PHARMACOLOGICAL AND FUNCTIONAL CHARACTERIZATION OF NOVEL EP AND DP RECEPTOR AGONISTS: DP1 RECEPTOR MEDIATES PENILE ERECTION IN MULTIPLE SPECIES

Vol. 179, No. 4, Supplement, Monday, May 19, 2008

802 VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) GENE THERAPY USING NANOMOLECULAR TRANSFECTION TECHNOLOGY IN COMBINATION WITH DAILY, ORAL SILDENAFIL NORMALIZES ERECTILE FUNCTION IN A DIABETIC RAT MODEL Joseph E Dall’Era*, Randall B Meacham, Soren N Carlsen, Sweaty Koul, Jesse Mills, Hari K Koul. Denver, CO. INTRODUCTION AND OBJECTIVE: Current treatment modalities for erectile dysfunction (ED), including oral phosphodiesterase-5 (PDE-5) inhibitors are ineffective in a large percentage of diabetic men. We have previously shown that intracavernosal gene therapy with VEGF in a diabetic rat model improves erectile function by improving endothelial FHOO IXQFWLRQ :H K\SRWKHVL]H WKDW XWLOL]DWLRQ RI D QDQRPROHFXODU transfection reagent to overexpress VEGF in the diabetic rat penis will enhance the response to oral PDE-5 inhibitors. METHODS: A total of 35, 6-month-old male Sprague-Dawley UDWVZHUHGLYLGHGLQWRJURXSVKHDOWK\FRQWUROVVWUHSWR]RWRFLQ67=  induced diabetics, diabetics treated by transfection with VEGF, diabetics WUHDWHGZLWKGDLO\RUDOVLOGHQD¿ODGPLQLVWUDWLRQDQGGLDEHWLFVWUHDWHGZLWK DFRPELQDWLRQRI9(*)WUDQVIHFWLRQDQGGDLO\VLOGHQD¿O7UDQVIHFWLRQ was performed using a linear, polyethylenimine nanomolecular transfection reagent. Functional studies using cavernous nerve stimulation were performed 3 weeks after the initiation of treatment. 7KHUDWVZHUHVDFUL¿FHGDQGSHQLOHWLVVXHFROOHFWHGIRUPROHFXODUDQG immunohistochemical studies. RESULTS: The mean maximal intracavernous pressure in WKHGLDEHWLFJURXSPP+J ZDVVLJQL¿FDQWO\ORZHUWKDQWKHQRUPDO controls (76 mm Hg) and the diabetic group receiving the VEGF gene therapy (47 mm Hg). The mean maximal ICPs for rats receiving daily VLOGHQD¿ORUDFRPELQDWLRQRI9(*)JHQHWKHUDS\DQGGDLO\VLOGHQD¿O were 51 mm Hg and 71 mm Hg, respectively. Expression of VEGF was FRQ¿UPHGZLWKSRO\PHUDVHFKDLQUHDFWLRQDQGLPPXQRKLVWRFKHPLVWU\ CONCLUSIONS: Transfection of VEGF using this nanomolecular technology in a diabetic rat model improves the UHVSRQVHWRGDLO\RUDOVLOGHQD¿O$FRPELQDWLRQRIWKHVHWZRWKHUDSLHV UHVXOWVLQDQHDUQRUPDOL]DWLRQRIHUHFWLOHIXQFWLRQLQWKLVDQLPDOPRGHO of diabetic ED. Source of Funding: None

803 PUTATIVE MECHANISMS OF SILDENAFIL’S PREVENTION OF PRIAPISM IN TRANSGENIC SICKLE CELL MICE: ROLE OF eNOS UNCOUPLING AND PHOSPHORYLATION Trinity J Bivalacqua*, Biljana Musicki, Hunter C Champion, Arthur L Burnett. Baltimore, MD. INTRODUCTION AND OBJECTIVE: Mechanisms responsible for “nitric oxide (NO) imbalance”, in reference to deranged NO signaling, in sickle cell-associated priapism are not fully understood. Our objective was to identify putative mechanisms by which phosphodiesterase type 5 (PDE5) inhibitor therapy, resulting in improved endothelium-derived NO biosynthesis, may treat recurrent priapism related to sickle cell disease. METHODS: Transgenic sickle cell (SS) mice, expressing KXPDQ VLFNOH KHPRJORELQ ZHUH XWLOL]HG )LYH JURXSV ZHUH XVHG   ZLOG W\SH :7    VLFNOH FHOO KHWHUR]\JRWHV 66    VLFNOH FHOO KRPR]\JRWHV66  :7VLOGHQD¿OPJNJSHUGD\[ZNV  DQG 66VLOGHQD¿O*URXSVXQGHUZHQWFDYHUQRXVQHUYHVWLPXODWLRQ (CNS) to assess erectile function. The frequency of erectile responses (erections/hr) pre- and post-stimulation was determined. Constitutive and inducible nitric oxide synthase (NOS), endothelial NOS (eNOS) uncoupling, total and phosphorylated eNOS (p-eNOS, Ser-1177), cGMP, and PDE5 activities were determined. RESULTS: Erectile responses to CNS and spontaneous HUHFWLRQVSUHDQGSRVW&16ZHUHVLJQL¿FDQWO\HQKDQFHG3 LQ66 -/- when compared to SS +/- and WT. cNOS, cGMP, and PDE5 activities were decreased (P<0.05) in SS-/- mice penes. iNOS activity was unchanged between groups. Active eNOS dimer vs. inactive monomer (ratio 1:10) was reduced in SS-/- mouse penes compared to WT and SS+/- mice (P<0.05). Additionally, p-eNOS (Ser-1177) was decreased 3 LQ66PLFHSHQHV66PLFHWUHDWHGZLWKVLOGHQD¿OKDGD

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reduction in spontaneous erections and reduced erectile responses, ZKLOHVLOGHQD¿OWKHUDS\KDGQRHIIHFWRQ:76LOGHQD¿OWKHUDS\UHVWRUHG cNOS, cGMP, PDE5 activity, prevented eNOS uncoupling, and directly phosphorylated eNOS (Ser-1177) in SS -/- mice penes. CONCLUSIONS: Priapic activity in SS -/- mice is associated ZLWKDUHGXFWLRQLQ1263'(H[SUHVVLRQDVZHOODVVLJQL¿FDQWH126 uncoupling and reduced p-eNOS (Ser-1177). Chronic PDE5 inhibitor therapy restored NO/cGMP/PDE5 signaling cascade in SS-/- mice penes via direct phosphorylation of eNOS (activated form of eNOS) DQGSUHYHQWLRQRIIXQFWLRQDOXQFRXSOLQJRIWKHH126HQ]\PH7KHVH PROHFXODU ¿QGLQJV VXJJHVW WKDW JOREDO G\VUHJXODWLRQ RI HQGRWKHOLDO derived NO occurs in sickle-cell disease related priapism, further highlighting the importance of NO in maintaining penile vascular homeostasis. Source of Funding: AUA Foundation Astellas USA Foundation MD/PhD Fellowship.

804 PHARMACOLOGICAL AND FUNCTIONAL CHARACTERIZATION OF NOVEL EP AND DP RECEPTOR AGONISTS: DP1 RECEPTOR MEDIATES PENILE ERECTION IN MULTIPLE SPECIES Nadia Brugger, Noel N Kim*, Gian Luca Araldi, Abdulmaged M Traish, Stephen Palmer. Rockland, MA, San Diego, CA, and Boston, MA. INTRODUCTION AND OBJECTIVE: Despite the widespread use of prostaglandin E1 DV DQ HI¿FDFLRXV WUHDWPHQW IRU PDOH HUHFWLOH dysfunction for more than two decades, research on prostanoid IXQFWLRQLQSHQLOHSK\VLRORJ\KDVEHHQOLPLWHG:HFKDUDFWHUL]HGWKH pharmacological and physiological activity of novel subtype-selective EP and DP receptor agonists. METHODS: Radioligand binding and second messenger DVVD\VZHUHXVHGWRGH¿QHUHFHSWRUVXEW\SHVSHFL¿FLW\RIWKH(3DQG '3DJRQLVWV)XQFWLRQDODFWLYLW\ZDVIXUWKHUFKDUDFWHUL]HGXVLQJLVRODWHG human and rabbit penile cavernosal tissue in organ baths. In vivo activity on erectile function was assessed in rabbits and rats. RESULTS: In organ bath preparations of human cavernosal tissue contracted with phenylephrine, EP2 and EP4-selective 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=0.23µM). There was no consistent correlation between the SKDUPDFRORJLFDOSUR¿OHUHFHSWRUELQGLQJDQGVHFRQGPHVVHQJHUDVVD\V  of the EP agonists and their effect on cavernosal tissue tone. In contrast, the DP1-selective agonist AS702224 (EC50=29nM) was more effective in relaxing human cavernosal tissue than either PGE1, PGD2 (EC50=58nM) or the DP agonist BW245C (EC50=59nM). 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 ZDVVLJQL¿FDQWO\KLJKHUWKDQYHKLFOHDORQH 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. Source of Funding: EMD Serono Research Institute and Boston University Institute for Sexual Medicine.

805 EARLY INDUCTION OF ERECTILE DYSFUNCTION BY ANGIOTENSIN II IN RAT Frederic Mampouma*, Taben M Hale, Serge Carrier, Denis deBlois. Montreal, QC, Canada. INTRODUCTION AND OBJECTIVE: Erectile dysfunction (ED) has been suggested to be an early indicator of cardiovascular disease. On the other hand, angiotensin II (AngII) receptor AT1 blockers, used for the treatment of hypertension, appear to improve erectile function. Whether elevated levels of AngII are involved in the pathophysiology of ED has not yet been addressed directly. The current study aims to