The Penile Erection Efficacy of a New Phosphodiesterase Type 5 Inhibitor, Mirodenafil (SK3530), in Rabbits with Acute Spinal Cord Injury

FULL PAPER Pharmacology

Ji-Youn JUNG1), Sang-Ki KIM1), Byeong-Soo KIM1), Seung-Ho LEE1), Young-Seok PARK1), So-Jung KIM1), Changsun CHOI2), Seong-Il YOON3), Jong-Suk KIM4), Sung-Dae CHO5), Gwang-Jin IM6), Soo-Min LEE6), Ji-Won JUNG7) and Yong-Soon LEE7)*

1)Department of Companion and Laboratory Animal Science, Kongju National University, Yesan, 2)Department of Food and Nutrition, College of Human Ecology, Chung-Ang University, Ansung, 3)Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 4)Department of Biochemistry, Institute of Medical Science, Chunbuk National University Medical School, Jeonju, 5)Department of Oral Pathology, School of Dentistry, Chonbuk National University, Jeonju, 6)Life Science Research Center, SK Chemicals, Suwon and 7)Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, Seoul, Korea

(Received 8 April 2008/Accepted 26 May 2008)

ABSTRACT. Mirodenafil (SK3530) is a new potent and selective inhibitor of cGMP-specific phosphodiesterase type 5 (PDE5). Recent clinical trials have demonstrated that mirodenafil is an effective treatment for erectile dysfunction. Its mechanism of action is enhancement of nitric oxide (NO) induced cGMP formation resulting in significant relaxation of the corpus cavernosum (CC). The aim of this study was to investigate the oral efficacy of mirodenafil in an acute spinal cord-injured rabbit model. Mirodenafil or sildenafil citrate was given orally to male rabbits with a surgical transection of the spinal cord at the L2-L4 lumbar vertebra or ischemic-reperfusion spinal cord injury (SCI). Erections were evaluated in a time-course manner by measuring the length of the uncovered penile mucosa. In the transection SCI model, penile erections were induced at 0.3, 1 and 3 mg/kg of mirodenafil but sildenafil only showed an erectile response at 3 mg/kg. The effects of 1 and 3 mg/kg of mirodenafil were significantly increased by intravenous injection of sodium nitroprusside (SNP), a nitric oxide donor. In the ischemic-reperfusion injury model, 3 mg/kg of either mirodenafil or sildenafil produced a penile erection response. After injection of SNP, the lengths of immediate penile erections were significantly increased in the 1 and 3 mg/kg mirodenafil and 3 mg/kg sildenafil groups. The onset of erectile activity was faster with mirodenafil than with sildenafil citrate. These results dem- onstrate that mirodenafil may be useful for treating erectile dysfunction in patients with a spinal cord injury. KEY WORDS: mirodenafil, penile erection, phosphodiesterase inhibitor, SK3530, spinal cord injury. J. Vet. Med. Sci. 70(11): 1199–1204, 2008

The advent of phosphodiesterase type 5 (PDE5) inhibi- receive effective treatment. The process of care model des- tors as oral therapy for erectile dysfunction (ED) ushered in ignates counseling, vacuum pump, and PDE 5 inhibitors a revolution in clinical management of this condition. Oral such as sildenafil as first-line options for ED in the primary ED medications influence the complex neurological, vascu- care setting [12]. lar and humoral process underlying penile erection. PDE5 From the neurological perspective, penile erection is inhibitors influence local regulatory mechanisms of erec- caused by a change in activity of efferent autonomic path- tion, potentiating the smooth muscle relaxing effects of ways to erectile tissues and somatic pathways to perineal nitric oxide (NO) on resistance arteries and trabecular striated muscles [3]. The spinal cord contains the cell bod- smooth muscle within the corpus cavernosum [4]. ies of autonomic and somatic motoneurons that innervate Across the world, approximately 152 million men have peripheral targets. Sympathetic outflow is mainly anti-erec- ED, including about 31 million men in Europe and 18 mil- tile, sacral parasympathetic outflow is pro-erectile and lion men in the US [6, 24]. With anticipated increases in the pudendal outflow, through contraction of perineal striated median ages of Western industrialized societies and popula- muscles, enhances an already present erection [13, 18]. The tion growth in developing nations, the number of cases is parts of the spinal network that control erection consist of projected to increase by about 170 million worldwide over neurons in the spinal midbrain and forebrain nuclei after the next 25 years. A wide variety of treatment options are mating, a condition that recruits and sensory genital affer- now available for patients with ED, ranging from hormone ence [11]. In all mammals studied so far, the main pro-erec- replacement, counseling, non-invasive devices and oral tile peripheral pathway is represented by the pelvic and therapies to injectable agents, penile implants and vascular cavernous nerve. Electrical stimulation applied on the sac- surgery. As a result, almost all men with ED of any type can ral root or pelvic and cavernous nerves in conscious and anesthetized animals elicits, depending on the recordings *CORRESPONDENCE TO: LEE, Y.-S., Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National Univer- performed, visible penile erection, increased penile volume, sity, San 56–1 Shillim-Dong, Kwanak-gu, Seoul 151–742, South engorgement of the penis with blood, increased blood leak- Korea. age from the sectioned penis and increased intracavernous e-mail: [email protected] or [email protected] pressure [31]. 1200 J-Y. JUNG ET AL.

Spinal cord injury (SCI) results in injury to both neural The aim of this study was to assess the effect of a selec- and vascular elements. The extent to which the vascular tive PDE5 inhibitor, mirodenafil (SK3530), in rabbits with injury contributes to secondary pathogenesis is dependent acute spinal cord injury after oral administration and to on not only the initial disruption of blood vessels, leading to compare it with sildenafil as a positive control. prominent intraparenchymal hemorrhage, but also progres- sive disruption of the blood-spinal cord barrier coincident MATERIALS AND METHODS with infiltration of inflammatory cells. These events influence both acutely and chronically injured spinal cords and Test materials: Mirodenafil (SK3530) was synthesized define, in part, the extent of functional recovery. Erectile by SK Chemicals (Suwon, South Korea) with >99.8% purity dysfunction is a common complication in spinal cord injury as determined by both HPLC and potentiometric titrations in patients. Experimental models of spinal cord injury include glacial acetic acid. The mirodenafil was stable for at least contusions, compression, ischemia and crush injuries. Each 12 weeks at both ambient temperature and under accelerated model generates injuries that mimic certain clinical aspects conditions (20°C, 75% RH). Sodium nitroprusside (SNP) of mechanical damage and the ensuing posttraumatic was obtained from Sigma-Aldrich Chemical Co. (St. Louis, ischemia. Researchers have also used either transection or MO, U.S.A.). Sildenafil citrate (Viagra®) was purchased hemisection to study injury. Transection and hemisection from Pfizer Inc. (Pfizer, New York, NY, U.S.A.). are less clinically relevant but offer the distinct advantage of Mirodenafil and sildenafil were dissolved in saline for consistent reproducibility. The most sensitive organ is the administration. spinal cord, and ischemia can produce paraparesis or para- Animals: The studies were performed in accordance with plegia after operation on the thoracic aorta. In this experi- the “Guide for the Care and Use of Laboratory Animals” ment, spinal cord injury was induced by the ischemic- from the National Institute of Health. New Zealand white reperfusion and transection method. rabbits (Yonam Laboratory Animals, Cheonan, South SK Chemicals has recently developed a PDE5 inhibitor, Korea) weighing an average of 2.5–3.0 kg were used in this mirodenafil (SK3530), for use as a potential oral drug for study. The animals were acclimatized for a week and treatment of ED. Mirodenafil, 5-ethyl-2-{5-[4-(2-hydroxy- housed individually. During all experiments, the animals ethyl)-piperazine-1-sulfonyl]-2-propoxy-phenyl}-7-propyl- were kept in a standard laboratory conditioned room (tem- 3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (Fig. 1), is a perature 23 ± 2°C, humidity range 40–70%, 12 hr light/dark new potent and selective inhibitor of cGMP-specific PDE5 cycle) and were allowed free access to food and UV-steril- that has pKa value of 5.99 and log partition coefficient ized tap water ad libitum. (octanol/water) of 3.67 [20]. It differs from sildenafil in the Surgical procedure for the transection SCI model: Five N- or O-side chain and has a dihydropyrrole ring instead of rabbits per group underwent the same anesthetic manage- a pyrazole ring. These modifications confer a more potent ment, and an ear vein catheter (22 gauge) was placed to and selective inhibitory effect on PDE5 to mirodenafil than administer additional medications and intravenous fluids. those of sildenafil [26]. Pharmacokinetic data for mirodena- Briefly, the lumbodorsal fascia region was exposed and cut fil suggests that it is relatively well absorbed in the gas- at the midline of the spinous processes after a dorsal skin trointestinal tract and shows linear pharmacokinetics over incision of the L2-L4 region. The paraspinous muscle was the investigated dose range [32]. Based on unpublished then separated from the spinous process and lamina. Bone data, mirodenafil for which is currently under phase III clin- extending out laterally from the spinous process was ical investigation, appears to be safe and effective for treat- removed with a rongeur (laminectomy). Once the spinal ment of male ED [25, 26, 32]. cord was exposed, spinal cord transection was performed using fine cut scissors or a blade between the L2-L3 and L3- L4 region. A single dose of 30 mg/kg of ceftazimine was then injected intramuscularly. Surgical procedure for the ischemic-reperfusion SCI model: Acute spinal cord injury was induced by the aortic cross-clamping method as described previously [27, 30]. Five rabbits were used in each dosing group. Each animal was anesthetized with an intramuscular injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). An ear vein catheter (22 gauge) was placed for administration of additional medications and intravenous fluids. Heparin at a dose of 150 IU/ kg was given intravenously and allowed to circulate for 5 min followed by infusion of 0.9% NaCl solution at a rate of 25 ml/hr. The abdomen was sterilely prepared and draped. A midline laparotomy was performed and the vicera were reflected to the right. The abdominal aorta was then Fig. 1. Chemical structure of mirodenafil (SK3530). clamped with vascular clamps. One clamp was positioned PENILE ERECTION EFFICACY OF MIRODENAFIL 1201 just below the left renal artery and another was placed at the aortoiliac bifurcation. Each animal underwent 40 min of spinal cord ischemia. Collective clamping of the great vessels produced spinal cord ischemia in the rabbits and after 40 min all control animals were rendered paraplegic [9, 22]. Moreover, the reliability and reproducibility of this procedure were good as previously reported [1]. Neurologic examination: Twenty-four and forty-eight hours after the procedures, neurologic status was evaluated using Tarlov’s scoring system. The criteria were as follows: T0, spastic paraplegia with no movement of the hind limbs; T1, spastic paraplegia with slight movement of the hind limbs; T2, good movement of the hind limbs but unable to stand; T3, able to stand, but unable to walk normally; and T4, complete recovery. Fig. 2. Effects of oral administration of mirodenafil or sildenafil Penile erection test: Penile erection testing was per- on length of the exposed penile mucosa in the transection SCI formed in conscious rabbits [9, 27]. The animals showing a model. SNP was injected intravenously 60 min after drug Tarlov’s score of T0 were used for the penile erection test. administration. Penile erection was evaluated at 24 hr after the surgical procedures. After oral administration of either mirodenafil or sildenafil citrate at the dose of 0.3 mg/kg, 1 mg/kg or 3 mg/ into the ear vein of the animal at 1 mg/kg, then measurement kg (n=5), the length of the uncovered penile mucosa was of the uncovered penile mucosa was performed every 1 min measured with a sliding digital caliper. SNP was dissolved for up to 30 min. in saline (0.2 mg/ml) and administered into the ear vein of When given orally, doses of 0.3, 1 and 3 mg/kg the animal at 1 mg/kg 60 min after drug administration. mirodenafil induced a penile erection for the first 60 min. Measurements of the uncovered penile mucosa were made The minimal effective dose of mirodenafil was 0.3 mg/kg every minute for up to 90 min. The mean length of the (Fig. 2). Sildenafil showed no erectile response for the first exposed penile mucosa in each group was calculated. 60 min at 0.3 and 1 mg/kg. Weak response exposing the Histopathological examination of the spinal cord: The uncovered penile mucosa was observed at 3 mg/kg. The animals were sacrificed at 48 hr after completion of penile onset of erection with mirodenafil (0.3 mg/kg, 12 min; 1 mg/ erection test and their lumbar spinal cords were removed. kg, 7 min; 3 mg/kg, 17 min) was faster than with sildenafil The tissue samples were fixed in 10% formalin and embed- (3 mg/kg, 26 min). ded in paraffin with a routine follow-up procedure. Sections When SNP was given intravenously after 60 min, admin- were cut at a thickness of 4 µm and stained with hematoxy- istrations of 1 and 3 mg/kg mirodenafil were significantly lin and eosin and methyl green stains for light microscopic potentiated (Fig. 2). Immediately after injection of SNP, examination. Morphologic evaluation of the spinal cords both 1 and 3 mg/kg mirodenafil produced a penile erection was done blindly. The histopathological findings were with a peak mean length of 17.3 ± 2.7 mm and 15.7 ± 2.3 graded from 1 to 3 [21]. Grade 1 indicated the appearance mm, respectively. These effects were clearly stronger than of a normal spinal cord. Grades 2 and 3 denoted swollen the sildenafil-induced penile erection (3 mg/kg, 4.9 ± 1.1 axons with occasional necrotic neurons and many necrotic mm). neurons, respectively. Effect of mirodenafil on penile erection in the ischemic- Statistical analysis: Statistical significance was assessed reperfusion SCI model: After oral administration of using the Student’s t-test. A value of P<0.05 compared with mirodenafil or sildenafil, penile erections were observed at the control was considered statistically significant. a dose of 3 mg/kg. However, no other groups showed a response for the first 60 min (Fig. 3). The onset of action for RESULTS mirodenafil and sildenafil occurred at 5 min and 9 min, respectively. The maximum mean lengths of penile erection Effect of mirodenafil on penile erection in the transection at 3 mg/kg of mirodenafil and sildenafil were 4.1 ± 0.6 mm SCI model: Mirodenafil or sildenafil citrate (0.3, 1 or 3 mg/ and 3.3 ± 0.4 mm, respectively. After administering SNP, kg) was given orally to male rabbits with a surgical transec- the lengths of penile erections were significantly increased tion of the spinal cord, and then their erections were evalu- at 1 mg/kg mirodenafil, 3 mg/kg mirodenafil and 3 mg/kg ated in a time-course manner by measuring the length of the sildenafil. Mirodenafil induced penile erections with maxi- uncovered penile mucosa every 1 min for up to 60 min. mum mean lengths of 7.5 ± 0.8 mm at 1 mg/kg and 7.1 ± 0.5 Sixty min after drug administration, SNP was administered mm at 3 mg/kg dose. Sildenafil showed its maximum erec- 1202 J-Y. JUNG ET AL.

stronger penile erectile response than with SNP alone. A higher dose, 3 mg/kg mirodenafil, alone induced erections and was immediately potentiated by SNP. Oral administration of 3 mg/kg sildenafil alone induced erections in the transection and ischemic-reperfusion models, and co- administration of SNP with 1 or 3 mg/kg of sildenafil induced penile erections that were stronger than SNP treatment alone. Histopathological examination of the spinal cord: For histopathological examination, the lumbar spinal cords applied mirodenafil or sildenafil were obtained from each rabbit. Neuronal degeneration was observed in the lumbar spinal cords, including shrunken neurons, nissl substance disappearance and neuronal necrosis (Fig. 4). The grades of the lesions were 2 or 3 in all rabbits.

DISCUSSION

Fig. 3. Effects of oral administration of mirodenafil or sildenafil Spinal cord injury (SCI) is known as one of the main eti- on length of the exposed penile mucosa in ischemic-reperfusion ological factors relevant to erectile dysfunction. It has been SCI model. SNP was injected intravenously 60 min after drug estimated that 10–19% of ED has a neurogenic origin [14]. administration. Statistical significance (p<0.05) for the mucosal Spinal cord injury is one of the main causes of neurogenic lengths of the mirodenafil-treated animals at the time of maximum erection compared with the sildenafil-treated animals is ED and results in ED in 50% of cases of lumbar injury. In indicated by asterisks (*). some men with a spinal injury, only a reflexogenic erection remains [2]. SCI prevents effective release of NO from the parasympathetic nonadrenergic, noncholinergic (NANC) tile response of 5.2 ± 0.6 mm at 3 mg/kg. At the same dose, nerves, which prevent the initiation of erection [7]. the erectogenic potential of mirodenafil was significantly In this study, the rabbit spinal cord injury model was better than that of sildenafil. Statistical analysis was con- induced by ischemic-reperfusion and transection method. ducted at the time point of maximum erection in the While in vitro techniques using corpus cavernosal tissues mirodenafil treated groups. All three doses of mirodenafil permit initial screening of appropriate pharmacological were significantly different compared with sildenafil at the activity, it is always important to verify that the required p<0.05 level, as indicated by asterisks in Fig. 3 (*). activities can be documented in vivo [16]. Penile erection Quantification of erectogenic potential of mirodenafil receives afferent information conveyed by somatic and vis- using area under the curve (AUC): The time course of ceral fibers originating from the penis and perigenital area penile erections, expressed as the area under the curve, after [19]. Furthermore, it receives direct descending projections oral administration of mirodenafil or sildenafil followed by from a collection of neurons present in the brainstem and intravenous injection of SNP is shown in Table 1. The table hypothalamic nuclei, which are considered premotor neu- shows that the erectogenic effect of mirodenafil was poten- rons. The spinal network that controls penile erection and tiated in the transection model. In the ischemic-reperfusion its relations with afferent fibers from the periphery and model, oral doses of 0.3 mg or 1 mg/kg alone did not induce descending projections from supraspinal nuclei have only a penile erection; co-administration of SNP resulted in a begun to be elucidated [23].

Table 1. Time course of erection response after oral administration of mirodenafil or sildenafil in rabbits with SCI Dose Transection model Ischemic-reperfusion model (mg/kg) (AUCd)/mm × min) (AUC/mm × min) –SNPa) +SNPb) Totalc) –SNP +SNP Total Control 0 0 16.94 16.94 0 4.88 4.88 Mirodenafil 0.3 166.24 13.92 180.16 0 12.19 12.19 1 163.87 267.05 430.92 0 65.98 65.98 3 148.47 226.00 374.47 27.51 45.25 72.76 Sildenafil 0.3 0 17.05 17.05 0 5.48 5.48 1 0 15.26 15.26 0 13.08 13.08 3 13.72 17.88 31.6 28.54 40.16 68.7 a) 0 to 60 min; b) 60 to 90 min; c) 0 to 90 min; d) Time (min) × length of uncovered penile mucosa (mm). PENILE ERECTION EFFICACY OF MIRODENAFIL 1203

erection by targeting the PDE5 isoenzyme [29]. These agents amplify the NO-sGC-cGMP signaling pathway underlying ED largely irrespective of etiology. The NO released from the nonadrenergic noncholinergic neurons is not the only mechanism involved in erection [5]. The fact that endothelial cells constitutively express endothelial nitric oxide synthase (eNOS) and can release NO, which diffuses to adjacent smooth muscle cells and causes them to relax [8], partly supports the reason why mirodenafil alone could induce a penile erection in the SCI model without sexual stimulation or extrinsic NO administration in our study. This suggests that the relaxing action of mirodenafil on the corpus cavernosum prior to administration of SNP may be mediated by the NO released from the endothelium. At the same dose, these effects of mirodenafil alone were clearly stronger than sildenafil alone in inducing penile erection in the absence of an NO-drive. In this study, the efficacy of mirodenafil was potentiated by intravenous injection of SNP, a nitric oxide donor. Potentiation of the effect by a nitric oxide donor implies that mirodenafil can enhance erectile activity during sexual arousal. Under the experimental conditions in this model, a nitric oxide donor, such as SNP, may replace the naturally released NO and enhance the efficacy of mirodenafil. Although the constitutive release of NO by the penile endothelium may have assisted mirodenafil to cause erection without sexual stimulation or extrinsic NO, the fact that the erectogenic effect of mirodenafil was markedly and immediately potentiated by SNP suggests that mirodenafil may enhance sexual stimulation mediated erection. Fig. 4. Histopathological findings of the lumbar spinal cord As a result, the efficacy of mirodenafil was stronger than (L6). (a) Control and (b) ischemic-reperfusion SCI model. The that of sildenafil in spinal cord injury models in regard to the neuronal degeneration in the ischemic-reperfusion SCI model peak length of the uncovered penile mucosa and onset time exhibited disappearance of Nissl substance (thick arrow), of action. These findings suggest that mirodenafil may be shrunken cells (thin arrow) and neuronal necrosis (methyl useful for the treatment of erectile dysfunction in patients green staining). The scale bar represents 100 µm. with a spinal cord injury.

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