Pharmacological and Functional Characterization of Novel EP and DP Receptor Agonists: DP1 Receptor Mediates Penile Erection in Multiple Species

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Pharmacological and Functional Characterization of Novel EP and DP Receptor Agonists: DP1 Receptor Mediates Penile Erection in Multiple Species Nadia Brugger, PhD,* Noel N. Kim, PhD,† Gian Luca Araldi, PhD,* Abdulmaged M. Traish, PhD,†‡ and Stephen S. Palmer, PhD§ *EMD Serono Research Institute—Medicinal Chemistry, Rockland, MA, USA; †Boston University School of Medicine—Urology, Boston, MA, USA; ‡Boston University School of Medicine—Biochemistry, Boston, MA, USA; § EMD Serono Research Institute—Reproductive Biology, Rockland, MA, USA DOI: 10.1111/j.1743-6109.2007.00676.x

ABSTRACT

Introduction. Despite the widespread use of prostaglandin E1 as an efficacious treatment for male erectile dysfunction for more than two decades, research on prostanoid function in penile physiology has been limited. Aim. To characterize the pharmacological and physiological activity of novel subtype-selective EP and DP receptor agonists. Methods. Radioligand binding and second messenger assays were used to define receptor subtype specificity of the EP and DP agonists. Functional activity was further characterized using isolated human and rabbit penile cavernosal tissue in organ baths. In vivo activity was assessed in rabbits and rats by measuring changes in cavernous pressure after intracavernosal injection of receptor agonists. Main Outcome Measures. Receptor binding and signal transduction, smooth muscle contractile activity, erectile function. Results. In organ bath preparations of human cavernosal tissue contracted with phenylephrine, EP2- and EP4selective agonists exhibited variable potency in causing relaxation. One of the compounds caused mild contraction, and none of the compounds was as effective as PGE1 (EC50 = 0.23 mM). There was no consistent correlation between the pharmacological profile (receptor binding and second messenger assays) of the EP agonists and their effect on cavernosal tissue tone. In contrast, the DP1-selective agonist AS702224 (EC50 =29 nM) was more effective in relaxing human cavernosal tissue than either PGE1, PGD2 (EC50 = 58 nM), or the DP agonist BW245C (EC50 =59 nM). In rabbit cavernosal tissue, PGE1 and PGD2 caused only contraction, while AS702224 and BW245C caused relaxation. Intracavernosal administration of AS702224 and BW245C also caused penile tumescence in rabbits and rats. For each compound, the erectile response improved with increasing dose and was significantly higher than vehicle alone. Conclusions. These data suggest that AS702224 is a potent DP1-selective agonist that causes penile erection. The DP1 receptor mediates relaxation in human cavernosal tissue, and stimulates pro-erectile responses in rat and rabbit. Thus, rabbits and rats can be useful models for investigating the physiological function of DP1 receptors. Brugger N, Kim NN, Araldi GL, Traish AM, and Palmer SS. Pharmacological and functional characterization of novel EP and DP receptor agonists: DP1 receptor mediates penile erection in multiple species. J Sex Med 2008;5:344–356. Key Words. Prostaglandin D2; Prostaglandin E1; Organ Bath

Introduction

P

rostaglandins constitute a family of bioactive lipids that are important regulators of both normal homeostatic and pathological processes.

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Through autocrine or paracrine mechanisms, prostaglandins bind specific G-protein-coupled receptors (GPCRs) to mediate their varied physiological responses [1,2]. Through their ability to regulate blood flow and connective tissue metabolism, © 2007 International Society for Sexual Medicine

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DP1 Agonists are Pro-Erectogenic prostanoids have been postulated to play a critical role in male sexual function. The composition of the cavernosal (erectile) tissue and the tone of the vascular smooth muscle within the penis are critical determinants of erectile function [3,4]. The vasodilatory prostanoids PGE2 and PGD2 are both synthesized by penile cavernosal tissue, and exert their effects through functional EP and DP receptors [5–10]. The pharmacological and molecular characterization of EP and DP receptors is complicated by the existence of multiple receptor subtypes and splice variants that couple to different G-proteins. In human penile corpus cavernosum, EP1, EP2, EP3-type I, EP3-type II, EP4, and DP receptors have been detected [6,7,9,10]. EP1 (Gq-coupled) and EP3-type I (Gi-coupled) receptors mediate smooth muscle contraction by stimulating phospholipase C (inositol phosphate pathway) or inhibiting adenylyl cyclase, respectively [1,2]. In contrast, EP2, EP3-type II, and EP4 receptors are coupled to Gs-protein and stimulate adenylyl cyclase to mediate smooth muscle relaxation [1,2]. Present evidence is consistent with coupling of DP receptors to Gs-protein with activation of adenylyl cyclase [1]. Recently, the Gi-protein-coupled CRTH2 (chemoattractant receptor-homologous molecule expressed on T helper type 2 cells) has been identified as another PGD receptor, prompting limited use of the DP2 nomenclature [1]. For consistency, the Gs-coupled DP receptor has been termed the DP1 receptor. Historically, the lack of receptor subtypeselective agonists and antagonists has limited the ability of investigators to study the biological effects of each receptor subtype [11]. Recently, we discovered a series of novel EP receptor agonists that were orally active in several animal models of normal reproductive physiology or in inflammatory diseases (unpublished results, S.S. Palmer, EMD Serono Research Institute, Rockland, MA, USA). These compounds exhibited high affinity for EP2 and EP4 receptors, stimulated cAMP production, and effectively relaxed both vascular and nonvascular smooth muscle. At the same time, relatively minor chemical modifications to these molecules resulted in the synthesis of very potent DP1 receptor agonists. The goal of this study was to characterize the pharmacological and physiological activity of these novel EP and DP receptor subtype-selective compounds in the context of penile cavernosal tissue relaxation and erectile function in human, rabbit, and rat. In addition to identifying candidate drugs for treating male erec-

tile dysfunction, the activities of these recently developed compounds serve as useful probes for revealing new insights into the regulation of penile erection by prostaglandin receptors. Materials and Methods

Receptor Binding Assays Receptor binding was performed on membranes prepared from HEK293 cells expressing cloned EP1, EP2, EP3, EP4, or DP1 receptors. For EP receptors, a 100-mL reaction mixture containing 20 mg of membrane was mixed with [5,6,8,11, 12,14,15 (n)-3H]prostaglandin E2 ([3H]PGE2) (Perkin Elmer, Norwalk, CT, USA), along with increasing concentrations of test compounds in a final concentration of 1% DMSO. The compounds were diluted in 25-mM MES, 10-mM MgCl2, 1-mM EDTA, pH 6 (binding buffer), containing 4% DMSO. [3H]PGE2 was added at concentrations equal to previously determined Kd values for EP1 (3 nM), EP2 (8 nM), EP3 (2 nM), and EP4 (2 nM). For the EP2 and EP4 receptor membrane reactions, 500 mg of wheat germ agglutinin scintillation proximity assay beads (Amersham, Piscataway, NJ, USA) was added to the wells. All reactions were incubated at room temperature, with shaking, for 1 hr. The EP2 and EP4 reactions were quantitated on a Topcount reader (Packard Instrument Co., Meriden, CT, USA), whereas the EP1 and EP3 reactions were terminated by filtration through glass fiber (GF/C) Unifilter plates (Whatman, Florham Park, NJ, USA) that were previously soaked in 0.5% PEI (Sigma, St Louis, MO, USA). Unifilter plate wells were then washed four times with 200 mL of binding buffer and were dried for 30 min at 50°C. After sealing the bottom of the plates, 100 mL of scintillation cocktail (Ultima gold XR, Packard, Meriden, CT, USA) was added in the wells, and filters were incubated for 1 hour at room temperature, and the radioactivity remaining on filters was measured using a Topcount plate counter (Packard). Membranes (20 mg/well) prepared from HEK293 cells expressing human DP1 receptor were incubated with 2.5 nM [5,6,8,9,12,14,153 H(N)]prostaglandin D2 ([3H]PGD2) (Perkin Elmer) in binding buffer (10-mM BES/KOH, pH 7.4; 10-mM MnCl2), for 1 hour at room temperature. Binding constants were determined by adding increasing concentrations of test compound or reference compounds, dissolved in DMSO (1% final v/v), to solution containing [3H]PGD2 and membranes. Nonspecific binding J Sex Med 2008;5:344–356

346 was determined with 10 mM of unlabeled PGD2 (Cayman, Ann Arbor, MI, USA). Reactions were terminated by filtration through GF/C glass fiber Unifilter plates (Whatman) that were previously soaked in 0.5% PEI. Unifilter plate wells were then washed four times with 200 mL of binding buffer and were dried for 30 min at 50°C. After sealing the bottom of the plates, 100 mL of scintillation cocktail (Ultima gold XR, Packard) was added in the wells, and filters were incubated for 1 hour at room temperature, and the radioactivity remaining on filters was measured using a Topcount plate counter.

Production of cAMP by EP2, EP4, and DP1 Production of cAMP in response to prostanoid compounds was measured in HEK293 cells transfected with EP2, EP4, or DP1 receptor, respectively. The cells were plated at a density of 20,000 cells/well in 96-well plates, 1 day prior to the assay. Stimulation was carried out in assay buffer (phenol red-free DMEM/F12, containing 0.1% bovine serum albumin, 0.1-mM isobutylmethylxanthine, and 1% penicillin-streptomycin) for 60 min with increasing doses of test molecules. Following stimulation, cells were lysed, and cAMP in the lysate was measured using a cAMP chemiluminescent assay kit (Tropix, Bedford, MA, USA) as per manufacturer’s instructions. Cavernosal Tissue Procurement Human tissue was procured from consenting patients through a protocol approved by the Institutional Review Board for Human Studies at the Boston University Medical Center. All cavernosal tissue was obtained from men (45–70 years old) undergoing penile prosthesis implantation surgery. Etiologies of erectile dysfunction included radical prostatectomy, pelvic trauma, generalized peripheral vascular disease, and Peyronie’s disease. Tissue from diabetic patients were excluded. Segments of corpora cavernosa that are routinely removed during the procedure were immediately placed in chilled (4°C) physiologic salt solution (PSS) and were transported to the laboratory. Tissue strips, measuring approximately 3 ¥ 3 ¥ 10 mm, were cut and prepared for organ bath studies. Nonhuman tissue was obtained from adult male New Zealand White rabbits (4.0–4.5 kg, Pine Acres, Norton, MA, USA). The animals were sedated and anesthetized with a subcutaneous injection of xylazine (5 mg/kg), followed by intramuscular administration of ketamine (40 mg/kg) and acepromazine (0.75 mg/kg). After lidocaine J Sex Med 2008;5:344–356

Brugger et al. (2% solution) infiltration, the chest cavity was opened, and the animals were euthanized by exsanguination through the thoracic aorta. The penis was excised and cleaned by removing the corpus spongiosum and urethra. Corpus cavernosum tissue strips were dissected away from the surrounding tunica albuginea and were prepared for organ bath studies. All animal studies were approved by the Institutional Animal Care and Use Committee at the Boston University School of Medicine.

Preparation of Drug Stock Solutions for Organ Bath and in vivo Studies PGE1, PGD2, BW245C, BW A868C, and BAYu3405 (Cayman Chemical Co.) were stored at -20°C in solid form until the day of use. Stock solutions were made by adding 1 mL of 70% DMSO to a vial containing 1 mg of compound. All novel test compounds were dissolved in 1 mL of 70% DMSO, divided into 100-mL aliquots, and stored at -20°C until use. For dose responses in organ baths, stock solutions were diluted with 70% DMSO to make the highest concentration, and then serially diluted with 2% DMSO for all other doses. In a typical dose response curve, the concentration of DMSO remained below 0.1% in the bath up to 10-6 M of prostanoid and did not exceed 0.47% at 10-5 M of prostanoid. For in vivo studies, all compounds were dissolved in 40% propylene glycol. Organ Bath Studies Human or rabbit cavernosal tissue strips were mounted onto a fixed support with silk ties and were attached to a tension transducer (model FT03, Grass-Telefactor, Astro-Med, Inc., West Warwick, RI, USA) with a rigid metal wire. After mounting, the tissue strips were immersed in 25 mL baths of PSS (118.3 mM NaCl, 4.7 mM KCl, 0.6 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, 0.026 mM CaNa2EDTA, and 11.1 mM glucose). The solution was gassed with 5% CO2, 20% O2, and 75% N2 (Middlesex Gases and Technologies, Inc., Everett, MA, USA) to attain a pH of 7.4, and the temperature was maintained at 37°C. All tissue strips were treated with 3-mM indomethacin to inhibit endogenous prostanoid production and stretched incrementally (1 g tension/stretch). The tissue strips were periodically contracted with 1-mM phenylephrine and washed with fresh PSS. When the amplitude of the phenylephrine-induced contraction was within 10% of the previous contraction, this tension was considered optimal for

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DP1 Agonists are Pro-Erectogenic isometric contraction. For dose responses, all tissue strips were extensively washed with fresh PSS and then contracted with 1-mM phenylephrine. After a stable tone was achieved, the tissue strips were exposed to increasing concentrations of vehicle, or EP or DP agonists by cumulative addition. At the end of each experiment, all tissue strips were treated with 10-mM papaverine and 10-mM nitroprusside to induce maximal relaxation.

In vivo Studies Adult male New Zealand White rabbits (4.0– 4.5 kg, Pine Acres) and Sprague-Dawley rats (250 g, Crl:CD strain, Charles River Laboratories, Wilmington, MA, USA) were used for in vivo studies. Animals were anesthetized with an intramuscular injection of ketamine (35 mg/kg for rabbits and 50 mg/kg for rats) and xylazine (5 mg/kg for rabbits and 8 mg/kg for rats), and were secured in the supine position. The carotid artery was exposed through a midline neck incision and was cannulated with an angiocatheter (20 gauge for rabbits and 24 gauge for rats). The angiocatheter was connected to a pressure transducer (Transpac IV, Abbott Laboratories, North Chicago, IL, USA) to measure systemic arterial blood pressure (SAP). To measure intracavernosal pressure (ICP) in rabbits, a 21-gauge minicatheter filled with heparinized saline was inserted into the cavernosal body near the base of the penis and was connected to a second pressure transducer. A 23-gauge minicatheter was used for rats. Intracavernosal and systemic arterial pressure were continuously recorded by means of pressure channel amplifiers in the Transonic BLF21D flowmeter (Transonic Systems, Inc., Ithaca, NY, USA) and Windaq software (Dataq Instruments, Akron, OH, USA). For both rabbits and rats, all test compounds were administered by intracavernosal injection to deliver the indicated dose in 0.1-mL total volume. For each animal, the response to vehicle was obtained prior to test compound. Administration of DP agonists began with the lowest dose and proceeded to higher doses, allowing 20–30 minutes between each dose. Data Analysis For organ bath studies, all responses were expressed as a percentage of relaxation caused by papaverine and nitroprusside. EC50 values were calculated using Prism software (GraphPad, San Diego, CA, USA). The total amount of relaxatory response over the range of drug concentrations tested was determined by the area over the plotted

curves (AOC). For erectile function studies, because penile ICP is ultimately limited by the systemic arterial pressure (SAP), all erectile responses were normalized by calculating the ratio of ICP/SAP. Using these normalized response curves, the area under the curve (AUC) was determined for each response using Windaq and Microsoft Excel (Microsoft Corp, Redmond, Washington, USA) software. In many instances, intracavernosal administration of DP agonists caused prolonged erectile responses that did not return to baseline before the next dose was administered. Thus, for all responses, the AUC was determined for a standard duration of 300 seconds. For final analysis of data, parameters were compared using analysis of variance (anova). If the anova P value was less than 0.05, paired posttest comparisons were carried out using the Tukey–Kramer test. All data were expressed as mean ⫾ SEM. Results

Pharmacological Characterization of Novel EP and DP Receptor Agonists Receptor-binding assays for over 520 EP2/EP4 agonists were determined, and a subset of the compounds used in these experiments was selected for evaluation in cavernosal tissue tension assays. The binding affinity (Ki) and cellular activity (EC50) for this subset of EP receptor agonists is shown in Table 1. Each compound exhibited greater selectivity for EP2 or EP4 receptors, compared with EP1 or EP3 receptors, and this chemical series demonstrated specificity for EP2/EP4 receptors relative to DP, FP, TP, and a set of GPCRs. In HEK cells that were not transfected with EP receptors, the EP receptor agonists were without effect in binding assays or cAMP stimulation. During the course of defining the properties of the EP pharmacophore, several molecules demonstrated increased activity on DP1 receptors. The activities of the best four molecules generated in this program are shown in Table 2. Compared with PGD2, AS702222 and AS702224 demonstrated very similar binding affinities for DP1 and DP2 receptors, with similar or improved ability to stimulate cAMP production in transfected cell systems. Compared with the reference molecule BW245C, AS702224 had very similar in vitro pharmacology. Functional Assessment of EP2 and EP4 Receptor Agonists by Organ Bath Assays Based upon cAMP assays, an array of agonists with varying EP2 and EP4 activities were evaluated for J Sex Med 2008;5:344–356

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Table 1 Chemical structures and biochemical properties of EP2/EP4-selective agonists evaluated for corpus cavernosal relaxation h-EP2 Compound

Structure

Ki(nM)

h-EP4 EC50(nM)

Ki(nM)

EC50(nM)

O COOH

PGE2

HO

24

1,973

4

49

53

97

5

0.3

141

41

2

1

1,932

482

177

32

9

36,630

26

1

3

0.3

5,326

3,611

29

0.15

18

23

183

21

56

30

1,977

312

HO

COOH

O N

AS701715

COOH

O N

AS701666

HO

COOH

O

AS701766

N

0.02

HO

O

AS701931

O O S N

COOH

182

N COOH

O

AS701724

N

HO COOH

O

AS701753

N N

Br HO

AS701740

COOH

O N

O

AS701919

N

S

COOH

their ability to relax human cavernosal tissue strips and compared with PGE1. In isolated human penile cavernosal tissue strips contracted with the a1-adrenergic receptor agonist phenylephrine, J Sex Med 2008;5:344–356

PGE1 caused substantial relaxation at doses ⱖ1 mM (from 60% to >80% relaxation), irrespective of the etiology of erectile dysfunction. Cumulative addition of vehicle had no effect on

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DP1 Agonists are Pro-Erectogenic Table 2 Chemical structures and biochemical properties of DP agonists evaluated for corpus cavernosal relaxation h-DP1 Compound

Structure

Ki(nM)

EC50(nM)

4.10

0.30

1.76

0.15

2.41

0.07

2.80

0.03

OH COOH

PGD2 O HO

COOH AS702222

O

N HO

COOH AS702224

O

N HO O

BW245C

HN O

COOH N HO

phenylephrine-induced contraction. Interestingly, none of the EP2/EP4-selective agonists were as effective as PGE1 in relaxing human cavernosal tissue (Figure 1 and Table 3). Further, there was no consistent association with EP receptor subtype specificity and efficacy in relaxing cavernosal tissue. For example, the most efficacious compound was AS701931 (EC50 = 1.7 mM). With respect to the EC50 values for cAMP stimulation, this compound activated EP2 receptors more efficiently than EP4 receptors by 20-fold. However, a compound with a similar receptor subtype selectivity profile (AS701919) had an EC50 of 63 mM in the organ bath assay, comparable with an EP4-selective compound (AS701753). Compounds with similar activation profiles for EP2 and EP4 receptors (AS701740 and AS701724) caused moderate relaxation or contraction. Limited experiments were performed on isolated tissue strips of rabbit penile corpus cavernosum. In contrast to human cavernosal tissue responses, PGE1 caused only contraction in rabbit cavernosal tissue strips. Surprisingly, most EP agonists did elicit slight to moderate relaxation responses in rabbit cavernosal tissue (data not

Figure 1 Effect of EP2 and EP4 receptor agonists on human penile cavernosal tissue tone. Organ bath preparations of isolated human penile cavernosal tissue strips were contracted with 1-mM phenylephrine. After the contractile response was stabilized, tissue strips were exposed to increasing concentrations of EP2 or EP4 receptor-selective agonists. Data are expressed as a percentage of phenylephrine-induced tone (mean ⫾ SEM). All dose responses were conducted in the presence of 3-mM indomethacin to inhibit the synthesis of endogenous prostanoids. Agonists are grouped in each panel by their relative potency, as determined by receptor-binding assays (see Tables 1 and 3).

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Table 3 Efficacy of EP agonists in relaxing corpus cavernosum tissue. Each EP agonist was evaluated in the organ bath assay, and the EC50 and area over the curve (AOC) were determined from relaxation response curves (e.g., Figure 1). Absent EC50 and positive AOC value indicates that a contractile response was obtained. For human tissue, dose responses were performed in “N” patients using two to four tissue strips from each patient Human tissue

Relative potency for EP2*

Compound

EC50

AOC

N

Subtype selectivity

EC50

Ki

PGE1 AS701766 AS701753 AS701715 AS701666 AS701724 AS701740 AS701919 AS701931

0.23 mM 259.6 mM 64.86 mM 16.64 mM 22.36 mM — 13.85 mM 62.66 mM 1.69 mM

-370 -174 -84 -72 -139 +108 -81 -54 -235

6 5 3 6 5 5 6 5 4

ND EP4 EP4 EP4 EP4 EP4 — EP2 EP2

ND 24,100 18,055 359 41 5 1 0.1 0.05

ND 11 183 11 71 9 0.12 0.03 0.0008

*Relative potency is expressed as the ratio of EP2/EP4 for EC50 and Ki parameters in Table 1. Thus, AS701666 has a 41-fold larger EC50 and a 71-fold larger Ki for EP2 relative to EP4, indicating a higher preference for EP4 receptors. Similarly, AS701919 has a 10-fold smaller EC50 and a 33-fold smaller Ki for EP2 relative to EP4, indicating a higher preference for EP2 receptors. ND = not determined.

shown). In addition, the most efficacious compounds (AS701766 and AS701666) had EC50 values that were 2–3 orders of magnitude lower than in human tissue strips and exhibited preferential activity with EP4 receptors. However, similar to human tissue, no consistent correlation was observed between tissue relaxation parameters and receptor subtype affinity or potency of the EP agonists.

Functional Assessment of DP Receptor Agonists by Organ Bath Assays In human cavernosal tissue strips, the naturally occurring prostanoid PGD2 caused relaxation up to 0.3 mM, and then caused contraction at higher concentrations (Figure 2). DP1 receptor agonists BW245C and AS702224 caused dose-dependent relaxation with similar potency and no apparent contractile activity in human tissue (Table 4 and Figure 2). The extent of relaxation (maximal effect) was greater for AS702224 than BW245C, as reflected by the higher AOC value (Table 4). Importantly, the efficacy of both AS702224 (EC50 = 29 nM) and BW245C (EC50 = 59 nM) was greater than PGE1 (EC50 = 230 nM). In isolated tissue strips of rabbit penile corpus cavernosum, PGD2 caused only contraction within the range of doses tested (Figure 3). However, AS702224 (EC50 = 6.6 mM) and BW245C (EC50 = 0.6 mM) both caused dose-dependent relaxation in rabbit cavernosal tissue (Figure 3 and Table 4). While these DP1-selective agonists were less effective in relaxing rabbit tissue than human tissue, the EC50 and AOC values obtained in rabbit tissue indicate that they produced better responses J Sex Med 2008;5:344–356

than many of the EP agonists in either rabbit or human tissue. Similar to the human tissue studies, cumulative addition of vehicle did not change rabbit cavernosal tissue tone.

In vivo Assessment of Erectile Activity The response profile for DP1 receptor agonists in the organ bath studies formed the rationale for further in vivo evaluation of these compounds in the rabbit. Because the rat has also been used extensively for studying erectile physiology, we also evaluated the in vivo efficacy of DP1 receptor agonists in eliciting erection in rats. In rabbits, intracavernosal injection of BW245C or AS702224 caused either sustained periods of penile tumescence or transient erectile episodes that lasted for 1–3 minutes. In one rabbit, repeated and transient episodes of spontaneous penile tumescence were recorded after intracavernosal administration of the highest dose of AS702224. In a separate animal, intracavernosal injection of the highest dose of AS702224 resulted in the recording of arterial pressure waves that were clearly transmitted into the cavernosal compartment, indicating that the penile resistance arteries were fully dilated. Compared with vehicle, BW245C and AS702224 consistently caused dose-dependent increases in the ICP, as reflected by the peak response or the AUC (Figure 4A–D). Although PGD2 did not cause relaxation of rabbit cavernosal tissue in the organ bath assay (Figure 3), intracavernosal administration of PGD2 did result in erectile activity in rabbits. However, unlike the monophasic response seen with BW245C and AS702224 (increase followed

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Table 4 Relative efficacy of DP receptor agonists in relaxing human and rabbit corpus cavernosum. Each DP agonist was evaluated in the organ bath assay, and the EC50 and area over the curve (AOC) were determined from relaxation response curves (see Figures 2 and 3). Absent EC50 and positive AOC values indicate that a contractile response was obtained. For human tissue, dose responses were performed in “N” patients using two to four tissue strips from each patient. For rabbit studies, dose responses were performed in “N” tissue strips. Human tissue

Rabbit tissue

Compound

EC50

AOC

N

EC50

AOC

N

PGD2 BW245C AS702224

58.8 nM* 59.6 nM 29.2 nM

-288 -462 -519

5 3 6

— 0.60 mM 6.66 mM

+40 -326 -220

4 5 5

*EC50 is determined only from the relaxation phase of the PGD2 response curve (see Figure 2).

by plateau), PGD2 caused multiphasic ICP responses with multiple “peaks and valleys” or stepwise increases prior to reaching a sustained plateau phase. The mean increase in AUC for PGD2 approached but did not reach statistical significance when compared with vehicle (anova P value = 0.0574; Figure 4E,F). Intracavernosal injection of BW245C and AS702224 in rats also resulted in robust erectile activity that was significantly greater than vehicle (Figure 5). The dose-dependent response observed for both DP1 agonists was consistent in all rats evaluated.

Figure 2 Effect of DP receptor agonists on human penile cavernosal tissue tone. Organ bath preparations of isolated human penile cavernosal tissue strips were contracted with 1-mM phenylephrine and were exposed to increasing concentrations of DP receptor agonists. Data are expressed as a percentage of phenylephrine-induced tone (mean ⫾ SEM). All dose responses were conducted in the presence of 3-mM indomethacin to inhibit the synthesis of endogenous prostanoids. Additional data are summarized in Table 4.

Figure 3 Effect of DP agonists on rabbit penile cavernosal tissue tone. Organ bath preparations of isolated rabbit penile cavernosal tissue strips were contracted with 1-mM phenylephrine and were exposed to increasing concentrations of DP receptor agonists. Data are expressed as a percentage of phenylephrine-induced tone (mean ⫾ SEM). All dose responses were conducted in the presence of 3-mM indomethacin to inhibit the synthesis of endogenous prostanoids. Additional data are summarized in Table 4.

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Figure 4 Erectile response to DP receptor agonist administration in rabbits. Penile intracavernosal blood pressure (ICP) and systemic arterial blood pressure (SAP) was recorded in anesthetized rabbits. Vehicle or increasing concentrations of BW245C (N = 6), AS702224 (N = 6), or PGD2 (N = 5) were administered by intracavernosal injection, allowing 20–30 minutes between each injection. Data are expressed as the mean ⫾ SEM of the response peak (ICP/SAP ratio; left panels) and the area under the curve (AUC; right panels). For all responses, the AUC was determined for a standard duration of 300 seconds. Responses were analyzed by analysis of variance and posttest comparisons were carried out using the Tukey– Kramer test (*P < 0.05 vs. vehicle; † P < 0.05 vs. 1 and 2.5-mg doses).

Effects of DP1 Agonists on Blood Pressure To investigate a possible systemic vascular response, PGD2 was administered to the rabbits intravenously. Intravenous administration of PGD2 (100 mg) caused an initial transient decrease (mean response = -5.9 ⫾ 2.3 mm Hg) followed by a sustained elevation of systemic blood pressure (mean response = +8.7 ⫾ 5.7 mm Hg). In rabbits, no overt changes in systemic blood pressure were noted after intracavernosal administration of any dose of AS702224 or BW245C. A gradual and modest decline in systemic blood pressure over the course of each study was noted in most but not all rabbits. However, this decline is commonly observed in anesthetized animals even when no vasodilators are administered. The effect of AS702224 on systemic blood pressure was further examined by intravenous administration. At a dose of 100 mg, intravenous AS702224 had no significant impact on systemic blood pressure. In contrast, rats exhibited significant declines in systemic blood pressure that ultimately attenuated the ICP response. However, it should be noted that identical doses (1–10 mg) and injection J Sex Med 2008;5:344–356

volumes (100 mL) were used in both rabbits and rats. Thus, the concentration of DP agonists would have been higher in rats. Discussion

Studies examining the vasodilatory action of prostanoids in penile tissue have almost exclusively been focused on the effects of EP receptor agonists. Because of the widespread use of intracavernosal and intraurethral PGE1 formulations for the treatment of erectile dysfunction, there is continued interest in the role of EP receptors in erectile physiology. Given the pharmacological characterization of EP2 and EP4 receptors being coupled to adenylyl cyclase activation, agonists with superior subtype selectivity for these receptors over other contractile prostanoid receptors (EP1, EP3, FP, IP, and TP) would appear to be excellent candidates as pro-erectile agents with greater potency and efficacy. However, in our study, none of the EP2/EP4-selective AS compounds was as effective as PGE1 in relaxing human cavernosal tissue. These data suggest that

DP1 Agonists are Pro-Erectogenic

Figure 5 Erectile response to DP receptor agonist administration in rats. Penile tumescence in response to intracavernosal administration of vehicle or increasing concentrations of BW245C (N = 4) or AS702224 (N = 3) was recorded (see Figure 4) in anesthetized rats. Data are expressed as the area under the curve (AUC; mean ⫾ SEM). For all responses, the AUC was determined for a standard duration of 300 seconds. Responses were analyzed by analysis of variance and posttest comparisons were carried out using the Tukey–Kramer test (*P < 0.05 vs. vehicle; †P < 0.05 vs. 1-mg dose).

neither EP2 nor EP4 individually mediates the relaxation response to PGE1. It is possible that both receptor subtypes act in an additive or synergistic fashion to produce the response to PGE1. However, even the AS compounds that activated EP2 and EP4 receptors with similar potency did not approach the efficacy of PGE1 in relaxing human cavernosal tissue. In a previous study, expression of messenges ribonucleic acid for both EP3-type I (adenylyl cyclase inhibiting) and EP3-type II (adenylyl cyclase stimulating) receptors was detected in human corpus cavernosum [10]. Thus, it remains unclear whether PGE1 mediates its vasodilatory effects in the penile corpus cavernosum by acting exclusively through EP2/EP4 receptors or whether the

353 activation of other prostanoid receptors is necessary. Nevertheless, our data indicate that functional responses of corpus cavernosal tissue to PGE1 are species-dependent (relaxatory in human but not rabbit), and suggest that any given EP receptor subtype is not likely to be solely responsible for prostanoid-mediated relaxation of this tissue. The consistent activity of BW 245C and the novel DP1 receptor agonist AS702224 in pharmacological, ex vivo, and in vivo assays suggests that the DP1 receptor is present in penile tissue of rats and rabbits, as well as humans. More specifically, these data suggest that DP1 receptors mediate cavernosal smooth muscle relaxation by stimulating the production of cAMP in multiple species to initiate and sustain penile erection in the absence of neural stimulation. In contrast, the endogenous prostanoid PGD2 induced dose-dependent relaxation and contraction in human cavernosal tissue, and only contraction in rabbit cavernosal tissue. Because PGD2 binds both DP1 and DP2 receptors, this suggests that DP1 receptors may be more prevalent in human cavernosal tissue, whereas DP2 receptors may be more prevalent in rabbit cavernosal tissue. The antagonistic actions of DP1 and DP2 receptors on smooth muscle contractility may account for the decreased extent of relaxation (lower AOC value) induced by PGD2 when compared with the DP1-selective agonists. Despite the poor contractile response observed in the organ bath assays, it is interesting to note that intracavernosal administration of PGD2 in rabbits did elicit penile tumescent activity. While this erectile response approached but did not reach statistical significance over vehicle-induced tumescence, the mean response values were comparable with those obtained with intracavernosal BW245C. The distribution of prostanoid receptors may be different in human penile resistance arteries, compared with human cavernosal tissue [8]. Thus, it is possible that PGD2 may have different reactivity in the blood vessels of the penile circulation (either arteries or veins). PGD2 may have caused vasoconstriction in the draining venules of the penile circulation to initiate penile tumescence. Alternatively, retrograde perfusion of PGD2 from the injection may have caused arterial dilation to initiate penile erection. While there is no data on the reactivity of penile blood vessels to PGD2, the multiphasic ICP responses observed in rabbits, coupled with the fact that intravenous administration of PGD2 causes a sustained elevation of systemic blood pressure, suggest that mulJ Sex Med 2008;5:344–356

354 tiple and opposing responses may be mediated by the various DP receptor subtypes in various blood vessels and cavernosal tissue in the rabbit. Similar considerations of species-specific, differential receptor distribution between penile blood vessels and cavernosal tissue may also be relevant for EP receptors. While we did not observe rabbit cavernosal tissue relaxation in response to PGE1 in our assays, limited studies have demonstrated penile tumescence after intracavernosal injection of PGE1 in rabbits [12,13]. These erectile responses were suppressed by a cAMP-dependent protein kinase antagonist. These findings, in conjunction with our DP1 agonist data, highlight the possibility of inconsistent activity between ex vivo and in vivo assays when studying prostanoid compounds. The distribution of prostanoid receptor subtypes in penile tissues of various species remains unknown. Thus, isolated cavernosal tissue strip reactivity is not necessarily predictive of erectile response in vivo. In studying prostanoid regulation of penile physiology, the use of multiple assay systems is recommended. Systemic blood pressure remained largely unchanged after administration of DP1-selective agonists. No overt changes in arterial blood pressure were noted in rabbits after administration of any dose of AS702224 (either intracavernosal or intravenous). Thus, while the influence of anesthesia may mask the cardiovascular response to a vasoactive drug, it is unlikely that intracavernosal injection of AS702224 would have a significant impact upon systemic blood pressure in the rabbit. Because identical doses and volumes were used in both rabbits and rats, the hypotensive response observed in rats is probably due to the higher resulting concentration of DP1 agonist in a smaller animal with lower blood volume. Also, it is likely that more of the drug would have leaked out of the cavernosal sinusoids and into the general venous circulation during intracavernosal injection because of the smaller capacity of the rat penis. Nevertheless, significant in vivo pro-erectile activity was induced by AS702224 in both rabbits and rats. If animal size and blood volume were taken into consideration for dosing, drug concentrations that are adequate to elicit penile erection are unlikely to cause adverse effects on systemic blood flow. Further development of DP1 agonists as intracavernosal agents may provide an effective therapeutic option for the treatment of men with erectile dysfunction. This strategy seems particuJ Sex Med 2008;5:344–356

Brugger et al. larly advantageous given the greater functional potency of DP1-selective agonists over PGE1 in relaxing human cavernosal tissue. In addition, intracavernosal or intraurethral PGE1 formulations may cause mild to severe discomfort at a rate of approximately 10% of all self-administration events in a significant population of men (32–58%) with erectile dysfunction [14–18]. In particular, men with diabetes or those who have had radical prostatectomy surgery appear to be the most susceptible to experiencing pain or discomfort upon PGE1 administration [19–22]. Because of the numerous and diverse biological actions of prostanoids, the molecular mechanisms involved in pain remain unclear. However, all four EP receptor subtypes have been implicated in various models of hyperalgesia (mechanical, thermal, inflammatory, and neuropathic) in various sensory nerves [2,23–31]. In contrast, PGD2 has been shown to inhibit allodynia caused by intrathecal administration of PGE2 or the peptide nociceptin/ orphanin FQ [32,33]. Thus, DP1-selective agonists may offer an alternative to PGE1 with less potential to cause discomfort or pain associated with intracavernosal injection. In summary, two different nonhuman species have been identified as useful models for investigating the role of DP1 receptors in erectile physiology. In addition, intracavernosally administered AS702224 is a potent DP1-selective agonist that may prove efficacious in initiating penile erection in humans. Further development of DP1 receptor agonists as intracavernosal agents may potentially provide a more desirable alternative to intracavernosal PGE1.

Nomenclature

PGE PGD EP DP FP IP TP cAMP HEK BES DMEM DMSO EDTA MES PEI

prostaglandin E prostaglandin D PGE receptor PGD receptor prostaglandin F receptor prostaglandin I (prostacyclin) receptor thromboxane receptor adenosine 3′:5′-cyclic monophosphate human embryonic kidney cell line N,N-Bis(2-hydroxyethyl)-2aminoethanesulfonic acid Dulbecco’s modified Eagle’s medium dimethyl sulfoxide ethylene diamine tetracetic acid 2-(N-morpholino)ethanesulfonic acid polyethyleneimine

355

DP1 Agonists are Pro-Erectogenic Acknowledgments

We thank Drs. Irwin Goldstein and Ricardo Munarriz for procuring the human cavernosal tissue used in these studies. We also acknowledge Andrew Nisbet and Abdullah Armagan for their assistance with the in vivo studies. This work was supported by a grant from the Serono Reproductive Biology Institute, Serono International S.A. and by funds from the Institute for Sexual Medicine at Boston University. Corresponding Author: Stephen S. Palmer, PhD, EMD Serono Research Institute, Reproductive Biology, 1 Technology Place, Rockland, MA 02370, USA. Tel: 781-681-2795; Fax: 781-681-2910; E-mail: stephen. [email protected] Conflict of Interest: N. Brugger, G. Araldi, and S.S. Palmer are employees of Serono, the company that developed the compounds used in this study. N. Kim and A. Traish received grant support from Serono to perform this research. References

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