CN101244257A - Application of type II collagen allosteric peptide for treating rheumatoid arthritis with nasal mucosa medicine administration - Google Patents
Application of type II collagen allosteric peptide for treating rheumatoid arthritis with nasal mucosa medicine administration Download PDFInfo
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- CN101244257A CN101244257A CNA2007101798362A CN200710179836A CN101244257A CN 101244257 A CN101244257 A CN 101244257A CN A2007101798362 A CNA2007101798362 A CN A2007101798362A CN 200710179836 A CN200710179836 A CN 200710179836A CN 101244257 A CN101244257 A CN 101244257A
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Abstract
The invention provides a nasal mucosa administration approach using II type collagen altered peptide to cure rheumatoid arthritis, the II type collagen altered peptide nasal mucosa administration specific can inhibit abnormal immune reaction of the rheumatoid arthritis, and control the development of the disease radically aiming at the original tache of disease incidence.
Description
Technical field
The present invention relates to the application of II Collagen Type VI allosteric peptide for treating rheumatoid arthritis with nasal mucosa medicine administration.
One, background technology
Rheumatoid arthritis (RA) be a kind of serve as the general autoimmune disease of main performance with chronic aggressive arthropathy.Prevalence in China is 0.34%, and there are nearly 5,000,000 patients in the whole nation, and disability rate is up to 93%.This sick pathogenic process is a kind of process that is subjected to antigen to drive " exciting a chain immunoreation ".The immunologic injury of infectant and autoimmune response mediation is the basis that the rheumatoid arthritis morbidity and the state of an illness develop.Antigen polypeptide is expressed by intravital antigen presenting cell, and activation T lymphocyte, cause inflammatory mediators such as release, immunoglobulin, chemotactic factor and the free radical generation of cytokine to increase, and then cause vasculitis, synovial hyperplasia, the characteristic pathological change of rheumatoid arthritis such as cartilage and osteoclasia.
At present, still there is not the medicine of fundamentally controlling rheumatoid arthritis.Clinically mainly by respite symptoms such as NSAID (non-steroidal anti-inflammatory drug) such as ibuprofen and diclofenacs.Slowly acting on antirheumatic such as methotrexate and leflunomide etc. then is that DNA is synthetic extensively to suppress immunoreation by suppressing, and then suppresses arthritis.Therefore, these medicines are not the initial link that acts on morbidity in treating rheumatoid arthritis, are difficult to control targetedly pathological changes, cause whole body and arthropathy constantly to make progress, and finally disable.And, since the extensive immunosuppressive action of these medicines, bone marrow depression, and untoward reaction such as abnormal liver function are more, cause a lot of patients can not accept these Drug therapys.
The inventor can be in order to II Collagen Type VI (CII) the allosteric peptide of treatment rheumatoid arthritis through having discovered for many years.Studies have shown that CII allosteric peptide can suppress HLA-DR β 1 molecule and T cell to antigenic identification and the autoimmune response that causes therefrom, checks the generation and the development of primary disease from the key link of rheumatoid arthritis morbidity.Among the application CII allosteric peptide is carried out treating rheumatoid arthritis with nasal mucosa medicine administration.
Two, summary of the invention
The nasal mucosa medicine administration approach that the purpose of this invention is to provide CII allosteric peptide treatment rheumatoid arthritis.
Term used herein " CII allosteric peptide " refer in the CII263-272 polypeptide any one or a plurality of aminoacid modify after still can with HLA-DR β 1 bonded polypeptide.The method that is used for modified amino acid is well known to those skilled in the art.
Among the present invention applied CII allosteric peptide mainly be alanine (A) with short-side chain replace continuously TXi Baoshouti in the CII263-272 polypeptide (TCR) recognition site and and the relevant specific amino acids of HLA-DR β 1 affinity, synthetic many CII263-272 allosteric peptides of design, allosteric peptide and HLA-DR β 1 molecule have affinity, but are not discerned by TXi Baoshouti.
Mutual identification between HLA-DR β 1, antigen polypeptide and TCR three molecules and combination are the key links of rheumatoid arthritis abnormal immune reaction.CII allosteric peptide can the competitive inhibition pathogenic antigens effect and the T cell to antigenic identification, check the intravital abnormal immune reaction of rheumatoid arthritis patients, thereby the progress of control morbidity and pathological changes reaches the purpose of fundamentally treating rheumatoid arthritis.
Verified, the 70-74 amino acids among the HLA-DR β 1 contains one section common sequences QK/RAA (being Gln-Lys/Arg-Arg-Ala-Ala, glutamine-lysine/arginine-arginine-Ala-Ala)
(1), this section common sequences is relevant with the formation of HLA-DR beta 1-6 antigen engagement groove, is the function amino acid of its conjugated antigen.The 71st positively charged in this sequence amino acids (Lys or Arg) that studies have shown that in a large number of the inventor and Wucherfennig etc. is the bonded key position of antigen
(2-7)
By X-ray diffraction technology discovering to HLA-DR β 1-antigen dimer crystalline texture, the multiple antigenic peptides that HLA-DR β 1 (DR4/DR1) molecule relevant with rheumatoid arthritis combines is all extremely similar in configuration, comprising degeneration II Collagen Type VI (CII) and heat shock protein etc.
(8-10)From the stereochemical structure (Fig. 1) of these peptides as seen, the side chain of Phe263 (P1), Glu266 (P4) and Gly271 (P9) stretches to HLA-DR β 1 molecule in left side, in all or part of embedding antigen engagement groove.Other amino acid whose side chain then stretches to a side or (TXi Baoshouti) direction opposite with HLA-DR β 1, stimulates the T cell activation.The P1 of CII polypeptide, P4, P9 side chain embed in " pocket " of HLA-DR β 1 conjugated antigen as seen from Figure 2.The 71st amino acids (Lys71) positively charged on electronegative P4 (Glu) and the HLA-DR β 1 is adjacent, forms the polar bond of high-affinity.Therefore, Glu266 may be the key amino acid of CII polypeptide in conjunction with DR β 1.Research afterwards proves that further CII mainly combines with TXi Baoshouti by Gln267, Gly268, Pro269 and Lys270, causes t cell activation.This shows that main and HLA-DR β 1 bonded aminoacid are Phe263 in the CII polypeptide, Gly265, Glu266 and Gly271, Glu272, and mainly and the bonded aminoacid of TXi Baoshouti be Gln267, Gly268, Pro269, Lys270 (table 1).
The table 1.CII in TXi Baoshouti (TCR) and HLA-DR β 1 bonded aminoacid
*F=Phe (phenylalanine), K=Lys (lysine), G=Gly (glycine), E=Glu (glutamic acid),
Q=Gln (glutamine), P=Pro (proline), R=Arg (arginine), A=Ala (alanine)
Based on above research basis, the inventor replaces in the CII polypeptide with alanine (A) or glycine (G) and reaches and the relevant specific amino acids of HLA-DR β 1 affinity with the TCR combination, has synthesized many CII allosteric peptides.But these CII allosteric peptide specificitys are in conjunction with HLA-DR β 1, and affinity increases, and the competitive inhibition autoantigen combines with HLA-DR β's 1.Thereby suppress by the autoimmune response of HLA-DR β 1 mediation and the pathological processes such as inflammatory mediator release that cause thus.
The zoopery in our early stage finds that CII allosteric peptide subcutaneous injection and intravenous injection all can suppress the arthritis of experimental arthritis, alleviates the damage of its radiology and pathology.But drug administration by injection inconvenience in clinical practice is also uneconomical.The via intranasal application administration has advantages such as absorption is rapid-action, bioavailability is high, easy to use.Therefore, in embodiments of the invention, the outer the strongest peptide sequence of inhibitory action: the FKGEAGPAAE of our preferred body.Use nasal mucosa medicine administration approach treatment experimental arthritis rat model, and obtain good therapeutic effect, prompting CII allosteric peptide can pass through nasal mucosa medicine administration, thereby the activation of T cell is suppressed, and reaches treatment rheumatoid arthritis purpose.
Description of drawings
Fig. 1 .CII polypeptide and HLA-DR β 1 bonded crystal structure.The side chain of the P1 of CII polypeptide, P4 and P9 embeds the antigen of HLA-DR β 1 in conjunction with in " pocket ".Lys on P4 (Glu, electronegative) and the HLA-DR β 1
71(positively charged) forms the polar bond point (Immunity 7:473-81,1997) of high-affinity.
Fig. 2 .CII allosteric peptide nasal mucosa medicine administration is to the inhibitory action of CIA rat arthritis.CII allosteric peptide treatment group arthritis score be starkly lower than PBS group (
*And CII prototype peptide treatment group arthritis score and PBS group does not relatively have obvious significant difference (P＞0.05) P＜0.05).
The influence that Fig. 3 .CII allosteric peptide nasal mucosa medicine administration changes the CIA rat body weight.Allosteric peptide treatment group rat body weight increasing degree will be higher than PBS treatment group and prototype peptide treatment group (P＜0.05).
The treatment of Fig. 4 .CII allosteric peptide is to the influence of CIA pathology damage.Visible significantly articular cartilage of prototype peptide treatment group (b) and PBS treatment group (c) and bone erosion and joint space narrow down, and the rarely seen inflammatory cell infiltration of allosteric peptide treatment group (a).
Fig. 5 .CII allosteric peptide is to the influence of CIA rat anti CII IgG antibody and hypotype thereof.The allosteric peptide can reduce anti-CIIIgG2a type antibody titer (P＜0.05), and antagonism CII IgG and IgG1 type antibody titer do not make significant difference (P＞0.05).
Further specify the present invention by the following examples, but the scope that the following example does not limit the present invention in any way.
The specific embodiment
Embodiment 1: the design of polypeptide and synthetic
Aforesaid research work
(4-7)Verified, the 267-270 amino acids mainly combines with TXi Baoshouti in the CII polypeptide, and activated T cell.In the test of the present invention at first solid phase method synthesized CII263-272 prototype peptide and used alanine and replace corresponding amino acid whose CII allosteric peptide (table 2).For the absorbance, the availability that improve polypeptide and heighten the effect of a treatment, the aminoterminal of every peptide is linked to each other with the tetradecanoic acid gene in synthetic, in order to polypeptide to intracellular transport.Used decapeptide all contains 4 above hydrophilic residues in this research, as lysine (K) and glutamic acid (E), is easy to dissolving and absorption.These peptides do not contain methionine (M), tryptophan (W) or easy deamination, the decarboxylized aspartic acid (N) etc. of easy degraded.Therefore, the good stability of peptide, and the effect that is easy to enter its interference of performance T cell recognition in the cell.
The design of table 2. non-T cell conjugating peptide
The F=phenylalanine, K=lysine, G=glycine, E=glutamic acid, Q=glutamine, A=alanine, P=proline
Studies show that the binding ability of above-mentioned CII263-272 allosteric peptide and HLA-DR β 1 strengthens, and the activation capability of T cell significantly is lower than CII 263-272 prototype peptide, and can suppress the t cell immune response that CII263-272 stimulates.
Embodiment 2: CII allosteric peptide nasal mucosa medicine administration of the present invention is to the inhibitory action of experimental arthritis
In causing arthritic immunoreation, antigen-presenting cell is by the antigen induction t cell activation, and impel the propagation of T cell, cause that intravital CII antibody of animal and cytokine (for example IFN-γ) level increases, and then cause the characteristic pathological change of rheumatoid arthritis.So, can understand non-T cell conjugating peptide to arthritic inhibitory action by the level of the intravital cytokine of test experience animal.Simultaneously, also can make comprehensive judge to curative effect of medication by pathological section from histological angle.
In zoopery scheme of the present invention, with the collagen induced arthritis (CIA) that cattle CII induces the Lewis rat, with CII allosteric peptide rats with arthritis is treated then, find that the allosteric peptide can obviously suppress rat arthritis, the arthroncus degree is alleviated, (seeing part as a result).
(1) foundation of experimental arthritis animal model:
Experimental arthritis model among the present invention has adopted internationally recognized collagen induced arthritis (CIA) model.Laboratory animal is an inbred line Lewis rat.This experiment is all raised in The People's Hospital of Peking University zoopery center (SPF level) with rat, and rat is closed in the polystyrene cage that is lined with wood wool, keeps 12 hours the daytime/circulate, make it and can freely drink water and pickuping food night.The rat that is used to test is the 6-8 female rats in age in week, body weight 120 ± 10g.The whole programs that relate to animal in the experiment are all undertaken by China's management of laboratory animal orderliness.
Buy cattle II Collagen Type VI (lot number: C1188) from Sigma company, dissolve the solution that is made into 4mg/ml, the isopyknic complete freund adjuvant of reuse (Sigma, the U.S.) emulsifying with anhydrous glacial acetic acid, inject 150 μ l emulsions in the root of the tail portion of every Lewis rat, injected dose is 300 μ l/.This Success in Experiment has been set up the arthritis model of Lewis rat.
(2) to arthritic assessment:
Inducing the back with CII 12-14 days, ankle joint and interphalangeal joint swelling appear in 70% rat.With the level evaluation arthritis of keeping the score of 0-16, every of four claws is kept the score by 0-4, wherein 0 minute=joint does not have redness; 1 minute=joint is slightly red and swollen; 2 minutes=joint moderate redness; 3 minutes=joint severe redness; 4 minutes=joint severe swelling and can not bearing a heavy burden, or joint deformity appears.The joint scoring addition in morbidity joint, best result is 16 minutes.
(3) CII allosteric peptide nasal mucosa medicine administration is to the treatment of CIA rat:
The arthritis performance appearred in 12-14 days in the Lewis rat behind initial immunity, have 21 rats morbidities.The rat of morbidity is divided into 3 groups, 7 every group at random.Begin to give respectively allosteric peptide, prototype peptide and PBS nose the same day in falling ill and splashed into treatment.Allosteric peptide and prototype peptide dosage are 100 μ g/ time (polypeptide is dissolved in PBS, and concentration is 2.5mg/ml), every day 1 time, and administration next day that successive administration changing into after 5 days, administration is 12 times altogether.Matched group gives nose at every turn and splashes into PBS 40 μ l.Observe the variation of rat body weight every day, and make arthritis score to arthritic joint number takes place with aforesaid arthritis appraisal procedure.Initial immunity was put to death animal after 38 days, left and took peripheral blood, and the ELISA method detects the variation of anti-CII-IgG antibody and hypotype thereof, detected the concentration of the respective fine intracellular cytokine in its serum with IFN-γ and IL-10ELISA test kit (Bender company, Austria); Simultaneously, get the back joints of foot, fixing, HE dyeing is done in decalcification, section.
(4) use the SPSS software kit and do statistical analysis.
(5) result: CII allosteric peptide nasal mucosa medicine administration of the present invention is to the inhibitory action of experimental arthritis
With CII 30 female Lewis rats are carried out arthritic inducing, arthroncus in various degree appearred in 21 rats in the time of the 12nd day to the 14th day as a result.Appearring the arthritic same day, with rat at random be divided into 3 groups, give allosteric peptide, prototype peptide and PBS nose respectively and splash into treatment, and mark to arthritic joint number takes place with aforesaid arthritis appraisal procedure.
The result shows that arthritis score did not have significant difference between each was organized before the treatment.The treatment of allosteric peptide organizes that the rat arthroncus alleviates gradually in 2 weeks after treatment, and the arthroncus of prototype peptide treatment group and PBS matched group increases the weight of gradually.Experiment finishes row back, back ankle joint pathological examination, and the result shows that prototype peptide and PBS treatment group rat ankle joint exist tangible synovial hyperplasia and pannus to form, and articular cartilage and subchondral bone matter are destroyed obviously, and the severe patient articular cavity disappears, and ankylosis occurs.And allosteric peptide treatment group the rat ankle joint only inflammatory cell infiltration and the synovial hyperplasia of degree of taking a favourable turn, articular cartilage and subchondral bone matter are destroyed not obvious.The allosteric peptide significantly reduces the titre of anti-CII IgG antibody 2a hypotype in the CIA rat body, and reduces gamma interferon (IFN-γ) level in the serum.These results prove that the treatment of CII allosteric peptide nasal mucosa medicine administration can alleviate the autoimmune inflammation of CIA rat, suppresses the arthritic degree and the course of disease, and this polypeptide nasal mucosa medicine administration may have therapeutical effect to rheumatoid arthritis.
Table 3. is respectively organized changes of cytokine in the serum of rat treatment back
*Compare with matched group, CII allosteric peptide can reduce IFN-γ level (P＜0.05) in the CIA rat blood serum, and to the not obviously influence (P＞0.05) of IL-10 level.
Sequence table
＜110〉The People's Hospital of Peking University
＜120〉application of II Collagen Type VI allosteric peptide for treating rheumatoid arthritis with nasal mucosa medicine administration
<140>
<141>2007-11-14
<160>2
<170>PatentIn?version?3.1
<210>1
＜211〉10 (aminoacid)
＜212〉peptide
＜213〉artificial sequence
＜223〉synthetic
<400>1
Phe?Lys?Gly?Glu?Gln?Gly?Pro?Lys?Gly?Glu
<210>2
＜211〉10 (aminoacid)
＜212〉peptide
＜213〉artificial sequence
＜223〉synthetic
<400>2
Phe?Lys?Gly?Glu?Ala?Gly?Pro?Ala?Ala?Glu
Claims (3)
1. replace in the CII263-272 peptide sequence with alanine (A) or glycine (G) and reach the specific amino acids relevant and synthetic CII allosteric peptide with HLA-DR β 1 affinity with the TCR combination.
2. the application of the CII allosteric peptide of claim 1 in the nasal mucosa medicine administration dosage form of preparation treatment rheumatoid arthritis.
3. the CII allosteric peptide nasal mucosa medicine administration of claim 1 is in the application of treatment rheumatoid arthritis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107073076A (en) * | 2014-11-11 | 2017-08-18 | 株式会社日皮 | Immune activation agent, cellular immunity activator and T cell multiplication agent |
| CN115671253A (en) * | 2021-07-30 | 2023-02-03 | 河北菲尼斯生物技术有限公司 | Application of SE-DR affinity peptide in preparing medicine for treating rheumatic diseases |
-
2007
- 2007-12-19 CN CNA2007101798362A patent/CN101244257A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107073076A (en) * | 2014-11-11 | 2017-08-18 | 株式会社日皮 | Immune activation agent, cellular immunity activator and T cell multiplication agent |
| CN115671253A (en) * | 2021-07-30 | 2023-02-03 | 河北菲尼斯生物技术有限公司 | Application of SE-DR affinity peptide in preparing medicine for treating rheumatic diseases |
| CN115671253B (en) * | 2021-07-30 | 2024-02-27 | 河北菲尼斯生物技术有限公司 | Application of SE-DR affinity peptide in preparation of medicines for treating rheumatism |
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Open date: 20080820 |