CN116919982A - Pharmaceutical composition for treating arthritis and application thereof - Google Patents
Pharmaceutical composition for treating arthritis and application thereof Download PDFInfo
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- CN116919982A CN116919982A CN202210360895.4A CN202210360895A CN116919982A CN 116919982 A CN116919982 A CN 116919982A CN 202210360895 A CN202210360895 A CN 202210360895A CN 116919982 A CN116919982 A CN 116919982A
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- hyaluronic acid
- sodium salt
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- 206010003246 arthritis Diseases 0.000 title claims abstract description 24
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 182
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a pharmaceutical composition for treating arthritis, comprising: (1) crosslinked hyaluronic acid microspheres; and (2) non-crosslinked hyaluronic acid or a salt thereof. The cross-linked hyaluronic acid microsphere is formed by crosslinking two functionalized and modified hyaluronic acid or sodium salt thereof through thiol click chemistry, and is further mixed with the non-cross-linked hyaluronic acid or salt thereof after granulation to form a compound hyaluronic acid hydrogel preparation, and the hydrogel can be directly used for treating arthritis or combined with other medicines.
Description
Technical Field
The invention relates to the field of medicine, in particular to a pharmaceutical composition compounded by cross-linked and non-cross-linked hyaluronic acid and application thereof in osteoarthritis treatment.
Background
Arthritis, particularly osteoarthritis, is a common degenerative joint disease that occurs in weight bearing joint areas such as knee, hip, shoulder and metatarsophalangeal joints. Osteoarthritis patients are mostly old people over 50 years old, common clinical manifestations are joint pain, swelling, cartilage abrasion, bone spur hyperplasia and the like, and severe cases can have joint deformation and limited movement, and the life quality of patients is greatly affected. Along with the aging of population and the continuous aggravation of obesity rate, the incidence of osteoarthritis is continuously rising, and the statistics shows that the national osteoarthritis patients in 2015 reach 1.5 hundred million, and the people have the trend of younger, so that the treatment of arthritis becomes a serious problem to be solved urgently.
The existing clinic treatment on osteoarthritis focuses on conservative treatment so as to relieve inflammatory reaction, pain, improve joint function and delay disease development, and is combined with comprehensive treatment such as exercise, physiotherapy, drug treatment, surgery and the like. Common pharmaceutical treatments include analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids, and polysaccharide lubricant protectors such as hyaluronic acid for intra-articular injection.
Osteoarthritis pathological changes are mostly caused by cartilage degeneration, excessive wear of articular surface cartilage and destruction of cartilage tissue induce bone hyperplasia of subchondral bone to form osteophytes, causing joint pain, deformity and dysfunction. The normal intra-articular joint synovial fluid has a buffering and lubricating effect, plays a key role in protecting cartilage tissues, and the hyaluronic acid is taken as an important component of the joint synovial fluid, so that the synovial fluid can be ensured to have good viscoelasticity, and has extremely important significance in maintaining normal physiological functions of the synovial fluid. In joint synovial fluid of osteoarthritis patients, the molecular weight, concentration and viscoelasticity of hyaluronic acid are obviously reduced, so that the injection of hyaluronic acid in joint cavities, in particular high molecular weight hyaluronic acid, can directly enhance the lubrication function of joint synovial fluid and relieve synovial fluid inflammatory reaction, cartilage injury and pain.
The existing hyaluronic acid intra-articular cavity injection can be divided into a crosslinking type and a non-crosslinking type. Natural hyaluronic acid is easily decomposed by hyaluronidase in vivo, has short half-life in vivo and short duration of therapeutic effect. The property that the active ingredient is stored for a long time in the joint cavity has certain advantages in the aspect of long-term curative effect of treating arthritis due to the fact that the degradation speed of the crosslinked hyaluronic acid is low. However, the preparation of the conventional crosslinked hyaluronic acid has various limitations and potential safety hazards, and the mechanical properties of the crosslinked hydrogel system are in and out of the normal synovial tissue. Therefore, how to combine the advantages of high molecular weight hyaluronic acid and crosslinked hyaluronic acid and prepare the composite hydrogel preparation with good biocompatibility, low degradation speed and stable mechanical property is a problem to be solved at present.
Disclosure of Invention
In order to solve the above-mentioned problems, an object of the present invention is to provide a hydrogel in which cross-linked and non-cross-linked hyaluronic acid is compounded with arthritis, particularly osteoarthritis. The hydrogel has the advantages of both crosslinking type hyaluronic acid and non-crosslinking type hyaluronic acid, and after injection, the crosslinking type hyaluronic acid provides mechanical strength and structural support, so that the hydrogel has remarkably increased duration time in a cavity and realizes long-acting treatment effect; the non-crosslinked hyaluronic acid provides similar viscoelasticity, cohesiveness and film forming property as the sliding liquid in the joint cavity, and the small molecular part generated after degradation can also accelerate the healing process of the cartilage defect part; and certain self-assembly characteristics exist between the crosslinked hyaluronic acid particles and the uncrosslinked hyaluronic acid, so that excellent mechanical and biological properties are realized.
Aiming at the purposes, the technical scheme of the invention is as follows:
in one aspect, the invention provides a pharmaceutical composition for treating arthritis, the pharmaceutical composition comprising:
(1) Crosslinked hyaluronic acid microspheres; and
(2) Non-crosslinked hyaluronic acid or a salt thereof.
In the pharmaceutical composition provided by the invention, the crosslinked hyaluronic acid microsphere is formed by crosslinking vinyl functional hyaluronic acid or sodium salt thereof and mercapto functional hyaluronic acid or sodium salt thereof.
Preferably, the crosslinked hyaluronic acid microsphere is made by a method comprising the steps of:
(1) Mixing vinyl functional hyaluronic acid or sodium salt thereof and mercapto functional hyaluronic acid or sodium salt thereof in water, normal saline or buffer salt solution to obtain cross-linked hyaluronic acid;
(2) Sieving and granulating the crosslinked hyaluronic acid.
Wherein the vinyl functional hyaluronic acid OR its sodium salt has a modification of-COOH OR-OH contained in a part of the repeating unit side chain to-COOR OR-OR, R being-R 1 -C(O)-CH＝CH 2 Wherein:
R 1 is a chemical bond;
R 1 is- (CH) 2 ) m -O-, wherein m = 1-5, and (CH 2 ) m Any one of CH 2 Optionally substituted with-OH; or (b)
R 1 is-C (O) - (CH) 2 ) p -C(O)-O-(CH 2 ) m -O-, wherein p=1-3, m=1-5, and (CH 2 ) m Any one of CH 2 Optionally substituted with-OH.
Preferably, -COOH contained in the side chain of the repeating unit in the vinyl-functional hyaluronic acid or sodium salt thereof is modified to be-COOR; alternatively, the-OH group contained in the side chain of the repeating unit is modified to the-OR group. Preferably, m=2-3. Preferably, p=2-3.
More preferably, the vinyl-functional hyaluronic acid or sodium salt thereof has a molecular weight of 10-2000kDa, preferably 200-800kDa, more preferably 400-600kDa, even more preferably 400-500kDa, e.g. 450kDa.
According to a specific embodiment of the present invention, the vinyl-functional hyaluronic acid or sodium salt thereof comprises a structure as shown in any of the following:
and wherein the thiol-functional hyaluronic acid OR a sodium salt thereof has a moiety wherein-COOH OR-OH contained in the side chain of the repeating unit is modified to-COOR ' OR-OR ', and R ' is-R 2 -C(O)-(CH 2 ) q -S-(CH 2 ) t -SH, wherein q=1-3, t=1-5, and (CH 2 ) q And (CH) 2 ) t Any one or more CH 2 Optionally by-OH, -NH 2 or-CH 3 Substitution, and wherein:
R 2 is a chemical bond; or (b)
R 2 Is- (CH) 2 ) y -O-, wherein y = 1-5, and (CH 2 ) y Any one of CH 2 Optionally substituted with-OH.
Preferably, -COOH contained in the side chain of the repeating unit in the mercapto-functional hyaluronic acid or the sodium salt thereof is modified to be-COOR'; alternatively, the-OH group contained in the side chain of the repeating unit is modified to be the-OR'. Preferably, q=2-3. Preferably, t=3-4. Preferably, y=2-3.
More preferably, the thiol-functional hyaluronic acid or sodium salt thereof has a molecular weight of 10-2000kDa, preferably 200-800kDa, more preferably 400-600kDa, even more preferably 400-500kDa, e.g. 450kDa.
According to a specific embodiment of the present invention, in the pharmaceutical combination provided by the present invention, the thiol-functional hyaluronic acid or sodium salt thereof comprises a structure as shown in any one of the following:
preferably, in step (1), the vinyl-functional hyaluronic acid or sodium salt thereof and/or the mercapto-functional hyaluronic acid or sodium salt thereof is in the form of a solution in water, physiological saline or a buffer salt solution (e.g. a buffer salt solution with an osmotic pressure in the range of 270-350 mOsm/L), such as citrate, formate, acetate, phthalate, citrate, phosphate buffer.
According to a specific embodiment of the present invention, the concentration of the vinyl-functionalized hyaluronic acid solution is 0.5-2%, preferably 1% -2%, in terms of mass (g) volume (ml) ratio concentration; and/or the concentration of the thiol-functionalized hyaluronic acid solution is 0.5-2%, preferably 1% -2%, in terms of mass (g) volume (ml) ratio concentration.
Preferably, the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are mixed in a mass ratio of 1:0.1 to 0.1:1, preferably 1:1.
Preferably, in step (2), the cross-linked hyaluronic acid is granulated by passing through a 50-400 mesh screen, preferably a 100 mesh screen.
Preferably, in the pharmaceutical composition, the non-crosslinked hyaluronic acid or salt thereof has a molecular weight of 50-2000kDa, preferably 100-1400kDa, more preferably 450-1200kDa, still more preferably 800-1200kDa, e.g. 1000kDa.
Preferably, in the pharmaceutical composition, the mass ratio between the cross-linked hyaluronic acid microspheres and the non-cross-linked hyaluronic acid or salt thereof is 95:5-80:20, preferably 95:5-90:10.
Further, other drugs for treating arthritis, such as stem cells, anti-inflammatory agents or fibrinogen, are also included in the pharmaceutical compositions provided by the present invention. The other drug may be contained in crosslinked hyaluronic acid microspheres, mixed with either of the vinyl-functional hyaluronic acid or sodium salt thereof and the thiol-functional hyaluronic acid or sodium salt thereof prior to crosslinking.
Preferably, the stem cells are human or other mammalian-derived mesenchymal stem cells, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, more preferably adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells. According to a specific embodiment of the invention, the stem cells are adipose tissue-derived mesenchymal stem cells.
Preferably, the anti-inflammatory agent may be a non-steroidal anti-inflammatory agent (e.g., a COX-2 inhibitor), an opioid, or the like. According to a specific embodiment of the invention, the non-steroidal anti-inflammatory drug is diclofenac sodium.
Alternatively, the pharmaceutical compositions provided herein may be used in combination with other drugs for the treatment of arthritis.
In another aspect, the present invention provides a method of preparing the pharmaceutical composition, the method comprising:
(1) Mixing the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof in water, physiological saline or a buffer salt solution (e.g. a buffer salt solution having an osmotic pressure in the range of 270-350 mOsm/L) to obtain cross-linked hyaluronic acid;
(2) Sieving and granulating the crosslinked hyaluronic acid to obtain crosslinked hyaluronic acid microspheres; and
(3) Mixing the crosslinked hyaluronic acid microspheres with the non-crosslinked hyaluronic acid or salt thereof.
Preferably, in step (1) of the preparation method, the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are prepared into solutions having a concentration of 0.5-2%, preferably 1% -2%, respectively, in terms of mass (g) volume (ml) ratio concentration, and then mixed and crosslinked. Such as citrate, formate, acetate, phthalate, citrate, phosphate buffer.
Preferably, the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are mixed in a mass ratio of 1:0.1 to 0.1:1, preferably 1:1.
Optionally, the preparation method further comprises: in step (1), the vinyl-functional hyaluronic acid or sodium salt thereof and/or the mercapto-functional hyaluronic acid or sodium salt thereof is mixed with the other drug for treating arthritis, and then mixed and crosslinked.
Preferably, in step (2) of the preparation method, the crosslinked hyaluronic acid is granulated by passing through a 50-400 mesh sieve, preferably a 100 mesh sieve.
Preferably, in step (3) of the preparation method, the crosslinked hyaluronic acid microspheres are mixed with the non-crosslinked hyaluronic acid or salt thereof in a mass ratio of 95:5-80:20, preferably 95:5-90:10.
In yet another aspect, the invention provides the use of said pharmaceutical composition in the manufacture of a medicament for the treatment of arthritis.
Preferably, the arthritis is osteoarthritis. Wherein the osteoarthritis has symptoms such as pain at the joint, limited joint movement, excessive wear of the articular cartilage, and the like. Preferably, the medicament is an injection, preferably an intra-articular injection.
Compared with the prior art, the invention provides a hydrogel preparation compounded by cross-linked hyaluronic acid and non-cross-linked hyaluronic acid. The cross-linked hyaluronic acid is a hydrogel three-dimensional network structure formed by mixing vinyl and mercapto functional hyaluronic acid or sodium salt thereof and then performing chemical cross-linking through thiol clicking; the gel is subjected to sieving treatment and granulation, and then is physically mixed with natural hyaluronic acid to form the composite hydrogel preparation. Experiments prove that the composite hydrogel preparation provided by the invention has the advantages of high molecular weight hyaluronic acid, good viscoelasticity and good buffering property, has the advantages of quick crosslinking and slow degradation speed of crosslinked hyaluronic acid, and can be used for treating osteoarthritis. Without being limited by any theory, the hydrogel particle component has certain mechanical strength, and can play a certain functional supporting role in the joint cavity after injection. Meanwhile, the hyaluronic acid hydrogel in the form of cross-linked particles can remarkably increase the duration time of the hyaluronic acid hydrogel in a cavity, so that the long-acting treatment effect is realized; the hyaluronic acid component with medium and high molecular weight has similar viscoelasticity, cohesion and film forming property as the sliding liquid in the joint cavity, not only plays a role in lubricating the joint, but also can accelerate the healing process of cartilage defect parts after degradation. In addition, the crosslinked hyaluronic acid microparticles have certain self-assembly characteristics with the non-crosslinked hyaluronic acid. The non-crosslinked phase can generate certain self-assembly entanglement with a crosslinked structure while the lubricating colloidal particle moves, so that excellent mechanical and biological properties are realized.
In particular, the composite hydrogel preparation is obviously superior to the in-vitro degradation of non-crosslinked hyaluronic acid in the in-vitro enzymolysis time. And the hyaluronic acid is added in proportion, the self-assembly effect between the crosslinked phase and the non-crosslinked phase ensures that the composite product is more similar to the viscoelasticity and the buffering property of the natural joint synovial fluid, and meanwhile, the degradation speed of hyaluronic acid molecules is slowed down by virtue of the stable space structure of the crosslinked hyaluronic acid, the mechanical property of the joint synovial fluid close to normal is ensured, and the long-term treatment effect of the joint synovial fluid is promoted. The composite hydrogel preparation can be used for intra-articular injection and has great application prospect in treating arthritis.
In addition, the cross-linked and non-cross-linked hyaluronic acid composite hydrogel preparation provided by the invention not only can be directly used for treating osteoarthritis, but also can be further combined with other reagents such as fibrinogen, so that the treatment effect, such as cartilage injury repair, is optimized.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: rabbit knee joint model injection saline, alzhi, L5-A2-B2, M5-A2-B2, H5-A2-B2 five weeks after rabbit knee joint rough map.
Fig. 2: in vitro slow release profile of complex hyaluronic acid hydrogel with and without diclofenac sodium.
Detailed Description
The pharmaceutical composition provided by the invention is a composite hydrogel preparation prepared as follows: preparing vinyl and mercapto functional hyaluronic acid respectively; mixing two kinds of functionalized hyaluronic acid in proportion and then carrying out chemical crosslinking; granulating the obtained crosslinked hyaluronic acid; the obtained microsphere is mixed with high molecular weight hyaluronic acid in proportion to prepare the composite hydrogel preparation.
In addition, the pharmaceutical composition provided by the invention can be further combined with other treatment means.
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below were commercially available products or were obtained by synthesis according to the already-owned patents unless otherwise specified.
In the examples, compounds A1, A2, A3 shown below are taken as examples of vinyl-functionalized hyaluronic acid:
examples of thiol-functional hyaluronic acids are compounds B1, B2, B3 shown below:
compounds A4, A5 and B4, B5 shown below were used as control examples of two functionalized hyaluronic acids, respectively:
examples1 preparation of Compound hyaluronic acid hydrogel
Vinyl-functional hyaluronic acids A1, A2, A3, A4, A5 having a molecular weight of 450kDa and mercapto-functional hyaluronic acids B1, B2, B3, B4, B5 were dissolved in physiological saline at a mass volume concentration (w/v) of 1%, respectively. After complete dissolution, the cross-linked hyaluronic acid gel with the concentration of 1% is correspondingly prepared according to the same mass ratio by the named number, and the cross-linked hyaluronic acid gel is named as A1-B1, A2-B2, A3-B3, A4-B4 and A5-B5 respectively. The obtained crosslinked hyaluronic acid gel is granulated by a 100-mesh screen, and the mass ratio of crosslinked particles to uncrosslinked hyaluronic acid is 95: 5. 90:10 and 80:20, respectively mixing non-crosslinked low molecular weight (100 kDa), medium molecular weight (450 kDa) and high molecular weight hyaluronic acid (1000 kDa) to obtain composite hydrogel preparation, and the composition of the sample is shown in Table 1.
TABLE 1 composition of composite hydrogel formulations
Examples2 rheological mechanical property test of composite hyaluronic acid hydrogel
With reference to the inherent mechanical properties of joint synovial fluid in a human body, the optimal range of the elastic modulus of the hydrogel is 100-500Pa, and the optimal range of the viscous modulus of the hydrogel is 30-100Pa.
The hydrogel samples prepared as in example 1 were placed on a rheometer at room temperature and peristaltic scanned with the rheometer under a fixed frequency mode (1-20 Hz) and the values (Pa) of the elastic modulus and viscous modulus of the resulting hydrogels were examined. The results are shown in Table 2.
TABLE 2 rheological mechanical Property test results of composite hydrogel formulations
As can be seen from the data in the table, three groups A1-B1, A2-B2 and A3-B3 in the hydrogel sample prepared in example 1 all obtain mechanical performance attributes similar to the joint sliding hydrodynamic performance after being compounded with non-crosslinked hyaluronic acid with high, medium and low molecular weights in different proportions. Wherein, under the same mixing proportion of cross-linked phase and non-cross-linked phase, the non-cross-linked phase with high molecular weight can obviously improve the viscoelasticity of the biphasic hydrogel. On the other hand, a greater proportion of the non-crosslinked phase, while slightly increasing the viscous modulus, also significantly reduces the elastic modulus. The modulus of the bidirectional hydrogel obtained after the combination of the A4-B4 groups is too high, and the modulus of the A5-B5 groups is too low, so that the bidirectional hydrogel is not suitable for intra-articular injection.
Examples3 swelling Performance test of Compound hyaluronic acid hydrogel
In order to prevent the hydrogel system from being excessively swollen to cause compression to surrounding tissues, the optimal range of the swelling ratio of the hydrogel is 100 to 120%.
The hydrogel group samples of example 2, which were up to the rheological property, were selected, weighed after crosslinking was completed, and the initial weight (W 0 ) The hydrogel samples were then immersed in isotonic phosphate buffer and incubated at 37℃with shaking at constant temperature (about 150 rpm). After 24 hours the hydrogel samples were taken out and weighed (W t ) The hydrogel swelling ratio (W t /W 0 x 100%). The results are shown in Table 3.
TABLE 3 results of swelling Property test of composite hydrogel formulations
From the data obtained in the table, it can be seen that the addition of a higher proportion of non-crosslinked phase hyaluronic acid leads to a significant increase in the swelling ratio of the biphasic system, which also leads to the remaining 80, apart from H20-A1-B1 and H20-A2-B2: the swelling ratios of the 20 groups each exceeded the upper limit of the optimum swelling range of 120%. While the remaining hydrogel groups exhibit good swelling properties in the presence of a relatively low proportion of non-crosslinked phase.
Examples4 external degradation of composite hyaluronic acid hydrogel
To ensure that the hydrogel plays an effective role in vivo, the system design expects the hydrogel to degrade in vitro for 6-12 months.
The fresh hydrogel samples of examples 2 and 3, which were satisfactory, were weighed in a permeable isolation chamber with a semipermeable membrane and the initial weight (W 0 ) The isolation chamber containing the hydrogel sample was then immersed in phosphate buffer and incubated at 37℃with shaking at constant temperature (about 150 rpm). The isolation chamber was taken out at a fixed time point for weighing (W t ) Until the hydrogel is completely degraded (W t -W 0 =0), record dropAnd (5) a solution period. The results are shown in Table 4.
TABLE 4 in vitro degradation test results of composite hydrogel formulations
Experimental results show that the in vitro degradation rate of the composite hydrogel can be accelerated to different degrees by a high proportion of non-crosslinked hyaluronic acid component; on the other hand, the high molecular weight non-crosslinked hyaluronic acid can prolong the degradation time in vitro compared with the low molecular weight non-crosslinked hyaluronic acid. By combining the results of examples 2 to 4, L5, M5, H5 and H10 for the A1-B1 crosslinked phase consisted of non-crosslinked phases; l5, M5, H5 and H10 non-crosslinked phases for the A2-B2 crosslinked phase; the hydrogel system formed by the M5 and H5 non-crosslinked phases of the A3-B3 crosslinked phases meets the optimal attribute conditions.
Examples5 composite hyaluronic acid hydrogel animal experiment results
1. Sample preparation:
experimental groups were prepared with A2-B2 compounded with non-crosslinked phases of different molecular weights:
1) Experimental group L5-A2-B2: example Low molecular weight non-crosslinked composite crosslinked hyaluronic acid hydrogel (non-crosslinked hyaluronic acid molecular weight 100 kDa)
2) Experimental group M5-A2-B2: in the examples, molecular weight non-crosslinked composite crosslinked hyaluronic acid hydrogel (non-crosslinked hyaluronic acid molecular weight 450 kDa)
3) Experimental group H5-A2-B2: example high molecular weight non-crosslinked composite crosslinked hyaluronic acid hydrogel (non-crosslinked hyaluronic acid molecular weight 1000 kDa)
4) Positive control group: alzhi (Alzhi)
5) Negative control group: physiological saline
2. Grouping of experimental animals:
30 healthy New Zealand white rabbits are taken, weighing about 2kg, and are randomly divided into 5 groups of 6 rabbits each.
3. Preparation and administration of osteoarthritis models
An arthritis lesion model was produced by transection of the anterior cruciate ligament, and 0.3mL of samples L5-A2-B2, M5-A2-B2, H5-A2-B2 were injected into the joint cavity of the rabbits of the experimental group, respectively, for 5 weeks after the completion of the molding, 0.3mL of physiological saline was injected into the joint cavity of the rabbits of the negative control group, and 0.3mL of Alzhi was administered to the control group, during which the animals were continuously observed.
4. Sample collection and detection
Animals were sacrificed 5 weeks after injection of samples, alzhi or physiological saline, joint fluids were taken out for observation and identification of total protein content and glucan content therein, joint tissue was generally observed, joint tissue was fixed, paraffin sections were prepared, HE and safranine fast green staining were performed respectively, and the degree of articular cartilage damage was evaluated by observation under a microscope.
5. The results are shown in FIG. 1:
1) According to the OARSI macroscopic score, the experimental groups L5-A2-B2, M5-A2-B2, H5-A2-B2 and the control group are obviously different from the negative control group, and the treatment effect of the composite hydrogel of the embodiment is proved to be excellent.
2) The joint tissue section staining results show that the comparison of the experimental groups L5-A2-B2, M5-A2-B2, H5-A2-B2 and the control group with the negative control group is obviously different. The results show that the experimental group H5-A2-B2 has better curative effects on cartilage structure, cell morphology and the like than the control group and the negative control group, and has more obvious effect on relieving cartilage degeneration.
ExamplesApplication of 6-compound hyaluronic acid hydrogel in drug slow release
100mg of diclofenac sodium was dissolved in 2mL of physiological saline for injection (5%, w/v). After that, thiol-functionalized hyaluronic acid B2 and vinyl-functionalized hyaluronic acid A2 were dissolved at a concentration of 1% using 1mL of a solution of diclofenac sodium, respectively, and after the dissolution was completed, the two solutions were vortexed and mixed uniformly.
Sieving with 100 mesh sieve after chemical crosslinking, granulating, mixing with low molecular weight (100 kDa), medium molecular weight (450 kDa) and high molecular weight (1000 kDa) hyaluronic acid according to the ratio of crosslinked microparticles to non-crosslinked hyaluronic acid of 95:5, mixing uniformly again, and performing in vitro slow release experiment to verify the wrapping and slow release effects of in situ crosslinked hyaluronic acid hydrogel on medicine.
Drug release was followed by High Performance Liquid Chromatography (HPLC). The experimental procedure was as follows:
1. sample preparation:
experimental groups were prepared with A2-B2 compounded with non-crosslinked phases of different molecular weights:
1) Experimental group L5-A2-B2: L5-A2-B2 composite hyaluronic acid hydrogel containing diclofenac sodium
2) Experimental group M5-A2-B2: M5-A2-B2 composite hyaluronic acid hydrogel containing diclofenac sodium
3) Experimental group H5-A2-B2: diclofenac sodium-containing H5-A5-B5 complex hyaluronic acid hydrogel, blank set prepared in example 1:
4) Blank L5-A2-B2: L5-A2-B2 composite hyaluronic acid hydrogel
5) Blank M5-A2-B2: M5-A2-B2 composite hyaluronic acid hydrogel
6) Blank H5-A2-B2: H5-A2-B2 composite hyaluronic acid hydrogel
Samples of either the experimental or blank 100 μl were dispensed into 1.5 ml centrifuge tubes for use.
2. Experimental grouping:
the experimental samples were 30 in total, with 5 for each experimental group, blank group.
3. Sample collection and detection:
after gel formation, the experimental samples were each added with PBS (1 mL/tube) and incubated at 50rpm in a constant temperature shaker at 37 ℃. The supernatant from the sample tube was removed at a predetermined sample collection time point, replaced with fresh PBS (1 mL/tube), and the supernatant was filtered and assayed for drug content using HPLC.
4. The results are shown in FIG. 2:
the in vitro slow release experiment results prove that the compound hyaluronic acid hydrogel L5-A2-B2, M5-A2-B2 and H5-A2-B2 can effectively control the release speed of the medicine, and the medicine can be continuously and slowly released within 20-30 days. Wherein the high molecular weight non-crosslinked component can slow the release rate of the drug to achieve a longer time controlled release effect.
ExamplesTreatment of osteoarthritis by combining hyaluronic acid hydrogel with active ingredient
Referring to example 6, a proper amount of fibrinogen was thoroughly mixed with a vinyl-and mercapto-functional hyaluronic acid solution to prepare a crosslinked hyaluronic acid hydrogel containing bioactive components. Sieving and granulating the crosslinked hydrogel by a 100-mesh sieve, mixing with low-molecular weight, medium-molecular weight and high-molecular weight hyaluronic acid according to the ratio of 95:5, and uniformly mixing again to obtain the composite hyaluronic acid hydrogel preparation containing active ingredients.
1. Sample preparation:
experimental groups were prepared with A2-B2 compounded with non-crosslinked phases of different molecular weights:
1) Experimental group L5-A2-B2-C:0.3mL L5-A2-B2 composite hydrogel containing active fibrinogen
2) Experimental group M5-A2-B2-C:0.3mL M5-A2-B2 composite hydrogel containing active fibrinogen
3) Experimental group H5-A2-B2-C:0.3mL H5-A2-B2 composite hydrogel containing active fibrinogen
4) Positive control group: 0.3mL Alzhi
5) Negative control group: 0.3mL physiological saline
2. Grouping of experimental animals:
30 healthy New Zealand white rabbits are taken, weighing about 2kg, and are randomly divided into 5 groups of 6 rabbits each.
3. Preparation and administration of osteoarthritis models
An arthritis lesion model was produced by anterior cruciate ligament transection, and 0.3mL of the experimental group L5-A2-B2-C, M5-A2-B2-C, H-A2-B2-C sample was injected into the joint cavity of the experimental group rabbit at four weeks after the completion of the molding, 0.3mL of physiological saline was injected into the joint cavity of the negative control group rabbit, and 0.3mL of Alzhi was administered weekly for 5 weeks in the control group, during which the animals were continuously observed.
4. Sample collection and detection
Animals were sacrificed 5 weeks after injection of samples, alcian or physiological saline. And (3) cutting joint tissues for fixation, preparing paraffin sections, respectively carrying out HE (high-speed) and safranine solid-green staining, and observing and evaluating the damage and repair degree of the joint cartilage under a microscope.
5. Cartilage repair outcome assessment:
1) According to the OARSI macroscopic score, the experimental group L5-A2-B2-C, M5-A2-B2-C, H5-A2-B2-C and the control group are significantly different from the negative control group, wherein the experimental group L5-A2-B2-C and M5-A2-B2-C perform better than the control group. Experiments prove that the composite hydrogel of the embodiment has excellent effect of treating arthritis, and has better treatment effect after adding fibrinogen and other active substances.
2) The joint tissue section staining results show that compared with the negative control group, the experimental groups L5-A2-B2, M5-A2-B2 and H5-A2-B2 have better curative effects on cartilage surface repair, chondrocyte morphology and the like, and have obvious effects on relieving cartilage degeneration. The experimental groups L5-A2-B2 and M5-A2-B2 are better in cartilage injury repair compared with other groups, and the results prove that the fibrinogen addition can optimize the treatment effect of the composite hydrogel on arthritis and the injury repair effect of cartilage.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.
Claims (14)
1. A pharmaceutical composition for treating arthritis, the pharmaceutical composition comprising:
(1) Crosslinked hyaluronic acid microspheres; and
(2) Non-crosslinked hyaluronic acid or a salt thereof.
2. The pharmaceutical composition according to claim 1, wherein the crosslinked hyaluronic acid microspheres are formed by crosslinking vinyl-functional hyaluronic acid or a sodium salt thereof and mercapto-functional hyaluronic acid or a sodium salt thereof;
preferably, the crosslinked hyaluronic acid microsphere is made by a method comprising the steps of:
(1) Mixing vinyl functional hyaluronic acid or sodium salt thereof and mercapto functional hyaluronic acid or sodium salt thereof in water, normal saline or buffer salt solution to obtain cross-linked hyaluronic acid;
(2) Sieving and granulating the crosslinked hyaluronic acid.
3. The pharmaceutical composition according to claim 1 OR 2, wherein the vinyl-functionalized hyaluronic acid OR sodium salt thereof has a modification of-COOH OR-OH contained in a side chain of a part of the repeating units to-COOR OR-OR, and R is-R 1 -C(O)-CH＝CH 2 Wherein:
R 1 is a chemical bond;
R 1 is- (CH) 2 ) m -O-, wherein m = 1-5, and (CH 2 ) m Any one of CH 2 Optionally substituted with-OH; or (b)
R 1 is-C (O) - (CH) 2 ) p -C(O)-O-(CH 2 ) m -O-, wherein p=1-3, m=1-5, and (CH 2 ) m Any one of CH 2 Optionally substituted with-OH.
4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the-COOH contained in the side chain of the repeating unit in the vinyl-functional hyaluronic acid or sodium salt thereof is modified to the-COOR; alternatively, the-OH contained in the side chain of the repeating unit is modified to the-OR;
preferably, m=2-3; preferably, p=2-3;
more preferably, the vinyl-functional hyaluronic acid or sodium salt thereof has a molecular weight of 10-2000kDa, preferably 200-800kDa, more preferably 400-600kDa, even more preferably 400-500kDa, e.g. 450kDa;
more preferably, the vinyl-functional hyaluronic acid or sodium salt thereof comprises a structure as shown in any of the following:
5. the pharmaceutical composition according to any one of claims 1 to 4, wherein the thiol-functional hyaluronic acid OR sodium salt thereof has a moiety of the recurring unit side chain of-COOH OR-OH modified to-COOR ' OR-OR ', R ' is-R 2 -C(O)-(CH 2 ) q -S-(CH 2 ) t -SH, wherein q=1-3, t=1-5, and (CH 2 ) q And (CH) 2 ) t Any one or more CH 2 Optionally by-OH, -NH 2 or-CH 3 Substitution, and wherein:
R 2 is a chemical bond; or (b)
R 2 Is- (CH) 2 ) y -O-, wherein y = 1-5, and (CH 2 ) y Any one of CH 2 Optionally substituted with-OH.
6. The pharmaceutical combination according to any one of claims 1 to 5, wherein the-COOH contained in the side chain of the repeating unit in the thiol-functional hyaluronic acid or sodium salt thereof is modified to the-COOR'; alternatively, the-OH contained in the side chain of the repeating unit is modified to the-OR';
preferably, q=2-3; preferably, t=3-4; preferably, y=2-3;
more preferably, the thiol-functional hyaluronic acid or sodium salt thereof has a molecular weight of 10-2000kDa, preferably 200-800kDa, more preferably 400-600kDa, even more preferably 400-500kDa, e.g. 450kDa;
more preferably, the thiol-functional hyaluronic acid or sodium salt thereof comprises a structure as shown in any of the following:
7. the pharmaceutical combination according to any one of claims 1 to 6, wherein in step (1) the vinyl-functional hyaluronic acid or sodium salt thereof and/or the mercapto-functional hyaluronic acid or sodium salt thereof is in the form of a solution in water, physiological saline or a buffered saline solution;
preferably, the concentration of the vinyl-functionalized hyaluronic acid solution is 0.5-2%, preferably 1% -2%, in terms of mass (g) volume (ml) ratio concentration; and/or the concentration of the thiol-functionalized hyaluronic acid solution is 0.5-2%, preferably 1% -2%, in terms of mass (g) volume (ml) ratio concentration.
8. The pharmaceutical composition according to any one of claims 1 to 7, wherein in step (1), the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are mixed in a mass ratio of 1:0.1 to 0.1:1, preferably 1:1;
preferably, in step (2), the cross-linked hyaluronic acid is granulated by passing through a 50-400 mesh screen, preferably a 100 mesh screen.
9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the molecular weight of the non-crosslinked hyaluronic acid or salt thereof in the pharmaceutical composition is 50-2000kDa, preferably 100-1400kDa, more preferably 450-1200kDa, further preferably 800-1200kDa, such as 1000kDa;
preferably, in the pharmaceutical composition, the mass ratio between the cross-linked hyaluronic acid microspheres and the non-cross-linked hyaluronic acid or salt thereof is 95:5-80:20, preferably 95:5-90:10.
10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition further comprises an additional drug for the treatment of arthritis;
preferably, the other drug is a stem cell, an anti-inflammatory or fibrinogen;
preferably, the other drug is contained in the crosslinked hyaluronic acid microsphere.
11. A process for preparing the pharmaceutical composition of any one of claims 1 to 10, the process comprising:
(1) Mixing the vinyl functional hyaluronic acid or sodium salt thereof and the mercapto functional hyaluronic acid or sodium salt thereof in water, normal saline or buffer salt solution to obtain crosslinked hyaluronic acid;
(2) Sieving and granulating the crosslinked hyaluronic acid to obtain crosslinked hyaluronic acid microspheres; and
(3) The crosslinked hyaluronic acid microspheres are mixed with non-crosslinked hyaluronic acid or a salt thereof.
12. The preparation method according to claim 11, wherein in step (1) of the preparation method, the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are prepared into solutions having a concentration of 0.5-2%, preferably 1% -2%, respectively, in terms of mass (g) volume (ml) ratio concentration, and are mixed and crosslinked;
preferably, the vinyl-functional hyaluronic acid or sodium salt thereof and the mercapto-functional hyaluronic acid or sodium salt thereof are mixed in a mass ratio of 1:0.1 to 0.1:1, preferably 1:1.
13. The production method according to claim 11 or 12, characterized in that the production method further comprises: in step (1), the vinyl-functional hyaluronic acid or sodium salt thereof and/or the mercapto-functional hyaluronic acid or sodium salt thereof is mixed with the other drug for treating arthritis, and then the two are mixed and crosslinked;
preferably, in step (2), the cross-linked hyaluronic acid is granulated through a 50-400 mesh screen, preferably a 100 mesh screen;
preferably, in step (3), the cross-linked hyaluronic acid microspheres are mixed with the non-cross-linked hyaluronic acid or salt thereof in a mass ratio of 95:5-80:20, preferably 95:5-90:10.
14. Use of a pharmaceutical composition according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment of arthritis;
preferably, the arthritis is osteoarthritis, preferably with symptoms such as pain at the joint site, limited joint movement, excessive wear of the articular cartilage, etc.;
preferably, the medicament is an injection, preferably an intra-articular injection.
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| CN117298355A (en) * | 2023-11-28 | 2023-12-29 | 常州百瑞吉生物医药股份有限公司 | Coated hyaluronic acid gel composition and preparation method and application thereof |
| CN118994656A (en) * | 2024-08-20 | 2024-11-22 | 徐州医科大学附属医院 | Thiol-ene crosslinked hyaluronic acid gel compound and preparation method and application thereof |
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| CN117298355A (en) * | 2023-11-28 | 2023-12-29 | 常州百瑞吉生物医药股份有限公司 | Coated hyaluronic acid gel composition and preparation method and application thereof |
| CN117298355B (en) * | 2023-11-28 | 2024-03-08 | 常州百瑞吉生物医药股份有限公司 | Coated hyaluronic acid gel composition and preparation method and application thereof |
| CN118994656A (en) * | 2024-08-20 | 2024-11-22 | 徐州医科大学附属医院 | Thiol-ene crosslinked hyaluronic acid gel compound and preparation method and application thereof |
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