1 INTRODUCTION Necessity of the thesis Knee osteoarthritis is a common skeletal musculoskeletal condition, which is one of the main causes of decreased mobility and inactivity The disease has expensive treatment costs and ineffective effects while many serious adverse effects create a burden on patients and society The search for safe and effective medicines of medicinal origin that can treat this disease is very necessary, especially in a country with a rich source of medicinal materials and a long-standing traditional medicine like in Vietnam TD0015 consists of 21 medicinal herbs that have been reported by many studies around the world for their anti-inflammatory, analgesic and anti-degenerative effects Thus, confirming the effectiveness of treatment as well as providing scientific evidence on the safety of products when combining these herbs is necessary, scientific and practical issue It brings new options for treating osteoarthritis In order to confirm the effectiveness of treatment as well as provide scientific evidence for product safety, the study titled "Experimental study on the safety and effectiveness of TD0015 hard pills for the treament of knee osteoarthritis " was carried out Objectives Determinate acute toxicity and subchronic toxicity of TD0015 hard pills on the experiment Evaluate the anti-inflammatory and analgesic effects of TD0015 hard pills on the experiment Evaluate the effect of TD0015 hard pills on rat model of knee osteoarthritis The novelty of the thesis The thesis is the first study to establish an animal model of knee osteoarthritis in rats in Vietnam This is a relatively new empirical model in the world, employing Kim Joon Ki's method, which allows the evaluation of the symptom-relieving effects in osteoarthritis such as inflammation, pain, movement limitation, and cartilage damage On this basis, TD0015 has displayed the anti-inflammatory, analgesic effects and the ability to inhibit articular cartilage destruction Additionally, the results have also suggested that the mechanism of action is reducing the specific cytokine indicators in osteoarthritis, including interleukin-1β and TNF-α, thereby making the basis for clinical trial studies on the effectiveness of TD0015 in the treatment of knee osteoarthritis The meaning of scientific and practical subjects The research results of the thesis have contributed to confirming the safety and treatment effects of TD0015 knee osteoarthritis in experiments, creating a premise to continue clinical trials This is a scientific basis that develops an effective treatment for knee osteoarthritis from available natural materials Thesis outline The thesis has 139 pages, including the following sections: Introduction (2 pages), Overview (36 pages), Subjects and research methods (18 pages), Results (42 pages) including 34 tables, charts, 38 figures,discussion (38 pages), Conclusions (2 pages), Recommendations (1 page) The thesis has 177 English and Vietnamese references Chapter OVERVIEW 1.1 Knee osteoarthritis Osteoarthritis is a lesion of the entire joint, including the damage of cartilage, subchondral bones, ligaments, joint muscles, synovial membranes, along with some morphological changes such as joint space narrowing, osteophyte formation, and fibrosis in subchondral bones A high proportion of osteoarthritis occurs in the knee Synovitis is considered a key factor in the pathogenesis of osteoarthritis The damage of subchondral bone also plays an important role in this condition, manifested by bone remodeling that occurs primarily in the early stages of osteoarthritis This can make the subchondral bone less capable of absorbing and dispersing the force of action, combined with an increase in bone mass to increase the force transmitted through the joint, therefore, leading to increased joint damage The main treatment options include non-pharmacological treatment, drug therapy (pharmacotherapy), and surgical treatment 1.2 Drugs for osteoarthritis Current osteoarthritis medications include symptomatic medications and disease-modifying osteoarthritis drugs In particular, anti-inflammatory and analgesic drugs play a vital role in relieving symptoms quickly 3 1.2.1 Anti-inflammatory and analgesics drugs The AAOS recommends that patients with symptomatic knee OA and risks of gastrointestinal disorders (age ≥ 60 years, multiple co-morbidities, a history of peptic ulcer disease or bleeding, concomitant use of corticosteroids and/or anticoagulants) should use one of the following medications for pain relief: Paracetamol (no more than 4g daily); Topical NSAIDs; Non-selective oral NSAIDs with gastroprotective drugs; COX-2 selective inhibitors 1.2.2 Disease-modifying osteoarthritis drugs Disease-modifying osteoarthritis drugs include glucosamine, chondroitin sulfate, diacerein, avocado-soybean unsaponifiable, and hyaluronic acid The effects of these drugs lasted long (average 1-2 months after use) and maintained even after stopping treatment (after a few weeks to 2-3 months) Glucosamine exists in the body as a major substrate in proteoglycan biosynthesis - a compound necessary to maintain cartilage integrity Although the mechanism of action is not clear, glucosamine has been shown to reverse the inflammatory and destructive effects of IL-1 on cartilage and degenerative cartilage Chondroitin sulfate is a polysaccharide that inhibits several cartilage-digesting enzymes, especially metalloprotease, improving pain and cartilage structure Diacerein is an inhibitor of cytokines such as IL-1 by reducing the number and sensitivity of IL-1 receptors to cartilage in cells; decreasing TNF-α levels, and reducing the production of cytokines, NO, and MMPs, which damage cartilage cells and synovial membranes However, diacerein is associated with a significant risk of diarrhea in patients Hyaluronic acid has the effects of covering and lubricating the surface of articular cartilage, preventing the loss of proteoglycan.However, the evidence for its improvement of motor function and pain relief is controversial Besides, some other drugs, such as inhibitors of matrix metalloproteinases (MMPs); ADAMTS inhibitors; iNOS synthesis inhibitor; monoclonal antibodies to IL-1β (canakinumab), antibodies to TNF-α (adalimumab, infliximab), are under investigation to further understand the molecular mechanism of osteoarthritis 1.3 Overview of TD0015 hard pills 1.3.1 TD0015 TD0015 consists of 21 ingredients: Cortex Phellodendri amurensi (2,26g), Radix Paeoniae lactiflorae (0,77g), Radix Rehmanniae glutinosae (0,7g), Cortex Eucommiae (0,47g), Poria cocos (0,47g), Radix Codonopsis pilosulae (0,34g), Radix Angelicae sinensis (0,34g), Rhizoma Anemarrhenae (0,31g), Flos Pruni persicae (0,26g), Radix Ledebouriellae seseloidis (0,23g), Herba Loranthi (0,23g), Radix Gentianae macrophyllae (0,23g), PericarpiumCitri reticulatae perenne (0,22g), Rhizoma Ligustici wallichii (0,17g), Radix Angelicae pubescentis (0,17g), Radix Glycyrrhizae (0,12g), Ramulus Cinnnamomi (0,08g), Herba Asari (0,08g), Radix Achyranthis bidentatae (0,03g), Plastrum Testudinis (2,97g), mixed bone (0,7g) 1.3.2 Studies on the effects on TD0015 in the treatment of OA Many components in TD0015 have been reported to reduce inflammation, relieve pain, and improve cartilage structure in osteoarthritis, which includes Cortex Phellodendri amurensi, Cortex Eucommiae, Ramulus Cinnnamomi, Poria cocos, Herba Asari, Radix Angelicae pubescentis Hoang Thi Thang et al in 2017 also reported the effects of TD0015 on relieving pain, improving the cervical spine’s range of motion, reducing cervical scapulohumeral syndrome, and improving of patients’ movement when combining the electroacupuncture method with TD0015 Chapter SUBJECTS AND METHODOLOGY 2.1 TD0015 preparation TD0015 was manufactured as hard pills according to the quality standards of Sao Thai Duong Joint Stock Company, Vietnam The preparation was packed as grams per sachet The predicted clinical dose of TD0015 is sachets per day (equivalent to 10 grams per day), with the treatment duration of consecutive months 2.2 Subjects: Swiss mice, Wistar rats 2.3 Methodology 2.3.1 Acute and subchronic toxicity of TD0015 in animals 2.3.1.1 Acute oral toxicity of TD0015 in mice: Litchfield – Wilcoxon’s method The acute oral toxicity study of TD0015 was conducted according to the general guidelines for methodologies on research and evaluation of traditional medicine of WHO Mice weighing 20 ± 2g were randomly divided into groups, 10 per group TD0015 was administered from the highest non-lethal dose to the lowest dose that killed 100% mice Mice were fasted for 12 hours before receiving the medication, ad libitum access to water The number of dead mice was recorded during the first 72 hours, and the general conditions of mice were evaluated within days after taking the drug If any mouse died, the autopsy was conducted to assess organ damage 50% mortality (LD50) was calculated according to the mortality rate within the first 72 hours 2.3.1.2 Subchronic oral toxicity of TD0015 in rats: WHO guidelines Rats were randomized into groups (n = 10), taking the drugs orally for 90 days: - Group (control): sterile distilled water 10 ml/kg b.w/day - Group 2: TD0015 1,2g/kg body weight/day - Group 3: TD0015 3,6g/kg body weight/day Parameters for follow-up at baseline (D0), after 30 days (D30), 60 days (D60) and 90 days (D90), included: general status, body weight Parameters for hematopoietic functions: number of red blood cells, average red cell volume, hemoglobin content, hematocrit, leukocyte counts, leukocyte formula and platelet counts Evaluation of liver functions through the determination of certain metabolites in the blood: albumin and total cholesterol Evaluation of liver damage by quantitative enzyme activity in blood: ALT, AST Evaluation of glomerular filtration function by quantifying creatinine concentration in blood Histopathology: After 90 days of treatment, the rats were operated to evaluate the whole body 30% of rats in each group were randomly examined for liver and kidney structure 2.3.2 The analgesic effect of TD0015 Adapting from the method of Ezeja Mi (2011) 2.3.2.1 The analgesic effect of TD0015 using Hot plate Mice were randomized into groups (n = 10), taking the drugs orally for days: - Group (control): sterile distilled water 20 ml/kg b.w/day - Group 2: codeine phosphate 20 mg/kg body weight/day - Group 3: TD0015 2,4g/kg body weight/day - Group 4: TD0015 7,2g/kg body weight/day Time to reaction to temperature was measured in each mouse before the experiment and one hour after taking codeine/TD0015 Place the animals on the hot plate maintained at 55 ± 10C Time to reaction was counted from the moment animals were put on the hot plate to when they licked their hind legs Reaction time to the heat stimulation was compared between before and after taking codeine/TD0015 2.3.2.2 The analgesic effect of TD0015 using Dynamic Plantar Aesthesiometer Mice were randomized into groups (n = 10), same study design as above Measure the response time to pain and the force to inflict pain in rats before taking codeine/TD0015 and one hour after taking codeine/TD0015 Reaction time to the pain stimulation was compared between before and after taking codeine/TD0015 2.3.2.3 The analgesic effect of TD0015 using acetic acid Mice were randomized into groups (n = 10), same study design as above Codeine was replaced with aspirin 150mg/kg On the last day, hour after taking aspirin/TD0015, 0.2 mL of 1% acetic acid was injected into the abdominal cavity of the animals Count the number of cramps in each rat in 5-minute intervals for 30 minutes after acetic acid injection 2.3.3 The anti-inflammatory effect of TD0015 Adapting from the method of Kim Kyung Soo (2008) 2.3.3.1 The acute anti-inflammatory effect of TD0015 * Carrageenin-induced rat paw oedema model Rats were randomized into groups (n = 10), taking the drugs orally for days - Group (control): sterile distilled water 10 ml/kg b.w - Group 2: aspirin 200 mg/kg body weight/day - Group 3: TD0015 1,2g/kg body weight/day - Group 4: TD0015 3,6g/kg body weight/day On day 5, hour after taking aspirin/TD0015, inflammation was by induced by injecting 0.05ml of 1% carrageenin into the back of the rat’s right paw Measure the paw volume with specialized equipment at the following timepoints: before inducing inflammation (V0); hours after the onset of inflammation (V2), hours (V4), hours (V6) and 24 hours (V24) Results were calculated using the Fontaine's formula * Carrageenin-induced peritonitis model Rats were randomized into groups (n = 10), same study design as above On day 5, hour after taking aspirin/TD0015, induce peritonitis in rats with carrageenin solution of 0,05g + formaldehyde of 1,5 ml, mixed sufficiently in 100ml of physiological saline, inject 1ml/100g into the abdominal cavity After 24 hours, open the rat's abdominal cavity to suck the inflamed exudate, measure the volume, count the number of white blood cells/ml of the exudate and quantify the protein in the exudate 2.3.3.2 The chronic anti-inflammatory effect using asbestos-induced granuloma model Mice were randomized into groups (n = 10) - Group (control): sterile distilled water 20 ml/kg b.w/day - Group 2: methylprednisolon 10 mg/kg body weight/day - Group 3: TD0015 2,4g/kg body weight/day - Group 4: TD0015 7,2g/kg body weight/day Inducing chronic inflammation by implanting 6mg sterilized asbestos fibers with 1% carrageenin (drying 120oC for hour), in the neck of each mouse After transplanting granulomas, mice were given orally distilled water or methylprednisolone/TD0015 continuously for 10 days On day 11, mice were sacrificed The granulomas were removed and weighed Randomly select granulomas per group to perform histology The remaining granulomas were dried at 56°C for 18 hours Weigh the granulomas after they were dried 2.3.4 The anti-osteoarthritis effect of TD0015 Adapting from the methods of Kim (2012), Calado (2015) Rats were randomized into the following groups (n = 10) Group 1A and 1B (negative control): sterile distilled water 10 ml/kg body weight/day Group 2A and 2B (positive control): sterile distilled water 20 ml/kg body weight/day Group 3: diclofenac 3mg/kg body weight/day Group 4: TD0015 1,2g/kg body weight/day Group 5: TD0015 3,6g/kg body weight/day Rats were housed in assigned groups under laboratory conditions weeks before study entry Rats in group to group were induced osteoarthritis using the method of Kim et al, which was injecting MIA solution 3mg/joint into the right knee joint of each rat The control group was injected with physiological saline acting as solvent into the right knee joint of each animal The volume injected was 50µl/joint After MIA injection, rats were orally given water and diclofenac/TD0015 for consecutive weeks, once a day The evaluated indicators include: 2.3.4.1 Diameter of the knees Knee diameter must be measured by an electric measurement Knee diameter was measured by one researcher at all times to ensure consistency The parameter was measured at the following timepoints: day 0, day 3, day 5, day 7, day 14, day 21, day 28, day 35, day 42 of the treatment period The outcome is the change in knee diameter at each timepoint 2.3.4.2 The analgesic, improving knee joint movement effects of TD0015 * The analgesic effect using Randall Selitto method The Analgesy meter 7200 was used to measure pain at the right knee of animals in all groups, before the study and every week after MIA injection for weeks, comparing among groups * The effect of improving knee joint movement using Dynamic Plantar Aesthesiometer Measure the response time to pain and the force to inflict pain in animals, at the right hind paw of the rats before the study and every week after MIA injection for weeks, comparing among groups This experiment assessed the analgesic effects and the right knee movement skills of the rats * The effect of improving knee joint movement using Hot plate Training phase (3 weeks): rats were acquainted with the Hot plate times, with a 1-week interval Efficiency evaluation phase: Knee movement was assessed once at the beginning of the study and every week after MIA injection for weeks, using the following indicators: * Time to jump off the hot plate: Place the animal on the hot plate (55 ± degrees C) The response time to heat stimulation, which causes the rat to jump off the hot plate (in seconds), is calculated from the time the rat is placed on the hot plate until the animal jumps off and escapes from the Hot plate * The height achieved when the rats jump from the hot plate: the maximum height achieved when the animals successfully escape from the hot plate, touch the hind paws on to the pipe wall and cling to the edge of the pipe wall to escape * The number of times the rats jump from the hot plate: A momentum jump is when the rat jumps but fails because of limited movement of the knee joint Calculate the number of failed jumps for each rat and compared among groups Rat activities were recorded via a camera system to ensure accuracy and uniformity 2.3.4.3 Cytokines IL-1β and TNF α are specific indicators that were quantified in rats’ serum after weeks of MIA injection using ELISA technique, using IL-1β and TNFα KIT for rats, Cloud Clone Corp (USA) 2.3.4.4 Histopathology of knee joints * After weeks of MIA injection: Rats in Group 1B, 2B were anesthetized, right knee joints were collected and preserved in a 10% formaldehyde solution * After weeks of MIA injection: Evaluation on rats of all groups after weeks of MIA injection and medication The rats were anesthetized and the right knees were removed, preserved in a 10% solution of formaldehyde The degree of osteoarthritis was assessed on histopathological specimens, based on lesion scores by Janusz and Al Saffar methods 2.4 Statistical analysis: Data were collected and analyzed using Excel 2013 and SPSS 20.0 software, with appropriate statistical algorithms (Student’s t-test, Paired t-test, Mann-Whitney U test) P-value < 0.05 was considered as statistical significance Chapter RESULTS 3.1 Acute and subchronic toxicity of TD0015 in animals 3.1.1 Acute oral toxicity in mice Table 3.1 Correlation between TD0015 dose and mice mortality Group n Group Group Group Group 10 10 10 10 Dose of TD0015 (g/kg) 15,0 22,5 30,0 37,5 Percentage of death (%) 0 0 Other toxicity signs None None None None Mice were administered up to 37,5g/kg of body weight In all mice, no signs of toxicity or death were observed within the critical 72 hours post-administration and to the end of day Hence, the LD50 evaluated by the Litchfield – Wilcoxon method could not be determined 3.1.2 Subchronic oral toxicity in rats 3.1.2.1 Body weight and clinical observation During the experiment, rats in all groups displayed normal activities, well consumption, bright eyes, silky hair, dry feces Animal 10 weight in all groups increased compared to before the study but the increase of groups taking TD0015 was lower than in the control group, especially after 90 days (p 0.05) After the treatment, animals administered TD0015 at doses had significantly lower cholesterol levels compared to the control group and compared to before treatment (p 0,05 > 0,05 > 0,05 1,57 ± 0,13 1,52 ± 0,23 1,50 ± 0,22 > 0,05 > 0,05 > 0,05 1,36 ± 0,12 1,14 ± 0,22 1,10 ± 0,13 > 0,05 < 0,05 < 0,05 p > 0,05 > 0,05 > 0,05 < 0,05 3.1.2.4 Effects on liver damage There was no difference in the concentration of AST and ALT enzymes in TD0015 administered groups compared with the control group, and between before and after taking medication during the study period (p> 0.05) 3.1.2.5 Effects on kidney function TD0015 at doses did not affect the creatinine concentration in rat blood after 30 days, 60 days and 90 days of continuous drug administration (p > 0.05) 3.1.2.6 Effects on histopathology of liver and kidney - Liver histopathology: In all groups, the liver structure was not reversed The central venous areas of the hepatic lobes and the portal 11 spaces were not fibrotic, with no inflammation and no increased bile duct production The hepatocytes were normal or with very mild degeneration There was no difference in liver microscopic structure between groups of TD0015 and the control group - Kidney histopathology: In all groups, glomerular morphology, and structure were within normal limit, no fibrosis, no proliferation Renal parenchyma was not inflamed or necrotic, with normal stroma, without the penetration of inflammatory cells 3.2 The analgesic effect of TD0015 3.2.1 The analgesic effect of TD0015 using Hot plate Codeine demonstrated a markedly long-lasting effect in response to temperature compared to before treatment ,and compared to the control group (p 0.05) 3.2.2 The analgesic effect of TD0015 using Dynamic Plantar Aesthesiometer TD0015 at doses taken for consecutive days significantly increased the force to inflict pain and pain response time on the pain threshold meter (p