CN110721308A - Pharmaceutical compound preparation for treating osteoarthritis and preparation method thereof - Google Patents

Pharmaceutical compound preparation for treating osteoarthritis and preparation method thereof Download PDF

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CN110721308A
CN110721308A CN201911136558.1A CN201911136558A CN110721308A CN 110721308 A CN110721308 A CN 110721308A CN 201911136558 A CN201911136558 A CN 201911136558A CN 110721308 A CN110721308 A CN 110721308A
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黄石书
宁宁
陈佳丽
李佩芳
吴迪伟
尤炫合
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West China Hospital of Sichuan University
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Abstract

The invention discloses a pharmaceutical compound preparation for treating osteoarthritis, which is preferably a compound injection, and comprises a nerve oriented factor Sema and a sodium hyaluronate injection, wherein the concentration of the nerve oriented factor Sema in the sodium hyaluronate injection is 4-9 mu g/mL, and in a preferred embodiment, the nerve oriented factor Sema is selected from Sema3a protein. Clinical test results prove that the injection prepared by combining the protein of the nerve-targeting factor Sema3a and the sodium hyaluronate injection has better treatment effect on patients with early degenerative osteoarthritis than the single treatment of the sodium hyaluronate injection.

Description

Pharmaceutical compound preparation for treating osteoarthritis and preparation method thereof
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a pharmaceutical compound preparation for treating osteoarthritis and a preparation method thereof.
Background
Osteoarthritis (OA) is a synovial joint disease characterized by cartilage loss and bone reaction around joints, which is also known as osteoarthropathy, degenerative osteoarthropathy, hypertrophic or proliferative arthritis, and is the most common rheumatic disease worldwide. Osteoarthritis is the degenerative damage of articular cartilage and reactive hyperplasia of articular margin and subchondral bone caused by a plurality of factors such as aging, obesity, strain, trauma, congenital abnormality of joints, joint deformity and the like. Osteoarthritis is the most common joint disease in middle-aged and elderly people, investigation shows that the prevalence rate of people over 60 years old can reach 50%, the prevalence rate of people over 75 years old can reach 80%, and the disability rate of the disease is as high as 53%, and investigation shows that osteoarthritis becomes a disability head killer of old people.
Osteoarthritis mostly has hidden diseases and slow progress, mild and moderate dull pain can occur in affected joints intermittently, the osteoarthritis can be induced or aggravated due to excessive activities, climate change and other reasons, symptoms and signs of severe patients are obvious and not continuously relieved, and even joint deformity and dysfunction occur.
The goal of osteoarthritis treatment is to alleviate symptoms, delay joint structural changes, maintain joint function, and improve quality of life. The OA treatment guidelines established by ACR in 2000 and the recommendations of the european college of rheumatology (EULAR) for knee OA treatment are basically three aspects, namely non-drug therapy, surgical therapy. Among them, non-steroidal anti-inflammatory drugs (NSAIDs) are a class of nonsteroidal drugs with different chemical structures and having anti-inflammatory, analgesic, antipyretic, etc. functions in drug therapy, and are commonly used for the treatment of osteoarthritis. Notably, some trials have observed that certain NSAIDs inhibit cartilage synthesis,accelerated cartilage degradation, and in addition, oral nsaids often cause gastrointestinal problems (especially in elderly patients), often with renal, hepatic and other functional abnormalities for prolonged use. In addition to NSAIDs, steroid drugs are frequently used in osteoarthritis treatment because they can rapidly improve symptoms, but frequent use of steroid drugs may damage joints, so steroid injection therapy should be carefully considered. Second, intra-articular injection using hyaluronic acid injection, a visco-elastic supplementation therapy, is becoming more and more common in the treatment of osteoarthritis conditions, marketed in china
Figure BDA0002279736980000021
The preparation for intra-articular injection contains sodium hyaluronate derived from flos Celosiae Cristatae, and has average molecular weight of 800,000 daltons. It has effects of relieving pain, anti-inflammatory properties, improving joint mobility, temporary absence of severe adverse drug reactions, etc., but it cannot inhibit the progression of osteoarthritis and is only effective for early osteoarthritis.
The Semaphorin protein family is a group of secreted or membrane-associated protein families characterized by cysteine-rich regions, originally discovered as neurite-directing factors affecting neural development, and is a large class of secreted or transmembrane glycoprotein molecules involved in the regulation of many important physiological processes in the body, including regulation of neuronal axonal development, angiogenesis, bone differentiation, cardiovascular development, and the like. They are classified into type 8 according to their composition characteristics, in which some of the family protein molecules participate in the regulation of Immune function and are thus named Immune Semaphorin, such as Semaphorin 4D (Sema4D, also known as CD100), Semaphorin3A (Sema3A), and Semaphorin 7A (Sema7A), and the like. Among them, Sema3A was first discovered and is one of the most classical Sema family members that regulates and directs the growth of nerve axons. In recent years, it has been found that Sema3A has a role in guiding neurites, and is closely related to various pathophysiological phenomena in the body, such as cell migration, tumor growth, angiogenesis, bone metabolism, and immunoregulation.
In the non-patent document, "expression and significance of Semaphorin3A in serum and peripheral blood mononuclear cells of patients with rheumatoid arthritis" the expression level of Semaphorin3A (Sema3A) in peripheral blood and serum of patients with Rheumatoid Arthritis (RA) is mentioned, and the role of Sema3A in the pathogenesis of RA is studied. The results show that the serum Sema3A level of the RA patients is obviously higher than that of healthy people, and the serum Sema3A level is positively correlated with platelet count, ESR, IgM, rheumatoid factors and HRF-IgG.
Non-patent literature 'research progress of Sema3A, a potential target of action in bone remodeling' proves that Sema3A has the functions of promoting osteoblast generation and simultaneously inhibiting osteoclast generation, fracture healing is closely related to nerve fiber growth into callus, and a nerve growth guide factor Sema3A can regulate bone mass metabolism by regulating sensory nerve fiber growth.
The patent document "a method for promoting osteogenic differentiation and inhibiting adipogenic differentiation of adipose-derived stem cells using Sema3A gene" discloses a method for promoting osteogenic differentiation and inhibiting adipogenic differentiation of adipose-derived stem cells using Sema3A gene. The invention constructs a recombinant plasmid containing a Sema3A gene sequence, a Sema3A lentiviral vector is obtained by transfecting a Sema3A recombinant plasmid and an auxiliary packaging plasmid into 293T cells, the Sema3A gene is introduced into adipose-derived stem cells by the lentiviral vector under the assistance of polybrene, osteogenic differentiation of the adipose-derived stem cells is remarkably promoted, meanwhile, differentiation of the adipose-derived stem cells to the adipogenic direction is inhibited, and the introduction of the Sema3A gene can better apply the adipose-derived stem cells to promotion of bone defect healing and implant bone union.
Although the prior art discloses the use of Sema3A for the treatment of bone related diseases, and is directed only to bone treatment, cartilage is not involved. Sodium hyaluronate is one of the medicines with the best effect and the most widely applied arthritis symptom relief in the market at present, has certain approval on the curative effects of protecting joints and delaying or inhibiting joint degeneration, but has obvious joint swelling and discomfort after injection due to large injection dosage. In the invention, Sema3A and sodium hyaluronate are combined to prepare a compound preparation for treating articular cartilage degeneration and injury caused by multiple factors such as aging, obesity, strain, trauma, joint congenital abnormality, joint deformity and the like, and side effects caused by single treatment of sodium hyaluronate can be avoided.
Disclosure of Invention
The invention aims to provide application of the nerve-targeting factor Sema in preparing a compound preparation for treating osteoarthritis, and also aims to provide a compound injection for treating osteoarthritis and a preparation method thereof.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides the use of the nerve-targeting factor Sema in the preparation of a compound preparation for treating osteoarthritis, wherein the sequence of the nerve-targeting factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve targeting factor Sema is a variant of SEQ ID No. 1, wherein the difference of the variant from SEQ ID No. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
Preferably, the nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown as SEQ ID NO. 1.
The amino acid sequence of SEQ ID NO. 1 of the present invention is as follows:
Figure BDA0002279736980000041
Figure BDA0002279736980000051
the preparation raw materials of the compound preparation comprise: the nerve targeting factor Sema is prepared from Sema and sodium hyaluronate injection, wherein the concentration of the Sema in the sodium hyaluronate injection is 4-9 mu g/mL.
The sequence of the nerve targeting factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve targeting factor Sema is a variant of SEQ ID No. 1, wherein the difference of the variant from SEQ ID No. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
Preferably, the nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown as SEQ ID NO. 1.
Preferably, the compound preparation is preferably an injection.
In a second aspect, the present invention provides a combination for use in the treatment of osteoarthritis, the combination comprising: the nerve targeting factor Sema is prepared from Sema and sodium hyaluronate injection, wherein the concentration of the Sema in the sodium hyaluronate injection is 4-9 mu g/mL.
Preferably, the concentration of the nerve guide factor Sema in the sodium hyaluronate injection is 4-6 mu g/mL.
In a preferred embodiment of the invention, the concentration of the nerve-targeting factor Sema in the sodium hyaluronate injection is 4 μ g/mL.
The sequence of the nerve targeting factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve targeting factor Sema is a variant of SEQ ID No. 1, wherein the difference of the variant from SEQ ID No. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
Preferably, the nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown as SEQ ID NO. 1.
In the present invention, the Sema3a protein may be selected from a natural protein or a recombinant protein, and preferably, the Sema3a protein is a human recombinant protein.
The sodium hyaluronate injection can be prepared by a commercial way or a laboratory, the concentration of the sodium hyaluronate injection is 10mg/mL, and the solvent is PBS. In a preferred embodiment of the invention, the sodium hyaluronate injection is from the commercial route.
Preferably, the compound preparation for treating osteoarthritis is an injection, and the injection comprises: the nerve targeting factor Sema is sodium hyaluronate injection, and the concentration of the nerve targeting factor Sema in the sodium hyaluronate injection is 4-9 mug/mL.
Preferably, the concentration of the nerve guide factor Sema in the sodium hyaluronate injection is 4-6 mu g/mL.
In a preferred embodiment of the invention, the concentration of the nerve-targeting factor Sema in the sodium hyaluronate injection is 4 μ g/mL.
The nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown as SEQID NO. 1.
Preferably, the injection also comprises one or the combination of two of an antioxidant, a bacteriostatic agent, an analgesic and a pH regulator.
The antioxidant is selected from one or more of sodium sulfite, sodium metabisulfite, sodium bisulfite, sodium thiosulfate and vitamin C.
The bacteriostatic agent and the analgesic agent are selected from one or the combination of two of chlorobutanol and benzyl alcohol.
The pH regulator is one or more selected from tartaric acid-tartrate, citric acid-sodium citrate, disodium hydrogen phosphate-sodium dihydrogen phosphate, sodium hydroxide, and hydrochloric acid.
In a third aspect, the invention provides a preparation method of a compound injection for treating osteoarthritis, which comprises the following steps:
(1) preparing liquid, namely adding the nerve guidance factor Sema into the sodium hyaluronate injection according to a certain proportion to prepare liquid medicine with a certain concentration;
(2) activated carbon treatment, namely adding activated carbon for needles, the amount of which is 0.1 to 1.0 percent of the total amount of the solution, into the liquid medicine for adsorption treatment;
(3) primarily filtering, namely filtering the liquid medicine by using filter paper;
(4) filtering to remove bacteria, and removing bacteria and heat source with microporous membrane with pore diameter of 0.22 μm;
(5) subpackaging and encapsulating to obtain the injection.
Preferably, the concentration of the nerve guidance factor Sema in the step (1) is 4-9. mu.g/mL, more preferably 4-6. mu.g/mL, and most preferably 4. mu.g/mL.
Preferably, the activated carbon in the step (2) is dried and activated for 3 to 4 hours before being used.
Preferably, the filter material used in the step (3) is silk cloth or a filter plate.
Preferably, the filtration method in the steps (3) and (4) adopts pressure filtration or reduced pressure filtration, and in the present invention, the filtration adopts pressure filtration.
Preferably, the injection prepared in step (5) is in a unit dosage form, and each 2.5ml of sodium hyaluronate injection contains 10 μ g of nerve targeting factor Sema.
The sequence of the nerve targeting factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve targeting factor Sema is a variant of SEQ ID No. 1, wherein the difference of the variant from SEQ ID No. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
Preferably, the nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown as SEQ ID NO. 1.
The invention has the beneficial effects that: the traditional treatment technical means can only relieve osteoarthritis symptoms, but at present, FDA and China still do not have a drug approved for preventing or slowing disease progression, and the nerve-oriented factor Sema-based drug compound injection taking sodium hyaluronate injection as a carrier can prevent and slow osteoarthritis progression. And Sema3A and sodium hyaluronate are combined to prepare a compound injection for treating articular cartilage degeneration injury, and side effects of joint swelling, long pain time and the like caused by single treatment of sodium hyaluronate can be avoided.
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FIG. 1 quantitative MRI scattergram of clinical test of Sema3a protein compound injection
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 preparation of Compound injection
S1: preparing a solution, namely dissolving 40 mu g of Sema3a human recombinant protein in 10mL of sodium hyaluronate injection, and sufficiently shaking and dissolving to prepare a Sema3 a-sodium hyaluronate compound injection with the Sema3a protein concentration of 4 mu g/mL;
s2: activated carbon treatment, namely adding activated carbon for needles, the amount of which is 0.2 percent of the total amount of the solution, into the liquid medicine for adsorption treatment;
s, 3: primarily filtering, namely filtering the liquid medicine by using filter paper;
s4: filtering to remove bacteria, and removing bacteria and heat source with microporous membrane with pore diameter of 0.22 μm;
s5: the liquid medicine is subpackaged by 2mL and encapsulated to obtain the injection.
EXAMPLE 2 preparation of Compound injection
The preparation method is the same as that in example 1, except that 60. mu.g of Sema3a human recombinant protein is taken in step S1 and dissolved in 10mL of sodium hyaluronate injection, and the Sema3a protein is prepared with the concentration of 6. mu.g/mL by fully shaking and dissolving.
EXAMPLE 3 preparation of Compound injection
The preparation method is the same as that of example 1, except that in step S1, 90 μ g of Sema3a human recombinant protein is taken and dissolved in 10mL of sodium hyaluronate injection, and the Sema3a protein is prepared with the concentration of 9 μ g/mL by fully shaking and dissolving.
Effect example 1 quality control of injection
The quality of the injection 1-3 prepared by the invention is tested, the test items comprise the properties of the injection, pH value, allergy test, abnormal toxicity, sterility test, heat source test and long-term toxicity test, and the detection method comprises the following steps:
the characteristics are as follows: the injection containing the Sema3a protein and the sodium hyaluronate prepared by the invention is a colorless clear liquid, and is qualified if the injection is a colorless clear solution, has no impurities and no precipitates in visual inspection.
pH value: and (3) detecting the pH value of the injection to be detected according to a detection method of the appendix VIH of the second part of the Chinese pharmacopoeia 2000 edition, and if the pH value is 6.5-7.5, detecting to be qualified.
And (3) allergy test: 6 healthy qualified guinea pigs with the weight of 250-350g are used in each group, 0.5mL of injection to be tested is injected into each abdominal cavity, 3 times of continuous injection are carried out, on the 14 th day after the last injection, 1mL of injection to be tested is injected intravenously, and within 15 minutes after the injection, if the 6 guinea pigs have anaphylactic reaction, such as two or more of hair erection, dyspnea, sneeze, retching or cough over 3 sounds, or one of phenomena of rumble, convulsion, collapse or death, the guinea pigs are judged to be positive, and if the anaphylactic reaction does not occur, the guinea pigs are qualified.
Abnormal toxicity: according to the method specified in appendix XIC of the second part of the 2000 th edition of the Chinese pharmacopoeia, 6 healthy mice with the weight of 200-250g are used in each group, 0.5ml of injection to be tested is injected intravenously, the death condition of the mice is observed within the specified time, and if no toxic reaction occurs, the mice are qualified.
And (4) sterile inspection: the detection is carried out according to the detection method specified in appendix XIH of the second part of the 2000 th edition of Chinese pharmacopoeia, the injection to be detected is added into 100mL0.9 percent sterile sodium chloride solution, the mixture is shaken up and treated by a membrane filtration method, and the detection is carried out according to the specification, and the detection is qualified if the mixture is sterile.
Pyrogen test: according to the method specified in appendix XID of the second part of the 2000 th edition of Chinese pharmacopoeia, the quantity of bacterial endotoxin (or pyrogen) contained in the injection to be tested is determined by using the rabbits, the injection dose is 0.5mL per 1kg of the body weight of the rabbits, and if the pyrogen reaction does not occur, the test is qualified.
Long-term toxicity assay: 6 healthy mice with the weight of 200-250g are used in each group, 0.5ml of injection to be tested is injected intravenously, the injection is continuously administrated for three months, hematuria examination is carried out 2 days after each administration, and if the abnormal conditions occur, the examination is qualified.
The test results of 3 groups of injections prepared according to the present invention are shown in the following table.
TABLE 1 quality test results for injections
Group of Traits pH value Allergy test Abnormal toxicity Sterility testing Heat source inspection Long term toxicity
Example 1 Qualified Qualified Qualified Qualified Qualified Qualified Qualified
Example 2 Qualified Qualified Qualified Qualified Qualified Qualified Qualified
Example 3 Qualified Qualified Qualified Qualified Qualified Qualified Qualified
The quality inspection data prove that the prepared Sema3a protein and sodium hyaluronate compound injection has the safety meeting the requirement of injection, no toxic effect and higher safety when applied to human bodies.
Effect example 2 clinical drug efficacy test
The clinical research scheme is drawn up according to the relevant requirements of the quality management standard of the clinical test of the medicine, is approved by the ethical committee of the western hospital of Sichuan university, and is documented in the clinical test institution of the medicine of the western hospital of Sichuan university.
1. The purpose of the test is as follows: the clinical efficacy and safety of the compound injection based on the Sema3a protein and sodium hyaluronate for treating patients with early degenerative osteoarthritis are evaluated.
2. The inclusion criteria were as follows:
① patients diagnosed with knee osteoarthritis based on the american college of rheumatology clinical classification criteria;
② the study lateral knee was graded as Kellgren-Lawrence grade 1 or 2, and the study lateral knee was graded no further than the study lateral.
③ Chronic pain persists for at least 1 month;
④ mean osteoarthritis index (WOMAC) pain subtotal score determined on a 0-10 point scale at week 0 (item 5) meets regimen requirements;
⑤ patients greater than or equal to 40 years old and less than 70 years old, and voluntarily sign an informed consent with sufficient knowledge;
⑥ to be able to walk without wheelchair support.
3. Exclusion criteria were as follows:
① patients diagnosed with secondary knee osteoarthritis;
② patients with other diseases/disorders that result in impaired knee joint function;
③ patients with osteoarthritis in the contralateral knee joint and requiring intra-articular drug therapy for pain relief;
④ patients not amenable to treatment with drugs (e.g., patients with surgically indicated indications);
⑤ patients who received one or more of the following treatments within 2 weeks prior to the first use of the study drug:
1) knee joint motion treatment on both sides or any side;
2) the traditional Chinese medicine is used for treating knee osteoarthritis;
⑥ patients who received one or more of the following treatments within 4 weeks prior to the first use of the study drug:
1) local injection of glucocorticoid or anesthetic at knee joint on two sides or any side, and external glucocorticoid treatment;
2) oral, suppository or intravenous formulations of glucocorticoids;
⑦ received treatment with a hyaluronic acid injection of the knee joint (bilateral or bilateral) within 24 weeks prior to the first use of study medication;
⑧ ipsilateral hip or ankle joint pathology;
⑨ rheumatic diseases or conditions;
⑩ history of bilateral or unilateral lower limb arthroplasty;
Figure BDA0002279736980000121
patients who cannot be evaluated for clinical performance;
Figure BDA0002279736980000122
skin diseases or skin infections around the joint site, with the risk of causing injection infections;
severe heart disease, renal failure, hematological diseases, and diabetes;
Figure BDA0002279736980000124
a history of hypersensitivity to any component of the investigational drug (IMP);
Figure BDA0002279736980000125
patients who developed pregnancy, suspected pregnancy, lactating women or patients who refused to use contraceptive measures during the study (male or female);
Figure BDA0002279736980000126
the current disease has severe liver damage or has a history of severe liver damage;
the current disease has malignant tumor or the history of malignant tumor within 5 years before screening;
Figure BDA0002279736980000132
patients receiving additional IMP treatment within 24 weeks prior to the first study drug use;
Figure BDA0002279736980000133
the researcher or assistant researcher decides that other patients who are not suitable for participation in the study.
4. The test procedure was as follows:
① patients with primary degenerative gonarthritis who met the inclusion criteria were recruited by clinical outpatient clinics, numbered and randomly assigned into 4 groups of 5 persons each.
② quantitative magnetic resonance imaging examination (MRI) was performed on their tested knee joints before treatment, and the examination data was expressed as a Region Of Interest (ROI) area.
③ the Sema3a protein-containing injection prepared in examples 1-3 was injected into the knee joint cavities of the patients in groups 1-3, once a week, for a total of 5 injections, and group 4 was a control group, and the subjects in the control group were injected with sodium hyaluronate injection according to the pharmaceutical instruction.
After 5 weeks (one treatment period) of treatment, quantitative MRI was performed on the knee joint of the patient, and the recovery of the distal femur cartilage of the patient was obtained by quantitative MRI before and after the treatment, and the ROI growth rate (%) was calculated as (after-treatment ROI-before-treatment ROI)/before-treatment ROI × 100.
④ the results of the experiment are shown in the following table:
TABLE 2 clinical efficacy data for compound injections based on Sema3a protein and sodium hyaluronate
Figure BDA0002279736980000131
Figure BDA0002279736980000141
By measuring T2Changes in the cartilage content were observed as relaxation times, T after and before treatment2Higher growth rates, by comparison, indicate better recovery of articular cartilage in the subject. The recovery condition of distal cartilage of femur of a subject can be obtained by comparing the compound injection prepared in examples 1-3 with the sodium hyaluronate injection, and the treatment effect of the combined Sema3a protein and sodium hyaluronate on patients with early degenerative osteoarthritis is better than that of the single preparation of the sodium hyaluronate injection.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
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<120> pharmaceutical compound preparation for treating osteoarthritis and preparation method thereof
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Claims (10)

1. The application of nerve oriented factor Sema in preparing a compound preparation for treating osteoarthritis is disclosed, wherein the sequence of the nerve oriented factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve-targeting factor Sema is a variant of SEQ ID NO. 1, wherein the difference of the variant from SEQ ID NO. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
2. The use according to claim 1, wherein said nerve targeting factor Sema is selected from the Sema3a protein, and the amino acid sequence of said Sema3a protein is as shown in SEQ ID No. 1.
3. A combination formulation as claimed in claim 1 for use in the treatment of osteoarthritis, the combination formulation comprising: the nerve targeting factor Sema is prepared from Sema and sodium hyaluronate injection, wherein the concentration of the Sema in the sodium hyaluronate injection is 4-9 mu g/mL.
4. The compound preparation of claim 3, wherein the concentration of the nerve-targeting factor Sema in the sodium hyaluronate injection is 4-6 μ g/mL.
5. The combination preparation of claim 4, wherein the nerve targeting factor Sema is selected from Sema3a protein, and the amino acid sequence of the Sema3a protein is shown in SEQ ID NO. 1.
6. The pharmaceutical combination according to any one of claims 1-5, wherein the pharmaceutical combination for treating osteoarthritis is an injection.
7. The pharmaceutical composition of claim 6, wherein the injection further comprises one or a combination of two of an antioxidant, a bacteriostatic agent, an analgesic and a pH regulator.
8. A method for preparing the compound injection for treating osteoarthritis according to claim 6, comprising the following steps:
(1) preparing liquid, namely adding the nerve guidance factor Sema into the sodium hyaluronate injection according to a certain proportion to prepare liquid medicine with a certain concentration;
(2) activated carbon treatment, namely adding activated carbon for needles, the amount of which is 0.1 to 1.0 percent of the total amount of the solution, into the liquid medicine for adsorption treatment;
(3) primarily filtering, namely filtering the liquid medicine by using filter paper;
(4) filtering to remove bacteria, and removing bacteria and heat source with microporous membrane with pore diameter of 0.22 μm;
(5) subpackaging and encapsulating to obtain the injection.
9. The method according to claim 8, wherein the concentration of the nerve guidance factor Sema in step (1) is 4 to 9 μ g/mL;
the activated carbon in the step (2) is dried and activated for 3-4 hours before use.
The filter material used in the step (3) is silk cloth or a filter plate.
The filtration method in the steps (3) and (4) adopts pressure filtration or reduced pressure filtration;
the injection prepared in the step (5) is a unit dosage form, and each 2.5ml of sodium hyaluronate injection contains 10 mu g of nerve targeting factor Sema.
10. The method according to claim 8, wherein the sequence of the nerve targeting factor Sema is selected from one of the following amino acid sequences:
1) the amino acid sequence of the nerve targeting factor Sema is all or part of the sequence shown in SEQ ID NO. 1;
2) the amino acid sequence of the nerve targeting factor Sema is a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity degree with SEQ ID NO. 1;
3) the difference between the amino acid sequence of the nerve targeting factor Sema and the sequence shown in SEQ ID NO. 1 is not more than 5, 4, 3, 2 or 1 amino acid;
4) the amino acid sequence of the nerve-targeting factor Sema is a variant of SEQ ID NO. 1, wherein the difference of the variant from SEQ ID NO. 1 comprises a substitution, deletion and/or insertion of one or more amino acid residues of the sequence or at least one N-/C-terminal extension.
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