CN115137717A

CN115137717A - Application of disulfiram medicament in treating osteoarthritis

Info

Publication number: CN115137717A
Application number: CN202210640855.5A
Authority: CN
Inventors: 于寅; 刘怿斯; 陈飞; 王刚
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-10-04
Also published as: WO2023236229A1

Abstract

The invention provides an application of disulfiram medicament in treating osteoarthritis. The disulfiram drug is used to enhance or induce subchondral bone recanalization following injury. The disulfiram drug is used for improving the function of chondrocytes. The improvement in chondrocyte function specifically includes improvement in trabecular average thickness, improvement in trabecular number, improvement in trabecular resolution, and improvement in bone density or bone mineral density parameters. The disulfiram drug is used for treating osteoarthritis caused by injury. The injury includes an injury to a knee, hip, ankle, or elbow joint. The disulfiram medicament can be used for treating osteoarthritis, enhancing or inducing subchondral bone recatalsis and restoring joint bone beds, so that the disulfiram medicament is newly applied and has wide application prospect.

Description

Application of disulfiram medicament in treating osteoarthritis

Technical Field

The invention relates to the technical field of medicines, in particular to application of a disulfiram medicament in treating osteoarthritis.

Background

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration, destruction, and hyperosteogeny of articular cartilage, is a serious medical problem, and is the fourth leading disability disease in our country and the third leading disability disease in europe and america.

Subchondral bone is the bone bed of the joint on which the articular cartilage is located. Traditionally, osteoarthritis (OA) is considered the wear and tear of articular cartilage, but recent evidence suggests that subchondral bone disorders and synovial inflammation can initiate and lead to disease progression.

Osteoarthritis is characterized by a variety of diseases in which inflammation, immune and central nervous system dysfunction play a central role in overall joint damage, damage progression, pain and disability. Subchondral bone sclerosis and thickening are one of the main causes of osteoarthritis. The subchondral bone is a transition part at the joint of the osteochondral, is arranged between soft tissue and hard tissue, is used for absorbing stress in the process of loading the joint, and can cause micro fracture at the joint of the osteochondral and collapse of bone when abnormal loading occurs, and then the subchondral bone hardening phenomenon occurs. Many experiments have demonstrated that subchondral bone recanalization or sclerosis occurs before degeneration of articular cartilage whose integrity depends on the biomechanical properties of the underlying bone bed, and therefore subchondral bone sclerosis may be the initiating factor in the onset of OA.

Disulfiram is commonly used for treating tumors in the prior art, and the symptom of osteoarthritis is improved by injecting unloaded biogel, and the unloaded biogel does not contain therapeutic drugs, so the effect of treating osteoarthritis is limited.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the application of the disulfiram medicament in treating osteoarthritis.

In order to realize the purpose, the invention adopts the technical scheme that:

the disulfiram nano-medicament is applied to treating osteoarthritis.

Further, the disulfiram drug comprises a drug comprising disulfiram or a disulfiram derivative and a delivery system comprising PLGA and PLGA-b-PEG.

Further, the mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 25-50:25-50:10.

Further, the mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 45:45:10.

further, the disulfiram drug is used to enhance or induce subchondral bone recanalization following injury.

Further, the disulfiram drug is used for improving chondrocyte function.

Further, the improvement of chondrocyte function specifically comprises improvement of the average trabecular thickness, improvement of trabecular number, improvement of trabecular resolution, and improvement of bone density or bone mineral density parameters.

Further, the disulfiram drug is used for treating osteoarthritis caused by injury.

Further, the injury includes an injury to a knee, hip, ankle, or elbow joint.

Further, the disulfiram drug is introduced into a focal site by means of local injection.

The invention provides a new application of disulfiram drug for treating osteoarthritis, and experimental data of the invention show that the disulfiram drug is true and effective in the aspect of treating osteoarthritis, can improve the functions of chondrocytes, specifically comprises the improvement of the average thickness of trabeculae, the improvement of the number of trabeculae, the improvement of the separation degree of the trabeculae, the improvement of parameters such as bone density or bone mineral density, and the like, can also be used for treating osteoarthritis caused by injury, has great significance, and provides a new and wide application prospect for the disulfiram drug.

Drawings

In fig. 1, a is a graph of the toxicity test results of disulfiram drug on chondrocytes, and B is a graph of the improvement effect of different concentrations of disulfiram drug on OA chondrocytes.

FIG. 2 is a micro-CT image of various groups of bone joints 21 days after the rat iodoacetic acid osteoarthritis model is established and after 4 weeks of treatment with different compositions.

Fig. 3 is a comparison of subchondral bone recanalization important parameter measurements 21 days after the rat iodoacetic acid osteoarthritis model was established, 4 weeks after treatment with different compositions.

FIG. 4 is a graph showing the expression of OA-associated inflammatory factor.

FIG. 5 is a graph showing the comparison of the expression level of OA chondrocyte-associated inflammatory protein.

FIG. 6 is a comparison chart of the expression of inflammation-associated protein 1L-1 beta and ASC detected by immunofluorescence.

Fig. 7 is a graph comparing the number of chondrocytes to normal number at 1 to 3 days of injection of a drug-loaded scaffold.

FIG. 8 is a histological analysis of H & E staining and Safranin-O staining of tibial plateau and femoral condyle in OA animals.

FIG. 9 is a graph showing the expression evaluation of Aggrecan and Collagen II after immunohistochemical staining of knee joint tissue sections.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1: the present embodiments provide the use of a disulfiram drug comprising disulfiram and/or a disulfiram derivative for the treatment of osteoarthritis. The disulfiram drug can be used to enhance or induce subchondral bone recanalization following injury. The disulfiram drug can be used for improving chondrocyte function; the improvement of chondrocyte function specifically includes improvement of trabecular average thickness, improvement of trabecular number, improvement of trabecular separation, and improvement of bone density or bone mineral density parameters. The disulfiram drug is used for treating osteoarthritis caused by injury; the injury includes an injury to a knee, hip, ankle, or elbow joint. The disulfiram drug can be introduced into the focal site by means of local injection.

The disulfiram drug may be prepared in the form of nanoparticles, and may for example comprise a drug comprising disulfiram or a disulfiram derivative and a delivery system comprising PLGA and PLGA-b-PEG. The mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 25-50:25-50:10, preferably, the mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 45:45:10.

however, since the release rate of the nanoparticle form drug is too fast and the sustained release effect is limited, the effect of directly using the nanoparticle form drug for in vivo treatment is poor, and the nanoparticle form drug can be used as an encapsulating drug, for example, the nanoparticle form drug can be encapsulated by gel to enhance the sustained release effect. The present application is only illustrative of the novel use of disulfiram for the treatment of osteoarthritis and does not require the specific form of the disulfiram.

At present, disulfiram medicaments are mainly used for treating tumors, and the inventor finds that disulfiram and derivatives thereof have a very good curative effect on treating osteoarthritis in long-term research, so that the disulfiram and derivatives thereof have a wider application range.

Firstly, a drug toxicity experiment is carried out on the disulfiram drug, as shown in A in figure 1, the toxicity experiment of the disulfiram drug on cartilage cells is carried out by CCK-8, the abscissa in the figure is the concentration of the disulfiram drug, the ordinate is the absorbance value after CCK detection, and the survival number of the cartilage cells can be judged according to the absorbance. As can be seen from the figure, when the concentration of disulfiram is below 100ng/ml, the survival number of chondrocytes is larger, i.e. the toxicity of the drug is smaller in the concentration range.

As shown in B of fig. 1, the effect of different concentrations of disulfiram drug treatment after interleukin 1 β induced chondrocyte to inflammatory chondrocyte (OA) is shown, where NC is the normal group, and it can be seen from the graph that when the concentration of disulfiram drug is 50ng/ml, the data is closest to that of NC group, i.e. the treatment effect is the best at this concentration, and at concentrations of 1ng/ml, 10ng/ml and 100ng/ml, the survival number of chondrocytes is increased compared to that without the addition of disulfiram drug, indicating that the treatment effect can be achieved at the concentration of 100ng/ml or less. However, at a concentration of 200ng/ml, the number of viable chondrocytes is reduced compared to that obtained without the addition of disulfiram, and this concentration range is not effective for treatment.

Next, the therapeutic effect of disulfiram was visually observed by means of bone joint pictures, as can be observed from fig. 2, the femoral condyle and tibial plateau of the OA group could observe the characteristics of OA, such as erosion and osteophyte formation, while the normal cartilage in the NC (normal) group presented a flat, smooth surface without any defects and osteophyte formation. However, after 4 weeks of intervention with disulfiram drug (nanoparticles) or drug-loaded scaffolds (gel-loaded nanoparticles) in the experimental group, it was observed that cartilage damage was significantly reduced, as well as osteophytes and articular surface roughness were also significantly reduced. Therefore, the disulfiram medicament has certain effect on treating cartilage injury, and the medicament-carrying bracket has better curative effect.

The therapeutic effect on rat traumatic arthritis proves that the disulfiram medicament is effective in treating osteoarthritis and improving the function of chondrocytes, and specifically comprises the following steps:

establishing a rat iodoacetic acid osteoarthritis model: the method comprises the steps of adopting athymic rats of 8 months old, performing intraperitoneal injection to pentobarbital for anesthesia, taking a supine position, exposing a knee joint, and injecting 100ul iodoacetic acid (1 g/L) into the knee joint of the rat, wherein after 2 weeks of operation, joint capsules swell, the joint surface is dull, after 4 weeks of operation, joint cartilages are yellow, small cracks appear in the joints, and bone spurs and joint ligament adhesion can be touched in the 6 th week.

Rat iodoacetic acid osteoarthritis model the composition treatments: 21 days after the rat iodoacetic acid osteoarthritis model is established, the composition (no-load microgel, disulfiram drug or drug-loaded scaffold) is used for treating for 2 weeks and 4 weeks, and the key parameters of joint states such as the average thickness of bone trabeculae, the number of bone trabeculae, the separation degree of bone trabeculae, bone density/bone mineral density and the like are measured. The specific detection results are shown in fig. 7.

Fig. 3 is the determination of important parameters for subchondral bone remodeling 21 days after the rat iodoacetic acid osteoarthritis model was established and after 4 weeks of treatment, wherein fig. 3A is the tb.th (trabecular mean thickness) determination, fig. 3B is the tb.n (trabecular number) determination, fig. 3C is the tb.sp (trabecular number separation) determination, fig. 3D is the BMD (bone density/bone mineral density) determination, and fig. 3E is the BV/TV (relative bone volume/bone body integral number) determination. The components are as follows: NC stands for a normal rat group, OA stands for an ioacetic acid rat osteoarthritis model group at 21 days, gelMA stands for an unloaded microgel treatment group, NPs/DSF stands for a disulfiram drug treatment group, and GelMA-NPs/DSF stands for a drug-loaded stent treatment group.

It can be seen from FIG. 3A that the average trabecular thickness of the OA group was decreased compared to that of the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group were increased compared to that of the OA group, and were close to that of the NC (normal) group. It can be seen from FIG. 3B that the trabecular number of the OA group is decreased compared with that of the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group are increased compared with the OA group, and are close to the NC (normal) group. It can be seen from FIG. 3C that the trabecular separation number of the OA group was increased compared to that of the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group were decreased compared to the OA group, and were close to the NC (normal) group. It can be seen from FIG. 3D that the OA group had a decreased bone density/bone mineral density compared to the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group were increased compared to the OA group and were close to the NC (normal) group. It can be seen from FIG. 3E that the relative bone volume/bone volume fraction of the OA group is decreased compared to that of the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group are increased compared to that of the OA group, approaching that of the NC (normal) group.

The results show that the disulfiram medicament has the effects of treating osteoarthritis and improving the function of chondrocytes, and the medicament-carrying bracket has better slow release effect compared with the disulfiram medicament, so that the treatment and improvement effects of the medicament-carrying bracket are more obvious.

As shown in FIG. 4, the factor expressions of five inflammatory genes related to bone joints, MMP13, 1L-6, 1L-1 beta, NLRP3 and Aggrecan, are shown in the following three groups: NC stands for normal group, and factor expression of normal medium is added; 1L-1 beta represents a modeling group, and refers to factor expression after 24 hours of 1L-1 beta treatment and OA modeling; 1L-1 beta + DSF 50ng/ml represents the treatment group, meaning that 1L-1 beta represents the factor expression of the treatment group after two hours of treatment with 50ng/ml disulfiram drug. As can be seen, the factor expression of 1L-1 beta for the model building group was significantly increased as compared with that of the NC group, while the factor expression of 1L-1 beta + DSF 50ng/ml for the treatment group was significantly decreased as compared with that of 1L-1 beta for the model building group, and was close to that of the NC group. Thus, the disulfiram drug with the concentration of 50ng/ml can improve osteoarthritis.

As shown in FIG. 5, the expression level of OA chondrocyte-related inflammation protein was measured by ELISA in the presence or absence of disulfiram drug. Three groups of related inflammatory proteins are TNF- α, 1L-6 and 1L-1 β, respectively. Wherein OA represents an osteoarthritis group and OA +50ng/ml DSF represents a disulfiram drug treatment group with a concentration of 50 ng/ml. As can be seen from the figure, the expression level of related inflammatory proteins after being treated by the disulfiram drug with the concentration of 50ng/ml is reduced according to the OA group. Thus, the disulfiram drug with the concentration of 50ng/ml can improve osteoarthritis.

As shown in FIG. 6, in order to detect the expression of inflammation-associated protein 1L-1 beta and ASC by immunofluorescence, the green color in the color chart shows that the OA inflammation group is expressed, but the OA inflammation group is not expressed in the treatment group and the normal group. It is demonstrated that disulfiram drug is effective in treating osteoarthritis.

Figure 7 is a graph of the effect of drug-loaded scaffolds on chondrocyte numbers at 1-3 days of injection. Wherein NC-D1 represents the number of normal chondrocytes on the first day, NC-D2 represents the number of normal chondrocytes on the second day, NC-D3 represents the number of normal chondrocytes on the third day, gelMA-D1 represents the number of chondrocytes one day after injection of the drug-loaded scaffold, gelMA-D2 represents the number of chondrocytes two days after injection of the drug-loaded scaffold, and GelMA-D3 represents the number of chondrocytes three days after injection of the drug-loaded scaffold. As can be seen, the drug-loaded scaffold had no growth stimulating or growth inhibiting effect on chondrocyte growth.

As shown in FIG. 8, the histological analysis of H & E staining and Safranin-O staining was performed on the tibial plateau and femoral condyle of OA animals. Wherein OA represents an osteoarthritis group, gelMA represents an unloaded micro gel sphere treatment group, DSF/NPs represents a disulfiram drug nanoparticle treatment group, and GelMA-DSF/NPs represents a drug-loaded stent treatment group. As can be seen from the color chart, according to the histopathological examination thereof, osteogenesis was green, cartilage was red, and OA histone polysaccharide (red) content was decreased. It can be seen that the articular cartilage layer in group OA has rough surface, integrity failure, partial matrix fibrosis, and visible fibrous granulation tissue filling; and significant reduction in OA-associated cartilage deformability (smooth articular surface), cartilage destruction and proteoglycan loss were seen compared to the drug-loaded scaffold treatment group.

FIG. 9 shows a graph of Aggrecan and Collagen II expression evaluation after immunohistochemical staining of knee joint tissue sections. Aggrecan also be called proteoglycan, and macromolecular proteoglycan existing in extracellular matrix of connective tissue is a main structural macromolecule of cartilage; collagen II, also known as type II bone glue, is the main organic component of cartilage and joints, is rich in amino acids which are particularly needed by bone connective tissue, can help the regeneration of human cartilage tissue, and is widely applied to the evaluation of cartilage repair. As can be seen from the figure, the positive expression protein in the treatment group is obviously increased compared with the OA group, and the agrececan and Collagen II in the drug-loaded stent treatment group are both obviously increased. The morphology and number of chondrocytes also increased relative to the OA group. According to the results, the disulfiram medicament nano-particles, the medicament-carrying micro gel spheres and the medicament-carrying bracket can remarkably relieve the inflammatory injury of osteoarthritis to cartilage, effectively treat osteoarthritis and repair cartilage.

The experimental data can show that the disulfiram medicament has certain effectiveness in treating osteoarthritis, a new application scene is opened for the disulfiram medicament, and the application of the disulfiram medicament is wider.

According to the embodiment of the invention, the drug-loaded scaffold is introduced in a mode of local injection such as a joint cavity, so that the recatalsis of subchondral bone is promoted, and the aim of treating osteoarthritis is further achieved. Experimental detection proves that the drug-loaded scaffold can induce and enhance subchondral bone remodeling, improve joint state key parameters such as trabecular average thickness, trabecular number, trabecular separation degree and bone density/bone mineral density, and prove that disulfiram loaded injectable microgel can induce and enhance subchondral bone remodeling, thereby treating osteoarthritis. The drug-loaded stent provides a brand-new, safe and effective treatment method for treating osteoarthritis, has great significance in treating osteoarthritis, and has wide application prospect.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above examples only express preferred embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of disulfiram in the treatment of osteoarthritis.

2. The disulfiram drug for use in the treatment of osteoarthritis as claimed in claim 1 wherein: the disulfiram drug comprises a drug and a drug delivery system, wherein the drug comprises disulfiram or a disulfiram derivative, and the drug delivery system comprises PLGA and PLGA-b-PEG.

3. The disulfiram drug for use in the treatment of osteoarthritis as claimed in claim 2 wherein: the mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 25-50:25-50:10.

4. the disulfiram drug for use in the treatment of osteoarthritis according to claim 2 or 3, characterized in that: the mass ratio of the PLGA, the PLGA-b-PEG and the medicament is 45:45:10.

5. the use of a disulfiram drug according to claim 1 for the treatment of osteoarthritis characterized in that: the disulfiram drug is used to enhance or induce subchondral bone recanalization following injury.

6. The disulfiram drug for use in the treatment of osteoarthritis as claimed in claim 1 wherein: the disulfiram drug is used for improving the function of chondrocytes.

7. The disulfiram drug of claim 6 for use in the treatment of osteoarthritis characterized in that: the improvement in chondrocyte function specifically includes improvement in trabecular average thickness, improvement in trabecular number, improvement in trabecular resolution, and improvement in bone density or bone mineral density parameters.

8. The use of a disulfiram drug according to claim 1 for the treatment of osteoarthritis characterized in that: the disulfiram drug is used for treating osteoarthritis caused by injury.

9. The disulfiram drug for use in the treatment of osteoarthritis as claimed in claim 8 wherein: the injury includes an injury to a knee, hip, ankle, or elbow joint.

10. The use of a disulfiram drug according to claim 1 for the treatment of osteoarthritis characterized in that: the disulfiram medicament is introduced into a focus part by a local injection mode.