Nghiên cứu độc tính và tác dụng điều trị suy giảm sinh dục đực của viên hoàn cứng TD0014 trên thực nghiệm tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY MAI PHUONG THANH EXPERIMENTAL STUDY ON THE SAFETY AND EFFECTIVENESS OF TD0014 HARD PILLS FOR THE TREATMENT OF MALE SEXUAL DYSFUNCTION Specialty: Pharmacology and Toxicology Code: 62720120 SUMMARY OF DOCTORAL THESIS HANOI – 2019 The study was completed at: HANOI MEDICAL UNIVERSITY Scientific supervisors: Assoc Prof Ph.D Pham Thi Van Anh Assoc Prof Ph.D Nguyen Trong Thong Reviewer 1: Reviewer 2: Reviewer 3: The doctoral thesis was publicly defended in front of the University Council at Hanoi Medical University on The thesis can be found at: - National Library of Vietnam - The Library of Hanoi Medical University INTRODUCTION Male sexual dysfunction includes disorders of desire, erectile dysfunction, abnormal ejaculation, orgasmic dysfunction and failure of detumescence; these conditions may appear alone or in combination with each other Researching and seeking drugs for the treatment of male sexual dysfunction originating from medicinal herbs are of great interest to physicians TD0014 is a preparation of herbal medicines which comprises thirty-two medicinal plants and deer velvet The composition of TD0014 has several medicinal herbs that have been studied and used since ancient times in traditional folk medicine as an aphrodisiac However, no studies have provided reliable shreds of evidence of their effects on reproductive functions, or toxicity when combining them in TD0014 Therefore, the study titled "Experimental study on the safety and effectiveness of TD0014 hard pills for the treatment of male sexual dysfunction" was carried out to: Determine acute and subchronic toxicity of TD0014 in experimental animals Evaluate androgenic activity and effects on erectile function of TD0014 in experimental animals Evaluate the effects of TD0014 on sodium valproate-induced reproductive decline in male rats Necessity of the thesis Male sexual dysfunction is a common disorder associated with a wide range of physical and psychological conditions Although this disease is not fatal, does not require emergency management but greatly affects the morale and quality of life of patients Research and development of preventive and therapeutic agents for male sexual dysfunction have been becoming a special concern of world medicine Treatment of male sexual dysfunction according to modern medicine is highly effective in improving symptoms However, one must be conscientious of its adverse effects Following the current trends in medicine, the treatment approach based on the theory of traditional medicine that has many advantages such as reducing the economic burden, convenience, friendliness, and fewer side effects has deployed in many countries around the world, including Vietnam, a country with long-standing traditional medicine and is considered to have great potential for medicinal materials in Southeast Asia and in the world with the source of rich and diverse resources The widespread use of herbal medicines, nevertheless, requires scientific verification of their indications and effects by use of modern medical analysis Therefore, studies of toxicity and effects of medicinal herbs, herbal remedies or products derived from medicinal plants such as TD004 which has used in the market as dietary supplement are necessary, scientific and practical New contributions from the thesis TD0014 is a preparation derived from over thirty natural ingredients When combining many medicinal ingredients in a preparation, the most worrying issue is the interaction between the ingredients in the preparation process as well as in the metabolism in the body, which can increase toxicity, decrease or loss of effects The study has shown positive results with high safety and good effectiveness in treating male sexual dysfunctions of TD0014 hard pills on the experiment, which considered a new contribution of the thesis These results are the basis for further preclinical and clinical trials which will provide the scientific foundation for using TD0014 in treating male sexual dysfunction Tests of androgenic activity have been conducted by a number of domestic research institutes with a variety of preparations, but mostly on castrated male rats Evaluation of the androgenic activity of TD0014 in weanling male rats can also be considered a new contribution of the thesis Thesis outline The thesis consists of 153 pages, including introduction (2 pages), overview (44 pages), object and methods of research (12 pages), results (43 pages which comprises 37 tables, 16 figures, 10 charts), discussion (49 pages), conclusion (2 pages), proposal (1 page) There are 194 English and Vietnamese references Chapter OVERVIEW 1.1 Overview of male sexual dysfunction according to modern medicine Male sexual dysfunction includes disorders of desire, erectile dysfunction, abnormal ejaculation, orgasmic dysfunction and failure of detumescence; these conditions may appear alone or in combination with each other According to modern medicine, methods used to treat male sexual dysfunction include testosterone replacement therapy and treatments for one of the most common symptoms of the disease which is erectile dysfunction 1.1.1 Testosterone replacement therapy Testosterone treatment aims to restore testosterone levels to the physiological range in men with consistently low levels of serum testosterone and associated symptoms of androgen deficiency The aim is to improve quality of life, sense of well-being, sexual function, muscle strength and bone mineral density Current testosterone therapies include implants, intramuscular injections, oral formulations, transdermal delivery systems, transbuccal delivery systems, and intranasal testosterone The risks of testosterone replacement therapy depend on age, life circumstances and other medical conditions, include exacerbation of prostate cancer, male breast cancer, worsening benign prostatic hyperplasia, hepatotoxicity and liver tumor, polycythemia, an increased risk of obstructive sleep apnea and congestive heart failure, infertility, and skin diseases With these risks, TRT is contraindicated in men with known or suspected androgen-dependent carcinoma of the prostate or of the male mammary gland, severe lower urinary tract symptoms due to benign prostatic hyperplasia, past or present liver tumors, severe chronic cardiac failure (NYHA IV), severe sleep apnoea, hematocrit > 54%, and male infertility-active desire to have children 1.1.2 Drugs for erectile dysfunction Lifestyle modifications, oral phosphodiesterase-5 (PDE5) inhibitors, intracorporal injections, topical medication, surgery, vacuum devices, and acupuncture are some of the treatments available today for erectile dysfunction, among which PDE inhibitors are the first-line treatment in patients with erectile dysfunction PDE5 inhibitors are indicated for treatment of men with erectile dysfunction, which is the inability to achieve or maintain a penile erection sufficient for satisfactory sexual performance In order for them to be effective, sexual stimulation is required There are currently four widely approved PDE5 inhibitors and are the first choice in treating erectile dysfunction, including sildenafil, tadalafil, vardenafil, and avanafil; in addition, a number of other PDE5 inhibitors are licensed in certain countries, such as udenafil and mirodenafil designated in Korea, or lodenafil used in Brazil All PDE5 inhibitors are contraindicated in patients with a known hypersensitivity to any component of the agents; patients who are taking any form of organic nitrates, regularly or intermittently; patients who have loss of vision in one eye because of non-arteritic anterior ischaemic optic neuropathy (NAION), regardless of whether this episode was in connection or not with previous PDE5 inhibitor exposure They should not be used in men for whom sexual activity is inadvisable (e.g patients with severe cardiovascular disorders such as unstable angina or severe cardiac failure), severe hepatic impairment, hypotension (blood pressure < 90/50 mmHg), recent history of stroke or myocardial infarction and known hereditary degenerative retinal disorders such as retinitis pigmentosa (a minority of these patients have genetic disorders of retinal phosphodiesterases) 1.2 Overview of the mode of action of medicinal herbs for male sexual dysfunction A great number of traditional herbal medicine have been used to treat male sexual dysfunction, especially erectile dysfunction, applied singly or in the form of compound formulas Most of the herbal remedies are used empirically and thereby are not convincing In this condition, many studies are being conducted to investigate the underlying mechanism of those herbs using modern biotechnology Figure The effects of male sexual stimulation of the main bioactive compounds of medicinal plants 1.3 Herbal formula TD0014 preparation The major ingredients of the herbal formula are obtained from thirty-three natural products: Tribulus terrestris (4.00g), Chrysanthemum sinense (1.83g), Prunus persica (1.14g), Vigna cylindrica (1.14g), Eurycoma longifolia (0.69g), Sophora japonica (0.57g), Dioscorea persimilis (0.43g), Dioscorea tokoro (0.40g), Polygonum multiflorum (0.40g), Citrus deliciosa (0.34g), Polyscias fruticosa (0.34g), Tinospora sinensis (0.29g), Chaenomeles lagenaria (0.29g), Passiflora foetida (0.29g), Zizyphus sativa (0.29g), Rehmannia glutinosa (0.23g), Angelica sinensis (0.23g), Alisma plantago-aquatica L var orientalis Samuelsson (0.23g), Achyranthes bidentata (0.23g), Schizandra chinensis (0.23g), Morinda offcinalis (0.23g), Rosa laevigata (0.23g), Allium sativum (0.20g), Lycium sinense (0.17g), Glycyrrhiza uralensis (0.14g), Panax ginseng (0.11g), Ligusticum wallichii (0.11g), Cistanche tubulosa (0.11g), Atractylodes macrocephala (0.11g), Radix Codonopsis (0.11g), Cuscuta sinensis (0.11g), Psoralea corylifolia (0.06g), Cornu Cervi parvum (7.2mg) The composition of TD0014 has several medicinal herbs that have been shown to enhance sexual activity in many preclinical and clinical studies, including Tribulus terrestris, Eurycoma longifolia, Angelica sinensis, Morinda offcinalis, Lycium sinense, Panax ginseng, Ligusticum wallichii, Cistanche tubulosa, Cuscuta sinensis, Psoralea corylifolia Chapter OBJECTS AND RESEARCH METHODS 2.1 Polyherbal formula TD0014 preparation TD0014 was manufactured as hard pills according to the quality standard of Sao Thai Duong Joint Stock Company, Vietnam The preparation was packed in 7.5 grams per sachet The predicted human dose of TD0014 is sachets in divided doses (equivalent to 15 grams of raw medicinal materials/day) 2.2 Animals: Swiss mice, Wistar rats 2.3 Research methods 2.3.1 Acute and subchronic toxicity of TD0014 in animals 2.3.1.1 Acute oral toxicity The acute oral toxicity study of TD0014 was conducted according to the general guidelines for methodologies on research and evaluation of traditional medicine of WHO and determined lethal dose of 50% by the Litchfield-Wilcoxon method Adult male mice were separately divided into groups of 10 animals After an overnight fast, each dose of TD0014 was administered orally from the highest nonlethal dose to the lowest one that killed 100% mice The animals were observed for signs of toxicity and mortality for the first critical 72 hours and thereafter daily for days The oral median lethal dose (LD50) was calculated according to the number of mice mortality within the first 72 hours 2.3.1.2 Subchronic oral toxicity The subchronic oral toxicity study of TD0014 was carried out according to the general guidelines for methodologies on research and evaluation of traditional medicine of WHO The toxicity study was carried out using thirty-three adult male rats The rats were divided into three groups of 11 animals per group Group I served as the vehicle control and received ml/100 g b.wt sterile distilled water daily while groups II and III were administered TD0014 at the dose of 1.8 and 5.4 g/kg b.wt daily in appropriate volume of distilled water for 90 days On the first day (D0) and at the end of day 30 (D30), D60, and D90, rats in all group were weighed and collected blood samples from femoral vein for determining haematological parameters (red blood cell, haemoglobin concentration, hematocrit, mean capsular volume, total white blood cells and white blood cell differential, platelet count) and biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total cholesterol, total bilirubin, creatinine) At the end of the experiment, 30% of rats of each group were made unconscious by cervical dislodgement, then the internal organs (liver, kidney) were removed and observed for any gross lesions These organs were preserved in a fixation medium of 10% buffered formalin for histopathological study 2.3.2 Androgenic activity of TD0014 in immature male rats The androgenic activity of TD0014 evaluated by the Hershberger assay has been adapted and standardized according to OECD guidelines 2.3.2.1 Androgenic effects of TD0014 in the castrated male rats For the experimental, 45 peripubertal male rats were used with aged between 42 and 50 days After castration by removing both testes and epididymides and full recovery in seven days, the rats were separated into groups of animals each - Group I: Intact + distilled water - Group II: Castrated + distilled water - Group III: Castrated + testosterone at dose of 0.4 mg/kg - Group IV: Castrated + TD0014 at dose of 1.8 g/kg - Group V: Castrated + TD0014 at dose of 5.4 g/kg) The rats received testosterone by s.c injection or TD0014 by oral gavage, once daily for 10 consecutive days 24 hours after the last administration, the rats were sacrificed by exsanguination Blood samples from the carotid artery were collected for testosterone measurement The five androgen-dependent tissues: ventral prostate, seminal vesicles, levator ani-bulbocavernosus muscle (LABC), bulbourethral glands (Cowper), and glans penis were harvested and weighed 2.3.2.2 Androgenic effects of TD0014 in the weanling male rats Forty weanling 21-day old male rats were separated into groups of 10 animals each, treated daily for 10 consecutive days with distilled water (control; 10 mL/kg b.w orally), testosterone propionate (TP) s.c (androgenic control; mg/kg/day s.c.), or TD0014 at 1.8 and 5.4 g/kg by gavage The animals were killed 24 h after the last dose, followed by exsanguination Blood samples from the carotid artery were collected and stored until analyzed for testosterone concentration At necropsy, the reproductive organs (testis, epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator ani/bulbocavernosus muscles [LABC]) were excised, trimmed free of fat and connective tissues, and weighed 2.3.3 Effects of TD0014 on erectile function The experiment was performed according to the method of Gajbhiye et al (2015) Adult male rats were randomly separated into groups of animals each: 1) control, 2) sildenafil treatment, and 3) TD0014 treatment In each group, animals were administered per os one-time with either distilled water (10 ml/kg b.w.), or sildenafil (6 mg/kg b.w.), or TD0014 (1.8 g/kg b.w.) At hours after treatment, rats were anaesthetized with an intraperitoneal injection of ketamine at a dose of 25 mg/kg The corpus cavernosum were exposed To measure intracavernosal pressure (ICP), a heparinized 23-gauge butterfly needle was inserted into the proximal portion of the corpus cavernosum A bipolar electrical stimulator was placed on the ganglion to stimulate the cavernosal nerve for 60 seconds at V and 20 Hz for millisecond periods The cavernosal nerve stimulation was conducted times with a 10-minute interval between stimulations The mean systemic arterial pressure (MAP) was monitored simultaneously with ICP monitoring The right carotid artery was dissected and then PE-50 tubing was inserted into the carotid artery The catheter was connected to both a pressure transducer and an amplifier unit which was connected to a data acquisition module Before and after each electrical stimulation, ICP and MAP was recorded on a computer by Powerlab system record software Erectile function was evaluated by the following parameters: ICP before and after the electrical stimulation of the cavernous nerve (basal ICP and maximal ICP), time to the maximal ICP, response time to the elec- trical stimulation of the cavernous nerve, total ICP (ICP vs stimulation time, area under curve), MAP and maximal ICP/MAP ratio 2.3.4 Effects of TD0014 on sodium valproate-induced reproductive decline in male rats The experiment used sodium valproate (SVP) to cause reproductive toxicity in male rats according to a model described by Nishimura et al (2000) 2.3.4.1 Protective role of TD0014 The adult male rats were randomly divided into the four groups The control group (Group 1, negative control) was given distilled water orally Another group (Group 2, posittive control) received SVP (500 mg/kg, oral) for weeks and served as toxic control The remaining two groups (Group and 4) received TD0014 orally at doses of 1.8, and 5.4 g/kg respectively along with SVP for weeks On 5th week, one male rat was randomly coupled with two untreated virgin females for weeks At the end of 7th 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 - 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 weeks to cause reproductive toxicity, then distilled water or TD0014 was continuously administered orally for 10 days: - Group 1: not given SVP for weeks, distilled water 10 mL/kg/day for 10 days - Group 2: given SVP for weeks, distilled water 10 mL/kg/day for 10 days - Group 3: given SVP for weeks, TD0014 1.8 g/kg/day for 10 days - Group 4: given SVP for 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 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, Chisquare test) A p value