Effect of TD0014 on intracavernous pressure elicited with electrical stimulation of the cavernous nerve in male rats

JOURNAL OF MEDICAL RESEARCH<br /> <br /> EFFECT OF TD0014 ON INTRACAVERNOUS PRESSURE<br /> ELICITED WITH ELECTRICAL STIMULATION OF THE<br /> CAVERNOUS NERVE IN MALE RATS<br /> Mai Phuong Thanh1, Pham Thi Van Anh1, Nguyen Trong Thong1,<br /> Nguyen Thi Huong Lien 2<br /> 1<br /> <br /> Department of Farmacology, Hanoi Medical University, 2Sunstar Joint Stock Company<br /> <br /> This study aimed to determine the effects of an herbal formula named TD0014 on the electrostimulationinduced erection in a rat model. Eighteen sixteen-week-old male Wistar rats were used. The intracavernous<br /> pressure and the arterial blood pressure were simultaneously monitored during cavernous nerve<br /> electrical stimulation before and after the administration of TD0014 (1.8 g/kg). Statistical analysis was<br /> performed on measurements of maximal intracavernous pressure (MIP), mean arterial blood pressure<br /> (MAP) and the MIP/MAP. The administration of TD0014 resulted in a significant increase in the baseline<br /> level of intracavernosal pressure (ICP) before stimulation in comparison to the control group (P < 0.05).<br /> The findings also showed that the maximal ICP and the area under the curve (AUC time × ICP curves)<br /> in TD0014 group were greater than distilled water group, however, this difference was not significant.<br /> TD0014 had no significant effect on the mean arterial pressure. These results indicated that the herbal<br /> formulation TD0014 may have positive and selective effects on improving erectile functions in male rats.<br /> <br /> Keywords: TD0014, cavernous nerve stimulation, intracavernous pressure, rat<br /> <br /> I. INTRODUCTION<br /> <br /> Erectile dysfunction (ED) is a term<br /> recommended by a panel of experts in<br /> 1992 to replace the term “impotence” [1].<br /> ED is when a man is unable to get and/or<br /> keep an erection. It is not a disease, but<br /> a symptom of some other problem, either<br /> physiological, psychological or a mixture<br /> of both [2]. Although physiological ED<br /> itself is not life threatening, it is a strong<br /> predictor of high-mortality diseases such as<br /> cardiovascular disease [3]. ED does directly<br /> Corresponding author: Mai Phuong Thanh, Department<br /> of Pharmacology, Hanoi Medical University<br /> Email: maiphuongthanh@hmu.edu.vn<br /> Received: 05 June 2017<br /> Accepted: 16 November 2017<br /> <br /> 36<br /> <br /> and negatively impact the quality of life of<br /> afflicted men and their spouse [4]. Recently<br /> developed phosphodiesterase type-5 (PDE5) inhibitors have been widely used as firstline therapeutics to treat ED [5]. PDE5 is<br /> an enzyme found primarily in the smooth<br /> muscle of the corpus cavernosum that<br /> selectively cleaves and degrades cGMP<br /> to 5′-GMP. PDE5 inhibitors are similar in<br /> structure to cGMP; they competitively bind<br /> to PDE5 and inhibit cGMP hydrolysis. The<br /> increased amounts of cGMP enhance the<br /> effects of NO a potent vasodicator. This<br /> increase in cGMP in the smooth muscle cells<br /> is responsible for prolonging an erection [6].<br /> Although large, multicenter clinical trials<br /> have shown the efficacy and tolerability of<br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> these drugs in patients with ED of various<br /> etiologies and a broad range of severity;<br /> however, 30% to 35% of patients fail to<br /> respond. The use of PDE - 5 inhibitors may<br /> result in several side effects, including visual<br /> disturbances, headache, facial flushing,<br /> rhinitis, and indigestion. Other treatments<br /> for ED include penile injection therapy or<br /> penile implants. However, such methods<br /> are invasive and irreversible, and are<br /> therefore not widely used [7]. Thus, there<br /> is a continued need for the development of<br /> new noninvasive and effective therapies to<br /> treat patients with ED.<br /> Despite the remarkable developments<br /> of modern medicine, many people are<br /> still favorably disposed towards herbal<br /> medicines owing to the aggressive<br /> treatment protocols, toxicity, and drug<br /> tolerance associated with modern therapies.<br /> The widespread use of herbal medicines,<br /> however, requires scientific verification of<br /> their effects.<br /> TD0014 is a preparation which<br /> comprises of thirty-three medicinal plants.<br /> The composition of TD0014 has several<br /> medicinal herbs that have been studied<br /> and used since ancient times in traditional<br /> folk medicine as an aphrodisiac [8 - 15].<br /> However, no studies have shown reliable<br /> evidence of their effect on reproductive<br /> function, or toxicity when taken in TD0014.<br /> Therefore, the purpose of this study was to<br /> explore the impact of TD0014 on erectile<br /> capacity in adult male rats.<br /> <br /> II. MATERIALS AND METHODS<br /> 1. Materials<br /> JMR 111 E2 (2) - 2018<br /> <br /> Herbal formula TD0014 preparation<br /> TD0014 was manufactured as hard pills<br /> according to the quality standard of Sunstar<br /> Joint Stock Company, Vietnam. The major<br /> ingredients of the herbal formula are obtained<br /> from thirty-three plants (per 7.5g of serving):<br /> Tribulus terrestris (4 g), Chrysanthemum<br /> sinense (1.83 g), Prunus persica (1.14<br /> g), Vigna cylindrica (1.14 g), Eurycoma<br /> longifolia (0.69 g), Sophora japonica (0.57g),<br /> Dioscorea persimilis (0.43 g), Dioscorea<br /> tokoro (0.4 g), Polygonum multiflorum (0.4<br /> g), Citrus deliciosa (0.34 g), Polyscias<br /> fruticosa (0.34 g), Tinospora sinensis<br /> (0.29 g), Chaenomeles lagenaria (0.29 g),<br /> Passiflora foetida (0.29 g), Zizyphus sativa<br /> (0.29 g), Rehmannia glutinosa (0.23 g),<br /> Angelica sinensis (0.23 g), Alisma plantagoaquatica<br /> L. var. orientalis Samuelsson<br /> (0.23 g), Achyranthes bidentata (0.23 g),<br /> Schizandra chinensis (0.23 g), Morinda<br /> offcinalis (0.23 g), Rosa laevigata (0.23<br /> g), Allium sativum (0.2 g), Lycium sinense<br /> (0.17 g), Glycyrrhiza uralensis (0.14 g),<br /> Panax ginseng (0.11 g), Ligusticum wallichii<br /> (0.11 g), Cistanche tubulosa (0.11 g),<br /> Atractylodes macrocephala (0.11 g), Radix<br /> Codonopsis (0.11 g), Cuscuta sinensis (0.11<br /> g), Psoralea corylifolia (0.06 g), Cornu Cervi<br /> parvum (7.2 mg). The experimental animals<br /> drank the test drug mixed with pure water.<br /> Animal groups<br /> Wistar male rats (250 - 300 g), 16 weeks<br /> of age, were used in our study. They were<br /> housed in groups of six rats per cage in a 24<br /> - hour air - conditioned room with access to<br /> standard certified rodent diet and water ad<br /> libitum. They were acclimated to housing for<br /> at least 1 week prior to investigation.<br /> 37<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> The rats were randomly separated into 3 groups of 6 animals each: 1) control, 2) sildenafil<br /> treatment, and 3) TD0014 treatment. In each group, animals were administered per os onetime with either distilled water (10 ml/kg b.w.), or sildenafil (6 mg/kg b.w.), or TD0014 (1.8 g/kg<br /> b.w.). 2 hours after treatment, erectile function was evaluated by measuring intracavernosal<br /> pressure (ICP) and maximal ICP/mean arterial pressure (MIP/MAP).<br /> 2. Methods<br /> Measurement of intracavernosal pressure<br /> Rats were anaesthetized with an intraperitoneal injection of ketamine at a dose of 25 mg/<br /> kg (Rotexmedica, Germany). The penis was dissected and the corpus cavernosum and crus<br /> were exposed in a supine position. A low-midline incision was made to access the pelvis, and<br /> the pelvic ganglion lateral to the right prostate was exposed. The penile skin was degloved<br /> and the corpus cavernosum was identified. To measure ICP, a heparinized 23-gauge butterfly<br /> needle was inserted into the proximal portion of the corpus cavernosum. A bipolar electrical<br /> stimulator was placed on the ganglion to stimulate the cavernosal nerve for 60 seconds at 5 V<br /> and 20 Hz for 2 millisecond periods. The cavernosal nerve stimulation was conducted 3 times<br /> with a 10-minute interval between stimulations. Before and after each electrical stimulation, ICP<br /> was recorded on a computer by Powerlab system record software. ICP data was normalized<br /> by mean systemic arterial pressure (MAP) and the MAP was monitored simultaneously with<br /> ICP monitoring. The right carotid artery was dissected via a midline cervical incision under<br /> a microscope, and then PE-50 tubing was inserted into the carotid artery. The catheter was<br /> connected to both a pressure transducer and an amplifier unit which was connected to a data<br /> acquisition module. MAP was recorded on a computer by Powerlab system record software.<br /> Statistical analyses<br /> Data was analyzed employing with the Labchart pro software and Microsoft Excel 2010.<br /> All data is presented as mean ± standard deviation. Statistical significance was determined<br /> by Student's t-test and p < 0.05 were considered to be significant.<br /> <br /> III. RESULTS<br /> **<br /> <br /> * p < 0.05; ** p < 0.01; *** p < 0.001; compared with control (Student’t-test)<br /> Figure 1. Intracavernous pressure (ICP) before and after electrical stimulation of the<br /> cavernous nerve in rats from each experimental group<br /> 38<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> In sildenafil group, before and after the electrical stimulation of the cavernous nerve,<br /> the ICP significantly increased compared to the control group. In the flaccid state, TD0014<br /> elevated the ICP level which was statistically different compared to the control animals<br /> (p < 0.05). In addition, the ICP after stimulation also increased in TD0014 group, however, it<br /> was not statistically significant.<br /> <br /> **<br /> ΔΔΔ<br /> <br /> **p < 0.01 vs. control rats (Student’t-test); ∆∆∆p < 0.001 vs. sildenafil rats ((Student’t-test)<br /> Figure 2. Effect of TD0014 on time to the maximal ICP and response time<br /> to the electrical stimulation of the cavernous nerve<br /> Observing in Figure 2, the parameter of time to the maximal ICP was not different amongst<br /> treatment groups. The treatment with sildenafil increased the response time to electrical<br /> stimulation (p < 0.01). The animals treated with TD0014 were not able to extend the response<br /> time.<br /> Table 1. Total ICP (ICP vs stimulation time, area under curve) in treatment rats and<br /> control rats.<br /> Treatment<br /> Group 1 (control group)<br /> <br /> Total ICP (mmHg*s)<br /> 1375.06 ± 822.02<br /> <br /> Group 2 (sildenafil)<br /> <br /> 2213.70 ± 1077.94*<br /> <br /> Group 3 (TD0014)<br /> <br /> 1722.96 ± 710.00<br /> <br /> *p < 0.05, response significantly different from control rats<br /> In Table 1, statistical analysis indicated that sildenafil group showed significantly greater<br /> total ICP compared with the control group. TD0014 group exhibited an increase in total ICP,<br /> but it was not statistically significant.<br /> The effect of sildenafil and TD0014 on blood pressure is shown in Table 1. After sildenafil<br /> administration, MAP decreased by approximately 10 mmHg, but this was not a significant<br /> difference to the control (p > 0.05) MIP/MAP increased significantly compared with distilled<br /> water group and TD0014 group (p < 0.05 an p < 0.01 respectively). There was no statistical<br /> JMR 111 E2 (2) - 2018<br /> <br /> 39<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> differences in MAP and MIP/MAP values between the TD0014 group and the control group.<br /> Table 2. Effect of TD0014 on maximal ICP/MAP ratio<br /> Treatment<br /> <br /> MAP (mmHg)<br /> <br /> MIP/MAP<br /> <br /> Group 1 (control group)<br /> <br /> 106.34 ± 18.08<br /> <br /> 0.40 ± 0.11<br /> <br /> Group 2 (sildenafil)<br /> <br /> 96.43 ± 13.76<br /> <br /> 0.56 ± 0.14*<br /> <br /> Group 3 (TD0014)<br /> <br /> 110.67 ± 5.05<br /> <br /> 0.44 ± 0.09∆∆<br /> <br /> *p < 0.05 as compared with control; ∆∆p < 0.01 as compared with sildenafil<br /> <br /> IV. DISCUSSION<br /> Penile erection is a hemodynamic<br /> process which penile arteries, penis and<br /> penile venous system induced by neuroendocrine regulation [16]. Change in<br /> ICP induced by penile cavernous nerve<br /> stimulation is often used as an objective<br /> assessment index for the impact of drugs<br /> on erectile function in vivo [17].<br /> Stimulation of penile cavernous nerve<br /> could activate nitric oxide synthase activity<br /> of non-adrenergic and non-cholinergic<br /> nerve endings, promote production and<br /> release of NO. No leads increased to cGMP<br /> concentration, which induces the relaxation<br /> of smooth muscle and reults in erection [18].<br /> Sildenafil is a potent and selective inhibitor<br /> of cyclic guanosine monophosphate (cGMP)<br /> specific phosphodiesterase type 5 (PDE5)<br /> which is responsible for the degradation<br /> of cGMP in the corpus cavernosum of the<br /> penis. Therefore, increased cGMP within<br /> the corpus cavernosum can lead to smooth<br /> muscle relaxation [19]. The results of Fig 1,<br /> Fig 2, and Tab 1 clearly demonstrated the<br /> beneficial effects of sildenafil on erectile<br /> dysfunction, as shown by the ability to<br /> significantly increase ICP before and after<br /> <br /> 40<br /> <br /> electrostimulation (53% and 25% increase,<br /> respectively), prolong response time with<br /> stimulation, and increase total ICP (61%<br /> increase) compared to the control group.<br /> In this study, the administration of<br /> TD0014 at 1.8 g/kg/day showed an<br /> improvement of ICP level before the<br /> start of electrostimulation, which means<br /> the herbal preparation has a pro-erectile<br /> activity. Subsequently TD0014 can improve<br /> the ICP value during erection. After<br /> electrostimulating the cavernosal nerve,<br /> compared to the control group, TD0014<br /> tended to have an elevation in the maximal<br /> ICP (12% increase), total ICP (25%<br /> increase), and a prolonged response time<br /> to the stimulus, i.e prolong the duration of<br /> penile tumescence, however, these results<br /> showed no signitificant differences (p > 0.05).<br /> Thus, in the current study, TD0014 given<br /> once before nerve stimulation markedly<br /> increased baseline ICP, slightly increased<br /> both the MIP and the response time to<br /> the stimulus. These results suggested that<br /> TD0014 may be beneficial for patients with<br /> erectile dysfunction, and can improving the<br /> patient's sexual life.<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br />