Experimental study on the safety and effectiveness of td0014 hard pills for the treatment of male sexual dysfunction

ve control) received SVP (500 mg/kg, oral) for 7 weeks and served as toxic control. The remaining two groups (Group 3 and 4) received TD0014 orally at doses of 1.8, and 5.4 g/kg respectively along with SVP for 7 weeks. On 5 th week, one male rat was randomly coupled with two untreated virgin females for 2 weeks. At the end of 7 th week, all rats were weighed and killed by exsanguination. Following parameters were calculated: - Male rats: the weights of testes and accessory sexual organs (glans penis, epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator ani/bulbocavernosus muscles [LABC]), serum testosterone level, semen analysis (sperm counts, sperm motility, sperm viability and sperm morphology), histopathology of testis. 8 - Female rats: pregnancy rate. 2.3.4.2. Restorative role of TD0014 Adult male rats were randomly divided into four groups. The animals were given SVP at the dose of 500 mg/kg/day for 7 weeks to cause reproductive toxicity, then distilled water or TD0014 was continuously administered orally for 10 days: - Group 1: not given SVP for 7 weeks, distilled water 10 mL/kg/day for 10 days. - Group 2: given SVP for 7 weeks, distilled water 10 mL/kg/day for 10 days. - Group 3: given SVP for 7 weeks, TD0014 1.8 g/kg/day for 10 days. - Group 4: given SVP for 7 weeks, TD0014 5.4 g/kg/day for 10 days. After 10 days of treatment, one male rat was randomly coupled with two untreated virgin females for 2 weeks. At the end of pairing period, parameters of male and female rats were determined similarly to the study of protective effects of TD0014. 2.4. Statistical analysis Data were analyzed by Excel 2010 and SPSS 22.0 software, using appropriate statistical algorithms (Student's t-test, Paired t-test, Mann-Whitney U test, Chi- square test). A p value<0.05 was considered as statistically significant. Chapter 3 RESULTS 3.1. Acute and subchronic toxicity of TD0014 in animals 3.1.1. Acute toxicity of TD0014 Table 3.1. Correlation between TD0014 dose and mice mortality Group (n = 10) Dose of TD0014 (g/kg) Percentage of dead (%) Other abnormal findings Group 1 22.50 0 None Group 2 33.75 0 None Group 3 45.00 0 None Group 4 56.25 0 None In all-male testing animals, signs of neither toxicity nor death among the mice were observed within the critical 72 hours post-administration and to the end of the seventh day. Hence the LD50 evaluated by the Litchfield - Wilcoxon method could not be determined (orally). 3.1.2. Subchronic toxicity of TD0014 3.1.2.1. Body weight and clinical observation Neither deaths nor obvious clinical signs of toxicity in the rats were observed for all groups. Physical observation of the treated rats throughout the experimental period indicated that none of them showed signs of toxicity in their skin, eyes, or 9 behavioral changes, diarrhea, tremors, sleep, and coma. Normal body weight gains were observed during the study period compared to the control group. 3.1.2.2. Hematological analysis Daily oral administration of TD0014 for 90 days produced no effect on all hematological parameters. The results show that none of the groups differed significantly when compared to the control for all parameters. 3.1.2.3. Biochemical analysis None of the biochemical parameters were affected by the oral administration of TD0014 for 90 days. There was no statistical difference in the concentration of the indicator enzymes of liver cell damage (AST, ALT) in the group treated with TD0014 compared to the group treated with distilled water. No significant changes in liver function parameters (albumin, total cholesterol, total bilirubin) and glomerular filtration function parameters (creatinine) were noted. Table 3.2. Effects of TD0014 on serum transaminase levels AST (UI/L) ALT (UI/L) Control (n = 11) 1.8 g/kg (n = 11) 5.4 g/kg (n = 10) Control (n = 11) 1.8 g/kg (n = 11) 5.4 g/kg (n = 10) D0 112.91 ± 25.04 108.64 ± 15.52 121.00 ± 24.31 60.00 ± 18.07 62.18 ± 13.55 59.00 ± 7.76 D30 110.27 ± 10.62 103.82 ± 26.04 117.36 ± 30.40 50.64 ± 7.12 51.00 ± 11.74 50.91 ± 8.84 p (paired t-test) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 D60 101.55 ± 7.06 113.36 ± 20.21 112.55 ± 17.46 53.45 ± 9.13 58.45 ± 14.08 61.64 ± 10.68 p (paired t-test) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 D90 104.73 ± 22.00 106.73 ± 17.70 107.90 ± 17.04 66.00 ± 12.24 60.91 ± 11.78 60.40 ± 14.71 p (paired t-test) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 3.1.2.4. Histopathology study - Gross pathologic observations: Liver and kidney did not show any abnormal changes in texture, shape, size or color compared to the control. There was no sign of necrosis or lesion was appreciated on the organs of all treated groups. - Light microscopy of liver: There were no significant histopathological presentations observed in the groups treated with distilled water and treatment groups. The microscopic examination of liver sections of rats showed the normal architecture of structural units of the liver, the hepatic lobules, formed by cords of hepatocytes separated by hepatic sinusoids. No portal inflammation was seen. - Light microscopy of kidney: There were no adverse histopathological presentations observed in all the treatment groups. The microscopic architecture 10 of sections of kidney in treated groups had a similar appearance to that of the controls in which renal corpuscles maintaining their normal size of urinary space and normal tubular structures are examined. No necrosis was observed. 3.2. Androgenic activities of TD0014 in immature male rats Table 3.3. Effects of TD0014 on weights of accessory sexual organs and serum testosterone levels of the castrated male rats Parameters Groups (n = 9) Intact + distilled water Castrated + distilled water Castrated + testosterone 0.4 mg/kg Castrated + TD0014 1.8 g/kg Castrated + TD0014 5.4 g/kg Weight (mg/100g b.wt) Glans penis 28.2 ± 7.8 21.0 ± 5.8 * 42.2 ± 4.2 ### 22.3 ± 4.5 22.6 ± 6.3 Seminal vesicles 19.7 ± 5.7 8.7 ± 2.1 *** 81.6 ± 19.2 ### 9.3 ± 2.4 8.2 ± 2.6 Ventral prostate 22.2 ± 6.4 5.0 ± 1.6 *** 34.2 ± 8.3 ### 8.9 ± 2.8 ## 17.7 ± 4.2 ### Cowper's glands 8.4 ± 1.9 1.3 ± 0.2 *** 10.7 ± 2.6 ### 1.9 ± 0.6 # 1.6 ± 0.3 # LABC 104.1 ± 19.2 44.6 ± 9.0 *** 139.6 ± 30.2 ### 39.8 ± 9.2 36.5 ± 9.8 Testosterone (nmol/L) 1.817 ± 0.491 0.120 ± 0.038 *** 3.098 ± 0.975 ††† 0.166 ± 0.031 † 0.366 ± 0.113 ††† *p<0.05; ***p<0.001 compared with group I (intact + distilled water) (Student’t-test) # p<0.05; ## p<0.01; ### p<0.001 compared with group II (castrated + distilled water) (Student’t-test) With regard to castrated controls, rats exposed to the plant extracts showed a significant increase in the relative weights of the ventral prostate and the Cowper’s glands, and a significant increase in the serum testosterone levels. On the weanling animals, TD0014 at the dose of 5.4 g/kg caused a significant increase in the weights of Cowper’s glands, LABC, and prostate of rats, while TD0014 at the dose of 1.8 g/kg caused only a significant increase in the weight of Cowper’s glands. TD0014 treatment resulted in a significant increase in the serum testosterone levels. 11 Table 3.4. Effects of TD0014 on weights of reproductive organs and serum testosterone levels of the weanling male rats Parameters Groups (n = 10) Control Testosterone 1.0 mg/kg TD0014 1.8 g/kg TD0014 5.4 g/kg Weight (mg/100g b.wt) Testis 901.8 ± 180.9 786.5 ± 159.1 891.6 ± 117.9 893.7 ± 162.9 Seminal vesicles 24.1 ± 6.7 215.8 ± 48.4 *** 19.5 ± 5.6 21.9 ± 5.7 Epididymis 127.6 ± 24.0 252.2 ± 35.4 *** 130.1 ± 30.0 123.8 ± 22.6 Ventral prostate 24.4 ± 8.0 100.2 ± 17.6 *** 21.7 ± 5.1 37.6 ± 7.9 ** Cowper's glands 3.4 ± 0.7 21.2 ± 3.0 *** 4.8 ± 1.3 ** 4.3 ± 0.8 * LABC 55.0 ± 11.0 194.6 ± 23.4 *** 46.0 ± 10.2 70.8 ± 15.4 * Testosterone (nmol/L) 0.087 ± 0.002 15.343 ± 1.939*** 0.235 ± 0.089*** 0.293 ± 0.062*** *p<0.05; **p<0.01; ***p<0.001 compared with control (Student’t-test) 3.3. Effects of TD0014 on erectile function * p < 0.05; ** p < 0.01; *** p < 0.001 compared with control (Student’t-test) Chart 3.1. Intracavernous pressure (ICP) before and after electrical stimulation of the cavernous nerve in rats from each experimental group Before stimulation: TD0014 elevated the basal ICP level which was statistically different compared to the control animals. After stimulation: TD0014 group exhibited an increase in maximal ICP, total ICP, and response time to the electrical stimulation, but they were not statistically significant compared with distilled water group. There was no statistical 0 10 20 30 40 50 60 70 Before stimulation After stimulation (maximal ICP) IC P ( m m H g ) Control Sildenafil TD0014 * ** *** 12 differences in MAP and maximal ICP/MAP values between the TD0014 group and the control group. Table 3.5. Effects of TD0014 on time to the maximal ICP and response time to the electrical stimulation of the cavernous nerve, and maximal ICP/MAP ratio Groups (n = 6) Time to the maximal ICP (s) Response time to stimulation (s) MAP Maximal ICP/ MAP Control 38,833 ± 20,614 109,441 ± 50,721 106,34 ± 18,08 0,40 ± 0,11 Sildenafil 40,016 ± 18,290 158,902 ± 47,607** 96,43 ± 13,76 0,56 ± 0,14*** TD0014 38,819 ± 14,245 114,196 ± 17,298 110,67 ± 5,05 0,44 ± 0,09 **p<0.01; ***p<0.001 compared with control *p<0.05 compared with control Chart 3.2. Effect of TD0014 on total ICP (ICP vs stimulation time, area under curve) 3.4. Effects of TD0014 on sodium valproate-induced reproductive decline in male rats 3.4.1. Protective effects 3.4.1.1. Effects on the weight of organs The weight of organs, including testes, accessory sexual organs (glans penis, epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator ani/bulbocavernosus muscles [LABC]), and several other organs (liver, kidney, adrenal glands) in the SVP treated group were significantly reduced as compared to the control group. A daily oral administration of TD0014 for 7 consecutive weeks was followed by a significant increase in relative weights of the testes, some accessory organs (TD0014 at the dose of 5.4 g/kg caused a significant increase in the weights of glans penis, seminal vesicles, epididymis, and Cowper’s glands of rats; TD0014 at the dose of 1.8 g/kg caused only a significant increase in the weight 0 500 1000 1500 2000 2500 Control Sildenafil TD0014 Total ICP (mmHg*s) * 13 of glans penis and epididymis), and adrenal glands when compared to SVP- intoxicated control group. 3.4.1.2. Effects on sperm analysis Table 3.6. Protective effects of TD0014 on the size of seminiferous tubule, sperm count and viability in SVP-intoxicated rats Groups Size of seminiferous tubule (pixell) Sperm count (10 6 /mL) Sperm viability (%) Negative control 452.74 ± 55.12 144.74 ± 18.73 93.71 ± 2.50 Positive control 326.09 ± 38.81*** 3.83 ± 1.17*** 59.83 ± 12.73*** TD0014 1.8 g/kg 371.97 ± 25.62 ▲ 18.33 ± 5.85 ▲▲▲ 51.67 ± 15.11 TD0014 5.4 g/kg 462.20 ± 58.25 ▲▲▲ 106.20 ± 33.13 ▲▲▲ 88.70 ± 6.18 ▲▲▲ ***p<0.001 compared with negative control (Student’t-test) ▲ p<0.05; ▲▲▲ p<0.001 compared with positive control (Student’t-test) Table 3.7. Protective effects of TD0014 on sperm morphology in SVP-intoxicated rats Groups n Normal (%) Abnormal (%) Head Midpiece Tail Negative control 8 59.00 ± 6.98 14.57 ± 4.20 10.00 ± 1.63 16.43 ± 5.32 Positive control 6 29.17 ± 1.17 *** 31.33 ± 6.98 *** 20.83 ± 3.76 *** 18.67 ± 3.44 TD0014 1.8 g/kg 6 37.83 ± 11.07 24.17 ± 5.38 14.50 ± 3.39 ▲ 23.50 ± 6.41 TD0014 5.4 g/kg 10 40.90 ± 12.62 ▲ 18.70 ± 5.03 ▲▲▲ 16.00 ± 4.64 ▲ 24.40 ± 7.12 ***p<0.001 compared with negative control (Student’t-test) ▲ p<0.05; ▲▲▲ p<0.001 compared with positive control (Student’t-test) Chart 3.3. Protective effects of TD0014 on sperm motility in SVP-intoxicated rats When compared with positive intoxicated rats, TD0014 with all tested doses significantly increased the size of seminiferous tubule and sperm count, while the 0% 20% 40% 60% 80% 100% Negative control Positive control TD0014 1.8 g/kg TD0014 5.4 g/kg Immotile In situ motility Slow motility Rapid motility 14 percentage of sperm viability and abnormality were only improved at the dose of 5.4 g/kg TD0014. The percentage of immotile sperm was 100% in SVP-intoxicated rats. The semen of male rats received TD0014 at the dose of 1.8 g/kg had motile sperms, however, the spermatozoa have non-progressive motility, i.e it moved only in situ. The male rats exposed to multiple oral dose of TD0014 at 5.4 g/kg/day were significantly improved the speed of movement of spermatozoa with the presence of rapid and slow progressive sperm. 3.4.1.3. Effects on serum testosterone level and histopathological of testis Normal histological structure of seminiferous tubules filled with mature sperms Atrophied seminiferous tubules and edema with absence of sperms Partial improvement of the germinal epithelium of seminiferous tubules, but their lumen still wide and not full of sperm cell lineage Seminiferous tubules with a narrow lumen filled by sperm cell lineage Figure 3.1. Protective effects of TD0014 on photomicrographs of testes Table 3.8. Protective effects of TD0014 on serum testosterone in SVP-intoxicated rats Groups n Testosterone (nmol/L) Group 1: Negative control 8 4,17 ± 1,22 Group 2: Positive control (SVP) 6 1,35 ± 0,44*** Group 3: SVP + TD0014 (1.8 g/kg) 6 4,30 ± 1,10 ▲▲▲ Group 4: SVP + TD0014 (5.4 g/kg) 10 5,64 ± 1,03 ▲▲▲≠ ***p<0.001 compared with negative control (Student’t-test); ▲▲▲p<0.001 compared with positive control (Student’t-test); ≠p<0,05 compared with TD0014 at low dose (Student’t-test) Oral administration of SVP (500 mg/kg) to rats during 7 weeks of the experimental period significantly decreased serum testosterone when compared Negative control Positive control TD0014 at 1.8 g/kg TD0014 at 5.4 g/kg 15 with the normal control group. Co-administration of TD0014 with SVP significantly increased serum testosterone when compared with the intoxicated control group, in a dose-dependent fashion. 3.4.1.4. Effect on pregnancy rate of female rats The positive control group had only 1/20 pregnant female rats (pregnancy rate was 5%) and a marked decrease compared to the negative control group (60%). There was no difference in the pregnancy rate of the low-dose TD0014 group (10%) and the positive control group. The pregnancy rate of the high-dose TD0014 group (30%) was significantly higher than that of the positive control group. 3.4.2. Restorative effects 3.4.2.1. Effects on the weight of organs The weight of organs, including testes, accessory sexual organs (glans penis, epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator ani/bulbocavernosus muscles [LABC]), and several other organs (liver, kidney, adrenal glands) in the SVP treated group were significantly reduced as compared to the control group. A daily oral administration of TD0014 at the dose of 5.4 g/kg for 10 consecutive days was followed by a significant increase in relative weights of the testes and 3 accessory organs (seminal vesicles, epididymis, and LABC) of rats; TD0014 at the dose of 1.8 g/kg caused only a significant increase in the weight of 2 accessory sexual organs (seminal vesicles, LABC) when compared to SVP-intoxicated control group. TD0014 at both doses tended to increase adrenal gland weight. 3.4.2.2. Effects on sperm analysis Table 3.9. Restorative effects of TD0014 on the size of seminiferous tubule, sperm count and viability in SVP-intoxicated rats Groups Size of seminiferous tubule (pixell) Sperm count (10 6 /mL) Sperm viability (%) Sperm speed (μm/s) Negative control 429.70 ± 20.00 161.78 ± 24.15 71.67 ± 6.67 54.46 ± 4.91 Positive control 380.87 ± 15.20*** 60.44 ± 16.48*** 58.22 ± 10.03** 38.91 ± 6.56*** TD0014 1.8 g/kg 405.82 ± 21.57 ▲ 98.44 ± 19.82 ▲▲▲ 58.89 ± 14.42 49.97 ± 12.55 ▲ TD0014 5.4 g/kg 405.70 ± 15.84 ▲ 107.00 ± 25.62 ▲▲▲ 67.00 ± 4.21 ▲ 47.01 ± 9.15 ▲ ***p<0.001 compared with negative control (Student’t-test) ▲ p<0.05; ▲▲▲ p<0.001 compared with positive control (Student’t-test) TD0014 at both doses significantly increased size of seminiferous tubule, sperm count and sperm motility, simultaneously decreased sperm abnormality. 16 Sperm viability tended to increase compared to positive control group with the presence of TD0014, however a statistically significant difference was only observed in the high dose TD0014 group. Table 3.10. Restorative effects of TD0014 on sperm morphology in SVP-intoxicated rats Groups (n = 9) Normal (%) Abnormal (%) Head Midpiece Tail Negative control 56.83 ± 4.12 19.67 ± 1.21 10.33 ± 2.07 13.17 ± 1.17 Positive control 44.14 ± 3.67*** 26.43 ± 2.88*** 14.29 ± 2.63* 15.14 ± 1.86* TD0014 1.8 g/kg 51.67 ± 4.59 ▲▲ 22.50 ± 4.93 11.00 ± 1.41 ▲ 14.83 ± 1.94 TD0014 5.4 g/kg 49.75 ± 5.12 ▲ 23.88 ± 2.36 11.75 ± 1.67 ▲ 14.63 ± 2.45 *p<0.05; ***p<0.001 compared with negative control (Student’t-test) ▲ p<0.05; ▲▲ p<0.01 compared with positive control (Student’t-test) 3.4.2.3. Effects on serum testosterone level and histopathological of testis Normal histological structure of seminiferous tubules filled with mature sperms Mild fluid retention in the interstitial tissue, fewer sperms in the germinal epithelium of the seminiferous tubule Mild fluid retention in the interstitial tissue, fewer sperms in the seminiferous tubule lumen Thick germinal epithelium of the seminiferous tubule containing a full range of sperm cell lineage Figure 2. Restorative effects of TD0014 on photomicrographs of testes TD0014 with all tested doses significantly increased serum testosterone level, in a dose dependent manner, when compared with positive intoxicated rats. Negative control Positive control TD0014 at 1.8 g/kg TD0014 at 5.4 g/kg 17 Table 3.11. Restorative effects of TD0014 on serum testosterone in SVP-intoxicated rats Groups n Testosterone (nmol/L) Group 1: Negative control 9 4.17 ± 1.22 Group 2: Positive control (SVP) 9 1.35 ± 0.44*** Group 3: SVP + TD0014 (1.8 g/kg) 9 4.30 ± 1.10 ▲▲▲ Group 4: SVP + TD0014 (5.4 g/kg) 9 5.64 ± 1.03 ▲▲▲≠ ***p<0.001 compared with negative control (Student’t-test); ▲▲▲p<0.001 compared with positive control (Student’t-test); ≠p<0.05 compared with TD0014 at low dose (Student’t-test) 3.4.2.4. Effect on pregnancy rate of female rats There were no pregnant female rats in the positive control group after the pairing period. The pregnancy rate in the low- and high-dose TD0014 group was 11.1% and 16.7%, respectively. No differences in pregnancy rate were observed in TD0014-treated groups. Chapter 4 DISCUSSION 4.1. Acute and subchronic toxicity of TD0014 in animals 4.1.1. Acute toxicity The acute toxicity study of TD0014 indicated no changes in the behavior and in the sensory nervous system responses in the animals. Also no adverse gastrointestinal effects were observed in the male mice used in the experiment with up to 56.25 g/kg b.wt. The median acute toxicity value (LD50) of the extract was estimated to be more than 56.25 g/kg body weight. According to Ghosh (1984) and Klaasen et al. (1995), TD0014 could be classified as being non toxic, since the LD50 by oral route was found to be above 15 g/kg body weight. 4.1.2. Subchronic toxicity 4.1.2.1. Body weight and clinical observation Daily treatment with both doses of TD0014 to male Wistar rats for a period of 90 days did not show any toxicity related morbidities and mortalities. The body weight changes serve as a sensitive indicator of the general health status of animals. The weight gains were observed in all animals administered with TD0014. It can be stated that TD0014 did not interfere with the normal metabolism of animals as corroborated by the nonsignificant difference from animals in the vehicle control group. 4.1.2.2. Hematological analysis Evaluation of hematological parameters can be used to determine the extent of the deleterious effect of TD0014 on the blood of an animal. A blood count test was undertaken for all the TD0014-treated and control groups and the results show no significant effects. The insignificant effect of TD0014 on red blood indices (total red blood cells, hemoglobin concentration, hematocrit, mean corpuscular volume), 18 total white blood cells and white blood cell differential, platelet count indicates that TD0014 did not affect functions of the hematopoietic system. 4.1.2.3. Biochemical analysis Liver and kidney function analysis is very important in the toxicity evaluation of drugs and plant extracts as they are both necessary for the survival of an organism. Liver is a primary destination for any toxic substance entered to the body, especially through gastrointestinal route, the liver suffers first. Because of its wide range of functions, any abnormal change in the liver will definitely affect complete metabolism of an animal. High levels of transaminases are reported in liver diseases or hepatotoxicity. The non-significant change of these enzymes between the control and treated group animals after 90 days administration indicated that TD0014 did not cause adverse toxic effect or hepatic damage on the liver. Any abnormal change in total bilirubin, albumin, and total cholesterol might be due to reduced functions of the liver. Thus, the insignificant change in serum concentration of total bilirubin, albumin and total cholesterol in the TD0014-treated and control group further confirmed that TD0014 did not impair the hepatocellular functions at any of the doses tested. Kidney is a sensitive organ, whose function is known to be affected by a number of factors such as drugs including phytochemicals of plant origin that ultimately lead to renal failure. Creatinine is excreted by glomerular filtration and the clearance is dependent on the rate at which it is removed from the blood by the kidneys, therefore, an increase in the plasma creatinine level suggests kidney damage specifically renal filtration mechanism. In the present study, change in plasma creatinine level TD0014-treated groups showed non-significant differences indicating a normal renal function. 4.1.2.4. Histopathology study Histopathological examinations of liver and kidneys harvested from treated and control animals provide information to strengthen the findings on biochemical and heamatological parameters. The microscopic examination revealed that none of the organs from TD0014-treated rats showed any alteration in cell structure, inflammation or any unfavourable effects when viewed under the light microscope using multiple magnification powers. Since there are no significant increases observed in liver and kidney parameters, therefore strongly suggests that there are no obvious detrimental effects or morphological disturbances caused by the daily oral administration of TD0014 for 90 days. In light of these findings, we may conclude that TD0014 is not toxic in all the doses studied herein and did not produce any toxic signs or evident symptoms at acute and subchronic oral toxicity. Looking for the toxicity information of the natural ingredients of TD0014, we found that most of these herbs had an LD50 19 value above 2 g/kg and were classified by GHS as unlikely to present acute hazard, simultaneously long-term use on experimental animals also did not cause adverse effects on the structure and function of some internal organs. 4.2. Androgenic activities and effects of TD0014 on erectile function 4.2.1. Androgenic activities of TD0014 The Hershberger Bioassay is a short-term in vivo screening assay for androgen agonists which is based on the changes in weight of several androgen-dependent tissues on the day after treatme