CN101448474A - Compositions and methods for inhibiting adhesions - Google Patents

Abstract

The present invention provides compositions and methods for inhibiting adhesions. The methods involve administering solutions containing hydrogel precursors such as polysaccharide derivatives, e.g., derivatives of hyaluronic acid, cellulose, or dextran, to a subject at a site where adhesions may form, e.g., as a consequence of surgery, injury, or infection. The hydrogel precursors, e.g., polysaccharide derivatives, become crosslinked following their administration to form a hydrogel that maintains tissue separation. In certain embodiments of the invention one or both solutions contains particles, e.g., polymeric nanoparticles or microparticles, so that a composite hydrogel containing the particles is formed. The solution(s), particle(s), or both, may contain a biologically active agent such as an agent that contributes to inhibiting adhesions. The biologically active agent may be covalently attached to a hydrogel precursor.

Description

The compositions and the method that suppress adhesion

Related application

The application's case is advocated the U.S. Provisional Patent Application case U.S.S.N.60/791 of application on April 12nd, 2006 according to 35 U.S.C. § 119 (e), 362, the U.S.S.N.60/857 of application on November 8th, 2006, the U.S.S.N.60/901 of application on February 13rd, 557 and 2007,241 priority, all described patents all are incorporated herein by reference.

Government supports

Working portion as herein described is subjected to the support of NIH (National Institutes of Health) permission (GM073626).U.S. government can enjoy some right to the present invention.

Technical field

Do not have

Background technology

Adhesion is the connection between the tissue, organ or other anatomical structure that are separated from each other usually.It normally is made of the fabric strip of cicatrix sample tissue and generally is to stimulate the back to produce such as operation, damage or infection etc.Because postoperative intestinal adhesion is inevitable with such as severe complications such as abdominal part and pelvic pain, infertile and intestinal obstruction, so it is common and serious potentially incident.According to estimates, 80% abdominal operation meeting causes adhesion, causes people's health and economy to be sustained a great loss.The adhesion that operation back, pelvic cavity district forms is the leading reason that causes operation back pelvic pain, infertile and small intestinal obstruction.Wound and infection, the wound in abdominal part or the pelvic cavity zone and infect and also can cause adhesion especially.

At least, the multiple pharmacology of test prevention or treatment adhesion and in animal model based on the method for barrier, and several methods based on barrier have been used for commercial use.These methods comprise such as can absorb adhesion barrier (Interceed (TC7) Absorbable Adhesion Barrier) ((the Johnson ﹠amp of Johson ﹠ Johnson because of special crack (TC7); Johnson)) and Film ( Membrane) product of (gene enzyme company (Genzyme Corp.)).Many existing barrier devices are to be made of cross-linked polysaccharides or glycosaminoglycans to small part.

Hyaluronic acid (HA) has been caused extensive concern (this people such as (Burns) of Berne as the material of reaching these purposes, prevent tissue injury and tissue adhesion (Prevention of tissue injuryand postsurgical adhesions by precoating tissues with hyaluronic acid solutions.), operations research magazine (Journal of Surgical Research) 1995 by organizing with the hyaluronic acid solution precoating; 59:644-652; Parker people such as (Peck), polymer solution and thin film (Polymer solutions and films as tissue-protective andbarrier adjuvants.) as organization protection and barrier adjuvant, Di Zejia GS (diZerega GS) compiles, operation on peritoneum (Peritoneal Surgery.) New York (New York): Springer Verlag (Springer), 2000. 499-520 pages or leaves; And Rogers people such as (Rodgers), reproduce adhesion with hyaluronic acid behind the rabbit operation on peritoneum and form (Reproduction of adhesion formation withhyaluronic acid after peritoneal surgery in rabbits.) reproduction and contraception (Fertil Steril.) 1997; 67 (3): 553-558, described document is incorporated herein by reference separately).HA connects D-glucuronic acid (GlcUA) and β-1 by β-1,4, the linear polysaccharide that 3N-acetyl group-D-glycosamine (GlcNAc) disaccharide unit constitutes, and be the common component of mammalian cell epimatrix.The native form of HA is bio-compatible, biodegradable and relative non-immunogenic.Although it has these desirable characteristics, the existing method based on HA that is used to suppress postoperative intestinal adhesion new life or recurs has distinct disadvantage.For instance, when using with the solution form, the effectiveness of HA removes fast because of the abdominal cavity and suffers damage that (Suo Ni people such as (Sawhney) is used for optimization (Optimization of photopolymerized bioerodible hydrogel properties for adhesionprevention.) the biomedical material research magazine (J.Biomed.Mater.Res.) 1994 of the photopolymerization bioerosion hydrogel character of Film with Preventing Adhesion; 28:831-838, it is incorporated herein by reference).Have separately or with the HA solid composite of (for example) of other combination of materials prepared sheet form that goes out and to comprise following defective: be difficult to use thin film (for example, be difficult to handle, exsiccant thin film adheres on the glove, the pliability deficiency, need to remove), incompatible with laparoscopic procedure and be lower than required effect.Therefore, need to suppress the improvement compositions and the method for adhesion in the affiliated field.

Summary of the invention

The invention provides the compositions and the method that suppress adhesion.Although described compositions and method can be used for suppressing formation, progress or the recurrence of any position adhesion in the body, expect that it will be particularly useful for suppressing peritoneal adhesion.

Purpose of the present invention be to provide can be used for suppressing adhesion development, progress and/or recurrence be suitable for the polysaccharide derivates of in-situ polymerization in vivo, for example HA, cellulose or glucan derivative.Another purpose of the present invention be to provide suppress that adhesion forms, the method for progress and/or recurrence, thereby its be by throw with after being applied to histologic lesion or damage location rapidly original position be cross-linked with each other form suppress that adhesion forms, the polysaccharide derivates of the hydrogel of progress and/or recurrence realizes.

Another purpose of the present invention is to provide polysaccharide derivates and its combination, and it will crosslinked and gelling rapidly in helping the time range that its original position uses.Thereby another purpose of the present invention is to provide generation to be provided the method such as gelling time and isoparametric control of half-life by the formed hydrogel of cross-linked polysaccharides.

Another purpose of the present invention is to provide hydridization polysaccharide based aquagel compositions, and wherein said compositions is delivered in the body bioactivator (for example, therapeutic agent) in the mode that continues according to circumstances.In certain embodiments, bioactivator is an antiinflammatory, for example glucocorticoid (for example, prednisone (prednisone), dexamethasone (dexamethasone), budesonide (budesonide)) and non-steroidal antiinflammatory (for example, ibuprofen (ibuprofen), aspirin (aspirin)).In certain embodiments, bioactivator is a fibrinolytic agent, such as plasminogen activator or streptokinase.Can according to circumstances these medicaments be incorporated in polymeric material or the substrate into prolongation or controlled release for described medicament.In certain embodiments, described medicament and hydrogel or a kind of hydrogel precursor are directly linked.

Another purpose of the present invention is to provide hydrogel composition, and it contains and according to circumstances bioactivator is delivered to intravital granule.On the one hand, the invention provides a kind of method that suppresses adhesion, it comprises following steps: with position in first hydrogel precursor and throwing of second hydrogel precursor and the individual body; Wherein said first and second hydrogel precursors are cross-linked to form hydrogel after being in contact with one another, and wherein said hydrogel suppresses adhesion.First and second hydrogel precursor can one or more solution form provide.In certain embodiments of the invention, hydrogel precursor is a polysaccharide derivates.On the one hand, the invention provides a kind of method that suppresses adhesion, it comprises following steps: with position in throwing of first polysaccharide derivates and the individual body; With second polysaccharide derivates is thrown with individual body in described position, wherein after described first and second polysaccharide derivates was in contact with one another, described polysaccharide derivates was cross-linked to form hydrogel, and wherein said hydrogel suppresses adhesion.In certain embodiments of the invention, first polysaccharide derivates comprises first functional group and second polysaccharide derivates comprises second functional group, and described first and second functional groups formation covalent bond that reacts to each other under physiological condition.In certain embodiments of the invention, a functional group is that hydrazides and a functional group are aldehyde.In certain embodiments of the invention, polysaccharide derivates is the HA derivant.In certain embodiments of the invention, a kind of polysaccharide derivates is that HA derivant and another polysaccharide derivates are cellulose derivative (for example, carboxymethyl cellulose (CMC), hydroxypropyl emthylcellulose (HPMC), methylcellulose (MC)).In some other embodiment of the present invention, a kind of polysaccharide derivates is that HA derivant and another polysaccharide derivates are glucan derivative.In certain embodiments, the present invention comprise with the solution that comprises first polysaccharide derivates throw with individual body in the position; With described position in second solution throwing that will comprise second polysaccharide derivates and the described individual body.

In certain embodiments of the invention, at least a polysaccharide derivates comprises non-polysaccharide part.In other embodiments, at least a hydrogel precursor is non-polysaccharide polymer.

On the other hand, the invention provides a kind of compositions that comprises hyaluronic acid (HA) derivant of solution form, the concentration of wherein said HA derivant is greater than 5mg/ml.In another embodiment, the invention provides a kind of compositions that comprises hyaluronic acid (HA) derivant of solution form, the concentration of wherein said HA derivant is greater than 10mg/ml.In another embodiment, the invention provides a kind of compositions that comprises hyaluronic acid (HA) derivant of solution form, the concentration of wherein said HA derivant is greater than 15mg/ml.In another embodiment, the invention provides a kind of compositions that comprises hyaluronic acid (HA) derivant of solution form, the concentration of wherein said HA derivant is greater than 25mg/ml.In certain embodiments of the invention, the concentration of HA derivant is less than or equal to 100mg/ml.In other embodiment of the present invention, the concentration of HA derivant is between 50mg/ml and 75mg/ml.

On the other hand, the invention provides a kind of hydrogel that comprises crosslinked HA derivant, wherein said hydrogel has at least 10 days half-life under the situation that has the 10U/ml hyaluronidase.In certain embodiments, the invention provides a kind of HA-cellulose, HA-glucosan or other polysaccharide derivates of HA-, wherein the gained hydrogel is not so good as corresponding HA-HA hydrogel sensitivity to hyaluronidase.In certain embodiments, the invention provides a kind of compositions that comprises the cellulose or the glucan derivative of solution form, the concentration of wherein said cellulose or glucan derivative is greater than 5mg/ml, greater than 10mg/ml, greater than 15mg/ml or greater than 25mg/ml.

On the other hand, the invention provides a kind of compositions, it comprises first polysaccharide derivates and a plurality of granule.Polysaccharide derivates can be HA derivant, cellulose derivative or glucan derivative.

On the other hand, the invention provides a kind of compositions, it comprises first and second polysaccharide derivates and a plurality of granule.In certain embodiments of the invention, first and second polysaccharide derivates is cross-linked to form hydrogel.Described granule can contain bioactivator.In certain embodiments, bioactivator is antiinflammatory (for example, dexamethasone, prednisone, budesonide, ibuprofen, an aspirin etc.).In other embodiments, bioactivator is fibrinolytic agent (for example plasminogen activator, a streptokinase).

The present invention provides a kind of method that suppresses adhesion in addition, it comprises following steps: with a plurality of granules and at least a polysaccharide derivates throw with individual body in the position, wherein separately or with first polysaccharide derivates of second polysaccharide derivates combination throw with after be cross-linked to form and hold back described particulate hydrogel.In certain embodiments of the invention, described method comprises throws and first and second polysaccharide derivates, and wherein at least a derivant is the HA derivant.In certain embodiments of the invention, at least a derivant is a cellulose derivative.In certain embodiments of the invention, at least a derivant is a glucan derivative.In certain embodiments of the invention, at least a derivant comprises non-polysaccharide part.

The present invention provides a kind of method that suppresses adhesion in addition, and it comprises following steps: first solution that will comprise first polysaccharide derivates is thrown and the interior position of individual body; Throw and described position with second solution that will comprise second polysaccharide derivates, any one or two kinds ofly in the wherein said solution comprise a plurality of granules, and wherein said polysaccharide derivates throw with after be cross-linked to form the hydrogel of trapped particles.In certain embodiments of the invention, first solution comprises a HA derivant and second solution comprises the 2nd HA derivant.In certain embodiments of the invention, first solution comprises the HA derivant and second solution comprises cellulose derivative.In certain embodiments of the invention, first solution comprises the HA derivant and second solution comprises glucan derivative.In certain embodiments of the invention, first solution comprises the HA derivant and second solution comprises another polysaccharide derivates.

On the other hand, the invention provides a kind of method with position in granule throwing and the body, it comprises: the compositions that will comprise granule and one or more hydrogel precursors is thrown and described position, and wherein said one or more hydrogel precursors form granule is retained in wherein hydrogel.In certain embodiments of the invention, at least a hydrogel precursor is a polysaccharide derivates.

Description of drawings

The scanning electron micrograph of Fig. 1 .HAX gel.Scale=10 μ m.

Fig. 2. mesothelial cell's vigor under the situation that has HAX gel (20mg/ml).White bars and grey bar are indicated at ordinary culture medium respectively and are contained the cell of growing in the culture medium of 10U/ml hyaluronidase.Data are intermediate value and the 25th percentage point and the 75th percentage point (n=4).

Fig. 3. (A) be applied to HAX on impaired stomach wall and the caecum.The arrow indication adheres to the gel of site of administration.(B) in the animal of HAX treatment, do not observing adhesion.(C) in untreated contrast, observe 3 minutes adhesion.The AW=stomach wall.

Fig. 4. histological examination.(A) from the adhesion (5 *) of untreated animal.Arrow is indicated impaired myocyte; (B) from the feature image (10 *) of the adhesion of another untreated animal.Note indication inflammation and Fibrotic high cell mass band.(C) the hang oneself no adhesion stomach wall (20 *) of animal of 20mg/ml HAX treatment.Note the blue coating on the lumen side.The AW=abdominal wall muscle, the Sm=smooth muscle.

Fig. 5. (A) in the 10U/ml hyaluronidase, discharge glucuronic acid (n=5) during the degraded HAX gel.(B) under the situation that has hyaluronic acid (HA) and monomer whose component, produce tPA by the mesothelial cell.

(A) concentration of Fig. 6 .HA-CHO and (C) Mw to macroscopical gel degradation effect of kinetics.Mw and the concentration (mg/ml) of legend indication HA-ADH or HA-CHO.Indicated value is the meansigma methods and the standard deviation of four measuring value.

Fig. 7. (A) PLGA nano-particle, (B) lyophilizing HAX gel and (C) the SEM picture of lyophilizing hybrid gel.

Fig. 8. mesothelial cell's vigor under the situation that has hybrid gel (20mg/ml HAX, PLGA nano-particle).White bars and grey bar are indicated at ordinary culture medium respectively and are contained the cell of growing in the culture medium of 10U/ml hyaluronidase.Data are intermediate value and the 25th percentage point and the 75th percentage point (n=4).

Fig. 9. injected back 2 days or 7 days remaining hybrid gels of intraperitoneal (with the arrow indication).(A) 10mg/ml HAX+20mg/ml PLGA nano-particle, (B and C) 20mg/ml HAX+20mg/ml PLGA.(A) illustration in is showed and the isolating hybrid gel of peritoneum.The B=intestinal, the AW=stomach wall.

Figure 10. (A) be applied to hybrid gel on impaired stomach wall and the caecum.The arrow indication adheres to the hybrid gel of site of administration.(B) in the animal of hybrid gel treatment, do not observing adhesion.The AW=stomach wall.

Figure 11. histological examination.(A) hybrid gel (200 *) that reclaims from 7 days rabbit of postoperative.Note foamy macrophage.(B) with the feature image (400 *) of the similar foamy macrophage of gel residue seen in the injection back 7 days mice (illustration).(C) the hang oneself no adhesion abdominal wall muscle (200 *) of rabbit of hybrid gel treatment; (D) at the feature image (400 *) of intra-operative through the abdominal wall muscle surface that hybrid gel covers.Note the foaminess of macrophage.AW=is through the scratch abdominal wall muscle.

Figure 12. show the chemical constitution of various synthetic polysaccharide derivates.(A) HA-ADH, (B) HA-ALD, (C) CMC-ALD (R=CH ₂COOH or H), HPMC-ALD (R=CH ₂CH (OH) CH ₃Or H) or MC-ALD (R=CH ₃Or H).

Figure 13 illustrates the device that can be used for throwing and contain the solution of crosslinkable polysaccharide derivates.

Figure 14 illustrates the multichannel device that can be used for throwing and contain the solution of crosslinkable polysaccharide derivates.

Figure 15 illustrates the multiple-string hookup that can be used for throwing and contain the solution of crosslinkable polysaccharide derivates.

Figure 16 is the bar diagram that represents the ability of HA derivant and the crosslinked formed multiple hydrogel inhibition adhesion of cellulose derivative.

Figure 17. the degradation kinetics in the 10 units/ml hyaluronidase of 37 ℃ of following hydrogels in PBS.The volume of hydrogel and the ratio of initial volume were represented with percentage ratio when the volume of hydrogel (%) was each time point.Data are mean+SD (n=4).

Figure 18. analyze the influence of measured aldehyde polymer (HA-CHO, CMC-CHO, MC-CHO and HPMC-CHO) pair cell vigor by MTT.(A) cultivate mesothelial cell after 3 days with polymer.(B) cultivate macrophage (J774.A1 cell line) after 2 days with polymer.Data are mean+SD (n=4).

Figure 19. the peritoneum of 1 all mices behind the injection water gel.(A) HAX: no residue.(B) HA-CMC: the shallow layer of noting gel-like material.(C) HA-MC: note the residual materials of recruitment, show that tweezers are immersed in it down.

Figure 20. the prevention of peritoneal adhesion in the rabbit scratch model.(A) adhesion brings out.Note the hemorrhage surface of abdominal-wall defect (arrow) and caecum.(B) 1 all backs are in the visible adhesion of section of the animal for the treatment of through normal saline.(C) in the animal of HA-MC treatment, there be not adhesion after 1 week.

Figure 21. the microphotograph of the tissue that damage 1 week of back reclaims in the damaged intestinal scratch model of rabbit sidewall.(A) abrasive cross section in the animal of normal saline treatment.Caecum chamber (CE) is in the upper left corner of picture.The striped muscle of caecum smooth muscle and abdominal muscles tissue (AM) merges.Amplification 100 *.(B) hydrogel from reclaiming through the animal of HA-MC treatment has inflammatory cells (being mainly macrophage and lymphocyte).Amplification 100 *.(C) position of abdominal-wall defect in the animal of HA-MC treatment.Described damaged re-epithelialize (arrow) has callus lower floor (being mainly fibroblast).Amplification 400 *.(D) normal untreated parietal peritoneum.Mesothelium (arrow) is positioned on connective tissue (CT) and the abdominal muscle.

Figure 22. (A) DX, (B) DX-CHO, (C) CMDX, (D) CMDX-ADH, (E) CMC and (F) chemical constitution of CMC-CHO.

Figure 23. (A) DX, (B) CMD and (C) the FT-IR spectrum of CMD-ADH.

The expanding volume of Figure 24 .37 ℃ of following hydrogel in the PBS buffer.Measured value is mean+SD (N=4).

Figure 25. the gelling time of hydrogel.Measured value is mean+SD (N=5).

The expanding volume of Figure 26 .37 ℃ of following hydrogel in the PBS buffer.Measured value is mean+SD (N=4).

Be immersed in the picture of back 5 days hydrogels in the PBS buffer under Figure 27 .37 ℃.(A)70kDa-CMDX-DX(5％(w/v)/6％(w/v))。(B)70kDa-CMDX-CMC(5％(w/v)/6％(w/v))。

Figure 28. analyze the influence of measured not modified and synthetic polymer (DX, CMC, CMDX-ADH, CMC-CHO and DX-CHO) pair cell vigor by MTT.Data are mean+SD (n=4).(A) cultivate mesothelial cell after 3 days with polymer.The data of CMC-CHO is described in the above-mentioned example 12.(B) cultivate macrophage cell line J774.A1 after 2 days with polymer.

Figure 29. the peritoneum of 2 all mices behind the injection 70kDa-CMDX-DX.

Figure 30. the peritoneum of the adhesion prevention test of carrying out with rabbit scratch model.Bring out 1 week of operation back in adhesion and carry out laparotomy.(A-1、A-2)70kDa-CMDX-DX(2％(w/v)/5％(w/v))。The CMDX-DX gel worsens peritoneal adhesion.(B)70kDa-CMDX-CMC(5％(w/v)/6％(w/v))；(C-1，C-2)500kDa-CMDX-CMC(4％(w/v)/6％(w/v))。The CMDX-CMC gel reduces peritoneal adhesion.The result of control experiment quotes from aforementioned research.

Figure 31. histology's picture of the intestinal scratch model experiment that the rabbit sidewall is damaged.(A) the CMDX-DX gel is bonded at (5 *) on the caecum bag 14.(B) enlarged drawing (40 *) of the adhesive surface of figure A.(C) stomach wall (5 *) that under CMDX-DX gel situation, reclaims.(D) the normal stomach wall (5 *) under 500kDa-CMDX-CMC gel situation.

Figure 32. the synthetic sketch map that contains the cross-linked-hyaluronic acid hydrogel (HAX-DEX) of dexamethasone.Final hydrogel is to form by aldehyde derivatization hyaluronic acid is mixed with hyaluronic acid-adipic acid two hydrazides-succinic acid dexamethasone (drawing hatched chemical compound).

Figure 33. analyze the human mesothelial cell's who cultivates with the variable concentrations synthetic polymer measured vigor by MTT.Data are meansigma methods and standard deviation.

Figure 34. the time-histories of dexamethasone concentration in discharging culture medium.Data are meansigma methods and standard deviation.

The time-histories of the expanding volume of Figure 35 .HAX and HAX-DEX.Data are meansigma methods and standard deviation.

Figure 36. dexamethasone to former generation mouse macrophage TNF-α and the influence of the generation of IL-6.Value is the meansigma methods of twice measurement.

Figure 37. the time-histories of former generation mouse macrophage generation IL-6.Data are meansigma methods and standard deviation.

Figure 38. the time-histories of former generation mouse macrophage generation TNF-α.Data are meansigma methods and standard deviation.

Figure 39. inject the hydrogel that took out in back 2 days.(A) stripped HAX-DEX hydrogel.(B) the representative haematoxylin painted HAX-DEX gel in Yihong (top) and lower floor's muscle (bottom) sample, 20 *.Two samples of inflammatory cell in (C and D) HAX gel and around the HAX gel, be respectively 20 * and 40 *.Blueness material relatively uniformly is gel (G).

Figure 40. the scanning electron micrograph of lyophilizing budesonide-HAX.Scale=100 μ m.

The dissolubility of Figure 41 .37 ℃ of following budesonide in normal saline.(A) phase solubility figure.(B) pass in time budesonide concentration and the precipitation quality.Data are meansigma methods and standard deviation (n=4).

Figure 42. the in vitro release dynamics of budesonide among budesonide-HAX.Data are meansigma methods and standard deviation (n=4).

Figure 43. (A) loss in weight (being expressed as percentage ratio) after the laparotomy second time with respect to initial body weight.(B) has the percentage ratio of the animal of various adhesion scores.0 minute=no adhesion; 2 minutes=by the separable tissue adhesion of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.(C) the area summation (cm of 2 minutes and adhesion in 3 fens ²).The loss in weight and adhesion area all are expressed as intermediate value and the 25th percentage point and the 75th percentage point (n=6). ^*The significant difference of expression and normal saline contrast, and With

Significant difference between each group that expression is compared.Contrast and HAX through the normal saline treatment organize self-reference (ref) [tPA].

Figure 44. tissue and the normal structure of the animal of the budesonide of hanging oneself-normal saline treatment.(A) stomach wall surface (200 *); (B) caecum surface (200 *); (C) caecum surface (400 *).SK: stomach wall skeletal muscle; SM: visceral smooth muscle; Me a: cortex.

Figure 45. inject the hydrogel that took out in back 2 days.(A-D) general appearance of Xie Pouing.(A) HAX of original position.Note inflammation and tangible vascularity.(B) budesonide-HAX of original position.(C) can not with the isolating stripped HAX of skin.(D) from the isolating budesonide-HAX of skin except that the little cortex of having a mind to retain (small rind).The painted part in (E and F) haematoxylin Yihong (all be 40 *).(E)HAX。Note a large amount of inflammatory responses.(F) budesonide-HAX.Note not existing relatively of inflammation.In E and F, the light blue material in the inner chamber is a hydrogel, and it is centered on by esosiophiliccapsule.

The specific embodiment

Definition

Term " angiogenesis inhibitor " and " anti-angiogenic agent " are used interchangeably in this article referring to suppress or to reduce the medicament of one or more processes relevant with angiogenesis, and described process includes, but is not limited to endothelial cell proliferation, endotheliocyte survival, endothelial cell migration, precursor and is divided into endotheliocyte and capillary tube formation.

As used herein, " anti-infective " is meant any material that suppresses one or more infectious pathogens (for example virus, antibacterial, fungus, protozoacide, anthelmintic, trematodiasis or other parasite) propagation.Anti-infective in vitro (that is, in the cell culture), in vivo represent in (that is, when throwing) or the two and suppress active with animal that infection risk is arranged or infection animal.Anti-infective preferably has the activity of inhibition in vivo with the dosage of treatment tolerance.

As used herein, " antiinflammatory " is meant any material that suppresses one or more inflammation diseases million or symptom.

As used herein, " aqueous medium " meaning refers to contain the liquid medium of water and the solvent (for example, dimethyl formamide, dimethyl sulfoxine and hydrocarbon alcohol, glycol or glycerol) that one or more can be miscible with water according to circumstances.Aqueous medium can contain at least 50 volume %, 60 volume %, 70 volume %, 80 volume %, 90 volume % or more water.Should be appreciated that aqueous medium can contain multiple dissolving, disperses or be suspended in material wherein.

From context, may be obvious that (will 100% situation except that described numeral) unless otherwise mentioned or in addition, otherwise be usually included in the numeral in 5% scope of either direction (being greater than or less than described numeral) of described numeral about the term " about " of numeral above probable value.

" bio-compatible " is meant with employed amount in employed position nontoxic in fact and also can not cause or cause the material of significantly harmful or adverse influence (for example, unacceptable immunity or inflammatory response, unacceptable scar tissue formation etc.) to receptor's health in employed position to receptor's cell.

" biodegradable " meaning refers to for example to pass through hydrolysis under physiological condition, and/or by natural biological process (such as the enzyme effect that exists in cell or the body), and/or such as processes such as dissolving, dispersions physics and/or chemolysis are forming the material of less chemical substance in cell or individual body, described less chemical substance usually can be by body metabolism and is used according to circumstances and/or drain or otherwise handle.The biodegradable chemical compound is preferably bio-compatible.For purposes of the present invention, think that molecular weight is biodegradable because of amount of monomer reduces the polymer that reduces in time in vivo.

" bioactivator " is for having any chemical compound or the medicament of required biological activity (for example treat, diagnose and/or prevent characteristic) in vivo, or its pharmaceutically acceptable salt.Should be appreciated that, may need from granule and/or hydrogel, to discharge described medicament so that its performance biological activity.Bioactivator includes, but is not limited to as used herein therapeutic agent.Bioactivator can be (but being not limited to) artificial or naturally occurring micromolecule, peptide or polypeptide, immunoglobulin, for example antibody, nucleic acid etc.And unrestriction ground, hormone, somatomedin, medicine, cytokine, chemotactic factor, thrombin and endogenous blood coagulation inhibitor etc. also are bioactivator.

Term " endoscope " meaning refers to minor diameter tubulose instrument, and it uses optical fiber usually, in the otch of design with the insertion health, is used for visual during the Minimally Invasive Surgery program and operation.Described term comprises " peritoneoscope ", its through design so that tissue in the abdomen pelvic cavity and organ are visual and can operate; " arthroscope ", its through design so that organizing in the joint space is visual and can operate etc.

As used herein, " fibrinolytic agent " is meant any material that directly or indirectly causes fibrin degradation.

" HAX hydrogel " is the hydrogel that is formed by crosslinked HA derivant.

" hybridized hydrogel " is for comprising the composite aquogel of granule and cross-linked polysaccharides derivant.

" hydrogel " is for comprising the three-dimensional netted thing of the hydrophilic polymer that contains big water gaging.Hydrogel can for example contain 30%, 40%, 50%, 60%, 70%, 80%, 90% or even the water of volume more in w/w." hydrogel precursor " is the polymer that is partially soluble in the aqueous medium at least and can be cross-linked to form hydrogel.

" inhibition adhesion " thus quantity, scope and/or the order of severity of the adhesion that occurs decreased when being meant the order of severity (for example, density or anti-machinery or chemical depletion) that throwing and compositions and/or performing a programme make adhesion quantity, adhesion scope (for example area) and/or adhesion with respect to no described dispensing.Compositions or program can suppress adhesion formation or growth after the stimulation that promotes adhesion; Can suppress the adhesion progress; And/or can suppress adherence recurrence after its spontaneous regression or behind machinery or the chemical depletion.

" original position " meaning refers to that hydrogel is formed at the position that needs hydrogel in fact but not is formed at other places and subsequently it is applied to the position that needs it.For instance, for purposes of the present invention, think on the individual health or body in the forming of hydrogel " original position ".

" liposome " is for containing the artificial microcosmic spheroidal particle that forms in the aqueous compartment of bioactivator by double-layer of lipoid (or multilamellar) is enclosed.

" the local peritoneum dispensing of prolongation " is meant and throws and one or more compositionss, so that area equals at least apart from the part of the interior area of damaging part border 10cm distance in the described composition in its entirety contact peritoneum, for example thus the polysaccharide derivates that in described compositions, exists crosslinked after, area equals to be covered by hydrogel layer apart from a part of peritoneum of the interior area of damaging part border 10cm distance at least.Undermined position can be any position of obviously jeopardizing peritoneum entity or function, and for example operative incision, damage, infection cause the position (for example, inflammation, transudate etc.) that visible entity changes in the peritoneum.The area that is covered can for example adjoin with damaging part and around damaging part, perhaps can be positioned on the apparent surface of peritoneum.For instance, operative incision so that parietal peritoneum that infringement can be in the stomach wall are impaired, and the area that is covered can be on the visceral peritoneum relative with damaging part.

" full peritoneum dispensing (Pan-peritoneal administration) " be meant throw with one or more compositionss so that described compositions throw with after integral body contact peritoneum substantial portion (for example, at least 10% peritoneal surface is long-pending), thereby for example the polysaccharide derivates that in described compositions, exists crosslinked after, make that at least 10% peritoneal surface is long-pending to be covered by hydrogel layer.

" granule " is meant that wisp, fragment or smallclothes material and its include, but is not limited to aggregated particles, biodegradable granule, abiotic degradable granule, single emulsion particle, two emulsion particle, cohesion matter, liposome, micron particle (microparticle), nano-particle (nanoparticle), macroscopic particles, bead, crystal, aggregation, complex, through grinding, mill or through substrate, crosslinking protein or the polyoses grain (comprising the granule that comprises HAX) of otherwise damage.Granule can be made of one matter or multiple material.In certain embodiments of the invention, granule is not a virion.

Term " peritoneum " is meant the serosity shape film of arranging along abdomen pelvic cavity wall, and it extends to the upper surface of pelvic floor from phrenic inner surface.In addition, peritoneum to small part covers various abdomen pelvic organs, for example intestinal, stomach, liver, kidney, adrenal gland, spleen, bladder, uterus, ovary and fallopian tube.Fluid film lubricates peritoneal surface and promotes internal organs relative to each other or with respect to stomach wall or tub wall to move freely usually.Term " peritoneal adhesion " is meant the adhesion that takes place in the peritoneal cavity.Peritoneal adhesion makes organ or tissue be connected with each other or connects together with abdomen pelvic cavity wall.

" Film with Preventing Adhesion " be meant before forming adhesion, throw with or administering therapeutic compositions and/or program so that reduce the probability that will respond particular injury, stimulation or condition of illness formation adhesion.Should be appreciated that the probability that " Film with Preventing Adhesion " do not need adhesion to form is reduced to zero.But say that " Film with Preventing Adhesion " is that the probability of instigating particular injury or stimulating posterior synechiae to form significantly reduces clinically, for example responds specific adhesion and promotes the adhesion incidence rate of damage, condition of illness or stimulation or quantity significantly to reduce clinically.

" micromolecule " is meant natural existence or artificial the generation, and () organic compound for example, via chemosynthesis, it has low relatively molecular weight and is not protein, polypeptide or nucleic acid.Usually, micromolecule has the molecular weight less than about 1500g/mol.In addition, micromolecule has a plurality of carbon-carbon bonds usually.

" dissolubility " is meant the amount of substance of (for example) formation saturated solution in the solvent that is dissolved in designated volume under specified temp and pH value.Dissolubility can for example use and shake a bottle dissolving method (ASTM:E 1148-02, be used to measure the standard method of test (Standard Test Method for Measurements of Aqueous Solubility) of aqueous solubilities, manual of standards (Book of Standards), the 11.05th volume) measure.Can be between 3.0 and 9.0, for example between between 4.0 and 8.0, between between 5.0 and 8.0, between 6.0 and 8.0, for example between 6.5 and 7.6, for example between 6.8-7.4, for example be 7.0 or the pH value of any intermediate value of above-mentioned scope measure dissolubility down.Can be between 20 and 40 ℃, for example about 25-37 ℃, for example test dissolubility under the temperature for any intermediate value of about 37 ℃ or above-mentioned scope.For instance, can be at about pH7.0-7.4 and about 37 ℃ of following dissolubility of measuring.

As used herein, " individuality " be meant intend to transmit medicament (for example) to its to reach the individuality of experiment, diagnosis and/or therapeutic purposes.Preferred individuality is a mammal, especially domestic mammal (for example Canis familiaris L., cat etc.), primate or the mankind.Be in doctor or other health care personnel individuality under nursing and can be described as " patient ".

" continue discharge composite " for comprise bioactivator as a kind of in its component and comprise in addition one or more according to circumstances part because the entity structure of composite effectively makes described therapeutic agent continue the compositions related of other component, element or the structure that discharge.Continue to be released in a period of time, for example at least 2,3,4,5 or 6, at least 1,2,4 or 6 weeks, reach about 1,2,3,4,6,8,10,12,15,18 or 24 months in continuously or the release or the transmission that intermittently take place.

" therapeutic agent " that is also referred to as " medicine " be meant herein throw with individual with treatment disease, disease or other approve clinically to the deleterious condition of illness of individuality or reach the medicament of prevention purpose, and the treatment of its disease to health, disease or condition of illness or prevention have clinically effect significantly.Therapeutic agent includes, but is not limited to medicament listed in the following document: American Pharmacopeia (United States Pharmacopeia, USP), Gourde(G) graceful (Goodman) and (Gilman) therapeutic pharmacological basis (The Pharmacological Basis of Therapeutics), the 10th edition, (Mike Lao Xier (McGraw Hill)), 2001; Ka Zhuoen B. (Katzung, B.) (volume) basis and clinical pharmacology (Basic andClinical Pharmacology), Mike's Lao Xier/Appleton and Lange (Appleton; Lange); The 8th edition (on JIUYUE 21st, 2000); Handbook on doctor's table (Physician ' s Desk Reference) (Thomson publishing company (ThomsonPublishing)), and/or the Clinics and Practices of Merck handbook (The Merck Manual of Diagnosis and Therapy), the 17th edition (1999) or the 18th edition (2006) of publishing thereafter, mark H. Bill (Mark H.Beers) and Robert Bel examine (Robert Berkow) (volume), Merck publishing company (Merck Publishing Group), or under the situation of animal, Merck veterinary's handbook (The Merck Veterinary Manual), the 9th edition, Cann C.A. (Kahn, C.A.) (volume), Merck publishing company (Merck Publishing Group) is in 2005.

As used herein, " treatment adhesion " be meant to follow and be used to destroy or reduce the scope of adhesion or the program of the order of severity, throw with or use the compositions and/or the program of the order of severity of the probability that can reverse, alleviate, alleviate and/or suppress the adhesion progress and/or the order of severity or can reduce adherence recurrence and/or adherence recurrence." treatment adhesion " also refers to throw and or uses the progress of one or more symptoms (for example, pain, intestinal obstruction, infertile) that can reverse, alleviate, alleviate, suppress adhesion or reduce the probability of its recurrence and/or the compositions and/or the program of the order of severity.Therefore, " treatment adhesion " relate in damage or formed adhesion after stimulating after promptly throw with or administering therapeutic compositions and/or program.

" tumor " is meant the lump by the caused abnormal structure of excessive cell differentiation.Tumor can be optimum (non-carcinous) or pernicious (carcinous)." tumor " comprises the disease that excessively is divided into feature with hematopoietic cell.Described disease comprises pernicious and the preceding blood disorder that cancerates, such as leukemia, lymphoma, myeloma and myeloproliferative disease.Any diagnosing tumour in the method for multiple technologies approvals be can use, physical diagnosis, imaging study, histopathology (for example, pair cell or tissue samples are carried out), biochemical test etc. comprised.The specific limiting examples of tumor (for example comprises sarcoma, carcinoma of prostate, breast carcinoma, carcinoma of endometrium, neoplastic hematologic disorder, leukemia, hodgkin's and non-Hodgkin lymphomas (non-Hodgkin ' s lymphoma), multiple myeloma and other plasma cell disease, myeloproliferative disease), the cerebral tumor (for example, low level astrocytoma, a modification astrocytoma, glioblastoma multiforme, oligodendroglioma and ependymoma) and gastrointestinal stromal tumor (GIST).Sarcoma comprises osteosarcoma, ewing's sarcoma (Ewing ' ssarcoma), soft tissue sarcoma and leiomyosarcoma of stomach.Other example of malignant tumor comprises minicell and nonsmall-cell lung cancer, renal carcinoma (for example, renal cell carcinoma), hepatocarcinoma, cancer of pancreas, esophageal carcinoma, colon cancer, rectal cancer, gastric cancer, breast carcinoma, ovarian cancer, bladder cancer, carcinoma of testis, thyroid carcinoma, a cancer and neck cancer, thyroid carcinoma etc.As used herein, " tumor " comprises the transfer of primary tumor.

" viscosity " is meant the measurement for liquid consistency or anti-liquid fluidity under assigned temperature.Can use known several different methods and Instrument measuring viscosity in the affiliated field.For instance, weighing polymer and subsequently it being dissolved in the appropriate solvent at first.Solution and viscometer are put into water bath with thermostatic control.Make and obtain thermal balance in the solution.Subsequently liquid is put on the top portion scale designation on the viscometer.Recording solution is from overhead stream time of part scale designation on earth.Can according in the ASTM manual of standards (ASTM Book of Standards) about the putting into practice of dilute polymer viscosity (ASTM D2857) the 08.01st volume (in June, 2005) or comprise the viscosity of the solution of polymer about the relevant ASTM standard test of particular polymers.Can be between 20 and 40 ℃, for example about 25-37 ℃, for example test dissolubility under the temperature for any intermediate value of about 37 ℃ or above-mentioned scope.For instance, can be at about pH 7.0-7.4 and about 37 ℃ of following dissolubility of measuring.

I. the Antiblock compositions that comprises original position crosslinkable polysaccharide derivates

It is reported that adhesion is that the inflammatory process by complexity causes, wherein keep isolating tissue often to be connected with each other in vivo usually because of operation wound, damage or infection.These adhesions (comprising the adhesion that is caused by other reason) are the main cause such as intestinal obstruction and severe complication such as infertile.Other adhesion related complication comprises chronic abdomen pelvic pain, urinary tract obstruction and urinary dysfunction.Suspection plays effect in adhesion forms inflammatory process comprises that bonding, the macrophage of activation, fibrin deposition and the adjacent tissue of neutrophil cell accumulation and damaged tissues are invaded, fibroblast proliferation in the zone, collagen deposition, angiogenesis and the generation of lasting adhesion organization.Current Therapeutic Method comprises and uses steroidal and non-steroid antiinflammatory drug thing and relate to the multiple method based on barrier of using thin film or the film of attempting to keep separate tissue.Yet the effect of these methods is limited.

The invention provides the compositions and the method that prevent and/or treat adhesion.The present invention partly is the discovery generation by the inventor: some polysaccharide (for example, hyaluronic acid (HA) or cellulose derivative) when throw with the solution form with body in a position (for example, the position of tissue injury or infringement) the original position time (, offer medicine in vivo position or near the position of offeing medicine in the body) is cross-linked with each other and forms the hydrogel that suppresses adhesion.First aspect the invention provides a kind of method that suppresses adhesion, and it comprises following steps: with position in throwing of first polysaccharide derivates and the individual body; With second polysaccharide derivates is thrown with individual body in described position, wherein after described first and second polysaccharide derivates was in contact with one another, described polysaccharide derivates was cross-linked to form hydrogel, and wherein said hydrogel suppresses adhesion.Throw with before, polysaccharide derivates is dissolved in the solution.Solution in fact side by side can be thrown with individual.Can be before dispensing solution be mixed to form single solution, in the case, preferably take place essence crosslinked before with its throw with.Mainly illustrate the derivant of HA, cellulose and glucosan herein, and the present invention is encompassed in the compositions that suppresses adhesion and the method and use other polysaccharide and its derivant and non-polysaccharide polymer hydrogel precursor herein in described other compositions and the method.

Hydrogel is to form between tissue that between tissue that may be in contact with one another in addition or the structure and for example can therefore develop adhesion in wound healing process or structure.Therefore, hydrogel is used to separate tissue or the structure that has experienced damage, wound, external environment condition exposure or any other types of damage.Therefore the present invention provides a kind of tissue or structure of making to keep isolating method, and it comprises following steps: with position in throwing of first polysaccharide derivates and the individual body; With second polysaccharide derivates is thrown with individual body in described position, wherein after described hydrogel precursor is in contact with one another, described first and second polysaccharide derivates is cross-linked to form hydrogel, and wherein said hydrogel is between tissue or structure that the need maintenance is separated from each other.

Hydrogel inhibition tissue or structure adhere to and suppress the development of cicatrix shape fabric strip between described tissue or the structure mutually.Can promote to stimulate (that is any incident that, increases the probability of adhesion formation, progress and/or recurrence) back to throw and solution in adhesion.Adhesion promotes the example of stimulus object to comprise operation, damage and infects.Hydrogel degradation in vivo and therefore need not to remove.

The invention provides the hydrogel that is cross-linked to form by with first polysaccharide derivates and second polysaccharide derivates, wherein said first polysaccharide derivates is different with second polysaccharide derivates.For instance, first polysaccharide derivates can be the HA derivant that comprises first functional group, and second polysaccharide derivates can be the cellulose derivative that comprises second functional group.In addition for instance, first polysaccharide derivates is the HA derivant that comprises first functional group, and second polysaccharide derivates is the glucan derivative that comprises second functional group.First and second functional group can be selected from amine, amide, aldehyde, ester, hydroxyl or hydrazides.

The present invention provides the hydrogel that is cross-linked to form by with first polysaccharide derivates and second polysaccharide derivates in addition, wherein said first polysaccharide is identical with second polysaccharide, and wherein said first polysaccharide derivates comprises first functional group and described second polysaccharide derivates comprises second functional group, and wherein said first and second functional group can be cross-linked with each other.Polysaccharide can for example be HA, cellulose, glucosan or the derivant of any one.

Can use multiple polysaccharide derivates.In certain embodiments of the invention, at least a polysaccharide derivates is the HA derivant.In certain embodiments of the invention, two kinds of polysaccharide derivates all are the HA derivant.Therefore, the invention provides a kind of method, its (i) will comprise the solution of a HA derivant and throw and the interior position of individual body; The solution that (ii) will comprise the 2nd HA derivant throw with individual body in described position, wherein after described solution was in contact with one another, described first and second HA derivant was cross-linked to form hydrogel, and wherein said hydrogel suppresses adhesion and forms.In certain embodiments of the invention, polysaccharide is not by the polysaccharide of degrading for endogenous enzyme spcificity for the mankind.Do not wish to be bound by any theory, the hydrogel that is formed by the derivant of described polysaccharide to small part can have in vivo than by the long half-life of the formed hydrogel of HA derivant.

In certain embodiments of the invention, at least a polysaccharide derivates is a cellulose derivative.For instance, in certain embodiments of the invention, first polysaccharide derivates is that the HA derivant and second polysaccharide derivates are cellulose derivative.

In certain embodiments of the invention, at least a polysaccharide derivates is a glucan derivative.For instance, in certain embodiments of the invention, first polysaccharide derivates is that the HA derivant and second polysaccharide derivates are glucan derivative.

HA (being also referred to as hyaluronan or hyaluronate) is for containing the not branch polysaccharide of the repetition disaccharide subunit that is made of N-acetyl group-D-glycosamine and D-glucuronic acid.(referring to Laurent T.C. (Laurent, T.C.) (volume)., the biology of hyaluronan and derivant thereof and medical application (Biology and Medical Applications of Hyaluronan and ItsDerivatives), London: Portland publishing house (London:Portland Press), 1998).The structure of HA is as shown in hereinafter.

As used herein, term " hyaluronic acid (HA) " is meant HA and any salt thereof, for example hyaluronate sodium, potassium hyaluronate, hyaluronic acid magnesium, calcium hyauronate etc.Term " HA derivant " is meant the HA by the native form chemical modification shown in above.Modification can comprise interpolation or produce new functional group (for example, amine, amide, aldehyde, ester, hydroxyl, hydrazides etc.), in the case, claims HA " through functionalized ".The variable-scaleization of modified disaccharide subunit, and can select the degree of modifying so that control such as characteristics such as gelling time, half-life, hardness.Some modifies the backbone structure that keeps natural HA, and at least some sugar rings are opened in other modification.For instance, at least some sugar rings of glucuronic acid part are opened in some modification.

The first and second HA derivants of the present invention comprise first and second functional groups respectively, and it reacts to each other and forms the covalent bond that first and second HA derivant is linked together.Therefore solution is used with liquid form and it is in contact with one another, and be blended together before being in contact with one another before dispensing or during dispensing or after the dispensing according to circumstances or during contact.The crosslinked formation of capacity makes liquid be transformed into semisolid or gellike state.

In certain embodiments of the invention, use the HA derivant comprise at least two different functional groups, wherein said functional group under physiological condition, react to each other form crosslinked.Thereby can select functional group that it is not reacted to each other in fact, up to the physiological condition that is exposed to pH value, temperature and/or salinity.Therefore, should be appreciated that the present invention does not need two the HA derivants that can distinguish mutually, but can use the one matter that comprises a plurality of different functional groups that can be crosslinked.

Multiple different HA derivant can be used among the present invention.Suitably the key character of derivant is, first and second functional group must be with capacity reaction and enough rapid, thereby forms hydrogel in a period of time scope of permission after solution is in contact with one another.In certain embodiments of the invention, hydrogel is that solution is in contact with one another back (for example dispensing back) between between 1-3 second and 5 minutes, between between 1-3 second and 3 minutes, between between 1-3 second and 60 seconds, between forming between 1-3 second and 30 seconds or in the time between 1-3 second and 15 seconds.Usually with solution throw with body in mix before the position, or with solution mix with its to the throwing at a position in the body with carry out simultaneously.For instance, can use multitube injection device (for example, the multitube syringe) to throw and solution, wherein each solution is being thrown and preceding all being contained in independent accepter or the pipe.Solution can and/or be thrown in throwing and process and be in contact with one another afterwards.Preferred derivant is under physiological condition, and (for example) is crosslinked in the aqueous environments of the pH value between 6.0 and 8.0.

Suitably another key character of HA derivant is that gained hydrogel itself should obviously not impel adhesion development, inflammation or other undesirable effect.The multiple suitable HA derivant of organizing binding agent or binding agent has been proposed.Compare with HA derivant of the present invention, described derivant can make adhesion problem worse but not with its solution.Proposed the HA derivant of multiple support as tissue regeneration, it can be the cell growth and infiltration provides suitable environment.Yet for the purpose that suppresses adhesion, the environment that strengthens cellular infiltration and/or propagation is undesirable.The present invention identifies that to be suitable for original position rapidly crosslinked and form the polysaccharide derivates of the hydrogel that suppresses adhesion, for example HA derivant.

Can use the method for multiple crosslinkable polysaccharide derivates and the described derivant of formation.In certain embodiments of the invention, polysaccharide derivates is cross-linked with each other and need not independent cross-linking agent, and for example first and second derivants comprise the functional group of the formation covalent bond that reacts to each other.In certain embodiments of the invention, polysaccharide derivates reacts to each other and produces nontoxic, bio-compatible product, for example water.In certain embodiments of the invention, two kinds of polysaccharide derivates are not all by using cross-linking agent to modify.In certain embodiments of the invention, polysaccharide derivates is crosslinked under the situation that does not need light.

In aspect the present invention is one or more, can use multiple HA derivant.In certain embodiments of the invention, by locating to form active ester at the carboxyl (COOH) of glucuronic acid part and carrying out subsequently at one end to contain nucleophilic group and contain through the replacement of the side chain of protection functional group and introduce functional group at the other end, for example, described in following document: United States Patent (USP) the 6th, 630, No. 457, it is incorporated herein by reference; With Bu Bite P. (Bulpitt, P.) and Ai Shiliman D. (Aeschlimann, D.), (1999) biomedical material research magazine (J.Biomed.Mater.Res.), 47,152-169, it is incorporated herein by reference.Described method can for example be used to produce the HA derivant that comprises amine or aldehyde functional group.Can use I-hydroxybenzotriazole (HOBT) or N-hydroxy thiosuccinimide and use carbodiimides (such as EDC) to form the active ester of HA for coupling subsequently.Can comprise hydrazine and reactive amines (such as ethylenediamine) with the amine of the ester reaction that forms with HOBT immediately, it has pKa value in proper range, and consequently it is not protonated under acid ph value (for example, about 5.5 to 7.0).The use of N-hydroxy thiosuccinimide makes can carry out coupling under about pH value of 7.0 to 8.5, thereby allows to use primary amine.These methods can be used for carrying out following reaction:

HA-COOH+H ₂N-R—>HA-CO-NH-R (I)，

HA-COOH+R′-NH-R—>HA-CO-NR′—R (II)。

R and R ' can be any in the multiple part, and such as hydrogen, alkyl, aryl, alkylaryl or aryl alkyl, it can contain the hetero atom such as oxygen, nitrogen and sulfur.But side chain branch or not branched and can be saturatedly maybe can contain one or more Multiple Bonds (multiple bond).The carbon atom of side chain can be continuous, or can separate through one or more functional groups, such as oxygen atom, ketone group, amino, oxygen base carbonyl etc.Side chain can replace through aryl moiety or halogen atom, or can be in whole or in part by forming such as ring structures such as cyclopenta, cyclohexyl, suberyl.Side chain can have functional end-group for crosslinked, described functional group such as aldehyde, amine, aromatic yl azide, hydrazides, maleimide, sulfydryl, ester, carboxylate, imide ester, hydroxyl etc.Therefore, can use the method to produce to comprise in the multiple different functional groups any HA derivant.

Be applicable to that the carbodiimides that forms the HA derivant can provide as follows: R-N=C=N-R ', (III).

R and R ' can be any in (for example) multiple part as indicated above.For instance, R and R ' can be independently selected from the group that is made up of following each group: hydrogen, have the alkyl of 1-25 carbon atom and comprise and be substituted alkyl, alkoxyl, aryloxy group, aryloxy alkyl etc.For instance, R and R ' can be alkyl, cycloalkyl, aryl or it is substituted form.In certain embodiments of the invention, use the temperature in about 20-80 ℃ scope for example to dissolve in carbodiimides in the aqueous medium down to small part.Exemplary carbodiimides comprises EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimides); ETC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimides methiodide); N, N '-dicyclohexyl carbodiimide; N-pi-allyl-N '-(beta-hydroxyethyl) carbodiimides; N-(alpha-alpha-dimethyl aminopropyl)-N '-tert-butyl group carbodiimides; N-(alpha-alpha-dimethyl aminopropyl)-N '-(β-bromine pi-allyl) carbodiimides; 1-(3-dimethylaminopropyl)-3-(6-benzoyl-amido hexyl) carbodiimides and N-cyclohexyl-N '-β-(4-methyl morpholine) ethyl carbodiimides (CMC) etc.

In certain embodiments of the invention, side chain comprises two hydrazides.The HA derivant that can formation as indicated above comprises two hydrazides.Also can use other method that is used to form the HA derivant that comprises two hydrazides functional groups.For instance, United States Patent (USP) the 5th, 616, No. 568 a kind of two hydrazides that utilize of (it is incorporated herein by reference) teaching make HA functionalized to form the method for two hydrazide groups-HA.At first two hydrazides are added among the HA, add carbodiimides subsequently.Described reaction can be carried out under the pH value of about 4-0 and can be as shown in hereinafter:

HA---COOH+H ₂N---NH---CO---A---CO---NH---NH ₂(two hydrazides) (IV)

(carbodiimides)

HA---CO---NH---NH---CO---A---CO---NH---NH ₂(two hydrazide groups-HA) (V).

Multiple two hydrazides can be used for above-mentioned reaction, wherein A represents the variable interval base.For instance, A can be alkyl, assorted alkyl, is substituted alkyl, is substituted assorted alkyl etc., and wherein these terms are as United States Patent (USP) the 5th, 616, use with illustration described in No. 568.In certain embodiments of the invention, two hydrazides have following formula:

NH ₂NHCO(CH ₂) _n，CONHNH ₂ (VI)，

N=1 to 18 wherein.

For instance, two useful hydrazides comprise succinic acid (succinic acid) (n=2), adipic acid (adipic acid) (n=4), suberic acid (suberic acid) (n=6), oxalic acid (ethanedioic acid) (n=0), maleic acid (malonic acid) (n=1), glue acid (1,3-propanedicarboxylic acid) (n=3), jambulol (1,5-pentanedicarboxylic acid .) (n=5), azelaic acid (Azelaic Acid) (n=7), sebacic acid (decanedioic acid) (n=8), dodecanedioic acid (n=10), brassylic acid (tridecandioic acid) two hydrazides (n=11) etc., n=20 at the most.Suitable carbodiimides is in above discussing.

Be showed in hereinafter and will be referred to herein as HA-ADH through the structure of the functionalized suitable HA derivant of two hydrazides (adipic acid two hydrazides).The position (that is the carboxyl place of glucuronic acid part) of HA is modified in the arrow indication.

In certain embodiments of the invention, be not by modifying the carboxyl of glucuronic acid part, forming the HA derivant that comprises aldehyde functional group but form aldehyde radical by oxidation glucuronic acid hydroxyl partly.Can use multiple oxidant, for example periodate, for example Potassium metaperiodate. (KIO ₄), sodium metaperiodate (NaIO ₄) or HIO ₄Other oxidant comprises permanganate, chromate or two chromate.

This structure through oxidation HA derivant is showed in hereinafter and will be referred to herein as HA-CHO.The site that the arrow indication is modified.

Should be appreciated that in any in above-mentioned modification protocols, only a part of sugar moieties is modified among the HA.The scope of modifying can change.For instance, in certain embodiments of the invention, between 5% and 99-100% between associated sugars part (for example, the glucuronic acid part under the situation in above-mentioned modification) modified.In certain embodiments of the invention, the associated sugars part between 10% and 75% is modified.Can be by the scope of several different methods control modification.For instance, temperature, pH value and the time that reaction is carried out can become with reagent (for example, carbodiimides, amide, two hydrazides etc.) concentration.For realizing higher degree of modification, can use excessive dressing agent, for example two hydrazides and/or carbodiimides.For instance, in one embodiment, two hydrazides that 10-100 is doubly excessive add in the solution that comprises HA, and/or subsequently that 2-100 is doubly excessive carbodiimides reagent adds in the reactant mixture.In certain embodiments of the invention, select the value of these parameters so that realize higher relatively degree of modification, for example make between 50% and 99-100% between associated sugars partly modify.For instance, the associated sugars between 50% and 80% is partly modified.Yet, thereby keep enough low degree of modification to make solution keep suitable fluid state, rather than the thickness so that be not easy to handle and inject of becoming too.In certain embodiments of the invention, modified between the associated sugars part between 10% and 30% or between 30% and 50%.

Can react to each other crosslinked as indicated above by making the derivant that comprises different functional groups through functionalized HA derivant.For instance, (i) comprise aldehyde a HA derivant can with the 2nd HA derivatives reaction that comprises amine; (ii) comprise active ester (such as the NHS ester) a HA derivant can with the 2nd HA derivatives reaction that comprises amine; (iii) comprise maleimide a HA derivant can with the 2nd HA derivatives reaction that comprises sulfydryl; (iv) comprise hydrazides a HA derivant can with the 2nd HA derivatives reaction that comprises aldehyde etc.In certain embodiments of the invention, the HA derivant is linked together by other key except that disulfide bond.In an embodiment who merits attention especially, first solution contains the HA derivant that comprises through the functionalized glucuronic acid part of two hydrazides, and second solution contains the HA derivant that forms aldehyde radical at the glucuronic acid hydroxyl place partly of HA through oxidation.As schematic presentation hereinafter, first and second HA derivant is cross-linked to form hydrazone compound, wherein R ₁And R ₂The expression HA (or in certain embodiments of the invention the expression another polysaccharide, such as cellulose derivative) part.

Inventor of the present invention confirms, and hydrogel that original position form crosslinked by some HA derivant even all will develop the ability (referring to example) that also represents significant inhibition adhesion under the condition of adhesion at 100% individuality under the situation that does not have hydrogel.Surprisingly, some hydrogel (wherein natural HA backbone structure changes through the glucuronic acid epoxidation) that comprises the HA derivant can suppress adhesion and manifest low cytotoxicity and good bio-compatible, even but expectability destroys natural HA backbone structure may make compositions inspire inflammation and/or tool immunogenicity in vivo.

One aspect of the present invention is following discovery: can be for example concentration by changing polysaccharide derivates in the solution and/or molecular weight control some important parameter by the formed hydrogel of cross-linked polysaccharides derivant (such as the HA derivant), such as gelling time and degradation rate.One aspect of the present invention relates to following discovery: the high concentration of HA derivant can molecular weight and/or the degree of modification of modified HA realize by suitable selection in the solution.Specifically, find,, can increase by modifying the accessible concentration of the HA derivant that HA produced by reducing the molecular weight of not modified HA derivant.Therefore, the dissolubility of the HA derivant that is produced by the HA that modifies lower molecular weight is higher than by the dissolubility that higher molecular weight HA is carried out the derivant that identical modification produced, can reach higher concentration thus, and can not make too thickness and be not easy to handle and injection of compositions.The invention provides a kind of compositions that comprises the HA derivant of solution form, the concentration of wherein said HA derivant is greater than 5mg/ml, for example up to 150mg/ml.In certain embodiments, the concentration of HA derivant is higher than 10mg/ml.In other embodiments, the concentration of HA derivant is higher than 15mg/ml or is higher than 25mg/ml.Described concentration can be at least 26mg/ml, 30mg/ml, 40mg/ml, 50mg/ml etc. at least at least at least.For purposes of the present invention, the concentration that is higher than the HA derivant of 25mg/ml is called " high concentration ".The present invention provides a kind of compositions that comprises the HA derivant of solution form in addition, and the concentration of wherein said HA derivant is between 50mg/ml and 100mg/ml, for example between 50mg/ml and 75mg/ml.Described solution preferably has enough low viscosity, so it is easy to handle, and for example is easy to injection thus.The HA derivant can be in the HA derivant mentioned above any.For instance, the HA derivant can be HA-ADH or HA-CHO.In certain embodiments of the invention, the HA derivant is dissolved in the aqueous medium.The present invention provides the method for making hydrogel in addition, and it comprises is in contact with one another first solution that comprises a HA derivant and second solution that comprises the 2nd HA derivant, and at least a in the wherein said solution have a higher H A derivatives concentration.Hydrogel can be used for any purpose as herein described, and comprises bioactivator and/or granule according to circumstances.

The present invention provides in addition by first solution that comprises a HA derivant being contacted with second solution that comprises the 2nd HA derivant and make the formed hydrogel of described derivant crosslinked (in-situ cross-linked according to circumstances), in wherein said first solution in the concentration of a HA derivant, described second solution concentration of the 2nd HA derivant or the two all be higher than 5mg/ml, be higher than 10mg/ml, be higher than 15mg/ml or be higher than 25mg/ml.In certain embodiments of the invention, the concentration of the 2nd HA derivant or the two is between 50mg/ml and 100mg/ml, for example between 50mg/ml and 75mg/ml in the concentration of a HA derivant, second solution in first solution.Described in example, be surprisingly found out that the hydrogel that is formed by the solution that comprises at least a high concentration HA derivant represents " gelling time " (meaning refers to that the HA derivant is cross-linked to form the required time of gel after being in contact with one another) that (i) shortens; The (ii) degradation rate of Jiang Diing and the half-life (meaning refers to that the hydrogel weight in wet base reduced for 50% required time) of increase thus; Or the gelling time that (iii) shortens and the degradation rate of reduction.For instance, when the concentration of HA-ADH and HA-CHO solution respectively when 20mg/ml is increased to 75mg/ml and 30mg/ml, the half-life of the hydrogel that is formed by crosslinked first and second HA derivant (HA-ADH and HA-CHO) was increased to 11 days from 5 days, and when described concentration increased to 75mg/ml and 60mg/ml, the described half-life was increased to 22.5 days or 51 days (referring to example).Therefore, the invention provides the hydrogel that forms by crosslinked HA derivant, wherein said hydrogel have surpass 10 days, half-life between about 10 days and about 50 days for example.

Should be appreciated that, can use multiple distinct methods to measure gelling time and degradation rate.Suitable method is provided in herein.Yet, should be appreciated that, although it is the employed ad hoc approach of this paper is applicable to the effect that quantize to change concentration and/or molecular weight, irrelevant with the ad hoc approach that is used to assess these parameters about the general discovery of control gelling time and degradation rate.Although should also be clear that and use specific HA derivant explanation these aspects of the present invention, be not to limit the invention to these specific derivatives by any way.The present invention provides the compositions that comprises such as other polysaccharide derivates such as cellulose and glucosans in addition, and the concentration of wherein said polysaccharide derivates is described about the HA derivant as mentioned.Example 12 and 13 vide infra.Described derivant can include, but is not limited to functionalized cellulose or functionalized cellulose derivative.Spendable other derivant includes, but is not limited to functionalized glucosan and functionalized glucan derivative.Also can use other natural and synthetic polysaccharide and derivant thereof to prepare compositions of the present invention.Therefore, the present invention includes with this paper described similar and as be applicable to the compositions and the method for other hydrogel precursor (other polysaccharide derivates that for example comprises derivant described herein, for example cellulose derivative and glucan derivative) about the HA derivant.

The remarkable ability that is suppressed adhesion by the formed hydrogel of in-situ cross-linked polysaccharide derivates is not limited to the HA derivant.Inventor of the present invention confirms that the hydrogel that is formed in position by crosslinked some HA derivant and some cellulose derivative has similar adhesion inhibitory action (example 11 and 12).For instance, also suppress adhesion and represent good bio-compatible (example 11 and 12) by any formed hydrogel in crosslinked HA derivant and the multiple cellulose derivative (wherein the structure of native cellulose main chain changes by the oxosugar ring) at intraperitoneal.

The present invention provides a kind of method that suppresses adhesion in addition, and it comprises (i) with position in throwing of HA derivant and the individual body; (ii) with described position in cellulose derivative throwing and the individual body, wherein said HA derivant and cellulose derivative are cross-linked to form hydrogel after being in contact with one another, and wherein said hydrogel suppresses adhesion.HA derivant and cellulose derivative can be dissolved in the independent solution of throwing in fact simultaneously and individuality.

Cellulose is β-interconnective linear polymer of D-glucopyranose units (card Mead (Kamide), cellulose and cellulose derivative: characterization of molecules and its application (Cellulose And Cellulose Derivatives:MolecularCharacterization and Its Applications), like to think only your (Elsevier), 2005).Term " cellulose derivative " is meant the cellulose by its native form chemical modification.In certain embodiments of the invention, polysaccharide is a cellulose derivative, such as methylcellulose (MC), carboxymethyl cellulose (CMC), hydroxy methocel (HMC), hydroxypropyl cellulose (HPC), hydroxyethyl-cellulose (HEC) or hydroxypropyl emthylcellulose (HPMC), wherein one or more OH groups are replaced through OR, and wherein R represents any in the multiple part.Should be appreciated that some but non-all modified (referring to Figure 12) are arranged in any glucose moiety in the cellulose derivative mentioned above.In general, can use and above prepare the crosslinkable cellulose derivative about the described similar mode of HA derivant.For instance, cellulose or cellulose derivative are modified to form functionalized cellulose derivative, it comprise can with suitable covalently bound functional group of second functional group (for example, the functional group on the HA derivant), such as amine, amide, aldehyde, ester, hydroxyl or hydrazides.For instance,, can form aldehyde radicals by some hydroxyls in the oxidizing glucose part and form the cellulose derivative that comprises aldehyde functional group about as described in the HA as this paper.Be called MC-CHO, CMC-CHO and HPMC-CHO (referring to Figure 12) in this article by the formed MC of hydroxyl formation aldehyde radical, the CMC of oxidizing glucose part and the derivant of HPMC.Similar name can be used for other cellulose derivative.

The invention provides a kind of compositions that comprises the cellulose derivative of solution form, the concentration of wherein said cellulose derivative is greater than 5mg/ml, for example up to 150mg/ml.In certain embodiments, the concentration of cellulose derivative is higher than 10mg/ml.In other embodiments, the concentration of cellulose derivative is higher than 15mg/ml, is higher than 20mg/ml or is higher than 25mg/ml.For purposes of the present invention, the concentration that is higher than the cellulose derivative of 25mg/ml is called " high concentration ".Described solution preferably has enough low viscosity, so it is easy to handle, and for example is easy to injection thus.Cellulose derivative can be any in the cellulose derivative described herein.For instance, cellulose derivative can be MC-CHO, CMC-CHO and HPMC-CHO.

In certain embodiments of the invention, use HA derivant that comprises two hydrazides functional groups and the cellulose derivative that comprises aldehyde radical to form hydrogel.For instance, first solution can comprise HA-ADH and second solution can comprise MC-CHO, CMC-CHO or HPMC-CHO.HA and cellulose derivative are cross-linked to form following hydrazone compound: HA-CMC, HA-HPMC and HA-MC.

In addition, inventor of the present invention shows that also the hydrogel that is formed in position by crosslinked some HA derivant and some glucan derivative has similar adhesion inhibitory action (example 13).For instance, the formed hydrogel of glucan derivative that has been changed by the structure of crosslinked HA, cellulose or other glucan derivative and natural glucosan main chain also suppresses adhesion and represents good bio-compatible (example 13) at intraperitoneal.

The present invention provides a kind of method that suppresses adhesion in addition, and it comprises (i) with position in HA or cellulose derivative throwing and the individual body; (ii) with described position in glucan derivative throwing and the individual body, wherein said HA or cellulose derivative and glucan derivative are cross-linked to form hydrogel after being in contact with one another, and wherein said hydrogel suppresses adhesion.HA or cellulose derivative and glucan derivative can be dissolved in throw in fact simultaneously with individual independent solution in.

Glucosan is a kind of branch polysaccharide of complexity.Glucosan comprises many glucose moieties that are joined together to form straight chain via α 1 → 6 glucosides binding.Branch is begun by α 1 → 3 binding usually, but it also can be from α 1 → 2 or α 1 → 4 binding.The structure of glucosan linear fraction is illustrated among Figure 22.Term " glucan derivative " is meant the glucosan from its native form chemical modification.In certain embodiments of the invention, polysaccharide is a glucan derivative, and wherein one or more OH groups are replaced through OR, and wherein R represents in the multiple part any.In other embodiments, glucan derivative is the aldehyde derivatives that contains of glucosan periodate processing.Should be appreciated that some but non-all modified (referring to Figure 22) are arranged in any glucose moiety in the glucan derivative mentioned above.In general, can use and above prepare the crosslinkable glucan derivative about HA or the described similar mode of cellulose derivative.For instance, glucosan or glucan derivative are modified to form functionalized cellulose derivative, it comprise can with suitable covalently bound functional group of second functional group (for example, the functional group on the HA derivant), such as amine, amide, aldehyde, ester, hydroxyl or hydrazides.For instance, about as described in HA and the cellulose, can form the glucan derivative that comprises aldehyde functional group to form aldehyde radical as this paper by some hydroxyls in the oxidizing glucose part.Hydroxyl by the oxidizing glucose part is referred to herein as DX-CHO (referring to Figure 22) to form the formed glucan derivative of aldehyde radical.Derivant through the Sensor Chip CM 5 (CMDX) of hydrazides base group modification is called CMDX-ADH (referring to Figure 22).

The invention provides a kind of compositions that comprises the glucan derivative of solution form, the concentration of wherein said glucan derivative is greater than 5mg/ml, for example up to 150mg/ml.In certain embodiments, the concentration of glucan derivative is higher than 10mg/ml.In other embodiments, the concentration of glucan derivative is higher than 15mg/ml, is higher than 20mg/ml or is higher than 25mg/ml.For purposes of the present invention, the concentration that is higher than the glucan derivative of 25mg/ml is called " high concentration ".Described solution preferably has enough low viscosity, so it is easy to handle, and for example is easy to injection thus.Glucan derivative can be any in the glucan derivative described herein.For instance, glucan derivative can be DX-CHO or CMDX-ADH.

In certain embodiments of the invention, use HA or cellulose derivative that comprises two hydrazides functional groups and the glucan derivative that comprises aldehyde radical.For instance, first solution can comprise HA-ADH and second solution can comprise DX-CHO.HA and glucan derivative are cross-linked to form HA-DX.In certain embodiments of the invention, use cellulose derivative that comprises two hydrazides functional groups and the glucan derivative that comprises aldehyde radical.For instance, first solution can comprise CMC-ADH and second solution can comprise DX-CHO.In addition for instance, first solution can comprise CMDX-ADH and second solution can comprise CMC-CHO.Cellulose and glucan derivative are cross-linked to form CMC-DX.In certain embodiments of the invention, use the glucan derivative that comprises two hydrazides functional groups and another glucan derivative that comprises aldehyde radical.For instance, first solution can comprise CMDX-ADH and second solution can comprise DX-CHO.Cellulose and glucan derivative are cross-linked to form CMDX-DX.

Should be appreciated that in any of the foregoing description, only a part of available functional groups is crosslinked on first and second polysaccharide derivates.Can control crosslink density by for example suitably selecting the molecular weight of polysaccharide derivates.Exemplary crosslink density is about 1 * 10 ⁶To about 1 * 10 ⁸Mol/cm ³Scope in.In certain embodiments, crosslink density is in 3-50 * 10 ⁷Mol/cm ³Scope in.

In certain embodiments of the invention, polysaccharide derivates throw with before form the hydrogel micron particle but not with the solution form throw with.Hydrogel particle can be suspended in the liquid medium and throw and position to the body.

In certain embodiments of the invention, at least aly be suitable for the polysaccharide derivates that in-situ cross-linked formation suppresses adhesion and/or be used for the compositions of other purpose described herein and comprise a part that contains non-polysaccharide polymer, for example, described polysaccharide derivates comprises the polysaccharide or derivatives thereof covalently bound with one or more non-polysaccharide polymers.The non-polysaccharide meaning refers to that described polymer contains in weight, quantity or the two less than 1% sugar monomer, and for example described polymer is gone up substantially and do not contained sugar.In certain embodiments of the invention, non-polysaccharide partly comprise by weight between 1% and 10%-90% between polymer, and/or between 1% and 10%-90% between monomer be non-sugar monomer.Connection can betide arbitrary position in the polysaccharide chain, for example places of the sugar moieties of the positioned internal of arbitrary end of described chain or generation linearity or apparatus derivatorius.Non-polysaccharide polymer can be in the multiple polymers any, for example when can be as any non-polysaccharide polymer of hydrogel precursor when covalently bound with polysaccharide derivates.Usually, described polymer is a hydrophilic polymer, and promptly it is in glassware for drinking water affinity and its water soluble.Suitable non-polysaccharide polymer include, but is not limited to polyethers (such as Polyethylene Glycol (PEG) or polypropylene glycol (PPG)), polyethylene glycol oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polypeptide (such as, gelatin, poly-(1-glutamic acid), polylysine (PLL)) and these materials in any derivant or comprise in these materials any concatenator, admixture or complex.For instance, in one embodiment, at least a polysaccharide derivates is the HA derivant of covalently bound PEG or PEG derivant, and wherein HA part or peg moiety comprise first functional group.Second polysaccharide derivates can be any polysaccharide derivates that comprises with the functional group of first functional group reactions.For instance, in one embodiment, the HA derivant is PEG-HA-ADH, and second polysaccharide derivates comprises the functional group of reacting with two hydrazides, for example HA-CHO.The multiple PEG derivant that comprises the suitable functional group that promotes the formation covalent bond is on sale in the market.For example referring to, the advanced Pegylation 2005-2006 catalogue (Nektar Advanced Pegylation2005-2006 Product Catalog) of Nei Keta, Nei Keta treats company (Nektar Therapeutics), California San Carlos (SanCarlos, CA), it describes multiple described chemical compound.

In certain embodiments of the invention, use the first and second crosslinkable hydrogel precursors, wherein a kind of hydrogel precursor comprises such as polysaccharide derivates such as HA, cellulose or glucan derivatives or by forming such as polysaccharide derivates such as HA, cellulose or glucan derivatives, and another hydrogel precursor comprises non-polysaccharide polymer or forms (that is being sugar less than 1 weight % polymer or less than 1% monomer) by non-polysaccharide polymer.Any derivant be can include, but is not limited in polyethers (such as Polyethylene Glycol (PEG) or polypropylene glycol (PPG)), polyethylene glycol oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polypeptide (such as, gelatin, chitosan or poly-(1-glutamic acid)) and these materials with the exemplary non-polysaccharide polymer that polysaccharide derivates is cross-linked to form hydrogel or any concatenator, admixture or complex comprised in these materials.In exemplary embodiments, the first crosslinkable hydrogel precursor is that the HA-ADH and the second crosslinkable hydrogel precursor are the PEG derivant that comprises aldehyde radical.

Although described polysaccharide derivates in this article in detail to illustrate the present invention, in other embodiment of the present invention, thereby hydrogel is to produce the hydrogel that suppresses adhesion by in-situ cross-linked two kinds of non-polysaccharide polymers to form.Each non-polysaccharide polymer comprises functional group, the wherein said functional group formation covalent bond that can react to each other.Suitable functional group is for above about the described functional group of cross-linked polysaccharides derivant.Exemplary non-polysaccharide polymer comprises mentioned above contain or modified to contain the polymer for crosslinked suitable functional group.

II. the compositions that comprises crosslinkable polysaccharide derivates and bioactivator

The present invention provides compositions as indicated above in addition, thereby wherein hydrogel is to produce the hydrogel that comprises bioactivator by the cross-linked hydrogel precursor to form.For instance, contain first polysaccharide derivates solution, contain the solution of second polysaccharide derivates or the two comprises one or more bioactivators.Therefore, contain one or more bioactivators by the formed hydrogel of crosslinked first and second polysaccharide derivates.In some embodiments of the invention, each self-contained different bioactivator of described solution.The hydrogel of Xing Chenging contains two or more bioactivator thus.Perhaps, can will contain the solution of first and second hydrogel precursors and one or more contain one or more bioactivators separately other solution combination.In another embodiment, bioactivator is added to immediately by in the formed compositions of first and second solution of combination.

Polysaccharide derivates can for example be HA derivant, cellulose derivative, glucan derivative etc.In certain embodiments of the invention, first and second polysaccharide derivates is the HA derivant.In certain embodiments of the invention, first and second polysaccharide derivates is a cellulose derivative.In certain embodiments of the invention, first and second polysaccharide derivates is a glucan derivative.In certain embodiments of the invention, first polysaccharide derivates is that the HA derivant and second polysaccharide derivates are cellulose derivative.In certain embodiments of the invention, first polysaccharide derivates is that the HA derivant and second polysaccharide derivates are glucan derivative.In certain embodiments of the invention, first polysaccharide derivates is that the cellulose derivative and second polysaccharide derivates are glucan derivative.In certain embodiments of the invention, hydrogel is to be formed by polysaccharide derivates more than three kinds or three kinds.Any combination crosslinkable of HA derivant, cellulose derivative, glucan derivative, other polysaccharide derivates or other polymer forms hydrogel of the present invention.

By comprising any bioactivator in the formed hydrogel of cross-linked polysaccharides derivant.Therefore the invention provides the hydrogel that contains in the multiple bioactivator any.The present invention provides a kind of solution in addition, its contain such as in polysaccharide derivates such as HA, cellulose or glucan derivative and the multiple bioactivator any.

The present invention provides a kind of preparation to comprise the method for the hydrogel of bioactivator in addition, described method comprises following steps: first solution that comprises first polysaccharide derivates and second solution that comprises second polysaccharide derivates are in contact with one another, at least a bioactivator that comprises in the wherein said solution, and wherein said first and second polysaccharide derivates comprise the functional group that is cross-linked with each other.The present invention provides a kind of preparation to comprise the method for the hydrogel of bioactivator in addition, described method comprises following steps: first solution that comprises first polysaccharide derivates, second solution and the bioactivator that comprises second polysaccharide derivates are in contact with one another, and wherein said first and second polysaccharide derivates comprise the functional group that is cross-linked with each other.Bioactivator is the solution form according to circumstances.In certain embodiments of the invention, by being thrown with individuality, described solution prepares hydrogel, and wherein said hydrogel precursor is cross-linked with each other and forms the hydrogel of encapsulation bioactivator.

In certain embodiments of the invention, bioactivator is a therapeutic agent.Useful bioactivator kind comprises anti-infective, antiinflammatory, antiproliferative (for example cytotoxic agent), antitumor agent (promptly, suppress or prevent the medicament of malignant cell propagation and/or inhibition or prophylaxis of tumours growth or diffusion), analgesic (being lenitive medicament), antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant (promptly, suppress or prevention thrombosis but do not dissolve the medicament of existing thrombosis, be also referred to as antithrombotic agent), protein hydrolytic reagent or strengthen proteoclastic medicament (some of them also for antithrombotic agent), free radical scavenger, antioxidant, (for example, the anti-TGF-P agent of fibroid repair inhibitors, Beracilline, pentoxifylline (pentoxifyllin)) etc.Other useful therapeutic agent kind comprises hypnotic, tranquilizer, tranquilizer, anticonvulsant, muscle relaxant, spasmolytic, sympathomimetic, painstaking effort tubing medicine (for example, anti-arrhythmic, positive inotropic medicament) etc.In certain embodiments of the invention, bioactivator is not an anesthetis.In certain embodiments of the invention, bioactivator is not an antiproliferative.In certain embodiments, bioactivator is an antiinflammatory.In certain embodiments, bioactivator is the non-steroidal antiinflammatory.In other embodiments, bioactivator is steroidal antiinflammatory (for example glucocorticoid, a corticosteroid).

The a certain amount of bioactive substance of preferred interpolation, described amount are that medicine and pharmacology are effectively measured when the solution that will comprise polysaccharide derivates and/or non-polysaccharide polymer is in right amount thrown and is individual, and it can change.Described medicament can suppress maybe can not suppress adhesion.Should be appreciated that some medicaments not only belong to a class and can have multiple mechanism of action.Do not plan the tabulation of particular agent is restricted to the member of a classification.

The classification that is used for the present invention's exemplary anti-infective is a quinolinones; beta-lactam (for example; penicillin (penicillin) or cephalosporin (cephalosporin)); carbapenem (carbapenem); aminoglycoside; macrolide; lincosamide (lincosamide); the ketone lactone; tetracycline; glycylcycline; lincomycin oxazolidone; amide alcohol (amphenicol); ansamycin (ansamycin); polymyxin (polymyxin); ammonia first ring element (aminomethlycycline); lincosamide; streptogramin (streptogramin); 2, the 4-di-amino-pyrimidine; nitrofuran; sulfonamide; sulfone; rifabutin (rifabutin); dapsone (dapsone); peptide; glycopeptide and nucleoside analog.In certain embodiments of the invention, the medicament of anti-infective for having broad spectrum of activity at the various bacteria species.In certain embodiments, described medicament is effective to one or more gram-positive bacteriums (gram positive bacteria) species, one or more gram negative bacteria species or the two.In certain embodiments of the invention, described medicament to may the contaminated operation wound or the antibacterial or the fungus of damage effective.For instance, described medicament is effective to the antibacterial or the fungus that see usually on the skin or in the gastrointestinal tract.

The example of spendable specific anti-infective includes, but is not limited to erythromycin (erythromycin), nafcillin (nafcillin), cefazolin (cefazolin), imipenum (imipenem), aztreonam (aztreonam), gentamycin (gentamicin), Sulfamethoxazole (sulfamethoxazole), vancomycin (vancomycin), ciprofloxacin (ciprofloxacin), trimethoprim (trimethoprim), rifampicin (rifampin), metronidazole (metronidazole), clindamycin (clindamycin), teicoplanin (teicoplanin), mupirocin (mupirocin), azithromycin (azithromycin), clarithromycin (clarithromycin), ofloxacin (ofloxacin), lomefloxacin (lomefloxacin), norfloxacin (norfloxacin), nalidixic acid (nalidixic acid), Sparfloxacin (sparfloxacin), pefloxacin (pefloxacin), amifloxacin (amifloxacin), Gatifloxacin (gatifloxacin), Moxifloxacin (moxifloxacin), Gemifloxacin (gemifloxacin), enoxacin (enoxacin), fleroxacin (fleroxacin), minocycline (minocycline), linezolid (linezolid), temafloxacin (temafloxacin), tosufloxacin (tosufloxacin), clinafloxacin (clinafloxacin), sulbactam (sulbactam), clavulanic acid (clavulanic acid), amphotericin B (amphotericin B), fluconazol (fluconazole), itraconazole (itraconazole), ketoconazole (ketoconazole) and nystatin (nystatin).

The exemplary kind that is used for the present invention's antiinflammatory comprises multiple non-steroidal antiinflammatory (for example, Cycloxygenase-1 inhibitor), corticosteroid, glucocorticoid and antiinflammatory antibody or polypeptide.

Exemplary antiinflammatory comprises prednisone, dexamethasone, fluorometholone (fluorometholone), prednisolone (prednisolone), methylprednisolone (methylprednisolone), clobetasol (clobetasol), halogen Beta rope (halobetasol), hydrocortisone (hydrocortisone), triamcinolone acetonide (triamcinolone), betamethasone (betamethasone), fluocinonide (fluocinolone), fluorine west a kind of apple moral (fluocinonide), loteprednol (loteprednol), medrysone (medrysone), rimexolone (rimexolone), celecoxib (celecoxib), folic acid (folic acid), diclofenac (diclofenac), diflunisal (difiunisal), Lip river, Fino sweet smell (fenoprofen), flurbiprofen (flurbiprofen), indomethacin (indomethacin), ketoprofen (ketoprofen), meclofenamic acid salt (meclofenamate), first chlorine side hydrochlorate (meclofamate), piroxicam (piroxicam), sulindac (sulindac), Diplosal (salsalate), nabumetone (nabumetone), oxaprozin (oxaprozin), Tolmetin (tolmetin), hydroxychloroquine sulfate (hydroxychloroquine sulfate), sieve husband Cox (rofecoxib), Embrel (etanercept), Yin Fulimei (infliximab), leflunomide (lefiunomide), naproxen (naproxen), oxaprozin (oxaprozin), piroxicam (piroxicam), Salicylate (salicylates), valdecoxib (valdecoxib), sulfasalazine (sulfasalazine), methylprednisolone (methylprednisolone), ibuprofen (ibuprofen), budesonide (budesonide), meloxicam (meloxicam), acetic acid Methyllprednisolone (methylprednisolone acetate), sodium aurothiomalate (gold sodium thiomalate), aspirin (aspirin), azathioprine (azathioprine), triamcinolone acetonide acetate (triamcinolone acetonide), dextropropoxyphene napsilate/acetyl aminophenol (propxyphene napsylate/apap), folate (folate), nabumetone (nabumetone), diclofenac (diclofenac), ketorolac (ketorolac), piroxicam (piroxicam), etodolac (etodolac), diclofenac sodium (diclofenac sodium), diclofenac potassium (diclofenac potassium), oxaprozin (oxaprozin), methotrexate (methotrexate), minocycline (minocycline), tacrolimus (tacrolimus) (FK-506), sirolimus (sirolimus) (rapamycin (rapamycin)) and forms of rapamycin analogs, Phenylbutazone (phenylbutazone), Diclofenac Sodium/Misoprosrol (diclofenac sodium/misoprostol), acetyl aminophenol (acetaminophen), indomethacin (indomethacin), glucosamine sulfate/chrondroitin (glucosaminesulfate/chondroitin) and ciclosporin (cyclosporine) or its analog etc.Exemplary non-steroidal antiinflammatory (NSAID) comprises celecoxib, diclofenac, diflunisal, etodolac, Salicylate, Lip river, Fino sweet smell, ibuprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, first chlorine side hydrochlorate, meclofenamic acid salt, meloxicam, naproxen, piroxicam, sulindac, Diplosal, nabumetone, aspirin, oxaprozin and Tolmetin.Exemplary steroidal antiinflammatory comprises dexamethasone, fluorometholone, prednisolone, loteprednol, medrysone, prednisone, Methyllprednisolone, cortisone, budesonide, rimexolone, clobetasol, halogen Beta rope, hydrocortisone, triamcinolone acetonide, betamethasone, fluocinonide and fluorine west a kind of apple moral.Other antiinflammatory that merits attention comprise antibody in conjunction with TNF-α (for example, Yin Fulimei,

) and as the polypeptide of soluble TNF-α receptor (for example, Embrel,

).

Other example of antiinflammatory for the anti-inflammation drugs that suppresses cytokine (cytokine suppressive anti-inflammatory drug, CSAID); Anti-TNF alpha antibodies (for example disclose No. the 20040126372nd, case referring to the U.S., it is incorporated herein by reference); CA2/ Yin Fulimei (chimeric anti-TNF alpha antibodies; Sen Tuoke company (Centocor)); IL-4 (antiinflammatory cytokine; IL-10, IL-10 and/or IL-4 agonist (for example, agonist antibody or micromolecule); The IL-1 receptor antagonist; TNF-bp/s-TNF (soluble TNF is conjugated protein); Phosphodiesterase IN type inhibitor; Thalidomide (thalidomide) and Thalidomide related drugs; Leflunomide (leflunomide); Tranexamic acid (tranexamic acid) and other plasminogen activation inhibitor; PGE1, tenidap (tenidap), anti-IL-12 antibody; Anti-IL-18 antibody; Interleukin 11; Interleukin-13; The Interleukin-17 inhibitor; Gold; Penicillamine; Chloroquine (chloroquine); Hydroxychloroquine; Chlorambucil (chlorambucil); Cyclophosphamide (cyclophosphamide); Ciclosporin; The lymph nodes of body as a whole irradiation; Antithymocyte globulin; Anti-CD 4 antibodies; The CD5-toxin; Oral administration and peptide and collagen protein; Lobenzarit Disodium (lobenzarit disodium); ICAM-1 short interfering rna (siRNA) or antisense oligonucleotide; The complementary receptor 1 of solubility.

The angiogenesis inhibitor that is used for the present invention comprises the medicament (being referred to herein as " anti-VEGF agent ") of inhibition or antagonizing vessel endothelial cell growth factor (ECGF) (VEGF) or its receptor.Useful medicament for example comprises antibody, antibody fragment and the nucleic acid with merit iso series or vegf receptor in conjunction with one or more VEGF.Described combination can for example suppress one or more VEGF with the interaction between merit iso series and its receptor.Avastin (Avastin) (Genentech company (Genentech)) is a kind ofly also (to comment in Ferrara N. (Ferrara, N.) endocrinology comment (Endocr Rev.), 25 (4): 581-611, in 2004) in conjunction with the total length humanized antibodies of VEGF.Lu Sen is a kind of combination and the humanized antibodies's fragment that suppresses VEGF A (VEGF-A) for this (Lucentis) (Genentech company).(dagger-axe moral sieve J. (Gaudreault J.) waits the people, ophthalmology's research and vision (Invest Ophthalmol.Vis.Sci.) 46,726-733 (2005) and list of references wherein).Ma Kugen (Macugen) (Pfizer (Pfizer), Anna Imtech (Eyetech)) is a kind of combination and suppresses VEGF ₁₆₅VEGF nucleic acid ligands (being also referred to as fit) (United States Patent (USP) the 6th, 051, No. 698).Fit or antibody all can be used among the present invention with these and other of merit iso series in conjunction with one or more VEGF.

Other angiogenesis inhibitor comprises various endogenous or synthetic peptides, such as his spit of fland (combstatin) of angiostatin (angiostatin), Ai Ruisitan (arresten), bank statin (canstatin), bur, Endostatin (endostatin), thrombostondin (thrombospondin) and tumor chalone (tumstatin).Other angiogenesis inhibitor molecule comprises Thalidomide and its angiogenesis inhibitor derivant, such as iMiD (Ba Misi A (Bamias A), season is the European international medical journal of Prologis (DimopoulosMA.) (Eur JIntern Med.) 14 (8) not: 459-469,2003; Robin Bartlett JB (Bartlett JB), De Ruiji K (Dredge K), Dai Leixi AG. (Dalgleish AG.) cancer is commented on (Nat Rev Cancer.) 4 (4) naturally: 314-22,2004).

The exemplary kind of antiproliferative comprises alkylating agent; sulfonic acid alkane ester; aziridine; Ethylenimine and methyl melamine (methylamelamine); chlormethine; antibiotic; antimetabolite; folacin; purine analogue; pyrimidine analogue; androgen; androgen antagonist; anti-adrenal gland; galactoside; estrogen antagonist; taxane; platinum analogs; microtubule inhibitor (for example microtubule depolymerization agent or stabilizing agent); topoisomerase enzyme inhibitor; histone deacetylase (HDAC) inhibitor; the aggregation inhibitor; proteasome inhibitor; short apoptosis agent; inhibitors of kinases; radiosiotope; animal; plant or bacteriotoxin etc.

The particular instance that belongs to the medicament of these kinds comprises alkylating agent, such as thio-tepa (thiotepa) and cyclophosphamide (cyclosphosphamide); Sulfonic acid alkane ester is such as busulfan (busulfan), an improsulfan (improsulfan) and piposulfan (piposulfan); Aziridine is such as benzo DOPA (benzodopa), carboquone (carboquone), malt yeast DOPA (meturedopa) and outstanding benefit crust (uredopa); Ethylenimine and methyl melamine comprise altretamine (altretamine), tretamine (triethylenemelamine), triethylenephosphoramide (trietylenephosphoramide), triethylene thiophosphoramide (triethiylenethiophosphoramide) and trihydroxy methyl melamine (trimethylolomelamine); Chlormethine (nitrogen mustard) is such as chlorambucil (chlorambucil), chlornaphazine (chlornaphazine), chromium phosphamide (cholophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), nitrobine hydrochloride (mechlorethamine oxidehydrochloride), melphalan (melphalan), novembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), NSC-34462 edema of the legs during pregnancy (uracil mustard); Nitrourea (nitrosurea) is such as carmustine (carmustine), NSC-178248 (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and Ranimustine (ranimnustine); Antibiotic is such as Acker Lenno mycin (aclacinomysin), D actinomycin D (actinomycin), Ossir mycin (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamycin (calicheamicin), the Carlow is than star (carabicin), carminomycin (carminomycin), carzinophillin (carzinophilin), chromomycin (chromomycinis), actinomycin D (dactinomycin), daunomycin, detorubicin (detorubicin), 6-diazo-5-oxo-L-nor-leucine, doxorubicin (doxorubicin) (amycin (Adriamycin)), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycin), mycophenolic acid (mycophenolic acid), nogalamycin (nogalamycin), Olivomycin (olivomycin), peplomycin (peplomycin), rich for sieve mycin (potfiromycin), fast mycin (puromycin), Kui is drawn mycin (quelamycin), rodorubicin (rodorubicin), rufocromomycin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), a left side is gentle than star (zorubicin); Antimetabolite is such as methotrexate and 5-fluorouracil (5-FU); Folacin is such as Di Nuotening (denopterin), methotrexate, Pteropterin (pteropterin), trimetrexate (trimetrexate); Purine analogue is such as NSC-118218 (fludarabine), Ismipur, ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue is such as ancitabine (ancitabine), azacitidine (azacitidine), 6-sulfur azoles pyrimidine, carmofur (carmofur), cytosine arabinoside (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), azauridine (floxuridine), 5-FU; Androgen is such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), mepitiostane (mepitiostane), Testolactone (testolactone); Androgen antagonist, androgen receptor antagonists for example is such as spironolactone (spironolactone), flutamide (flutamide), finasteride (finasteride); Antiadrenergic drug is such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); Folic acid supplement (folic acid replenisher) is such as folic acid; Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid (aminolevulinicacid); Amsacrine (amsacrine); Shellfish Qu Buxin (bestrabucil); Bisantrene (bisantrene); Yi Da Qu Xian (edatraxate); De Fufaming (defofamine); Demecolcine (demecolcine); Diaziquone (diaziquone); Yi Funixin (elfornithine); Elliptinium acetate (elliptinium acetate); Etoglucid (etoglucid); Ganite (Fujisawa). (gallium nitrate); Hydroxyurea (hydroxyurea); Lentinan (lentinan); Luo Nidaning (lonidainine); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Pyrrole pellet not (mopidanmol) not; Buddhist nun Qu Yining ((nitraerine); Pentostatin (pentostatin); Egg amine nitrogen mustard (phenamet); Pirarubicin (pirarubicin); ZUYECAO acid (podophyllinic acid); 2-ethyl hydrazides; Procarbazine (procarbazine); Razoxane (razoxane); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triaziquone (triaziquone); 2,2 ', 2 ＂-RA3; Urethane (urethan); Vindesine (vindesine); Dacarbazine (dacarbazine); Mannomustine (mannomustine); Dibromo mannitol (mitobronitol); Mitolactol (mitolactol); Pipobroman (pipobroman); Melon Xi Tuoxin (gacytosine); Galactoside (" Ara-C "); Thiophene is for group; Taxane, for example paclitaxel (paclitaxel) and docetaxel (doxetaxel); Chlorambucil; Gemcitabine (gemcitabine); The 6-thioguanine; Purinethol; Methotrexate; Platinum analogs is such as cisplatin (cisplatin) and carboplatin (carboplatin); Platinum; Etoposide (VP-16); Ifosfamide (ifosfamide); Ametycin; Mitoxantrone (mitoxantrone); Vincristine (vincristine); Vincaleucoblastine (vinblastine); Vinorelbine (vinorelbine); Nvelbine (navelbine); Nuo Fantelong (novantrone); Teniposide (teniposide); Daunomycin; Aminopterin-induced syndrome (aminopterin); Cut tumor and reach (xeloda); Ibandronate (ibandronate); CPT-11; Topoisomerase enzyme inhibitor RFS 2000; Diphtheria toxin, diphtherotoxin (diphtheria toxin); Ricin (ricin); Pseudomonal toxin A (pseudomonas toxin A); Conotoxin (conotoxin); Er Fujiajiniaoansuan (difluoromethylornithine (DMFO)); Tretinoin; Ai Sibo mycin (esperamicin); Ka Xitabin (capecitabine); HER-2 antibody (

); Inhibitors of kinases is such as lattice

And pharmaceutically acceptable salt, acid or the derivant of above-mentioned any material.

Be applicable to that protein hydrolytic reagent among the present invention comprises the component of tissue plasmin activator (tPA)/fibrinolysin cascade.The component of tPA/ fibrinolysin cascade comprises the plasminogen activator, such as tissue plasmin activator (tPA) and its variant, plasminogen and fibrinolysin.Plasminogen activator (PA) comes the catalysis plasminogen to be converted into the serine protease of fibrinolysin (Vasari (Vassalli) 1991) for obtain two chains that keep the disulfide bond bridging to connect by the single peptide bond of cracking (R561-V562).Fibrinolysin is a kind of effective serine protease, and its main substrate in vivo is fibrin (protein component of thrombosis).Exist under the fibrinous situation, tPA stimulates the plasminogen activation.Fibrinolysin has multiple substrate and direct or indirect many other protein of cracking, comprises the most of protein seen in the ECM.As used herein, " directly " meaning finger protein enzyme with interact through cracked polypeptide entity, and " indirectly " meaning finger protein enzyme not with interact through cracked polypeptide entity, but itself and another molecule (for example, another protease) interact the direct or indirect more described polypeptide of cracking of described another molecule.Fibrinolysin can also activated metal protease precursor.The metalloproteases ECM molecule of degrading again.Metalloproteases also can be used among some embodiment of the present invention.

Two kinds of PA in mammal, have been identified, tectotype PA (tPA) and urokinase type PA (uPA).The major physiological function of PA is to cause thromboembolism by the fibrinolysin that plasminogen is activated into fibrin degradation.The tPA that is used for the present invention can be from any species, but for the mankind are thrown with, preferred usually human tPA or its variant of using.In the embodiment that merits attention especially, bioactivator is tPA.Described in example 10, the hydrogel that contains tPA represents the ability of the inhibition adhesion of increase under the viscous adhesion situation of (such as can be in the adhesion that forms after the pilosity infringement incident).TPA and its useful variant (comprising the variant with improved characteristic) are described in the following patent: United States Patent (USP) the 6th, 284, No. 247, the 6th, 261, No. 837, the 5th, 869, No. 314, the 5th, 770, No. 426, the 5th, 753, No. 486, the 5th, 728, No. 566, the 5th, 728, No. 565, the 5th, 714, No. 372, the 5th, 616, No. 486, the 5th, 612, No. 029, the 5th, 587, No. 159, the 5th, 520, No. 913, the 5th, 520, No. 911, the 5th, 411, No. 871, the 5th, 385, No. 732, the 5th, 262, No. 170, the 5th, 185, No. 259, the 5th, 108, No. 901, the 4th, 766, No. 075, the 4th, 853, No. 330 and amortize in other patent of Genentech company limited.(but unrestricted) for instance, the protease domain of tPA variant can have variation with respect to naturally occurring tPA, and/or with respect to naturally occurring tPA, can have one or more amino acid whose disappearances at the N-terminal place.The tPA variant can have one or more other glycosylation sites with respect to naturally occurring tPA, and/or can have destruction will be present in the variation of the glycosylation site among the naturally occurring tPA usually in eukaryotic cell (for example mammalian cell) when expressing.The half-life that can useful characteristic includes, but is not limited to increase, the activity of increase, increase to fibrinous affinity or specificity etc.

At En Tezi gene database (Entrez Gene database) ((the National Center for Biotechnology Information of American National biotechnology information centre, NCBI)) give human tPA gene I 5327 in, and the gene bank accession number of full length amino acid, mRNA and gene order is respectively AAA98809, K03021 and NM_000930.Yet, it should be noted that described in No. the 4th, 853,330, (for example) United States Patent (USP) the preferred mature form that uses the tPA that lacks signal peptide sequence, or its variant.

Have tPA and normally secrete, and activate by the form of two chains of the generation of the specific site in the proteolytic cleavage polypeptide chain with the form of single chain protein matter as member's chymotrypsin protein enzyme family serine protease.For plasminogen, the form of strand and two chains is all had an activity, but two chain forms is active higher.Fibrinolysin produces positive feedback loop thus with the form that strand tPA activates into two chains.The strand of tPA or two chain forms or its combination all can be used among the present invention.

On sale and approved is used for throwing with human with at various condition of illness in the market for tPA and its variant.For instance, alteplase (

, Genentech company, St. Francis, south, California (South San Franciso, CA)) is recombinant human tPA.Reteplase (Reteplase) (

Bai Linge-Mannheim (Boehringer Mannheim), the gloomy Toro of Luo Shi (Roche Centoror)) be the non-glycosylated form of reorganization of human tPA, the genetic engineering modified one-tenth of wherein said molecule contains 355 in 527 aminoacid of urporotein.For how for general enzyme (Tenecteplase) (

Genentech company) be 527 amino acid whose glycoprotein derivants of human tPA, the difference of itself and natural human tPA is to have three aminoacid replacement.These replacements reduce plasma clearance, increase fibrin combination (and increasing fibrin-specific thus) and the increase resistance to plasminogen activator inhibitor-1 (PAI-1).Anistreplase (Anistreplase) (anistreplase

, SmithKline Beecham (SmithKline Beecham)) and be another commercially available human tPA.

Other plasminogen activator comprise streptokinase (

) and urokinase

The two all is commercially available.

Other Proteolytic enzyme reinforcing agent that is used for the present invention comprises the tPA activator, such as vampire (Desmodusrotundus) salivary plasminogen activator (DSPA) desmoteplase (Desmoteplase) (Pa Long company (Paion), Germany (Germany)), it derives from saliva (Li Bailatuo GT people such as (Liberatore GT), the vampire plasminogen activator (desmoteplase): unique fibrinolysin (the Vampire bat salivaryplasminogen activator (desmoteplase): apoplexy (Stroke.) in February, 2003 a unique fibrinolytic enzyme that does not promoteneurodegeneration.) that does not promote neural degraded of vampire; 34 (2): 537-43; It is incorporated herein by reference).Characterize out four kinds of distinct protease and be called vampire salivary plasminogen activator (DSPA).Total length vampire plasminogen activator (DSPAI) for the variant of further investigation and its represent with human tPA greater than 72% consensus amino acid sequence.Yet two important function differences are conspicuous.At first, DSPA exists with the single chain molecule form that can't be cracked into 2 chain forms.Secondly, as if the catalytic activity of DSPA is decided on the fibrin cofactor.Such as Rui Sipu enzyme (rescupase) ( the safe company ( Grunenthal ) of Glan ) and the urokinase fibrinolytic protein activator of fento lyase ( microplasmin ) ( a kind of pyrolysis product of plasminogen ) also can be used among each embodiment of the present invention.A Feipu enzyme ( Alfimeprase ) ( Nu Waluo company ( Nuvelo ) ) is for being used for another Proteolytic enzyme reinforcing agent of the present invention. ( fibrolase ) ( ( Agkistrodon contortrix contortrix ) ) ( CF. ( Toombs CF. ) ( 2001 ) ： ( Alfimeprase：pharmacology of a novelfibrinolytic metalloproteinase for thrombolysis. ) ( Haemostasis. ) 31 ( 3-6 ) ：141-7, ) 。 The direct cracking fibrin of these enzymes.Itself also can be used for nevin fibrinolytic enzyme among the present invention.Sbphylokinase (staphylokinase) and streptodornase (streptodornase) are also useful.

In some embodiments of the invention, throwing and fibrinolysin or miniature fibrinolysin (mini-plasmin) substitute tPA, or except that throw with tPA also throwing and fibrinolysin or miniature fibrinolysin.Also can use multiple other medicament with class plasmin activity.In general, described material can cracking typical case's fibrinolysin substrate, such as synthetic substrate S-2251 ^TM(it is the branch division color substrate that is generally used for fibrinolysin and active plasminogen for the Caro lucky Instrumentation Laboratory (Chromogenix-Instrumentation Laboratory) that rubs, Milan, ITA (Milan, Italy)).Also can use to have active other medicament of class tPA, for example it can the cracking plasminogen and to make its activation with the similar mode of tPA.

Lumbrukinase (Lumbrokinase) is for deriving from fibrinolysin or one group of enzyme of the positive earthworm of Lumbricus powder (Lumbricus rubellus).For example referring to, gene (the PI239 of relevant coding Lumbrukinase, gene bank registration number AF433650 number) Ke Long report (Pueraria lobota people such as (Ge), (2005) finish expression (Cloning of thrombolytic enzyme (lumbrokinase) fromearthworm and its expression in the yeast Pichia pastoris.) protein expression and purification (Protein ExprPurif.) in July, 2005 in the red saccharomyces pastorianus from the clone of the thrombolytic enzyme (Lumbrukinase) of Lumbricus and its at yeast; 42 (1): 20-8, it is incorporated herein by reference).Also useful (tall (the Cho of isolating other fibrinolytic protein enzyme from Lumbricus, people such as IH), (2004) are from the purification of six kinds of fibrinolytic serine proteases of the positive earthworm of Lumbricus powder and sign (Purification and characterization of six fibrinolytic serine-proteases fromearthworm Lumbricus rubellus.) Biochemistry and Molecular Biology magazine (J Biochem Mol Biol.) March 31 in 2004; 37 (2): 199-205, it is incorporated herein by reference).Also use nattokinase (nattokinase).

Isolated multiple fibrinolysin also can use from various anthelmintics, insecticide and parasite.For instance, crosslinked (Zha Waluowa L. (the Zavalova of unstability enzyme (destabilase) (a kind of enzyme that is present in the Hirudo) hydrolysis of fibrin, L.), (1996) are from coding specificity cracking endo-type-ε of Hementaria officianalis (Hirudo medicinalis) (γ-Glu)-Lys isopeptide bond and help gene (Genes from the medicinal leech (Hirudo medicinalis) codingfor unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptidebonds and help to dissolve blood clots.) MGG (Mol Gen Genet.) 253 (1-2): the 20-5 of thrombolytic not common enzyme; People such as Zha Waluowa L., (2002) from Fibrinogen-Fibrin system regulation agent (the Fibrinogen-fibrinsystem regulators from bloodsuckers.) biochemistry (Moscow) (Biochemistry (Mosc)) 67 (1) of Hirudo: 135-42, it is incorporated herein by reference separately).

In some embodiments of the invention, throw with fibrinolysin and substituted tPA originally, or except that throwing and tPA, also throw and plasminogen.

Other protein hydrolytic reagent or strengthen proteoclastic medicament and comprise papain, chain egg enzyme (papase), pepsin, trypsin, chymase and hyaluronidase.

Other medicament with anticoagulant or anti-thrombosis activity comprises that heparin (heparin), hirudin (hirudin), ancrod (ancrod), dicoumarol (dicumarol), Xin Qima (sincumar), iloprost (iloprost), L-arginine, two are than Mi Da () dipyramidole) and other platelet function inhibitor, polyethers (such as polyethylene glycol oxide) etc.

Free radical scavenger or antioxidant comprise vitamin A, vitamin E, allopurinol (allopurinol), superoxide dismutase, dimethyl sulfoxine, catalase (catalase), trimetazidine (tremetazidine), ascorbic acid (vitamin C), methylene blue (methylene blue), La Zhaluode (lazaroid), manganese-desferrioxamine (mangan-desferoxamine).

Analgesic comprises local anesthetic (for example, sodium channel blockers); The central action agent is such as anaesthetic (for example opiate, such as morphine (morphine)); The non-steroidal antiinflammatory; Pyrazolone and salicyclic acid derivatives; Acetaminophen (acetyl aminophenol); Tramadol (tramadol) etc.Use the Drug therapy neuropathic pain of not thinking some other kinds of analgesic usually, for example tricyclic antidepressants and some anticonvulsant etc.

In certain embodiments of the invention, bioactivator is the medicament by RNAi interference mechanism inhibition of gene expression.RNAi is a kind of by containing common about 17-29 nucleotide long, for example long double-stranded part of about 19-21 nucleotide and the endogenous cell sequence-specific gene silencing mechanism of the short nucleic acid initiation given prominence to of one or two strand according to circumstances, (perfume blocks P. (Shankar to a chain of wherein said double-stranded part corresponding to target gene and with its complementation, people such as P.), JAMA. (2005) 293 (11): 1367-73, it is incorporated herein by reference).Can cause that the medicament (being referred to herein as the RNAi agent) of gene silencing comprises short interfering rna (siRNA) and can process the molecule that produces siRNA in cell by RNAi, such as short hairpin RNA (shRNA).Multiple RNAi agent can cause the sequence-specific degraded of mRNA or suppress translation.In one embodiment, the RNAi agent is the siRNA that comprises two complementary nucleic acid chains, wherein chain and target gene complementation, and long and each chain comprises that 1-3 nucleotide grows to wherein said chain for about 19-23 nucleotide 3 ' and outstanding.Should be appreciated that, need not between RNAi agent and the target gene or RNAi agent duplex two parts between best complementary, as long as exist enough complementarity to take place to allow hybridization.Complementary degree usually will be at least 15 continuous nucleotides at least 80%, at least 90% or more.In certain embodiments, the RNAi agent contains the duplex that at least 19 nucleotide length have 0,1,2 or 3 mispairing, and the hybridization of chain and target gene forms the duplex that at least 19 nucleotide length have 0,1,2 or 3 mispairing in the wherein said chain.Should be appreciated that, although endogenous synthetic RNAi agent normally is made of RNA, but the RNAi agent of using chemosynthesis to produce also can comprise one or more deoxyribonucleotides or nucleotide analog, modified backbone structure etc. except that the ribonucleotide that is connected by phosphodiester bond, can comprise that maybe one or more deoxyribonucleotides or nucleotide analog, modified backbone structure etc. substitute the ribonucleotide that is connected by phosphodiester bond.

The invention provides a kind of novel method that the RNAi agent is delivered to individuality, described method comprises following steps: first and second hydrogel precursors and RNAi agent are thrown with individual, wherein said hydrogel precursor is cross-linked to form hydrogel behind throwing and described individuality, wherein said hydrogel encapsulation RNAi agent.The present invention provides the compositions that contains the RNAi agent in addition.Described compositions can be hydrogel or contains in the solution of hydrogel precursor as herein described any.Hydrogel composition can be used for the arbitrary place with RNAi agent localized delivery a plurality of positions in the body, for example abdomen pelvic cavity, joint space, maincenter or peripheral nervous system or its part (for example, brain, spinal cord, peripheral nerve).By diffusion in the middle of gel or along with gel degradation comes to discharge the RNAi agent from hydrogel.

Can throw and any RNAi agent.In certain embodiments of the invention, the RNAi agent is the therapeutic agent of above-mentioned any kind of.In an important embodiment, the RNAi agent has the adhesion inhibitory action.For instance, the RNAi agent can suppress to encode short angiogenesis, inspire the expression of gene of the polypeptide that inflammation or short fibrin form.

Usually make one or more solution be in contact with one another before and/or before throwing and the described solution bioactivator is added in the described solution.For instance, before the solution that will contain a HA derivant is loaded in the device that is ready to use in throwing and described solution, bioactivator is added in the described solution.Can with bioactivator and HA derivant simultaneously, in eclipsed interval or successively (meaning refers to dissolve a kind of material and then adds another material) be dissolved in the liquid medium.Solution is mixed or stir to guarantee the uniform distribution of described medicament.The total amount of employed bioactivator can change.For instance, after described medicament being added in first or second solution, the concentration of described medicament can be at 1 μ g/ml in the scope of 1.0g/ml.In certain embodiments of the invention, described concentration is between between 10 μ g/ml and the 100mg/ml or between 100 μ g/ml and 10mg/ml.In certain embodiments of the invention, after described medicament being added in first or second solution, the concentration of described medicament between between 0.1nM and the 10mM, for example between 1nM and 1mM, for example in the scope between 10nM and 100nM.Bioactive agent concentration in the formed hydrogel in the crosslinked back of first and second polysaccharide derivates will be decided on its concentration in each solution and the relative volume of employed solution.The formation of hydrogel will be held back bioactivator, and it slowly discharges from hydrogel by diffusion and/or along with the hydrogel material vivo degradation.

In other embodiment of the present invention, one or more polysaccharide derivates (for example HA derivant) have the bioactivator covalently bound with it.Can use in the several different methods any between polysaccharide derivates and bioactivator, to form covalent bond.Bioactivator and polysaccharide derivates can comprise the functional group that can react to each other or modified to comprise the functional group that can react to each other, for example, and as mentioned about first and second described functional group of HA derivatives reaction.Perhaps, can use with difunctionality or Heterobifunctional cross-linking agent.Be used for linking with crosslinked universal method and be described in following document: ＂ crosslinked (CrossLinking) ＂, Pierre's Si chemical technology library (Pierce Chemical Technical Library) is from website URL Www.piercenet.comAvailable and be published in 1994-95 Pierre Si catalogue at first and wherein quoted list of references, king SS (Wong SS), protein links and cross-linking chemistry (Chemistry of Protein Conjugation andCrosslinking), CRC publishing company (CRC Press Publishers), Boca Raton (Boca Raton), 1991; With G.T. He Mansen (G.T.Hermanson), biological connecting technology (Bioconjugate Techniques), the academic publishing company limited (Academic Press, Inc.), 1995.For instance, according to certain embodiments of the invention, use the difunctionality cross-linking reagent with bioactivator and polysaccharide derivates coupling such as HA derivant, cellulose derivative or glucan derivative.In general, the difunctionality cross-linking reagent contains two reactive groups, thereby a kind of mode of covalently bound two target groups is provided.Reactive group in the chemical crosslinking reagent belongs to variety classes usually, such as succinimido ester, maleimide, pyridine radicals disulfide and iodo-acetamide.In certain embodiments, use such as reagent Acibenzolars such as dicyclohexyl carbodiimide (DCC) or DCI for follow-up binding.

Perhaps, as indicated above, can make functional group on the bioactivator and the functional group's direct reaction on the polysaccharide derivates.If bioactivator does not contain suitable functional group, under can using so in the field any in the known several different methods add described functional group.For instance, if bioactivator is a polypeptide, lysine residue can be added so or terminal amine provides amido.Perhaps, can be modified so that it comprises cysteine residues polypeptide, thereby sulfydryl is provided.

The biological activity that can not make described medicament that is connected of preferred bioactivator and polysaccharide derivates is reduced to useful below horizontal or obviously disturb the crosslinked of first and second polysaccharide derivates.May need to use different functional groups to connect bioactivator and make two kinds of polysaccharide derivates carry out cross-linking reaction.

In the embodiment that merits attention especially, with dexamethasone or other glucocorticoid and HA, cellulose or glucan derivative binding.Polysaccharide derivates can contain non-polysaccharide polymer part, for example peg moiety.In the embodiment that another merits attention especially, dexamethasone and HA or HA derivant are linked.In the embodiment that another merits attention especially, dexamethasone and cellulose or cellulose derivative are linked.In the embodiment that another merits attention especially, dexamethasone and glucosan or glucan derivative are linked.

In the embodiment that merits attention especially, with ibuprofen or other NSAID and HA, cellulose or glucan derivative binding.Polysaccharide derivates can contain non-polysaccharide polymer part, for example peg moiety.In the embodiment that another merits attention especially, NSAID is linked with HA or HA derivant.In the embodiment that another merits attention especially, NSAID is linked with cellulose or cellulose derivative.In the embodiment that another merits attention especially, NSAID is linked with glucosan or glucan derivative.

In certain embodiments of the invention, therapeutic agent and polysaccharide derivates is covalently bound via the key of facile hydrolysis under physiological condition and/or easy enzymolysis.Unsettled binding comprises ester, amide, carboxylic acid amide esters, thioesters, anhydride, urea, carbonic ester, semicarbazones, hydrazone, oxime, iminocarbonic ester, phosphate ester, phosphorus piperazine, urethane (urethane) and acid anhydride key.Being easy to cracked other binding in vivo comprises and containing through polypeptide endogenous or identification of protease that external source provides and cracked site.Exemplary protease comprises serine protease, aspartyl protease, acid protease, alkaline protease, metalloproteases (for example matrix metalloproteinase), carboxypeptidase, amino peptidase, cysteine peptidase etc.The cracking site of these protease has been known in the affiliated field.

It is the compositions (hydrogel and the solution that comprises bioactivator) that comprises the polysaccharide derivates of non-polysaccharide polymer part that the present invention provides at least a hydrogel precursor in addition.The polysaccharide derivates that comprises non-polysaccharide polymer part can be in the described derivant of part i any.Polysaccharide derivates comprises the polysaccharide or derivatives thereof covalently bound with one or more non-polysaccharide polymers usually.

In other embodiments, the invention provides a kind of hydrogel that comprises one or more bioactivators, wherein said hydrogel is to be formed by crosslinked two kinds of non-polysaccharide polymers.Usually non-polysaccharide polymer is dissolved in as mentioned about in the described solution of polysaccharide derivates, one or both in the wherein said solution contain bioactivator.By for example described solution being thrown with individuality it is in contact with one another.Each non-polysaccharide polymer comprises functional group, the wherein said functional group formation covalent bond that can react to each other.Suitable functional group is for above about the described functional group of cross-linked polysaccharides derivant.Exemplary non-polysaccharide polymer comprises that part i is described and contains for crosslinked suitable functional group or can be modified to contain the polymer for crosslinked suitable functional group.

III. comprise polysaccharide derivates and particulate hybrid composition

The present invention provides a kind of polysaccharide derivates and a plurality of grains of composition of comprising in addition.For instance, the invention provides an a kind of HA derivant and a plurality of grains of composition of comprising.Polysaccharide derivates comprises the functional group that can form covalent bond with second functional group.In certain embodiments of the invention, described compositions comprises liquid medium (for example aqueous medium), first polysaccharide derivates and a plurality of granule.Can or be scattered in the described medium described particle suspending.In certain embodiments of the invention, described compositions is by making the crosslinked hydrogel of making of first and second polysaccharide derivates (the wherein any one or two kinds of HA of can be derivant).For instance, make to comprise the HA derivant and comprise a plurality of particulate first solution in addition and contact with second solution that comprises second polysaccharide derivates.In certain embodiments of the invention, second polysaccharide derivates is the 2nd HA derivant.First and second HA derivant reacts to each other forming covalent cross-linking betwixt via functional group, thereby form granule is retained in wherein hydrogel.In certain embodiments of the invention, second polysaccharide derivates is a cellulose derivative.In certain embodiments of the invention, second polysaccharide derivates is a glucan derivative.HA derivant and cellulose or glucan derivative react to each other forming covalent cross-linking betwixt via functional group, thereby form granule are retained in wherein hydrogel.It should be noted that the present invention is not the restriction that is subjected to forming the method for the hydrogel of trapped particles by any way.

In certain embodiments, the invention provides a kind of hydrogel precursor and a plurality of grains of composition of comprising, wherein said hydrogel precursor can contact the back with second hydrogel precursor and form hydrogel in the time between 1 second and 5 minutes under physiological condition for example.In the embodiment that merits attention especially, individuality is thrown with after, hydrogel is between 1 second and 5 minutes, for example between between 1 second and 1 minute, between forming between 1 second and 30 seconds or in the time between 1 second and 15 seconds.In certain embodiments of the invention, hydrogel precursor comprises non-polysaccharide polymer part or it is non-polysaccharide polymer.

Can be with any is incorporated in the compositions in the multiple granule, for example incorporate into and comprise hydrogel precursor (such as polysaccharide derivates, for example HA derivant, cellulose derivative or glucan derivative) liquid medium in, and therefore incorporate into by in the made hydrogel of crosslinked first and second hydrogel precursor (for example polysaccharide derivates or non-polysaccharide polymer).Described granule can for example be polymerization micron particle or nano-particle or liposome.

Can use biodegradable various polymer (for example, bio-compatible polymer) to make granule.Described polymer can be straight chain, side chain or cross-linked homopolymer, copolymer (comprising block copolymer).Suitable bio-compatible polymer (some are wherein arranged is biodegradable) for example comprises poly-(lactide), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid), poly-(glycolic), poly-(lactic acid-copolymerization-glycolic), polycaprolactone, Merlon, polyesteramide, poly-(beta-amino ester), polyanhydride, poly-(amide), poly-(aminoacid), Polyethylene Glycol and its derivant, poe, polyacetals, polybutylcyanoacrylate, polyether ester, poly-(dioxy Ketohexamethylene), poly-(alkylidene alkylates), the copolymer of Polyethylene Glycol and poe, Biodegradable polyurethane.Other polymer comprises poly-(ether), such as polyethylene glycol oxide, poly-(ethylene glycol) and poly-(four methylene oxygen ether); Polyvinyl-poly-(acrylate) and poly-(methacrylate) (such as methyl methacrylate, ethyl methacrylate, other alkane ester of methacrylic acid, hydroxyethyl methylacrylate), acrylic acid and methacrylic acid, and other, such as poly-(vinyl alcohol), poly-(vinylpyrrolidone) and poly-(vinyl acetate); Poly-(ammonia ester); Cellulose and its derivant are such as alkyl, hydroxyalkyl, ether, ester, NC Nitroncellulose and various cellulose acetate; Poly-(siloxanes) etc.Other polymeric material comprises the material that has material based on natural, such as polysaccharide (for example, alginic acid-chitosan, agarose, hyaluronic acid), gelatin, collagen protein and/or other protein, with and composition thereof and/or modified form.Chemistry or the biologically-derived thing (for example, the replacement of chemical group, interpolation are for example by conventional alkyl, alkylidene, hydroxylating, oxidation and other modification of making of one of ordinary skill in the art) of also containing in the polymer disclosed herein any.In addition, can use admixture, graft polymers and the copolymer (comprising block copolymer) of arbitrary these polymer.Copolymer can contain the different monomers subunit of various ratios.For instance, the copolymer that comprises monomer A and monomers B can contain between monomer A between 5% and 95% and the monomers B between 5% and 95% (wherein said percentage ratio is meant in the percentage ratio of amount of monomer and adds up to 100%).Should be appreciated that have some need use suitable initiator or cross-linking agent in these polymer so that polymerization.

Other exemplary polymer comprises cellulose derivative (such as carboxymethyl cellulose), polyurethanes or polyureas, crosslinked poly-(vinyl acetate) etc., the ethylene-vinyl ester copolymer, ethylene-vinyl caproate copolymer, ethylene-vinyl propionate ester copolymer, ethylene-vinyl butyrate copolymer, ethylene-valeric acid vinyl ester copolymers, ethylene-trimethylace tonitric vinyl ester copolymers, ethylene-diethacetic acid vinyl ester copolymers, ethylene-3 Methylbutanoic acid vinyl ester copolymers, ethylene-3-3-acid dimethyl vinyl ester copolymers and ethylene-vinyl benzoate copolymer, or its mixture.

Can encapsulate or be retained in wherein or be adsorbed on lip-deep bioactivator release etc. required granule degradation rate to be provided and/or to make to being selected such as features such as crosslinked and monomer concentrations.

In certain embodiments of the invention, described granule itself is to be made of crosslinked polysaccharide derivates (for example HA, glucosan and/or cellulose derivative).

The micron particle and the nano-particle that are used for the present invention can have multiple size.In general, micron particle will have 500 microns or lower, for example between between 1 and 500 micron, between between 50 and 500 microns, between between 100 and 250 microns, between between 20 and 50 microns, between the diameter between 1 and 20 micron, between 1 and 10 micron etc., and nano-particle will have less than 1 micron, for example between between 10nm and the 100nm, between between 100nm and the 250nm, between between 100nm and the 500nm, between between 250nm and the 500nm, between the diameter between 250nm and the 750nm, between 500nm and 750nm.If the granule by any preparation in the said method has the size range that exceeds required scope, can for example use screen cloth sorting granule so.Particulate size (for example, diameter) or shape can be in fact evenly or its size and/or shape can be inhomogeneous.It can spherical in shapely in fact maybe can have other shape, for example cylindrical, ellipsoid or pyramid, and in the case, relative dimensions will be for the longest straight-line dimension but not diameter.

Known any method manufacturing in the field under nano-particle or micron particle can use includes, but is not limited to spray drying, is separated, single emulsifying and two emulsifying, solvent evaporation, solvent extraction and simple and complex coacervation.Also can use granulation, extrude and/or the round as a ball graininess polymeric compositions of making.In certain embodiments of the invention, use multilayer particle.For instance, described granule can contain one or more layers of core and coating core.Core and layer can be made by same material or different materials.Be used for making particulate material and method and be depicted in for example following document: United States Patent (USP) the 4th, 272, No. 398, it is incorporated herein by reference; The 6th, 428, No. 815, it is incorporated herein by reference; List of references wherein.

Can change and be used to prepare particulate condition to obtain having the granule of required size or characteristic (for example, hydrophobicity, hydrophilic, formalness, " viscosity ", shape etc.).Prepare particulate method and employed condition (for example, solvent, temperature, concentration, air velocity etc.) also by deciding forming of therapeutic agent and/or polymeric matrix.

Can use in the multiple conventional liposome preparation technology any to prepare the liposome that is used for the present invention, such as ultrasonic Treatment, chelating dialyse, homogenize, the solvent infusion adds and extrudes, freeze-thaw is extruded, microemulsified etc.These technology etc. are discussed in for example following patent: United States Patent (USP) the 4th, 728, No. 578, UK Patent Application case G.B.2193095A, United States Patent (USP) the 4th, 533, No. 254, the 4th, 728, No. 575, the 4th, 737, No. 323, the 4th, 753, No. 788 and the 4th, 935, No. 171, it is incorporated herein by reference separately.Also can see Gregory Ya Jisi G. (Gregoriades, G.) (volume), liposome technology (Liposome Technology), 1-3 volume, CRC, Boca Raton (Boca Raton), 1984; Gregory Ya Jisi G. (Gregoriades, G.) (volume) is as the liposome (Liposomes as DrugCarriers) of pharmaceutical carriers, (the John Wiley of John Wei Li publishing company; Sons), strange Chester (Chichester), 1988,1984; Lars gram D.D. (Lasic, D.D.), liposome: acquire application (Liposomes:From Physics toApplications) from physics, like to think only your (Elsevier), Amsterdam (Amsterdam), 1993; (Martin is F.) with Lars gram D. (Lasic, D.) (volume) hidden liposome (Stealth Liposomes), CRC, Boca Raton (Boca Raton), 1995 for Martin F.; (Woodle is M.C) with Si Tuomu G. (Storm, G.) (volume) for Wood M.C, long circulating liposomes: old medicine, new therapy (Long Circulating Liposomes:Old Drugs, New Therapeutics), Springer (Springer), Berlin (Berlin), 1997; (Torchilin is V.P.) with Wei Sige V. (Weissig, V.) (volume) for Tuo Qilin V.P, liposome. hands-on approach (Liposomes.Practical Approach), Oxford University Press (OxfordUniversity Press), Oxford (Oxford), 2003.

The material that can be used for preparing liposome of the present invention comprises known any material or its combination that is suitable for making up liposome of one of ordinary skill in the art.Employed lipid can be natural or synthetic source.Described material includes, but is not limited to lipid, such as cholesterol, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidic acid, phosphatidylinositols, defat lipid (lysolipid), fatty acid, sphingomyelins, glycosyl sphingolipid, glycolipid (glucolipid, glycolipid), thioester, have lipid, polymerizable lipid and its combination that amide, ether and ester are connected fatty acid.

Compositions can contain a plurality of particle swarms, and wherein said group is to be made and/or its characteristic (such as size or shape) difference by the same material of different materials or different ratios.

The ratio that contains the weight of granule density in the solution of polysaccharide derivates (for example HA derivant, cellulose derivative or glucan derivative) and described particulate weight and polysaccharide derivates can change.For instance, the solution that contains polysaccharide derivates can contain the granule between 100 μ g/ml and 10g/ml, for example between between 1mg/ml and the 1.0g/ml or the granule between 1mg/ml and 100mg/ml.The ratio of particle weight and polysaccharide derivates weight can for example change in 100:1 at 1:1001 in the solution.For instance, described ratio can perhaps between 1:2 and 2:1, for example be about 1:1 between between 1:10 and the 10:1 or between 1:5 and 5:1.

In certain embodiments of the invention, bioactivator and granule associate with physics mode, and in other embodiment of the present invention, described granule does not have bioactivator associating with it.Physics mode associates and can be covalently or non-covalently.Association can be specificity or nonspecific.Preferred physics mode associate for when granule is handled and with its with solution in the polysaccharide derivates combination time keep stable and at least compositions is being thrown with individuality before keep stablizing and continue after this one section and can change and the association of controllable time period according to circumstances usually.Can be via one or more noncovalent interaction mechanism (such as ionic interaction, hydrogen bond, hydrophobic interaction etc.) with bioactivator and particle association.For instance, one or more medicaments can be held back, embedding, seal or be packaged in the granule.Described granule can be full of described medicament and/or described medicament is adsorbable on described particulate surface.Can be by diffusion or along with granule degradation in vivo release medicine from granule.Release can take place in a few hours, a couple of days, several weeks, several months etc.

Described granule can contain any one or more bioactivator mentioned above, arbitrary therapeutic agent for example mentioned above.In some embodiments of the invention, contain the solution of polysaccharide derivates or the hydrogel that forms by crosslinked first and second polysaccharide derivates can contain at least two kinds of distinct particle swarms.Described group can about in a plurality of parameters any and differ from one another.For instance, making described particulate material can be different, the bioactivator difference that described granule contains, and its size is not equal.For instance; in one embodiment; first group comprises the granule that contains first bioactivator or by the granulometric composition that contains first bioactivator, and second group comprises the granule that contains second bioactivator or by the granulometric composition that contains second bioactivator.In certain embodiments of the invention, any one or two kinds of in the polysaccharide derivates is the HA derivant.In certain embodiments of the invention, first solution contains a HA derivant and first particle swarm, and second solution contains the 2nd HA derivant and second particle swarm.First and second HA derivant is crosslinked to cause forming the hydrogel that contains first particle swarm and second particle swarm.In certain embodiments of the invention, first solution contains the HA derivant and first particle swarm, and second solution contains the cellulose derivative and second particle swarm.In certain embodiments of the invention, first solution contains the HA derivant and first particle swarm, and second solution contains the glucan derivative and second particle swarm.HA derivant and cellulose or glucan derivative be crosslinked to cause forming the hydrogel that contains first particle swarm and second particle swarm.In any one, first particle swarm in first and second solution and the second particle swarm concentration can be through selecting to be implemented in final granule density required in the hydrogel in these embodiments.In some embodiments of the invention, some (but non-whole) granules comprise bioactivator.Certainly, each solution can contain multiple variable grain group, and for example 2,3,4 or more variable grain groups.

Apparent as one of ordinary skill in the art, can in the granule forming process or after forming, biological agent be loaded in the described granule.Can hold back by polymeric matrix, embedding or encapsulation bioactivator or bioactivator is sealed in the liposome aqueous core in the heart, but and/or bioactivator coated particle surface.The granule of biologically active agent can be scattered in the whole polymeric matrix.Bioactivator preferably constitutes the granule between about 0.01 weight % and 90 weight %, for example constitutes about 1 weight % arrives about 70 weight % to about 80 weight % or about 10 weight % granule.In certain embodiments, bioactivator constitutes about 10%-20 weight % or about 30%-50 weight % granule.One of ordinary skill in the art will understand, and in the process of selecting suitable polymer and manufacture method, select with the stable compatible material and the method for bioactivator quite important.For instance, need avoid to cause the processing temperature of described medicament essence degraded or degeneration (it can cause bioactive forfeiture).Thereby also need test composition to guarantee in the required time section, to discharge a large amount of described medicaments.

In certain embodiments of the invention, that bioactivator and grain fraction (for example polymer or lipid composition) is covalently bound.Can use for forming in the described covalently bound several different methods any.Bioactivator can comprise can with the functional group of functional group reactions on polymer or the lipid.Can to polymer or lipid, bioactivator or the two is modified so that it comprises suitable functional group.Perhaps, can use cross-linking agent.Correlation technique is with above similar about covalently bound bioactivator and the described method of polysaccharide derivates.Preferred described connection can not make the biological activity of described medicament be reduced to useful below horizontal or interference granule formation.In certain embodiments of the invention, via facile hydrolysis under physiological condition and/or easily the key of enzymolysis is covalently bound with bioactivator and particulate polymer or lipid composition.Described bond cleavage is separated meeting release bioactive agent or promotion particle release bioactivator from granule.

In certain embodiments of the invention, except that one or more bioactivators, described granule contains one or more other medicaments, for example excipient, buffer agent, spheronizer material (spheronizing agent), filler, surfactant, disintegrating agent, binding agent, plasticizer or coating materials.Other medicament itself can not have remarkable biological agent, but the biological agent of scalable bioactivator.Exemplary materials is described in United States Patent (USP) the 5th, 846, and in No. 565, described patent is to be incorporated herein by reference.Suitable medicament for example comprises carbohydrate, aminoacid, fatty acid, surfactant, salt, metal ion and increases long-pending agent and it is for known to the one of ordinary skill in the art.The amount of employed excipient can be based on the ratio (by weight) of excipient and bioactivator.For aminoacid, fatty acid, salt and carbohydrate (such as sucrose, lactose, mannitol sugar, glucosan and heparin), in certain embodiments of the invention, the ratio of carbohydrate and bioactivator is between about 1:10 and about 20:1.For surfactant, in certain embodiments of the invention, the ratio of surfactant and bioactivator is between about 1:1000 and about 1:20.

In certain embodiments, other medicament medicament of the release characteristics of particle release bioactivator (being referred to herein as " release regulator ") for a change.The kinetics that contains the particle release bioactivator of bioactivator and release regulator is different from and does not contain release regulator and the kinetics of other identical particle release bioactivator.Kinetics any change in can be in many ways.For instance, release regulator can postpone to discharge or increase release.Discharging in the part or all of time period of taking place, rate of release can increase or reduce.For instance, the existence of release regulator can reduce or prevent initial " pulse " effect (wherein discharging the bioactivator of larger proportion granule contacts back one period short period with liquid in).Some granule with required time section after liquid contacts in discharge mass efficient load, but when time point after a while, possibly can't continue to discharge other medicament.Release regulator can change from the required time of the bioactivator of particle release prescribed percentage.For instance, release regulator can increase or reduce the required time of bioactivator all to be discharged on release 10%, 25%, 50%, 75%, 90% or the sill.For instance, pot life increases or reduces between the factor between .1 and 10 times.Exemplary release regulator comprises lyophobic dust, and hydrophobic surfactant for example is such as poloxamer (poloxamer).Other exemplary release regulator is phospholipid, cholesterol, polymethacrylates, sugar, protein, acrylate block copolymer (such as strange E-100 of You Te (Eudagrit E-100) and related compound) and zinc.Release regulator can be the medicament that forms the hole or fill the hole in the polymeric matrices in polymeric matrices.

The present invention provides a kind of compositions in addition, and it comprises the micron particle that (a) comprises one or more cross-linked polysaccharides derivants (for example HA derivant); (b) a plurality of nano-particle.Micron particle can be suspended in the medium and directly be applied to position in the body, for example be applied to peritoneum.Nano-particle can contain bioactivator.Therefore, described compositions can comprise the micron particle that comprises the cross-linked polysaccharides derivant, wherein said micron particle encapsulation nano-particle.In one embodiment of the invention, prepare the micron particle that comprises one or more HA derivants by in comprising the oil and the continuous phase of emulsifying agent, adding the solution of a HA derivant, bioactivator and the 2nd HA derivant successively and it being homogenized.Homogenize is to carry out 1-20 minute with 1000-9000rpm.Subsequently 40-50 ℃ with the emulsion stirred overnight with evaporation water from decentralized photo.With micron particle washed with isopropyl alcohol 3-6 time, evaporation residue isopropyl alcohol subsequently.Can before little encapsulation, bioactivator be packaged in the nano-particle in advance.Nano-particle can be polymer/nanoparticle or liposome.

In another embodiment of the present invention, can prepare micron particle by spray drying HA derivant, bioactivator and release regulator.

It is the compositions that comprises the polysaccharide derivates of non-polysaccharide polymer part (hydrogel and comprise hydrogel precursor and a plurality of particulate solution) that the present invention provides at least a hydrogel precursor in addition.The polysaccharide derivates that comprises non-polysaccharide polymer part can be in the described derivant of part i any.Polysaccharide derivates comprises the polysaccharide or derivatives thereof covalently bound with one or more non-polysaccharide polymers usually.Granule can be in the granule mentioned above any.

In other embodiments, the invention provides a kind of a plurality of particulate hydrogels that comprise, wherein said hydrogel is to be formed by crosslinked two kinds of non-polysaccharide polymers.Usually non-polysaccharide polymer is dissolved in as mentioned about in the described solution of polysaccharide derivates, one or both in the wherein said solution contain granule.By for example described solution being thrown with individuality it is in contact with one another.Each non-polysaccharide polymer comprises functional group, the wherein said functional group formation covalent bond that can react to each other.Suitable functional group is for above about the described functional group of cross-linked polysaccharides derivant.Exemplary non-polysaccharide polymer comprises that part i is described and contains for crosslinked suitable functional group or can be modified to contain the polymer for crosslinked suitable functional group.Granule can be in the granule mentioned above any.

It should be noted that described granule provides in the solution that contains hydrogel precursor in above-mentioned arbitrary embodiment, or can independent solution or dried forms provide.

IV. use

The compositions and methods of the invention serve many purposes, and include, but is not limited to prevention and treatment adhesion and throwing and therapeutic agent.In using for these any, hydrogel precursor (for example polysaccharide derivates) can provide and can be dissolved in before dispensing in the suitable liquid medium (for example water or normal saline) by dried forms (for example lyophilizing).Perhaps, hydrogel precursor (for example polysaccharide derivates) can the solution form provide.Can with preceding bioactivator and/or granule (comprising one or more bioactivators according to circumstances) be added in the dry polymer or solution of not commensurability (deciding on using) in throwing.Perhaps, comprising polysaccharide derivates and bioactivator or grains of composition can dried forms provide and adds in the liquid medium subsequently.

A. the pattern of offeing medicine

As indicated above, the invention provides several different methods, its comprise with hydrogel precursor throw with individual body in the position, wherein said hydrogel precursor throw with after be cross-linked to form hydrogel.Hydrogel precursor normally with the solution form throw with.Described solution any in can be in many ways throw with.For instance, can use multitube injection device (for example, multitube syringe, bivalve applicator (dual valve applicator)) that multiple solution is delivered to desired location in fact simultaneously.In certain embodiments of the invention, described device comprises the multiple solution of temporary transient injection to blended chamber takes place wherein and before dispensing.The hydrogel precursor of throwing with the independent solution form that is in contact with one another is in vivo contained in the present invention.Throwing and two or more solution are in fact simultaneously contained in the present invention.These two kinds of solution can be in contact with one another in throwing and process.The present invention is also contained by throwing the single solution that forms with multiple solution by each self-contained hydrogel precursor and is thrown and described multiple solution.Described solution consequently seldom takes place crosslinked in throwing and process or crosslinked and described compositions does not take place to keep fluid state usually in throwing combination not long ago with it.For instance, in the method for the inhibition adhesion that comprises following steps: (a) first hydrogel precursor that will comprise first polysaccharide derivates throw with individual body in the position; (b) second hydrogel precursor that will comprise second polysaccharide derivates or non-polysaccharide polymer throw with individual body in described position, the present invention includes following examples: wherein hydrogel precursor is dissolved in the solution so that it not long ago was in contact with one another with it and/or mixes mutually throwing.The present invention also comprises following examples: wherein hydrogel precursor is dissolved in the independent solution, with described solution through one or more such as about 1-60 second, for example 1-30 second, 5-20 second, about 10 seconds discrete or continuous time section throws in fact simultaneously and.The present invention also comprises following examples: wherein with hydrogel precursor with independent solution form throw with, make its throw with after be in contact with one another.Can be by such as in the several different methods such as coextrusion in solution each pipe from the multitube injection device any thrown and solution in fact simultaneously.In the embodiments of the invention of throwing with compositions more than three kinds or three kinds, any two or two or more compositions can throw in fact simultaneously with.Compositions can through the single time period or through a plurality of discrete time sections throw with, its can be considered separately independent throw with.A plurality of discrete time sections can take place in several minutes, a few hours etc.For instance, full peritoneum throw with or complicated vertebra or cranium operation can relate to the repeatedly discrete dispensing of going through a few hours.

Figure 13 illustrates the illustrative arrangement that is used to throw with solution.Figure 14 illustrates and can be used for throwing and the multichannel device that reaches four kinds of different components (for example four kinds of different solutions).Some solution contain hydrogel precursor, for example polysaccharide derivates; And other solution need not to contain hydrogel precursor.For instance, some solution can contain granule.In addition, can under the situation that does not have liquid, granule be loaded in the passage of described device, and throw with process in it is mixed with solution.Figure 15 illustrates the two-tube device that links with droplet-shaped apparatus for converting (such as aerosol apparatus or nebulizer).Graphic right side is divided into the enlarged drawing of the part of described device.Described device is specially adapted to compositions is thrown rapidly and relatively large zone (for example for full peritoneum is used).

Perhaps, can use indivedual injection devices (syringe, conduit, sleeve pipe etc.), only be careful make solution throwing and the time or throw and be in contact with one another soon afterwards and get final product.Can use endoscope, for example peritoneoscope, arthroscope etc.For instance, it is suitable having multichannel observation instrument.In one embodiment, use double injection peritoneoscope or arthroscope.In one embodiment, described compositions is to use under imaging guiding (for example fluoroscopic guidance).In another embodiment, first and second solution can be mixed in the container and subsequently simply it is toppled over, sprays or is sprayed on the desired location.Can throw with after the soln using blend tool mixed or utilize distribution instrument (for example, scraper sample instrument) distribution.

In certain embodiments of the invention, the solution that comprises hydrogel precursor (for example polysaccharide derivates, such as the HA derivant) comprises detectable substance.But described material vision-based detection, for example dyestuff or coloring agent; Perhaps can detect (for example, described material can be radioactive substance) by another way.Preferred detectable substance is to health harmless (unless it is inevitable with the toxic material to normal cell and undesirable cell, such as antiproliferative or antitumor agent for mechanism of action).The existence of detectable substance make to throw individuality with compositions be easier to differentiate institute throws and material and therefore can be used for guiding offeing medicine.Some detectable substance can its thrown with after be used to follow the trail of polysaccharide derivates, for example monitor its degraded.

B. prevent and treat adhesion

Can throw with compositions of the present invention with treatment or Film with Preventing Adhesion under any situation in multiple type of surgery.The limiting examples of the operative procedure that the compositions and methods of the invention are suitable for comprises abdomen pelvic cavity, ophthalmology, orthopedic, gastrointestinal, breast, cranium, head and neck, cardiovascular, gynecological, joint (for example arthroscope), urology department, shaping, reproduces, flesh and bone and neuromuscular are performed the operation.Specific abdominal part program for example comprises the operation of removing or repairing one or more abdomen pelvic organs or its part.Example comprises stomach, intestinal (for example, duodenum, jejunum, ileum, colon, rectum), vermiform appendix, gallbladder, liver, kidney, bladder, urethra and prostatic operation.The abdomen operation on pelvis comprises that also hernia is repaired, aneurysm is repaired and the peritoneal adhesion cracking.Gynecological's program comprises the infertile operation that treatment is caused by diseases of fallopian tubes (for example, the adhesion that links to each other with ovary, fallopian tube and pili).Described operation comprises salpingostomy, salpingolysis and ovary synechotomy.Gynecilogical operation comprises that ovariectomy, uterectomy, removal endometriosis, prevention form adhesion, treatment ectopic pregnancy, uterus or bottom muscular tumor etc. again.Other operation comprises the operation of treatment incontinence or vaginocele.Obstetric operation comprises Caesarian section.Flesh and osseous surgery comprise lumbar vertebra plate excision, lumbar discectomy art, flexor tendon operation, spinal fusion and joint replacement or repairing.The neuromuscular operation comprises and is used to repair nerve, extracts neural or freely holds back neural operation.The breast operation that relates to sternotomy can cause forming adhesion between heart or large artery trunks and thoracic cavity epithelial layer.The breast operation comprises esophagus operation, lung operation (for example lung subtracts perhaps tumor removal), by-pass operation, aneurysm is repaired and the cardiac valve replacement operation.Operation also comprises the operation that is used for diagnostic purpose with the operation of implanting in multiple prosthese or the implantable device (such as defibrillator) any and execution through carrying out.The cranium operative procedure comprises the operation at tumor, epilepsy of the multiple program of needs.These programs can cause the adhesion that relates to skull, meninges and/or cortex usually.Ocular surgical is used operation, glaucoma filtering surgery and the lacrimal drainage system program that comprises at stravismus.

Therefore in certain embodiments of the invention, throwing and described compositions are with treatment or prevention peritoneal adhesion.In other embodiment of the present invention, throw with described compositions with treatment or prevention pleural adhesion, for example between the lobe of the lung and/or the fiber adhesion between pleura visceralis and the parietal pleura.In other embodiment of the present invention, throw the adhesion that relates to pericardium (for example, visceral pericardium, pericardium viscerale or pericardium parietale, fiber pericardium) with compositions with treatment or prevention.In other embodiment of the present invention, throwing and compositions are with treatment or pre-epidural adhesion preventing (relating to the adhesion of dural epidural space).

Usually, will be in the time that produces first otch throwing and described compositions when finishing surgical closure or last endoscopic tools being taken out in individual body certain is some between time of (what take place later on is as the criterion).Compositions can be thrown and the individuality that does not before develop adhesion, maybe it can be thrown and gives the individuality that has developed adhesion.In one embodiment, described compositions is thrown and the individuality that just experiences the program that reduces the adhesion that is pre-existing in.For instance, described program can be used compositions of the present invention and recur with Film with Preventing Adhesion subsequently with machinery or the chemical cracking or the destruction of the adhesion that is pre-existing in.

Throw with the cumulative volume of giving individual compositions and can change based on multiple factor, described factor is mainly the zone of plan water gel layer overlies, whether thickness, throwing and position and the compositions of required hydrogel comprises therapeutic agent.Exemplary volume is between .1ml and 5000ml, for example between between .5ml and the 1000ml, between between 1ml and the 500ml, between 10ml and 100ml etc., should be appreciated that these volumes are approximation and comprise all subranges.

Can throw throws with described compositions so that institute and material or damage the part that (for example operative incision or damage) position or covering are positioned at the epithelial surface (for example peritoneum, pleura, pachymeninx) of damaging part offside by the covering of its hydrogel that forms.The zone that is covered can or be positioned at around the structural zone relative with damaging part and from damaging part or be positioned at the structural zone relative with the damaging part one section variable distance that stretches out at damaging part.For instance, the zone that is covered can stretch out up to about 1,2,3,4 from damaging part, 5cm or longer average distance.By throw and the material or the gross area that hydrogel covered that forms by described material can be at about 0.1cm ²The gross area that arrives about peritoneum is (for example, up to about 1.5-2.0m ²) scope in.For instance, the gross area that is covered can be at about 1.0cm ²To about 1.0m ², for example about 5.0cm ²To about .5m ²Scope in.A plurality of zone of dispersions that compositions can be applied to adjacent area or separate by zone without covering.

Comprising under the situation of grains of composition, the particulate volume and weight that is transmitted also can change and will on throw and the cumulative volume of solution and solution in particulate concentration decide.Can be adjusted to transmit required total particle volume or weight these parameters.In certain embodiments of the invention, granule is passed to individuality with the amount between .1mg/kg and 2g/kg.Exemplary amount is between 1mg/kg and 1000mg/kg, for example in the scope between 5mg/kg and 700mg/kg.The particulate amount of being transmitted need not can absolute magnitude to represent based on the weight of individuality.Exemplary amount is between .1mg and 500g, for example between between 1mg and the 100g or in the scope between 10mg and 1g.

C. drug delivery

The compositions that arbitrary hydrogel of formation as indicated above contains therapeutic agent (associating with physics mode with granule according to circumstances) can be used for treating multiple disease, disease and condition of illness or is used to prevent purpose.In certain embodiments of the invention, hydrogel makes therapeutic agent continue to discharge.The independent throwing of hydrogel precursor and medicament with the described medicament of valid density can be provided in a period of time, if this for time that will cause at the described medicament of throwing under the situation that does not have hydrogel precursor with same amount at least 2,3,5,10,20 or more times and/or under the situation that does not cause inappropriate side effect, use hydrogel precursor to make and can give and higher accumulated dose.In certain embodiments of the invention, come sustained release speed by the speed of control hydrogel and/or granule degraded.Therefore, the invention provides a kind of compositions that comprises the hydrogel precursor and the therapeutic agent of solution form, wherein said hydrogel precursor provides for any hydrogel precursor as herein described and with any concentration range as herein described.

Any position in compositions throwing and the individual body can be included, but is not limited to the position of above being discussed in the situation that suppresses adhesion.For instance, compositions can be thrown with peritoneum to treat disease or disease or the treatment general disease that mainly in the abdomen pelvic cavity, manifests.The peritoneum drug delivery is the noticeable selection that is used for the treatment of the multiple therapeutic agent of general disease, and this has high surface area to small part owing to peritoneum and can be used for absorbing medicament.In certain embodiments of the invention, the compositions that will comprise anti-infective is used for the treatment of the probability of infection or for example preventative reduction post-operative infection.The patient that experience may jeopardize the operative procedure of intestinal wall especially has infection (for example peritonitis) risk.In one embodiment, during abdominal operation or afterwards, compositions is thrown and individual abdomen pelvic cavity.Compositions can be applied to any position of abdomen pelvic cavity, directly be applied to one or more abdomen organs and/or adjacent tissue or be applied to the parietal peritoneum that roughly is positioned at abdomen organ's opposite position, so that form the visceral peritoneum and the isolating hydrogel of parietal peritoneum that will cover organ.Can use and prolong local peritoneum dispensing or full peritoneum throwing of warp and compositions.

In another embodiment, use compositions of the present invention to throw and antitumor agent.Can throw with antitumor agent and be arranged in the tumor of abdomen pelvic cavity, for example tumor of abdominal part or pelvic organ with treatment.In one embodiment, before exenterate is arranged in the tumor of abdomen pelvic cavity, during or afterwards compositions is thrown with individual.Peritoneum is thrown and compositions entirely.Do not wish to be subjected to the restriction of any theory, but throwing can reduce the development of transfer with compositions of the present invention and/or the peritoneum of inhibition tumor is sent out.In another embodiment, compositions is thrown in the abdomen pelvic cavity with the individuality of suffering from tumor.Described compositions makes antitumor agent continue to discharge.For instance, can discharge described medicament through at least 1,2,3,4,6 or 8 weeks or longer time.Compositions can be applied to tumor organ of living in or can use more widely.Therefore, the invention provides the method that a kind of treatment is suffered from tumor or the individuality of the risk that develops tumor is arranged, it comprises throws compositions of the present invention with individual, and wherein said compositions comprises antitumor agent.Can use any compositions that comprises at least a hydrogel precursor as herein described (for example polysaccharide derivates, such as the HA derivant), wherein said at least a hydrogel precursor throw with individuality after be cross-linked to form hydrogel.Described therapeutic agent can associate with physics mode with arbitrary class granule described herein.Tumor can be positioned at the abdomen pelvic cavity.

In another embodiment, use compositions of the present invention that therapeutic agent is thrown and individual joint.Individuality for example can be suffered from arthritis.The exemplary therapeutic agent that is suitable for throwing with the joint space comprises antiinflammatory and analgesic.Therefore, the invention provides a kind of treatment and suffer from the method for individuality of the risk of the disease or the condition of illness that influence the joint or disease that the influence on development joint is arranged or condition of illness, it comprises throws compositions of the present invention and described joint, and wherein said compositions comprises through selecting to treat or to prevent the therapeutic agent of described condition of illness.Can for example compositions be expelled in the Synovial cavity.Can use any compositions that comprises at least a hydrogel precursor as herein described, wherein said at least a hydrogel precursor is cross-linked to form hydrogel in throwing and individual back with another hydrogel precursor.Therapeutic agent can associate with physics mode with arbitrary class granule described herein.

Can use any throwing and compositions in the number of ways such as Intradermal for example, subcutaneous, intramuscular.Any bodily tissue all can be used as the reservoir that comprises one or more hydrogel precursors (for example polysaccharide derivates) and a plurality of grains of composition, wherein said hydrogel precursor throw with after be cross-linked to form hydrogel.

D. hydrogel forms fast

As indicated above, one aspect of the present invention forms benefit and the research and development of appropriate combination thing and the method that realization quick in situ hydrogel forms that hydrogel provides for understanding by quick cross-linked hydrogel precursor original position.Any embodiment of the present invention can utilize the compositions that contains hydrogel precursor to implement, and described hydrogel precursor is being in contact with one another the back between 1-5 and between 60 seconds, between 1-5 and between 30 seconds, form hydrogel between 1-15 and between 20 seconds or between 1-5 and in the time between 10 seconds.Any embodiment of the present invention can utilize the compositions that contains hydrogel precursor to implement, and described hydrogel precursor is in contact with one another the back between 1-5 and between 60 seconds, between 1-5 and between 30 seconds, form hydrogel between 1-15 and between 20 seconds or between 1-5 and in the time between 10 seconds at the solution that contains hydrogel precursor.Any embodiment of the present invention can utilize the compositions that contains hydrogel precursor to implement, and described hydrogel precursor is being thrown with individual afterwards between 1-5 and between 60 seconds, between 1-5 and between 30 seconds, form hydrogel between 1-15 and between 20 seconds or between 1-5 and in the time between 10 seconds.

V. pack or test kit

The present invention also provides packing or test kit, and it comprises one or more compositionss as described herein in container.For instance, container can comprise the HA derivant of drying (for example lyophilizing) form or solution form.If the HA derivant is to provide with dried forms, the product packing can comprise the container with appropriate solvent or diluent (for example Injectable sterile water) so.Packing also can comprise the bulletin that links to each other with container, it typically is the form of government organs' defined of production, use or the sale of managed care device, medicine and/or biological medicine, described thus bulletin reflect described mechanism ratify described compositions be used for human or throwing for animals with treatment adhesion disease or except that one or more indications of treatment adhesion or treat one or more indications but not treatment adhesion (for example, prevention or treat post-operative infection).Also can comprise the relevant description of using described medicament or compositions.Described description can comprise about rebuild HA solution, to the loading that wherein adds granule, transfer device, throwing and appropriate amount and information such as pattern.

In certain embodiments of the invention, packing will contain a plurality of individual containers, and it contains the HA derivant of dried forms or solution form separately.For instance, first container contains a HA derivant and second container contains the 2nd HA derivant.Thereby the first and second HA derivants can provide the hydrogel that formation has required feature when it is in contact with one another by scheduled volume in solution.Described packing also can comprise one or more containers that contain at the bioactivator of throwing and before treating to make up with the HA derivant.

In certain embodiments, packing contains other polysaccharide derivates, such as cellulose or glucan derivative.In certain embodiments, comprise the combination of the HA, cellulose and/or the glucan derivative that are used to form required hydrogel.Described packing also can comprise other polymer, such as synthetic polymer.Packing also can comprise protein.

Medical packaging also can comprise and contain and will be included in the particulate accepter in the solution with polysaccharide derivates (for example HA, cellulose or glucan derivative).Perhaps, accepter can contain for example derivant and the granule of predetermined ratio.Described granule can contain bioactivator.

A plurality of containers form of the single larger container of deadend (for example plastics or foamed polystyrene plastics (styrofoam) box) relatively provide.

Packing can comprise device or the accepter that contains the solution of polysaccharide derivates (for example HA, cellulose or glucan derivative) for preparation.Described device can be for example for measuring or mixing arrangement.

Packing can comprise the device that is used to throw with compositions of the present invention.Illustrative arrangement comprises syringe, for example multitube syringe, conduit, endoscope, arthroscope, peritoneoscope.Endoscope, arthroscope or peritoneoscope can have a plurality of passages to allow throwing and multiple indivedual solution.Other device that can comprise is thrown and the endoscopic tools of compositions of the present invention or the adnexa of laparoscopic tool for convenient.Certainly, described device also can provide separately.

Example

Example 1: the preparation of derivatives of hyaluronic acids and cross-linked hydrogel and sign

Materials and methods

Hyaluronic acid: hyaluronic acid (HA, nominal 1.36MD: high MW and 490kD: be medium MW) available from enzyme company (Genzyme Corporation) (Cambridge, Massachusetts (Cambridge, MA)).HA (nominal 50kD: be that (Lifecore Biomedical, Inc.) (SIKA (Chaska, MN)) is looked in the Minnesota State available from life sciences biological medicine company limited low MW).All other reagent are all available from Sigma-A De Ritchie company (Sigma-Aldrich) (St. Louis (St.Louis, MO, USA)).

The hyaluronic preparation of crosslinkable: the synthetic original position crosslinkable HA derivant of method (the merchant X (JiaX) that follows previous report, Klum is won G (Colombo G), Pa Dela R (Padera R), bright lattice R (Langer R), the in-situ cross-linked hyaluronic acid of gram Chinese DS. (Kohane DS.) make the sciatic nerve blocking-up prolong (Prolongation of sciatic nerveblockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004; 25 (19): 4797-4804, it is incorporated herein by reference).In simple terms, under pH6.8 and room temperature, react under the situation that has 1-ethyl-3-carbodiimides (EDC) and I-hydroxybenzotriazole (HOBt) by the adipic dihydrazide that makes HA (unless otherwise mentioned, otherwise for medium MW) and 30 times of molar excess and to prepare HA-adipic dihydrazide (HA-ADH).By thorough dialysis and ethanol precipitation purified product.By at room temperature making HA (unless indicate in addition, otherwise for high MW) prepare HA-aldehyde (HA-CHO) in 2 hours with equimolar sodium metaperiodate reaction in the dark place.Come cessation reaction by adding ethylene glycol.By thorough dialysis purified product.With the purified product lyophilizing and be stored under 4 ℃.Use gel permeation chromatography (GPC) to measure the molecular weight (MW) of crosslinkable HA derivant.Utilize water hydrogel linear columns (Ultrahydrogel Linear column) (water generation company (Waters), Massachusetts Penelope Milford (Milford, MA)) and the 0.05M acetate aqueous solution that contains 0.2M NaCl (pH 6.7) are carried out GPC as mobile phase (0.8ml/min).Utilize a series of pulullan polysaccharides (pullulan) standard substance preparation MW calibration curve.Use according to the method for being reported ¹H NMR analyzes (for HA-ADH, the Mercury of Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A. (Varian) (Mercury) 300MHz) (merchant X (Jia X), Klum is won G (Colombo G), Pa Dela R (PaderaR), bright lattice R (Langer R), the in-situ cross-linked hyaluronic acid of gram Chinese DS. (Kohane DS.) make the sciatic nerve blocking-up prolong (Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004; 25 (19): 4797-4804, it is incorporated herein by reference) and aldehyde analysis (for HA-CHO) (gram Chinese DS. (Kohane DS), sharp general M (Lipp M), gold Buddhist nun RC (Kinney RC), Anthony DC (Anthony DC), Louis DN (Louis DN), Luo Dan N people such as (Lotan N) is contained bio-compatible (Biocompatibility oflipid-protein-sugar particles containing bupivacaine in the epineurium.) the biomedical material research magazine (J Biomed Mater Res) 2002 of lipid-protein-sugared granule in epineurium of bupivacaine; 59 (3): 450-459, it is incorporated herein by reference) the mensuration degree of modification.

The sign of HAX hydrogel: the in-situ gelling measure of time of hydrogel is as follows.With magnetic stirring bar (polytetrafluoroethylene fluorocarbon resin (Teflon fluorocarbon resin), 5 * 2mm, Fei Xue scientific ﹠ technical corporation (Fisher Scientific)) is put in the center of normal saline solution (20mg/ml) droplet of 100 microlitre HA-ADH in the Pi Shi culture plate (Petri dish).Subsequently 100 microlitre HA-CHO solution (20mg/ml) are added in the HA-ADH dropping liquid, and use PC-320 type healthy and free from worry (Corning) hot plate/agitator with the 155rpm agitating solution.Time when forming solution fully with the isolating solid globules of described tray bottom is considered as gelling time.The result is reported to the meansigma methods and the standard deviation of 4 independent measurement values.(JEOL JSM 6060, (JEOL USA, Inc.), Massachusetts Pi Baidi (Peabody, MA)) observes the form of lyophilizing HAX gel to U.S. JEOL company limited by scanning electron microscope.In liquid nitrogen after the cooling with the lyophilised gel fragmentation to expose the structure of gel inside.Before observation, utilize the sample of palladium and gold (100 dusts are thick) dash coat fragmentation.

The statistical analysis of example 1-5 since data not always followed normal distribution distribute, so it is expressed as intermediate value and the 25th percentage point and the 75th percentage point.(Chicago, Illinois State city (Chicago, IL)) uses Mann-Whitney U test (Mann-Whitney U-test), Kruskal-Wallis test (Kruskal-Wallis test) or the expense accurate check of snow (Fisher ' s exact test) to carry out statistical inference to use SPSS software.Think that the p value that goes up less than 0.05 at two-tailed test (2-tailed test) is remarkable for statistics.

The result

Characterize crosslinkable HA derivant HA-adipic dihydrazide (HA-ADH) and HA-aldehyde (HA-CHO) by multiple distinct methods.The MW of original HA and modified HA is summarized in the table 1.Peak average MW (Mp) slightly increases and modifies post polymerization thing polydispersity at ADH and increases above 2 times.Aldehyde (CHO) is modified and is caused MW significantly to reduce.After CHO modifies, the MW of HA (high MW) and HA (medium MW) is reduced to 188 and 253kD from nominal 1.36MD and 478kD respectively, this (merchant X (Jia X) that conforms to previous report, Klum is won G (ColomboG), Pa Dela R (Padera R), bright lattice R (Langer R), the in-situ cross-linked hyaluronic acid of gram Chinese DS. (Kohane DS.) make the sciatic nerve blocking-up prolong (Prolongation of sciatic nerve blockade by in situ cross-linkedhyaluronic acid.) biomaterial (Biomaterials) 2004; 25 (19): 4797-4804, it is incorporated herein by reference).This result shows that 2 hours oxidation reaction is lured the obvious cracking of sugar ring into, and irrelevant with original MW. ¹H NMR and aldehyde analysis result show that respectively 52.9 ± 4.7% of ADH and HA links 16.6 ± 4.8% aldehyde radicals formation (n=7) in (n=4) and each the HA repetitive.

Form the HAX gel rapidly two kinds of HA derivant contact backs.Described in method, under the situation of two kinds of components of continuous stirring, HAX gel (20mg/ml) forms in 3.5 ± 0.6sec.Utilize SEM to check the form (Fig. 1) of crosslinked HAX gel after the lyophilizing.Cross-linked hydrogel has continuous circular or polygonal hole, be typical cross-linked hydrogel (merchant X (Jia X), Bo Dike J A (Burdick J A), Ke Bole J (Kobler J), Clifton RJ (CliftonRJ), Roseau Butterworth base JJ (Rosowski JJ), carry special this SM (Zeitels SM) people of etc.ing, the synthetic and sign of original position crosslinkable hyaluronic acid based aquagel and be used for the regenerated potential application of vocal cords (Synthesis and Characterizationof in Situ Cross-Linkable Hyaluronic Acid-Based Hydrogels with Potential Application forVocal Fold Regeneration.) macromolecule (Macromolecules) 2004; 37 (9): 3239-3248, it is incorporated herein by reference) have a 10-20 μ m diameter.

The general introduction of the molecular weight of the original and modified HA of table 1.

1. provide by manufacturer

2. peak mean molecule quantity

3. weight average molecular weight

4. number average molecular weight

5. polydispersity index=Mw/Mn

Example 2: the bio-compatible of HAX hydrogel in vitro

Materials and methods

In vitro cell viability analysis: (ATCC CRL-9444) is incubated at and contains Yi Ershi salt (Earle ' s salt), L-glutaminate and 2.2g/L sodium bicarbonate and be supplemented with in the culture medium 199 of 3.3nM epidermal growth factor, 400nM hydrocortisone, 870nM insulin, 20mM HEPES and 10% hyclone with human mesothelial cell.With 25 generation of the 5th generation to the cell be used for following research.The mesothelial cell is inoculated in the 1ml culture medium in 24 well culture plates with the density of 50,000 cells in every hole.After cultivating whole night, culture medium is replaced with fresh culture or fresh culture with 10U/ml hyaluronidase, and sterile preparation 100 μ l cylindrical (diameter: 5mm, highly: 5.1mm) HAX gel (20mg/ml) and it is added in each hole.Exist under the situation of hydrogel cultivate different time after, utilize MTT assay kit (Sai Ertaite of Pu Luomaige company 96 non-radioactive cell proliferation analyses (PromegaCellTiter 96 Non-Radioactive Cell Proliferation Assay)) evaluation cell viability.The result is reported at the intermediate value of the standardized measured absorbance of the absorbance of undressed control cells (absorbance of standardization cell viability %=100 * grow under having the situation of the sample cell in the culture medium/grow in the absorbance of the cell in the culture medium) and the 25th percentage point and the 75th percentage point.

The result

Measure in vitro cytotoxicity (Di Zejia GS (diZerega GS) peritoneum, peritoneum healing and the adhesion formation (Peritoneum of HAX gel to the mesothelial cell, peritoneal healing, and adhesion formation.), Di Zejia GS (diZerega GS) compiles, operation on peritoneum (Peritoneal Surgery.) New York (New York): Springer Verlag (Springer), 2000. 3-37 pages or leaves; It is incorporated herein by reference).Growth reaches 3 days in the culture medium of the 10 units per ml hyaluronidases that have or do not exist degraded HAX gel existing under the situation of 100 μ l 20mg/ml HAX gels (referring to method) to make these cells.As utilize MTT to analyze institute's evaluation, and the vigor of cell that was exposed to HAX after 3 days under having the situation of hyaluronidase is slightly (16%) reduction (p=0.042), and the existence of HAX does not have statistics appreciable impact (Fig. 2) to the cell viability in arbitrary group.In vitro analysis of cell proliferation shows, the HAX gel is all compatible with the mesothelial cell under non-degradation condition (ordinary culture medium) and degradation condition (culture medium that contains the 10U/ml hyaluronidase).

Example 3: by in-situ cross-linked HAX gel for prevention peritoneal adhesion

Materials and methods

The HAX gel in vivo use the scheme of ratifying about animal care according to committee of the Massachusetts Institute of Technology (Massachusetts Institute ofTechnology Committee), abide by the nursing of relevant laboratory animal and the NIH of use and instruct (NIH announces #85-23, revision in 1985) nursing animal.With female albefaction rabbit (Spain hare (Oryctolagus cuniculus); New Zealand white (New Zealand White), Ke Wensi company (Covance), (3 ± 0.5kg) as animal pattern for Pennsylvania Hei Zeerdun (Hazleton, PA)).Use ketamine (Ketamine) (35mg/kg, intramuscular) and xylazine (Xylazine) (5mg/kg, intramuscular) to lure anesthesia into; Use throw via endotracheal tube and oxygen in 1-3% isoflurane (isoflurane) realize keeping.In whole process, use aseptic technique.In the whole surgery process, lactated Ringer's solution (lactated Ringer ' s solution) is provided and continues to monitor vital signs to animal.Produce the long midline incision of 10cm along the white line on the stomach wall (linea alba), and open peritoneum.According to the method for reporting in the document with modification (auspicious tower H difficult to understand (Orita H), good fortune card savart M (Fukasawa M), this W of gill base (Girgis W), the inhibition of tissue adhesion in Di Zejia GS. (diZerega GS.) the standard rabbit model: utilize the tissue plasmin activator to carry out intraperitoneal treatment (Inhibition of postsurgical adhesions in astandardized rabbit model:intraperitoneal treatment with tissue plasminogen activator.) International Journal of Fertility (Int J Fertil) 1991; 36 (3) .172-177, it is incorporated herein by reference) bring out peritoneal adhesion.On the stomach wall of right side, it is damaged to begin to cut the 3 * 4cm that comprises parietal peritoneum and one deck muscle (about 1mm is thick) from distance center line 1cm.Subsequently, caecum is being gone up external 7 bags (beginning to the 12nd bag from the 6th bag of ileocecus (ileocecal junction) far-end) that turn to mesentery side (anti-mesenteric side), it is being separated and using aseptic operation to brush under the two-way scratch 80-160 causing bleeding.

20 animals are assigned randomly to following experimental group: (i) not treatment (n=12); (ii) utilize the crosslinked HA of 10ml to cover through open abdomen and abrasive caecum surface (n=8).Before using, be dissolved in the physiological saline solution with material sterilization 2 hours and with it by the ultraviolet-sterilization illumination.Gel precursors solution (5ml HA-ADH (20mg/ml) and 5mlHA-CHO (20mg/ml)) is put into the asepsis injector that 10ml independently links to each other with hundred special bivalve applicators (Baxter dual valve applicator), and by No. 15 pin coextrusion.Liquid precursor begins gelling immediately, meets the shape of using the zone.Visual inspection, gelling is finished in less than 3 minutes: hydrogel does not flow when surpassing described time point.

After the treatment affected area, utilize 2-0 to treasure good suture (Ethilon) and closed peritoneum of 3-0 fenaminosulf suture (Dexon) and stomach wall respectively.Utilize 2-0 to treasure the closed skin of good suture.Animal is waken up and freely take food and water.Postoperative 8 hours is through subcutaneous throwing and buprenorphine (Buprenorphine) 0.02-0.03mg/kg.

In 1 week after the program, utilize intravenous 100mg/kg pentobarbital sodium (sodium pentobarbital) that animal is implemented euthanasia.The amending method that uses the method for being reported is to adhesion scoring (this JW of Berne (Burns JW), this Jenner K (Skinner K), examine special J (Colt J), shed woods A (Sheidlin A), the gloomy R of Blang (Bronson R), Ya Kebai Y people such as (Yaacobi Y) is by preventing tissue injury and tissue adhesion (Preventionof tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acidsolutions.) operations research magazine (Journal of Surgical Research) 1995 with hyaluronic acid solution precoating tissue; 59:644-652, it is incorporated herein by reference).0 minute=no adhesion, 1 minute=utilize gravity to adhere to by isolating tissue; 2 minutes=can adhere to by the isolating tissue of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.The fixation of tissue that will be reclaimed by postmortem is embedded in the paraffin in 10% formalin (formalin), section also use standard technique with haematoxylin and Yihong dyeing for histological examination.

The result

Described in method, make 20 animals receive caecum scratch and part celiotomy incision.In first week after surgery, lose 5.7 ± 4.6% body weight without 12 animals (contrast) of HAX gel for treating.12 10 (83%) rabbits development adhesions in 3 fens (Fig. 3 C) of having merely hit.In these 10 rabbits, 6 directly develop adhesion on the opposite of celiotomy incision, and the adhesion of 2 development relates to the incision edge, and the adhesion of 1 development relates to a plurality of intestinal sections; In a back animal, there is tangible local hemorrhage in intra-operative.1 rabbit development adheres to the center line abdominal part and sews up, and promptly it is irrelevant with the stomach wall through cutting.

In first week after surgery, lose 9.0 ± 7.4% body weight (compare with untreated contrast, p is not remarkable) through 8 animals of 10ml HAX gel for treating.Only 2 animals (25%) development adhesion in 3 fens (compare with untreated contrast, p=0.019 takes the snow Precision Test).There are 4 not represent adhesion (Fig. 3 B) in 8 animals.1 animal has the adhesion (1 minute) of the Gravity Separation utilized.2 animals are in the development adhesion (2 minutes and 3 minutes) of abdominal part suture site, and 1 animal development adhesion in 3 fens between abrasive bag and not abrasive ileocecus.Yet, do not relate to impaired stomach wall.It should be noted that described 2 minutes and 1 adhesion in 3 fens relates to the suture site of not using the HAX gel.Similarly, another adhesions in 3 fens between two rings of intestinal also only relate to one through treating the surface.At when dissected, the HAX gel rubber material still is present on the therapentic part (impaired stomach wall and/or abrasive caecum surface).Macroscopy, the amount and the elasticity of described material obviously reduce.

Will be apparent from data from the untreated animal, the damage of stomach wall is played crucial effects for the development adhesion.As if the influence of caecum galled spots not too obvious.Heal in a week and do not retain any obvious sign in the abrasive caecum surface that adhesion does not relate to, and adhesion is usually directed to scratch and not abrasive bag.In addition, in caecum scratch and stomach wall is not impaired or only slight abrasive some pilot studies in, the incidence rate of adhesion extremely low (data not shown).

For light microscopy, the sample of obtaining from the adhesion position of untreated animal represents the close attachment of myenteron sarcocyte and abdominal wall muscle tissue, has the different-thickness between inflammation and fibrosis and the evidence (edema, little deep dyed color cell, central nucleus) (Fig. 4 A and 4B) of muscle injury.By contrast, the sample of obtaining from the damage location through the treatment animal of no adhesion represents the blue dyeing material coating, and light to moderate inflammatory cell (major part is for macrophage and some neutrophil cells are arranged) soaks into (Fig. 4 C).From the sample through the adhesion position of the animal of HAX treatment of two development adhesions have with from the identical outward appearance in the adhesion position of untreated animal.In sample, do not observe coating herein, from these two animals; It should be noted that these adhesions betide uncoated position.

This result of experiment is summarized in the table 2.The result shows that the HAX gel reduces the in vivo effect of peritoneal adhesion.When comparing the sickness rate of adhesion in 3 fens (it is the firm connection that can only pass through cutting and separating), the ability of gel for prevention adhesion is especially obvious; Rudimentary adhesion is the viscosity of different stage but not the adhesion of operation in the related meanings.Development adhesion in 3 fens in 10 (83%) 7 days is after surgery arranged in the animal in 12 untreated matched groups.Histological examination confirms that adhesion in 3 fens is to be caused by inflammation and fibrosis.By contrast, in being applied to the animal of HAX gel for treating of damaged part, 8 utilizations have only 2 (25%) adhesion in 3 fens to occur.Two adhesions in 3 fens seen in the treatment group relate to sew up the incision or two caecum surfaces between, this all is not with the position of HAX treatment.If the adhesion at uncoated position is got rid of outside analyzing, the incidence rate of adhesion in 3 fens is respectively 82% (11 have merely hit 9) and 0% (6 are not all had) in matched group and the treatment group so.

It should be noted that and the analysis showed that crosslinked HAX gel of the present invention bio-compatible in peritoneum.Before confirmed bio-compatible (the merchant X (Jia X) in perineurium of this system, Klum is won G (Colombo G), Pa Dela R (Padera R), bright lattice R (Langer R), the in-situ cross-linked hyaluronic acid of gram Chinese DS. (Kohane DS.) make the sciatic nerve blocking-up prolong (Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronicacid.) biomaterial (Biomaterials) 2004; 25 (19): 4797-4804, it is incorporated herein by reference).Yet the biocompatibility in a tissue may not be predicted the biocompatibility in peritoneum.For instance, although polymeric microspheres is at perineurium (gram Chinese DS. (Kohane DS), sharp general M (Lipp M), gold Buddhist nun RC (Kinney RC), Anthony DC (Anthony DC), Louis DN (Louis DN), Luo Dan N people such as (Lotan N) is contained bio-compatible (Biocompatibility oflipid-protein-sugar particles containing bupivacaine in the epineurium.) the biomedical material research magazine (J Biomed Mater Res) 2002 of lipid-protein-sugared granule in epineurium of bupivacaine; 59 (3): 450-459, it is incorporated herein by reference) and the interior bio-compatible of many other tissues, but it tends to cause adhesion (gram Chinese DS. (Kohane DS) at intraperitoneal, carry this JY (Tse JY), she is Y difficult to understand (Yeo Y), Pa Dela R (Padera R), Shu Baina M (Shubina M), bright lattice R (LangerR) is for the biodegradable polymerization microsphere and nano-particle (Biodegradable polymericmicrospheres and nanospheres for drug delivery in the peritoneum.) biomedical material research magazine (J Biomed Mater Res) the 2005:In press of intraperitoneal drug delivery, and it is incorporated herein by reference).

Table 2: the assessment of peritoneal adhesion

Example 4:HA catabolite is to the influence of tPA and PAI-1 generation

Confirmed that the balance between fibrinolysis and the fibrinolysis activity is important (Buddhist gram K (Falk K) for the development pole that mediates adhesion, the P of Bu Jie Qwest (Bjorquist P), the M of this Tom Qwest (Stromqvist M), Huo Mudaer L. (Holmdahl L.) reduces tentative adhesion formation (Reduction of experimental adhesion formation by inhibition of plasminogen activatorinhibitor type 1.) Britain operation magazine (British Journal of Surgery) 2001 by suppressing 1 type plasminogen activator inhibitor; 88:286-289; The guest reaches MM (Binda MM), Mo Linnasi CR (Molinas CR), the PR of Ke Ning section (Koninckx PR.) reactive oxygen species and adhesion form: clinical meaning (the Reactive oxygen species and adhesion formation:clinical implications in adhesion prevention.) human reproduction (Human Reproduction) 2003 of adhesion prevention; 18 (12): 2503-2507, described document is incorporated herein by reference separately).The serous coat fibrinolysis mainly is subjected to the mesothelium of t-PA and PAI to discharge regulation and control, and (orange red is L (Tietze L) now, the strange A of Ai Bairui (Eibrecht A), Si Qierte C (Schauerte C), Kroes is breathed out fragrant B (Klosterhalfen B), Ah not 's Tacchinardi B (Amo-Takyi B), Ji Halun J people such as (Gehlen J), by transforminggrowthfactor-(TGF-β 1), tumor necrosis factor (TNF-α) and interleukin 1 β (IL-1 β) regulate the short fibrin hydrolysis and the antifibrin hydrolysis properties (Modulation of pro-and antifibrinolytic properties of human peritoneal mesothelial cells bytransforming growth factor beta1 (TGF-beta1)) of human peritoneal mesothelium cell, tumor necrosis factor alpha (TNF-alpha) andinterleukin 1beta (IL-1beta) .) thrombosis and hemostasis (Thromb Haemost) 1998; 79 (2): 362-370, it is incorporated herein by reference).Whether the catabolite of having studied HAX can cause that may reduce adhesion by the HAX gel forms the tPA that causes and the change of PAI-1 mesothelium output.Make stable discharge (Fig. 5 A) of described catabolite at the crosslinked HA gel of in the 10U/ml hyaluronidase, cultivating under 37 ℃.The mesothelial cell cultivated in being supplemented with or not being supplemented with 100 μ l normal saline or 100 μ l contain in the 1ml culture medium of normal saline of a kind of (D-glucuronic acid or N-acetyl group-D-glycosamine) in 20mg/ml HA (490kD or 50kD MW) or two kinds of monomer components of HA.Fig. 5 B demonstration and untreated or compare through the contrast of normal saline treatment utilize the tPA output among the mesothelial cell of the hyaluronic acid of different MW and monomers grow to have the remarkable and minimum reduction of statistics (p=0.15).Difference statistics between the group of HA or monomer treatment is remarkable (p=0.112) not.Similarly, any treatment is to the influence of PAI-1 output all not obvious (data not shown).Do not wish to be bound by any theory, believe that significantly being reduced in of adhesion formation of utilizing the HAX gel to cause to a great extent may be owing to the barrier function of HAX gel.Yet, because the etiology that adhesion forms is subjected to the mediation of many biological events, so the potential solubility leaching (solubility HA, monomer) of HAX gel can not increase tPA output or influence the probability that the fact of PAI-1 content in vitro can not be got rid of biotic influence.

Example 5: monomer concentration and molecular weight are to the influence of HAX gel degradation

Materials and methods

The degraded of HAX gel in the hyaluronidase: the HAX gel that preparation is made up of the HA-ADH and the HA-CHO of the various concentration with various molecular weights, and monitor the degraded of gel in the hyaluronidase in time.Prepare the HAX gel in the 2ml microcentrifugal tube by using turbine mixer that the 150 μ l HA-ADH of various concentration and Mw and 150 μ l HA-CHO are mixed in immediately, and subsequently with it in cultivation in hyaluronidase (50U/ml is in PBS) under 37 ℃.When putting at the fixed time, remove the hyaluronidase buffer fully, and remain the weight in wet base of HAX gel through weight analysis determining.The figure of gel quality affects %/original gel weight in wet base and time relation when being plotted as the result with each time point.

The result

For the potentiality of evaluation, study the influence of various gel strengths by the characteristic of further optimization of the crosslink density that changes substrate and control HAX gel.Above employed HA viscosity is extremely strong, and is difficult to dissolve the HA that is higher than 20mg/ml.Therefore, for increasing HA concentration, prepare the precursor of low Mw.For this reason, by 50kD HA (but not 490kD) preparation HA-ADH and by 490kD HA (but not 1.36MD) preparation HA-CHO.This allows allotment 75mg/mlHA-ADH and 60mg/ml HA-CHO.For macroscopical gel quality affects of quickening the gel degradation process and observe in the reasonable time section changes, use the 50U/ml hyaluronidase to carry out degradation experiment.

The HAX gel of all concentration all expands at first and degrades with given pace (this decides on concentration (Fig. 6 A) and molecular weight (Fig. 6 B)) subsequently.When the concentration of HA-ADH and HA-CHO solution respectively when 20mg/ml is increased to 75mg/ml and 30mg/ml, the time (" half-life ") of hydrogel weight in wet base reduction by 50% was increased to 11 days from 5 days, and when described concentration increased to 75mg/ml and 60mg/ml, the described time was increased to 22.5 days (Fig. 6 A).(note in compare back one, having only the concentration of HA-CHO to change.) when the concentration of HA-ADH and HA-CHO keeps constant, utilize the half-life of the gel that the HA-CHO obtained by higher Mw makes long (and 1.36MD HA-CHO greater than 50 days 22 days with respect to 490kD HA-CHO; Fig. 6 B).These the experiment in, with the HAX gel pouring in microcentrifugal tube, wherein said gel only a side to the hyaluronic acid enzymatic solution.Therefore, the absolute half-life shown here may be not directly related with other experiment (all surface of wherein said gel all is exposed to enzymatic solution) described herein.

The result who is provided in this example shows that the HAX gel is in case formation promptly presents persistent entity barrier, and its can last for days several weeks (concentration and molecular weight on gel component are decided, referring to Fig. 6) is up to finally being for example endogenous hyaluronic acid enzymatic degradation.Thereby can according to described method by change these parameters controllably regulate the degraded time-histories the fact make native system be suitable for application-specific, this optionally the barrier function time span and decide.

Example 6: the preparation and the sign of hydridization HA/ nano-particle hydrogel

Materials and methods

Material hyaluronic acid (HA, 1.36MDa and 490kDa) is available from gene enzyme company (GenzymeCorporation) (Cambridge, Massachusetts (Cambridge, MA)).Poly-(lactic acid-copolymerization-glycolic) (Mw 90,000 for PLGA, lactide: Acetic acid, hydroxy-, bimol. cyclic ester=65:35) are that (Cambridge, Massachusetts (Cambridge, MA)) obtains from Arco Mu Si company (Alkermes).Polyvinyl alcohol (PVA, Mw 6000) is available from gloomy this company limited of Berli (Polysciences, Inc.) (Wellington, Binzhou (Warrington, PA)).Unless indicate in addition, otherwise all other reagent are all available from Sigma-A De Ritchie company (Sigma-Aldrich) (St. Louis (St.Louis, MO, USA)).

Synthetic original position crosslinkable HA derivant described in the preparation of crosslinkable hyaluronic acid and PLGA nano-particle such as the example 1.Prepare blank PLGA nano-particle by single emulsification method.PLGA 200mg is dissolved in the 3:2 mixture of 5ml dichloromethane and dimethyl sulfoxine (DMSO).Polymer solution is directly added among 20ml 5% PVA.Use ultrasonoscope (Wei Baisaier series VC-250 type (Vibracell VC-250), ultrasound wave and the (Sonics of Materials Co., Ltd subsequently; Materials Inc.), Connecticut State Dan Boni (Dunbury, CT)) homogenizes mixture and reaches 1 minute to produce oil-in-water emulsions.Add to formed emulsion in the 100ml distilled water and stirred overnight at room temperature.Under reduced pressure remove residual solvent.By using L8-70M ultrahigh speed centrifuge (Beckman company (Beckman), California Fulton (Fullerton, CA)) and SW28 upset bucket type rotor (swinging bucket rotor) with 25,000rpm collected nano-particle in centrifugal 20 minutes, and by make its pass through ultrafilter membrane (the Ao Tesaier Amy agree YM100 (Ultracel Amicon YM100), Millipore Corp. (Millipore), the Massachusetts is than Le Lika (Billerica, MA)) be further purified, subsequently lyophilizing.(U.S. Brooker Hai Wen instrument company (Brookhaven Instruments Corporation), New York Huo Tesiweier (Holtsville, NY)) measures particle diameter to utilize ZetaPALS type zeta potential analyser (zeta potential analyzer).

The preparation of hydrogel prepares the cross-linked-hyaluronic acid hydrogel (" HAX ") of no nano-particle by the solution that mixes 20mg/ml gel precursors (HA-ADH and HA-CHO).By being mixed in, 20mg/ml gel precursors solution prepares the compound HAX gel (" hydridization ") that contains the PLGA nano-particle in the 20mg/ml PLGA nano granule suspension.

Scanning electron microscope analysis is checked the configuration of surface of PLGA nano-particle and the internal structure of lyophilizing hydrogel by scanning electron microscope analysis.Make hydrogel by being blended in the equal-volume precursor solution for preparing in the distilled water.Subsequently with hydrogel lyophilizing and cooling back fragmentation in liquid nitrogen.(JEOLJSM 6320, U.S. JEOL company limited, Massachusetts Pi Baidi (Peabody, MA)) observation with palladium and gold (100 dusts are thick) dash coat and use scanning electron microscope with sample.

Gelling time is added the HA-ADH solution (HA-ADH and PLGA nano-particle are 20mg/ml or 10mg/ml in normal saline) that 100 μ l contain the PLGA nano-particle in 8 * 35mm vial to, described vial contains magnetic stirring bar (polytetrafluoroethylene fluorocarbon resin (Teflon fluorocarbon resin), 5 * 2mm, Fei Xue scientific ﹠ technical corporation (Fisher Scientific)).The HA-CHO solution (HA-CHO and PLGA nano-particle all are 20mg/ml or 10mg/ml in normal saline) that subsequently 100 μ l is contained the PLGA nano-particle adds in the described bottle, and use healthy and free from worry PC-320 type hot plate/agitator with the 155rpm agitating solution up to forming hybrid gel.Time when forming solution fully with the isolating solid globules of described tray bottom is considered as gelling time.For relatively, also test the formation of HAX gel.The result is reported to the meansigma methods and the standard deviation of 4 independent measurement values.

The rheological characteristic test prepares cylindrical HAX and hybrid gel by using 1ml syringe and hundred special bivalve applicators (Baxter dual valve applicator) that 20mg/ml gel precursors solution is added to be sandwiched in two rubber molds between the slide glass.Prepared hydrogel's diameter and thickness are respectively 8mm and 3.5mm.Subsequently gel is transferred to AR1000N type flow graph (TA instrument company (TA Instruments), in the Delaware State Newcastle (New Castle, DE)) for rheological measurement.All experiments all are at room temperature to use the parallel-plate of 8mm diameter to carry out.Measure shear modulus G by creep test and stress scans test (stress sweep test).For creep test, make hydrogel experience constant shear stress (5,10,20 or 40Pa) reach 90 seconds and it was replied 90 seconds.After about 60 seconds, strain reaches steady state value in each creep and return phase.G is determined as the inverse of strain (return phase latter stage reading) counter stress slope of a curve.For the stress scans test, be applied to the interior oscillatory stress of 1-100Pa scope with constant frequency (0.1Hz).The elastic modulus G that will obtain under 40Pa ' as the approximation of G, this is near 0 because of viscous modulus G ＂.The result is reported to the meansigma methods and the standard deviation of 4 independent measurement values.

The statistical analysis of example 6-8 is reported to meansigma methods and standard deviation with the rheological measurement value, and uses student t check (Student t-test) to be compared.Since the cell culture data not always followed normal distribution distribute, so it is expressed as intermediate value and the 25th percentage point and the 75th percentage point; Also report mark in this way.For this reason, (Chicago, Illinois State city (Chicago, IL)) uses Mann-Whitney U test (Mann-Whitney U-test), Kruskal-Wallis test (Kruskal-Wallis test) or the expense accurate check of snow (Fisher ' s exact test) to carry out statistical inference to use SPSS software.Think that the p value that goes up less than 0.05 at two-tailed test (2-tailed test) is remarkable for statistics.

The result

The average diameter of the sign PLGA nano-particle of hybrid gel and zeta potential are respectively 278.4 ± 18.7nm and-18.1 ± 4.0mV.By scanning electron microscope analysis, particle size distribution is at 50nm (Fig. 7 A) in the scope of the relative broad of 300nm.Lyophilizing hybrid gel substrate has the continuous hole of diameter in 5 to 10 μ m (Fig. 7 B) scope, the hole of its HAX gel of can comparing (Fig. 7 C).Hybrid gel has rough surface (Fig. 7 B illustration), shows that nanoparticles embedded is in gel-type vehicle.As described herein, the HAX gel is that as one of advantage of adhesion barrier it is in-situ cross-linked with the treatment adhesion in scope between can be in due course.Incorporating into of polymer/nanoparticle whether disturb gelling for evaluation, the HAX gel is compared with the gelling time of hybrid gel.Two system's gellings rapidly after mixing, and do not have statistics or in fact significant difference (table 3) between the two.Similarly, wish to determine whether nano-particle can influence the mechanical property of gel.Modulus of shearing is not subjected to nano-particle to influence (table 3).

Table 3.HAX gel and the gelling time of hybrid gel and the comparison of modulus of shearing

Example 7: the biocompatibility of hydridization HA/ nano-particle hydrogel

Materials and methods

The hydridization of preparation described in hydrogel such as the example 6 HA/ nano-particle hydrogel.

(ATCC, (hero company is (among the Invitrogen CRL-9444) to be incubated at the culture medium 199 that contains Yi Ershi salt (Earle ' ssalt), L-glutaminate and 2.2g/L sodium bicarbonate and be supplemented with 3.3nM epidermal growth factor, 400nM hydrocortisone, 870nM insulin, 20mM HEPES and 10% hyclone with human mesothelial cell for analysis of cell proliferation.Cell is inoculated in the 1ml culture medium in 24 well culture plates with the density of 50,000 cells in every hole.Whole night, cylindrical hybrid gel of 100 μ l or normal saline are added in each hole.Exist under the situation of hydrogel cultivate different time after, utilize MTT assay kit (Sai Ertaite of Pu Luomaige company 96 non-radioactive cell proliferation analyses (PromegaCellTiter 96 Non-Radioactive Cell Proliferation Assay)) evaluation cell viability.The result is reported at the standardized measured absorbance of the absorbance of undressed control cells (absorbance of standardization cell viability %=100 * grow under having the situation of the sample cell in the culture medium/grow in the absorbance of the cell in the culture medium) intermediate value and the 25th percentage point and the 75th percentage point.

The result

Utilize MTT analyzing evaluation hybrid gel to the in vitro influence of mesothelial cell's vigor.Make cell growth under the situation that has the cylindrical hybrid gels of 100 μ l (20mg/ml HAX+20mg/ml PLGA nano-particle) (measuring 3.5mm * 8mm diameter) reach 3 days.This is to carry out in the culture medium that has or do not exist 10 units per ml hyaluronidases (enzyme of a kind of HAX of degraded).(in matched group, the denominator that this provides the standardization vigor adds 100 μ l standard normal saline and substitutes gel.) cell viability keeps good when cultivating the 1st day and the 3rd day, show that the existence of this and hyaluronidase has nothing to do.There is no the significant difference of statistics between the group shown in Fig. 8.

Example 8: by the adhesion of in-situ cross-linked hydridization HA/ nano-particle gel for prevention mouse model

Materials and methods

The hydridization of preparation described in hydrogel such as the example 6 HA/ nano-particle hydrogel.

The scheme that the in vivo application of hybrid gel is ratified about animal care according to committee of the Massachusetts Institute of Technology (Massachusetts Institute ofTechnology Committee), abide by the nursing of relevant laboratory animal and the NIH of use and instruct (NIH announces #85-23, revision in 1985) nursing animal.

Injection model uses the male SV129 mice of heavy 20-35g in the mouse peritoneum.The aseptic hybrid gel of 1ml is injected in the peritoneum via the single perforation in the left lower quadrant.Before using, be dissolved in the physiological saline solution with material sterilization 2 hours and with it by the ultraviolet-sterilization illumination.The HA-ADH 0.5ml (10mg/mlHA-ADH and 20mg/ml nano-particle) that will have the PLGA nano-particle puts into the asepsis injector that independent 1ml links to each other with hundred special bivalve applicators with HA-CHO 0.5ml (10mg/ml contains the 20mg/ml nano-particle), and by No. 20 pin coextrusion.Injection back 2 or 7 days utilizes carbon dioxide that animal is implemented euthanasia.The existence of checking adhesion in the animal whether and the hybrid gel residue whether as seen.

The fixation of tissue that histological examination will be reclaimed by postmortem is embedded in the paraffin in 10% formalin, and section also uses standard technique with haematoxylin and Yihong (H﹠amp; E) dyeing.

The result

The research and development research and development of the new method of peritoneum drug delivery cause extensive concern with the method that prevents and/or treats peritoneal adhesion and other purpose with drug delivery in peritoneum.Yet clearance rate hinders the drug delivery of peritoneum faster.Attempt to determine whether and to improve the peritoneum drug delivery by using polymer base controlled-release technology.Also study peritoneum and use poly-(lactic acid-copolymerization-glycolic) (PLGA) micron particle of (a kind of biodegradable polymer that has good biocompatibility usually that is usually used in medicine controlled releasing) and adaptability of nano-particle.With the diameter of 10 to 100mg dosage is that the 90kDaPLGA micron particle of 5 to 250 μ m is injected into (every group of n=3-5 only) in the Muridae peritoneum.Find injection back 2 all polymer residues and adhesion than high rate (for example, the 5 μ m micron particle of 50mg cause adhesion in 83% animal).The histology discloses chronic inflammation, and foreign-body giant cell is outstanding and particle diameter greater than 5 μ m.By 54,57 and the 5 μ m microspheres (gamma-radiation) made of 10kDa PLGA cause few adhesion (16.7%) and residue incidence rate similarly.The nano-particle of 90kDa PLGA (265nm) also can cause the adhesion (6.3% animal) of much less, and this may be because it was completely cut off from the intraperitoneal removing and spleen and liver in 2 days, notices foamy macrophage in spleen and liver.These experiments show that micron particle of being tested and nano-particle all can't be provided for the acceptable polymeric medicine transmission system of peritoneum separately.Suppose that the compound water congealing colloid system for the intraperitoneal drug delivery that granule is retained in the original position crosslinkable hyaluronic acid gel also will serve as the barrier that suppresses adhesion formation when allowing effectively to use the polymer-matrix drug delivery.Specifically, suppose otherwise to cause under the particulate situation that adhesion forms that the near small part of hydrogel prevents to cause that through the granule of sealing adhesion forms.Under the particulate situation that will otherwise remove rapidly from intraperitoneal, hydrogel is retained in intraperitoneal with described granule.

The biocompatibility of injection model remains on intraperitoneal for check HAX with nano-particle and prevents the supposition that adhesion that the existence by high molecular PLGA causes forms simultaneously in the mouse peritoneum, and 10 or the 20mg/ml HAX that contain 20mg PLGA nano-particle with 1ml inject mouse peritoneum.The previous animal that has been presented at injection nano-particle under the situation that does not have HAX left peritoneum in 2 days, retain few polymer residues, and had spleen and the foamy macrophage (data does not show) that becomes greatly, decolours usually.

Inject postmortem in back 2 days when (table 4), the form with discrete block keeps in the injection site to contain the hybrid gel of 10mg/ml HAX, and described block is easy to separate with abdominal contents on every side (Fig. 9 A).The hybrid gel that contains 20mg/ml HAX seems the stronger abdominal contents that adheres to, and still is easy to and contacts organ and separate, but it retains some residues (Fig. 9 B).Adhesion not to be noted or spleen size or color unusual all in arbitrary group.Inject and put to death the hydridization group that another contains 20mg/ml HAX in back 7 days.4 mice loyalties have 3 to see gel (Fig. 9 C), and gel quality affects can be comparable to the gel quality affects that reclaims after 2 days.In this group, see adhesion single situation under, visible few gel or can't see gel, this shows that possible intracavity gel injection has penetrated intestinal (and can infer subsequently and remove) in feces.In addition, spleen seems very normal.Light microscopy for the dyed gel slide glass that reclaims from the abdominal cavity of many animals is noticed foamy macrophage, shows exist (Figure 11 B, the illustration) of nano-particle through keeping.In liver or spleen, do not see foamy macrophage.As in all figure of Fig. 9, finding out, around many gels, notice the vascularity of increase.

The biocompatibility of hybrid gel in table 4. mouse model

HAX(mg)	10	20	20
				PLGA nano-particle (mg)	20	20	20
Apart between when dissected	2	2	7
				n	4	4	4
Adhesion	0	0	1
				Adhesion %	0	0	25
The existence of remaining gel	4	4	3

Example 9: by the peritoneal adhesion of in-situ cross-linked hydridization HA/ nano-particle gel for prevention rabbit adhesion model

Materials and methods

HydrogelPreparation hydridization HA/ nano-particle hydrogel described in example 6.

The scheme that the in vivo application of hybrid gel is ratified about animal care according to committee of the Massachusetts Institute of Technology (Massachusetts Institute ofTechnology Committee), abide by the nursing of relevant laboratory animal and the NIH of use and instruct (NIH announces #85-23, revision in 1985) nursing animal.

The rabbit sidewall is damaged-and caecum scratch model is female albefaction rabbit (Spain hare (Oryctolagus cuniculus); New Zealand white (New Zealand White), Ke Wensi company (Covance), (3 ± 0.5kg) as animal pattern for Pennsylvania Hei Zeerdun (Hazleton, PA)).Described in example 3, bring out tissue adhesion.In simple terms, after opening peritoneum, begin that from distance center line 1cm the right side stomach wall is produced 3 * 4cm and comprise the damaged of parietal peritoneum and one deck muscle (about 1mm is thick) along the long midline incision of the 10cm of the white line on the stomach wall.Subsequently, use the mesentery side of aseptic operation brush 80-160 time, cause bleeding from the 6th bag of ileocecus far-end to the 12nd bag of two-way scratch caecum.

20 animals are assigned randomly to following experimental group: (i) not treatment (n=12); (ii) utilize 10ml crosslinking hybrid gel to cover through open abdomen and caecum surface.The HA-ADH (20mg/mlHA-ADH and 20mg/ml nano-particle) that 5ml is had the PLGA nano-particle puts into the 10ml independence asepsis injector that links to each other with hundred special bivalve applicators with 5ml HA-CHO (20mg/ml HA-CHO and 20mg/ml nano-particle), and by No. 15 pin coextrusion.Liquid precursor begins gelling immediately, meets the shape of using the zone.Visual inspection, gelling is finished in less than 3 minutes: hydrogel does not flow when surpassing described time point.

Described in example 3, take post-operative care.In 1 week after the program, utilize pentobarbital sodium that animal is implemented euthanasia.Follow the modified method reported to adhesion scoring (this JW of Berne (Burns JW), this Jenner K (Skinner K), examine special J (Colt J), shed woods A (Sheidlin A), the gloomy R of Blang (Bronson R), Ya Kebai Y people such as (Yaacobi Y) is by preventing tissue injury and tissue adhesion (Prevention of tissue injuryand postsurgical adhesions by precoating tissues with hyaluronic acid solutions.) operations research magazine (Journal of Surgical Research) 1995 with hyaluronic acid solution precoating tissue; 59:644-652, it is incorporated herein by reference).The no adhesion of expression in 0 fen, expression in 1 fen can utilize the tissue of Gravity Separation to adhere to; Expression in 2 fens can adhere to by the isolating tissue of blunt dissection; And the adhesion that expression in 3 fens needs sharp weapon to dissect.(whether it comprises the suture site also to note the position of adhesion; The position and the quantity of related caecum bag in the adhesion) and the weight change.

The fixation of tissue that histological examination will be reclaimed by postmortem is embedded in the paraffin in 10% formalin, and section also uses standard technique with haematoxylin and Yihong (H﹠amp; E) dyeing.

The result

The abdominal part sidewall of rabbit is damaged-caecum scratch model in described in the biocompatibility and effect such as method of hybrid gel, make 8 rabbits accept laparotomy, wherein with the caecum scratch and cut one section adjacent stomach wall.(the 20mg nano-particle is in 10ml 20mg/ml HAX) is applied on the damaged part with hybrid gel.During 1 week back postmortem (table 5), its loss in weight can be comparable to matched group (identical damage, treatment, n=12).Intermediate value adhesion mark obviously less (p=0.002, Mann-Whitney U test in the treatment group; 0.001, take accurately check of snow).Although 83.3% animal development adhesion in 3 fens (can only pass through the firm connection of cutting and separating) (example 3) in the matched group, the development of neither one treatment group 3 minutes adhesions (p＜0.001, Mann-Whitney U tests; P=0.001 takes accurately check of snow).Compare with 12 animals of matched group 2 (16.7%), have in 8 animals of treatment group 5 (62.5%) do not represent fully tissue adhere to (Figure 10 B) (0.04, Mann-Whitney U test; P=0.035 takes accurately check of snow).

In 3 animals of development adhesion in 2 fens, 1 just on the caecum surface and near the adhesion between open abdomen of suture site, 1 just through the scratch caecum with do not abrade between the caecum, and 1 just through the scratch caecum with do not abrade between the caecum and caecum is surperficial and near the impaired stomach wall of suture site between develop 2 adhesions in 2 fens.Therefore, take place that many positions for covering without hybrid gel are arranged in the position of these adhesions in 2 fens.During postmortem, notice hybrid gel still at its position of using, but quantity and mechanical property reduce obviously.In some cases, the caecum surface that is covered by the residue of hybrid gel keeps the initial abrasive black blood (old blood) of trace.During light microscopy, in dyed gel residue slide glass, notice and infer the foamy macrophage (Figure 11 A and B) that contains polymer fragment; Be also noted that free copolymer (bright spot).In liver or spleen, do not see the foamy macrophage (not shown).On the surface of the impaired stomach wall of no adhesion of using hybrid gel, also see foamy macrophage (Figure 11 C and D).

Table 5: the assessment of peritoneal adhesion in the rabbit model

^*In intermediate value and the bracket the 25th percentage point and the 75th percentage point.

Described data show that hybrid system has than low cytotoxicity for the peritoneal mesothelium cell in vitro; Bio-compatible in peritoneum in vivo; And inherently can Film with Preventing Adhesion.With regard to Film with Preventing Adhesion, its at least with HAX similar (example 3).Incorporate among the HAX basically nano-particle into gelling time or modulus of shearing and do not have influence gel systems.The shortage of cell described in the existence by foamy macrophage in the gel residue and liver and spleen (wherein it is noticed in the mice with the nano-particle injection of the suitable quality that does not have HAX) can find out that HAX successfully remains on the time that intraperitoneal reaches Therapy lasted with nano-particle.HAX also can prevent the polymer formation adhesion that kept.

The gel systems that described original position forms is easy to use with double syringe or similar device.Gelling time allows the user that gel is applied to ad-hoc location and can not overflow in adjacent area relatively fast.Discuss as mentioned, can change gelling time by changing polymer concentration and/or molecular weight or crosslink density.Therefore, can be easy to inject this system that uses by peritoneoscope or transdermal.Potential use is not limited to peritoneum and can be used for throwing and multiple therapeutic agent (comprising medicament that suppresses adhesion formation and/or the medicament with other required effect).

Hybrid gel for prevention rabbit sidewall damaged-peritoneal adhesion of caecum scratch model is very effective.When comparing the sickness rate of adhesion in 3 fens (it is the firm connection that can only pass through the sharp weapon anatomical isolation), this is especially obvious.Compare with 83.3% incidence rate (example 3) in the untreated animal, in the animal of hybrid gel treatment, do not have adhesion in 3 fens.Similarly, compare with 17% of matched group, 62.5% through the treatment animal do not represent adhesion.3 examples organized adhesion (can separate by blunt dissection) to come across near between place, suture site or two the caecum surfaces (wherein 1 not scratch) in 2 minutes, that is, all zones that relate to adhesion all cover without hybrid gel.Herein, gel is to be applied in the border of damage location.The systemic application of striding large surface area can further increase effect.Generally speaking, hydridization HAX-nano-granular system described herein appears as suitable intraperitoneal drug delivery system and effective barrier of prevention of postoperative adhesion.

Example 10: the peritoneal adhesion that repeats the laparotomy model by the in-situ cross-linked HA gel for prevention that contains tPA

Materials and methods

The optimization of gel strength prepares HA-ADH and HA-CHO as mentioned above.Utilize the HA-ADH with different Mw of variable concentrations (comprising that maximum can reach concentration) and the formulations prepared from solutions HAX gel of HA-CHO.By use turbine mixer with 150 μ l HA-ADH and 150 μ l HA-CHO solution are mixed in the 2ml microcentrifugal tube and subsequently with its under 37 ℃ in hyaluronidase (50U/ml is in PBS) in cultivation prepare the HAX gel.When putting at the fixed time, remove the hyaluronidase buffer fully, and remain the weight in wet base of HAX gel through weight analysis determining.Repeat the laparotomy model and use female albefaction rabbit (Spain hare (Oryctolagus cuniculus); New Zealand white (NewZealand White)) (3 ± 0.5kg).Execution as indicated above is laparotomy and post-operative care for the first time.The back execution of 1 week is laparotomy for the second time.When checking before the 2nd laparotomy, following animal is got rid of from the 2nd laparotomy: (i) body weight loss surpasses 15% owing to the 1st laparotomy; (ii) eating disorder.When carrying out laparotomy, as described above to adhesion scoring dissolving (cutting) subsequently.Use aseptic brush, unidirectional scratch again is before through open abdomen 50 times and two-way caecum surface 150-200 time that abrades again between the 6th and the 12nd bag, up to hemorrhage of acquisition (bleeding bed).

30 animals are assigned to experimental group at random and test material is applied to affected area.Operator is to character the unknown of described material.Be dissolved in respectively in the 5ml physiological saline solution with HA-ADH and HA-CHO sterilization 2 hours and with it by ultraviolet-sterilization illumination.As described above, utilize the bivalve applicator that the 10ml gel is applied to damaged part.In contrast, make a treated animal not receive treatment, and another group of received 10ml normal saline.

After the treatment affected area, utilize 2-0 to treasure good suture (Ethilon) and closed peritoneum of 3-0 fenaminosulf suture (Dexon) and stomach wall respectively.Utilize 2-0 to treasure the closed skin of good suture.Animal is waken up and freely take food and water.In the postoperative 48 hours, with 8-12 hour at interval through subcutaneous throwing and buprenorphine 0.02-0.03mg/kg.1 week was put to death animal by the intravenous injection pentobarbital sodium after the 2nd laparotomy.In two ways adhesion is marked: (i) quality score of adhesion is as follows: the amending method that uses the method for being reported is to adhesion scoring (this JW of Berne (Burns JW), this Jenner K (Skinner K), examine special J (Colt J), shed woods A (Sheidlin A), the gloomy R of Blang (Bronson R), Ya Kebai Y people such as (Yaacobi Y) is by preventing tissue injury and tissue adhesion (Preventionof tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acidsolutions.) operations research magazine (Journal of Surgical Research) 1995 with hyaluronic acid solution precoating tissue; 59:644-652, it is incorporated herein by reference).0 minute=no adhesion, 1 minute=utilize gravity to adhere to by isolating tissue; 2 minutes=can adhere to by the isolating tissue of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.If have a plurality of adhesions, select score the higher person so as representative score with different scores; (ii) measured 2 fens or the area of adhesion in 3 fens to be used for adhesion Fixed AmountAssessment.(whether it comprises the suture site to note the position of adhesion; The position and the quantity of related caecum bag in the adhesion) and the weight change.The fixation of tissue that will be reclaimed by postmortem is embedded in the paraffin in 10% formalin, and section is also dyeed for histological examination with haematoxylin and Yihong.

The result

Viscosity can stop the concentration of the HA solution of previous use to increase (especially HA-ADH).For further increasing concentration, use the HA of low Mw to prepare modified HA.As shown in table 6, maximum actual concentrations (concentration that can utilize suction pipe or syringe to handle) increases with the reduction of the Mw of HA.

The Cmax of the modified HA solution of table 6.

HA-ADH *	Concentration max(mg/ml)	HA-CHO *	Concentration max(mg/ml)
				10kD	180	50kD	150
50kD	75	490kD	60
				490kD	20	1360kD	60

^*Should notice that listed Mw is the nominal Mw of original HA.

All have the optimization of gel strength the HAX gel that increases concentration and all continue the time (table 7) more much longer than 20mg/ml HAX gel.It should be noted that the longest gel of persistent period is not the maximum concentration gel.Although have high density, the gel of being made up of low Mw HA (that is, having many breach on the main chain) seems to have mass loss faster in the latter half of degraded.In the HA combination of being tested, 75mg/ml HA-A (50kD) and 60mg/ml HA-B (1.36MD) provide the HAX gel of degraded the most slowly.(t in the 50u/ml hyaluronidase _1/2: 51 days).To be called HAX by the gel that 75mg/mlHA-ADH (50kD)+60mg/ml HA-CHO (1.36MD) forms _Hx

The table 7.HAX half-life of gel in the 50u/ml hyaluronidase

HA-ADH	HA-CHO	T in the 50u/ml hyaluronidase 1/2(my god) a
			490k，20mg/ml	1.36M，20mg/ml	5
490k，20mg/ml	50k，150mg/ml	11
			50k，75mg/ml	50k，150mg/ml	27
10k，180mg/ml	50k，150mg/ml	17
			50k，75mg/ml	1.36M，60mg/ml	51 b
50k，75mg/ml	490k，60mg/ml	22.5

A. the hydrogel weight in wet base reduces for 50% time

B. measure by extrapolation

Repeat this model of laparotomy Study of model and produce extensive, reproducible adhesion.Because the abundant adhesion of its generation is very big challenge for treatment, thus itself and be of little use.Repeat the laparotomy model and make that can assess candidate material prevents severe adhesion and/or recur the effect of adhesion (commercially available prod only represents limited action to it).In addition, this model makes and can differentiate even than the more effective material of material described in the previous examples.

After the 1st laparotomy, 29 (96.7%) development adhesion in 3 fens (with part adhesion in 2 fens) is arranged in 30 animals.Described adhesion relates generally to the stomach wall of incision and through abrasive caecum bag, also relates to usually not abrading caecum (mainly around ileocecus, because its position makes it can contact the peritoneal wall of incision).In the 1st when week after the 1st laparotomy, animal loses-6.1 ± 3.9% body weight.By sharp weapon or blunt dissection dissolve accretions carefully.Dissolving causes the degree of depth damage of stomach wall and subsequently hemorrhage usually.Through abrasive caecum is easier to be more hemorrhage than initial scratch again.If without any treatment close incisions, when detecting once more after 1 week, 100% (n=6) developed adhesion in 3 fens.Adhesion is not limited to damaged location, and relates between stitching thread, not impaired caecum surface and/or caecum on the stomach wall, between caecum proximal colonic surface.The intermediate value gross area of adhesion is 12.7 (25%:94,75%:16.6) cm ²Between the survival period in 1 week, animal loses-3.5 ± 7.4% body weight after the 2nd laparotomy.

The in vivo effect of hydrogel is used and is repeated laparotomy model measurement tPA-HAX _HxThe in vivo effect of gel and with it with the HAX gel with contain the hybrid gel of PLGA nano-particle (granule of 1:1: effect HA derivant ratio (w/w)) is compared.As other contrast, also throw the tPA-HAX of heat treatment inactivation with (i) tPA _HxGel; (ii) do not exist the tPA solution group of HAX to annotate.Experimental program is summarized in down:

The result is summarized in the table 8.In simple terms, the most effective by the tPA-HAX that uses high concentration HA derivant to realize for the adhesion in the dual damage model of prevention with high crosslink density (" hx ").Although HAX (hx) (no tPA) is not too effective, the reduction that tPA (tPA solution is applied on the damaged tissues) causes the adhesion area is annotated by non-activity tPA-HAX (hx) and group.Do not wish to be bound by any theory, this effect may be relevant with the non-active ingredient in the activating enzymes (tPA, gene enzyme company).The activating enzymes that contain 100mg tPA also contain 3.5g L-arginine, 1g phosphoric acid, polysorbate80 (＜11mg).Therefore, these medicaments all are suitable for indivedual or make up and forgive in hydrogel of the present invention.

Generally speaking, HAX and hybrid gel can be realized the reduction (about 17%) of the relative appropriateness of the middle-and-high-ranking other adhesion of this model, and the HAX that contains tissue plasmin activator (tPA) makes the incidence rate of high-level adhesion obviously reduce by 60% and make the surface area of these adhesions reduce 100 times.Importantly, described treatment benefit is to obtain not causing under the hemorrhage situation of general, and the hemorrhage subject matter that has become the free tPA of use that is reported of described general.

Table 8: the effect that contains the HAX gel of tPA

1.tPA be to use (2.2mg activating enzymes+L-arginine+phosphoric acid+polysorbate 80) with the form that gene enzyme company is provided.

2.tPA through boiling 20 minutes inactivations.Utilize ELISA to determine the destruction of protein configuration.

Example 11: the peritoneal adhesion that the hydrogel that prevention is formed by in-situ cross-linked HA and cellulose derivative causes

This case description is by the research of the original position crosslinkable hydrogel of HA and cellulose derivative (such as CMC (carboxymethyl cellulose), MC (methylcellulose) and HPMC (hydroxypropyl emthylcellulose)) formation.

Materials and methods

The preparation of HA-ADH, HA-CHO, CMC-CHO, MC-CHO, HPMC-CHO: described scheme is substantially the same with the scheme that is used for synthetic HA-CHO mentioned above.

The preparation of disk hydrogel: use double syringe with the aqueous solution of 2wt% HA-ADH and 2wt% HA-CHO, CMC-CHO, HPMC-CHO or MC-CHO in being clipped in two rubber molds between the slide glass.Prepared hydrogel's diameter and thickness are respectively 1.2cm and 3.5mm.

The result

Synthetic and the sign of CMC-CHO, HPMC-CHO and MC-CHO confirms successfully synthetic by NMR and FT-IR.Weight average molecular weight M by gpc measurement _wBe 10 ³To 10 ⁷The degree of modification of HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO is about 50%.

The gelling time of HA-CMC, HA-HPMC and HA-MC: by forming multiple hydrogel with HA-ADH and different aldehyde polysaccharide (such as CMC-CHO, MC-CHO and HPMC-CHO) are crosslinked.As shown in table 9, the gelling time of HAX (being formed by crosslinked HA-ADH and HA-CHO), HA-HPMC, HA-MC and HA-CMC shows that it is for in-situ cross-linked adaptability in about 3-18 scope of second.

The shear modulus G of HA-CMC, HA-HPMC and HA-MC: the measured G-value of being measured by flow graph is showed in the table 9, and HA-HPMC represents relative higher value with HA-MC.

Gelling time and the modulus of shearing of table 9.HA-CMC, HA-HPMC and HA-MC

Hydrogel is expelled in the peritoneum and will be expelled in the mouse peritoneum by the 1ml hydrogel that 0.5ml 2wt% HA-ADH and 0.5ml 2wt% CMC-CHO (or MC-CHO, HPMC-CHO) form.After in being expelled to mouse peritoneum, these hydrogel biocompatibility are good.Injected back 4 days, and found to be the HA-HPMC gel of adhesion block form.On the other hand, HA-CMC and HAX are in the diffusion of whole intraperitoneal and cover all organs.HA-MC has intermediate structure/denseness.In injection 2 weeks of back, almost there is not residue in the animal of injection HAX.The HA-HPMC gel still kept when 2 weeks.The HA-CMC gel also keeps and covers internal organs with skim.As shown in table 10, the injection of these gels can not cause peritoneal adhesion.

Table 10: the sickness rate of adhesion

Hydrogel	The 4th day	The 1st week	The 2nd week	The 3rd week	Amount to
						HAX	0/2	0/1	0/1	0/1	0/5
HA-CMC	0/3	0/1	0/1	0/6	0/6
						HA-HPMC	0/2	0/1	1/1	0/1	1/5
HA-MC	0/2	0/1	0/1		0/4

Also test the ability that suppresses the adhesion of the rabbit adhesion model described in the example 3 by crosslinked HA derivant and the formed composite aquogel of cellulose derivative.In simple terms, with HA-ADH together with CMC-CHO, MC-CHO or HPMC-CHO throw with to form 2wt%HA-CMC, HA-MC, HA-HPMC hydrogel.The normal saline injection is compared.As shown in table 11, HA-CMC, HA-MC and HA-HPMC represent good peritoneum and adhere to preventive effect.

Table 11: rabbit test result (every group of 4 rabbits)

Example 12: the hydrogel by in-situ cross-linked hyaluronic acid (HA) and cellulose derivative prevents peritoneal adhesion

Foreword

Operation posterior peritoneum adhesion can causing pelvic pain, intestinal obstruction and infertile (Di Zejia G.S. (DiZerega, G.S.), operation on peritoneum (Peritoneal Surgery.) 1999. New York: Springer (New York:Springer); It is incorporated herein by reference).At the multiple film barrier of commercial research device, obtain in various degree success (Di Zejia G.S. (and DiZerega, G.S.), operation on peritoneum (Peritoneal Surgery.) 1999. New York: Springer (New York:Springer); It is incorporated herein by reference).Because the gel that is formed by two kinds of different polymer in situ of simple mixing is easy at room temperature operate and does not need radiating light source or poisonous chemical cross-linking agent, thus described for this purpose gel cause people interest (Jones D.B. (and Johns, D.B.); Rogers K.E. (Rodgers, K.E.); Tang Naxiu W.D. (Donahue, W.D.); Qi Wopusi T.C (Kiorpes, T.C); Di Zejia G.S. (diZerega, G.S.), reduce adhesion (Reduction of adhesion formation bypostoperative administration of ionically cross-linked hyaluronic acid.) fertility and sterile (FertilSteril) 1997 by the back throwing of performing the operation with the ionomer hyaluronic acid; 68 (1): 37-42; Lee H. (Li, H.); Liu Y.C (Liu, Y.C); Easypro X.Z. (Shu, X.Z.); Gray S.D. (Gray, S.D.); Prestwich G.D. (Presrwich, G.D.), the synthetic and biological assessment (Synthesis and biological evaluation of a cross-linkedhyaluronan-mitomycin C hydrogel) of crosslinked hyaluronan-ametycin hydrogel. biomacromolecule (Biomacromolecules) 2004; 5 (3): 895-902; Liu Y.C (Liu, Y.C); Lee H. (Li, H.); Easypro X.Z. (Shu, X.Z.); Gray S.D. (Gray, S.D.); Prestwich G.D. (Prestwich, G.D.), the crosslinked hyaluronan hydrogel that contains ametycin reduces operation postabdomen adhesion (Crosslinked hyaluronan hydrogels containing mitomycin C reducepostoperative abdominal adhesions.) fertility and sterile (Fertil Steril) 2005; 83:1275-1283; Europe S.H. (Oh, S.H.); Gold nurse J.K. (Kim, J.K.); Song K.S. (Song, K.S.); Promise and S.M. (Noh, S.M.); Gill S.H. (Ghil, S.H.); Excellent gram S.H. (Yuk, S.H.); Lee J.H. (Lee, J.H.), utilize the sol-gel transition behavior to restrain mixture prevention of postoperative tissue adhesion (Prevention ofpostsurgical tissue adhesion by anti-inflammatory drug-loaded pluronic mixtures with sol-geltransition behavior.) biomaterial research magazine A (J Biomed Mater Res A) 2005 by the general stream Buddhist nun who is loaded with anti-inflammation drugs; 72 (3): 306-16; Yi Ou Y. (Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogelsprevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; The special P. of boolean's skin (Bulpitt, P.); Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemical modification of hyaluronic acid:Preparation of functionalizedderivatives and their use in the formation of novel biocompatible hydrogels.) the biomedical material research magazine (J Biomed Mater Res) 1999 of novel bio-compatible hydrogel; 47 (2): 152-169; Merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciatic nerveblockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): 4797-4804, described document is incorporated herein by reference separately).It is easy to be applied on the affected area usually, especially when being difficult to simple sheet covers or all the more so when described zone is very big.

Hyaluronic acid (HA) be described application the good candidate material (Jones D.B. (and Johns, D.B.); Rogers K.E. (Rodgers, K.E.); Tang Naxiu W.D. (Donahue, W.D.); Qi Wopusi T.C (Kiorpes, T.C); Di Zejia G.S. (diZerega, G.S.), reduce adhesion (Reductionof adhesion formation by postoperative administration of ionically cross-linked hyaluronicacid.) fertility and sterile (Fertil Steril) 1997 by the back throwing of performing the operation with the ionomer hyaluronic acid; 68 (1): 37-42; Lee H. (Li, H.); Liu Y.C (Liu, Y.C); Easypro X.Z. (Shu, X.Z.); Gray S.D. (Gray, S.D.); Prestwich G.D. (Presrwich, G.D.), the synthetic and biological assessment (Synthesis and biological evaluationof a cross-linked hyaluronan-mitomycin C hydrogel) of crosslinked hyaluronan-ametycin hydrogel. biomacromolecule (Biomacromolecules) 2004; 5 (3): 895-902; Liu Y.C (Liu, Y.C); Lee H. (Li, H.); Easypro X.Z. (Shu, X.Z.); Gray S.D. (Gray, S.D.); Prestwich G.D. (Prestwich, G.D.), the crosslinked hyaluronan hydrogel that contains ametycin reduces operation postabdomen adhesion (Crosslinked hyaluronan hydrogels containing mitomycinC reduce postoperative abdominal adhesions.) fertility and sterile (Fertil Steril) 2005; 83:1275-1283; Yi Ou Y. (Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogelsprevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; The special P. of boolean's skin (Bulpitt, P.); Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemical modification of hyaluronic acid:Preparation of functionalizedderivatives and their use in the formation of novel biocompatible hydrogels.) the biomedical material research magazine (J Biomed Mater Res) 1999 of novel bio-compatible hydrogel; 47 (2): 152-169; Merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciatic nerveblockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): 4797-4804, described document is incorporated herein by reference separately), this is because HA is at intraperitoneal bio-compatible (Di Zejia G.S. (DiZerega as everyone knows, G.S.), operation on peritoneum (Peritoneal Surgery.) 1999. New York: Springer (New York:Springer), it is incorporated herein by reference) and chemical crosslinking HA hydrogel (HAX) can prevent peritoneal adhesion in the rabbit model (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesionsin a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference).Hyaluronic acid through endogenous hyaluronidase (Ke Nipu P.A. (and Knepper, P.A.); Method Berman A.I. (Farbman, A.I.); Tai Sier A.G. (Telser, A.G.), hyaluronic degraded in external source hyaluronidase and the lagophthalmos (ExogenousHyaluronidases And Degradation Of Hyaluronic-Acid In The Rabbit Eye.) ophthalmology research and vision (Investigative Ophthalmology and Visual Science) 1984; 25 (3): 286-293; It is incorporated herein by reference) and hydroxyl (Suo Te L (Soltes, L.); Man Diqi R. (Mendichi, R.); Gram root G. (Kogan, G.); Si Qile J. (Schiller, J.); Si Tankewaka M. (Stankovska, M.); Arnold J. (Arnhold, J.), reactive oxygen species is for Degradation (the Degradative action of reactive oxygen species onhyaluronan.) biomacromolecule (Biomacromolecules) 2006 of hyaluronan; 7 (3): 659-668; Outstanding N. (Yui, N.); Kano T. difficult to understand (Okano, T.); Sakurai well Y. (Sakurai, Y.), the reactive degraded of the inflammatory of cross-linked hyaluronic acid gel (Inflammation Responsive Degradation Of Cross-Linked Hyaluronic-Acid Gels.) controlled release magazine (J Control Rel) 1992; 22 (2): 105-116, described document is incorporated herein by reference separately) degraded.Confirmed that the HAX gel degrades in fact in 1 week, intraperitoneal retain the amount of obvious minimizing gel (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels preventpost-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference).Yet, to decide on the order of severity or the area of damage, design can will be useful at the hydrogel of intraperitoneal last much longer.Suppose with HA with can be in human body other bio-compatible polysaccharide heterozygosis of enzymatic degradation can when keeping the good biocompatibility of HA, not slow down degraded.Also known such as carboxymethyl cellulose (CMC) (Ai Jinsi T.E. (and Elkins, T.E.); Berli R.J. (Bury, R.J.); Auspicious special J.L (Ritter, J.L.); Icepro F.W. (Ling, F.W.); Ao Kasi R.A. (Ahokas, R.A.); Huo Musi C.A. (Homsey, C.A.); Malin blocks L.R. (Malinak, L.R.), in rat 1, give birth to and sterile (Fertil Steril) 1984 by carboxymethylcellulose sodium solution Film with Preventing Adhesion (Adhesion Prevention By Solutions Of Sodium Carboxymethylcellulose InThe Rat.1.); 41 (6): 926-928; Liu L.S. (Liu, L.S.); Burger R.A. (Berg, R.A.), the adhesion barrier of carboxymethyl cellulose and polyethylene glycol oxide pluralgel (Adhesion barriersof carboxymethylcellulose and polyethylene oxide composite gels.) biomedical material research magazine (J Biomed Mater Res) 2002; 63 (3): 326-332; Lille C.M. (Lehr, C.M.); Bai Wasite J.A. (Bouwstra, J.A.); This peculiar E.H. (Schacht, E.H.); Jia Yinge H.E. (Junginger, H.E.), in vitro assessment (Invitro Evaluation OfMucoadhesive Properties Of Chitosan And Some Other Natural Polymers.) the Inpharm magazine (International Journal of Pharmaceutics) 1992 of the mucosa blocking characteristics of chitosan and some other natural polymers; 78 (1): 43-48; Jessica Lynch R.E. (Leach, R.E.); This J.W. of Berne (Burns, J.W.); David E.J. (Dawe, E.J.); This Meath crust uncle M.D. (SmithBarbour, M.D.); Moral M.P. (Diamond covers in Dell, M.P.), utilize hyaluronate/carboxymethyl cellulose gel to reduce tissue adhesion formation (Reduction of postsurgical adhesion formation in the rabbituterine horn model with use of hyaluronate/carboxymethylcellulose gel.) fertility and sterile (FertilSteril) 1998 in the rabbit uterus angle model; 69 (3): 415-418); Described document is incorporated herein by reference separately) and carboxylic propyl methocel (HPMC) (Lille C.M. (Lehr, C.M.); Bai Wasite J.A. (Bouwstra, J.A.); This peculiar E.H. (Schacht, E.H.); Jia Yinge H.E. (Junginger, H.E.), in vitro assessment (Invitro Evaluation Of Mucoadhesive Properties Of ChitosanAnd Some Other Natural Polymers.) the Inpharm magazine (International Journal of Pharmaceutics) 1992 of the mucosa blocking characteristics of chitosan and some other natural polymers; 78 (1): 43-48; Described document is incorporated herein by reference) etc. cellulose derivative have excellent biological compatibility at intraperitoneal.Methylcellulose (MC) is still unknown at endoperitoneal biocompatibility, but according to reports, the mixture of MC and HA in intrathecal injection liquid bio-compatible (ancient handkerchief tower D. (and Gupta, D.); The special C.H. of tower (Tator, C.H.); Xiao Qite M.S. (Shoichet, M.S.), be used for the hyaluronan of localized delivery in the sheath of impaired spinal cord and fast gelation injectable admixture (the Fast-gelling injectable blend of hyaluronan and methylcellulosefor intrathecal localized delivery to the injured spinal cord.) biomaterial (Biomaterials) 2006 of methylcellulose; 27:2370-2379, it is incorporated herein by reference).

Synthesized the in-situ cross-linked injection aquagel that constitutes by HA and cellulose derivative (such as CMC, HPMC and MC).Also characterize these hydrogels in vitro, study its for the cytotoxicity of cell culture with and biocompatibility in the Muridae peritoneum.At last, also study the effectiveness that it is used for preventing the rabbit model peritoneal adhesion.

Materials and methods

Synthesizing of polymer and hydrogel

Reagent: HA (M _w=490kDa and 1.4MDa) available from gene enzyme company (Cambridge, Massachusetts (Cambridge, MA)).CMC (production code member: C4888), HPMC (production code member: H9262), MC (production code member: M0387), hyaluronidase, adipic dihydrazide (ADH), 1-ethyl-3-[3-(dimethylamino) propyl group]-carbodiimides (EDC), hydroxybenzotriazole (HOBt), sodium metaperiodate, ethylene glycol, tert-butyl carbazate (t-BC), sodium bicarbonate, sodium chloride and acetic acid is all available from Sigma-aldrich corp (St. Louis, the Missouri State (St.Louis, MO)).To be used as the standard substance of gel permeation chromatography (GPC) available from the pulullan polysaccharide of Showa Denko K. K (Showa Denko) (Japan).

The preparation of aldehyde polymer:As shown in figure 12,1.4MDa HA, CMC, HPMC and MC are modified into aldehyde form (being respectively HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO).Described scheme is similar to scheme (gram Chinese DS (Kohane DS), the general M in lining (Lipp M), golden Buddhist nun RC (Kinney RC), Anthony DC (Anthony DC), the Louis DN (Louis DN) that before is used for HA-CHO; Luo Dan N people such as (Lotan N) is contained biocompatibility (Biocompatibility oflipid-protein-sugar particles containing bupivacaine in the epineurium.) the biomedical material research magazine (J Biomed Mater Res) 2002 of lipid-protein-sugared granule in epineurium of bupivacaine; 59 (3): 450-459; Gram Chinese DS (Kohane DS), carry this JY (Tse JY), Yi Ou Y (Yeo Y), Pa Dela R (Padera R), Shu Baina M (Shubina M), bright lattice R. (Langer R.) are used for biodegradable polymerization microsphere and nanometer spheroid (Biodegradable polymericmicrospheres and nanospheres for drug delivery in the peritoneum.) the biomedical material research magazine (J Biomed Mater Res) 2005 that the peritoneum Chinese medicine transmits: publish; Ao Ruita H (Orita H), Fu Kasiwa M (Fukasawa M), Kirghiz Republic W (Girgis W), the inhibition of tissue adhesion in Di Zejia GS. (diZerega GS.) the standard rabbit model: utilize the tissue plasmin activator to carry out the international fertility of intraperitoneal treatment (Inhibition of postsurgical adhesions in astandardized rabbit model:intraperitoneal treatment with tissue plasminogen activator.) magazine (Int J Fertil 1991); 36 (3): 172-177, described document is incorporated herein by reference separately).In simple terms, 1.5g HA, CMC, HPMC or MC are dissolved in the 150ml water, add the 802mg sodium metaperiodate subsequently, and stirred 2 hours.Add 200 μ l ethylene glycol with cessation reaction, and relative immediately water dialysis mixture.With the purified product lyophilizing and in 4 ℃ of storages down.

The preparation of hydrazides polymer:Use previous described method with 490kDa HA be modified into adipic dihydrazide HA (HA-ADH) (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronicacid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; The special P. of boolean's skin (Bulpitt, P.); Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemical modification of hyaluronic acid:Preparation offunctionalized derivatives and their use in the formation of novel biocompatible hydrogels.) the biomedical material research magazine (J Biomed Mater Res) 1999 of novel bio-compatible hydrogel; 47 (2): 152-169; Merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciaticnerve blockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): 4797-4804, described document is incorporated herein by reference separately).

The preparation of disk hydrogel: use double syringe (hundred special companies (Baxter), this state Deere of Illinois takes moral (Deerfield, IL)) and the 2wt%HA-ADH among the PBS and the 2wt%HA-CHO among the PBS, CMC-CHO, HPMC-CHO or MC-CHO are expelled to are clipped in two rubber molds between the slide glass.Prepared hydrogel's diameter and thickness are respectively 1.2cm and 3.5mm.Hereinafter respectively these cross-linked hydrogels are called HAX, HA-CMC, HA-HPMC and HA-MC.

The sign of polymer and hydrogel

The sign of polymer:Carry out the D of 10mg/ml HA-ADH, HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO ₂O solution ¹(Varian technology company (Varian): excellent Buddhist nun carries 300 type spectrophotometers (Unity 300 spectrophotometer) to H-NMR, California Paro Austria many (Palo Alto, CA)) spectrum analysis.As the X.Q. that goes into business (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongationof sciatic nerve blockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): described in the 4797-4804, after aldehyde polymer (HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO) reacts with t-BC, it is analyzed.Measurement is at D ₂The polymer that reacts with t-BC among the O ¹H-NMR spectrum.

Use the molecular weight of gpc measurement polysaccharide.Post is water generation hydrogel linear columns (Ultrahydrogel Linear) (water generation company (Waters), Massachusetts Penelope Milford (Milford, MA)), and refractive index (RI) is by refractometer (Wyatt Technology: OPTILAB DSP (Wyatt Technology:OPTILAB DSP), the holy tower Barbara in California (Santa Barbara, CA)) detects.Mobile phase is the mixture of 0.05M sodium and 0.2M sodium chloride (pH=6.7), and its flow rate is 0.8ml/min.Pulullan polysaccharide (Sai Duolisi (Shodex), pulullan polysaccharide standard substance P5-P800 (Pullulan Standards P5-P800), Japan) is used as the molecular weight standard product.

Hydrogel characterizes:Measure gelling time by following scheme.100 microlitre HA-CHO, CMC-CHO, HPMC-CHO or MC-CHO aqueous solution are added in the 100 microlitre HA-ADH aqueous solutions, on the Pi Shi culture plate, use hot plate/agitator (healthy and free from worry PC-320 type (Corning:Model PC-320), New York Corning Incorporated (Corning, NY)) utilizes magnetic stirring bar to mix with 155rpm.Gelling time is the time of mixture when becoming bead; Each sample is measured 4 times.

(AR1000 of TA instrument company (TA Instruments:AR1000), knob Karst, De Lahua state (New Castle, Delaware)) measures the modulus of shearing of prepared disc gel to utilize flow graph.Disc gel immersed reach 5 days among the PBS and make its expansion reach balance.Carry out creep and lax test with different shear stresses.Applied shear stress 3 minutes, be subsequently 3 minutes lax.In each the measurement, reach constant and it gets back to zero at lax testing period chien shih subsequently at creep test period strain value.Slope by the linear relationship between the stress and strain calculates shear modulus G.The R of the fit line between the stress and strain ²Value is higher than 0.95.

In PBS, measure the expansible time-histories of gel dish under 37 ℃ in the gravimetric analysis mode.In PBS, soak the weight W of measuring hydrogel after the gelling after 5 days _sW _sInitial weight W with hydrogel after the gelling just _iExpansion ratio Q be with Q=W _s/ W _iCalculate.

Degradation kinetics is measured as follows: cultivate 4 water-setting lacquer disk(-sc)s in the 10 units per ml hyaluronidases under 37 ℃ in PBS.When each time point, the gel dish is weighed, and replace the hyaluronic acid enzymatic solution.Measure and reach 14 days.The ratio of the volume of hydrogel and initial volume when measuring each time point (volume of hydrogel (%)).

Cytotoxicity analysis

Use human mesothelial cell (ATCC:CRL-9444, Virginia Malthus (Manassas, VA)) and macrophage cell line J774.A1 (ATCC:TIB-67 ^TM) analyze (Pu Luomaige company (Promega), the in vitro cell viability of state of Wisconsin Madison (Madison, WI)) research under the situation that has HA-CHO, CMC-CHO, MC-CHO and HPMC-CHO by MTT.

Under 37 ℃ in 5% CO ₂In, the mesothelial cell is grown in complete growth medium (GIBCO: contain Yi Ershi BSS (Earle ' s BSS), 0.75mM L-glutaminate and 1.25g/L sodium bicarbonate and be supplemented with the culture medium 199 of 3.3nM epidermal growth factor, 400nM hydrocortisone, 870nM insulin, 20mM HEPES and 10% hyclone) and keep.Make macrophage the DMEM culture medium (GIBCO: the DMEM catalog number (Cat.No.) 10569-010 with 10% hyclone) growth and keep.With 5 * 10 ⁴Individual cell is put into each hole of 24 well culture plates, and under 37 ℃ in 5% CO ₂The middle cultivation whole night replaced culture medium subsequently with the culture medium that contains variable concentrations HA-B, CMC-B, HPMC-B and MC-B.After adding these materials under mesothelial cell's the situation the 3rd day or under the situation of J774.A1 cell the 2nd day, carry out MTT and analyze.100 microlitre tetrazolium solution are added in each hole and at 37 ℃ to descend to cultivate 4 hours.Use 1ml detergent solution will be dissolved by the purple Jia Za (formazan) that the active wire plastochondria produces and pass through the plate reader (SpectraMax of molecular device company 384 types (Molecular Devices:SpectraMax384) at 570nm subsequently, (Union City, CA)) reads associating city, California.With absorbance about each hole standardization without the cell of polymer treatment.

In vivo experiment

Animal care according to the Massachusetts Institute of Technology (Massachusetts Institute of Technology) is nursed all animals with use committee's (Animal Care and Use Committee) scheme of being ratified and the nursing principle of laboratory animal (NIH announces #85-23, revision in 1985).

Hydrogel is expelled in the mouse peritoneum

The SV129 mice of heavy 25g be available from tower Kornic Systems Corp. (New York breathe out moral grandson (Hudson, NY)) and with its fraction set with the 6AM-6PM stable breeding that circulates daytime-night.

Shine 2 hours with polymer sterilization by UV, subsequently it is dissolved in the normal saline with 2wt% concentration.Utilize 50mg/kg ketamine and 10mg/kg xylazine to lure anesthesia into, and produce the 5mm skin incision, expose translucent stomach wall.Stomach wall is passed in No. 24 remaining needles (Terumo company: Se Fuxi venous detaining needle (Terumo:Surflash I.V.Catheter), Japan) placement, and be blown into the 0.3ml air to determine the location.Make the remaining needle 1cm that advances subsequently, and use double syringe applicator (hundred special companies (Baxter), this state Deere of Illinois takes moral (Deerfield, IL)) injection 0.5ml aldehyde polysaccharide (HA-CHO, CMC-CHO, HPMC-CHO or MC-CHO) and 0.5ml HA-ADH.

Inject back 4 days, 1 week, 2 weeks and 3 week the back put to death mices, and the existence of assessment residue and adhesion.It is unknown which kind of treatment the dissector receives to indivedual mices.When needing sampling,, be fixed in 10% formalin and use standard technique to handle for histologic analysis (the painted slide glass in haematoxylin Yihong) to the abdominal contents sampling.

By the rabbit sidewall damaged-preventive effect of intestinal scratch model evaluation peritoneal adhesion

Bring out peritoneal adhesion (Yi Ou people such as (Yeo), original position crosslinkable hyaluronic acid gel prevention rabbit model operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels prevent post-operativeabdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 as described; 27:4698-4705, it is incorporated herein by reference).Use ketamine (intramuscular 35mg/kg) and xylazine (intramuscular 5mg/kg) to make female albefaction rabbit (Spain hare (Oryctolagus cuniculus); New Zealand white (New Zealand White), Ke Wensi company, Pennsylvania Hei Zeerdun (Hazleton, PA)) (3 ± 0.5kg) anesthesia, and use the 1-3% isoflurane in the equilibrium oxygen to keep.Produce the long midline incision of 10cm along white line, and open peritoneum.Bring out peritoneal adhesion by on the stomach wall of right side, producing damaged and scratch caecum 7 bags of 3 * 4cm up to obtaining hemorrhage surface.

4 animals of each experimental group random assortment: (i) normal saline; (ii) utilize the crosslinked HA-CMC of 10ml, HA-HPMC or HA-MC to cover through open abdomen with through scratch caecum surface.Before using, be dissolved in the physiological saline solution with material sterilization 2 hours and with it by the ultraviolet-sterilization illumination.(5ml HA-ADH (20mg/ml) and 5ml CMC-CHO, HPMC-CHO or MC-CHO (20mg/ml)) puts into the 10ml independence asepsis injector that links to each other with the double syringe applicator with gel precursors solution, and by No. 15 pin coextrusion.Liquid precursor begins gelling immediately, meets the shape of target region.

Give as described above the postoperative animal care (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkablehyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference).In 1 week after the program, utilize intravenous 100mg/kg pentobarbital sodium that animal is implemented euthanasia.Use the method for being reported that adhesion is marked: 0 minute=no adhesion, 1 minute=utilize gravity to adhere to by isolating tissue; 2 minutes=can adhere to by the isolating tissue of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.Also measured 2 fens and the area of adhesion in 3 fens.As indicated above sample of tissue and the preparation of being paid close attention to is used for histologic analysis.

Statistical analysis

By ANOVA student t check analysis data then.Adhesion score between each hydrogel and the contrast is carried out the inferior rank test of Wilcock (Wilcoxon rank-sum test).Utilize card radar lattice to draw

(Si Niji software company (Synergy Software)) carries out statistical test.It is significant that p value＜0.05 is considered to statistics.

The result

Synthetic and the sign of HA-ADH, HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO

By the methene proton of adipic dihydrazide determine HA-ADH synthetic (having doublet) at 1.62ppm place tool singlet and at 2.25ppm and 2.38ppm place (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situcross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in arabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; The special P. of boolean's skin (Bulpitt, P.); Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemical modification ofhyaluronic acid:Preparation of functionalized derivatives and their use in the formation ofnovel biocompatible hydrogels.) the biomedical material research magazine (J Biomed Mater Res) 1999 of novel bio-compatible hydrogel; 47 (2): 152-169; Merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronicacid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): 4797-4804, described document is incorporated herein by reference separately).Calculate the degree of modification by the peak area of N-acetyl group-D-glycosamine residue (singlet) of HA and ratio at the peak area of the methene proton of 1.62ppm place adipic dihydrazide at the 2.0ppm place; Degree of modification is 48.4%.

For analyzing the aldehyde radical that forms by oxidation reaction, make aldehyde polymer and t-BC reaction, carry out subsequently ¹H-NMR analyzes.In each aldehyde modified polysaccharide, the chemical shift (the unimodal and 1.43ppm place at 1.20ppm place unimodal) of the tert-butyl group appears, show successfully synthetic HA-CHO, CMC-CHO, HPMC-CHO and MC-CHO.The M of HA _wAnd M _w/ M _nBe 1432kDa and 5.2.The M of CMC, HPMC and MC _w1MDa.The M of aldehyde polymer _wBetween 109 and 239kDa between, this is less than the M of HA-ADH _w(table 12.1).

The molecular weight of the modified polymer of table 12.1.

* before reported (gram Chinese DS. (Kohane DS), sharp general M (Lipp M), gold Buddhist nun RC (Kinney RC), Anthony DC (Anthony DC), Louis DN (Louis DN), Luo Dan N people such as (Lotan N) is contained bio-compatible (Biocompatibility oflipid-protein-sugar particles containing bupivacaine in the epineurium.) the biomedical material research magazine (J Biomed Mater Res) 2002 of lipid-protein-sugared granule in epineurium of bupivacaine; 59 (3): 450-459, it is incorporated herein by reference).

M _w: weight average molecular weight; M _n: number average molecular weight.

The in vitro physicochemical characteristics of original position hydrogel

HA-ADH and aldehyde modified cellulose derivant all form gel (table 12.2) in the scope in acceptable blink.The gelling time of HA-CMC is obviously grown (p＜0.0001 between HA-CMC and other aldehyde polysaccharide) than the time of all the other materials.

The physical characteristic of table 12.2. cross-linked hydrogel

^*Measure when in phosphate buffered saline, soaking 5 days

Data are mean+SD (n=4)

Hydrogel expands, and soaks to reach balance and maintenance constant (data does not show) in ensuing 4 days in back 1 day in PBS.From start to finish, expansion ratio (table 12.2) ordering is as follows: HAX, HA-CMC〉HA-MC (p＜0.001)〉HA-HPMC (p=0.0053).

Modulus of shearing (table 12.2) G of HAX is less than the modulus of shearing (p＜0.05) of HA-CMC, HA-HPMC or HA-MC.The modulus of shearing of HA-CMC is less than the modulus of shearing (p＜0.05) of HA-HPMC or HA-MC.The significant difference (p=0.93) that does not have G between HA-MC and the HPMC.

The degradation kinetics of hyaluronic acid enzymatic solution

The degradation kinetics of hydrogel in the hyaluronic acid enzymatic solution there are differences (Figure 17).HAX degraded the fastest (when the 1st day and the 2nd day with respect to HA-CMC, p＜0.001).HAX and HA-CMC degrade to the 4th day and the 5th day the time respectively fully.By contrast, HA-MC and HA-HPMC last the also not degraded fully of 2 weeks.A possible reason of these differences is that the crosslinking degree of HA-MC and HA-HPMC is than HAX height.Another reason is not for expanding as HAX is the same with HA-CMC with HA-HPMC owing to HA-MC, so transparent papery acid enzyme can't effectively be diffused into wherein.

Polymer is to the influence of mesothelial cell and macrophage vigor

Under the situation that has multiple concentration aldehyde polymer, cultivate the mesothelial cell.For all polymer, cell viability all exists dose dependent to reduce.0.3% (w/v) down HA-CHO and MC-CHO do not make cell viability reduce (p〉0.05) (Figure 18 A), and HPMC-CHO (p=0.0011) and CMC-CHO (p=0.044) cause the less reduction of cell viability.Under higher concentration, HA-CHO represents the less reduction of cell viability, and cellulose derivative represents more reduction: cultivate that the ranking compositor of cell viability is HA-CHO after 3 days〉CMC-CHO〉MC-CHO〉HPMC-CHO (under 0.9% (w/v) for any pairing p＜0.01).

The polymer that aldehyde is modified also represents the dose-dependent effects (Figure 18 B) to the macrophage cell viability.In this example, do not find the difference of cell viability between HA and the cellulose derivative.Although there are some significant differences between the chemical compound of some concentration, generally speaking the cell viability between each group is similar.

The biocompatibility of original position hydrogel in the mouse peritoneum

With 1ml gel precursors injection mice (n=4 to 6).In ensuing three weeks, put to death animal and form (table 12.3) with the evaluation adhesion with predetermined time interval.In all 20 animals, only 1 adhesion occurs between bladder and other internal organs.Knownly there are cicatrix and grumeleuse, so think that described adhesion is caused by the direct wound during the gel injection at the adhesion position.

Table 12.3. adhesion behind the intraperitoneal injection water gel in mice

Can't measure the amount of remaining gel in the abdominal cavity.During macroscopy, seem to inject in the animal of HA-MC the hydrogel residual volume than much more (Figure 19) in other animal.After 3 weeks, the HAX complete obiteration, and discovery is the HA-HPMC of the less volume of discrete gel.HA-CMC only keeps with the thin layer that covers internal organs.The peritoneum of all samples and viscera tissue are learned normal.

The prevention of peritoneal adhesion

In rabbit, bring out adhesion (table 12.4) by scratch caecum and one section adjacent stomach wall of incision.In control animal, use normal saline and replace.The development adhesion (Figure 19 B) in big zone of all animals in the normal saline group.In the group of HA-CMC (p=0.0011), HA-MC (p＜0.0001) and HA-HPMC (p＜0.0001) treatment, the area of adhesion greatly reduces.For described parameter, there is not the significant difference of statistics between each gel.The significant difference that the adhesion score reduces only comes across HA-MC (p=0.023) (Figure 19 C).It should be noted that 2 adhesions in 3 fens are to be formed on the tangent line of abdominal part center line (that is, to use the outside in the zone of gel) in the rabbit of HA-HPMC treatment.Describe elsewhere HAX for the prevention peritoneal adhesion effectiveness (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Shellfish Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situcross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in arabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705).

Table 12.4. hydrogel is for the effectiveness of peritoneal adhesion in the prevention rabbit

The animal weight loss is worked as interior loss of week after being meant operation.The adhesion area is the gross area of 2 minutes and adhesion in 3 fens.Weight changes and the adhesion area is to represent (every group of n=4) with mean+SD.

The histologic analysis at adhesion position manifests in the tissue that connects caecum and stomach wall and has fibroblast and inflammatory cells (Figure 20 A) in the animal of normal saline treatment.In the animal of crosslinkable gel for treating, in the hydrogel residue, find neutrophil cell and macrophage (Figure 20 B).Under the situation of Film with Preventing Adhesion, damage location re-epithelialize (Figure 20 C), but compare with normal stomach wall, the fibroblast in the lower floor is remarkable (Figure 20 D) still.Utilize all three kinds of cellulose derivatives to observe similar results.

Discuss

Hydridization HA-cellulose derivative hydrogel provided herein is applicable to peritoneum.Its physicochemical characteristics that comprises gelling time, mechanical strength, water content, expansion kinetics and degradation kinetics all is suitable for desired use.This is confirmed by intra-operative good treatment characteristic and biological achievement.Although precursor polymer represents certain cytotoxicity in vitro, there is no tangible local toxicity in vivo.A kind of possible crosslinked fast few free precursor that retains that is interpreted as.The optimum character of these composites represents by its biocompatibility in Muridae and rabbit model, but long-term safety and effect still remain to be confirmed.At last, hydrogel represents the remarkable effect that reduces adhesion formation.Notice, be present in the region exterior that many adhesions in the rabbit all are positioned at administering therapeutic.Therefore, still unsolved major issue is as described hereinly to use in the mode that is confined to damage location for these material the bests, still more extensive using, or be to be administered to whole peritoneum.

There is the commercially available material that related substances is formed that has that is used to prevent peritoneal adhesion.Suitable

(gene enzyme company) is that (moral people such as (Diamond) covers in Dell for HA and CMC and preformed hydrogel thin slice that 1-(3-dimethylaminopropyl)-the 3-ethyl-carbodiimide hydrochloride is cross-linked to form, reduce adhesion by suitable good fortune film (HAL-F) after the myomectomy: single blind, perspective, multiple center clinical study (Reduction of adhesions afteruterine myomectomy by Seprafilm membrane (HAL-F): A blinded at random, prospective, randomized, multicenter clinical study.) fertility and sterile (Fertility And Sterility) 1996; 66 (6) .904-910; Florian Kringe J. (Kling, J.), the suitable good fortune of gene enzyme company obtains FDA and sells (Genzyme ' s Seprafilm gets FDAmarketing nod.) the natural biological technology (Nature Biotechnology) 1996 of approval; 14 (5): 572-572; Described document is incorporated herein by reference separately).Yin Te ((the Johnson ﹠amp of Johson ﹠ Johnson; Johnson)) be oxidized regenerated cellulose pre-formed sheet (Pa Jidasi K. (and Pagidas, K.); Tu Landi T. (Tulandi, T.), ringer's lactate, influence (Effects Of RingerLactate, Interceed (Tc7) the And Gore-Tex Surgical Membrane On Postsurgical AdhesionFormation.) fertility and sterile (the Fertility And Sterility) 1992 that with film tissue adhesion are formed because of the operation of special crack (Tc7) and GORE-TEX; 57 (1): 199-201, it is incorporated herein by reference).These devices are (suitable at cross-linking chemistry, the described crosslinked molecule that discharges with device of the present invention Discharge carbodiimides, and material as herein described discharges water) and material composition aspect there are differences.Yet the most significant difference is that these materials must use with the form of solid thin-sheet, and the composite that this paper provided is that in-situ gelling forms under the situation of not using cross-linking agent.

But with regard to the type on the type of the device that is easy to the property used application of water gel and possible medicable surface, in-situ crosslinking aquogel has the benefit that is better than conventional barrier device.The material that this paper provided has multiple physicochemical characteristics.Use the hydrazides form (but not HA-ADH) of cellulose derivative for example to come further influencing characterisitic by making the gained gel even enzymatic hydrolysis being had more resistance.Be also noted that there is significant difference in the cost between cellulose derivative and the hyaluronic acid.

In the gel that this paper tested, the HA-MC gel is the most effective for the prevention peritoneal adhesion.This can in vitro degraded be than HA-CMC and HAX slowly and therefore to have the fact of the barrier effect that more prolongs relevant in the hyaluronidase (a kind of enzyme that is present in the peritoneum) with HA-MC.The difference of the effectiveness of various hydrogel Film with Preventing Adhesion is attributable to unknown inherent bioactive difference, and this situation with HA is identical.Can change the effectiveness of Film with Preventing Adhesion by the physicochemical characteristics of further optimization gel.

The physicochemical characteristics of inspected hydrogel comprises gelling time, mechanical strength, water content, expansion kinetics and degradation kinetics.These characteristics are interrelated, and depend on the characteristic of prepolymer to a great extent, such as the concentration in viscosity, electric charge, configuration, dissolubility, degree of modification, the solution, mixing convenience etc.The difference of performance may be to be caused by these parameters between the various polymer, but our result does not make us discern described mechanism.

Relation between known expansion ratio, polymer concentration and the modulus of shearing (this K.S. of Anxi (Anseth, K.S.); Bao Man C.N. (Bowman, C.N.); The rich Richard Pipes L. (BrannonPeppas that exerts, L.), mechanical property (Mechanical properties of hydrogels and their experimental determination.) biomaterial (Biomaterials) 1996,17 (17): the 1647-1657 of hydrogel and its measuring; Richard Pipes N.A. (Peppas, N.A.); Wheat riel E.W. (Merrill, E.W.), as cross-linking polyvinyl alcohol hydrogel (CrosslinkedPolyvinyl-Alcohol) the Hydrogels As Swollen Elastic Networks. of expansion elastomeric network) journal of applied (J ApplPolym Sci) 1977; 21 (7): 1763-1770, described document is incorporated herein by reference separately), above the physicochemical characteristics of being studied shows that the crosslinking degree of HA-HPMC and HA-MC may be than HAX and HA-CMC height.Higher crosslink density may partly be by MC-CHO and HPMC-CHO is not electronegative thus hydrazides polymer and aldehyde polymer between Coulomb repulsion cause less than the fact of utilizing HA-CHO and CMC-CHO.This explanation conforms to than HA-CMC gelling with HA-HPMC soon with HA-MC.

Conclusion

The hybridized hydrogel of HA as herein described and cellulose derivative can be used and crosslinked rapidly via double syringe, shows the property used that is easy in abdominal and Laparoscopic clinical setting.Although the cellulose derivative that aldehyde is modified represents certain cytotoxicity in vitro, in the Muridae peritoneum, there is excellent biological compatibility.These composites are effective for the peritoneal adhesion of the damaged model of prevention rabbit caecum damage-sidewall.

Example 13: the in-situ cross-linked injection aquagel of glucosan base that is used to prevent peritoneal adhesion

Foreword

Peritoneal adhesion is the caused serious consequence of abdominal part and operation on pelvis, and can cause serious pain, intestinal obstruction and infertile.Be easy to be applied to peritoneum and can be very effective by the in-situ cross-linked gel that mixes two kinds of polymer formation.Such as hyaluronic acid (HA), represented good biocompatibility at intraperitoneal through biomaterials such as oxidized cellulose and cellulose derivatives.Glucosan is the another kind of noticeable basic material that is used for the original position cross-linkable matrix.Glucosan (DX) be glucose moiety mainly by α-1, the polysaccharide that the 6-binding connects.Used 40kDa and 70kDa glucosan prevention angiemphraxis clinically, as plasma extender be used for anticoagulant therapy.Confirmed that glucosan has biocompatibility at intraperitoneal.20th century the eighties, use 32% solution of 70kDa glucosan to prevent peritoneal adhesion clinically, but owing to exist successfully simultaneously and unsuccessful clinical trial, so stopped using glucosan.

In this research, synthetic novel glucosan base injection aquagel prevents peritoneal adhesion.At room temperature, make the Sensor Chip CM 5 (CMDX) modified through hydrazide group (CMDX-ADH) with crosslinked through aldehyde group modified DX or CMC (DX-CHO or CMC-CHO).Characterize the gained hydrogel in vitro, the research prepolymer for the cytotoxicity of cell culture and its for prevention rabbit sidewall damaged-intestinal scratch model in the effectiveness of peritoneal adhesion.

Materials and methods

Synthesizing of polymer and hydrogel

Reagent: 70kDa (production code member: D4751), 500kDa (production code member: D1037) and 2MDa (production code member: D5376) from the glucosan of Leuconostoc mesenteroides (Leuconostoc mesenteroide), CMC (production code member: C4888), adipic dihydrazide (ADH), 1-ethyl-3-[3-(dimethylamino) propyl group]-carbodiimides (EDC), hydroxybenzotriazole (HOBt), sodium metaperiodate, ethylene glycol, tert-butyl carbazate (t-BC), monoxone, sodium hydroxide, sodium chloride and hydrochloric acid are all available from Sigma-aldrich corp.

The preparation of Sensor Chip CM 5 (CMDX):Utilize preceding method to become Sensor Chip CM 5 (CMDX) [paper of answering (Ying ' s paper) and biological concatenator (Bioconjugate) 1997] with 70kDa and 500kDa are glucan-modified.In simple terms, the 10g glucosan is dissolved in the 100ml distilled water whole night, and adds 24.0g sodium hydroxide and 30.2g monoxone subsequently.Make solution at 70 ℃ of following backflow 145min, utilize 6N hydrochloric acid with its pH=7.0 that neutralizes rapidly, to distill water dialysis 3 days and lyophilizing subsequently.Productive rate is 75-80%.

The preparation of adipic dihydrazide Sensor Chip CM 5 (CMDX-ADH):With with as the identical scheme of the previous adipic dihydrazide hyaluronic acid of being reported 70kDa and 500kDa CMDX are modified into adipic dihydrazide CMDX (CMDX-ADH).

The preparation of aldehyde glucosan (DX-CHO) and carboxymethyl cellulose (CMC-CHO):2MDa DX, 70kDaDX and CMC are modified into aldehyde 70kDa DX (70kDa DX-CHO), 2MDa DX (2MDa DX-CHO) and aldehyde CMC (CMC-CHO) respectively.Scheme is identical with previous report.

The preparation of disk hydrogel: use double syringe (hundred special companies (Baxter), this state Deere of Illinois takes moral (Deerfield, IL)) and 2wt% CMDX-ADH PBS buffer solution and 2wt% aldehyde polymer (such as DX-CHO and CMC-CHO) PBS buffer solution are expelled to are sandwiched in two rubber molds between the slide glass.Prepared hydrogel's diameter and thickness are respectively 1.2cm and 3.5mm.These disk hydrogels are called CMDX-DX and CMDX-CMC.

The sign of polymer and hydrogel

The sign of polymer:Use ¹H-NMR (Varian technology company (Varian): excellent Buddhist nun carries 300 type spectrophotometers (Unity 300 spectrophotometer)) spectrum analysis is determined synthetic.Measure D ₂10mg/ml CMDX, CMDX-ADH, DX-CHO and CMC-CHO among the O.Next, the t-BC of 10 times of molar excess is added among the DX-CHO and CMC-CHO polysaccharide in the pure water, and make aldehyde radical and t-BC reaction.After to water dialysis and lyophilizing, at D ₂Measure through the polymer of reaction among the O ¹H-NMR spectrum.It is elementary composition to measure to carry out elementary analysis.

Behind the KBr sheet of each polymer of preparation, measure the FT-IR spectrum of CMDX, CMDX-ADH, DX-CHO and CMC-CHO.

CMDX-ADH is carried out elementary analysis.

The sign of hydrogel:The scheme of passing through to be reported is measured gelling time.Using healthy and free from worry PC-320 type hot plate/agitator to use under the stirring rod stirring on the Pi Shi culture plate, 0.1ml DX-CHO or CMC-CHO aqueous solution are added in the 0.1ml CMDX-ADH aqueous solution with 155rpm.Solution mixture is become the measure of time 5 times of hydrogel bead.

In the PBS buffer, measure the expansible time-histories of prepared disc gel under 37 ℃ in the gravimetric analysis mode.In the PBS buffer, soak the weight W of measuring hydrogel after the gelling after 5 days _sW _sInitial weight W with hydrogel after the gelling just _iExpansion ratio Q be with Q=W _s/ W _iCalculate, and draw the figure of hydrogel.

Cytotoxicity analysis

Using human mesothelial cell is (CRL-9444:ATCC) and macrophage cell line J774.A1 (TIB-67 ^TM: ATCC) analyze the in vitro cell viability of (Pu Luomaige company (Promega)) research under the situation that has DX, CMC, CMDX-ADH, DX-CHO and CMC-CHO by MTT.Scheme is identical with previous report [HA-CMC paper].In simple terms, under 37 ℃ in 5%CO ₂In, the mesothelial cell is grown in complete growth medium (have Yi Ershi BSS (Earle ' s BSS), 0.75mM L-glutaminate and 1.25g/L sodium bicarbonate and be supplemented with the culture medium 199 of 3.3nM epidermal growth factor, 400nM hydrocortisone, 870nM insulin, 20mM HEPES and 10% hyclone) and keep.Make macrophage in the DMEM with 10% hyclone (Gibco catalog number (Cat.No.) 10569-010), grow and keep.After adding material, in the mesothelial cell, the 2nd day the time, carry out MTT and analyze the 3rd day the time or in macrophage.Have only DX-CHO to upset MTT and analyze, therefore before just beginning analysis, utilize fresh culture to replace culture medium.Make described value standardization by control experiment (in cell, not adding material).

Hydrogel is expelled in the mouse peritoneum

Scheme is identical with previous method.(Buddhist gram K (Falk K), the P of Bu Jie Qwest (Bjorquist P), the M of this Tom Qwest (Stromqvist M), Huo Mudaer L. (Holmdahl L.) reduces tentative adhesion formation (Reduction of experimental adhesion formation by inhibitionof plasminogen activator inhibitor type 1.) Britain operation magazine (British Journal of Surgery) 2001 by suppressing 1 type plasminogen activator inhibitor; 88:286-289, it is incorporated herein by reference).The SV129 mice of heavily about 25g is purchased White Tower Kornic Systems Corp. (Taconic), and (moral grandson (Hudson is breathed out in New York, NY)), and use double syringe (hundred special companies (Baxter), this state Deere of Illinois takes 1.0ml CMDX-DX gel injection that moral (Deerfield, IL)) will be made of 0.5ml CMDX-ADH (5% w/v) and 0.5ml DX-CHO (2% w/v) via remaining needle in peritoneum.Laparotomy was implemented to mice in 2 weeks in the injection back.The dissector is to which kind of the hydrogel the unknown of each injected in mice.Evaluate the existence of adhesion by the dissector.

By the rabbit sidewall damaged-preventive effect of intestinal scratch model evaluation peritoneal adhesion

Scheme is identical with previous method.Referring to example 12.With female albefaction rabbit (Spain hare (Oryctolaguscuniculus); New Zealand white (New Zealand White), Ke Wensi company (Covance), (3 ± 0.5kg) as animal pattern for Pennsylvania Hei Zeerdun (Hazleton, PA)).12 animals are assigned randomly to following experimental group: (i) 70kDa-CMDX-DX gel (n=4), (ii) 70kDa-CMDX-CMC gel (n=4) and (iii) each 10ml of 500kDa-CMDX-CMC gel (n=4).

Utilize the scratch under the 80-160 of damaged and 7 bags of caecum of 3 * 4cm on the stomach wall of right side to bring out peritoneal adhesion.(5ml CMDX-ADH (be 50mg/ml or be 40mg/ml for 500kDa-CMDX-ADH for 70kDa-CMDX-ADH) and 5ml DX-CHO (25mg/ml) or CMC-CHO (60mg/ml)) puts into the 10ml independence asepsis injector that links to each other with hundred special bivalve applicators (Baxter dual valve applicator) with gel precursors solution, and by No. 15 pin coextrusion.

In 1 week after the program, animal is implemented euthanasia.Following adhesion is marked: 0 minute=no adhesion, 1 minute=utilize gravity to adhere to by isolating tissue; 2 minutes=can adhere to by the isolating tissue of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.The fixation of tissue that will reclaim by postmortem in 10% formalin, and with haematoxylin and Yihong dyeing for histological examination.

Statistical analysis is mainly by student t check analysis data.Before the t check, carry out ANOVA.Adhesion score between each hydrogel and the contrast is carried out the inferior rank test of Wilcock (Wilcoxon rank-sum test).Following distribution class variable: 0 minute=1,1 minute=2,2 minutes=3 and 3 minutes=4.Utilize the Ka Leidage pressgang

(Si Niji software company (Synergy Software)) carries out all statistical test.It is significant that p value＜0.05 is considered to statistics.

The result

Synthetic and the sign of CMDX, CMDX-ADH, DX-CHO and CMC-CHO

By FT-IR (Figure 23) and ¹H NMR (Figure 24) determines synthesizing of CMDX, CMDX-ADH.Two kinds glucosan (70kDa and 500kDa) come to the same thing.

During FTIR, as shown in Figure 23, glucosan is at 1650cm ^-1Has the absorbance peak value when [paper of sharp promise (Lino ' s paper)].By at 1608cm ^-1The modification of CMDX is determined at the peak that Shi Xianxian is new (the C=O elongation of reflection carboxyl).Pass through 1664cm in addition ^-1The time the new peak C=O of amide groups (reflection elongation) show modification to CMDX-ADH.

Glucosan be lower than 3.0ppm and be higher than 5.2ppm the zone in no NMR peak.In CMDX, new peak comes across 5.13ppm.In CMDX-ADH, new peak comes across 2.09 and 2.32ppm (doublet, 4H, N-(CH ₂)-C) and 1.60ppm (singlet, 4H, C-(CH ₂)-C).

By the elementary analysis of CMDX-ADH, the percentage by weight of carbon, hydrogen and nitrogen be respectively 44.4%, 6.6% among the 70kDa CMDX-ADH and 9.9% and 500kDa CMDX-ADH in 44.7%, 6.3% and 9.7%.Is 44% among 46% among 70kDa CMDX-ADH and the 500kDa CMDX-ADH by the Ratio Estimation adipic dihydrazide of nitrogen and carbon to the degree of modification of CMDX-ADH.

The FT-IR of DX-CHO is identical with previous disclosed data with the NMR analysis result, determines synthetic.Such as report, produce CMC-CHO and characterized.

The in vitro characteristic of original position hydrogel

The gelling time of all hydrogels all is (Figure 25) of concentration dependent, so all hydrogels all can be modified the acquisition fast gelation time under prepolymer and the hydrazides modification prepolymer at the aldehyde of higher concentration.Under each concentration, the gelling time of CMDX-DX gel is all than CMDX-CMC gel short (Figure 25).

The CMDX-DX gel is in the gelling after-contraction, and CMDX-CMC expands after gelling, therefore for example the expanding volume of 70kDa-CMDX-CMC (5%/6%) than CMDX-DX (5%/6%) high 3 times (Figure 26).Very different (Figure 26 B): the CMDX-DX of the physical appearance of hydrogel are light yellow and show slightly opaque, and 70kDa-CMDX-CMC is transparent and clarification.The expansion of 500kDa-CMDX-CMC (5%/6%) is between 70kDa-CMDX-CMC and the expansible centre of CMDX-DX.

The biocompatibility of original position hydrogel in vitro analyzing

Not modified raw material represents the smallest cell toxicity in cell culture, but some modification can influence cell viability.

Under the situation that has the uncrosslinked prepolymer CMDX-ADH of multiple concentration, DX-CHO and CMC-CHO, cultivate mesothelial cell (Figure 28 A).CMDX-ADH and CMC-CHO represent slight dose dependent toxicity, and the toxicity of DX-CHO is then much higher.

The assemble mode of the cytotoxic effect in the macrophage similar (Figure 28 B).The aldehyde derivatives cytotoxicity of glucosan is extremely strong, and CMC-CHO also is like this.Even 70kDa-and 500kDa-CMDX-ADH do not have influence to the macrophage vigor yet under high concentration.Not modified CMC and DX under all concentration can increase cell viability.

Analyzed as can be known by the MTT about two kinds of cell lines, the CMDX-ADH biocompatibility is splendid, and can expect that also its hydrogel is also extremely strong at endoperitoneal biocompatibility.

The peritoneal adhesion prophylactic function of original position hydrogel

Through all 4 rabbit development adhesions of CMDX-DX treatment, and the adhesion area is greater than control experiment (p=0.0027, table 13.1, Figure 30 A-1).Find to separate the hydrogel fragment of grumeleuse form at intraperitoneal, it is retained in the adhesion sometimes.Therefore, although this gel is present in the damage location place, it does not serve as barrier.As observed in the test of expanding, it is light yellow and firm that gel pieces is.It firmly adheres to tissue (Figure 30 A-2), and is different with other in-situ cross-linked gel of having studied.

Table 13.1

By contrast, find that 70kDa-and 500kDa-CMDX-CMC intersperse among in the whole peritoneum, but part keeps with the form of being close to block sometimes, as shown in Fig. 9 B.The pale red color and luster of material is caused (Figure 30 C-2) by blood contamination.What compared with the control, two kinds of hydrogels caused all that adhesion forms area obviously reduces (table 1, Figure 30 B, C-1) (for 70kDa-and 500kDa-CMDX-CMC, p＜0.0001).

It is much smaller that the intermediate value adhesion of two groups of CMDX-CMC gets in proportion by subtraction untreated contrast or the CMDX-DX gel score of any one, can't represent significance,statistical but sample-size is too little.That is compiled relatively obtains 0.009 p value from the data of two CMDX-CMC group.The intermediate value adhesion score of two groups of CMDX-CMC is similar and do not have the significant difference of statistics.

During histologic analysis, the CMDX-DX gel effectively adheres to normal and impaired caecum surface (Figure 30 A), and inflammatory cells obviously is infiltrated on (Figure 30 B) in lower floor's connective tissue.And by contrast, the mesothelium (Figure 30 C) that reclaims in the animal of CMDX-CMC treatment has the connective tissue lower floor (Figure 30 D) that greatly thickens.

The peritoneal injection of CMDX-DX gel

CMDX-DX can't Film with Preventing Adhesion be attributable to Film with Preventing Adhesion effect shortage or owing to the direct effect that causes adhesion.For whether evaluation material itself is harmful, give and 4 mice CMDX-ADH and DH-CHO through peritoneal injection by double syringe, thereby form CMDX-DX in original position.In injection 2 weeks of back, put to death animal and check its abdominal cavity.CMDX-DX can not cause peritoneal adhesion but find (Figure 29) in firmly adhering to structural firm light yellow grumeleuse.

Discuss

Glucosan has high biocompatibility and is to be suitable for the lower cost materials that peritoneum is used.Synthetic two kinds need not the glucosan based aquagel that the low-molecular-weight cross-linking agent can form herein.Although the two total CMDX-ADH part, it has distinct characteristic.

The gelling time of CMDX-DX is than the gelling time much shorter of CMDX-CMC.There are some possible reasons for this difference, comprise the degree of modification of viscosity, hydrazides or aldehyde etc., but main cause may be water miscible difference between glucosan and the CMC.Because in the research formerly, the gelling time of 3wt/vol% 500kDa-CMDX-CMC gel is extremely slow, as 65.8 ± 5.0sec; And the gelling time of 2wt/vol% 490kDa-HA (hyaluronic acid)-CMC gel is exceedingly fast, as 18.5 ± 1.7sec.The degree of modification of HA-ADH and CMDX-ADH and molecular weight much at one, so the dissolubility of DX and CMC extremely a little less than.On the other hand, because CMDX-ADH is synthetic by identical DX with DX-CHO, so the gelling time of CMDX-DX gel is exceedingly fast.Polymer with same polymer main chain can easily mix mutually.

After the gelling, CMDX-DX shrinks and the CMDX-CMC expansion.This can the DX-CHO neutral be explained by the Coulomb repulsion between the CMC-CHO negative charge.

The performance of two kinds of material prevention peritoneal adhesions exists significantly different.CMDX-DX increases the weight of adhesion, and the CMDX-CMC Film with Preventing Adhesion.Cytotoxicity as the DX-CHO shown in vitro may cause that the effect of its Film with Preventing Adhesion lacks.Yet described toxicity is that the exposure of experience 2 days (under the situation of macrophage) or 3 days (for the mesothelial cell) occurs.To exist sufficiently high free CMDX-DX content to cause tissue injury after can not concluding crosslinked (cost several seconds) thus.Cross-linked material itself can be harmful to.

Although realized immense success by the in-situ crosslinking aquogel as the barrier device, its degradation time is of short duration relatively, and is especially all the more so based on hyaluronic hydrogel.Yet the barrier device remains on original position and forms the unknown still of required time span to avoid adhesion.In the case, can find the abundant residues material when advantage of susceptible of proof CMDX-DX is postmortem, show to have degradation kinetics slowly.This slow degraded may only be arranged in the fact of liver basically owing to the glucanase of high relatively polymer concentration, low relatively expanding volume and the glucosan of degrading.

Conclusion

The cross-linked hydrogel of the carboxymethyl cellulose that Sensor Chip CM 5 that hydrazides is modified and aldehyde are modified represents the effect of preventing peritoneal adhesion.The degradation rate slowly of these gels and low cost show its possible effect aspect the prevention peritoneal adhesion.

Example 14: the original position crosslinkable of hyaluronic acid and dexamethasone links the antiphlogistic activity of hydrogel

Foreword

Operation posterior peritoneum adhesion can cause pain, intestinal obstruction and infertile (Di Zejia G.S. (DiZerega, G.S.), operation on peritoneum (Peritoneal Surgery.), Springer (New York:Springer) New York, 1999; It is incorporated herein by reference).Used multiple polysaccharide based aquagel barrier system to come Film with Preventing Adhesion, and obtain success (Di Zejia G.S. (DiZerega in various degree, G.S.), operation on peritoneum (Peritoneal Surgery.) Springer (New York:Springer) New York, 1999; It is incorporated herein by reference).Can be potential by the in-situ cross-linked hydrogel that forms as barrier (Jones D.B. (Johns, D.B.), Rogers K.E. (Rodgers, K.E.), Tang Naxiu W.D. (Donahue, W.D.), general this T.C (Kiorpes of Cole, T.C) and Di Zejia G.S. (diZerega, G.S.) throw and ionomer hyaluronic acid minimizing adhesion formation (Reduction of adhesion formation by postoperativeadministration of ionically cross-linked hyaluronic acid.) fertility and sterile (Fertility AndSterility) 68 (1997) 37-42 by postoperative, it is incorporated herein by reference), (Lee H. (Li, H.), Liu Y.C (Liu, Y.C), X.Z. (Shu relaxes, X.Z.), Gray S.D. (Gray, S.D.) and the Preece tie up strange G.D. (Prestwich, G.D.) synthetic and biological assessment (Synthesis and biologicalevaluation of a cross-linked hyaluronan-mitomycin C hydrogel.) biomacromolecule (Biomacromolecules) 5 (2004) 895-902 of crosslinked hyaluronan-ametycin hydrogel, it is incorporated herein by reference), (Liu Y.C (Liu, Y.C), Lee H. (Li, H.), X.Z. (Shu relaxes, X.Z.), Gray S.D. (Gray, S.D.) and the Preece tie up strange G.D. (Prestwich, G.D.) the crosslinked hyaluronan hydrogel that contains ametycin reduces postoperative abdominal adhesion (Crosslinkedhyaluronan hydrogels containing mitomycin C reduce postoperative abdominal adhesions.) fertility and sterile (Fertility And Sterility) 83 (2005) 1275-1283, it is incorporated herein by reference), (Europe S.H. (Oh, S.H.), gold nurse J.K. (Kim, J.K.), Song K.S. (Song, K.S.), promise river S.M. (Noh, S.M.), Kiel S.H. (Ghil, S.H.), but excellent S.H. (Yuk, S.H.) and Lee J.H. (Lee, J.H.) restrain mixture prevention of postoperative tissue adhesion (Prevention of postsurgical tissue adhesion by anti-inflammatory drug-loaded pluronicmixtures with sol-gel transition behavior.) biomedical material research magazine A (J Biomed Mater ResA) 72 (2005) 306-16 by the general stream Buddhist nun who is loaded with anti-inflammation drugs with sol-gel transition behavior, it is incorporated herein by reference), (according to Europe Y. (Yeo, Y.), sea thunder C (Highley, C), ripple Lars E. (Bellas, E.), dust holder T. (Ito, T.), agate Renyi R. (Marini, R.), bright lattice R. (Langer, R.) and gram Chinese D. (Kohane, D.) postoperative abdominal adhesion (Insitu cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions ina rabbit model.) biomaterial (Biomaterials) 27 (2006) 4698-4705 in the original position crosslinkable hyaluronic acid gel prevention rabbit model, it is incorporated herein by reference), and comparable pre-formed sheet is easier to use.Specifically, usually hyaluronic acid (HA) derivant is used for peritoneum.The HA hydrogel that the in-situ chemical that adds or do not add medicine is modified is used for this purpose (Jones D.B. (Johns, D.B.), Rogers K.E. (Rodgers, K.E.), Tang Naxiu W.D. (Donahue, W.D.), general this T.C (Kiorpes of Cole, T.C) and Di Zejia G.S. (diZerega, G.S.) throw and ionomer hyaluronic acid minimizing adhesion formation (Reduction of adhesion formation by postoperative administration ofionically cross-linked hyaluronic acid.) fertility and sterile (Fertility And Ste rility) 68 (1997) 37-42 by postoperative, it is incorporated herein by reference), (Lee H. (Li, H.), Liu Y.C (Liu, Y.C), X.Z. (Shu relaxes, X.Z.), Gray S.D. (Gray, S.D.) and the Preece tie up strange G.D. (Prestwich, G.D.) synthetic and biological assessment (Synthesis and biological evaluation of a cross-linkedhyaluronan-mitomycin C hydrogel.) biomacromolecule (Biomacromolecules) 5 (2004) 895-902 of crosslinked hyaluronan-ametycin hydrogel, it is incorporated herein by reference), (Liu Y.C (Liu, Y.C), Lee H. (Li, H.), X.Z. (Shu relaxes, X.Z.), Gray S.D. (Gray, S.D.) and the Preece tie up strange G.D. (Prestwich, G.D.) the crosslinked hyaluronan hydrogel that contains ametycin reduces postoperative abdominal adhesion (Crosslinked hyaluronan hydrogels containingmitomycin C reduce postoperative abdominal adhesions.) fertility and sterile (Fertility AndSterility) 83 1275-1283, it is incorporated herein by reference), (according to Europe Y. (Yeo, Y.), sea thunder C (Highley, C), ripple Lars E. (Bellas, E.), dust holder T. (Ito, T.), agate Renyi R. (Marini, R.), bright lattice R. (Langer, R.) and gram Chinese D. (Kohane, D.) postoperative abdominal adhesion (Insitu cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions ina rabbit model.) biomaterial (Biomaterials) 27 4698-4705 in the original position crosslinkable hyaluronic acid gel prevention rabbit model, it is incorporated herein by reference).Recently, confirmed by the crosslinked aldehyde of hyaluronan-and original position crosslinkable hydrogel (special P. (Bulpitt of boolean's skin of constituting of the HA that modifies of hydrazides, P.) and Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemicalmodification of hyaluronic acid:Preparation of functionalized derivatives and their use in theformation of novel biocompatible hydrogels.) biomedical material research magazine (Journal OfBiomedical Materials Research) 47 (1999) 152-169 of novel bio-compatible hydrogel, and it is incorporated herein by reference); (merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.) and gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronic acid.) biomaterial (Biomaterials) 25 (2004) 4797-4804 by in-situ cross-linked hyaluronic acid, it is incorporated herein by reference) for prevention rabbit sidewall damaged-peritoneal adhesion in the caecum scratch model is effectively (according to Europe Y. (Yeo, Y.), sea thunder C (Highley, C), ripple Lars E. (Bellas, E.), dust holder T. (Ito, T.), agate Renyi R. (Marini, R.), bright lattice R. (Langer, R.) and gram Chinese D. (Kohane, D.) postoperative abdominal adhesion (Insitucross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in arabbit model.) biomaterial (Biomaterials) 27 (2006) 4698-4705 in the original position crosslinkable hyaluronic acid gel prevention rabbit model, it is incorporated herein by reference).

The ability of the bio-compatible barrier that most of described device all utilizes described device to serve as between healing stage to separate perished surface.In this article, it is unusual hydrogel to be modified the Pathophysiology that forms with direct solution adhesion.It is reported that the inflammation meeting impels the formation peritoneal adhesion.Except that the potential disorganization effect of inflammation itself, inflammatory cells discharges such as tumor necrosis factor-alpha (TNF-α), interleukin-1 and 6 cytokines such as (IL-1 and 6).These cytokine inductions mesothelial cell produces plasminogen activator inhibitor-1 and 2 (PAI-1 and PAI-2), it reduces the activity of plasminogen activator (PA), slow down fibrin degradation (Wo Weier S.A. (Whawell, S.A.), Scott Ke Baimu D.M. (Scottcoombes, D.M.), Wei Pangde M.N. (Vipond, M.N.), special S.J. (the Tebbutt of Taibo, S.J.) and Simpson J.N. (Thompson, J.N.) the human peritoneal mesothelium cell of tumor necrosis factor mediation discharges plasminogen activator inhibitor-1 (TumorNecrosis Factor-Mediated Release Of Plasminogen-ActivatorInhibitor-1 By Human Peritoneal Mesothelial Cells.) Britain operation magazine (British Journal OfSurgery) 81 (1994) 214-216, it is incorporated herein by reference), (Wo Weier S.A. (Whawell, S.A.) and Simpson J.N. (Thompson, J.N.) the human mesothelial cell of cytokine induction discharges plasminogen activator inhibitor-1 (Cytokine-Induced Release Of Plasminogen-Activator Inhibitor-1 By HumanMesothelial Cells.) Europe operation magazine (European Journal Of Surgery) 161 (1995) 315-318, it is incorporated herein by reference), (denapon Si Baige V.W.M. (Vanhinsbergh, V.W.M.), bohr K.A. (Bauer, K.A.), gram Nice tower T. (Kooistra, T.), Krafft C (Kluft, C), Dorje Ward G. (Dooijewaard, G.), Harry Hillman M.L. (Sherman, M.L.) and knob Wen Huiwen W. (Nieuwenhuizen, W.) Fibrinolytic progress-tissue type plasminogen activator between the tumor necrosis factor incorporating period among the mankind, 1 type plasminogen activator inhibitor and fibrin (former) catabolite follow increase (Progress Of Fibrinolysis DuringTumor-Necrosis-Factor Infusions In Humans-Concomitant Increase In Tissue-TypePlasminogen-Activator, Plasminogen-Activator Inhibitor Type-1, And Fibrin (Ogen) Degradation Products.) hematology (Blood) 76 (1990) 2284-2289, it is incorporated herein by reference), (Muller base I.K. (Mullarky, I.K.), Si Dabei F.M. (Szaba, F.M.), Burger human relations K.N. (Berggren, K.N.), khoum that L.W. (Kummer, L.W.), Wei Erhaimu L.B. (Wilhelm, L.B.), Pa Lunte M.A. (Parent, M.A.), Johnson L.L. (Johnson, L.L.) and this close thunder S.T. (Smiley, S.T.) tumor necrosis factor and IFN-but not between hemorrhage or pathogen load indication infection period the level (Tumor necrosis factor alpha and gamma interferon, but not hemorrhage or pathogen burden, dictate levels of protective fibrin deposition during infection.) of preventative fibrin deposition infect and immunology (Infection AndImmunity) 74 (2006) 1181-1188, it is incorporated herein by reference), (Muller base I.K. (Mullarky, I.K.), Si Dabei F.M. (Szaba, F.M.), Burger human relations K.N. (Berggren, K.N.), khoum that L.W. (Kummer, L.W.), Wei Erhaimu L.B. (Wilhelm, L.B.), Pa Lunte M.A. (Parent, M.A.), Johnson L.L. (Johnson, L.L.) and this close thunder S.T. (Smiley, S.T.) tumor necrosis factor and IFN-but not level (the Tumor necrosis factor alpha andgamma interferon of preventative fibrin deposition between hemorrhage or pathogen load indication infection period, but not hemorrhage or pathogen burden, dictate levels of protective fibrindeposition during infection.) infect and immunology (Infection And Immunity) 74 (2006) 1181-1188).These processes can promote adhesion to form.The minimizing that pro-inflammatory cytokine discharges can strengthen the prevention to peritoneal adhesion.For this reason, several studies personnel have tested the effectiveness of anti-inflammation drugs to peritoneal adhesion, comprise non-steroid antiinflammatory drug (NSAID), such as ibuprofen (Europe S.H. (Oh, S.H.), gold nurse J.K. (Kim, J.K.), Song K.S. (Song, K.S.), promise river S.M. (Noh, S.M.), Kiel S.H. (Ghil, S.H.), but excellent S.H. (Yuk, S.H.) and Lee J.H. (Lee, J.H.) restrain mixture prevention of postoperative tissue adhesion (Prevention of postsurgical tissue adhesion by anti-inflammatory drug-loadedpluronic mixtures with sol-gel transition behavior.) biomedical research magazine A (J Biomed MaterRes A) 72 (2005) 306-16 by the general stream Buddhist nun who is loaded with anti-inflammation drugs with sol-gel transition behavior, it is incorporated herein by reference), (Ben Teman B.G. (Bateman, B.G.), knob thunder W.C, Jr. (Nunley, W.C, Jr.) and Ke Qiyin J.D. (Kitchin, J.D.), the 3rd edition, utilize ibuprofen prevention of postoperative peritoneal adhesion (Prevention of postoperative peritoneal adhesions with ibuprofen.) fertility and sterile (Fertil Steril) 38 (1982) 107-8, it is incorporated herein by reference), (western village K. (Nishimura, K.), middle village R.M. (Nakamura, R.M.) and Di Zejia G.S. (diZerega, G.S.) the biochemistry assessment of post-surgical trauma reparation: utilize the adhesion of ibuprofen prevention intraperitoneal to form (Biochemical evaluation of postsurgicalwound repair:prevention of intraperitoneal adhesion formation with ibuprofen.) operations research magazine (J Surg Res) 34 (1983) 219-26, it is incorporated herein by reference), (Rogers K. (Rodgers, K.), Kirghiz Republic W. (Girgis, W.), Di Zejia G.S. (diZerega, G.S.), Bradley agree K. (Bracken, K.) and auspicious fine jade L. (Richer, L.) suppress the international fertility of tissue adhesion (Inhibition ofpostsurgical adhesions by liposomes containing nonsteroidal antiinflammatory drugs.) magazine (Int J Fertil) 35 (1990) 315-20 by the liposome that contains non-steroid antiinflammatory drug, it is incorporated herein by reference), (Li Gelande E.K. (LeGrand, E.K.), Rogers K.E. (Rodgers, K.E.), and Kirghiz Republic W. (Girgis, W.), Kemp Lip river J.D. (Campeau, J.D.) and Di Zejia G.S. (it is incorporated herein by reference for Dizerega, the G.S.) effect of the plain agent of non-steroid antiinflammatory drug and antithrombotic (Comparative efficacy of nonsteroidal anti-inflammatory drugs andanti-thromboxane agents in a rabbit adhesion-prevention model.) research property operation magazine (J InvestSurg) 8 (1995) 187-94 relatively in the rabbit adhesion prophylaxis model), (Lee J.H. (Lee, J.H.), and high A.K. (Go, A.K.), Europe S.H. (Oh, S.H.), (Lee is K.E.) with excellent gram S.H. (Yuk for Lee K.E., S.H.) be loaded with ibuprofen PLLA-PEG diblock copolymer thin film organize tissue adhesion potentiality (Tissue anti-adhesionpotential of ibuprofen-loaded PLLA-PEG diblock copolymer films.) biomaterial (Biomaterials) 26 (2005) 671-8, it is incorporated herein by reference); And glucocorticoid, such as dexamethasone (Huo Keer M. (Hockel, M.), Ou Te S. (Ott, S.), uncommon graceful U. (Siemann, U.) and Ke Sier T. (Kissel, T.) utilize lasting intraperitoneal dexamethasone prevention rat peritoneum adhesion (Prevention Of Peritoneal Adhesions In The Rat With Sustained IntraperitonealDexamethasone Delivered By A Novel Therapeutic System.) Annales ChirurgiaeEtGynaecologiae 76 (1987) 306-313 that transmit by novel therapy system, it is incorporated herein by reference), (Bu Kenmaier C.C. (Buckenmaier, C.C.), Pu Sitelei A.E. (Pusateri A.E.), Halley Si R.A. (Harris, R.A.) and hertz S.P. (Hetz, S.P.) use relatively tissue adhesion treatment (Comparison of antiadhesivetreatments using an objective rat model.) U.S. surgeon magazine (American Surgeon) 65 (1999) 274-282 of target rat model, it is incorporated herein by reference), (bitter Cook is bank T. (Kucukozkan now, T.), Er Suoyi B. (Ersoy, B.), yogurt D. (Uygur, D.) and sweet many degree C. (Gundogdu, C.) pass through sodium cromoglicate after the operation on pelvis, dexamethasone, normal saline and aprotinin Film with Preventing Adhesion (Prevention of adhesions by sodiumchromoglycate, dexamethasone, saline and aprotinin after pelvic surgery.) ANZ operation magazine (ANZ J Surg) 74 (2004) 1111-5, it is incorporated herein by reference).These have incorporated in the host material, described host material such as liposome (Rogers K. (Rodgers, K.), Kirghiz Republic W. (Girgis, W.), Di Zejia G.S. (diZerega, G.S.), Bradley is agree K. (Bracken, K.) and auspicious fine jade L. (Richer, L.) suppress the international fertility of tissue adhesion (Inhibition of postsurgical adhesions by liposomescontaining nonsteroidal antiinflammatory drugs.) magazine (Int J Fertil) 35 (1990) 315-20 by the liposome that contains non-steroid antiinflammatory drug, it is incorporated herein by reference), poly-(L-lactic acid) (PLLA)-Polyethylene Glycol (PEG) diblock copolymer thin film (Lee J.H. (Lee, J.H.), high A.K. (Go, A.K.), Europe S.H. (Oh, S.H.), Lee K.E. (Lee, K.E.) and excellent gram S.H. (Yuk, S.H.) be loaded with ibuprofen PLLA-PEG diblock copolymer thin film organize tissue adhesion potentiality (Tissue anti-adhesion potential of ibuprofen-loaded PLLA-PEG diblockcopolymer films.) biomaterial (Biomaterials) 26 (2005) 671-8, it is incorporated herein by reference), the mixture of poloxamer and alginate hydrogel (Europe S.H. (Oh, S.H.); Gold nurse J.K. (Kim, J.K.); Song K.S. (Song, K.S.); Promise and S.M. (Noh, S.M.); Gill S.H. (Ghil, S.H.); Excellent gram S.H. (Yuk, S.H.) and Lee J.H. (Lee, J.H.), utilize the sol-gel transition behavior to restrain mixture prevention of postoperative tissue adhesion (Prevention of postsurgical tissue adhesion byanti-inflammatory drug-loaded pluronic mixtures with sol-gel transition behavior.) biomaterial research magazine A (J Biomed Mater Res A) 72 (2005) 306-16 by the general stream Buddhist nun who is loaded with anti-inflammation drugs, it is incorporated herein by reference) and gather (Acetic acid, hydroxy-, bimol. cyclic ester-copolymerization-lactide) (PLGA) micron particle (Huo Keer M. (Hockel, M.), water S. (Ott, S.), Harry Hillman U. (Siemann, U.) and Qi Saier T. (Kissel, T.) utilize peritoneal adhesion (Prevention Of Peritoneal Adhesions In The Rat WithSustained Intraperitoneal Dexamethasone Delivered By A Novel Therapeutic System.) (Annales Chirurgiae Et Gynaecologiae) 76 (1987) 306-313 in the lasting intraperitoneal dexamethasone prevention rat of transmitting by novel therapy system, it is incorporated herein by reference).Find, the biocompatibility of hydrogel based system in peritoneum usually than hydrophobic polymer device (for example device that constitutes by PLGA) strong (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Rich Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.) and gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronicacid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference), (gram Chinese D.S. (Kohane, D.S.), this J.Y. (Tse of the base of a fruit, J.Y.), Yi Ou Y. (Yeo, Y.), Pa Dela R. (Padera, R.), (Shubina is M.) with bright lattice R. (Langer for Shu Binna M., R.) be used for the biodegradable polymerization microsphere of intraperitoneal drug delivery and nanometer spheroid (Biodegradable polymeric microspheres and nanospheres for drug delivery in theperitoneum.) biomedical material research magazine A part (Journal Of Biomedical Materials ResearchPart A) 77A (2006) 351-361, it is incorporated herein by reference), but because low-molecular-weight drug diffuses through hydrogel matrix rapidly, so have faster release dynamics.A kind of method of this problem of controlling is for linking (mark Luo De A.D. (McLeod with micromolecule and hydrogel, A.D.), holder human relations Dinon L. (Tolentino, L.) and Tuo Zeer T.N. (Tozer, T.N.) stable state pharmacokinetics (GIucocorticoid-Dextran Conjugates As Potential Prodrugs ForColon-Specific Delivery-Steady-State Pharmacokinetics In The Rat.) biological pharmacology and the drug deposition (Biopharmaceutics in glucocorticoid-glucosan concatenator potential prodrug-rat of transmitting as colon-specific; Drug Disposition) 15 (1994) 151-161, it is incorporated herein by reference), (mark Luo De A.D. (McLeod, A.D.), Fred D.R. (Friend, D.R.) and Tuo Zeer T.N. (Tozer, T.N.) glucocorticoid-glucosan concatenator is as potential prodrug-hydrolysis rat gastrointestinal tract content (GIucocorticoid-Dextran Conjugates As Potential Prodrugs For Colon-Specific Delivery-Hydrolysis In Rat Gastrointestinal-Tract Contents.) pharmaceutical science magazine (JournalOfPharmaceutical Sciences) 83 (1994) 1284-1288 of colon-specific transmission, it is incorporated herein by reference), (all S.Y. (Zhou, S.Y.), prunus mume (sieb.) sieb.et zucc. Q.B. (Mei, Q.B.), Liu L. (Liu, L.), Guo X. (Guo, X.), Qiu B.S. (Qiu, B.S.), Zhao D.H. (Zhao, D.H.) and Cao C.H. (Cho, C.H.) transmission of glucocorticoid concatenator in rat gastrointestinal tract and its treatment (Delivery of glucocorticoid conjugate in ratgastrointestinal tract and its treatment for ulcerative colitis.) Acta Pharmacologica Sinica (ActaPharmacologica Sinica) 22 (2001) 761-764 for ulcerative colitis, it is incorporated herein by reference), (huge Y.N. (Pang, Y.N.), open Y. (Zhang, Y.) and open Z.R. (Zhang, Z.R.) the synthetic of enzyme dependency prodrug is used for assessment (Synthesis of an enzyme-dependent prodrug and evaluation of itspotential for colon targeting.) world's gastroenterology magazine (World Journal Of Gastroenterology) 8 (2002) 913-917 of the potentiality of colon target with it, it is incorporated herein by reference), (Po Yani T. (Pouyani, T.) and Prestwich G.D. (Prestwich, G.D.) functional derivative-pharmaceutical carriers of hyaluronic acid oligosaccharide and novel biomaterial (Functionalized Derivatives of Hyaluronic-Acid Oligosaccharides-Drug Carriers and NovelBiomaterials.) biological concatenator chemistry (Bioconjugate Chemistry) 5 (1994) 339-347, it is incorporated herein by reference), (Prestwich people such as (Prestwich), hyaluronic controlled chemistry is modified: hydrazide derivatives synthetic, use and biodegradation (Controlled chemical modification of hyaluronic acid:synthesis, applications, and biodegradation of hydrazide derivatives.) controlled release magazine (Journal OfControlled Release) 53 (1998) 93-103, it is incorporated herein by reference), (the strange people such as (Rajews ki) of the clean thinking of thunder, the enzymatic of polymerization prodrug-hyaluronic hydrocortisone ester and non-enzymatic hydrolysis (EnzymaticAnd Nonenzymatic Hydrolysis Of A Polymeric Prodrug-Hydrocortisone Esters OfHyaluronic-Acid.) international pharmacopedics magazine (International Journal Of Pharmaceutics) 82 (1992) 205-213, it is incorporated herein by reference), (Ai Fute people such as (Everts), use E-to select plain directed immune concatenator to be delivered to (Selective intracellulardelivery of dexamethasone into activated endothelial cells using an E-selectin-directedimmunoconjugate.) Journal of Immunology (Journal Of Immunology) 168 (2002) 883-889 in the active endotheliocyte in the dexamethasone selecting cell, it is incorporated herein by reference), (Mel Ji Te people such as (Melgert), make dexamethasone targeting Kupffer cell: to rat hepatitis and Fibrotic influence (Targeting dexamethasone to Kupffer cells:Effects on liverinflammation and fibrosis in rats.) hepatology (Hepatology) 34 (2001) 719-728, it is incorporated herein by reference).These concatenator may command release dynamics, but concatenator polymer itself can be removed from intraperitoneal rapidly.

Hydrogel matrix discharges anti-inflammation drugs and hydrogel is diffused into ectoperitoneal problem in order to solve, design and synthesize in-situ cross-linked characteristic of combination and the banded hydrogel of medicine (Bo Yani T. (Pouyani, T.) and Prestwich G.D. (Prestwich, G.D.) biological chemistry (Bioconjugate Chemistry) 5 (1994) 339-347 that link of the functional derivative-pharmaceutical carriers of hyaluronic acid oligosaccharide and novel biomaterial (FunctionalizedDerivatives of Hyaluronic-Acid Oligosaccharides-Drug Carriers and Novel Biomaterials.), it is incorporated herein by reference).Link and through hydrazide group or aldehyde group modifiedly make it can pass through hydrazone key and the crosslinked HA of other HA thereby produced via ester bond connection and effective synthetic glucocorticoid agonist dexamethasone.In this article, characterize these materials and also confirm its antiphlogistic activity in vitro and in vivo.

Materials and methods

Material

Dexamethasone, succinic anhydrides, ethanol, 1-[3-(dimethylamino) propyl group]-3-ethyl-carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine, N-hydroxy-succinamide (NHS), dicyclohexyl carbodiimide (DCC), anhydrous propanone, dimethyl sulfoxine (DMSO), adipic dihydrazide (ADH), hydroxybenzotriazole (HOBt), sodium metaperiodate, ethylene glycol, tert-butyl carbazate, sodium chloride, phosphoric acid and sodium is all available from aldrich corp (Aldrich).HA (Mw=490kDa or 1.36MkDa) is available from gene enzyme company.

Method

Synthesizing of hyaluronic acid-adipic dihydrazide (HA-ADH) and hyaluronic acid-aldehyde (HA-ALD)

Carry out synthetic (Yi Ou people such as (Yeo) as described, original position crosslinkable hyaluronic acid gel prevention rabbit model operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels prevent post-operativeabdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 27 (2006) 4698-4705, it is incorporated herein by reference), (special P. (the Bulpitt of boolean's skin, people such as P.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its be used to form novel bio-compatible hydrogel purposes (New strategy forchemical modification of hyaluronic acid:Preparation of functionalized derivatives and theiruse in the formation of novel biocompatible hydrogels.) biomedical material research magazine (J BiomedMater Res) 47 (1999) 152-169 its be incorporated herein by reference), (merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.) and gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciaticnerve blockade by insitu cross-linked hyaluronic acid.) biomaterial (Biomaterials) 25 (2004) 4797-4804 by in-situ cross-linked hyaluronic acid, it is incorporated herein by reference).HA-ADH and HA-ALD are respectively synthetic by 1.36MDa HA and 490kDa HA.

Synthesizing of dexamethasone-succinate (dex-suc)

This program is based on previous report (huge Y.N. (Pang, Y.N.), open Y. (Zhang, Y.) and open Z.R. (Zhang, Z.R.) the synthetic of enzyme dependency prodrug is used for assessment (Synthesis of anenzyme-dependent prodrug and evaluation of its potential for colon targeting.) world's gastroenterology magazine (World Journal Of Gastroenterology) 8 (2002) 913-917 of the potentiality of colon target with it, it is incorporated herein by reference), (Ai Fute (Everts), wealthy gram R.J. (Kok, R.J.), Ai Sijisiduote S.A. (Asgeirsdottir, S.A.), the lucky special B.N. (Melgert of mayer, B.N.), Mu Lunna T.J.M. (Moolenaar, T.J.M.), healthy and free from worry G.A. (Koning, G.A.), Fan Luyin M.J.A. (van Luyn, M.J.A.), prunus mume (sieb.) sieb.et zucc. gill D.K.F. (Meijer, D.K.F.) and Mo Lunna G. (Molema, G.) use E-to select plain directed immune concatenator to be delivered to (Selective intracellular delivery of dexamethasone into activated endothelialcells using an E-selectin-directed immunoconjugate.) Journal of Immunology (Journal Of Immunology) 168 (2002) 883-889 in the active endotheliocyte in the dexamethasone selecting cell, it is incorporated herein by reference).Under the nitrogen, 2.455g dexamethasone (6.25mmol), 10.52g succinic anhydrides and 0.795g 4-dimethylaminopyridine are dissolved in the 400ml anhydrous propanone.Under the room temperature with solution stirring whole night.Behind the evaporation acetone, white crystal is dissolved in the 36ml ethanol, adds the 84ml pure water subsequently gradually.Solution was kept 2 days down at 4 ℃, and be settled out white needle-like crystals.With its filtration and drying under reduced pressure.Carry out 2 (productive rates: 90-95%) of this redeposition.

Synthesizing of N-hydroxy-succinamide dexamethasone-succinate (NHS-dex-suc)

This program is based on previous report (Po Yani T. (Pouyani, T.) and Prestwich G.D. (Prestwich, G.D.) functional derivative-pharmaceutical carriers of hyaluronic acid oligosaccharide and novel biomaterial (FunctionalizedDerivatives of Hyaluronic-Acid Oligosaccharides-Drug Carriers and Novel Biomaterials.) biological concatenator chemistry (Bioconjugate Chemistry) 5 (1994) 339-347, it is incorporated herein by reference).1.3930g dex-suc, 0.3355g N-hydroxy-succinamide (NHS) and 0.6040mg dicyclohexyl carbodiimide (DCC) are dissolved in the 80ml acetone, and at room temperature stirred 16 hours, produce the white crystals precipitation.By the filtered and recycled crystal, evaporative removal acetone also obtains exsiccant white crystal, and promptly uses (productive rate: 90-95%) without being further purified.

Synthesizing of hyaluronic acid-adipic dihydrazide-dexamethasone-succinate (HA-DEX)

This program is based on previous report (Po Yani T. (Pouyani, T.) and Prestwich G.D. (Prestwich, G.D.) functional derivative-pharmaceutical carriers of hyaluronic acid oligosaccharide and novel biomaterial (FunctionalizedDerivatives of Hyaluronic-Acid Oligosaccharides-Drug Carriers and Novel Biomaterials.) biological concatenator chemistry (Bioconjugate Chemistry) 5 (1994) 339-347, it is incorporated herein by reference).100mg HA-ADH is dissolved in 13.33ml NaHCO ₃In the buffer (pH=8.5).125.8mgNHS-dex-suc is dissolved among the 26.67ml DMF (dimethyl formamide).Pouring NHS-dex-suc solution into HA-ADH solution through 30 minutes also at room temperature stirred 18 hours.In 300ml acetone, make the polymer redeposition, subsequently to water dialysis 3 days.The purified product lyophilizing is also stored (productive rate: 70-80%) down in 4 ℃ subsequently.

The preparation of disk hydrogel (HAX and HAX-DEX)

Preparation HA-DEX and the crosslinked disk hydrogel (HAX-DEX) of HA-ALD.(hundred special companies (Baxter), this state Deere of Illinois takes moral (Deerfield, IL)) and 2% (w/v) aqueous solution of HA-DEX and HA-ALD is expelled to is sandwiched in two rubber molds between the slide glass to use double syringe.Prepared hydrogel's diameter and thickness are respectively 1.2cm and 3.5mm.Prepare the crosslinked disk hydrogel (HAX) of HA-ADH and HA-ALD in the same manner.

The sign of polymer

Dexamethasone and dexamethasone-succinate are dissolved in d ₆Among-the DMSO, and HA-ADH and HA-DEX be dissolved in D ₂Among the O, and pass through ¹H-NMR spectrum (Varian technology company (Varian): excellent Buddhist nun carries 300 type spectrophotometers (Unity 300 spectrophotometer)) is analyzed.

By using Atlantis (Atlantis) dC18 analytical column (dC18,4.6 * 250mm, particle diameter 5 μ m) the synthetic and purity of high performance liquid chromatography (Agilent technology company (Agilent technologies) 1100 series) checking dex-suc, NHS-dex-suc, HA-ADH and HA-DEX.Mobile phase is acetonitrile and NaH ₂PO ₄/ H ₃PO ₄The mixture of buffer (60/40, pH 3.8).Flow velocity is 1ml/min and measures UV absorbance (Hewlett-Packard (Hewlett Packard): G1314A) at 246nm.As report, connect the base back by amount (the Ai Fute people such as (Evertss) of HPLC analysis at the basic hydrolysis succinate with the link coupled dexamethasone of HA, use E-to select plain directed immune concatenator to be delivered to (Selective intracellular delivery ofdexamethasone into activated endothelial cells using an E-selectin-directedimmunoconjugate.) Journal of Immunology (Journal Of Immunology) 168 (2002) 883-889 in the active endotheliocyte in the dexamethasone selecting cell, it is incorporated herein by reference).

The sign of hydrogel

Such as report measure gelling time (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Rich Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.); Gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkablehyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 27 (2006) 4698-4705 in the original position crosslinkable hyaluronic acid gel prevention rabbit model, it is incorporated herein by reference).In simple terms, using under the magnetic bar stirring, 0.1ml 2% (w/v) HA-ALD aqueous solution is added in 0.1ml 2% (w/v) HA-DEX or the HA-ADH aqueous solution, and measure the time that mixture becomes the hydrogel bead.

(AR1000 of TA instrument company (TA Instruments:AR1000), knob Karst, De Lahua state (New Castle, Delaware)) measures the modulus of shearing of disc gel to utilize flow graph.Preparation disk hydrogel as indicated above makes it expand 5 days in PBS buffer (pH=7.4) subsequently, and measures expanding volume in the gravimetric analysis mode.Carry out creep and lax test with different shear stresses.Slope by the linear relationship between the stress and strain calculates shear modulus G.The R of the fit line between the stress and strain ²Value is higher than 0.95.

Vigor is analyzed

Use human mesothelial cell system (CRL-9444:ATCC) to use MTT to analyze (Pu Luomaige company (Promega)) and be determined under the situation that has HA, HA-ADH, HA-ALD and HA-DEX in vitro cell viability.Under 37 ℃ in 5%CO ₂In, make cell in complete growth medium (have Yi Ershi BSS (Earle ' s BSS), 0.75mM L-glutaminate and 1.25g/L sodium bicarbonate and be supplemented with the culture medium 199 of 3.3nM epidermal growth factor, 400nM hydrocortisone, 870nM insulin, 20mM HEPES and 10% hyclone), grow and keep.With 5 * 10 ⁴Individual cell put into each hole of 24 well culture plates and under 37 ℃ in 5% CO ₂The middle cultivation whole night.Culture medium is replaced with the culture medium that contains variable concentrations HA, HA-ADH, HA-DEX or HA-ALD.Added behind these materials the 3rd day, and carried out MTT and analyze.100 microlitre tetrazolium solution are added in each hole and at 37 ℃ to descend to cultivate 4 hours.Use the 1ml detergent solution to dissolve by the purple Jia Za that the active wire plastochondria produces and to measure by plate reader (SpectraMax384 of molecular device company type (Molecular Devices:SpectraMax 384)) at 570nm subsequently.At the hole standardization absorbance that test material is not added in the culture medium.

Dexamethasone discharges and the HAX-DEX degradation kinetics

The discoid HAX-DEX hydrogel of use 2% as indicated above (w/v) gel precursors formulations prepared from solutions.GIBCO catalog number (Cat.No.) 10569-010), have 0.5% a BSA (bovine serum albumin: DMEM aldrich corp) or have 10% FBS (hyclone: among DMEM GIBCO catalog number (Cat.No.) 10082-147), and cultivated 8 days down at 37 ℃ discoid hydrogel is soaked in 4ml DMEM (modified her the Ge Shi culture medium (Dulbecco ' s Modified EagleMedium) of Du Beikashi:.When the 1st day, the 2nd day, the 3rd day and the 5th day culture medium is replaced fully with fresh culture and, add in the cell subsequently-80 ℃ of storages.

Measure the degraded time-histories of HAX and HAX-DEX disk in the gravimetric analysis mode.Measure the weight W of hydrogel when putting in the some time _sWith expanding volume Q (%) with Q=W _s/ W _iCalculate, wherein W _iInitial weight for hydrogel.

From mice former generation macrophage preparation

(moral grandson (Hudson, NY)) is breathed out in New York to the C57/B16 mice available from tower Kornic Systems Corp..(DIFCO experimental apparatus company (DIFCO Laboratories), Detroit, the state of Michigan (Detroit, MI)) is expelled in the abdominal cavity with the aseptic thioglycolic acid saline solution of 2ml 3% (w/v).Injected back 4 days, and passed through CO ₂Mice is implemented euthanasia and injects the ice-cold PBS buffer that contains 5mM EDTA of 6ml.After stirring peritoneum with tweezers, sucking-off contains the solution of macrophage.Cell is placed among the ice-cold on ice DMEM immediately, with after scouring, counting and coated plate.Obtain every mice about 10 ⁷Individual cell.To contain 5 * 10 among the DMEM of 10% (v/v) FBS ⁵Individual cell add in each holes of 96 well culture plates and under 37 ℃ in 5%CO ₂The middle cultivation whole night.

The macrophage that is excited by lipopolysaccharide (LPS) produces cytokine

By removing non-adherent cell with 20 microlitres/hole PBS buffer.The dexamethasone that subsequently 100 μ l is had pre-culture medium (2.2.9 part) of cultivating of the DMEM of 10%FBS and 100 μ l and hydrogel or a concentration known adds in the adhesion macrophage in each hole.Under 37 ℃ in 5% CO ₂The middle cultivation after 24 hours, (Sigma company (Sigma), St. Louis, the Missouri State (St.Louis, MO) catalog number (Cat.No.) L-4391) adds in each hole to reach the ultimate density of 100ng/ml with 25 μ l 900ng/mlLPS.After cultivating 16 hours again, the collection culture medium is also stored up to analysis under-80 ℃.Measure TNF-α, IL-6 and the concentration of dexamethasone in described culture medium by ELISA (enzyme connects the immunoadsorbent analysis).The ELISA test kit of TNF-α, IL-6 and dexamethasone is respectively available from R﹠amp; D system company (Dorset (DuoSet) catalog number (Cat.No.) DY406), (the mice IL-6ELISA MAX of Paribas company (BioLegend) ^TMOrganize (getting Lars (Deluxe)) and Ni Aoji company limited (Neogen Corporation).

In vivo experiment

The scheme of ratifying about animal care according to committee of the Massachusetts Institute of Technology (Massachusetts Institute of Technology Committee), abide by the nursing of relevant laboratory animal and the NIH of use and instruct (NIH announces #85-23, revision in 1985) nursing animal.Utilize the male SV129 mice of the 2-3% isoflurane counterweight 25-30g in the oxygen to implement anesthesia.Scrape the back center line off and clean with 70% isopropanol water solution.In the center line of back, inject 1mlHAX-DEX down with double syringe (in the syringe 0.5ml HA-DEX is arranged, 0.5ml HA-ALD is arranged in another syringe) percutaneous.Injected back 2 days, and put to death animal and use standard technique to handle tissue for histologic analysis.

Statistical analysis

By the strange t check of Wei Er (Welch ' s t-test) (the t check that has unequal variance between each group) analytical data.Be the relatively effect of culture medium in the release dynamics test, before the strange t check of Wei Er, carry out ANOVA.Between different time points with same medium and gel comparison in use paired t-test.Utilize Ka Leidage

(Si Niji software company (Synergy Software)) carries out all statistical test.It is significant that p value＜0.05 is considered to statistics.

The result

The sign of polymer (HA-ADH and HA-DEX)

All material all is synthetic according to the scheme among Figure 32.

Respectively by NMR spectrum 4.48 and the chemical potential in-migration of the 21-methene proton of 4.70ppm to 4.78 and 5.03ppm place dexamethasone determine that dex-suc's is synthetic.In addition, the chemical shift of 24-methylene and 25-methene proton appears at the 2.59ppm place.The elution time of dexamethasone, dex-suc and the NHD-dex-suc that measures by HPLC is respectively 4.9min, 5.5 and 7.9min.By HPLC, the efficient that dexamethasone changes into dex-suc and NHS-dex-suc is respectively〉99% and 96%.

By ¹H-NMR spectrum is determined synthesizing of HA-ADH; The 48.4%D-glucuronic acid residue modified by ADH (Yi Ou Y. (and Yeo, Y.); The sharp C in sea (Highley, C); Rich Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, R.) and gram Chinese D. (Kohane, D.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogelsprevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 27 (2006) 4698-4705 in the original position crosslinkable hyaluronic acid gel prevention rabbit model, it is incorporated herein by reference).This is that ratio by the integrated value at the peak of the methyl proton of the integrated value at the peak of the methyl proton of the ADH of 1.62ppm place residue and the N-of 2.00ppm place acetyl group-D-glycosamine residue calculates.

By HPLC and ¹H-NMR determines the synthetic HA-DEX by HA-ADH.Measure by HPLC, HA-DEX has UV absorbance peak value under 246nm, and HA-ADH does not then have.The time of staying of HA-DEX is 3.0min.Utilize NMR spectrum, the synthetic HA-DEX by HA-ADH is also determined in the appearance that connects the chemical shift of basic 24-methylene and 25-methene proton by succinate in the dex-of the 2.63ppm place succinate.The integrated value at peak of methene proton that is connected base by 2.63ppm place succinate is definite with the ratio of the integrated value at the peak of the methyl proton of the N-of 2.00ppm place acetyl group-D-glycosamine, and 13.1% D-glucuronic acid residue is connected radical reaction with the succinate of NHS-dex-suc.Thus, calculate: 35.3% (=48.4%-13.1%) the ADH residue connects the base modification without succinate, and 27% (=13.1%/48.4%) HA-ADH residue and NHS-dex-suc reaction.

Preliminary study confirms that this HA-dexamethasone product discharges most of dexamethasone in the short period section, because chemical compound has efficient, so be undesirable on biology.In addition, the experience of utilizing some polymer systems (for example, poly-(lactic acid-copolymerization-glycolic) microsphere (gram Chinese D.S. (Kohane, D.S.), this J.Y. (Tse of the base of a fruit, J.Y.), Yi Ou Y. (Yeo, Y.), Pa Dela R. (Padera, R.), Shu Binna M. (Shubina, M.) and bright lattice R. (Langer, R.) be used for the biodegradable polymerization microsphere of intraperitoneal drug delivery and nanometer spheroid (Biodegradable polymericmicrospheres and nanospheres for drug delivery in the peritoneum.) biomedical material research magazine A part (Journal Of Biomedical Materials Research Part A) 77A (2006) 351-361, it is incorporated herein by reference) show that the hydrophobicity that increases this material may be unfavorable to its biocompatibility that is used for some required purposes (for example peritoneum).Therefore, reduce the dexamethasone load by thorough dialysis.When HPLC analyzed end-product, the percentage ratio with the link coupled HA-ADH of dexamethasone in the end-product only was 0.1-0.2% (n=3), shows that nearly all HA-ADH discharges because of succinate connects basic hydrolysis in dialysis procedure.Confirm as the turbidity that in solution, increases by material, even this extremely low dexamethasone also has influence to the dissolubility of described concatenator in water to the final degree of modification of HA.This end-product is called as HA-DEX hereinafter.

The sign of hydrogel (HAX and HAX-DEX)

(22.3 ± 0.5sec) than HAX long (3.5 ± 1.0 for the gelling time of HAX-DEX; N=4 in each group, p＜0.0001), estimate that this is to reduce by 27% because can be used for the quantity of crosslinked hydrazides group on the HA-ADH because of the dexamethasone modification.

In the PBS buffer, soak after 5 days, when in PBS when crosslinked, HAX and HAX-DEX compare with initial volume and expand 206.2 ± 14.9% and 235.6 ± 36.9% (n=4, p=0.21).

The shear stress values of HAX and HAX-DEX is respectively 32.4 ± 17.2Pa (n=4) and 22.1 ± 13.5Pa (n=4), but difference not on the statistics significantly.These values show two kinds of hydrogel alterable height shapes, and show that HAX-DEX is with crosslinked with the roughly the same degree of HAX.

Synthetic polymer is to the influence of mesothelial cell's vigor

HAX and HAX-DEX gel discharge polymer segments between its degradative phase, such as HA-ADH, HA-ALD or HA-DEX.Carrying out MTT analyzes with the influence of research synthetic polymer to mesothelial cell's vigor.Not modified HA, HA-ALD and HA-ADH (being n=4) represent the less reduction (Figure 33) of cell viability.(for not modified HA and HA-ADH, under all concentration, p＜0.005 compared with the control.For HA-ALD, at 0.3,0.6,0.9 and 1.5% time, p is respectively 0.4,0.07,0.017 and 0.003).HA-DEX (n=4) causes bigger reduction (for example, the comparison between HA-ADH and the HA-DEX of mesothelial cell's vigor; P under all concentration＜0.005) (Figure 33).

The time-histories of the release dynamics of dexamethasone and hydrogel volume in the cell culture medium

The HAX-DEX gel is cultivated in cell culture medium (DMEM).When the 1st day, the 2nd day, the 3rd day, the 5th day and the 8th day, change culture medium and as method described in, use the concentration (Figure 34) of the dexamethasone that the ELISA measurement discharged.In independent group, use culture medium: 0.5% bovine serum albumin (BSA) or 10% hyclone (FBS) as more representative physiology's associated fluid.Under all three kinds of situations, first three day release of dexamethasone is to take place with constant relatively speed, reduces gradually subsequently.The dexamethasone total amount that is discharged is respectively 0.94 ± 0.20 μ g among the DMEM; Have 1.33 ± 0.39 μ g among the DMEM of BSA; With 1.02 ± 0.41 μ g among the DMEM with FBS, this respectively with DMEM in discharge altogether 17.8 ± 3.8% through link dexamethasone, have among the DMEM of BSA altogether 25.3 ± 7.4% through link dexamethasone and have among the DMEM of FBS altogether 19.3 ± 7.8% corresponding through linking dexamethasone.Therefore, during release experiment, about 20% dexamethasone discharges with the free drug form because of the cracking that succinate connects base.When these experiments finished, the gel-free block retained; Infer that all the other 80% dexamethasone are to discharge with the banded form of polymer segments.The GPC of the culture medium that is discharged also confirms the existence through the macromole of dexamethasone derivatization (macromer), is impossible but directly measure the amount that discharges in this way.Dexamethasone concentration during except that the 5th day in the BSA culture medium is higher than pure DMEM (p=0.031) and the FBS culture medium (p=0.009), when putting at any time in the different culture medium concentration of dexamethasone all do not have the significant difference of statistics.

HAX and HAX-DEX hydrogel expand in cell culture medium and degraded (Figure 35) thereupon.The two all represents expansion in a couple of days.Just expanding in the time of the 1st day does not exist the significant difference of statistics between HAX and the HAX-DEX, but during by the 3rd day, HAX-DEX expands greatly (p＜0.05) than HAX in DMEM+BSA and DMEM+FBS.Therefore the degradation rate of HAX-DEX is faster than HAX, and the volume of HAX-DEX gel significantly reduces the 5th day the time.

The antiinflammation of HAX-DEX

Dexamethasone makes that the output dose dependent of IL-6 and TNF-α reduces (Figure 36) in the macrophage that lipopolysaccharide (LPS) stimulates.Under the situation of no dexamethasone in the LPS stimulated cells concentration of IL-6 and TNF-α be respectively 773 ± 13ng/ml and 11723 ± 87pg/ml (n=4).Be lower than 10 ^-9The dexamethasone concentration of M is to the not influence of output of described two kinds of molecules.

Be the antiinflammatory effectiveness of research HAX-DEX, the former generation peritoneal macrophages that utilizes that the release culture medium that is produced by HAX in the experiment in aforementioned part and HAX-DEX handles that LPS stimulates.After being exposed to peritoneal macrophages, measure the concentration of IL-6 in the described culture medium (Figure 37) and TNF-α (Figure 38).Figure 36 and Figure 37 and 38 relatively disclose, the culture medium that is obtained by the release experiment of utilizing HAX can obviously not weaken the output of IL-6 or TNF-α.By contrast, being exposed to the culture medium that is obtained by HAX-DEX can make the two produce obviously reduction.Last 3 days respectively with HAX and compared, in HAX-DEX, see peritoneal macrophages the statistics of IL-6 (Figure 37) and TNF-α (Figure 38) output is significantly suppressed with 5 days.

Biocompatibility and tissue reaction

Percutaneous is injected 1ml HAX or HAX-DEX (n=4 separately) down in male SV129 mice.Injected back 2 days, and scraped off to animal enforcement euthanasia and with it.After the dissection, as via finding out in skin and the subcutaneous tissue, the profile boundary of HAX-DEX bag is more clear than HAX.Although the HAX gel is toughness slightly, it is mobile much better than and be diffused in the tissue plane.The HAX-DEX gel is easy to remove (extraction) (Figure 39 A) with the form of discrete entities from its capsule bag.The HAX-DEX gel is more clear than HAX gel.These discoveries form contrast with the in vitro result (wherein the HAX-DEX degraded is faster than HAX) above.All dyed parts of HAX-DEX gel smear all show and almost completely do not have leukocyte infiltration, and have only 1 HAX gel smear to have fine and close neutrophil cell and macrophage is assembled.In addition, the tissue and the remaining gel at gel-organizational interface place are presented in cellular infiltration much better than among the HAX than in the HAX-DEX gel (Figure 39 B-D).

Discuss

Describe herein: the synthetic and sign that discharges crosslinked HA hydrogel through linking dexamethasone.This system is characterised in that and will makes and be easy to use and retain at intraperitoneal.Hydrogel is the low dexamethasone that loads in earth polar consciously.As indicated above, a problem is that the excessive hydrophobicity of substrate will weaken the biocompatibility of HA substrate.In addition, the dexamethasone efficacy of a drug is strong, and it comprises and weakens wound healing, immunosuppressant, hypertension, gastrointestinal hemorrhage etc.Therefore, dosage minimizes for making general action or making the minimized while of splitting of contiguous wound can provide local antiphlogistic activity very important.Even utilize described extremely low useful load, the medicine that hydrogel also can discharge valid density clinically lasts a couple of days, and can not take place and will discharge suddenly in a large number by the dexamethasone that dialysis is fallen is caused.On the contrary, compare Cytotoxic appropriate increase with HAX by HAX-DEX and can determine these importances about the consideration of useful load.Yet, should notice that known cross-linked hydrogel slowly degrades, in vivo so in vitro the concentration of the free HAX-DEX of Huo Deing unlikely occurs.

The multidigit research worker has been reported the effectiveness (Huo Keer M. (Hockel that the steroidal glucocorticoid receptor agonist forms in the prevention peritoneal adhesion, people such as M.), utilize lasting intraperitoneal dexamethasone prevention rat peritoneum adhesion (Prevention Of Peritoneal Adhesions In The Rat With Sustained IntraperitonealDexamethasone Delivered By A Novel Therapeutic System.) Annales Chirurgiae EtGynaecologiae 76 (1987) 306-313 that transmit by novel therapy system, it is incorporated herein by reference), (Bu Kenmaier C.C. (Buckenmaier, people such as C.C.), use relatively tissue adhesion treatment (Comparison ofantiadhesive treatments using an objective rat model.) U.S. surgeon magazine (AmericanSurgeon) 65 (1999) 274-282 of target rat model, it is incorporated herein by reference), (Cook Ke Ziken T. (Kucukozkan, people such as T.), pass through sodium cromoglicate after the operation on pelvis, dexamethasone, normal saline and aprotinin Film with Preventing Adhesion (Preventionof adhesions by sodium chromoglycate, dexamethasone, saline and aprotinin after pelvicsurgery.) ANZ operation magazine (ANZ J Surg) 74 (2004) 1111-5, it is incorporated herein by reference).According to reports, these chemical compounds all are to work by reducing mechanism such as mesothelial cell and peritoneal macrophages generation cytokine.In this article, by measuring two kinds of cytokines of effectiveness research that dexamethasone discharges.TNF-α is the important cytokine (Huo Mudaer L. (Homdahl that acute inflammation and peritoneal adhesion form, L.) and Allen Iverson M.L. (Ivarsson, M.L.) cytokine, condense altogether and effect (the The role of cytokines of fibrinolysis in peritoneal tissues is repaired, coagulation, and fibrinolysis in peritonealtissue repair.) Europe operation magazine (European Journal OfSurgery) 165 (1999) 1012-1019, it is incorporated herein by reference), (Munster that S.E. (Mutsaers, S.E.) mesothelial cell: its structure, function and its effect (Mesothelial cells:Their structure, function and role in serosal repair.) respiratory disease in serous coat is repaired are learned (Respirology) 7 (2002) 171-191, and it is incorporated herein by reference).It stimulates the mesothelial cell to secrete multiple amboceptor: plasminogen activator inhibitor (PAI) (Wo Weier S.A. (Whawell, people such as S.A.), the human peritoneal mesothelium cell of tumor necrosis factor mediation discharges plasminogen activator inhibitor-1 (Tumor Necrosis Factor-Mediated Release Of Plasminogen-ActivatorInhibitor-1 By Human Peritoneal Mesothelial Cells.) Britain operation magazine (British Journal OfSurgery) 81 (1994) 214-216, it is incorporated herein by reference), (Wo Weier S.A. (Whawell, S.A.) and Simpson J.N. (Thompson, J.N.) the human mesothelial cell of cytokine induction discharges plasminogen activator inhibitor-1 (Cytokine-Induced Release Of Plasminogen-Activator Inhibitor-1 By HumanMesothelial Cells.) Europe operation magazine (European Journal Of Surgery) 161 (1995) 315-318, it is incorporated herein by reference), it slows down fibrosis (denapon Si Baige V.W.M. (Vanhinsbergh, V.W.M.), bohr K.A. (Bauer, K.A.), gram Nice tower T. (Kooistra, T.), Krafft C (Kluft, C), Dorje Ward G. (Dooijewaard, G.), Harry Hillman M.L. (Sherman, M.L.) and knob Wen Huiwen W. (Nieuwenhuizen, W.) Fibrinolytic progress-tissue type plasminogen activator between the tumor necrosis factor incorporating period among the mankind, 1 type plasminogen activator inhibitor and fibrin (former) catabolite follow increase (ProgressOf Fibrinolysis During Tumor-Necrosis-Factor Infusions In Humans-Concomitant IncreaseIn Tissue-Type Plasminogen-Activator, Plasminogen-Activator Inhibitor Type-1, AndFibrin (Ogen) Degradation Products.) hematology (Blood) 76 (1990) 2284-2289, it is incorporated herein by reference), (Muller base I.K. (Mullarky, people such as I.K.), tumor necrosis factor and IFN-but not level (the Tumor necrosis factoralpha and gamma interferon of preventative fibrin deposition between hemorrhage or pathogen load indication infection period, but not hemorrhage or pathogen burden, dictate levels ofprotective fibrin deposition during infection.) infect and immunology (Infection And Immunity) 74 (2006) 1181-1188, it is incorporated herein by reference); IL-1, IL-6 (Munster that S.E. (Mutsaers, S.E.) mesothelial cell: its structure, function and its effect (Mesothelial cells:Their structure in serous coat is repaired, function and role in serosal repair.) respiratory disease is learned (Respirology) 7 (2002) 171-191, it is incorporated herein by reference) and the prostaglandin (sharp N. (Topley of Top, N.), Peterson M.M. (Petersen, M.M.), Mackenzie R. (Mackenzie, R.), knob Buddhist script written on pattra leaves A. (Neubauer, A.), Si Tanlianwo E. (Stylianou, E.), Ka Weier V. (Kaever, V.), Davis M. (Davies, M.), Cole Si G.A. (Coles, G.A.), fine jade Leix A. (Jorres, A.) and WILLIAMS-DARLING Ton J.D. (Williams, J.D.) human peritoneal mesothelium cell prostaglandin synthetic-cytokine induction Cycloxygenase courier Rna (Human Peritoneal Mesothelial CellProstaglandin Synthesis-Induction Of Cyclooxygenase Messenger-Rna By PeritonealMacrophage-Derived Cytokines.) international kidney periodical (Kidney International) 46 (1994) 900-909 that obtain by peritoneal macrophages, it is incorporated herein by reference), it quickens peritoneal inflammation; IL-8 (Munster that S.E. (Mutsaers, S.E.) mesothelial cell: its structure, function and its effect (Mesothelial cells:Their structure in serous coat is repaired, function and role in serosal repair.) respiratory disease is learned (Respirology) 7 (2002) 171-191, it is incorporated herein by reference), monocyte chemoattractant protein-1 (MCP-1) (Munster that S.E. (Mutsaers, S.E.) mesothelial cell: its structure, function and its effect (Mesothelial cells:Their structure in serous coat is repaired, function and role in serosal repair.) respiratory disease is learned (Respirology) 7 (2002) 171-191, it is incorporated herein by reference) etc., it induces neutrophil cell and monocyte recruitement.Therefore, the activity of inhibition TNF-α has the potential value that suppresses peritoneal adhesion.IL-6 is produced by the various kinds of cell that comprises macrophage, fibroblast and mesothelial cell.IL-1 and TNF-α induce its generation (people such as (Topley) in the dose dependent mode in the mesothelial cell, the synthetic interleukin-6 of human peritoneal mesothelium cell-be subjected to IL-1-β and Tnf-α to induce (Human PeritonealMesothelial Cells Synthesize Interleukin-6-Induction By Il-1-Beta And Tnf-Alpha.) international kidney periodical (Kidney International) 43 (1993) 226-233, it is incorporated herein by reference).It has the multiple effect that adhesion forms that relates to, comprise and stimulate the mesothelial cell to secrete PAI (Wo Weier S.A. (Whawell, people such as S.A.), the human mesothelial cell of cytokine induction discharges plasminogen activator inhibitor-1 (Cytokine-InducedRelease Of Plasminogen-Activator Inhibitor-1 By Human Mesothelial Cells.) Europe operation magazine (European Journal Of Surgery) 161 (1995) 315-318, and it is incorporated herein by reference); Suppress mesothelial cell's propagation (people such as (Lanfrancone) of Lan Fu Netac, human peritoneal mesothelium cell produces many cytokines (granulocyte colony-stimulating factor [Csf], granulocyte-mononuclear cell-Csf, macrophage-Csf, interleukin-1 [Il-1] and I1-6) and through Il-1 activation and stimulate with growth (Human Peritoneal Mesothelial Cells Produce ManyCytokines (Granulocyte Colony-Stimulating Factor[Csf], Granulocyte-Monocyte-Csf, Macrophage-Csf, Interleukin-1[Il-1], And Il-6) And Are Activated And Stimulated To GrowBy Il-1. hematology (Blood) 80 (1992) 2835-2842, it is incorporated herein by reference); Discharge with induction of vascular endothelial growth factor (VEGF), promote angiogenesis (gram Chinese people such as (Cohen) thus, interleukin-6 induction of vascular endothelial growth factor sublist reaches (Interleukin 6 induces the expression of vascular endothelialgrowth factor.) journal of biological chemistry (Journal of Biological Chemistry) 271 (1996) 736-741, it is incorporated herein by reference), as TNF α or TGF-β.

Confirmed the biocompatibility of HA based substrate in the peritoneum and in fact it is used to prevent adaptability (Yi Ou people such as (Yeo), original position crosslinkable hyaluronic acid gel prevention rabbit model operation postabdomen adhesion (the In situcross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in arabbit model.) biomaterial (Biomaterials) 2006 of peritoneal adhesion; 27:4698-4705, it is incorporated herein by reference).Dexamethasone is selected for because of its effectiveness definitely and HA substrate links, and therefore might utilize the minimum biological optimum substrate-wish that especially avoiding making it to have hydrophobicity realizes given inflammatory effects of changing possibly.The biocompatibility of mediator is very important: one studies show that, by hydrophobic polymer poly--the microsphere with low dexamethasone load that DL-Acetic acid, hydroxy-, bimol. cyclic ester-copolymerization-lactide (PLGA) constitutes makes the adhesion aggravation, and the microsphere with higher dexamethasone load reduces adhesion and forms.This shows, effect (the gram Chinese people such as (Kohane) that dexamethasone is used for Film with Preventing Adhesion is offset in the effect that causes adhesion of polymeric microspheres (a kind of attested subsequently characteristic), be used for biodegradable polymer microsphere and nanometer spheroid (Biodegradable polymeric microspheres and nanospheres fordrug delivery in the peritoneum.) (Journal of Biomedical Materials Research Part A) 77A (2006) 351-361 that the peritoneum Chinese medicine transmits, it is incorporated herein by reference).Prepolymer HA-DEX may cause than the low cell viability of employed other precursor macromolecule of this paper, but importantly, should note the pharmacotoxicological effect of known dexamethasone and the character of analysis, the direct toxic action so this can reflect antiproliferative effect.In addition, the concentration of the uncrosslinked HA-DEX of possibility will be more much lower than the concentration of mixing this place test of back.In fact, described result shows the biocompatibility of the HAX-DEX biocompatibility of HAX of can comparing, and causes even slighter inflammatory response.

Conclusion

As suppress macrophage TNF-α and IL-6 and produce institute's confirmations, the original position crosslinkable binding hydrogel of hyaluronic acid and dexamethasone has suitable navigability and release biology effective dexamethasone.In vivo, the HAX-DEX gel soaks into the inflammatory cells lower than HAX gel and associates.

Example 15: utilize the original position crosslinkable hyaluronan hydrogel prevention peritoneal adhesion of transmitting budesonide

Foreword

This case description contains the in-situ cross-linked hyaluronic acid gel (barrier device) of glucocorticoid receptor agonist budesonide.Selecting budesonide is because known its inflammation effect in adhesion forms.Selecting hyaluronic acid is because known its biocompatibility in peritoneum.Use double syringe to use the system that comprises two kinds of cross-linkable precursor liquid, thereby in less than 10 seconds, be formed with elastic force and persistent hydrogel.The severe that the rabbit peritoneal adhesion forms repeat sidewall damaged-secondary damage of caecum scratch model after, this composite or reference substance are administered to damaged part.In the big adhesion of all development in the animal of normal saline treatment.With the budesonide in the normal saline or do not have in the animal of hydrogel treatment of budesonide, adhesion formation and area slightly alleviate.In the animal of the treatment of the budesonide in hydrogel, the incidence rate and the area of adhesion obviously reduce.In rat during subcutaneous injection, represent budesonide in the hydrogel and compare independent hydrogel inflammation and reduce.Generally speaking, the budesonide in the hyaluronic acid gel is suitable and very effective for the adhesion in the prevention severe repeated trauma model.It is a kind of potential promising system of tissue adhesion prevention; Therefore, the present invention's effectiveness of containing approval barrier device can greatly be strengthened by simultaneous drug delivery.

Peritoneal adhesion is can be at operation wound or infect that the lasting tissue between the structure is connected in abdominal part that the back forms and the pelvic cavity.The incidence rate of tissue adhesion is up to 80% and cause serious clinical effectiveness usually, such as pain, infertile or intestinal obstruction (Di Zejia GS (diZerega GS) peritoneum, peritoneum healing and adhesion formation (Peritoneum, peritoneal healing, andadhesion formation.), Di Zejia GS (diZerega GS) compiles, operation on peritoneum (Peritoneal Surgery.) New York (New York): Springer Verlag (Springer), 2000. 3-37 pages or leaves; It is incorporated herein by reference).In the trial of Film with Preventing Adhesion, the multidigit research worker has been used the medicament of intervening the critical events of adhesion in forming, and (Di Zejia GS (diZerega GS) peritoneum, peritoneum healing and adhesion form (Peritoneum, peritoneal healing, and adhesionformation.), Di Zejia GS (diZerega GS) compiles, operation on peritoneum (Peritoneal Surgery.) New York (NewYork): Springer Verlag (Springer), 2000. 3-37 pages or leaves; It is incorporated herein by reference).Pathogenetic inflammatory component that adhesion forms has become target commonly used (the diligent bank T in bitter Cook (Kucukozkan T) of the pharmacotherapy that uses multiple steroidal antibiotic medicine, Esso is with B (Ersoy B), outstanding lattice D (Uygur D), pass through sodium cromoglicate after Gan Duodu C. (GundogduC.) operation on pelvis, dexamethasone, normal saline and aprotinin Film with Preventing Adhesion (Prevention ofadhesions by sodium chromoglycate, dexamethasone, saline and aprotinin after pelvicsurgery.) ANZ operation magazine (ANZ J Surg) 2004; 74 (12): 1111-1115; Huo Keer M. (Hockel, M.), Ou Te S. (Ott, S.), uncommon graceful U. (Siemann, U.), (Kissel T.) utilizes lasting intraperitoneal dexamethasone prevention rat peritoneum adhesion (Prevention Of Peritoneal Adhesions InThe Rat With Sustained Intraperitoneal Dexamethasone Delivered By A Novel TherapeuticSystem.) the Annales Chirurgiae Et Gynaecologiae 1987 that transmits by novel therapy system to Ke Sier T.; 76:306-313; Bu Kenmaier C.C. (Buckenmaier, C.C.), Pu Sitelei A.E. (Pusateri A.E.), Halley Si R.A. (Harris, R.A.), (Hetz S.P.) uses relatively tissue adhesion treatment (Comparison of antiadhesivetreatments using an objective rat model.) U.S. surgeon magazine (American Surgeon) 1999 of target rat model to hertz S.P.; 65:274-282; Horse riel J (Maurer J), effect (The effect of aqueous progesterone on operative adhesion formation.) fertility and sterile (Fertil Steril) 1983 that Bang Nawente L. (Bonaventura L.) progesterone aqueous solution forms in postoperative intestinal adhesion; 39 (4): 485-489; Jia Zannijia A (Gazzaniga A), James J (JamesJ), uncle Xiao J (Shobe J), the prevention of peritoneal adhesion in Ou Puhaimu E. (Oppenheim E.) rat.The effect of dexamethasone, methylprednisolone, promethazine and human plasmin (Prevention of peritoneal adhesions in the rat.The effects of dexamethasone, methylprednisolone, promethazine, and human fibrinolysin.) surgery's archives (Arch Surg) 1975; 110 (4): 429-432; Failure (Failure of intraperitoneal adjuncts to improve the outcome ofpelvic operations in young women.) U.S.'s obstetrics and gynecology magazine (Am J Obstet Gynecol) 1985 with Jansen R. (Jansen R.) intraperitoneal adnexa improvement young woman operation on pelvis; 153 (4): 363-371, all documents all are incorporated herein by reference).Yet the effectiveness of these medicament Film with Preventing Adhesion is in animal model (Jia Zannijia A (Gazzaniga A), James J (James J), uncle Xiao J (ShobeJ), the prevention of peritoneal adhesion in Ou Puhaimu E. (Oppenheim E.) rat.The effect of dexamethasone, methylprednisolone, promethazine and human plasmin (Prevention of peritoneal adhesions in the rat.The effects ofdexamethasone, methylprednisolone, promethazine, and human fibrinolysin.) surgery's archives (Arch Surg) 1975; 110 (4): 429-432, it is incorporated herein by reference) and clinical trial (failure (Failure of intraperitoneal adjunctsto improve the outcome of pelvic operations in young women.) U.S.'s obstetrics and the gynecology magazine (Am J Obstet Gynecol) 1985 of Jansen R. (Jansen R.) intraperitoneal adnexa improvement young woman operation on pelvis; 153 (4): 363-371, it is incorporated herein by reference) in and inconsistent, especially (prevention (Prevention of postoperative formation and reformation of pelvic adhesions.) the Te Er Tener KH (Treutner KH) that forms and form again after the operation of Larsen B. (Larsson B.) pelvic adhesion all the more so when Film with Preventing Adhesion recurs, the V of section in this khoum is general (Schumpelick V) editor peritoneal adhesion (PeritonealAdhesions.) Berlin: Springer-Fu glug Te Luo publishing house (Berlin:Springer-Verlag Telos), 1997. the 331-334 page or leaf, it is incorporated herein by reference).

The limited effectiveness of medicine quick removing from peritoneum can causing intraperitoneal drug administration.Make medicine keep to make its biological agent maximization than the suitable transmission system of high local concentrations.In this example, selected the drug delivery system of original position crosslinkable hyaluronan hydrogel (HAX) as anti-inflammatory compound.Formerly in the example, confirmed HAX for prevention rabbit sidewall damaged-peritoneal adhesion in the caecum scratch model has good effectiveness, and this and nano-particle exist irrelevant (Yi Ou Y (Yeo Y) people of etc.ing, postabdomen adhesion (the In situcross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in arabbit model.) biomaterial (Biomaterials) 2006 of performing the operation in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; With Yi Ou Y. (Yeo, Y.), rely on T. (Ito, T.), rich Lars E. (Bellas, E.), sharp CB (the Highley in sea, CB), and Ma Ruini R. (Marini, R.), gram Chinese DS (Kohane, DS), the original position crosslinkable hyaluronic acid gel that contains polymer/nanoparticle (In situ cross-linkable hyaluronic acid hydrogels containing polymericnanoparticles for preventing post-operative abdominal adhesions.) surgery's record event (Ann Surg) 2006 reports of the postabdomen adhesion that is used for preventing performing the operation, two pieces of documents all are incorporated herein by reference).Budesonide has the effective glucocorticoid activity (handbook on doctor's table (Physicians ' Desk Reference): Thomson PDR (Thomson PDR) of the dexamethasone of can comparing, 2006, it is incorporated herein by reference) and after whole body absorbs, change into non-activity metabolite (handbook on doctor's table (Physicians ' Desk Reference): Thomson PDR (Thomson PDR) rapidly, 2006, it is incorporated herein by reference).Use strict repeated trauma animal model to confirm the tissue adhesion activity of budesonide in this example.Use bio-compatible HAX can significantly improve its tissue adhesion activity, thus the adhesion in the animal that prevention major part is fully tested.

Materials and methods

The preparation of original position crosslinkable HA derivant

Follow the synthetic original position crosslinkable HA derivant of the method for before being reported and analyzed (Yi Ou Y. (and Yeo, Y.); The sharp CB in sea (Highley, CB); Rich Lars E. (Bellas, E.); Support T. (Ito, T.); Agate Renyi R. (Marini, R.); Bright lattice R. (Langer, people such as R.), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesionsin a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; The special P. of boolean's skin (Bulpitt, P.); Ai Siqiman D. (Aeschlimann, D.), the hyaluronic New Policy of chemical modification: the preparation of functional derivative and its are used to form purposes (New strategy for chemical modification ofhyaluronic acid:Preparation of functionalized derivatives and their use in the formation ofnovel biocompatible hydrogels.) the biomedical material research magazine (J Biomed Mater Res) 1999 of novel bio-compatible hydrogel; 47:152-169; With merchant X.Q. (Jia, X.Q.); Klum win G. (Colombo, G.); Pa Dela R. (Padera, R.); Bright lattice R. (Langer, R.); Gram Chinese D.S. (Kohane, D.S.), prolong sciatic nerve blocking-up (Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronicacid.) biomaterial (Biomaterials) 2004 by in-situ cross-linked hyaluronic acid; 25 (19): 4797-4804, all described documents are incorporated herein by reference separately).In simple terms, prepare HA-adipic dihydrazide (HA-ADH) by the carboxyl in adipic dihydrazide and the HA main chain is linked, and prepare HA-aldehyde (HA-CHO) by HA and sodium metaperiodate are reacted.

The preparation of budesonide-normal saline and budesonide-HAX

At first budesonide is dissolved in the ethanol to make the stock solution of 8.2mg/ml.By being added in the 10ml normal saline, the 0.16ml stock solution prepares budesonide-normal saline.For budesonide-HAX, 0.08ml budesonide stock solution is added to respectively among 5ml HA-ADH (20mg/ml) and the 5ml HA-CHO (20mg/ml).By using hundred special double syringes to prepare budesonide-HAX gel via two kinds of precursor solutions of same outlet eluting.Budesonide-normal saline and budesonide-HAX contain the 0.13mg/ml budesonide.

The sign of budesonide-HAX

As discussed previously, the in-situ gelling time of at room temperature measuring budesonide-HAX, (Yi Ou people's original position crosslinkable hyaluronic acid gels such as (Yeo) prevented operation postabdomen adhesion (In situ cross-linkable hyaluronicacid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the rabbit model; 27:4698-4705, it is incorporated herein by reference).In simple terms, preparation 20mg/ml HA-ADH and HA-CHO solution in normal saline.As indicated above, budesonide is added in each solution to reach 0.13mg/ml.Under continuous stirring, 100 microlitre HA-ADH solution are mixed with 100 microlitre HA-CHO solution.Time when forming solution with the isolating solid globules of described tray bottom is considered as gelling time.Check the form of lyophilizing budesonide-HAX internal structure by scanning electron microscope analysis (SEM).With budesonide-HAX gel lyophilizing and cooling back fragmentation in liquid nitrogen.With sample palladium and gold (150 dusts are thick) dash coat and use scanning electron microscope (JEOL JSM6320, U.S. JEOL company limited, Massachusetts Pi Baidi (Peabody, MA)) observation.

The measurement of the dissolubility of budesonide in normal saline

Measure maxima solubility (Jenner promise AR (Gennaro AR) editor of budesonide in normal saline as described, Lei Mingdun: pharmacy theory and practice (Remington:The Science and Practice of Pharmacy.) the 20th edition, philadelphia, pa (Philadelphia, PA): Donald Lippincott William Si ﹠amp; (the LippincottWilliams. ﹠amp of Louis Wilkins operating room; Wilkins), 2000, it is incorporated herein by reference).At first, the budesonide with recruitment mixes with normal saline so that 25mg/ml to be provided in described system the budesonide concentration to 0.002mg/ml.Under 37 ℃ with mixture continuous stirring 24 hours, and subsequently with 12, centrifugal 5 minutes of 000rpm so that solution be separated with solid.Measure the solution concentration of middle budesonide mutually by high performance liquid chromatography (HPLC).Measure the saturation solubility of budesonide in normal saline by extrapolation linear section (its slope is 0) to the y axle.

In independent experiment, preparation budesonide as indicated above-normal saline 0.13mg/ml is divided into the aliquot of 1ml, and cultivates down in 37 ℃ under continuous stirring.When fixed time interval, obtain budesonide-normal saline aliquot and with 12, centrifugal 5 minutes of 000rpm analyzes for HPLC to isolate the 0.8ml supernatant.The potential residue 0.2ml that precipitates budesonide that contains is dissolved in the 0.8ml acetonitrile and utilizes HPLC to be analyzed.

Budesonide release dynamics in vitro

Discoid budesonide-the HAX of preparation in being sandwiched in two rubber molds between the glass slide.Prepared hydrogel's diameter and thickness are respectively 8mm and 3.5mm (about 150 μ l).Budesonide-HAX gel weighed and put into Ai Benduofu pipe (eppendorf tube), to wherein adding the 1ml phosphate buffered saline (PBS) that contains the 10U/ml hyaluronidase, and under stirring continuously, cultivate down at 37 ℃.Behind brief rotary weakening, take a sample, and replace the 0.5ml fresh culture discharging culture medium 0.5ml.To discharge that sample is freezing to be analyzed up to HPLC.

The HPLC of budesonide analyzes

Chromatographic system is by the HPLC solvent transmission system that is equipped with automatic sampler and UV detector (1100 series, Agilent technology company (Agilent Technologies), California Paro Austria many (Palo Alto, CA)) composition.Analytical column is Atlantis dC18 (dC18; 4.6 * 250mm; Particle diameter 5 μ m).Mobile phase is 0.1% acetic acid of 30:70 and the mixture of acetonitrile, and flow velocity is 1ml/min.5 μ l samples are expelled on the post of pre-balance, wash 10min with mobile phase subsequently.The UV detector is arranged on 248nm.By with concentration relevant the obtain calibration curve of the peak area in the chromatogram with the budesonide standard substance.The time of staying: 6.1min.Detection limit: 0.2 μ g/ml.

Budesonide-HAX in vivo uses

The scheme of ratifying about animal care according to committee of the Massachusetts Institute of Technology (Massachusetts Institute of Technology Committee), abide by the nursing of relevant laboratory animal and the NIH of use and instruct (NIH announces #85-23, revision in 1985) nursing animal.

The subcutaneous administration of budesonide-HAX

Utilize the 2-3% isoflurane in the oxygen that male Sprague's-Dao Li rat (Sprague-Dawley rat) (320g-420g) is implemented to anaesthetize.By double syringe (in the syringe 0.5ml HA-ADH is arranged, 0.5ml HA-CHO is arranged in another syringe) injection 1ml budesonide-HAX or HAX.Preparation as indicated above contains the budesonide-HAX of 0.13mg/ml budesonide.Therefore, giving the total amount with the budesonide of rat is 0.3-0.4mg/kg.Replace the budesonide stock solution to prepare HAX (negative control) by adding ethanol.To be expelled in the center line of back under budesonide-HAX or the HAX percutaneous.Inject back 2 days (n=5) or 5 days (n=4), put to death animal to check tissue reaction.Use standard technique to handle tissue for histologic analysis.

Prevent peritoneal adhesion by budesonide-HAX

As described in [tPA] in the previous research, bring out female albefaction rabbit (Spain hare (Oryctolaguscuniculus), New Zealand white (New Zealand White)) (peritoneal adhesion in 3 ± 0.5kg) via repeating laparotomy.In simple terms, carry out caecum scratch and celiotomy incision to bring out newborn adhesion (Yi Ou people such as (Yeo), original position crosslinkable hyaluronic acid gel prevention rabbit model operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels preventpost-operative abdominal adhesionsin a rabbit model.) biomaterial (Biomaterials) 2006; 27:4698-4705, it is incorporated herein by reference).1 week back is carried out laparotomy for the second time and is introduced other damage with the cutting adhesion and in the position identical with damage in the first time laparotomy.Remove the excessive blood of injury region, and use hundred special double syringes that 10ml budesonide-normal saline or budesonide-HAX are applied to scratch zone (n=6) more subsequently.Two kinds of composites all contain 1.3mg budesonide (0.44mg/kg).Operator is about characteristic the unknown of treatment.

As employing post-operative care as described in the other places (Yi Ou people such as (Yeo), operation postabdomen adhesion (In situ cross-linkable hyaluronic acid hydrogels preventpost-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference).Put to death animal by the intravenous injection pentobarbital sodium after the 2nd laparotomy.(this JW of Berne people such as (Burns JW) is by preventing tissue injury and tissue adhesion (Prevention of tissue injuryand postsurgical adhesions by precoating tissues with hyaluronic acid solutions.) operations research magazine (J Surg Res) 1995 with hyaluronic acid solution precoating tissue to the adhesion scoring for the amending method of the method that use is reported; 59:644-652, it is incorporated herein by reference): 0 minute=no adhesion; 1 minute=utilize the gravity will isolating tissue adhesion; 2 minutes=can pass through the isolating tissue adhesion of blunt dissection; 3 minutes=adhesion that needs sharp weapon to dissect.If there is multiple adhesion, select higher score as representative score with different scores.Measured 2 fens or the area of adhesion in 3 fens for the qualitative assessment of adhesion.The appraiser is about treatment the unknown that each animal received.The fixation of tissue that will be reclaimed by postmortem is embedded in the paraffin in 10% formalin, and section is also dyeed for histological examination with haematoxylin and Yihong.

Statistical analysis

Will be from the data of in vivo testing with meansigma methods and the 25th percentage point and the 75th percentage point of expression, this is because its not followed normal distribution distribution always, and (Chicago, Illinois State city (Chicago, IL)) uses Mann-Whitney U test or utilizes the accurate check of expense snow to carry out statistics and infer after Kruskal-Wallis test to use SPSS software.Think that the p value less than 0.05 is remarkable for statistics on two-tailed test.

The result

The formation of budesonide-HAX gel

After under continuous stirring, mixing, the solution of two kinds of HA derivants (20mg/ml) (the two all contains the 0.13mg/ml budesonide) forms budesonide-HAX gel in 4.1 ± 1.7sec, the gelling time similar (3.9 ± 1.1sec, p=0.72 when two tail t check) of this HAX gel during with no budesonide.Under SEM (Figure 40), lyophilizing budesonide-HAX gel represents porous network (typical case of cross-linked hydrogel) (merchant X (Jia X), Bo Dike J A (BurdickJ A), Ke Bole J (Kobler J), Clifton RJ (Clifton RJ), Roseau Butterworth base JJ (Rosowski JJ), carry special this SM (Zeitels SM) people of etc.ing, the synthetic and sign of original position crosslinkable hyaluronic acid based aquagel and be used for the regenerated potential application of vocal cords (Synthesis and Characterization of in Situ Cross-Linkable HyaluronicAcid-Based Hydrogels with Potential Application for Vocal Fold Regeneration.) macromolecule (Macromolecules) 2004; 37 (9): 3239-3248; With Yi Ou Y (Yeo Y), Bo Dike JA (Burdick JA), sea sharp CB (Highley CB), agate Renyi R (Marini R), bright lattice R (Langer R), the peritoneum of gram Chinese DS. (KohaneDS.) chitosan and UV crosslinkable chitosan is used (Peritoneal application of chitosan andUV-cross-linkable chitosan.) biomedical material research magazine (J Biomed Mater Res) 2006; 78A (4): 668-675; Two pieces of documents all are incorporated herein by reference).

The dissolubility of budesonide in normal saline

Classify according to American Pharmacopeia (United States Pharmacopoeia), budesonide " in fact soluble " Yu Shuizhong (dissolubility:＜0.1mg/ml) (American Pharmacopeia, Rockwell MD (Rockville, MD): American Pharmacopeia committee company limited (United States Pharmacopeial Convention, Inc.)).For the soluble fraction of budesonide in the assessment composite that this paper studied, measure its dissolubility in normal saline under 37 ℃.Not commensurability budesonide powder is dissolved whole night down at 37 ℃.The concentration of budesonide is issued to stablely in the solution that is separated with undissolved solid at 0.027mg/ml, and this is defined as at the saturation solubility (Figure 41 A) of 37 ℃ of following budesonides in normal saline.

Select 0.44mg/kg as in vivo research (the bitter Cook diligent bank T (Kucukozkan T) of budesonide dosage for the experiment of the previous use of basis dexamethasone Film with Preventing Adhesion, Esso is with B (Ersoy B), outstanding lattice D (Uygur D), pass through sodium cromoglicate after Gan Duodu C. (Gundogdu C.) operation on pelvis, dexamethasone, normal saline and aprotinin Film with Preventing Adhesion (Prevention of adhesions by sodium chromoglycate, dexamethasone, saline andaprotinin after pelvic surgery.) ANZ operation magazine (ANZ J Surg) 2004; 74 (12): 1111-1115; Huo Keer M. (Hockel, M.), Ou Te S. (Ott, S.), uncommon graceful U. (Siemann, U.), (Kissel T.) utilizes lasting intraperitoneal dexamethasone prevention rat peritoneum adhesion (Prevention OfPeritoneal Adhesions In The Rat With Sustained Intraperitoneal Dexamethasone DeliveredBy A Novel Therapeutic System.) the Annales Chirurgiae Et Gynaecologiae 1987 that transmits by novel therapy system to Ke Sier T.; 76:306-313; With Bu Kenmaier C.C. (Buckenmaier, C.C.), Pu Sitelei AE (Pusateri AE), Halley Si RA (Harris, RA), (Hetz SP) uses relatively tissue adhesion treatment (Comparison of antiadhesivetreatments using an objective rat model.) U.S. surgeon magazine (American Surgeon) 1999 of target rat model to hertz SP; 65:274-282, all described documents all are incorporated herein by reference).In these researchs, the dosage of dexamethasone arrives in the scope of 4mg/kg 0.33.The glucocorticoid of known budesonide is renderd a service suitable (general action is stronger 40 times than cortisone) (handbook on doctor's table (Physicians ' desk reference): Thomson PDR (Thomson PDR) with dexamethasone, 2006, it is incorporated herein by reference), so the lower limit of employed dexamethasone dosage range is suitable in the dosage of the employed budesonide of this paper and other research.For the dosage with 10ml budesonide-normal saline or budesonide-HAX offers the 3kg rabbit, so prepare budesonide-normal saline and the budesonide-HAX that contains the 0.13mg/ml budesonide.Be the change of the solution concentration that is determined at 37 ℃ of following budesonides, budesonide-normal saline is cultivated down at 37 ℃, and the analytical solution phase that passs in time.Soon, budesonide-normal saline promptly contains 0.13 ± 0.004mg/ml budesonide (the total budesonide in=98.5 ± 3.5% system) at solution in mutually after the preparation.When cultivating down for 37 ℃, the concentration of budesonide reduced rapidly in 2 hours, reached 0.034 ± 0.001mg/ml (the total budesonide in=25.2 ± 0.7% system) (Figure 41 B) in 12 hours.In residue 0.2ml, reclaim the residue (Figure 41 B) of precipitation form.

Budesonide release dynamics in vitro

Contain 10 units per ml hyaluronidases (its provide with previous research in impaired peritoneum in the suitable in vitro gel degradation speed of gel degradation speed) PBS in check the budesonide release dynamics (Yi Ou Y. (Yeo of HAX, people such as Y.), operation postabdomen adhesion (In situ cross-linkablehyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705; With Yi Ou Y. (Yeo, Y.); Support T. (Ito, T.); Rich Lars E. (Bellas, E.); The sharp CB in sea (Highley, CB); Agate Renyi R. (Marini, R.); Gram Chinese DS (Kohane, DS), be used for preventing the performing the operation original position crosslinkable hyaluronic acid gel that contains polymer/nanoparticle (Insitu cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions ina rabbit model.) surgery's archives (Ann Surg) 2006 reports of postabdomen adhesion, described two pieces of documents all are incorporated herein by reference).For making the release of the dissolved budesonide of release dynamics reflection, the amount of incorporating medicine into is lower than its saturation solubility (0.027mg/ml).

62.6 ± 6.8% expection budesonide in 24 hours (Figure 42) discharges from the HAX gel.Even after gel is degraded fully, do not have yet and significant further discharge.Can explain loading and the total difference between the burst size by the loss of degraded HAX gel between sampling date (it is for semi-solid).Therefore this degraded in second day of gel has only a small amount of loose gel pieces to the still can observe in the time of 8 days.

Budesonide-HAX in vivo uses

Prevent peritoneal adhesion by budesonide-HAX

Be the tissue adhesion effect of assessment budesonide-normal saline and budesonide-HAX, use the repeated trauma model.Since bring out than conventional sidewall damaged-adherent model that caecum scratch model is stronger represents facility and confirms that very effectively HAX itself compares improved tissue adhesion effect, so using described model is quite noticeable (Yi Ou Y. (Yeo, people such as Y.), operation postabdomen adhesion (In situ cross-linkablehyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006 in the original position crosslinkable hyaluronic acid gel prevention rabbit model; 27:4698-4705, it is incorporated herein by reference).

As indicated above, scratch of rabbit caecum and generation sidewall is damaged.After 1 week, the development of 100% animal 2 minutes and/or adhesion in 3 fens.Abrade again with these adhesion dissolvings and with caecum and sidewall damage location.Budesonide-normal saline or budesonide-HAX are applied on the affected area again.Historical control is to be provided by the animal from independent normal saline of the usefulness of the experiment of carrying out simultaneously (n=6) or HAX (n=6) treatment separately.After 1 week, animal is implemented euthanasia for postmortem.Two groups are experienced the similar loss in weight (Figure 43 A) (p=0.378) between survival period; The two is suitable with situation with the historical control group of independent normal saline or HAX treatment (n=6, p=0.272, Kruskal-Wallis test) separately.Development adhesion in 3 fens (Figure 43 B) in the animal of 83% usefulness budesonide-normal saline treatment.Although the matched group no difference of science of statistics of adhesion score and normal saline treatment, the adhesion area roughly reduces by 3 times (p=0.01).

Budesonide among the HAX obviously reduces adhesion score and area, even is better than the situation of the animal of budesonide treatment.Adhesion in 67% test animal obtains prevention fully.In residue animal (n=2), the adhesion area is 1.6 and 4.6cm ², its contrast [tPA] that is significantly less than normal saline treatment (p=0.003), budesonide-normal saline (p=0.046) and HAX[tPA] (p=0.022).

During histological examination, the sample of obtaining from adhesion is the fibrous connective tissue that connects caecum smooth muscle and stomach wall skeletal muscle layer.In budesonide-normal saline and budesonide-HAX group, observe the surface regeneration epithelium (Figure 44) of healing caecum suitable and stomach wall with unaffected peritoneal surface.The thickness of lower floor's granulation tissue and cell are formed and are not had notable difference between two groups.

The tissue reaction of subcutaneous budesonide-HAX

Be the antiinflammation of checking budesonide-HAX, there is or does not exist the HAX of budesonide in male Sprague's-Dao Li rat (Sprague-Dawley rat) subcutaneous injection, thereby form discrete swelling.Injected back 2 days and 5 days, and gathered gel by the dissector of the unknown.After the gross anatomy, the HAX gel presents the inflammation (Figure 45 A) of quite big degree, and the translucent or opaque thicker organized layer of covering the high vascularization of implant, its can't with lower floor's skin anatomical isolation (Figure 45 B).Therefore by contrast, the budesonide gel represents the vascularization and the inflammation of much less, can find out gel (Figure 45 C) clearly and may be obvious that gel (Figure 45 D) with the surrounding tissue anatomical isolation under most of situation.Histological assessments discloses the contrast between back 2 days two kinds of hydrogels of injection, thus its to be easy to be unknown observer's difference.In the sample that does not have budesonide, the space that is occupied by hydrogel is by inflammatory cells, especially neutrophil cell is fallen into oblivion (Figure 45 E).In having the sample of budesonide, inflammatory response greatly reduces, thereby makes water-setting agent be kept perfectly to a great extent (Figure 45 F).In the time of the 5th day, can not utilize each group of gross anatomy or histological examination difference.

Discuss

Confirm herein and can significantly improve the tissue adhesion effect of budesonide by using the drug delivery system that can keep higher local drug concentration at the perished surface place.

Budesonide is in fact water-fast chemical compound, and the saturation solubility in normal saline is 0.027mg/ml under 37 ℃.The part that exceeds the budesonide-normal saline of described dissolubility boundary precipitated (Figure 41 B) rapidly through 2 hours.Therefore, the budesonide-normal saline that is applied to peritoneum is actually the mixture of saturated solution and budesonide precipitation suspension.If precipitation is retained in the peritoneum, so described precipitation itself can be served as the reservoir of continuous drug release.As comparing with the contrast of normal saline treatment, budesonide-normal saline obviously reduces the adhesion area, but the frequency of adhesion in 3 fens there is no different with contrast.Medicinal effectiveness is subjected to that the restriction of relative low dosage and/or budesonide-normal saline remove rapidly from peritoneum be possible.Yet when making up with HAX, same dose is but highly effective.

In rat model, the localized sustained transmission of using the dexamethasone that the PLGA micron particle carries out for Film with Preventing Adhesion than the effective (Huo Keer M. (Hockel of dexamethasone crystal suspension, M.), Ou Te S. (Ott, S.), uncommon graceful U. (Siemann, U.), (Kissel T.) utilizes lasting intraperitoneal dexamethasone prevention rat peritoneum adhesion (Prevention Of Peritoneal Adhesions In The Rat With Sustained IntraperitonealDexamethasone Delivered By A Novel Therapeutic System.) the Annales Chirurgiae EtGynaecologiae 1987 that transmits by novel therapy system to Ke Sier T.; 76:306-313, it is incorporated herein by reference), this situation just appears when using the micron particle that higher dosage dexamethasone (4mg/kg) is provided in a large number but have only.A small amount of micron particle makes adhesion aggravate.Confirmed before that the PLGA micron particle itself brought out peritoneal adhesion (gram Chinese DS (Kohane DS), carry this JY (Tse JY), Yi Ou Y (Yeo Y), Pa Dela R (Padera R), Shu Baina M (Shubina M), bright lattice R. (Langer R.) are used for biodegradable polymerization microsphere and nanometer spheroid (Biodegradable polymericmicrospheres and nanospheres for drug delivery in the peritoneum.) the biomedical material research magazine (J Biomed Mater Res) 2006 that the peritoneum Chinese medicine transmits; 77A (2): 351-361, it is incorporated herein by reference).The adhesion prophylactic activity of dexamethasone is offset in a kind of short adhesion effect meeting that is interpreted as mediator of these reports.The effectiveness of system described herein can partly not cause the true of adhesion owing to mediator itself and in fact it has inherent tissue adhesion activity (Yi Ou people such as (Yeo), original position crosslinkable hyaluronic acid gel prevention rabbit model operation postabdomen adhesion (In situ cross-linkablehyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.) biomaterial (Biomaterials) 2006; 27:4698-4705, it is incorporated herein by reference).

The usefulness of subcutaneous experiment is that the antiinflammation of budesonide gel may continue 2 days but be less than 5 days at least, and the persistent period that budesonide discharges in this and the ex vivo experiment is roughly the same.Therefore, this brief relatively drug release phase seems soluble existence and do not have result difference between the hydrogel of budesonide.This equates following viewpoint: the critical events that adhesion forms normally takes place in pro-2-3 days, and shows that the persistent period of the drug release that the adhesion prevention is required may be extremely short.

Although many methods of adhesion prevention are failed, in laboratory animal, there is noticeable efficacy data in clinical experiment.Reason that works may for: it is to assess in the animal model of allowing relatively, wherein adhesion prevents relatively easy (Wei Siman DM (Wiseman DM.) animal adhesion model: design, variable and dependency (Animaladhesion models:design, variables, and relevance.), Di Zejia GS (diZerega GS) editor, operation on peritoneum (Peritoneal Surgery) New York: Springer (New York:Springer), 2000. the 459-476 page or leaf, it is incorporated herein by reference).This is to use a reason that has more challenging repeated trauma model.

Budesonide-HAX is easy to preparation and handles, under the situation that has blood and peritoneal fluid effectively and can use via laparotomy or endoscope.

Equivalent and category

One of ordinary skill in the art will use only a kind of normal experiment to recognize the many equivalents that maybe can determine specific embodiment of the present invention as herein described.Category of the present invention does not plan to be limited to above-mentioned embodiment, but as described in the claims of enclosing.

In claims, unless do opposite explanation or apparent by context in addition in addition, otherwise can mean multiple such as the article of " " and " described ".Unless do opposite explanation or apparent by context in addition in addition, think and between one group of one or more member, comprise " or " claims or embodiment satisfy one, a plurality of or all members of described group and be present in, be used for given product or method or otherwise relevant with given product or method.The present invention includes a member of strictly described group be present in, be used for given product or method or otherwise with given product or the relevant embodiment of method.The present invention also comprise a plurality of or all members of described group all be present in, be used for given product or method or otherwise with given product or the relevant embodiment of method.In addition, should be appreciated that the present invention is contained will introduce all changes, combination and change in another claim from one or more claim or from one or more qualifications of embodiment relevant portion, key element, clause, exemplary term etc.For instance, can be to any claim correct of deciding on another claim so that it comprises one or more qualifications for the treatment of as in any other claim that a basic claim decides that see.In addition, when right requires a kind of compositions of narration, unless obviously will cause and conflict or contradiction unless do indication or one of ordinary skill in the art in addition, otherwise should be appreciated that to comprise for any purpose disclosed herein and use described method for compositions, and comprise according to the either party's legal system in known other method in preparation method disclosed herein or the affiliated field and be equipped with method for compositions.For instance, should be appreciated that any compositions of the present invention all can be used for suppressing any position and/or by this paper discussed or affiliated field in the adhesion that causes of known any reason form, make progress and/or recur.Should also be clear that any compositions according to preparation method for compositions disclosed herein preparation all can be used for suppressing any position and/or by this paper discussed or affiliated field in the adhesion that causes of known any reason form, make progress and/or recur.In addition, the compositions according to any method preparation of preparation compositions disclosed herein is contained in the present invention.

In key element is under the situation about providing with tabulation with for example Ma Kuxi group (Markush group) form, should be appreciated that each subgroup that also discloses described key element and can remove any key element from described group.It shall yet further be noted that term " comprises " plans for open and allow and forgive other key element or step.Should be appreciated that, in general, mention when comprising specific factor, feature, step etc. in the present invention or each side of the present invention, some embodiment of the present invention or each side of the present invention by or form by described key element, feature, step etc. basically.For succinct purpose, these embodiment do not specify with these speech in this article.Therefore, for the various embodiments of the present invention that comprise one or more key elements, feature, step etc., the present invention also provide by or the embodiment that forms by these key elements, feature, step etc. basically.

When given range, comprise end points.In addition, should be appreciated that, unless do indication in addition or one understanding from context and/or one of ordinary skill in the art is apparent, otherwise can take any particular value in the scope described in the different embodiments of the invention with the value that scope is represented, unless in addition clear indication in the context, otherwise be accurate to 1/10th of described scope lower limit unit.Should also be clear that, unless do indication in addition or one understanding from context and/or one of ordinary skill in the art is apparent, otherwise can take any subrange in the specified scope with the value that scope is represented, wherein the endpoint table of described subrange is shown as and reach and 1/10th of described scope lower limit unit identical levels of precisions.

In addition, should be appreciated that any specific embodiment of the present invention can get rid of clearly from one or more claim.Any embodiment, key element, feature, application or the aspect of the present composition and/or method (for example, arbitrary class covalent bond, arbitrary class bioactivator or the particular agent between any hydrogel precursor, any polysaccharide derivates or non-polysaccharide polymer (for example any HA derivant or cellulose derivative), any molecular weight ranges, any cross-linking agent, the hydrogel precursor, any particle diameter and/or material are formed, any purpose of any dosing way or position, throwing and compositions etc.) all can be got rid of outside one or more claim in office.For instance, in certain embodiments of the invention, bioactivator is not an antiproliferative.For brief purpose, get rid of all not clearly statements in this article of all embodiment of one or more key elements, feature, purpose or aspect.

Claims (60)

1. method that suppresses adhesion, described method comprises following steps:

First hydrogel precursor is thrown and is given position in the individual body; With

With described position in throwing of second hydrogel precursor and the described individual body,

Wherein said first hydrogel precursor is cellulose derivative or glucan derivative; Wherein said second hydrogel precursor is a glucan derivative; Wherein described first with after described second hydrogel precursor is in contact with one another, described hydrogel precursor is cross-linked to form hydrogel; And wherein said hydrogel suppresses adhesion.

2. method according to claim 1, wherein said first hydrogel precursor comprises first functional group; Wherein said second hydrogel precursor comprises second functional group; And wherein said first and described second functional group formation covalent bond that under physiological condition, reacts to each other.

3. method according to claim 1, at least a in wherein said first hydrogel precursor or described second hydrogel precursor comprise non-polysaccharide part.

4. method according to claim 1, wherein said cellulose derivative are selected from the group that is made up of MC derivant, CMC derivant and HPMC derivant.

5. method according to claim 1, wherein said first hydrogel precursor are that CMC derivant and described second hydrogel precursor are the Sensor Chip CM 5 derivant.

6. method according to claim 1, wherein said second hydrogel precursor are that CMDX-ADH and described first hydrogel precursor are selected from the group that is made up of MC-CHO, CMC-CHO and HPMC-CHO.

7. method according to claim 1, wherein said second hydrogel precursor are that CMDX-ADH and described first hydrogel precursor are CMC-CHO.

8. method according to claim 1, wherein said first hydrogel precursor are that first glucan derivative and described second hydrogel precursor are second glucan derivative.

9. method according to claim 8, wherein said first glucan derivative are that CMDX-ADH and described second glucan derivative are CMDX-CHO.

10. method according to claim 1, wherein said hydrogel precursor be with the solution form throw with.

11. method according to claim 1, wherein said hydrogel precursor be with endoscope's mode or use syringe throw with.

12. method according to claim 1, wherein said hydrogel are to be in contact with one another the back at described hydrogel precursor to form in 1 and 100 second.

13. method according to claim 1, wherein said hydrogel precursor come down under the situation that does not have free cross-linker to throw with.

Throw and at least a solution that comprises cellulose derivative 14. method according to claim 1, wherein said method comprise, the concentration of wherein said cellulose derivative is greater than 5mg/ml.

Throw and at least a solution that comprises cellulose derivative 15. method according to claim 1, wherein said method comprise, the concentration of wherein said cellulose derivative is greater than 25mg/ml.

Throw and at least a solution that comprises cellulose derivative 16. method according to claim 1, wherein said method comprise, the concentration of wherein said cellulose derivative is greater than 50mg/ml.

Throw and at least a solution that comprises glucan derivative 17. method according to claim 1, wherein said method comprise, the concentration of wherein said glucan derivative is greater than 5mg/ml.

Throw and at least a solution that comprises glucan derivative 18. method according to claim 1, wherein said method comprise, the concentration of wherein said glucan derivative is greater than 25mg/ml.

Throw and at least a solution that comprises glucan derivative 19. method according to claim 1, wherein said method comprise, the concentration of wherein said glucan derivative is greater than 50mg/ml.

20. method according to claim 1, its be additionally contained in throw with described first and described second hydrogel precursor before, destroy the step of the adhesion that described position exists.

21. method according to claim 1, wherein said method comprise to throw and bioactivator with the solution or the independent solution form of hydrogel precursor formation.

22. method according to claim 21, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

23. method according to claim 21, wherein said bioactivator are antiinflammatory.

24. method according to claim 23, wherein said antiinflammatory are the non-steroidal antiinflammatory.

25. method according to claim 24, wherein said non-steroidal antiinflammatory is selected from the group that is made up of following: celecoxib (celecoxib), diclofenac (diclofenac), diflunisal (diflunisal), etodolac (etodolac), Salicylate (salicylate), Lip river, Fino sweet smell (fenoprofen), ibuprofen (ibuprofen), flurbiprofen (flurbiprofen), indomethacin (indomethacin), ketoprofen (ketoprofen), ketorolac (ketorolac), first chlorine side hydrochlorate (meclofamate), meclofenamic acid salt (meclofenamate), meloxicam (meloxicam), naproxen (naproxen), piroxicam (piroxicam), sulindac (sulindac), Diplosal (salsalate), nabumetone (nabumetone), aspirin (aspirin), oxaprozin (oxaprozin) and Tolmetin (tolmetin).

26. method according to claim 23, wherein said antiinflammatory are the steroidal antiinflammatory.

27. method according to claim 26, wherein said steroidal antiinflammatory is selected from the group that is made up of following: dexamethasone (dexamethasone), fluorometholone (fluorometholone), prednisolone (prednisolone), loteprednol (loteprednol), medrysone (medrysone), prednisone (prednisone), Methyllprednisolone (methylpredisolone), budesonide (budesonide), cortisone (cortisone), rimexolone (rimexolone), clobetasol (clobetasol), halogen Beta rope (halobetasol), hydrocortisone (hydrocortisone), triamcinolone acetonide (triamcinolone), betamethasone (betamethasone), fluocinonide (fluocinolone) and fluorine west a kind of apple moral (fluocinonide).

28. method according to claim 1, wherein said first and described second hydrogel precursor be by weight the ratio between 1:10 and 10:1 throw with.

29. method according to claim 1, wherein at least a described hydrogel precursor has bioactivator covalently bound with it.

30. method according to claim 29, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

31. method according to claim 29, wherein said bioactivator are antiinflammatory.

32. method according to claim 31, wherein said antiinflammatory are the non-steroidal antiinflammatory.

33. method according to claim 32, wherein said non-steroidal antiinflammatory is selected from the group that is made up of following: celecoxib, diclofenac, diflunisal, etodolac, Salicylate, Lip river, Fino sweet smell, ibuprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, first chlorine side hydrochlorate, meclofenamic acid salt, meloxicam, naproxen, piroxicam, sulindac, Diplosal, nabumetone, aspirin, oxaprozin and Tolmetin.

34. method according to claim 31, wherein said antiinflammatory are the steroidal antiinflammatory.

35. method according to claim 34, wherein said steroidal antiinflammatory is selected from the group that is made up of following: dexamethasone, fluorometholone, prednisolone, loteprednol, medrysone, prednisone, Methyllprednisolone, budesonide, cortisone, rimexolone, clobetasol, halogen Beta rope, hydrocortisone, triamcinolone acetonide, betamethasone, fluocinonide and fluorine west a kind of apple moral.

36. method according to claim 1, its comprise in addition with a plurality of granules with described hydrogel precursor throw with, thereby described granule is retained in the hydrogel that is formed by crosslinked described hydrogel precursor.

37. method according to claim 36, wherein said granule is to comprise nano-particle (nanoparticle) or the micron particle (microparticle) that is selected from by the material of the following group that forms: poly-(lactide), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid), poly-(glycolic), poly-(lactic acid-copolymerization-glycolic), polycaprolactone, Merlon, polyesteramide, poly-(beta-amino ester), polyanhydride, poly-(amide), poly-(aminoacid), Polyethylene Glycol and its derivant, poe, polyacetals, polybutylcyanoacrylate, polyether ester, poly-(dioxy Ketohexamethylene), poly-(alkylidene alkylates), the copolymer of Polyethylene Glycol and poe, the admixture of Biodegradable polyurethane and arbitrary aforementioned polymer or copolymer, and liposome.

38. method according to claim 36, wherein said granule is being thrown with preceding being to exist in solution with hydrogel precursor.

39. method according to claim 36, wherein said granule comprises bioactivator.

40. according to the described method of claim 39, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

41. according to the described method of claim 39, wherein said bioactivator is an antiinflammatory.

42. a compositions, it comprises:

(a) cellulose derivative;

(b) glucan derivative; With

(c) a plurality of granules.

43. according to the described compositions of claim 42; wherein said granule is to comprise nano-particle or the micron particle that is selected from by the material of the following group that forms: poly-(lactide); poly-(Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactic acid); poly-(glycolic); poly-(lactic acid-copolymerization-glycolic); polycaprolactone; Merlon; polyesteramide; poly-(beta-amino ester); polyanhydride; poly-(amide); poly-(aminoacid); Polyethylene Glycol and its derivant; poe; polyacetals; polybutylcyanoacrylate; polyether ester; poly-(dioxy Ketohexamethylene); poly-(alkylidene alkylates); the copolymer of Polyethylene Glycol and poe; the admixture of Biodegradable polyurethane and arbitrary aforementioned polymer or copolymer, and liposome.

44. according to the described compositions of claim 42, wherein said granular biological degradable.

45. according to the described compositions of claim 42, wherein said granule comprises bioactivator.

46. according to the described compositions of claim 45, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

47. according to the described compositions of claim 45, wherein said bioactivator is an antiinflammatory.

48. according to the described compositions of claim 42, wherein said compositions is a hydrogel, wherein said cellulose derivative and described glucan derivative are cross-linked with each other.

49. according to the described compositions of claim 42, wherein said cellulose derivative or described glucan derivative have bioactivator covalently bound with it.

50. according to the described compositions of claim 49, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

51. one kind granule thrown method with position to body, it comprises:

The compositions that will comprise granule, first hydrogel precursor and second hydrogel precursor throw with to described position;

Wherein said first hydrogel precursor is cellulose derivative or glucan derivative; Wherein said second hydrogel precursor is a glucan derivative; And wherein said first holds back described particulate hydrogel with described second hydrogel precursor throwing to form with the back.

52. according to the described method of claim 51, wherein said compositions is to throw and, at least a granule that contains in the described solution with the form of one or more solution.

53. according to the described method of claim 51, wherein said granule comprises bioactivator.

54. according to the described method of claim 51; wherein said granule is to comprise nano-particle or the micron particle that is selected from by the material of the following group that forms: poly-(lactide); poly-(Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactic acid); poly-(glycolic); poly-(lactic acid-copolymerization-glycolic); polycaprolactone; Merlon; polyesteramide; poly-(beta-amino ester); polyanhydride; poly-(amide); poly-(aminoacid); Polyethylene Glycol and its derivant; poe; polyacetals; polybutylcyanoacrylate; polyether ester; poly-(dioxy Ketohexamethylene); poly-(alkylidene alkylates); the copolymer of Polyethylene Glycol and poe; the admixture of Biodegradable polyurethane and arbitrary aforementioned polymer or copolymer, and liposome.

55. a method of throwing and giving individuality with bioactivator, it comprises following steps:

The compositions that will comprise bioactivator, first hydrogel precursor and second hydrogel precursor throw with to the position; Wherein said first hydrogel precursor is cellulose derivative or glucan derivative; Wherein said second hydrogel precursor is a glucan derivative; And wherein said first forms the hydrogel of wherein holding back described bioactivator with described second hydrogel precursor.

56. according to the described method of claim 55, wherein said bioactivator and hydrogel precursor are covalently bound.

57. according to the described method of claim 55, wherein said bioactivator is a therapeutic agent, and it is selected from the group that is made up of following: anti-infective, antiinflammatory, antiproliferative, antitumor agent, antioxidant, angiogenesis inhibitor, immunosuppressant, immunomodulator, anticoagulant, protein hydrolytic reagent, the proteoclastic medicament of enhancing, free radical scavenger, antioxidant, fibroid repair inhibitors and RNAi agent.

58. according to the described method of claim 55, wherein said bioactivator is an antiinflammatory.

59. according to the described method of claim 55, wherein said bioactivator and granule associate with physics mode.

60. according to the described method of claim 55; wherein said bioactivator and nano-particle or micron particle associate with physics mode; described nano-particle or micron particle comprise poly-(lactide); poly-(Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester); poly-(lactic acid); poly-(glycolic); poly-(lactic acid-copolymerization-glycolic); polycaprolactone; Merlon; polyesteramide; poly-(beta-amino ester); polyanhydride; poly-(amide); poly-(aminoacid); Polyethylene Glycol and its derivant; poe; polyacetals; polybutylcyanoacrylate; polyether ester; poly-(dioxy Ketohexamethylene); poly-(alkylidene alkylates); the copolymer of Polyethylene Glycol and poe; the admixture of Biodegradable polyurethane and arbitrary aforementioned polymer or copolymer, and liposome.

Images