Molecular Mechanisms That Could Contribute to Prolonged Effectiveness of PDE5 Inhibitors to Improve Erectile Function

Home , Cyclic nucleotide, PDE5 inhibitor

REVIEW Molecular mechanisms that could contribute to prolonged effectiveness of PDE5 inhibitors to improve erectile function

SH Francis, GZ Morris and JD Corbin

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA

Cyclic guanosine monophosphate (cGMP) in penile vascular smooth muscle cells (VSMC) plays a key role in promoting penile erection. Phosphodiesterase-5 (PDE5) in VSMC breaks down cGMP to counter this effect. Sildenafil (Viagra), vardenafil (Levitra) and tadalafil (Cialis), treatments for erectile dysfunction, inhibit PDE5 action. Many men with erectile dysfunction have improved erectile function after plasma inhibitor concentration falls below therapeutic levels. Maximum effect plus onset and duration of action of inhibitor determines its efficacy. The rate and extent of cellular drug accumulation and efflux of drug from smooth muscle cells plus persistence of drug effects in these cell impact these parameters. We propose possible molecular mechanisms that could account for prolonged action of PDE5 inhibitors including (1) persistence of biochemical effects after inhibitor is cleared from cells, and (2) retention of drug in VSMC beyond plasma clearance. International Journal of Impotence Research (2008) 20, 333–342; doi:10.1038/ijir.2008.4; published online 17 April 2008

Keywords: PDE5 inhibitors; erectile dysfunction; prolonged drug action; sildenafil; vardenafil; tadalafil

Introduction which is abundant in VSMC, specifically breaks down cGMP to the inactive 50-GMP at its catalytic The central role of cyclic guanosine monophosphate site and also binds cGMP at nonhydrolytic allosteric (cGMP) signaling in the erectogenic response of sites in its regulatory domain;8–10 cGMP binding to the penile tissues to sexual stimuli is now well allosteric sites increases activity of the catalytic site.11–13 established.1,2 Nitric oxide (NO) released from both The cellular cGMP level in corpus cavernosum is nonadrenergic noncholinergic nerve terminals and primarily dictated by the balance between the rate of endothelial cells of the penis activates a NO- cGMP synthesis by guanylyl cyclases and cGMP sensitive guanylyl cyclase to increase conversion breakdown by PDE5. Under healthy physiological of GTP to cGMP (Figure 1). Cyclic GMP binds with conditions, cGMP elevation in penile tissues in high affinity to cGMP-dependent protein kinase response to sexual stimulation is sufficient to cause (PKG) and activates this enzyme to cause phosphor- selective vasodilatation of the penile vasculature ylation of numerous cellular proteins that are and engorgement of the organ. This pathway can involved in modulating the level of cellular calcium be compromised in a number of ways including and calcium sensitivity of calcium-regulated path- decreased blood flow due to structural and functional ways.3–5 The consequences of this effect, that is, deterioration of the peripheral vascular bed and/or cGMP activation of PKG activity, are central to the nerves, insufficient production of NO by neurons contractile state of penile vascular smooth muscle and/or endothelium, excessive breakdown of cGMP cells (VSMC) and the erectile response to sexual by PDE5, decreased compliance of the VSMC and stimuli (Figure 1).1,6,7 Phosphodiesterase-5 (PDE5), penile connective tissue or a combination of these processes. As can be seen in Figure 1, deficiencies in several other sites of the signaling pathway are also possible. In some of these scenarios, cGMP elevation in Correspondence: Professor SH Francis, Department of the VSMC may not be adequate to provide for a Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 21st and Garland, sufficient degree of penile tumescence thereby resulting Nashville, TN 37232-0615, USA. in erectile dysfunction (ED). However, inhibition of E-mail: [email protected] PDE5 activity would favor greater accumulation Received 21 December 2007; revised 30 January 2008; of cGMP and facilitate the erectile response in many accepted 30 January 2008; published online 17 April 2008 of these conditions.7,14–16 Prolonged effect of PDE5 inhibitors SH Francis et al 334 EROTIC STIMULUS PDE5 INHIBITOR I settings, and in some instances use of a different dosing regimen, may mandate closer consideration NEURONS & ENDOTHELIUM of the pharmacokinetics of these medications and cG cG cG cG characteristics reported by patients currently using GTP PDE5 the products. PDE5 inhibitors other than sildenafil, cGMP 5’-GMP vardenafil and tadalafil have also either already NITRIC GUANYLYL OXIDE CYCLASE been launched or are in clinical trials in world cG 19 PKG ATP markets. The pharmacokinetics of sildenafil, vardenafil P-PROTEINS PROTEINS and tadalafil have been well documented overall.

PI The onset of action, maximum effect and duration of Phosphatases action of PDE5 inhibitors are among many pharma- Lower Ca++ & Desensitize to Ca++ cokinetic factors that must be considered for optimal efficacy of the medications as well as ease and safety Smooth Muscle Relaxation of use by patients. Among many such factors, the Penile Erection rate of accumulation, extent of accumulation and rate of efflux of drug in cells will impact these Figure 1 Cartoon depiction of cyclic guanosine monophosphate (cGMP)-signaling pathway in smooth muscle cell in penile parameters. There are now numerous reports that vasculature. Nitric oxide (NO) released from nonadrenergic many individuals who use PDE5 inhibitor medica- noncholinergic nerve terminals in the penis and endothelial cells tions experience an improvement in the erectile lining the vasculature in response to erotic stimuli diffuses into response long after the plasma level of the respective the smooth muscle cell where it activates an NO-sensitive drug is below what is considered to be a therapeutic guanylyl cyclase causing increased synthesis of cGMP (shown 20–24 as CG in the white circle) from GTP. Cyclic GMP is broken down level. In this report, we will discuss molecular at the catalytic site of phosphodiesterase-5 (PDE5) shown as the mechanisms that could account for prolonged action open V in the black oval and binds to allosteric sites in the of PDE5 inhibitors. regulatory domain of PDE5 (shown on a different portion of the Sildenafil, vardenafil, and tadalafil have high black oval). Cyclic GMP binds to and activates cGMP-dependent ¼ 4, 0.1–0.4 and 2 nM, protein kinase (PKG) shown as the black rectangle, thereby inhibitory affinity (IC50 promoting a cascade of phosphorylation events resulting in respectively) for the catalytic site of PDE5 compared lowering of intracellular calcium, desensitization to calcium to the B600- to 25 000-fold lower affinity of B 25–28 signaling, smooth muscle relaxation and penile erection. Phos- the natural substrate, cGMP (Km 2.5 mM). phorylation of proteins by PKG is counterbalanced by phospho- PDE5-selective inhibitors compete with cGMP for protein phosphatases, which dephosphorylate the proteins. PDE5 access to the catalytic site and are therefore inhibitors (shown as I in the white diamond), such as sildenafil 12,28,29 (Viagra), vardenafil (Levitra) and tadalafil (Cialis), have high known as ‘competitive inhibitors’. The three affinity for the PDE5 catalytic site thereby blocking cGMP access inhibitors do not interact with the nonhydrolytic to the site and fostering cGMP accumulation. The allosteric allosteric cGMP-binding sites on PDE5 (Figure 1).29 cGMP-binding sites of PDE5 and PKG are distinct from each other and from the catalytic site of PDE5, as indicated by the different shapes of the sites that interact with cGMP so that PDE5 inhibitors interact only with the PDE5 catalytic site. Prolonged action of PDE5 inhibitors in treatment of ED Use of PDE5 inhibitors to treat disease By all accounts, efficacy of action of sildenafil, PDE5 is the target of three potent PDE5-selective vardenafil or tadalafil to enhance the erectile inhibitors (sildenafil, (Viagra), vardenafil (Levitra) response appears to compare well. The time (Tmax) and tadalafil (Cialis)), which are commercially required to reach maximum plasma concentration available in many countries as oral treatment for (Cmax) and the time of onset of action vary slightly ED.17 Sildenafil (marketed as Revatio) has also been with tadalafil being slightly slower than sildenafil or approved in the United States and Europe as an oral vardenafil. However, the drugs differ substantially treatment of pulmonary arterial hypertension; the in the rate of clearance from plasma; t1 of plasma 2 three drugs are currently being investigated for clearance is B4 h for sildenafil or vardenafil potential use in treatment of a number of other compared to 18 h for tadalafil. Effect of sildenafil medical problems including cardiac hypertrophy or vardenafil in men is optimum at B0.5–1 h, which associated with heart failure, Raynaud’s disease, approximates the time of peak plasma concentration premature ejaculation, priapism, Peyronie’s disease, (Cmax). Plasma concentration of these drugs wanes recovery from stroke, cardiac reperfusion injury, with t1 of B4–5 h so that plasma drug level would be 2 intermittent claudication, benign prostate syndrome low after 12 h (B13% of Cmax). However, studies and other lower urinary tract symptoms, overactive indicate that men still have facilitated erections 12 h bladder, female sexual dysfunction, cognitive func- after dosing.20,30,31 Likewise, patients who have 18 tions and adjustment of the circadian rhythm. taken tadalafil, which has a t1 of B18 h, have 2 Increased use of these drugs in a number of medical reported improved erectile function at 36 h when

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 335 plasma concentration of the drug would be B25% of or without concomitant elevation of cGMP will first 17 Cmax. The prolonged effectiveness of these medica- be considered. Ordinarily, elevation of cGMP and tions adds flexibility to initiation of sexual activity for activation of PKG set in motion a cascade of cellular patients with ED and could reduce frequency of events, and action of PDE5 counters this effect by dosing. If this pattern applies to other tissues (for hydrolyzing the nucleotide. In tissues that have example, the pulmonary vasculature), it could mean been studied, evidence suggests that the action of that less frequent dosing regimens might be used to PDEs accounts for the major portion of the capacity bring about the desired therapeutic goal. This could of the cell to reduce cyclic nucleotide levels.32,33 result in lower exposure of the patient to medication The presence of PDE5 inhibitors blocks action of and cost savings for the therapy. PDE5 and enhances cGMP accumulation and cGMP The time course of drug action is generally signaling. It is not known if the level of cGMP thought to closely correlate with plasma level of achieved in the presence of PDE5 inhibitors is the drug. According to this model, clearance of a similar to that which occurs under physiological drug from plasma, exit of drug from the tissue and conditions or if cGMP accumulates to a much higher cessation of drug action in that tissue would level. In porcine coronary artery smooth muscles, a essentially coincide. However, prolonged action of threefold elevation of cGMP produces maximum potent PDE5 inhibitors does not fit this model well. relaxation.34 PDE5 is the major cGMP-hydrolyzing Several biochemical mechanisms might explain the PDE in corpus cavernosum8 so that as long as PDE5 apparently prolonged effect of PDE5 inhibitors. (1) If inhibitors are present, a significant cGMP level is the inhibitor is cleared from tissue in parallel with likely to persist in this tissue even after the erection its clearance from plasma, then the persistence of its has subsided due to other influences. If so, PKG biological effects must be due to prior exposure of is likely to remain activated to some extent for the tissue to inhibitor perhaps in combination with several hours. persistent activation of the cGMP-signaling pathway The long duration of the increased action of PKG during the time of exposure (that is, cellular described above could elicit effects not experienced ‘memory’ of the cGMP-signaling experience). More- under most physiological circumstances since PDE5 over, if the drug has been cleared from the cell, PDE5 action in the absence of inhibitors would be activity would be restored and cGMP level would be predicted to quickly return cGMP and activity of predicted to be near basal level (Figure 2a). (2) PKG to near basal level. The effects of phosphoryla- Alternatively, if drug is not cleared in parallel with tion reactions mediated by PKG are generally clearance from plasma, that is, drug is retained (or thought to be rapidly counterbalanced by the action sequestered) in VSMC of the corpus cavernosum of phosphoprotein phosphatases. Even in the face of and breakdown of cGMP by PDE5 remains largely declining cGMP and decreased PKG activity, rever- blocked (Figure 2b), a low level of other cGMP- sal of the effects of PKG phosphorylations may differ hydrolyzing PDEs present in these cells could lower among the phosphoproteins; some may be rapidly cGMP somewhat. This would foster (1) sustained dephosphorylated and returned to their original cGMP binding to allosteric cGMP-binding sites on functional state, others may be protected against PDE5 to enhance affinity for inhibitors, (2) sustained rapid dephosphorylation by their location in the cell cGMP binding to PKG and activation of the cGMP- or by being complexed with other proteins, thereby signaling pathway and (3) increased cellular cGMP sustaining the original effect of increased cGMP. In that is not readily hydrolyzed. In this latter scenario, fact, little is known about the true consequences and cellular concentrations of PDE5 inhibitor and cGMP time courses of the action of either of these path- would be elevated to some extent, which would ways. If activation of PKG and/or effects of PKG- result from specific sequestration of each of these mediated phosphorylations are not fully reversed in ligands, that is, inhibitor bound tightly to the PDE5 a timely fashion (Figure 2a), subsequent elevation of catalytic site and cGMP bound tightly to allosteric cGMP by erotic stimuli may produce a heightened cGMP-binding sites in PKG and PDE5. These erectile response even in the absence of PDE5 possible explanations for prolonged effects of inhibitors. A prolonged effect of phosphorylation PDE5 inhibitors on the erectile response will be events on cellular protein function could occur by a considered herein. These hypotheses are based on number of mechanisms including (1) prolonged established molecular mechanisms known to be phosphorylation of target proteins in the cell associated with PDE5 and PKG although the perhaps due to slow dephosphorylation by considerations do not exclude other mechanisms. phosphoprotein phosphatases, (2) structural changes such as conversion between forms that have different levels of activity, (3) alterations in protein levels due to alteration in gene expression, Persistent consequences of activation of the mRNA translation and/or stability, and/or protein cGMP-signaling pathway turnover rate, (4) translocation of proteins between The possibility of a ‘memory’ effect following cellular compartments and (5) increased sensitivity exposure of tissue to PDE5 inhibitors either with to cGMP and/or the cGMP-signaling pathway.

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 336 Potential Mechanisms for Prolonged Effect of PDE5 Inhibitor After Inhibitor is Cleared From the Cell Very Low Plasma Inhibitor I NITRIC PDE5 OXIDE GTP Low 5’-GMP cGMP GUANYLYL CYCLASE PKG ATP

P-PROTEINS PROTEINS PROLONGED EFFECTS OF PKG SUBSTRATE PHOSPHORYLATION: ? Prolonged Phosphorylation ? Structural Changes Prolonged Effect on ? Increased Sensitivity Penile Erection After to cGMP Exposure to PDE5 ? Altered Protein Levels ? Translocation Inhibitors

Very Low Cellular Inhibitor

Figure 2 Potential processes that could contribute to persistence of the effects of activation of the cyclic guanosine monophosphate (cGMP)-signaling pathway in vascular smooth muscle cells (VSMC) in presence of phosphodiesterase-5 (PDE5) inhibitors. Following administration of PDE5 inhibitor and sexual stimulation, high cGMP level in the VSMC may be achieved and sustained for several hours because PDE5 breakdown of cGMP would be blocked. (a) If inhibitor is cleared from the cell in parallel with clearance from the plasma, PDE5 activity would be restored, cGMP would be lowered and protein kinase (PKG) activation would decline (as indicated by the white arrows). However, prolonged effects of PKG phosphorylation of proteins that occurred when the cGMP-signaling pathway was very active may persist. (b) If inhibitor is retained in the cell long after it has been largely cleared from plasma, PDE5 activity would continue to be blocked (indicated by the white arrow) and high level of cGMP resulting from earlier sexual stimulation would be preserved since PDE5 is the main cGMP-hydrolyzing PDE in penile tissue. Continued activation of PKG would sustain phosphorylation events mediated by this enzyme including phosphorylation of PDE5 (indicated by P) and PKG autophosphorylation indicated by P that renders both enzymes more sensitive to cGMP effects.

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 337 PKG-mediated phosphorylation of PDE5 produces individual is chronically medicated. The conse- an apparent conformational change in the enzyme quences of short-term changes in the activity of the and increases affinity for cGMP at its allosteric cGMP-signaling pathway may differ significantly sites and for inhibitors/cGMP at its catalytic from that of a more sustained activation and warrant site.11,35–37 Cyclic GMP-activated PKG undergoes careful investigation. autophosphorylation, which slightly elevates the enzyme’s activity even in the absence of cGMP and increases its sensitivity to subsequent increases in cGMP (Figure 3).38–41 Autophosphorylation also Retention of PDE5 inhibitors in VSMC after causes an apparent conformational change in the clearance from plasma enzyme and promotes higher affinity for cGMP The possibility that PDE5 inhibitors may be retained binding compared to unphospho-PKG, thereby in VSMC and promote improved erectile response retaining cGMP at the allosteric cGMP-binding even after they are cleared from the plasma is also sites;38,42 since the cGMP-binding allosteric sites of plausible (Figures 2b and 4). Sildenafil, vardenafil PKG are positively cooperative,43 this would tend to and tadalafil are not metabolized in the VSMC; for ‘prime’ PKG for full activation by a subsequent small reversal of the effect of these medications in tissue, increase in cGMP. Some proteins that have been the drugs must dissociate from PDE5, diffuse through phosphorylated by PKG may subsequently be cova- the cytosol to the plasma membrane, traverse the lently modified by other posttranslational modifications plasma membrane and be transported to the liver for such as oxidation, ubiquitination or phosphorylation by modification and clearance by the action of cyto- other kinases that could prolong or otherwise impact chromes. Drugs such as these that have relatively the consequences of cGMP signaling. short plasma half-lives, bind with high affinity to Changes in the level of proteins that are substrates intracellular receptors such as PDE5, but are not for PKG could occur by numerous processes. PKG- degraded in the target cell, are in a somewhat unique mediated phosphorylation targets transcription fac- class that has not been studied extensively. Unanti- tors that regulate gene expression,44,45 which could cipated cellular pharmacokinetic characteristics that alter levels of proteins downstream of PKG. In other affect cellular clearance may apply to these drugs. instances, phosphorylation can target proteins for The entry and accumulation of PDE5 inhibitors in breakdown via the ubiquitination pathway.46,47 cells are expected to be impacted by the level of Cyclic GMP activation of PKG in some cells elicits PDE5. Accordingly, cells that are abundant in PDE5 translocation of the enzyme from the cytosol to are predicted to selectively acquire and retain the specific subcellular locations and phosphorylation inhibitor compared to cells with little or no PDE5 of target proteins within that compartment.44,45,48–50 (Figures 4a–c). Following absorption of the drugs Activation of PKG has also been shown to elicit into the blood stream, the inhibitor is transported to redistribution of certain proteins, for example, the VSMC surface by the systemic circulation. RhoA,51 within the cell or insertion of proteins into Initially, absence of the drug in the cytosol creates membranes thereby altering function.52,53 Some a large chemical gradient for the compound between cellular processes affected by PKG activity, for the exterior and interior of the cell; this would example, changes in the polymerization of fibrillar facilitate drug entry into the cell (Figure 4a). Since proteins or insertion of proteins into membranes the inhibitors are bound with very high affinity to may be slow to reverse. These are important PDE5, it is predicted that as soon as an inhibitor considerations when applying pharmacological regi- enters the cell, it will almost instantaneously be mens that foster elevation of cGMP and which may bound by PDE5, that is, largely sequestered from the be sustained for several hours or even days if the cytosol, and free inhibitor in the cytosol will remain

cG cG Phospho-PKG cGMP Proteins ATP cG P Pi Nitric Guanylyl PP1 Oxide Cyclase cG cG P -Proteins GTP Unphospho-PKG

Physiological Effects

Figure 3 Autophosphorylation of protein kinase (PKG) increases activity and affinity for cyclic guanosine monophosphate (cG). Elevation of cGMP increases PKG autophosphorylation. Autophosphorylated PKG has higher affinity for cGMP (as indicated by the heavier lines and position of cGMP in the model) and higher basal activity in the absence of cGMP. Both autophosphorylated and unphosphorylated PKG are fully activated by cGMP binding and carry out phosphorylation of cellular proteins.

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 338 High Plasma Inhibitor I very low, thereby maintaining the gradient. Inhibitor Low Intracellular Inhibitor I Low Intracellular cGMP binding to PDE5 will increase the rate of accumulation of inhibitors since, following inhibitor influx I I into cells, efflux of cellular inhibitor should be PDE5 I slowed because of the low amount of free inhibitor I I PDE5 I in the cytosol (Figure 4a). Furthermore, a significant PDE5 portion of the inhibitor molecules that dissociate

I from PDE5 would likely rebind to the enzyme before I I leaving the cell. These factors would facilitate PDE5 PDE5 I PDE5 accumulation of inhibitor in the cell commensurate I with cellular level of PDE5. According to this model, PDE5 Not Saturated I accumulation of inhibitor in the cell would be I With Inhibitor I saturable and determined by concentration of I intracellular PDE5. When all of the PDE5 catalytic sites are filled (saturated), concentration of free High Plasma Inhibitor I inhibitor in the cytosol would eventually approach High Intracellular Inhibitor I High Intracellular cGMP that in plasma (Figure 4b), but total inhibitor in the

I cell would be significantly impacted by the level of P cG PDE5 in that cell so that total inhibitor in the I P cG PDE5 I I cell ¼ inhibitor bound to PDE5 þ free inhibitor in the PDE5 I I cytosol. The prolonged action of the commercialized I P cG PDE5 inhibitors reported in some patients is also P cG I II I PDE5 predicted to result from the same processes, that is, PDE5 P cG I the combined effects of (1) high level of PDE5 in I I I PDE5 P cG VSMC and (2) high affinity of PDE5 for these I PDE5 inhibitors. The basal affinity of PDE5 for the I PDE5 Saturated with I inhibitors, which is intrinsically high (KD ¼ 0.1– Inhibitor and cGMP 4nM), can increase further in response to elevation I 12 I of cGMP as would occur with sexual stimulation and/or prolonged exposure to the respective drugs.54 As discussed below, this increased affinity I Low Plasma Inhibitor is caused by allosteric binding of cGMP to PDE5, Low Intracellular Inhibitor increased phosphorylation of the enzyme and prolonged perturbation of the catalytic site of the P cG 9,11,12,55 I P cG enzyme by elevated cGMP and inhibitor. PDE5 I I PDE5 Therefore, a few hours after ingestion of a PDE5 inhibitor either in the presence or absence of P cG sexual arousal, plasma inhibitor concentration P cG II I PDE5 would be high and it is predicted that the catalytic PDE5 P cG site of PDE5 in VSMC would be saturated with I PDE5 P cG inhibitor and that the affinity of PDE5 for the I PDE5 inhibitor would be significantly higher than in the Inhibitor and cGMP Still Associated with basal state (Figure 4b). This higher affinity binding PDE5 would foster continued association of the inhibitor with PDE5 and retention of the inhibitor in the Figure 4 Cartoon models depicting theoretical accumulation VSMC. 56,57 and retention of potent phosphodiesterase-5 (PDE5) inhibitors in In simplest terms, VSMC can be considered to vascular smooth muscle cells (VSMC). (a) Black circles with a V resemble dialysis bags containing both unbound and insert represent the form of PDE5 that has lower affinity for PDE5-bound inhibitors. As plasma inhibitor de- inhibitor. Inhibitor, represented by the diamond marked I, binds to the catalytic site on PDE5 to block cyclic guanosine monophosphate clines, free inhibitor in the cytosol of the cell would (cGMP) breakdown. (b) Binding of inhibitor, phosphorylation, and be expected to decline in parallel. However, the cGMP binding to allosteric sites indicated as CG in a white circle on population of inhibitor molecules bound in a PDE5- PDE5 induce a conformational change (as indicated by the elongated inhibitor complex may decline more slowly (Figures oval compared to the black circles in Figure 4a) and increase affinity of the enzyme for inhibitor as indicated by the embedding of the 4c and 5) because escape of the inhibitor from the inhibitor in the black oval. Favored interaction between inhibitor cell is not exclusively dependent on the rate of and PDE5 is indicated by the heavy arrows. In this situation, free diffusion of that molecule through the membrane to inhibitors in the plasma and cytosol are equal. (c) Despite essential the plasma. Rather, PDE5 molecules may signifi- clearance of inhibitor from plasma surrounding the cell, significant cantly interfere with inhibitor exit. Inhibitors are amount of inhibitor remains bound to PDE5 inside the cell and loss of inhibitor from the cell is slowed. reversibly bound to PDE5 so that the complex is in a dynamic equilibrium, that is, the inhibitor is

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 339 High Affinity cGMP P cG cG PDE5 PDE5 k Slow Exit Very High Affinity I cG P cG cG k PDE5 P I I PDE5 I PDE5 PDE5 NITRIC 5’-GMP OXIDE cG cG cG I cGMP PKG Time PKG ATP PKG PKG P-PROTEINS PROTEINS P P cG I PKG I I PDE5 PDE5 PDE5 Lower Cytosolic Ca cG Desensitize to Ca Very High Affinity Very High Affinity Very High Affinity Figure 6 Processes that affect affinity of phosphodiesterase-5 Prolonged Effect of PDE5 Inhibitors (PDE5) for inhibitors. on Penile Erection

subunit concentration) and KD is the equilibrium Figure 5 Cartoon depiction of features affecting longevity of the dissociation constant of the PDE5-inhibitor com- phosphodiesterase-5 (PDE5) complex with potent inhibitors. 56 B À7 Potent inhibitors are reversibly bound to PDE5. The ratio of the plex. Assuming that PDE5 is 5 Â 10 Min 8 rate of inhibitor dissociation (koff) from the PDE5-inhibitor VSMC (unpublished data) and that KD of unpho- À10 12 complex and the rate at which it is bound or rebound (kon)is sphorylated PDE5 for vardenafil is 4 Â 10 M, called the KD and reflects the affinity of the enzyme for the intracellular binding of inhibitor to PDE5 would inhibitor. With very high affinity interactions, association with PDE5 is favored in this equilibrium so that much of the inhibitor decrease the rate of efflux of this inhibitor by a factor rebinds quickly shortly after it dissociates from the enzyme and ofB1500 compared to the rate of efflux in cells with PDE5 activity is low as indicated by the white arrow. no PDE5. This factor would be 41500 if the affinity of the PDE5 catalytic site for inhibitors such as constantly dissociating from the enzyme at a certain vardenafil is increased by cellular events such as rate (koff) and being rebound at a certain rate (kon) binding of cGMP to the PDE5 allosteric sites, (Figure 5). The ratio of koff to kon (called KD) reflects phosphorylation of PDE5, prolonged exposure of the affinity of the enzyme for the inhibitor. With the protein to inhibitor or other processes. high-affinity inhibitors, association with PDE5 is While PDE5 has an intrinsically high affinity for favored in this equilibrium so that much of the sildenafil, vardenafil and tadalafil, modifications of inhibitor rebinds quickly after it dissociates from the the protein have been shown to further enhance enzyme. The high concentration of PDE5 would also affinity for some or all of these inhibitors, creating foster rebinding of the inhibitor because the on-rate very high affinity binding (Figure 6); these include of inhibitor binding is affected by concentration of cGMP binding to PDE5 allosteric sites and catalytic both inhibitor and PDE5. site, phosphorylation of the enzyme by PKG which As some of the inhibitor escapes the cell and is is facilitated by either inhibitor binding to PDE5 removed from the vicinity of the cell by the catalytic site or cGMP binding to allosteric sites on circulation, there would be ‘open’ PDE5 catalytic PDE5, exposure of the enzyme to inhibitor for sites so that not only would rebinding of the several hours or a combination of these effects.9,11– inhibitor occur with the same PDE5, but also other 13,35,55 Because cGMP binding to allosteric cGMP- PDE5 molecules would be available to intercept and binding sites on PDE5 and PKG is also fostered bind the inhibitor (Figure 5), thereby further under these circumstances and protected against impeding escape from the cell. Therefore, an breakdown,58,59 the sequestration of cGMP in these inhibitor molecule would have a much higher sites would result in higher cellular concentration of probability of rebinding to free sites on PDE5 than cGMP compared to the basal state and might provide of leaving the cell. Such a dissociation and re- a reservoir of cGMP for subsequent release, thereby association scenario of the drug for PDE5 could altering efficiency derived from a subsequent small potentially occur many times before clearance of the increase in the second messenger signal.60 drug from the cytosol is achieved. This could result in concentration and retention, or ‘trapping’, of the drug in VSMC, thus extending its duration of action. The effect of these processes to slow the t1 of 2 Selective distribution of ligands inhibitor efflux from VSMC can be quantified by considering the concentration of PDE5 in cells along The characteristics that we have considered pertain- with the affinity of PDE5 for the inhibitor. The effect ing to selective partitioning of PDE5 inhibitors may of PDE5 to slow inhibitor exit from the cell would be be more common for other drugs than are generally proportional to 1 þ P/KD, where P is the concentra- appreciated. Results from studies of the tissue tion of inhibitor-binding sites on PDE5 (PDE5 distribution of two compounds, chlordecone

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 340 (a pesticide) and hexafluoroacetone, which are as to increase their maximum effect. These topics unrelated to PDE5 inhibitors, support this type of will be discussed elsewhere. model.61 Both are hydrophobic compounds that selectively accumulate in liver.61–63 Evidence suggests that this selective hepatic sequestration most likely represents high affinity, but reversible, bind- Concluding remarks ing of the drug to glutathione and/or glutathione transferase, which are particularly abundant in liver Two possible explanations for continued effects of compared to other tissues. Maximum binding of PDE5 inhibitors in corpus cavernosum well after the chlordecone in liver closely relates to concentration inhibitor level in plasma has fallen to a subther- of glutathione in that tissue. Compounds that apeutic level are discussed herein. First, even if are chemically very similar to chlordecone or PDE5 inhibitor is efficiently cleared from the cell, hexafluoroacetone (mirex and acetone, respectively) effects resulting from PKG-mediated autophosphor- but lack high affinity for glutathione transferase do ylation and heterophosphorylation (phosphoryla- not selectively partition to the liver.64 Therefore, in tion of other cellular proteins) may be slow to some instances, drugs that bind with high affinity to reverse particularly if cGMP is elevated in the cells intracellular receptors that are particularly abundant for an extended period of time. In particular, in certain cell types preferentially accumulate in sustained autophosphorylation of PKG would keep those cells; it would then be predicted that potent the enzyme in a partially active state and also PDE5 inhibitors would reach higher concentrations sensitize the enzyme to subsequent increases in in cells where PDE5 is particularly abundant. cGMP, perhaps facilitating an erectile response Other model systems predict slow cellular re- despite a modest elevation of cGMP in response to moval of compounds that are tightly bound to a subsequent erotic stimulus. Second, the high intracellular proteins, thereby providing something affinity of PDE5 for inhibitors, the high concentra- like a ‘memory device’ for prolonged effects tion of PDE5 in tissues such as the corpus mediated by molecules even after extracellular cavernosum and rebinding of inhibitors to PDE5 concentration of the ligand has dropped substan- are predicted to prolong action beyond the time tially.56,65 This is supported by known character- predicted from clearance of these medications from istics of many high-affinity, slowly dissociating plasma. If PDE5 inhibitors are retained in the cell protein–ligand complexes. The exit of ribose from beyond the time of clearance from plasma, cGMP a dialysis bag is significantly slowed when the level and PKG activity may remain high in penile ribose-binding protein from Escherichia coli, which VSMC since PDE5 is the main cGMP-hydrolyzing is involved in chemotactic responses, is present.65 PDE in this tissue, and it would continue to be Similarly, dialysis of the regulatory subunit of blocked by the presence of the inhibitors. These cAMP-dependent protein kinase in complex with considerations have important medical and finan- cAMP for 5 days does not remove cAMP from the cial implications for regimens used for treatment of dialysis bag.57 PKGIa to which a trace amount of ED, pulmonary arterial hypertension and other [3H]cGMP has been added prior to gel filtration maladies associated with PDE5 action and warrant retains the ligand despite prolonged absence of studies to address the basis for sustained effective- unbound [3H]cGMP in the milieu.66 A similar effect ness of PDE5 inhibitors beyond the time predicted is observed for the complex between [3H]cGMP and from plasma clearance. the isolated R domain of PDE525 and for the complex between hexoses (glucose and galactose) and a 67 galactose-binding protein. These events are not Acknowledgments due to irreversibility of the interaction between the ligand and the protein because addition of a This work was supported by NIH DK40029. competing high-affinity ligand shows rapid ex- change and displacement of the first ligand. Despite continual dissociation of ligands from their targets, those that bind with high affinity are likely to References rapidly rebind, thereby prolonging the functional lifetimes of the complexes and presumably biologi- 1 Burnett AL. Nitric oxide regulation of penile erection: biology cal effects resulting from those complexes. and therapeutic implications. J Androl 2002; 23: S20–S26. 2 Burnett AL. The role of nitric oxide in erectile dysfunction: implications for medical therapy. J Clin Hypertens 2006; 8: 53–62. 3 Hofmann F. The biology of cyclic GMP-dependent protein Other considerations kinases. J Biol Chem 2005; 280: 1–4. 4 Lincoln TM, Dey N, Sellak H. Invited review: cGMP-dependent protein kinase signaling mechanisms in Many of the same mechanisms would be predicted smooth muscle: from the regulation of tone to gene expression. to hasten onset of action of PDE5 inhibitors as well J Appl Physiol 2001; 91: 1421–1430.

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 341 5 Sausbier M, Schubert R, Voigt V, Hirneiss C, Pfeifer A, Korth M cGMP-specific phosphodiesterase. J Biol Chem 1998; 273: et al. Mechanisms of NO/cGMP-dependent vasorelaxation. 6460–6466. Circ Res 2000; 87: 825–830. 27 Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, 6 Francis SH, Corbin JD. Phosphodiesterase-5 inhibition: the Naylor AM et al. Sildenafil: an orally active type 5 cyclic molecular biology of erectile function and dysfunction. Urol GMP-specific phosphodiesterase inhibitor for the treatment of Clin North Am 2005; 32: 419–429. penile erectile dysfunction. Int J Impot Res 1996; 8: 47–52. 7 Corbin JD, Francis SH. Molecular biology and pharmacology of 28 Ballard SA, Gingell CJ, Tang K, Turner LA, Price ME, Naylor PDE-5-inhibitor therapy for erectile dysfunction. J Androl AM. Effects of sildenafil on the relaxation of human corpus 2003; 24: S38–S41. cavernosum tissue in vitro and on the activities of cyclic 8 Gopal VK, Francis SH, Corbin JD. Allosteric sites of phospho- nucleotide phosphodiesterase isozymes. J Urol 1998; 159: diesterase-5 (PDE5). A potential role in negative feedback 2164–2171. regulation of cGMP signaling in corpus cavernosum. Eur J 29 Turko IV, Ballard SA, Francis SH, Corbin JD. Inhibition of Biochem/FEBS 2001; 268: 3304–3312. cyclic GMP-binding cyclic GMP-specific phosphodiesterase 9 Francis SH, Zoraghi R, Kotera J, Ke H, Bessay EP, Blount MA (type 5) by sildenafil and related compounds. Mol Pharmacol et al. Phosphodiesterase 5: molecular characteristics relating 1999; 56: 124–130. to structure, function, and regulation. In: Beavo JA, Francis 30 Gupta M, Kovar A, Meibohm B. The clinical pharmacokinetics SH, Houslay MD (ed). Cyclic Nucleotide Phosphodiesterases of phosphodiesterase-5 inhibitors for erectile dysfunction. in Health and Disease. CRC Press: Boca Raton, 2006, J Clin Pharmacol 2005; 45: 987–1003. pp 131–164. 31 Valiquette L, Montorsi F, Hellstrom WJ, Giuliano F, Homering 10 Corbin JD, Francis SH. Cyclic GMP phosphodiesterase-5: M, Taylor T et al. Penetration and maintenance of erection target of sildenafil. J Biol Chem 1999; 274: 13729–13732. with vardenafil: a time-from-dosing analysis. Can J Urol 2005; 11 Corbin JD, Turko IV, Beasley A, Francis SH. Phosphorylation 12: 2687–2698; discussion 2699. of phosphodiesterase-5 by cyclic nucleotide-dependent pro- 32 Barber R, Butcher RW. The quantitative relationship between tein kinase alters its catalytic and allosteric cGMP-binding intracellular concentration and egress of cyclic AMP from activities. Eur J Biochem/FEBS 2000; 267: 2760–2767. cultured cells. Mol Pharmacol 1981; 19: 38–43. 12 Blount MA, Beasley A, Zoraghi R, Sekhar KR, Bessay EP, 33 Mercapide J, Santiago E, Alberdi E, Martinez-Irujo JJ. Francis SH et al. Binding of tritiated sildenafil, tadalafil, or Contribution of phosphodiesterase isoenzymes and cyclic vardenafil to the phosphodiesterase-5 catalytic site displays nucleotide efflux to the regulation of cyclic GMP levels in potency, specificity, heterogeneity, and cGMP stimulation. aortic smooth muscle cells. Biochem Pharmacol 1999; 58: Mol Pharmacol 2004; 66: 144–152. 1675–1683. 13 Corbin JD, Blount MA, Weeks II JL, Beasley A, Kuhn KP, Ho 34 Jiang H, Colbran JL, Francis SH, Corbin JD. Direct evidence for YS et al. [3H]sildenafil binding to phosphodiesterase-5 is cross-activation of cGMP-dependent protein kinase by cAMP specific, kinetically heterogeneous, and stimulated by cGMP. in pig coronary arteries. J Biol Chem 1992; 267: 1015–1019. Mol Pharmacol 2003; 63: 1364–1372. 35 Bessay EP, Zoraghi R, Blount MA, Grimes KA, Beasley A, 14 Corbin JD, Francis SH. Pharmacology of phosphodiesterase-5 Francis SH et al. Phosphorylation of phosphodiesterase-5 is inhibitors. Int J Clin Pract 2002; 56: 453–459. promoted by a conformational change induced by sildenafil, 15 Kuthe A. Phosphodiesterase 5 inhibitors in male sexual vardenafil, or tadalafil. Front Biosci 2007; 12: 1899–1910. dysfunction. Curr Opin Urol 2003; 13: 405–410. 36 Francis SH, Chu DM, Thomas MK, Beasley A, Grimes K, 16 Stief CG. Phosphodiesterase inhibitors in the treatment of Busch JL et al. Ligand-induced conformational changes in erectile dysfunction. Drugs Today 2000; 36: 93–99. cyclic nucleotide phosphodiesterases and cyclic nucleotide- 17 Francis SH, Corbin JD. Molecular mechanisms and pharma- dependent protein kinases. Methods 1998; 14: 81–92. cokinetics of phosphodiesterase-5 antagonists. Curr Urol Rep 37 Francis SH, Grimes KA, Liu L, Thompson WJ, Corbin JD. 2003; 4: 457–465. Phosphorylation of isolated human PDE5 regulatory domain 18 Sandner P, Hutter J, Tinel H, Ziegelbauer K, Bischoff E. PDE5 increases cGMP-binding affinity and induces an apparent inhibitors beyond erectile dysfunction. Int J Impot Res 2007; conformational change. J Biol Chem 2001; 277: 47581–47587. 19: 533–543. 38 Smith JA, Francis SH, Walsh KA, Kumar S, Corbin JD. 19 Salem EA, Kendirci M, Hellstrom WJ. Udenafil, a long-acting Autophosphorylation of type Ibeta cGMP-dependent protein PDE5 inhibitor for erectile dysfunction. Curr Opin Investig kinase increases basal catalytic activity and enhances allos- Drugs 2006; 7: 661–669. teric activation by cGMP or cAMP. J Biol Chem 1996; 271: 20 Moncada I, Jara J, Subira D, Castano I, Hernandez C. Efficacy 20756–20762. of sildenafil citrate at 12 h after dosing: re-exploring the 39 Busch JL, Bessay EP, Francis SH, Corbin JD. A conserved therapeutic window. Eur Urol 2004; 46: 357–360; discussion serine juxtaposed to the pseudosubstrate site of type I cGMP- 360–351. dependent protein kinase contributes strongly to autoinhibi- 21 Gingell C, Sultana SR, Wulff MB, Gepi-Attee S. Duration of tion and lower cGMP affinity. J Biol Chem 2002; 277: action of sildenafil citrate in men with erectile dysfunction. 34048–34054. J Sex Med 2004; 1: 179–184. 40 Hofmann F, Gensheimer HP, Gobel C. Autophosphorylation of 22 Youn JM, Feldman RA, Auerbach SM, Kaufman JM, Garcia CS, cGMP-dependent protein kinase is stimulated only by Shen W et al. Tadalafil improved erectile function at twenty- occupancy of one of the two cGMP binding sites. FEBS Lett four and thirty-six hours after dosing in men with erectile 1983; 164: 350–354. dysfunction: US trial. J Androl 2005; 26: 310–318. 41 Hofmann F, Gensheimer HP, Gobel C. cGMP-dependent 23 Porst H, Padma-Nathan H, Giuliano F, Anglin G, Varanese L, protein kinase. Autophosphorylation changes the character- Rosen R. Efficacy of tadalafil for the treatment of erectile istics of binding site 1. Eur J Biochem 1985; 147: 361–365. dysfunction at 24 and 36 h after dosing: a randomized 42 Chu DM, Francis SH, Thomas JW, Maksymovitch EA, Fosler controlled trial. Urol 2003; 62: 121–125; discussion 125–126. M, Corbin JD. Activation by autophosphorylation or cGMP 24 Shabsigh R, Seftel AD, Rosen RC, Porst H, Ahuja S, Deeley MC binding produces a similar apparent conformational change in et al. Review of time of onset and duration of clinical efficacy cGMP-dependent protein kinase. J Biol Chem 1998; 273: of phosphodiesterase type 5 inhibitors in treatment of erectile 14649–14656. dysfunction. Urol 2006; 68: 689–696. 43 Corbin JD, Doskeland SO. Studies of two different intrachain 25 Thomas MK, Francis SH, Corbin JD. Characterization of a cGMP-binding sites of cGMP-dependent protein kinase. J Biol purified bovine lung cGMP-binding cGMP phosphodiesterase. Chem 1983; 258: 11391–11397. J Biol Chem 1990; 265: 14964–14970. 44 Broderick KE, Zhang T, Rangaswami H, Zeng Y, Zhao X, Boss 26 Turko IV, Francis SH, Corbin JD. Potential roles of conserved GR et al. Guanosine 30,50-cyclic monophosphate (cGMP)/ amino acids in the catalytic domain of the cGMP-binding cGMP-dependent protein kinase induce interleukin-6

International Journal of Impotence Research Prolonged effect of PDE5 inhibitors SH Francis et al 342 transcription in osteoblasts. Mol Endocrinol 2007; 21: phosphodiesterase-5 (PDE5) catalytic site to higher affinity by 1148–1162. PDE5 inhibitors. J Pharmacol Exp Ther 2008, In press. 45 Pilz RB, Broderick KE. Role of cyclic GMP in gene regulation. 56 Silhavy TJ, Szmelcman S, Boos W, Schwartz M. On the Front Biosci 2005; 10: 1239–1268. significance of the retention of ligand by protein. Proc Natl 46 Lu Z, Liu D, Hornia A, Devonish W, Pagano M, Aacd Sci USA 1975; 72: 2120–2124. Foster DA. Activation of protein kinase C triggers 57 Corbin JD, Sugden PH, West L, Flockhart DA, Lincoln TM, its ubiquitination and degradation. Mol Cell Biol 1998; 18: McCarthy D. Studies on the properties and mode of action of 839–845. the purified regulatory subunit of bovine heart adenosine 30: 47 Kuo WL, Duke CJ, Abe MK, Kaplan EL, Gomes S, Rosner MR. 50-monophosphate-dependent protein kinase. J Biol Chem ERK7 expression and kinase activity is regulated by the 1978; 253: 3997–4003. ubiquitin–proteosome pathway. J Biol Chem 2004; 279: 58 Kotera J, Francis SH, Grimes KA, Rouse A, Blount MA, Corbin 23073–23081. JD. Allosteric sites of phosphodiesterase-5 sequester cyclic 48 Wyatt TA, Lincoln TM, Pryzwansky KB. Vimentin is transi- GMP. Front Biosci 2004; 9: 378–386. ently co-localized with and phosphorylated by cyclic GMP- 59 Kotera J, Grimes KA, Corbin JD, Francis SH. cGMP-dependent dependent protein kinase in formyl-peptide-stimulated neu- protein kinase protects cGMP from hydrolysis by phospho- trophils. J Biol Chem 1991; 266: 21274–21280. diesterase-5. Biochem J 2003; 372: 419–426. 49 Pryzwansky KB, Wyatt TA, Lincoln TM. Cyclic guanosine 60 Corbin JD, Kotera J, Gopal VK, Cote RH, Francis SH. monophosphate-dependent protein kinase is targeted to Regulation of cyclic nucleotide levels by sequestration. In: intermediate filaments and phosphorylates vimentin in Bradshaw RH (ed). Handbook of Signaling. Academic Press: A23187-stimulated human neutrophils. Blood 1995; 85: New York, 2004, pp 465–470. 222–230. 61 Belfiore CJ, Yang RS, Chubb LS, Lohitnavy M, Lohitnavy OS, 50 Pryzwansky KB, Wyatt TA, Nichols H, Lincoln TM. Compart- Andersen ME. Hepatic sequestration of chlordecone and mentalization of cyclic GMP-dependent protein kinase in hexafluoroacetone evaluated by pharmacokinetic modeling. formyl-peptide stimulated neutrophils. Blood 1990; 76: Toxicology 2007; 234: 59–72. 612–618. 62 Borzelleca JF, Lester D. Acute toxicity of some perhalogenated 51 Murthy KS, Zhou H, Grider JR, Makhlouf GM. Inhibition of acetones. Toxicol Appl Pharmacol 1965; 7: 592–597. sustained smooth muscle contraction by PKA and PKG 63 Gillies PJ, Rickard RW. Toxicokinetics of [14C]hexafluoroace- preferentially mediated by phosphorylation of RhoA. Am J tone in the rat. Toxicol Appl Pharmacol 1984; 73: 23–29. Physiol 2003; 284: G1006–G1016. 64 Belfiore A, Costantino A, Frasca F, Pandini G, Mineo R, 52 Golin-Bisello F, Bradbury N, Ameen N. STa and cGMP Vigneri P et al. Overexpression of membrane glycoprotein stimulate CFTR translocation to the surface of villus enter- PC-1 in MDA-MB231 breast cancer cells is associated with ocytes in rat jejunum and is regulated by protein kinase G. Am inhibition of insulin receptor tyrosine kinase activity. Mol J Physiol Cell Physiol 2005; 289: C708–C716. Endocrinol 1996; 10: 1318–1326. 53 Zhu CB, Hewlett WA, Francis SH, Corbin JD, Blakely RD. 65 Willis RC, Furlong CE. Purification and properties of a ribose- Stimulation of serotonin transport by the cyclic GMP binding protein from Escherichia coli. J Biol Chem 1974; 249: phosphodiesterase-5 inhibitor sildenafil. Eur J Pharmacol 6926–6929. 2004; 504: 1–6. 66 Wolfe L, Francis SH, Landiss LR, Corbin JD. Interconvertible 54 Blount MA, Zoraghi R, Bessay EP, Beasley A, Francis SH, cGMP-free and cGMP-bound forms of cGMP-dependent Corbin JD. Conversion of phosphodiesterase-5 (PDE5) catalytic protein kinase in mammalian tissues. J Biol Chem 1987; 262: site to higher affinity by PDE5 inhibitors. J Pharmacol Exp 16906–16913. Ther 2007; 323: 730–737. 67 Richarme G, Kepes A. Release of glucose from purified 55 Bessay EP, Blount MA, Zoraghi R, Beasley A, Grimes KA, galactose-binding protein of Escherichia coli upon addition Francis SH et al. Phosphorylation increases conversion of of galactose. Eur J Biochem/FEBS 1974; 45: 127–133.

International Journal of Impotence Research