CN101123977A - Compositions for induction of a therapeutic response - Google Patents

Abstract

Compositions for attracting specific cells to an in vivo site and for stimulating the attracted cells and local resident cells to achieve a desired therapy are described. In one embodiment, a composition for initiating and promoting repair and regeneration of tissue is described. In another embodiment, a composition for inducing a cytotoxic response to tumor cells is described. The compositions are comprised of drug reservoirs containing one or more therapeutic agents effective (1) to attract one or more desired cells to the tissue site; (2) to stimulate activity, e.g., proliferation, differentiation, and/or release of biological factors that promote a desired activity, in the attracted cells; and (3) to prolong survival of the attracted cells and, if desired, local resident cells. A device for administering the composition at a desired site is also described.

Description

Be used for the compositions that inductive treatment is replied

Invention field

The present invention relates to compositions, its be used for that specific cells is attracted to a certain position in the body and be used to stimulate the cell that is attracted and partial permanent cell to reach desired therapeutic.More specifically, the present invention relates to compositions, it is used for cell and tissue regeneration that (1) makes tissue site impaired or injured in the body, and perhaps (2) induce the cellullar immunologic response to cancer.Said composition is deposited on impaired tissue site or tumor locus can attract essential cell effectively, under the situation of tissue repair, make cell and tissue regeneration, thereby in impaired tissue regions, promote accurate normal function and blood flow, perhaps, under the situation of immunotherapy for cancer, influence the destruction of tumor tissues.The present invention also provides a kind of equipment that is used for compositions is deposited to an expection position in the body.

Background of invention

The loss function of deterioration, necrosis and secondary or fibrosis, for example, this paper is commonly referred to as " histologic lesion " or " tissue injury ", may be caused by disease or injury.For example, myocardial necrosis and fibroblast proliferation may be caused by the ischemia that coronary occlusion causes.In addition, the ischemia that causes of apoplexy can cause that brain worsens and be downright bad.Tissue injury can be the part of multiple disease also, comprises diabetes, multiple sclerosis, sclerosis, renal failure etc.Damage also can be interrupted causing by the tissue that surgical operation or body injury cause.

Body provides tissue repair mechanism, relates to the interaction between coagulation process and the immune composition.Process of tissue reparation was divided into for three stages usually: (1) inflammation; (2) propagation; (3) reinvent.Although these stages are defined as different incidents, they occur as a continuum, and to move on to the tissue repair point in another stage from a stage be artificial.

Yet there is not or is not enough to influence the recovery fully of tissue in the endogenous tissue repair mechanisms in some histologic lesions.For example, the injury to aixs cylinder in the central nervous system causes permanent lesion, and wherein, the neuron that is used for the recovery organization function can not be regenerated.Type i diabetes, liver cirrhosis, congestive heart failure, skeletal muscle atrophy, parkinson disease, spinal cord injury are for causing other example of function of organization's loss.Can cause organ dysfunction all or local the forfeiture from the death of the critical organ tissue of disease injury or genetic defect, and afunction often is can not repair with irreversible.

The ischemia incident is another reason of considerable histologic lesion.Cause the ischemia incident of histologic lesion to cause myocardial infarction, apoplexy, skeletal muscle infraction and other disease.Current Therapeutic Method all can not cure these symptoms or promote injured, for example, downright bad, forfeiture or Fibrotic not function tissue regeneration.

Therefore, need to promote the mechanism of tissue regeneration strongly.Particularly be desirable to provide compositions and the method that can rebuild tissue, its can with int tissue bond with promote in impaired tissue regions or near blood flow, and the accurate normal function of permission tissue.

Also need to be used for the treatment of the improved compositions and the method for cancer.Cancer can appear in the many tissues and organ of body, and is usually directed to primary tumor, and it can discharge by blood vessel or the metastatic cell of lymphsystem.When in a single day tumor forms, remove tumor and need under the prerequisite of not murdering the patient, destroy or remove all malignant cells.Current, typically treat primary tumor and transfer by excision, chemotherapy, radiotherapy or immunotherapy.Operative treatment is often owing to not accessible more intraorganic tumor mass are obstructed, as brain, liver, lung and pancreas.Also be difficult to eradicate each cancerous cell, this is because surgical operation can not be removed each shifts, but and the treatment of kill cancer cell also be deleterious to normal cell.If the minority cancerous cell is residual, they can be bred makes palindromia (resergence), and does not resemble normal cell, and they may be to being used to resist their therapeutic agent generation Drug resistance.Therefore, surgical operation, chemotherapy or radiotherapy all can not provide persistent immunity to palindromia.Stimulating immune system provides a kind of damaging less but more effective secular cancer treatment method with identification and destruction of cancer cells.Zoopery provides the immunne response evidence to tumor, and has shown that the T lymphocyte is the amboceptor of tumour immunity.Based on immunne response is better understood, provide new immunization therapy strategy to the understanding progress of the related molecule of antigen presentation and T cell activation.Although the immunotherapy of cancer has shown the result with prospect in many animal tumors system, but the inconsistent (Janeway of the successful result of the clinical trial of immunotherapy, C.et a/., Immunobiology:The Immunesystem in Health and Disease, 5th Edition, Garland Science Publishing, 2001).

Tumor-infiltrated lymphocyte (TIL) is regarded as the cytotoxic agent of immunocompetence host to the tumor cell of offering discernible tumor antigen usually.Yet tumor cell has developed the immunity identification of many strategies with the prevention host.Therefore, need a kind of mechanism, it can promote tumor antigen identification at primary malignant tumor or metastasis site, and forms the cytotoxic response to tumor cell.Also need to promote the infiltration of cytotoxic cell to tumor.Particularly need to promote to pass through these phenomenons of the distant place tumor mass of surgical operation or radiotherapy in the treatment.

Summary of the invention

On the one hand, the present invention includes the compositions that is used for cellular response, it comprises first reagent that can effectively the cytotaxis of one or more expections be arrived tissue site; Can stimulate second reagent of described cell activity effectively; And the 3rd reagent that can influence the survival of described cell effectively.In one embodiment, compositions is used to promote cell and/or tissue regeneration, and described first reagent can be attracted to tissue site with one or more stem cell, CFU-GM and accesory cell (accessory cells) effectively.In another kind of embodiment, described compositions is used to induce the immunne response to tumor, and described first reagent can attract the cell of the one or more T-of being selected from lymphocytes, macrophage, segmented cell, antigen presenting cell and natural killer cell effectively.

In one embodiment, described second reagent can stimulate the activity that is selected from propagation and differentiation effectively.

In various embodiments, what be used for that inductive treatment replys is organized as skeletal muscle, liver, pancreas, brain, cardiac muscle, central nervous system or tumor tissues.

In one embodiment, the described cardiac muscular tissue (cardiac tissue) that is organized as, described first reagent can attract to be selected from the cell of circulation blood monocyte, systemic vascular cellulation or circulation tremulous pulse cellulation; Described second reagent can stimulate releasing factor from described cell, generates to promote angiogenesis and/or tremulous pulse; Described the 3rd reagent can influence the survival of the deutero-macrophage of circulation blood monocyte that is arranged in cardiac muscular tissue.

In one embodiment, described first reagent is selected from macrophage chemical attraction albumen (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5, active normal T cellular expression and excretory cytokine (RANTES) of regulating, the Fu Shi factor (Fraktalkines), macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide (N-farnesyl peptides), complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-β), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1) or macrophage cluster stimulating factor (M-CSF).

In another kind of embodiment, described second reagent is selected from macrophage chemical attraction albumen (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5, tumor necrosis factor (TNF)-a, TNF-β, active normal T cellular expression and excretory cytokine (RANTES) of regulating, the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide (N-farnesylpeptides) complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-β), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1), macrophage cluster stimulating factor (M-CSF) and lipopolysaccharide.

In other embodiments, described the 3rd reagent is selected from GM-CSF, G-CSF, CSF-1 and M-CSF.

In another kind of embodiment,, described compositions replys but being designed to the treatment of induced tumor.In this embodiment, described first reagent is selected from IL-8, MIG, IL-12, MCP-1 ,-2 ,-3 ,-4 ,-5, MIP-1 α, MIP-1 β, MIP-1 γ and RANTES.Described second reagent is selected from GM-CSF and IL-12.Described the 3rd reagent is selected from MIG, platelet factor 4, MCP-1 ,-2 ,-3 and MIP-1 γ.

These reagent discharge from said composition simultaneously, perhaps discharge continuously in another kind of embodiment.The drug storage warehouse that contains plurality of reagents can comprise biodegradable and nonbiodegradable by polymer formation.In other embodiments, this drug storage warehouse contains a targeting part, as the cell adhesion molecule that is connected with the outer surface of drug storage warehouse.

Described drug storage warehouse compositions can be made into a kind of dosage form that for example is selected from Emulsion, gel, pasty state or liquid.

On the other hand, the present invention contains a kind of method of replying at specific tissue site inductive treatment of being used for, its by in selected tissue site or near the aforesaid compositions that comprises one or more drug storage warehouses of deposition, this drug storage warehouse contains one or more can induce the expection therapeutic agent of replying effectively.In brief, described drug storage warehouse comprises one or more reagent, and this reagent (i) effectively is attracted to tissue site with one or more stem cell that are selected from CFU-GM, accesory cell, T-lymphocyte, macrophage, segmented cell, antigen presenting cell or natural killer cell; (ii) stimulate the one or more cells that are attracted, make it have a kind of activity that is selected from propagation and differentiation; The (iii) survival of the one or more cells that are attracted in the influence tissue.

In one embodiment, the described cardiac muscular tissue that is organized as, and described deposition can promote tremulous pulse to generate and/or angiogenesis effectively.

In another kind of embodiment, the described tumor mass that is organized as, described deposition can be attracted to tumor with cytotoxic cell effectively.

On the other hand, the present invention includes a kind of method that is used to promote cell and/or tissue regeneration.This method is included in the selected tissue site or near deposition contains one or more drug storage warehouses of one or more therapeutic agents, and this therapeutic agent (i) effectively is attracted to tissue site with one or more stem cell, CFU-GM and accesory cell; (ii) directly stimulate cell and extracellular matrix components local and that be attracted, from local and the cell that is attracted, discharge the biological reagent that can promote cell and/or tissue regeneration; And (iii) influence that be attracted and survival local regenerative cell in the described tissue.

In one embodiment, contain one or more biological reagents at the sedimentary drug storage warehouse of tissue site.

In another kind of embodiment, administration composition is with the parenchymal tissue of regeneration of damaged.A kind of example of tissue is a cardiac muscular tissue, for example, promotes tremulous pulse to generate and/or angiogenesis there.

At a kind of myocardium embodiment that regenerates that is used for, the biological reagent that comprises in drug storage warehouse comprises (i) first reagent, can attract to be selected from the cell of circulation blood monocyte, systemic vascular cellulation and circulation tremulous pulse cellulation effectively; (ii) second reagent can stimulate to discharge the factor that promotes that angiogenesis and/or tremulous pulse generate effectively; And (iii) the 3rd reagent, can influence the survival of the deutero-macrophage of circulation blood monocyte that in cardiac muscular tissue, exists effectively.

In one embodiment, described first reagent is peptide, can attract CFU-GM, stem cell effectively, and preferred accesory cell.Described first reagent can be macrophage chemical attraction albumen (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5, active normal T cellular expression and excretory cytokine (RANTES) of regulating, the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide (N-farnesyl peptides) complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-β), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1) or macrophage cluster stimulating factor (M-CSF), and the suitable fragment of function.

In one embodiment, described second reagent can stimulate cell and/or the local regenerative cell who is attracted effectively.The example of second reagent comprises macrophage chemical attraction albumen (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5, tumor necrosis factor (TNF)-a, TNF-β, active normal T cellular expression and excretory cytokine (RANTES) of regulating, the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide (N-farnesylpeptides) C5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-β), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1), macrophage cluster stimulating factor (M-CSF), lipopolysaccharide, and the suitable fragment of function.

In one embodiment, the described the 3rd biological reagent is selected from GM-CSF, G-CSF, CSF-1, M-CSF, and the suitable fragment of function.

Other example of the biological reagent that comprises in drug storage warehouse comprises that fibroblast growth factor (FGF, FGF-1, FGF-2), TGF-α, insulin like growth factor (IGF-1), angiogenin-1, angiopoietin-2, VEGF (VEGF), VEGF constitute thing such as VEGF-2, VEGF165 and VEGF121, platelet derived growth factor (PDGF)-A, PDGF-B, PDGF-BB, endothelium mitogenesis somatomedin, and the suitable fragment of function.

Expection adopts the tissue of this method treatment to comprise, for example, and skeletal muscle, liver, pancreas, brain, cardiac muscle and central nervous system.

In one embodiment, described drug storage warehouse has the bio-ligand that is connected to this drug storage warehouse outer surface.The example of part comprises cell adhesion molecule.

In another kind of embodiment, described drug storage warehouse is made by biodegradable polymer or nonbiodegradable polymer.In another kind of embodiment, described drug storage warehouse comprises into the bubble lipoid, perhaps is made into a kind of dosage form that is selected from Emulsion, gel, pasty state and liquid.

In certain embodiments, described drug storage warehouse can deposit to interstice, and they have mobility there.

On the other hand, the present invention comprises a kind of compositions that is used to promote cell and/or tissue regeneration, and it comprises first reagent that can effectively one or more stem cell, CFU-GM and accesory cell be attracted to described tissue; Can stimulate second reagent of described cell and/or local regenerated cell activity effectively; And the 3rd reagent that can influence the survival of described cell effectively.

In one embodiment, reagent discharges from compositions simultaneously.In another kind of embodiment, reagent discharges from compositions continuously

In another embodiment, reagent is wrapped in the spherical drug storage warehouse.In certain embodiments, described drug storage warehouse can have a bio-ligand that is attached to outer surface.The example of part is cell adhesion molecule.

On the other hand, the present invention includes a kind of being used for immunocyte is attracted to a position to promote tumoricidal compositions.This method is included in the selected position or near deposition contains one or more drug storage warehouses of one or more therapeutic agents, and this therapeutic agent (i) effectively is attracted to tissue site with one or more lymphocytes, macrophage, antigen presenting cell (comprising dendritic cell), inflammatory cell, natural killer cell, CFU-GM and accesory cell; (ii) directly stimulate cell and extracellular matrix components local and that be attracted, and from described part and the cell that is attracted, discharge the Cytotoxic biological reagent that can promote cancerous cell; And (iii) influence that be attracted and survival local regenerative cell in the described tissue, and promote immunogenic response to cancerous cell.

In one embodiment, the biological reagent that drug storage warehouse contains can be realized following function: (i) with lymphocyte, macrophage, antigen presenting cell (comprising dendritic cell) and inflammatory cell, comprise that polymorphonuclear (polymoronuclear) the leukocyte chemical attraction from blood flow, lymphatic vessel and near tissue enters the middle section of tumor mass; (ii) stimulate tumor antigen to offer lymphocyte by antigen presenting cell, to form the systemic cellullar immunologic response (with the form of cytotoxic lymphocyte and natural killer cell) of all tumor cells in the facedown body, to comprise the tumor cell in other tumor mass that is present in the tumor cell that receives compositions and body; And (iii) strengthen that be attracted and the survival original cell in part, and reply exempting from service of cancerous cell is former promoting, comprise and impel cytotoxicity and natural killer T IL to be transported to tumor mass, thereby cause tumor cell destruction.

In one embodiment, realize the chemical attraction of pair cells by one or more chemical attractant that comprise in drug storage warehouse, chemical attractant is selected from IL-8, MIG, IL-12, MCP-1 ,-2 ,-3 ,-4 ,-5, MIP-1 α, MIP-1 β, MIP-1 γ and RANTES.

In another kind of embodiment, described drug storage warehouse comprises the stimulant of a kind of GM-CSF of being selected from or IL-12.

In another embodiment, in described drug storage warehouse, be used to promote to be attracted the survival of cell and be used to promote that treatment the 3rd reagent of replying of exempting from service to tumor is to be selected from MIG, platelet factor 4, MCP-1 ,-2 ,-3 or one or more reagent of MIP-1 γ.

On the other hand, the present invention also comprises a kind of equipment that is used to transmit compositions, particularly transmits aforesaid compositions.Described equipment comprises a catheter shaft, and it has the inner chamber that therefrom extends and end at the port; At least the remote domain of local compliance, a local at least hard near-end; A single track part, it is arranged near the catheter shaft far-end and has the seal wire junction surface; A user interface, far-end that has and near-end are rigidly connected to the catheter shaft near-end at the interface far-end; And a shell, the near-end that it is connected to the interface is used to hold piston mobile.

In one embodiment, described equipment can also comprise a pin, and it is arranged in the catheter shaft of deployed condition not to small part, and can launch from the catheter shaft of deployed condition.Described pin can be for straight or crooked.

In another kind of embodiment, the seal wire junction surface comprises that also an intracavity paves (endoluminal paving) equipment, is used for along tube wall or body cavity deposition composition.In this embodiment, intracavity is paved the flexible cup that equipment has elongation, and its center cavity that has is used for receiving compositions from the catheter shaft port.In addition, in this embodiment, the catheter shaft port is arranged to the equipment of paving in the adjacent cavities and compositions is sent to intracavity pave equipment.Intracavity is paved equipment can comprise hole and a following hole at least one, is used for seal wire is introduced by cup.Preferably, this intracavity is paved equipment and is used the spiral type seal wire.

Described equipment also can comprise a component pipe, extends through a remote domain to small part.Described component pipe can comprise a thermoplastic elastomer (TPE) and wire braid.In one embodiment, the near-end of described component Guan Yuzhen contact.In another kind of embodiment, described component pipe adheres to the near-end of pin.The catheter shaft near-end can also comprise the axial region of a reinforcing, with contacting of user interface far-end.

Described equipment can also comprise at least one attached pipe, and it passes through the axle proximal end region to small part and extends.In one embodiment, described equipment contains at least two attached pipes, to give the proximal end region support structure or the stability of catheter shaft.

Described remote domain and proximal end region can connect together in transition region.In one embodiment, this transition region is formed by the thermoplastic elastomer (TPE) of the hot briquetting of fusing, across near and remote domain.In another kind of embodiment, transition region contains a joint, is connected the far-end of described component pipe and the near-end of at least one attached pipe.

Described medicine transfer equipment can also comprise at least one balloon, is arranged on the far-end of single track part.In one embodiment, at least one balloon comprises at least two balloons that are arranged on the single track opposite side partly.In another kind of embodiment, at least one described at least one balloon has biconial when expanding.Described equipment can also comprise one or more passages in catheter shaft, it is used for introducing and removing fluid, especially commutes the fluid of balloon.

On the other hand, the present invention contains a kind of transfer equipment that is used to transmit compositions, it comprises (i) catheter shaft, it has the inner chamber that therefrom extends to port, at least the remote domain of local compliance, at least the near-end of local stiffness, it is arranged on the transition region between end and the near field, and at the reinforcing axial region of near-end, (ii) a single track part is arranged to the far-end of adjacent pipes axle and is had the seal wire junction surface, (iii) user interface, it has far-end and near-end, and is rigidly connected to the catheter shaft near-end at the interface far-end, and (iv) shell that is connected to the interface near-end that is used to hold piston mobile.Described equipment contains a component pipe that extends through near axis area and stop in transition region to small part.Described equipment contains at least one attached pipe, and it extends through the proximal end region of catheter shaft to small part.

In conjunction with the accompanying drawings, by reading following detailed description of the present invention, will understand these and other objects of the present invention and feature more fully.

Description of drawings

Fig. 1 is for causing the panorama sketch of the tissue regeneration process on the cellular level owing to deposition compositions according to the present invention;

Fig. 2 is the surface and the cross section stravismus detail drawing of a kind of drug storage warehouse of imitateing of using in compositions;

Fig. 3 is a kind of plane graph of the drug storage warehouse depositing device of imitateing;

Fig. 4 is the plane detail drawing of the catheter shaft of equipment among Fig. 3;

Fig. 5 A-5C is the detail drawing of catheter shaft distal portions, has shown that seal wire in the single track part engages (Fig. 5 A) and is used for the needle point (Fig. 5 B, 5C) of penetrate tissue;

Fig. 6 A-6B is by line 6A-6A among Fig. 5 C and the lateral cross figure of 6B-6B;

Fig. 7 A-7B has shown the embodiment of balloon shape conduit, and it has biconial balloon profile (Fig. 7 A) and two spheric balloons (Fig. 7 B);

Fig. 7 C-7D is the enforcement illustration of balloon shape conduit, has shown the position of biconial balloon (Fig. 7 C) and two spheric balloons (Fig. 7 D) of the pin that penetrate tissue in the blood vessel is provided;

Fig. 8 A-8B is the cross-sectional view that the pin of a-quadrant among Fig. 4 is contracted (Fig. 8 A) and stretches out (Fig. 8 B) from catheter shaft;

Fig. 9 A is the cross-sectional view in B zone among Fig. 4;

Fig. 9 B is the cross-sectional view by Fig. 9 A center line F-F;

Fig. 9 C-9D is the cross-sectional view that is similar to Fig. 9 B, but has biconial balloon (as described in Fig. 7 A) in the equipment or have two spheric balloons (as described in Fig. 7 B);

Figure 10 is the longitudinal cross-section in D zone among Fig. 4;

Figure 11 is the longitudinal cross-section in C zone among Fig. 4;

Figure 12 is the longitudinal cross-section in E zone among Fig. 4;

Figure 13 has illustrated that use equipment deposits to the bolus of drug storage warehouse preparation in the cardiac muscle at ischemia position;

Figure 14 A-14C has shown a kind of embodiment for choosing, and wherein, the spiral type seal wire is used to equipment be advanced and locate the pin that is used for penetrate tissue;

Figure 15 is the top view full figure of the user interface of equipment;

Figure 16 A and 16B are the side view (Figure 16 A) and the oblique view (Figure 16 B) of user interface shown in Figure 15;

Figure 17 A-17B is the oblique view of user interface, shown at its extracting position near-end (Figure 17 A) and at its extracting position and half-twist; And

Figure 18 A-18D is the multiple view that is used for the equipment of paving along the intracavity of intravital pipe or chamber wall deposition composition.

Detailed Description Of The Invention

I. definition

" tissue " used herein refers in the body or an external complete organ, an organ fragment, or two or more cell.

" parenchymal tissue " used herein refers to specific functional organization, and it is different from specific organ.For example, the parenchymal tissue of heart is a cardiac muscle, is made up of cardiac muscle fiber and vascular system thereof.In addition, the parenchymal tissue of liver mainly is made up of hepatocyte.

Reversible change in response to multiple damage in " deterioration " phalangeal cell and the tissue.These damages comprise and are exposed to harmful reagent (chemistry with biology) and comprise in pressure and the extremely cold very hot physical factor.Be exposed to above-mentioned damage for a long time or consumingly and can cause cell death and necrosis.

" necrosis " the morphological change after the phalangeal cell death in the cell.Therefore, " downright bad tissue " comprises cell downright bad in the morphology.

" histologic lesion " used herein refers to multiple damage one or more influences to tissue; These influences comprise parenchymal tissue's cell deterioration, cell death, necrosis, tissue disappearance and fibroblastic or neuroglial cicatrix.

" stem cell " used herein is often referred to a kind of specific cell type, and it has self renewal and generates the specific ability of specific cell type.Although the most cells of body as myocardial cell or Skin Cell, carry out specific function, and stem cell is just carried out function up to receiving the signal that is divided into specific cells.Stem cell can carry out self-replication, and also can be evolved into the mature cell with characteristic morphology and specific function.Typically, stem cell reaches it at them and produces intermediary cell type before breaking up fully.This intermediate cell is called as precursor or CFU-GM.Stem cell can pass through the mitosis self renewal, and also can (pass through mitosis) and generate the CFU-GM can be divided into the accurate functional organization cell component in the individuality of growing up.Stem cell used herein comprises adult stem cell, and it is undifferentiated cell, spreads all over the individual body of growing up and is distributed in the multiple differentiated tissue, comprises the stem cell of hemopoietic, substrate an and matter of peripheral blood, blood and derived from bone marrow.For example, the hematopoietic stem cell that is present in the bone marrow of growing up can be filled cerebral cortex with well differentiated Purkinje cell neuron, and it is important (Wagers et al., Science, 297:2256 (2002)) for normal cortex neural line function.

As used herein, " CFU-GM " refers to participate in the cell of health tissues regenerative process." CFU-GM " is from the mitotic daughter cell of stem cell.These cells are different from stem cell by bear different task in atomization (being stimulated by plurality of reagents), and it produces the part differentiation family of cell line.These cell lines bear the specific phenotypic specific cells in differentiation and that the have common described tissue fully essence at last again.For example, α, the β of myocardial cell, skeleton sarcoplast, pancreas, gamma cells, hepatocyte, central nervous system's neuron, macroglia, oligodendroglia, can come from bone marrow stem cell and local tissue-derived adult stem cell with microglia, and be retained in the differentiated tissue of certain organs.Described tabulation only is an illustrative, and as restriction, but in these cells each all has the ability that starts tissue regeneration.Other example of CFU-GM is the local noble cells in the brain of growing up, and it is for being positioned at the daughter of the nervous system adult stem cell in the specific region (for example, dentate gyrus of Hippocampus and inferior chamber district).The cell migration of these part differentiation is to the outlying position of brain.At described outlying position, these cells promptly can be changed into the particular type that the neuron that breaks up fully also can be changed into glial cell.The function that neuron is brought into normal play in the neural circuit road.Glial cell is fulfiled the distinctive differentiation function of neuroglia class.For example, oligodendroglia generates myelin, and it is a kind of basic extracellular matrix components among the CNS, but the neuronic aixs cylinder of electric insulation.Satellite cell is the group cell of skeletal muscle fiber, and typically is positioned at the muscle fiber near surface of differentiation.Satellite cell enters mitosis and merges to form the multinuclear muscle fiber of differentiation.By suitable chemical attractant, CFU-GM can attracted to the particular organization zone of being studied, with the regenerative process of beginning health tissues.

" accesory cell " reference used herein and the regenerated cell of parenchyma, but it is not parenchyma, stem cell or parenchyma CFU-GM; The synthetic justacrine of accesory cell can stimulate stem cell and CFU-GM biotic factor, for example, marrow stromal cell, the function of performance accesory cell, generation can prevent the hepatocyte growth factor (HGF) of Neuron Apoptosis, and the nerve growth factor (NGF), neurotrophic factor 4 (NT4) and brain derived neurotrophic factor (BDNF), the propagation of all these stimulating neuronal CFU-GM and differentiation, and the apoptosis in neuronal of inhibition differentiation; The synthetic justacrine of accesory cell is to the necessary extracellular matrix components of functional framework of parenchymal tissue; The biological reagent of extracellular matrix can be modified and be moulded to the synthetic justacrine of accesory cell, to promote impaired tissue regeneration essence; The example of accesory cell is including, but not limited to following: the tissue macrophages of (a) coming the self-loopa blood monocyte; (b) marrow stromal cell, it can generate Interstitial cell in regenerating tissues for it; (c) be positioned at central nervous system's microglia.

It is used herein that " stimulation " or stimulating cell or the local regenerative cell who is attracted " refers to induce multiple activity and the phenomenon relevant with the irriate cell.These activity and phenomenon comprise: (i) in-house mobility increases; (ii) increase (for example, discharging stem cell from bone marrow enters blood flow and enter tissue at a distance subsequently) from a mobility who is organized into another tissue; (iii) cell adhesion is arrived other cell, extracellular matrix and cell adhesion part; (iv) stimulate duplicating and mitosis of DNA; (the v) synthetic and secretion of stimulating cytokine; (vi) stimulate the synthetic and secretion of chemotactic factor; (the vii) synthetic and secretion of irritation cell epimatrix composition; (viii) suppress apoptosis; (ix) influence (for example, antioxidation) of prevention degeneration reagent; (x) the synthetic and secretion of the factor of stimulation degradable extracellular matrix (for example, stimulate the release matrix metalloproteases by tissue macrophages, so that the span in extracellular matrix, the arteriole of the ischemia damage that is used to regenerate and be used for cell migration); (xi) induce cell between noble cells to connect the plasticity of framework; For example, known base cell derived factor 1-α (SDF-1 α) and interleukin-6 (IL-6) can directly act on the CNS neuron of differentiation, so that neurotoxicity infringement generation regeneration is being caused when replying that synapse connects rearrangement; (xii) promote that CFU-GM and differentiation of stem cells are function parenchyma completely; (xiii) modulate with control regenerated parenchymal tissue in the differentiation of multiple different cell types; (xiv) discharge the biological reagent that can promote that angiogenesis and/or tremulous pulse generate from the cell that is attracted; (xv) promotion stem cell, CFU-GM, accesory cell or local regenerative cell's propagation; And/or (xvi) the synthetic and secretion of stimulating factor, cytokine, the described factor can increase the cytotoxic response of the macrophage of the killer cell of lymphocyte, B cell, dendritic cell, natural killer cell, lymphokineactivation and cancerous cell.

Term " is placed on " and term " deposition " with by " is placed on " or " deposition ", and the compositions in specific tissue regions is used, refer to expect in the position or near the described compositions of introducing.Said composition can directly be placed on the target site in the tissue, perhaps can be placed near the body cavity of target tissue site.For example, the tissue site that is positioned over heart be included in pericardial cavity (space between the inner membrance of wall and parcel heart) interior deposition composition and in heart tissue (cardiac muscle) or on deposition composition.

When reagent not only can discharge active substance from the cell moderate stimulation, and also can induce CFU-GM generation mitosis, CFU-GM differentiation, the differentiation of coordinating different cell lines or engineered cells epimatrix the time, claiming this reagent pair cell to have " directly " influences.

" angiogenesis " used herein refers to cause that the endotheliocyte that forms new capillary network forms.

" tremulous pulse generation " used herein refers to the growth in situ of tremulous pulse, and it is fed to ischemic tissue, tumor or inflamed sites by propagation endothelium and smooth muscle cell from the small artery connective tissue (connection) of previous existence with blood.

" ischemic tissue ", " being in ischemic tissue " refer to tissue, tumor or inflamed sites experience blood supply insufficiency or are in blood supply insufficiency.

" ischemia " or " ischemia incident " refers to specific cell, tissue or organ blood supply insufficiency.The result that blood supply reduces is to organ or tissue's oxygen supply deficiency (histanoxia).Secular histanoxia may cause the injury to affected organ or tissue.

" anoxia " in fact refers to not exist fully oxygen in organ or tissue, if talk about for a long time, it can cause cell, organ or tissue's death.

" histanoxia " or " hypoxic condition " refers under this condition, and the oxygen supply that cell, organ or tissue accept is insufficient.

II. the compositions that is used for tissue regeneration and cancer immunization therapy

On the one hand, the present invention contains a kind of compositions, it is suitable for vivo medicine-feeding to a tissue site, this position is p cell degeneration or cell and tissue die or necrosis, tissue disappearance or downright bad back fibroblast or neuroglial cicatrix, these perhaps all influences perhaps are in any one in these influences, any two or more or whole danger.More briefly, said composition is applicable to damage back or is in tissue in the damage.Described compositions comprises first reagent, can effectively at least one stem cell and CFU-GM be attracted to the tissue injury zone; Second reagent can stimulate to be attracted cell activity effectively; With the 3rd reagent, can influence the survival that is attracted cell effectively, and preferred the influence from the survival of the cell that is attracted cell differentiation.In certain embodiments, described first reagent also will attract accesory cell.As will be described further, first, second and the 3rd reagent depend on types of organization and attracted to the stem cell or the CFU-GM at described position, and optional accesory cell.Above-mentioned all cells is autologous.All cells are transferred in the tissue site that is studied by the pipeline of normal presence in body, and it comprises the gap in blood and lymphatic vessel system, cerebrospinal fluid system and the body tissue.

As implied above, compositions can be widely used in multiple damage back or is in tissue in the damage danger, and below the compositions that is exclusively used in particular organization example is provided.In order to describe said composition in detail comprehensively, provide a kind of compositions example that is deposited on the cerebral tissue position in conjunction with Fig. 1.Yet, as from following discloses clear and definite, compositions of describing in detail in Fig. 1 and process are the example that damages in the cerebral tissue, and general generalized concept can be applicable to any impaired tissue.

After Fig. 1 has illustrated the claimed compositions of in cerebral tissue position deposition, the tissue regeneration process of the simplification on a kind of cellular level.Impaired cerebral tissue generally with 10 expressions, can be the result of ischemia incident, as apoplexy or disorder, and as Ah's Mohs (Alzheimer) disease, multiple sclerosis, meningoencephalitis, or because physical damnification.Compositions is deposited near tissue site 10 or this position, to promote regeneration impaired or dead tissue.In this embodiment, compositions adopts the form of drug storage warehouse, and as drug storage warehouse 12,14,16, it is designed to contain and discharges selected therapeutic agent.As noted above, described drug storage warehouse contains first therapeutic agent, can effectively stem cell and/or CFU-GM be reached randomly, and accesory cell is attracted to tissue site; Second therapeutic agent can stimulate to be attracted cell activity effectively; And the 3rd therapeutic agent, can influence effectively and be attracted, randomly, the survival of regenerated complete noble cells.In certain embodiments, as will be described further, same reagent promptly can be used as chemical attractant, for example, as first therapeutic agent, can be used as stimulant and/or survival reinforcing agent again.That is, in some cases, a kind of single agents can provide one or more functions of first, second and the 3rd reagent.

In specific explanation, described drug storage warehouse 12,14,16 contains first reagent, and it is as the chemical attractant of stem cell, such as the stem cell of circulation derived from bone marrow, as near the cell 18 the capillary tube 20.The reaction of the chemotactic factor gradient of setting up as the chemical attractant that drug storage warehouse is discharged, near the tissue stem cell enters by the capillary endothelial migration is shown in cell 22.The cell that is attracted can comprise stem cell and/or CFU-GM, can help the cell of tissue regeneration as mononuclear cell and other.Near accesory cell, as with the represented macrophage of macrophage 24, be attracted to tissue regions by the chemotactic factor gradient, shown in 25.In addition, stem cell near the tissue or the stem cell that comes from specific source of human stem cell position are moved to tissue site by this gradient effect.For example, for brain, adult neural stem cell as cell 26, comes from dentate gyrus or the inferior chamber district of Hippocampus, attracted to this zone.Is that near the concentration of drug storage warehouse sedimentary stem cell and/or CFU-GM increase with cytotaxis to the whole result in the described drug storage warehouse by chemotactic factor, and in some cases, the concentration of accesory cell increases, as shown in Figure 1.

In certain embodiments, the drug storage warehouse in the compositions comprises surface ligand, and it is used for interacting with cell that be attracted or that be present in tissue site.Fig. 1 has illustrated this feature, and stem cell such as cell 28,30 are attracted to the cell surface of drug storage warehouse 14.The part that the surface connects has multiple function, and can be the cell that attracted to the damaged tissues position connecting portion is provided, so that the stimulus object biotic factor is offered cell surface receptor with the function effective and efficient manner, and/or the degeneration of prevention biotic factor.Select part according to the tissue of being treated with to the demand that is attracted cell.The example of part comprises cell adhesion molecule, extracellular matrix components, and segment.Other example of the part that is used for particular organization below is provided.

Being deposited on drug storage warehouse in the compositions of tissue site contains and is useful on the reagent that release can stimulate the cell that is attracted to have an effect effectively.Fig. 1 has shown the cytokine 32 that discharges from drug storage warehouse 16, induced dry-cell 34 is divided into pyramidal neurons 36.The stem cell that is connected to drug storage warehouse 14 is induced to differentiate into glial cell 38 (spider cell) by the suitable stimulant in the drug storage warehouse 14.In addition, stimulate to be attracted to this regional macrophage, produce cytokine 42 as macrophage 40 to produce chemotactic factor and cytokine.When stimulant existed, other cell was induced propagation, and for example, under the stimulation of cytokine 48, nervous system stem cell 46 propagation are daughter cell 50,52.Daughter cell can further break up, and for example is divided into pyramidal neurons, and is as shown in arrow 54.

Although not shown among Fig. 1, described drug storage warehouse also comprises a kind of reagent, and it can allow the average life of regenerated complete noble cells in cell that the survival that is attracted cell or prolongation are attracted and the damaged tissues effectively.Suitable reagent should change according to types of organization and the cell that is attracted, and below provided the embodiment that is used for multiple tissue.

According to another feature of described compositions, drug storage warehouse can move from initial deposition site and be dispersed in the organization space.This feature has been described in Fig. 1, and arrow 27 is indicated moving of drug storage warehouses 12.Typically, this moving in tissue will occur with the result that CONCENTRATION DISTRIBUTION, buoyancy and/or fluid move.

Fig. 2 is the detail drawing of drug storage warehouse 12 interior a-quadrants among Fig. 1.In this embodiment, drug storage warehouse 12 is porous, shown in hole 60.As will be described further, other embodiment of contemplated composition does not comprise the porous drug storage warehouse that is used to hold therapeutic agent.Therapeutic agent is comprised in the hole of drug storage warehouse,, in hole 60, still also can be contained in the zone between the hole as cytokine 62, and as the solid area of drug storage warehouse, for example, in the intermolecular space of drug storage warehouse compositions.For the cell that is attracted is retained near the drug storage warehouse, the cell adhesion part as cellular adhesion peptide 68, can be merged in the drug storage warehouse substrate, thereby the part in these peptides is exposed on the drug storage warehouse surface, shown in 66.Among other embodiment of the drug storage warehouse of expection, the drug storage warehouse surface scribbles the compositions that comprises cellular adhesion peptide, and peptide will be exposed to be attached to cell surface receptor.

In a word, (i) with stem cell and/or CFU-GM and selectively effectively to contain the deposition of compositions of one or more therapeutic agents, accesory cell is attracted to damage back or is in tissue regions in the damage, (ii) stimulate the cell that is attracted, for example, stem cell, CFU-GM and/or accesory cell, and/or local regenerated cell, to start or to participate in a series of incidents that cause tissue regeneration, and the survival that (iii) promotes the cell that is attracted in this zone and other cell, to prolong the activity and the life-span of regenerating tissues.The accurate normal function of functional organization's regeneration tolerable tissue, and in a preferred embodiment, regenerated tissue will combine with near the int health tissues the damage field.Described compositions promotes tissue regeneration by injecting the health tissues cell of living at tissue regions cicatrix and impaired.

As intelligible from above content, described compositions is widely used for the regeneration of damaged tissues, and especially impaired parenchymal tissue.To further explain the present invention by being suitable for the regenerated particular composition of particular organization now, these particular organizations are caused by the impaired institute of i or I, comprise cardiac muscle, skeletal muscle, liver, pancreas, the central nervous system kidney of unifying.

A. the tissue after ischemia or be in and be used to promote the regenerated compositions of parenchymal tissue in the ischemic tissue

In one embodiment, compositions comprises the drug storage warehouse that contains one or more selecteed therapeutic agents, is used for the damaged tissues regeneration that is caused by the ischemia incident.The disease that treatment is caused by ischemia is always a field of medicaments of actively probing into, and especially in western countries, there, ischemic heart disease is always the leading factor that causes mortality rate.Myocardial ischaemia was caused by unexpected obstruction of blood vessel structure in unexpected obstruction of the main coronary artery that can cause cardiomyocyte cell death and the heart supply area.Similarly, the main angiemphraxis of other organ and tissue can cause the key cell death of this tissue and the serious degeneration of ability that this tissue is kept its normal correct function.For example, cerebral ischemia, wherein, the arterial blood that flows to brain is obstructed or is dropped to the subcritical level, can cause of short duration ischemia outbreak and apoplexy.Although myocardial ischemia and cerebral ischemia are two kinds of more common ischemia forms, it also damages other body tissue, as eye, kidney, liver, body lower limb, intestinal, skin and repulsion flap etc.

Be used to control ischemic fresh approach and comprise and attempt to stimulate the growth of new capillary network and the growth of collateral vessel, this generates by blood vessel and tremulous pulse and realizes.Angiogenesis is the new interior rubber-insulated wire blood vessel of finger-type one-tenth composition capillary network typically.Tremulous pulse generates and to be different from angiogenesis, and it refers to the mitosis by endothelium and smooth muscle cell, with existing side arteriole growth and transform the function tremulous pulse as, forms the theca externa of elastic thin layer and conjunctive tissue.The vascular smooth muscle cell layer provides the intensity and the integrity of vasomotion control and structure.With arteriole and capillary network contrast.Capillary bed is to carry out nutrition in the tissue and the gas clearing house is essential.Yet because its diameter is less, capillary tube can not satisfy the needs of tissue to a large amount of blood flows all the time.This demand is often only satisfied by larger-diameter tremulous pulse.

The strategy that is used to stimulate angiogenesis and tremulous pulse to generate concentrates on the regional area that essential biological peptide is sent to ischemic tissue.For example, transmit basic fibroblast growth factor (bFGF) and change, shown (Laham R.J.etal., Clin.Cardiol.22 (Supp.1): 16-19, (1999) of improving of blood flow to impaired tremulous pulse.Administration VEGF (VEGF) has improved myocardium blood flow and has been proposed for promotion vascular tissue repairs (EP-A-506477).The biotic factor that other is proposed is used to promote angiogenesis and/or tremulous pulse to generate comprises placental growth factor, and (WO 01/57181; WO 01/56593) and cluster stimulating factor (WO 99/17798).Also described a kind of method in the prior art, it relates to packs therapeutic agent into mononuclear cell to stimulate tremulous pulse generation (WO 00/60054).In addition, has ischemia limbs (Isner, J.M.et al., Lancet, 348:370-374, (1996)) and the patient of myocardial ischaemia in cause angiogenesis other method adopted the tremulous pulse gene transfer (Losorda of ph VEGF, D.W.et al., Circulation, 98 (25): 2800-2804, (1996)).

Shown by transplanting strategy that myocardial cell or skeleton sarcoplast replace scar tissue zone downright bad in heart and the skeletal muscle and obtained some success (Leor aspect the survival of part transplanted cells making, J.et al., Circulation, 94 (Supp.11): 331-336, (1996); Murry et al., J.Clin.Invest., 98:2512-2523, (1996); Taylor et al., Nat.Med., 4:929-933, (1998); Tomita et al., Circulation, 100 (Supp.11): 247-256, (1999); Menusche et al., Lancet, 357:279, (2000)).Yet they are failed to recombinate and carry out cardiac muscle or the skeletal muscle and the crown or peripheral artery of the health of function with the health tissues of living jointly.

These methods by cell transplantation and promotion angiogenesis and tremulous pulse generation reflect in the early stage trial aspect ischemic Therapeutic Method.Although some challenging discoveries are arranged, these methods all can not return to significant degree with the blood flow in ischemia zone or the structural intergrity of cell, so that improve the physiological property in ischemia zone clinically.Therefore, press for all the time and angiogenesis, tremulous pulse generated and parenchyma returns to and can be used for clinical significance degree.

This paper provides and has been used for promoting the tissue or the parenchymal tissue in tissue after the ischemia or the hypoxic tissue that are in ischemia to recover and sanguimotor compositions and method.As the therapeutics feature, the compositions of description can stimulate angiogenesis, tremulous pulse generation and cardiac muscle and skeletal muscle fiber regeneration effectively.

1. be used to promote the regenerated therapeutic agent of parenchyma regeneration, revascularization and/or tremulous pulse

As noted above, compositions contains one or more therapeutic agents, can attract stem cell and/or CFU-GM effectively, and selectively attract accesory cell, has an effect with irritation cell, and influences the survival of cell.For the compositions that is designed for treating ischemic tissue, therapeutic agent more specifically is chosen to (i) with the circulation hemocyte, comprise medullary cell, stem cell and CFU-GM and mononuclear cell (as accesory cell) are attracted to the ischemia position or are in ischemic tissue, (ii) stimulate the multiple phenomenon in the described cell, comprise that mitosis takes place, the mobility, connect, differentiation, release can promote angiogenesis, the biological reagent that tremulous pulse generates, and be myocardial cell and skeletal muscle fiber, and (iii) influence the macrophage of monocyte derived with bone marrow and differentiation of stem cells, the myocyte and the muscle fiber of differentiation, myocyte's CFU-GM, sarcoplast, the survival of the endotheliocyte in smooth muscle cell and the tissue.As everyone knows, mononuclear cell comes from the myeloid progenitor of derived from bone marrow and belongs to monokaryon phagocyte system.These cells are a class leukocyte, and circulation also can enter organization space by the endodermis migration from blood flow in blood, and their maturations are macrophage there.The microorganism of macrophage phagocytic and digestion invasion and allochthon and impaired and cell aging.These cells also help arteriole is transformed into tremulous pulse by extracellular proteinase, and it opens passage in the extracellular matrix promoting the propagation and the migration of smooth muscle cell, and the tremulous pulse that holds transformation.As used herein, " mononuclear cell " refers to other cell of mononuclear cell and demonstration and the same or analogous biological agent of mononuclear cell.

Myocardial cell and skeleton sarcoplast come from bone marrow stem cell and the stem cell that is retained in the muscle.They differentiate from muscle stem cell similar with sarcoplast and that be called as satellite cell.Satellite cell enters mitosis and several merging to form the differentiation muscle fiber.These cells can attracted to the tissue regions of being studied by suitable chemical attractant, are in the regenerative process of the healthy muscular tissue in the ischemia zone with the zone neutralization of beginning ischemia.

Therefore, a class therapeutic agent that is used for ischemic tissue that comprises in the present composition is a biological reagent, can be effectively granulation promoting CFU-GM, precursor endothelium and the smooth muscle cell of circulation blood monocyte, bone marrow stem cell, derived from bone marrow be attracted to tissue on the line.Described reagent can be the signalling molecule, as chemical attractant, can effectively the deutero-granulation promoting CFU-GM of circulating monocytic cell, bone marrow stem cell, bone marrow and muscle, precursor endothelium and smooth muscle cell be attracted to tissue regions and from circulation, move to tissue regions.The chemical attractant that is suitable for comprises, but be not limited to, chemotactic factor, as macrophage chemical attraction albumen (MCP-1, MCP-2, MCP-3, MCP-4, MCP-5), RANTES (normal T cellular expression of activatory adjusting and excretory cytokine), the Fu Shi factor, macrophage inflammatory protein (MIP)-1-a and MIP-1-β, N-farnesyl-peptide (N-farnesyl peptides) complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), stem cell factor (SCF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), platelet-derived somatomedin AB and BB (PDGF-AB, BB), leukaemia inhibitory factor (LIF), and the function of these chemical attractant equates segment.Also can expect and be used for the deutero-granulation promoting CFU-GM of mononuclear cell, bone marrow stem cell, bone marrow and muscle as attractant, precursor endothelium and smooth muscle cell be transforming growth factor (TGF-β), interleukin and cluster stimulating factor, as granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1) and macrophage cluster stimulating factor (M-CSF), and the suitable fragment of function.

Except the deutero-granulation promoting CFU-GM of mononuclear cell, bone marrow stem cell, bone marrow and muscle, precursor endothelium and smooth muscle cell, wish that also chemical attractant can be suitable for attracting other cell that relates in angiogenesis and/or tremulous pulse generative process, perhaps chemical attractant can optionally attract these other cells specially.Circulating cells also relates to angiogenesis, tremulous pulse and generates, and muscle cell is grown the leukocyte that comprises other, platelet, the leukocyte precursor of derived from bone marrow, the stem cell and the vascular endothelial cell CFU-GM of derived from bone marrow.Vascular smooth muscle cell (VSMC) is broken up from multiple precursor, comprises bone marrow stem cell, macrophage and the partial Interstitial cell of organizing.Expection as stimulus object be used for smooth muscle cell and endotheliocyte for GM-CSF, G-CSF, M-CSF, SCF and platelet derived growth factor (PDGF-BB).

Table 1 has been summed up the multiple somatomedin that cardiac muscle and Skeletal Muscle Cell and precursor thereof are had stimulation.These factors are suitable as chemical attractant, or as the reagent of further breeding and break up some cell.

Table 1

The factor	Factor full name	Active
			bFGF	Basic fibroblast growth factor	Induce myoblastic propagation and migration; Stimulate one-tenth flesh with Interstitial cell reorganization; Promote the sarcoplast differentiation
HGF	Hepatocyte growth factor	Induce myoblastic propagation and migration; Increase the cell number in sarcoplast propagation and the increase myotube; Strengthen the transfer ability of myogenous cell
			IGF-1	Insulin like growth factor 1	Promote the survival of skeletal myoblast
IGF	Insulin like growth factor	Induce myocyte's CFU-GM generation mitosis of cardiac muscle and be divided into myotube; Promote the myoblastic survival of skeleton and be divided into myotube
			EPO	Erythropoietin	Suppress skeleton sarcoplast apoptosis; Between the idiophase, stimulate sarcoplast propagation with the amplification progenitor cell populations; Play a role at muscle development with in repairing
LIF	Leukaemia inhibitory factor	Strengthen nervimuscular activity; Strengthen nervus motorius and sensorineural survival; Increase anathrepsis; Mitosis promoting takes place and the skeletal myoblast differentiation
			NGF	The nerve growth factor	Sarcoplast propagation, fusion are to form the active stimulus of myotube

Another therapeutic agent in the compositions is a kind of reagent that can stimulate release can promote the biological reagent that angiogenesis and/or tremulous pulse generate, and this paper claims " stimulant ".Will be appreciated that this stimulant can be identical reagent with the reagent that is used for mononuclear cell, stem cell and granulation promoting CFU-GM are attracted to research organization of institute, or different reagent.Stimulant can stimulate in mononuclear cell, stem cell or other tissue on the line or near angiogenesis and tremulous pulse cellulation effectively, to discharge the biologic artifact of one or more participation angiogenesis, tremulous pulse generation and muscle cell regenerative process.Except that mononuclear cell, the cell that relates to angiogenesis, tremulous pulse generation and muscle cell regenerative process comprises leukocyte precursor, the stem cell of derived from bone marrow, vascular endothelial cell, the vascular smooth muscle cell of endotheliocyte, mastocyte, lymphocyte, granulocyte, leukocyte, platelet, derived from bone marrow and precursor thereof.

In addition, wish that stimulant also can influence tremulous pulse generation, angiogenesis and the regeneration of muscle parenchymal tissue by directly acting on mononuclear cell, other accesory cell, stem cell and the myocyte's CFU-GM that directly acts on these cells.As shown in table 1, stimulate the cardiac muscle and the propagation of Skeletal Muscle Cell precursor and break up by many in the known described reagent to form regenerated cardiac muscle and skeletal muscle fiber, it implements its influence by directly acting on object.Do not stimulating under middle biotic factor generation and the excretory prerequisite muscle essence of can regenerating.Similarly, many reagent that stimulate angiogenesis and tremulous pulse to generate are implemented its influence by directly acting on vascular endothelial cell and CFU-GM thereof and vascular smooth muscle cell and precursor thereof.

Stimulant can be chemical compound biology or abiotic, its stimulate from macrophage and cell other blood vessel origin and the tremulous pulse origin produce blood vessel origin with the bioactive molecule tremulous pulse origin, perhaps it stimulates propagation and the survival and the myoblastic fusion of differentiation of stem cells and muscle cell.The example of biological reagent comprises, but be not limited to, chemotactic factor, as macrophage chemical attraction albumen (MCP-1, MCP-2,, MCP-3, MCP-4, MCP-5), tumor necrosis factor (TNF-a, TNF-β), RANTES (normal T cellular expression of activatory adjusting and excretory cytokine), the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α and MIP-1-β, and the function of these reagent equates segment.Also expection as stimulant be interleukin, cluster stimulating factor, as granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1) and macrophage cluster stimulating factor (M-CSF), platelet derived growth factor (PDGF-AB and PDGF-BB), fibroblast growth factor (b-FGF) substantially, insulin like growth factor (IGF-1), the nerve growth factor (NFG), and the function of any of these reagent equates segment.Lipopolysaccharide (LPS) is the derivant of some bacteria cell walls, also is suitable for as effective stimulus object, as lacks the similar molecule of LPS in the poisonous zone of molecule, and it has the macrophage-stimulating effect of LPS.What also expection was used as stimulant is angiogenin-1 and-2 (Ang-1 and Ang-2), hepatocyte growth factor (HGF), leukotriene B4, complement activation product C 3a and C5a, leukaemia inhibitory factor (LIF), erythropoietin (Epo), U-18496 and transforming growth factor (TGF-β).

Stimulate angiogenesis, tremulous pulse generation and the regenerated reagent of muscle essence except that can effectively circulating monocytic cell, bone marrow stem cell, bone marrow and muscle derived granulation promoting CFU-GM, precursor endothelium and smooth muscle cell being attracted to the tissue of being studied and passing through directly effect or pass through the secretion inducing bioactive agents, compositions can also comprise a kind of reagent, it can influence the cell survival in the tissue of being paid close attention to effectively, and described cell has participated in the regeneration of vascular system and muscle parenchymal tissue.For example, prolong from the survival of the monocytic macrophage that attracted to tissue regions and prolong that the survival with the cell tremulous pulse origin other blood vessel origin will promote and prolong the required reagent that relates to angiogenesis and/or tremulous pulse generation of release in the tissue regions.Compositions also can comprise a kind of reagent, and it can increase the number that participates in angiogenesis and tremulous pulse generation and can be divided into the circulating stem cell of cardiac muscle and Skeletal Muscle Cell effectively.Can influence the circulation blood monocyte and be arranged in the monocyte derived of tissue macrophage, and other helps angiogenesis and tremulous pulse to generate and the biological reagent that increases the cell survival of stem cell number comprises GM-CSF, G-CSF, CSF-1, M-CSF, IGF-1, Ang-1, and the function reagent and the fragment thereof that equate.

Compositions also can comprise the biological reagent that one or more relate to angiogenesis and tremulous pulse generative process.These reagent typically other cell from macrophage and ischemic tissue discharge, but also can be used as the part of therapeutics compositions.The factor that relates to the generation of angiogenesis and/or tremulous pulse comprises fibroblast growth factor (FGF, FGF-1, FGF-2), TGF-a, insulin like growth factor-1 (IGF-1), angiogenin-1 (Ang-1), angiopoietin-2 (Ang-2), VEGF (VEGF), its function equal fragments, VEGF constitutes thing such as VEGF-2, VEGF165 and VEGF121, platelet derived growth factor-A (PDGF-A) and/or PDGF-B and/or PDGF-BB, Placenta Hominis derivative growth factor (PIGF) and other endothelium mitotic growth factor, or its function equal fragments.

Compositions also can comprise a kind of reagent that helps circulating monocytic cell, macrophage or other cell are attached to tissue regions.Described reagent also can play a role with macrophage and described drug storage warehouse in conjunction with or closely is connected, make the approaching reagent of macrophage from drug storage warehouse release.In one embodiment, compositions also comprises cell adhesion molecule, as ICAM, VCAM, PECAM; VE-adheres to son (cadherin); Fn Fiberonectin (fibronectin) segment is as RGD and REDV; Synthetic adhesin polypeptide is as VAPG and KQAGDV; The heterogeneous mixture of extracellular matrix molecule and collagen, Fn Fiberonectin, heparin, dextran, Hai Lan, laminin and from the treated extracellular matrix (ECM) of animal origin.As described below, described reagent is connected with described drug storage warehouse in many ways, comprises described reagent is sandwiched or the drug storage warehouse of packing into coated surfaces or reagent is connected to the drug storage warehouse outer surface.

2. cardiac muscle regeneration

As intelligible,, can realize treating and be in the ischemia danger or the cardiac muscular tissue after the ischemia by adopting compositions described herein from above discussion.Except that since infraction or in the wind-induced ischemia, cardiac muscular tissue is subjected to the infringement from other symptom easily, as inflammation, deleterious, metabolic and born etiologic cardiomyopathy, and congestive heart failure.The caused cardiac muscular tissue of symptom that the aforesaid example that is suitable for the compositions of ischemia cardiac muscular tissue also can be used for the treatment of except that ischemia is impaired.In addition, the compositions that is used for the treatment of damaged myocardium can design according to following discussion.

As noted above, compositions of the present invention need have the biotic factor of three kinds of functions, and chemical attraction stem cell, CFU-GM and accesory cell stimulate described cell and strengthen described cell and the survival of the noble cells of regeneration cardiac muscle.The compositions that is exclusively used in treatment cardiac muscular tissue will comprise the attraction CFU-GM, and it comprises the myocardial cell stem cell of myocardium sarcoplast (myocardial cell), bone marrow stem cell, hematopoietic stem cell, marrow stromal cell, existence.In process of tissue reparation, also relate to endotheliocyte, vascular smooth muscle cell and precursor thereof.Therefore, compositions will comprise at least a factor as the chemical attractant of one or more described CFU-GM.Many examples of chemical attractant have more than been provided.

Compositions also will comprise and at least aly be used to stimulate CFU-GM to have an effect, for example, and the factor of differentiation and/or propagation.Suitable stimulating factor including but not limited to stem cell factor, insulin like growth factor-and-2 (IGF-1, IGF-2), transforming growth factor-a ,-β (TGF-a, TGF-β), heparin associative list skin growth factor class somatomedin (HB-EGF) and U-18496.

Compositions also will comprise at least a factor, and it is had an effect to promote CFU-GM and differentiation offspring's thereof survival, and the example of stimulant is including, but not limited to GM-CSF, G-CSF, CSF-1, M-CS and IGF-1.

Compositions also will comprise the cell adhesion molecule part, and it is attached to the cell surface conglutnin on stem cell (particularly VLA4) and the CFU-GM.The example of part is vascular cell adhesion molecule 1 (VCAM-1).Adhere to by this part and to have guaranteed stem cell is navigated to compositions deposition site in the damaged tissues.

3. skeletal muscle regeneration

In another kind of embodiment, compositions is designed to repair skeletal muscle by generating muscle fiber.Compositions is applicable to the regeneration of impaired skeletal muscle, and for example, (Duchenne ' s) muscular dystrophy, physics extruding injury, obstructive peripheral diseases and other damage cause because heat injury, Du Xing.

The CFU-GM that relates to repair process comprises bone marrow stem cell, hematopoietic stem cell, marrow stromal cell, skeletal muscle satellite cell, sarcoplast, peripheral blood cells and muscle CFU-GM.Is to realize by the compositions that introducing contains chemical attractant these cytotaxis to the skeletal muscle damaging part.The example of attractant comprises somatomedin, as basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-3 (TGF-β), platelet derived growth factor (PDGF), platelet derived growth factor-AB (PDGF-AB), platelet derived growth factor-BB (PDGF-BB), interleukin 8 (IL-8), tumor necrosis factor-a (TNF-a), leukotriene B4, Fn Fiberonectin (Fn) segment, complement activation product C 3a and C5a, leukaemia inhibitory factor (LIF) and active normal T cellular expression and excretory cytokine (RANTES) of regulating.

Compositions also will comprise the factor that cell that at least a stimulation is attracted is had an effect.The factor that is fit to comprises, but be not limited to insulin like growth factor-I (IGF-I), insulin like growth factor-II (IGF-II), basic fibroblast growth factor (bFGF), the nerve growth factor (NGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), platelet derived growth factor-BB (PDGF-BB), transforming growth factor (TGF-β), erythropoietin (EPo), human leukemia inhibitory factor (hLIF) and U-18496.

Compositions also will comprise one or more factors of the noble cells survival that can prolong CFU-GM and regeneration parenchymal tissue.The factor that can prolong the cell survival in the skeletal muscle tissue comprises, insulin like growth factor-I (IGF-I), insulin like growth factor-II (IGF-II), platelet derived growth factor-BB (PDGF-BB) and erythropoietin (EPO).

Compositions also will comprise the cell adhesion molecule part, and it is attached to the cell surface conglutnin on stem cell and the CFU-GM.The example of part comprises vascular cell adhesion molecule 1 (VCAM-1), basement membrane and Fn Fiberonectin segment (Fn).Adhere to by this part and to have guaranteed stem cell is navigated to compositions deposition site in the damaged tissues.Also known basement membrane can be stablized and keep as above the activity as the various kinds of cell factor of the stimulus object factor of expection, and has an effect these cytokine parts are offered the receptor on the stem cell and the surface of CFU-GM, to produce optimum stimulation.

4. the tissue regeneration in the liver

Liver is the target that numerous disease is attacked.These diseases comprise infectious, autoimmunity and not infectious process, as chemical toxicity.The example of infectious disease comprises: (i) viral hepatitis, for example, hepatitis A, B, C, D and G, and (ii) parasitics hepatitis, for example, Schistosoma mansoni, Schistosoma haematobium and Schistosoma japonicum.(Harrison′s Principles ofInternalMedicine，Fauci et al.eds.，1998，pgs 1660-1725)。Influence the noninfectious example of liver, comprise autoimmune disease, as (i) autoimmune hepatitis and (ii) constitutional gallbladder sclerosis.(Harrison′s Principles of Internal Medicine，Fauci et al.eds.，1998，pgs 1701-1709)。No matter the attack to liver is infectious, autoimmunity or not infectious, if keep the infringement of liver is not treated, will cause cicatrix or fibrosis.Be sclerosis in fibrosis latter stage.From pathology, sclerosis is defined as large-scale fibrosis in the liver, and it has the pass with formation regeneration.For many types, if not whole chronic hepatic injurys, be hardened to final common approach, and cumulative typically.Although in many cases, liver has huge self-regeneration ability, but hepatic injury can suppress or abolish this regeneration capacity.Therefore, the present invention expects that deposition composition is to influence the regeneration of hepatic tissue.

The stem cell and the CFU-GM that relate to liver regeneration comprise hepatocyte, medullary cell, liver precursor stem cell, liver adult stem cell, liver epithelial solencyte and ovum.By introducing contain chemical attractant compositions and with these cytotaxis to the tissue injury position.The example of attractant comprise granulocyte cluster stimulating factor (G-CSF), interleukin 8 (IL-8), stroma cell derivative factor (SDF-1), stem cell factor (SCF), sulphuric acid polysaccharide such as flucoidan and above-mentioned be used for cardiac muscle, skeletal muscle be used to attract stem cell and CFU-GM all chemical attractant to promote that angiogenesis and/or tremulous pulse generate.

Compositions also will comprise the factor that cell that at least a stimulation is attracted is had an effect.The factor that is fit to comprises, but be not limited to insulin like growth factor-1 (IGF-I), insulin like growth factor-11 (IGF-II), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), activin-B, interleukin-6 (IL-6), tumor necrosis factor (TNF), fat polysaccharide (LPS), transforming growth factor-a (TGF-a), transcription factor comprises the nuclear factor (NFKB) that is used for B cell kappa chain, STAT3, AP-1, C/EBP regulating liver-QI bundle swashs thing (HSS).

Compositions also will comprise one or more factors of the survival of the noble cells that can prolong CFU-GM and regenerated liver parenchyma tissue.Can prolong that the factor of cell survival comprises interleukin-6 (IL-6) and tumor necrosis factor (TNF) in the hepatic tissue.

5. the tissue regeneration in the pancreas

The human pancreas is a kind of gland that comprises external secretion and endocrine tissue, and secretion and endocrine tissue that the external secretion tissue relates to digestive enzyme relate to generation insulin, glucagon, somatostatin and pancreatic polypeptide.Insulin and glucagon are had an effect together with the generation and the metabolism of control glucose.Endocrine pancreas comprises the Langerhans islets of langerhans, and it is the aggregation of polypeptide hormone, produces extensively to spread all over the cell of glandular tissue and be maximum at the pancreas afterbody.Typically, whole islet tissues only account for about 1 or 2 percentage ratio of pancreas quality.

Islet tissue comprises at least three kinds of cell type that function is different: A (or " a ") cell can make glucagon, B (or " β ") cell is made insulin, and D (or " δ ") cell can be made the PP cell of the 3rd island hormone, somatostatin and secretion pancreatic polypeptide.The B cell is the abundantest in three types the island cell.Insulin promotes cell, and especially muscle cell is to the absorption of glucose, and prevention is stored in glycogen in liver and the muscle by excessive damage.

The 4-5% of diabetes affects whole world population, and be modal metabolic disturbance.The individual amount that diagnosis suffers from diabetes increases fast, and especially in developed country, and disorder often causes accessory complication, as retinopathy, nephropathy, neuropathy and cardiovascular disease.II type (irrelevant with insulin) diabetes are modal diabetes form, and the case of diagnosis is more than 90%, and causing jointly by insulin resistance, pancreatic beta cell dysfunction or the two.The disorderly part of β cell function is because to the increase in demand of insulin and the β cell can not be corresponding to producing and the biologically active insulin of secretion capacity causes.I type (insulin is correlated with) diabetes are destroyed by the autoimmunity of the insulin that produces the β cell and are caused, cause insulin deficit.For two types diabetes, existing therapeutic scheme may need administration every day insulin.These therapeutic schemes are not make us full, and this is because they can not be cured, and great majority be not enough to prevent next with the diabetes complications associated with arterial system.

The strategy that replacement can produce the pancreatic beta cell of insulin concentrates on allogenic islet cells and stem cell transplantation.All transplanting that come from the donor that is different from receptor need the long term administration immune suppressant drug, transplanting host disease with the repulsion of prevention transplanted tissue, be infective bacterial and viral infection screening graft donor, and the easy trouble body constitution of other weak disease is carried out genescreen.

The infringement of pancreatic tissue may be caused by diabetes.Therefore, the present invention expects that deposition composition influences the regeneration or the reparation of pancreatic tissue.

Relate to the regenerated cell of impaired pancreatic tissue and comprise β cell, island stem cell, island CFU-GM and pancreatic duct stem cell.By introducing contain chemical attractant compositions and with these cytotaxis to the histologic lesion position.The example of attractant comprises cell derived factor-1 (SDF-1), stem cell factor (SCF), flucoidan and the extracellular matrix (ECM) of VEGF (VEGF), granulocyte cluster stimulating factor (G-CSF), interleukin 8 (IL-8), substrate.

Compositions also will comprise at least a factor that stimulates the cell that is attracted to have an effect.The factor that is fit to comprises, but be not limited to the nerve growth factor (NGF), basic fibroblast growth factor (bFGF), VEGF (VEGF), insulin like growth factor-I (IGF-I), insulin like growth factor-II (IGF-II), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), keratinocyte growth factor (KGF), activin-A, extendin-4, growth hormone, prolactin antagonist, human placental lactogen, nicotine, β tunicin (BTC), leukaemia inhibitory factor (LIF), macrophage migration inhibitory factor (MIF), the islets of langerhans factor-1 (Is1-1), pancreas duodenum homology frame-1 (Pdx-1), pancreas duodenum homology frame-4 (Pdx-4), pancreas duodenum homology frame-6 (Pdx-6), cdx-2, Nkx-6 and HNF (HNF-1 α).

Compositions also will comprise one or more factors of the survival of the noble cells that can prolong stem cell, CFU-GM, regenerating tissues.The factor example that can prolong cell survival in the pancreatic tissue is insulin like growth factor-1 (IGF 1), the nerve growth factor (NGF) and interleukin-6 (IL-6).

Compositions also can comprise the part that can be incorporated into stem cell and CFU-GM surface.The expection of these parts increases the regeneration effect by increasing near the cell number as the stimulating factor of the part of compositions.But knownly take place and differentiation in conjunction with stem cell and CFU-GM cell adhesion part mitosis promoting to extracellular matrix.Suitable part comprises N, R, E-cadherin and other cell adhesion molecule (CAM), comprises nerve cell adhesion molecule (NCAM).

Tissue regeneration among 6 central nervous system

Brain and spinal cord tissue also can be caused by multiple situation and situation, comprise infection (as various antibacterials and viral meningitis), angiopathy (as hemorrhagic and ischemic stroke), degenerative disease (as multiple sclerosis, parkinson, Ah's Mohs disease) and physical damnification, comprise cerebral concussion, laceration of brain and the pressure and the crush injury of spinal cord.

Therefore, in one embodiment, the present invention expection utilizes said composition regeneration damage back or is in brain and myeloid tissue in the damage.Particularly, said composition will comprise first reagent that can attract important cells effectively, including, but not limited to microglia, oligodendroglia, nervous system adult stem cell, neuron, bone marrow (BM) cell, accesory cell (AC), smooth muscle cell (SMC), bone marrow stromal cell (mSC), marrow hemopoietic stem cells (hSC) and astrocyte.In these cells some can be moved by blood brain barrier (BBB) and/or are present in the cerebral tissue.Known attraction accesory cell is basic to the response brain injury.Known marrow stromal cell (mSC), myeloid element (hSC), other bone marrow (BM) cell, microglia, astrocyte and mononuclear cell macrophage enter injured tissues zone and as accesory cell (AC), generate the various kinds of cell factor and other biotic factor, the mitosis of direct induced dry-cell of this factor and CFU-GM takes place, and CFU-GM is divided into functional neuroglia and neuron.Reagent can attract one or more above-mentioned cells effectively, comprises hepatocyte growth factor (HGF), macrophage chemical attractant protein 1-1 (MCP-1), stroma cell derivative factor-1a (SDF-1a), stroma cell derivative factor-1 β (SDF-1 (β), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1 (IL-1) and platelet derived growth factor-AB (PDGF-AB).

Second reagent that is included in the compositions can stimulate the multiple phenomenon that is attracted in the cell, it comprises: (I) proliferating stem cells and CFU-GM, (ii) be divided into functional parenchyma, and (iii) produce multiple cytokine and other biological reagent that stimulates proliferation, breaks up and regulate and coordinate the differentiation between regenerating nerve colloid and the neuron.The example of stimulant includes, but are not limited to neurotrophic factor 3 (NT3), neurotrophic factor derived from brain (BDNF), the nerve growth factor (NGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), the proteinic NEP1-40 inhibitor of Nogo, neurotrophic factor 4 (NT4), beta-mercaptoethanol (β-ME), human leukemia inhibitory factor (hLIF), tretinoin (RA), interleukin-1 (IL-1), interleukin-6 (IL-6), platelet derived growth factor-AB (PDGF-AB), transforming growth factor-a (TGF-a), stem cell factor (SCF), VEGF (VEGF), insulin, forskolin, valproic acid, heparin, heparinoid, glycosylated cells inhibitor-C and phorbol myristic acid acetate (TPA).

In addition, also contemplated composition comprises and directly acts on histiocyte epimatrix and the blood-brain barrier, to allow the reagent of growing fully of regenerated nervous system parenchymal tissue.These reagent can be realized neuronic plasticity, wherein, are different from the damaged tissues those, and it realizes parenchymal tissue's regeneration by the neuroid that reconnects in the structure.Realize that the regenerated example agents of these compositions comprises the NEP1-40 inhibitor and the VEGF A (VEGF-A) of neurotrophic factor 3 (NT3), chondroitin A BC (chABC), Nogo protein bound.

Compositions also will comprise one or more factors that prolong the cell survival of stem cell, CFU-GM and/or differentiation.Can prolong that the factor of cell survival comprises in the cerebral tissue, neurotrophic factor derived from brain (BDNF), the nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor-AB (PDGF-AB), glycosylated cells inhibitor-C, beta-mercaptoethanol (β-ME), butylated hydroxyanisol (BHA), dimethyl sulfoxine (DMSO), hepatocyte growth factor (HGF), the nerve growth factor, neurotrophic factor 4 (NT4), butylated hydroxy-methylbenzene (BHT) and human leukemia inhibitory factor (hLIF).

Compositions also can comprise the part that can be attached to stem cell, CFU-GM and other cell surface.Expect that the cell number of stimulating factor of vicinity of the ingredient that these parts can be by increasing compositions increases the regeneration effect.But known stem cell and CFU-GM are attached to the cell adhesion portion mitosis promoting of extracellular matrix to be taken place and differentiation.Suitable part comprises laminin and vascular cell adhesion molecule 1 (VCAM-1).

7. the tissue regeneration of kidney

In another kind of embodiment, the present invention includes the tissue regeneration of kidney.Multiple disease can cause the infringement to excess of the kidney matter tissue, the blood vessel soccer star nephritis, toxicity nephropathy and the pyelonephritis that cause as atheromatosis, renal veins thrombosis, thrombosis of renal artery, thrombosis, diabetic nephropathy, multiple cause of disease.As the result of infringement, be a kind of serious symptoms by the acute or chronic kidney renal failure that causes that descends, it can cause that the filtration of kidney, absorption again, endocrine and homeostasis function are significantly or completely lose.

Therefore, the compositions that deposition contains therapeutic agent can attract those can break up or breed cell to cell effectively, the part or all of function that is provided by kidney can be provided, and can produce the kidney of functional nephrocyte or regenerating functional, expect all or part of like this.The stem cell of kidney can be worked to forming renal tubular cell.The cell of known bone marrow matrix (mSC) can move to the damaging part that is caused by glomerulonephritis and the Interstitial cell structure is provided from bone marrow by peripheral blood, and when the regeneration kidney, it is required (basic functional units of kidney) for complete kidney Elementary Function.Hematopoietic stem cell can be divided into mesangial cell.By reagent such as interleukin 11 (IL-11) and steel factor (steel factor) promotion differentiation and propagation.Deposition contains chemical attractant, has the reagent that can promote cell differentiation and propagation simultaneously, and a kind of combination of agents thing that strengthens stem cell, CFU-GM survival attracts to be divided into stem cell or the CFU-GM that forms the nephridial tissue cell, the cell of differentiation will cause impaired nephridial tissue reparation, tDy, for example, the multiple nephrocyte of regenerating is as mesangial cell, podocyte, epithelial cell, smooth muscle cell and endotheliocyte.

8. tissue regeneration compositions and stem cell transplantation

Stem cell transplantation is widely studied as a kind of Therapeutic Method, and it can be used for treating multiple disease, as leukemia and multiple myeloma.In another embodiment of the invention, expectation is with stem cell transplantation, and deposition contains chemical attractant, irritation cell differentiation or the factor of propagation and the compositions that strengthens the factor of described cell survival that is useful on stem cell and CFU-GM.In this embodiment, select the factor according to disease and/or by the tissue of being treated.The factor that is fit to is selected and can be determined according to the guidance that provides in the above description.

B. be used for immunocyte is attracted to the compositions of tumor locus

On the other hand, the invention provides compositions, it passes through the cytotoxicity of tumor infiltrating lymphocyte (TIL) promotion to tumor cell in primary tumor and/or metastatic tumor.Described compositions when being deposited on primary tumor or metastasis site, promoting the tumor degeneration and finally causes tumor destruction.Compositions can by other natural reservoir (of bird flu viruses) effectively the toxic general immunity of irritation cell reply, stimulation reply tumor antigen in directed primary tumor or the metastatic tumor.Said composition contains one or more drug storage warehouses, be applicable to and be deposited on tumor locus, described drug storage warehouse contains (i) and is used for the lymphocytic chemical attractant of T, comprise cytotoxicity and helper T cell, dendritic and natural killer cell, their CFU-GM and accesory cell, as eosinocyte and tumoricidal macrophage; But the (ii) reagent of irritation cell activation and differentiation and propagation; And (iii) can strengthen cell survival effectively and the reagent of immunity is provided.

More specifically, compositions contains one or more therapeutic agents, and (i) attracts lymphocyte (comprising lymphocytic all subclass of natural T lymphocyte and NK cell), macrophage, segmented cell and antigen presenting cell (comprising dendritic cell) with the infiltration tumor mass and directly enter alive and tumor cell necrosis effectively; (ii) stimulate the inflammatory response in the tumor mass, it offers tumor antigen to lymphocyte, and ends at cytotoxicity and the lymphocytic propagation of NK cell (NK) that generally can be used for the tumor outbreak in body; And the survival that (iii) promotes to be attracted cell, allow cytotoxicity in the immune host and NK cell lymphocyte are let pass into tumor mass, be used for directly approaching and kill tumor cell.

In one embodiment, can attract effectively one or more cells to one or more reagent of tumor locus comprise GM-CSF, interleukin 12 (IL-12), TCA4 (SLC), mononuclear cell chemical attraction albumen (MCP), by the inductive monokine of INF γ (MIG) tumor necrosis factor (TNF), macrophage inflammatory protein (MIP), stroma cell derivative factor-1a (SDF-1a), stroma cell derivative factor-1 β (SDF-1 β), RANTES and interleukin-1 (IL-1).Preferred chemical attraction reagent is the chemical attraction reagent that comprises IL-8, MIG, IL-12, MCP-1 ,-2 ,-3 ,-4 ,-5, MIP-1a, MIP-1 β, MIP-1 γ and RANTES.

The multiple phenomenon of cell moderate stimulation that described second reagent can be attracted, it comprises: (i) propagation T lymphocyte, the killer cell that comprises cytotoxicity and helper T cell, dendritic and natural killer cell, lymphokineactivation, their CFU-GM and accesory cell are as eosinocyte and tumoricidal macrophage and CFU-GM thereof; (ii) be divided into the activatory immunity and the accesory cell of function; And (iii) producing the various kinds of cell factor and other biological reagent, it stimulates proliferation, breaks up and regulates and coordinates differentiation between the immune system CFU-GM, promotion immune and the survival of accessory cell and the cytotoxicity kill capability of enhance immunity cell.The example of stimulant includes, but are not limited to, interleukin-1 (IL-1), interleukin II (IL-2), interleukin-6 (IL-6), interleukin 7 (IL-7), interleukin 12 (IL-12) and GM-CSF.

Described the 3rd reagent that is used for cancer immunization therapy in the compositions is a kind of can promote that be attracted and local cell survival, to strengthen the reagent that cytotoxicity and NK cell lymphocyte move into tumor mass.For this feature, suitable reagent comprises MIG, platelet factor 4, MCP-1 ,-2 ,-3 and MIP-1 γ.

C. drug storage warehouse

Aforesaid one or more reagent are expected and the drug storage warehouse form of reagent are deposited on the tissue regions of being studied to contain.Drug storage warehouse contains and discharges therapeutic agent, with the attraction and the activation of promotion stem cell, CFU-GM and preferred accesory cell, and accesory cell, CFU-GM and the survival of differentiated tissue's parenchyma fully.As will be described, drug storage warehouse can be designed and synthesize in required kinetics mode and discharge related reagent.

The first kind of drug storage warehouse that can imitate is a kind of macropore drug storage warehouse, comprises the biological and chemical inert particulate with interconnected pores.Open for worn-out to microparticle surfaces in the hole, is used for the outside and inner space, hole of microgranule and is communicated with.The microgranule example that is used to form this macropore drug storage warehouse for example is described in the U.S. patent 5,135,740, and it is incorporated herein by reference.

These microgranules typically in the liquid liquid systems formation with suspension polymerization exist.Substantially, the solution and the water unmixing that contain monomer and polyalcohol catalyst.Miscible but be included in the solution with solution with the atent solvent of not mixing of water.Solution is suspended in the aqueous solution subsequently, and it comprises additive such as surfactant and dispersant usually to promote to form suspension or Emulsion.In case suspension is confirmed as having the discrete droplets of desired size, polymer will be typically by adopting increase temperature or radiation that the reactant activation is affected.In case finish polymerization, from suspension, reclaim the solid particle that produces.Microgranule is solid-state spherical porous structure, and polymer forms around inert fluid, thereby forms pore network.Atent solvent, it occupies the hole of microgranule as giving birth to the reagent hole or pore-forming.Living hole reagent is removed the back and endoporus can be used for filling curative, as described below.

The macropore microgranule also can prepare by solvent evaporation from biodegradable or nonbiodegradable polymer.For solvent evaporation process, with the expection polymer dissolution in organic solvent and solution pour into subsequently one deck have the expection particle size sodium chloride crystal (Mooney, et al., J.Biomed.Mater.Res.37:413-420, (1997)).Usually adopt the evaporative removal solvent, and the solid polymer that generates is immersed in the water,, generate porous polymer drug storage warehouse with elimination sodium chloride.Alternatively, sodium chloride crystal can be by dispersed with stirring in polymer solution, to obtain the homodisperse of sodium chloride crystal.Subsequently, disperse thing dropwise to be expressed in the non-solvent of polymer, stir simultaneously to center on sodium chloride crystal precipitation polymers drop.By filtering or polymer particles that centrifugal collection is solid-state and immersing subsequently in the water, obtain porous polymer drug storage warehouse with elimination sodium chloride.The substitute that is appreciated that sodium chloride comprises any nontoxic water soluble salt or low-molecular-weight water-soluble polymer, and it can be filtered to produce the porosity of expection.

In the research of carrying out for support the present invention, as described in Example 1, prepared the polymer drug storage warehouse.Form the microgranule of average-size 20um from butyl methacrylate, vinyl ethylene glycol dimethacrylate and polyvinyl alcohol.The microgranule porosity is 80%.Embodiment 3A describes another kind of embodiment in detail, wherein, but has prepared the solid-state spherical particle of the polymer of biological corrosion, and it also contains GM-CSF.

Microgranule can great changes have taken place dimensionally, and diameter is about 0.0001 micron-Yue 100 microns, preferred about 0.001 micron-Yue 40 microns.Hole dimension in the microgranule also can great changes have taken place, and final size depends on the chemical characteristic of employed polymer.Typical total pore volume is the about 10cc/g of about 0.01-, the about 6cc/g of preferably about 0.1-; Average pore diameter is about 0.000001 micron-Yue 3.0 microns, preferred about 0.000001 micron-Yue 1.0 microns.

Microgranule is equipped with aforesaid one or more therapeutic agent, for example pass through described agent dissolves (necessary words in aqueous solution, be buffered to suitable pH), and mix up to all liquid and absorbed to generate free-pouring powder by microgranule by stirring microgranule and aqueous solution.Microgranule can be frozen dry to remove the water in the microgranule, the therapeutic agent of remaining lyophilized form in the microgranule of hole subsequently.Embodiment 2 has described a kind of research, wherein, and in the porous polymer particles of single biological reagent being packed into by microgranule and biological reagent solution are mixed.After the solution absorption, microgranule to remove water, is stayed in the microparticle pores biological reagent by lyophilizing.Embodiment 2 has also described preparation and has contained the drug storage warehouse that biological reagent makes up.

Embodiment 3 has described another research, wherein, but has prepared the polymer spheres of the biological corrosion that contains one or more biological reagents.But the microgranule of this biological corrosion is formed by the common Acetic acid, hydroxy-, bimol. cyclic ester of DL-lactide and contains a kind of single agents GM-CSF, or the combination of reagent G-CSF and RANTES.Be appreciated that the technology of preparing of describing can be used for the single agents or the reagent composition of any expection are clipped in the porous or solid-state microgranule in embodiment 2 and 3.

A kind of process of carrying therapeutic agent in microgranule secretly for choosing is at first, before described reagent is sandwiched the macropore microgranule, therapeutic agent to be joined in the polymer solution, as the copolymer (Pluronic of polyethylene oxide-polypropylene oxide-polyethylene oxide ^), other water-soluble polymer as polyvinylpyrrolidone (polyvinylpyrilidone), polyvinyl alcohol or other any nontoxic water-soluble polymer, comprises biopolymer, as collagen, cellulose, hyaluronate etc.The aqueous solution of curative-polymer is stayed in the aforesaid small porous particle subsequently.This confession is selected process to provide around freeze dried therapeutic agent and is coated with polymer, and it can be used for the reagent that prevention is mingled with when being transmitted and is subjected to the attack of enzyme, and allows further control release reagent from small porous particle.As can be understood, this polymer by selection coating curative can strengthen the control that reagent is discharged.The release of controlling described reagent is also by selecting small porous particle size, hole dimension and the reagent of packing in microgranule to realize.In addition, be appreciated that also one of several reagent of staying in the small porous particle can scribble polymer, with the release of this kind reagent that slows down.Be appreciated that also that before sandwiching small porous particle one or more reagent in the small porous particle can scribble one or more different coat polymers, discharge with the difference that produces each reagent from microgranule.

Another drug storage warehouse that is used for the present composition is microcapsule and microgranule, wherein contains or be dispersed with the therapeutic agent of expection.Microcapsule and microgranule pharmacy and medicine transmit industry be known (for example referring to Baker, R.W., CONTROLLED RELEASE OF BIOLOGICALLYACTIVE AGENTS, John Wiley﹠amp; Sons, NY, 1987; Ranade V.andHollinger, M., DRUG DELIVERY SYSTEMS, CRC Press, 1996).Microcapsule is typically referred to as the drug storage warehouse of active agent, and it is aggregated thing film shell and centers on.Microgranule is typically referred to as whole system, and wherein, therapeutic agent spreads all over microgranule.Yet, many preparations are arranged between these two kinds of definition, as the microcapsule piece, and this preparation also is applicable to this paper.

Microcapsule and microgranule can be from biodegradable or nonbiodegradable polymer manufacture.Microcapsule can adopt several different methods to form at an easy rate, comprise cohesion, interfacial polymerization, solvent evaporation and physics method for packing (, Baker, R.W., CONTROLLED RELEASE OFBIOLOGICALLY ACTIVE AGENTS, John Wiley﹠amp; Sons, NY, 1987).Adopt many technology well known in the art to prepare microgranule, a kind of plain mode is exactly that the thin polymer film that only will contain dispersive therapeutic agent pulverizes and is suitable size.Spray-dired particular therapeutic agent is another kind of method from polymer solution.The particular step that is used to encapsulate bioactive agents is disclosed among U.S. patent 4,675,189 and the U.S. patent application No.20010033868, and it is incorporated herein by reference.

Another transmission carrier that is used for this compositions is the polymer gel preparation.A kind of polymer that is particularly useful for this gel preparation is polyox-yethylene-polyoxypropylene block copolymer (Pluronic ).These copolymers demonstrate opposite hot gel behavior, have the excellent drug release characteristics, and have hypotoxicity.Copolymer gel is as the function of temperature and polymer concentration, wherein, and aqueous gel when solution is heated.Gel has (25 ℃) low viscosity under the room temperature, but viscosity increases when body temperature (37 ℃).

For forming compositions, required therapeutic agent combines in liquid with polymer, preferred aqueous solutions.Solution can pass through suitable transfer equipment, and catheter drug delivery is to target site as described below.When being sent to warmer environment, the gelling localization occurring and deposit therapeutic agent at the expection position.

Be used to prepare other the suitable polymer that transmits drug storage warehouse and include, but are not limited to collagen (Pieper, J.S.et al., Biomaterials, 21:1689-1699 (2000)); Cellulose (Grassl, E.D.et.al., J.Biomed.Mater.Res., 60:607-612 (2002)); Yaupon gel (Ramamurthi, A.et al., J.Biomed.Mater.Res., 60:196-205 (2002)); Deutero-dextran (Letourneur, D.et al., J.Biomed.Mater.Res., 60:94-100 (2002)): heparin alginate (Laham, R.et al., Circulation, 100:1865-1871 (1999)); Alginate (US patent 6,238,705; 6,096,344); And chochleates (US patent 6,403,056).The drug storage warehouse that contains required therapeutic agent for example, is also adopted lyophilizing to remove subsequently by the aqueous solution for preparing described reagent and polymer and desolvates to produce the lyophilized form of biological reagent in a kind of drug storage warehouse by these material preparations.Directly administration of lyophilized powder, or be suspended in once more in any suitable liquid, as low viscous flow body, viscogel or ointment or Water-In-Oil or oil in water emulsion.Suspending agent can be water or non-water and select according to the multiple factor, for example, on carrier, make described drug storage warehouse elapsed time and be sent to the time of tissue and the biological reagent release dynamics of expection.

Yet another kind of suitable drug storage warehouse is a liposome.Liposome is spheric lipid carrier, and size and is made up of one or more lipid bilayers that water-containing space is incapsulated between 0.01 and 10 micron.Multiple amphoteric lipid is used to form bilayer, as phospholipid (for example referring to U.S. patent 5,013,556).Lipid molecular be arranged into its polar head-group towards water and with the hydrophobic hydrocarbon afterbody in the contiguous bilayer another, therefore form the concentricity bimolecular lipid lobule that separately contains water spacer that gathers.

Therapeutic agent can be clipped in the moisture clearance space of liposome, or in the lipid bilayer, depends on the physico-chemical property of described reagent and lipid bilayer compositions.Discharging described reagent from liposome can make to measure according to the selection of lipid bilayer composition.

The described drug storage warehouse of the therapeutic agent that contains in an embodiment of the present invention, has the biological reagent that is connected with described drug storage warehouse outer surface.For example, in the drug storage warehouse of liposome, outer liposome surface comprises the polar head-group of the phospholipid of forming lipid bilayer.The polar head-group of phospholipid part can derivatization before and after liposome forms, to comprise biological reagent, as cell adhesion molecule.Biological reagent can be directly connected to lipid head base, or connects by polymeric arms.Two kinds of methods, i.e. the molecule and the molecule that is connected by the space arm of the surface of liposome connection are described in the prior art (for example referring to U.S. patent 5,013,556; Zalipsky, S., polyethylene glycol-lipid conjugates in Lasic, D.and Martin, F., Eds.STEALTH LIPOSOMS, CRC Press, 1995; WO 98/16202; WO94/21281).

Be appreciated that biological reagent also can be connected to the outer surface of aforesaid drug storage warehouse at an easy rate.For example, the outer polymer shell of microcapsule or microgranule can be processed, so that some living polymer part to be provided, to be used for subsequent reaction and to connect required therapeutic agent to the surface.What alternatively, therapeutic agent can be at microcapsule or microgranule is surperficial coated.In this manner, in case compositions is deposited on the target tissue, therapeutic agent then exposes at once.

Also will understand, make described drug storage warehouse biology or synthetic polymer can be selected according to its chemical composition, perhaps it can be derived to allow drug storage warehouse and biological reagent to participate in certain type physical attraction, so that therapeutic agent is remained on the surface.In this manner, in case compositions is deposited on the target tissue, therapeutic agent then exposes at once.

As mentioned above, described drug storage warehouse contains one or more therapeutic agents, as chemical attractant, stimulant and/or survival reinforcing agent.Discharging one or more reagent can make to measure by selecting described drug storage warehouse and preparation compositions.For example, in an embodiment, wherein, two kinds of reagent are stayed in the described drug storage warehouse, and described reagent can be made into to be used for to discharge simultaneously or continuously.If the therapeutic agent that is sandwiched in wherein has visibly different permeability to the polymer of formation capsule external coating or to the polymer that forms matrix of microparticles, then the release from microcapsule or microgranule will be successive.Also can be for example from the continuous release of microgranule and microcapsule, contain the thin layer of different polymer by preparation, one or more therapeutic agents are dispersed in the multiple thin layer or are clipped between each thin layer and realize.Continuously the embodiment that discharges for example is disclosed in the U.S. patent 5,472,708,5,260,069.

Realize that the other method that discharges continuously is two or more drug storage warehouse colonies of administration, wherein, first colony is designed to discharge first reagent with certain speed, and second is designed to discharge second and the reagent of back with different speed with the colony of back.Under the situation of polymer drug storage warehouse, by changing polymer composition, polymer thickness, particle size; Perhaps, under the situation of liposome drug storage warehouse, select and carrier dimensions, be easy to obtain different release rates by lipid.

Embodiment 4 has described the external IL-12 that discharges from drug storage warehouse, this drug storage warehouse includes biodegradable polymer, DL-lactide Acetic acid, hydroxy-, bimol. cyclic ester altogether.In brief, the drug storage warehouse that contains IL-12 is placed in the container that contains buffer salt and to discharge IL-12 determined as the function of time in this salt.After 11 days, discharge 60% IL-12.

As will be described further, in use, aforesaid any drug storage warehouse, for example, the bolus as little, be deposited into, or required tissue site on (referring to embodiment 5 and 6).For example, the bolus of drug storage warehouse can be deposited into several millimeters of cardiac muscles from the tube wall of placing the deposition conduit.Described drug storage warehouse can move by backlash freedom in tissue.Each drug storage warehouse is mainly used in angiogenesis, tremulous pulse generates and muscle cell regeneration, and when it is mobile in tissue, can promote these processes to repeat at diverse location.By this way, adopt the minimally damage, can obtain a kind of relative wide range of therapeutic effect.

Also as described below, for using in cardiac muscle, a kind of selection that is used to transmit described drug storage warehouse is by with the broken coronary arterial wall of minor diameter acupuncture.The another kind of selection is exactly to be injected directly into cardiac muscle in operation process aspect the bag heart.A kind of confession that is used to be injected directly into cardiac muscle select approach be percutaneously (by horizontal blood vessel path) by the endocardium of cardiac chambers.In skeletal muscle, drug storage warehouse can puncture by the percutaneous needles of minor diameter and be transmitted.Described drug storage warehouse also can be during PTCA, support or catheter treatment (artherectomy) process, or is transmitted by percutaneous by the percutaneous step that is used for placing described drug storage warehouse separately.In some cases, it is favourable that percutaneous is placed described drug storage warehouse, and this is because use the blood vessel imaging technology to make the location more accurate simultaneously.

Another feature that drug storage warehouse of the present invention provides is the mobility after depositing to therapentic part.Realize its mobility by making the gravity that has less than the drug storage warehouse of gap body fluid to small part.This will allow described drug storage warehouse floating and mobile with fluid in tissue fluid.

III. use method for compositions

A. promote the method for tissue regeneration

On the other hand, the present invention contains a kind of method, and it is used for after damage or is in tissue downright bad and infringement promoting tissue regeneration.This method expection promptly, is not united the therapeutic scheme of other medicines, operation or intervention as a kind of main therapeutic modality.Yet this method can be used together with other therapeutic modality.For example, comprise that the compositions in the drug storage warehouse contains one or more therapeutic agents, as mentioned above, can be deposited, to strengthen hemoperfusion to ischemic tissue around the narrow zone of being treated in support placement and balloon postangioplasty.Compositions also can be united transmission with other therapeutic agent that uses in the treatment coronary artery disease, form reagent as antithrombotic.When also being expected at surgical operation or any minimally damage process or in the tissue of being studied or near during the deposition described drug storage warehouse.

In one embodiment, promote tissue repair, angiogenesis and/or tremulous pulse to generate to realize tissue regeneration by being deposited on one or more drug storage warehouses that selected tissue site contains one or more therapeutic agents, this drug storage warehouse is (i) other cell of attracting the circulation blood monocyte, helping angiogenesis and tremulous pulse to generate effectively, or the progeny cell of stem cell and differentiated tissue thereof; (ii) stimulate to discharge biological reagent, it promotes angiogenesis, tremulous pulse to generate, and cardiac muscle or skeletal muscle fiber (muscle cell regeneration) are broken up in infiltration fully; And by directly acting on mononuclear cell, stem cell, CFU-GM and helping angiogenesis, tremulous pulse generation and regenerated other cell of muscle cell to realize these phenomenons; And (iii) in this tissue, influence the circulation blood monocyte, deutero-macrophage, other cell that helps angiogenesis and tremulous pulse to generate, the survival of stem cell and differentiation progeny cell thereof.For example, contain the drug storage warehouse of chemical attractant such as MCP-1 and GM-CSF, be deposited on and be in ischemic position or ischemia rear, prolonged the survival of circulation blood monocyte and deutero-macrophage.MCP-1 is used to attract to circulate blood monocyte to tissue regions and the factor that stimulates the mononuclear cell/deutero-macrophage that is attracted to discharge to relate to angiogenesis and/or tremulous pulse to generate.Attract and stimulation is used to make the sophisticated peripheral mononuclear cells of tissue macrophages, and mononuclear cell moves subsequently and enters clearance space, start discharge its directly influence to form capillary tube and transform arteriole be the signal factor of tremulous pulse.The existence of GM-CSF has strengthened the time-to-live of macrophage, has prolonged the cycle that discharges biotic factor from the macrophage that recovers.By this way, in the tissue of target site, promoting angiogenesis and/or tremulous pulse to generate.

For example, the chemical attractant that drug storage warehouse contains, as stem cell factor (SCF), it attracts circulating stem cell in tissue, or G its have the M-CSF of the ability that increases the circulating stem cell number, be deposited on ischemia position on the line or ischemia rear.SCF is used for attracting circulation and permanent stem cell to tissue regions, and GM-CSF is used to increase the stem cell number that enters the zone.Somatomedin promotes that differentiation of stem cells is a sarcoplast, and it merges with cardiac muscle and the skeletal muscle tissue of regenerating.By this way, promote that the muscle cell around target site is regenerated in the tissue.

Embodiment 5 has described and has used the drug storage warehouse that contains GM-CSF, MCP-1 and RANTES, be used for cell and tissue regeneration after rabbit ischemia incident, M-CSF for example, MCP-1 and RANTES have the activity as chemical attractant, be used to relate to the cell of cell regeneration and, be used for breaking up and breeding these cells as stimulus object.

As another embodiment, described drug storage warehouse also can be made into to comprise cell adhesion molecule, as ICAM, to promote mononuclear cell or other blood vessel origin and the cell adhesion tremulous pulse origin to tissue regions.Cell adhesion molecule can be attached to the outer surface of one or more drug storage warehouses, or is included in and is used in the described drug storage warehouse discharging.Adhesion molecule appears at the plasma membrane of macrophage, fibroblast and smooth muscle cell as the part that is used for conglutnin and similar species.Adhesion molecule also attract macrophage to described drug storage warehouse and can physical bond to described drug storage warehouse to guarantee that macrophage is in required zone and macrophage farthest is exposed to from the molecule signalling factor of described drug storage warehouse release.

Except chemical attractant, stimulant and survival promoting agent, drug storage warehouse can randomly contain other reagent that relates to angiogenesis, tremulous pulse generation and muscle cell regenerative process.For example, be expected at and comprise somatomedin or cytokine in the drug storage warehouse, with participate in a series of angiogenesis, tremulous pulse generates and the muscle cell regenerative process, perhaps stimulates or change some incident in this process.

As noted above, the release dynamics of one or more therapeutic agents that sandwiched can and prepare described drug storage warehouse by selection and control.Described drug storage warehouse is used for attracting, keeps, stimulates and strengthens survival, propagation and differentiated progenitor cells, including, but not limited to peripheral mononuclear cells, myocardial cell and skeleton sarcoplast, these cells discharge the multiple signalling factor that relates to tissue regeneration subsequently, and these factors can comprise or not comprise angiogenesis and/or tremulous pulse generates.Because described drug storage warehouse discharges biological reagent in the controlled time, can keep the local concentration quilt of described reagent, so that target tissue is replied.Equally, multiple biological reagent has different target cells replys, and the kinetics mode that they can be different is released, and can reach the valid density of required reagent during tissue is replied.By this way, relating to angiogenesis, tremulous pulse generation and the regenerated various kinds of cell of muscle cell colony can be adjusted and coordinate, to promote forming functional neovasculature, cardiac muscle and periphery muscle in being in dangerous tissue.For example, stimulate proliferation and be arranged in the vascular endothelial cell of new arteriole or small artery inner chamber, can coordinate with the propagation and the mobility of stimulated vascular smooth muscle cell, to form the flesh layer, cover vascular endothelial cell. in an embodiment of the present invention, the drug storage warehouse that is deposited is used for attracting, stimulates and promote monocytic survival that when stimulating, the appropriate time of mononuclear cell in angiogenesis and/or tremulous pulse generative process discharges the suitable factor.In another kind of embodiment, described drug storage warehouse also contains reagent, and it has replenished the factor concentration that discharges from the mononuclear cell that recovers, be used to participate in and strengthen the formation of new arteriole.

In another kind of embodiment, the present invention expected a kind of during regeneration muscular tissue the method for regenerating nerve, deposition is equipped with the drug storage warehouse of suitable therapeutic and biological reagent, hope is in the growth of exciting nerve of ischemia rear region.

In Therapeutic Method, described drug storage warehouse can be deposited on tissue site by multiple technologies.For example, adopt and inject pin, described drug storage warehouse can directly inject in the cavity of tissue or contiguous target tissue.Drug storage warehouse can be implanted by operation separately or with armarium such as support.

Described drug storage warehouse also can be close to target site to cavity wall by the thin layer that makes the viscosity pasty state, hydrogel or other polymer support substrate that contain described drug storage warehouse, or the contiguous target tissue of other body cavity, be deposited to tissue site as pericardial sac around the epicardial surface of heart.Interim wall part when the polymer support of formation pasty state or gel can discharge biological reagent for biodegradable and/or conduct.The intracavity tiled system is at for example U.S. patent No.5, is described in 328,471.Below describe the intracavity that is designed for this compositions and paved the specific embodiment of equipment.

As embodiment, the drug storage warehouse that is made into pasty state can be deposited in the pericardium or in the cerebrospinal fluid space, be used for generating and the tremulous pulse generation at cerebral infarction or ischemia zone induction of vascular.The Concentraton gradient of being set up by described drug storage warehouse will attract the cell with the tremulous pulse origin of blood vessel origin to enter tissue from blood flow.Alternatively, pasty state or gel can be applied near the tube wall in pipe restricted area (obstructions) or the territory, healthy area under control, be transported to reality with realization through tube wall or potential ischemia position

Described drug storage warehouse also can adopt conduit to be deposited, and is such as described in more detail below.It is well known in the art that conduit is generally, and is suitable as the conduit of direct infusion medicament and ballon catheter with drug treatment medicine ability.In a preferred embodiment, used to have and to have thrust tissue or tube wall are used for placing the structure of described drug storage warehouse in clearance space conduit.For example, a kind of device description of the little pin with the factor that is used to transmit the blood vessel origin is at US 6,152, and is in 141, incorporated herein by reference.The equipment of another example has below been described.

The drug storage warehouse of Shi Yonging also can be used for the wall transmission in conjunction with the pharmaceutical compositions of routine in the methods of the invention.Under the situation of successive conduit transmission, drug storage warehouse will combine with acceptable fluid carrier, for example, and with preparations such as sterilized water, isobaric salt, glucose solutions.Said preparation can contain the acceptable auxiliary substance of pharmacy, as commonly used in pharmaceutical formulations, comprises buffer agent, tension adjustment agent, as sodium acetate, sodium lactate, sodium chloride, and potassium chloride and calcium chloride etc.The common method that is used for preparing these pharmaceutical formulations is described in Remington ' s PHARMACEUTICALSCIENCES (Mack Publishing Co., Philadelphia, Pa., 1985).

Compositions transmits a period of time, is enough to the physiological effect that reaches required, that is, promote around the angiogenic growth of the tissue of target site and regenerate muscle cell.Usually, the biotic factor total amount that is transmitted will change according to organization factors, patient's characteristic and required effect.For specific patient, determine that by the attending doctor suitable dosage instructions about how to take medicine are good.Because correct dosage varies with each individual, and depends on age and general health status, conventional " dosage-titration " method of using of doctor; That is, the dosage of taking to the patient during beginning is lower than the required level of replying that produces expection, and increases dosage gradually up to reaching required effect.

Also will understand, except that selected biotic factor, the preparation that contains described drug storage warehouse can comprise other reagent.For example, preparation can comprise anticoagulant and antithrombotic agent, as heparin, and low-molecular-weight heparin etc.

B. can promote the treatment method for cancer of cellullar immunologic response

On the other hand, the present invention includes a kind of method that is used to promote to the cellullar immunologic response of patient's cancerous cell.Described method is included in primary tumor or the transfer or near deposition contains one or more combination of agents things, and (i) attracts one or more lymphocytes, macrophage, dendritic cell, natural killer cell, CFU-GM and accesory cell to tissue site effectively; (ii) stimulate to directly act on cell and extracellular matrix components local and that be attracted, and discharge the Cytotoxic biological reagent that can promote cancerous cell from described part and the cell that is attracted; And (iii) influence that be attracted and survival local regenerative cell in this tissue, the permanent immunity of cancerous cell is replied promoting.

As mentioned above, the compositions that comprises the drug storage warehouse that contains one or more therapeutic agents is deposited by being injected directly into primary tumor or entering the remote position of being infected by tumor cell by conduit.Compositions can be transmitted with other chemotherapeutant that is used for the treatment of cancer.When also being expected at surgical operation or in any minimally damage process, described drug storage warehouse is deposited in the tissue of being studied or near.Aforesaid preparation is suitable for this aspect too.

Embodiment 6 has described the purposes of compositions.Said composition comprises biodegradable polymer drug storage warehouse, and it contains and is useful on the IL-12 that makes tumor degeneration in the Mus.As mentioned above, IL-12 is as the chemical attractant of lymphocyte, macrophage, antigen presenting cell (comprising dendritic cell) and inflammatory cell.IL-12 also as the stimulus object of these cells, breeds, breaks up and/or antigen presentation with promotion.These activities cause finally can cause replying of tumor degeneration.

IV. the equipment that is used for the administration example compositions

On the other hand, the invention provides the equipment that is used at selected tissue site deposition drug storage warehouse.The various embodiments of equipment has been described in conjunction with Fig. 3-18.

Fig. 3 is a kind of plane graph of imitateing equipment, is used to deposit aforesaid drug storage warehouse.Equipment 110 comprises a catheter shaft 112, a user interface 114 and a shell that is used to store piston 116.Fig. 4 has at length shown catheter shaft 112, and wherein, this axle extends between far-end 118 and axle reinforcing portion 120.Axle reinforcing portion proximal subscribers interface 14 (Fig. 3) also are designed to the kinking during use of prevention axle, as described later.Catheter shaft also comprises a single track seal wire junction surface 122, and it has contiguous seal wire mouth 124, is used for seal wire is introduced part 122.The flexible axial region 126 of tip can move in swan-neck, have stronger inflexible paraxial 128, allow to promote and to reverse the tip axial region.

Fig. 5 A has shown the detail drawing of catheter shaft 112 tips parts.Demonstrate the flexible axial region 126 and the single track seal wire junction surface 122 of catheter shaft tip.Catheter shaft rides on the seal wire 130 by only partly engaging at single track.Limited joint between seal wire and the axle is by reversing steer distal end farthest in small-bore container.Axle ends at pin 132 places that are used at tissue site deposition drug storage warehouse.In one embodiment, shown in Fig. 5 B and 5C, broken being organized in the clearance space of acupuncture deposits.Fig. 5 B has shown the conduit axial region 126 of the tip flexibility that is positioned at main body pipe 134.Axle engages with seal wire 130 at single track seal wire part 122 places.Pin 132 ends at oblique needle point 136 places that can puncture tube wall, to enter clearance space and to place drug storage warehouse.Fig. 5 C has shown the identical device that is arranged in pipe 134, but rotates oblique pin 132 to puncture tube wall in the another location that Fig. 5 B shows.

Fig. 6 A-6B is for by line 6A-6A and 6B-6B among Fig. 5 C, shown single track part 122 (Fig. 6 A) and near the lateral cross figure in the zone (Fig. 6 B) of single track part.In Fig. 6 A, single track part 122 is located in the pipe 134.Seal wire 130 partly engages with single track and is arranged in guidewire lumen 138.Can see the axle inner chamber 140 of catheter shaft, and in axle inner chamber 140, be provided with the pin 132 that defines pin inner chamber 142, be used to transmit drug storage warehouse.The tip axial region 126 that limits axle inner chamber 140 wherein, can be seen in the zone that has shown close single track seal wire part in Fig. 6 B.Pin 132 is located in the inner chamber 140 and limits pin inner chamber 142.The seal wire 130 that is in this position in equipment does not engage binding with catheter shaft.

Fig. 7 A-7B has illustrated another embodiment of equipment, and wherein, the balloon of tip is used for the positioning catheter point, with the form penetrate tissue of straight needle.Fig. 7 A demonstrates and leads 130 and engage with the single track part 122 at the flexible axial region 126 tip places that are positioned at equipment.Balloon 144 is installed in the acra of single track part, is biconial (as shown) during expansion.As can be appreciated, make distal end of catheter be placed into tube wall, allow straight needle 146 to puncture tube wall, so that deposition drug storage warehouse in the nigh organization space with acute angle to balloon inflation.Fig. 7 B has shown a kind of embodiment for choosing, and wherein, two spheric balloons 148,150 are located at the opposite side of single track part 122.The balloon that shows with swelling state is placed into tube wall with distal end of catheter with acute angle, allows straight needle 146 to puncture tube wall.

Fig. 7 C and 7D have shown the above two kinds of embodiment that have the biconial that is located in the blood vessel and two spheric balloons respectively.In Fig. 7 C, be appreciated that the 144 restricted effects of 134 pairs of balloons of tube wall, thereby make pin 146 puncture tube wall with angle a.Fig. 7 D demonstrates two spheric balloons 148,150 and is limited by tube wall, thereby makes pin 146 puncture tube wall with angle a.

Fig. 8 A is the cross-sectional view of regional A among Fig. 4.Single track part 122 comprises guidewire lumen 138 and axle inner chamber 142.Pin 146 with splay end 46a is positioned at an inner chamber 142.Seal wire 130 is located in the inner chamber 138.Fig. 8 B has shown identical cross-sectional view, but pin 146 extends far-end 118 from catheter shaft, is used for placing drug storage warehouse at required tissue site.This pin shows with the deployed condition with angle a.Be appreciated that this angle can in very large range change, depend on the prefabricated curvature of pin itself and the angle of spread that realizes by part of appliance such as balloon, as in Fig. 7 A-7D, discussing.

Fig. 9 A is the cross-sectional view of area B among Fig. 4.The flexible conduit axial region 126 of tip defines the axle inner chamber 140 that pin 146 wherein is installed.Component pipe 152 is near pin 146, and it contains thermoplastic elastomer (TPE) 154 and wire braid 156.Interface 158 between component pipe and the pin is as the contact point of two elements, and can be at layout setting adhesives or other link.

Fig. 9 B wherein, can see that pin 146 is located in the inner chamber 140 of catheter shaft 126 for the cross-sectional view by Fig. 9 A center line F-F.Fig. 9 C and 9D are for having biconial balloon (Fig. 7 A) or having the cross section of same position in the equipment of two spheric balloons (Fig. 7 B).The equipment that contains one or more balloons will have one or more passages, as the passage among the passage among Fig. 9 C 158 or Fig. 9 D 160,162, be used for that the control media that fluid typically is angiography is introduced balloon and be used for inflation, and remove fluid and be used for venting.

Figure 10 is the longitudinal cross-section figure of region D among Fig. 4, from the flexible axial region 126 of the tip of conduit on stronger inflexible paraxial 128 transition region 164.Transition region 164 is formed by the hot briquetting thermoplastic elastomer (TPE) of fusing, across the zone between flexible tip axle and rigidity are paraxial.The component pipe 152 that is arranged in the flexible shaft inner chamber is connected with the joint of preferably being made up of metal such as rustless steel 166.168 places are connected by any suitable parts the connector at the described component pipe and the first terminal 166a place at the interface, as adhesive bond, welding, welding.For example, reinforce attached pipe 169 and realizes interior rigid of near axis area 128 by adding along the inwall that forms paraxial 128 elastic tube wall 170.Substantially, adopt bonding or other any suitable parts, by thermosetting thermoplastic elastomer body component on attached pipe attached pipe 169 is connected with paraxial and is fixed on 167 places, interface, allow elastomer-attached duct member works to have elasticity.

Attached pipe 172 is in abutting connection with the joint at the second terminal 166b place.Attached pipe 172 is located in the inner chamber that is limited by attached pipe 169, and plays a role as the storage barrel of syringe, and it is filled with and will be deposited to the drug storage warehouse preparation of tissue site.For example, attached pipe can be filled with freeze dried drug storage warehouse raw material or liquid drug storage warehouse suspension, is used for moving through the component pipe by piston and enters the pin inner chamber and finally enter the tissue of expection.Figure 11 has illustrated this one side of equipment in further detail.

Figure 11 is the longitudinal cross-section of zone C among Fig. 4, has shown the position of the piston 176 that can move in attached pipe 172.As described in following Figure 15-17, piston 176 can move by the mechanical mechanism in the user interface, and conduct can be by the continuous metal axle of attached pipe slip.Piston 176 can be formed by rustless steel, and scribbles politef at far-end, is free to slide to allow piston as lubricant.Piston slides by attached pipe, and preferably produces tight seal, to realize mobile drug storage warehouse preparation.Polytetrafluorethylecoatings coatings is also guaranteed tight seal as the subordinate sleeve pipe.

Figure 12 is the longitudinal cross-section of area E among Fig. 4, demonstrates the axle reinforcing portion 120 (Fig. 4) that just is positioned at user interface 114 (Fig. 3) tip.Outstanding pipe fitting 178 is positioned in the user interface, and is used to make a hardening with the location of control appliance better with lay.Particularly, reinforcing that provides by parts 178 and hardening eliminated axle in this zone owing to reverse the kinking that causes with flexing.

Before describing user interface in detail, a kind of equipment of imitateing is provided among Figure 13, be used for drug storage warehouse is for example deposited to, produce the cardiac muscle at the ischemia position of the inaccessible tip of ischemic atherosclerosis.As we can see from the figure, the tip flexible shaft part 126 of equipment 110 is arranged in the sagging coronary artery 180 of left front.Equipment enters by the coronary artery guide pipe 182 that is arranged in the main coronary artery 184 in left side.The caval vein of aortic arch, inside and the main coronary ostium in left side are shown as 186,188,190 respectively.Equipment arrives the ischemia positions by 130 guiding of crown seal wire by atheroma inaccessible 192 or is in position 194 in the ischemia danger.Pin 132 slave units launch and the bolus 196 of drug storage warehouse preparation are deposited to the ischemia position.

In aforesaid apparatus embodiments, the straight seal wire of a routine (element 130) is used to equipment is directed to required tissue site.A kind of for the choosing embodiment in, shown in 14A-14C among the figure, the spiral type seal wire be used to make equipment advance and positioning needle with penetrate tissue.Figure 14 A has shown pipe 200 and the spiral type seal wire 202 that places container intracavity.This one side according to the present invention, catheter device inserts container by following the spiral type seal wire, as shown in Figure 14B.For ease of observing, Figure 14 C has omitted container, has shown that catheter device is connected single track part 122 at its flexible far-end by spiral type seal wire 202 joints.Straight needle 146 extends from catheter shaft far-end 118, as shown in Figure 14B, has certain angle to pass tube wall by means of the spiral type seal wire.Be appreciated that tube wall is passed in the torsion angle that can change in the spiral type seal wire with change angle.

Now with reference to Figure 15-17 user interface shown in Figure 4 120 is described.Figure 15 has shown the whole top view of the user interface 120 of preferred embodiment.This interface comprises tip part 220, and it is attached to catheter shaft (Figure 12) in outstanding pipe fitting 178 place's rigidity.In the opposite end of user interface is nearly part 222, it is rigidly connected to attached pipe inside (element 172 among Figure 10), component pipe (element 152 among Fig. 9 A) and pin are generically and collectively referred to as " needle assembly " and can allow the user to control pin, comprise from catheter shaft launch or prolong, with institute withdraw an interior punctured position and the rotation of pin.Nearly part 222 also can allow by making before the sliding plunger and then control transmits described drug storage warehouse preparation (element 76 of Figure 11).Grommet 224 is attached to the nearly part of user interface, and it provides strain relief (also being described among Fig. 4) for piston shell 116.The groove grip area 226,228 that has that lays respectively at the near of user interface and tip part can be rotated with control appliance by the user.Particularly, rotating handles zone 228 can allow to reverse and be applied to conduit.Moving handle zone 226 may command needle assemblys and rotation pin.

Figure 16 A and 16B are the side view (Figure 16 A) and the oblique view (Figure 16 B) of user interface shown in Figure 15.The deposition that is arranged on the nearly part 222 of user interface with actuator button 230 for the user movably so that piston 176 advances (for sightless, referring to the element 76 among Figure 11 in the drawings).Press down deposition and engage with a pair of roller or sliding clamp with the actuator button, but so that piston-advance and distribute the repeated deposition thing of drug storage warehouse preparation.Be positioned on the user interface tip part needle assembly lock actuator button 232 for the user movably.Press down button 232 and decontrol nearly part 222, can make its rotation, advance or withdraw and rotate, advance or withdraw corresponding to described pin.Be in its button of stretching out the position fully 232, for example, when not being pressed down, nearly part and needle assembly lock onto and tip part and the corresponding position of catheter shaft.Needle assembly lock actuator button 232 is seated depression 234 places, opens assembly with prevention owing to neglecting.Pointer- type indicator 236a, 236b are located on tip and the nearly part, with the tip pin part of the relative rotation degree that shows pin and the inclination direction with respect to catheter shaft.

Figure 17 A-17B is the oblique view of user interface among Figure 15-16, demonstrates to be positioned at extracting position (Figure 17 A) and in the nearly part of its extracting position half-twist (Figure 17 B).The nearly part that is positioned at extracting position is corresponding to withdraw the fully acra of catheter shaft of pin.Among the superincumbent figure, the user interface of demonstration is in the position that needle assembly is extended, and pin is unfolded so that at tissue site deposition drug storage warehouse preparation.Pin body assembly 238 is rigidly connected to nearly part and slidably moves and rotation in the tip part.As shown in Figure 17 B, the rotation of nearly part is corresponding to the rotation of pin.The rotation degree can adopt pointer- type indicator 236a, 236b to watch.

Figure 18 A-18D has illustrated that a kind of intracavity paves equipment, is used for along tube wall or body cavity deposition composition.This equipment contains the flexible cup 350 of elongation a little, has central cavity, is used for receiving compositions by supply pipe 352.Supply pipe is preferably integrally formed with flexible cup, and 354 places stop in the hole, and as shown in Figure 18 A, compositions enters flexible cup by this hole from supply pipe.Cup has the surface of opening, and is used for along the tube wall deposition composition and closes rear section (referring to Figure 18 C) and sidewall deposits along wall guaranteeing.

In cup, be provided with additional holes 356,358, be used for cup is introduced and drawn to seal wire 360.Can see that in Figure 18 B (front view), 18C (rearview) and 18D (being inserted into container) spiral type seal wire 360 inserts the last hole 356 and the following hole 358 of cup.If necessary, can suitable seal mechanism be set, to eliminate or to be reduced in the compositions disappearance of infall at the infall of thread eye 356,358.

As shown in Figure 18 D, the spiral type seal wire contacts with blood vessel 362 rigidity.In use, flexible cup is controlled automatically and is moved along spiral type seal wire physics by the user or at near-end as single track portion.When cup when single track moves, in the open cup that compositions is introduced into inner tubal wall contacts.Mobile cup is sprinkled upon a layer composition on the tube wall, in essence, is " to pave " tube wall with compositions.Adopt piston to move aforementioned drug storage warehouse preparation and can realize supply the drug storage warehouse compositions that is used to pave.Finish the equipment of paving moving in the cup by the drug storage warehouse preparation being continued move and push along its path.

How to realize multiple purpose of the present invention and feature as can be seen from the above description.The compositions that comprises in drug storage warehouse is deposited over to be needed in the particular organization of the therapeutics effect space.Described drug storage warehouse can move freely by tissue cavity, to discharge the biological reagent that multiple key cell can be sucked the target tissue chamber.In order to be used for tissue repair and regeneration, drug storage warehouse is designed to discharge the attractant that can cause stem cell, accesory cell and/or CFU-GM inclination tissue cavity, and CFU-GM and accesory cell depend on specific types of organization.By drug storage warehouse deposition, some stem cell (comprise bone marrow hematogenesis with Interstitial cell) can be not to be to be attracted in the multiple different tissue indiscriminately, for example, and liver, skeletal muscle and brain.The example of CFU-GM comprises the endotheliocyte from local vascular, comprise capillary tube and large artery trunks and vein, myeloid endotheliocyte CFU-GM, smooth muscle cell from local organization, circulation smooth muscle cell CFU-GM is from the fibroblast CFU-GM of local organization or blood, near myofibrillar satellite cell, from the nervous system adult stem cell of cerebral cortex specific region, contiguous sarcoplast and other cell.Accesory cell comprises the mononuclear cell macrophage, is attracted from peripheral blood.Similarly can be attracted from the source, part the rare phagocyte accesory cell of particular organization, for example, from the Kupffer cell of liver with from the microglia of brain.In order to be used for the tumor degeneration, described drug storage warehouse is designed to discharge and causes lymphocyte, macrophage, segmented cell, the reductive attractant of antigen presenting cell and/or natural killer cell.In tumor, exist these cells to trigger a series of incidents that can cause the tumor degeneration at last.

In addition, described drug storage warehouse contains the biological reagent that is useful on release, the cell that its stimulation is attracted is done multiple basic thing, comprise and cause proliferative cell epimatrix (ECM) raw material, as collagen and elastin laminin, to cause degeneration and to transform ECM, synthetic cell adheres to amboceptor, signalling molecule between synthetic cell, be divided into cell with specific function (for example, the contractility of contractility and management myocyte CFU-GM, smooth muscle cell, form the ability that the cell of particular type is connected for endotheliocyte, neuron and rhabdium).Discharge different biological reagents by phasing, multi-mode ground, active successive stages can be coordinated to synthesize, break up to drug storage warehouse by different cell masses.

Drug storage warehouse also discharges the biological reagent that can influence differentiation model, function, static and/or apoptosis in critical cell mass in addition.Discharge suitable reagent by drug storage warehouse and can in the elementary event that fibrosis necrosis or postnecrotic zone is returned to accurate normal function, promote control and coordination based on cell.

V. embodiment

Following examples have illustrated preparation of compositions described herein and purposes, and are absolutely not to be used to limit the scope of the invention.

Embodiment 1

Preparation polymerization drug storage warehouse

Following composition is introduced reactor: 12 gram butyl methacrylates, 8 gram vinyl ethylene glycol dimethacrylates, 0.2 gram benzoyl peroxide are dissolved in the 80 gram toluene, and 16 gram polyvinyl alcohol are dissolved in the 400 gram distilled water.Adopt the material in the mechanical agitator stirred vessel, so that form the organic facies float at aqueous phase.Increasing mixing speed is about 20 μ m to reach average droplet size.Then, with mixture heated to 70 ℃ and kept this temperature 3 hours, to finish polymerization process and to form solid-state micropore drug storage warehouse microgranule.

Collect solid particle by filtering, and clean for several times to remove suspending agent with hot water.The micropore microgranule is suspended in the water once more, and in float, injects steam to remove toluene from microgranule.After removing all toluene, water cleans microgranule, filters and drying.Analyze the particle size and the porosity of solid-state microporous polymer microsphere microgranule.The average particle size of micropore drug storage warehouse is 25 μ m, and the average particle porosity is 80%.

Embodiment 2

Preparation has the compositions of biological reagent

Following cytokine and somatomedin are packed into according in the prepared polymerization drug storage warehouse of the method for describing among the embodiment 1.With freeze dried interleukin 12 (IL-12), granular leukocyte macrophage cluster stimulating factor (GM-CSF), interferon-(IFN-γ), active normal T cellular expression and excretory (RANTES) that regulates, TCA4 (SLC), tumor necrosis factor-a (TNF-a), granulocyte cluster stimulating factor (GCSF) and macrophage chemical attraction albumen-1 (MCP-1) are packed in the microgranule, as the microgranule that contains a kind of single cell factor or somatomedin, perhaps as the microgranule drug storage warehouse that contains at least a cytokine or cytokine and growth factor combination.To expect that as follows reagent or agent combination pack in the microgranule.Clean 1mg drug storage warehouse microgranule with anhydrous isopropyl alcohol, filter and vacuum drying.Freeze dried cytokine of 100 μ g or somatomedin are dissolved in the buffer salt solution, and filter this solution with the filter of 0.2 μ m.Filtrate is mixed with drug storage warehouse microgranule through cleaning and filtering, and is stirred to solution and is adsorbed by microgranule.To remove water, freeze dried cytokine or somatomedin are stayed in the drug storage warehouse microgranule microgranule lyophilizing.

For loaded with cytokine combination, combinations of. growth factors or combination of cytokines and somatomedin, adopted similar step.For example, freeze dried IL-12 of 100 μ g and the freeze dried GM-CSF of 100 μ g are dissolved in the buffer salt solution.Solution filtered and mix, be adsorbed up to solution with polymerization drug storage warehouse through cleaning-drying.Freeze-drying particle is stayed in the micropore microgranule freeze dried IL-12 and GM-CSF.

Embodiment 3

The drug storage warehouse of GM-CSF and G-CSF and RANTES is equipped with in preparation

A. the microgranule that has GM-CSF

With the freeze dried GM-CSF ultra-sonic dispersion of 100 μ g in the dichloromethane solution of 50/50 copolymer of 1cc 4% (the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether).Behind the cytokine homodisperse, the polymer solution that will contain dispersive cytokine is poured in the excessive precipitation solvent, as petroleum ether.The spherical droplets that contains the polymer solution of dispersive GM-CSF is precipitated as the solid-state spherical polymer microgranule that contains GM-CSF.By filtering collection solid particle and lyophilizing to remove residual solvent.

B. the microgranule that has G-CSF and RANTES

But preparation contains the biological corrosion microsphere of combination of cytokines, combinations of. growth factors or cytokine and combinations of. growth factors by the following method.With the freeze dried G-CSF of 100 μ g and the freeze dried RANTES ultra-sonic dispersion of 100 μ g in the dichloromethane solution of 4% 50/50 copolymer (the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether).Behind the cytokine homodisperse, the polymer solution that will contain dispersive cytokine is poured in the excessive precipitation solvent, as petroleum ether.The spherical droplets that contains the polymer solution of dispersive G-CSF and RANTES is precipitated as the solid-state spherical polymer microgranule that contains G-CSF and RANTES.By filtering collection solid particle and lyophilizing to remove residual solvent.

Embodiment 4

Release in vitro IL-12 from the drug storage warehouse compositions

According to the step of describing among the embodiment 2 with the IL-12 drug storage warehouse microgranule of packing into.The IL-12 that mensuration discharges from biodegradable drug storage warehouse has adopted the IL-12 that cultivates microgranule and adopted the enzyme linked immunosorbent assay analysis method analysis release that is exclusively used in IL-12 in buffer salt solution.The release dynamics of IL-12 demonstrated and discharged 60% IL-12 in 11 day.

Embodiment 5

The compositions that is used for cell and tissue regeneration

Will be from a sagging main branch coronarius ligation in New Zealand white rabbit left front, because the tremulous pulse tip causes myocardial infarction to obstruction of artery.After blocking for 2 weeks, micropore drug storage warehouse bolus and the biodegradable microsphere that contains GM-CSF, MCP-1 and RANTES as according to preparation as described in the embodiment 3B, are sent to invagination zone periphery through femoral artery.Adopt the drug storage warehouse depositing device of describing among Fig. 3 and 13 that micropore drug storage warehouse bolus and biodegradable microsphere are sent to the invagination zone.One treated animal accepts to contain the drug storage warehouse of GM-CSF, MCP-1 and RANTES.Another treated animal is organized as control, only accepts the blank drug storage warehouse of factor-containing not or somatomedin.As the function of time, the increase of maximum blood flow is measured tremulous pulse and is replied degree during increase by monitoring angiogram upside tremulous pulse size of corpse and number and the vasodilation.Employing is estimated mononuclear cell from the isolated cell of animal of being treated and is infiltrated the degree in drug storage warehouse zone and the apoptosis (survival) that is attracted cell.In addition, adopt tissue-estimating and had a multipotency mononuclear cell stem cell that enters the drug storage warehouse zone through circulation from bone marrow.

Embodiment 6

In the drug storage warehouse compositions body that is used for the tumor degeneration, discharge IL-12

To the injection 1 * 10 down of every male and female BALB/c Corium Mus ⁶The CT-26 cell of living, a kind of colon tumor cell line.Allow tumor cell to form entity with solid tumour morphology and microstructure.Measured a gross tumor volume in per 2 days.Determine tumor mass after 14 days, animal is divided into two groups.Organize as control for one group, another winding is subjected to inject in the single tumor drug storage warehouse that 150 μ g contain IL-12.Measure once control group in per 4 days and accepted one group gross tumor volume of the drug storage warehouse treatment that IL-12 fills.Being observed gross tumor volume in the quilt animal for the treatment of reduces.

Although invention has been described according to specific embodiment, it will be apparent for a person skilled in the art that: without departing from the scope of the present invention, can make various changes and modification.

Claims (27)

1. compositions that is used for cellular response, it comprises

Effectively with first reagent of one or more expection cytotaxis to tissue site;

Stimulate second reagent of this cytoactive effectively; And

Influence the 3rd reagent of this cell survival effectively.

2. according to the compositions of claim 1, wherein, described compositions is used to promote cell and/or tissue regeneration, and described first reagent is attracted to described tissue site with one or more stem cell, CFU-GM and accesory cell effectively.

3. according to the compositions of claim 1 or 2, wherein, the activity that described second reagent stimulates proliferation effectively or breaks up.

4. according to the compositions of claim 3, wherein, described tissue is selected from skeletal muscle, liver, pancreas, brain, cardiac muscle or central nervous system.

5. according to the compositions of claim 3, wherein, the described cardiac muscular tissue that is organized as, described first reagent attracts to be selected from the cell of circulation blood monocyte, systemic vascular cellulation or circulation tremulous pulse cellulation; Described second reagent stimulation releasing factor from described cell generates to promote angiogenesis and/or tremulous pulse; Described the 3rd agents influence is arranged in the survival of the deutero-macrophage of circulation blood monocyte of cardiac muscular tissue.

6. according to the compositions of claim 5, wherein, described first reagent is selected from macrophage chemical attraction albumen (MCP)-1, MCP-2, MCP-3, MCP-4, MCP-5, active adjusting, normal T cellular expression and excretory cytokine (RANTES), the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide, complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-β), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1) or macrophage cluster stimulating factor (M-CSF).

7. according to the compositions of claim 5, wherein, described second reagent is selected from macrophage chemical attraction albumen (MCP)-1, MCP-2, IVICP-3, MCP-4, MCP-5, tumor necrosis factor (TNF)-a, TNF-D, active adjusting, normal T cellular expression and excretory cytokine (RANTES), the Fu Shi factor, macrophage inflammatory protein (MIP)-1-α, MIP-1-β, N-farnesyl-peptide, complement activation product C 5a, leukotriene B4, platelet activating factor (PAF), transforming growth factor (TGF-beta), interleukin, granular leukocyte macrophage cluster stimulating factor (GM-CSF), granulocyte cluster stimulating factor (G-CSF), cluster stimulating factor-1 (CSF-1), macrophage cluster stimulating factor (M-CSF) and lipopolysaccharide.

8. according to the compositions of claim 5, wherein, described the 3rd reagent is selected from GM-CSF, G-CSF, CSF-1 or M-CSF.

9. according to the compositions of claim 5, wherein, one or more in described first, second or the 3rd reagent are selected from fibroblast growth factor (FGF, FGF-1, FGF-2), TGF-a, insulin like growth factor (IGF-1), angiogenin-1, angiopoietin-2, VEGF (VEGF), VEGF and constitute thing, as VEGF-2, VEGF165 and VEGF121, platelet derived growth factor (PDGF)-A, PDGF-B, PDGF-BB, Placenta Hominis derivative growth factor (PIGF) or endothelium mitotic growth factor.

10. according to the compositions of claim 1, wherein, described compositions is used to induce the immunne response to tumor, and described first reagent can attract the cell of the one or more T-of being selected from lymphocytes, macrophage, segmented cell, antigen presenting cell or natural killer cell effectively.

11. according to the compositions of claim 10, wherein, described first reagent is selected from IL-8, MIG, IL-12, MCP-1 ,-2 ,-3 ,-4 ,-5, MIP-1 α, MIP-1 β, MIP-1 γ or RANTES.

12. according to the compositions of claim 10 or 11, wherein, described second reagent is selected from GM-CSF or IL-12.

13. according to the compositions of claim 12, wherein, described the 3rd reagent is selected from MIG, platelet factor 4, MCP-1 ,-2 ,-3 or MIP-1 γ.

14. according to each compositions in the claim 1,2 or 10, wherein, described reagent discharges from compositions simultaneously.

15. according to each compositions in the claim 1,2 or 10, wherein, described reagent discharges from compositions continuously.

16. according to each compositions in the claim 1,2 or 10, wherein, described reagent is wrapped in the spherical drug storage warehouse of being made up of polymer.

17. according to the compositions of claim 16, wherein, described drug storage warehouse has outer surface, and bio-ligand links to each other with described outer surface.

18. according to the compositions of claim 17, wherein, described part is cell adhesion molecule.

19. according to the compositions of claim 16, wherein, described compositions is included in many medicine drug storage warehouses, makes the dosage form that is selected from Emulsion, gel, pasty state or liquid.

20. one kind is used for the compositions of replying at specific tissue site inductive treatment, comprises one or more drug storages that contain each compositions in the with good grounds claim 1,2 or 10.

21. a compositions that is used to prepare the medicine that can reply at specific tissue site inductive treatment, it comprises one or more drug storage warehouses that contain each compositions in the with good grounds claim 1,2 or 10.

22. a medicine transfer equipment is used for transmitting according to claim 1,2 or 10 each compositionss, it comprises:

Catheter shaft, it has the inner chamber that extends to port, the remote domain of local compliance at least, the near-end of local stiffness at least, and at the reinforcing axial region of near-end;

At least one attached pipe, it extends through a proximal end region to small part;

The single track part, it is arranged to the far-end of adjacent pipes axle and has the seal wire junction surface;

User interface, it has far-end and near-end, and is rigidly connected to the catheter shaft near-end at the interface far-end;

Be connected to the shell of interface near-end, be used to hold a piston mobile.

23. according to the medicine transfer equipment of claim 22, it comprises also to small part and is positioned at the pin of catheter shaft remote domain with deployed condition not that it launches with deployed condition from catheter shaft.

24. according to the medicine transfer equipment of claim 23, wherein, described pin is selected from straight needle or curved pin.

25. according to the medicine transfer equipment of claim 22, wherein, described seal wire junction surface comprises that also an intracavity paves equipment, it is used for along tube wall or body cavity deposition composition.

26. according to the medicine transfer equipment of claim 25, described intracavity is paved equipment and had a flexible cup that has the elongation of center cavity, it is used for receiving compositions from the catheter shaft port.

27. according to the medicine transfer equipment of claim 26, described intracavity is paved equipment and had hole and a following hole at least one, it is used for introducing seal wire by cup.

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