阅读说明：本技术 一种能够治疗骨关节炎的可注射的温度敏感水凝胶 (Injectable temperature sensitive hydrogel capable of treating osteoarthritis ) 是由 张涛 刘纯 曹旭 于 2020-06-11 设计创作，主要内容包括：本发明涉及一种能够治疗骨关节炎的可注射的温度敏感水凝胶,其包括：胶原蛋白、海藻酸盐、透明质酸、钙盐以及用于形成碱性环境的盐,其中所述透明质酸的体积分数不小于24%。本发明还涉及一种药物组合物,其包含上述能够治疗骨关节炎的可注射的温度敏感水凝胶和药学上可接受的载体。本发明的水凝胶具有良好生物相容性及可降解性；在不使用交联剂的情况下,兼具有良好可注射性、流动性以及良好固化性能,可有效保护关节软骨状及改善软骨下骨状态。(The present invention relates to an injectable temperature sensitive hydrogel capable of treating osteoarthritis, comprising: collagen, alginate, hyaluronic acid, calcium salt and salt for forming alkaline environment, wherein the volume fraction of hyaluronic acid is not less than 24%. The present invention also relates to a pharmaceutical composition comprising the above injectable temperature sensitive hydrogel capable of treating osteoarthritis and a pharmaceutically acceptable carrier. The hydrogel has good biocompatibility and degradability; under the condition of not using a cross-linking agent, the injection-molding liquid has good injectability, fluidity and curing performance, and can effectively protect articular cartilage and improve the state of subchondral bone.)

1. An injectable temperature-sensitive hydrogel capable of treating osteoarthritis, comprising: collagen, alginate, hyaluronic acid, calcium salt and salt for forming alkaline environment, wherein the volume fraction of hyaluronic acid is not less than 24%.

2. The temperature-sensitive hydrogel of claim 1, wherein the calcium salt is calcium pyrophosphate, hydroxyapatite, or a combination thereof.

3. The temperature-sensitive hydrogel gel of claim 1, wherein the salt for forming an alkaline environment is sodium bicarbonate.

4. The temperature-sensitive hydrogel according to any one of claims 1 to 3, wherein the hyaluronic acid is a hyaluronic acid of natural origin.

5. The temperature-sensitive hydrogel according to any one of claims 1 to 3, wherein the collagen is a collagen of natural origin.

6. The temperature-sensitive hydrogel of claim 5, wherein said collagen comprises type I collagen or type II collagen.

7. The temperature-sensitive hydrogel of any one of claims 1 to 3, wherein the temperature-sensitive hydrogel does not comprise a crosslinker.

8. The temperature-sensitive hydrogel according to any one of claims 1 to 3, wherein the temperature-sensitive hydrogel has fluidity.

9. A pharmaceutical composition comprising the temperature-sensitive hydrogel of any one of claims 1-8 and a pharmaceutically acceptable carrier.

Technical Field

The present invention relates to osteoarthritis treatment, and more particularly to an injectable temperature sensitive hydrogel capable of treating osteoarthritis.

Background

Subchondral bone is located below cartilage and mainly comprises subchondral cortex end plates, trabecular bone structures, blood vessels and trabecular gap structures, and has the main biological functions of absorbing stress, buffering shock, maintaining joint shapes, providing nutrition for cartilage and removing metabolites. Subchondral bone and articular cartilage form a tightly combined functional unit called the "osteochondral node" that maintains the balance of the intra-articular environment as a functional entity. The trabecular bone of the subchondral bone plays an important role in shock absorption and support in the normal joint, and may also play an important role in the nutrient supply and metabolism of cartilage. The pathological changes in the presence of bone marrow edema (BML) in the subchondral bone region are mainly characterized by the following three points: bone matrix edema, fibrous tissue hyperplasia, inflammatory cell infiltration. It is mainly due to the massive perfusion of subchondral bone tissue. Biopsy findings were performed on areas of subchondral bone with altered BML: the trabecular spaces of the bone widen, with a large volume of edema fluid filling between them, regional liponecrosis, fibrovascular hyperplasia, and the formation of reactive new bone with varying degrees. Subchondral bone has been identified as a potential target for the treatment of OA.

Osteoarthritis (OA) is characterized by degenerative breakdown of cartilage, subchondral bone sclerosis, bone marrow edema in subchondral bone regions, osteophyte formation at joint margins, synovial lesions, contracture of joint capsules, ligament relaxation, muscle atrophy, and the like. For osteoarthritis patients, cartilage repair technologies in the prior art are difficult to take effect. At present, the treatment focus at home and abroad is mainly on symptomatic drug treatment, the aim is to relieve pain, relieve inflammation, delay cartilage degeneration, improve functions and avoid or reduce deformity, but the curative effect is not ideal, and finally patients have to select artificial joint replacement, so that the cost is high, and disastrous complications such as amputation caused by postoperative thrombosis and postoperative infection exist, so that an effective treatment scheme for osteoarthritis becomes a serious challenge for global clinicians.

Disclosure of Invention

In one aspect, the present invention provides an injectable temperature-sensitive hydrogel capable of treating osteoarthritis, comprising: collagen, alginate, hyaluronic acid, calcium salt and salt for forming alkaline environment, wherein the volume fraction of hyaluronic acid is not less than 24%.

In some embodiments, wherein the calcium salt is calcium pyrophosphate, hydroxyapatite, or a combination thereof. In some embodiments, wherein the salt for forming an alkaline environment is sodium bicarbonate.

In some embodiments, wherein the hyaluronic acid is of natural origin. In some embodiments, wherein the collagen comprises type I collagen or type II collagen. In some embodiments, wherein the collagen is naturally derived collagen.

In some embodiments, the temperature-sensitive hydrogel does not comprise a crosslinking agent. In some embodiments, the temperature-sensitive hydrogel has fluidity.

In another aspect, the present invention provides a pharmaceutical composition comprising any one of the hydrogels described above and a pharmaceutically acceptable carrier.

In a further aspect the present invention provides the use of any one of the hydrogels as described above in the manufacture of a medicament for the treatment of osteoarthritis.

In some embodiments, the osteoarthritis is traumatic arthritis, degenerative osteoarthritis, or rheumatoid arthritis.

The above-mentioned hydrogels are suitable for injection. In a particularly preferred embodiment, the formulation of the invention is for subchondral bone injection. As shown in FIG. 1, subchondral bone has a cellular structure, and the hydrogel of the present invention has a certain fluidity to better fill the area and to be cured in situ after injection. The gel of the present invention may be administered by direct injection into the defect, wherein the gel is preferably formed in situ. In a preferred embodiment of the invention, the administration of the subchondral bone injection is performed in a joint selected from the group consisting of hip, knee, elbow, wrist, ankle, spine, foot, finger, toe, hand, shoulder, rib, scapula, thigh, shin, ankle and a joint along a spinal condyle. In another preferred embodiment, the administration of the subchondral bone injection is performed in a joint of the hip or knee.

Those skilled in the art will appreciate that in order to achieve in situ retention of the injectable gel at the subchondral bone, the contradictory problems of gel flowability and curability need to be addressed. Sufficient fluidity is required because the gel formulation needs to be injected and filled in the subchondral bone region, but sufficient curing strength is required to provide sufficient mechanical strength to reside in the subchondral bone in order to achieve gel retention in situ in the subchondral bone region. The hydrogel of the present invention solves this conflict in the prior art by providing a hydrogel that has both sufficient fluidity and sufficient curing strength throughout its formulation. Meanwhile, the inventor of the present invention found through a large number of experiments that by using the hydrogel of the present invention with a hyaluronic acid volume fraction of not less than 24%, the use of a cross-linking agent commonly used in the prior art, which is generally toxic due to the residue of the cross-linking agent and unreacted monomers, can be avoided. Also, both the collagen and hyaluronic acid of the present invention may be of natural origin rather than being chemically modified, thus eliminating the need for complex chemical modification steps. Therefore, the hydrogel can obviously improve the biological safety, has strong development applicability and better medical prospect.

The hydrogels of the present invention are capable of providing sufficient fluidity to achieve subchondral bone injection and filling of the honeycomb structure in the subchondral bone region without the use of a cross-linking agent, while still maintaining sufficient curing properties to achieve curing and in situ retention in the subchondral bone region. By filling the hydrogel of the present invention with both fluidity and curing properties, it is possible to remove edema fluid and inflammatory substances, for example, in the subchondral bone region from the region, thereby alleviating or treating the progression of osteoarthritis. In addition, the hydrogel of the present invention also has at least one or more of the following advantages: has good biocompatibility and degradability; can promote bone repair.

Drawings

Fig. 1. subchondral bone is in a honeycomb structure.

Fig. 2 (a) a hydrogel prepared according to example 1; (B) behavior of the hydrogel prepared according to example 1 after gelling.

FIG. 3 (A) safranin fast green staining pattern of articular cartilage; (B) in the ACLT osteoarthritis rat model, the OSRSI score of rat cartilage is improved after the subchondral bone is given hydrogel treatment. Scale 1mmp<0.05。

Detailed Description

The term "alginate" refers to any form of alginate. One of the forms that can be used in the context of the present invention is sodium alginate.

The term "collagen" is a naturally occurring protein, and type I collagen as well as type II collagen are preferred forms that can be used in the context of the present invention. In addition, other forms of collagen may be employed in the present invention.

The term "temperature-sensitive hydrogel" is an environmentally-sensitive smart gel that exists in a liquid or semi-solid form prior to storage and administration, is exposed to the physiological state of the body after administration, undergoes a phase transition at the site of administration immediately to a semi-solid gel state. Temperature sensitive hydrogels can sense changes in temperature to produce functional effects. When the environmental temperature changes, the volume of the hydrogel changes, and when a certain critical area is reached and exceeded, the volume changes suddenly in a volume-phase transition called gel, and the corresponding temperature is called phase transition temperature. The phase transition temperature of the hydrogels of the invention is preferably 37^oC。

In the context of the present invention, the term "treatment" refers to any beneficial effect on the progression of a disease, including attenuation, reduction and reduction or remission of disease progression after the onset of the disease.

The term "pharmaceutically acceptable" is intended to encompass any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered. For example, for intra-articular administration, the gel may be formulated in unit dosage form for injection in a carrier such as, but not limited to, saline, dextrose solution.

The term "osteoarthritis" as used herein means the most common form of arthritis. The term "osteoarthritis" includes primary osteoarthritis and secondary osteoarthritis (see, for example, merck manual, 17 th edition, page 449). Osteoarthritis may be caused by cartilage breakdown. A small amount of cartilage may be shed, causing pain and swelling in the joint between the bones. Over time, the cartilage may wear away completely, causing the bone to rub against the bone. Osteoarthritis can affect any joint, but generally involves the hands, shoulders, and weight bearing joints, such as the hips, knees, feet, and spine. In a preferred example, the osteoarthritis may be knee osteoarthritis or hip osteoarthritis. The term especially includes forms of osteoarthritis, which are classified according to the OARSI classification system as grade 1 to grade 4 or grade 1 to grade 6. The skilled person will understand the osteoarthritis classification used in the art, in particular the OARSI assessment system (also known as oocha).

The term "patient" is intended to include, but is not limited to, mammals such as humans, dogs, cows, horses, sheep, goats, cats, mice, rabbits or rats. More preferably, the patient is a human.

EXAMPLE 1 preparation of hydrogels

1. Preparation of stock solutions

1) Type I collagen stock: 5 mg/ml type I collagen was prepared using HCl pH = 2;

2) sodium alginate stock solution: preparing 2.5% w/v sodium alginate by using ultrapure water;

3) hyaluronic acid stock solution: preparing a 10 mg/ml hyaluronic acid solution using ultrapure water;

4) calcium pyrophosphate stock solution: preparing a 2 mg/ml calcium pyrophosphate solution using HCl pH = 2;

5) sodium bicarbonate stock solution: supersaturated sodium bicarbonate stock solutions were prepared using ultrapure water to a final concentration of 7.4 g/L.

2. Preparation of hydrogel (hydrogel)

a) 1ul of calcium pyrophosphate and 10 mg of hydroxyapatite particles were added per 5 ml of type I collagen stock solution;

b) according to 60 ul NaHCO₃100 ul of sodium alginate, 240ul of hyaluronic acid stock solution, and 600 ul of collagen stock solution prepared in step (a) (i.e., containing proportionally added calcium pyrophosphate and hydroxyapatite particles) were sequentially added with each component (after each component was added, the mixture was vortexed sufficiently to mix the components uniformly), and 1 ml of hydrogel was prepared (as shown in fig. 2A, a photograph of the hydrogel of the present invention).

3. Verification of gelling

Placing the hydrogel in 37^oC cell incubator incubate for 30 minutes, the hydrogel will undergo sol-gel transition and become solid, but because the inside contains 24% hyaluronic acid, therefore can not gel sufficiently, still keep certain mobility (figure 2B).

Example 2 Effect of the gel of the invention on osteoarthritis

SD rats were divided into 2 groups: model group (model), treatment group (gel-treat). Animals were subjected to anterior cruciate ligament resection surgery to establish an osteoarthritis model. Namely, the SD rat was anesthetized and fixed on the platform, and the surgical leg was bent at 90 °. The skin on the surface of the knee is cut by a scalpel, and the subcutaneous fascia is separated bluntly, so that subcutaneous blood vessels are avoided. The femoral medial muscle is cut from the joint of the patellar ligament formed by the femoral medial muscle, the intramuscular blood vessel of the femoral medial muscle is avoided, and bleeding is avoided. The patella is then pushed laterally, exposing the knee joint space. Pulling the fat pad under the patella by using the micro forceps, and shearing off the anterior cruciate ligament. Then the wound is sutured and the iodine tincture is applied externally. After operation, the animals are noticed to recover and put back into cages for feeding. The post-operative model group was not treated, and the treatment group was administered with the hydrogel prepared as in example 1 injected at subchondral bone. At 10 weeks after administration, articular cartilage of the model group and that of the treatment group were obtained, respectively. Articular cartilage was stained with safranin fast green and scored for OARSI-modified mankin scales.

As shown in fig. 3, the OSRI score of rats was significantly improved after the hydrogel treatment of the present invention. The hydrogel can obviously protect the articular cartilage.