MXPA99005535A - Wound healing formulations containing human plasma fibronectin - Google Patents

Abstract

Se producen soluciones no tamponadas, acuosas, que contienen concentraciones elevadas de fibronectina y otros promotores de curación de heridas, que pueden luego utilizarse para hacer formulaciones de gel y crema tópicas con concentraciones elevadas de promotores de curación de heridas. Las formulaciones se utilizan para la curación de heridas cutáneas. Las formulaciones proporcionan liberación lenta y tiempo de contacto incremento de la fibronectina y de otros promotores de curación de heridas al sitio de herida, conduciendo a la absorción efectiva de los promotores.

Description

FORMULATIONS FOR WOUND HEALING CONTAINING HUMAN PLASMA FIBRONECTIN RELATED APPLICATION This application TS a Continuation in Part of the Application of United States No, of Series 08 / 488,253, filed on 7 d? June 1995 for Formulations for Wound Healing that Contain Human Plasma Fibronectin.

FIELD OF THE INVENTION The present invention ST relates to topical dosage forms containing human plasma fibronectin for use in promoting wound healing. In particular, the invention ST relates to the healing d? chronic venous ulcers, BACKGROUND OF THE INVENTION Fibronectin is a large glycoprotein containing about 5% carbohydrate. The characteristic form of plasma fibronectin is a 440,000 dalton disulfide-linked dimer, each subunit having a molecular weight of approximately 220,000 daltons. Normally found in plasma at a concentration of approximately 300 ug / ml, fibronectin is extracted and purified using a method described by Hynes1. Plasma fibronectin is also known by several different names, including cold insoluble globulin, antigelatin factor, cell fixation, cell dispersion factor, and opsonic alpha2 surface bond glycoprotein. These names reflect the biological activities of fibronectin such as cell recruitment, opsonization of particulate debris and promotion of wound contraction. Reviews on the structure and activities of fibronectin have been published in many places2-3. The healing of wounds is usually divided into three phases: the inflammatory phase, the proliferating phase and the reodelation phase. Fibronectin has been reported to be involved in each stage of the wound healing process, particularly creating a platform to which the invading cells can attach. Initially, many mediators, such as fibronetin and fibrinogen, are released at the site of the wound. Fibronectin promotes the migration of inflammatory cells to the wound and waste phagocytosis through monocytes. Subsequently, angiogenesis and reepithelialization occur. At this stage, fibronectin exerts chemotactic activity on the endothelial cells, and promotes the migration of epithelial cells and fibroblasts to the basement membrane. Fibronetin also appears to be an essential component of the remodeling phase where it plays a major role in the organization of Collagen fibrils. The fibrillar collagen finally forms fibrous bundles that greatly improve the tensile strength of the tissue, leading to wound closure. Topically applied plasma fibronectin has been reported as being useful for increasing the healing rate of wounds such as corneal wounds4,5 and leg ulcers6, however, no one has described a topical carrier suitable for use in treating wounds. that can ensure the delivery of an effective amount of fibronetin, a major limiting factor in developing an effective topical dosage form of a drug is not only to have an active drug, but also to have a formulation that allows the passage of the active drug from the carrier (cream, ointment, gel, etc.) to the delivery site (which in the case of the present invention is a skin wound). Very active drugs, such as growth factors, have no therapeutic value if the topical formulation does not allow the drug to move from the semi-solid carrier to the wound. Therefore, it would be highly desirable to develop a formulation that maximizes the contact time of fibronectin with the wound and also controls the release of fibronectin to the wound, thereby leading to high absorption values. The present invention provides Delivery system in the form of aqueous gels and a cream.

SUMMARY OF THE INVENTION The present invention provides aqueous gel formulations and a cream formulation containing fibronectin and its use for the delivery of an effective wound healing amount of fibronectin to a wound site. The gel formulation comprises a soluble polymer in water, pharmaceutically acceptable, which is prepared from an effective amount of fibronectin. Examples of said compounds include; vinyl polymers, e.g., polyacrylic acid; Epoxymers of polyoxyethylene-polyoxypropylene block, v. gr. poloxamer; and Cellulose derivatives, v.gr ,, hydroxypropylcellulose (HPC), The polymer provides viscosity values between 50,000 and 1,000,000 cps at room temperature. The cream formulation is prepared from a commercially available cream base, ie, Scheringt® base (Schering Canada Inc., Point-Claire, Quebec), which has viscosity values between 60,000 to 80,000 cps at room temperature. Many advantages are attributed to these dosage forms. The gel and cream formulations of the present invention release effective amounts of a wound healing promoter. Other advantages of the gel formulations include: ability to maintain wound moisture (which results from the high water content of the gels), ease of application and removal (by washing) of the wound. They also provide a fresh feeling when topically applied which increases the patient's comfort, The slow release system of the gel formulations of the present invention provides prolonged release of fibronectin to the wound site, This property of these formulations allows less frequent application to the wound, resulting in less disturbance of the healing process, These formulations maintain fibronectin delivery for up to 24 hours; but in accordance with the kinetic data obtained from permeation studies, a therapeutic program of "two eces a day" is a preferred embodiment of the present invention, the formulation of the dosage forms ß - Thoughtful topics for the incorporation of fibronectin must respect various quality criteria. All components of the preparation, including solvent, gelling agent and preservative must be non-toxic for the wound and compatible with the drug. The final product should promote the optimal release of the drug to its site of action, be of adequate consistency to, improve the contact time of the drug with the wound and be sterile. Preferred formulations of this invention can be used with other promoters of healing of wounds having a composition similar to fibronectin, such as simulated-sized proteins (extracellular matrix proteins such as troponin, laminin, vitronectin, fibrinogen) or smaller size (such as peptides, including growth factors), promoters may be used of wound healing of humans and other mammals in formulations for veterinary applications. Preferred formulations can be correlated with the evaluation results of these formulations using an in vitro diffusion cell system consisting of a rigid receptor containing a de-epithelialized skin sample, the de-epithelialized side facing upwards towards a donor compartment and the dermal side oriented downward towards a receptor compartment, the de-epithelialized skin sample is prepared by removing a section of 8 μm from the epidermal surface of the skin using an der atome (1 / 10,000 scissor scale) and the dermal side was carefully cleaned of any Adherent subcutaneous tissues and / or blood vessels, The receptor compartment is connected to a circulation circuit board, with the buffer temperature maintained at 37 ° C while the skin surface is around 32 ° C. Preferred formulations will have a "value Ábs "of more than 7, 8, preferably at least 13.40, A preferred method for preparing the gels of the invention is to concentrate human fibronectin in demineralized water, which contains the polymerization promoter (NaOH) at a pH of about 0.1 to about 11.0. For higher concentrated fibronectin gels (0.5-1.0%), it is preferable to lyophilize fibronectin. In both cases, the resulting solutions have a pH of about 8.0 to about 11.0. When a carbomer gel is to be prepared, a pH of about 9.0 is preferred, Thus, it is possible to produce fibronectin solutions Highly concentrated, not precipitated without using buffers, such as saccharides or stabilizers (v, g., albumin), Concentrated fibronectin solutions, 2 mg / ml to 10 mg / ml, can be achieved safely using these methods, The same method can be used to produce highly concentrated aqueous formulations of other wound healing promoters and other proteins generally insoluble in water. The appropriate fibronetin solution is mixed with a concentrated solution of gelling agent. The two solutions are mixed by multiple exchanges under pressure using devices, such as syringes, which do not vigorously agitate the mixture in order to avoid precipitation of fibronectin. The mixing devices are connected by an adapter device, BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates a cumulative uptake of radiolabelled fibronectin over time from various gel formulations containing carbomer Carbopol ™ P-934 (0.375%), poloxamer Pluronic (R) F127 (20.0%) , sodium carboxymethylcellulose (CMC 3.0%). and of the control (phosphate buffered saline), Figure 2 illustrates the skin absorption of radiolabeled fibronectin from various dosage forms and control (buffered saline, with phosphate) at time = 1-2 hours, Figure 3 illustrates the electrophoresis of human plasma (FN) embedded in an earbomer gel © Carbopol'1"(Carbopol (s) P-934 carbomer 0.375% + chlorocresol 0.1%) after 0, 2, 6 and 8 months , Section A, Recovery of FN after a gelatin binding test, Ection E: Integrity of FN after 240 days of gel storage at 4aC, It should be noted that in section B. the resolution of the band is affected by the presence of talis contaminants such as carbomer Carbopolt®) in the sample, Figure 4 shows a trace of dermal absorption with viscosity from different topical preparations, Figure 5 demonstrates the absorption values in the skin by using concentrations in Increasing fibronectin. in carbomer gel containing 0.28% carbomer and the control (phosphate buffered exit solution), Figure 6 illustrates the effect of two different concentrations of earbomér © on fibronectin absorption values in de-epithelialized skin, both formulations of carbero ero contain 0.2% fibronectin; the control is phosphate buffer solution.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides dosage forms that are specially formulated for, the therapeutic use of fibronectin as a topical wound healing promoter. Dosage forms selected for topical applications should ideally release large amounts of fibronectin, be sterile and non-toxic to the wound. Various factors such as the physical and chemical properties of the glycoprotein as well as criteria for clinical use should be considered when composing these formulations. Among these limitations, the main one is related to the solubility of fibronectin in water that is low and, therefore, mitigates against the preparation of concentrated gels or creams. Fibronectin is only slightly soluble in water and can be precipitated at concentrations as low as 5 g / ml in aqueous solution, its solubility is also affected by changes in pH and low temperatures, in the same way, formulations that require dispersion of dust Polymer in the fibronectin solution under agitation can not be easily prepared since precipitation of the glycoprotein can occur, low agitation, fibronectin can aggregate and form long mats of insoluble material, the viscosity must be optimal in order to allow a Sufficient adherence to the wound, so good release capabilities. Temperatures above. 60eC that are frequently required to provide sterile preparations, denaturalize the fibronectipa, since a terminal sterilization process can not be performed on the final product, the preparation of concentrated bases of vehicles without fibronectin is usually unavoidable, portions of these sterile bases can be They are diluted with a defined amount of a fibronectin solution. To achieve adequate dispersion of fibronectin towards semi-solid dosage forms, an incorporation step involving agitation, which may lead to precipitation of the drug, is often required. The gelling agents such c © m © carbómer © Carbop © l! R) and poloxamer © can circumvent this problem because they are sterilized before gelation under a viscous shape similar to liquid, a highly concentrated preparation of carbómer © of Carb © pol (H) is prepared and subjected to aut © keyAs described below, the fibronectin solution that also contains the polymerization promoter (NaOH) is then mixed in syringes with the carbomer © base of Carbopol (S), building the gel during the dispersion of the drug towards the same. ©, in the © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © À © © À © © À © © À © À © À © À © À © À © À © À © À © À © À © À © 2007 company is performed at 4 SC using a 0.22 μm filter, a non-toxic, non-sensitizing condom, compatible with the components of the formulation is added to the dosage form in a preferred embodiment of the invention, all conditions above are respected in the preferred dosage forms described in detail below. An amount effective for wound healing of human plasma fibronectin for use in the present invention is within the range of about 0.05 to about 1.0%, preferably about 1.0%, fibronectin is isolated from human plasma using a gelatin-Sepharose affinity chromatography method, in this method, gelatin is covalently coupled to Sepharose 4B after activation of CNBr. The binding capacity for human plasma fibronectin © provided by this system is >; 1 mg / ml gel, homologous human plasma fibronectin © autologous fibronectin obtained from recombinant DNA technology can be used in the present invention1-7, En cas © of using homologous plasma fibronectin, lots of different donors that have to be tested for atypical antibodies, hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV), human T cell lymphotropic virus (HTLV), cytomegalovirus (CMV) and syphilis, These tests they have to be made in the donors just before the donation and 6 months later, Between tant ©, special steps must be taken in order to inactivate the potential viruses, an inactivation method that uses tri (n-butyl) f © sf t © / Tween-80 or tri (n-butyl) phosphate / Triton X-100 (solvent / detergent method) must be performed in all plasma donations7,8, In the gel formulation for topical wound healing, the viscosity may be inside of the scale of 50,000 to 1,000,000 cps, more preferably between 100,000 and 650,000 cps. In the cream formulation, the viscosity may be within the range of 60,000 to 80,000 cps. All viscosity values are in centipoise (cps) c © m is measured using a Brookiield viscometer, Tests were performed at 0.5 rpm and at room temperature, In one embodiment of the present invention, the gel formulation may comprise 0.25 to 1.0% in weight of polyacelic acid that has a molecular weight of about 740,000 to 5,000,000, In a preferred embodiment, the polyacrylic acid is present at 0.35 to 0.75% by weight and has a viscosity of about 350,000 cps. The pH of the acidic polyacrylic gel should be within the range of 5 to 8 and more preferably between 6.5 and 7.5. The polymer of polyacrylic acid, also known as carbomer ©, is sold at low temperatures. the. Carbopol brand (R). The carb © © (R) Carbomer preferred grade © is P-934. In the embodiment, the gel formulation may comprise 18 to 35% by weight of polyoxyethylene block-polymer © © © xipr © pilen © having a molecular weight of about 2,000 to 13,000, In a preferred embodiment, the polyoxyethylene-polyoxypropylene block copolymer is present at 18 to 25% by weight and has a viscosity of about 450,000 cps at room temperature. The pH of the block copolymer gel should be within the range of 6 to 8 and more preferably between 6.5 and 75. The polyoxyethylene-polyoxypropylene block copolymer, also known as poloxamer, is sold under the Pluronic tide "", The degree of poloxamer © Pluronie (R) preferred is F-127 (p? loxamer © 407), In a further embodiment, the gel formulation may comprise 1 to 5% cellulose derivative, which may be hydroxypropylcellulose (HPC) and has a viscosity of about 25,000 to 150,000 cps, HPC has a molecular weight of about 370,000 to 1,150,000, in a preferred embodiment, cellulose derivative is present at from 2.0 to 4.0% in weight and has a viscosity of about 150,000 cps for HPC, the cellulose derivatives used in the present invention are commonly known in © Klucel by HPC; The preferred grade is Klucel-HF, In a further embodiment, a cream formulation is prepared from a commercially available cream base, i.e., Schering (R) base, This cream base (oil in water emulsion) contains ceteth-20, ceteestearil® alcohol, chloroeres®, mineral oil, monobasic sodium phosphate, phosphoric acid, sodium hydroxide, water and white petrolatum. The viscosity of the preparation can be modified by varying the water content and polyethyleneglic ©! The formulations of the present invention contain an aqueous phase in combination with a protein and, thus, are prone to attack by bacteria and fungi. Microbial growth not only makes the formulation lose without it being a danger of potential toxicity and a source of infection for patientsEven though microbial growth is less likely to be dangerous when it is used in a topical preparation, it is especially important to preserve the topics that patients have to apply to broken, inflamed skin. The viscosity degradation reported with some polymers when © are exposed to microbial contamination is also important. Thus, a preservative should be added to the preparation to ensure long-term sterility and stability. The present invention provides gels comprising a preservative selected from phenol-containing compounds of para-hydroxybenzoate. In one modality The gel formulation can contain 0.1 to 0.2% by weight of chlorocresol, a derivative of phenol or 0.01 to 0.3% by weight of p-hydroxybenzoate, methyl and propylparaben. In embodiment, the cream formulation contains 0.1 to 0.2% in weight of chloreresol. Stabilizers may be added to the formulation to provide stable compositions of fibronectin. They can help to conserve biological activities on an extended term basis and can improve the water solubility of fibronectin. Among these agents, albumin, disaccharides such as serum, and cyclic ligosaccharides such as cyclodextrins are stabilizers of choice, These agents can be used either alone or in combination, albumin. human is preferable in terms of antigenicity and should be free of microbial contamination, Cyclodextrins of the group beta (7 glucose units) are of choice and hydr © xipr © pil-beta-cyclo © dextrin is preferred. can comprise 0.01 to 0.1% in albumin weight, preferably 0.01 to 0.05%, and / or 0.5 to 5.0% by weight of sucrose, preferably 3.0 to 5.0 %; and / or 1.0 to 10% by weight of hydroxypropyl-beta-ciel dextrin, preferably 2.0 to 5.0%. Some authors have suggested that the protease activity in some chronic wounds may cause the degradation of adhesion proteins such as fibronectin and prevent the cell adhesion necessary for normal wound closure9. The metalloproteinases and proteases of serum have been identified in chronic wound fluid9'10 and fibronectin has been reported to be highly sensitive to leaching by proteases11. The protection of fibronectin integrity can be achieved by the addition of inhibitors of protease in the dosage form. The present invention also provides formulations which may comprise a metalloprotease inhibitor such as EDTA and / or a serum protease inhibitor such as aprotinin (Trasylol (R), Miles) with this target in view. In one embodiment, the dosage form may comprise 0.01 to 1.0% in EDTA pes and / or 1.5 to 45.0 Inh U% in aprotinin pes wherein 1 Inh U = 26 units of Kallikrein inhibitor, The formulations of the present invention can be applied to the wound site by any appropriate means that ensures that the wound surface will be completely covered, For example, it can be applied directly to the wound site or used to coat fibers of a gauze. The following examples are intended to illustrate additional aspects of the invention and they should be considered as limiting their scope in any way, such as to form a bandage of wound healing that can then be placed on a wound.

EXAMPLE 1 Isolation of fibronectin from human plasma 1) A sterilization step is necessary for all donations of homologous plasma. In order to inactivate the potential viruses, a sterilization procedure using the solvent / detergent method is used. 1% tri (n-butyl) phosphate (TNBP) and 1% Triton X-100 are added to the plasma for 6 hours at 242C. After that, soy bean oil is added to the plasma and allowed to mix for minus 30 minutes in order to extract TNBP, the residual Triton will be removed by dialysis, This first step is skipped if autologous plasma is used, 2) Primer © a column of gelatin-Sepharose 4B is previously washed with a solution of Tris-HCl in order to balance the gel, 3) The plasma is diluted (1: 1). ) with a solution of Tris-HCl and is pumped through the column in the presence of fluoride © of phenyl ethylsulfonyl © 0.001 M (MPSF) for about 15 hours at 4ßC, 4) The column lueg © is washed three times in order to elute plasma proteins not specifically bound to the gel. All the washing steps are carried out using a solution of Tris-HCl pH 7.5, a 1 M solution of Ncl is added to the second washing step to elute the contaminant, 5) The fibronectin elution is carried out performed using 0.1 M Na acetate + 1 M KBr solution, 6) Then two dialysis steps are performed to remove the contaminants (Triton X-1QQ, Kbr, Na acetat ©), Dialysis is performed against PBS respectively and sterile water, 7) The solution is concentrated by ultrafiltration under nitrogen pressure, 8) La. filtration of terminal sterilization using a 0.22 um filter is performed to ensure sterility, 9) Aliquots are placed in fractions and frozen at -20aC until incorporated into the topical dosage form, EXAMPLE 2 Polyacrylic acid gels Polyacrylic acid (carbomer ©) gels (carbomer Garb © p <l &R), BF Goodrich), Carbomer © is a polymer derived from acrylic acid, It is a polymer of elevated molecular weight (740,000 to 5,000,000) which gels when neutralized by strong alkalis (NaOH) or amines (triethanolamine). It forms gels at relatively low concentrations, that is to say so low c © m 0.25%, and its viscosity is greatly reduced by the addition of electrolytes, The preferred degree of acid © p © liacrlic © is carbómer © de Carb © p © lÍR) 934-P at concentrations ranging from 0.35 to 0.75% (w / w), lower concentrations are insufficient to promote adhesion to the wound and higher concentrations reduce the release of fibronectin from the gel, The viscosity of the peliaerlic acid gels is stable between pH 6 to 8 with a preferred pH scale between 6.5 to 7.5. The viscosity is reduced in the presence of strong electrolytes. The polyacrylic acid gel containing 0.2% fibrenectin (weight / weight), 0.375% Carb © pol (R) 934-P carbomer, and 0.1% elocresol was prepared as follows: dissolved Gl © r © cres © l (1.0 g) in warm distilled water (95 ml) (65aC) under slow stirring, when chlorocresol is completely dissolved, the solution is cooled to room temperature while After stirring, Carb © p © l (R) 934-P carbomer was added, slowly dispersing it on the surface of the solution, and mixed with a paddle-type stirrer for about 3 hours. This dispersion was then autoclaved to provide a sterile, concentrated gel base (3.75% w / w). A solution of fibronectin material, 2.2 mg / ml (90 ml), was filtered through a 0.22 μm acetate filter. A polymerization promoter, sodium hydroxide, was added to the fibronectin solution in an amount which will neutralize a 10 g portion of the dispersion to 3.75% of the carbomer © of Carb © p © l (R), ie 1250 ul, of 3M NaOH, the solution of fibronectin material and carbomer dispersion of Carbop © lÍR) were mixed into syringes taking care to avoid introducing air bubbles and avoiding contamination by working in an aseptic environment, such as under a laminar flow hood, Generally, syringes are used, and multiple exchanges are applied under pressure. An adapter device, such as a female Luer connection, can be used to connect the syringes or the exchange apparatus. Vigorous agitation is minimized in order to avoid precipitation of fibreneetin. This preparation provides a clear, preserved gel. (100 g) fibronectin free of microorganisms with viscosity of around 350,000 cps. This gel formulation was applied twice a day on leg ulcers in a study pil © t in humans and showed an improved regimen of wound healing without any adverse effect. In a preferred embodiment, concentrated solutions of fibronectin, up to at least 10 mg / ml, can be prepared. These solutions can be used to prepare a concentrated fibronectin gel. In order to prepare the fibreneetin gel, the The following ingredients should be added in sequence, and are required for the preparation of 10 grams of gel, varying in flbronectin concentrations of 0.5 to 1.0%. First, the pH of 9.8 grams of demineralized water is adjusted to about 8.0 to about 11.0 with 0.094 grams of 3M NaOH, then lyophilized fibronetin is dissolved in demineralized water, pH 8.0 - 11.0 in amounts that vary from 0.5 to 1.0 grams. When a carbomer gel is to be prepared, a pH of 9.0 is preferred for the demineralized water. In a final procedure, add 0.028 grams of carbomer to the mixture, EXAMPLE 3 Copolymer gels © of polyoxyethylene-polyoxypropylene block © Copolymer-block polymers of polyoxyethylene-polypropylene © (p © © xámer ©) (poloxámer © Pluronict®), BASF Wyandotte), The degree were prepared Poloxamer © is preferred by Pluronic (R) F-127 at concentrations ranging from 18 to 25% (w / w). Poloxámer © de Plur © nic (R) F-127 is a low molecular weight polymer (2,000 to 13,000) that exhibits thermal gelation characteristics. The gelaeion occurs when the concentration reaches 18% poloxamer. The viscosity of the poloxamer is proportional to the concentration of the polymer, tip® of polymer used (molecular weight) and temperature, the fluid to 4SC, the polymer © gels at temperatures Increasing, providing high viscosity values at room temperature, In contrast to carbomer Carb © pÍl), the addition of ions improves the viscosity of the. Preparation, Concentrated aqueous solutions (20 to 30%) have been reported to demonstrate a dramatic increase in viscosity when heated from 4aC to body temperature, Additionally, if the ionic strength of the solution increases, the viscosity increases more rapidly With increasing temperature, various grades are available, but the gradient F-127 is the least toxic and the gelation may be at lower concentrations. The poloxamer gels prepared in this invention are solutions of low viscosity at 4aC and are They gel quickly when heated at body temperature.

A pà © là ©merà © gel containing (w / w) 0.2% fibronectin and 20% poloxa er © of Plur © nie (R> F-127 was prepared as follows; 2,2 mg / ml fibronectin (80 ml) was filtered through a 0 22 m acetate filter Pluronic poloxamer "" F-127 (20 g) was added to 80 ml of the fibronectin solution and allowed to dissolve without agitation at 4aC for about 3 days The resulting solution (100 g) is a liquid-like one, The gelation occurs instantaneously when the solution comes into contact with the wound, A sterilization filtration process performed at 4aC could also be applied to the final solution if sterile polexammer powder can not be obtained, the viscosity varies from n © detectable values to 4BC at 450,000 cps at room temperature, EXAMPLE 4 Cellulose Derivative Gels Hydroxypropylcellulose (HPC) gels were prepared. In order to illustrate this type of formulations, the preparation of a 3% HPC gel is described as follows, hydroxipr © pilcelul © sa Klucel-HF at concentrations ranging from 2 to 4% (w / w), A gel formulation containing (w / w) of 0.1% fibronectin, 3% HPC and parabens was prepared c © m follows; Methylparaben (0.05 g) and propylparaben (0.02 g) were dissolved in warm deionized water (94 ml). The HPC powder was sterilized using a dry heat sterilization process, then HPC (6 g) was dispersed in this solution and allowed to mix with a tipped paddle for about 3 hours. This provides a sterile concentrated gel base (6% w / w). A section of fibronectin material 2 mg / ml (50 ml) was filtered through a 0.22 μm acetate filter. The fibronectin solution (50 ml) was then slowly added to a portion (50 g) of This concentrated base using the low speed arrow of the agitator. This provides a preserved gel (100 g) with viscosity of approximately 150,000 cps, EXAMPLE 5 Cream Formulation A cream formulation containing (w / w) 0.1% fibronectin, sterile cream base (ScheringRI base), Schering) and chloroeresol 0.1% was prepared c © m followed: a solution of fibronectin material 2 mg / ml (50 ml) was filtered through a 0.22 μm acetate filter. The fibronectin solution (50 ml) was then slowly added to a portion (50 g) of the cream base using the low speed arrow of a shaker, 2? This provides a preserved cream (8100 g) with a viscosity of around 70,000 cps, EXAMPLE 6 Release kinetics of different topical dosage forms The effectiveness of each topical formulation to release fibronetin was evaluated using an in vitro diffusion cell system. Permeation studies were all performed on de-epithelialized breast and abdominal skin samples obtained from peen and abdominal reduction lipoctomy surgeries, An 8-μm section was separated from the epidermal surface of the skin using a dermatome (1 / 10,000 scissor scale) and the dermal lad was carefully cleansed of any adherent and subcutaneous subcutaneous tissues. / or blood vessels. De-epithelialized human skin was used in order to reproduce the pathological condition found in chronic venous ulcers where the epidermis layer is absent. The selected diffusion cell system consisted of a rigid receptor containing the skin sample, the de-epithelialized © © directed upwards towards the donor compartment and the dermal ief facing downwards toward the receiving compartment.

The receptor compartment was connected to a circuit of circulating buffer. The buffer temperature was maintained at 37ffiC while the surface of the skin was at around 32aC. Each analysis was performed on a 0.64 cm2 skin sample using an aliquot of 100 uL of topical 1Si-fibronectin formulation sample. After the experiment, the skin was separated from the diffusion cell, washed 10 times with a volume of water of 8 ml by washing, and was analyzed to determine its content of radioactivity in gamma radioactivity counter. The total amount absorbed (dermis + receptor compartment) divided by the applied dose gave the percentage of absorption. This diffusion cell system has provided excellent reproducible intra- and inter-experimental results. All dosage forms were made in salt-free solution since the viscosity values could have been influenced by the presence of electrolytes. For example, the viscosity values of carbomer gels are reduced in the presence of strong electrolytes in contrast to the poloxamer gels that are more viscous when electrolytes are added to the. preparation. Several authors have compared percutaneous absorption studies using in vitr © and in viv © techniques to establish the reliability of results using these methods13-11-15. These comparisons have clearly shown that in vitro studies can accurately reflect the stay alive. The statistical analysis applied to our experiments has shown a good correlation value between studies performed on the skin obtained from different sources. These data have shown that e_. The skin reagents had no effect on the results. Percutaneous absorption studies are usually performed on intact skin and are designed to evaluate the release of a substance from a topical vehicle and its absorption through the skin barrier. principal, that is, the stratum cornea. In skin lesions, the barrier effect on the stratum cornea is absent. With this pathological condition, only the diffusion of the dermatological vehicle will be a major determinant for the subsequent penetration of the drug into the dermis. The diffusion cell system described above is an appropriate in vitro model for cutaneous ulcers, the kinetic data of the Fibronectin release from various dosage forms were obtained at 4, 12 and 24 hours. Table 1 summarizes these data for 5 = 12 hours. The control consisted of 125T-fibronectin in phosphate buffered saline. pH 7.4. The liposomes used in the carbomer © Carbopol (R) 934 P (1%) + liposome formulation (15%) (Lipogel) were made from Liposo as Pro-lipos © mas (Pr © -lipo 3090 SH (MR). Mayer, France). The cellulose derivatives are identified as CMC for sodium carboxymethylcellulose and HPC for hydroxypropylcellulose. The base Dermabase (R) (Borden, Ltee., Don Mills, Ontario, Canada) and base Seherig (R) s n cream bases available in the market and were diluted 1: 1 for these experiments. The symbol () refers to the concentration of components and "Abs value" to the percentage of fibronectin radio-tagged © found in the dermis after a 12-hour exposure time.

TABLE 1 Formulation () Abs value Contr ©! 24.75% Lipogel 3.70% Base Dermabase "" (1 = 1) 5 - 80% CMC 3% 6.70% Carbopol Carbomer (R) + Glycerol (Carbogly) 0.375% / 10% 7.80% Base Schering (R) (1: 1) 9.90% P © © xámer © Plur © nic! R) F-127 20% 12.80% Carbómer © Carbopol (R) 934 P 0.375% 13.40% HPC 3% 15.209! Figure 1 traces kinetic data of three forms of gel dosage and control solution over time. From this graph it can be seen that the absorption process tends to be more important between time vO and 12 hours than between time 12 and 24 hours, suggesting that two applications per day could release more fibronectin than a program once a day, Figure 2 illustrates cutaneous absorption of radiolabeled fibronectin of various dosage forms and control at time = 12 hours, Dunnett's statistical test ST used to statistically identify significant differences between the carbomer gel of Carbopol "" and other formulations, This test has also shown significant differences between Lipogel, Carbogly and Carbopol carbomer gel "", it turns out that it can be correlated with those of effectiveness obtained during chemical tests (see Example 8), The effectiveness of the carbomer gel formulation of Carbopol (R ) is particularly surprising since Carbopol carbomer gel "" has a higher degree of viscosity than many of the other formulations studied. Also noteworthy are the differences in Abs value between the Carbopol carbomer gel "" and CMC formulations since both share the same degree of viscosity, Figure 4 shows that there is not always a clear relationship between viscosity and absorption, when consider some of the preparations for which the viscosity values were determined. For example, the base Dermabase "" which has a relatively low viscosity (119,000 cps) when compared to the Cárbopol carbomer gel "" (411,300 cps) presents low release capacities (5.80%) when compared to the carbomer gel of Carbopol "" (13.40%), Figure 5 demonstrates that absorption values higher than 13.40% can be obtained using 0.28% of carbomer and higher concentrations of fibronectin, Figure 6 directly compares the absorption values obtained in the diffusion-cell skin cell system for carbomer gels of 0.28% and 0.375%, both of which have 0.2% fibronectin, In Figures 5 and 6, the amount of fibronectin was measured using ELISA procedures, A polystyrene microtiter plate is incubated in 100 ul of different fibronectin samples in carbonate / bicarbonate buffer a 50 mM, pH 9.6 at 4aC overnight. A solution of 5% bovine serum albumin (BSA) in T in PBS, pH 7.5, was used as a blocking buffer for 30 minutes at 37aC. wash four times with Tween PBS pH 7, 5 and 100 ul of rabbit anti-FN, produced by methods well known in the art, diluted 1 / 100,000 in 0.5% PBS Tween pH 7.4, added and plate incubated at 37eC for 1 hour, After rinsing four times with PBS buffer, 100 ul horseradish peroxidase conjugated rabbit (Jackson Immunoresearch Laboratories, Inc., PA) diluted 1 / 100,000 in 0.5% of PBS Tween pH 74, are added and the plate is incubated at 37BC for 1 hour, excess conjugate is removed from the completely washed, and the peroxidase bound to the wells is detected by the addition of ABTS (2, '-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) in citrate buffer pH 4.6 containing 0.015% of hydrogen peroxide. The reaction is followed by increases in absorbance at 410 nm compared to a conventional fibronectin peroxidase reaction, EXAMPLE 7 Stability of fibronectin gel Activity T biological integrity of fibronectin macrostructure in ST gel formulations evaluated (Figure 3), Tests were performed on a gel sample containing (w / w) 0, 2% fibronec ina, 0.375% Carbopol carboment "" P-934, and 0.1% chlorocresol, The sample was kept at 4eC for 32 Weeks. Electrophoresis techniques were used to determine the fibronectin macrostructure integrity in gel. After the gel sample was dissolved in 1M NaCl + Tris-HCl solution pH 6.8-7.4, it was allowed to migrate in a 7, 5% acrylamide gel in accordance with the Laemmli method ("Denaturation (SDS) of discontinuous gel electrophoresis"; Laemmli gel method, "pages; 10.2.4 - 10.2.9, Current Protocols in Molecular Biology (1994), Compared with a fresh conventional solution (column 0), the results showed that about 100% fibronectin can be identified around the 220,000 band (column B) indicating that very little degradation occurs, if any, Biological activity was assessed using an affinity chromatography test, in-bond gelatin is one of these biological activities which can be easily secured relative After a sample of ST gel dissolved in a solution of 1M NaCl, a known amount of this ST viscous solution placed T? an Eppendorf tube in the presence of gelatin Sepharose 4B and then ST som? The contents were further rinsed with a fresh solution of 1M NaCl, subjected to centrifugation and the supernatant discarded in order to remove contaminants such as Carbopol carbomer and chlorocresol. and came the dissolution of the gel. Fibronectin was eluted from the gßlatina-sepharose 4B using a solution of 1 M KBr. The fraction collected was allowed to migrate in a gel d? 7.5% acrylamide d? compliance with the Laemmli method. The band then ST? Valued with respect to its fibronectin content using an assay? Densitometric scanning, The collected sample could also be evaluated spectrophotometrically using optical density at a lambda wavelength = 280 nm, Compared with a freshly prepared fibronestin gel (column 0), it can VTGST that a large amount (80%) of fibronectin was recovered of the sample d? gel formulation (column 8 months) indicating that the gelatin d bonding activity? Glycoprotin can be preserved for a long time? a period d? prolonged time in this dosage form, EXAMPLE 8 Clinical tests; treatment of chronic ulcers d? leg Five clinical trials (pilot studies) were conducted to investigate the usefulness of different dosage forms containing exogenous human plasma fibronectin in the treatment d? chronic venous ulcers d? lower limbs, In these tests, autologous plaque fibrinogen was used and patients with ulcers qu? were resistant to conventional therapy for at least three months ST selected, The specific objective of the first experiment was to determine the effectiveness of fibrinoctin topically applied to a wound healing promoter. We included seven patients in this study and received instructions? "flood" the wound area with a solution of fibronectin, 1 mg / ml (0.1%) in PBS (phosphate buffered saline) twice a day, After two more regular application d? That solution, five d? these ST patients had a dramatic decrease in the size of their wound, specifically at least 75% d? reduction of the integrated surface area, A second experiment was designed to evaluate the effectiveness of a semisolid dosage form containing 0.1% fibrontin weight, encapsulated in 15% liposomes, which in turn were incorporated into a carbom of Carbopol "" (1%) known as Lipogel, The hypothesis was that if the contact time of the glycopropin with the wound could be improved, a faster decrease in the healing time could be observed theoretically. Six patients were included in this. study and had what? Apply the formulation to your wound twice a day, None presented a substantial decrease in size d? his wound during the following three months of regular treatment, In an effort to improve the way d? dosage, an experiment was undertaken to evaluate the therapeutic potential of a topical gel formulation containing (w / w) 0.2% fibrontin incorporated in 0.375% of Carbopol carbomer "" and 10% d? glycerol (Carbogly). The glycerol had been added to the formulation in order to take advantage of its humectant effect that could be beneficial to the wound. Eleven patients were recruited for this study and they also had to apply the gel twice a day. Among these patients, 27% had a regression of more than 50% and its size d? wound after d? three months of treatment, The results of the permsation studies can explain, at least in part, what might have happened in previous experiments, Figure 2 shows that preparations such as Lipogel and Carbopol carbomer "" + glycerol does not lead to high values d? absorption. In contrast, 0.375% Carbopol carbomer "" without glycerol provides significantly higher absorption values (p <0.001), This glycerol effect was not observed with formulations containing fibronectin above 0.2%, The solution used in the first exp0rimento is identified as the control in this graph. This last preparation provides the highest release capabilities but does not represent a formulation that could be useful for patients due to its fluid consistency. Considering these results, ST investigated in eight patients a formulation containing 0.2% d? fibronectin (w / w) in 0.375% d? carbomer d? Carbopol "". without glycerol. In accordance with clinical studies and d? permeation, this formulation is the preferred carrier using Carbopol carbomer that is available for the use of fibroticctin in topical wound healing. Preliminary data showed that 50% of the patients studied had a regression of more than 505 of their wound size within of three months of treatment, including two complete responses (100% cure) that occurred within the first eight weeks of treatment, A fifteen clinical trial comprising 40 patients was conducted with the formulation containing 0.25 fibronectin (p / p) Carbopol carbomer <; R) without glycerol, In this clinical trial, patients were stratified d? compliance with the duration of ulcer d? skin (age of the ulcer) at the beginning d? the test, In patients with ulcers d? 6 months or less in duration, the formulation qu? contains 0.2% fibronectin (w / w) in 0.375% Carbopol carbomer "" no fu? superior to placebo. The placebo was Carbopol carbomer "without glycerol and without containing fibrontin, However, in patients with ulcers of 7 months or more duration in the time of randomization, the formulation of fibronectin containing 0.2% fibronectin ( p / p) in 0.375% carbomer d? Carhapol "" without glycerol was clearly superior, After 20 weeks d? treatment, 58% of the treated group (7/12) had a reduction in wound size of at least 755 after 20 weeks d? Treatment, Only 25% (1/4)? n the placebo group had this beneficial effect, Above all, patients treated with place (n = 4) ST deteriorated an average wound size increasing by approximately 60% in size, In the group treated with fibrontin (n = ll), the average wound size decreased to about 30% after 20 weeks of treatment. The present invention also provides other formulations that are as useful as this using the permeation study described in Example 5 or a model system for testing the various formulations, EXAMPLE 9 Case Reports To illustrate the efficacy of the formulation qu? contains fibronectin and 0.375% carbomer Carbopol (R) 934-P (w / w), we present two specific cases of venous ulcer d? leg, chronic. These cases are of interest in that the first case was highly resistant to conventional therapy and the second case was a large ulcer. Factors such as duration and surface area have been identified by various authors as playing a major role in the prognosis of venous ulcer, Case 1 A 37-year-old ST woman presented with a ten-year history of chronic venous ulcer of the right lower limb, Her medical history was not significant, except for four episodes of phlebitis.

The last episode occurred during pregnancy and eventually resulted in an ulcer. The review of the medical treatments that were tested revealed the use d? Topical antiseptics, elastic stockings, and skin graft without any positive results. The patient presented to our clinic with a wound with pain of 1.60 cm2. Despite the fact that his ulcer was relatively small, he seemed highly resistant to therapy. Six weeks after starting the application of fibronectin gel. A reduction of 92% was observed? his size of wound, The full r? pitelialization was observed after a course of treatment of ten weeks, A follow-up visit scheduled a month later did not reveal deterioration in the condition of his wound.

Case 2 A 39-year-old man presented with a seven-month history d? chronic venous ulcer of the left leg. His medical history was not significant except for a saphenectomy of the left lower limb doc? years before Topical ST antibiotics were prescribed to the patient? Without any effect of the size of your wound, We presented in our clinic with an ulcer of 10.5 em2 result d? a local trauma. Lymphedema of the left lower limb was important and a large crusted necrotic layer bordered the wound, The occupation of the paci? Nt? it restricted him to standing for long periods of time. even when this situation probably worsened his wound condition, it could not be eliminated, After d? four weeks d? Regular application of a placebo gel and normal saline, the size of the wound increased to 21.5 cm2 as a consequence of local debridement. The placebo gel comprised 0.375% Carbopol carbomer "" 934-P, 0.1% chlorocresol, purified water and NaOH to adjust the pH Active treatment with Carbapol carbomil gel "" containing fibronectin started at this time, the maximum wound size was scored six weeks later (37.5 cm2), revealing an ulcer greater than initially assumed. The process d? wound healing occurred between weeks six and eight and was completed after 21 weeks d? active treatment A follow-up visit scheduled a month later did not reveal deterioration in condition d? his wound. Even though the present invention has been described in relation to particular modalities d? the same, many other variations and modifications and other uses will be evident to those experienced in the field. Therefore it is understood that numerous variations can be made d? the invention that are well within the scope and spirit d? This invention as described in the appended claims, REFERENCES 1 - Hynes, R. O,, Methods for identification of fibronectin (cap, 2. page 12), IN: Fibronectins New York: Sptinger-Verlag, 1990, 2 - Hyn? S, RO, Methods for identification of fibronectin (Chapter 2, pages 7-23) and Healing d? Hernia, inflammation and fibrosis (Chapter 14, pages 349-64), IN: Fibronectins NTW York: Springer-Verlag, 1990, 3 - Brotchi ?. H., Wakeield, D. Fibronectin: Structure, function and significance in wound healing; Australas J Dermatol 1990; 31: 47-56 4 - Nishida, T., Nakagawa, S ,, Awata, T, et al .. Rapid preparation of eye drops of autologous purified fibrinogen from d? patient plasma, Jpn J > Gphthalmol 1982: 26: 416-24. 5 - Phan, T.M. , Foster. C.S. , Boruchoff, S.A. and col ,, Topical fibronectin in the treatment of persistent corneal epithelial defects of trophic ulcers. Am J. Qphthalmol 1987; 104: 494-501, 6 - Wysocki, A., Baxter, C.R ,, Bergstresser, P.R, et al. Topical fibronectin therapy for treatment of a patient with chronic stasis ulcers. Arch Dermatol 1988; 124: 175-77. 7 - Edwards, CA Piet, M.P.J., Chin, S. et al ,. Treatment of tri (n-butyl) phosphate / detergent from therapeutic and experimental licensed blood derivatives. Vox Sang 1987; 52: 53-9. 8 -. 8 - Horowitz, B., Bonomo. R,, Prihce. A.M. and col, Plasma treated with solvent / detergent: an inactivated substitute d? virus for fresh frozen plasma. Blood 1992; 79: 826-31. 9 - Grinnell, F. - Ho. CH,, Wysocki, A .. Degradation of fibronectin and vitronectin in fluid d? Chronic wound: Analysis by cell secant, immunosecant and cell adhesion assays. J. Invest D? Rmatol 1992; 98: 410-6, 10 - Chen. W, Y, J., Rogers. TO, . , Lydon, M.J., Characterization of biological properties d? wound fluid collected during early stages d? wound healing J Invest Dermatol 1992; 99: 559-64 11 - Berman, M., Mansßau. E, Law, M. et al., Ulceration is correlated with the degradation of fibrin and fibronectin on the corneal surface. Invest Ophthalmol Vis Sci 1983; 24; 1358-66. 12 - Horowitz, B., Chang. M.D.Y .. Preparation of fibrontin for therapeutic administration; IN: D.F, Mosher (? D,), Fibronectin, pages 441-55, San Diego. Academic Press 198, 13 - Franz, T.J., Percutaneous Absorption On the relevance of in vitro data, J Invest D? Rmatol 1975: 64: 190-95. 14 - Bronaugh, R.L., Stewart, R.F ,, Congdon, E.R. et al, Methods for in vitro percutaneous absorption studies. I: Comparison with in vivo results. Toxicol Appl Pharmacol 1982; 62: 474-80, 15 - Bronaugh. R.L., Stewart. R, F. , Methods for in vitro percutaneous absorption studies IV; The flow through diffusion rate. J Pharm Sci 1985: 75: 64-67,

Claims (1)

1. CLAIMS 1, - A method for preparing an unbuffered aqueous solution! not stabilized, which contains at least 2 mg / ml d? fiber that comprises step d? add purified fibronectin to basic water. 2, - The method of claim 1, wherein the water is demineralized water, 3- The method of claim 1. wherein the fibronectin is human plasma fibronectin. 4, - The method of claim 1, wherein the purified fibronectin is freeze-dried. - A method for preparing an unbuffered aqueous solution d? fibronectin. which comprises the steps of: a) adjusting the pH of mineralized water of around d? 8.0 to approximately 11.0 and b) add purified fibrontin to the water of step a, 6, - The method d? claim 5, wherein the unbuffered aqueous solution contains about 2 mg / ml at approximately? 10 mg / ml d? fibronectin, 7 - The method of claim 5, wherein the fibronectin is human plasma fibronectin. 8. The method of claim 5, wherein the purified fibrontin is lyophilized. 9. - A method for preparing an unbuffered aqueous solution d? fibronectin comprising the steps of: a) adjusting the pH d? demineralized water at approximately 9.0 and b) add purified fibronectin to water from step a. 10. A method for preparing an unbuffered aqueous solution containing around d? 2 mg / ml to approximately 10 mg / ml d? a protein d? extracellular matrix comprising the steps of: a) adjusting the pH of uninflated water from about 8.0 to about 11.0 and b) adding purified extracellular matrix protein to the water from step a. 11. A method for preparing an unbuffered aqueous solution containing about 2 mg / ml to about 10 mg / ml of a growth hormone comprising the steps of a) adjusting the pH d? demineralized water of around 8.0 to about 11.0 and b) add hormone d? purified growth to water from step a, 12. An unbuffered aqueous solution containing about 2 mg / ml to about 10 mg / ml of a wound healing promoter. 13. - The unbuffered aqueous formulation according to claim 12, wherein the healing promoter d? wound is selected from the group that consisted ? n fibronectin, thrombospondin. laminin, vitronectin. fibronogeho or growth factors. 14. A pharmaceutical formulation comprising at least about 0.5% to about 1.0% by weight of fibrontin. based on the total weight of the pharmaceutical formulation, comprising: a) a solution d? unbuffered aqueous fibronectin, produced according to claim 5, and b) a pharmaceutically acceptable carrier, 15, - The pharmaceutical formulation of claim 14, wherein the pharmaceutically acceptable carrier TS a water soluble pharmaceutical polymer, having a viscosity d? 50,000 to about 1,000,000 cps at room temperature. 16, - The pharmaceutical formulation of claim 14, further comprising at least one extracellular matrix protein selected from the group consisting of thrombospondin, laminin, vitronectin and fibrinogen, 17, - The pharmaceutical formulation of claim 14, comprising furthermore at least one growth factor, 18. An aqueous gel formulation for curing chronic wounds, comprising about 0.28% carbomerous and about 0.5% to about 1.0% fibronectin weight, based on the total weight of the formulation d? gel.