【Animal modeling】-Selective cathepsin K inhibitor MIV-711 reduces joint pathology in animal models of osteoarthritis
动物造模,关节炎
z-bo
2022-02-04
389
Background: Osteoarthritis (OA) is a common musculoskeletal disease characterized by damage to the joint structure, leading to symptoms such as joint stiffness and pain. Osteoarthritis affects the entire joint, including cartilage and subchondral bone degeneration and synovial inflammation. The disease is characterized by cartilage degradation, and the role of subchondral bone in the occurrence and development of osteoarthritis has received more and more attention in recent years. A high level of subchondral bone displacement indicates a narrowing of the joint space in patients with osteoarthritis. Bone wear, which reflects the loss and flattening of subchondral bone, may be due to insufficient bone mass, and is related to bone marrow disease, cartilage damage and structural abnormalities. The change of subchondral bone seems to occur in the early stage of OA, possibly before cartilage damage. However, it is also clearly recognized that there are extensive interactions between subchondral bone and articular cartilage, and early biomechanical changes may affect another corresponding part. Cathepsin K is mainly expressed by osteoclasts and is a key enzyme involved in bone resorption through the lysis of type I collagen. Cathepsin K is also expressed in cartilage cells in cartilage. It can cleave proteoglycans and type II collagen, the main component of cartilage matrix. In the case of compact osteogenesis imperfecta, the disease is caused by inhibiting cathepsin K gene mutations and exhibits abnormally dense bone (osteosclerosis). This effect is reproduced in transgenic mice lacking cathepsin K. Selective cathepsin K inhibitors increase bone mineral density in postmenopausal osteoporosis and postmenopausal osteoporosis patients. Odanacatib, the most advanced cathepsin K inhibitor, reduced the incidence of vertebral and hip fractures in postmenopausal women in a phase III clinical study. Although a large number of studies have proven that cathepsin K inhibitors can have beneficial effects on osteoporotic bone, the effects of cathepsin K on subchondral bone and articular cartilage are poorly understood. The data available in preclinical models of joint degeneration supports the positive role of cathepsin K in the disease process, which is confirmed by experimental cathepsin K inhibitor studies. The cathepsin K inhibitor sb553484, despite its poor selectivity, exerts chondroprotective effects on dogs with partial meniscus excision. The selective cathepsin K inhibitor l-006235 reverses anterior cruciate ligament transection (ACLT) rabbit subchondral bone loss. However, neither of these two compounds have progressed to clinical development. MIV-711 is a potent and selective proteinase K inhibitor currently used in phase II clinical OA treatment. Patients with knee osteoarthritis who received MIV-711 daily treatment for 6 months showed joint structural benefits. Compared with patients who received placebo, magnetic resonance imaging (MRI) assessed the cartilage thinning of the affected knee joint. This article summarizes the pathological effects of MIV-711 on ACLT rabbit joints and partial meniscus resection dogs. The clinically relevant risks of MIV-711 dose used in animal models are considered safe.
Method: Rabbit Anterior Cruciate Ligament Tectome (ACLT): Male New Zealand rabbits, 32. At the beginning of the experiment, the animals were about 9 months old and weighed 3.7-4.5 kg. They were randomly divided into four groups according to their body weight. Preoperative intravenous butorphanol (0.5-1 mg/kg, intramuscular injection) for analgesia, and glycopyrrolate (0.1 mg/kg) 15 minutes before induction. Ketamine (20-40 mg/kg, intramuscular injection) and xylazine (5-10 mg/kg, intramuscular injection) mixed anesthesia, followed by placement of a tracheal tube. Isoflurane maintains general anesthesia. Cefazolin (40 mg/kg, intravenous injection) was administered during the perioperative period, and after tracheal extubation, yobine was given to reverse xylazine. Fentanyl patches (25 µg/h) are placed on the incision area of the skin to provide postoperative pain relief. Under aseptic conditions, the incision on the inside of the right knee is 1.5-2.5 cm. The patella is laterally dislocated and the knee joint is fully flexed. The anterior cruciate ligament (ACL) is exposed and cut by an appropriate blade. For animals in the sham operation group, ACL was exposed but not cut. The joints are rinsed with sterile saline and closed. The muscle and skin are sutured and closed with surgical glue. Cyclomethanol (0.5-1 mg/kg, intramuscular injection) provides postoperative analgesia, and the animals are placed in single cages.
Experimental design and sample collection: In the sham operation group and one week after ACLT, the animals received oral MIV-711 or excipients for 7 consecutive weeks at a dose of 4ml/kg. The four groups are: (1) Sham surgery + excipient group; (2) ACLT surgery + excipient group; (3) ACLT surgery + MIV-711, 30umol/kg group and (4), ACLT surgery + MIV -711, 100umol/kg group. The dose and dosing interval are based on normal New Zealand rabbit cathepsin K titer and pharmacokinetics. The study dose of rabbit ACLT was selected to be the same as the well-tolerated exposure range of humans, effectively reducing the biomarkers of bone resorption and cartilage degradation in healthy subjects. Twelve 1 ml blood samples were collected from each animal in a staggered manner to cover as many time points as possible for determination. Blood samples were collected on day 1, day 10, and week 4 and week 7. The samples were collected in EDTA coated tubes and kept on ice. The plasma is separated by centrifugation (2000g at 4°C for 3-5 minutes) and then stored frozen at -70°C until analysis. All animals collected urine before surgery, and biomarker measurements were taken before surgery, day 1, day 10, and week 4 and week 7 before administration. Urine is stored frozen (-70--80°C) until analysis.
Micro-computed tomography (uCT): uCT scans were performed from all animal femurs and tibias to observe bone and cartilage respectively. With an isotropic voxel resolution of 36 μm, an exposure time of 300 milliseconds, 2000 views and 1 frame of image data are obtained. Each sample was scanned twice, once for bone data and once for soft tissue data. After the bone scan, Hexabrix was used to contrast-enhance the stained cartilage of the knee. VHLab segmented the region of interest (ROI) (osteophytes, cartilage and subchondral bone) for the femur and tibia of each sample. A region spanning 9 mm of the femoral articular surface and 6.5 mm of the tibia was selected as the anteroposterior direction of the segmented cartilage and subchondral bone. Then multiply the voxel count by the cube pixel resolution to obtain volume measurements of different ROIs. Calculate the distance from the bone to the outer surface of the articular cartilage. Generate an image and convert the distance to a color map to observe the thickness along the length of the cartilage.
Canine partial meniscus resection surgery: 30 adult female beagle dogs, weighing 7.5-11.3 kg, about 6-8 months old, these dogs were domesticated for at least 7 days before starting the study. At least 12 hours before the operation, a 2.5 mg patch of fentanyl transdermal patch was placed on the ventral skin of each dog's tail and fixed in place with tape. The analgesic duration of fentanyl patch is 72 to 80 hours, and the analgesic effect is about 25μg/h. Preoperative subcutaneous administration of Weichangning (0.005-0.02 mg/kg), acepromazine (0.01-0.05 mg/kg) and morphine (0.5-1.0 mg/kg). Anesthesia was induced by intravenous injection of propofol (2-6 mg/kg); each dog was intubated with trachea and isoflurane was used to maintain anesthesia. Shave the hair of the surgical department and scrub with a sterile gauze containing chlorhexidine. A local anesthetic block of lidocaine and bupivacaine (1.5 mg) was injected into the incision to provide local anesthesia. Lay a hole towel and perform surgery. Make a 2 cm medial parapatella incision and expose the meniscus. The anterior part of the medial meniscus is removed, the medial collateral ligament is incised, and the meniscus attachment is removed, causing articular cartilage damage. Use a hemostatic forceps to stop the bleeding. The meniscus injury is caused by the resection of the medial collateral ligament. Cut through the meniscus to the full thickness, eliminating about 1/2 of the meniscus. The synovial membrane and skin are closed by sutures. Animals are closely monitored throughout the recovery period and postoperative period. Animal vocalization, exercise, stirring observation, heart rate and respiration rate are monitored. After about 72 hours, the fentanyl transdermal patch was removed. After the fentanyl patch was removed, no dogs showed non-weight-bearing limp.
Experimental design and sample collection: Dogs receive 30ug/ml MIV-711 or excipient (n=15) every morning. The dose and dosing interval of MIV-711 are based on the selection of the potency of the dog MIV-711 cathepsin K enzyme (KI = 1.5 nmol/L) and the normal dog PK. The administration was started the day before the operation and lasted 28 days. Each dog was fasted for at least 12h before administration and was given food 4h after administration. To assess the exposure of MIV-711, blood was collected 1, 7, and 28 days after administration. The drug was administered before the oral administration and 1, 2, 4, 8 and 24h after the oral administration. The samples were centrifuged at 2-8°C for 30 minutes at 3000g for 10 minutes, plasma was separated and stored at 70°C until analysis. Collect as much synovial fluid of the dog as possible before the operation, and compare it with the synovial fluid of the contralateral knee of the operation and the operation. Use a syringe to sample directly. The synovial fluid is transferred to a test tube containing EDTA. Centrifuge the sample, collect the supernatant and store it at ~70°C until analysis. Urine samples were collected 1 to 3 days before surgery, before treatment (baseline specimen), 4 to 7 days after treatment (day 7), and 26 to 28 days after treatment (day 28). Collect through the metabolic cage, if no urine is collected, assist in urination, the collection volume is 15ml. Centrifuge urine for 10 minutes at 3000 rpm at 2-8°C. Collect the urine supernatant and store it at ?70°C until analysis.
Macro scoring: All animals were euthanized on the 29th day. Acepromazine was injected for sedation, and pentobarbital sodium (88 mg/kg or more) was injected for euthanasia. Open the left knee, measure, describe and photograph the disjointed and diseased medial tibia and femur. Use the photos to determine the percentage of lesion area on the medial tibia for each. The total cartilage lesions are measured (length × width) and described as shallow, medium, and deep. According to the measurement of the lesion, the general subjective cartilage degeneration score of the femoral head on the medial and lateral sides of the tibia: 0: normal, 1: surface degeneration up to 10 square millimeters, 2 surface degeneration greater than 10 square millimeters, 3: moderate degeneration up to 15 square millimeters, 4: moderate degradation greater than 15 square millimeters. The subjective score of each area is used to generate a subjective overall index of cartilage degeneration for each area of each animal. The actual measurement of cartilage lesions is derived based on the length × width × depth score: (1=mild, 2=moderate, 3=severe).
Assessing biomarkers and MIV-711 exposure: Routine CTX-I experiments to assess bone resorption. The CTX-I (type I collagen carboxyl terminal peptide) and CTX-II (type II collagen carboxyl terminal peptide) were measured in rabbits and kynuria. The concentration of urinary helix peptide (HP-1) in rabbits was determined. The level of CTX-II in canine synovial fluid was measured.
Results: The effect of MIV-711 on rabbit ACLT model and the pharmacokinetic study of MIV-711 in rabbits: A single administration of MIV-711 was 30 and 100 umol/kg, and Cmax was 0.25 and 1.8 umol/L, respectively. The area under the curve (AUC) of the low and high dose groups at 0-24 h was 0.78 and 7umol H/L, respectively.
The effect of MIV-711 on the bone resorption marker HP-1: 7 days after the operation, the level of HP-1 in the sham operation control group increased about 1.8 times. Compared with the baseline value, ACLT rabbit urine HP-1 levels increased 2.3-2.4 times after surgery. Ten days after administration, the level of HP-1 in ACLT+ vehicle group was higher than that in sham operation group. After 4 weeks of MIV-711 administration, the levels of HP-1 in the ACLT and sham operation groups were similar.
The effect of MIV-711 on the cartilage degradation biomarker CTX-II: baseline urine CTX-II levels are similar. During the whole experiment, the urine CTX-II levels of the sham operation group and ACLT animals were similar. MIV-711 dose-dependently reduced CTX-II levels.
The effect of MIV-711 on the subchondral bone structure: MIV-711 significantly reduces the total thickness of the femoral subchondral bone plate after surgery. The total thickness of the bone plates of the two groups receiving MIV-711 was the same as that of the sham operation group.
The effect of MIV-711 on the structure of articular cartilage: ACLT causes thickening of articular cartilage, especially in the posterior medial condyle cartilage, while focal cartilage thinning or complete erosion in some areas, especially in the medial subcondylar area. MIV-711 attenuates ACLT-induced increase in cartilage swelling.
The effect of MIV-711 on the model of partial meniscus resection in dogs: The effect of MIV-711 on biomarkers: MIV-711 reduces the levels of CTX-I and CTX-II.
Conclusion: The selective cathepsin K inhibitor MIV-711 reduces the joint pathology of the experimental model of OA and reduces the biomarker of bone resorption and cartilage degradation. Similar exposures and changes in biomarker levels support MIV-711 as a potential treatment for arthritis.
