CN104257659A - Application of epidermal growth factor receptor blocking agent to preparation of medicine for treating osteoarthritis - Google Patents

Application of epidermal growth factor receptor blocking agent to preparation of medicine for treating osteoarthritis Download PDF

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CN104257659A
CN104257659A CN201410414758.XA CN201410414758A CN104257659A CN 104257659 A CN104257659 A CN 104257659A CN 201410414758 A CN201410414758 A CN 201410414758A CN 104257659 A CN104257659 A CN 104257659A
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chitosan
egf
gefitinib
osteoarthritis
group
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欧阳宏伟
吴岩
熊思
王俊娟
邹晓辉
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Zhejiang University ZJU
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin

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Abstract

The invention discloses application of an epidermal growth factor receptor blocking agent to preparation of a medicine for treating osteoarthritis. The epidermal growth factor receptor blocking agent is gefitinib or erlotinib. Gefitinib/chitosan microspheres are favorable in dispersion and smooth in surface, has the drug slow-release performance and mouse articular cavity biocompatibility, can effectively relieve mouse osteoarthritis, promotes the expression of mouse osteoarthritis knee joint collagen II and inhibits the expression of MMP-13; besides, gefitinib is a clinical medicine, is widely used for treating the non-small cell lung cancer, and obtains excellent clinical curative effects, so that the extremely high economic cost and the long research and development period, which are required for medicine research and development, are reduced and shortened respectively; and owing to the dosing way of sustained-release medicine delivery, the local medicine concentration can be improved while corresponding side effects caused by whole-body medicine application are reduced, and a clinical application prospect is achieved.

Description

The application of EGF-R ELISA blocker in preparation treatment medicine for treating arthritis
(1) technical field
The present invention relates to a kind of application of EGF-R ELISA blocker, the particularly application of EGF-R ELISA blocker in preparation treatment medicine for treating arthritis.
(2) background technology
Osteoarthritis (osteoarthritis, OA) is a kind of chronic joint disease being feature with the degeneration of articular cartilage, destruction and hyperosteogeny.Primary disease is multiple after the middle age.Domestic preliminary survey display, the total prevalence rate of osteoarthritis is about 15%, and the prevalence of 40 years old crowd is 10%-17%, within more than 60 years old, then reaches 50%.And more than 75 years old in crowd, 80% suffers from osteoarthritis.The final disability rate of this disease is 53%.The most common with arthralgia, hyperosteogeny and limitation of activity clinically.The morbidity of osteoarthritis is without region and race difference.Age, obesity, inflammation, wound and inherited genetic factors may be relevant with the generation of primary disease.
According to U.S.'s knee joint osteoarthritis treatment guidelines, the medicine that may be used for osteoarthritis at present has corticosteroid hormone, and acetaminophen and epoxide hydrolase-2 press down agent.These medicines are all treat for the symptom of osteoarthritis.Trace it to its cause and be that we are not enough for the Pathophysiology understanding of osteoarthritis, along with the development of molecular biology and translational medicine in recent years, there is deep understanding to the morbidity pathogenesis of osteoarthritis, especially participated in the blocker of osteoarthritis primary signal pathways.TGF-β, PI3K-mTOR, the blocker of the associated signal paths such as Wnt and inflammatory factor is widely used in the treatment of osteoarthritis, but be only only limitted to the infrastest stage, distant from final clinical practice, even if finally may be used for clinical, also need to pay huge economic cost, and these signal paths are all unidirectional to the adjustment of osteoarthritis, namely mainly block the factor destroying cartilage.In recent years, find that EGF-R ELISA (EGFR, Epidermal Growth Factor Receptor) signal path take part in the generation of cartilage development and osteoarthritis, this receptor expresses rising in the OA sample of animal and human; The part TGF-α of EGFR receptor suppresses the synthesis of collagen II and increases the secretion of MMP-13 simultaneously.Therefore, can think that the receptor ginseng of state of activation plays an important role in the developing of OA.And block this receptor existing cartilage both can have been protected can to promote that it carries out self-regeneration.
Gefitinib can effectively block EGFR receptor thus improve the survival rate of cancer patient in nonsmall-cell lung cancer, and because this medicine is chemotherapeutics, its treatment that whether may be used for bone cartilage related disorder becomes the focus of concern.Basic research shows, gives SD rat oral gavage and the thickness of growth plate cartilage within 7 days, can be made to double; The analog Erlotinib of gefitinib effectively can block the synovial membrane inflammation of rheumatoid joint; Gefitinib participates in the bone remoulding of subchondral bone, and above-mentioned research shows that gefitinib may be used for the treatment of bone cartilage related disorder.Gefitinib systemic administration can produce corresponding side effect, therefore improves its route of administration and mode, not only can reduce the generation of side effect, and can improve curative effect.Chitosan is the deacetylation product of chitin; it can be absorbed by the body to utilize has good biocompatibility with human tissue organ and cell; nontoxic; there is biological degradability; the oligochitosan produced in degradation process does not accumulate in vivo; almost non-immunogenicity, being therefore widely used in biological medicine is good Thermosensitive Material Used for Controlled Releasing of Medicine.Due to the various advantages of chitosan sustained-release microsphere, prepare the treatment that gefitinib/chitosan sustained-release microsphere can be effective to osteoarthritis.
(3) summary of the invention
The object of the invention is to provide a kind of new opplication of EGF-R ELISA blocker, i.e. the application of EGF-R ELISA blocker in preparation treatment medicine for treating arthritis.The present invention, for the tyrosine kinase inhibitor gefitinib of EGFR, provides the preparation method of gefitinib/chitosan microball, and specify that the effect of gefitinib/chitosan microball in mice osteoarthritis.
The technical solution used in the present invention is:
The invention provides the application of a kind of EGF-R ELISA blocker in preparation treatment medicine for treating arthritis, described EGF-R ELISA blocker is gefitinib or Erlotinib.
Further, described EGF-R ELISA blocker is applied with the form of EGF-R ELISA blocker/chitosan microball.
Further, described EGF-R ELISA blocker/chitosan microball is prepared as follows: (1) chitosan solution: with glacial acetic acid aqueous dissolution chitosan, be mixed with chitosan solution; (2) emulsification system: add EGF-R ELISA blocker in chitosan solution, mix homogeneously, stirring and emulsifying under 40 DEG C of waters bath with thermostatic control, 800-1000r/min condition, forms emulsification system; The consumption of described chitosan solution is in chitosan mass, and described chitosan and EGF-R ELISA blocker mass ratio are 100:1-800:1 (preferred 400:1); (3) microsphere: add glutaraldehyde in emulsification system, keep 40 DEG C, stir crosslinked, after reaction terminates, leave standstill 30min, after complete layering, remove upper liquid, lower floor's granule uses petroleum ether and washed with isopropyl alcohol respectively, then lyophilization, obtains EGF-R ELISA blocker/chitosan microball.
Further, the volumetric concentration of described glacial acetic acid aqueous solution is 2%.
Further, in described chitosan solution, the mass concentration of chitosan is 0.01-0.1g/ml (preferred 0.02g/ml).
Further, described lyophilization is lyophilizing at-109 DEG C.
Further, the volume addition of described glutaraldehyde counts 2.0-15ml/g (preferred 12.5ml/g) with the quality of chitosan in step (2) chitosan solution.
In addition, the present invention also provides a kind of EGF-R ELISA blocker/chitosan microball, described EGF-R ELISA blocker/chitosan microball is prepared as follows: (1) chitosan solution: be the glacial acetic acid aqueous dissolution chitosan of 2% by volumetric concentration, is mixed with the chitosan solution (preferred 0.02g/ml) of 0.01-0.1g/ml; (2) emulsification system: add EGF-R ELISA blocker in chitosan solution, mix homogeneously, stirring and emulsifying under 40 DEG C of waters bath with thermostatic control, 800-1000r/min condition, forms emulsification system; The consumption of described chitosan solution is in chitosan mass, and described chitosan and EGF-R ELISA blocker mass ratio are 100:1-800:1 (preferred 400:1); Described EGF-R ELISA blocker is gefitinib or Erlotinib; (3) microsphere: add glutaraldehyde in emulsification system, keep 40 DEG C, stir crosslinked, after reaction terminates, leave standstill 30min, after complete layering, remove upper liquid, lower floor's granule uses petroleum ether and washed with isopropyl alcohol respectively, then lyophilization, obtains EGF-R ELISA blocker/chitosan microball; The volume addition of described glutaraldehyde counts 2.0-15ml/g (preferred 12.5ml/g) with the quality of chitosan in step (2) chitosan solution.
EGF-R ELISA blocker of the present invention/chitosan microball quality consumption is 0.88-7.04 × 10 -1mg/Kg body weight (preferably 3.52 × 10 -1mg/Kg), namely EGF-R ELISA blocker quality consumption is 2.2-17.6 × 10 -4mg/Kg body weight (preferably 8.8 × 10 -4mg/Kg).
EGF-R ELISA blocker/chitosan microball of the present invention (preferred gefitinib/chitosan microball) with the caused osteoarthritis modelling verification of mice anterior cruciate ligament of knee joint excision its to the intervention effect of osteoarthritis.
Gefitinib chemical name of the present invention is: N-(the chloro-4-fluorophenyl of 3-)-7-methoxyl group-6-(3-morpholine propoxyl group) quinazoline-4-amine, molecular formula is: C 22h 24clFN 4o 3, molecular weight is: 446.90.
Two (2-the methoxyethoxy)-4-quinolinamine hydrochlorate of Erlotinib chemical name of the present invention: N-(3-acetylene phenyl)-6,7-, molecular formula: C 22h 23n 3o 4hCl, molecular weight: 429.90.
At present, the clinical medicine for osteoarthritis treatment concentrates on the symptom improving patient mostly, and the Preclinical Drug that osteoarthritis has an intervention effect is mainly suppressed to its inflammatory process or suppresses the secretion of destructive enzyme.Along with basic researchers builds consensus in osteoarthritis Mechanism Study, the overexpression of extracellular matrix synthesis suppression and digestive enzyme is related in the pathogenic process of osteoarthritis, the present invention is from gefitinib, Erlotinib is started with to the synthesis of collagen II in osteoarthritis forming process and the secretion of MMP-13, first cutting bone property of mice anterior cruciate ligament of knee joint arthritis model is set up, by carrying out corresponding histological score to mice knee joint pathologic examination after joint cavity injection gefitinib/chitosan microball, observe Mouse cartilage disorganization degree, and the expression of collagen II and MMP-13, and by blank group, the comparison of model group and gefitinib/chitosan microball medication group, study gefitinib/chitosan microball joint cavity injection to the arthritic preventive and therapeutic effect of Mouse Bone, inquire into gefitinib lapses to mechanism impact on osteoarthritis, show that gefitinib is by promoting the synthesis of collagen II and suppressing the secretion of MMP-13 to suppress the pathological process of osteoarthritis, thus provide experimental basis for the further clinical practice of this medicine.
Domestic and international utilization chitosan has obtained the result with using value as slow-released carrier, main manifestations is that chitosan microball has good biocompatibility and good slowly releasing effect thus obviously reduces the consumption of medicine and improve therapeutic effect.Chitosan microball is that experimentation of the present invention provides necessary condition.
C57BL/6J mice 24 extracts 18 kneed anterior cruciate ligaments of excision mice and carries out osteoarthritis modeling, and be divided into osteoarthritis group after modeling, chitosan microball group, gefitinib/chitosan microball group, residue mice is sham operated rats.Be left intact after about 6 the lower limb modelings of osteoarthritis group, about 6 the lower limb modelings of chitosan microball group start the chitosan microball of joint cavity injection not drug containing after 1 week, every 3 days once, gefitinib/chitosan microball group about 6 lower limb modeling starts joint cavity injection gefitinib/chitosan microball after 1 week, every 3 days once, test after 8 weeks and put to death all mices, observe each group of mice knee joint pathology Histological change and carry out corresponding histological score.Result shows: gefitinib/chitosan microball good dispersion, smooth surface, there is the performance of slow releasing pharmaceutical, there is mice articular cavity biocompatibility, effectively alleviate mice osteoarthritis, promote the expression of mice osteoarthritis knee joint collagen II and suppress the expression of MMP-13.
Beneficial effect of the present invention is mainly reflected in: epidermal growth factor blocker of the present invention/chitosan microball good dispersion, smooth surface, there is the performance of slow releasing pharmaceutical, there is mice articular cavity biocompatibility, effective alleviation mice osteoarthritis, promotes the expression of mice osteoarthritis knee joint collagen II and suppresses the expression of MMP-13; In addition, gefitinib, Erlotinib are a kind of clinical applications, are widely used in the treatment of nonsmall-cell lung cancer and achieve good clinical efficacy, decrease the economic cost of the costliness required for medicament research and development and very long R&D cycle; The route of administration of sustained-release administration, can reduce systemic administration and produce corresponding side effect, have potential applicability in clinical practice while raising local drug concentration.
(4) accompanying drawing explanation
Fig. 1 is chitosan microball scanning electron microscope phenogram.
Fig. 2 is gefitinib/chitosan microball scanning electron microscope phenogram.
Fig. 3 is the external elution profiles figure of gefitinib/chitosan microball.
Fig. 4 is Erlotinib/chitosan microball scanning electron microscope phenogram.
Fig. 5 is the external elution profiles figure of Erlotinib/chitosan microball.
Fig. 6 is the HE colored graph of articular cavity synovial tissue, and A is sham operated rats, and B is osteoarthritis group, and C is chitosan sustained-release microsphere group, and D is gefitinib/chitosan microball group.
Fig. 7 is sham operated rats, osteoarthritis group, the inflammatory score of chitosan sustained-release microsphere group and gefitinib/Chitosan microspheres group articular cavity synovial tissue.
Fig. 8 is cartilaginous tissue section p-EGFR immunohistochemical staining figure, and A is sham operated rats, and B is osteoarthritis group, and C is chitosan sustained-release microsphere group, and D is gefitinib/chitosan microball group.
Fig. 9 is the sarranine-alcian blue colored graph of cartilaginous tissue section, and A is sham operated rats, and B is osteoarthritis group, C is chitosan sustained-release microsphere group, D is dosage group in gefitinib/chitosan microball, and E is gefitinib/chitosan microball low dose group, and F is gefitinib/chitosan microball high dose group.
Figure 10 is sham operated rats, osteoarthritis group, international osteoarthritis EASD (Osteoarthritis Research Society International, the OARSI) scoring of chitosan microball group and gefitinib/chitosan microball group cartilaginous tissue.
Figure 11 is cartilaginous tissue Collagen content in slice II immunohistochemical staining figure, and A is sham operated rats, and B is osteoarthritis group, and C is chitosan sustained-release microsphere group, and D is gefitinib/chitosan microball group.
Figure 12 is cartilaginous tissue section MMP-13 immunohistochemical staining figure, and A is sham operated rats, and B is osteoarthritis group, and C is chitosan sustained-release microsphere group, and D is gefitinib/chitosan microball group.
Figure 13 is the collagen II expression of cell.
Figure 14 is the MMP-13 expression of cell.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment of the present invention experiment material used:
Laboratory animal: C57BL/6J mice in female 4 week age 24 SPF level Zhejiang University animal experimental center provide.
Experimental drug:
Gefitinib (Iressa, Iressa): AstraZeneca pharmaceutical Co. Ltd of Britain lot number: J20070047
Chitosan: Bai Ao bio tech ltd, Shanghai lot number: 050106
Main agents:
Experimental instrument and equipment:
The preparation of embodiment 1 microsphere and sign detect
The present invention adopts the method for emulsion polymerisation to prepare chitosan microball: first, is the glacial acetic acid aqueous dissolution chitosan of 2%, is mixed with the chitosan solution of 0.02g/ml by volumetric concentration.Secondly, synthesis emulsification system: measure chitosan solution 20ml, add 1mg gefitinib wherein, mix homogeneously is stand-by, stirs in 40 DEG C of waters bath with thermostatic control, and in whole emulsion process, mixing speed remains on 800-1000rpm/min, to make system even, form emulsification system.Then, in emulsification system, add 5ml cross-linking agent glutaraldehyde, keep 40 DEG C, stirring reaction.After cross-linking reaction terminates, leave standstill 30min, after complete layering, remove upper liquid, lower floor's granule uses petroleum ether and washed with isopropyl alcohol 3 times respectively, and then lyophilizing at-109 DEG C, obtain yellow gefitinib/chitosan microball, scanning electron microscope as shown in Figure 2.Not add gefitinib in contrast under similarity condition, prepare chitosan microball, scanning electron microscope as shown in Figure 1.
The sustained release performance of medicine: microsphere 40mg to be measured is placed in 100ml shake flask volume, and adds 20ml deionized water wherein.Be 1h, 3h, 6h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 84h, 96h at sampling time point, take out the aqueous solution of malic acid (2-hydroxyl succinic acid) that 2ml sample adds 400 μ l (0.07mg/ml), then be that the isopropyl alcohol of 90% is (containing 0.75molL by volumetric concentration -1hydrochloric acid) standardize solution is to 10ml, and sampling spectrophotometer measures absorbance at wavelength 350nm place, the results are shown in Figure shown in 3.
Embodiment 2
Change gefitinib in embodiment 1 into Erlotinib, other operations are with embodiment 1, and obtain Erlotinib/chitosan microball, as shown in Figure 4, sustained release performance as shown in Figure 5 for scanning electron microscope.
The application of embodiment 3 gefitinibs/chitosan microball
1. Animal Model:
1. anaesthetize: calculate, with 1% pentobarbital sodium intraperitoneal anesthesia by 40mg/kg.
2. row mice knee joint Skin sensitization test in an aseptic environment, uses 2% iodophor disinfection, cuts the otch of about 0.5cm size along patellar ligament medial border, successively cut and expose knee joint, cut off anterior cruciate ligament of knee joint under the microscope with microsurgery instruments, sterilize immediately, sew up.Sham operated rats only exposes disinfection and stitching after knee joint.
2. laboratory animal grouping:
40 C57BL/6J mices are randomly drawed 36 and carry out model foundation, are divided at random 6 groups (often organizing 6): osteoarthritis group, chitosan microball group, gefitinib/chitosan microball low dose group (1 × 10 after modeling -1mg/Kg), dosage group (4 × 10 in gefitinib/chitosan microball -1mg/Kg), gefitinib/chitosan microball high dose group (7 × 10 -1mg/Kg), sham operated rats.
3. administering mode:
First group (sham operated rats): normally raise;
Second group (osteoarthritis group): normally raise;
3rd group (chitosan microball group): modeling 1 week, injects 20 μ l chitosan microballs (prepared by embodiment 1) every 2 days knee joints.
4th group (in gefitinib/chitosan microball dosage group): modeling 1 week, injects 20 μ l gefitinib/chitosan microballs (prepared by embodiment 1) every 2 days knee joints.
4. specimen is taked
Test after 8 weeks and put to death whole mice, get after knee joint is placed on and fixes 48 hours in 10% formalin solution, with 4% ethylenediaminetetraacetic acid (EDTA) decalcification 4 weeks, through dehydration, specimens paraffin embedding slices, HE and sarranine-alcian blue dyeing, carry out liver histopathological analysis routinely.Row collagen I I, MMP-13 and p-EGFR SABC detects simultaneously.
5. histological score method:
(1) gonitis scoring: to often organizing the rear inflammatory conditions observing synovial tissue of kneed paraffin section capable HE dyeing.Loose synovial lining cell, the proliferative state of substrate and the gathering of inflammatory cell are as score basis, and there are not above-mentioned 3 factors is 0 grade, is slightly 1 grade, and moderate is 2 grades and be seriously 3 grades.
(2) OARSI marking system: to often organizing the scoring carrying out cartilage after the capable sarranine of kneed paraffin section-alcian blue dyes.Normal cartilage is 0 point; Glycoprotein lose and cartilage surface complete be 0.5 point; Cartilage surface is coarse is 1 point; Loss is organized to be 2 points perpendicular to the crack of cartilage surface or a small amount of softness of any ratio; It is 3 points that cartilage loses 1 – 25% reaching cartilage thickness; It is 4 points that cartilage loses 25 – 50% reaching cartilage thickness; It is 5 points that cartilage loses 50 – 75% reaching cartilage thickness; Cartilage is lost and reached more than 75% of cartilage thickness is 6 points.
6. statistical method
Statistical procedures: continuous data all represents with x ± s, adopts SPSS13.0 statistical analysis software to process.
Compare between many groups and compare employing variance analysis between any two.P<0.01 thinks there is remarkable significant difference between group between two, and P<0.05 thinks there is significant difference between group between two.
7. experimental result
7.1 chitosan sustained-release microsphere features
7.1.1 the form of chitosan microball: 2 kinds of chitosan microballs of preparation are in talking yellow powder.It is spherical that microsphere prepared by scanning electron microscopic observation is class substantially, smooth surface, and size is even, and dispersion better.Chitosan microball (see Fig. 1) and gefitinib/chitosan microball (see Fig. 2), size is similar, and packaging medicine does not affect the form of chitosan, size and dispersion.
7.1.2 the external release of gefitinib/chitosan microball detects
The vitro drug release situation (see Fig. 3) of gefitinib/Chitosan microspheres.In first 60 hours, drug releasing rate is very fast, and then mild gradually, by 96 hours, gefitinib all discharged.
7.1.3 the biocompatibility of chitosan microball and inflammatory score: sham operated rats synovial tissue is normal, without loose lining cell *, matrix components hypertrophy and inflammatory cell infiltration (see A in Fig. 6).Osteoarthritis group (see B in Fig. 6) and chitosan microball group (see C in Fig. 6) lining cell * are loose, stromal cell hyperplasia and inflammatory cell infiltration obvious.In gefitinib/chitosan microball, the synovial tissue of dosage group (see D in Fig. 6) is similar to normal group, no significant difference, and possible reason is the inflammation that gefitinib can suppress synovial membrane.Inflammatory score result display (see Fig. 7), osteoarthritis group and chitosan microball group difference meaningless (P<0.01).Gefitinib/chitosan microball group and osteoarthritis group or chitosan microball group difference meaningful (P<0.01).
7.2 mice knee joint pathology histological indications
7.2.1 mice knee joint p-EGFR ImmunohistochemistryResults Results: sham operated rats (see A in Fig. 8) can observe the positive expression of this receptor, shows that the normal physiological function that it is maintaining cartilage plays a crucial role.The expression of osteoarthritis group (see B in Fig. 8), apparently higher than sham operated rats, shows to make its up-regulated under the pathology of osteoarthritis stimulates, suppresses this receptor may can alleviate the order of severity of osteoarthritis.P-EGFR is expression no significant difference in chitosan microball group (in see Fig. 8 C) with osteoarthritis group, illustrate that the pathological process of chitosan microball to osteoarthritis itself is without intervention effect, also illustrate that chitosan can not aggravate the deterioration of osteoarthritis simultaneously.And the expression of this receptor in gefitinib/chitosan microball group (in see Fig. 8 D) is obviously less than chitosan microball group or osteoarthritis group, simultaneously close to the expression of sham operated rats, the p-EGFR that obviously can be suppressed high expressed in osteoarthritis pathogenic process by chitosan microball slow release gefitinib can be thought.
7.2.2 mice knee joint sarranine-alcian blue dyeing and OARSI appraisal result: this experiment adopts excision anterior cruciate ligament of knee joint to produce osteoarthritis model, the coloration result of osteoarthritis group (see B in Fig. 9) shows obvious cartilage defect, the loss of cellular mast and extracellular matrix, shows that this model can be used for the intervention effect of drugs to osteoarthritis.The performance of chitosan microball group (see C in Fig. 9) is similar to the performance of osteoarthritis group, and OARSI appraisal result (see Figure 10) no difference of science of statistics.Gefitinib/chitosan microball group (see D in Fig. 9) is without cartilage defect, compare with sham operated rats (see A in Fig. 9), although the crack of vertical cartilage surface can be seen, but cartilaginous tissue is obviously better than osteoarthritis group or chitosan microball group, the dosage along with gefitinib increases effect and strengthens (see E and F in Fig. 9).OARSI appraisal result (see Figure 10) shows gefitinib/chitosan microball group and osteoarthritis group or chitosan microball group and has obvious significant difference (P<0.01), shows that gefitinib obviously can suppress the destructive process of osteoarthritis cartilage.
7.2.3 mice knee joint Col II ImmunohistochemistryResults Results: chondrocyte matrix is made up of collagen and chitosan, any factor can destroying its synthesis and structure can cause osteoarthritis.Sham operated rats (see A in Figure 11) can observe the high expressed of cell peripheral collagen, shows that the normal physiological function that it is maintaining cartilage plays a crucial role.The expression of osteoarthritis group (see B in Figure 11) is starkly lower than sham operated rats, shows to make collagen degradation under the pathology of osteoarthritis stimulates.Collagen is expression no significant difference in chitosan microball group (in see Figure 11 C) with osteoarthritis group, illustrate that the pathological process of chitosan microball to osteoarthritis itself is without intervention effect, also illustrate that chitosan can not aggravate the deterioration of osteoarthritis simultaneously.And the expression of collagen in gefitinib/chitosan microball group (in see Figure 11 D) is obviously more than chitosan microball group or osteoarthritis group, simultaneously close to the expression of sham operated rats, the collagen that obviously can be promoted low expression in osteoarthritis pathogenic process by chitosan microball slow release gefitinib can be thought.Testing unique intervention factor due to this is medicine gefitinib, therefore, can think that this medicine can promote the expression of extracellular matrix collagen by suppressing the activated state of EGFR, thus suppress the development of osteoarthritis.
7.2.4 mice knee joint MMP-13 ImmunohistochemistryResults Results: research in recent years shows this digestive enzyme its important function in the generation evolution of osteoarthritis.Sham operated rats (see A in Figure 12) can observe the positive expression of this digestive enzyme, shows that the normal physiological function that it is maintaining cartilage plays a crucial role.The expression of osteoarthritis group (see B in Figure 12), apparently higher than sham operated rats, shows to make its up-regulated under the pathology of osteoarthritis stimulates, suppresses this digestive enzyme may can alleviate the order of severity of osteoarthritis.MMP-13 is expression no significant difference in chitosan microball group (in see Figure 12 C) with osteoarthritis group, illustrate that the pathological process of chitosan microball to osteoarthritis itself is without intervention effect, also illustrate that chitosan can not aggravate the deterioration of osteoarthritis simultaneously.And the expression of this digestive enzyme in gefitinib/chitosan microball group (in see Figure 12 D) is obviously less than chitosan microball group or osteoarthritis group, simultaneously close to the expression of sham operated rats, can think the MMP-13 that obviously can be suppressed high expressed in osteoarthritis pathogenic process by chitosan microball slow release gefitinib, testing unique intervention factor due to this is medicine gefitinib, therefore, can think that this medicine can suppress brokenly the expression of active row factor MMP-13 by suppressing the activated state of EGFR, thus suppress the development of osteoarthritis.
The application of embodiment 3 Erlotinib in Mouse cartilage cell
(1) experiment material
Cell culture: C57BL/6 newborn mice 4 is only provided by Zhejiang University's Experimental Animal Center, newborn C57BL/6 mice, disconnected neck is put to death, bilateral knee articular cartilage is cut under aseptic condition, put into culture dish, for several times, with the soft tissue around microforceps removal articular cartilage and osseous tissue under stereomicroscope, 1 × PBS is rinsing again to immerse rinsing in 1 × PBS.After 2.5g/L trypsinization 15min, again remove excess tissue around articular cartilage.Shred after 1 × PBS rinsing, add 0.1% II Collagenase Type digestion 4h, after digestion, cell suspension is filtered with 150 order stainless steel filtering nets, the centrifugal 5min of 1000r/min, abandon supernatant, add the DMEM/F12 culture medium containing volume fraction 10% hyclone, dual anti-(penicillin 100U/mL, streptomycin 100U/mL), blood counting chamber counts, with 1 × 10 8the density of/L is inoculated in culture bottle, is placed in CO 2(temperature 37 DEG C, saturated humidity, volume fraction 5%CO is cultivated in incubator 2).48h backsight cell attachment situation changes culture fluid, and every couple of days changes liquid 1 time later.Through the chondrocyte in the 1-3 generation obtained of going down to posterity for this experimentation.
Experimental agents is prepared: Erlotinib (Erlotinib, Tarceva, Erlotinib) Shanghai company limited of Roche Group, 25mg Erlotinib sheet is dissolved in aseptic distilled water and is settled to 25ml, concussion mixing after the complete disintegrate of medicine, the centrifugal 15min of 12000rpm also gets supernatant as storage liquid.
Experiment reagent: transforming growth factor TGF-α buys from Sigma company; DMEM/F12 culture medium, trypsin and II Collagenase Type are from Gibco company; Hyclone and PBS are from HyClone company; TRIZOL is from Invitrogen company; R T-PCR test kit and premix Ex TaqTM test kit is from TAKARA company.
Instrument: Thermo Forma CO2 incubator, 3111USA; Low temperature desk centrifuge, 5145R Eppendorf; PCR instrument, Eppendorf; Aseptic operating platform, safe and sound company of Su Jing group; Mx3000P quantitative real time PCR Instrument, Stratagene company.
(2) experimental technique
Cell culture and grouping: chondrocyte adopts DMEM/F12 culture medium culturing to 60% density containing volume fraction 10% hyclone, dual anti-(penicillin 100U/mL, streptomycin 100U/mL) to be used for Experiment intervention in 6 orifice plates.Cell experiment is divided into four groups: be respectively blank group, Erlotinib group, TGF α group and TGF α+Erlotinib group.Blank group is left intact, Erlotinib group only adds 10ng/ml Erlotinib in the medium, TGF α group is the 10ng/ml TGF-α (aseptic double-distilled water preparation) adding 2 μ l in culture medium, TGF α+Erlotinib group 10ng/ml Erlotinib pretreatment adds 2 μ l 10ng/ml TGF-α after 30 minutes cultivates, and detects the collagen II of all cells and the expression of MMP-13 after 48h.
RT-PCR detects: extract the chondrocyte RNA through experiment process with Trizol respectively, and illustrate according to Takara company test kit and carry out reverse transcription and amplification, amplification condition is as follows: 94 DEG C of degeneration 40s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, and last 72 DEG C extend 10min.Experiment repetition 3 times.
(3) experimental result: Erlotinib obviously can reverse the low expression of collagen II caused by TGF-α, and higher than blank group, and simple Erlotinib can not cause the reduction (see Figure 13) of collagen II; Erlotinib obviously can reverse the MMP-13 high expressed caused by TGF-α, and simple Erlotinib can not cause the rising (see Figure 14) of MMP-13.

Claims (9)

1. the application of EGF-R ELISA blocker in preparation treatment medicine for treating arthritis.
2. apply as claimed in claim 1, it is characterized in that described EGF-R ELISA blocker is gefitinib or Erlotinib.
3. apply as claimed in claim 1, it is characterized in that described EGF-R ELISA blocker is applied with the form of EGF-R ELISA blocker/chitosan microball.
4. apply as claimed in claim 3, it is characterized in that described EGF-R ELISA blocker/chitosan microball is prepared as follows: (1) chitosan solution: with glacial acetic acid aqueous dissolution chitosan, be mixed with chitosan solution; (2) emulsification system: add EGF-R ELISA blocker in chitosan solution, mix homogeneously, stirring and emulsifying under 40 DEG C of waters bath with thermostatic control, 800-1000r/min condition, forms emulsification system; The consumption of described chitosan solution is in chitosan mass, and described chitosan and EGF-R ELISA blocker mass ratio are 100:1-800:1; (3) microsphere: add glutaraldehyde in emulsification system, keep 40 DEG C, stir crosslinked, after reaction terminates, leave standstill 30min, after complete layering, remove upper liquid, lower floor's granule uses petroleum ether and washed with isopropyl alcohol respectively, then lyophilization, obtains EGF-R ELISA blocker/chitosan microball.
5. apply as claimed in claim 4, it is characterized in that the volumetric concentration of described glacial acetic acid aqueous solution is 2%.
6. apply as claimed in claim 4, it is characterized in that the mass concentration of chitosan in described chitosan solution is 0.01-0.1g/ml.
7. apply as claimed in claim 4, it is characterized in that described lyophilization is lyophilizing at-109 DEG C.
8. apply as claimed in claim 4, it is characterized in that the volume addition of described glutaraldehyde counts 2.0-15ml/g with the quality of chitosan in step (2) chitosan solution.
9. EGF-R ELISA blocker/chitosan microball, it is characterized in that described EGF-R ELISA blocker/chitosan microball is prepared as follows: (1) chitosan solution: be the glacial acetic acid aqueous dissolution chitosan of 2% by volumetric concentration, be mixed with the chitosan solution of 0.01-0.1g/ml; (2) emulsification system: add EGF-R ELISA blocker in chitosan solution, mix homogeneously, stirring and emulsifying under 40 DEG C of waters bath with thermostatic control, 800-1000r/min condition, forms emulsification system; The consumption of described chitosan solution is in chitosan mass, and described chitosan and EGF-R ELISA blocker mass ratio are 100:1-800:1; Described EGF-R ELISA blocker is gefitinib or Erlotinib; (3) microsphere: add glutaraldehyde in emulsification system, keep 40 DEG C, stir crosslinked, after reaction terminates, leave standstill 30min, after complete layering, remove upper liquid, lower floor's granule uses petroleum ether and washed with isopropyl alcohol respectively, then lyophilization, obtains EGF-R ELISA blocker/chitosan microball; The volume addition of described glutaraldehyde counts 2.0-15ml/g with the quality of chitosan in step (2) chitosan solution.
CN201410414758.XA 2014-08-21 2014-08-21 Application of epidermal growth factor receptor blocking agent to preparation of medicine for treating osteoarthritis Pending CN104257659A (en)

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CN105349644A (en) * 2015-11-06 2016-02-24 林进 Application of TSPAN15 for preparing osteoarthritis diagnostic preparations
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CN105349644A (en) * 2015-11-06 2016-02-24 林进 Application of TSPAN15 for preparing osteoarthritis diagnostic preparations
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