CN1697647A - Liquor and method for restraining ache, inflammation and cartilage degradation - Google Patents

Liquor and method for restraining ache, inflammation and cartilage degradation Download PDF

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Publication number
CN1697647A
CN1697647A CNA038031361A CN03803136A CN1697647A CN 1697647 A CN1697647 A CN 1697647A CN A038031361 A CNA038031361 A CN A038031361A CN 03803136 A CN03803136 A CN 03803136A CN 1697647 A CN1697647 A CN 1697647A
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cartilage
guiding
chondroprotective agents
delivery system
chondrolysis
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G·A·德莫普洛斯
P·P·帕尔默
J·M·赫尔兹
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Omeros Corp
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Omeros Medical Systems Inc
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Abstract

Methods and compositions for inhibiting articular cartilage degradation. The compositions preferably include multiple chondroprotective agents, including at least one agent that promotes cartilage anabolic activity and at least one agent that inhibits cartilage catabolism. The compositions may also include one or more pain and inflammation inhibitory agents. The compositions may be administered systemically, such as to treat patients at risk of cartilage degradation at multiple joints, and suitably may be formulated in a carrier or delivery vehicle that is targeted to the joints. Alternatively the compositions may be injected or infused directly into the joint.

Description

Be used for inhibition of pain, the solution of inflammation and cartilage degradation and method
The cross reference of related application
The application requires the U.S. Provisional Application No.60/353 of submission on February 1st, 2002,552 priority, U.S. Provisional Application No.60/353,552 is the U. S. application No.10/031 that submitted on January 18th, 2002,546 continuation is partly applied for, U. S. application No.10/031,546 is American National phase application of the international application No.PCT/US00/19864 of appointment U.S. of submitting on July 21st, 2000, international application No.PCT/US00/19864 requires the U.S. Provisional Application No.60/144 of submission on July 21st, 1999,904 priority, U.S. Provisional Application No.60/144, the 904th, the U. S. application No.09/839 that submit to April 20 calendar year 2001,633 continuation is partly applied for, U. S. application No.09/839,633 is that the continuation of the international application No.PCT/US99/26330 of appointment U.S. of submitting on November 5th, 1999 is partly applied for, international application No.PCT/US99/26330 requires the U.S. Provisional Application No.60/107 of submission on November 5th, 1998, the priority of the international application No.PCT/US99/24625 of the appointment U.S. that on October 20th, 256 and 1999 submitted to, international application No.PCT/US99/24625 requires the U.S. Provisional Application No.60/105 of submission on October 20th, 1998,026 priority requires the interests of these priority at this according to 35 USC 119 (e) and 120 parts.
Invention field
The present invention relates to be used to protect the therapeutic combination and the method for articular cartilage.
Background of invention
Cause the disease and the situation of intraarticular cartilage destruction to cause significant publilc health concern, consider that particularly the demographic class hour of aged tendency of population is all the more so.Articular cartilage has been represented the complication system of many different moleculars.In arthritis, relate to number of mechanisms in the articular cartilage degeneration as rheumatoid arthritis (RA) and osteoarthritis (OA).OA is a kind of non-inflammation arthritis, and it is the most common form of joint disease, and is the deputy factor that causes early retirement and deformity that is only second to cardiovascular disease.Some individualities show OA in single or a limited number of joint, for example may be because traumatic damage unexpected or operation.Many other individualities are owing to the relevant wearing and tearing of old and feeble or secular motion or vocational activity and tear and stand multiarticulate OA.RA is the most common form of inflammatory arthritis, has influenced 3% women and 1% male.Most of RA patients have the little joint of multi-joint symptom, particularly hands, elbow, wrist and shoulder.
The destruction of transparent articular cartilage is the sign of OA and disabling property RA.Although many Therapeutic Method can provide the alleviation of symptom, do not prove that also any therapeutic scheme can stop the progress of articular cartilage degeneration.The progressive failure of articular cartilage has caused the irreversible impaired of joint motions with losing.These changes of articular cartilage are osteoarthritis (OA) and the common final pathology affair of rheumatoid arthritis (RA).
The cartilage destruction process may be relevant with the surgical operation in joint or initial by it.Arthroscopy is a kind of surgical procedures, and it passes a little otch that covers in top skin and the joint capsule, will insert with the photographing unit that the far-end light source links to each other with video-frequency monitor in the joint (for example, knee joint, shoulder etc.) on the anatomy.Pass similar otch, also surgical operating instrument can be placed in the joint, under the guidance of arthroscope radiography, use.Because the raising of arthroscopy technical staff level, more and more surgical procedures that once undertaken by " open " surgery operating technology, the method for present available arthroscopy is finished.Such operation comprises that for example, kneed part meniscectomy and ligament are rebuild, the acromioplasty of shoulder and rotator muscle running-on debridement and synovectomy of elbow.Because the expansion of surgery indication and the exploitation of minor diameter arthroscope, the arthroscopy of wrist and ankle have also become conventional inspection.
In the process of arthroscopy each time, with the continuous lavation joint of physiology irrigating solution (for example normal saline or lactated Ringer's solution), make the joint capsule expansion, remove the operation fragment, thereby intraarticular radiography more clearly is provided.Marshall's United States Patent (USP) 4,504,493 discloses a kind of equimolar glycerine water solution, and it is a kind of non--conductivity and clear and bright irrigating solution of optics that is used for arthroscopy.Traditional physiology irrigating solution can not provide analgesia, the effect of antiinflammatory or anti-cartilage degradation.
Summary of the invention
The invention provides a kind of being used for reduces or prevents the destructive method and composition of joint articular cartilage, comprises the combination of the Chondroprotective agents of using two or more metabolic activities.The metabolic activity agent comprises, but be not limited to, directly or indirectly effect and regulate or change the chemical compound of biology, biochemistry or the biophysics state of cell, the reagent that comprises the current potential that changes cytoplasma membrane, the part combination or the enzymatic activity of cell receptor, the interior or localized enzyme in extracellular of cell, protein-protein interaction, RNA-protein-interacting, or DNA-protein-interacting.In one aspect of the invention, provide pharmaceutical composition based on the metabolic activity Chondroprotective agents of at least two kinds of combination of agents that act on the different molecular target simultaneously.
Representative Chondroprotective agents comprises, for example: (1) il-1 protein family comprises IL-1 β for example, the receptor antagonist of IL-17 and IL-18; (2) tumor necrosis factor (TNF) receptor family comprises, for example, and the antagonist of TNF-R1; (3) agonist of interleukin 4,10 and 13 receptors; (4) TGF-beta receptor superfamily comprises, BMP-2 for example, the antagonist of BMP-4 and BMP-7; (5) inhibitor of COX-2; (6) map kinase family comprises, for example, and the inhibitor of p38 map kinase; (7) matrix metalloproteinase (MMP) protein family comprises, for example the inhibitor of MMP-3 and MMP-9; (8) NF-kB protein family comprises, for example with the dimeric inhibitor of the compound p50/p65 of I κ B; (9) nitricoxide synthase (NOS) family comprises, for example the inhibitor of iNOS; (10) agonist of integrin receptor and antagonist, for example, α vβ 3The agonist of integrin; (11) inhibitor of Protein kinase C (PKC) family; (12) protein tyrosine kinase family comprises, for example the inhibitor of src subfamily; (13) regulator of Protein-tyrosine-phosphatase; (14) inhibitor of albumen src homology 2 (SH2) domain.Other Chondroprotective agents comprises other somatomedin, as insulin like growth factor (as IGF-1) and fibroblast growth factor (as bFGF).
In a kind of preferred embodiment, at least a reagent is cytokine or the growth factor receptors agonist that anti-inflammatory activity directly is provided and/or promotes the cartilage anabolic process, be also referred to as " anabolic agent " herein, at least a second reagent is to suppress the chondrolysis metabolic process and also can suppress short scorching process, is also referred to as the receptor antagonist or the enzyme inhibitor of " chondrolysis metabolic poison " or " catabolism inhibitor " herein.The term of Shi Yonging " Chondroprotective agents " comprises anabolic agent and chondrolysis metabolic poison herein.
In this embodiment of the present invention, at least a Chondroprotective agents is antiinflammatory/anabolism cytokine, and its function is to prevent the effect of proinflammatory cytokine in the joint, promotes that cartilage matrix is synthetic and suppresses substrate degradation.These receptor stimulating agents comprise, for example, specific antiinflammatory and anabolism cytokine, as interleukin (IL) agonist (for example, IL-4, IL-10 and IL-13) and the transforming growth factor-beta superfamily (for example, TGF β and BMP-7) the specificity member, insulin like growth factor (for example, IGF-1) and fibroblast growth factor (for example, bFGF).At least the second kind of Chondroprotective agents is selected from class inhibition and (for example reduces short scorching molecule target activity or the receptor antagonist of expressing or enzyme inhibitor; the IL-1 receptor antagonist; TNF-α receptor antagonist; cyclooxygenase-2 inhibitor; map kinase inhibitor; nitricoxide synthase (NOS) inhibitor, and nuclear factor κ B (NF κ B) inhibitor).Second kind of Chondroprotective agents also can be selected from and suppress the metabolic matrix metalloproteinase of chondrolysis; suppress the metabolic cell adhesion molecule of chondrolysis; comprise integrin agonist and integrin antagonist; suppress signal transmission inhibitor in the metabolic cell of chondrolysis; the inhibitor that comprises inhibitors of protein kinase C and protein tyrosine kinase inhibitor and the metabolic SH2 domain of inhibition chondrolysis.
Articular cartilage is a kind of extracellular matrix of specialization, and it is by the articular chondrocytes generation of metabolic activity and keeps.The extracellular matrix of normal health keep the dynamic equilibrium between mixing of reflection biosynthesis speed and matrix components, and their deterioration velocity and afterwards from the cartilage to the synovial fluid in loss between dynamic equilibrium.Yet, the regulatory mechanism of matrix body homeostasis not fully aware of also, they very clearly change in IJD and reply articular trauma, so that the destruction speed of substrate surpasses the synthesis rate of new matrix components.Usually, think that the homeostasis of substrate represented the dynamic equilibrium between catabolic cytokine and anabolic cytokine (comprising somatomedin) effect.The best of breed that is used for the therapeutic agent of cartilage protection passes through to promote synthesis rate, suppresses to destroy speed simultaneously and moves dynamic substrate balance, thereby make anabolic process reach maximum, and promote to repair.
Catabolic cytokine works to the specific receptor on the chondrocyte as IL-1 β and TNF-α, produces to induce MMPs, MMPs wherein can induce the degeneration of substrate, and degenerate is by anabolic cytokine, and as TGF-β, BMP-2 and IGF-1 suppress.Therefore, not best for repair of cartilage only, because also need anabolic agent to induce or promote the biosynthesis and the assembling of substrate generation composition based on the Therapeutic Method that suppresses catabolic process (as the combination of MMP inhibitor and IL-1 antagonist).The second, cause the destructive catabolism cytokine of cartilage matrix (IL-1, TNF, IL-17, IL-18, LIF) multiformity shows that the activity of all catabolism cytokines of blocking-up is unpractical.On the contrary, only rely on and use anabolic agent, as IGF-1, the method for BMP-2 or BMP-7 is not best, and this is because it can not resist the counter regulation effect of catabolism cytokine.TGF-β, BMP-2 and IGF-1 also can act on specific receptor, produce matrix components to induce chondrocyte, and it is by IL-1 β, TNF α, IL-17 and LIF suppress.Therefore, the optimal treatment combination that is used for cartilage protection is made up of at least a anabolic agent and the metabolic inhibitor of a kind of chondrolysis.
In one aspect of the invention, use multiple Chondroprotective agents for patient by the whole body approach with articular cartilage degeneration danger.The plurality of reagents that general is used comprises the reagent and the metabolic reagent of at least a inhibition chondrolysis of at least a promotion cartilage anabolic activity.Every kind of reagent all is capacity, so that when sending the joint that is delivered to the patient with solution, provides effectively combination of treatment, thereby suppresses the chondrolysis metabolic process and promote the cartilage anabolic process.In addition, can use the reagent of one or more inhibition of pain and/or inflammation with Chondroprotective agents.When the patient had the dangerous of degenerative joint simultaneously or suffers from degenerative disorders in a plurality of joints, it was can be preferred that the general of multiple Chondroprotective agents is used.
In order to make unfavorable or unwanted general action minimum, aspect of the embodiment of systemic delivery of the present invention, a kind of therapeutic strategy is to send to pass combination of agents in carrier that is directed at the joint or delivery vectors.In a kind of embodiment of preferred guiding; at least a anabolism Chondroprotective agents and/or at least a catabolism inhibition Chondroprotective agents; and preferred anabolism Chondroprotective agents and catabolism inhibition Chondroprotective agents; can be encapsulated in delivery vectors, in nanosphere.Guiding antibody or antibody fragment are coupled to nanosphere.Antibody or antibody fragment are specific to and are positioned at the IA antigenic determinant that is directed to.A kind of Therapeutic Method of the present invention comprises the compositions to these one or more Chondroprotective agents that are directed to, seal of patient's systemic administration with cartilage degradation danger, preferably by in the blood vessel, intramuscular, subcutaneous or suck and to use.
In another aspect of this invention, provide the compositions that general is used that is used for that comprises multiple Chondroprotective agents, wherein comprised the reagent and the metabolic reagent of at least a inhibition chondrolysis of at least a promotion cartilage anabolic activity.In addition, one or more reagent of inhibition of pain and/or inflammation can be included in the compositions.The dosage of all reagent that comprise all is enough to when systemic administration to provide in the joint cartilage protection therapeutical effect.Also provide preparation to comprise the method for the medicine of the described compositions that is used for the treatment of the patient that cartilage degradation danger is arranged.
For the compositions that described general is used is directed at the joint; at least a anabolism Chondroprotective agents and/or at least a catabolism inhibition Chondroprotective agents; and preferred anabolism Chondroprotective agents and catabolism inhibition Chondroprotective agents; be encapsulated in delivery vectors; in nanosphere, described nanosphere is coupled to specific antibody or the antibody fragment that is positioned at IA antigenic determinant.Also provide a kind of preparation to be used for the treatment of the method for the patient's that cartilage degradation danger is arranged medicine; described medicine comprises the Chondroprotective agents of sealing; described Chondroprotective agents is coupled to antibody or antibody fragment, and described antibody or antibody fragment are directed at and are positioned at IA antigenic determinant.
At different aspect of the present invention; can prepare in the pharmacy effective carrier comprise a kind of or preferred multiple metabolic activity Chondroprotective agents and one or more are used for the reagent of inhibition of pain, inflammation etc.; or the more preferably combination of the plurality of reagents of anabolic agent and catabolism inhibitor, be used for directly being delivered to patient's joint at intraarticular.Although the systemic delivery of cartilage protection compositions of the present invention is preferred for the disease or the situation in a plurality of joints of influence, in other cases, the part of compositions of the present invention is sent and passed can be preferred.Described situation can comprise that treatment suffers from the cartilage degradation character condition that only influences simple joint or minority joint or the patient of disease, peri-operation period is used operation or the relevant program of intervention with the joint, or works as unwanted side effect and can use under the relevant situation with general.This part of the present invention send pass aspect, described compositions is (to comprise and be used for the treatment of the cartilage degradation disease by intra-articular injection, as osteoarthritis or rheumatoid arthritis) or through infusion and local sending passed, the peri-operation period (that is, before operation, in the operation and/or operation back) that is included in surgery arthroscope program is used.
This part of the present invention is sent and is passed the aspect a kind of solution is provided, and it is formed in the mixture of the plurality of reagents of the low concentration in the physiology electrolysis carrier fluid, is used for the medium of local inhibition of pain, inflammation and cartilage degradation.The present invention also provides a kind of being used for directly to send the method that is delivered to operative site at peri-operation period with the irrigation solution that contains these reagent, described solution acts locally on receptor and enzyme level at operative site, so that at this position restriction pain, inflammation and cartilage degradation.Because local peri-operation period of the present invention send the method for passing, can use other to send the reagent of the dosage of the method for passing (that is, intravenous, intramuscular, subcutaneous and oral) to reach required curative effect to be lower than.
Anti-pain in the solution and/or antiinflammatory and/or anti-cartilage degradation agent comprise the reagent that is selected from polytype receptor antagonist and agonist and enzyme activator and inhibitor, and every type is to suppress and/or the molecular matrix of cartilage degradation works by different pain and/or inflammation.
Except anti-cartilage degradation agent, compositions of the present invention can comprise anti-pain and/antiinflammatory.Be used for the representative reagent of inhibition of pain and/or inflammation, comprise, for example (1) 5-hydroxytryptamine receptor antagonist; (2) 5-hydroxytryptamine receptor agonist; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitor; (6) tachykinin receptor antagonists comprises neurokinin 1And neurokinin 2The receptor subtype antagonist; (7) calcitonin-gene-related peptide (CGRP) receptor antagonist; (8) interleukin receptor antagonist; (9) inhibitor of activated enzyme in the route of synthesis of arachidonic acid metabolite comprises (a) phosphatase inhibitors, comprises PLA 2Isoform inhibitor and PLC isoform inhibitor, (b) cyclooxygenase-2 inhibitor and (c) lipoxygenase inhibitor; (10) prostaglandins receptor antagonist comprises eicosanoid EP-1 and EP-4 receptor subtype antagonist and thromboxan receptor subtype antagonist; (11) LTRA comprises leukotriene B 4Receptor subtype agonist and leukotriene D 4The receptor subtype agonist; (12) opioid receptor agonist, comprise μ-, δ-and κ-opiate receptor subtype agonist; (13) P 2The purinoceptor antagonist comprises P 2XReceptor antagonist and P 2YReceptor antagonist; (14) calcium-channel antagonists.Above-mentioned each reagent is all as antiinflammatory and/or as anti-injury (promptly anti-pain or analgesia) agent.Selective reagent need be adjusted according to specific application from the chemical compound of these types.
The present invention also provides a kind of method for preparing medicine, the mixed in one aspect of the invention dilution irrigating solution that is made into of this medicine, and for arthroscopy operating period, the wash one's hands joint part at art position, particularly patient of continuous irrigation uses.Send in this part of the present invention and to pass in the embodiment, this method need be dissolved at least a anti-cartilage degradation agent and preferred one or more anti-pain/antiinflammatories in the physiological electrolyte carrier fluid, for the application of some anti-cartilage degradation agent, the concentration of every kind of reagent preferably is no more than about 100,000nM, more preferably no more than about 25,000nM, and be most preferably not exceeding about 10,000nM.
Part of the present invention send the method for passing the aspect can be at inhibition of pain, during the treatment or diagnostic procedure of inflammation and cartilage degradation, directly provides the sending of dilution combination of multiple receptor antagonist and agonist and enzyme inhibitor and activator to pass to wound or operative site.Because this solution active component is the local in a continuous manner operation tissue that directly applies to, therefore with same medicine oral, produce the required dosage of therapeutical effect when intramuscular, subcutaneous or intravenous administration and compare, these medicines can effectively use by extremely low dosage.Terminology used here " part " speech comprises medicine in wound or other operative sites and application on every side, get rid of oral, subcutaneous, intravenous and intramuscular administration.Terminology used here " continuously " comprises with frequent interval, use incessantly, use repeatedly, and remove short interruption and use with uninterruptedly using introducing other medicines or preparation or the operating equipment, like this, can be at wound or the local predetermined concentration that keeps substantial constant of operative site.
The advantage that the reagent low dosage is used has three.The most important is usually not limit the systemic side effects of these medicinal applications.Secondly, in the solution of the present invention, the reagent of selecting for special applications has high degree of specificity to its medium and mediation target that plays a role of relying.This species specificity is to rely on used hanging down to be kept to amount of reagent.Last point, the cost of these active agents in each operation process is very low.
These reagent through the advantage of lavation or other fluid mode topicals are: (1) topical can guarantee the concentration known of target site, and no matter metabolism between the patient, the variability of blood flow etc.(2) because be the mode of passing of directly sending, can obtain to treat concentration immediately, like this, improve for the control of amount of reagent.(3) the active agent part directly is applied to wound or operative site, can reduce these medicines basically by the caused degraded of whole body process (crossing and two metabolism excessively as head), if these medicines are with oral, intravenous, subcutaneous, or the intramuscular administration, then this degraded may appear.This situation is especially suitable to those albumen and peptide class active agent, and this class reagent can be by rapid metabolism.Therefore, topical can use the chemical compound or the reagent that can not adopt other administrations in treatment.For example, some reagent of following kind of apoplexy due to endogenous wind are peptides: bradykinin receptor antagonists; Tachykinin receptor antagonists; Opioid receptor agonist; The CGRP receptor antagonist; Interleukin receptor antagonist; The TNF receptor antagonist; TGF-beta receptor agonist; BMP-2 and BMP-7 receptor stimulating agent; IL4, IL10 and IL-13 receptor stimulating agent; With integrin receptor agonist and antagonist.Although that part of reagent that needs continuous supplementation to be degraded, guaranteeing being enough to keep receptor share or the saturated topical therapeutic concentration of enzyme is kept all the time in the whole surgery process, the part of wound or operative site is sent continuously and is passed the degraded or the metabolism that can make medicine and reach minimum degree.
According to this respect of the present invention, this solution of peri-operation period local application can produce preferential analgesia, antiinflammatory and cartilage protection effect in whole surgical procedures.Term used herein " peri-operation period " speech comprises in the process to be used, and crosses use in conjunction in Cheng Qian and the process, use in conjunction behind the process N-process, and cross Cheng Qian, use in conjunction behind the process N-process.In order farthest to obtain preferential antiinflammatory, analgesia (for some application), and cartilage protection (for some application) effect were preferably in the Cheng Qian, all used solution of the present invention behind the process N-process.By before tangible local operation wound stimulation occurring, occupy the target receptor or make target enzyme inactivation or activation, the special approach of reagent scalable in the solution of the present invention is with preferential inhibition target pathology process.If preferentially suppressed them before inflammatory mediator and process generation histologic lesion, its advantage is compared with the administration after the damage beginning so, and is more remarkable.
Verified, by using the pain that solution that the present invention contains plurality of reagents suppresses more than one, inflammation or cartilage degradation medium can obviously reduce inflammation and the degree of pain, and the cartilage protection effect ought to be provided.Irrigating solution of the present invention comprises the combination of medicine, and every kind of solution all can act on multiple receptor or enzyme.Like this, the combination that multiple medicine can resist pathological process simultaneously effectively comprises pain and inflammation, and the equilibrated forfeiture of cartilage.Effect that it is generally acknowledged these reagent is synergitic, and this is that multiple receptor antagonist of the present invention and inhibition agonist use with combining form, and the effect of comparing disproportionate increase with the effect of single agents is provided.The synergism of the several reagent of the present invention is discussed by embodiment, describes those reagent below in detail.
Irrigating solution with respect to present use, peri-operation period uses solution of the present invention should cause operative site pain and inflammation clinically, reach obviously alleviating of cartilage degradation, reduce behind the corrective surgery demand thus to analgesic (as opiate), in due course, can make patient's operative site movable as early as possible.As for surgeon and operation room work personnel aspect, compare with traditional irrigating solution, do not need extra effort just can use solution of the present invention.For the best cartilage protection of this respect of the present invention, before the surgical operation, during and/or directly use solution of the present invention afterwards to the joint.
In another aspect of this invention, provide the compositions that comprises anabolism promoter and catabolism inhibitor that is used to protect cartilage.Like this, described combination has caused a kind of state, it is characterized in that: the cartilage anabolic activity that equals or exceeds the chondrolysis metabolic activity; Keep cartilaginous tissue so that keep existence, or increase the cartilage volume; Perhaps increase the degraded that also reduces cartilage matrix by the synthetic cartilage matrix of articular cartilage simultaneously.
The accompanying drawing summary
Now will be by embodiment, and, set forth the present invention in more detail with reference to corresponding accompanying drawing, wherein:
Fig. 1 is the graphic summary of chondrocyte, and it has shown that the molecule target and the signal that cause inflammatory mediator generation and cartilage metabolism to move transmit flow of information.External signal is by many cell surface receptors family, comprise cytokine receptor, as il-1 (IL-1) receptor family and tumor necrosis factor (TNF) receptor family, TGF-beta receptor family and integrin and the integration carried out are assembled on the common intracellular signal pipeline of major protein group of molecules comprising, protein molecular wherein is treatment target (map kinase, the PKC that is included in the medicine in the solution of the present invention, tyrosine kinase, SH2 albumen, COX, PLA2 and NF-6B).The chondrocytes expressed of the many inducible genes products of activation may command of these signal pipelines, comprise IL-1, TNF-α, IL-6, IL-8 and stromelysin (MMP-3), and other medium (nitric oxide (NO) and PGE2), it can cause inflammation and/or cartilage degradation, or the propagation of the synthetic and chondrocyte of substrate molecule.
Fig. 2 provides synovial cell's graphic summary, and its demonstration causes inflammatory mediator produces and cartilage metabolism changes molecule target and signal to transmit flow of information.External signal is by the family of many cell surface receptors, comprise cytokine receptor, as il-1 (IL-1) receptor family and tumor necrosis factor (TNF) receptor family, g protein coupled receptor, as Kallidin I, histamine and 5-hydroxy tryptamine hypotype, the integration of carrying out with integrin is assembled on the common intracellular signal pipeline of major protein group of molecules comprising, protein molecular wherein is the treatment target (map kinase that is included in the medicine in the solution of the present invention, PKC, tyrosine kinase, SH2 albumen, COX, PLA2 and NF-6B).The synovial cell of the many inducible genes products of activation may command of these signal pipelines expresses, and comprises IL-1, TNF-α, and IL-6, IL-8 and stromelysin (MMP-3), it can cause inflammation and/or cartilage degradation.
Fig. 3 is chondrocyte and synovial cell's common signal pipeline figure, and it comprises is responsible for causing inflammation and/or cartilage degradation, the signal of interest albumen of " cross (talk) (crosstalk) " between proinflammatory cytokine approach and GPCR activated receptor approach.
Fig. 4 is chondrocyte and synovial cell's common signal pipeline figure, and it comprises the signal of interest albumen of being responsible for " cross (talk) " between proinflammatory cytokine approach and GPCR activated receptor approach.Identified in the preferred chondrocyte protection of the present invention solution the specific molecular site of some drugs effect.
Fig. 5 is the figure that is present in molecule target among chondrocyte or the synovial cell, and it promotes the anabolic reaction of cartilage.Identified in the preferred cartilage protection solution of the present invention the specificity site of some drugs effect.
Fig. 6 is the figure that is present in chondrocyte or synovial cell's molecule target, and it promotes the catabolic reaction of cartilage.Identified in the preferred cartilage protection solution of the present invention the specificity site of some drugs effect.
Fig. 7 be with il-1 (IL-1,10U/ml) excite whole night after, regulate the graphical representation of the PGE2 that agonist produces in the synovial fluid culture by G-albumen.With histamine (100 μ M, hollow post), or Kallidin I (1 μ M, solid post) stimulates the specified number of times of this culture, is discharged into PGE2 in the culture supernatants according to embodiment 6 described mensuration.Shown value is the mean+SD that derives from representative test, and with respect to baseline PGE2 that unprovoked culture produced and calibrate.
Fig. 8 suppresses the graphic representation that PGE2 produces in the synovial fluid culture by ketoprofen.Under (representing with ■) being arranged or do not have situation that the ketoprofen of (with " △ " or " " expression) prescribed concentration participates in, excite this culture whole night with IL-1 (10U/ml).After one day, in the culture supernatants of handling whole night with ketoprofen, measure PGE2, clean remaining culture, ketoprofen with prescribed concentration was cultivated 10 minutes, then under the continued presence of specified amount ketoprofen, measurement is replied and is used histamine (100 μ M subsequently,) or Kallidin I (1 μ M, △) 3 minutes PGE2s that produce of invasion and attack, data presented is normalized to respectively available from the maximization of each agonist and replys, and is expressed as the mean+SD from three experiments of several different cell lines.
Fig. 9 is under the situation that the G-of IL-1 that prescribed concentration is arranged and interpolation protein-coupled receptor ligand participates in, and the 16th hour, ketoprofen was to the graphical representation of the IL-6 effect that produced by the synovial fluid culture.Be with or without under the 0.75 μ M ketoprofen participation situation, have following additional receptors ligand: 1) 1.0 μ M activate the isoproterenol (ISO) of cyclic adenosine monophosphate approach, or 2) in the test somatomedin of one of histamine (HIS) of 100 μ M activation IP3/ calcium approach, IL-1 with prescribed concentration (0.3,1.0 and 3.0pg/ml) cultivated this culture 16 hours.Collect culture supernatants, replaced once with the fresh medium aliquot that contains identical agonist additive every 8 hours.Then handle, collection is equivalent to processing in every 8-16 hour supernatant medium once then, and analyzes IL-6.
Detailed description of the preferred embodiments
The invention provides the method and composition that is used to protect cartilage.In first kind of embodiment, provide a kind of in the joint local application comprise the method for the first kind of reagent and the metabolic second kind of combination of agents thing of at least a inhibition chondrolysis of at least a promotion cartilage anabolic activity.In the first aspect of the embodiment of the present invention, described compositions is to send by injection said composition and part in the joint to pass, and described compositions comprises a kind of delivery vectors of lasting release.In the second aspect of the embodiment of the present invention, described compositions comprises liquid lavation carrier, and send in the peri-operation period part in operation or intervention program and be delivered in the joint.
In second kind of embodiment of the present invention, provide a kind of and used the first kind of reagent comprising at least a promotion cartilage anabolic activity and the method for the metabolic second kind of combination of agents thing of at least a inhibition chondrolysis to patient's general.
In the third embodiment of the present invention, provide a kind of and used the first kind of reagent comprising at least a promotion cartilage anabolic activity and/or the method for the metabolic second kind of combination of agents thing of at least a inhibition chondrolysis, at least a joint that is directed in the wherein said reagent to patient's general.Before describing these embodiments in detail, without wishing to be bound by theory, set forth according to cartilage protection principle of the present invention.
I. the ultimate principle of cartilage protection
Nearest progress in the biochemistry of inflammation and cartilage destruction and molecular biology are understood hints the effect of many endogenous cell factors.The multiple pro-inflammatory mediator that participates in the forfeiture of generation cartilage in inflamed joints is cytokine, TNF-α, IL-1, IL-6 and IL-8.The level that these proinflammatory cytokine numbers raise appears in the knee joint synovial fluid of acute injury fast, and the level that should raise continues at least 4 week (Cameron in the patient, M.L. wait the people, Synovialfluid cytokine concentrations as possible prognosticindicators in the ACL-deficient knee ", Knee Surg.SportsTraumatol.Arthroscopy 2:38-44 (1994)).These cytokines are produced by several activatory cell types in the joint partly, comprise synovioblast, synovial membrane macrophage and chondrocyte.Pathophysiology activity in the local cytokine mediated acute and chronic inflammation situation that produces, and be metabolic important autocrine of chondrolysis and paracrine medium.These effect of cytokines are characterised in that they cause the ability of multiple action to different cellular targets and they are with positive coorperativity or negative cooperative mode and the interactional ability of other cytokines.IL-1 and TNF-α are particular importances, because they are the initial cartilage destruction effect by destroying balance between the homergy also, and the activity of the tissue depressant (TIMP) by regulating intrinsic protein (as matrix metalloproteinase (MMP)) and metalloproteases and the destruction of initial cartilage matrix composition.The balance of the height regulation and control between the active medium that chondrocyte is worked has been represented in the cytokine control of cartilage homeostasis, and this decision is the generation substrate degradation or repairs.
Joint injury often produces the inflammatory reaction of involving synovial tissue in the joint space, and can cause the degraded of articular cartilage.Rapid change in human knee joint synovial membrane and the cartilage metabolism is described (Cameron, people such as M.L. see above (1994) in joint injury and arthroscope operation; Cameron, M.L. wait the people, " The natural history of theanterior cruciate ligament-deficient knee:Changes insynovial fluid cytokine and keratan sulfate concentrations ", Am.J.Sports Med.25:751-754 (1997)).Sharply increase in the knee joint synovial fluid in specific proinflammatory cytokine level being seen acute inflammation stage after anterior cruciate ligament (ACL) breaks (reaching 2-4 the order of magnitude).Because matrix metalloproteinase (MMP) is created in the also significant change of generation in the cartilage matrix concentration as the excess of collagenase and stromelysin-1, (Lohmander raises in this concentration patient's behind acute injury the synovial fluid, L.S. wait the people, Temporal patterns of stromelysin-1 tissue inhibitor, andproteoglycan fragments in human knee joint fluid after injuryto the cruciate ligament or meniscus ", J.Orthopaedic Res.12:21-28 (1994)).Variation in cytokine relevant with cartilage degradation and the cartilage matrix labelling (as Dan Baijutang) temporarily is maximum, but the time period of sustainable prolongation (3 months-1 year) slowly reduces and still is higher than and damages preceding baseline values.
Wound owing to arthroscope operation self can cause significant post-operation inflammatory, and this has reflected the extra struvite activation of cell in the joint, comprises the rise of cyclo-oxygenase-2 and other proinflammatory cytokines.Patient with disruptive remarkable ratio of ACL (60-90%) shows among the 10-15 that after damage the radiography in the knee joint indication of osteoarthritis (OA) changes (Cameron, people such as M.L. see above (1994)).Thereby, the change in the inflammatory conditions that initial joint injury and the compound action of operation wound can be inducing sustained and the relevant cartilage matrix metabolism, it seems that this be to cause subsequently in articular cartilage development to be degenerated changing and the causative factor of osteoarthritis early-stage development.The magnitude of this health problem is basic, and this is because the 1996 overall estimate numbers of being only the arthroscope program of carrying out in the U.S. are exactly 1,800,000, and has annual 10% estimated growth rate approximately.Thereby, the degraded of a kind of pharmaceutical methods with articular cartilage in the prevention joint need be provided.
Although postoperative is painful and inflammation is considered to significant clinical problem, present arthroscope operating medicine is learned therapeutic scheme and is only related to the acute postoperative analgesia.Existing operative treatment mode does not solve the inductive chronic inflammatory condition of postoperative and to the needs of the cartilage destruction in the joint that suppresses operative treatment.Therefore, clearly need to develop a kind of effective integration pharmacotherapy, this therapy will solve the acute and chronic aspect of painful and inflammation, and the pathological change of cartilage metabolism in that damage and joint operative treatment.
First embodiment of this aspect according to the present invention; providing a kind of send compositions in the medicine effective carrier of passing to reduce or prevents the destructive method of articular cartilage by directly using intraarticular to patient's joint; said composition comprises that a kind of Chondroprotective agents of or preferred multiple metabolic activity suppresses the reagent of misery and/or inflammation together with one or more; as previously described; perhaps be preferably the combination of the Chondroprotective agents of two or more metabolic activities, at least a of the anabolic process of at least a promotion cartilage of this reagent and this reagent is the inhibitor of chondrolysis metabolic process.The reagent of metabolic activity comprises, but be not limited to chemical compounds that all directly or indirectly regulated or changed biology, biochemistry or biophysics's situation of cell, comprise the part that changes plasmalemma potential, cell receptor in conjunction with or enzymatic activity, cell in or the reagent of the localized enzyme in extracellular, protein-protein interaction, RNA-protein-interacting or DNA-protein-interacting.For example, this reagent can comprise in the receptor stimulating agent of initial signal transduction cascade, the receptor antagonist that suppresses signal transduction pathway, the cell or the activator of exoenzyme and inhibitor and adjusting transcription factor and the bonded reagent of DNA.
Suitable Chondroprotective agents comprises as (1) il-1 protein family, comprises IL-1 β for example, the receptor antagonist of IL-17 and IL-18; (2) tumor necrosis factor (TNF) receptor family comprises, for example, and the antagonist of TNF-R1; (3) agonist of interleukin 4,10 and 13 receptors; (4) TGF-beta receptor superfamily comprises, BMP-2 for example, the antagonist of BMP-4 and BMP-7; (5) inhibitor of COX-2; (6) map kinase family comprises, for example, and the kinase whose inhibitor of p38MAP; (7) matrix metalloproteinase (MMP) protein family comprises, for example the inhibitor of MMP-3 and MMP-9; (8) NF-kB protein family comprises, for example with the dimeric inhibitor of the compound p50/p65 of I κ B; (9) one nitricoxide synthase (NOS) families comprise, for example the inhibitor of iNOS; (10) agonist of integrin receptor and antagonist, for example, α vβ 3The agonist of integrin; (11) inhibitor of Protein kinase C (PKC) family; (12) protein tyrosine kinase family comprises, for example the inhibitor of src subfamily; (13) regulator of Protein-tyrosine-phosphatase; (14) inhibitor of albumen src homology 2 (SH2) domain.Be used for other suitable Chondroprotective agents of the present invention and comprise other somatomedin, as insulin like growth factor (as IGF-1) and fibroblast growth factor (as bFGF).
First embodiment of the present invention provides uses the local materia medica method of sending combined therapy joint damage or operative treatment of the Chondroprotective agents of passing, to realize maximum treatment benefit.Second embodiment of the present invention provides the combination of using Chondroprotective agents by general that the materia medica method of treatment is provided.The application of Chondroprotective agents combination has overcome the limitation of prior treatment method; should existing Therapeutic Method depend on and use single reagent, the change between the synthetic and degraded of catabolic process had taken place to help in this process to block polyfactorial cartilage destruction process.The compositions and methods that this aspect of the present invention utilizes combination simultaneously the different molecular target to be worked uniquely; with the anabolism that promotes cartilage with suppress imbalance or over-drastic chondrolysis metabolic process; with realization the maximum of inflammatory process is suppressed and keep the stable state of cartilage, thereby realize the cartilage protection effect in the joint.The inhibition of individual molecule target or known mechanism of inducing cartilage destruction (catabolism), as inhibition to il-1 (IL-1) and IL-1 receptors bind, may not the suitableeest, this be because, for example, TNF-α shares many eclipsed short inflammation and chondrolysis metabolic function by its unique receptor-mediated effect and IL-1, and TNF-α also is considered to the main medium of cartilage destruction in the joint.Similarly, when not having the inhibition catabolic process, utilize the pharmaceutical agent that only strengthens the cartilage anabolic process not resist the suitablelyyest be present in the catabolism factor of damaging in the joint.
Specifically, one aspect of the present invention provides the Chondroprotective agents pharmaceutical composition of metabolic activity, and said composition is based at least two kinds of combination of agents that simultaneously different molecule targets worked.In a representative embodiment, at least a reagent is cytokine or the growth factor receptors agonist that anti-inflammatory activity directly is provided and/or promotes the cartilage anabolic process, and at least the second kind of reagent is receptor antagonist or the enzyme inhibitor that suppresses short inflammation and/or chondrolysis metabolic process.The representative drugs compositions comprises at least a reagent of taking from anti-inflammatory/anabolism cytokine kind, and this reagent suppresses the effect of proinflammatory cytokine in the joint on function, promotes that cartilage matrix is synthetic and suppresses substrate degradation.These receptor stimulating agents comprise, but be not limited to specific anti-inflammatory and anabolism cytokine, as special member (as TGF β and BMP-7), insulin like growth factor (as IGF-1) and the fibroblast growth factor (as bFGF) of interleukin (IL) agonist (as IL-4, IL-10 and IL-13) and transforming growth factor-beta superfamily.At least a kind of taking from receptor antagonist or enzyme inhibitor of second kind of reagent, this receptor antagonist or enzyme inhibitor suppress or reduce the active or expression of short scorching molecule target (as IL-1 receptor antagonist, TNF-α receptor antagonist, cyclooxygenase-2 inhibitor, map kinase inhibitor, nitric oxide synthetase (NOS) inhibitor and nuclear factor κ B (NF κ B) inhibitor).The reagent of metabolic activity comprises the functional agonist and the antagonist of the receptor that is positioned at cell surface, and the inhibitor of the enzyme of membrane-bound or cell exocrine (as stromelysin and collagenase).In addition, many reagent needles comprise that to the new target as localized enzyme and transcription factor in the cell of transduction and integration surface receptor signal the inhibitor of enzyme NOS, COX-2 and mitogen-activated protein kinase (MAPK) and protein-dna interact as the inhibitor of transcription factor NF κ B.This method makes it possible to by promoting the anabolic process that cytokine drives simultaneously and suppressing the integrity that catabolic process is kept cartilage.
Compositions in the preferred embodiment of the invention has constituted a kind of new Therapeutic Method by making up the multiple materia medica reagent that not isoacceptor and/or enzyme molecule target are worked.Up to now, the materia medica strategy concentrates on the medicine of developing high specific, and indivedual receptor subtypes and enzyme isoforms that this medicine conducts the reaction of neurotransmitter and hormone for mediation to individual signal have selectivity.
In addition, although single receptor subtype or enzyme deactivation, the activation of other receptor subtypes or enzyme and the signal conduction of gained as a result often can trigger the cascade effect.This has explained and has used single receptor-specific medicine to block significant difficulty in the pathophysiological processes that multiple signal transmitting medium (as cytokine, somatomedin or eicosanoid) works in this process.Therefore, it may be invalid only being directed at specific indivedual receptor subtypes or isotype.
Opposite with the standard method of materia medica treatment, the Therapeutic Method of this compositions is based on following ultimate principle, promptly the medicine that simultaneously different molecule targets is worked to be combined in the activity that suppresses four corner in the development of pathophysiology situation be highly effective.In addition, specific receptor subtype is opposite with only being directed at, and compositions comprises the medicine (referring to Fig. 1) that is directed at the general molecular mechanism that works in the different cytophysiology processes that comprise in misery, inflammation and cartilage degradation evolution.The cascade of extra receptor and enzyme is minimized.In these pathophysiology approach, compositions suppresses the cascade of " upstream " and " downstream ".
The example that " upstream " suppresses is the cyclo-oxygenase antagonist in painful and inflammation are recovered.Cyclo-oxygenase (COX 1And COX 2) conversion of arachidonic acid is to the struvite and transformation biosynthetic intermedium PGH of nocuity medium, this medium comprises prostaglandin, leukotriene and thromboxane.The cyclooxygenase-2 inhibitor blocking-up forms these struvite and " upstreams " the nocuity medium.This strategy has been got rid of interactional needs between the prostaglandins product of the hypotype of 7 kinds of descriptions of blocking-up prostaglandin receptoroid and COX bio-chemical pathway.Similarly " upstream " inhibitor is to press down enzyme peptide, promptly a kind of kallikrein inhibitor.Kallikrein is a kind of serine protease, and high-molecular weight kininogen is with generation Kallidin I, the i.e. important medium of misery and inflammation in the cutting blood plasma.By suppressing kallikrein, press down the enzyme peptide and suppressed the synthetic of Kallidin I effectively, thereby provide effective " upstream " to these inflammatory mediators to suppress.
Compositions of the present invention also can be used " downstream " inhibitor with control pathophysiology approach.Used in being contained in gradual articular cartilage degeneration among the synovial cell and chondrocyte preparation of multiple inflammatory cytokine (as IL-1 β and TNF α) processing, map kinase inhibitor has produced the cartilage protection effect.The p38 map kinase is the transportation point in the signal transduction path of multiple catabolism cytokine, and its inhibition has prevented the rise of the cellular products of multiple mediation cartilage degradation.Therefore; map kinase inhibitor provides significant advantage by " downstream " cartilage protection effect is provided in the recovery of arthritis, should " downstream " cartilage protection effect do not rely on physiology's combination of cytokine receptor agonist of the cartilage homeostasis of initial change.
II. the part of cartilage protection compositions is sent and is passed
The particular preferred embodiment that the present invention is used for the solution of cartilage protection and operative procedure comprises the combination of agents that simultaneously the different molecular target is worked; with the anabolism that promotes cartilage with suppress imbalance or over-drastic chondrolysis metabolic process; with realization the maximum of inflammatory process is suppressed and keep the stable state of cartilage, thereby realize the cartilage protection effect in the joint.
Lavation of one embodiment of this invention and injection-type solution are a kind of or preferred multiple Chondroprotective agents and randomly one or more miseries and/or the dilute solution of inflammation inhibition reagent in physiology's carrier.This carrier is a liquid solution, be used to comprise biocompatible solvent, suspension, aggretion type and non-polymeric type gel, paste and ointment herein, and the delivery vectors that continues release, as microparticle, spherula or the nano-particle of forming by albumen, liposome, saccharide, synthetic organic compound or inorganic compound.Carrier preferably can comprise the electrolytical aqueous solution of physiology, as normal saline or newborn acidifying Ringer's solution.
Send in each surgical solution of passing embodiment in part of the present invention, realizing for necessary concentration of desired therapeutic effect and the dosage, reagent is included in liquid or the fluid solution with low concentration and carries out the part with low dosage and send and pass with respect to the systemic medication application process.As used herein, " liquid " or " fluid " is intended to comprise pharmaceutically acceptable biocompatible solvent, suspension, aggretion type and non-polymeric type gel, paste and ointment.Carrier preferably can comprise the electrolytical aqueous solution of physiology, as normal saline or newborn acidifying Ringer's solution.Send by other drug route of administration (being intravenous, subcutaneous, intramuscular or oral) reagent of passing similar dosage with the therapeutic effect that obtains to be equal to be impossible or unpractiaca, this be because the medicine that general is used can carry out that head crosses and two metabolism.The concentration of each reagent can be based in part on its receptor dissociation constant K dOr enzyme suppresses constant K iDetermine.As used herein, the term dissociation constant be intended to comprise for its separately agonist-receptor or the equilibrium dissociation constant of antagonist-acceptor interaction and for its separately activator-enzyme or the balance of inhibitor-enzyme interacting suppress constant.Preferably with 0.1-10,000 takes advantage of K dOr K iLow concentration comprise each reagent.Preferably with 1.0-1,000 takes advantage of K dOr K iConcentration comprise each reagent, and most preferably about 100 take advantage of K dOr K iConcentration comprise each reagent.Send in the part and to pass the site when not having metabolic conversion, can adjust with the explanation dilution these concentration as required.Selection is used for the accurate reagent of solution and the concentration of reagent changes according to specific application, and is as mentioned below.
Solution according to an aspect of the present invention can comprise single or multiple Chondroprotective agents; be preferably multiple Chondroprotective agents; wherein at least a be the anabolism Chondroprotective agents and at least a be the chondrolysis metabolic poison, comprise that perhaps two kinds of Chondroprotective agents of low concentration and misery and/or inflammation suppress combination of agents.Yet, because the synergism of aforementioned plurality of reagents and the needs that extensively suppress cartilage destruction and randomly blocking-up misery and inflammation are preferably used plurality of reagents.
Multiple drug regimen can carry out the part and send and pass by intra-articular injection or by inculcating, be included in the operation arthroscope program separately or pass sending of link coupled contiguous program with sending of continuing of postoperative and pass (being before the art and/or in the art and/or postoperative), as by the pump regulated or use and continue the delivery vectors that discharges.The delivery vectors that continue to discharge can include, but are not limited to microparticle, spherula or the nano-particle be made up of albumen, liposome, saccharide, synthetic organic compound or inorganic compound.Thereby, in some embodiments, the invention provides separately or with analgesia and/or anti-inflammatory agent through injection or inculcate and send the agent combination of passing.By in when damage or tightly (as peri-operation period) directly send and pass quick initial effect that Chondroprotective agents realizes and had before the initial program triggering is with afterreaction its potentiality that suppress in part thereafter, thereby local tissue damage and the forfeiture of cartilage have subsequently preferentially been limited.
Advantage of the present invention comprises: the multifactorial disease that 1) in acute and/or chronic condition, is directed at cartilage destruction because of the composition of medicine treatment; 2) Chondroprotective agents combination can with anti-inflammatory and analgesia agent combination; 3) partial the sending of drug regimen (applicable) passed the instantaneous treatment concentration that has realized Chondroprotective agents in the joint; 4) (applicable) contiguous program use irrigating solution with the required scope of treatment in the arthroscope operative procedure in the joint, provide levels of drugs continue keep; 5) (for this embodiment of the present invention) part is sent to pass and is compared the minimizing that allows in the total drug dose and the frequency of taking medicine with systemic delivery; 6) (for this embodiment of the present invention) is directed at sending of localized site to the joint and passs and avoided general toxicity and reduced side effect; With 7) (for this embodiment of the present invention) arthrotropic direct part send and passs peptide and the albumen that makes it possible to use new pharmaceutically active, comprising cytokine and somatomedin, may not be that treatment is gone up useful if this peptide and albumen are limited to the general route of administration.
Because molecule and cell mechanism to the effect that these Chondroprotective agents limited; when in irrigating solution (with the combination of other Chondroprotective agents or with other anti-painful and anti-inflammatory agent combinations described herein) carry out Perioperative Application of Artificial or in addition through inculcating or inject when directly being applied to the joint, these chemical compounds are expected can show the cartilage protection effect.Particularly, these reagent are when send when passing to expect it is effective medicine by irrigating solution in the arthroscope operative procedure.The Chondroprotective agents of each metabolic activity all can be preferably send with one or more other Chondroprotective agents combinations and is delivered in the joint space, comprises small-molecule drug, peptide, albumen, reorganization chimeric protein, antibody, oligonucleotide or gene therapy vector (virus with non-virus).For example, a kind of medicine such as MAPK inhibitor can be to the relevant cell of the fluid gap any and joint and the structure that comprises the normal function that participates in the joint or present joint owing to pathological conditions play a role.These cells and structure include, but are not limited to: the synovial cell comprises A type fibroblast and Type B macrophage; The cartilaginous element in joint such as chondroblast and chondrocyte; Cell with bone photo closes comprises periosteum cell, osteocyte, osteoblast, osteoclast; Inflammatory cell comprises lymphocyte, macrophage, mastocyte, mononuclear cell, eosinophilic granulocyte; With other cells, comprise endotheliocyte, smooth muscle cell, fibroblast and neurocyte; And above-mentioned combination.
This aspect of the present invention also provides the preparation of active therapeutic agent, this active therapeutic agent can strengthen that sending of Chondroprotective agents be passed, taken in, stability or pharmacokinetics be used for medicine introduced or the preparation that is applied to the joint send and passs.This preparation can include, but are not limited to microparticle, spherula or the nano-particle be made up of albumen, liposome, saccharide, synthetic organic compound or inorganic compound.The invention provides the combination of Chondroprotective agents or a kind of or preferred multiple Chondroprotective agents and one or more anti-miseries and/or sending of anti-inflammatory agent and pass, they exist as mixture (as one group of spherula of sealing one or more reagent) discrete in multiple pharmaceutically active substance in the homogeneous carrier (as the single spherula of sealing) or the single delivery vectors.The example of preparation molecule includes, but are not limited to hydrophilic polymer, polycation (as protamine, spermidine, polylysine), the peptide that material can be directed at particular cell types or synthetic part and antibody, gel, slow release property substrate (promptly continuing to send carrier, solubility and the insoluble granule passed) and unlisted preparation composition.
On the one hand; the invention provides the combination of the combination of two or more Chondroprotective agents or a kind of or preferred multiple Chondroprotective agents and one or more anti-miseries and/or anti-inflammatory agent, send by irrigating solution and the part of containing the preserved material of medicine individually or with the combination of one or more anti-miseries and/or anti-inflammatory agent and pass, this irrigating solution and preserved material are delivered to ill tissue or joint to treat effective low concentration and exist and make medicine directly to send.Contain the preserved material of medicine or irrigating solution can be before art and/or in the art and/or postoperative use with operative procedure, or can use with the irrelevant other times of operative procedure.Medicine send the general method (as intramuscular, intravenous, subcutaneous) of passing the medicine (with higher accumulated dose) of higher concentration need be applied to the patient to realize significant treatment concentration in target joint.General is used also can cause high concentration in the tissue except that target joint, this is unexpected, and depends on dosage and can cause deleterious side effect.These general methods make medicine carry out two and cross metabolism and degraded fast, thus the persistent period of having limited effective treatment concentration.Because Chondroprotective agents combination (having or do not have one or more anti-misery and/or anti-inflammatory agent) is directly by inculcating or lavation is applied to the joint, so need not vascular perfusion to take medicine to target tissue.This significant advantage makes it possible to carry out sending than low part for the treatment of effective accumulated dose of multiple Chondroprotective agents and passs.
A. the method for passing is sent in the part
Solution of the present invention can be applicable to multiple operation/interference program, comprises operation, diagnosis and treatment technology.The combination of Chondroprotective agents of the present invention can be used by injection or lavation.For injection solution, can will depend on the characteristic of active agent in the patient that will treat, the solution and specific method of application with the amount of the active component that produces single dosage form with carrier material combination and change.Yet, the given dose level for any particular patient that is appreciated that will depend on multiple factor, the activity that comprises applied specific compound, patient's age, body weight, general health, sex and diet, the time of using, the approach of using, the seriousness of excretory speed of drug regimen and the specified disease for the treatment of.
Injection-type preparation such as aseptic injection type are moisture or contain oil suspension and can use suitable dispersant or wetting agent and suspending agent preparation according to technique known.Aseptic injection type preparation also can be aseptic injection type solution or the suspension in atoxic parenteral acceptable diluent or the solvent, as the solution in the propylene glycol.In carrier and the solvent water, Ringer's solution and isoosmotic sodium chloride solution arranged applicable acceptance.In addition, aseptic fixedly oil is routinely as solvent or suspension media.For this purpose, the oil of any biocompatible be can use, synthetic monoglyceride or diglyceride comprised.In addition, fatty acid such as oleic acid also can be used in the preparation of injection.
Injection of the present invention can be used with the arthroscope operation, perhaps can use in any time that the doctor who instructs patient care needing to be defined as.
Irrigating solution of the present invention can be used by peri-operation period in the arthroscope operation in anatomy joint.As used herein, term " peri-operation period " comprises before application in the program, the program and the application in the program, program in and before the application and program after the program, in the program and the application after the program.This solution is preferably used before program and/or after the program and in the program.This program is utilized physiology's lavation fluid routinely, and as normal saline or newborn acidifying Ringer's solution, this fluid is applied to surgical site by the well-known technology of those skilled in the art.Method of the present invention comprises the lavation fluid that substitutes conventional application with anti-misery/anti-inflammatory of the present invention/cartilage protection irrigating solution.Irrigating solution was applied to wound or surgical site before program is initial, preferably before tissue injury, and the duration of running through program continuously, with painful and inflammation and cartilage degradation of blocking-up preferentially.As general used herein, term " lavation " means with flow of liquid lavation wound or anatomical structure.Term " application " is intended to comprise lavation and other local methods of introducing solution of the present invention, is incorporated into surgical site as the solution with gel form, makes gel solution remain on this site then in whole procedure.As general used herein, term " continuously " is also intended to comprise following situation, wherein there is frequency to repeat and frequent lavation wound with the predetermined treatment local concentration that is enough to keep agents useful for same, and comprise following application, wherein have the intermittence of the essential lavation fluid flow of surgical technic to stop.
Be when not having metabolic conversion, to send the reagent concentration of passing above, to realize exposure level predetermined on the surgical site to the surgical site part to the listed concentration of each reagent in the solution of the present invention.Be appreciated that to regulate with explanation the drug level in the given solution and send dilution when passing.The solution concentration in the above-mentioned embodiment is not regulated with explanation metabolic conversion or the dilution that distributed by total health, this is that these situations of having avoided of passing are sent in the part because use on the contrary with oral, intravenous, subcutaneous or intramuscular.
The arthroscope technology that can adopt solution of the present invention comprises reconstruct, acromioplasty, rotor cover debridement, synovectomy of elbow and the wrist and the ankle joint endoscope of part meniscus and ligament in the knee joint with nonrestrictive example.Irrigating solution can be seen the intraarticular that does not block to be enough to the making expansible flow velocity of joint capsule be applied to the joint continuously in art so that the residue of removing operation is also feasible.
In this arthroscope technology, provide among the suitable arthroscope irrigating solution embodiment 1-3 hereinafter of inhibition cartilage degradation and control misery and inflammation.
Send in each solution of passing this invention is intended to the part, realizing for necessary concentration of desired therapeutic effect and the dosage, reagent is included in the solution with low concentration and carries out the part with low dosage and send and pass with respect to the systemic medication application process.Sending the reagent of passing similar dosage by other drug route of administration (being intravenous, subcutaneous, intramuscular or oral) is impossible with the therapeutic effect that obtains to be equal to, this is to cross metabolism because the medicine that general is used can carry out head mistake and two, and often removes from systemic circulation fast.
When finishing arthroscope or " opening " joint (as knee joint, shoulder etc.) program, the conventional intra-articular injection of practice of the present invention and opiate and local anesthetic should be distinguished.The solution of this aspect of the present invention is used to run through inculcating continuously so that the preferential inhibition to painful and inflammation to be provided of operative procedure.On the contrary, realize that with the SE of the local anesthetic of present application the necessary high concentration of therapeutic efficiency can cause dark general toxicity.
After finishing program of the present invention, may need to use identical Chondroprotective agents and/or misery and/or the inflammation inhibitor of the irrigating solution with being used for surgical site of injecting or using higher concentration in addition, with substituting or replenishing as opiate.May also need after operative procedure, send the solution of passing effective dose, thereby agglomerate solution is remained on after operative procedure in patient's the synovial membrane capsule to the joint.
As mentioned before, compositions of the present invention also can be suitable for being injected directly in the anatomy joint, and said composition comprises multiple Chondroprotective agents, comprises preferably at least a catabolism inhibitor and at least a anabolism promoter.This reagent is preferred the selection, and comprises each reagent with enough amounts, and the treatment effective composition is provided when passing in the patient joint to send in the solution part, to suppress the chondrolysis metabolic process and to promote the cartilage anabolic process.This compositions can be carried out local injection and be suffered from chronic condition such as osteoarthritis or rheumatoid arthritis so that cartilage protection effect, this patient to be provided to the patient, or suffers from acute situation as the wound from operation or accidental damage.A kind of suitable compositions of local injection provides in the table 4 hereinafter.
III. the general of cartilage protection compositions is used
Embodiment of the present invention have been sent in the part of cartilage protection compositions so far and have been described aspect passing, as by intra-articular injection.Local injection is described as having several advantages that general is used that are better than, and comprises avoiding of systemic side effects.Although the part of the present composition is sent and passed in many examples is preferred, it is unpractiaca for some cartilage degradation situations.This is especially true for the patient who suffers from chronic cartilage degradation disease, and a plurality of sites are in the danger of cartilage degradation simultaneously in this disease, this disease such as rheumatoid arthritis, multi-joint osteoarthritis and other Polyarthropathies.For this patient, to it each or most of ill site (as the joint) injection cartilage protection compositions be painful, unpractiaca, expensive or in addition treatment forbid.The above-mentioned part that is used for is sent the cartilage protection compositions of passing can be adapted to pass through the general approach according to another aspect of the present invention to use.The systemic delivery of the present composition is suitable for, and has a plurality of patients that are in the site in the cartilage degradation danger but be not limited to treatment.Except that the multi-joint osteoarthritis and rheumatoid arthritis further discussed hereinafter, this aspect of the present invention can be used for treating other non-inflammation and inflammation arthritide, include, but are not limited to neuropathic arthropathy, acute rheumatic fever, ochronosis, systemic lupus erythematosus (sle), juvenile rheumatic arthritis, arthritic psoriasis, ankylosing spondylitis and other spondyloarthropathies and crystalline arthrosis.
A. arthritis mechanism
After understanding the mechanism that comprises in the articular cartilage degraded of rheumatology arthrosis (as rheumatoid arthritis (RA) and osteoarthritis (OA)), can better understanding be arranged to this aspect of the present invention.RA is the inflammatory arthritis of most common form, influences about 3% women and 1% male.Most patients has multiple and common symmetric joint is involved, especially the little joint of hands, elbow, wrist and shoulder.OA is the arthrosis of most common form, and is the reason that causes early retirement and deformity that is only second to cardiovascular disease.The destruction of HAC is the characteristics of OA and disabling property RA.
Although but various Therapeutic Method relief of symptoms, treatment has not proved and can slow down the destructive progress of articular cartilage.In OA, can there be the inhibition of normal cartilage cell function or the composing type impotentia of these cells, so that the degradation rate of the reparation speed of substrate and increase coupling.Various cytokines and inflammatory mediator have shown the imbalance that can form in the chondrocyte complex functionality, perhaps selectively increase the catabolism of cartilage matrix by the enzyme that raises various matrix degradations, and this enzyme comprises matrix metalloproteinase (comprising collagenase).
Treat the disease that comprises cartilage degradation for optimization ground, expection needs to stimulate anabolic process to suppress the metabolic therapeutic scheme of chondrolysis simultaneously.Thereby the Therapeutic Method that preamble is described to be used for suppressing the cartilage destruction of arthrosis can be used for the treatment of arthritis situation, and as OA and RA, this Therapeutic Method is based on the combination of cartilage anabolism reagent and chondrolysis metabolic poison.As indicated above, the consideration of practice requires to use these therapeutic agents and this disease that influences a plurality of sites in the health is simultaneously carried out best treatment by general.
B. combination of agents
This aspect of the present invention thereby provide comprises that the compositions and the general of Chondroprotective agents combination use this method for compositions.As mentioned before, the reagent that is directed at different receptors or molecule target is used for polyfactorial method.Therapeutic combination of the present invention preferably includes the Chondroprotective agents and the metabolic reagent of at least a inhibition chondrolysis of at least a promotion cartilage anabolic activity.This combination expection makes the limit optimization, and expection is better than only solving the conventional therapy of cartilage damage and the research that exploitation recently only solves the synthetic medicine of cartilage.The suitable anabolism promoter and the catabolism inhibitor that are used for local application above are being described, and expection also can be used in this general compositions.Being appreciated that the present invention is above-mentioned send the aspect of the compositions of passing and advantage also to be applicable to general embodiment of the present invention on applicable degree about the part.
Cartilage protection compositions of the present invention can suitably comprise one or more following anabolism promoter, with as nonrestrictive example: interleukin (IL) agonist (as IL-4, IL-10, IL-13 agonist); The member of transforming growth factor-beta superfamily comprises TGF-beta-agonists (as TGF β 1, TGF β 2, TGF β 3) and bone morphogenetic protein agonist (as BMP-2, BMP-4, BMP-6, BMP-7); Insulin like growth factor (as IGF-1) and fibroblast growth factor (as bFGF); And the fragment, disappearance, interpolation, aminoacid replacement, sudden change and the modification that keep the biological property of these naturally occurring reagent.
Cartilage protection compositions of the present invention can suitably comprise one or more following chondrolysis metabolic poisons, with as nonrestrictive example: the IL-1 receptor antagonist; TNF-α receptor antagonist; the cyclo-oxygenase-2 specific inhibitor; map kinase inhibitor; nitric oxide synthase inhibitor activity; the nuclear factor kB inhibitor; matrix metallo-proteinase inhibitor; suppress the metabolic cell adhesion molecule of chondrolysis (comprising integrin agonist and integrin antagonist); suppress signal conduction depressant drug (comprising inhibitors of protein kinase C and protein tyrosine kinase inhibitor) and the metabolic SH2 domain of inhibition chondrolysis inhibitor in the metabolic cell of chondrolysis.
As described about previous embodiments, at least a chondrolysis metabolic poison in general anabolism reagent/catabolism inhibitor combination can be and suppresses the metabolic soluble recepter of chondrolysis, as solubility IL-1 receptor or soluble tumor necrosis factor receptor.Specific example comprises human IL-1 receptor, soluble tumor necrosis factor receptor and the chimeric rhTNFR:Fc of recombinant soluble.The example that is used for the soluble tumor necrosis factor receptor that the present invention integrates comprises and is disclosed in the U.S. Patent number 5 of authorizing people such as Jacobs, 605, functional TNF-alpha-2 antagonists in 690, and the example that is used for the present invention's the human IL-1 receptor of solubility comprises that those are disclosed in the U.S. Patent number 6 of authorizing people such as Thompson, 159, in 460, herein the disclosure content clearly is incorporated herein by reference.This aspect according to the present invention is used for comprising with the catabolism inhibitor likely especially of the anabolism agent combination of systemic delivery the inhibitor of IL-1ra, TNFR1-IgG1 fusion rotein and matrix metalloproteinase.
As described further below, the cartilage protection compositions also can comprise one or more miseries and/or inflammation inhibitor or other treatment agent.The example that is applicable to the cartilage protection compositions of systemic delivery provides among the embodiment 5-20 hereinafter.
C. systemic delivery
This aspect of the present invention needs the systemic delivery of Chondroprotective agents and potential anti-inflammatory and/or analgesics or other treatment agent, to provide therapeutical effect in site, a plurality of joint.As used herein, term " systemic delivery " and " general is used " are intended to comprise, but be not limited to oral, intramuscular, subcutaneous, intravenous, suction, Sublingual, mouthful, partial, endermic, nose with other methods of application, these modes can cause sending one or more sites that the reagent of passing is distributed to needs therapeutical effect effectively.The preferred systemic delivery approach of this compositions is intravenous, intramuscular, subcutaneous and suction.The accurate general route of administration that is appreciated that the reagent of selecting to be used for particular composition of the present invention will partly be considered the sensitivity of this reagent pair metabolic conversion approach relevant with given route of administration and determine.For example, the approach that is adapted to pass through most except that oral of Toplink reagent is used.
Compositions of the present invention can be carried out general with the interval of determining and used on periodic basis, to keep the therapeutical effect of expection level.For example, compositions can every 2-4 week by using as subcutaneous injection.Dosage will consider that the various factors that can influence the reagent composition effect determines by the doctor.These factors comprise the cartilage site that is intended to treat, the size in joint (if suitably), the amount of the cartilaginous tissue that treat, the site of cartilage infringement, the situation of the cartilage that damages during treatment, patient's age, sex and body weight and other clinical factors.The dosage of each independent reagent will change as the function of existence that comprises other anabolism in the compositions and catabolism inhibitor and any drug delivery carriers (as continuing suitable delivery vectors) and characteristic.In addition, scalable dosage is with the explanation frequency of administration with send variation in the PK (pharmacokinetic) profile of the reagent of passing.Progress in individual treatment can be monitored by well known to a person skilled in the art several different methods, comprises clinical estimation, radiography and nuclear magnetic resonance, computed tomography, biochemical biomarker and arthroscope estimation.
D. delivery vectors and guiding
Making the compositions of various general route of administration carry out composite methods is suitable in this compositions.If Chondroprotective agents and the inflammation that comprises and painful inhibitor or other treatment agent are suitably compound in physiology's carrier or delivery vectors, with as being applicable to given general route of administration, as previously described.In one aspect of the invention, the systemic delivery of these combination of agents things or its arbitrary composition or multiple composition can mix in the drug delivery carriers or with this drug delivery carriers and make up, as delivery vectors and/or the stock that continues to discharge.
As used herein, term " delivery vectors " is intended to comprise that all contain, coupling or carry the structure of therapeutic agent, as nanosphere and other nano-particle, spherula and other microparticles, micelle and liposome, comprise the carrier that forms by albumen, lipid, saccharide, synthetic organic compound or inorganic compound.The preferred delivery vectors of the general compositions that is used for the present invention's guiding that further describes hereinafter is " granule ", should " granule " be intended to comprise nanosphere and other nano-particle, spherula and other microparticles, micelle and other carriers, but not comprise the more not preferred liposome of also describing hereinafter.Term " send delivery system " and means delivery vectors and one or more to contain or link coupled therapeutic agent.
Term " continue discharge system " means a kind of delivery system that send, and this send delivery system to provide any or all to mix the secular, enhanced of reagent or sending of regulating passed, persistent period or availability.The example of sustained release system comprises, but be not limited to the microparticle, spherula, nano-particle, albumen, liposome, saccharide, synthetic organic compound, inorganic compound and the injection-type hydrogel that contain medicine, as U.S. Patent Application Serial Number 09/861 by people such as Jun Li, 182 is described, herein it clearly is incorporated herein by reference.The system of the lasting release that other drug is suitable is known; and pass Chondroprotective agents applicable to sending at consistent relatively treatment level according to the present invention; thereby when comparing, can reduce side effect, and the longer continuous action time is provided with the agglomerate systemic delivery of reagent.
As used herein, term " stock " means and a kind ofly send the drug delivery system of passing in the site of needs effect or away from the site of needs effect, and this system provides and has been used for continuing stocking of the therapeutic agent that discharges.
Single reagent can be compound in uniform homogeneous blend, can be the mixing of independent microparticle, spherula etc. or use, and perhaps can simultaneously and use separately.
Minimize in order to make to be harmful to unwanted systemic effect, in one aspect of the invention, a kind of therapeutic strategy is to send to pass reagent composition in delivery vectors, and this delivery vectors preferably is directed at site or a plurality of site, particularly joint of containing cartilage in the health.As used herein, " delivery vectors of guiding " is to can be used for the medicine systemic delivery and delivery vectors that change, thereby the medication amount that arrives joint or required local action site arrive than the delivery vectors of using non-change or when not having delivery vectors the medication amount in joint or required local action site bigger (, because preferably molecule, cell or the anatomical structure with the joint is relevant for the delivery vectors of guiding, so medicine preferably is positioned the joint).Similarly, " guiding send delivery system " is the delivery vectors that contains the guiding of one or more medicines." medicine of guiding " means directly and is connected or link coupled therapeutic agent with target structure.As used herein, in the joint or required local action site have medicine that the general of " preferential interaction " uses in the joint or required local action site show bigger pharmacologic activity than other sites of great majority in health.
1. Dao Xiang ultimate principle
In OA, can there be the inhibition of normal cartilage cell function or the composing type impotentia of these cells, so that the degradation rate of the reparation speed of substrate and increase coupling.Various cytokines and inflammatory mediator have shown the imbalance that can form in the chondrocyte complex functionality, perhaps selectively increase the catabolism of cartilage matrix by the enzyme that raises various matrix degradations, and this enzyme comprises matrix metalloproteinase (comprising collagenase).Thereby the forfeiture of cartilage cell epimatrix (CEM) integrity is the result of the dynamic unbalance between the synthesizing activity of cartilage anabolic process and the catabolic activity that causes degrading.
Cytokine, somatomedin and the general of other biological bioactive molecule are used normal relevant with serious adverse.For example, the pathology effect is used with other factors relevant with the TGF-β 1 of general.In addition; the systemic delivery of unprotected (exposing) polypeptide often is subjected to the restriction owing to the protein reagent stability problem of the quick degraded of human cytokines in the circulation and inactivation, and this unprotected polypeptide is preferred often for previously described multi-joint arthrosis.
Among OA and the RA damage of articular cartilage may with the synthetic of the catabolism factor in the microenvironment of joint with discharge relevant.Research has in the past proved that proinflammatory cytokine (as IL-1) and inducible genes (as NO synzyme, COX-2, MMP) express at inflammatory arthritis rash patient's synovial membrane camber.Similarly, these media and gene often are expressed in the chondrocyte of OA-influence.In two kinds of situations, the joint microenvironment of specialization defines just influences the pathophysiology of articular cartilage situation environment fatefully.For this reason, expectation is led to the treatment molecule, so that it preferably positions and its expection target in the joint space is played a role.The catabolism inhibition Chondroprotective agents that the anabolism Chondroprotective agents general used for the cartilage protection in joint and/or general use is provided in this aspect of the present invention and preferably both are directed at the mechanism in joint.
Thereby preferably sending the approach of passing is the action site that these protein factors is directed at the joint.In order to realize clinical practice, need these reagent be sent the safety method of passing in the patient joint in the mode that continues and localize.Also need a kind of biological degradability pharmaceutical carrier, this pharmaceutical carrier can be protected when outside the joint and stable general is used the anabolism factor and/or catabolism inhibitive factor provide the unique method that drug delivery carriers is directed at the joint simultaneously.
Cartilage anabolism somatomedin is by body part is excretory power media to be arranged with small amount in the joint, to cause partial biologically in articular cartilage.Under normal physiological conditions, produce suitable anabolism somatomedin by the synovial cell in the chondrocyte in the cartilage and other articulation structures, to serve as the essential signal of the cartilage of keeping healthy and stable state by influencing the cartilage matrix metabolism.
2. antibody targeted delivery vectors
The cartilage protection compositions that the present invention preferably leads comprises at least a anabolism promotion property Chondroprotective agents and/or at least a catabolism inhibition Chondroprotective agents in the delivery vectors that is contained in guiding, and is preferably anabolism promotion property Chondroprotective agents and catabolism inhibition Chondroprotective agents.The delivery vectors of guiding preferably comprises granule, and most preferably comprises nano-particle, and this granule and most preferred nano-particle are sealed at least a and be preferably whole Chondroprotective agents.Granule is by being directed at the joint with link coupled guidance quality antibody of this granule or antibody fragment, this antibody or fragment for be positioned in the joint (promptly with respect to other optimum seeking sites of great majority in the health be expressed in the joint, preferably highly be expressed in the joint, and most preferably be defined in expression in the joint) the antigenicity determinant be specific.The Chondroprotective agents that is also referred to as the antibody targeted granule of " immune particle of guiding " herein and seals herein is that general is used.The compositions of part guiding is absorbed by the joint.Remaining composition is drained and/or metabolism.In the joint, guidance quality antibody or fragment combine with target antigen.Along with the past of time; granule is degraded in the joint; thereby with the mode local Chondroprotective agents of passing treatment concentration that send in the joint that continue to discharge in the section at the fixed time; so that the cell (as the primary cell in joint) that will regulate is carried out partial effect, this cell comprises synovial cell and chondrocyte.Therapeutic agent can spread or be discharged in the synovial fluid, and to be incorporated into the cell surface of articulation structure subsequently, to absorb or enter in the cell of articulation structure, or directly to being present in cytokine and/or the protease-producing strain effect in the synovial fluid.
The compositions of this aspect of the present invention guiding can be directed at according to the present invention in patient's the joint, and need not to know and cause arthropathic specific molecular pathology.The guidance quality antibody molecule guarantees that the reagent of sealing preferably is positioned in the joint, and more preferably is positioned the contiguous place of articular cartilage composition or combines with this composition.
Thereby, this aspect of the present invention provides that struvite, non-inflammation is suffered from a kind of pharmaceutical preparation treatment or other involve the arthropathic patient's in one or more joints method by using, this pharmaceutical preparation comprises the drug delivery carriers of guiding, preferably contains cartilage anabolism reagent and anti-catabolism reagent.This patient can suffer from OA, RA or other arthrosiss, as non-inflammation and inflammatory arthritis rash, include, but are not limited to neuropathic arthropathy, acute rheumatic fever, ochronosis, systemic lupus erythematosus (sle), juvenile rheumatic arthritis, arthritic psoriasis, ankylosing spondylitis and other spondyloarthropathies and crystalline arthrosis.The patient that the Treatment and composition for of this aspect guiding of the present invention is particularly suitable for suffering from osteoarthritis.The systemic delivery that is directed at agent combination in the carrier in joint makes it possible to treat this situation, and makes harmful and unwanted systemic effect minimizes.
A. the feature and the evaluation that hit of joint
The invention provides the method and composition of drug targeting in the joint, and describe preferred target in the joint in detail, this target comprises the relevant antigenic determinant of molecule, cell and tissue with articular cartilage and other articulation structures.The example of this target is selected from: collagen comprises II Collagen Type VI and accessory V, VI, IX, X and XI Collagen Type VI; Dan Baijutang comprises big aggregation Dan Baijutang, aggrecan, decorin, disaccharidase catenin polysaccharide, fibromodulin and lumican; The stromatin of cartilage oligomeric, glycoprotein-39; Dan Baijutang chondroitin sulfate and glycosaminoglycans; Macrophage synovial cell and fibroblast synovial cell; Chondrocyte.
In a preferred embodiment, the immune particle of guiding irreversibly act on, with reversible mode carry out in conjunction with or combine with the special component of articular cartilage (also being known as hyaline cartilage) in the joint.Other molecule targets in the joint can comprise the composition of articular chondrocytes epimatrix, as cartilage specificity collagen, comprise II, V, VI, IX, X and XI Collagen Type VI, aggrecan and other are little is rich in leucic Dan Baijutang, comprises decorin, disaccharidase catenin polysaccharide, fibromodulin and lumican.Dan Baijutang is the high molecular weight component of albumen and polysaccharide, and spreads all in the vertebrate structure organization, but also is present on the cell surface.Polysaccharide unit glycosaminoglycans (GAG) in the Dan Baijutang is acidity two glycopolymers that contain the derivant of amino sugar glycosamine or galactosamine, and is useful target.Stromatin of cartilage oligomeric (COMP) and the glycoprotein-39 (HC-gp39) that is also referred to as YKL-40 are useful target equally.
Articular cartilage contains collagen dissimilar in several heredity, and this collagen can be used as molecular marker in the present invention, and immune particle comprises that corresponding antibody can adhere to this labelling, is delivered to antibody combining site thereby make the therapeutic agent of sealing to send.The main collagen of articular cartilage is the 90%-95% that the II Collagen Type VI accounts for joint or the total collagen content of hyaline cartilage, and forms the fibrous structure of the cross strap that is shown by ultramicroscope.The II Collagen Type VI also is the unique and specific labelling of hyaline cartilage.People such as Hollander, J.Clin.Invest.93:1722 (1994); Freed, people such as L, Exp.Cell Res.240:58 (1998).It is development crosslinked between the fibril of covalency that the main extracellular of the tropocollagen molecule after betiding fibril and forming is modified.Described and the bonded antibody of II Collagen Type VI specificity epitope.Kafienah, people such as W., Tissue Engineering 8:817-826 (2002); Kolettas, people such as E, Rheumatology 40:1146-1156 (2001).II Collagen Type VI and the associated epitope in articular cartilage thereof have been represented preferred target of the present invention.
For example, the II Collagen Type VI monoclonal antibody isotype IgG of called after clone 6B3 1(Linsenmayer, people such as T.F., Biochem.Biophys.Res.Commun.92 (2): 440-6 (1980)) identification has the α 1 (II) and α 3 (XI) chain of identical primary structure.In Western blot, the lathyritic II Collagen Type VI TC behind this mAb and the usefulness mammal collagenase digesting AThe fragment reaction.It also with the reaction of the II Collagen Type VI of pepsin digestion.Its epitope mapping is in the triple helix of II Collagen Type VI, and demonstration and I type and III Collagen Type VI no cross reaction.The Bromine cyanide. of II Collagen Type VI (CNBr) peptide immunoblotting shows the reaction of this mAb and CB11 fragment, and this fragment is the site of immunogenicity epi-position on the complete II type molecule.
In another embodiment, the II Collagen Type VI of application human cartilage specific C NBr-cutting has been developed monoclonal antibody (the isotype IgG of II Collagen Type VI as immunogen 1) (Miller, E.J., Biochemistry 11:4903-4909 (1972); Glant, people such as T.T., Histochemistry 82:149-158 (1985a); British Journal ofHaematology 90:757-766 (1995)).This mAb commercial can available from ChemiconInternational (Temecula, CA), with the mankind and the reaction of cattle II Collagen Type VI of the dissolved and CNBr-of pepsin cutting.Do not observe cross reaction with I, III, V and IX Collagen Type VI.In a preferred embodiment of the present invention, the antibody of II Collagen Type VI is that the dissociation constant of 0.1-10nM combines with the target epi-position with the scope.
Accessory articular cartilage collagen also has contribution to the structure of substrate on the amount, and serves as useful target of the present invention.For example, a kind of non-protofibre collagen of weak point is IX Collagen Type VI (contain glycosaminoglycan chains, thereby also think Dan Baijutang) and II Collagen Type VI fibril covalent bond, and helps fibriilarly to interconnect or make fibril to combine with other substrate molecules.The XI Collagen Type VI is a kind of accessory fibril collagen, can participate in controlling the fibriilar diameter of II type.Other collagens comprise that V-type and VI type also can form part substrate.The less important collagen of these articular cartilage also can be brought into play function, this immune particle and the suitable antibodies of the immune particle target that instructs as antibody.
In another embodiment of the invention, be contained in dissimilar Dan Baijutang in the articular cartilage and can be used among the present invention with as the particulate molecular marker of binding immunoassay, thereby make therapeutic agent can send the site that is delivered to antibodies.In articular cartilage, Dan Baijutang has been formed the second largest part in the solid phase, accounts for the 5%-10% of net weight.The Dan Baijutang of cartilage matrix mainly is made up of big aggregation (50%-85%) and big non-aggregation (10%-40%) Dan Baijutang.Also there is different small protein polysaccharide.
The Dan Baijutang of the contributive cartilage of material character to tissue is big high molecular monomer (molecular weight 1-4 * 10 the most significantly 6).Big Dan Baijutang structurally is made up of the protein core of the extension with several zoness of different: have the N-stub area of 2 spherical structure territories (G1 and G2), be rich in the domain of keratan sulfate; The long domain that is rich in chondroitin sulfate, this domain also contains some keratan sulfates that are dispersed in and neutral oligonucleotide chain; Spherical structure territory G3 with the C-end.Aggregation forms by the many Dan Baijutang monomers in G1 spherical structure territory and hyaluronate chain combination.Each Dan Baijutang-hyaluronic acid sat linkage is stable by independent ball-joint albumen (molecular weight 41,000-48,000).The abundance than size (200-400nm) and chain that is rich in the zone of chondroitin sulfate in the chondroitin sulfate proteoglycan aggregation makes it become the preferred target of the immune particle of this aspect guiding of the present invention.
Provide by HC gp-39 with the link coupled antibody of immune particle of the present invention or its segmental extra target.In the joint, the HC gp-39 fragment with suitable antigenicity character also is enough to drug delivery carriers is led.
Immune particle can lead with antibody or its fragment, irreversiblely to react with the ad hoc structure of synovium of joint, to combine (modal) or combination in reversible mode.Specialization joint cell as preferred target comprises 2 kinds of main synovial cell's types, i.e. macrophage synovial cell (A type) and fibroblast synovial cell (Type B).
With the link coupled antibody of immune particle of the present invention or its segmental extra target be chondrocyte.The multiple albumen that is present in cell surface and can serves as the epi-position of cell guiding of the known expression of these cells.
In another embodiment of the invention, the preferred target of the drug delivery system of the chondroitin sulfate proteoglycan representative guiding relevant with articular cartilage.Described with the specific epi-position of chondroitin sulfate proteoglycan is bonded and be used for monoclonal antibody of the present invention.Morgan Jr., people such as A., Hybridoma 1:27-36 (1981); Schrappe, people such as M., Cancer Res.52:3838-3844 (1992); Schrappe, people such as M., Cancer Res.51:4986-93 (1991).The chondroitin sulfate proteoglycan monoclonal antibody that a kind of such example is the mouse anti mankind, called after clone 9.2.27 (IgG 2aIsotype).9.2.27 the chondroitin sulfate proteoglycan core glycoprotein and 210 of the sophisticated 250kDa of antibody recognition, 220 and the precursor polypeptide of 240kDa.The mouse anti mankind's aggrecan monoclonal antibody is promptly cloned 2A2.1 and also is suitable for the present invention, and commercial can available from United States Biological (Swampscott, MA).This antibody does not react with the chondroitin sulfate join domain.Transmission electron microscope shows that it is incorporated in the N-end portion of chondroitin sulfate adhering zone.
B. the guiding of the new epi-position relevant with cartilage degradation
In normal adult cartilage, do not exist or exist with low-down level, but at some stage of RA or OA raises or more the cartilage biomolecule composition of high expressed also can serve as the drug delivery system of the present invention's guiding target.The preferred target relevant with the cartilage degradation situation is the new epi-position that comes across on the articular cartilage that is diagnosed as OA, RA or other degenerative arthropathies patient, as the new epi-position of aggrecan or other chondroproteoglycans, and especially immunolocalization in the new epi-position of this patient's articular cartilage shallow-layer.
In one aspect of the invention, guidance quality antibody or antibody fragment and II Collagen Type VI or the segmental new epi-position of II Collagen Type VI or cleavage site specificity combine, particularly by matrix metalloproteinase (MMP)-1,3,8 or 13, or other members of MMP protein family, or the member who takes off integrin and metalloproteases (ADMATS) protein family with thrombospondin primitive acts on the new epi-position or the cleavage site of generation alone or in combination.ADMATS is further described in patent application WO 00/04917, EP 0 823 478 and United States Patent (USP) 5,811,535 and Tang, and people such as B. are among FEBS Lett.445 (2-3): the 223-225 (1999).
The antibody that is directed at the defined epitope that the specific region by II Collagen Type VI structure limits can be used in the guidance quality compositions of the present invention.These structural region parts are important in by the cartilage that betides the II Collagen Type VI degradation effects among OA and the RA.The degeneration of II Collagen Type VI causes the fragment of collagen protein, and this fragment discharges from cartilage matrix and comes across in the synovial fluid, moves in the circulation, and removes by urine.For having maximum effectiveness, compositions of the present invention is directed at the epi-position on the fragment of still relevant with cartilage matrix physics rather than release.
Each collagenase, MMP-1 (EC 3.4.24.7), MMP-8 (EC 3.4.24.34) and MMP-13 (EC 3.4.24.-) all have the ability of fibril formation property II Collagen Type VI of cutting triple helix, thereby produce big (3/4-length) amino terminal fragment and less (1/4-length) carboxyl terminal fragment.Kafienah, people such as W., Biochem.J.331:727-732 (1998).Their all initial 3/4 and 1/4 fragments of cutting specific Gly-Leu/Ile key with generating feature.Specific collagenase isotype is relevant with the forfeiture of the pathology of cartilage.Billinghurst, people such as R., J.Clin.Invest.99:1534-1545 (1997).For example, in an OA animal model, the focus area of collagenase 1 and collagenase 3 is positioned the extracellular matrix of OA injury site in the knee joint, meets with the cutting of the collagen of 3/4-1/4.Collagenase 3 albumen also are rich in the middle tibia cartilage in diseased joints.Huebner, people such as J., Arthritis ﹠amp; Rheumatism 41:877-890 (1998).
In from synovial membrane, synovial fluid and cartilage sample among the people who suffers from RA and OA, detected collagenase 1 (MMP-1).(MMP-13 is (to cut the II Collagen Type VI than collagenase 1 fast 5-10 speed doubly for collagenase 3.Noteworthy is to have identified MMP-13 in synovial membrane in the people who suffers from RA and OA and the human OA cartilage.
Fibril collagen can destroy by helical cut and cause degeneration, perhaps destroys by the cutting of end peptide to cause crosslinked removal.Two research has confirmed the existence of active collagenase in the cartilage by the polyclonal antiserum of the new epitope specificity of collagen that generates to the epi-position hidden in the II Collagen Type VI spiral zone with by collagenase, and this epi-position of hiding makes collagen separate supination in the cutting owing to collagenase and obtains exposure.These find to show that collagenases 1, collagenase 3 or both all participate in the cartilage degradation relevant with various arthritides.Thereby, generated from the specific II Collagen Type VI degraded of collagenase cutting and can be used for the new epi-position of specific guide of the present invention to the useful antibody targeted property in aspect.These epitope mappings are in terminal 3/4 fragment sequence of the N-of α 1 (II) chain, and known this sequence contains the epi-position (by antibody COL2-3/4m identification) to AH12L3 and CB11B.
In arthritide, the catabolism that the chondroproteoglycan aggrecan increases is the main pathological process that causes articular cartilage degeneration.The forfeiture of Sulfated glycosaminoglycans subsequently jeopardizes the function and the structural intergrity of cartilage matrix, and this Sulfated glycosaminoglycans is the inherent composition of aggrecan molecule.Over time, this process causes cartilage degradation.The original position degraded of aggrecan is included in the Proteolytic enzyme process of the cutting that is arranged in the specific peptide bond of core protein.The enzymatic activity that the contributive fullest of this process is characterized is the result of metalloproteases specific function.Original position aggrecan hydrolysis by matrix metalloproteinase (MMP) has obtained broad research.Yet, be fully recognized that at present that the major protein enzyme of being responsible for aggrecan original position degraded in the articular cartilage is aggrecan enzyme (aggrecanase), its two kinds isotypes of identifying recently are the members that take off integrin and metalloproteases (ADisintegrin And Metalloproteinase with Thrombospondinmotif (ADAMTS)) gene family with thrombospondin primitive.There is the monoclonal antibody technique of identifying the new epi-position on aggrecan or the aggrecan catabolite.In addition, another aspect of the present invention is the bonded monoclonal antibody of new epi-position or its fragment on application and aggrecan or the aggrecan fragment, to be directed at the nano-particle of the combination that contains anabolism promoter and catabolism inhibitor.
The catabolism and the forfeiture of the aggrecan of articular cartilage in the mainly responsible arthritis arthrosis commitment of aggrecan enzyme determined in temporary transient research.Although be successive, this process is most probably before the collagen decomposition metabolism.At the late stage of lysis, when the collagen decomposition metabolism takes place, there is the evidence of degraded of the MMP-mediation of remaining aggrecan in the small scale cartilage.
The monoclonal antibody technique of the new epi-position of catabolism on the proteolytic degradation product is identified in the possibility development and application, to identify for different substrates degradability enzyme family unique and distinctive specific cleavage site.In these researchs, characterized several monoclonal antibodies of specifically identifying the new epi-position of catabolism (as the N-of Proteolytic enzyme cleaved products or the new epi-position of C-terminal amino acid sequence generation), this new epi-position is generated by the effect of (IGD) domain between the bead of active aggrecan enzyme (or MMP) to aggrecan.These antibody have been used to monitor aggrecan and have been connected proteic proteolysis.In addition, those skilled in the art will recognize that following antibody can be used for immune particle is directed at the joint, and do not deviate from scope of the present invention, the new epi-position on the stromatin catabolite that this antibody recognition generates in the chondrolysis metabolic process.
In another aspect of the present invention, guidance quality antibody or antibody fragment combine with aggrecan or the segmental new epi-position of aggrecan or cleavage site in the cartilage specifically, particularly by MMP-1,3,8 or 13, or other members of MMP protein family, or the member of ADMATS protein family acts on the new epi-position or the cleavage site of generation alone or in combination.The example of the monoclonal antibody of different structure epi-position or new epi-position is 8-A-4 or BC-3 in identification aggrecan or the aggrecan fragment.MAb 2-B-6 has been used to survey a large amount of aggrecan catabolites, and this product is by generating at aggrecan enzyme, MMP or other proteolytic activities along many sites of the core protein of aggrecan.The Sulfated unsaturated chondroitin sulfate disaccharide of 4-that MAb 2-B-6 identification and these core protein fragments are adhered to.Relevant antibody MAb3-8-3 also has been used to identify the different deglycosylated aggrecan metabolite that contains the Sulfated chondroitin sulfate oligosaccharide of 6-.MAb BC-3 identification is by aminoacid sequence alanine-arginine, and the N-end that glycine (ARGxx...) limits is epitope sequences newly, and this sequence is generated by the aggrecan enzyme catabolism in the IGD domain of aggrecan.
What ADAMTS-4 gene (Genbank NM-005099) and ADAMTS-5 gene (Genbank007038) coding had thrombospondin primitive-4 and 5 takes off integrin and metalloproteases, and this enzyme is the member of ADAMTS protein family.The member of family has several different albumen members, comprises pre-peptide region, metalloproteases domain, takes off integrin spline structure territory and thrombospondin 1 type (TS) primitive.The single member of this family is variant in the TS of C-end primitive number, and some have unique C-end structure territory.By the terminal TS primitive of the azymia C-of ADAMTS-4 gene code.Enzyme by the ADAMTS-5 gene code contains the terminal TS primitive of 2 C-, and the function of performance aggrecan enzyme is with cutting aggrecan, promptly a kind of main chondroproteoglycan.Thereby these two kinds of enzymes all are responsible for generation (Tortorella, people such as M., J.Biol.Chem.275 (33): the 25791-25797 (2000) of new epi-position on the degraded of aggrecan and aggrecan and the aggrecan fragment; Tortorella, people such as M., J.Biol.Chem.275 (24): 18566-18573 (2000); Abbaszade, people such as I., J.Biol.Chem.274 (33): 23443-23450 (1999)).
Treat in another embodiment of the human cartilage in the OA situation in the present invention, application-oriented in the antibody of the new epi-position labelling of the early stage biochemistry of the OA that is called 3-B-3 (-).3-B-3 (-) epi-position is the relevant phenotypic alternation of OA-of chondroitin sulfate (glycosaminoglycans) chain end of aggrecan.
C. guidance quality antibody feature
As used herein, term " antibody " is intended to comprise the immunologic competence part (promptly contain and combine with antibody specificity or the molecule of immunoreactive antigen binding site) of immunoglobulin molecules and immunoglobulin molecules.This term description immunoglobulin, no matter it is natively or synthetic preparation.The albumen that comprises antibody can be derived from natural origin, or partly or entirely synthetic preparation.The example of antibody comprises all immunoglobulin hypotypes and Fab and F (ab ') 2, scFv, Fv, dAb, Fd fragment and be disclosed in United States Patent (USP) 5,534, the fragment in 254.ScFv refers to the minimum binding structural domain of the strand of immunoglobulin molecules.Term " antibody " also means following antibody, and this antibody is brought into play function at ECS, at the plasma membrane of cell or in the intracellular region territory of cell (as kytoplasm or nuclear), to regulate expression of gene or the activity that one or more regulate cartilage metabolisms.Being used for preferred antibody of the present invention comprises humanized, chimeric and the human monoclonal antibody.
In the situation of antibody, term " fragment " refers to any aminoacid sequence of the polypeptide portion of any above-mentioned guiding, and this sequence has the common relevant composition of source, structure and the Mechanism and FunctionsDNA suitable with complete antigen for the purpose of the present invention's guiding.Antibody convenes the application that is also intended to comprise this antibody fragment with method described herein in the compositions.
The preferred embodiment of the invention is used humanization or human antibodies or its fragment that is covalently attached on nanosphere or other particle surfaces, has sealed the curative Chondroprotective agents of the present invention in this nanosphere or other granules.Antibody or its fragment are preferably as the guidance quality molecule on the particle surface; in this granule, sealed curative Chondroprotective agents; this is essential in vivo enough stable because of them, and shows that minimum quilt contains the potentiality of the serum of cell drain body protein from the particle surface removal.Expect that human monoclonal antibody completely or humanized murine antibody are that the therapeutic agent that the most useful guidance is applied to human patients send the joint of passing guidance quality antibody type, wherein this antibody combines with any molecule of cartilage cell epimatrix or the cell in joint, and this is because this patient does not generate immunoreation after using.
For example, the nucleotide sequence in nucleotide sequence that mouse monoclonal antibody can be by the Mus Fv zone of will encoding (promptly containing antigen binding site) and coding human constant domain zone and Fc zone carries out genetic recombination and carries out chimeric.The residue of some Mus also remaines in the frame construction territory, human variable region to guarantee correct target site associativity feature.Can recognize that the humanized antibody that is used for leading has the immunoreactive advantage that reduces antibody in the host receptor or polypeptide, and can be used for increasing the intravital half-life and reduce harmful immunoreation puting together antibody on nano-particle or other containment particle surfaces.
Antibody or its analog that application has complete human characteristic are extremely beneficial with the treatment human patients.Can adopt with those and be disclosed in United States Patent (USP) 6,075,181,6,235,883 and 6,492,160 and patent application EP 1 167 537 A1 in similar methods, herein its disclosure clearly is incorporated herein by reference.This method had been used to generate the complete human antibodies of multiple anti-human IL-8 and EGF-R ELISA in the past.In the most preferred embodiment of the present invention, antibody or antibody fragment are that the dissociation constant of 0.1-10 nanomole combines with the target epi-position with the scope.
Although human antibodies or humanized antibody are preferably used among the present invention with the treatment human patients completely, also should understand method and composition of the present invention and also be used for the veterinary and use other mammals (as horse and Canis familiaris L.) to treat easy trouble degenerative joint.
What D. be used to lead send the Chondroprotective agents of passing
The preferred aspect of the embodiment of the present invention's guiding will comprise at least a Chondroprotective agents of sealing or comprising in nano-particle or other delivery vectors, adhered to guidance quality antibody, antibody fragment or other guidance quality structures on this nano-particle or other delivery vectors.Reagent that seal or that comprise can be any disclosed herein anabolism Chondroprotective agents or catabolism inhibitor, and this reagent has chemistry or the architectural feature that makes it seal or to comprise in the nano-particle of selecting or other delivery vectors.When in addition, other generals are used highly easily by (as the albumen and the peptide) of metabolic degradation if or the general that do not lead use then cause the reagent of harmful side effect to be preferred for leading.For example; disclosed hereinafter each class anabolism Chondroprotective agents (member who comprises transforming growth factor (TGF)-beta superfamily; comprise TGF-beta-agonists and bone morphogenetic protein agonist; insulin like growth factor and fibroblast growth factor) and hereinafter disclosed some catabolism inhibitor (interleukin-1 receptor antagonist and TNF-α receptor antagonist) be albumen, and be very suitable for equally sending and passing with the encapsulated form of guiding.
The encapsulation compositions that the present invention most preferably leads is included in anabolism Chondroprotective agents or the catabolism inhibition Chondroprotective agents of sealing in the immune particle (or granule of other guiding) of identical guiding, and both are preferably albumen by reagent.Selectively, each reagent can be sealed separately, and can use two kinds of (or above two kinds) type-directed particulate mixture, perhaps more not simultaneously or send the reagent of passing two or more guiding successively separately, to cause in the joint, having this reagent simultaneously.
In some instances, only anabolism Chondroprotective agents or catabolism inhibitor can be sealed, and perhaps a kind of reagent is not relevant with undesired systemic side effects.In this example, a kind of reagent can send with the form of sealing guiding and pass, and another kind of reagent send with the non-encapsulated form of non-guiding and pass, they or send together with the same dose form of mixture and to pass or send separately and pass.
All advantages of providing by combinations thereof are provided; although it is preferred not as using anabolism reagent and catabolism inhibitor simultaneously that the sending of the single Chondroprotective agents of sealing in being directed at the antigenic immune particle that is positioned in the joint (anabolism promote property or catabolism inhibition) passed, and also is possible.
E. reagent seals
The size of material is to determine whether it can see through the synovial membrane capillary wall and move to the principal element in joint from systemic circulation.It has been generally acknowledged that the particulate maximum gauge that can move through the synovial membrane capillary wall is 50 nanometers.Yet some can be transported by transcytosis up to the granules of polystyrene of the lecithin bag quilt of 240 nanometers the diameter that studies show that of synovial membrane capillary permeability.The present invention has overcome the restriction that the synovial membrane permeability barrier applies by preferably adopting the conditional granule of sealing of a class size distribution (as nano-particle (being preferably nanosphere)).
The dosage form that the present invention continue to discharge can comprise have dispersive therapeutic agent therein microparticle and/or nano-particle, perhaps can comprise the therapeutic agent of pure preferred crystalline solid form.
The therapeutic dosage form of this aspect of the present invention can have any configuration that is suitable for continuing release.
The present invention preferably continues to discharge the therapeutic dose form and has following size, biodegradation and biocompatible feature.
The delivery system that send of the present invention's guiding preferably utilizes size to be limited to the nano-particle of diameter for about 5 nanometers-Yue 750 nanometers, and about 10-is about, and 500 nanometers are preferred, and about 200 nanometers of most preferably about 20-.If confirm to cause permeability enough in the morbid state, so as an alternative useful but more preferably size be the about 100 microns microparticle of the about 1-of diameter, about 1-is preferred for about 25 microns, about 10 microns of most preferably about 1-.
But preferred granule is biodegrade and the biological degradability structure that preferably discharges the medicine that loads at about 1-in about 150 days time period with the therapeutic level, and this time period was preferably about 7-about 60 days, and about 14-was most preferred in about 30 days.It will be appreciated by those skilled in the art that medicine can take place by the combination including, but not limited to the physical process of diffusion and degraded from the release of nano-particle and microparticle, and can be described by the dynamic process of complexity, this dynamic process is unique for each carrier formulation and anabolism and anti-catabolism therapeutic agent.
Preferred granule is biocompatible with the joint target tissue and the local physiological environment of application dosage form, comprises producing biocompatible biodegradable product.Suitable compositions comprises from the biodegradable particles of natural polymer preparation, comprises hyaluronan (hyaluronan), chitosan, collagen, gelatin and alginate.These natural polymers can with other combination of polymers to generate the copolymer pellet of forming by as chitosan and gelatin.Synthetic biological degradability poly-(alpha-hydroxy esters) successfully is used to prepare the microparticle that mixes protein for treatment agent such as human growth hormone as polylactic acid (PLLA), polyglycolic acid (PGA) and copolymer p LGA.The example that is applicable to the particulate another kind of Biodegradable polymer of preparation the present invention guiding is amphipathic ABA triblock copolymer, as poly(ethylene oxide): poly-(3-hydroxybutyric acid): poly(ethylene oxide).
When the polymer nano granules system of selecting the selected Chondroprotective agents of the present invention to use, the suitable biological activity of guaranteeing entrapped drug also is important.
The application of the Biodegradable polymer nanosphere of guiding has the advantage that the different release characteristics of selection are provided for the therapeutic agent of sealing.For some drug regimens, the suitableeest release dynamics is made up of dual dispose procedure, and wherein each active component shows that different lasting release characteristics is to provide the suitableeest pharmacokinetics in the joint.Those skilled in the art will recognize based on disclosure herein; to be different from the suitableeest release dynamics of nano-particle or microparticle for each independent medicine, and also will be medication amount, granular size and other functions by the definite plysiochemical character of granulometric composition that loads in preparation in the granule.Can regulate to obtain the suitableeest therapeutic concentration in synovial fluid and the intraarticular gap, to realize required therapeutic concentration the quantitative rate of release in the encapsulated particles of each medicine from the joint.For example, can carry out in vitro study to characterize the dual release dynamics of sustained release formulation, wherein each composition (as anabolism medicine and catabolism inhibitor) is showed lasting release in during 7-30 day.Each drug release is gone into amount in the synovial fluid, and to carry out quantitative methods be that those skilled in the art are well-known, and comprise the measurement to radiolabeled drugs.Selectively, may adhere to fluorescence or other optics reporter molecules medicine with the preparation labelling.One skilled in the art will recognize that there are many indirect quantitative approachs that as ELISA or mass spectrography, this method all is specific for each reagent.
Since actual, be difficult to realize similar release dynamics for two kinds of different substantially medicines of size, and many anabolism and catabolic combination are also like this.For example, in embodiment preferred of the present invention, expection send pass be characterised in that molecular weight be the catabolism inhibitor of 200-500 such as p38 map kinase inhibitor (for example, SB203580, MW=377) and length be anabolism reagent such as the human IL-10 of 160 aminoacid and MW with about 18Kda.The independent control that each reagent is continued rate of release can realize by changing particulate structure composition and/or generating two or more immune particle mixture.In mixture, one group of granule is a homogenizing about the anabolism reagent of sealing, and on the same group granule is not a homogenizing about the catabolism inhibitor of sealing.Two groups of blended granules change in forming at its size and polymer separately, if but suitably, then be characterised in that the rate of release that its active agent is similar, or be consistent rate of release, thereby make each seal the local therapeutic effects optimization of reagent respectively.
Liposome is not the preferred delivery vectors of systemic delivery Chondroprotective agents according to the present invention of being used to lead.Send delivery system to compare with nanosphere with the granule that other continue to discharge, liposome has the short half-life in blood circulation.The liposome medicament conjugate can be retained in liver and the spleen, thereby causes the release of liposome destruction and active agent.Reagent thereby distribute with the activated state general, rather than be protected before in being positioned the joint.It is not highly to continue that reagent send the release of delivery system from the liposome that leads, and the immune particle that has than guiding send delivery system lower localization.Owing to these reasons, the application of granule (as nanosphere) is highly preferred with respect to the application of liposome.Even so; but for the anabolism Chondroprotective agents of systemic delivery or comprise the Chondroprotective agents combination of anabolism reagent; its camber need lead, and the liposome of guiding proves suitable, and provides with respect to exposed medicine and send the advantage of passing.
F. the antibody and the coupling of sealing reagent
Make guidance quality antibody and the nano-particle that continue to discharge comprise chemical cross-linking agent and isodigeranyl function cross-linking compounds (i.e. " connector ") by representativeness " coupling " method that key covalently or non-covalently is connected, this cross-linking agent and chemical compound react with the reactive group (as hydroxyl, amino, acylamino-or sulfydryl) of guidance quality antibody and be present in nano-particle or other guidance quality carrier surfaces on other reactive groups (having similar chemical characteristic) between form key.The key that forms between guidance quality antibody and granule or other delivery vectors can include, but are not limited to following: peptide bond, disulfide bond, thioester bond, amido link and thioether bond.
Continue to discharge and directly puting together of dosage form and guidance quality albumen (antibody) can destroy the molecule of guiding or the identification of cell by the guidance quality antibody of modifying.Part interlayer attachment techniques is to realize continuing to discharge the useful alternative method of adhering to of dosage form and guidance quality binding proteins (antibody).These technology can comprise that application does not form one-level peptide or protein shell in conjunction with the albumen of targeted cell population.Binding proteins is combined, so that the granule as a result with functional binding proteins/peptide to be provided with one-level peptide or protein shell.A kind of exemplary part sandwich method comprises that avidin or Succ-PEG-DSPE pass through above about described functional group of " directly " associated methods and granule covalent attachment.Binding proteins is biotin (as by Acibenzolar, hydrazides, iodoacetal, maleimide amino or the similar functional group) derivatization by functionalization, is preferably MIN.Part (being the biotin of binding peptide or albumen/functionalization) takes place with the biotinylated albumen/peptide that adheres to by using saturation capacity of avidin/Succ-PEG-DSPE one-level protein shell.
3. selectable guidance quality and preferential effectiveness send the method for passing
Chondroprotective agents among the present invention and combination thereof are led, or realize that in the joint this reagent or compositions are with respect to the additive method of other preferential effectiveness in site also within the scope of the present invention.For example, known two kinds of main synovial cell's types are the albumen that macrophage synovial cell (A type) and fibroblast synovial cell (Type B) and chondrocyte can be expressed multiple uniqueness, this albumen is present in these cell surfaces, and can serve as the epi-position of specific cells guidance quality.The reagent proteic or that preferentially be expressed in inflammation or the diseased joints that selection is directed at preferential expression in the joint can preferentially increase local action, and unwanted systemic effect is minimized, and this reagent is preferably the combination of anabolism promoter and catabolism inhibitor.
As mentioned above, molecule target in the joint can comprise the composition of cartilage cell epimatrix, as cartilage specificity collagen, comprise II, IX, X, XI Collagen Type VI, aggrecan and other are little is rich in leucic Dan Baijutang (as decorin, disaccharidase catenin polysaccharide, fibromodulin and lumican).Comprise equally being the stromatin (COMP) of cartilage oligomeric and the glycoprotein-39 (HC-gp39) that is also referred to as YKL-40 as target.The re-set target that is used for this aspect of the present invention is a biochemistry cartilage labelling, and this labelling is not present in the normal adult cartilage, or only exists with low-down level, but is present in some stage of RA or OA.Similarly, the selection of Chondroprotective agents also can be preferentially increases local actions with respect to other zones of health, and this reagent is preferably the anabolism promoter of the receptor-specific that raises in ill or inflammatory conditions and the combination of catabolism inhibitor.
As mentioned above, can the reagent sealed or plurality of reagents be directed at one or more structures in the joint with corresponding guidance quality antibody (or antibody fragment) coupling by making the reagent sealed.This antibody also can be applicable to the key and the exposed coupling of medicine own that cuts in the local joint environment potentially, to realize the arthrotropic guiding of medicine that general is used and to send and pass.Similarly; can select the Chondroprotective agents that is included in anabolism of the present invention promotion property and the catabolism inhibition compositions; so that its remainder with respect to health preferentially acts in the site in the joint, thereby preferentially bring into play their effects to synovial cell and/or chondrocyte and/or extracellular matrix components.
E. select the method for delivery vectors and dosage
The specific nanosphere system and guidance quality antibody or the fragment that are used to send the reagent of passing selection according to the present invention, and the accurate loading or the dosage of the therapeutic agent that comprises in the compositions of guiding can be analyzed definite according to the present invention.Analytical method comprises that the patient to this diagnostic test of needs uses and contains the antibody labeling nanosphere (or granule of other guiding) of sealing therapeutic agent, and makes the patient carry out the position of imaging analysis with the nanosphere of determining to contain medicine.Then, the degree of deposition can be determined by using imaging analysis in the patient joint.Imaging analysis is well-known in the medical domain, and unrestrictedly comprises x-ray analysis, nuclear magnetic resonance (MRI) or computed tomography (CT).In a preferred embodiment, joint guidance quality antibody can be with carrying out labelling by the detectivity reagent of imaging in the patient.For example, guidance quality antibody can carry out labelling with contrast agents such as barium, and this reagent can be used for the x-ray analysis, perhaps carries out labelling with magnetic contrast agents such as gadolinium chelate compound, and this reagent can use MRI or CT surveys.Other labelling reagent unrestrictedly comprise radiosiotope as 99Tc.Except that imaging analysis, can from the patient, obtain vivisection to determine particulate existence and concentration in the joint (as synovial tissue).
Part of the present invention is sent and is passed embodiment and be described aspect concentration of treatment agent suitable when passing is sent in the part, and this concentration is enough to send in the part inhibition or the therapeutic effect that site (as the joint) provides predeterminated level of passing.When systemic delivery, needs are used higher reagent concentration or dosage.This systemic doses and/or concentration are that the active agent that causes providing enough in the required site of potential cartilage degradation after any metabolic conversion process is necessary, and the local therapeutic effects of desired level need be realized in this site.Especially, the suitable therapeutic of systemic doses and/or concentration and preferred levels cause providing active agent with following concentration level in localized site (as the joint), and this concentration level is respectively in the therapeutic and preferred concentration range for of passing sent in aforementioned part.
Delivery system is sent in lasting release for guiding, in compositions, comprise enough reagent dosage or loading, to cause in joint or action site, can in required lasting deenergized period, realizing the predetermined local concentration that continues in the deenergized period of required therapeutical effect level.Thereby, comprise enough reagent dosage or loading, causing the reagent of sealing by the scheduled volume of joint picked-up, thereby the metabolic conversion of reagent before the local environment that arrives joint or joint sealed in explanation.The reagent of sealing that arrives this scheduled volume in joint will be according to the disclosure that comprises herein, thereby when nanosphere or other are sealed when sending the delivery system degraded, reagent discharges in the local action site, to be provided at the local concentration in this reagent therapeutic concentration range at required lasting deenergized period (, more preferably being 1 day-2 week) as during 1 day-4 week.
IV. be used to suppress the reagent of cartilage degradation
Below be the example categories that is applicable to the Chondroprotective agents in the compositions of the present invention, the illustrative drug of each apoplexy due to endogenous wind.Although without wishing to be bound by theory, think that the selection reason of classes of agents makes the reagent work obtain illustrating.
1. il-1 (IL-1) receptor antagonist
Interleukin I L-1 exists with two kinds of forms, IL-1 α and IL-1 β, and it is the polypeptide that derives from the isolated genes product, this isolated genes product has a similar immunomodulating and short scorching function spectrum.IL-1 is the polypeptide of a kind of 17kD, and it can act on or be produced by the many cell types in the joint, comprises synovioblast and macrophage, chondrocyte, endotheliocyte and mononuclear cell and macrophage.Evidence show, play an important role in the pathophysiology damage of arthritis that IL-1 occurs in the damage joint and articular cartilage.
It is by two kinds of IL-1 receptors (IL-1R) that these two kinds of form cartilages damage effect of cytokines, one of them mediation of I type IL-1 or II type IL-1 receptor.The IL-1 receptor structurally is clearly, and belongs to the separation superfamily that exist for feature of immunoglobulin in conjunction with the territory.These receptors have and the similar amino acid homology thing of other receptor that contains the immunoglobulin territory.Bigger I type IL-1 receptor is neutralized in the fibrocyte at the T cell expresses, and less II type IL-1 receptor is at the B cell, and mononuclear cell is expressed in neutrophil and the medullary cell.
II type IL-1 receptor can combine with the IL-1 β of high-affinity, but the combination of Il-1 β can not cause intracellular signal transduction, and this is because it and I type IL-1 receptors bind.On the contrary, the precursor that the II receptor can be used as solubility IL-1 binding factor works, and it comes off out from cell, and this soluble recepter works as physiological IL1 beta antagonists.Naturally occurring IL-1 is conjugated protein to be described, and it is outside corresponding with solubility II receptor.
Cloned and checked order with the IL-1 receptors bind, naturally occurring secretion soluble ligand, it optionally is called the IL-1 receptor antagonist, and (sIL-1RA, IL-1Ra IL-1ra), and find this part albumen of 22kD at least of encoding.IL-1Ra suppresses combining of IL-1 α and IL-1 β and I and II type IL-1 receptor competitively.Il-1Ra is a kind of pure receptor antagonist, because it can not activate the theca cell signal transduction mechanism relevant with the IL-1 receptor with the combination of receptor.Though the binding affinity of this albumen and IL-1Rs is higher, but still need be excessive, 10-100 molal quantity doubly suppresses to express the IL-1 biological response of I type IL-1R cell.Known, the cell that produces IL-1Ra comprises mononuclear cell, neutrophil, macrophage, synovial cell and chondrocyte.Verified, IL-1Ra can suppress PGE 2Synthetic, proinflammatory cytokine and MMPs induce, and the generation of nitrogen oxide.Secreting type IL-1R discharges in tentative inductive inflammatory phase mesosome.Importantly, IL-1Ra expresses in synovial tissue, and is present in normal person's the synovial fluid.In the patient of knee damage, the acute phase of the level of IL-1Ra after damage increases suddenly in the synovial fluid, is reduced to also lower than the normal level in inferior-acute and chronic states subsequently.Thereby proof IL-1Ra plays physiological effect in the replying of joint injury.
It has been generally acknowledged that IL-1 is that reigning cartilage damages cytokine, because it is by the ability that chondrocyte and synovial cell stimulate digestive enzyme and proinflammatory cytokine to produce, it can play a role in the damage in joint.And IL-1 β still is the inhibitor of a kind of effective inhibition chondrocyte synthetic proteins polysaccharide and collagen.At cellular level, the replying of the synovioblast that IL-1 is beta induced comprises PGE2, the increase of collagenase and other neutral protease product, and proinflammatory cytokine, the rise of IL-6 and IL-8.
IL-1, it is present in arthrosis patient's the joint fluid, stimulates chondrocyte: 1) make enzyme, as stromelysin, fibroblast and neutrophilia collagenase, and the synthetic quantity of plasminogen activator improves 2) inhibitor-1 of inhibition plasminogen activator and synthesizing of TIMP.And IL-1 β still is a kind of effective, and matrix components such as II Collagen Type VI and the synthetic inhibitor of proteoglycan, II Collagen Type VI wherein are the main form of collagen in the articular cartilage.Imbalance between protease and inhibitor level causes the increase of active protease amount.This increase combines with the biosynthetic inhibition of substrate, causes cartilage degradation.In zooscopy, IL-1 is expelled to causes in the rabbit knee that proteoglycan gets rid of from articular cartilage.
Because IL-1 is a kind of important, relate to the pathogenetic cytokine of chronic synovitis and cartilage degradation, the effect that therefore reduces its generation or block it is a kind of new suitable Therapeutic Method that is used to alleviate synovial membrane inflammation and the cartilage protection effect is provided.Can utilize the Therapeutic Method of various antagonism agonist IL-1 and its natural membranes bind receptor interphase interaction, these methods comprise: 1) naturally occurring, specific IL-1 acceptor inhibitor comprises IL-1Ra and solubility IL-1 receptor; 2) anti-IL-1 Abs; 3) micromolecule antagonist, it can be peptide peptide or non-.
The ability of blocking this important cytokine effect can act in the joint the various kinds of cell type (for example, synovioblast and chondrocyte), thereby suppress pathological effect subsequently, as inflammatory cell infiltration in the joint, synovial hyperplasia, synovial cell's activation and cartilage infringement and the synthetic inhibition of cartilage matrix.The IL-1 receptor antagonist should be blocked the propagation of IL-1 inflammatory response, disturbs lysis thus.The treatment effectiveness of many IL-1 receptor antagonists is confirmed by the animal model of inflammation and arthritis (RA and OA).Behind subcutaneous injection Il-1Ra or intra-articular injection solubility I type IL-1R, RA patient's clinical disease improves.
The effect of IL-1 β and IL-1Ra depends on their local concentrations separately.In the sliding diaphragm-operated supernatant of RA, the level of IL-1 β than the horizontal Senior Three of IL-1Ra doubly.Therefore, the idiopathic generation of local I L-1Ra is not enough to suppress the effect of IL-1 β, this excessive because of having (10-100 doubly), the Il-1Ra molecule of big molal quantity could suppress the inductive biological respinse of IL-1 in the cell, the IL-1R of wherein said cellular expression I type.Therefore, the IL-1 that IL-1Ra of high dose is used for the human volunteer of blocking-up trouble RA in the body.The IL-1Ra that the part is present in the synovial membrane provides a kind of negative signal, the process in its downward modulation synovitis to small part IL-1 mediation, and as leukocyte gathering in Inflamed tissue, collagenase and PGE that the synovial cell produces 2IL-1Ra is injected directly in the joint of Canis animals ACL model, and uses with gene transfection and in people's synovioblast, be the based gene Therapeutic Method, the cartilage protection effect of susceptible of proof IL-1Ra IL-1Ra.
The invention discloses proteic part of IL-1 soluble recepter and whole body and send and pass, described IL-1 soluble receptor body protein is made up of the extracellular domain of IL-1R, and the IL-1 cytokine molecule in can binding soln.Particularly, by embodiment, in U.S. Pat 5,319; soluble human IL-1 receptor (shuIL-1R) polypeptide that contains aminoacid sequence 1-312 is disclosed in 071 and US5,726,148; it can be selected from the antiinflammatory class with one or more, anti-pain class, or the drug regimen of cartilage protection class.Optionally, disclosed in 071 as U.S. Pat 5,319, the part of the fusion rotein of being made up of the sIL-1R binding domain polypeptide or whole body send to pass and can promote the cartilage protection effect.In addition, also disclose United States Patent (USP) 5,817, the part of disclosed IL-1 receptor antagonist or whole body send and pass in 306, to be used for the present invention.Verified, the shuIL-1R soluble recepter can be in conjunction with the IL-1 with millimicro affinity.When under various inflammation or pathophysiological condition, the medicine part being used for joint tissue; the IL-1R soluble recepter of treatment valid density; send as the part of shuIL-1R and to pass and to be undertaken by it is injected directly in the joint; or (for example with irrigating solution; during the arthroscope surgical procedures) form carry out; this lavation can with one or more cartilage protection medicines as Chondroprotective agents disclosed herein, anti-inflammatory drug, or anti-pain medication is used in combination.The IL-1 that this treatment preferentially suppresses collagenase-1 and stromelysin-1 generation stimulates.Use complete diverse ways; this method is based on the gene transmission of the 1 type soluble recepter that is used for local IL-1 of generation and/or TNF-α; it has been found that; the existence of these cytokine soluble recepters can be in the arthritis acute inflammation stage of antigen induction, for the knee joint of rabbit provides protection.
Be suitable for use as Chondroprotective agents of the present invention with high-affinity and the bonded IL-1 receptor antagonist of people I type IL-1 receptor-specific peptide (11-15 aminoacid).These little peptides are compared with the bigger reorganization IL-1 soluble recepter or the IL-1ra that recombinates, and more favourable part can be provided, and comprise synthetic difficulty or ease and cost, and the ability of infiltration biological barrier.Two kinds of peptides the most effective based in vitro effects are: Ac-FEWTPGWYQJYALPL-NH 2(AF12198, IC 50=0.5-2nM) Ac-FEWTPGWYQJYNH 2(AF11567).AF11567 is the truncate translation of AF12198, and it lacks 4C-end residue, and low slightly with respect to the affinity of I type IL-1 receptor, but it has similar 2.3-2.6 hour plasma half-life.As if when via the administration of intravenous infusion whole body, relatively poor dissolubility and metabolism have fast suppressed the effect of AF12198.Send the method for passing by direct part, as be expelled in the IA joint space, or it is included in be suitable for then can partly overcoming these restrictions in surgery irrigating solution of the present invention or other transfusion.The embodiment that is suitable for IL-1 receptor antagonist of the present invention is as follows.Send for all parts and to pass (that is, injection, infusion and lavation) and whole body send (comprising by using the delivery system of the guiding) mode of passing, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Described concentration expection is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 1
The treatment of interleukin-1 receptor antagonist and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??rshuIL-1R ??0.2-2000 ??200
??rhIL-1ra ??0.2-2000 ??200
Anti-IL1 antibody ??0.2-2000 ??200
??AF12198 ??0.2-2000 ??200
??AF11567 ??0.2-2000 ??200
2. tumor necrosis factor (TNF) receptor antagonist
TNF-α, a kind of cytokine that is mainly produced by activated macrophage has multiple biological agent, comprises some by the receptor-mediated gene transcription adjusting of specificity T NF, and immunoregulatory activity.At first, cloned two kinds of not isoacceptors that are known as TNF-R1 and TNF-2, and found that it produces as soluble recepter.
Receptor in this family is single transmembrane protein, has a lot of congeners at their extracellular domain, and their relatively short born of the same parents' internal areas then have considerably less sequence homology thing.The effect of TNF is by producing with the bonded factor of cell surface receptor, and in fact, it is present in all cell types of having studied.People have identified and have cloned two receptors.One of them acceptor type is known as TNFR-II (or A type or 75kDa), 439 the amino acid whose transmembrane proteins of encoding, and have the molecular weight of 75kDa.Second acceptor type is known as TNFR-I (or Type B or 55kDa), has the molecular weight of 55kDa, and 426 the amino acid whose transmembrane proteins of encoding.TNFR1 comprises born of the same parents' internal area, and it can pass through NF-kB pathway excitation signal.
Two receptors of TNF have all shown the high-affinity in conjunction with TNF α.People have separated soluble TNF acceptor (sTNFR), and confirm that it is owing to coming off of membrane-bound receptor extracellular domain occurs.People have identified two types TNFRs, and with its called after sTNFRI (TNF BPI) and sTNFRII (TNF BPII).And verified, the form of these these soluble recepters all can be represented the clipped form of above-mentioned two kinds of TNFR types.
TNF α plays an important role in cell sequence that inflammatory response and cartilage damage and molecular events.The short scorching effect overlaid of the many effects of TNF α and IL-1.The short scorching effect of TNF α is to stimulate proinflammatory cytokine, comprises IL-1, the release of IL-6 and IL-8.TNF-α also can induce matrix metalloproteinase from neutrophil, discharges in fibroblast and the chondrocyte, thereby makes cartilage degradation, and part is to be undertaken by the stimulation of collagenase.In addition, TNF-α also can raise the COX-2 in normal person's articular chondrocytes and the synovioblast, causes the increase of PGE2 product.
This cytokine and IL-1 are considered to excite and produce the pathological effect of articular cartilage, comprise the leukocyte infiltration, synovial hyperplasia, synovial cell's activation, cartilage infringement and the synthetic inhibition of cartilage matrix.Particularly, during synovial membrane inflammation, can see the increase of TNF-alpha levels in the synovium of joint liquid, and the increase of the TNF α of synovial cell's generation.Therefore, whole body send to be passed, and comprises the delivery system that send of guiding; or the local irrigating solution of passing that send; soluble TNF α receptor in transfusion or the injection can be in conjunction with free TNF α, and as the antagonist of TNF receptor in the surrounding tissue and work, thereby the cartilage protection effect is provided.
The invention describes the purposes of functional TNF-alpha-2 antagonists; it can be by the removing of effective free ligand; or it is direct with receptor autophosphorylation; emulative interaction and born of the same parents block the interaction of part and its related film receptor is outward used separately or is provided the cartilage protection effect with other agent combination.Also can utilize various Therapeutic Method to come the antagonism agonist, TNF-α, and the interaction between its natural membranes bind receptor, these methods comprise: 1) naturally occurring specificity T NF-alpha active inhibitor comprises the use of soluble TNF-α receptor; 2) use of anti-TNF-Alpha antibodies; 3) use of micromolecule antagonist, this antagonist can be peptides peptide or non-.
The invention discloses the proteic purposes of chimeric soluble recepter (CSR), the extracellular domain of TNF receptor wherein, the activity that it has in conjunction with the TNF molecule is covalently bound to a territory of IgG molecule.Particularly, disclosed in 851 by first embodiment as U.S. Pat 5,447, can use the chimeric polyeptides (reorganization chimera) of the extracellular domain that contains the outer polypeptide of TNF receptor born of the same parents, the outer polypeptide of born of the same parents wherein is connected with the CH3 district with the CH2 of mice IgG1 heavy chain polypeptide.Verified this chimeric TNF soluble recepter (in U.S. Pat 5,447,851 in also be called " chimeric tnf inhibitor ") can combine with the TNF-α of high-affinity, and it is very effective as the bioactive inhibitor of TNF-α.
In addition, second embodiment is chimeric fusion constructs, and it is made up of the ligand binding domain of TNF receptor and Fc antibody (being also referred to as Fc fusion soluble receptor) part, with respect to its manufactured comes out of TNF-α receptor.The invention also discloses U.S. Pat 5,605,690 described soluble TNF acceptors: Fc fusion rotein, or the purposes of arbitrary modified forms.Have now and studies confirm that this soluble TNF acceptor: the Fc fusion rotein maintains the high binding affinity to TNF α.
In being used as the soluble recepter scope of pharmacology's antagonist, the term soluble recepter comprises, but be not restricted to: aminoacid sequence or the corresponding soluble recepter of its soluble fragments that (1) and natural (endogenous) produces, this aminoacid sequence or its fragment are made up of the membrane receptor extracellular domain of total length, (2) recombinant soluble receptor, it is a total length, the truncate of naturally occurring receptor amino acid sequence or partial sequence, it keeps the ability in conjunction with associated ligands, retains biological activity and analog thereof, (3) chimeric soluble recepter, the recombinant soluble receptor that it is made up of truncate or partial sequence, this sequence is outer corresponding in conjunction with the part in territory with total length receptor amino acid sequence born of the same parents, and the total length receptor amino acid sequence by oligomer (for example is, aminoacid) attached to the IgG polypeptide (for example, IgG hinge and Fc territory on) the corresponding sequence of a part, its retains biological activity and in conjunction with the ability of associated ligands.
Solubility, ligand binding domain is natural outside the born of the same parents of cytokine receptor is present in the body fluid, and relates to the bioactive adjusting of cytokine.Many hematopoietic cytokine receptors (Il-1R, IL-4R, IL-6R, the natural existence of solubility clipped form TNFR) had been reported.For example, soluble TNF R is present in health volunteer's the urine and serum with the concentration of about 1-2ng/ml.Because lack signal transduction functionality, these cytokines are conjugated protein to be because the complete alternative splicing of receptor sequence (film-combining form) mRNA, or owing to the proteolytic cleavage of receptor membrane combining form and release produce.Though effect is not confirmed fully that also they can be used as the physiology antagonist of its complementary endogenous cell factor and play a role in the body of these solubility truncated receptors.This antagonism be because the removing of (1) free ligand, this removing is by carrying out with the combination of its relevant soluble recepter, reduced effective Cf of membrane-bound receptor, (2) effect of cytokines with just produce after cell surface receptor combines.
TNF-α soluble recepter is the natural agonist as TNF-R1 and TNF-R2, plays a role by vying each other with these cell surface receptors of free ligand common set body.Pharmaceutically, the TNF soluble recepter can be used as antagonist, plays a role by its ability that reduces the free ligand bioavailability, rather than suppresses mechanism (that is, with membrane receptor on the endogenic ligand competition of common binding site) by competition and play a role.With the TNF soluble recepter of treatment effective dose join in the joint can be effectively in and the biological activity of part.Give in the body test of recombinant soluble receptor verified its inflammation-inhibiting ability of replying, and can be used as antagonist and work.
In the present invention; be suitable for use as Chondroprotective agents; and with other Chondroprotective agents; anti-pain and/or antiinflammatory combination; thereby the reagent that suppresses the cartilage damage comprises soluble TNF R; people's chimeric polyeptides (reorganization chimera), it comprises the extracellular domain of the TNF-α receptor (p80) that partly links to each other with human IgG1's Fc, and anti-TNF-Alpha antibodies.Send (that is, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 2
The treatment of TNF-receptor antagonist and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??sTNFR ??0.1-2000 ??200
Chimeric rhTNFR:Fc ??0.1-2000 ??200
Anti-TNF-Alpha antibodies ??0.2-2000 ??200
3. interleukin-2-receptor agonist
Some cytokine is the signal glycoprotein, and it is the important medium that synovitis and cartilage damage.Recently cartilage infringement mechanism analysis is shown, not only the abswolute level of short scorching major cytokine IL-1 is extremely important in the decision cartilage injury, and the cytokine of cartilage homeostasis control also is by catabolism and anabolic adjusting cytokine, and the domination of the balance between the anabolism somatomedin.If being equilibrated in the inflammatory conditions that helps IL-1 β of IL-1 β and IL-1Ra product changes, it will help the morbidity that chronic inflammatory disease and cartilage damage so, as taking place behind the knee joint surgical operation.Effective treatment reagent that can suppress the proinflammatory cytokine generation of arthritis position comprises anti-inflammatory cytokines, IL-4, IL-10 and IL-13.These cytokines are significantly damaged with external articular cartilage in the ameliorate body, and this point plays a role to various approach by them and realizes, described approach refers to and alleviates the ballistic approach of IL-1.Therefore, anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 can reduce inflammation effectively by following method: the 1) generation of minimizing proinflammatory cytokine, 2) reduce natural anti-inflammatory cytokines, as the generation of IL-1Ra, recently, IL-4 is confirmed in vivo.
IL-4 can alleviate the inflammatory process in rheumatoid arthritis (RA) the patient synovial membrane.In the rheumatoid synovial membrane, IL-4 can suppress the generation of the proinflammatory cytokine that caused by the synovial membrane sheet, thereby suppresses synovial cell's propagation and reduce the absorption again of bone.IL-4 can also promote direct cartilage protection effect by suppressing synthesizing of person joint's chondrocyte matrix metalloproteinase-3 (MMP-3).The cell culture system of using person joint's chondrocyte is used to assess generation and metalloproteases-1 (TIMP-1) role of IL-4 to the inductive MMP-3 of IL-1.It is found that IL-4 can suppress the MMP-3 albumen of IL-1 stimulation and the activity of enzyme.In addition, IL-4 also suppresses the inductive MMP-3 mRNA of IL-1.Inducing of iNOS can be by IL-4, and IL-10 and IL-3 suppress.Therefore, in IJD, can see the protective medium that IL-4 damages as a kind of joint and work.
In addition, IL-4 has supported the effect of cartilage protection to the balance role of IL-1 adjusting cytokine levels.IL-4 and IL-10 all can suppress the inflammatory cytokine that produced by freshly prepd rheumatoid synovial cell.Though the independent use of each interleukin all produces effect, but the combination of IL-4 and IL-10 can be worked in coordination with inhibition IL-1 and the IL-6 of TNF-α stimulation and the generation of IL-8, and the not influence of the viability of pair cell.IL-4 is joined the generation that can increase IL-1Ra in the synovial membrane culture, and reduce the generation of IL-1 β.Recently reported with IL-4 and carried out interior therapeutic, it promotes alleviating of rat test arthritic symptom by the balance that difference acts on IL-1 β/IL-1Ra.The total numerous characteristics of another kind of cytokine IL-13 and IL-4, it also induces the IL-1Ra in the RA synovial membrane.Therefore, the whole body of IL-4 and IL-13 compositions or local sending are passed the therapeutic value that can provide collaborative.
IL-10 has numerous characteristics, and this shows that it is a kind of good candidate material that cartilage damages that suppresses.It suppresses the release of IL-1 and TNF-α, and stimulates the generation of TIMP-1, suppresses MMP-2 simultaneously.Recently report the generation of IL-10 in the RA synovial membrane, and described the characteristic of IL-10 antiinflammatory action.IL-10 can suppress the IL-1 β that use synovial membrane sheet is produced in the stripped RA model, but its inhibition degree than IL-4 is little.
Using non--partial approach to send in the arthritis animal model of the delivery cell factor, had been found that the protection effect that IL-4 and IL-10 damage cartilage.In the collagen-induced arthritis model of murine, IL-4 and IL-10 combined therapy can produce huge improvement.Except the inhibition of the visible inflammation of naked eyes, can also alleviate cellular infiltration in the synovial tissue with IL-4 and IL-10 combined therapy, and cause the remarkable protection effect that cartilage is damaged.In addition, the mRNA level of TNF-α and IL-1 can highly be suppressed in synovial tissue and articular cartilage.On the contrary, the mRNA level of IL-1 receptor antagonist (IL-1Ra) then continues to raise, and its hint protection mechanism may be relevant with the inhibition generation of TNF-α and IL-1, and with the equilibrated rise of IL-1Ra/IL-1.These data are consistent with the main effect of IL-10 in the endogenous of inflammatory response and articular cartilage damage suppresses, and the combined therapy of IL-4 and Il-10 has shown potential therapeutic value.
The effect of endogenous IL-4 and IL-10 and these cytokines were studied in the commitment of murine streptococcus cell wall (SCW) arthritis model that macrophage relies on the therapeutic effect of arthritis and cartilage damage.And confirmed that endogenous IL-10 works in the arthritic adjusting of SCW.The adding of exogenous IL-10 has further enlarged the inhibition effect of endogenous IL-10.Use the compositions of IL-4 and IL-10 can find a more significant effect.This combination can make swelling alleviate, and makes the synthetic increase of chondrocyte proteoglycan.With the combination treatment of IL-4 and IL-10, can reduce the level of TNF-α basically, just as with separately treatment of IL-10, but also cause the remarkable reduction of IL-1 β level in the synovial membrane, a kind of additional potential clinical effect.Generally speaking; these data and IL-4 and IL-10 by whole body or locally send that thereby to be delivered to the effect that the joint prevents that cartilage from damaging consistent, and show that the compositions that contains IL-4 and IL-10 can provide than being used alone the bigger effective therapeutic value of reagent as Chondroprotective agents.
Severe combined immunodeficiency (SCID) mice is assessed when being expelled in the human rheumatoid synovial membrane in IL-4 or the IL-10 body as model, to the effect of cartilage degradation and mononuclear cell (MNC) recruitment.People IL-4 (rhIL-4,100ng with reorganization; RhIL-10,100ng), the compositions of IL-4 and IL-10, or TNF-α (1000U), or phosphate buffered saline (PBS) weekly twice are expelled in the human rheumatoid synovial membrane and cartilage that derives from five rheumatoid arthritis patients around altogether.It is found that the compositions of people IL-4 and IL-10 can suppress the morbidity and the cartilage degradation of people synovial tissue, prove that these interleukin agonist have the cartilage protection effect.
The people IL-13 that cloned and checked order, and it has the numerous characteristics of IL-4.The homology of IL-13 and IL-4 is approximately 25%.As IL-4, IL-13 can reduce the proinflammatory cytokine that is produced by synovial fluid mononuclear cells, comprises IL-1 and TNF-α.IL-13 has shown antiinflammatory action in the body, thereby has the treatment effectiveness in the treatment of articular cartilage damage.
As IL-4, IL-10 and IL-13 agonist compounds effective comprise naturally occurring people IL-4, IL-10 and IL-13, the people IL-4 (rhIL-4) that recombinates, rhIL-10, with rhIL-13 and partial sequence thereof, or peptide sequence, it is by using recombinant DNA technology to be configured, with identification IL-4, IL-10 and IL-13 receptor, and can lip-deep these receptors of active cell.It is particularly including by anti-Fc acceptor portion and anti-IL-4, and is anti--IL-10, and the polyspecific molecule of anti-il-13 acceptor portion composition, and wherein at least a portion is to use recombinant DNA technology to construct.Under with the situation of interleukin agonist as the medicine agonist, term interleukin agonist comprises, but be not restricted to: (1) peptide sequence, it is corresponding with aminoacid sequence or its fragment that natural (endogenic) produces, (2) recombinant interleukin, it is the truncate or the partial sequence of the naturally occurring interleukin aminoacid sequence of total length, it keeps the ability in conjunction with associated receptor, and retains biological activity, and analog, (3) chimeric interleukin, a kind of recombinant polypeptide, it is by forming with the corresponding truncate of the part of full length amino acid sequence or partial sequence, wherein full length amino acid sequence be by oligomer (for example, aminoacid) attached to the corresponding sequence of a part in IgG polypeptide (for example, IgG twisting district and Fc territory) on, it keeps the ability in conjunction with associated receptor, and retains biological activity.
Below the embodiment that is suitable for interleukin agonist of the present invention is listed in.Send (for example, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 3
The treatment of interleukin agonist and preferred concentration
Chemical compound The part is sent and is passed Preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??rhuman?IL-4 ??0.5-5,000 ??5-500
??rhuman?IL-10 ??0.5-5,000 ??5-500
??rhuman?IL-13 ??0.5-5,000 ??5-500
4. transforming growth factor-beta superfamily agonist
Transforming growth factor-beta (TGF-β) superfamily member is 25kD, can influence the multiple-effect multifunctional protein of various cell functions, and known its relates to the reparation of tissue and reinvent.In many cases, it can improve the interaction of cell and extracellular matrix (ECM), and increases gathering of ECM by secretion and the generation that stimulates ECM albumen and protease inhibitor.TGF-β and other cytokine have collaborative interaction, show anti-inflammatory activity usually.People have identified the multiple isoform of TGF-β, its total similar amino acid sequence homology.In people's tissue, had been found that TGF-β 1, TGF-β 2 and TGF-β 3, though their binding affinity difference, they all play a role in mammalian cell.
The TGF-beta superfamily member is a chondrocyte proliferation, effective regulator that differentiation and extracellular matrix gather.In the cartilage organ cultures, the metabolism of TGF-β 1 scalable proteoglycan and synthesizing by rabbit articular chondrocytes stimulation collagen and glycosaminoglycans.In addition, TGF-β 1 can increase the expression of TIMP in person joint's chondrocyte, and the expression of downward modulation IL-1 receptor in articular cartilage.
Bone morphogenetic protein (BMPs) is the cell growth, the multifunctional regulator of differentiation and programmed cell death, and it belongs to transforming growth factor (TGF-β) superfamily.Having identified the mammal bmp protein family member who surpasses a dozen, can be many groups with its subclassification according to their structure wherein.BMP-2 and BMP-4 are closely similar each other.BMP-5, BMP-6, BMP (OP)-1 (also claiming BMP-7), similar on structure each other with OP-2/BMP-8.The growth variability factor (GDF)-5 (also claiming cartilage deutero-morphogenetic proteins-1), GDF-6 (also claiming cartilage deutero-morphogenesis protein-2) forms another relevant group with GDF-7.With BMP-2, BMP-4, BMP-6 and OP-1/BMP-7 form contrast, the formation of its inductor internal skeleton and cartilage, and GDF-5, GDF-6 and GDF-7 can more effectively induce cartilage and tendon-spline structure (Wolfman etc., 1997).
The TGF-beta superfamily member is by bringing into play their effect with two types serine/threonine kinase receptors bind, and the two is all very important (Massague, 1998) for signal transduction.The II receptor is to form active kinases, and it makes the bonded I receptor of part change phosphorylation.The I receptor activates substrate in the born of the same parents, and as Smad albumen, and it takes place by the specific mechanism of this intracellular signal transduction.Isolated seven kinds of different I receptors of mammal, it is called as activin acceptor sample kinases (ALK)-1-ALK7 at first.IA type bmp receptor (BMPR-IA or ALK-3) and IB type bmp receptor (BMPR-IB or ALK-6) are structurally similar each other, and specifically in conjunction with BMPs and II receptor.Proved that ALK-2 can be in conjunction with activin, but up-to-date data show, it is the BMPs that determines, for example, and the I receptor of OP-1/BMP-7 (Macias-Silva etc., 1998).ALK-1 is structurally closely similar with ALK-2, but does not also know its physiology part.ALK-5 and ALK-4 are respectively the I receptors of TGF-β (T β R-I) and activin (ActR-IB).ALK-7 and ALK-4 and ALK-5 are structurally closely similar, but still do not have to determine its part.
Be suitable for cartilage protection solution of the present invention, the agonist of naturally occurring TGF-β and bmp agonist and synthetic or people's reorganization (rh) can interact with above-mentioned arbitrary bmp receptor.Term used herein " TGF-β and bmp agonist " comprises its fragment, disappearance, and addition, amino acid replacement, sudden change and modification, it has kept the biological nature of naturally occurring TGF-β and bmp agonist part.TGF-β or bmp agonist can use separately, or use with other TGF-beta superfamily member synergistic combination as anabolic cartilage reagent (becoming reparation cartilage or that promote cartilage matrix), or are used in combination with the metabolic inhibitor of blocking-up chondrolysis.
The I receptor can play II receptor downstream composition.The specificity of I receptor intracellular signal is to be determined by the specificity district in the serine/threonine kinase territory, and it is called as the L45 ring.Therefore, the structure of the L45 of BMPR-IA/ALK-3 and BMPR-IB/ALK-6 (BMPR-I group) ring is mutually the same, and their transducible similar cell signals.Equally, T β R-1/ALK-5, ActR-IB/ALK-4, and the L45 of ALK-7 (T β R-I group) ring is mutually the same, and they can activate similar substrate (Chen etc., 1998).The L45 ring of ALK-1 and ALK-2 (ALK-1 group) is obviously different with other I receptor, but they can activate the similar substrate of organizing to BMPR-1 (Armes etc., 1999) of I receptor.
Various albumen can be from TGF-β and BMP serine/threonine kinase receptor transduction signal.Among them, the molecule of being furtherd investigate is the albumen of Smad family.Identified a plurality of different Smad albumen in the mammal, and these albumen have been divided into three subgroups, that is, and the Smads of receptor-adjusting (R-Smads), common gametophyte Smads (Co-Smads) and inhibition Smads.R-Smads derives from the complex with Co-Smads, and is directly activated by the I receptor, and transfers in the nuclear.The different aggressiveness of Smad directly or by other DNA-is conjugated protein combines with DNA, thereby regulates transcribing of target gene.Smad1, Smad5 and Smad8 are activated by BMPs, Smad2 and Smad3 are activated by TGF-β.For example, with the Smad4 combination, the Smad2 that plays a role as Co-Smad is transferred in the nuclear, and it activates the gene transcription of mediation TGF biological agent therein.Smad6 and Smad7 are structurally irrelevant with other Smads, and work to suppress Smads.But proved BMPs inductor outer and the interior new cartilage of body and the formation of bone, and regulated the growth and the differentiation of chondrocyte.And these albumen also relate to the repair of cartilage process.Various researchs have confirmed that BMPs also can promote and be held in the phenotype of cartilage, its be by them chicken appendage bud cell culture and fetus rat chondroblast, and rabbit and the synthetic ability of cattle articular chondrocytes moderate stimulation proteoglycan are represented.BMPs is confirmed by transgenic method for cartilage and osteoplastic importance, has wherein studied the rejecting of specific b MP gene.
A member of BMP family, BMP (OP-1 or BMP-7) is under normal and the pathological conditions, seems particular importance as the cartilage homeostasis during the repair of cartilage.OP-1 is unique member of BMP family seemingly, with the deutero-morphogenetic proteins of cartilage of being expressed by adult's articular chondrocytes.(Chubinskaya，S.，J.Histochemistry?andCytochemistry?48：239-50(2000))。OP-1 is purification from bone matrix at first, and can induce the formation of cartilage and bone.Clone people's OP-1 gene, and prepared bioactive reorganization OP-1 homodimer.The people OP-1 that recombinates can stimulate the synthetic of aggrecan and II Collagen Type VI by the vitro human articular chondrocytes.It can also hinder IL-1 to the metabolic illeffects of these chondrocytes, and the cattle cartilage injury of blocking-up CH-296 mediation.This effect can be by the proteoglycan of research recombined human OP-1 to person joint's chondrocyte, and the effect that PGE2 and IL-1 receptor antagonist produce is confirmed that wherein person joint's chondrocyte is to cultivate under the situation that has il-1 β to participate in.Can effectively overcome the synthetic downward modulation of the beta induced proteoglycan of low dosage IL-1 with OP-1 handler articular chondrocytes.And, discover that OP-1 can stimulate the substrate set of hyaluronan and CD44 human chondrocytes to require the synthetic of other molecule.To OP-1 in the ripe cartilage of people, regulate and express studies have shown that OP-1 in cartilage protection with the effect in repairing, and can be with OP-1 as the therapeutic agent that promotes cartilage anabolism and person joint's repair of cartilage.
During the position, OP-1 (BMP-7) induces the formation of cartilage and bone in being implanted to intravital skeleton or outside the skeleton.The effect of OP-1 healing holostrome skin articular cartilage damage is promptly passed the articular cartilage of rabbit knee and is bored two adjacent holes by following method research.OP-1 causes the healing of articular cartilage and the regeneration of articular surface, and it comprises the cell of similar ripe articular chondrocytes.
These data show, in order to prevent that behind surgery operating wound human cartilage from degenerating and the organism homeostasis of maintenance articular cartilage, the preferred embodiment that the present invention puts into practice used solution comprises the whole body or the topical application of TGF-beta superfamily member, preferred TGF β 2, BMP-7 (OP-1) or BMP-2, or the equivalent agonist that plays a role by same receptor, it is used by these parts.Whole body or part are sent and are passed and can carry out with one or more drug regimens, wherein this medicine be the chondrolysis metabolic process inhibitor (for example, map kinase inhibitor, MMP inhibitor or nitric oxide synthase inhibitors) and/or other be used for the reagent of inhibition of pain and inflammation.
In using TGF-β and the situation of bmp agonist as the medicine agonist, term TGF-β and bmp agonist comprise, but be not restricted to: (1) peptide sequence, it is corresponding with aminoacid sequence or its fragment that natural (endogenous) produces, (2) reorganization TGF-β s and BMPs, it is a total length, the truncate or the partial sequence of naturally occurring TGF-β and BMP aminoacid sequence, it keeps the ability in conjunction with their each autoreceptors, and remain with biological activity and analog thereof, (3) chimeric TGF-β s and BMPs, the recombinant polypeptide that it is made up of truncate or partial sequence, this truncate or partial sequence are corresponding with the part of full length amino acid sequence, this full length amino acid sequence by oligomer (for example, aminoacid) be attached to that (for example, IgG twisting district and Fc territory on) the corresponding sequence of a part, it remains with in conjunction with the ability of associated receptor and biological activity with the IgG polypeptide.
Below the embodiment that is suitable for TGF-β of the present invention and bmp agonist is listed in.Send (that is, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Be used to send the surgical operation solution treatment range of concentrations that is delivered to the joint to assess from (Kd) constant value that dissociates of each part of its associated receptor.And for special cell type and tissue, these values are different, are the embodiment of BMP-4 below.Have 125I-BMP-4 has disclosed specificity in conjunction with test, the existence of high affinity combined sites, and it has the dissociation constant of 110pM, and each cell has about 6000 receptors.Therefore, when 11nM BMP-4, it is maximum that the combination of part reaches, and effectively receptor is occupied (saturated) fully.The existence of BMP-4 functional receptor in former generation articular chondrocytes is proved.
Table 4
The treatment of TGF-β and BMP-receptor stimulating agent and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??TGF-β1 ??0.05-500 ??0.5-100
??TGF-β2 ??0.05-500 ??0.5-100
??BMP-2 ??0.1-2000 ??1-200
??BMP-4 ??0.1-2000 ??1-200
??BMP-7(OP-1) ??0.1-2000 ??1-200
5. cyclo-oxygenase-2 (Cox-2) inhibitor
Though nonsteroidal and-inflammatory drug (NSAIDs) is widely used as antiinflammatory, also do not study its therapeutic use especially as Chondroprotective agents.The direct molecule target of NSAID medicine is first enzyme in the prostaglandin route of synthesis, and it is known as prostaglandin endoperoxides synzyme or fatty acid cyclo-oxygenase.Two kinds of correlation forms of cyclo-oxygenase had been described, the characteristic of cyclo-oxygenase-1 or 1 type (COX-1) and cyclo-oxygenase-2 (COX-2).These isozymes are known as prostaglandin G/H sythase (PGHS)-1 and PGSH-2.The rate-limiting step during but the equal catalysis prostaglandins of these two kinds of enzymes forms, wherein the formation of prostaglandins is the conversion of arachidonic acid to PGH2.COX-1 is present in platelet and the endotheliocyte, and has primary activity.By comparison, at the endotheliocyte in joint, macrophage has been identified COX-2 in fibroblast and other cell, and its expression is by proinflammatory cytokine, and is inductive as IL-1 and TNF-α.
In inflamed joints, the expression of COX-2 is raised, and the activity of COX-2 increases along with its rise, causes the synthetic increase of prostaglandin, and prostaglandin wherein is present in inflammatory arthropathy patient's the synovial fluid.The cell source of prostaglandin in the joint (PGs) comprises activatory chondrocyte, A and Type B synovial cell and wellability macrophage.Comprise gene expression by important cell function in the joint metabolism of PGs adjusting, the synthetic and propagation of extracellular matrix.Because COX-2 is in the inflammation joint tissue, or after being exposed to inflammation (for example, causing by damage or surgical wound) medium, express, therefore expect that the use of cox 2 inhibitor can provide antiinflammatory and cartilage protection activity.
Joint injury and arthroscope surgical procedures can excite the cartilage of inflammatory arthropathy to damage.Chondrocyte is only cell type in the articular cartilage, and by endogenous inflammatory mediator, comprises the release of PGs and participates in the damage of they self substrate.Studies have shown that the COX-2 gene expression in normal person's articular chondrocytes, the synthetic release with PG of albumen is by cytokine, comprises IL-1, and TNF-α and IL-6 are inductive rapidly.The level of mRNA can be behind cytokine induction be detected in 2 hours earlier, reached higher level in 6 hours, and had at least 72 hours very long expression persistent period.Equally, the activatory cell culture studies of people synovial cell IL-1 α and TNF-α shows that the expression of COX-2 and the product of PGE2 (PGE2) significantly increase.Use various NSAIDS, can eliminate inductive PGE2 as ketoprofen treatment and reply.In the cultured chondrocytes system, specific cox 2 inhibitor compound N S-398 can prevent the increase by the PGE2 product of cytokine induction, that the level of COX-1 then keeps is stable (Morisset, S., 1998, J.Rheumatol.25:1146-53).Thereby, infer that blocking the PG generation by the activation chondrocyte can provide the cartilage protection effect, chondrocyte wherein is relevant with the expression of COX-2.
NSAIDS is normally used for the treatment of osteoarthritis or rheumatoid arthritis patients, but they are then also disputable to the metabolic effect of articular cartilage in these arthritis diseases.For example, be successful with NSAIDs clinical treatment rheumatoid arthritis and osteoarthritis for reducing inflammation.Yet; some does not have optionally NSAIDs to COX-2; mainly be Salicylate and sorbitrate, can promote osteoarticular cartilage to damage by the synthetic proteoglycan of infringement chondrocyte, other NSAIDs be then by stimulating repair of cartilage to have some cartilage protection effect.Many researchs confirm that all NSAIDS is very little or not effect to the effect of cartilage.Because the medicine that is used for the treatment of synovial membrane behind traumatic joint injury and the surgery operating wound and cartilage injury at present seldom, thereby need set up each NSAID and act on the special performance that causes the pathophysiological mechanism that cartilage damages.
Because pharmacology's difference of these two kinds of COX isozymes, thereby developed isozyme-specificity (selectivity) cyclooxygenase-2 inhibitor that is used for anti-inflammatory treatment, and in the arthritis model, tested the part of these identical cox 2 inhibitors.Yet, cox 2 inhibitor is to the synthetic and degeneration of cartilage proteoglycan, and IL-1 in the synovial membrane, IL-6, the interaction in vitro of the generation of IL-8 and prostaglandins shows, significant variation can take place to the effect that interleukin and eicosanoid in cartilage in the body and the synovial membrane produce in some NSAIDs, and the comprehensive function of these parameters can influence the effect of cox 2 inhibitor to the cartilage integrity.For example, some NSAIDS is owing to the synthetic joint injury of rheumatoid arthritis of quickening of generation that improves proinflammatory cytokine or inhibition cartilage proteoglycan.Yet although the clinical effectiveness of COX-2 specific inhibitor may change, the inhibition of COX-2 typically alleviates synovitis, and alleviates the danger that cartilage damages.
Carried out various biochemistrys, cell, and animal experiment are with the relative selectivity of assessment COX-1 and COX-2 homotype inhibitor.Usually, defining standard optionally is to suppress the ratio of constant (or COX-2/COX-1) with respect to the COX-1/COX-2 that given biochemistry or test cell line system obtain.This optionally ratio can be used for illustrating the absolute IC that different enzymatic activitys suppresses 50Value, it obtains (for example, the platelet and the macrophage system of stably express recombined human COX isozyme) between microsome and test cell line system.And the inhibition simulation of COX-2 is by cartilage protection (inhibition) cytokine, and as the inhibitory action that IL-4 excites, COX-2's is synthetic in its downward modulation born of the same parents.Selectivity contrast more than 45 NSAIDs and cox 2 inhibitor has shown COX-2 putting in order to the COX-1 relative selectivity: DuP697＞SC-58451=celecoxib＞nimesulide=meloxicam=piroxicam=NS-398=RS-57067＞SC-57666＞SC-58125＞flosulide＞etodolac＞L-745,337＞DFU-T-614 has the IC of 7nM-17 μ M 50Value.
According to selective COX-2-inhibitor 2; molecule and cytosis mechanism as celecoxib and rotecoxib; and according to zooscopy, expectation is when being directly used in the joint with these chemical compounds with irrigating solution or injection form peri-operation period, and these chemical compounds can show the cartilage protection effect.Particularly, the expectation cox 2 inhibitor is a kind of so effective medicine, and it send with the form of irrigating solution at the arthroscope intra-operative and passs, or before surgical procedures or other joint injury, among or afterwards, be injected directly in the joint.
The embodiment that is suitable for cox 2 inhibitor of the present invention lists below.Send (for example, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 5
The treatment of cyclooxygenase-2 inhibitor and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
rofecoxib (MK966) ????0.3-30,000 ????30-3,000
SC-58451 ????0.3-30,000 ????30-3,000
celecoxib(SC-58125) ????0.3-30,000 ????30-3,000
meloxicam ????0.5-50,000 ????50-5,000
nimesulide ????0.5-50,000 ????50-5,000
diclofenac ????0.3-30,000 ????30-3,000
NS-398 ????0.3-30,000 ????30-3,000
L-745，337 ????0.2-100,000 ????20-10,000
RS57067 ????0.2-100,000 ????20-10,000
SC-57666 ????0.2-100,000 ????20-10,000
flosulide ????0.2-100,000 ????20-10,000
6.MAP inhibitors of kinases
Mitogen-activated albumen (MAP) kinases is a histone serine/threonine kinase, it is to reply various born of the same parents' external stimulues, and performance being activated as the time spent from the cell surface transduction signal to nuclear.This map kinase cascade is a kind of main intracellular signal pipeline, and it is from somatomedin, and hormone and inflammatory cytokine transmit to middle early gene.Combine with other signal pipeline, these activatory mitogen-activator protein-kinases (MAPKs) change the activity and the phosphorylation state of transcription factor respectively, and the propagation of finally regulating cell, the variation and with cell replying to environmental stress.For example, the member of MAPK family (p38) is from potential proinflammatory cytokine, the alpha mediated main biochemical signals pipeline of IL-1 and TNF-, thus cyclo-oxygenase-2 (COX-2) in the irriate macrophage induced by relating to the cis acting factor that the COX-2 genetic transcription regulates.
The member of map kinase class reagent is by at least three tame group compositions, known their sequence, and the size of activation ring, and all different by the activation of born of the same parents' external stimulue, and they participate in different signal transduction pathways.Main member in the map kinase family comprises extracellular signal-regulated kinase (ERKs), ERK1 and ERK2 (being respectively p44MAPK and p42MAPK); Stress activated protein kinase 1 (SAPK1) family, it also is known as JNK or jun N-end kinases family; With p38MAP kinases family, it also is called as stress activated kinases 2/3 (SAPK-2/3).The P38 kinases is by stress be activated, is foremost proinflammatory cytokine.In p38 family, have four different congeners (isoform or isozyme) at least, the title of its standard is respectively SAPK2a, SAPK2b, SAPK2d, SAPK3, or p38 α, β, δ (SAPK4) and γ.Being used for map kinase inhibitor of the present invention can interact with above-mentioned arbitrary map kinase or its combination.For the specificity map kinase inhibitor, can in the concentration range of broad, change by the test method of vitro enzyme purification and the inhibition constant of test cell line method sign, and prove its function in this uses.The activation of p38 map kinase is by the dual phosphorylation mediation of threonine and serine residue.Proved with TNF α and IL-1 and handled all (in 5 minutes) increase phosphorylations and activate the p38 map kinase rapidly of cell.
Previous work has confirmed can to suppress the p38 map kinase specifically by micromolecular inhibitor, and (Nature 372:739-746 (1994) also produces the antiinflammatory action of biochemistry level in various animal models for Lee, J. etc.Cuenda and partner (Cuenda, A. etc., FEBSLett.264:229 (1995)) prove, chemical compound, SB203580[4-(4-fluorophenyl)-2-(4-methyl sulfinyl phenyl)-5-(4-pyridine radicals) imidazoles] vitro inhibition p38 (IC 50=0.6 μ M), suppresses the activation of the MAPK of activated protein kinase-2, and stop phosphorylation in the body of heatshock protein (hsp) 27 when replying IL-1 and cellular stress.The kinases selectivity that can suppress the SB203580 of p38 is by at least 15 other protein kinases, comprise PKC, PKA, (Lee, the J.Pharmacol.Ther.82:389-397 (1999)) that the faint inhibition that src and receptor serine kinase enzyme family are external or the failure of inhibition are confirmed.In cell research, cultivate IL-1 capable of blocking and inductive enzyme phosphorylation of TNF-α and the generation of IL-8 afterwards with SB203580 in advance.It send the preferred effect of passing inhibitor during being supported in surgical procedures.
Used specificity to suppress the SB203580 research p38 mitogen-effect of activated protein kinase (MAPK) in the biochemistry inflammatory response that causes cartilage to damage of this enzyme.IL-1, it is by p38 MAPK Selective Control, effect be to regulate prostaglandin H synthase-2 (COX-2), metalloproteases, and IL-6 (Ridley, S etc., 1997, J.Immunol.158:3165-73).In human fibroblasts and vascular endothelial cell, SB203850 suppresses (IC 50=0.5 μ M) phosphorylation of hsp 27 (the active indicant of a kind of p38 MAPK) in the inductive fibroblast of IL-1, and can not influence the activated protein kinase approach of other known IL-1 (p42/44 MAPK, p54 MAPK/c-Jun N-holds kinases).In addition, SB203580 can significantly suppress the IL-6 (during 1 μ M, about 30-50%) that IL-1 stimulates, and does not have IL-8 to produce from human fibroblasts and endotheliocyte.
Importantly, SB203580 suppresses consumingly that IL-1 excites, and the prostaglandin of fibroblast and human endothelial cell produces.It is relevant with the inductive inhibition of mRNA with COX-2 albumen.PGE 2Help to increase expression of MMPs mRNA, described matrix metalloproteinase is the important medium of cartilage degradation.When by cytokine and the activation of born of the same parents' external stimulue, synovioblast and chondrocyte all can be expressed the COX-2 gene high-levelly.The MAPK inhibitor is owing to its inhibition activity to map kinase provides cartilage protection activity, and wherein this map kinase is expressed in these and other cell type.
When general or local when being used for the joint tissue of various inflammatories or Pathophysiology disease, expectation MAPK inhibitor is effective as Chondroprotective agents.In many bodies, all described the characteristic of SB203580 in the pharmacological model, and proved that it had secular activity when oral administration.SB203580 can suppress the collagenase-1 of IL-1 generation and the stimulation of stromelysin-1, and does not influence the synthetic of TIMP-1.In addition, SB203580 can prevent collagenase-1 that IL-1-excites and the increase of stromelysin mRNA.In the cartilage damage model, the proteoglycan that short-term IL-1 excites absorbs heavily that synthetic inhibition is not influenced by SB203580 with proteoglycan, but can prevent secular collagen infringement.In addition, SB203580 can effectively suppress NO production in IL-1 inductive cattle articular cartilage explant and the chondrocyte (Badger 1998).These observation in vitro provide foundation for the cartilage protection activity of map kinase inhibitor, wherein this can by the map kinase general directly or topical administration in these joint tissues.
The p38 map kinase relates to the inductive cytokine-expressing of TNF-, and the generation (Beyaert, R. etc., EMBO are (1996) J.15:1914-23) of this medicine proinflammatory cytokine capable of blocking that plays a role as p38 map kinase activity inhibitor.Handle cell with TNF α and can activate p38 MAPK approach, this is confirmed by the increase of p38 MAPK self phosphorylation and the activation of substrate protein thereof.Can block the activation of MAPK of TNF α inductive activated protein kinase-2 and the phosphorylation of hsp27 fully with the SB203580 pretreatment cell.Under the same conditions, SB203580 also can suppress the synthetic expression that reaches reporter gene of the inductive IL-6 of TNF α fully, and it is to be driven by the minimal promoter that contains two kinds of NF-6B factors.Therefore, these researchs and the correlational study of other p38 inhibitor proved inhibitor, as SB203580 and FR133605 selectivity to p38 MAPK effect, its optionally hinder TNF-α and IL-1 inductive, with the activation of cartilage degradation proteins associated.Therefore, the map kinase signal pipeline that comes selectivity to suppress these important proinflammatory cytokines by the kinases that suppresses the receptor downstream has proved that map kinase inhibitor can provide the cartilage protection effect.
In the animal model of a lot of cytokines inhibition and inflammation disease, assessed SB203580.Confirmed that it is a kind of effective, has 15-25mg/kg IC 50The inhibitor that inflammatory cytokine produces in the rat of value and the mice body.50mg/kg has therapeutic activity for the SB203580 of amount of reagent in the collagen-induced arthritis of DBA/LACJ mice, it can significantly suppress the inflammation of pawl.When with 30 and 60mg/kg/ days dosage p.o. when giving SB203580, can in the inductive arthritis of Lewis rat adjuvant, observe antarthritic activity.Additional evidence is with respect to having 0.6 μ M IC 50Bone absorb heavily that beneficial effect obtains.
In a word, various biochemistrys, cell and zooscopy show that p38 MAPK plays an important role in the replying of IL-1 and TNF-α regulating, and it relates to some inflammatory response gene, as the adjusting of COX-2 mRNA level.The preparation of p38 inhibitor proinflammatory cytokine capable of blocking also suppresses the generation of MMPs, and has confirmed to suppress the damage of collagen in the cartilage explant.
The proinflammatory cytokine that the blocking-up of MAPK inhibitor is important, as the purposes of IL-1 and TNF-α effect for the many cell types in the joint, comprise synovioblast, the effect that macrophage and chondrocyte are all useful, thereby inhibition pathological effect afterwards, as the infiltration of inflammatory cell to the joint, the propagation of synovial membrane, synovial cell's activation and the damage of cartilage.Therefore, the MAPK inhibitor should disturb lysis thus by the propagation of aforementioned cytokine blocking-up inflammatory response.
Below the embodiment that is suitable for MAPK inhibitor of the present invention is listed in.Send (that is, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for listed reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 6
The treatment of map kinase inhibitor and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??SB?203580 ??0.5-50,000 ??50-5,000
SB 203580 iodine ??0.5-50,000 ??50-5,000
??SB?202190 ??0.2-20,000 ??20-2,000
??SB?242235 ??0.2-10,000 ??20-1,000
??SB?220025 ??0.2-10,000 ??20-1,000
??RWJ?67657 ??0.3-30,000 ??30-3,000
??RWJ?68354 ??0.9-90,000 ??90-9,000
??FR133605 ??1-100,000 ??10-10,000
??L-167307 ??0.5-50,000 ??50-5,000
??PD?98059 ??0.1-10,000 ??10-1000
??PD?169316 ??1-100,000 ??10-10,000
7. the inhibitor of matrix metalloproteinase
Articular cartilage damage is a joint disease, as a common trait in osteoarthritis and the rheumatoid arthritis, but also occurs in after the joint injury.Pathophysiology ground can be observed the structural failure of proteoglycan and collagen, the biological activity characteristic of its infringement cartilage.The keeping of the extracellular matrix of normal health can reflect that matrix components mixes and biosynthesis speed between balance, their deterioration velocity and subsequently from the cartilage to the synovial fluid in loss.Various protease all have the potentiality that cartilage is split, and it relates to degenerative process, particularly matrix metalloproteinase.
Matrix metalloproteinase (MMPs), or matrixins are the zinc endopeptidase of at least 15 of gangs, and it plays a role outside born of the same parents, and play an important role in histopathology is degenerated.Table 23 provides present name of MMP member and selectivity title.A lot of MMPs are all by altitude mixture control, and much all are not primary expressions in normal structure.Yet proinflammatory cytokine as IL-1 and TNF-α, excites transcript and expression.By the imbalance that activation and rise produced of the MMPs that makes tissue deterioration is in the synovial membrane inflammation answering that continues after chronic inflammatory diseases and the joint injury, the main reason factor of cartilage damage process.In the patient of knee meniscus injury or first Anterior Cruciate Ligament Ruptures, studied the metabolism of cartilage matrix.Stromelysin-1 (MMP-3), collagenase increases in tissue inhibitor of metalloproteinase (TIMP-1) and the segmental concentration of the proteoglycan people's knee synovial fluid after traumatic knee joint damage.Temporarily, these values increase immediately, surpass reference levels, and keep raise (10 times of increases) significantly in the time more than 1 year.These variations may promote the increase of proteoglycan fragment concentrations, and it is after the knee ligament damage, and is observed in synovial fluid.
Table 7
Matrix metalloproteinase
??MMP Alternative title EC number Substrate
??MMP-1 The collagenase of matter between collagenase fibroblast collagenase ??EC3.4.24.7 Collagen (I, II II, VII, and X); Gelatin; Aggrecan; The versican of hyaluronidase-processing; Proteoglycan connects albumen; Big tenascin-C; α 1-antitrypsin/-α 1Protease inhibitor (α 1-AT)；α 1Antichymotrypsin (α 1-ACHYM)； ??α 2M; Rat α 1M; Gestation district albumen; Rat α 1I3(α 1-inhibitor 3); Ovostatin; Nestin; MBP; GST-TNF/TNF peptide; L-selects; IL-1 β; Serum amyloid A; IGF-BP5; MMP-2; MMP-13
??MMP-2 72-kDa gelatinase gelatin enzyme A IV Collagen Type VI enzyme neutrophilia gelatinase ??EC3.4.24.24 Collagen (I, IV, V, VI, X, XI, and XIV); Gelatin; Elastin laminin; Fibronectin; EHS-laminin., Kallinin; Gelatin-3; Aggrecan; Decorin; The versican of hyaluronidase-processing; Proteoglycan connects albumen; Osteonectin; MBP; GST-TNF/ TNF peptide; IL-1 β; A β 1-40；Aβ 10-20； ??APP 695；α 1-AT; Prolysyl oxidase fusion rotein; IGF-BP5; IGF-BP3; FGF R1;
MMP-1；MMP-9；MMP-13
MMP-3 Stromelysin-1 Transin ?EC?3.4.24.17 Collagen (III, IV, V, IX); Gelatin; Assemble proteoglycan; The versican of versican and hyaluronidase-processing; Perlecan; Decorin; Proteoglycan connects albumen; Big tenascin-C; Fibronectin; Laminin; Nestin; Osteonectin; Elastin laminin; Casein; α 1-ACHYM; Antithrombase-III; α 2M; Ovostain; The P material; MBP; GST TNF/TNF peptide; IL-1 β; Serum amyloid A; IGF-BP3; Fibrinogen and commissure fibrin; Plasminogen; MMP 1 " superactivation " MMP-2/TIMP-2 complex; MMP-7; MMP-8; MMP 9; MMP-13
MMP-7 Matrilysin PUMP ?EC?3.4.24.23 Collagen iv and X; Gelatin; Aggrecan; Decorin; Proteoglycan connects albumen; Fibronectin and laminin; Solubility fibronectin fibril; Enactin; Big and little tenascin-C; Osteonectin; β 4 integrins; Elastin laminin; Casein; Transferrins; MBP; α 1-AT; The GST-TNF/TNF peptide; Plasminogen; MMP-1; MMP-2; MMP-9; MMP-9/TIMP-1
MMP-8 Neutrophil collagenase collagenase I ?EC?3.4.24.34 Collagen (I, II, III, V, VII and X); Gelatin; Aggrecan; α 1-AT；α 1-ACHYM； α 2-antifibrinolysin; Fibronectin
MMP-9 92kDa gelatinase gelatinase B ?EC?3.4.24.35 Collagen (IV, V, VII, X and XIV); Gelatin; Elastin laminin; Galectin-3; Aggrecan; The versican that hyaluronidase is handled; Proteoglycan connects albumen; Fibronectin; Enactin; Osteonectin; α 1-AT；MBP；GST-TNF/TNF
Peptide; IL-1 β; A β 1-40 Plasminogen
MMP-10 stromelysin-2 EC?3.4.24.22 Collagen (III, IV and V); Gelatin; Casein; Aggrecan; Elastin laminin; Proteoglycan connects albumen; MMP-1; MMP-8
MMP-11 stromelysin-3 People's enzyme: α 1-AT；α 2M; Casein, laminin, fibronectin, gelatin, collagen iv and carboxymethylated transferrins
MMP-12 macrophage Metalloclastase Collagen iv; Gelatin; Elastin laminin and κ-elastin laminin; Casein; α 1-AT; Fibronectin; Vitronectin; Laminin; Enactin; The proteoglycan monomer; GST-TNF; MBP; Fibrinogen; Fibrin; Plasminogen
MMP-13 collagenase-3 Collagen (I, II and III, IV, IX, X and XIV); Gelatin, α 1-ACHYM and PAI-2; Aggrecan; Perlecan; Big tenascin-C; Osteonectin; MMP-9
MMP-14?MT-MMP-1 Collagen (I, II and III); Gelatin, casein, κ-elastin laminin; Fibronectin, laminin, vitronectin and proteoglycan; Big tenascin-C, enactin; α 1-AT，α 2M；GST-TNF；MMP-2；MMP-13
MMP-15?MT-MMP-2 Fibronectin, big tenascin-C, enactin, laminin, aggrecan, perlecan; GST-TNF; MMP-2
The enzyme of MMP family is from human chondrocytes and synovial cell, as excretory in the synovioblast.In addition, use in situ hybridization, people's synovial membrane can synthesize stromelysin-1 and collagenase.Stromelysin-1 (MMP-3) can make all components of cartilage matrix degenerate.Prove chondrocyte on evidence by making the enzyme of substrate degeneration, the release of collagenase-3, and help cartilage degradation.Secrete in the cell of MMPs with the form of hiding, outside born of the same parents, be activated, and suppressed by the tissue depressant of metalloproteases (TIMPs).Balance between MMPs and TIMPs activity is very important for the maintenance of complete cartilage matrix.In pathological conditions, under the condition as osteoarthritis and rheumatoid arthritis, many researchs have all proved the rising of MMPs amount, and it causes the imbalance between MMPs and TIMPs, and this imbalance is considered to the observed reason of damaging to cartilage.
MMPs is by cytokine, regulates as il-1 (IL-1) and somatomedin, and it acts on chondrocyte and synovial cell, thereby improves the generation of their protease.Other proinflammatory cytokine, as IL-6, IL-8 and TNF-α 2Also can raise the generation of substrate-degeneration enzyme.
It causes cartilage to damage, and it is normally assessed by the division of the loss of sulfated glycosaminoglycans (GAGs) and collagen.IL-1, it is present in arthrosis patient's the joint fluid, stimulates the synthetic enzyme of chondrocyte, as stromelysin, fibroblast and neutrophilia collagenase, and the amount of plasminogen activator increases.In addition, IL-1 suppresses the inhibitor-1 of plasminogen activator and synthesizing of TIMP, also suppresses the synthetic of matrix components such as collagen.Imbalance between inhibitor and enzyme level causes the amount of active protease to increase, and combines with the biosynthetic inhibition of substrate, causes cartilage degradation.
Use the cartilage section as external model, the cartilage lesion that verified collagenase inhibitors can excite by rheumatoid synovioblast (RSF) inhibition IL-1 or IL-8.Collagenase inhibitors, 1,10-neighbour-phenanthrenone and phosphoramidon have suppressed 10-150 μ M concentration RSF cell basically and have penetrated in the cartilage in the mode that concentration relies on.Cytokine has proved that to the excretory selectively acting of protease the degenerative joint of synovial membrane fibroblast-like cells mediation is a kind of process of altitude mixture control.Therefore, expectation is local suppresses the ability that proteinase activity in the joint and relevant joint matrix degenerate and can suppress the cartilage damage process.The effect of inhibitor in limited vitro system shows that it is effective as Chondroprotective agents that the treatment intervention of passing the synthetic MMP inhibitor with suitable pharmacokinetics is sent in the whole body part.
The embodiment that is suitable for MMP inhibitor of the present invention comprises U-24522 ((R, S)-N-[2-[2-(hydroxyl amino)-2-oxygen ethyl]-4-methyl isophthalic acid-oxygen amyl group]-the L-leucyl-L-phenylalaniamid), peptide such as MMP inhibitor I and MMP-3 inhibitor, bigger albumen, as TIMP-1 or its fragment, and it is listed in the following table: send for all parts and pass (that is injection,, infusion and lavation) form, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred and most preferable concentrations that contains listed reagent irrigating solution, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 8
The treatment and the preferred concentration of matrix metalloproteinase (MMPs) inhibitor
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
U-24522 ????0.2-2,000 ????20-200
Minocycline ????30-500,000 ????300-3,000
MMP inhibitor I 4-Abz-Gly-Pro-D-Leu-D-Ala-NHOH ????0.3-3,000 ????3-600
MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH 2 ????0.5-5,000 ????5-500
Rhuman?TIMP1 ????0.5-5,000 ????5-500
Rhuman?TIMP2 ????0.5-3,000 ????3-600
8. the inhibitor of nuclear factor κ B (NF κ B)
Short inflammation and cartilage injury's cellular pathways is outer and intracellular signal mechanism adjusting by born of the same parents, and it is that new therapeutic part and systemic drug send the target of passing.Utilize by proinflammatory cytokine il-1 (IL-1), but activating transcription factor, and the complete molecular signal mechanism of nuclear factor κ B (NF κ B) is defined relatively poorly.Yet the important molecule relevant with the intracellular signal of gene transcription level is short scorching transcription factor, (NF κ B).The activity of NF κ B is mediated by gang's transcription factor subunit, and it can combine with the DNA of homodimer or heterodimer form.Because be known as the combination of the inhibition subunit of I κ B, these subunits are present in the cell cytoplasm with inactive form typically.The NF κ B that the activation of IL-1 receptor and other extracellular signal can cause the degraded of I κ B and association separating from the inhibitor, transfer in the nuclear then.NF κ B relates to the inductive expression of IL-1, and can increase the short expression of scorching COX-2 albumen in the RA synovioblast.
NF κ B is with its basis that act as the common downstream signal factor as the evaluation of important molecule target, and this common downstream signal factor scalable is many damages the gene expression of the relevant important inflammatory mediator of approach with arthritis and cartilage.Many genes (COX-2, collagenase, IL-6, replying all IL-8) arranged by promoter, and this promoter comprises the NF κ B promoter factor.The activation of NF κ B can mediate among the synovial cell, and is many for very important proteic the inducing of inflammatory process, as cytokine, and cell-adhesion molecule, metalloproteases and other participate in the albumen that Prostaglandins and Leukotrienes (COX-2) produces among the synovial cell.Thereby this transcription factor is represented very important target on a kind of physiology, and it directly aims at people's synovioblast in treatment, person joint's chondrocyte, and other joint cell replying damage.
Particularly, the exposure of reference's rheumatoid synovioblast (RSF) on interleukin-11 β (IL-1 β) causes the common rise of phospholipase A2 (PLA2) and the induction type cyclo-oxygenase (COX-2) of 85kD.These two enzymes all can promote PGE subsequently 2, a kind of biosynthesis of main inflammatory mediator in the joint.Oligonucleotide trapper and antisense are used to confirm that NF κ B has participated in the adjusting of prostaglandins metabolic enzyme.Use the mRNA of antisense oligonucleotide antagonism NF κ B, can cause and the bonded minimizing of COX gene promoter.
Hymenialdisine, a kind of natural marine product, recently verified it be the activation of NF κ B, and the PGE2 product that the RSF that stimulates of IL-1 suppresses relies on the inhibitor that mode (IC50=1 μ M) exposes with concentration.The specificity of molecule target is by the use analog, aldisine, and inhibitors of protein kinase C, and RO32-0432, it is inactive, proves.Activated direct effect is remarkable minimizing by the NF κ B that combines with typical κ B consensus sequence motif to Hymenialdisine to the inductive NF κ of IL-1 B, and the inhibition that the p65 of irriate moves from the cytosol of processing cell is confirmed.As the inhibitor of desired a kind of NF κ B transcriptional regulatory, COX-2 that the RSF that hymenialdisine-handles can not transcription response IL-1 or the mRNA of PLA2.Therefore, can be observed the decline of protein level of these enzyme inductions and the reduction that produces the inducibility of PGE2.In addition, interleukin-8 (IL-8) product that IL-1-stimulates, known its is that a kind of NF κ B-regulates incident, also suppress by hymenialdisine, and the inductive VEGF of IL-1, a kind of non--gene that NF κ B-regulates, generation be not exposed to the influence of hemenialdisine.Therefore, hymenialdisine can suppress the formation of PGE2 in the synovioblast that IL-1-stimulates by it to the activated inhibitory action of NF κ B.It is for defining it as new inhibitor, and the effect of blocking-up NF κ B in arthritis and cartilage injury provides the foundation.
Below the embodiment that is suitable for NF kB inhibitor of the present invention is listed in.Send (that is, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for listed each reagent, provide the preferred and most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 9
The treatment of NF kB inhibitor and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
Caffeic acid phenylethylester (CAPE) ??1-100,000 ??50-20,000
??DM-CAPE ??0.5-50,000 ??50-5,000
The SN-50 peptide ??0.1-100,000 ??100-20,000
??Hymenialdisine ??1-100,000 ??100-10,000
??Pyrolidone?dithiocarbamate ??1-50,000 ??50-10,000
9. nitric oxide synthase inhibitors
Nitric oxide (NO) is in a kind of born of the same parents of being distributed widely in and the outer medium of born of the same parents, and it relates to the pathophysiological mechanism of some linked groups's disease.NO is by gang's enzyme, and the NO synthase forms from the L-arginine, and this enzyme is localized in the born of the same parents.Three kinds of isoforms of the NO synthase that can check order have been cloned.Endotheliocyte NO synthase (ecNOS) and brain NO synthase (bNOS) are active basically.The isoform that the NO synthase is different, induced NOS (iNOS) can comprise in the chondrocyte and finding in the various kinds of cell type.Seldom, but it is raised when replying pro-inflammatory mediator such as IL-1 β and TNF-α under basic condition for it.Recent discovery shows that IL-1 is a kind of stimulant of very effective chondrocyte NO synthase, and IL-1 plays a role by the level that it raises iNOS.At intraarticular, chondrocyte is the main source of NO, and is considered to mediate the multiple effect of IL-1 in the inflammatory arthropathy by the inductive chondrocyte iNOS of proinflammatory cytokine.
The medicine that specificity suppresses chondrocyte induction type NO synthase (iNOS) has the therapeutical effect that prevents because of the cartilage injury of joint injury (for example, the surgical procedures in joint) generation.The evidence of supporting this useful therapeutic effect is based on many basic researches, this basic research assessed various iNOS suppress the ability of the induction type NO synthase activity in the chondrocyte of cultivating and the osteoarthritis patient cartilage explant.One compounds is called as the isothiourea that S-replaces, and is a kind of effective, the biosynthetic inhibitor of NO in the cartilage.The effective sex ratio N of S-methyl isothiourea and S-(amino-ethyl) isothiourea G-monomethyl-high 2-4 of L arginine doubly than the high 5-10 of aminoguanidine doubly, compares N ω-nitro-L-arginine and N ω-nitro-L-arginine methyl ester is high more than 300 times.These isothiourea chemical compounds can provide a kind of and effectively the iNOS in the cartilage be had the inhibitor of relative specificity, thus be suitable for whole body of the present invention or part send and pass (Jang, D., 1996, Eur.J.Pharmacol.312-341:347).
Use vitro system,, thereby can assess the treatment effectiveness of the cartilage protection of NO synthase inhibitor as the effect of isolating chondrocyte definition to cartilage matrix.Pass through N G-monomethyl-L-arginine (L-NMMA), a kind of disclosed NO synthase inhibitor suppresses the generation of endogenous NO, can suppress the gelatinase that chondrocyte produced, collagenase and stromelysin by IL-1 β-stimulation.The generation that suppresses NO also can partly reduce the increase of lactate product, and wherein the lactate product occurs from the exposure of chondrocyte on IL-1 β.Handle cartilage fragment with L-NMMA, can make IL-1 β suppress the synthetic effect of glycosaminoglycans and reverse, suppress the MMP activity that IL-1 β stimulates, increase the generation of IL-1 receptor antagonist (IL-1ra).NO also can pass through to reduce the generation of TGF-β 1 and synthesizing of indirect regulation proteoglycan, and wherein TGF β 1 is owing to the exposure of chondrocyte on IL-1 β produces.It also can prevent the increase of the TGF β 1 of autocrine-stimulation, thereby eliminates the anabolic action of the cytokine in this cartilage.
New aminoguanidine has been compared in a research, S-methyl isothiourea (SMT), inhibiting effectiveness when S-amino-ethyl isothiourea (AETU), L-NMMA and NG-nitro-L-arginine methyl ester (L-NAME) no inhibitor produce with NO recombined human IL-1 β response protein polysaccharide is synthetic.Different culture systems is replied IL-1 β invasion and attack in concentration dependence mode, and wherein the NO product increases greatly, and the synthetic quilt of proteoglycan suppresses significantly.Above-mentioned no inhibitor (1-1000 μ M) has suppressed the NO that chondrocyte produced that handles with IL-1 β, and it syntheticly has a significant effect to what recover proteoglycan in the chondrocyte.Therefore, if use the generation of treatment valid density and dosage NO capable of blocking, can prevent the synthetic of IL-1 β Profilin polysaccharide so.
The NO synthase is to express in the cartilage that obtains from arthritis patient joint.In rheumatoid arthritis or osteoarthritis patient, the level that can be observed nitrite in the synovial fluid increases, and has proved that the important source of NO product among these patients is to derive from articular cartilage.In addition, L-NIL, a kind of lasting whole body of effective iNOS inhibitor send and pass the development that can alleviate the tentative OA of Canis familiaris L. (being inductive by the dissection of ACL), and cause IL-1 β, PGE 2, the basic decline of NO and MMP product.These discoveries hinted NO be a kind of effectively, the regulator of IL-1 β effect, and help the pathophysiology of joint disease.
Therefore; the result shows that specific NO synthase inhibitor is the effective novel drugs of clinical treatment synovitis in these external and bodies; and when itself and one or more drug regimen whole body or part being sent when passing; the cartilage protection effect can be provided; one or more medicines wherein are selected from the antiinflammatory in the joint that is used for the treatment of joint that surgical treatment crosses or other damage; cartilage protection and anti-pain class medicine.
Below the embodiment that is suitable for NO synthase inhibitor of the present invention is listed in.Send (that is, injection, infusion and the lavation) form of passing for all parts, it is that treatment is effective that the best of each suitable reagent is given amount of reagent and/or concentration.As an embodiment, for each listed reagent, provide the preferred concentration and the most preferable concentrations of the irrigating solution that contains listed reagent, expect that this concentration is that treatment is effective.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.In one embodiment, the preferred NO synthase inhibitor that is used for solution of the present invention is 1400W ((N-3-(amino methyl) benzyl) ethanamidine), and is optionally a kind of, slowly, the iNOS inhibitor of combining closely, diphenyleneiodinium and 1,3-PBIT.
Table 10
The treatment of nitric oxide synthase inhibitors and preferred concentration
Chemical compound The part is sent and is passed Most preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
??N G-monomethyl-L-arginine ??50,50,000 ??3,000
??1400W-diphenyleneiodium ??0.1-1,000 ??1-20
The S-methyl isothiourea ??1-1,000 ??10-100
S-(amino-ethyl) isothiourea ??1-1,000 ??10-100
??L-N 6-(1-imino group ethyl) lysine ??1-1,000 ??10-100
??1，3-PBITU ??0.5-500 ??5-100
2-ethyl-2-thiopseudourea ??2-20,000 ??20-2,000
10. cell adhesion molecule
The agonist of 10a integrin and antagonist
Integrin is the heterodimer that is positioned on the plasma membrane, and it comprises the α and the β subunit of binding partner, and this part is that extracellular matrix (ECM) composition maybe can be other big albumen, as collagen, laminin, vitronectin, bone connect albumen (OPN) and fibronectin (FN).The degeneration of cartilage matrix is according to these cells and the interactional mechanism of ECM, is regulated by chondrocyte.The chondrocyte expression of gene is, partly, controls by cells contacting, and this cell is in conjunction with the interaction that comprises the ECM composition in integrin and the chondrocyte surrounding.Therefore, the integrin on the chondrocyte relates to the control of cartilage homeostasis, and on behalf of a class, this family's receptor can prevent the target of the treatment of cartilage degradation.
Human chondrocytes can be expressed a series of by different α and β subunit, comprises α 1β 1, α 5β 1, α V β 1Measure the integrin receptor that less subunit is formed with other.α V β particularly importantly wherein 3Integrin, known its in conjunction with OPN.The α V β of blocking-up monoclonal antibody (mAb) LM609 3Complex-specific function works as the agonist with part OPN similar form.It can reduce a lot of short scorching and cartilage damage media, as IL-1, and the generation of NO and PGE2.Therefore, agonist mAb LM609 has the present invention of being suitable for to use.
In addition, two kinds of peptide mimicses have been studied, MK-383 (Merck) and RO-4483 (Hoffmann-LaRoche) in clinical in the II phase.Because these all are micromolecule, so their half-life is shorter, usefulness is higher.As if yet they and the interactional specificity of other closely-related integrin are less.These peptide mimicses also are suitable for the present invention.
Table 11
The treatment of integrin and preferred concentration
The kind of reagent: The part is sent and is passed Preferred part is sent and is passed
Treatment concentration (μ g/ml) Concentration (μ g/ml)
Integrin:
??αVβ 3?mAB?LM?609 ??0.05-5,000 ??5-500
Echiststin (echistatine) ??0.1-10,000 ??100-1,000
11. it is anti--chemoattractant
Anti-chemoattractant can prevent the chemotaxis of inflammatory cell.The representative embodiment of the anti-chemotactic target that can play a role comprises, but is not restricted to F-Met-Leu-Phe receptor, IL-8 receptor, MCP-1 receptor and MIP-1-I/RANTES receptor.Very early after deliberation the medicine in this type of reagent, but they are applicable to that situation of the present invention is not also theorized.
12. intracellular signal inhibitor
12a. kinases inhibitor
I. protein kinase (PKC) inhibitor
Play an important role in the cell surface signal transduction of Protein kinase C (PKC) in many physiological process.The PKC isozyme can be used as the downstream target and is activated, and this target is caused by the g protein coupled receptor (for example, 5-hydroxy tryptamine, Kallidin I etc.) or the initiate activation of proinflammatory cytokine.These acceptor species all play an important role in the mediation cartilage degradation.
The molecular cloning analysis shows that PKC exists as a big nation that is made up of at least 8 subspecies (isozyme).The structure of these isozymes and connection receptor activation are different substantially with the mechanism of the variation that specific cell propagation is replied.The specificity isozyme can be expressed at various cell types, comprises the synovial cell, chondrocyte, neutrophil, medullary cell, and smooth muscle cell.Therefore the inhibitor of PKC influences the signal pipeline of various kinds of cell type probably, unless this inhibitor has the isozyme specificity.Therefore, the inhibitor of measurable PKC is effectively for the activation of blocking-up synovial cell and chondrocyte, and may have that the blocking-up neutrophil activates and with the antiinflammatory action of postadhesion.Described a lot of inhibitor, initial report shows, the IC of 50 μ M 50Can be used for suppressing the activity of calphotin C.G-603 (also being Go 6976) is a kind of new, effective pkc inhibitor, and it has 2-10 μ M IC for some 50The PKC isoform of value has the selectivity of height.List these and other drug concentrations below, GF109203X also is Go 6850 or bisindolylmaleimidesfor (buying from Warner-Lambert), believes that they are applicable to the present invention.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 12
Cartilage damages the treatment concentration and the preferred concentration of inhibitor
Reagent type The part is sent and is passed Preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
Inhibitors of protein kinase C:
Calphotin C ??0.5-50,000 ??100-5,000
??GF?109203X ??0.1-10,000 ??1-1,000
??G-6203(Go?6976) ??0.1-10,000 ??1-1,000
Ii. the inhibitor of protein tyrosine kinase
Though exist huge difference between a lot of receptor tyrosine kinase (RTK) family members, the signaling mechanism of these receptors has many common features.Biochemistry and molecular genetic studies show that part and combining of RTK extracellular domain can activate the catalytic activity (see figure 5) of the inherent tyrosine kinase of born of the same parents' internal area rapidly.Active increase causes the tyrosine-specific phosphorylation of substrate in many born of the same parents, and substrate comprises a consensus sequence motif in the born of the same parents wherein.Therefore, it causes the activation of approach cascade in a lot " downstream " signaling molecule and born of the same parents, approach cascade scalable phospholipid metabolism in these born of the same parents wherein, arachidonate metabolism, protein phosphorylation effect (comprising the mechanism except that protein kinase), calcium mobilization and transcription activating (see figure 2).The activity of the growth factor dependency tyrosine kinase of RTK cytoplasm domain is the main mechanism that intracellular signal produces, and it causes cell proliferation.Therefore, inhibitor has the effectiveness of this signal of blocking-up, and prevents the activation of synovial cell and chondrocyte thus.
Any one of various tyrosine phosphorylation inhibitory action chemical compounds is as tyrosine kinase activity (the external IC in the 0.5-1.0 μ M scope 50) specific inhibitor all have effectiveness, this is because they are to the almost not effect of other protein kinase and other signal transduction system.Only have sub-fraction tyrosine phosphorylation inhibitory action chemical compound to obtain from commercial, the suitable concentration that is used for these reagent of the present invention is listed below.In addition, reported D-82041 DEISENHOFEN, and confirmed its potential inhibitory action to the many protein tyrosine kinases of src superfamily, the suitable concentration that is used for this reagent of the present invention is listed below.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 13
The treatment of inhibitor and preferred concentration
Reagent type The part is sent and is passed Preferred part is sent and is passed
Treatment concentration (nM) Concentration (nM)
Kinases inhibitor
Lavendustin A ????10-100,000 ????100-10,000
Tyrphostin AG1296 ????10-100,000 ????100-10,000
Tyrphostin AG1295 ????10-100,000 ????100-10,000
D-82041 DEISENHOFEN ????1-100,000 ????10-1,000
PD?158780 ????0.1-10,000 ????10-500
PD?174265 ????0.1-10,000 ????10-500
12b. the regulator of intracellular protein tyrosine phosphatase
Contain the src-congener 2Non--transmembrane protein the tyrosine phosphatase (PTPases) in SH2 territory is known, claims that according to nomenclature they are SH-PTP1 and SH-PTP2.In addition, SH-PTP1 is considered to PTP1C, HCP or SHP.SH-PTP2 is considered to PTP1D or PTP2C.Equally, SH-PTP1 in the hematopoietic cell of all pedigrees neutralizes all variation stages all with high level expression, and identified that the SH-PTP1 gene is responsible for the phenotype of motheaten (me) mice, and provide the basis of prediction inhibitor effect, the interaction of this inhibitor itself and cell substrate capable of blocking.Stimulate neutrophil can cause the activation of tyrosine kinase with Chemotactic Peptide, this tyrosine kinase can mediate neutrophilia and reply (Cui, et al., 1994 J.Immunol) and the activity of PTPase, and by reversing the activity of regulating agonist induction in the effect of the activated tyrosine kinase of initial cell stimulation period.Can stimulate the active reagent of PTPase to have potential treatment as the antiinflammatory medium uses.
These identical PTPase can also regulate the activity of some RTKs.They seem the kinase whose effect of reverse balance activated receptor, thereby can represent the target of important drugs.In vitro tests confirms, injection PTPase insulin stimulating capable of blocking, the phosphorylation of tyrosyl residue on the intrinsic protein.Therefore, but the effect of the active activator Reverse Activity of PTPase RTK receptor in restenosis believed it to be used for solution of the present invention.In addition, with those cell adhesion molecule homoplastics, receptor-connection PTPase also can be used as the outer part of born of the same parents and works.The bonded functional outcome of part and extracellular domain also is not defined, but this hypothesis in conjunction with scalable endocellular phosphorus phytase activity be reasonably (Fashena and Zinn, 1995, Current Biology, 5,1367-1369).This effect adhesion by other cell surface adhesion molecule (NCAM) mediation capable of blocking, and a kind of antiinflammatory action is provided.So far also do not develop the medicine that is used for these purposes.
The inhibitor in territory 12c.SH2 (src congener 2 territories)
The SH2 territory is to identify in the src subtribe of protein tyrosine kinase (PTKs) at first, is non-catalytic protein sequence, and forms (Cohen etc., 1995) by about 100 aminoacid of preserving in various signal transducers.The SH2 territory plays a role as phosphotyrosine-binding molecule, and conclusive protein-protein associate (Pawson, Nature, 573-580,1995) in the signal transduction pathway in the mediated cell thus.Particularly, the effect that has clearly defined the SH2 territory is extremely important for the signal of receptor tyrosine kinase (RTK) mediation, as under the situation of platelet-derived somatomedin (PDGF) receptor.Autophosphorylation RTKs goes up the site of phosphoric acid tyrosine can play the SH2-protein binding site, and mediates the activation (see figure 2) (Carpenter, G., FASEB J.6:3283-3289,1992 of biochemical signals pipeline thus; Sierke, S. and Koland, J.Biochem.32:10102-10108,1993).The SH2 territory can be responsible for activatory growth factor receptors is coupled on the cell response, and this cell response comprises the change of gene expression and final cell proliferation.Therefore, predicted that the inhibitor of optionally blocking in the specificity RTKs that expresses on the synovial cell surface (get rid of IGFR and FGFR) activation can effectively block cartilage degradation after the arthroscopy process.
Identified at least 20 cytoplasmic proteins, it comprises the SH2 territory and plays a role in intracellular signal.The distribution in SH2 territory is not limited in a special albumen family, but can be at multiclass albumen, protein kinase, and the lipid kinases, phosphoprotein phosphatase, phospholipase finds in the albumen of control Ras and some transcription factor.The albumen of many SH2 of containing all has known enzymatic activity, other (Grb2 and Crk) then play a part " connector " and " conjugant " between cell surface receptor and " downstream " effector molecule (Marengere, L., etc., Nature 369:502-505,1994).Contain the SH2 territory, have enzymatic activity, and the proteic embodiment that is activated in signal transduction comprises, but be not restricted to the src subfamily (src (pp60 of protein tyrosine kinase C-src), abl, lck, fyn, fgr and other), phospholipase C γ(PLC γ), phosphatidyl-inositol 3-kinase (PI-3-kinases), p21-ras GTPase, its activator protein (GAP) and SH2-containing protein tyrosine phosphatase (SH-PTPases) (Songyang etc., Cell 72,767-778,1993).Because having from the activating cell surface receptor, these different SH2-albumen transmit signals to the important function of adding the interactional cascade of molecule; its final definition cell response; therefore block specificity SH2 protein binding, for example the inhibitor of c-src has potential therapeutic use in cartilage protection.
In addition, the adjusting of many immunity/inflammatory responses is by receptor-mediated, and this receptor is by containing the nonreceptor tyrosine kinase transmission signal in SH2 territory.(TCR) activates the T-cell by antigen specific T-cell receptor, can excite the signal transduction cascade that causes lymphokine secretion and T-cell proliferation.To reply be the increase of tyrosine kinase activity for the earliest a biochemistry after TCR activated.Particularly, the activation of neutrophil partly is to control by replying of cell surface IgG-R.The activation of these receptors can mediate did not identify activation with the tyrosine kinase in SH2 territory.Additional evidence shows that (lck, blk fyn) all participate in therefore can integrating the stimulus object that obtains from many independent receptor structures from the signal transduction pathway of cytokine and integrin receptor guiding many src-family kinases.Therefore, the inhibitor in specificity SH2 territory has the potentiality of the multiple neutrophil function of blocking-up, and plays the antiinflammatory medium.
The work that the medicine in SH2 territory is aimed in development is to carry out at external biochemistry and cellular level.This effort should be that successful, final medicine ought to be effective in practice of the present invention.
V. other reagent
Except above-mentioned Chondroprotective agents, the compositions of part of the present invention or systemic delivery can also comprise other therapeutic agent.For example, can comprise one or more antiinflammatories or analgesics (being also referred to as anti-pain agent herein).The suitable example of antiinflammatory and/or analgesics has a detailed description hereinafter.As another example, compositions of the present invention can comprise the antirheumatic (DMARDs) of one or more mitigate the disease, as methotrexate, sulfasalazine, gold compound, as oral gold, golden apple acid sodium and aurothioglucose, azathioprine, ciclosporin, antimalarial, steroidal class, colchicine, cyclophosphamide, hydroxychloroquine sulfate, leflunomide, minocycline and penicillamine.Antiinflammatory, analgesia and/or DMARD agent can be included in the compositions of the present invention, or can while or sequential ground separate administration.
Relate to the front at the systemic administration of the local application relevant, guiding with Chondroprotective agents and of the present invention many aspects of the benefit of using of plurality of reagents also be applicable to using of other reagent.Pain and the misery of alleviating operation back patient are in the clinical medicine, particularly the outpatient who carries out in the every year special focal area in the ever-increasing situation of quantity of performing the operation.The most widely used systemic agents, cyclooxygenase-2 inhibitor (for example, ibuprofen) and opioid (for example, morphine, fentanyl) have pronounced side effects, comprise gastrointestinal irritation/hemorrhage and respiration inhibition.In postoperative one period, the high rate of the nausea and vomiting relevant with opioid is a difficult problem especially.Thereby with the treatment postoperative pain avoid harmful side effect be the treatment reagent of purpose and be not easy the development, reason is that the molecule target of these reagent is dispersed throughout whole body, and mediates different physiological actions.Although inhibition of pain, the clinical needs of inflammation and cartilage degradation are apparent, but fail to develop effectively to transmit the inhibitor of pain, inflammation and cartilage degradation to amount of reagent, make disadvantageous systemic side effects reach minimum method simultaneously.As an example, frequently give routine (that is, intravenous, oral, the subcutaneous or intramuscular) method of opiate, usually, comprise serious respiration inhibition with significant adverse side effect with therapeutic dose, mood change, the nausea and vomiting of the mental fog and the degree of depth is relevant.
Research in the past is verified some endogenous reagent, as 5-hydroxy tryptamine (serotonine is called " 5-HT " herein sometimes), Kallidin I and histamine produce the ability of pain and inflammation.Sicuteri, F. etc., 5-hydroxy tryptamine-bradykinin be to the potentiation of people's pain receptor, life sciences (Life Sci) 4, pp.309-316 (1965); Rosenthal, S.R., histamine are as the chemical mediator of the skin pain sensation, and skin is studied magazine (J.Invest.Dermat.) 69, pp.98-105 (1977); Richardson, B.P. etc., the evaluation of 5-hydroxy tryptamine M-receptor subtype and a kind new medicine thing be to its specific blocking effect, nature (Nature) 316, pp.126-131 (1985); Whalley, E.T. etc., the effect of kassinin kinin agonist and antagonist, Naunyn-Schmiedeb pharmacological literature (Naunyn-Schmiedeb Arch.Pharmacol.) 36, pp.652-57 (1987); Lang, E. etc. are at the meticulous chemosensitivity that imports into of external arrogant Corium Mus skin.Neuro physiology magazine (J.Neurophysiol) 63, pp.887-901 (1990).
For example, the verified 5-HT that is used for people's vesicle (stripping off property skin) can cause by 5-HT 3The pain that receptor antagonist suppresses, Richardson etc., (1985).Equally, the peripheral applications bradykinin can produce the pain of being blocked by the bradykinin receptor antagonist.Sicuteri etc., 1965; Whalley etc., 1987; Dray, A., etc., bradykinin and inflammatory pain, neuroscience trend (Trends Neurosci.16, pp.99-104 (1993)).The peripheral applications histamine can produce the vasodilation that is suppressed by histamine receptor antagonists, pruritus and pain.Rosenthal, 1977; Douglas, W.W., " histamine and serotonine (5-hydroxy tryptamine) and antagonist thereof ": Goodman, L.S., Deng, ed., the pharmacological basis of treatment (The Pharmacologycal Basis of Thorapeutics), MacMillan Publishing Company, New York, pp.605-638 (1985); Rumore, M.M., et.al., the analgesic activity of antihistamine drug, life sciences (LifeSci) 36, pp.403-416 (1985).Verified, these three kinds of agonist (5-HT, Kallidin I and histamine) use in conjunction can show a kind of synergism that causes pain, produces a kind of lasting intensive pain signal.Sicuteri etc., 1965; Richardson etc., 1985; Kessler, W., etc., the excitement of the skin nervus centripetalis terminal that the external use in conjunction of inflammatory mediator causes and the modulating action of P material.Experimental brain research, (Exp.Brain Res.) 91, pp.467-476 (1992).
In vivo, 5-HT is arranged in platelet and axoneuron, and histamine is arranged in mastocyte, and Kallidin I then is at tissue injury, during pH change and the temperature change, produces from bigger precursor molecule.Because 5-HT can discharge from the platelet at tissue injury position in a large number, the level that causes blood plasma 5-HT is than the high 20 times of (Ashton of static level, J.H. etc., 5-hydroxy tryptamine is as suffering from the medium that rate of circulating flow changes in the angiostenosis disease Canis familiaris L. coronary artery, circulation (Circulation) 73, pp.572-578 (1986)), endogenous 5-HT may work in hyperpathia and the aspect of inflammation producing postoperative pain.In fact, hematoblastic activation can cause the stimulation of external periphery nociceptor.Ringkamp, M., etc., activatory human plasma platelet at stimulated in vitro rat skin nociceptor, neuroscience communication (Neurosci.Lett.) 170, pp.103-106 (1994).Equally, histamine and Kallidin I also can be discharged in the tissue during wound.Kimura, E. etc., the variation of Kallidin I level in the coronary sinus blood after the experimental coronary occlusion, the sick magazine of american heart (Am Heart J.) 85, pp.635-647 (1973); Douglas, 1985; Dray etc. (1993).
In addition, known prostaglandin also can cause pain and inflammation.Cyclooxygenase-2 inhibitor such as ibuprofen are generally used for non--surgical operation and postoperative environment, and the generation with the blocking-up prostaglandin reduces prostaglandin mediated pain and inflammation thus.Flower, R.J., etc., dipron and anti-inflammatory agent; Used medicine in the gout treatment is seen: Goodman, L.S., Deng the chief editor, the pharmacological basis of treatment (The Pharmacological Basis of Therapeutics), MacMillan Publishing Company, New York, pp.674-715 (1985).When cyclooxygenase-2 inhibitor uses according to a conventional method, relevant with some disadvantageous systemic side effects.For example, indomethacin or ketorolac have the generally acknowledged gastrointestinal and the adverse side effect of kidney.
As discussed, 5-HT, histamine, Kallidin I and prostaglandin can cause pain and inflammation.In the past twenty years, people understood and discussed these materials mediate its to surrounding tissue do the time spent by various receptors.Great majority research was carried out on rat or other animal models.But, all existing difference aspect pharmacology and the receptor sequence between the humans and animals.
In addition, at present the antagonist of these media is used for postoperative pain therapy.One class is called 5-HT and norepinephrine uptake antagonist, and the medicine that comprises amitriptyline has carried out oral, has obtained some successes of treatment chronic pain situation.But chronic mechanism with the acute pain state is considered to have very big different.In fact, use in two researchs of acute pain state of amitriptyline, proved that amitriptyline does not have lenitive effect at peri-operation period.Levine，J.D.et?al.，″Desipramine?Enhances?OpiatePostoperative?Analgesia，Pain?27：45-49(1986)；Kerrick，J.M.et?al.，″Low-Dose?Amitriptyline?as?an?Adjunct?to?Opioids?forPostoperative?Orthopedic?Pain：a?Placebo-ControlledTrialPeriod，″Pain?52：325-30(1993)。In these two researchs, all be oral administration.Second research has noticed and has produced lower overall comfort among the oral amitriptyline patient in fact after surgery that this may be because of the affinity of medicine to various kinds of amine receptor in the brain.
Outside the absorption of amitriptyline decapacitation blocking-up 5-HT and norepinephrine, or an effective 5-HT receptor antagonist.So, in the above-mentioned research, aspect alleviating postoperative pain, lack the curative effect this point and as if contradict with the lifting manipulation of endogenous 5-HT role in the acute pain.There are several reasons can explain that the amitriptyline of being found can not alleviate the reason of acute pain in these two researchs.(1). amitriptyline is brought into use in operation the last week in first research (Levine, etc., 1986), finish evening before that day up to operation, and in second research (Kerrick, etc., 1993), amitriptyline only uses after operation.So in tissue injury's phase of reality, just in the period that 5-HT will discharge, the level that is present in the amitriptyline in the operative site tissue is not clear.(2) amitriptyline is extensively metabolic by liver.Adopt the method for orally give, perhaps the concentration of amitriptyline can not remain on the activity that sufficiently high level is suppressed at the 5-HT that the operation back is discharged in second research in the operative site tissue in considerable time.(3) in view of there being multiple inflammatory mediator, and research proved the synergism between inflammatory mediator, therefore only blocks the inflammatory reaction that a kind of reagent (5-HT) is not sufficient to suppress to reply tissue injury.
There are several researchs verified, the histamine of high concentration (1%-3% solution-be 10-30mg/ml) 1(H 1) receptor antagonist has the effect of serving as surgical procedures local anesthetic.This anesthetic action is not via H 1Receptor-mediated, but since to the reason of neuron film sodium channel (similar) non-specific interaction to the effect of lignocaine.Owing to can produce the side effect (for example sedation) relevant, also histamine receptor antagonists is not applied topically under the perioperative environment at present with the histamine receptor antagonists of these height " anesthesia " concentration.
The synergism of the therapeutic combination of A. anti-pain and/or antiinflammatory reagent and Chondroprotective agents
In view of with the arthroscope therapeutic process after, the complexity of inflammation and cartilage homeostasis loss diseases associated process, with the multiplicity of the molecule target that relates to the blocking-up of unimolecule target or suppress, can not provide the suitable effects that prevents cartilage degradation and osteoarthritis development.In fact, also proof is ineffective in animal model for the zooscopy of many target directing different molecular receptors and/or enzyme, or also tells in clinical trial.Therefore, the different molecular target is worked and the local therapeutic combination that send the medicine of passing in the Therapeutic Method of cartilage protection be clinical effectively.Picture is following described, the principle of this concertedness molecule target therapy is from coming out understanding aspect the basic biochemical mechanism obtained progress inference in recent years, according to this mechanism, in the arthroscope process, synovial cell in synovial membrane and the cartilage and chondrocyte can transmit and integrate the stimulus object that exposes thereon.
Traditionally, the molecular switch of being responsible for cell signal is divided into main separation signal pipeline, each all comprises a different set of protein family, and its transducer as one group of special born of the same parents' external stimulue works, and mediates different cell responses.In this approach one of them, replied to produce contractility from neurotransmitter and hormone transduction signal by G-G-protein linked receptor (GPCRs), and this replying comprises inflammatory mediator, as the generation of PGE2.GPCRs by the proteic activation of trimerization G and with born of the same parents' internal target coupling (see figure 2).The signaling molecule embodiment that relates to by GPCR pathway activation synovial cell and chondrocyte is a histamine, Kallidin I, 5-hydroxy tryptamine and ATP.Second main path,, expressed and catabolic cytokine and other catabolism factor with regulator gene as the IL-1 transduction signal from proinflammatory cytokine by kinase cascade and NF-6B, comprises the generation of NO.
Can stimulate a class two receptoroids: GPCRs from the signal of neurotransmitter and hormone transmission, stride the film district by seven-spiral, or the ion channel of part-gate be formed." downstream " signal that derives from two kinds of receptors is at control kytoplasm Ca 2+Convergent (see figure 3) on the concentration.Each GPCR transmembrane receptor can activate a paraspecific trimerization G albumen, comprises G q, G iOr other.G qBut subunit activating phosphatase enzyme C γ, it causes the activation of Protein kinase C (PKC) and the increase (see figure 3) of cytosolic calcium levels.Successively, the rising of cellular calcium causes the activation of cPLA2, and the generation of arachidonic acid (AA).AA works as the substrate of COX in synovial cell and the chondrocyte, and it causes the generation of PGE2.The activation of PKC also can cause the activation of map kinase, and this map kinase can cause the activation of NF-B in cell and the tissue, and this activation excites by being exposed on the proinflammatory cytokine, and scalable relates to the increase of the metabolic protein gene expression of chondrolysis.
By different associated receptors, also regulating advolution on the cellular gene expression by IL-1 and the alpha mediated proinflammatory cytokine signal of TNF-.These signal transduction pathways that isoacceptor did not adopt use different kinases, and this kinases and receptor rather than signal pipeline are adjacent, at the advolution of map kinase level (Fig. 3 and 4).Signal transduction depends on the phosphorylation of residue in the kinase cascade, and kinases wherein comprises " downstream " enzyme, as the p38 map kinase.The activation of IL-1 receptor and TNF-receptor also causes stimulating map kinase, the common step (see figure 3) total with the link coupled GPCRs of Gq.Recognize now, ligand dependent " cross (talk) " but the transactivation kinase pathways, this kinase pathways is replied specificity GPCRs and cytokine, as the common stimulation of IL-1, causes the cell response (see figure 3) of system.Therefore, cause proinflammatory cytokine, iNOS, the combination of the selective depressant of the increase of COX-2 and MMPs gene expression, the transactivation (as illustrated in fig. 1 and 2) of its blocking-up shared signal pipeline is worked in coordination with and is played a role, thereby prevents inflammation and cartilage degradation behind the arthroscope operation process.
B. antiinflammatory and anti-pain agent
The antiinflammatory and/or the anti-pain agent that are used for the suitable type of the compositions and methods of the invention comprise: (1) 5-hydroxytryptamine receptor antagonist; (2) 5-hydroxytryptamine receptor agonist; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitor; (6) tachykinin receptor antagonists comprises neurokinin 1And neurokinin 2The receptor subtype antagonist; (7) calcitonin-gene-related peptide (CGRP) receptor antagonist; (8) interleukin receptor antagonist; (9) inhibitor of activated enzyme in the route of synthesis of arachidonic acid metabolite comprises (a) phosphatase inhibitors, comprises PLA 2Isoform inhibitor and PLC isoform inhibitor, (b) cyclooxygenase-2 inhibitor and (c) lipoxygenase inhibitor; (10) prostaglandins receptor antagonist comprises eicosanoid EP-1 and EP-4 receptor subtype antagonist and thromboxan receptor subtype antagonist; (11) LTRA comprises leukotriene B 4Receptor subtype agonist and leukotriene D 4The receptor subtype agonist; (12) opioid receptor agonist, comprise μ-, δ-and κ-opiate receptor subtype agonist; (13) P 2The purinoceptor antagonist comprises P 2XReceptor antagonist and P 2YReceptor antagonist; (14) calcium-channel antagonists.
Below be of the present inventionly to be intended to the description that the suitable medicine of passing that belongs to aforementioned every kind of antiinflammatory/anti-pain agent type is sent in the part, and the debita spissitudo that is used for solution.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.Although without wishing to be bound by theory, think that the selection reason of classes of agents makes the reagent work obtain illustrating.
Remove the cyclo-oxygenase inhibitor because selected special inhibitor may need outside the higher concentration, and every kind of medicine is preferably with 0.1-10,000 times to K dOr K iLow concentration be included in the solution.Preferably, every kind of medicine with 1.0～1000 times to K dOr K iConcentration, most preferably with 100 times to K dOr K iConcentration be included in the solution.Adjust these concentration as required, pass the site when not having metabolic conversion, be used for dilution to send in the part.As following illustrated, can select to be used for the definite reagent of solution according to the variation of application-specific, and the concentration of reagent.
1.5-seretonine receptor 5 antagonist
It is believed that 5-hydroxy tryptamine (5-HT) is by stimulating the 5-hydroxy tryptamine on the periphery nociception neuron 2(5-HT 2) and/or 5-hydroxy tryptamine 3(5-HT 3) receptor and algesiogenic.Most of research worker are agreed the receptor-mediated direct pain sensation (Richardson etc., 1985) that is produced by 5-HT of the 5-HT on the periphery nociceptor.Except that the pain that inhibition 5-HT causes, the 5-HT receptor antagonist also can suppress neurogenic inflammation by the activation that suppresses nociceptor.Barnes P.J. etc., the adjusting of neurogenic inflammation, pharmacology subject trend, 11, pp.185-189 (1990).But, in the research of rat ankle joint, then claim 5-HT 2Receptor is to cause the activated reason of the nociceptor that is caused by 5-HT.Grubb, B.D., etc., the research of the 5-HT receptor relevant, drug effect (Agents Action) 25, pp.216-18 (1988) with the nervus centripetalis that is positioned normal and inflammatory rat ankle joint.Therefore, 5-HT 2The activation of receptor also may play a role in periphery pain and neurogenic inflammation.
One of purpose of solution of the present invention is to block pain and multiple inflammatory process.Therefore, will discuss, in solution of the present invention, suitably adopt 5-HT as the back 2And 5-HT 3Receptor antagonist, or be used singly or in combination.Amitriptyline (Elavil TM) be a kind of suitable 5-HT receptor antagonist of the present invention that is used for.Amitriptyline uses for many years clinically as a kind of tranquilizer, and finds that it also has good effect, metoclopramide (Reglan to some chronic pain patient TM) use as the resisting emesis medicine clinically, but it is to 5-HT 3Receptor shows moderate affinity, and can suppress the effect of 5-HT to this receptor, so may suppress because 5-HT discharges caused pain from platelet.Therefore it also is suitable among the present invention.
The 5-HT that other are suitable 2Receptor antagonist comprises imipramine, trazodone, desipramine and ketanserin.The antihypertensive function of ketanserin has been used for clinical.Hedner, T., etc., novel 5-hydroxytryptamine antagonist ketanserin to experimental and clinical hypertensive effect, U.S.'s hypertension magazine (Am J.Hypertension, pp.317s-23s (Jul.1988)).The 5-HT that other are suitable 3Receptor antagonist comprises cisapride and ondansetron.The 5-hydroxy tryptamine that is fit to 1BReceptor antagonist comprises yohimbine, N-[methoxyl group-3-(4-methyl isophthalic acid-piperazinyl) phenyl]-2 '-methyl-4 '-(5-methyl)-1,2,4-oxadiazole-3-yl) [1, the 1-xenyl]-4-carboxylic acid amides (" GR 127935) and methiotepin.Used treatment concentration and the preferred concentration of these medicines listed in table 14 in the solution of the present invention.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 14
The treatment concentration and the preferred concentration of pain and/or inflammation inhibitor
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
5-hydroxy tryptamine 2Receptor antagonist:
Amitriptyline ??0.1-1,000 ??50-500
??MDL-11，939 ??0.1-1,000 ??50-500
??AMI-193 ??0.1-2,000 ??50-500
Desmethylimipramine ??0.1-1,000 ??50-500
Ketanserin ??0.1-1,000 ??50-500
5-hydroxy tryptamine 3Receptor antagonist:
A bent siron ??0.01-100 ??0.05-50
Metoclopramide ??10-10,000 ??200-2,000
Cisapride ??0.1-1,000 ??20-200
Ondansetron ??0.1-1,000 ??20-200
5-hydroxy tryptamine 1B(people 1D β ) antagonist:
??Isamoltare ??0.1-1,000 ??50-500
??GR?127935 ??0.1-1,000 ??10-500
Methiotepin (methiothepin) ??0.1-500 ??1-100
??SB216641 ??0.2-2,000 ??2-200
2.5-seretonine receptor 5 agonist
Known 5-HT 1A, 5-HT 1BAnd 5-HT 1DReceptor can suppress adenylate cyclase activity.Therefore, comprise the low dosage 5-hydroxy tryptamine 1A, 5-hydroxy tryptamine 1BAnd 5-hydroxy tryptamine 1DThe solution of the present invention of receptor stimulating agent should suppress the neuron of mediated pain and inflammation.Expectation is from 5-hydroxy tryptamine 1EAnd 5-hydroxy tryptamine 1FReceptor stimulating agent obtains identical effect, because these receptors also can suppress adenyl cyclase.
Buspirone is a kind of suitable 1A receptor stimulating agent for the present invention's application.Sumatriptan then is the 1A that suits, 1B, 1D and 1F receptor stimulating agent.Suitable 1B and 1D receptor stimulating agent are dihydroergotamine.Suitable 1E receptor stimulating agent is an ergometrine.The treatment concentration and the preferred concentration of these receptor stimulating agents are listed in table 15.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 15
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
5-hydroxy tryptamine 1AAgonist:
Buspirone ??1-1,000 ??10-200
??sumatriptan ??1-1,000 ??10-200
5-hydroxy tryptamine 1BAgonist:
Dihydroergotamine ??0.1-1,000 ??10-100
??sumatriptan ??1-1,000 ??10-200
??naratriptan ??1-1,000 ??10-200
??rizatriptan ??1-1,000 ??10-200
??zolmitriptan ??1-1,000 ??10-200
??L-694，247 ??1-1,000 ??10-200
5-hydroxy tryptamine 1DAgonist:
Dihydroergotamine ??0.1-1,000 ??10-100
??sumatriptan ??1-1,000 ??10-200
??naratriptan ??1-1,000 ??10-200
??rizatriptan ??1-1,000 ??10-200
??zolmitriptan ??1-1,000 ??10-200
??L-694，247 ??1-1,000 ??10-200
5-hydroxy tryptamine 1EAgonist:
Ergometrine ??10-2,000 ??100-1,000
5-hydroxy tryptamine 1FAgonist:
??sumatriptan ??1-1,000 ??10-200
3. histamine receptor antagonists
Histamine receptor is divided into histamine usually 1(H 1) and histamine 2(H 2) hypotype.The caused typical inflammatory reaction of administered peripherally histamine is via H 1Receptor-mediated.Douglas，1985。So solution of the present invention preferably includes histamine H 1Receptor antagonist.Promethazine (trade name Phenergan TM) be a kind of effective blocking-up H 1The resisting emesis agent commonly used of receptor, thereby be applicable to the present invention.What is interesting is that verified this medicine also has the local anesthesia effect, only produce the required concentration ratio blocking-up H of this effect 1The required concentration of receptor is wanted big several magnitude, therefore thinks that described effect is produced by different mechanisms.Histamine receptor antagonists concentration in the solution of the present invention is enough to suppress to relate to the activated H of nociceptor 1Receptor, but can not obtain " local anaesthesia " effect, thus eliminate some relevant systemic side effects.
The H that other are suitable 1Receptor antagonist comprises terfenadine, diphenhydramine, amitriptyline, pyrilamine and tripolidine.Because amitriptyline is as a kind of 5-hydroxy tryptamine 2Receptor antagonist also is effectively, so it has dual-use function in the present invention.These H 1Suitable treatment concentration and the preferred concentration of each listed in table 16 in the receptor antagonist.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 16
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Histamine receptor antagonists:
Promethazine ??0.1-1,000 ??50-200
Diphenhydramine ??0.1-1,000 ??50-200
Amitriptyline ??0.1-1,000 ??50-500
Terfenadine ??0.1-1,000 ??50-500
Pyrilamine ??0.1-1,000 ??5-200
??tripolidine ??0.01-100 ??5-20
4. bradykinin receptor antagonists
Bradykinin receptor is divided into Kallidin I usually 1(B 1) and Kallidin I 2(B 2) hypotype.Studies have shown that the acute periphery pain and the inflammation that are produced by Kallidin I are by B 2The hypotype mediation, and in chronic inflammatory disease, the pain that Kallidin I produces then is via B 1The hypotype mediation.Peikins, M.N. etc., Kallidin I B on two kinds of persistency hyperpathia of rat model 1And B 2Receptor antagonist des-Arg 9, [Leu 8The anti-nociception activity of]-BK and HOE 140.Pain (Pain) 53, pp.191-97 (1993); Dray, A., etc., Kallidin I and inflammatory pain, neuroscience trend (Trends Neurosci) 16, pp.99-104 (1993), these two pieces of documents all are incorporated herein by reference.
Bradykinin receptor antagonists is not used for clinical as yet at present.In these medicines some are peptides, therefore can not orally gives, because they may be digested.B 2Acute pain and inflammation that receptor antagonist blocking-up Kallidin I causes.Dray etc., 1993, B under the chronic inflammatory disease condition 1The receptor antagonist inhibition of pain.Deng, 1993; Dray et.al., 1993.So according to application, solution of the present invention preferentially comprises B 1And B 2In the receptor antagonist one or both.For example, acute and chronic joint disease all will be done arthroscopy, thereby the irrigating solution that is used for arthroscopy should comprise B 1And B 2Two kinds of receptor antagonists.
Be used for suitable bradykinin receptor antagonists of the present invention and comprise following Kallidin I 1Receptor antagonist: D-Arg-(Hyp 3-Thi 5-D-Tic 7-Oic 8[the des-Arg of)-BK 10] the derivant ([des-Arg of HOE 140 10] derivant, can buy from Hoechst pharmaceutical factory); And [Leu 8] des-Arg 9-BK.Suitable Kallidin I 2Receptor antagonist comprises: [D-Phe 7]-BK; D-Arg-(Hyp 3-Thi 5,8-D-Phe 7)-BK (" NPC 349 "); D-Arg-(Hyp 3-D-Phe 7)-BK (" NPC 567 "); And D-Arg-(Hyp 3-Thi 5-D-Tic 7-Oic 8)-BK (" HOE 140 ").Described more fully in the document of (1993) such as Perkins that these chemical compounds are introduced in front etc. (1993) and Dray.Suitable treatment and preferred concentration are shown in table 17.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 17
The treatment concentration and the preferred concentration of pain and/or inflammation inhibitor
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Kallidin I 1Receptor antagonist:
??[Leu 8]des-Arg 9-BK ??1-1,000 ??50-500
[the des-Arg of HOE 140 10] derivant ??1-1,000 ??50-500
??[Leu 9][des-Arg 10]Kalliden ??0.1-500 ??10-200
Kallidin I 2Receptor antagonist:
??[D-Phe 7]-BK ??100-10,000 ??200-5,000
??NPC?349 ??1-1,000 ??50-500
??NPC?567 ??1-1,000 ??50-500
??HOE?140 ??1-1,000 ??50-500
5. kallikrein inhibitor
Just as noted earlier, the peptide Kallidin I is the important medium of pain and inflammation.Kallidin I is as a kind of pyrolysis product, acted on high-molecular weight kininogen in the blood plasma and produced by kallikrein.Therefore there is the people to think that kallikrein is suppressing that Kallidin I produces and consequent pain and aspect of inflammation have therapeutic value.The kallikrein inhibitor that is suitable for the present invention's application is aprotinin.The suitable concentration of this medicine that uses for solution of the present invention is listed in following table 18.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 18
The treatment concentration and the preferred concentration of pain and/or inflammation inhibitor
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Kallikrein inhibitor:
Aprotinin ??0.1-1,000 ??50-500
6. tachykinin receptor antagonists
Tachykinin (TKs) is the peptide that is associated on gang's structure, comprises the P material, neurokinin A (NKA) and neurokinin B (NKB).Neuron is the main source of peripheral t Ks.Important systemic effect of TKs is a neural stimulation, and other effect comprises the vasodilation that epithelium relies on, plasma protein extravasation, the stimulation of mast cell degranulation and recovery and inflammatory cell.Maggi, C.A., general pharmacology is learned (Gen.Pharmacol.) 22, pp.1-24 (1991).In view of the above-mentioned physiological action combination that is mediated by the TK receptor activation, the TK receptor as target, is promoted that the method for pain relieving and treatment nerve inflammation is rational.
6a. neurokinin 1The receptor subtype antagonist
The P substance activating is called as NK 1The neurokinin receptor hypotype.The P material is a kind of 11 peptides that are present in the sensory nerve end.Known P material has after the activation of C-fiber, and the multiple effect that produces periphery inflammation and pain comprises vasodilation, plasma extravasation and mast cell degranulation.Levine, J.D., etc., peptide and the original nociceptor that imports into, Journal of Neuroscience (J.Neurosci) 13, p.2273 (1993).A kind of suitable P substance antagonist is ([D-Pro 9[spiro-gamma-lactam] Leu 10, Trp 11] physalaemin-(1-11)) (" GR 82334 ").Other are applicable to the present invention, act on NK 1The antagonist of receptor has: 1-imino group-2-(2-methoxy-phenyl)-ethyl)-and 7,7 biphenyl-4-hexahydroisoindoline ketone (3aR, 7aR) (" RP 67580 "); And 2s, 3s-cis-3-(2-methoxybenzyl amino)-2-benzhydryl quinuclidine (benzhydrylquinuclidine) (" CP 96,345 ").The suitable concentration of these reagent is listed in the table 19.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 19
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Neurokinin 1 receptor subtype inhibitor:
?GR?82334 ??1-1,000 ??10-500
?CP?96，345 ??1-10,000 ??100-1,000
?RP?67580 ??0.1-1,000 ??100-1,000
6b. neurokinin 2The receptor subtype antagonist
Neurokinin A is a kind of peptide, and it is collected in the sensory neuron with the P material, and it also can promote the inflammation and the pain sensation.The neurokinin A activation is referred to as NK 2Special neurokinin receptor.Edmonds-Alt, S., etc., the non-peptide neurokinin A of a kind of effective as selective (NK2) receptor antagonist, life sciences (Life Sci) 50:PL 101 (1992).Suitable NK 2The embodiment of antagonist comprises: ((S)-N-methyl-N-[4-(4-acetamido-4-ritalin subbase)-2-(3, the 4-dichloro-benzenes) benzamide (" (±)-SR 48968 "); Met-Asp-Trp-Phe-Dap-Leu (" MEN 10,627 "); And cyc (Gln-Trp-Phe-Gly-Leu-Met) (" L 659,877 ").The suitable concentration of these reagent is listed in table 20.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 20
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Neurokinin 2 receptor subtype antagonisies:
?MEN?10，627 ??1-1,000 ??10-1,000
??L?659，877 ??10-10,000 ??100-10,000
??(±)-SR?48968 ??10-10,000 ??100-10,000
7.CGRP receptor antagonist
Calcitonin-gene-related peptide (CGRP) also is a kind ofly to be collected in peptide in the sensory neuron with the P material, and it plays a role as vasodilation, and can strengthen the effect of P material.Brain, S.D. is etc., calcitonin-gene-related peptide (CGRP) with increase the caused inflammatory edema of cooperative effect of the mediators of vascular permeability.Britain pharmacology magazine (Br.J.Pharmacol.) 99, p.202 (1985).The embodiment of suitable CGRP receptor antagonist is I-CGRP-(8-37), the clipped form of CGRP.This peptide species suppresses the activation of CGRP receptor.The suitable concentration of this reagent is listed in table 21.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 21
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
The CGRP receptor antagonist:
??I-CGRP-(8-37) ??1-1,000 ??10-500
8. interleukin receptor antagonist
Interleukin is the peptide that cytokine is included in gang, is to be produced by leukocyte and other cell of replying inflammatory mediator.Interleukin (IL) may be effective periphery hyperalgesic agent.Ferriera, S.H., etc., interleukin-11 β conduct can be by a kind of strong hyperalgesic agent of tripeptide analog thing antagonism, nature (Nature) 334, p.698 (1988).A kind of embodiment of suitable IL-1 beta receptor antagonist is Lys-D-Pro-Thr, and it is an IL-1 β clipped form.This tripeptides suppresses the activation of IL-1 beta receptor.The suitable concentration of this reagent is listed in table 22.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 22
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Interleukin receptor antagonist:
??Lys-D-Pro-Thr ??1-1,000 ??10-500
9. act on the enzyme inhibitor in the arachidonic acid metabolite route of synthesis
9a. inhibitor of phospholipase enzymes
By phospholipase A 2(PLA 2) enzyme (cPLA 2, iPLA 2, sPLA 2) and the arachidonic acid that produces of phosphatase C (PLC) cause cascade reaction, it produces multiple inflammatory mediator, is called eicosanoid.In whole approach, many stages all can be suppressed, thereby reduce the generation of these inflammatory mediators.The embodiment that suppresses in these different phases is listed below.
Enzyme PLA 2The inhibitory action of isoform suppresses to discharge arachidonic acid from cell membrane, thus also just suppress the generation of prostaglandin and leukotriene, thus cause alleviating of inflammation and pain.Glaser, K.B., phospholipase A 2The regulation and control of all enzymes: selective depressant and pharmacology's potentiation thereof, progress in pharmacology (Adv.Pharmacol.32, p.31 (1995)).A kind of suitable PLA 2The embodiment of homotype enzyme agonist is Manoalide.The suitable working concentration of this reagent is listed in table 23.PLC γThe inhibitory action of isoform phosphatase also can cause the generation of prostaglandins and leukotriene to reduce, so also can cause pain and inflammation to alleviate.PLC γThe embodiment of isoform enzyme inhibitor is 1-[6-((17 β-3-methoxy female steroid-1,3,5 (10)-triolefins-17-yl) amino) hexyl]-1H-pyrroles-2, the 5-diketone.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 23
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Inhibitors of phosphatases:
??Manoalide ??100-100,000 ??500-10,000
Aristolochic Acid ??40-400,000 ??400-40,000
??ACA ??10-100,000 ??100-10,000
??HELSS ??6-6,000 ??60-6,000
9b. cyclooxygenase-2 inhibitor
NSAID (non-steroidal anti-inflammatory drug) (NSAIDs) is widely used as antiinflammatory, anti-heating, antithrombotic and analgesic.Lewis, R.A., prostaglandin and leukotriene are seen: rheumatology textbook, the third edition (Kelley W, N. waits the chief editor) p.258 (1989).The target molecule of these medicines is I type and II type cyclo-oxygenase (COX-1 and COX-2).These enzymes are also referred to as prostaglandin H synthase (PGHS)-1 (composing type) and-2 (induction types), but conversion of arachidonic acid is converted into PGH, and the latter is the intermediate in prostaglandin and the blood coagulation Cong alkane biosynthesis.The COX-2 enzyme is identified in macrophage and the fibroblast at endotheliocyte.This enzyme is induced by IL-1 and TNF-α, and raises its expression at inflammation part.The composition of COX-1 induced activity active and COX-2 all can cause producing the synthesizing of prostaglandin of pain and inflammation.
Many NSAIDs (diclofenacs of on market, selling at present, naproxen, indomethacin and ibuprofen etc.) the non-selective inhibitor of two kinds of isoform COX normally, but may demonstrate the selectivity bigger than COX-2 to COX-1, though this ratio changes to some extent according to different chemical compounds.With attempt to block native ligand and compare with the interaction of 7 kinds of described prostaglandins receptor subtypes, represent a kind of preferable therapeutic strategy with the way that COX-1 and inhibitor 2 blocking-up prostaglandin form.The antagonist of the prostaglandin receptoroid of being reported (EP-1, EP-2 and EP-3) is very rare, has only the blood coagulation Cong alkane A2 receptor antagonist of specificity high-affinity to be in the news.Wallace, J. and Cirino, G. pharmacology trend (Trends inPharm.Sci), 15, pp.405-406 (1994).
The treatment concentration and the preferred concentration of the used cyclooxygenase-2 inhibitor of solution are listed in table 24.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 24
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Cyclooxygenase-2 inhibitor:
Ketorolac ??100-10,000 ??500-5,000
Indomethacin ??1,000-500,000 ??10,000-200,000
9c. lipoxygenase inhibitor
The inhibition of lipoxygenase has suppressed to be considered to the leukotriene of inflammation and pain important medium, as leukotrienes B 4Generation.Lewis, R.A., prostaglandin and leukotriene are seen: rheumatology textbook, the third edition (Kelley W.N. waits the chief editor), p.258 (1989).An embodiment of lipoxygenase antagonist is 2,3,5-trimethyl-6-(12-hydroxyl-5,10-12 carbon diynyls)-1, and 4-benzoquinone (AA 861), its suitable concentration is listed in table 25.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 25
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Lipoxygenase inhibitor:
??AA?861 ??100-10,000 ??500-5,000
Caffeic acid ??500-50,000 ??2,000-20,000
10. prostaglandins receptor antagonist
The specificity prostaglandins that produces as arachidonic acid metabolite is by activating the proinflammatory effect that the prostaglandin receptoroid mediates them.The example of all kinds of specificity prostaglandins antagonisies has: eicosanoid EP-1 and EP-4 receptor subtype antagonist and thromboxan receptor subtype antagonist.A suitable prostaglandin E 2Receptor antagonist is also [b, f] [1,4] oxazole piperazine (oxazepine)-10 (11H)-carboxylic acid, 2-acetyl hydrazides (" SC19220 ") of 8-chlorodiphenyl.A kind of suitable thromboxan receptor subtype antagonist is [15-[1 α, 2 β (5Z), 3 β, 4 α]-7-[3-[2-(phenylamino)-carbonyl] diazanyl] methyl]-7-oxo bicyclo--[2,2,1]-heptan-the 2-yl]-5-enanthic acid (" SQ 29548 ").The suitable concentration of these reagent is listed in table 26.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 26
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Eicosanoid EP-1 antagonist:
??SC?19220 ??100-10,000 ??500-5,000
11. leukotriene receptor antagonist
Leukotriene (LTB 4, LTC 4And LTD 4) be the product of 5-lipoxygenase approach in the arachidonic acid metabolic, produce by the enzyme effect, have important biological property.Leukotriene relates to the many pathological states that comprise inflammation.How tame drugmaker all seeking specific antagonists, intervenes these pathological states as effective treatment means at present.Halushka, P.V., etc., pharmacology and toxicology comment yearbook (Annu.Rev.Pharmacol.Toxicol.) 29:213-239 (1989); Ford-Hutchinson, A. Immunological Identification comment (Crit.Rev.Immunol.10:1-12 (1990)).In comprising some immunocyte of eosinophilic granulocyte and neutrophil cell, find to have LTB 4Receptor.LTB 4With these receptors bind, cause chemotaxis and lysosome to discharge, thus the process of causing inflammation.With LTB 4The relevant signal transduction process of receptor activation comprises phospholipid base (P1) the metabolism stimulation that G-is protein mediated and the rising (see figure 2) of cellular calcium level.
A suitable leukotrienes B 4The example of receptor antagonist is SC (±)-(S)-7-(3-(2-encircles third methyl)-3-methoxyl group-4-[(methylamino)-carbonyl] phenoxy group) propoxyl group)-3,4-dihydro-8-propyl group-2H-1-.alpha.-5:6-benzopyran-2-propanoic acid (" SC53228 ").Be applicable to that this reagent concentration of the present invention lists in table 27.The leukotrienes B that other are suitable 4Receptor antagonist comprises [3-[-2 (7-chloro-2-quinolyl) vinyl] phenyl] [3-dimethylamino-3-oxopropyl] sulfenyl] methyl] thiohydracrylic acid (" MK 0571 ") and medicine LY 66071 and ICI 20,3219.MK 0571 also can be used as LTD 4The receptor subtype antagonist.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 27
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Leukotrienes B 4Antagonist:
??SC?53228 ??100-10,000 ??500-5,000
12. opioid receptor agonist
The activation of Opioid Receptors produces anti-nociception effect, so the agonist of these receptors is desired.Opioid Receptors comprise μ-, δ-and κ-opiate receptor hypotype.The μ receptor mapping is on peripheral sensory neuron end, and the activation of these receptors suppresses the sensory neuron activity.Basbaum, A.I., etc., is the opiate analgesics: the periphery target (the How Central is a Peripheral Target that how to become the center?, New England Journal of Medicine (N.Engl.J.Med), 325:1168 (1991).δ-and κ-receptor mapping in the orthosympathetic end that spreads out of, thereby can suppress the release inhibition of pain and the inflammation of prostaglandin.Taiwo, Y.O. etc., the hyperpathia that κ and δ opiate blocking-up sympathetic nerve rely on, Journal of Neuroscience (J.Neurosci) 11 volumes, p.928 (1991).The Opioid Receptors hypotype is the member of G-G-protein linked receptor superfamily.Therefore, all opioid receptor agonists interact and triggering signal by its related G-G-protein linked receptor.The example of suitable μ-opioid receptor agonist is fentanyl and Try-D-Ala-Gly-[N-MePhe]-NH (CH 2) 2OH (DAMGO).The example of suitable delta-opioid receptor stimulating agent is [D-Pen 2, D-Pen 5] enkephalin (" DPDPE ").The example of suitable κ-opioid receptor agonist is (trans)-3,4-two chloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-phenyl acetamide (" U 50,488 ").The suitable concentration that these reagent are every kind is listed in table 28.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 28
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
μ-opioid agonist:
??DAMGO ??0.1-100 ??0.5-20
??sufentanyl ??0.01-50 ??1-20
Fentanyl ??0.1-500 ??10-200
??PL017 ??0.05-50 ??0.25-10
The delta-opioid agonist:
??DPDPE ??0.1-500 ??1.0-100
κ-opioid agonist:
??U?50，488 ??0.1-500 ??1.0-100
13. purinoceptor antagonist
The outer ATP of born of the same parents by with P 2The interaction of purinoceptor plays signaling molecule.A main type of purinoceptor is P 2XPurinoceptor, it is permeable Na +, K +And Ca 2+The part control ion channel of inherent ion channel.The P that in sensory neuron, plays a role 2XReceptor to original nervus centripetalis send pass with pain sensation impression be important.Known ATP can make the sensory neuron depolarization, and is working aspect the nociceptor activation, because stimulate P from the ATP that damaged cell discharged 2XReceptor causes being subjected to the terminal depolarization of nerve fiber of sad evil.P 2XReceptor have highly strict distribution (Chen, C.C., etc., nature (Nature) V.377, pp.428-431 (1995) is because this receptor is expressed many equal receptors that can carry the pain stimulation thing in known these C-fibers by selectivity in the C-of the sensation that enters spinal cord fiber nerve.Therefore, P2X 3The localization and expression of this height strictness of receptor subtype makes these receptor subtypes become the fabulous target (seeing Fig. 3 and Fig. 7) of analgesic action.
Make the active purinoceptor of calcium, it belongs to G-protein receptor superfamily, is described on the surface of mammal articular chondrocytes.It is found that ATP stimulates and gives amount of reagent dependent, the instantaneous rising of calcium ion concentration in former generation of differentiation chondrocyte.The allos desensitization test confirms that after stimulating with ATP is preliminary, chondrocyte is not replied UTP.These results are consistent with the existence of the cell surface P2Y receptor of chondrocyte.In the chondrocyte that goes down to posterity, the inductive calcium mobilization of purine has shown the identical pharmacological profile about agonist sensitivity.ATP and UTP do not change the synthetic of cartilage matrix, and this synthesizing is to measure by the association rate that cartilage explant or elementary chondrocyte are incorporated in the glycosaminoglycans by [35S] sulfate.Do not changed by the substrate degeneration of measuring from cartilage explant release glycosaminoglycans by arbitrary agonist yet.Functional P2Y purinoceptor can make purine outside the born of the same parents in the existence on elementary articular chondrocytes surface, activates the metabolism of chondrocyte as the concentration of ATP.
Other research has been determined person joint's chondrocyte P1 and P2 purinoceptor expression of gene and has been described the distribution of ligand-mediated PGE2.P2Y2 receptor stimulating agent ATP and UTP stimulate a small amount of release of PGE2, and after personnel selection IL-1 α pretreatment, the horizontal concertedness of PGE2 raises.With after the IL-1 pretreatment, reply ATP and UTP and add fashionable PGE2 jointly and discharge and to be simulated by phorbol Semen Myristicae ethyl ester.The function of P2Y2 receptor is that the PGE2 that increases the IL-1 mediation discharges, and promotes IA pain and inflammation thus.Therefore, P2Y antagonist purposes in the present invention should prevent the activation by the inflammatory mediator of synovial cell and chondrocyte generation.
Be suitable for the P that the present invention uses 2X/ ATP purinoceptor antagonist comprises suramin and pyridoxal 5-phosphate (pyridoxylphosphate)-6-diphenyl diimide-2,4-disulfonic acid (" PPADS ").The suitable concentration of these reagent is listed in table 29.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 29
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
P2X and/or P2Y antagonist:
Suramin ??100-10,000 ??10,000-100,000
??PPADS ??100-10,000 ??10,000-100,000
14.Ca 2+Channel antagonist
Calcium-channel antagonists is one group of different pharmaceutical that can disturb calcium ion to stride membrane flux, and calcium ion is that to activate the cell effect of the neural inflammation of mediation needed.Calcium enters the synovial cell and chondrocyte is the committed step of mediation priming reaction in these cells.And, Kallidin I, histamine, 5-hydroxy tryptamine (SHT 2) and neurokinin receptor (NK 1And NK 2) in the neural inflammation of mediation, the effect in the signal transduction pathway comprises the increase of cellular calcium, thereby causes the activation of calcium channel on the serous coat.In many tissues, calcium-channel antagonists such as nifedipine can reduce the arachidonic acid that is caused by various stimulus object, the release of prostaglandin and leukotriene.Moncada, S., Flower, R. and Vane, J. sees: pharmacological basis (the 7th edition) the MacMillan Publ.inc.pp.660-5 (1995) of Goodman and GilmanShi treatment.
At last, calcium-channel antagonists and arbitrary tachykinin, histamine or brad ykinin antagonists are suppressing the collaborative effect of neural aspect of inflammation demonstration.Neurokinin receptor is firmly established in the effect of the neural aspect of inflammation of mediation.Neurokinin 1(NK 1) and neurokinin 2(NK 2) receptor (member of G albumen coupling superfamily) signal transduction pathway comprises that cellular calcium increases, thereby cause the activation of calcium channel on the serous coat.Equally, Kallidin I 2(BK 2) activation of receptor is relevant with the increase of cellular calcium level in synovial cell and the chondrocyte.Therefore, calcium-channel antagonists disturbs and to relate to the common mechanism that cellular calcium level that part enters by L type passage raises.This is a calcium-channel antagonists and to neurokinin, histamine, P 2Y and Kallidin I 2The interactional basis of concertedness between receptor antagonist.
The calcium-channel antagonists that is suitable for the present invention's use comprises nisoldipine, nifedipine, nimodipine, lacidipine, isradipine, amlodipine.The suitable concentration of these reagent is listed in table 30.Similarly, general compositions according to the present invention will be suitable for comprising the reagent of sufficient dosage or load, so as in the joint or action site obtain local concentration in the listed therapeutic domain.Delivery system is sent in lasting release for guiding, comprises the reagent of sufficient dosage or load in compositions so that at predetermined lasting deenergized period, in the joint or action site obtain local concentration in the listed therapeutic domain.
Table 30
The treatment of pain and/or inflammation inhibitor and preferred concentration
Types of agents The part is sent and is passed The part is sent and is passed
Treatment concentration (nM) Preferred concentration (nM)
Calcium-channel antagonists:
Nisoldipine ??1-10,000 ??100-1,000
Nifedipine ??1-10,000 ??100-5,000
Nimodipine ??1-10,000 ??100-5,000
Lacidipine ??1-10,000 ??100-5,000
Isradipine ??1-10,000 ??100-5,000
Amlodipine ??1-10,000 ??100-5,000
VI. embodiment
Be some some preparations of each side of the present invention (embodiment 1-3) that are suitable for lavation in some operative procedure below, and be used for whole body and send and pass, preparation as by intramuscular or subcutaneous injection (embodiment 4-11) is to use the summary of three clinical researches of reagent of the present invention subsequently.
Embodiment 1
The arthroscopy irrigating solution
Following compositions is applicable to the lavation in anatomy joint in the arthroscopy process.The same with described other irrigating solution of following embodiment, various medicines all are dissolved in and contain physiology electrolyte, in the carrier fluid as normal saline or lactate ringer's solution.
Types of agents Medicine Concentration (nM):
Map kinase inhibitor ??SB203580 ??200
Matrix metallo-proteinase inhibitor ??U-24522 ??200
The TGF-beta-agonists ??TGF-β2 ??200
Embodiment 2
The optional irrigating solution that is used for arthroscopy
Following compositions is during the arthroscopy process, is applicable to the selective preparation of anatomy joint lavation.
Types of agents Medicine Concentration (nM):
Map kinase inhibitor ??SB203580 ??200
Nitric oxide synthase inhibitors ??L-NIL ??1,000
The interleukin-2-receptor agonist ??IL-10 ??100
Embodiment 3
Optional irrigating solution
Following medicine and concentration range thereof in the solution of physiology carrier fluid are applicable to the present invention.
Types of agents Medicine Concentration (nM):
Map kinase inhibitor ??SB242235 ??200
Nitric oxide synthase inhibitors ??L-NIL ??10,000
The TGF-beta-agonists ??TGF-β2 ??100
Embodiment 4
Injection cartilage protection solution
Following compositions is suitable for being expelled in anatomical joint.Each medicine is containing physiology electrolyte, and is dissolved in the carrier fluid as normal saline or lactated Ringer's solution.Be suitable for giving the patient for the solution of amount of reagent 20ml.
Reagent type Medicine Concentration
The bmp receptor agonist ??BMP-7 ??100ng/ml
Nitric oxide synthase inhibitors ??1，3PBIT ??4.4μg/ml
The TGF-beta-agonists Pyrrolidine-dithiocarbamate ??16.4μg/ml
Embodiment 5
The cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site: (nM)
Map kinase inhibitor ??SB203580 ??200
Matrix metallo-proteinase inhibitor ??U-24522 ??200
The TGF-beta-agonists ??TGF-β2 ??200
Embodiment 6
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site (nM)
Map kinase inhibitor ??SB203580 ??200
Nitric oxide synthase inhibitors ??L-NIL ??1,000
The interleukin-2-receptor agonist ??IL-10 ??100
Embodiment 7
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site (nM):
Map kinase inhibitor ??SB242235 ??200
Nitric oxide synthase inhibitors ??L-NIL ??10,000
The TGF-beta-agonists ??TGF-β2 ??100
Embodiment 8
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site (nM):
Soluble TNF-α receptor (sTNFRII:Fc) ??Etanerocept ??(Enbrel TM， ??Immunex) ??250
Map kinase inhibitor ??SB203580 ??500
The TGF-beta-agonists ??TGF-β2 ??200
Embodiment 9
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site (nM):
IL-1 receptor antagonist (IL-1Ra) ??Anakinra ??(Kineret TM??Amgen) ??250
Map kinase inhibitor ??SB203580 ??500
The TGF-beta-agonists ??TGF-β2 ??200
Embodiment 10
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine to be will being included in the compositions in the concentration that the purpose site of action obtains following concentration, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine The concentration of action site (nM):
Map kinase inhibitor ??SB203580 ??500
?IGF-1 ??IGF-1 ??250
Bmp agonist ??BMP-2 ??200
Embodiment 11
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine is used with the dosage of pointing out, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine Dosage (mg/kg/ day):
Soluble TNF-α receptor (sTNFRII:Fc) ??Etanerocept ??(Enbrel TM， ??Immunex) ??0.5-1.0
Map kinase inhibitor ??SB203580 ??30-60
The TGF-beta-agonists ??TGF-β2 ??0.1-10
Embodiment 12
The optional cartilage protection compositions that is used for systemic delivery
Following cartilage protection compositions is applicable to for example passes through intramuscular or subcutaneous administration and systemic delivery.Each medicine is used with the dosage of pointing out, and is dissolved in the physiology carrier fluid or send in the delivery system.
Types of agents Medicine Dosage (mg/kg/ day):
IL-1 receptor antagonist (IL-1Ra) ??Anakinra ??(Kineret TM??Amgen) ??2.0
Map kinase inhibitor ??SB203580 ??30-60
The TGF-beta-agonists ??TGF-β2 ??0.1-10
Embodiment 13
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation in DL-poly (lactide-co-glycolide) (PLGA) nanosphere, the anti-people II of coupling Collagen Type VI monoclonal antibody on this nanosphere.This antibody targeted in articular cartilage the epi-position on the people II Collagen Type VI.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
Somatomedin ??IGF-1 ??250nM
Map kinase inhibitor ??SB220025 ??1000nM
The MMP inhibitor ??BB2516 ??(marimastat) ??200nM
Embodiment 14
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation in biodegradable PLA/PLGA copolymer nano ball, the anti-COLLECTIN HCL-P1 polysaccharide of coupling monoclonal antibody on this nanosphere.This antibody targeted in articular cartilage the new epi-position on the COLLECTIN HCL-P1 polysaccharide.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
Somatomedin ??IGF-1 ??250nM
Map kinase inhibitor ??SB220025 ??1000nM
The MMP inhibitor ??BB2516 ??(marimastat) ??200nM
Embodiment 15
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation in chitosan/gelatin nanosphere, the anti-people II of coupling Collagen Type VI monoclonal antibody on this nanosphere.This antibody targeted in articular cartilage the new epi-position on the people II Collagen Type VI.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
Map kinase inhibitor ??SB203580 ??500
Embodiment 16
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation in albumin nanometer ball, the anti-people II of coupling Collagen Type VI monoclonal antibody on this nanosphere.This antibody targeted in articular cartilage the new epi-position on the people II Collagen Type VI.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
The MMP inhibitor ??BB2516 ??200
Embodiment 17
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation in poly-(lactide-co-glycolide)/poly-(ethylene glycol) copolymer nano ball, the anti-people II of coupling Collagen Type VI monoclonal antibody on this nanosphere.This antibody targeted in articular cartilage the new epi-position on the people II Collagen Type VI.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
Map kinase inhibitor ??SB203580 ??500
Embodiment 18
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Bmp receptor agonist BMP-7 is encapsulated in poly-(lactide-co-glycolide) (PLGA) in the nanosphere, the anti-people II of coupling Collagen Type VI monoclonal antibody on this nanosphere.IGF receptor stimulating agent IGF-1 separately is encapsulated in chrondroitin-6-sulphuric acid/gelatin nanosphere, the anti-COLLECTIN HCL-P1 polysaccharide of coupling monoclonal antibody on this nanosphere.Be used for leading every type the antibodies of nanosphere on articular cartilage people II Collagen Type VI new epi-position and the new epi-position on the aggrecan.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
The IGF receptor stimulating agent ??IGF-1 ??500
Embodiment 19
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation is in albumin nanometer ball, and coupling is incorporated into the anti-people F (ab ') of II Collagen Type VI monoclonal antibody on this nanosphere 2Fragment.This F (ab ') 2Fragment is directed at the new epi-position on the people II Collagen Type VI.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
The MMP inhibitor ??BB2516 ??200
Embodiment 20
The cartilage protection drug delivery system of guiding
Following cartilage protection compositions for example is applicable to by intravenous, intramuscular, subcutaneous or suck and use and systemic delivery.Drug encapsulation is in albumin nanometer ball, and coupling is incorporated into anti-people's strand minimum of immunoglobulin molecules of II Collagen Type VI monoclonal antibody in conjunction with territory (scFv) on this nanosphere.The antibody targeted new epi-position on people II Collagen Type VI of this scFv.In compositions, comprise every kind of each medicine, obtain following concentration at the purpose action site when its concentration is enough to when nanosphere is degraded and reagent discharges in continuing deenergized period.
Types of agents The concentration of action site: (nM)
The bmp receptor agonist ??BMP-7 ??200
The MMP inhibitor ??BB2516 ??200
Research 1
The collaborative stimulation that quick PGE2 outburst produces when being exposed to IL-1 and GPCR agonist
Become the fiber-like synovial cell to have the characteristic of inflammatory cell, and the important regulator of arthritis and cartilage degradation seemingly.The feature that synovial cell's culture model system is used to describe cooperative interaction between IL-1 and acellular factor inflammatory mediator, it comprises that for the damage of mediation joint tissue the damage that occurs owing to the tissue injury during the arthroscope operation process is extremely important.Test with the adjusting of research G-G-protein linked receptor (GPCR) agonist (histamine, Kallidin I, isoproterenol), and describe the activity of ketoprofen in this system cytokine in people's synovioblast of being cultivated and prostaglandin product.Described and replied the PGE2 (PGE that il-1 (IL-1) stimulates 2), interleukin-6 (IL-6) and interleukin-8 (IL-8) induce kinetics.Studied after IL-1 excites, the GPCR part is strengthened the ability that cytokine produces.
In research 1-3, except as otherwise noted, use following test method and material.
1. Cell cultureSynovial tissue is from living through Clinical Research Center, the osteoarthritis patient of MacNeal Hospital joint replacement surgery obtains, and be sent in the Eagle ' s culture medium (DMEM) of breadboard Dulbecco ' s improvement, this culture medium contains penicillin (100 units/ml), streptomycin (100, and amphotericin B (0.25ug/ml) ug/ml).Dissect and the chopping synovial membrane with shears, and it is inoculated in the culture medium as explant, this culture medium is by containing L-glutamate, Glu (2mM), heat-killed hyclone (10%v/v), the DMEM composition of added with antibiotic.In the time of 37 ℃, at 5%CO 2Humid air in this culture of preservation.2-3 is in week, and adherent synovial cell grows from the explant the inside, and through trypsinize.Give inoculum feed supplement twice weekly, and converge.Test is to carry out on the cell that derives from passage 3-8.The test culture is inoculated on the ware of 35mm, and wherein the density in the 2ml culture medium is 7.5 * 10 3Cell/cm 2Culture is grown near the test meet, and contains 2.3 ± 0.3 * 10 5Individual cell (meansigma methods ± S.E.M., n=3), and the albumen (n=10) of 104 ± 13 μ g.This growth medium changes weekly twice.
2. Test is handledTest handle beginning before, change culture medium into tentative growth medium, it is by the DMEM that contains 2% heat-deactivation fetus calf serum, adds that above-mentioned L-glutamate, Glu and antibiotic form, so that cell is static.Second day, with 12-24 hour interval the IL-1 of specific concentration or other parts are joined in the condition growth medium and to excite culture.In some test, in order to analyze, after exciting with IL-1, the collection condition growth medium.Acute experiment is handled after this excites at interval and is carried out.From incubator, remove culture, with 2ml Locke ' s physiological buffer (LB compositions mM:NaCl, 154; KCl, 2.6; KH 2PO 4, 2.15; K 2HPO 4, 0.85; MgCl 2, 5; CaCl 2, 2; The D-glucose, 10; HEPES, 10; PH 7.4, BSA, and 0.1%w/v) aliquot is cleaned, then in 37 ℃ water-bath, with the additional LB aliquot balance that contains particular ligand 10 minutes.Remove this solution by suction, and in the time of 37 ℃, with containing the fresh buffer aliquot displacement certain time interval of specifying part.Typically, in 10 minutes cultivation space before process, add the medicine inhibitor, and agonist adds in the invasion and attack interval procedure that specific inhibitor all is present in 3 minutes.
3. The measurement of PGE2.After the designated treatment scheme is carried out, collect the aliquot (1ml) of culture supernatants, and freezing rapidly in liquid nitrogen.Store samples up to handling at-80 ℃.Use and PGE2 and E1 have the antibody of reactivity of equal value, analyze the aliquot of culture supernatants by the specified competitive capture radioimmunoassay procedure of maker (Sigma Chemical Co.).In order to quantize, use fixed concentration [ 3H] PGE2 and concentration increases gradually, competes the standard curve that PGE2 is drawn each algoscopy reliably.
4. The measurement of IL-6. the product of cytokine IL-6 also is to measure in the aliquot of supernatant culture medium, and this culture medium is-80 ℃ of freezing preservations.IL-6 is by sandwich ELISA, and the alkali phosphatase described with above-mentioned maker (Pharmingen) detects to be measured, and uses the standard curve of each pure recombined human cytokine preparation quantitative.Test determination is to carry out on the culture that duplicates.
Research 2
[ 3 H] thymidine mixes the test method with MTT
Synovial cell system normally assesses with respect to the ability that IL-1 is replied in amplification, its be with [ 3H] thymidine mixes and measures (Kimball ﹠amp; Fisher, 1988).Compare with the static culture in remaining on 2% serum, in this reagent, the most effective concentration of IL-1 can stimulate 10-20 doubly [ 3H] the mixing of thymidine (not marking data).
1. Data analysis.Immunoassay is normally carried out in the aliquot duplicating of each culture.Test determination carries out on twice or triple culture.Each test repeats at least two kinds of cell lines.(San Diego CA) carries out nonlinear regression curve and statistical analysis to use Graph-PAD Prism software.
2. Material.Cell culture: cell culture medium obtains from Sigma or Gibco/BRL.The fetus calf serum derive from Atlanta Biologicals Inc. (Norcross, GA).Medicine: recombination human interleukin-the 1st, (Cambridge MA) obtains from Genzyme.Ketoprofen is by Omeros Medical Systems, and (Seattle WA) provides Inc..Amitriptyline, the diphtheria element, 5-hydroxy tryptamine, isoproterenol, Kallidin I, histamine and PGE2 all derive from Sigma.The radio chemistry material: [ 3H] PGE2, be from American Radiolabeled Chemicals, (St.Louis MO) obtains Inc..All other reagent is bought from the normal business suppliers with highest purity.
The GPCR agonist, histamine and Kallidin I are measured in stimulation or before not stimulating IL-1 the effect that PGE2 among the people synovial cell produces, with the functional interaction between the assessment agonist, this agonist is by different types of receptor-mediated common drug effect.People's synovioblast of being cultivated is exposed to IL-10 (10U/ml) whole night, causes a large amount of risings of (4 hours) that postpone and the PGE2 product that continues, it can be measured by the increase of PGE2 in the measured by radioimmunoassay culture supernatants.In the IL-1 processing procedure (16-24 hour) that prolongs, the increasing of PGE2 product is gradually because the coordination of cPLA2 and COX-2, the expression (Crofford, 1984, Hulkower etc., 1984) of being raised.Reply the invasion and attack of histamine of maximum valid density (100 μ M) subsequently or Kallidin I (1, μ M) by being exposed to culture that IL-1 is excited whole night, its have additional fast (minute) and the generation of sturdy PGE2.Replying the representative data of the PGE2 generation time process of histamine or Kallidin I stimulation lists in Fig. 7.Under these conditions, compare the growth of HC 5-10 times PGE2 with the cell that the IL-1 that does not receive GPCR agonist addition excites.Kallidin I excites 10-15 growth doubly.The absolute magnitude of the PGE2 product that produced in the agonist invasion and attack procedure at simple 2 minutes is above exciting the amount that accumulation produces in the interval procedure at whole 18 hours IL-1.Can find out clearly that in Fig. 7 in the PGE2 preparation, the cracking of most HC appears in initial 2 minutes, because minimum additional accumulation is observed after 60 minutes.The PGE2 of Kallidin I-stimulation replys and is surpassing continuation increase in the cycle of identical time (2-doubly).Under the condition that does not have IL-1 to excite, natural synovial cell shows do not have detectable PGE2 product to reply independent stimulation with the GPCR agonist.Under the condition that IL-1 excites, histamine and Kallidin I are strengthened the release of PGE2 synergistically.
Use derives from osteoarthritis patient's cultivation synovioblast, we find proinflammatory cytokine, the effect that time dependence cooperative interaction between the IL-1 G-G-protein linked receptor relevant with physiology produces PGE2, and assessed the effect of target therapeutic agent.The GPCR agonist works by endogenous synovial membrane receptor, and increasing calcium and the inositolophosphate in the born of the same parents, and PKC signal pipeline suddenly enlarges the product of PGE2 in the cell rapidly to this receptor by coupling, and this cell excites with IL-1 in advance.The COX inhibitor weakens quick outburst that agonist causes and the long-term accumulated of PGE2 effectively.Therefore, be used for the different GPCR and the IL-1 approach synergism of intracellular signal transduction, produce fast or slowly, the long-term adjusting that PGE2 replys.
Produce the outburst of PGE2 fast, the synergism among the synovial cell between IL-1 and calcium-adjustings GPCRs partly is that the activation measured value of cPLA2 in many cell types illustrates by the quick growth of arachidonic acid release.Except the expression of inducing COX-2,, IL-1 also can increase the expression (Hulkower etc., 1994) of cPLA2.These two albumen provide the free arachidonic acid substrate of COX-2 jointly.The rise of the inductive important eicosanoid metabolic enzyme of IL-1, the GPCR part activates the ability combination that arachidonate discharges, and predicts that therefore it can be by the synthetic outflow that increases whole substrate of prostaglandin.CPLA2 is unique known PLA2, and it has the functional capability that the indication receptor is regulated, and may relate to the generation and the intracellular signal of arachic acid.Because cPLA2 is for whole work, increase calcium concentration and be activated, and the activation of bradykinin b 2 and histamine H1-receptor to be relevant with the activity of cellular calcium, this may be the key factor of regulating the outburst that quick agonist stimulates in the PGE2 product.Finally, very fast and the increase of the cytoplasmic calcium of moment excite by the activation of B2 or H1 receptor, it activates with cPLA2, the kinematic similarity that arachidonic acid release and observed PGE2 break out.
Research 3
Suppress the cracked formation of PGE2 by cyclooxygenase-2 inhibitor
The effect that ketoprofen, a kind of cyclooxygenase-2 inhibitor weaken PGE2 formation is by prolonging between exposure period, measures with the IL-1 co-cultivation; As shown in Figure 8, before GPCR agonist invasion and attack at interval subsequently, carry out simple preceding the cultivation.Exciting in the process of spending the night with IL-1, the formation of PGE2 has been eliminated in the adding of specific concentration ketoprofen, has the IC that determines by nonlinear regression analysis 50=4.5 ± 0.8nM (meansigma methods ± SEM, n=4 synovial cell system).Similar mensuration (not marking data) is with cyclooxygenase-2 inhibitor etodolac (IC 50=15.2 ± 4.6nM, n=4), ketorolac (2.2 ± 0.4nM, n=4), and indomethacin (3.2 ± 1.5nM n=2) carries out.
Fig. 8 has shown that also the cracked ketoprofen concentration dependent of agonist-excite PGE2 suppresses, and wherein IL-1 (10U/ml) the 100 μ M histamine (IC among the synovial cell who excites that spend the night are replied in the PGE2 cracking 50=3.4 ± 0.2nM, n=3) the peaceful kassinin kinin (IC of or 1 μ M 50=9.5 ± 2.0nM, invasion and attack n=3).Spend the night the ketoprofen inhibiting value observed between the IL-1 induction period relatively with these values and at PGE2.The inhibition of this result proof by the COX inhibitor begins to appear in the pretreatment in 10 minutes at interval before the GPCR agonist adds, but active directly retroactive inhibition is consistent with COX, rather than owing to changes relevant mechanism with prostaglandin regulatory enzyme expression.This inhibitory action immediately also is during the arthroscope surgical operation, send when being delivered to intraarticular with irrigating solution form part, and the effectiveness immediately of this medicine provides the foundation.
Research 4
The IL-6 that is produced by IL-1 and GPCR agonist induces inhibition with ketoprofen
The kinetics of inducing of replying interleukin-6 that IL-1 stimulates has been described.Synovial cell's culture is exposed, add histamine with IL-1 and handle with by inositoltriphosphoric acid salt (InsP3)/Protein kinase C pathway activation signal, or add the increase that isoproterenol activates cAMP in the born of the same parents.PGE2, the generation of IL-6 and IL-8 is after handling 1,2,4,6 and 24 hours, measures in culture supernatants.In this test, each processing carries out in isolating culture at interval.In above-mentioned disposition, the product of IL-6 is after exposing in 24 hours, increases by IL-1 is strong, but does not detect IL-6 at initial 6 hours at interval.The IL-6 product of replying IL-1 is not further increased by adding histamine, and histamine can not stimulate the generation of IL-6.IL-1 also can make IL-8 significantly increase (2000pg/ml), and it is to measure first at 6th hour that handles.The IL-8 product was at the 24th hour that is exposed to IL-1, and continuing increases significantly.
Detect inducing and the effect of GPCR agonist of cytokine that ketoprofen produces IL-1.But this scheme also test I L-1 to the inductive concentration dependent effect of IL-6 steady statue.Synovial cell's culture is exposed among the IL-1 and GPCR agonist of prescribed concentration.Collect culture supernatants, replaced with the fresh culture aliquot that contains identical agonist additive in per 8 hours.Measure the PGE2 in the supernatant, IL-6, and IL-8.
The data of IL-6 product are listed in Fig. 9, its under the situation that the IL-1 that prescribed concentration is arranged and additional part exist, the 16th hour (be equivalent to 8-16 hour processing at interval) output IL-6.Compare with independent IL-1, the adding of histamine or isoproterenol can not improve the generation of IL-6.When 1.0pg/ml IL-1, ketoprofen causes the inhibition of the IL-6 preparation that part (＜50%) IL-1 excites.In addition, ketoprofen can be suppressed at the generation of IL-6 in histamine or the common sample that stimulates of isoproterenol/IL-1.
Synovial cell's culture model system is used to describe synergistic feature between IL-1 and acellular factor inflammatory mediator, and it is extremely important for damage of regulating joint tissue, and described damage is included in the tissue injury that occurs in the arthroscope operation process.The result is summarized as follows: (1) IL-1 causes the PGE among the synovial cell that cultivates 2IL-6, increase greatly with IL-8, and but static culture can not produce these media of detection limit, and (2) PGE2 induces rapid appearance, and causes being discharged in the culture supernatants at the 4th hour PGE2, IL-8 discharged at the 6th hour, IL-6 discharge with longer interval and (3) after 24 hours IL-1 exposes, all these three media all keep raising in culture supernatants.
With they to PGE 2The effect of preparation is compared, and the GPCR agonist can not improve inductive IL-8 of IL-1 or IL-6, and can not increase the release of IL-8 and IL-6 after exciting with IL-1.As if inductive IL-8 of IL-1 and IL-6 can be by the PGE that follows 2Induce and be reinforced because ketoprofen has reduced these production of cytokines of replying IL-1.This result shows that it can provide curative cartilage protection effect when in operation process ketoprofen being sent when being delivered to the joint.
These results confirm, specificity G-coupled receptor signal pipeline and by the short scorching interaction of the synovial cell who produces between activating that stimulate of IL-1.The expectation similar mechanism is exercisable in chondrocyte.These interactions provide a kind of integration and adjusting synovial cell and the chondrocyte method that short inflammation is replied, and it depends on from the input of IA other endocrine or neurotransmitter receptor system.Potential clinical benefit and the principle that treatment is intervened emphasized in these discoveries, and this treatment intervention directs at the inhibition of G-G-protein linked receptor, and this receptor can pass through the calcium mobilization, phosphoinositide hydrolysis and PKC activation and mediation signal, and PGE in performing the operation with arthroscope 2The increase of product is relevant.These receptors that act on synovial cell and the chondrocyte comprise histamine H 1, Kallidin I, Substance P, 5HT2 and multipotency P2Y receptor.
Although have illustrated and described the preferred embodiments of the invention, should be appreciated that, can carry out multiple change and not leave the spirit and scope of the present invention disclosed scheme and method.For example, can find to strengthen or substitute disclosed reagent, the guiding antibody that disclosure herein is included and other Chondroprotective agents of delivery vectors, guiding antibody and delivery vectors.Therefore, the scope of the patent of mandate only is limited by the appended claims.

Claims (48)

1. drug delivery system that is used to protect the guiding of cartilage; comprise the multiple Chondroprotective agents that is included in the delivery vectors; described delivery vectors is coupled to specific antibody or the antibody fragment that is positioned at IA antigenic determinant; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
2. drug delivery system that is used to protect the guiding of cartilage; comprise multiple Chondroprotective agents; wherein at least a Chondroprotective agents is included in the delivery vectors that is directed at the joint; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; in the described multiple Chondroprotective agents each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
3. the drug delivery system of the guiding of claim 2, the delivery vectors of wherein said guiding is coupled to specific antibody or the antibody fragment that is positioned at IA antigenic determinant.
4. the drug delivery system of the guiding of claim 3, wherein the antigenic determinant of being led is on articular cartilage or in the articular cartilage.
5. the drug delivery system of the guiding of claim 4, wherein the antigenic determinant of being led is on the II of articular cartilage Collagen Type VI.
6. the drug delivery system of the guiding of claim 3, wherein the antigenic determinant of being led is on the chondrigen or on the chondroproteoglycan.
7. the drug delivery system of the guiding of claim 3, wherein in cell, molecule or the structure of the antigenic determinant of being led group on being selected from cell, molecule or the structure of organizing down or under being selected from: collagen comprises II Collagen Type VI and accessory V, VI, IX, X and XI Collagen Type VI; Dan Baijutang comprises big aggregation Dan Baijutang, aggrecan, decorin, disaccharidase catenin polysaccharide, fibromodulin and lumican; Cartilage oligo-substrate protein, glycoprotein-39; Dan Baijutang chondroitin sulfate and glycosaminoglycans; Macrophage synovial cell and fibroblast synovial cell; And chondrocyte.
8. the drug delivery system of the guiding of claim 3, wherein the antigenic determinant of being led is on synovial membrane or in the synovial membrane.
9. the drug delivery system of the guiding of claim 3, wherein the antigenic determinant of being led is epi-position or the new epi-position relevant with articular cartilage degeneration.
10. the drug delivery system of the guiding of claim 9, wherein epi-position that is led or new epi-position immunolocalization suffer from the patient's of osteoarthritis, rheumatoid arthritis or other degenerative joint disease articular cartilage shallow-layer in diagnosis.
11. the drug delivery system of the guiding of claim 9, wherein the antigenic determinant of being led is the II Collagen Type VI of articular cartilage or the new epi-position on the II Collagen Type VI fragment.
12. the drug delivery system of the guiding of claim 11, the cleavage site that the new epi-position immunolocalization that is wherein led generates in the effect alone or in combination of the enzyme by being selected from down group: matrix metalloproteinase (MMP)-1, MMP-3, MMP-8 and MMP-13, and other member of MMP family.
13. the drug delivery system of the guiding of claim 9, wherein epi-position that is led or new epi-position are on aggrecan, disaccharidase catenin polysaccharide or the decorin of articular cartilage.
14. the drug delivery system of the guiding of claim 9, wherein epi-position that is led or new epi-position are on the aggrecan or aggrecan fragment of articular cartilage.
15. the drug delivery system of the guiding of claim 14, the new epi-position immunolocalization that is wherein led is in the cleavage site that generates by the effect that belongs to the enzyme that takes off integrin and metalloproteases (ADAMTS) family and/or MMP family with thrombospondin primitive.
16. the drug delivery system of the guiding of claim 15, the cleavage site that the new epi-position immunolocalization that is wherein led generates in the effect alone or in combination by ADAMTS-4 and/or ADAMTS-5/11 enzyme.
17. the drug delivery system of the guiding of claim 3, wherein guidance quality antibody or antibody fragment are humanized, chimeric or human monoclonal antibodies.
18. the drug delivery system of the guiding of claim 2, wherein the anabolism Chondroprotective agents is included in the delivery vectors of guiding.
19. the drug delivery system of the guiding of claim 2, wherein the anabolism Chondroprotective agents is selected from and promotes the anabolic interleukin of cartilage (IL) agonist; Promote the member of the anabolic transforming growth factor-beta superfamily of cartilage, comprise TGF-beta-agonists and bone morphogenetic protein (BMP) agonist; Promote the anabolic insulin like growth factor of cartilage and promote the anabolic fibroblast growth factor of cartilage.
20. the drug delivery system of the guiding of claim 2, wherein the anabolism Chondroprotective agents is selected from IL-4, IL-10; IL-13; TGF β 1, TGF β 2, TGF β 3; BMP-2; BMP-4, BMP-6, BMP-7; IGF-1, bFGF and the fragment, disappearance, interpolation, aminoacid replacement, sudden change and the modification that keep the biological property of naturally occurring reagent.
21. the drug delivery system of the guiding of claim 2, wherein the anabolism Chondroprotective agents is selected from: promote the member of the anabolic transforming growth factor-beta superfamily of cartilage, comprise TGF-beta-agonists and bone morphogenetic protein (BMP) agonist; Promote the anabolic insulin like growth factor of cartilage and promote the anabolic fibroblast growth factor of cartilage.
22. the drug delivery system of the guiding of claim 2, wherein the metabolic inhibitor packages of chondrolysis is contained in the delivery vectors of guiding.
23. the drug delivery system of the guiding of claim 2, wherein the chondrolysis metabolic poison is selected from and suppresses the metabolic IL-1 receptor antagonist of chondrolysis, suppress the metabolic TNF-α of chondrolysis receptor antagonist, suppress the metabolic cyclo-oxygenase-2 specific inhibitor of chondrolysis, suppress the metabolic map kinase inhibitor of chondrolysis, suppress the metabolic nitric oxide synthase inhibitors of chondrolysis and suppress the metabolic nuclear factor kB inhibitor of chondrolysis.
24. the drug delivery system of the guiding of claim 2, wherein the chondrolysis metabolic poison is selected from and suppresses the metabolic matrix metalloproteinase of chondrolysis; Suppress the metabolic cell adhesion molecule of chondrolysis, comprise integrin agonist and integrin antagonist; Suppress signal transmission inhibitor in the metabolic cell of chondrolysis, comprise inhibitors of protein kinase C and protein tyrosine kinase inhibitor; With the inhibitor that suppresses the metabolic SH2 domain of chondrolysis.
25. the drug delivery system of the guiding of claim 2, wherein the chondrolysis metabolic poison comprises the reagent that is selected from the metabolic IL-1 receptor antagonist of inhibition chondrolysis and suppresses the metabolic TNF-α of chondrolysis receptor antagonist.
26. the drug delivery system of the guiding of claim 2, wherein anabolism Chondroprotective agents and chondrolysis metabolic poison all comprise albumen.
27. the drug delivery system of the guiding of claim 2, wherein anabolism Chondroprotective agents and chondrolysis metabolic poison are included in the delivery vectors of guiding.
28. the drug delivery system of the guiding of claim 2 comprises the delivery vectors of a plurality of guiding, wherein anabolism Chondroprotective agents and chondrolysis metabolic poison separately are included in in the delivery vectors of a plurality of guiding first and second.
29. the drug delivery system of the guiding of claim 28; wherein select the delivery vectors of first and second guiding, so that obtain for different in time release dynamics the anabolism Chondroprotective agents that is comprised and the chondrolysis metabolic poison that is comprised.
30. the drug delivery system of the guiding of claim 2, wherein Dao Xiang delivery vectors comprises the immune particle of guiding.
31. the guiding of claim 30 send delivery system, wherein Dao Xiang immune particle comprises nano-particle.
32. the guiding of claim 31 send delivery system, wherein the diameter of nano-particle is 5 nanometer to 750 nanometers.
33. the guiding of claim 31 send delivery system, wherein the diameter of nano-particle is 10 nanometer to 500 nanometers.
34. the guiding of claim 31 send delivery system, wherein the diameter of nano-particle is 20 nanometer to 200 nanometers.
35. the guiding of claim 31 send delivery system, wherein nano-particle is by the polymer formation that is selected from down group: hyaluronan, chitosan, collagen, gelatin, alginate, polylactic acid (PLLA), polyglycolic acid (PGA) and PLGA.
36. the guiding of claim 31 send delivery system, wherein nano-particle provided the lasting release of Chondroprotective agents during 1 day-4 week.
37. the guiding of claim 2 send delivery system, further comprise and be applicable to intravenous, intramuscular, subcutaneous or suck the carrier of using.
38. the guiding of claim 2 send delivery system, further comprise one or more other therapeutic agent.
39. the guiding of claim 2 send delivery system, further comprise one or more pain or inflammation inhibitor.
40. the guiding of claim 39 send delivery system, wherein pain or inflammation inhibitor are selected from the 5-hydroxytryptamine receptor antagonist, the 5-hydroxytryptamine receptor agonist, histamine receptor antagonists, bradykinin receptor antagonists, kallikrein inhibitor, tachykinin receptor antagonists, calcitonin-gene-related peptide (CGRP) receptor antagonist, interleukin receptor antagonist, the inhibitor of activated enzyme in the route of synthesis of arachidonic acid metabolite, cyclooxygenase-2 inhibitor,, lipoxygenase inhibitor, prostaglandins receptor antagonist, LTRA, opioid receptor agonist, purinoceptor antagonist, adenosine triphosphate (ATP) sensitive potassium channel opener and calcium-channel antagonists.
41. drug delivery system that is used to protect the guiding of cartilage; comprise multiple Chondroprotective agents; wherein at least a Chondroprotective agents is included in the delivery vectors of the molecule, cell or the structure that are directed at hyaline cartilage; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; in the described multiple Chondroprotective agents each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
42. drug delivery system that is used to protect the guiding of cartilage; comprise at least a Chondroprotective agents for the treatment of effective dose; described Chondroprotective agents is anabolism Chondroprotective agents or chondrolysis metabolic poison; it is included in the immune particle of guiding, and described granule is coupled to specific antibody or the antibody fragment that is positioned at IA antigenic determinant.
43. a drug delivery system that is used to protect the guiding of cartilage comprises the anabolism Chondroprotective agents for the treatment of effective dose, described anabolism Chondroprotective agents is included in the delivery vectors that is directed at the joint.
44. a drug delivery system that is used to protect the guiding of cartilage comprises the anabolism Chondroprotective agents for the treatment of effective dose, described anabolism Chondroprotective agents is included in the delivery vectors of the molecule, cell or the structure that are directed at hyaline cartilage.
45. a method that is used to protect patient's cartilage comprises:
To there being the patient who needs to send the drug delivery system of passing guiding; this drug delivery system comprises the multiple Chondroprotective agents that is included in the delivery vectors; described delivery vectors is coupled to specific antibody or the antibody fragment that is positioned at IA antigenic determinant; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
46. a method that is used to protect patient's cartilage comprises:
Giving simultaneously has the patient who needs to use multiple Chondroprotective agents; wherein at least a Chondroprotective agents is included in the delivery vectors that is directed at the joint; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; in the described multiple Chondroprotective agents each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
47. a method that is used to protect patient's cartilage comprises:
Giving simultaneously has the patient who needs to use multiple Chondroprotective agents; wherein at least a Chondroprotective agents is included in the delivery vectors that is directed at the new epi-position relevant with articular cartilage degeneration; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; in the described multiple Chondroprotective agents each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
48. a method that is used to protect patient's cartilage comprises:
Giving simultaneously has the patient who needs to use multiple Chondroprotective agents; wherein at least a Chondroprotective agents is included in the delivery vectors of the molecule, cell or the structure that are directed at hyaline cartilage; described multiple Chondroprotective agents comprises at least a anabolism Chondroprotective agents and at least a chondrolysis metabolic poison; in the described multiple Chondroprotective agents each all exists with the treatment effective dose; so that described multiple Chondroprotective agents had both suppressed the chondrolysis metabolism, promote the cartilage anabolism again.
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