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    • 专利摘要:
    The invention relates to microparticles comprising biocompatible polymers and epidermal growth factor. The invention also relates to the method for preparing said microparticles and to the use thereof to promote the healing of wounds.
    公开号:ES2540453A2
    申请号:ES201590050
    申请日:2013-12-02
    公开日:2015-07-09
    发明作者:Eusebio GAINZA LAFUENTE；Garazi GAINZA LUCEA；Oihane IBARROLA MORENO；Silvia VILLULLAS RINCÓN；Ángel DEL POZO PÉREZ；José Luís PEDRAZ MUÑOZ；Rosa María Hernández Martín；Manuela Igartua Olaechea；Alfredo GÓMEZ MENGOD
    申请人:Praxis Pharmaceutical SA；
    IPC主号:
    专利说明:

    P201590050
    05-27-2015 DESCRIPTION
    Microparticles with EGF, preparation and use procedure 5 Field of the invention
    The present invention belongs to the field of microencapsulation. Specifically, it refers to microparticles comprising biocompatible polymers and epidermal growth factor. It also refers to the procedure for preparing said
    10 microparticles and their use to promote wound healing. Background of the invention
    The aging of the population is associated with a progressive increase in
    15 chronic diseases and their complications. As a consequence of a combination of factors associated with age, such as venous insufficiency, occlusive arterial disease, obesity, immobility, deficiency of growth factors, diabetes mellitus, neurological defects and nutritional deficiencies, among others, there are wounds that significantly deteriorate the quality of life of patients, limiting their autonomy, in
    20 especially when they affect the lower extremities. The morphological changes of the skin in the elderly due to the passage of time, can favor a greater predisposition to mechanical forces, by pressure, friction or shear of small size or present a short period of time can generate lesions that, together with the healing problems associated with chronic diseases, make
    25 that these lesions do not heal, causing chronic wounds of difficult healing. Chronic wounds represent an important health problem from an epidemiological, economic and social point of view, associated with high morbidity in the elderly. Chronic wounds are a complex and heterogeneous group, although approximately 70% of them can be classified as pressure ulcers, diabetic or
    30 vascular; being less frequent those of inflammatory cause, tumor or by physical agents (for burns or radiation).
    Conventional chronic wound care includes therapies such as hyperbaric oxygen, negative pressure, surgical debridement or dressings. These therapies are
    35 palliative and unable to guarantee adequate tissue regeneration and wound closure. The topical application of growth factors has also been used for 2
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    improve healing, although with little success (Goldman R. Growth factors and chronic wound healing: past, present and future. Adv Skin Wound Care 2004; 17: 24-35).
    Epidermal Growth Factor (EGF) increases
    5 Epidermal cell proliferation and therefore, it is important for the healing process that depends on mitosis and the migration of dermal keratinocytes and fibroblasts. However, acute wounds can secrete proteolytic enzymes that significantly reduce the local bioavailability of EGF and therefore, its interaction time with the receptor. Therefore, for the EGF to exercise its
    10 mitogenic effect to achieve effective healing, continuous exposure of EGF with cells of at least 6-12 h is required. In this sense, a topical gel based on human recombinant epidermal growth factor (rhEGF), REGEN-D 150 ™, has been authorized in India for the treatment of grade I diabetic foot ulcers and II, to be applied
    15 twice a day until complete healing of the ulcer and that reduces the duration of healing. Similarly, in countries such as Argentina, Bolivia, Colombia, Cuba, Mexico and Venezuela, a rhEGF-based formulation, Heberprot-P, is marketed, which increases the granulation of high-grade diabetic foot ulcers after intralesional administration three times per week. .
    20 WO 2007/087759 describes a process of formulation of EGF in microspheres to prevent amputation of the diabetic foot, where the EGF is encapsulated with an encapsulation efficiency of 40-60% and the release rate of the EGF is 5 and 10 µg per day, for 14 days. Chu et al. (Nanotechnology promotes the
    25 full-thickness diabetic wound healing effect of recombinant human epidermal growth factor in diabetic rats ”, Wound Repair Regen 2010; 15: 499-505) describe nanoparticles with an EGF encapsulation efficiency of 85.6%, with an EGF content of 2%, which show a rapid release (initial burst) during the first hour and sustained for 24 hours. However, although the formulations described
    30 in Chu et al. they promote, in comparison with control groups, the proliferation of fibroblasts and the healing process in a diabetic rat model with deep wound induction, to achieve these results, they require a daily administration of the growth factor because the formulation used exerts a Sustained release of a maximum of 24 hours. Repeated administration involves a
    35 very important inconvenience for the patient, since, increasing the number of doses entails an increase in suffering adverse effects to the treatment, a greater 3
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    Probability of non-compliance with the same and in cases that are administered by injections in the wound, a rejection by the patient due to their painful sensation.
    Surprisingly, with the microparticles of the present invention, microparticles comprising EGF and polylactic-co-glycolic acid (PLGA) and alginate polymers, high encapsulation efficiencies of EGF (up to 88%) and sustained release of the EGF for at least 30 days allowing excellent healing results, even with a single administration. In addition, the rate of
    The sustained release of EGF is of the order of nanograms, which is also an advantage since prolonged exposures to higher concentrations of EGF can favor cell proliferation, which can be aggressive for the organism. Object of the invention
    The present invention relates to a microparticle (microparticle of the invention) comprising a PLGA polymer, an alginate polymer, and epidermal growth factor.
    In another aspect it refers to a pharmaceutical composition (pharmaceutical composition of the invention) comprising the microparticle of the invention and a pharmaceutically acceptable carrier.
    It also refers to the microparticle of the invention for use as a medicament.
    25 and to the microparticle of the invention for use to promote wound healing in an individual. It also refers to the pharmaceutical composition of the invention for use as a medicament and to the pharmaceutical composition of the invention for use to promote wound healing in an individual.
    In another aspect it refers to a kit comprising the microparticle of the invention or the pharmaceutical composition of the invention to promote wound healing.
    Finally, in another aspect it refers to a process for preparing the microparticle of the invention comprising the following steps: a. Addition of a solution of PLGA in an organic solvent (organic solution of PLGA) on a solution comprising alginate and factor of 4
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    epidermal growth (solution of EGF and alginate), and mixing of the organic solution of PLGA and the solution of EGF and alginate,
    b. Addition of the emulsion obtained in step a) on an aqueous solution
    comprising surfactant and sodium chloride (aqueous solution), and mixing the emulsion obtained in step a) and the aqueous solution,
    C. Addition of the emulsion obtained in b) on a solution comprising sodium chloride and calcium chloride (chloride solution), and mixture of the emulsion obtained in step b) and the chloride solution,
    d. Solvent extraction, and 10 e. Isolation of the microparticle. Brief description of the figures
    Figure 1. Scanning electron microscopy (SEM) photographs of the 15 microparticles (A) MP4 (x2500), (B) MP5 (x1000), and (C) MP5-γ (x1000).
    Figure 2. Graphical representation of the percentage of sustained release of EGF as a function of time in hours (A) and days (B).
    20 Figure 3. Micrographs of fibroblast cultures treated with (A) Serum free medium,
    (B) 15 ng / mL microparticles of EGF (C) 15 ng / ml free EGF.
    Figure 4. Photographs of wounds of rats treated with (A) Untreated control, (B) Vehicle control, (C) Empty microparticle control, (D) free EGF and (E) Microparticles with EGF.
    25 Figure 5. Micrographs of wound samples for the evaluation of the degree of reepithelialization at 7, 11 and 17 days of mice treated with (A) Untreated control, (B) Vehicle control, (C) Empty microparticle control, ( D) Free EGF and (E) Microparticles with EGF.
    30 Detailed description of the invention
    The present invention relates in a first aspect to a microparticle (hereinafter referred to as the microparticle of the invention) comprising a 35 PLGA polymer, an alginate polymer, and epidermal growth factor.
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    In the context of the present invention, the term "microparticle" refers to a free flow micrometric particle where solid, liquid or gaseous materials are coated with a film of polymeric or fatty material, having a size between 1 μm and 1000 μm. In a particular embodiment, the microparticle of the invention has a
    5 diameter in the range of 1 µm to 500 µm. More particularly, it has a diameter in the range of 1 µm to 100 µm, preferably the microparticle of the invention has a diameter in the range of 1 µm to 40 µm (Table 1).
    PLGA is a synthetic polymer formed by the association of the acid homopolymer
    Lactic acid (PLA) and glycolic acid (PGA), is highly biocompatible and toxicologically safe. In the context of the present invention, PLGA polymer is understood as any synthetic polymer formed by the association of the polymer of lactic acid and glycolic acid regardless of the proportion in which each of them takes part in the assembly. Alginate are natural copolymers of polysaccharides
    15 constituted by manuronic and glucuronic acids. Although synthetic polymers outperform natural ones in purity and reproducibility, the ultrapurification of alginates gives them low toxicity, being considered biocompatible.
    In a particular embodiment, the epidermal growth factor of the microparticle of
    The invention is the human recombinant epidermal growth factor (rhEGF). Said rhEGF can be obtained commercially (Peprotech, Promega, Pharmchem, etc.) or produced by recombinant DNA technology, as for example described in Marioka-Fujimoto et al. (Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli. J
    25 Biol Chem. 1991 Jan 25; 266 (3): 1728-32) or in patent application WO 91/18999 A1.
    In a particular embodiment, the microparticle of the invention comprises between 82.7% and 99.897% (w / w) of PLGA with respect to the total weight of the microparticle, between 0.003% and 1.5% (w / w) of EGF with respect to to the total weight of the microparticle and between 0.1% and 5% (w / w) of 30 alginate with respect to the total weight of the microparticle. In another particular embodiment, the microparticle of the invention comprises between 0.05% and 1% (w / w) of EGF with respect to the total weight of the microparticle and more particularly comprises 1% (w / w) of EGF with respect to to the total weight of the microparticle. In another particular embodiment, the microparticle of the invention comprises from 1% to 3% (w / w) of alginate with respect to the
    The total weight of the microparticle, and even more particularly, comprises 2% (w / w) of alginate with respect to the total weight of the microparticle. 6
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    In another particular embodiment, the microparticle of the invention optionally comprises human serum albumin (HSA) and polyethylene glycol (PEG). In a particular embodiment, the microparticle of the invention further comprises 0.1-10% (w / w) of HSA relative to the total weight of the microparticle and 0.1-0.8% (v / w) of PEG,
    5 preferably PEG400, with respect to the total weight of the microparticle. And even more particularly, 5% (w / w) HSA and 0.5% (v / w) PEG, preferably PEG400, with respect to the total weight of the microparticle.
    In another particular embodiment, the microparticle of the invention is lyophilized.
    In another particular embodiment, the microparticle of the invention described in the preceding paragraphs is embedded in a matrix in the form of a fibrin gel (fibrin matrix). The fibrin gel relies on the polymerization of fibrinogen and thrombin with calcium to form a fibrin network. The microparticles of the invention embedded in a matrix
    Fibrin 15 have the important advantage of being administered topically, for example, by applying them as dressings in the contour of the wound or in the same wound.
    As indicated above, EGF increases the proliferation and migration of epidermal and dermal cells and is therefore important for the process of
    20 healing. However, due to its short half-life in wounds, continuous exposure of EGF with cells is required to achieve effective healing. To solve this problem, the administered dose could not be increased excessively, since this factor, by favoring cell proliferation, can be aggressive for the organism, especially if it is administered systemically.
    Surprisingly, the microparticle of the invention is a sustained release system of EGF that releases EGF for at least 60 days (Fig. 2). A release of at least 60 days implies a sustained release for more than four times longer than the EGF microparticles described in the prior art. Further,
    One milligram of microparticles of the invention provides 0.2-2.5 µg of EGF / day in the rapid release phase (first 30 minutes, initial burst in which the EGF adhered to the surface of the microparticle is released). which allows activating the healing process, and sustained levels of 0.5-150 ng of EGF / day in the sustained release phase (slow release phase in which the EGF is released mainly by diffusion
    35 and erosion of the microparticle). The sustained release process allows reducing the dose thus avoiding adverse effects both associated with the dose of administration of the
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    EGF as those related to the injection of the formulation in the injured area. Examples of this are inflammation, infection, systemic effects associated with the administration of EGF (gastric alterations etc).
    Thus, in a particular embodiment, the microparticle of the invention defined according to any one of the particular embodiments described above is characterized in that the EGF is released for at least 30 days. In another particular embodiment, the microparticle of the invention is characterized in that the EGF is released from 30 to 120 days.
    In a particular embodiment, the microparticle of the invention defined according to any one of the particular embodiments described above is characterized in that the EGF is released for at least 60 days. In another particular embodiment, the microparticle of the invention is characterized in that the EGF is released from 60 to 120
    15 days.
    In a particular embodiment, the microparticle of the invention defined according to any one of the particular embodiments described above is characterized in that the EGF release rate is between 0.5 to 150 ng of EGF / mg of 20 microparticles / day in the phase of sustained release. In another particular embodiment, the release rate of EGF is between 0.9 to 100 ng of EGF / mg of microparticles / day in the sustained release phase. In another particular embodiment, the EGF release rate is between 0.9 to 50 ng of EGF / mg of microparticles / day in the sustained release phase, in another particular embodiment it is between 5 to 80 ng of EGF / mg from
    25 microparticles / day in the sustained release phase, and in another particular embodiment is between 5 to 50 ng of EGF / mg of microparticles / day in the sustained release phase.
    Other important advantages are that the microparticle of the invention has an encapsulation efficiency of 60-88% (Table 1) and that the biological activity of EGF in vitro is not affected even by the microencapsulation process (formation process of the microparticle), nor by sterilization by gamma radiation (Table 2). Thus, the microparticle of the invention can be sterilized by gamma radiation without its biological activity and its physicochemical properties being substantially affected. Gamma radiation sterilization offers the main advantage over others
    35 sterilization methods, the low reactivity that it induces on the material to be sterilized and the ease with which the process is controlled. 8
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    The EGF released from the microparticles of the invention is biologically active in vitro and is capable of reducing the area of a wound faster than the control groups in in vitro healing trials (Fig. 3). More advantageously, the EGF released from the microparticles of the invention maintains its biological activity in vivo and is capable of promoting wound healing in vivo both through intralesional applications (Fig. 4, Table 3) and topical in fibrin preparations ( Table 4). In addition, with the microparticles of the invention, a complete re-epithelialization of normal thickness is achieved (Fig. 5), the healing being similar to the physiological one. Finally, another important advantage of the microparticles of the invention is that the dose of administration of EGF
    10 microencapsulated is less than the required dose when free EGF is used and the administration is single (a single dose), which entails fewer side effects, both associated with the administration of EGF and those related to the injection of the formulation into the injured area (Example 6).
    All these characteristics make the microparticle of the invention sustainably release bioactive EGF in optimal amounts to activate the proliferation of epithelial cells without the EGF passing to other organs and tissues of the individual to be treated, allowing excellent results to be obtained. healing with single administrations, both in intralesional and topical applications.
    Therefore, a second aspect of the present invention relates to the use of the microparticle of the invention in the manufacture of a medicament. In a particular embodiment, it refers to the use of the microparticle of the invention in the preparation of a medicament for promoting wound healing in an individual. In a
    In particular embodiment, the medicament is applied by parenteral injection and in another particular embodiment, the medicament is applied by topical administration. In a particular embodiment, the wounds are diabetic foot ulcers, pressure ulcers and / or vascular ulcers.
    The present invention also relates to the microparticle of the invention for use as a medicament. It also refers to the microparticle of the invention for use to promote wound healing in an individual. In a particular embodiment, it refers to the microparticle for parenteral use to promote wound healing in an individual. And in another particular embodiment it refers to the microparticle
    35 for topical use to promote wound healing in an individual. In a particular embodiment, the wounds are diabetic foot ulcers, pressure ulcers and / or 9
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    vascular ulcers
    In the context of the present invention, the term "promoting wound healing" refers to accelerating, increasing, activating and / or initiating wound healing, 5 resulting in the wound healing in a manner similar to the physiological one being the mature and physiologically well-organized scar tissue. And the term "wound" refers to the damage produced in any part of the body of an individual including, but not limited to, burns, cuts, trauma, etc., as well as chronic conditions such as pressure ulcers, diabetic or vascular and associated chronic wounds. at 10 age. Pressure ulcers refer to a lesion located in the skin and / or underlying tissue, usually on a bony prominence, as a result of a pressure or pressure in combination with shear. Diabetic ulcer or diabetic foot ulcer refers to a clinical disturbance of neuropathic etiopathogenic basis and induced by sustained hyperglycemia, in which with or without coexistence of ischemia, and prior traumatic trigger, causes injury and / or ulceration of the foot, They may end up in amputation. Vascular ulcers include all ulcers caused by a disorder of the arterial or venous systems of the lower extremities. They are chronic ulcers usually located in the legs and constitute a group of lesions of special relevance, both clinically and
    20 epidemiological.
    The present invention relates in a third aspect to a pharmaceutical composition (pharmaceutical composition of the invention) comprising the microparticle of the invention described in the preceding paragraphs and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are widely known to those skilled in the art. In a particular embodiment of the invention, the vehicle comprises a 0.9% w / v aqueous NaCl solution, in another particular embodiment, the vehicle further comprises 0.1-1% w / v carboxymethylcellulose, preferably 0.3% carboxymethyl cellulose w / v, and in another particular embodiment, the vehicle further comprises
    30 0.1% Tween 20 v / v.
    In a particular embodiment the pharmaceutical composition of the invention further comprises one or several pharmacological agents selected from the group consisting of antiseptics, antibiotics, astringent, antifungal, antiviral, antihistamines,
    35 anti-inflammatories, collagen, vitamins, minerals and mixtures thereof.
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    In a fourth aspect, the present invention relates to the use of the pharmaceutical composition of the invention in the manufacture of a medicament. In a particular embodiment, it refers to the use of the pharmaceutical composition of the invention in the preparation of a medicament for promoting wound healing in a
    5 individual In a particular embodiment, the medicament is applied by parenteral injection and in another particular embodiment, the medicament is applied by topical administration. In a particular embodiment, the wounds are diabetic foot ulcers, pressure ulcers and / or vascular ulcers.
    The present invention also relates to the pharmaceutical composition of the invention for use as a medicament. It also refers to the pharmaceutical composition of the invention for use to promote wound healing in an individual. In a particular embodiment it refers to the pharmaceutical composition for parenteral use to promote wound healing in an individual. And in another
    Particular embodiment refers to the pharmaceutical composition for topical use to promote wound healing in an individual. In a particular embodiment, the wounds are diabetic foot ulcers, pressure ulcers and / or vascular ulcers.
    In a fifth aspect, the present invention relates to a kit comprising the
    The microparticle of the invention and / or the pharmaceutical composition of the invention to promote wound healing. In a particular embodiment the kit comprises microparticles or a pharmaceutical composition comprising them and also a device, preferably a syringe, for parenteral administration of the microparticle of the invention. Parenteral administration is carried out throughout the
    25 perimeter of the wound.
    In another particular embodiment, the kit comprises microparticles embedded in a fibrin matrix for topical administration in the contour of the wound or in the same wound. The dosage in dressings for topical administration can be carried out in several ways known to the expert. Thus, in a particular embodiment of the kit of the invention, the kit comprises three solutions, a fibrinogen solution, a solution with the microparticles of the invention and another thrombin solution. Also, the kit comprises a sterile mold to prepare the dressing. When the dressing is needed, the fibrinogen solution and microparticles are mixed, added to the mold and on
    35 these solutions are added thrombin solution. This mixture will be incubated at 37 ° C until it coagulates. In another particular embodiment, the kit comprises the fibrin gel that 11
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    It comprises the microparticles already prepared, which must then be stored at 4 ° C and used in less than 7-10 days.
    The present invention relates in a sixth aspect to a method (referred to hereinafter as the method of the invention) for preparing the microparticle of the invention described in the preceding paragraphs comprising the following steps:
    to. Addition of a solution of PLGA in an organic solvent (organic solution of PLGA) on a solution comprising alginate and epidermal growth factor (solution of EGF and alginate), and mixing of the solution
    10 organic PLGA and EGF and alginate solution,
    b. Addition of the emulsion obtained in step a) on an aqueous solution comprising surfactant and sodium chloride (aqueous solution), and mixing of the emulsion obtained in step a) and the aqueous solution,
    C. Addition of the emulsion obtained in b) on a solution comprising
    15 sodium chloride and calcium chloride (chloride solution), and mixture of the emulsion obtained in step b) and the chloride solution,
    d. Solvent Extraction, and
    and. Isolation of the microparticle.
    The mixing of steps a) and b) is carried out by means known to the person skilled in the art, among others, sonication, mechanical agitation, etc. In a particular embodiment, the mixing of step a) is carried out by ultrasonic application and the mixing of step b) is carried out on a paddle shaker.
    The organic solvent of the organic PLGA solution is any organic solvent that dissolves the PLGA and is immiscible with water, being selected in a particular embodiment, from the group consisting of dichloromethane, acetonitrile, ethyl acetate, acetone, chloroform and mixtures thereof. same. In another particular embodiment said solvent is dichloromethane and in another particular embodiment it is a mixture of dichloromethane and acetone
    30 (3: 1).
    The usable surfactant is known to the person skilled in the art. In a particular embodiment, the surfactant is selected from the group consisting of, among others, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sulfates of
    35 alkylpolyoxyethylene, sodium dioethylsulfosuccinate, quaternary ammonium compounds, for example cetyltrimethylammonium bromide and lauryl dimethylbenzylammonium chloride, ethers 12
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    polyoxyethylene fatty alcoholic esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters and polyvinyl alcohol (PVA) and mixtures thereof. In a particular embodiment, the surfactant used is PVA.
    In a particular embodiment of the process of the invention, the organic PLGA solution comprises 2-10% (w / v) PLGA relative to the total volume of the organic PLGA solution. In another particular embodiment, the PLGA solution comprises 35% (w / v) PLGA relative to the total volume of the organic PLGA solution. In another particular embodiment, the PLGA solution comprises 5% (w / v) PLGA relative to the
    10 total volume of organic PLGA solution. In another particular embodiment, the PLGA solution comprises 3% (w / v) PLGA relative to the total volume of the organic PLGA solution.
    In another particular embodiment, the EGF and alginate solution comprises between 0.0015% a
    0.75% (w / v) EGF with respect to the total volume of the EGF and alginate solution and 0.05% to 2.5% (w / v) alginate with respect to the total volume of the EGF solution and alginate. More particularly, the EGF and alginate solution comprises between 0.025% to 0.5% (w / v) EGF with respect to the total volume of the EGF and alginate solution and 0.5-1.5% (p / v) of alginate with respect to the total volume of the EGF solution and
    20 alginate. And even more particularly, the EGF and alginate solution comprises 0.5% (w / v) EGF with respect to the total volume of the EGF and alginate solution and 1% (w / v) alginate with respect to the Total volume of EGF and alginate solution.
    In another particular embodiment, the EGF and alginate solution further comprises 0.05-5%
    25 (w / v) of HSA with respect to the total volume of the EGF and alginate solution, more particularly 2.5% (w / v) of HSA, and 0.15-1.16% (v / v ) of polyethylene glycol (PEG) with respect to the total volume of the EGF and alginate solution, more particularly 0.73% (v / v) of PEG400.
    In another particular embodiment, the aqueous solution of step b) is a 5-10% NaCl solution (w / v) with respect to the total volume of the aqueous solution, and the chloride solution of step c) comprises 5 and 10% (w / v) of sodium chloride with respect to the total volume of the chloride solution and between 0.6 and 1.2 mM of calcium chloride. In another particular embodiment, the aqueous solution of step b) is a solution of NaCl at
    5 5% (w / v) with respect to the total volume of the aqueous solution, and the chloride solution of step c) comprise 5% by weight of sodium chloride with respect to the total volume of the 13
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    Chloride solution and 0.6 mM calcium chloride. In another particular embodiment, the aqueous solution of step b) is a solution of 5% NaCl (w / v) in an aqueous solution of 5% PVA (w / v).
    The solvent extraction (step d) is carried out by methods known to the person skilled in the art, such as by vacuum extraction, evaporation or extrusion. In a particular embodiment of the method of the invention, solvent extraction is carried out by evaporation. Finally, step e) of isolation of the microparticle is carried out by methods known to the person skilled in the art.
    10 matter, such as centrifugation, ultracentrifugation, tangential filtration, vacuum filtration or vacuum evaporation.
    In a particular embodiment, the microparticles isolated in step e) are optionally subjected to a lyophilization process. Optionally, said lyophilized microparticles are embedded in a matrix in the form of a fibrin gel (fibrin matrix). The fibrin gel is based on the polymerization of fibrinogen and thrombin with calcium to form a fibrin network, thus in a particular embodiment, the microparticles isolated in step e) are added to a fibrinogen solution to which a thrombin solution and immediately allowed to gel. The matrix in the form of fibrin gel is prepared by means known to the person skilled in the art, which are briefly based on the mixture of two separate solutions, one comprising fibrinogen and one comprising thrombin. Fibrinogen and thrombin solutions can be purchased commercially separately (for example, from Sigma-Aldrich) or in a commercial kit to produce fibrin gels, such as the
    25 kit of Tissucol ® Duo 5 mL (Baxter, SL, Valencia, Spain).
    In a final aspect, the present invention relates to the microparticle obtained by the process of the invention described above. It also refers to a composition comprising said microparticle and a pharmaceutically acceptable carrier. As well as the use of said microparticle or said pharmaceutical composition for the preparation of a medicament to promote wound healing in an individual. It also refers to the microparticle obtained by the process of the invention described above and to the pharmaceutical composition comprising it together with a pharmaceutically acceptable carrier for use to promote the
    35 wound healing in an individual. Finally, it also refers to the kit comprising said microparticle or said pharmaceutical composition for promoting the
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    wound healing Examples
    The following are concrete examples of embodiments of the invention that serve to illustrate the invention.
    EXAMPLE 1. Preparation of microparticles (MP)
    MP4 microparticle MP4 microparticles were prepared as detailed below. For the
    The first emulsion w1 / o (step a) of the method of the invention) was weighed 75 mg of PLGA and an organic solution of 5% PLGA (w / v) in dichloromethane and acetone (3: 1) was prepared. On the other hand, an aqueous solution was prepared with 0.1% commercial rhEGF (w / w), 5% HSA (w / w), 0.5% PEG400 (v / w) and 2 alginate % (w / w), with respect to the amount of PLGA. The emulsion is formed when the organic solution is poured onto the
    15 aqueous and ultrasound is applied for 15 seconds at an intensity of 50W (Branson® 250 sonicator). Then, this first emulsion was added over 15 mL of a 5% NaCl solution (w / v) in a 5% aqueous solution (w / v). This system was homogenized on a 500 rpm vane shaker (Biocote® Stirrer SS20) for 60 seconds, giving rise to the second emulsion w1 / o / w2 (emulsion
    20 resulting from step b)). This mixture was incorporated over 400 mL of a 5% NaCl (w / v) solution and 0.6 mM CaCl2 in MiliQ water and kept stirring for 30 minutes to facilitate evaporation of dichloromethane. Finally, to perform the isolation of the microparticles, the mixture was filtered in a vacuum pump using a 0.45 µm nylon filter. Afterwards, the filter was cleaned with MiliQ water and extracted
    25 the micorparticles that remained attached to it. Finally, the microparticles obtained using a Telstar® Freeze Dryer (LyoBeta) were lyophilized following the manufacturer's instructions.
    MP5 microparticle
    30 The protocol described for the MP4 microparticles was followed but the rhEGF load was increased from 0.1% to 1%, in order to correctly dose in vivo.
    MP5- microparticle (MP5 sterilized with gamma radiation)
    1% charged rhEGF microparticles (MP5) were sterilized with radiation
    35 gamma using 60Co as a radiation source. The process described in Igartua et al. (γ-Irradiation effects on biopharmaceutical properties of PLGA microspheres loaded 15
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    with SPf66 synthetic vaccine. European Journal of Pharmaceutics and Biopharmaceutics 2008 6; 69 (2): 519-526).
    EXAMPLE 2. Characterization of the microparticles
    5 Determination of the size and morphology of the microparticle The determination of the size and morphology of the microparticle was performed following the protocol described in Puras et al. (Encapsulation of Aβ1–15 in PLGA microparticles enhances serum antibody response in mice immunized by subcutaneous and intranasal routes, European Journal of Pharmaceutical Sciences 2011; 44 (3): 200–206).
    10 Briefly, the size of the microparticle was determined by laser diffraction, using the Coulter® Counter LS 130 (Particle Size Analyzer, Amherst) particle analyzer, according to Washington C. (Particle size analysis in pharmaceutics and other industried: theory and practice Taylor & Fancis 1992) and morphology was analyzed by scanning electron microscopy (Fig. 1 and Table 1).
    15 Determination of the surface load The determination of the surface load is carried out by measuring the zeta potential using the Zetasizer Nano series® equipment (Malvern Instruments user manual, 2009), as described in Salvador et al. (Combination of immune stimulating
    20 adjuvants with poly (lactide-co-glycolide) microspheres enhances the immune response of vaccines, Vaccine, 2012, 30 (3): 589–596) (Table 1).
    Determination of encapsulation efficiency (EE%) To determine the effectiveness of the microencapsulation process, first
    Instead, the EGF must be extracted from the microparticles and have it in solution. To do this, 1 mg of microparticles was weighed into an Eppendorf® and 400 µL of dimethylsulfoxide (DMSO) was added. With the help of a vortex, the mixture was stirred for 5 minutes at room temperature, thus, DMSO digests the polymer and enables the release of EGF. Next, 600 µL of ELISA diluent was added
    30 (composition: 0.05% Tween 20 and 0.1% BSA in DPBS). Once the EGF was extracted from the microparticles, it was quantified by ELISA (enzyme linked immunosorbent assay). For this, the commercial kit "human EGF ELISA development kit, Peprotech" (Human EGF Elisadevelopment kit protocol, Peprotech, 2011) was used according to the manufacturer's instructions.
    35 The encapsulation efficiency was determined by the following mathematical formula: 16
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    EE (%) = (Amount of EGF in the microparticle / Amount of initial EGF) x 100
    Table 1: Results of the physicochemical characterization of the microparticles of the invention. The mean ± standard deviation is shown.
    Microparticle Average size Potential Z (mV) Efficiency of (µm) EE encapsulation (%)
    MP4 15.47 ± 8.71 -23.60 ± 4.6 88.11 ± 1.51 MP5 12.18 ± 5.53 -25.50 ± 9.41 68.82 ± 1.50 MP5-γ 14 , 95 ± 6.00 -28.50 ± 7.67 60.01 ± 1.10
    As can be seen, the average size obtained ranges between 10 and 17 μm. Variations obtained in the particle size of the 10 different microparticles are not considered significant. The MP4, MP5 and MP5- microparticles have a smooth and uniform surface, without pores or irregularities. As for the surface charge, all formulations have very similar negative potentials, so that we cannot expect relevant differences in the biopharmaceutical behavior of the particles. The zeta potential obtained is very negative, so the particles will have a tendency to repel, avoiding aggregation. The zeta potential values close to +30 mV and 30 mV are normally considered stable ((Malvern Instruments user manual, 2009). Finally, in terms of encapsulation efficiency, the EE% of MP4 microparticles is 88.11 % As the EGF load increases from 0.1% (MP4) to 1% (MP5), the EE% decreases to 68.82%, as expected since it is known that the
    20 increase in EGF load means worse encapsulation. Likewise, when the microparticles are subjected to gamma radiation (MP5-γ), a slight decrease in encapsulation efficiency (60.01 ± 1.10%) is observed, a result that can be attributed to the degradation of the drug produced by the process of sterilization
    EXAMPLE 3. In vitro release assay The release profile of the EGF of the microparticles was carried out as described in Salvador et al. (Combination of immune stimulating adjuvants with poly (lactide-co-glycolide) microspheres enhances the immune response of vaccines, Vaccine, 2012, 30 (3): 589–596).
    30
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    The amount of EGF was quantified by ELISA following the procedure described in Example 2 for the determination of EE%. The results obtained were expressed by the cumulative percentage of EGF released with respect to 100% of the content in the microparticle sample as a function of time.
    5 The results obtained appear in Fig. 2, where it is shown that the differences in EGF release comparing the MP4, MP5 and MP5-γ microparticles are minimal. This test also included microparticles with the components of the state-of-the-art microparticles, that is, with PLGA and EGF.
    Surprisingly, the release of EGF from the microparticles of the invention occurs for at least 62 days, while microparticles with PLGA and EGF (MP1) do not have a sustained release over time, releasing the EGF encapsulated therein in less than a day. (Fig. 2A).
    EXAMPLE 4. In vitro study of the biological activity of EGF The biological activity of EGF was evaluated by adding EGF to a cell culture in the absence of serum. Cell proliferation, increased by the presence of the factor in the medium, was quantified using the CCK-8 assay (Cell Counting Kit-8, Sigma-Aldrich, Saint Louise) as described in Zhou et al (Effects of Leukemia
    20 Inhibitory Factor on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells. J Endod 2011 6; 37 (6): 819-824.).
    Knowing the concentration of EGF added to the cellular medium it is possible to determine the effective dose 50 (ED50, minimum dose necessary for half of the population 25 to proliferate). ED50 is directly related to the biological activity of EGF. A low ED50 value is indicative of increased biological activity of EGF.
    The samples that were analyzed were the following: -EGF standard lyophilized without encapsulation (lyophilized EGF). 30 -EGF extracted from microparticles in the release assay. (MP5) -EGF extracted from irradiated microparticles in the release assay. (MP5-γ)
    Table 2 shows the Effective Doses 50 (ED50) determined from the EGF standards used (lyophilized EGF) and those of the EGF extracted from the microparticles with a load of 1% EGF before and after subjecting them to the sterilization process .
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    Table 2.-ED50 and standard deviation (D.E.)
    Pg / ml concentration FROM.
    Lyophilized EGF 66.9510.67
    MP5 54.878.58
    MP5-γ 60.7811.75
    As shown in Table 2, there are no significant differences in activity
    5 EGF biological when undergoing lyophilization. Nor are differences observed when encapsulated in the microparticles of the invention, or when they are sterilized by gamma radiation. Thus, the microparticle of the invention can be sterilized by gamma radiation without its physicochemical properties being substantially affected.
    10
    EXAMPLE 5. In vitro healing test
    The in vitro healing test was carried out as described in Waltera et al. (Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays, Experimental Cell Research 15 2010, 316: 1271-1281), in particular the effect was analyzed in the following experimental groups: (A ) fresh serum-free medium, (B) 15 ng / mL rhEGF obtained from MP5 microparticles in Dulbecco's Modified Eagle Medium (DMEM) serum-free medium and (C) 15 ng / ml rhEGF-free in serum-free medium (DMEM). The average width of the gaps was calculated using ImageJ® software from the micrographs taken
    20 at 0, 5, 18 and 24 hours after the creation of the wound (Fig. 3).
    As can be seen in Fig. 3, the EGF released from the microparticles of the invention reduces the area of the wound faster than the control groups. In addition, it is seen that the cells treated with EGF do not undergo morphological changes, unlike those treated
    25 only with serum-free medium, which lose their fusiform and elongated morphology after 5 hours of treatment. It is demonstrated that the EGF released from the microparticle of the invention maintains its biological activity in vitro.
    EXAMPLE 6. Healing test in vivo
    30 Female Wistar rats were used to perform this assay and diabetes was induced following the protocol described in Li et al. (Research of PDGF-BB Gel on the Wound Healing of Diabetic Rats and Its Pharmacodynamics. J Surg Res 2008
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    3; 145 (1): 41-8.
    Wound induction was performed as described in Galiano and Michaels (Quantitative and reproducible murine model of excisional wound healing. Wound repair
    5 and regeneration 2004; 12 (4): 485-492). The experimental groups analyzed were the following: -Control group: Spontaneous scarring, without treatment. - Vehicle control group: administration of 500 μL of vehicle (0.3% carboxymethylcellulose and 0.1% tween 20 in 0.9% sodium chloride). Administration on the day of
    10 operation - Empty MP control group: administration of resuspended microparticles in 500 μL of vehicle. Administration of 1 dose on the day of the operation. - Free EGF group: administration of 75 μg free EGF dissolved in 500 μL of vehicle twice a week (days 0, 4, 7, 11, 14 and 17).
    15 - MP Group: EGF: administration of MP5 (corresponding to 75 μg of EGF) resuspended in 500 μL of vehicle. Administration of 1 dose on the day of the operation.
    In all cases, administration was made by dividing the dose through the center and the edges of the wound, administering the formulations subcutaneously, below 20 of the fascia of the panniculus carnosum.
    The macroscopic analysis was performed by taking photos of the wounds on days 0, 4, 7, 11, 14 and 17 graphically representing the evolution of the diameter of the wound as a function of time (Fig. 4, Table 3).
    25 Table 3.-Percentage of wound closure as a function of time in days. Data shown as mean ± standard deviation.
    Groups Day 4 Day 7 Day 11 Day 14 Day 17 experimental
    Control 14.34 ± 7.8250.76 ± 4.5879.60 ± 11.98 88.91 ± 5.60 94.40 ± 2.88
    Vehicle control 13.06 ± 8.8847.19 ± 6.5874.24 ± 9.9689.58 ± 4.34 95.43 ± 0.62
    MP control empty 14.98 ± 6.57 43.54 ± 10.8477.23 ± 7.4189.14 ± 0.89 95.13 ± 0.34
    Free EGF 25.44 ± 11.15 ●, ▲ 51.47 ± 10.2978.97 ± 4.4495.08 ± 1.03 97.00 ± 0.20
    MP: EGF 36.43 ± 5.45 *, # 61.06 ± 8.69◘, ●, ▼ 90.29 ± 3.60 ■ 97.31 ± 1.11 99.16 ± 0.49
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    * p <0.001 vs. control, vehicle control and MP control empty.
    #p <0.01 vs. EGF free.
    • p <0.01 vs. vehicle control
    ▲ p <0.05 vs. vehicle control and control. 5 ◘p <0.001 vs. MP control empty.
    ▼ p <0.05 vs control and free EGF.
    As shown in Table 3, the EGF released from the microparticle of the invention maintains its biological activity in vivo. Four days after the induction of the 10 wounds, in the groups treated with EGF either free or released from the microparticle (MP: EGF), the wound is reduced more than in the control groups. At 7 and 11 days, the MP: EGF group shows a statistically significant decrease in the area of the wound (61.06 ± 8.69%), compared to the free EGF (51.46 ± 10.29%). On days 7 and 11 after the wound, the wound closure rate of the MP: EGF 15 group is significantly higher than that of the other groups. It is important to highlight that the administration of free EGF is twice a week (75 µg dose of EGF) while the EGF released from the microparticle is applied only once. This is an important advantage since the number of repeated injections to be made in the area of the wound is reduced and the probability of suffering is reduced.
    20 adverse effects to treatment.
    Re-epithelialization For histological analysis, animals were sacrificed with CO2 on days 7, 11 and 17 (Fig. 5).
    25 The degree of re-epithelialization was measured following the criteria established in Sinha et al. (Effects of steel scalpel, ultrasonic scalpel, CO2 laser, and monopolar and bipolar electrosurgery on wound healing in guinea pig oral mucosa. Laryngoscope 2003; 113 (2): 228-236) The areas where the lesion was found were removed with the help of a scalpel, fixed in 10% formaldehyde for 24 hours and stained by
    30 hematoxylin-eosin staining. Re-epithelialization was studied from the seventh day.
    As shown in Fig. 5, in the group treated with microparticles with EGF and in the control group without treating re-epithelialization it is completed without irregularities on the day
    11. In the rest of the experimental groups, a complete re-epithelialization was not achieved until the end of the study. In addition, only in the MP group: EGF the re
    epithelialization was normal thickness, which does not occur when solutions are used 21
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    control or free EGF. This supposes an important advantage since with the EGF released from the microparticles of the invention the healing is similar to the physiological one being the scar tissue mature and physiologically well organized.
    5 EXAMPLE 7. Microparticles embedded in a fibrin matrix Preparation of the thrombin solution: Aliquot thrombin at a concentration of 50 U / mL in a solution of 0.1% BSA (w / v) and 0.025 mM CaCl2. at -20 ° C. Preparation of the fibrinogen solution: Weigh 30-60 mg of fibrinogen and add 1 mL of physiological serum (30 mg / mL). Leave at 37 ° C until dissolved.
    10 Fibrin gel gelling: Add 0.5 mL of the fibrinogen solution to a test tube where the rhEGF microparticles are. Add 0.125 mL of thrombin solution, quickly collect everything and add to a 24-well plate. Gel in an oven at 37 ° C and wait for it to gel. The fibrin gel thus prepared contains 1530 mg of fibrin.
    15 Table 4 shows the percentage of wound closure as a function of time in days. Data shown as mean ± standard deviation.
    Table 4. Percentage of wound closure and standard deviation. twenty
    Day 1 Day 4Day 8
    % close FROM. % closeFROM. % closeFROM.
    Control 0.000.005.045.719.867.94
    Vehicle control 0.000.001.756.9814.498.81
    MP control empty 0.000.007.354.369.635.85
    Free EGF 0.000.008.423.9619.945.21
    MP EGF 0.000.0022.107.7343.123.97
    Fibrin 0.000.005.923.4619.099.63
    MP EGF + fibrin 0.000.0017.911.7429.364.34
    As seen in Table 4, the EGF released from microparticles embedded in a fibrin matrix maintains its biological activity in vivo and is capable of promoting wound healing (Table 4).
    权利要求:
    Claims (13)
    [1]

    1. Microparticle comprising a polylactic-co-glycolic acid polymer (PLGA), an alginate polymer, and epidermal growth factor (EGF).
    Microparticle according to claim 1 wherein the epidermal growth factor is the human recombinant epidermal growth factor (rhEGF).
    [3]
    3. Microparticle according to claim 1 or 2 comprising between 82.7% and 99.897% (w / w) of PLGA, between 0.003% and 1.5% (w / w) of epidermal growth factor and between 0.1 % and 5% (w / w) of alginate, based on the total weight of the microparticle.
    Microparticle according to any one of the preceding claims, further comprising 0.1-10% (w / w) of human serum albumin and 0.1-0.8% (v / w) of polyethylene glycol with respect to the total weight of The microparticle
    [5]
    5. Microparticle according to any one of the preceding claims characterized in that the epidermal growth factor is released for at least 30 days.
    Microparticle according to any one of the preceding claims embedded in a fibrin matrix.
    [7]
    7. Pharmaceutical composition comprising a microparticle according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
    [8]
    8. Use of a microparticle according to any one of claims 1 to 6 for the preparation of a medicament.
    [9]
    9. Use of a microparticle according to any one of claims 1 to 6 for the preparation of a medicament for promoting wound healing in an individual.
    [10]
    10. Use according to the preceding claim, wherein the wounds are selected from the group
    25 formed by diabetic foot ulcers, pressure ulcers, vascular ulcers and combinations thereof.
    [11]
    11. Kit for promoting wound healing comprising a microparticle according to any one of claims 1 to 6.
    [12]
    12. Kit for promoting wound healing comprising a pharmaceutical composition according to claim 7.
    [13]
    13. Method for preparing a microparticle according to any one of claims 1 to 5 comprising the following steps:
    to. Addition of a solution of PLGA in an organic solvent (organic solution of PLGA) on a solution comprising alginate and EGF
    35 (EGF and alginate solution), and mixture of the organic PLGA solution and the EGF and alginate solution,
    2. 3

    b. Addition of the emulsion obtained in step a) on an aqueous solution comprising surfactants and sodium chloride (aqueous solution), and mixing of the emulsion obtained in step a) and the aqueous solution,
    C. Addition of the emulsion obtained in b) to a solution comprising chloride
    5 of sodium and calcium chloride (chloride solution), and mixture of the emulsion obtained in step b) and the chloride solution,
    d. Solvent Extraction, and
    and. Isolation of the microparticle.
    [14]
    14. A process according to the preceding claim wherein the organic solvent of the organic PLGA solution is a mixture of dichloromethane and acetone (3: 1).
    [15]
    15. The method according to any one of claims 13 or 14 wherein the aqueous solution of step b) comprises 5-10% NaCl (w / v) with respect to the total volume of the aqueous solution and the chloride solution of step c ) comprises 5-10% NaCl (w / v) by volume with respect to the total volume of the chloride solution and of
    15 0.6 to 1.2 mM CaCl2.
    [16]
    16. Process for the preparation of a microparticle according to claim 6 which comprises the following steps after the process steps according to any one of claims 13-15:
    F. Lyophilize the isolated microparticle in step e), and
    20 g Embed the lyophilized microparticle obtained in step f) in a fibrin matrix.
    24
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    同族专利:
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    WO2014083233A1|2014-06-05|
    ES2540453R1|2016-02-23|
    EP2737895A1|2014-06-04|
    ES2540453B1|2016-09-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5219998A|1990-06-04|1993-06-15|Levin Robert H|Yeast-derived epidermal growth factor|
    CN1720989A|2004-07-15|2006-01-18|深圳市清华源兴生物医药科技有限公司|Sustained release microsphere of epidermal growth factor, its preparation method and application|
    CU23388B6|2006-01-31|2009-07-16|Ct Ingenieria Genetica Biotech|PHARMACEUTICAL COMPOSITION OF MICROSPHERES TO PREVENT AMPUTATION OF THE DIABETIC FOOT|
    CU23526B6|2006-10-03|2010-05-19|Ct Ingenieria Genetica Biotech|METHOD FOR MORFOFUNCTIONAL RESTORATION OF PERIPHERAL NERVES IN DIABETIC NEUROPATHY|CN105287242A|2015-11-11|2016-02-03|浙江元研生物科技有限公司|Skin care product containing human stem cell growth factor and preparation method thereof|
    CU20190022A7|2019-03-18|2020-10-20|Centro De Ingenieria Genetica Y Biotecnologia Biocubafarma|PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF THE DIABETIC FOOT ULCER|
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