MXPA99011250A - Device and methods for wound treatment - Google Patents

Abstract

A method and a device for the treatment of wounds and promotion of wound healing and muscle regeneration. The method includes applying a composition including a non-biodegradable microsphere with a substantial surface charge to the subject. The device further includes a pharmaceutically acceptable carrier and a container for containing the composition and the carrier. The microspheres used in the present invention have been shown to promote wound healing and muscle regeneration both in vivo and in vitro.

Description

DEVICE AND METHODS FOR WOUND TREATMENT FIELD AND BACKGROUND OF THE INVENTION The present invention relates to the treatment of wounds and in particular, relates to a device and methods for accelerating wound healing and reinforcing the regeneration of muscle with microspheres as a therapeutic agent. Wound healing is a complex process involving factors such as cellular, extracellular matrix (ECM) components and the cellular environment. Basically, all wound healing involves the repair or replacement of damaged tissues. The precise nature of such repair or replacement depends on the tissues involved, although all these processes involve certain basic principles. To illustrate these principles, the healing of skin or skin wounds will be described, understanding that exposure could also be extended to all types of wound healing. The skin has several layers, including keratin, epidermis and dermis. If only the epidermis is damaged, as in most minor injuries, the keratinocytes move from the edge of the wound and eventually cover it, reforming the epidermis and keratin [D.R. Knighton and V.D. Fiegel, Inves t. Radi ol. , 26: 604-611, 1991].

P1705 / 99MX If all the layers of the skin are damaged or destroyed, new connective tissue, called granulation tissue, must first fill the space of the wound. This tissue is formed by the deposition of ECM components by fibroblasts, which move to the wound space [D.R. Knighton and V.D. Fiegel, Invest. Radi ol. , 26: 604-611, 1991]. It is currently believed that the deposition of these ECM components, such as collagen, is important to heal the wound. Indeed, the prior art shows that the strength of the healed wound ultimately depends on the deposition of collagen [Haukipuro, K., et al., Ann. Surg. , 213: 75-80, 1991]. In this way, the collagen deposit must be present at a sufficiently high level to give strength and support to the healed wound. All this multi-stage process must be completed to successfully achieve wound healing. If one or more of these components is missing, scarring is not performed, the skin is not repaired and the wound remains open. These open wounds can be easily infected, delaying further the healing process and leading to the formation of ulcers and sores on the skin. The process of wound healing is also inhibited in many patients by the presence of other complicating conditions, such as diabetes and advanced age. Patients with such conditions often have P1705 / 99MX skin wounds that ulcerate and refuse to heal or only heal slowly after a long period of time has passed. Various treatments have been used to accelerate the speed at which wounds heal. For example, U.S. Patent No. 4,772,591 discloses a method for accelerating the rate of wound healing by applying a combination of ascorbic acid, calcium, tyrosine or phenylalanine and anti-inflammatory substances to the wound. Similarly, U.S. Patent No. 4,590,212 discloses a method for applying acetaminophen to the wound. Many other patents have focused on other methods to accelerate the healing rate of a wound. However, none of these has proven effective. In an attempt to improve wound treatments, various pharmaceutical vehicles have been employed to deliver wound chemotherapeutic agents. Said vehicles are particularly required for skin wounds since these are usually either exposed to the air or covered with bandages or gauze. In any case, the therapeutic agent can be easily removed, for example, by rubbing. So, various creams, gels and powders have been used as pharmaceutical vehicles in an attempt to solve P1705 / 99 X this problem. An interesting group of pharmaceutical vehicles use microspheres, which are small microscopic particles made of various materials, including plastics and long-chain carbohydrates. Many applications of the microspheres as carriers for various therapeutic agents are known in the prior art. For example, U.S. Patent No. 5,264,207 discloses microspheres as a vehicle for a pharmaceutical or cosmetic substance. A composition containing the microspheres and the active substance is applied by the cutaneous route and the microspheres, in effect, allow said route of administration for the active substance. However, this reference does not show or suggest the use of the microspheres themselves as a therapeutic substance. Likewise, PCT Applications Nos. 096/13164 and W094 / 13333 both expose microspheres made of a material that catalyzes the production or release of certain therapeutic substances. PCT Application No. W096 / 13164 discloses polymeric nitric oxide adducts that release nitric oxide when applied directly to damaged tissue. PCT Application W094 / 13333 discloses particles that are chemically modified to have free radical activity in the wound environment. Again, no reference shows or suggests using P1705 / 99MX the microspheres themselves as a therapeutic substance, without chemical modification of the material of the microsphere. However, certain properties of the microspheres used as pharmaceutical vehicles were shown to influence the effect of the therapeutic substance itself. For example, the activation of cytotoxic T lymphocytes by means of class I alloantigen immobilized in latex microspheres was studied. Although class I alloantigen was clearly providing the stimulus by itself, the degree of cell stimulation was increased using particle sizes between 4 and 5 microns. [M.F. Mescher, J. Immunol. , 149: 2402-2405, 1992]. That increased stimulation could demonstrate surface contact requirements for cytotoxic T lymphocytes. In other words, the optimum particle size could increase the effect of class I alloantigen by providing an optimal surface area for cell contact. It must be emphasized, however, that these pearls were still vehicles for the active substance. Attempts have been made to exploit the apparent ability of certain particles to increase the effectiveness of active substances that promote wound healing. For example, U.S. Patent No. 3,842,830 discloses glass microparticles that act to promote P1705 / 99MX wound healing when applied directly to damaged tissue. U.S. Patent No. 5,092,883 discloses biodegradable positively charged dextran beads with similar ability to promote osteogenesis and healing of soft tissue lesions. However, none of these references show or suggest the promotion of muscle regeneration by administering microspheres to the wound. Furthermore, none of these references show microspheres that initially promote faster metabolism and cell proliferation, which still have a finite, limited effect, so that rapid metabolism and cell proliferation are not permanently induced. Such a limited effect is especially important for promoting wound healing, which requires an initial increase in cell metabolism and proliferation, followed by a cessation of said cellular activation after healing has occurred. Without an induction of such activation, wound healing will not take place. However, if the cellular activation does not stop after the scarring is practically complete, it can result in the formation of abnormal scars, as in the formation of keloids. In this way, there must be a balance between promotion and P1705 / 99MX the inhibition of cell metabolism and proliferation during wound healing. There is, therefore, the unmet medical need for a particular substance that can be applied directly to damaged tissue to promote healing, more so that it has self-limited effects and that it is practically non-toxic and that it can also promote muscle regeneration.

SUMMARY OF THE NINE It is an object of the present invention to provide a composition for treating a subject's wound. It is another object of the present invention to provide a composition containing an agent that has the ability to form multi-point contacts with cell membranes for the promotion of wound healing. It is still another object of the present invention to provide a microsphere as an agent of this type. It is still another object of the present invention to provide a device for containing the composition of the present invention. It is still another object of the present invention to provide methods for using the compositions of the present invention. These and other objects of the invention are P1705 / 99MX s describe in more detail in the following description, Figures and claims. The present invention relates to a composition, a device and a method for promoting wound healing using microspheres. Unexpectedly, microspheres of the particular size range described herein have the ability to promote wound healing without subsequent addition or inclusion of any drug or other therapeutic substance. Indeed, as described below, these microspheres do not degrade or undergo another chemical alteration to produce their therapeutic effect. In this way, these microspheres are the active ingredient in the compositions of the present invention, rather than simply acting as a vehicle for another distinct active ingredient. According to the teachings of the present invention, there is provided a composition, a method and a device for treating a wound of a subject. The composition basically consists of an agent that has the ability to form a multi-point contact with a cell membrane, the agent is practically non-biodegradable during the treatment period. Preferably, the agent is a microsphere having charged surface groups. According to one embodiment, the composition basically consists of an agent with charged surface groups, wherein the P1705 / 99MX load can be positive or negative. According to particular embodiments of the present invention, the material of the microsphere is selected from the group consisting of polystyrene, polystyrene derivative, polymethyl methacrylate (PMMA), silicone, polylysine, poly-N-ethyl-4-vinylpyridinium bromide and latex. According to certain embodiments of the present invention, the charged surface groups are selected from the charged groups consisting of polystyrene, polystyrene derivative, sulfate, poly-N-ethyl-4-vinylpyridinium bromide, protamine, protamine sulfate, protamine salts, polylysine and carboxyl. Also preferably, the microspheres have a diameter in a range between about 0.01 microns and 200 microns, more preferably in a range between about 0.1 microns and 100 microns and preferably superlative between about 0.1 and 20 microns. According to another preferred embodiment of the present invention, the composition also includes a pharmaceutically acceptable carrier for the microsphere. Preferably, the pharmaceutically acceptable carrier is an aqueous solution. Alternatively and preferably, the pharmaceutically acceptable carrier is a gel-forming material. More preferably, the gel-forming material includes methylcellulose. According to yet another preferred embodiment of the P1705 / 99MX present invention, the microsphere is present in a concentration in a range of approximately between 0.0001 percent and 1.5 percent, weight / weight. More preferably, the microsphere is present at a concentration in a range of approximately between 0.001 percent and 1.0 percent, weight / weight. With superlative preference the microsphere is present at a concentration in a range of approximately between 0.01 percent and 0.2 percent, weight / weight. According to yet another embodiment of the present invention, there is provided a composition for promoting muscle regeneration in a subject, the composition includes an agent that has the ability to form a multi-point contact with a muscle cell.; preferably a microsphere having a surface group with a substantial charge that can be positive or negative. According to yet another embodiment of the present invention, there is provided a device for treating a wound, which includes: (a) a composition that includes an agent that has the ability to form a multi-point contact with a cell membrane and a pharmaceutically acceptable carrier in which the agent is practically insoluble; and (b) a container for containing the composition. As exemplified, the vehicle is preferably selected from the group consisting of aqueous medium, vehicle P1705 / 99 X spray, ointment and bandage. According to another preferred embodiment of the present invention, the pharmaceutical carrier is selected from the group consisting of aqueous solution, gel-forming material, aerosol vehicle and ointment. Preferably, the pharmaceutical carrier is a gel-forming material. More preferably, the gel-forming material includes methylcellulose. With superlative preference, the container is a compressible tube. Alternatively and preferably, the pharmaceutical carrier is an aqueous solution. More preferably, the package is a sterile, substantially sealed package. Preferably , superlative, the sterile, substantially sealed package is an aerosol spray. According to another embodiment of the present invention, a device for promoting muscle regeneration is provided, which includes: (a) a composition that includes an agent that has the ability to form a multi-point contact with a cell membrane and a pharmaceutically acceptable carrier in which the agent is practically insoluble; and (b) a container for containing the composition. The method of the present invention may also be used cosmetically, to prevent the formation of excess scars on a cut or other skin wound such as facial skin and for P1705 / 99MX treat acne.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a device and method for promoting wound healing using microspheres. Unexpectedly, microspheres of the particular size range described herein promote wound healing without further addition or inclusion of any drug or other therapeutic substance. Indeed, as described below, these microspheres do not degrade or undergo another chemical alteration to produce their therapeutic effect. The structure of these microspheres includes a core and at least one type of charged surface group that is present at least on the outside of the core of the microsphere. Examples of materials suitable for the core include non-exclusive long-chain polymers such as polystyrene, polystyrene derivative, latex, poly-β-alanine, polylysine, poly-bromurb of N-ethyl-4-vinylpyridinium, polymethylacrylate (PMMA). ) and silicone. Preferably, the material is selected from the group consisting of polystyrene and polystyrene derivative. Examples of surface groups include non-exclusive poly-N-ethyl-4-vinylpyridinium bromide, protamine, protamine sulfate, P1705 / 99MX salts of protamine, polylysine, carboxyl, polystyrene and polystyrene derivative. More preferably, the surface group is selected from the group consisting of polylysine, polystyrene, polystyrene derivative and carboxyl. These surface groups may be present as part of the core of the microsphere or may be added later by chemical processes such as the derivation of the long chain polymer. Hereinafter, the term "derivation" refers to the process of chemical modification, which modifies or changes a molecule or a portion thereof. In this way, the structure of the microsphere includes a "core" and a "surface group", which are two separate elements, even when the list of possible species of each of these elements overlaps. For example, the polystyrene can be both a surface group and a core material of the microsphere. The microspheres produced from the polymer must be practically insoluble in aqueous medium and instead form a suspension or dispersion in said medium. To further clarify the parameters of the present invention, several terms should be defined. The term "wound" hereinafter includes any injury to any portion of a subject's body, including, in P1705 / 99MX non-exclusive form, acute states such as thermal burns, chemical burns, radiation burns, burns caused by excess exposure to ultraviolet radiation such as sunburn, damage to bodily tissues such as the perineum as a result of labor, including injuries suffered during medical procedures such as episiotomies, injuries induced by injuries including cuts, injuries suffered in automobile accidents or other mechanical accidents and those caused by bullets, knives and other weapons and post-surgical injuries, as well as chronic conditions such as pressure sores, bed sores, diabetes-related conditions and poor circulation and all types of acne. The areas of the body that can be treated with the present invention include, non-exclusively, skin, muscle and internal organs. Hereinafter, the term "subject" refers to humans or lower animals in which the invention is practiced. From here on, the term "promote" includes accelerating and intensifying. Hereinafter, "reducing scarring" includes preventing or decreasing the formation of excessive scarring such as keloids and hypertrophic scars, as well as decreasing the degree of scar tissue formation both externally, for example in the skin of the subject, and P1705 / 99MX internally, for example accessions. Finally, it should be noted that the method of the present invention may also be used cosmetically, to prevent excess scar formation in a cut or other wound in the skin, such as facial skin and to treat acne. In a cosmetic sense, the term "excess scar formation" includes any scarring that is undesirable or unacceptable from a cosmetic point of view. Although below the discussion refers to specific types of microspheres, it should be noted that this is not intended to be limiting in any way. Those skilled in the art will appreciate that these microspheres, more generally described as "agents," may be beads, particles or beads that are either solid or hollow. In preferred embodiments of the present invention, these agents are dispersed in a pharmaceutically acceptable carrier medium in which the agents are practically insoluble, such as a suspension in aqueous medium for example or in a non-aqueous medium such as an ointment or an aerosol spray. The form of the agents may be regular, for example spherical or elliptical or regular non-spherical forms; or the shape of the particles can be irregular, so that the surface is not a single continuous curve or so that the surface is not uniform.

P1705 / 99MX In addition, the agents can be a mixture of different polymers and can also be a mixture of different particles, beads or globules of different sizes. The agents can also have pores of different sizes. By way of example, the long chain polymer forming the agents, such as poly-β-alanine, can be crosslinked, which particularly favors the spherical shape of a microsphere, although such a shape can be obtained without crosslinking. An example of a manufacturing method for a cross-linked poly-ß-alanine microsphere is given in U.S. Patent No. 5,077,058 although it should be noted that this material would require subsequent derivatization to obtain a total surface charge of the microsphere. Alternatively, the particles may have irregular chaotic shapes, particularly if the polymer is not crosslinked. The particle can have any shape, for example, rolled, globular, extended and rolled randomly. Preferably, the polymer must not be biochemically reactive and must not be biodegradable. More preferably the polymer is practically non-biodegradable during the treatment period, so that it would remain undegraded during the period that is required for wound healing. Thereafter, the term "biodegradable" is P1705 / 99MX refers to agents that are not biodegradable during the treatment period which is the period required for wound treatment. At least, the agents must have the following properties: 1. They must have the ability to form multi-point contacts with cells or portions of cells in them, such as the outer cell membrane and molecules in this membrane; 2. Must have the ability to promote wound healing without significant chemical alteration or degradation; and 3. They should be practically insoluble in aqueous medium as body fluids and should form a suspension instead. These characteristics are important because as discussed below more closely, the effect of the agents of the present invention appears to be directly linked to the formation of multi-point contacts between the material of the agents and a portion of the cell such as the cell membrane. external, thereby forming an adherent surface for the cells to fix there. Such multi-point contacts are possible with many different polymers that allow charged groups to be accessible for interaction with molecules and portions of the outer cell membrane. In this way, although the description below focuses on a P1705 / 99MX type of agent, the microspheres, it is understood that the present invention covers any material that has the ability to form such multi-point contacts. As noted above, preferably the microsphere has a diameter in a range of approximately between 0.01 microns and approximately 200 microns, more preferably in a range of approximately 0.1 to 100 microns and preferably superlative between approximately 0.1 and 20 microns. Without wishing to be limited to any mechanism, it should be noted that these preferred ranges are the best size that allows the uptake of microspheres by macrophages that infiltrate the wound area. The microspheres appear to actually attract and activate the macrophages through contact with at least a portion of the macrophages, probably with the molecules of the outer cellular membrane of the macrophages. The anti-inflammatory and antibacterial effects observed for the microspheres are thus probably indirect effects, obtained by the activation of macrophages or other cells. Another important property of the microspheres is the charge of the surface groups. The total charge transported by certain preferred examples of microspheres was measured as Z or zeta potential by electrophoretic mobility (miliVolts) with a ZetaMaster (Malvern Instruments, P1705 / 99MX United Kingdom). The range of Z potentials determined in certain embodiments exemplified herein was between -29.58 mV and -79.76 mV. Hereinafter, the term "charged" refers to potential Z with an absolute value of at least about 1 V and preferably at least about 10 mV, either positive or negative. The microspheres in the tested suspensions do not aggregate, coalesce, aggregate or undergo irreversible agglomeration. Although the microspheres sedimented a little over time, they were easily resuspended with gentle agitation.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described here by way of example only, with reference to the accompanying drawings, wherein: Figure 1 is a graph showing the ability of the microspheres of the present invention to increase the activity of the phosphocycline creatine; Figure 2 is a graph illustrating the effect of the microspheres of the present invention on the synthesis of collagen; Figures 3A-3C illustrate the effect of the microspheres of the present invention in the myoblast form; P1705 / 99MX Figures 4A-4D illustrate the ability of microspheres to promote wound healing in rats; Figure 5 is a graph of the speed at which the wound area of Figure 4 decreases; Figures 6A and 6B compare the effect of the microspheres of the present invention on wound healing with tissue culture medium and saline solution in rats; Figures 7A-7D demonstrate the ability of the microspheres of the present invention to promote healing in a first human case study; Figures 8A and 8B further demonstrate the efficacy of the present invention in the human case study of Figures 7A-7D; Figures 9A and 9B demonstrate the efficacy of the present invention in a second case study in human; Figures 10A-10D show the effect of the present invention in a third case study in human; Figures HA and 11B show the efficacy of the present invention in a fourth case study in human; and Figure 12 shows the efficacy of a gel formulation of the present invention.

P1705 / 99MX DESCRIPTION OF THE PREFERRED MODALITIES The present invention is exemplified herein by the use of microspheres that can be used to promote wound healing in general, as well as muscle regeneration. Wound healing and muscle regeneration involve both the repair of damaged tissue and the replacement of lost tissue. The migration and proliferation of specific cell types must happen in an orderly and structured way, which can be easily differentiated from the unrestricted growth of malignant tissues such as solid tumors. In particular, cells involved in wound healing and muscle repair must first be activated to perform the roles that are required in the healing process. Although the exact mechanism is not known, structured cell growth, ordered in the proliferation that occurs in the healing of wounds clearly demonstrates the presence of a fairly organized regulatory process. As demonstrated in the Examples given below, the microspheres of the present invention do not appear to interfere with this complex, structured and organized process, since these microspheres clearly only accelerate the rate of the total healing process, as well as the stages within. of this process. However, unexpectedly the P1705 / 99MX microspheres of the present invention do not cause the cells to exhibit an unrestricted, continuous metabolic activation state, indicating that the normal regulatory process is not affected. In this way, the microspheres of the present invention do not cause unrestricted cell activation. Without limiting the present invention to a particular mechanism, the addition of microspheres with negatively charged groups may have a therapeutic effect on wound healing by serving as an additional surface for cell attachment and culture. One hypothesis for the effectiveness of the microspheres of the present invention is that the negatively charged groups allow the creation of multiple bonds between the solid surface of the microsphere and the cell membranes, which represent multi-point contacts between the material of the microsphere and the cellular membrane. The formation of these bonds causes changes in the distribution and state of membrane ligands, cytoskeletal reorganization, activation of intracellular signal transduction and other biochemical changes, ultimately leading to cell activation. Cell activation then leads to cell proliferation and the production of growth factors and collagen and other components of the extracellular matrix. It should be noted that the present invention need not depend on P1705 / 99MX some particular mechanism, since as shown below, these microspheres clearly have a beneficial effect for treatment and wound healing in vivo. Several different types of microspheres were tested in the Examples below. These microspheres were made of polystyrene, either with amino or carboxyl surface groups or without additional surface groups. The diameters of the microspheres varied between about 0.1 and 20 microns. The zeta potential of certain microspheres was also tested and it was shown that the size of the sphere and the type of surface groups clearly had an effect on the amount of total charge transported by each microsphere, which could have an important effect on the ability of the microsphere. the microsphere to promote wound healing. Although certain specific types of microspheres are illustrated, it is understood that many other types of related microspheres could be used if the following characteristics are satisfied. 1. Must have the ability to form multi-point contacts with cells or portions of cells in them; 2. Its mechanism of action must require chemical alteration or degradation; and 3. They must be practically insoluble in aqueous medium such as body fluids and in their P1705 / 99MX instead must form a suspension. Other preferable attributes include the following. First, the microspheres should preferably be made of material that is non-biodegradable during the treatment period, more preferably polystyrene. Second, the microspheres should preferably carry a substantial charge, more preferably a negative total charge. Although the size of the microspheres is less critical, preferably the microspheres should be between about 0.1 and 20 microns in diameter. Preferably, the microspheres must be derived with carboxyl surface groups, although other negatively charged groups can also be used. Thus, these types of microspheres are given for illustrative purposes only and do not imply that they are limited in any way. The principles and operation of the microspheres according to the present invention will be better understood with reference to the Examples, drawings and the accompanying description.

EXAMPLE 1 Effect of Microspheres on Phosphokinase Creatine The microspheres of the present invention clearly induced an initial increase in creatine phosphokinase (CFC) activity of P1705 / 99MX cultured myoblasts, as shown in Figure 1. However, after eight days, both the treated cells and the untreated cells demonstrated the same level of CFC activity, indicating that the induction of CFC activity increased by the microspheres of the present invention is temporary. The experimental method was the following. A rat embryonic skeletal muscle primary culture was prepared as described by Freshney [R. J. Freshney, Cul ture of Animal Cells, Willey, 1986, p. 117, 170-172]. Briefly, the muscles were dissected without skin and without bones and disintegrated by hot trypsinization (trypsin 0.25% at 36.5 ° C). Contamination by fibroblasts was reduced by pre-striating the cells for 1 hour in an incubator with 5% C0 at 37 ° C, since the fibroblasts adhere first to the tissue culture plates. The myoblasts were then seeded in 35 mm Petri dishes at a concentration of 50,000 cells per ml with 2 ml of medium (Dulbecco's modified Eagle medium: medium 199 in a ratio of 1: 4), enriched with antibiotics, horse serum 10% volume / volume and chicken embryo extract at 4% volume / volume. Chicken embryo extract was prepared from 10-day-old chicken embryos according to R. J. Freshney, Cul ture of Animal Cells, Willey, 1986. Antibiotics included amphotericin and gentamicin, P1705 / 99MXL diluted 1: 1000 from the initial standard concentration of 2.5 mg / ml. After 24 hours, the medium was decanted and replaced with fresh medium with a 20% volume / volume horse fetal serum content and 1% volume / volume chicken embryo extract. The cultured cells were then treated either with microspheres, beginning at the time of striatum, in medium for 4-8 days or with medium alone. The microspheres were either of carboxylated polystyrene of 1, 2 or 4.5 microns in diameter or of polystyrene alone of 4.5 microns in diameter. The concentration of microspheres was either 106 or 107 per ml of medium, obtaining similar results for both concentrations (not shown). After 4, 5, 6, 7 or 8 days of treatment, creatine phosphokinase activity was measured by a standard method ("Creatine Kinase", Worthington Enzyme Manual, Worthington Biochemical Corporation, Freehold, N.J., USA, 1972, pages 54-55). The results are shown in Figure 1, as Units of CFC activity per mg of total cellular protein. Figure 1 clearly demonstrates the ability of the microspheres of the present invention to induce an initial increase in creatine phosphokinase activity, as compared to control cells. After 4 days of treatment, the cells treated with microspheres P1705 / 99MX show an initial increase in CFC activity compared to control cells. This increase is particularly marked on days 5 and 6 of the treatment. However, on day 7, the CFC activity in the control cells begins to reach parity with that of the cells treated with microspheres. On day 8, both the control cells and the cells treated with microspheres show similar activity levels. Clearly the microspheres promote an initial increase in CFC activity in myoblasts, which stabilizes after 8 days of treatment. Said increased CFC activity is correlated with the biochemical maturation of myogenic cells. In this way, the microspheres promoted biochemical maturation of the cultured myoblasts.

EXAMPLE 2 Effect of Microspheres on Proliferation and Cell Fusion The microspheres of the present invention were shown to induce an initial increase in both cell proliferation and myoblast fusion, as compared to control cells (untreated), as shown below. Primary cultures of rat myoblasts were prepared in the same manner as described in Example 1 above, except that the cells were P1705 / 99MX cultured on coverslips. The treated cells were incubated with microspheres in medium, as described below more closely, while control cells only with medium. To determine the degree of cell proliferation, the cells were fixed in ethanol / acetic acid (3: 1) and then stained with hematoxylin-eosin. The stained cells were counted in an optical microscope. The mitotic index was calculated as the proportion of cells in mitosis counted per 1000 cells. For the cell proliferation test, polystyrene microspheres having sulfate surface groups with a diameter of 0.18 microns and a concentration of 107 microspheres / ml of medium were used. A 20-fold increase in the mitotic index was observed after treatment for 24 hours with microspheres compared to the control cells. Specifically, the mitotic index of the control cells was 1.25 + _ 0.7%, while that of the cells treated with microspheres was 24.6 + _ 1.0%. In this way, clearly, the microspheres promoted a large increase in the mitotic index of the myoblasts. The effect of the microspheres on myoblast fusion was also examined. The results are given in Table 1. In general, cells treated with microspheres exhibited P1705 / 99MX approximately a fusion speed of 150% compared to the controls. However, the extent of this effect depended on the type of microspheres and the duration of treatment. The types of microspheres that were tested are presented in Table 1. The diameter of the microspheres is given in microns under "Diameter". The superficial groups of in the polystyrene beads are given under "Surface Group". The polystyrene beads without any additional derivation are "polystyrene". The beads derived either with carboxyl or amino surface groups are described as "carboxy" and "amino", respectively. The concentration of beads is given as number of beads per ml of medium under "Concentration". The cells were prepared, fixed and stained in the same way as for the determination of myoblast proliferation rate, described above. The cells were striated at the density given in Table 1 as cells per ml of medium, in the "Initial Cells" column. Myoblast fusion measurements were made after a given number of days after treatment in "Days After Treatment". The degree of fusion is calculated as the proportion of nuclei within multinuclear or myosimplastic cells, related to the total number of nuclei within the field Microscopic P1705 / 99MX, given as "Fusion Ratio" for cells treated with microspheres and "Control Fusion" for untreated, control cells. At least 400 cores were counted for each experimental condition. The proportion of the degree of fusion in cells treated with microspheres and untreated, control cells is given as "Relative Effect". If no value is given for a particular space in Table 1, the value is the same as in the row above.

P1705 / 99MX Table 1. Effect of Microspheres in Myoblast Fusion As can be seen from Table 1, the different types of microspheres promoted myoblast cell fusion, although the scope of the effect depended on the diameter of the microsphere, the surface group in the microsphere, the number of days after treatment and the concentration. Fusion of myoblasts occurs when muscle tissue is formed during embryogenesis and is also a very important step in muscle regeneration and repair of damaged muscle tissue. In this way, the ability of the microspheres to promote such fusion clearly indicates the potential of these microspheres to promote muscle regeneration, as demonstrated below in Example 5.

EXAMPLE 3 Effect of Microspheres in Collagen Synthesis and Deposit As noted above in the Background section, the synthesis and deposition of collagen is an important step in the wound healing process. In addition, the amount of collagen deposited in the wound is an important determinant of the healing force. Thus, although the microspheres of the present invention clearly have a variety of effects on different types of cells, as demonstrated in the preceding and following Examples, P1705 / 99MX it is evident that an important determinant of the ability of a composition to promote wound healing is its effect on the synthesis and deposition of collagen. As shown in Figures 2A and 2B, the microspheres of the present invention clearly promote the synthesis of collagen by cultured fibroblasts. The largest effect is observed with Type I and Type II microspheres. The Type I microspheres had a diameter of 4.5 microns, were made of carboxylated polystyrene and had a Z potential of approximately -29.96 mV. Type II microspheres had a diameter of 0.49 microns, were made of polystyrene alone and had a Z potential of approximately -34.5 mV. The Type III microspheres had a diameter of 1.0 microns, were made of carboxylated polystyrene and had a Z potential of approximately -53.34 mV. The experimental method was as follows. Cultures of foreskin fibroblasts were grown in 75 cm2 plastic flasks (Corning Glass Works, Corning, NY) in Dulbecco's Modified Eagle Medium (DMEM) with a content of 4.5 mg / ml of glucose supplemented with 10% fetal bovine serum. in volume / volume, 2 mM L-glutamine, 50 μg / ml gentamicin sulfate and 2.5 mg / ml amphotericin B. The cultures were incubated at 37 ° C in 5% C02 until confluency. The fibroblasts were collected P1705 / 99MX using 0.25% trypsin solution / 0.05% EDTA and subcultures were made in 24 cavity plates at a density of 200,000 cells / well with the same medium for 24 hours, at which time the treated cells were incubated with microspheres of Type I, II or III. The control cells were incubated with medium alone. Collagen synthesis was measured as follows. The cultured fibroblasts were previously incubated in DMEM supplemented with 0.5% dialyzed fetal bovine serum for 24 hours. Cells were labeled with 3 μCi 2, 3-3H-proline or 3,4-3H-proline solution containing β-aminopropionitrile fumarate (BAPN) at a final concentration of 100 μM, in the presence (Figure 2A) or absence (Figure 2B) of 10 μM ascorbic acid as indicated. Ascorbic acid promotes the synthesis of collagen in fibroblasts and is an important factor of imulation. After 24 hours of incubation the reaction was terminated and the collagen was extracted from each cavity by the addition of 30 μl of cold acetic acid (0.5 M) containing pepsin (final concentration 0.5 mg / ml), followed by moderate agitation at room temperature for 4 hours. After centrifugation, the cell debris was discarded and 80 μl of collagen solution in 0.5 M acetic acid was added to the supernatant, with a P1705 / 99MX final collagen concentration of approximately 200 mg / ml. Collagen was precipitated from each supernatant by the addition of 0.4 ml of 5.2 M NaCl solution in 0.5 M acetic acid. After waiting 2 hours, the precipitated collagen was removed by centrifugation for 15 minutes at 15,000 rpm. The pellet was then resuspended in 750 μl of 10 mM TRIS buffer, pH 7.4 containing 1M NaCl. The collagen was precipitated by the addition of 750 μl of TRIS buffer, pH 7.4 containing 5M NaCl. After 2 hours the collagen was separated by centrifugation. It was redissolved in 0.5 M acetic acid and each sample was measured in a scintillation counter. The results are shown in Figure 2A and 2B, given as cpm per cavity. The data presented is an average of samples in quadruplicate. Both Type I and Type II microspheres had the ability to stimulate collagen synthesis above the level observed in control fibroblasts (untreated), both in the presence (Figure 2A) as in absence (Figure 2B) of ascorbic acid. Type I microspheres had a greater effect in relation to Type II microspheres in the presence of ascorbic acid, although the two types had a similar effect in the absence of ascorbic acid. Type III microspheres did not have a detectable effect on collagen synthesis either P1705 / 99MX in the presence or absence of ascorbic acid. A particularly interesting finding is that both Type I and Type II microspheres had an effect, while Type III microspheres did not, indicating that the specific size and material of the microspheres is important. In addition, both Type I and Type II microspheres caused an effect even in the absence of ascorbic acid, indicating that these two types of microspheres can intensify collagen synthesis even in the absence of other stimulating factors. Thus, clearly, both Type I and Type II microspheres have a substantial stimulatory effect on the synthesis of collagen.

EXAMPLE 4 Effect of Microspheres in the Form of Myoblasts Primary cell cultures of rat myoblasts were prepared in the same manner as described in Example 1 above. The cells were then incubated with polystyrene microspheres (treated cells) or without them (control cells) for 48 hours. The cells were then fixed in 1% glutaraldehyde in phosphate buffered saline for 1-4 days and rinsed with PBS (buffered saline solution of phosphate). The cells were then transferred to an acid solution P1705 / 99MX tannic 1% and guanidine hydrochloride 1% (ratio 1: 1) in PBS buffered saline phosphate) for 1 hour. The specimens were subsequently fixed at 0% 1% for 1 hour and dehydrated in graduated alcohol and 113 Freon at room temperature. The specimens were prepared on a slide, coated with gold and examined under a JEOL T-300 2 kV electronic scanning microscope. Figures 3A-3C illustrate the effect of the microspheres of the present invention in the form of myoblasts. The cell in Figure 3A has grown through the microscope, so that part of the cell surface is convex rather than flat. Figures 3B and 3C show pseudopodia extending from a portion of the cell in which the microsphere rests. The pseudopod of the cell in Figure 3C is particularly pronounced, showing that the microspheres clearly influence the shape of the myoblast. In addition, the formation and extension of a pseudopod obviously requires changes in the cytoskeletal structure, which shows that the microspheres also affect the cytoskeleton of the cell. The formation of said pseudopodia may be important for the migration of the cells to the wound area. In this way, the stimulation of said pseudopodia through the microspheres indicates its ability to promote another important step in the healing process of P1705 / 99MX wounds. EXAMPLE 5 Device and Method of Application The following description is a general device and method for the application of wound healing agents. The agents, for example microspheres, are preferably applied repeatedly to the wound to be treated. The frequency of application and the concentration applied depends on the severity of the symptoms and the response of the subject to the treatment. Those of ordinary skill in the art can easily determine optimal concentrations, dosing methodologies and repetition frequency. In the present study, the microspheres were applied to the wound to be treated approximately once or twice a day, although of course other application frequencies are possible. The method includes the step of administering agents such as microspheres, in a pharmaceutically acceptable vehicle in which the agents are practically insoluble, to a subject to be treated. Examples of pharmaceutically acceptable carriers include aqueous media for a suspension of the agents, non-aqueous media such as ointments, creams and aerosol generating material, as well as bandages soaked in the medium with the agents or otherwise containing them.

P1705 / 99MX As will be described below and in Example 9 in more detail, a particularly preferred pharmaceutically acceptable carrier is a gel-forming material such as methyl cellulose, so that the microspheres are in the form of a gel. Although methylcellulose is a particularly preferred example of such gel-forming materials, it is understood that other gel-forming materials could be used, especially if they were physiologically inert. The bandages can be occlusive or non-occlusive. In any case, agents that are in a pharmaceutically acceptable carrier can be described as a dispersion of agents. The agents are administered according to an effective dosing methodology, preferably until a predefined endpoint is reached, for example the absence of clinical symptoms in the subject. The closure of the wound being treated is an example of that final point. The device of the present invention includes a composition with one or more agents and a pharmaceutically acceptable carrier for the agents and a container for containing the composition. Preferably, the package is a sterile, substantially sealed package, such as a pump that disperses aerosol or a spray can. Alternatively and preferably, the package is a bandage substantially P1705 / 99MX sterile. Also alternatively and preferably, the package is a compressible tube or a pump that disperses gel. One of ordinary skill in the art will readily be able to select suitable containers for the composition depending on the properties of the vehicle that is preferred. Although the microspheres of the present invention can still be used without a vehicle, simply by placing them in the wound, extremely low concentrations of microspheres that are effective for wound healing are much easier to apply when presented in a pharmaceutical carrier. Preferably, the microspheres are present at a concentration in the range of between about 0.0001 percent and 1.5 percent, weight / weight. More preferably, the microspheres are present at a concentration in the range of between about 0.001 percent and 1.0 percent, weight / weight. With superlative preference, the microspheres are present at a concentration in the range of approximately between 0.01 percent and 0.2 percent, weight / weight. Such very low concentrations are the most efficient to store, transport and especially apply, when they are in a pharmaceutically acceptable vehicle and contained in a suitable container. An example of a preferred combination of P1705 / 99MX vehicle and container is an aqueous suspension or other liquid suspension contained within a pump that disperses aerosol. Another example of a particularly preferred combination of a vehicle and a container is a gel-forming material, such as methylcellulose or any other suitable gel forming substance, contained in a compressible tube. The gel form is particularly preferred because it increases the retention of the microspheres within the wound area, thereby allowing a small amount of the gel formulation to exert the same healing effect on the wound. Such an effect is demonstrated below in Example 9. In this way, the combinations of a vehicle and a container that are particularly preferred are clearly efficient and convenient for transport, storage and application of the compositions of the present invention, especially at these concentrations. of very low microspheres, which are preferred. Without taking into account the particular device used, the agents, such as microspheres, are preferably applied in a two-step procedure. The microspheres are first applied to the wound in a dispersion, by dripping, spraying, touching, washing or by any other suitable means of application. Preferably, they are allowed to pass between 30 seconds and 2 minutes before the second step, to P1705 / 99MX allow the microspheres to form an initial contact with the wound. Preferably, the second step includes applying to the wound an occlusive or non-occlusive dressing or other suitable cover soaked in the liquid suspension containing the microspheres. This substantially reduces or eliminates the absorption of the microspheres by the bandage or cover. This method was used in both rats and humans for wound healing, as described below in the Examples. The microspheres in the suspension do not aggregate, coalesce, aggregate or undergo irreversible agglomeration. Although the microspheres sedimented a little over time, they were easily resuspended with moderate agitation.

EXAMPLE 6 Promotion of Wound Healing in Rats with Microspheres As noted above in Examples 1-4, the microspheres of the present invention promote various cellular processes in vi tro that are important for wound healing. However, the effects in vivo and in vi tro do not always correlate. Therefore, in vi ve experiments were performed to evaluate the ability of microspheres to promote wound healing in rats. As it is shown in P1705 / 99MX Figures 4A-4D, the microspheres of the present invention clearly promote wound healing in rats. Figure 5 is a graph of the rate at which the area of the wound decreases, showing that the microspheres of the present invention increase the rate at which that decrease occurs. Finally, Table 2 shows that the microspheres promote muscle regeneration in rats. The experimental method was as follows. Male Wistar rats, weighing between 300 and 400 g, were anesthetized with nembutal (5 mg / kg body weight). An incision wound was made in the lateral parts of the Tibialis anterior muscle in the following manner. First, a longitudinal incision was made in the skin to expose the anterior Tibialis muscle. Then, the partial excision of this muscle was made by a cross section of the muscle fibers, along approximately half the width of the muscle. Then the piece that underwent the excision was cut from the muscle, leaving a gap of approximately 5 mm by 5 mm in the muscle. In all the rats the same amount of tissue that underwent excision (80 + 10 mg) was removed from exactly the same location in the muscle. The wound area was then prepared with 2 micron polystyrene microspheres in saline solution for treated rats and saline alone for control rats. The area of the wound was measured P1705 / 99MX between 3 and 15 days after the injury. Figures 4A-4D show pictures of wound areas prepared as described above. Figure 4A shows the wound of the control rat immediately after the injury, while Figure 4B shows the equivalent wound of the rat to be treated. Figures 4C and 4D show the same rats five days after the injury. The wound of the control rat was treated with saline alone and had not yet fully healed. In contrast, the wound of the treated rat, which was treated with microspheres, had completely healed. In this way, clearly the microspheres of the present invention promote more rapid healing of wounds. Figure 5 further illustrates the promotion of wound healing by the microspheres of the present invention. The wounds of the control rats eventually heal, but at a much slower rate than the wounds of the rats that were treated. In this way, the microspheres clearly increase the speed at which the area of the wound decreases and at which the wound heals. Slides were prepared for histological analysis by performing a biopsy puncture of the wound area. Rats were sacrificed 4, 5, 6, 7, 8, 9, 13 or 14 days after the lesion and biopsies were taken for histological examination. The number of P1705 / 99MX specialized myogenic cells incorporated into newly formed or repaired muscle fibers were counted by determining the number of "new" nuclei, which represent activated myogenic cells. The nuclei of these cells are large, basophilic nuclei with scattered chromatin and can be easily distinguished from existing myoblast nuclei. The results are given in Table 2.

P1705 / 99MX As shown in Table 2, the microspheres of the present invention clearly promote muscle regeneration, as determined by the number of "new" nuclei or incorporated in the muscle fibers. The fact that such measurements were made on histological samples taken from rats treated in vivo also indicates that the microspheres promote muscle regeneration in vivo as well as in vi tro. Finally, Figure 6 compares the effect of the microspheres of the present invention on wound healing with tissue culture medium and saline solution in rats. The wounds were induced in rats in the same manner as described above and the rats were treated with saline alone (Figure 6A, X), tissue culture medium alone (Figure 6B, K), saline plus microspheres (Figure 6A, 3) or tissue culture medium plus microspheres (Figure 6B, 3). The rats were photographed 4 days after the wound was made. As can be seen from Figures 6A and 6B, the microspheres had the ability to induce a much faster wound healing rate regardless of whether the vehicle was saline or tissue culture medium. In this way, the tissue culture medium has nothing to do with the effect of the microspheres of the present invention on the P1705 / 99MX wound healing.

EXAMPLE 7 Toxicity Studies of Microspheres No toxic effect was observed in preparation containing microspheres. The preliminary examination of rats that were treated at 65 and 180 days after the injury showed that none of the following organs showed signs of pathological changes: heart, liver, lungs, kidney, blood vessels, stomach, lymph nodes and brain. Experiments with fluorescently labeled microspheres showed that no signs of pathology were observed in treated rats. In addition, the microspheres did not penetrate any of the organs referred to above. No new growth was detected in the organs referred to above. Finally, the microspheres dispersed within the wound area but did not penetrate the regenerative muscle fibers. EXAMPLE 8 Effect of Microspheres on Wound Healing in Humans The in vi experiments described in Example 6 above clearly demonstrate that the microspheres of the present invention can promote wound healing and muscle regeneration in rats. In addition, the results of the P1705 / 99MX rat toxicity studies described in Example 7 show that the microspheres are practically non-toxic. Therefore, studies were conducted to determine the effect of the microspheres of the present invention on wound healing in humans. As described in detail below, the case studies demonstrate that microspheres clearly promote wound healing in humans. Preferably, the concentration of microspheres in a range of about 0.0001 percent and 1.5 percent, weight / weight, was used in an aqueous saline solution. More preferably, the concentration was in the range of between about 0.001 percent and 1.0 percent weight / weight. With superlative preference, the concentration was in the range of approximately between 0.01 percent and 0.2 percent weight / weight. The first case study was that of a 66-year-old woman with ulcers on her left leg who refused to heal. The patient also had cellulitis on the left leg and varicose veins on both legs. The ulcers on the inner thigh of the patient were treated with Milton 2% which is a corrosive chlorine salt in water. Ulcers of the patient's outer thigh were treated with microspheres of the present invention of 4.5 microns made from P1705 / 99MX polystyrene in tissue culture medium. Figure 7A shows the control wound on day 0, while Figure 7B shows the control wound after 4 months of treatment. Figure 7C shows the wound treated on day 0, while Figure 7D shows the wound treated after 4 months of treatment. Both the wounds treated with the microspheres of the present invention and those treated with Milton showed signs of infection and other healing difficulties during the following four months. However, at the end of the treatment period, the wounds that were treated with the microspheres had shown significant improvement. The size of the wound had diminished and the wounds were clean without signs of infection. In this way, even for wounds that were difficult to heal, due to complications such as infection, the microspheres of the present invention exhibited greater efficacy in promoting wound healing than currently available treatments. As a subsequent test, the wound that had served as control for Figure 7 above (Figures 7A and 7B) was treated with the same microspheres that were used to treat the wounds in Figures 7C and 7D. The results are shown in Figures 8A and 8B. Figure 8A shows P1705 / 99MX wound on day 0 of treatment with the microspheres, while Figure 8B shows the wound after 21 days of treatment. Clearly, the extent of the wound had diminished, even after such a short treatment period. In addition, the wound was superficial and was clean and no longer exuded. The second case study was of a 52-year-old woman who had a one year old infected wound on the front side of her left thigh. The wound was treated with Milton 1% for one week, debrided and then treated with the microspheres of Figures 7 and 8 for 10 days. Figure 9A shows the wound on day 0 of treatment, while Figure 9B shows the wound after 10 days of treatment. After 10 days, the wound showed significant improvement. It had diminished in extension to a small size it was clean and no longer produced exudations, as can be seen from Figure 9B. Although the wound did not close completely during the relatively short treatment period, its effects had improved significantly. The third case study was of a 19-year-old man who had been injured by a chemical spill in an industrial accident in the workplace. The chemicals in question, sulfides, caused serious burns and P1705 / 99MX blistering on the right side of the neck and in the right hand. During the first two days all wounds were treated with Silverol, a hydrogel with strong absorbency properties. Then, the wounds on the right forearm were treated with the microspheres of case studies 1 and 2, while the remaining wounds were treated with Silverol. The results are shown in Figures 10A and 10B (control wound on day 0 and day 5, respectively) and in Figures 10C and 10D (wounds treated on day 0 and day 5, respectively). After 5 days of treatment with microspheres, the condition of the wound in the forearm that was treated had improved significantly with respect to the remaining wounds, which were not treated with microspheres. The wound on the forearm had completely healed after 5 days of microsphere treatment. In contrast, the remaining wounds that were treated with Silverol had not healed completely. In this way, the microspheres clearly promoted wound healing, demonstrating greater efficacy than currently available treatments. The fourth case study was of a 52-year-old woman who had suffered second-degree burns to her buttocks from a hot bath. Wounds on the left gluteus were treated with Silverol, while those on the right gluteus P1705 / 99MX treated with the microspheres from the previous case studies. The results are shown, only for the wounds treated with microspheres, in Figures HA (treatment day 0) 11B (treatment day 7). Seven days after beginning treatment, the wounds in the right buttock that were treated with microspheres, had completely healed with good epithelial growth. In contrast, the wounds in the left gluteus, which were treated with Silverol, had not healed completely and closed relatively slowly. In this way, the microspheres had the ability to promote wound healing at a faster rate than conventional treatments. The fifth case study was of a 28-year-old woman who had suffered extensive and severe sunburn (data not shown). She was treated with microspheres from the previous case studies. The patient reported both a significant reduction in discomfort and rapid healing of the burn. In this way, the microspheres used in the method and device of the present invention can both alleviate discomfort and promote wound healing, although it should be noted that relief of discomfort is probably an indirect effect of the microspheres rather than direct analgesia. .

P1705 / 99MX Indeed, it is worth mentioning that the report of the above patient can only be inferred to include the apparent reduction in the feeling of discomfort from sunburn. This decreased discomfort probably does not demonstrate any ability of the microspheres that has a direct effect on the transmission of nerve impulses or actually directly modifies any of the many factors that lead to discomfort. Instead, this effect is probably quite indirect, presenting as a result of the activation of macrophages, which in turn have anti-inflammatory effects, which leads to the patient having a decrease in the feeling of discomfort. The sixth case study was of a 64-year-old man with diabetes mellitus and a post-surgical wound in his left leg that had not healed with conventional treatment eight months after surgery. The microspheres from the previous case studies were applied twice daily for 17 days. Before the treatment with the microspheres, the post-surgical wound was 7.5 cm by 8 cm and 6 cm deep, which is the depth at which the bone is located in that part of the leg and was marked by soft tissue gangrene and the bone. After three days of treatment with the microspheres, the gangrene P1705 / 99MX improved and was completely eliminated after eight days (data not shown). Granulation appeared in muscle and bone after three days of treatment. The bone regeneration was complete after ten days of treatment. On day 14, the wound was not closed but was covered with healthy color granulation. The depth of the wound had been reduced to almost 0.5 cm and the patient declared himself available for a skin graft surgery. The pain was also relieved almost immediately after the treatment began, but as noted in the fifth case study, this decreased discomfort was probably a rather indirect effect, rather than caused by a direct action of the microspheres. In this way, the microspheres clearly had the ability to successfully promote wound healing after conventional treatment had failed, even in the presence of complicating factors such as poor circulation associated with diabetes mellitus. From the six case histories associated with the extensive evidence obtained from the studies in rats, the use of agents, such as microspheres, according to the present invention has clearly demonstrated that it has greater efficacy for the promotion of wound healing and muscle regeneration that the treatments of the P1705 / 99MX prior art, currently available. The method and device promote, accelerate and intensify the healing of wounds as well as decrease the discomfort experienced by the subject. With respect to the decrease in discomfort, it should be noted that patients in the previous case studies also reported local reduction of pain and discomfort from the treated wound, particularly the patient suffering from sunburn, probably an indirect effect of the microspheres to through its (also indirect) anti-inflammatory action. Finally, although the data are not shown, an indirect bacteriostatic effect against wound infections by Pseudomonas species was also observed in humans. The mechanism of both the indirect anti-inflammatory action and the indirect bacteriostatic effect is not clear, but is probably the result of a cellular effect that involves the attraction and activation of macrophages. Regardless of the exact mechanism, the use of microspheres according to the present invention clearly represents a significant improvement in the treatment of wounds. In addition to these applications that were tested, it is considered that the microspheres of the present invention could be quite suitable for many different cosmetic applications. By P1705 / 99MX example, in addition to sunburn, the microspheres of the present invention are considered useful for the improvement or even the practically total cure of acne. The microspheres of the present invention could also be incorporated in shaving creams or gels, for the treatment of minor skin lesions such as scratches and small cuts associated with shaving. The microspheres of the present invention are also considered useful for the improvement or even virtually complete cure of any type of skin damage. The microspheres of the present invention are also considered useful for the improvement or even the almost total cure of hemorrhoids and intestinal ulcers, such as gastric ulcers, ulcers associated with ulcerative colitis and ulcers associated with Helicobacter pylori.

EXAMPLE 9 Gel-shaped Microspheres The liquid suspension of microspheres in aqueous solution, such as that tested in Example 8 above, was converted to the gel form by the addition of methylcellulose (Hoechst A.G., Germany) and another inert substance. The methylcellulose was used at a concentration in a range of between about 0.1 percent and 5 percent weight / weight. The gel-shaped microspheres were found quite P1705 / 99MX effective for wound healing, even smaller amounts of the gel formulation could still exert the same effect because the gel formulation helped retain the microspheres in the wound area. The experimental method was as follows. In this study, albino guinea pigs derived from Hartley weighing between 300 and 400 grams were used. All surgical procedures were performed under general anesthesia after the administration of ketamine hydrochloride (150 mg / kg, Parke-Davis, USA). After they were anesthetized, four skin segments of full thickness, bilaterally symmetrical, measuring 2 cm by 2 cm, two from the scapular region and two from the lumbar region were removed from the back of each animal by excision. After the surgery, the microspheres were applied in the form of a gel. Then bandages were applied to the wounds. The bandages were changed every 48 hours with general anesthesia, at which time all the wounds were washed with hot saline to eliminate any waste and any gel that remained inside the wound. All wounds were measured and photographed. Then the gel-shaped microspheres were applied again to the wounds and new bandages were applied. The gel formulation P1705 / 99MX was also put on the bandages. The four wounds in each animal were treated with the same concentration of microspheres in gel form. The concentration of microspheres was 0.0027 weight / weight in 2.5% methylcellulose. The microspheres themselves were made of polystyrene and were 4.3 microns in diameter. As a control, the wounds of some animals were treated with the same concentration of the same microspheres in a liquid suspension, which was also placed in the bandages. In both cases, microspheres were applied in the liquid suspension or in the gel formulation in a volume sufficient to practically fill the area of the wound, so that a smaller amount of the gel formulation was applied than the liquid suspension. The photographs were analyzed with the software program ImageMeasureMR (Phoenix Corp., Seattle, Washington, USA). The results were plotted as the variation of time in the fractional area of the wound or the closing rate, against the controls, and are shown in Figure 12. The time variation in the fractional area of the wound was calculated as A0- Ai / A0, where Ao was the area of the wound on day 0 and Ai was the area of the wound on day i. Clearly, both the wounds that were treated with the liquid suspension of microspheres and with the gel microsphere formulation healed at a substantial rate P1705 / 99MX similar, even when a decreased volume of the gel formulation was applied compared to the liquid suspension. It will be appreciated that the foregoing descriptions are intended only to serve as examples and that many other embodiments are possible within the spirit and scope of the present invention.

P1705 / 99MX

Claims (55)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A composition for treating a wound of a subject, the composition basically consists of a agent that has the ability to form a multi-point contact with a cell membrane, the agent is practically non-biodegradable during the period of treatment.
2. 2. The composition according to the claim 1, wherein the agent is a microsphere that presents surface groups with a substantial charge.
3. 3. The composition according to the claim 2, wherein the microsphere is made of a material selected from the group consisting of polystyrene, polystyrene derivative, polylysine, poly-bromide of N-ethyl-4-vinylpyridinium, polymethylacrylate and silicone.
4. 4. The composition according to the claim 3, wherein the microsphere is made of a material selected from the group consisting of polystyrene and polystyrene derivative.
5. 5. The composition according to the claim 2, wherein the surface group is selected from the group consisting of sulfate, poly-N-ethyl-4-vinylpyridinium bromide, protamine, sulphate P1705 / 99MX protamine, salts of protamine, polylysine, polystyrene, polystyrene derivative and carboxyl. The composition according to claim 5, wherein the surface group is selected from the group consisting of polylysine, polystyrene, polystyrene derivative and carboxyl. The composition according to claim 2, wherein the surface charge is practically negative. 8. The composition according to the claim 2, wherein the microsphere has a diameter in a range of approximately between 0.01 microns and 200 microns. 9. The composition according to claim 2, wherein the microsphere has a diameter in a range of between about 0.1 microns and 100 microns. The composition according to claim 9, wherein the microsphere has a diameter in a range of between about 0.1 microns and 20 microns. 11. The composition according to claim 2, further comprising a pharmaceutically acceptable carrier for the microsphere, the microsphere is practically insoluble in the vehicle. 12. The composition according to claim 11, wherein the pharmaceutically acceptable carrier is an aqueous solution. P1705 / 99MX 13. The composition according to claim 11, wherein the pharmaceutically acceptable carrier is a gel-forming material. The composition according to claim 13, wherein the gel-forming material includes methylcellulose. 15. The composition according to claim 1, wherein the wound is selected from the group consisting of burn, trauma, post-surgical, post-partum and chronic wound. 16. The composition according to claim 15, wherein the burn wound is selected from the group consisting of sunburn, chemical burn, radiation burn, and thermal burn. 17. The composition according to claim 15, wherein the chronic wound is selected from the group consisting of bed sores, pressure sores, injuries related to diabetes and related to poor circulation. The composition according to claim 1, wherein the wound that is located in a portion of the body of the subject is selected from the group consisting of skin, bone and muscle. 19. The composition according to the claim 18, where the composition also promotes muscle regeneration. 20. The composition according to the claim P1705 / 99MX 1, wherein the composition also promotes wound healing and substantially reduces scars and also relieves discomfort of the subject. The composition according to claim 1, wherein the microsphere is present at a concentration in a range of approximately between 0.0001 percent and 1.5 percent, weight / weight. 22. The composition according to the claim 21, wherein the microsphere is present at a concentration in a range of approximately between 0.001 percent and 1.0 percent, weight / weight. 23. The composition according to the claim 22, wherein the microsphere is present at a concentration - in a range of approximately between 0.01 percent and 0.2 percent, weight / weight. 24. A device for treating a wound, comprising: (a) a composition that includes: (i) an agent that has the ability to form a multi-point contact with a cell membrane to treat the wound, the agent is practically not biodegradable during the treatment period; and (ii) a pharmaceutically acceptable carrier for the agent, the agent is practically insoluble in the vehicle; and (b) a container for containing the composition. 25. The device according to the claim P1705 / 99HX 24, wherein the agent is a microsphere that presents surface groups with a substantial charge. The device according to claim 25, wherein the microsphere is made of a material selected from the group consisting of polystyrene, polystyrene derivative, polylysine, poly-N-ethyl-4-vinylpyridinium bromide, polymethylacrylate and silicone. The device according to claim 26, wherein the microsphere is made of a material selected from the group consisting of polystyrene and polystyrene derivative. 28. The device according to the claim 25, wherein the surface group is selected from the group consisting of sulfate, N-ethyl-4-vinylpyridinium poly-bromide, protamine, protamine sulfate, protamine salts, polylysine, polystyrene, polystyrene derivative and carboxyl. 29. The device according to claim 28, wherein the surface group is selected from the group consisting of polylysine and carboxyl. 30. The device according to claim 25, wherein the surface charge is practically negative. 31. The device according to the claim 25, wherein the microsphere has a diameter in a range of approximately between 0.01 microns and 200 microns. P1705 / 99MX 32. The device according to the claim 31, wherein the microsphere has a diameter in a range of approximately between 0.1 microns and 100 microns. 33. The device according to the claim 32, wherein the microsphere has a diameter in a range of approximately between 0.1 microns and 20 microns. 34. The device according to claim 25, wherein the wound is selected from the group consisting of burn, trauma, post-surgical, post-partum and chronic wound. 35. The device according to claim 34, wherein the burn wound is selected from the group consisting of sunburn, chemical burn, radiation burn, and thermal burn. 36. The device according to claim 34, wherein the chronic wound is selected from the group consisting of injuries related to diabetes and related to poor circulation. 37. The device according to claim 25, wherein the wound that is located in a portion of the subject's body is selected from the group consisting of skin and muscle. 38. The device according to claim 37, wherein the composition further promotes muscle regeneration. P1705 / 99MX 39. The device according to claim 25, wherein the agent further has the ability to promote wound healing, substantially reduce scars and alleviate discomfort. 40. The device according to the claim 25, wherein the pharmaceutical carrier is selected from the group consisting of aqueous solution, gel-forming material, aerosol vehicle and ointment. 41. The device according to claim 40, wherein the pharmaceutical carrier is a gel-forming material. 42. The device according to claim 41, wherein the gel-forming material includes methylcellulose. 43. The device according to the claim 41, wherein the container is a compressible tube. 44. The device according to claim 40, wherein the pharmaceutical carrier is an aqueous solution. 45. The device according to the claim 44, wherein the package is a sterile package, practically sealed. 46. The device according to the claim 45, wherein the sterile, virtually sealed package is an aerosol spray. 47. The device according to claim 44, wherein the package is a practically sterile bandage. P1705 / 99MX 48. A composition for treating a wound of a subject, the composition consisting basically of a microsphere that has the ability to form a multi-point contact with a cell membrane, the microsphere is practically non-biodegradable during the treatment period , the microsphere is made of a material selected from the group consisting of polystyrene, polystyrene derivatized with a fraction selected from the group consisting of amino and carboxyl, polylysine, poly-bromide of N-ethyl-4-vinylpyridinium, polymethylacrylate and silicone and the The material of the microsphere presents a surface group with a substantial charge. 49. The composition according to claim 48, wherein the surface group is selected from the group consisting of sulfate, protamine, protamine sulfate, protamine and carboxyl salts. 50. The composition according to claim 49, wherein the surface group is carboxyl. 51. The composition according to the claim 48, wherein the microsphere has a diameter in a range of approximately between 0.01 microns and 200 microns. 52. The composition according to claim 51, wherein the microsphere has a diameter in a range of between about 0.1 microns and 100 microns. 53. The composition according to the claim P1705 / 99MX 48, wherein the composition further comprises a pharmaceutically acceptable carrier for the microsphere, the microsphere is practically insoluble in the vehicle. 54. The composition according to the claim 49, wherein the wound is selected from the group consisting of burn, trauma, post-surgical, postpartum and chronic. 55. The composition according to claim 48, wherein the wound that is located in a portion of the subject's body is selected from the group consisting of skin and muscle. P1705 / 99MX