cyclic ade- nitrogen atoms in the heterocyclic ring system nosine monophosphate (cAMP) and of cyclic gua- (Figure 1). The structure of tadalafil departs entirely nosine monophosphate (cGMP). Therefore, a PDE from that of both sildenafil and vardenafil.1–3 inhibitor can elevate the intracellular concentration Biochemically, PDE5 inhibitors can be characterized of cAMP or cGMP depending on the particular by their potency and by their selectivity with respect substrate specificity of the respective PDE. to other PDEs. Both potency and selectivity influ- PDE5 is the predominant cGMP metabolizing PDE ence the pharmacodynamics, and the clinical in cavernosal tissue and in penile arteries, but it is properties of a PDE5 inhibitor. also localized in all vascular smooth muscle cells Potency is commonly expressed as IC50, or the (SMCs) as well as in platelets and other tissues, such drug concentration required to reduce the activity as the lung. Among the three pharmacologically and of the tested PDE by 50%. The lower the value of the biochemically characterized PDE5 inhibitors—silde- IC50, the higher the potency of the inhibitory agent. The reported values of IC50 can vary considerably depending on the enzyme preparation employed *Correspondence: E Bischoff, PhD, Bayer Pharma Re- and assay conditions (eg, the substrate concentra- search, Bayer Healthcare Wuppertal, PH-R-EU-CV-ll, Bldg tion). Consequently, only data obtained under 500, Wuppertal D-42096, Germany. identical analytical conditions can be compared. E-mail: [email protected] Sildenafil, vardenafil, and tadalafil are all potent PDE inhibitor potency and selectivity E Bischoff S12 Table 1 Substrate specificities and distributions of PDE families

PDE Substrate specificity Main tissue localization

1 cGMP4cAMP Brain, heart, vascular smooth muscle 2 cGMP ¼ cAMP Adrenal cortex, brain, heart, corpus cavernosum 3 cAMP/cGMP Heart, corpus cavernosum, liver pancreas, vascular smooth muscle, platelets 4 cAMP Lung, mast cells, vascular smooth muscle 5 cGMP Corpus cavernosum, lung, vascular smooth muscle, platelets, brain, esophagus 6 cGMP4cAMP Retina 7 cAMP44cGMP Skeletal muscle, T cells 8 cAMP Testis, thyroid 9 cGMP Broadly expressed, not well characterized 10 cGMP4cAMP Brain, testes 11 cAMP ¼ cGMP Skeletal muscle, prostate, liver, kidney, pituitary, testis

reports, vardenafil is identified as the most potent compound compared to sildenafil and tadalafil.4–7 In addition to potency, high selectivity determines the clinical relevance and therapeutic efficacy of any PDE5 inhibitor. Selectivity means that only PDE5 should be inhibited and no other PDE. Selectivity is defined as the ratio between the IC50 for a given PDE and the IC50 for PDE5. The greater that ratio is, the more selective the inhibitor for PDE5 compared to the comparative PDE. With some exceptions, all three PDE5 inhibitor compounds are selective. Table 2 summarizes the selectivity ratios of vardenafil and sildenafil.1 With respect to PDE6, the selectivity ratio of vardenafil is 15, compared to sildenafil with a selectivity ratio of approximately 7. This means that 15-fold higher concentrations of vardenafil and seven-fold higher concentrations of sildenafil are necessary to inhibit PDE6 in comparison to PDE5. With a selectivity ratio of 780 for PDE6, tadalafil is clearly more selective than either vardenafil or sildenafil.2,4 With respect to PDE1, selectivity of vardenafil is approximately 140, and about 350 with respect to PDE11. For both PDE1 and PDE11, sildenafil shares similar selectivity ratios with vardenafil at 40 and 200, respectively. Selectivity ratios with respect to other PDEs exceed 1000.1 Although extremely selective with respect to PDE1, tadalafil potently inhibits PDE11 (IC50 ¼ 37 nM), and therefore its selectivity ratio with respect to this PDE is only 5.6 Selectivity ratios also vary significantly between different scientific reports because they are calcu- lated from IC50 values. However, for sildenafil, vardenafil, and tadalafil, the rank order of selectivity Figure 1 Chemical structures of vardenafil, sildenafil, and tadalafil. Source: Haning H et al. Imidazo[5,1-f]triazin-4(3H)- remains the same. ones, a new class of potent PDE 5 inhibitors. Bioorg Med Chem 4 Lett 2002; 12(6): 865–868. For PDE1, the order of selectivity is: tadalafil vardenafil4sildenafil. For PDE6, the order of selectivity is: tadalafil4 PDE5-inhibitors and vardenafil displays the highest D potency. It inhibits PDE5 at concentrations 10-fold vardenafil sildenafil. 4 lower than does sildenafil and 13-fold lower than For PDE11, the order of selectivity is: sildenafil vardenafil4tadalafil. tadalafil. Its IC50 is 0.7 versus 6.6 nM for sildenafil and 9.4 nM for tadalafil.1 The scientific literature What could be the clinical relevance of the observed reports significantly different IC50 values, but in all selectivity profiles of these three compounds? With

International Journal of Impotence Research PDE inhibitor potency and selectivity E Bischoff S13 Table 2 Selectivity of PDE inhibitors based on human recombinant PDE preparations

a IC50 (nM) PDE5A PDE1 PDE2A PDE3B PDE4B PDE6 PDE7B PDE8 PDE9A PDE10A PDE11A

Vardenafil 0.89 121 410 000 2400 2055 11 4600 410 000 3370 1000 308 RatioX/5 1 136 410 000 2696 2308 15 5168 410 000 3786 1123 346 Sildenafil 8.5 350 410 000 410 000 3190 49 410 000 4100 000 410 000 3800 1725 RatioX/5 1 41 41000 41000 375 7.4 41000 41000 41000 447 203 Tadalafil 9.4 410000 410 000 410 000 410 000 n.d. 410 000 410 000 410 000 410 000 67 RatioX/5 1 41000 41000 41000 41000 41000 41000 41000 41000 7.1 a PDE6 based on bovine retina data. Source: Saenz de Tejada I, et al.1 respect to PDE1, selectivity may be clinically tadalafil will inhibit PDE11 (Table 1). None of the significant for several reasons. PDE1 is expressed three compounds is believed to inhibit other PDE in the brain, in myocardial cells, and in vascular families at therapeutic or supratherapeutic concen- SMCs. The three subtypes of PDE1—PDE1A, PDE1B, trations. and PDE1C—are all Ca2 þ -calmodulin activated. Indirectly, PDE5 inhibition may affect the phar- PDE1A and PDE1B affect cGMP to a greater degree macodynamics of PDE3. PDE3 is abundant in many than cAMP. PDE1C exerts an equivalent effect on tissues, including the myocardium, vascular smooth cGMP and cAMP, the Vmax for cAMP and cGMP muscle, platelets, hepatocytes, adipose tissue, pe- being similar. Nonselectivity of PDE5 inhibitors nile tissue and the pancreas. There is evidence to with respect to all PDE1 subtypes may induce suggest that PDE3 and PDE5 are the main PDE vasodilatation, flushing, and tachycardia. PDE1C isoforms involved in pulmonary artery relaxation.8 may play a significant role in the proliferation of The Vmax of PDE3 is 2–10-fold higher for cAMP SMC, because this subtype is highly expressed in compared with cGMP and is inhibited by physio- proliferating SMCs. To the contrary, nonproliferat- logically relevant concentrations of cGMP. If one ing cells exhibit only low levels of PDE1C expres- cell type expresses both enzymes, the cGMP- sion. It can be speculated that inhibition of PDE1C inhibited PDE3 and the cGMP-specific PDE5, even could produce beneficial effects due to its putative a perfectly selective inhibition of PDE5 could inhibition of SMC proliferation, an event that precipitate the inhibition of PDE3 indirectly, contributes importantly to the pathophysiology of due to increased cGMP levels induced by PDE5 atherosclerosis. inhibition.9 Therefore, if both PDE5 and PDE3 are PDE6 is only expressed in the retina and plays expressed in myocardial cells or in the Purkinje a decisive role in signal transduction of vision. fibers of the heart, an increase in heart rate and Inhibition of this enzyme can induce visual dis- a positive inotropic effect could result as a turbances, which have occurred at the highest consequence of PDE3-induced PDE5 inhibition. clinically applied dose of sildenafil and to a lesser However, experimental evidence increasingly extent with vardenafil. No visual disturbances have indicates that PDE5 is neither expressed in cardio- been reported with tadalafil use. myocytes nor in Purkinje fibers.10,11 Indirectly The physiological relevance of PDE11 has not yet induced vasodilation and inhibition of platelet been established. PDE11 is expressed in the liver, aggregation due to the colocalization of PDE5 and kidney, pituitary gland, in skeletal muscle, myocar- PDE3 in these cells appears possible, but is lacking dium, prostate, corpus cavernosum, and to a high in clinical relevance. As PDE3 is localized in penile degree in the testes. tissue, it cannot be excluded that elevated cAMP Musculoskeletal pain, in particular back pain, has also contributes to the erectogenic effect of PDE5 been reported during therapy for erectile dysfunc- inhibitors. tion (ED) with PDE5 inhibitors. The effects are more Based on 3 y of cardiovascular safety experience significant with tadalafil than with sildenafil or with sildenafil, important yet unanswered issues vardenafil. It has been speculated that back pain that should be addressed include: may be attributed to tadalafil inhibition of PDE11 or to tadalafil’s comparatively longer half-life. How- ever, to date, scant evidence exists to support these (1) Are there clinical implications to the intracel- hypotheses.2 lular incorporation of certain therapeutic com- The clinical relevance of selectivity-related issues pounds that might demonstrate high affinities depends in large part on the maximal plasma for PDE receptor sites? Potential test sources concentration of the respective compounds. In include the rod outer segment of the retina and therapeutic doses, the plasma concentrations of platelets. All therapeutically effective PDE sildenafil and vardenafil may reach the IC50 of inhibitors must be incorporated into the cell PDE6, a level not expected for tadalafil. Clearly, because all PDEs are localized in the cytoplasm

International Journal of Impotence Research PDE inhibitor potency and selectivity E Bischoff S14 and/or on intracellular membranes. Moreover, and potent PDE11 inhibitor might support currently compounds that are targeted to the CNS or to available data. On the other hand, we have known PDE6 in the retina must cross the blood–brain for some years of selective and potent PDE2 barrier or the blood–retina barrier. Compounds inhibitors, but as yet have no evidence to elucidate that bind irreversibly and with high affinity to the physiological role of the enzyme. Perhaps PDE enzymes are expected to be therapeutically PDE function can only be detected under patho- difficult to manipulate. Experimental evidence physiological conditions. In that case, interpreting exists to suggest that permanent inhibition of physiological function would require a respective PDE6 in the retina could precipitate apoptosis pathophysiological model. of these cells. Other targets could include the inhibition of platelet function via PDE inhibition in order to achieve anti-aggregatory effects. In this case as well, irreversible inhibition could be dangerous. It has been speculated References that permanent inhibition of PDE5 might be therapeutically useful not merely for a sympto- matic but for a curative treatment of ED. 1 Saenz de Tejada I et al. The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new However, clinical evidence has yet to be PDE5 inhibitor vardenafil. Int J Impot Res 2001; 13: 282–290. established. 2 Porst H. IC351 (tadalafil, Cialis): update on clinical experi- (2) Unlike tadalafil, vardenafil and sildenafil have ence. Int J Impot Res 2002; 14(Suppl 1): S57–S64. not been shown to cross-react with PDE11. This 3 Turko IV, Ballard SA, Francis SH, Corbin JD. Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodiesterase suggests that the catalytic domain of PDE11 (type 5) by sildenafil and related compounds. Mol Pharmacol differs in an important way from that of PDE5. 1999; 56: 124–130. Is there potential for drug development that 4 Saenz de Tejada I, Frutos JA, Gaudo M, Florio V. Comparative targets the unique substrate specificities of selectivity: profiles of tadalafil, sildenafil and vardenafil using PDE5 and PDE11 with the objectives of: an in vitro phosphodiesterase activity assay. Int J Impot Res 2002; 14(Suppl 4): S20–S32. 5 Corbin JD, Francis SH. Pharmacology of phosphodiesterase-5 (a) Identifying the physiologic function of inhibitors. Int J Clin Pract 2002; 277: 47581–47587. PDE11 and 6 Gbekor E et al. Selectivity of sildenafil and other phospho- (b) Applying pharmacologic therapies to iden- diesterase type 5 (PDE5) inhibitors against all human phosphodiesterase families. Eur Urol 2002; 42(Suppl 1): 63. tifiable PDE11-related disorders? 7 Kim NN et al. Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel Structurally, the most similar PDE enzymes are selective phosphodiesterase type 5 inhibitor. Life Sci 2001; 69: PDE5 and PDE6. Nevertheless, it is possible to 2249–2256. synthesize PDE inhibitors that are both potent and 8 Bardou M et al. Hypoxic vasoconstriction of rat main pulmonary artery: role of endogenous nitric oxide, potassium selective for each of the known PDE families and channels, and phosphodiesterase inhibition. J Cardiovasc for their subtypes as well, such as PDE1A, PDE1B, Pharmacol 2001; 38: 325–334. or PDE1C. However, it is not imperative that the 9 Stief CG. Phosphodiesterase inhibitors in the treatment of existence of a selective and potent inhibitor either erectile dysfunction. Drugs Today 2000; 36: 93–99. explains the physiologic function of the enzyme, 10 Moreland RB, Goldstein II, Kim NN, Traish A. Sildenafil citrate, a selective phosphodiesterase type 5 inhibitor. Trends or creates a new therapeutic principle. A detailed Endocrinol Metab 1999; 10: 97–104. description and a variety of biological data derived 11 McGrouther C et al. Biochemical in situ hybridisation (ISH) from a PDE11 knockout mouse12 shows that and immunohistochemical (ICH) characterisation of phospho- although the function of the enzyme could be diesterase type 5 (PDE5) expression in human corpus cavernosum (CC) and cardiac tissue. Int J Impot Res 2000; postulated from such an animal model, no clear 12(Suppl 1): S27. physiological role of the enzyme could be deduced 12 Burslem M, Harrow J, Lanfear I, Phillips SC. Modulation of from the experimental findings. Clearly, a selective PDE11A activity. Patent # EP 12 11 313 A2, 2002.

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