MXPA00008240A - Matrix protein compositions for wound healing - Google Patents

Abstract

Active enamel substances may be used for the preparation of a pharmaceutical or cosmetic composition for healing of a wound, improving healing of a wound, soft tissue regeneration or repair, or for preventing or treating infection of inflammation.

Description

COMPOSITIONS OF PROTEIN MATRIX FOR WOUND HEALING FIELD OF THE INVENTION The present invention relates to the uses of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix as therapeutic or prophylactic agents. The substances are active as wound healing, antibacterial and / or anti-inflammatory agents.

BACKGROUND OF THE INVENTION Matrices of enamel proteins such as those present in the enamel matrix are very well known as enamel precursors. Enamel proteins and enamel matrix derivatives have been previously described in patent literature to induce hard tissue formation (ie enamel formation, US Patent No. 4,672,032 (Slavkin)) or binding between hard tissues (EP -B-0 337 967 and EP-B-0 263 086). In this way, the prior art focuses exclusively on the regeneration of hard tissues, while the present application deals with the beneficial effects on the healing of wounds of REF .: 122565 soft tissue and anti-bacterial and anti-inflammatory effects that are unexpected discoveries.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based on the discovery that the enamel matrix, enamel matrix derivatives and / or enamel protein matrix (the term "an active enamel substance" is also used in the following for an enamel matrix) , an enamel matrix derivative or an enamel protein matrix) are beneficial agents for the improvement or good performance in the healing of wounds in soft tissues (ie non-mineralized tissues), such as collagen or tissues containing epithelium, including skin and mucosa, muscles, blood and lymph vessels, nerve tissues, glands, tendons, eyes and cartilage. As demonstrated in the experimental section here, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix specifically exert useful effects in the healing or prophylaxis of soft tissue wounds.

Therefore, the present invention relates to the use of a preparation of an active enamel substance for the preparation of a pharmaceutical or cosmetic composition i) for the healing of a wound, ii) to improve the healing of a wound and / or iii) for the regeneration and / or repair of soft tissue.

In another aspect, the invention relates to a method for improving the healing of a wound or to promote regeneration and / or repair of soft tissue, the method comprising administering, to an individual in need thereof, a therapeutically or prophylactically amount effective of an active enamel substance.

In addition, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix have been found to have anti-bacterial and / or anti-bacterial properties. anti-inflammatory drugs that can be used for the treatment of soft and hard tissue conditions (mineralized i.e.).

In other aspects, the invention relates to the use of A preparation of an active enamel substance for the preparation of a pharmaceutical composition for the prevention and / or treatment of an infection or an inflammatory condition. ^? ^^^^ ífe Wound healing Wounds and / or ulcers are usually found protruding from the skin or on a surface of the mucosa or as a result of an infarct on an organ ("attack"). A wound could be a result of a defect or injury or an underlying condition of the soft tissue. The regeneration of the experimentally induced experimental wounds has been previously described by the inventors and is not intended to be within the scope of the present invention. In the present context the term "skin" refers to the outermost surface of the body of an animal including a human and encompasses intact or almost intact skin, as well as a surface of injured skin. The term "mucosa" refers to an undamaged or damaged mucosa of an animal, such as a human and could be oral, buccal, aural, nasal, pulmonary, ocular, gastrointestinal, vaginal or rectal mucosa.

In the present context, the term "wound" represents a bodily injury with the rupture of the normal integrity of tissue structures. The term is also intended to encompass the terms "sore," "injury," "necrosis," and "ulcer." Normally, the term "sore" is a popular term for almost any lesion of the skin or mucosal membranes and the term "ulcer" is a local defect, or excavation, of the surface of an organ or tissue, which is produced by the scab formation of the necrotic tissue. In general, injury refers to any tissue defect. Necrosis refers to tissue death that results from infection, injury, inflammation, or heart attacks.

The term "wound" used in the present context represents any wound (see below for a classification of wounds) and at any particular stage in the healing process, which includes the stage before any healing has begun or even before a healing. Wound specifies how it is done in a surgical incision (prophylactic treatment).

Examples of wounds that can be prevented and / or treated in accordance with the present invention are, e.g. aseptic wounds, blunt wounds, incised wounds, lacerated wounds, non-penetrating wounds (ie wounds in which there is no break in the skin, but there is damage to the underlying structures), open wounds, penetrating wounds, perforation wounds, wounds puncture, septic wounds, subcutaneous wounds, etc. Examples of sores are bedsores, ulcers, chromium ulcer, cold sores, pressure ulcers, etc. Examples of ulcers are, eg, peptic ulcer, duodenal ulcer, gastric ulcer, gout ulcer, diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris (venous ulcer), sublingual ulcer, submucosal ulcer, symptomatic ulcer, trophic ulcer , tropical ulcer, venereal ulcer, eg caused by gonorrhea (including urethritis, endocervicitis and proctitis). The conditions related to the wounds or ulcers that could be successfully treated according to the invention are burns, anthrax, tetanus, gas gangrene, scarlet fever, erysipelas, beard psychosis, folliculitis, impetigo contagiosa or bullous impetigo, etc. There is often a certain overlap between the use of the terms "wound" and "ulcer" and "wound" and "sore" and, in addition, the terms are often used randomly. Therefore, as mentioned above, in the present context, the term "wound" encompasses the terms "ulcer", "injury", "sore" and "infarction", and the terms are used indiscriminately unless otherwise indicated contrary.

The types of wounds to be treated according to the invention also include i) general wounds, such as e.g., surgical, traumatic, infectious, ischemic, thermal, chemical and bullous wounds; ii) specific wounds for the oral cavity such as eg post-extirpation wounds, endodontic wounds especially in connection with the treatment of cysts and abscesses, ulcers and lesions of bacterial, viral or autonymological origin, mechanical, chemical, thermal wounds , infectious and lichenoid, herpes ulcers, aphthous stomatitis, acute necrotizing ulcerative gingivitis and burned mouth syndrome are specific examples; and iii) skin wounds such as, e.g., neoplasm, burns (e.g. chemical, thermal), lesions (bacterial, viral, autoimmune), bites and surgical incisions. Another way to classify wounds is as i) small tissue loss due to surgical incisions, minor abrasions and minor bites, or as ii) significant loss of tissue. The last group includes ischemic ulcers, pressure ulcers, fistula, lacerations, severe bites, thermal burns and wounds from donor sites (in soft and hard tissues) and heart attacks.

The healing effect of an active enamel substance has been found to be of interest in connection with the wounds that are present in the oral cavity. Such wounds could be bodily injuries or traumas associated with oral surgery including periodontal surgery, tooth extractions, endodontic treatment, insertion of tooth implants, application and use of dental prostheses, and the like. In the experimental section, the beneficial effect of an active enamel substance on such wounds has been demonstrated. In addition, a healing effect of the soft tissue has been observed.

In the healing of the oral cavity of wounds such as aphthous wounds, traumatic wounds or wounds associated with herpes, it is also improved after the application of an active enamel substance. Traumatic injuries and wounds associated with herpes, of course, can also be located in other parts of the body than in the oral cavity.

In other aspects of the invention, the wound to be prevented and / or treated is selected from the group consisting of aseptic wounds, infarcts, blunt wounds, incised wounds, lacerated wounds, non-penetrating wounds, open wounds, penetrating wounds, wounds of perforation, puncture wounds, septic wounds and subcutaneous wounds.

Other wounds which are of importance in connection with the present invention are wounds such as ischemic ulcers, pressure ulcers, fistula, severe bites, thermal burns and donor site wounds.

Ischemic ulcers and pressure ulcers are wounds that normally only heal very slowly and especially in such cases, it is of course of great importance for the patient an improved and faster healing. In addition, the costs involved in the treatment of patients suffering from such wounds are markedly reduced when the wound is improved and carried out more rapidly.

The wounds of donor sites are wounds that, e.g. they are presented in connection with the removal of hard tissue from one part of the body to the other part of the body e.g. in connection with the transplant. The wounds that result from such operations are very painful and therefore, improved healing is more valuable.

The term "skin" is used in a very broad sense that covers the epidermal layer of the skin and - in cases where the surface of the skin is more or less damaged - also the dermal layer of the skin. In addition to the stratum corneum, the epidermal layer of the skin is the outer layer (epithelial) and the deepest connective tissue of the skin is called the dermis.

Since the skin is the most exposed part of the body, it is particularly susceptible to various types of injuries such as, e.g., ruptures, cuts, abrasions, burns and frostbites or injuries that arise from various diseases. In addition, a lot of skin is often destroyed in accidents. However, due to the important barrier and the physiological function of the skin, the integrity of the skin is important for the welfare of the individual, and any cut or rupture represents a threat that must be established by the body to protect its continued existence.

In addition to skin lesions, lesions may also be present in all types of tissues (ie, soft and hard tissues). Soft tissue injuries including mucosal membranes and / or skin are especially relevant in connection with the present invention.

The healing of a wound on the skin or on a mucosal membrane undergoes a series of stages that result either in the repair or regeneration of the skin or mucous membrane. In recent years, regeneration and repair have been distinguished as two types of healing that could be presented. Regeneration could be defined as a biological process by which the architecture and function of the lost tissue are completely renewed. Repair, on the other hand, is a biological process by which the continuity of the broken tissue is restored by new tissues that do not duplicate the structure and function of the lost tissue.

The majority of wounds healed by repair means that the new tissue formed is structurally and chemically different from the original tissue (scar tissue). In the previous stage of tissue repair, a process that is almost always involved is the formation of a transient connective tissue in the area of tissue damage. This process begins with the formation of a new extracellular collagen matrix by the fibroblasts. This new extracellular collagen matrix is then the support for a connective tissue during the final healing process. The final cure is, in most tissues, a scar formation that contains the connective tissue. In tissues that have regenerative properties, such as, e.g., skin and bones, final healing includes tissue regeneration. -.i *. original. This regenerated tissue also frequently has some scar characteristics, e.g. a complication of a cured bone fracture.

Under normal circumstances, the body provides mechanisms for the healing of damaged skin or mucosa to restore the integrity of the skin or mucosal barrier. The repair process even for ruptures or minor injuries could take a period of time ranging from hours and days to weeks. However, in ulceration, the healing may be very slow and the wound may persist for a prolonged period of time, ie. months or even years The stages of wound healing normally include inflammation (usually 1-3 days), migration (normally 1-6 days), proliferation (usually 3-24 days) and maturation (usually 1-12 months). The curing process is a complex and well-orchestrated process that involves the migration, proliferation and differentiation of a variety of cell types, as well as the synthesis of matrix components. The curing process could be separated into the following three stages: i) Hemostasis and inflammation When platelets are present outside the circulatory system and exposed to thrombin and collagen, they become activated and aggregate. In this way, platelets initiate the repair process by adding and forming a temporary plug to ensure hemostasis and prevent the invasion of bacteria. Activated platelets initiate the coagulation and release system of growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factors (EGFs) and transformation growth factors (TGFs).

The first cells that invade the area of the wound are neutrophils followed by monocytes that are activated by macrophages.

The main role of neutrophils seems to be to clean the wound or defend the wound against contamination by bacteria and improve wound healing by removing dead cells and platelets. Infiltration of neutrophils ceases within about the first 48 hours, with the proviso that bacterial contamination in the wound is not present. Excess neutrophils are subjected to phagocytosis by tissue macrophages recruited from the circulation source of the monocytes found in the blood. Macrophages are thought to be essential for the efficient healing of wounds in that they are also responsible for the phagocytosis of pathogenic organisms and a clarification of tissue debris. In addition, they release numerous factors involved in subsequent cases of the healing process. Macrophages attract fibroblasts that begin the production of collagen. ii) Formation and re-epilation of granulation tissue Within 48 hours after the wound, the fibroblasts begin to proliferate and migrate into the wound space of the connective tissue at the edge of the wound. Fibroblasts produce collagens and glycosaminoglycans and inter alia the low oxygen tension in the wound stimulates the proliferation of endothelial cells. The endothelial cells give rise to the formation of a new capillary network.

The activators of collagenases and plasminogen are secreted from keratinocytes. If the wound is left undisturbed and well nourished with oxygen and nutrients, the keratinocytes will migrate over the wound. Keratinocytes are thought to only migrate over viable tissue and, therefore, keratinocytes migrate in the area below the dead tissue and the wound cortex.

The area of the wound is also decreased by contraction. iii) Dermal remodeling As soon as the re-epithesis is complete, tissue remodeling begins. This phase, which lasts several years, restores the strength of the wounded tissue.

All the healing processes mentioned above take considerable time. The speed of healing is influenced by the freedom of the wound from the infection, the general health of the individual, the presence of foreign bodies, etc. Some pathological conditions such as infection, maceration, dehydration, generally poor health and malnutrition can lead to the formation of a chronic ulcer such as, e.g., ischemic ulcers.

Until at least superficial healing has occurred, the wound remains at risk of re-infection or continued. Therefore, the faster the wound healing, the sooner the risk is removed.

In this way, any procedure that can influence the speed of wound healing or favorably influence wound healing is of great value.

In addition, since almost all tissue repair processes include the formation of premature connective tissue, a stimulation of this and subsequent processes are contemplated to improve wound healing.

In the present context, the term "clinical cure" is used to represent a situation where tissue disruption can not be observed visually and only discrete signs of inflammation are present, such as a light red tissue or a discretely swollen tissue. In addition, pain discomforts are not present when the organ is relaxed or not.

As mentioned above, the invention relates to the use of enamel matrix, enamel matrix derivatives and / or enamel protein matrix as a wound healing agent, i.e. an agent that accelerates, stimulates or promotes the healing of dermal or mucosal wounds. Therefore, an important use is also the use as regeneration of tissue and / or repair agents. In addition, due to the wound healing effect, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix have pain relief effect.

Traditionally, dry or wet to dry bandages have been used most commonly for wound care. These are being gradually replaced by humid environments that use occlusive bandages. To repair or successfully replace a damaged body part, the processes of wound healing, fibrosis and microbial invasion must balance with each other. Many tools available to protect the infection are in accordance with wound healing. Delayed wound healing or inflammation can exacerbate fibrosis. In addition, it has previously been suggested that growth factors such as epidermal growth factor (EGF), transforming growth factor-a (TGF-a), platelet-derived growth factor (PDGF), growth factors fibroblasts (FGFs) that include the acid fibroblast growth factor (a-FGF) and the basic fibroblast growth factor (β-FGF), the transforming growth factor β (TGF-β) and similar growth factors to insulin (IGF-1 and IGF-2), are drivers of the wound healing process and are frequently cited as promoters of wound healing; however, they can actually promote fibrosis, which, in turn, could impart successful healing. Although accelerated healing offers the main promise for reducing the risk of infection and the resulting inflammation that can lead to scarring, therapeutic attempts to accelerate the normal wound healing process have been established with relatively little success. This is probably because the repair process involves the normal complication of a number of factors, cf. previously.

For this purpose, the present inventors have observed that in several fibroblast cell cultures (embryonal, dermal, periodontal ligament derivative, fish or poultry), TGFβ1 is produced twice as such in cell cultures stimulated with EMDOGAIN® compared to cultures not stimulated when tested by, eg, ELISA in a sample of the culture medium (vi of Example 1 below). The increase is present after 24 hours of cultivation, but is pronounced more in the following days (days 2 and 3). After the second day, cell proliferation is also increased in cell cultures stimulated with EMDOGAIN®. A similar but less pronounced increase in TGFßl production is observed in human epithelial cells. As the TGFßl seems to be of central importance in the epi telization of the superficial wounds, these discoveries support the concept of the present invention.

In the oral cavity, the use of bandages is common. Such bandages are of the traditional type, e.g. Surgipads for stop bleeding and periodontal bandage Coe-Pack (Coe Laborato ies, the GC Group, Chicago, USA) in open wounds, Gaze administration in antibiotic solution is introduced into the extraction socket of the tooth and requires removal after a few days when the healing has begun. Rinsing with antiseptics such as chlorhexidine is used regularly after oral surgery. Sometimes, general or topical antibiotics are also prescribed.

In general, the following must be taken into specific precautions in connection with the treatment of wounds, such as, e.g., considerations of sterility, contamination problems, correct application of bandages / bandage, etc. that normally require that the treatment / application be performed by very capable nurses or similar. In this way, wound treatment often becomes a very expensive operation when the wound healing agent is to be applied several times daily. A desired reduction in the costs involved in the treatment of wound healing, therefore, is obtained when the frequency of application can be reduced or if the healing processes are improved, leading to a reduction in the period of time required to cure the wound. wound.

The present inventors have now found that the enamel matrix, enamel matrix derivatives and / or enamel protein matrix have wound healing properties. In addition, there are indications that the application of the enamel matrix, derived from the enamel matrix and / or enamel protein matrix leads to improved wound healing. Especially, the inventors have observed that after the application of the enamel protein matrix and / or enamel matrix derivatives, the stage of inflammation is shortened and the typical signs such as burning, redness, edema and pain are less to see ificable, and new tissues are formed more quickly. The time observed for wound healing (e.g., after surgery) is significantly shortened compared to surgery without the use of enamel matrix, enamel matrix derivatives and / or enamel protein matrix.

The therapeutic and / or prophylactic activity of the enamel matrix, derived from the enamel matrix and / or enamel protein matrix could, of course, be evidenced by the in vi ve tests using experimental animals or humans (see the experimental section of right here) . However, an indication of the efficiency and / or activity of the enamel matrix, derived from the enamel matrix and / or enamel protein matrix can be obtained by performing the relatively simple in vi tro tests such as, eg, the tests that involve cell cultures.

In addition, there are several parameters that could be used to evaluate a wound healing effect. These include: - Computer-assisted planimetry (evaluation of the opening speed of wound healing) - Laser Doppler effect image formation (evaluation of wound perfusion) - Tensicmetry (evaluation of the resistance of the wound) - Histopathology / cytology (microscopic evaluation of wounded tissues and fluids) Biochemistry (HPLC / RIA) (evaluation of various drugs and biochemical components of wounded tissue) - The ect rhodiagnostics (evaluation of the relationship between wound healing and innervation) Escint ilograf ia (radionuclide imaging of wounded tissue) In connection with the treatment of wounds / ulcers, debridement and cleanliness of the wound are of particular importance. It is believed that the cleaning and / or debridement of wounds / ulcers are a prerequisite for the healing process and, furthermore, when the wound healing agents are applied, such agents exert their effect on the fresh tissue and vital and not on dead tissue or contaminated tissue. Debridement of the necrotic tissue can be performed by at least four different methods: i) acid debridement, ii) mechanical debridement, iii) enzymatic debridement and iv) autolytic debridement.

Therefore, the present invention also relates to the use of a debridement method in combination with the use of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix for the healing or prevention of wounds. . Such a combination therapy involves the following two steps, namely i) a debridement method and ii) application of an enamel matrix, enamel matrix derivatives and / or enamel protein matrix and the two steps could be carried out as many times as desired in any appropriate order.

When the wound has been subjected to debridement, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix could be applied either directly on the wound or applied in the form of any suitable pharmaceutical composition such as, eg, a dry or wet healing bandage in which the enamel matrix has been incorporated, derived from the enamel matrix and / or enamel protein matrix. The enamel matrix, derived from the enamel matrix and / or enamel protein matrix, could, of course, also be applied in connection with the healing of the wound.

As will be discussed later, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix could be used as such or could be used in an appropriate preparation or pharmaceutical composition.

Effect of decreased infection In a further aspect of the present invention, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix are used as therapeutic or prophylactic agents having an antimicrobial effect. The enamel matrix, enamel matrix derivatives and / or enamel protein matrix exhibit diminishing infection properties.

In the present context, the term "diminishing effect of infection" refers to a treatment or preventive effect by an enamel matrix, enamel matrix derivatives and / or enamel protein matrix on an infection in a tissue of an individual when the tissue or the individual is treated with the enamel matrix, derived from the enamel matrix and / or protein matrix of esma 1 te.

The term infection refers to the invasion and multiplication of microorganisms in body tissues or accumulation in tissues, which could be clinically apparent or result in local cell injury due to competitive metabolism, enzymes, toxins, intracellular duplication or antigen response -ant icuerpo.

In accordance with the present invention, the infection to be prevented and / or treated could be caused by a microorganism. Microorganisms of interest according to the present invention include bacteria, viruses, yeast, molds, protozoa and r ic ke 11 s.

In the present context, the term "antibacterial effect" means that bacterial growth is suppressed or bacteria are destroyed. The term is not limited to certain bacteria, but encompasses, in general, any bacteria. However, the invention focuses on i) pathogenic bacteria that cause diseases in mammals including humans and / or ii) bacteria that are normally present in the body of mammals and that under certain conditions could cause undesirable conditions in the body.

Therefore, the invention relates to the use of a Active enamel substance for the prevention of or treatment of bacterial growth on the surface of the body, such as the skin, a surface of the mucosa or a nail or dental surface.

General and specific description of the bacterial conditions that will be counteracted.

The enamel matrix, derived from the enamel matrix and / or enamel protein matrix could be used for the treatment of an infection caused by bacteria together with or without the presence of an antimicrobial. The Gram-negative bacteria to be treated with the active enamel substance could be cocci such as Nei sseri a (e.g. N. Men ingi t i s, N. gonorrhea e and Aci n e t oba c t er rods, such as Ba c t e roi des (e.g. B.

^^. - ^ ^^ M ^ tA ^ im ^ t ^ »fragilis), Bordetella (eg B. pertussis, B. parapertussis), Brucella (eg B. melitentis, B. abortus Bang, B. suis), Campylobacter (eg C jejuni, C. coli, C. fetus), Citrobacter, Enterobacter, Escherichia (eg E. coli), Haemophilus (eg H. influenzae, H. parainf luenzae), Klebsiella (eg K. pneumoniae), Legionella (eg L. pneumophi la), Pasteurella (eg P. yersinia, P. multocida), Proteus (eg P. mirabilis, P. vulgaris), Pseudomonas (eg P. aerugmosa, P. pseudoma 1 lei, P. mallei), Salmonella (eg S enteritidis, S. infantitis S. Dublin S. typhi, S. for typhi, S. schottmul ler, S. chole'raesuis, S. typhimurium or any of the other 2,500 serotypes), Serratia (eg S. marscences, S. liquif aciens), Shigella (eg S. sonnel, S. flexneri, S. dysenteriae, S. boydii), Vibrio (eg V. cholerae, V. tor), and Yersinia (eg Y. enteroco 1 itica, Y. pseudotuber asses, Y. pestis). The Gram-positive bacteria to be treated with the active enamel substance could be cocci, such as Streptococcus (e.g. S. pneumoniae, S. viridians, S. faecalis, S. pyogenes), Staphylococcus (e.g. S. aureus, S. epidermis, S. saprophyt i cus, S. albus), and canes such as Actinomyces (e.g. A. israelli), Bacillus (e.g. B. cereus, B. subtilis, B. anthracis), Clostridium (e.g. C. botulinum, C. tetani, C. perfringens, C. difficile), Corynebacterium (e.g. C. diphtheriae), Listeria and Providence. Other bacteria that cause infection include Propionobacter ium acne and Pityosporon ovale.

The enamel matrix, derived from the enamel matrix and / or enamel protein matrix could also be used for the treatment of an infection caused by a spirochete such as, e.g., Borrelia, Leptospira, Treponema or Pseudomonas.

An antimicrobial to be used with the enamel matrix, enamel matrix derivatives and / or enamel protein matrix could be an antimicrobial having an antimicrobial action by inhibiting the synthesis of the cell wall, such as β-lactams and vancomycin, preferably penicillins, such as amdinocil ina, ampicillin, amoxicillin, azlocillin, bacampicillin, benzathine, pinicillin G, carbenicillin, cloxacillin, cyclacillin, di cloxacin 1, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin and ticarcillin; The drugs of the first generation cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin and cephradine, drugs of the second generation cefaclor, cefamandole, cefonicide, ceforanide, cefoxitin and cefuroxime, or the third generation drugs Cephaloperazone cephalosporins, cefotaxime, cefotetan, ceftazidime, ceftizoxime, ceftriaxone and moxalactam; carbapenend such as imipenem; or monobactams such as aztreonamo.

Other antimicrobial drugs with action by inhibiting the synthesis of proteins, such as chloramphenicol; other tetracyclines, preferably demeclocycline, doxycycline, methacycline, minocycline and oxytetracycline; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, e spec t i nomi c i na, streptomycin and tobramycin; polymyxins such as colistin, col ist imat at and polymyxin B and erythromycins and lincomycins; antimicrobials with action by inhibiting the synthesis of nucleic acids in particular sulfonamides, such as sulfacitin, sulfazine zin, sulphonyl sulfametizol and sulphipiin; trimethoprim, quinolones, novobiocin, pyrimethamine and rifampin.

In a specific embodiment of the invention, the infection is present in the oral cavity and the infection could be a bacterial condition.

Oral bacteria that are in contact are inhibited or otherwise combated. The examples (not conditions) include bacteria that cause cavities, e.g. Streptococcus mutans, Lactobacillus ssp. bacteria that cause periodontal disease e.g. Actinobacill us actinomycetemcomi tans, Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus micros, Campy wolf cte r (Fusobacter ia, Staphylococci), B. forsythus bacteria causing alveolitis etc., e.g. Staphylococcus, Actinomyces and Bacillus bacteria that cause periapical lesions, e.g Spi roche tes and all previous anti-inflammatory effects The present invention also relates to the uses of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix as therapeutic or prophylactic agents having an anti-inflammatory effect.

Various forms are used to suppress manifestations of inflammation, including the adrenocort icost eroids, the large group comprising the so-called non-steroidal anti-inflammatory drugs or NSAIDs, and drugs such as immunosuppressive agents. Adrenocorticosteroids, and especially glucocorticoids, have potent anti-inflammatory effects when used in pharmacological dosages. They specifically inhibit the previous vascular phase of the inflammatory process, decreasing the vascular permeability and in this way the migration of the granulocyte. Glucocorticoids also interfere with inflammatory and reparative processes, in that they inhibit the proliferation of mesenchymal cells and the production of extracellular macromolecules, including proteoglycans and collagen. It has been shown experimentally that glucocorticoids inhibit, example, the function of the macrophage, the production of ~ & amp; ^ to ^ ^ ^ ^ ^ humoral antibodies, cellular immunity and possibly the release of liposomal enzymes.

The severity of the damaged tissue may depend on the antigen / organism reaction of the organism, as well as the degree of retention of the inflammatory products in the affected area. The accumulation of mediators of local inflammation accelerates the process. In most cases, the process is slow, with immunoinflation of the tissue and the formation of granulation tissue containing inflammatory cells.

In the present context the term "anti-inflammatory effect" represents counteracting or suppressing the inflammation.

General description and specifies the type of inflammatory conditions that will be treated.

The inflammatory condition to be treated according to the present invention, of course, could be any inflammatory condition in / on any part of the body or any inflammatory condition present in the soft or hard tissue. In a modality of invention, the inflammatory condition is present in the ^ jtj ^ tíS ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ i ^ ij ^^^^ l ^. ^^^^^ oral cavity. Examples of conditions in the oral cavity are alveolitis, cheilitis, bone necrosis (after trauma), fractures.

In another embodiment of the invention, the inflammatory condition is present at a bone donor site. In a third embodiment of the invention, the inflammatory condition is present in an articular cavity. Examples of such inflammatory conditions are rheumatoid arthritis and related conditions.

Antibacterial anti-inflammatory In contrast to many antibiotic agents currently used, the enamel protein matrix will not compromise wound healing and rapid wound healing, but it does not leave room for chronic or long-lasting inflammation processes to develop. Also, the reorganization of the appropriate tissues, such as that described after the application of the enamel matrix derivatives on per iodontial defects, is clearly favored by rapid wound healing without bacteria or inflammatory reactions.

The application of the enamel matrix, derived from the enamel matrix and / or enamel protein matrix leads to the rapid healing of surgical incision wounds, possibly creating a surface that in contact with the bacteria inhibits its growth, but at the same time improves fibroblast migration and collagen synthesis. If the inflammatory stage is shortened, typical signs such as burning, redness, edema, and pain are less noticeable. 10 Enamel matrix, enamel matrix derivatives and enamel protein matrix The enamel matrix is a precursor for enamel 15 and could be obtained from any relevant natural source, i.e. a mammal in which the teeth are under development. An appropriate source is to develop teeth of sacrificed animals such as, e.g., calves, pigs or lambs. Another source is for example, fish skin. The enamel matrix can be prepared from teeth developed as previously described (EP-B-0 337 967 and EP-B-0 263 086). The enamel matrix is scraped and the derivatives of the enamel matrix are prepared, e.g. by extraction with aqueous solution such as a Tissue, a dilute acid or base or a water / solvent mixture, followed by size exclusion, deaeration or other purification steps, optionally followed by freeze drying. The enzymes could be deactivated by treatment with heat or solvents, in which case the derivatives could be stored in the liquid form without freeze drying.

In the present context, the enamel matrix derivatives are enamel matrix derivatives that include one or more of the enamel protein matrix or parts of such proteins, produced naturally by alternating binding or processing, or by enzymatic cutting or chemical of a protein of natural length, or by the synthesis of polypeptides in vi t ro oin vi vo (recombinant DNA methods or culture of diploid cells). Derivatives of the enamel protein matrix also include polypeptides or proteins related to the enamel matrix. The polypeptides or proteins could be linked to an appropriate biodegradable carrier molecule, such as polyamino acids or polysaccharides, or combinations thereof. In addition, the term derived from the enamel matrix also covers the analogous substances of synthesis.

Proteins are biological macromolecules made up of amino acid residues linked by peptide bonds. Proteins, like linear amino acid polymers, are also called polypeptides. Typically, the proteins have 50-800 amino acid residues and therefore have molecular weights in the range of about 6,000 to about several hundred thousand Daltons or more. Small proteins are called peptides or oligopeptides.

The matrix of enamel proteins are proteins that are normally present in the enamel matrix, i.e. the precursor for enamel (Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl 1: 282-91), or proteins that can be obtained by cutting such proteins. In general, such proteins have a molecular weight below 120,000 daltons and include ame logenins, not amelogenins, not proline-rich amelogenins, amelines (ameloblas t ina, sheatlina) and t uf t inas.

Examples of proteins for use according to the invention are amelogenins, non-amelogenins rich in proline, tuftelin, hair proteins, whey proteins, saliva proteins, amelin, tub ameloblas, sheatlin and derivatives and mixtures thereof. A preparation containing an active enamel substance for use according to the invention could also contain at least two of the aforementioned protein substances. A commercial product comprising amelogenins and possibly other protein matrices is labeled EMDOGAIN® (Biora AB).

In general, the major proteins of the enamel matrix are known as amelogenins. These constitute approximately 90% w / w of the protein matrix. The remaining 10% w / w includes non-amelogenins rich in proline, tuftelin, hair proteins, whey proteins and at least one salivary protein; however, other proteins such as, e.g., amelina (tub ameloblas, sheatlin) that have been identified in association with the enamel matrix could also be present. In addition, the various proteins could be synthesized and / or processed in several different sizes (i.e. different molecular weights). In this way, the dominant proteins in the enamel matrix, amelogenins, have been found to exist in several different sizes that together form supramolecular aggregates. They are markedly hydrophobic substances that under physiological conditions form aggregates. These can carry or be vehicles for other proteins or peptides.

Other protein substances that are suitable for use according to the present invention are also contemplated. Examples include proteins, such as proteins rich in proline and poly-proline. Other examples of substances that are contemplated as being suitable for use according to the present invention are aggregates of such proteins, enamel matrix derivatives and / or enamel protein matrix, as well as enamel matrix metabolites, derivatives of enamel matrix and / or enamel protein matrix. The metabolites could be of any size in the size range of the proteins to those of the short peptides.

As mentioned above, proteins, polypeptides or peptides for use according to the invention, typically have a molecular weight of at most about 120 kDa such as, eg, at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page electrophoresis.

The proteins for use according to the invention are normally presented in the form of a preparation, wherein the protein content of the active enamel substance in the preparation is in the range of about 0.05% w / w to 100% w / p such as, eg, about 5-99% w / w, about 10-95% w / w, about 15-90% w / w, about 20-90% w / w, about 30-90% w / w , about 40-85% w / w, about 50-80% w / w, about 60-70% w / w, about 70-90% w / w about 80-90% w / w.

A preparation of an active enamel substance for use according to the invention, it could also contain a mixture of active enamel substances with different molecular weights.

Proteins from an enamel matrix can divided into a high molecular weight part and a low molecular weight part, and it has been found that a well-defined fraction of the enamel protein matrix possesses valuable properties with respect to the treatment of peri-dental defects (i.e. per iodont ales). This fraction contains extractable acetic acid proteins, generally referred to as amelogenins and constitutes the low molecular weight part of an enamel matrix (see EP-B-0 337 967 and EP-B-0 263 086). 25 As discussed above, the low molecular weight part of an enamel matrix has an appropriate activity to induce binding between hard tissues in periodontal defects. In the present context, however, active proteins are not restricted to the low molecular weight part of an enamel matrix. At present, preferred proteins include enamel protein matrix such as amelogenin, amelin, tuftelin, etc. with molecular weights (as measured in vitro with SDS-PAGE) below about 60,000 Daltons, but proteins having a molecular weight above about 60,000 Daltons also have promising properties as candidates for wound healing, agents ant i -bact er ians and / or anti-inflammatories.

Therefore, it is contemplated that the active enamel substance for use according to the invention has a molecular weight of up to about 40,000 such as, e.g. a molecular weight of between about 5,000 and about 25,000.

Peptides are also within the scope of the present invention as described in WO 97/02730, i.e. peptides comprising at least one element of the sequence selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu) and which further comprises an amino acid sequence of which a strand of 20 amino acids is identical to a degree of at least 80% with a strand of amino acids having the same length, selected from the group consisting of the amino acid sequence in SEQ ID NO: 1 and a sequence consisting of amino acids 1 to 103 of SEQ ID NO: 1 and amino acids 6 to 324 of SEQ ID NO: 2.

By the term "sequence identity" is meant the identity in the amino acid sequence in the pair with respect to the identity and position of the amino acids of the peptides. A separation is counted as non-identity for one or more amino acids as appropriate.

Such peptides could comprise from 6 to 300 amino acids, at least 20 amino acids, at least 30 amino acids, such as at least 60 amino acids, at least 90 amino acids, at least 120 amino acids, at least 150 amino acids or at least 200 amino acids.

A method for the isolation of the enamel protein matrix involves the extraction of the proteins and the removal of the calcium and phosphate ions from the solubilized hydroxyapatite by an appropriate method, e.g. gel filtration, dialysis or ultrafiltration (see eg Janson, JC &Rydén, L. (Eds.), Protein purification, VCH Publishers 1989 and Harris, ELV &Angal, S., Protein purification methods - A practical approach, IRL Press, Oxford 1990).

A typical lyophilized protein preparation could contain mainly or exclusively up to 70-90% of amelogenins with a molecular weight (MW) between 40,000 and 5,000 Daltons, 10-30% is made of smaller peptides, salts and residual water. The main protein bands are at 20 kDa, 12-14 kDa and around 5 kDa.

Separating the proteins, e.g. by precipitation, ion exchange chromatography, preparative electrophoresis, gel penetration chromatography, reverse phase chromatography or affinity chromatography, the amelogenins of different molecular weights can be purified.

The combination of amelogenins of molecular weight, from a 20 kDa dominant compound to an aggregate of amelogenins with very different molecular weights between 40 and 5 kDa, and up to a dominant compound of 5 kDa could be varied. Another matrix of enamel proteins, such as amelin, tuftelin or proteolytic enzymes normally found in the enamel matrix, can be added and carried by the amelogenin aggregate.

As an alternative source of the enamel protein derivatives or matrix, synthetic routes well known to the person skilled in the art or the use of cultured cells or bacteria modified by recombinant DNA techniques could also be used (see , eg, Sambrook, J. et al .: Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).

Physicochemical properties of the enamel matrix, derived from the enamel matrix and the protein matrix of esma 1 t e.

In general, the enamel matrix, enamel matrix derivatives and enamel protein matrix are hydrophobic substances, i.e. less soluble in water, especially at elevated temperatures. In general, these proteins are soluble at non-physiological pH values and at a low temperature, such as about 4-20 ° C, while they will aggregate and precipitate at body temperature (35-37 ° C) and neutral pH.

The enamel matrix, derived from the enamel matrix and / or enamel protein matrix for use according to the invention, also includes an active enamel substance, wherein at least a part of the active enamel substance is in the form of aggregates or after the application in vi is capable of forming aggregates. The particle size of the aggregates is in the range of about 20 nm to about 1 μm.

It is contemplated that the solubility properties of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix are of importance in conjunction with the prophylactic and therapeutic activity of the substances. When a composition containing the enamel matrix, derived from the enamel matrix and / or enamel protein matrix (in the following also represented "active enamel substance" as a common term) is administered to e.g. a human, protein substances will precipitate because the pH usually prevails under physiological conditions. In this way, a layer of the enamel matrix, derived from the enamel matrix and / or enamel protein matrix is formed in ^ J ^ ÁÁ ^ ^^^^ £ j & * ^ go application site and this layer (which also may be a molecular layer in those cases where aggregates have been formed) is difficult to rinse off under physiological Furthermore, due to the substances bioadhesive properties (see below) the precipitated layer is firmly bound to the tissue also at the margin between the precipitated layer and the tissue. In this way, the protein layer covers the tissue on which the enamel matrix, enamel matrix derivatives and / or enamel protein matrix or components thereof have been applied and active enamel substances are maintained further. you for a prolonged period of time, ie it is not necessary to administer the active enamel substance at short intervals. In addition, the layer formed i n s i t u can almost be compared with an occlusive dressing, i.e. the formed layer protects the surrounding tissue in which the layer is formed. In the case of a wound tissue, an infected tissue or an inflamed tissue such a layer protects the tissue from further contamination from microorganisms present in the surroundings. In addition, the protein layer could exert its effect by direct contact with the tissue or with microorganisms present dent / on / in the tissue.

To allow a protein layer to form after application, it may be advantageous to incorporate a suitable buffer substance into a pharmaceutical or cosmetic composition of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix; the purpose of such a buffer substance could be to prevent the dissolution of the active enamel substance at the site of application.

Enamel matrix derivatives and enamel matrix proteins Enamel matrix is also observed (by the present inventors) to possess going bioadhesi properties, i.e. They have a capacity to adhere to the surfaces of the skin or mucosa. These properties are more valuable in conjunction with a therapeutic and / or prophylactic treatment at least for the following reasons: the prophylactically and / or therapeutically active substances can be maintained at the site of application for a prolonged period of time (ie i) the frequency of administration can be reduced, ii) a controlled release effect of the active substance is obtained and / or iii) a local treatment at the application site is improved) the substances could no-tim be suitable as vehicles for other prophylactically or therapeutically active substances, because a vehicle containing the enamel matrix, enamel matrix derivatives and / or enamel protein matrix can be formulated as a bioadhesive carrier (ie a novel carrier system). release of the bioadhesive drug on the bioadhesive properties of the enamel matrix, matrix derivatives enamel and / or enamel protein matrix).

Theories regarding the mechanism of action.

The enamel matrix is an example of an extracellular protein matrix that adheres to mineral surfaces as well as to protein surfaces. At physiological pH and temperature, the proteins form an insoluble supra-molecular aggregate (Fincham et al., In J. Struct. Biol., 1994 March-April; 112 (2): 103-9 and in J. Struct., Biol. 1995 July-August; 115 (1): 50-9), which is gradually degraded by proteolytic enzymes (it is presented in vi vo ein vi t ro with the proviso that the proteases have not been subjected to inactivation).

The recent observation that the enamel matrix Y. M * J * m * ?? utilb xlM '* 2. ' 1"- . ^ - «- .. ... t» »- y, • Sin p. - -: formed and presented temporarily during root formation and root cement can explain how the application of the enamel matrix, enamel matrix derivatives and / or enamel protein matrix promotes the regeneration of periodontal tissue. However, it is very surprising the observation that the present invention emphasizes that enamel matrix, enamel matrix derivatives and / or enamel protein matrix also have a positive effect on the healing of soft tissue defects such as wound healing . The same applies for observations regarding the anti-infective and anti-inflammatory effect.

In many species, the remains of the enamel matrix are found in the newly mineralized crown when a tooth is emerging in the oral cavity. It could be argued that a new tooth would be very vulnerable to the bacterial attack of the common oral bacteria, unless it had a natural protection during this initial phase.

The application of the insoluble enamel matrix, the enamel matrix derivatives and / or the enamel protein matrix with anti-bacterial and / or anti-inflammatory properties on a wounded surface will improve curing.

As demonstrated in the experimental section, the enamel matrix, enamel matrix derivatives and / or enamel protein matrix or protein aggregates hinder bacterial growth by contact inhibition, while exposed cells apparently react in the matrix of enamel as a normal environment that suppresses inflammatory responses.

According to the present invention, an enamel matrix, derived from enamel matrix and / or enamel matrix protein could be used for curative purposes, as well as for preventive purposes. In addition, an enamel matrix, derived from enamel matrix and / or enamel protein matrix could be used together with other active drug substances such as, e.g. anti-bacterial substances, anti-inf 1 ama, antiviral, antifungal or in combination with growth factors such as, eg, TGFβ, PDGF, IGF, FGF, keratinocyte growth factor or peptide analogues thereof ( it is believed that EGF promotes healing by improving the migration of epithelial cell cell division, in addition, EGF increases the number of fibroblasts in wounds resulting in increased collagen production). The enzymes - either inherently present in the enamel matrix or the preparation thereof or added - could also be used in combination with an enamel matrix, derived from enamel matrix and / or enamel protein matrix, especially proteases.

A preparation of the active enamel substance is usually formulated as a pharmaceutical or cosmetic composition. Such a composition could, of course, consist of the protein preparation or could additionally comprise a pharmaceutical or cosmetically acceptable excipient. The excipients especially suitable for use in the pharmaceutical or cosmetic compositions are propylene glycol alginate or hyaluronic acid or salts or derivatives thereof.

Pharmaceutical and / or cosmetic compositions.

In the following examples, suitable compositions containing the active enamel substances are given. Depending on the use of the active enamel substances, a composition could be a pharmaceutical or cosmetic composition. In the following term, "pharmaceutical composition" is also intended to encompass cosmetic compositions, as well as compositions belonging to the so-called gray area between pharmaceuticals and cosmetics, ie cosmeceuticals.

For administration to an individual (an animal or a human) the enamel matrix, derived from enamel matrix and / or enamel protein matrix (in the following also represented "active enamel substance") and / or a preparation of it is preferably formulated in a pharmaceutical composition containing the active enamel substance and, optionally, one or more other pharmaceutically acceptable excipients.

The compositions could be in the form of, e.g., solid, semi-solid or fluid compositions such as, e.g., bioabsorbent patches, potions, bandages, hydrogel dressings, hydrocolloid dressings, films, foams, lamellae, bandages, plasters, delivery devices, implants, powders, granules, granules, capsules, agarose or healthy quito beads, tablets, pills, pills, microcapsules, microspheres, nanoparticles, atomizers, aerosols, inhalation devices, gels, hydrogels, pastes, ointments, creams, syrups, suppositories, vagitories, toothpaste, solutions, dispersions, suspensions, emulsions, mixtures, lotions, mouthwashes, shampoos, enemas, cases containing e.g. two separate containers 10, wherein the first of the containers contains the active enamel substance optionally mixed with another active drug substance and / or pharmaceutically acceptable excipients and the second container contains an appropriate medium intended to be added to the first container before use , to obtain a composition ready for use; and in other appropriate forms such as, e.g., implants or implant coating or in the form suitable for use in connection with implantation or transplantation.

Compositions for application to the skin or mucosa are considered more important in connection with the present invention. In this way, a composition that rt_ a > »- < .¿ »* u & t- t t - * i *. . comprising the active enamel substance to be administered could be adapted for administration by any route, for example by topical (dermal), oral, buccal, nasal, aural, rectal or vaginal administration, or by administration to a body cavity such as , eg, a root of the tooth or a root canal of the tooth. In addition, a composition could be adapted for administration in connection with surgery, e.g. in connection with the incision inside the body to promote the healing of internal wounds and soft tissue damage.

As mentioned above, a composition of the active enamel substance could be appropriate for use during surgery, e.g. for the local application (e.g. in the oral cavity) in the form of a gel, film or as a rinsing solution or treatment with a paste or cream on the tissue or surfaces, to avoid bacterial attack. In connection with surgery or implantation in the root canal area of the tooth, a paste may be used to seal the cavity.

The compositions could be formulated according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J.C. Boylan, Marcel Dekker, Inc., New York, 1988.

As mentioned above, the application of a composition comprising an active enamel substance is intended for the skin or mucosa. Other applications, of course, could also be relevant such as, e.g., application in dentures, prostheses, implants and application to body cavities, such as the oral, nasal and vaginal cavities. The mucosa is preferably selected from the oral, buccal, nasal, aural, rectal and vaginal mucosa. In addition, the application could be directly on or over the wound or other soft tissue injuries.

In addition, the application within the dental / odontological area is also of great importance. Relevant examples are application to periodontal pockets (dental), to gingiva or gingival wounds or other wounds located in the oral cavity, or in connection with oral surgery.

It is further anticipated that, due to the bacterial properties of the active enamel substance described herein, it could be advantageously applied to the tooth or roots of the teeth for the prevention of caries and / or plaque. To support this use, it has been shown (Weinmann, JP et al: Hereditary disturbances of enamel formation and calcination, J. Amer. Dent. Ass. 32: 397-418; Sundell S, Hereditary amelogenesis imperfecta. study in a Swedish child population, Swed Dent J Suppl 1986; 31: 1-38) that teeth that develop imperfectly (amelogenesis imperfecta) and subsequently contain large amounts of amelogenins are remarkably resistant to caries.

A pharmaceutical composition comprising an active enamel substance serves as a drug delivery system. In the present context, the term "drug delivery system" represents a pharmaceutical composition (a pharmaceutical formulation or dosage form) which due to administration presents the active substance to the body of a human or animal. In this way, the term "drug delivery system" encompasses simple pharmaceutical compositions such as, e.g., creams, ointments, liquids, powders, tablets, etc. as well as more sophisticated formulations, such as aerosol devices, plasters, bandages, banda s, et c.

In addition to the active enamel substances, a pharmaceutical composition for use according to the invention could comprise pharmaceutically or cosmetically acceptable excipients.

A pharmaceutically or cosmetically acceptable excipient is a substance that is not substantially harmful to the individual to whom the composition is to be administered. Such excipient normally satisfies the requirements given by the national health authorities. Official pharmacopoeias such as e.g. the British Pharmacopoeia, the Pharmacopoeia of the United States and the European Pharmacopoeia set the standards for pharmaceutically acceptable excipients.

If a pharmaceutically acceptable excipient is suitable for use in the pharmaceutical composition, it is generally dependent on what type of dosage form is chosen for use for a particular type of wound. The following are examples of pharmaceutically acceptable excipients for use in different types of compositions for use according to the invention.

Next, a review of the pharmaceutical compositions relevant to the use according to the invention is given. The review is based on the particular administration route. However, it is appreciated that in those cases where a pharmaceutically acceptable excipient could be employed in different dosage forms or compositions, the application of a pharmaceutically acceptable excipient is not limited to a particular dosage form or a particular function of the excipient.

The choice of pharmaceutically acceptable excipients or excipients in a composition for use according to the invention and the optimum concentration thereof, in general, can not be predicted and must be determined on the basis of an experimental evaluation of such a composition. However, an expert in the art of pharmaceutical formulation can find guidance in e.g., "Remington's Pharmaceutical Sciences," 18th Edition, Mack Publishing Company, Easton, 1990.

Topical compositions For application to the mucosa or skin, the compositions for use according to the invention could conventionally contain non-toxic pharmaceutically acceptable carriers or excipients including microspheres and liposomes.

Compositions for use according to the invention include all types of solid, semi-solid and fluid compositions. The compositions of particular relevance are e.g. pastes, ointments, hydrophilic ointments, creams, gels, hydrogels, solutions, emulsions, suspensions, lotions, liniments, shampoos, jellies, soaps, bars, sprays, powders, films, foams, pads, sponges (eg collagen sponges), bandages (such as, eg, absorbent wound dressings), potions, plasters and transdermal delivery systems.

The pharmaceutically acceptable excipients could include solvents, buffering agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gel-forming agents, ointment bases, penetration enhancers, perfumes and skin-protecting agents. .

Examples of solvents are e.g. water, alcohols, vegetable and marine oils (eg edible oils such as almond oil, castor oil, cocoa butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil , peanut oil, poppy seed oil, rape seed oil, sesame oil, soybean oil, sunflower oil and tea seed oil), mineral oils, fatty oils, liquid paraffin, polyethylene glycols, propylene glycols , glycerol, pol ia 1 qui 1 if liquid loxanes and mixtures thereof.

Examples of buffering agents are e.g. citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphoric acid, diethylamine, etc.

Suitable examples of condoms for use in compositions are parabens, such as methyl p-hydroxybenzoate, ethyl, propyl, buty 1 paraben, isobutylparaben, i sopropi lparaben, potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol. , bronopol, bronidox, MDM hydantoin, iodopropini butylcarbamate, EDTA, benzalkonium chloride and benzyl alcohol or condom mixtures.

Examples of humectants are glycerin, propylene glycol, sorbitol, lactic acid, urea and mixtures thereof. í 3, *.? S. "» - '* - * my smos Examples of chelating agents are sodium EDTA and citric acid.

Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, cysteine and mixtures thereof.

Examples of emulsifying agents are gums that occur naturally, e.g. acacia gum or tragacanth gum; phosphatides that occur naturally, e.g. soy lecithin; sorbitan mono-oleate derivatives; wool fats; wool alcohols; esters of sorbitan; monoglycerides; fatty alcohols; esters of fatty acids (e.g. triglycerides or fatty acids); and mixtures thereof.

Examples of suspending agents are e.g. celluloses and cellulose derivatives such as, e.g., carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, Irish mustard, acacia gum, gum arabic, tragacanth and mixtures thereof.

Examples of gel bases, viscosity enhancing agents or components that are capable of taking the exudate from a wound are: liquid paraffin, polyethylene, fatty oils, silica or colloidal aluminum, zinc soaps, glycerol, propylene glycol, tragacanth, polymers of carboxyvinyl, magnesium-aluminum icates, Carbopol®, hydrophilic polymers such as, eg starch or cellulose derivatives such as, eg, carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, swollen hydrocolloids in water, Irish mustards, hyaluronate (eg that of hyaluronate optionally containing sodium chloride), and alginates including propylene glycol alginate .

Examples of ointment bases are e.g. beeswax, paraffin, cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), polyethylene glycols and condensation products between sorbitan esters of fatty acids and ethylene oxide, e.g. polyoxyethylene sorbitan mono-oleate (Tween).

Examples of water-emulsifying or hydrophobic ointment bases are paraffins, vegetable oils, animal fats, synthetic glycerides, waxes, lanolin and liquid polyalkylene glycans.

Examples of hydrophilic ointment bases are solid macrogols (polyethylene glycols).

Other examples of ointment bases are triethanolamine soaps, sulfated fatty alcohols and poly sorbates.

Examples of powdered components are: alginate, collagen, lactose, powder that is capable of forming a gel when applied to a wound (absorbs liquid / exude the wound). Normally the powders intended for the application in very open wounds must be sterile and the particles present must be micronized.

Examples of other excipients are polymers such as, carmellose, carmellose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acacia gum, gelatin, carbomer, emulsifiers such as vitamin E, glyceryl stearates, cetanil glucoside, mustard from Ireland, hyaluronates and alginates and cytosans.

Bandages and / or bandages are also important release systems for an active enamel substance. When the bandages are used as dosage forms, the active enamel substance could be mixed with the other materials / ingredients before or during the manufacture of the bandage or, the active enamel substance in some way, could be coated on the bandage e.g. immersing the bandage in a solution or dispersion of the active enamel substance or by atomizing a solution or dispersion of the active enamel substance on the bandage. Alternatively, the active enamel substance could be applied in the form of a powder to the bandage. The bandages could be in the form of absorb bandages. for wounds for application to exuding wounds. The bandages could also be in the form of hydrogel dressings (e.g., crosslinked polymers such as, e.g. Intrasite® containing carboxymethylcellulose ', propylene glycol or polysaccharides, disaccharides and proteins) or in the form of occlusive dressings such as, eg, alginates, healthy quito, hydrophilic polyurethane film, collagen sheets, plates, powders, foams or sponges ( eg polyurethane or silicone), hydrocolloids (eg carboxymethylcellulose, CMC), collagen and bandages based on hyaluronic acids including combinations of the same.

Bandages of alginate, healthy tissue and hydrocolloids take the exudate from the wound when they are placed on a wound. When they do this they produce an aqueous gel on the surface of the wound and this gel is believed to be beneficial for healing the wound, because it retains moisture from the wound.

It is also envisioned that the active enamel substance that could be incorporated into a tissue adhesive also comprises, e.g. fibrinogen and thrombin and optionally Factor XIII or other plasma coagulation factor, to provide hemostasis. The tissue adhesive could be prepared as a pre-mix of the active enamel substance, fibrinogen and optionally Factor XIII, the thrombin is added to the pre-mix immediately before the tissue adhesive is applied to the wound. Alternatively, the pre-mix of fibrinogen and the active enamel substance and optionally Factor XIII could be applied to the wound prior to the application of thrombin. In si t u, thrombin converts fibrinogen to fibrin, thus reproducing the coagulation process that occurs naturally in wound healing. The presence of the active enamel substance in the tissue adhesive could serve to accelerate the wound healing process as discussed above. A commercial product suitable for the inclusion of the active enamel substance is Tisseel®, a two-component fibrin produced by Immuno, AG, Vienna Austria.

In a toothpaste or mouthwash formulation or other formulation for application to the tooth or roots of the teeth, the active enamel substance could be present in a dissolved state in a vehicle of slightly acid pH or as a dispersion in a vehicle of neutral pH. It is anticipated that in use, the active enamel substance could form a protective layer on the surface of the tooth, whereby the adhesion of caries producing bacteria is avoided (see Example 4 below). In such dental care preparations, the active enamel substance could be formulated together with one or more other compounds having a caries preventive effect, particularly fluorine or other trace element, such as vanadium or molybdenum. At neutral pH, the trace element is believed to be linked to (eg by ionic bonds) or coupled into the active enamel substance, from which it is released to exert its caries-preventive effect when the active enamel substance dissolves at a pH of about 5.5 or less, eg due to the production of acid by the bacteria that produce cavities.

The compositions mentioned above for topical administration, are more suitable for application directly to wounds or could be appropriate for application to, or for introduction into, relevant body orifices, e.g. the rectal, urethral, vaginal, aural nasal or oral orifices. The composition could be applied simply directly on the part to be treated such as e.g., on the mucosa, or by any convenient route of administration.

The compositions that have proven to be of importance in conjunction with the topical application are those that have high toxic properties, i.e. the viscosity of the composition is affected e.g. by agitation, so that the viscosity of the composition at the time of administration can be reduced and, when the composition has been applied, the viscosity increases, so that the composition remains at the site of application.

Compositions for oral use or for application to the mucosa or skin.

The compositions suitable for use according to the invention could also be presented in the form of suspensions, emulsions or dispersions. Such compositions contain the active enamel substance in admixture with a dispersing or wetting agent, suspending agent and / or one or more preservatives and other pharmaceutically acceptable excipients. Such compositions may also be suitable for use in the release of the active enamel substance to e.g. an intact or damaged mucosa, such as by oral, buccal, nasal, rectal or vaginal administration or for administration to the skin or intact or damaged wounds.

Suitable dispersing agents or humectants are, for example, naturally occurring phosphatides, e.g., lecithin or soy lecithin; condensation products of ethylene oxide with e.g. a fatty acid, a long-chain aliphatic alcohol or a partial ester derived from fatty acids and a hexitol or a hexitol anhydride, for example polyoxyethylene stearate, polyoxyethylene sorbitol mono-oleate, polyoxyethylene sorbitan mono-oleate, etc.

Suitable suspending agents are, e.g., naturally occurring gums such as, e.g., acacia gum, xanthan gum or tragacanth gum; celluloses such as, e.g., sodium carboxymethyl cellulose, microcrystalline cellulose (e.g. Avicel® RC 591, methyl cellulose); alginates and chitosans such as, e.g., sodium alginate, et c.

The appropriate examples of condoms for use in the compositions according to the invention are the same as those mentioned above.

The compositions for use according to the invention could also be administered by the oral route.

Appropriate oral compositions could be in the form of a particulate formation or in the form of a solid, semi-solid or fluid dosage form.

Compositions for oral use include dosage forms such as, eg, powders, granules, granules, pads, tablets, capsules, effervescent tablets, chewable tablets, lozenges, immediate-release tablets and modified-release tablets, as well as fluid or liquid formulations. liquids such as, eg solutions, suspensions, emulsions, dispersions and mixtures. In addition, the composition could be in the form of powders, dispersible powders or granules suitable for the preparation of an aqueous suspension by the addition of a liquid medium such as, e.g. an aqueous medium With respect to solid dosage forms for oral (or topical) use, a composition for use according to the invention typically contains the active enamel substance and any additional active substances, optionally in admixture with one or more pharmaceutically acceptable excipients. . These excipients could be, for example, inert diluents or fillers, such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches, including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, etc. calcium sulfate or sodium phosphate; granulating and disintegrating agents, for example, cellulose derivatives, including microcrystalline cellulose, starches, including potato starch, croscarmellose sodium, alginates or alginic acid and chitosans; binding agents, for example, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pre-gelatin starch, cellulose - ^ Áy? ^? Y-m * »& & > - microcrystalline, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, polyvinyl acetate or polyethylene glycol; and chitosans; lubricating agents, including glidants and anti-adhesives, for example, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc.

Other pharmaceutically acceptable excipients may be dyes, flavoring agents, plasticizers, humectants, buffering agents, etc.

In these cases, when the pharmaceutical composition is in the form of a solid dosage form in the dosage form (eg a tablet or a capsule), the unit dosage form could be provided with a coating as one or more of the aforementioned coatings. later.

In cases where the composition is in the form of a tablet, capsule or a multiple unit composition, the composition or individual units or a tablet or capsule containing the individual units could be coated e.g., with a sugar coating, a film coating (eg based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers (Eudragit), polyethylene glycols and / or polyvinylpyrrolidone) or an enteric coating (eg based on methacrylic acid copolymer (Eudragit) , cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac and / or ethylcellulose). In addition, a delay time material such as, e.g., glyceryl monostearate or glyceryl distearate could be employed.

Rectal and / or vaginal compositions For application to the rectal or vaginal mucosa, appropriate compositions according to the invention include suppositories (emulsion or suspension type), enemas and rectal gelatin capsules (solutions or suspensions). Suitable pharmaceutically acceptable suppository bases include cocoa butter, esterified fatty acids, glycerinated gelatin and various water soluble or dispersible bases such as polyethylene glycols and polyoxyethylene sorbitan fatty acid esters. Various additives, e.g., enhancers or surfactants, could be employed.

Nasal compositions For application to the nasal mucosa (as well as to the oral mucosa), sprays and aerosols for inhalation, are appropriate compositions according to the invention. In a typical nasal composition, the active enamel substance is present in the form of a particulate formulation optionally dispersed in an appropriate vehicle. The pharmaceutically acceptable carriers and excipients and optionally other pharmaceutically acceptable materials present in the composition such as diluents, enhancers, flavoring agents, preservatives, etc. they are all selected in accordance with conventional pharmaceutical practice in a manner understood by those skilled in the art of pharmaceutical formulation.

Dosages of the enamel matrix, derived from the enamel matrix and enamel protein matrix In a pharmaceutical composition for use according to the invention in the skin or mucosa, an active enamel substance is generally present in a concentration ranging from about 0.01% to about 99.9% w / w. The amount of composition applied will normally result in a total protein amount per cm2 wound / skin / tissue area corresponding to approximately 0.01 mg / cm2 to approximately 20 mg / cm2 such as from approximately 0.1 mg / cm2 to approximately 15 mg / cm2. mg / cm2.

The applied amount of the composition depends on the concentration of the active enamel substance in the composition and the rate of release of the active enamel substance from the composition, but is generally in the range corresponding to the most 15-20 mg / cm2.

In cases where the active enamel substance is administered in the form of a fluid composition, the concentration of the active enamel substance in the composition is in the range corresponding to about 0.1 to about 50 mg / ml. Higher concentrations are in some cases desirable and may also be obtained such as a concentration of at least about 100 mg / ml.

When the composition is applied to the oral cavity, the following dosages are relevant: The areas of experimental defects (in monkeys) in the oral cavity typically have a size of about 4 x 2 x 5-6 mm corresponding to 50 μl or from about 0.025 to about 0.15 mg of total protein / mm2. It is usually applied up to 0.5 such as, e.g., 0.4, 0.3, 0.2 or 0.1 ml of a composition having a concentration of about 1-40 mg / ml such as, e.g., 5-30 mg / ml.

The areas of human defects in the oral cavity due to periodontal diseases typically have a size of approximately 5-10 x 2-4 x 5-10 mm which corresponds to approximately 200 μl and normally, to more than approximately 0.5- 1 ml, such as 0.2-0.3 ml per tooth, is applied of a composition having a concentration of about 1-40 mg total protein / ml such as, eg, 5-30 mg / ml is applied. 0.2-0.3 mg / ml corresponds to approximately 6 mg of protein per 25-100 mm2 or approximately 0.1 mg / mm2 if it is calculated only on the surface of the root. Normally, excessive volume is applied to allow coverage of all surfaces. Even a multilayer would only require a small fraction of the amounts mentioned above In general, approximately 0.1-0.05 ml as, e.g., about 0.15-0.3 ml or about 0.25-0.35 ml of a composition comprising the active enamel substance is applied in defect volumes in extraction socket (orifices after extraction of the tooth). The concentration of the active enamel substance in the composition is usually about 1-40 mg of total protein / ml such as, e.g., 5-30 ml / ml. When 0.3-0.4 ml of such composition is applied to the wisdom tooth, this volume corresponds to approximately 0.1 mg / cm2 (alveolus calculated as the cylinder with radius of 5 mm and height of 20 mm).

The concentration of the active enamel substance is a pharmaceutical composition dependent on the specific enamel substance, its potency, the severity of the disease to be prevented and the age and condition of the patient. The methods applicable to the selection of the relevant concentrations of the active enamel substance in the pharmaceutical composition are well known to one skilled in the art and could be performed according to well-established guidelines for good clinical practice (GCP) or regulations. of Investigational New Drug Exemption ("IND") as described in eg the International Standard ISO / DIS 14155 Clinical Investigation of Medical Devices, 1994 and ICI (International Commitee for Harmonization): Harmonized tripartite guideline for good clinical practice, Brookwood Medical Publications, Ltd, Surrey, UK, 1996. An expert in The art, by using the methods described in the manuals, guidelines and standard regulations as described above, as well as the general knowledge or common in the field, would be able to select the exact dosage regime to be implemented for. any active enamel substance and / or select other active substances and dosage form using simply routine experimentation procedures.

In other aspects, the invention relates to methods for i) preventing and / or treating wounds, ii) decreasing infection and iii) preventing and / or treating inflammation, the methods comprise administration to a mammal in need of such treatment of an effective amount of an active enamel substance.

As will be understood, the details and particularities 5 that relate to the use of an active enamel substance ai ^^^^^^^ t ^^ for the prevention and / or treatment of wounds will be the same as or the analogous ones for the details and particularities that refer to the other aspects of use (anti-bacterial and anti-bacterial aspects). inflammatories) and aspects of methods discussed above, and this means where appropriate, the above statements which relate to an active enamel substance, a preparation containing an active enamel substance, a pharmaceutical composition containing a substance of active enamel, the preparation of i) an active enamel substance, ii) a preparation containing an active enamel substance, iii) a pharmaceutical composition containing an active enamel substance, as well as the improved properties and uses, apply mutatis mutandis to all aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is further described with reference to the accompanying drawings in which: Fig. 1 is a graph showing the synthesis of DNA in human PDL cells stimulated with EMD or unstimulated cells; Fig. 2 is a graph showing the production of TGF-β1 in human PDL cells stimulated with EMD and unstimulated cells; Fig. 3 is a schematic drawing of a flow chamber and computer system used in the flow experiment described in Examples 3 and 4 below; Figs. 4, 5 and 6 are graphs showing the results of the three separate experiments showing the adhesion of A c t i n omyces vi s cos u s to glass plates treated with EMD and acetic acid, respectively; Figs. 7, 8 and 9 are graphs showing the results of three separate experiments showing the adhesion of Str ep t or co c cu cu m s t to glass plates treated with EMD and acetic acid, respectively; Fig. 10A is an X-ray photograph showing post operative damage after the removal of a wisdom tooth; Y Fig. 10B is an X-ray photograph showing regeneration of the periodontal ligament after treatment with EMD, as described in Example 12 below EXPERIMENTAL SECTION Materials and methods The enamel matrix derivative, EMDOGAIN®, from BIORA AB, S-205 12 Malm ?, Sweden, contains 30 mg of the frozen dried enamel protein matrix (in the following abbreviation for EMD) and 1 ml of solution of vehicle (propylene glycol alginate), are mixed before application, unless the protein and the vehicle are treated separately. The weight ratio is about 85/5/10 between the peaks of the main protein at 20, 14 and 5 kDa, respectively.

The heat treated EMDs are EMD that have been heated for 3 hours at about 80 ° C to inactivate the residual proteases.

The 20 kDa amelogenin protein and the tyrosine-rich amelogenin peptide (TRAP) of 5 kDa were isolated from EMD using HPLC gel penetration chromatography (TSK G-2000 SW equilibrated with 30% acetonitrile in 0.9% NaCl) and purified by reverse phase chromatography (Pro-RPC, HR 5/10, Pharmacia-Up j ohn, Sweden) using an acetonitrile gradient. The separated poly / poly peptides were then added in various amounts to the vehicle solution of EMDOGAIN®, unless separately tested.

Hyaluronic acid was HMT-0028 (MW 990,000) from Seikagaku Corporation, Tokyo, Japan.

Bacteria and yeast were first isolated from patients, classified by metabolic and antigenic properties according to standard procedures. The species of bacteria and yeast used are listed in the following table.

Albumin serum (bovine) and collagen type 1 (bovine) were obtained from Sigma, St. Louis, U.S.A.

The agar plates were all from "Brain Hart Infusion agar" by Difco, supplemented with red blood cells (100 ml per liter of agar).

EXAMPLES Example 1 Cell proliferation and production of TGF-ßl in PDL cells treated with EMDOGAIN® An EMD stock solution was prepared by dissolving one ampule (containing 30 mg of EMD) in 3 ml sterile filtered 0.1% HAc. 60 μl of the stock solution was added to 6000 μl of Dulbecco's Modified Eagle's Medium containing 10% calf lethal serum and 1% penicillin-streptomycin solution. 300 μl of the mixture was added to each well of the 96-well microtiter plates (NUNC A / S, Denmark, Cat. # 167008). 1000 Human Periodontal Ligament Cells (PDL) (obtained from the periodontal tissues of healthy humans of individuals who undergo premolar extractions for orthodontic reasons, and cultured substantially as described in Somerman et al., J. Den Tal Res. 67, 1998, pp. 66-70) were added to each well and incubated at 37 ° C, 5% C02 for 5 days.

The PDL cells used as control were cultured in Dulbecco's modified Eagle's Medium as described substantially above, but in the absence of EMD After incubation, the cells were subjected to a cell proliferation immunoprecipient by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) according to the manufacturer's instructions (Boehringer Mannheim, Cat. # 1647 229). In this procedure, BrdU is incorporated into the DNA of the growth cells instead of thymidine. The incorporation of BdrU by the ELISA test, and the amount of BrdU measured in the test is an indication of the speed of DNA synthesis and consequently the rate of cell proliferation of PDL cells.

The results shown in Fig. 1 show that PDL cells grown in the presence of EMD exhibit a significantly higher proliferation rate than PDL cells grown in the absence of EMD.

To 100 μl of the supernatant cells of the microtitre plate, 20 μl of IN HCl was added followed by incubation for 10 minutes at room temperature. The incubation mixture was neutralized with 20 μl of 1N NaOH / 0.5M HEPES. 100 μl of this mixture was added to 400 μl of a dilution buffer. 200 μl of the dilution was subjected to ELISA using the Quantikines ™ kit (# of Cat. DB 100) available from R &D Systems, UK, according to the manufacturer's instructions.

The results are shown in Fig. 2, showing a pronounced increase in the production of TGF-β1 in PDL cells incubated with EMD relative to PDL cells not incubated with EMD.

Example 2 Investigation of the growth of microorganisms in the presence of enamel matrix derivatives and enamel protein matrix.

The purpose of this example is to demonstrate the inhibitory influence of enamel matrix derivatives and enamel protein matrix on microbial growth.

The proteins used in this example were dissolved in phosphate buffered saline (PBS) with the pH adjusted to 5.5 with acetic acid. The microbes used were suspended in PBS, pH 6.8 at a final concentration that had an OD600 of 0.4. 50 microliters of EMDOGAIN® (30 mg of EMD in 1 ml of PGA) and 50 microliters of EMD, EMD treated with heat, fractions of EMD A, B, C and H (the 10 mg of the protein per ml of PBS buffer) were dripped on an agar plate and allowed to air dry on the top of the plate (9 cm in diameter, standard agar for the determination of added supplements for resistance as required by the individual microbes). A homogenous suspension of microbes (1 ml, OD280 = 0.5) was then added, atomizing the suspension on top of the agar plates, and the plates were incubated at 35 ° C for 3 days (aerobic cultures) or 14 days ( anaerobic cultures) in an atmosphere enriched with C02 or under the anaerobic condition according to the growth requirements of the individual. All crops were inspected daily. Type 1 collagen and serum albumin (both bovine) were tested under the same conditions as controls. Undiluted propylene glycollate (PGA-EMD vehicle), PBS buffer and hyaluronic acid (alternative HA-EMD vehicle) were also applied as negative controls.

The results are shown in Table 1. Only those derived from the enamel matrix or the matrix of the enamel proteins or derivatives inhibited the growth of some microbes. There were no signs of diffusion zones around the protein, indicating that the applied EMD proteins were added on the surface of the agar and that only the microbes in direct contact with the proteins were inhibited in growth. When they were shown they were harvested from the zones of inhibition and were cultivated in monocultures of liquid medium (LB broth with supplements) of the original microbes could be revived, suggesting that the active proteins are not microbicides. All the negative controls tested indicate that the non-specific mechanism did not influence the results.

TABLE 1 GROWTH (+/-) IN THE TOP OF THE TEST SUBSTANCE EMD fraction A: mainly amelogenin -26-20 kDa, EMD fraction B: proteins of -17-13 kDa, EMD fraction C: peptides of -10-5 kDa EMD fraction H: all proteins in EMD above 27 kDa of molecular weight. + indicates normal growth in microbes - indicates growth completely inhibited by microbes, +/- indicates some inhibition of growth when compared to negative controls.

All results in the table were recorded on the second day (aerobic cultures) or after five days (anaerobic cultures) of incubation.

These results show that proteins or peptides containing EMD, when allowed to aggregate on a surface, can inhibit the growth of certain Gram-negative rods and some Gram-positive cocci. Based on the characteristics of the basic behavior of the EMD proteins (ref jpc) and given that the effect is not microbicidal, a reasonable explanation for the effect observed in such aggregates forms an insoluble barrier that separates the microbes from the substrate or substrates from growth required.

Example 3 Effect of EMD on the adhesion rate of Act inomyces vi scosus in vitro Int rduction We explored the effect of EMD on the initial adhesion of Act inomyces viscosus, an oral organism that occurs widely in dental plaque, but is not commonly considered as associated with several periodontics. Although this organism could form aggregates with Porphyromona s gingiva 1 i s and in this way, could have an influence on the colonization of the root surface with potential pathogens, the Actinomyces spp. are found in relatively large proportions in healthy subgingival sites (these findings corroborate with those of Liljemark et al., Microbial, Immunol., 8, 1993, pp. 5-15, which found that, after periodontal treatment, the proportions of Act inomyces spp. Were significantly increased, Haffajee et al., J. Clin. Peridont, 24, 1997, pp. 767-776, concluded that the microbiological counts were relatively abundant in the subgmival plaque than in the subjects with a good response to the initial periodontal treatment of A. viscosus and T. denticola. materials Act inomyces viscosus HG85 was provided by Dr. A. J. van Winkelhoff (Dept. Of Oral Microbiology, ACTA). EMDOGAIN® was provided by BIORA (Malmó, Suencia). The RBS detergent was purchased from Fluka (Fluka Chemie AG, Buchs, Switzerland).

Bacterial growth and harvest A. viscosus was inoculated from the blood agar plates in intermittent culture in Schaedler's broth medium for 24 h at 37 ° C. This culture was used to inoculate a second culture in the Schaedler broth which was allowed to grow for 16 h. The cells were harvested by centrifugation (5 min at 6500 x g) and washed twice with demineralized water. Subsequently, the microorganisms were sonicated for 20 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to break the chains and bacterial aggregates. The sonication was done intermittently while cooling in a bath with ice and water. The cells were counted using a Bür ker-Tür ker cell counter.

Finally, A was suspended. vi s cos u s in the adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride and 1 mM calcium chloride, pH 6.8).

Coating of glass plates The glass plates were completely cleaned by sonication in 5% RBS detergent, rinsed extensively with tap water, washed in methanol and finally rinsing with distilled water. This procedure produced a water contact angle of zero degrees. The EMDs were dissolved in 0.01M acetic acid in a concentration of 7.5 mg / ml. The glass plates were divided into two halves using Teflon marker (DAKO A / S, Glostrup, Denmark). Acetic acid (0.01M) was applied to one side; 250 μg of EMD to the other. The plates were air dried in a flow chamber for 4-6 h.

Flow experiment The flow chamber and the computer system used in this experiment are shown schematically in the Fig. 3. Before each experiment, all the tubes and the flow chamber were filled with adhesion damper, care was taken that the system did not contain air bubbles. The coated glass plates were formed at the base of the flow chamber. The flow rate was set at 2.5 ml / min (which is comparable to the average flow velocity of human saliva). The bacterial suspension was circulated through the system for about 3-4 h, and the number of bacteria adhered to the substrate was counted. Three independent experiments were carried out. All experiments were performed with 3xl08 cells per 250 ml of adhesion buffer. During the experiment, the images were taken every 10-15 min at 6 predetermined sites on the EMD-coated and control plates. The channel height of the parallel flow chamber was 0.6 mm.

Analysis of results After counting the adhered cells in all the images, the results were transformed to bacteria per square centimeter. For each experiment, the final number of microorganisms per cm2 was used for statistical analysis (Student's t-test for paired observations using n as the number of experiments).

Results Experiment 1 (Fig. 4) showed a gradual increase in the amount of bound microorganisms particularly during the first 150 min of flow. After this time interval, the number of microorganisms bound to EMD had reached a plateau of 2.0x106 bacteria per cm2 that was approximately four times higher than on the acetic acid treated aspect of the glass plate.

Also in experiment 2 (Fig. 5), EMDs stimulated the adhesion of A very rapidly. I saw cos u s to the substrate. However, at the beginning of the experiment the effect was less clear. Perhaps this was due to a lower density of organisms used in the flow system. After 90 min the number of bacteria that bind to EMD increased gradually relative to the control, reaching a maximum of 1.4xl06 per cm2 after 3 h, a threefold increase.

The third experiment (Fig. 6) showed a stimulation of the adhesion of A. We saw the EMD coating after 5 min of flow. The EMD induced a rapid increase in the nr of organisms bound during the first 45 min. Subsequently, the adhesion continued progressively. Adhesion to the side treated with acetic acid showed a similar pattern, but with less adhesion of microorganisms. After 200 min, the adhesion to the coating to EMD was twice as high as on the surface treated with acetic acid.

In the 3 experiments, taken together, the difference was proved to be statistically significant (p <0.05).

From the results, it appears that EMD, used as a coating on a glass surface, has a considerable stimulating effect on the adhesion of A. vi s cos u s. Although it is not yet known what factors are responsible for this improved initial adhesion, it is assumed that the organism interacts with the proline residues that are richly present in the amelogenin component of the commercially available protein mixture. Bacterial adhesion is frequently determined by the recognition of protein-na-peptide and lect-carbohydrate lect. It is known that A. vi s cos us, with its type 1 fimbria, can be linked to proline-rich proteins, such as salivary proline-rich proteins (PRP's) and type I and II collagens.

The specific interactions between EMD and certain oral microorganisms could have important consequences for the composition of the biofilm in the oral cavity, since the ecology of the plaque could change. If the ecological changes could be made in the direction of the promoter organisms not associated with periodontal disease, the application of EMD could result in the important improvement of the periodontal condition, just by such action. This, of course, is very different from the other beneficial effects of EMD.

Example 4 Effect of EMD on the adhesion speed of S t rep ococcus m u ta n s in vi t ro Introduction There is a large body of evidence for a causative role of plaque organisms in the pathogenesis of oral diseases, such as periodontitis and caries. According to the current model of supragingival plaque formation, it is thought that S t rep t o co cc us ssp. They are important colonizers of the surface of the teeth. Subsequently, the plaque develops by bacterial growth and by additional accretion of other species of bacteria. This accretion can occur through the linkage of bacteria to bacteria or could be mediated by salivary molecules. The formation of plaque is also facilitated by the production of extracellular macromolecules. S. mutans is now considered to be one of the biofilm species that could guarantee the closest attention, due to its association with dental caries. Although several Gram positive bacteria (i.e. S. mutans) have been shown to cause alveolar bone loss in gnot obiotic animals, these microorganisms do not appear to be the main contributors to the ecology of periodontal pocket development. However, potentially pathological microorganisms must be able to evade host defense and immune mechanisms, as well as the destruction of the initiation of the woven host. materials Streptococcus mutans NS was kindly provided by Dr. H. van der Mei (Materia Technica, University of Groningen). EMDOGAIN® was provided by BIORA (Malmo, Sweden), the RBS detergent was purchased from Fluka (Fluka Chemie AG, Buchs, Switzerland) Bacterial growth and harvest S was inoculated m u ta n s of blood agar plates in intermittent culture in Todd Hewitt broth medium for 24 h at 37 ° C. This culture was used to inoculate a second culture in the Todd Hewitt broth medium, which was allowed to grow for 16 h. The cells were harvested by centrifugation (5 min at 6500 x g) and washed twice with demineralized water. Subsequently, the microorganisms were sonicated for 20 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to break the chains and bacterial aggregates. The cells were counted using a Bürker-Türker cell counter. Finally, S was suspended. m u t n s in the adhesion buffer (2 M potassium phosphate, 50 mM potassium chloride and 1 mM calcium chloride, pH 6.8).

Coating of glass plates The glass plates were completely cleaned by sonication in 5% RBS detergent, rinsed extensively with tap water, washed in methanol and finally rinsing with distilled water. This procedure produced a water contact angle of zero degrees. The DMEs were dissolved in 0.01 acetic acid at a concentration of 7.5 mg / ml. The glass plates were divided into two halves using Teflon marker (DAKO A / S, Glostrup, Denmark). Acetic acid (0.01M) was applied to one side; 250 μg of EMD to the other. The plates were air dried in a flow chamber for 4-6 h.

Flow experiment The flow chamber and the computer system used in this experiment are shown schematically in Fig. 3. Before each experiment, all the tubes and the flow chamber were filled with adhesion damper, care was taken that the system did not contained air bubbles. The coated glass plates were formed at the base of the flow chamber. The flow rate was set at 2.5 ml / min (which is comparable to the average flow velocity of human saliva). The bacterial suspension was circulated through the system for about 3-4 h, and the number of bacteria adhered to the substrate was counted. Three independent experiments were carried out. All experiments were performed with 3x10 ° cells per 250 ml of adhesion buffer. During the experiment, the images were taken every 10-15 min at 6 predetermined sites on the EMD-coated and control plates. The channel height of the parallel flow chamber was 0.6 mm.

Analysis of results After counting the adhered cells in all the images, the results were transformed to bacteria per square centimeter. For each experiment, the number end of microorganisms per cm2 was used for statistical analysis (Student's t-test for paired observations using n as the number of experiments).

Results 15 In each of the three experiments, the EMD showed an inhibitory effect on the adhesion rate of S. m u t n n s (Figs 7, 8, 9; p < 0.05). The inhibition amounted to approximately 40-70% when compared with controls treated with acetic acid The first experiment (Fig. 7) showed an inhibition of the amount of S after 10 min of flow. m u t a n s attached to the glass surface coated with EMD.

After 3 h, the adhesion was inhibited to approximately ^^^^^^^^ g * ¿^ ^ ^ ^^^^ ^ ^ ^^^^ j ^ j ^^^^ 60% control.

In the second experiment (Fig. 8) EMP started to inhibit the adhesion of S. mutans after 1 ^ h of flow. The number of S. mutans adhering to EMD reached a plateau of 0.5 million per cm2 after approximately 40 min of flow. After 3 ^ h, the accounts were approximately 25% compared to the controls.

The third experiment (Fig. 9) showed the inhibition of the adhesion of S. mutans under the influence of EMD from the beginning of the flow procedure. As in experiment 1, the inhibition increased to 60% of the control values after 3 h of flow.

The present study shows that EMDs have a significant inhibitory effect on the adhesion of S. mutans to glass surfaces. A possible explanation for this inhibition could be the presence of hydrophobic compounds in the EMD mixture. One of the proteins abundantly present in the mixture is amelogenin, a protein that contains, apart from an acidic hydrophilic C-terminal sequence, a hydrophobic center containing 100-300 residues enriched in proline, leucine, methionine and glutamine. Saito et al., Arch. Oral Biol. 42, 1997, pp. 539-545, found that the adherence of several strains of S. mutans to an immobilized hydrophobic protein (OAIS) was inhibited. The authors attribute the effect to the negative charge on the cell surface of the microorganisms (which is the case for S. mutans). Other surface characteristics could also be involved in the adhesion affected by the substrate. S. mutans contains an I / II surface antigen that has an N-terminal part particularly rich in alanine and includes random repeats. This region is predicted to be alpha-helical, adopting a coiled conformation, and could take into account the hydrophobicity of the cell surface associated with the expression of the I / I antigen.

Example 5 Effect of EMD on the growth of certain periopathogens The Prevotel intermediate and Porphyromonas gingivalis were pre-cultured for 10-16 hours at 37 ° C in thioglycollate broth supplemented with 0.5 mg / l of vitamin K and 5 mg / l of hemin in an aerobic atmosphere generated by GasPakPlus casings. in appropriate containers.

When the cultures reached an OD600 of 0.1-0.2 corresponding to the cell densities of 106-107 cfu (colony forming units) per ml, aliquots of 100 μl were extracted and the bacteria were precipitated by centrifugation. Bacteria were resuspended in 100 μl of a freshly prepared mixture of human serum and sterile saline, and suspensions containing 105-106 cells were transferred to sterile 1.5 ml Eppendorf tubes and mixed with (i) 100 μl of preparation of EMD (3 mg of EMD in 0.1 ml of PGA), (li) 100 μl of vehicle PGA or (iii) 100 μl of mixture of solution of serum / NaCl as the control of growth. 10 μl aliquots were taken for growth tests after 0, 3, 6 and 24 hours. The aliquots were serially diluted in sterilized 0.9% NaCl solution and 10 μl of the dilution steps were plated on Schaedler agar. The culture conditions were the same as for the precultures. The agar plates were incubated for 3-4 days and the cfus and cellular densities (cfu / ml) were subsequently calculated. All the experiments were repeated six times.

Results (given as cfu / ml in percent of the concentration at time 0). 1) Control crops at different time points 2) Crops in the presence of PGA vehicle at different time points 3) Crops in the presence of EMD at different points of time ^^^^^^^^ gj ^^ jf ^ The cultures were markedly inhibited by the presence of EMD compared to controls with the vehicle alone or without any addition of EMD.

Example 6 Investigation of the healing effect of EMDOGAIN® soft tissue wounds after periodontal surgery.

The purpose of this example is to show the influence of the enamel matrix derivatives and / or the enamel protein matrix on the healing of improved soft tissue wounds after periodontal surgery.

The experimental defects in the marginal periodontium of more than 50 teeth of Macaque monkeys were created by removing the dental cement, the periodontal membrane and the marginal alveolar bone at a cervico-apical distance of approximately 5 mm with a dental burr. Neither (the control) nor the enamel matrix derivative (obtained from EMDOGAIN® either as the non-reconstituted lyophilized powder or as the reconstituted composition) were then applied to the experimental defects. The concentration of the proteins in the reconstituted composition was about 5-30 mg / ml and the volume applied was in the range of about 0.1 to about 0.2 ml by default.

Wound healing was assessed visually during the following 8 weeks. In the defects where EMDOGAIN® was applied, good curing was present (there was no red color or swelling) and the plaque was insignificant after 2 weeks when the sutures were removed, good healing and little gingivitis after 5 weeks and curing without complications after 8 weeks, when the experiments were finished. In contrast, control defects showed inflammation with retractions and abundant plaque after 2 weeks, with severe retractions and gingivitis both after 5 weeks and after 8 weeks.

Example 7 Research on the wound healing effect of enamel matrix derivatives and enamel protein matrix after periodontal surgery.

The purpose of this example is to show the influence of enamel matrix derivatives and the enamel protein matrix on the rapid healing of wounds after periodontal surgery Fifty-five (55) patients who needed periodontal surgery were divided into two groups, one obtaining conventional surgery with the modified Widman flap technique (20 patients) and the other with the same procedure plus the application of EMDOGAIN® ( 35 patients) (the concentration was 30 mg protein / ml and approximately 0.3 ml was applied per tooth). None of the patients received antibiotics at the time of surgery, but all were instructed to use aseptic mouth wash (chlorhexidine) daily.

The active questioning of the patients was carried out at the time of removal of the sutures (1-3 weeks after surgery). While 3 (15%) of the control patients had post-surgical cases that required antibiotics, only one of the patients treated with EMDOGAIN® (3%) needed such treatment.

Example 8 Investigation of wound healing effect of enamel matrix derivatives and enamel protein matrix after tooth extraction.

The purpose of this example is to show the influence of enamel proteins / enamel matrix derivatives on wound healing after 3rd molar extraction.

Patients aged 30 years or older with molars of the third impacted or semi-impacted symmetrical jaw requiring the removal had a third molar extracted by the classical method, which involves lifting a vertical flap to perform the necessary osteoctomy and sectioning, while that the second was removed and the alveolus was filled with EMDOGAIN® before suturing. All patients received antibiotics (3 g of Amoxilicin or 1 g of Erythromycin) 1-2 hours before surgery and were given Ibuprofen (600 mg x 3) after surgery. Then they were instructed to rinse with Clothexidine (0.1%, 10 ml x 2) for 4 weeks.

The sutures were removed after 2 weeks. The curing of EMDOGAIN® and the control sites were evaluated by the patient and the dentist. In one center, 9 patients had extra-lateral extractions with / without EMDOGAIN®. One patient had slight irritation of the sutures at both sites, while another patient had severe pain only at the control site, but had no problems at the site treated with EMDOGAIN®. In a second center, three patients out of 6 had pain only at the control sites. Finally, in a third center, a patient had a serious case, alveolitis, which was diagnosed at the control site of a patient. The site treated with EMDOGAIN® was cured without problems. Another patient had slight irritation of the sutures at both extraction sites, but only the control site was inflamed and the pain and repeated irrigations with saline and the entry of pain killers were required.

These clinical results indicate that the application of EMDOGAIN® in the extraction socket after the extraction of the wisdom tooth can improve the healing and reduce the frequent painful swelling.

E j us 9 Investigation of the effect of enamel matrix derivatives and enamel matrix proteins in the healing of alveolitis sicca The purpose of this example is to show the influence of enamel / dermal matrix proteins on the healing of alveolitis sicca (dry bag) After the removal of an infected widow root, a male patient, aged 70, experienced severe pain and swelling relative to the extraction socket. When examined by this dentist, it became clear that he had developed a condition of alveolitis sicca, in which the initial clot had disintegrated and the wall of the alveolus bone was necrotic. The adjacent bone and soft tissues were inflamed.

The patient had a history of heart failure and was treated with the anticoagulant Marevan. As a result of his condition, peripheral blood circulation had been reduced. He also regularly smoked several cigarettes per day.

The alveolitis was treated in the traditional way with the removal of the necrotic bone and the induction of the new bleeding. Also, the gingiva was mobilized and a suture was applied to close the alveolus. The patient was then treated with penicillin (apocilin 660 mg, 2 tablets in the morning and in the evening for seven days) to counteract the infection and also instructed to rinse his mouth twice daily with a chlorhexidine solution. After five days, after finishing his antibiotic regimen, the patient showed after the dental clinic still complaints of severe pain. The inspection of the area of operation was performed visually and by palpitation and it was proved and showed that the alveolitis persisted and that necrotic bone was present. The rays X revealed bone destruction and necrosis all the way down the apical part of the alveolus. The area of operation was cleaned once more and the resulting bone lesion was filled with EMDOGAIN® (30 mg / ml, maximum 0.5 ml was applied), and a new suture was placed in the gingiva to close the alveolus. No additional treatment was instituted, but the patient was told to continue rinsing with chlorhexidine solution. Two days later the patient reported to the clinic that the pain and swelling had gone away. Clinical examination and removal of the suture a few weeks after the treatment * > ** _ &. * > * & * > »* With EMDOGAIN® revealed good healing without signs of necrotic tissue or inflammation and an intact gingiva with no red color or swelling covered the wound area. There was no bleeding or pain when palpated or tested. No odor or dirty taste or exudates could be observed. The patient reported no pain or other symptoms found. Example 10 Investigation of the prophylactic effect of enamel matrix derivatives and enamel protein matrix in alveolitis sicca The purpose of this example is to show the prophylactic effect of the enamel matrix derivatives and the enamel protein matrix to counteract the sicca alveolitis.

An 82-year-old female patient experienced a longitudinal root fracture of tooth 44. This tooth was a pillar of a bridge extending from tooth 35 to 46, and had undergone endodontic treatment several previous years. Clinically, the gingiva surrounding the tooth became inflamed and there was no gingival pocket in the entire apex of the tooth on the lingual side. X-rays showed several local periodontics of tooth 44 The patient had good oral hygiene, but due to a cardiac condition treated with Marevan (anti- coagulant), bleeding from the gingiva was easily triggered by the probe. Six months earlier the patient had had his 35 tooth surgically removed due to severe periodontitis. After such an operation, she experienced a long-lasting condition of the alveolitis sicca. She was very concerned that the removal of tooth 44 would not cause the same post-surgical complications she had experienced at the time. She was informed that the combination of her high age, treatment with Marevan and the infected root and the gingival pocket dramatically increased the risk of post-operative complications such as alveolitis, but that there was no alternative, but surgically removing the fragments of the root.

The patient agreed to have tooth 44 removed and, as an experiment, underwent prophylactic treatment with EMDOGAIN® to prevent the development of alveolitis sicca. The patient was anesthetized with incision and measurement of the buccal bone to allow the removal of the root fragment without releasing the bridge. After the removal, the free alveolus was mechanically cleaned with EMDOGAIN® (30 mg / ml, maximum 0.5 ml was applied) and the flap was replaced with a suture. On the same afternoon, the patient reported (by phone) prolonged bleeding from the area of operation (treatment with Marevan did not stop before surgery), but no other symptoms. When the suture was removed five days after the surgery, the soft tissue wound of operation had completely healed. The patient did not report any symptoms such as pain or swelling after the surgery and in general, he was very pleased with the treatment.

Example 11 Investigation of the effect of enamel matrix derivatives and enamel protein matrix on the wound of post-traumatic complications The purpose of this example is to show the influence of enamel / enamel matrix derivative proteins on the healing of post-rathmatics complications in a patient.

After an accident, a patient had the affected upper frontal tooth tied in an emergency clinic. The dentist found avital teeth 11 and 21, teeth 12 and 22 had mesio-incisal fractures of class I or II. The marginal gingiva was severely inflamed and poorly adhered to tooth surfaces. The patient had pain and complained of numbness, swelling and bad taste and smell. There was also evidence of periodontal ligament injury in the apical regions of teeth 11 and 21. Both of the central incisors were cleaned and the root was filled with freshly mixed Ca (OH) 2.

After 4 weeks, the healing of the wound was still judged as unsatisfactory. The condition had evolved into a chronic inflammation and the teeth were considered as lost. The standard treatment for this condition would be removal of all four incisors and replacement with a bridge or implants. However, the patient strongly opposed this treatment and as a last effort to save the teeth, a surgery of the gingival flap was performed on the four affected teeth (11, 12, 21, 22). Two ampoules of EMDOGAIN® (60 mg in 3 ml) were used.

At the most 2 ml of EMDOGAIN® (30 mg / ml) per tooth was applied with a syringe before the flaps were sutured with 7 stitches. Four of the sutures were removed 5 days after surgery. There was then a marked improvement in the subjective and clinical conditions. The patient no longer complained of pain, the feeling of numbness was gone and there was no smell or dirty taste of the affected area. After 2 weeks the remaining sutures were removed. The gingiva showed no signs of inflammation and the patient had no complaints. The gingiva was probed and had no signs of inflammation; it was firmly attached to the teeth and / or the alveolar bone, it was pink (not different from red as was observed in inflamed areas) and with normal interdental papilla (not swollen). In addition, a marked improvement was observed as the reappearance of the periodontal ligament in the affected parts of the tooth, and the deposition of the new alveolar bone as visualized by X-ray examination.

Example 12 Traumatic wound healing on neighboring teeth and nerves Case report A 39-year-old female patient experienced a severe pericoronitis around her lower left wisdom tooth (38). In the public dental clinic, the tooth was partially removed, leaving the apical half of the tooth in the jaw after a fracture of the iatrogenic root. With the pain, the patient was referred to a specialist in oral surgery the next day for the surgical removal of the root fragment.

Two days after the surgery, the patient went to see her regular dentist for control. The patient was swollen from her left side and showed a persistent and complete block of the left mandibular nerve. Clinical examination and X-ray photographs showed that during the surgery, severe damage was done by perforating the mandibular bone, the third apical of the distal root of teeth 37 and mandibular nerve channel (see X-rays, Fig. IA). The distal depth of the probe pocket in teeth 37 was 25 mm from the top of the crown of the teeth that was the apex of the distal root. In an effort to induce bone and nerve healing and regeneration of the lost periodontal ligament in tooth 37, the operation wound was opened and carefully cleaned. After debridement with saline from the exposed bone, the surface of the r ak 'yi, - & a • * & ** * - ^ • * • - * * * * "- ~ ** -. ** &- 1 _: _ yí-» _ distal root of 37 and the mandibular nerve was covered with EMDOGAIN® (30 mg / ml, applied in excess, ca. 1 ml) and the wound was sewn together with three sutures.The patient was instructed to rinse her mouth with a solution of chlorhexidine (Corsodyl®) twice a day for the next five days and in the On day five the prophylactic treatment with penicillin (Ampicillin, 660 mg x 4) was started.

After ten days, the patient returned for control and removal of the sutures. At this time, the swelling disappeared and the soft tissue healing was very good. However, complete anesthesia of the mandibular nerve persisted and the patient was informed that the prognosis for a ruptured nerve is, at best, uncertain. At this point, anesthesia made it impossible to prove the viability of tooth 37. Normally, root damage like the one presented here led to necrosis of the pulp and ankylosis of the tooth. To avoid these complications, endodontic treatment is indicated. However, to observe if the experimental treatment could promote a healing of the periodontal ligament, the patient agreed to leave the untreated tooth during this time. The patient was then registered for monthly checks.

Two months after the previous control, the patient had local hyperesthesia in his left lower lip, a sign of nerve healing. The soft tissue in region 37-38 was perfectly healed without healing. The X-rays also revealed the new bone formation in the extraction socket. The tooth 37 and the surrounding tissue still suffered from anesthesia.

Four months after the treatment, the anesthesia was removed and tooth 37 proved vital, but hyper sensitive, due to sensitivity tests to temperature and electricity. X-rays showed that the bone that fills the extraction socket was significant and there were no signs of periodontal regeneration in the distal root of tooth 37.

Five months after the initial treatment with EMDOGAIN®, the vitality of tooth 37 was proven normal. At this time, a complete regeneration of a functional periodontal ligament in the X-rays was evident (Fig. IB) and the newly formed alveolar bone of normal appearance had filled in the bone defects and the extraction of the alveolus. There were no signs of anisitis.

The distal depth of the bag probe in tooth 37 was again only 10 mm which was approximately 1 mm below the cement-enamel joint. After this check the patient was dismissed as completely cured and registered for ordinary calls at one year intervals.

Comments : Complete and rapid healing of traumatic wounds in neighboring teeth and nerves after surgical removal of the wisdom tooth is rare. Usually complications as severe as those reported above end with complete removal of the damaged tooth, or at least the endodontic removal of the tooth pulp and root filling followed by bone healing with ankylosis. A broken nerve usually takes 8 to 12 months to heal, if at all, and some regions with paresthesia often persist for several years. The rapid and good quality of the healing reported above is very unusual and should be considered a signal for EMDOGAIN® wound healing ability.

X-rays A: Patient two days after tooth removal 38. Observe the large distal defect in tooth 37 and the involvement of the mandibular canal. The distobuccal alveolar bone in tooth 37 was also removed during surgery.

B: Patient five months after surgery. Observe the sign of the complete functional periodontal ligament (hard lamina) in defect on the distal part of the root of tooth 37. There are no signs of ankylosis. The contour of the mandibular canal can now be observed and the extraction of the alveolus is completely filled with bone. Also note that the new distobucal alveolar bone forms around the tooth 37.

E xemployment 13 Investigation of the effect of the enamel matrix derivatives and the enamel protein matrix in the healing of ulcus cruris (venous ulcer) Patient 1 The patient was male, born in 1926 and had a history of recurrent thrombosis disease with poor post-tumult syndrome and recurrent venous ulcers. It was treated systemically with coumarin anticoagulant derivatives, and the ulcers were treated locally with Ciclodex (dextran monomer) BIOGAL and 3% boric acid solution.

At the time of initiation of treatment with EMDOGAIN®, the patient had a venous ulcer that had an oval size of 5 x 4 cm and a depth of 0.5 mm, which was in the granulation stage with very bad epithelialization.

The wound was disinfected with 3% H202, and 500 μl of EMDOGAIN was applied and atomized equally by means of a sterile bar. The EMDOGAIN® was left for 10 minutes in the air and then the wound was covered with Inadine (Johnson% Johnson) rayon bandage impregnated with 10% povidone-sodium odorant.

After 5 days, the epithelialization in the proximal part of the ulcer had been carried out and the ulcer decreased by 1.8 x 2.2 cm, and there was no collateral reaction (inflammation). EMDOGAIN® was not applied. After 12 days of additional epithelialization in the proximal part and the new epithelization in the lateral part were carried out in an area of approximately 2 x 2 cm. Almost half of the ulcer had healed. 400 μl of EMDOGAIN® was applied.

After 19 days, the additional epithelialization in the proximal and lateral parts of the ulcer had been carried out, but not in the distal part where the ulcer was instead of this deep (approximately 1 mm). More than half of the ulcer had healed. 300 μl of EMDOGAIN® was applied. Given the initiation of treatment with EMDOGAIN®, the patient did not feel any pain in the ulcer, the opposite of what he felt before the initiation of treatment. EMDOGAIN® was then applied once a week for up to 40 days (at 200 μl), and the ulcer was considered completely cured after 47 days.

Patient 2 The patient was female, born in 1949 and had varicose veins, chronic venous insufficiency and recurrent venous ulcers. The patient had a polyvalent allergy to pharmacists (drugs, medicines), as well as varicose eczema. The patient had previously been treated locally with Otosporin drugs (polymyxin B sulfate + neomycin sulfate + hydrocortisone) and a hydrocortisone compress.

At the time of initiation of treatment with EMDOGAIN®, her venous ulcer was 1 cm in diameter and 2 mm deep. 300 μl of EMDOGAIN® was applied.

After 5 days, the epithelialization was 2 mm around the wound (circumferentially) and there was no collateral reaction (inflammation). EMDOGAIN® was not applied. After 12 days, the size of the ulcer had decreased by approximately 2 mm in diameter, 100 μl of EMDOGAIN® was applied.

After 19 days, the ulcer was still approximately 2 mm in diameter, but the base was well granulated and the ulcer was not very deep (0.2 mm). 100 μl of EMDOGAIN® was applied.

The same patient had another ulcer on the other leg of approximately 0.1x1 cm. 200 μl of EMDOGAIN® was applied.

After 7 days, new epithelialization and good granulation was present in the background and the size had decreased to approximately 0.2 x 0.5 cm.

Then 100 μl of EMDOGAIN® was applied to each ulcer once a week until day 40, and the ulcers were considered completely cured after 47 days. No allergic reactions were observed with EMDOGAIN®.

Another ulcer had formed in the same leg, which had a size of approximately 0.5x0.3 cm to which 100 μl of EMDOGAIN® was applied.

Patient 3 The patient was female, born in 1929 and had deep vein thrombosis after erysipelas, and at the time of initiation of treatment with EMDOGAIN®, the patient showed a very large ulcus cruris that had a size of approximately 15 x 19 cm in the state of progression, the ulcer was considered almost disastrous after several treatments. 700 μl of EMDOGAIN® was applied in an area of approximately 3 cm from the upper margin.

After 7 days, epithelialization was not present, but the treated area was more transparent (-more structural) with small areas healed of granulation and there was no pain in this area or signs of progression. 700 μl of EMDOGAIN® was applied to the same area.

After 4 weeks, the patient developed an infection, which is thought to be caused by Ps e udom on s, in the distal part of the ulcer not treated with EMDOGAIN®. 700 μl of EMDOGAIN® was applied. The infection had disappeared after 7 days.

Patient 4 The patient was female, born in 1947 and had varicose veins with superficial thrombophlebitis after erysipelas, and at the time of initiation of EMDOGAIN® treatment, the patient had ulcus cruris that was approximately 2 x 0.8 cm in size with cleanliness, but without granulation in the base. 300 μl of EMDOGAIN® was applied.

After 7 days, the size of the ulcer had decreased to approximately 0.7 x 0.3 cm and the epithelization was present all around and the granulation in the background. 200 μl of EMDOGAIN® was applied, followed by 100 μl of EMDOGAIN® once a week for five weeks. The ulcer was considered completely cured after five weeks.

Example 14 EMDOGAIN® as an adjunct for non-surgical periodontal treatment in superficial superficial sites.

Objective The objective of the investigation was to evaluate if the application of EMDOGAIN® can improve the result of the healing of the non-surgical periodontal treatment. The specific help of this study was to evaluate the effect on flat surface sites.

Study design The study was carried out as a longitudinal 6-month longitudinal indi- vidual test. The study had a randomized design and double-shielded, split mouth, placebo controlled.

Suj ets 14 patients referred to the Periodontics Clinic, Department of Periodontology, University of Gdteborg, for the treatment of advanced periodontal disease.

Criterion for inclusion At least 3 sites of flat tooth surfaces in each of the 2 collateral quadrants with depth of the probe pocket = 5 mm, and at least a couple of sites with a probe depth of = 6 mm The selected teeth must have a vital pulp as determined by thermal or electrical stimulation or, if subjected to the treatment of the root canal, be asymptomatic and without technical issues.

Treatment After a baseline examination, all patients were given the case presentation and instructions on the control measures of the appropriate supragingi val plate. Graduation and planning of the root When the bleeding of the bags had ceased, 24% EDTA gel (obtained from Biora AB, Sweden) was applied in the bags for 2 minutes. The bags were then carefully irrigated with saline followed by the application of the test substance (EMDOGAIN®) or control (PGA gel).

Analysis The baseline examination, 1, 2, 3, 8 and 24 weeks after the examinations included the variables: 1. State of oral hygiene - presence / absence of plaque 2. Gingival condition Depth of the probe pocket 4. Level of attachment of the probe . Bleeding on the probe - presence / absence (15 seconds) 6. Hypersensitivity to dentine - after air blast stimulus (yes / no) 7. Degree of discomfort - recorded in weeks 1, 2 and 3 after examinations using a Visual Analogue Scale (VAS) < < of 10 cm.

PLATE: medium (sd) GINGIVAL INDEX: Media (sd) BLEEDING IN THE PATIENT PROBE: SUBJECTIVE EVALUATION; VAS annotation Week 1 Week 3 Conclusion ion The patients had fewer post-operative problems, less bleeding, less plaque and an improved gingival index. These results support the wound healing effect of EMDOGAIN®.

E xemplo 15 Pilot study of wound healing in pigs Introduction Objective The objective of this pilot study is to evaluate the healing process of wounds of thickness agietado in pigs, and to evaluate the effect of EMD in these wounds.

Reason for the choice of animal species The pig is selected as the test model because this species has provided a good model for the verification of wound healing in humans.

Materials and methods Animals The experiment will be carried out in 4 female SPF pigs (crossed from Danish country, Yorshire and Duroc). At the beginning of the acclimation period, the body weight of the animals will be approximately 35 kg.

A one-week acclimation period will be allowed, during which the animals will be observed daily to reject an animal that presents a poor condition. All observations will be recorded. accommodation The study will be carried out in a room for animals provided with filtered air at a temperature of 21 ° C ± 3 ° C, relative humidity of 55% ± 15% and air change of 10 times / hour. The room will be illuminated to give a cycle of 12 hours of light and 12 hours of darkness. The light will be from 06 to 18 h.

The animals will be housed individually in pens. Bed for the animals The bed for the animals will be made of softwood sawdust "LIGNOCEL H 3/4" by Hahn & Co., D-24796 Bredenbek- Kronsburg. Regular analyzes will be carried out for possible relevant contaminants.

Diet A commercially available pig diet, "Altromin 9033" by Chr. Petersen A / S, DK-4100 Ringsted will be offered (approximately 800 g twice daily). The analysis for the main nutritive components and the possible relevant contaminants are carried out regularly.

Water to drink Twice daily the animals will be offered 20 water to drink domestic quality. Analyzes are carried out regularly for possible relevant contaminants. ^^ ¡fa ^^ Zjtí Formation of wounds The wounds will be established on day 1. The animals will be anesthetized with Stresnil® Vet. Janssen, Belgium (40 mg of a zaperone / ml, 1 ml / 10 kg), and Atropin DAK, Denmark (1 mg of atropine / ml, 0.5 ml / 10 kg), given as a simple intramuscular injection followed by the i.v. from Hypnodil® Janssen, Belgium (50 mg of me t omida t / ml, approximately 2 ml).

A dor area will usually be shaved on the back side of the animal, washed with soap and water, disinfected with 70% ethanol, rinsed with saline, and finally dried with sterile gauze.

Eight wounds of cracked thickness (25 x 25 x 0.4 mm) will be made in the prepared area, 4 on each side of the spine, using an ACCU- Dermat om (GA 630, Aesculap®). The wounds will be numbered from 1 (more cranial) to 4 (more caudal) on the left side of the animal, and 5 (more cranial) to 8 (more caudal) on the right side of the animal.

Coagulated blood will be removed by sterilized gauze. Just before surgery, approximately 8 hours after the end of surgery, and where necessary, animals will then be given an intramuscular injection of Anorfin®, A / S GEA, Denmark (0.3 mg buprenorphine / ml, 0.04 ml / kg).

Dosage After the formation of the wounds, the wounds will be treated as follows: To EMD control At approximately 15 minutes before dosing, the EMD formulation will be prepared according to the instructions given by the manufacturer. The EMD formulation will be used within 2 hours after the preparation. For the wounds of treatment B, the EMDs will be applied as a thin layer on the surface of the wound. One vial of EMD will be used for 4 wounds.

Wound bandage The wounds will be bandaged with Tegaderm®. The bandages will be covered with a gauze bandage fixed with Fixomul®. The bandages, the gauze and the Fixomul® will be subject by means of a mesh-like body stocking, Bend-a-rate® (Tesval, Italy). Bandages will be observed on a daily basis.

The bandages will be changed on day 2 (all animals) and 3 (Nos. Of animals 3 and 4).

Before each change, the animals will be anesthetized with an intramuscular injection in the neck (1.0 ml / 10 kg body weight) of a mixture of Zoletil 50®Vet., Virbac, France (125 mg of tiletamine and 125 mg of zolazepam in 5 ml of solvent, 5 ml) Rompun®Vet. , Bayer, Germany (20 mg xylazine / ml, 6.5 ml) and Methadon® DAK, Nycomed DAK, Denmark (10 mg methadon / ml, 2.5 ml).

Observation of wounds Each wound will be observed and photographed on day 2 (all animals), 3 (all animals) and 4 (Nos. Of animals 3 and 4). The degree of exudation and inflammation will be evaluated. The appearance of the grafted epidermis will be described in detail.

Clinical signs All visible signs of health and any change in behavior will be recorded daily. Any deviation from the normal will be recorded with respect to start time, duration and intensity.

Body weight The animals will be weighed on arrival, on the day of the formation of wounds and at the end of the study.

Terminal observations On day 3 (approximately 56 hours after the formation of wounds), Nos. 1 and 2 will be sacrificed by a cut in the subclavian vein and the artery after a stunning blow with a screw gun.

On day 4 (approximately 72 hours after the formation of wounds), animals Nos. 3 and 4 were sacrificed by a cut in the subclavian vein and the artery after a stunning blow with a tanning gun.

Tissue sample Each wound will be cut freely with a block separated from the skeletal muscle tissue. If any adherence to the underlying skeletal muscle occurs, part of the muscle will be included in the material for fixation. Each block will be fixed in neutral 4% formaldehyde buffered with phosphate.

Histological preparation After the fixation of four representative samples of all the wounds, they will be coupled in paraffin, cut into nominal thicknesses of 5 μm and stained with hematoxylin and eosin. After staining the slides, they will be observed under the light of the microscope using a grid. This allows measurements of the total length of the wound and the length of the epithelial surface to be measured. This relationship will be expressed as a percentage of the wound covered by the epithelial cells by a slide. The mean values of each wound will be taken, after which the average values of the group will be calculated.

Be! The results will be processed to give the group mean values and standard deviations when appropriate. In addition, possible outcrops will be identified. Subsequently, each continuous variable will be tested for homogeneity of variance with the Bartlett test. If the variance is homogeneous, the analysis of variance will be carried out for the variable. If any significant differences are detected, possible intergroup differences will be verified with Dunnett's test. If the variance is heterogeneous, each variable will be tested for normality by the Shapiro-Wil k method. In the case of normal distribution, the possible differences of the intergroup will be identified with the Student's t-test. Otherwise, the possible differences of the intergroup will be verified by the Krus tal-Wallis test. If any significant intergroup difference is detected, the subsequent identification of the groups will be carried out with the Wilcoxon Rank-Surn test.

The statistical analysis will be done with SAS® procedures (version 6.12) described in "SAS / STAT® User's Guide, Version 6, Fourth Edition, Vol. 1 + 2", 1989, SAS Institute Inc., North Carolina 27513, USA .

LIST OF SEQUENCES < 110 > Biora AB < 120 > Compositions of protein matrix for wound healing. < 130 > 20542PC1 < 160 > 2 < 170 > FastSEC for Windows Version 3.0 < 210 > 1 < 211 > 407 < 212 > PRT < 213 > Rattus nor egicus < 400 > 1 Met Ser Ala Be Lys He Pro Leu Phe Lys Met Lys Gly Leu Leu Leu 1 5 10 15 Phe Leu Ser Leu Val Lys Met Ser Leu Wing Val Pro Wing Phe Pro Gln 20 25 30 Arg Pro Gly Gly Gln Gly Met Wing Pro Pro Gly Met Wing Ser Leu Ser 35 40 45 Leu Glu Thr Met Arg Gln Leu Gly Ser Leu Gln Gly Leu Asn Ala Leu 50 55 60 Ser Gln Tyr Ser Arg Leu Gly Phe Gly Lys Ala Leu Asn Ser Leu Trp 65 70 75 80 Leu His Gly Leu Leu Pro Pro His Asn Ser Phe Pro Trp He Gly Pro 85 90 95 Arg Glu His Glu Thr Gln Gln Pro Ser Leu Gln Pro His Gln Pro Gly 100 105 110 Leu Lys Pro Phe Leu Gln Pro Thr Wing Wing Thr Gly Val Gln Val Thr 115 120 125 Pro Gln Lys Pro Gly Pro His Pro Pro Met His Pro Gly Gln Leu Pro 130 135 140 Leu Gln Glu Gly Glu Leu He Wing Pro Asp Glu Pro Gln Val Wing Pro 145 150 155 160 Ser Glu Asn Pro Pro Thr Pro Glu Val Pro He Met Asp Phe Gly Asp 165 170 175 Pro Gln Phe Pro Thr Val Phe Gln He Wing His Ser Leu Ser Arg Gly 180 185 190 Pro Met Ala His Asn Lys Val Pro Thr Phe Tyr Pro Gly Met Phe Tyr 195 200 205 Met Ser Tyr Gly Wing Asn Gln Leu Asn Wing Pro Gly Arg He Gly Phe 210 215 220 Met Ser Ser Glu Glu Met Pro Gly Glu Arg Gly Ser Met Met Gly Tyr 225 230 235 240 Gly Thr Leu Phe Pro Gly Tyr Gly Gly Phe Arg Gln Thr Leu Arg Gly 245 250 255 Leu Asn Gln Asn Ser Pro Lys Gly Gly Asp Phe Thr Val Glu Val Asp 260 265 270 Ser Pro Val Ser Val Thr Lys Gly Pro Glu Lys Gly Glu Gly Pro Glu 275 280 285 Gly Ser Pro Leu Gln Glu Pro Pro Pro Asp Lys Gly Glu Asn Pro Wing 290 295 300 Leu Leu Ser Gln He Wing Pro Gly Wing His Wing Gly Leu Leu Wing Phe 305 310 315 320 Pro Asn Asp His He Pro Asn Met Wing Arg Gly Pro Wing Gly Gln Arg 325 330 335 Leu Leu Gly Val Thr Pro Ala Ala Ala Asp Pro Leu He Thr Pro Glu 340 345 350 Leu Ala Glu Val Tyr Glu Thr Tyr Gly Ala Asp Val Thr Thr Pro Leu 355 360 365 Gly Asp Gly Glu Wing Tnr Met Asp He Thr Met Ser Pro Asp Thr Gln 370 375 380 Gln Pro Pro Met Pro Gly Asn Lys Val His Gln Pro Gln Val His Asn 385 390 395 400 Wing Trp Arg Phe Gln Glu Pro 405 < 210 > 2 < 211 > 324 < 212 > PRT < 213 > Rattus norwegicus < 4 0 0 > 2 Met Lys Pro Asn Ser Met Glu Asn Ser Leu Pro Val Pro His Pro 1 5 10 15 Leu Pro Ser Gln Pro Ser Leu Gln Pro His Gln Pro Gly Leu Lys Pro 20 25 30 Phe Leu Gln Pro Thr Wing Wing Thr Gly Val Gln Val Thr Pro Gln Lys 35 40 45 Pro Gly Pro His Pro Pro Met His Pro Gly Gln Leu Pro Leu Gln Glu 50 55 60 Gly Glu Leu He Wing Pro Asp Glu Pro Gln Val Wing Pro Ser Glu Asn 65 70 75 80 Pro Pro Thr Pro Glu Val Pro He Met Asp Phe Gly Asp Pro Gln Phe 85 90 95 Pro Thr Val Phe Gln He Wing His Ser Leu Ser Arg Gly Pro Met Wing 100 105 110 His Asn Lys Val Pro Thr Phe Tyr Pro Gly Met Phe Tyr Met Ser Tyr 115 120 125 Gly Ala Asn Gln Leu Asn Ala Pro Gly Arg He Gly Phe Met Ser Ser 130 135 140 Glu Glu Met Pro Gly Glu Arg Gly Ser Pro Met Gly Tyr Gly Thr Leu 145 150 155 160 Phe Pro Gly Tyr Gly Gly Phe Arg Gln Thr Leu Arg Gly Leu Asn Gln 165 170 175 Asn Ser Pro Lys Gly Gly Asp Phe Thr Val Glu Val Asp Ser Pro Val 180 185 190 Ser Val Thr Lys Gly Pro Glu Lys Gly Glu Gly Pro Glu Gly Ser Pro 195 200 205 Leu Gln Glu Pro Pro Asp Lys Gly Glu Asn Pro Wing Leu Leu Ser 210 215 220 Gln He Wing Pro Gly Wing His Wing Gly Leu Leu Wing Phe Pro Asn Asp 225 230 235 240 His He Pro Asn Met Wing Arg Gly Pro Wing Gly Gln Arg Leu Leu Gly 245 250 255 Val Thr Pro Ala Ala Ala Asp Pro Leu He Thr Pro Glu Leu Ala Glu 260 265 270 Val Tyr Glu Thr Tyr Gly Wing Asp Val Thr Pro Pro Leu Gly Asp Gly 275 280 285 Glu Ala Thr Met Asp He Thr Met Ser Pro Asp Thr Gln Gln Pro Pro 290 295 300 Met Pro Gly Asn Lys Val His Gln Pro Gln Val His Asn Ala Trp Arg 305 310 315 320 Phe Gln Glu Pro It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (53)

1. The use of a preparation of an active enamel substance, characterized in that it is for the preparation of a pharmaceutical or cosmetic composition for the healing of a wound in the skin or mucosa.

2. The use of a preparation of an active enamel substance, characterized in that the preparation of a pharmaceutical or cosmetic composition is used to improve the healing of a wound on the skin or mucosa.

3. The use of a preparation of an active enamel substance, characterized in that the preparation of a pharmaceutical or cosmetic composition for the regeneration or repair of the skin or mucosa is used.

4. The use according to any of claims 1-3, characterized in that the wound occurs in the mucosa in the oral cavity.

5. The use according to any of claims 1-3, characterized in that the wound is a bodily injury or trauma associated with oral surgery that includes periodontal surgery, dental extraction or extractions, endodontic treatment, insertion of dental implants, application and use of dentures.

6. The use according to any of claims 1-3, characterized in that the wound is selected from the group consisting of aseptic wounds, blunt wounds, incisional wounds, lacerated wounds, non-penetrating wounds, open wounds, penetrating wounds, puncture wounds, puncture wounds, septic wounds, infarcted and subcutaneous wounds.

7. The use according to any of claims 1-3, characterized in that the wound is selected from the group consisting of ischemic ulcers, pressure ulcers, fistulas, severe bites, thermal burns and wounds at the donor site.

8. The use according to any of claims 1-4, characterized in that the wound is an aphthous wound, a traumatic wound or a wound associated with herpes.

9. The use of a preparation of an active enamel substance, characterized in that it is used for the preparation of a pharmaceutical composition for the prevention and / or treatment of an infection.

10. The use according to claim 9, characterized in that the infection is caused by a microorganism.

11. The use according to claims 9 or 10, characterized in that it is used for the prevention of or treatment of bacterial growth on a mucosal surface.

12. The use according to claim 9 or 10, characterized in that it is used for the prevention of or treatment of bacterial growth on a nail surface or a tooth.

13. The use according to claim 9 or 10, characterized in that the infection occurs in the oral cavity.

14. The use according to claim 13, characterized in that it is used for the prophylaxis and / or treatment of a bacterial condition in the oral cavity.

15. The use according to any of claims 9-14, characterized in that the infection is caused by bacteria causing cavities, e.g. Streptococcus mutans; bacteria that cause periodontal disease e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus micros, Campylobacter (Fusobacteria, Staphylococci), B. Forsythus; bacteria that cause alveolitis, etc., e.g. Staphylococcus, Actinomyces and Bacillus; and bacteria that cause periapical lesions, e.g. Spirochetes

16. The use according to any of claims 9-11, characterized in that the bacteria are present in the skin.

17. The use of a preparation of an active enamel substance, characterized in that it is used for the preparation of a pharmaceutical composition for the prevention and / or treatment of an inflammatory condition of the mucosa or the skin.

18. The use according to claim 17, characterized in that the inflammatory condition occurs in the oral cavity.

19. The use according to claim 17, characterized in that the inflammatory condition occurs in a bone donor site.

20. The use according to any of the preceding claims, characterized in that the active enamel substance is enamel matrix, enamel matrix derivatives and / or protein matrix 1t e.

21. The use according to any of the preceding claims, characterized in that the active enamel substance is selected from the group consisting of enamelins, amelogenins, non-amelogenins, non-amelogenins rich in proline, amelines (ameloblast ina, sheatlin), tuftelins, and derivatives of them and mixtures thereof.

22. The use according to any of the preceding claims, characterized in that the active enamel substance has a molecular weight of at most about 120 kDa such as, eg, at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page electrophoresis.

23. The use according to any of the preceding claims, characterized in that - the preparation of an active enamel substance containing a mixture of enamel active substances with different molecular weights.

24. The use according to any of the preceding claims, characterized in that the preparation of an active enamel substance comprises at least two substances selected from the group consisting of amelogenins, non-amelogenins rich in proline, tuftelin, hair proteins, whey proteins, salivary proteins, amelina, ameloblas tina, sheatlina, and derivatives thereof.

25. The use according to any of the preceding claims, characterized in that the active enamel substance has a molecular weight of up to about 40,000.

26. The use according to any of the preceding claims, characterized in that the active enamel substance has a molecular weight of between about 5,000 and about 25,000.

27. The use according to any of the preceding claims, characterized in that the major part of the active enamel substance has a molecular weight of about 20 kDa.

28. The use according to any of the preceding claims, characterized in that at least a part of the active enamel substance is in the form of aggregates or after application i n vi is capable of forming aggregates.

29. The use according to claim 26, characterized in that the aggregates have a particle size of about 20 nm to about 1 μm.

30. The use according to any of the preceding claims, characterized in that the protein content of the active enamel substance in the preparation is in a range of about 0.05% w / w to 100% w / w such as, eg, about 5- 99% w / w, approximately 10-95% w / w, approximately 15-90% w / w, approximately 20-90% w / w, approximately 30-90% w / w, approximately 40-85% w / w , about 50-80% w / w, about 60-70% w / w, about 70-90% w / w, or about 80-90% w / w.

31. The use according to any of the preceding claims, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

32. The use according to claim 31, characterized in that the pharmaceutically acceptable excipient is propylene glycol alginate.

33. The use according to claim 31, characterized in that the pharmaceutically acceptable excipient is hyaluronic acid or salts or derivatives thereof.

34. The use according to any of claims 1-33 of EMDOGAIN® or any of the proteins or peptides, characterized in that they are contained for the healing of wounds in the skin or mucosa.

35. A method for improving the healing of a wound in the mucosa or skin, characterized in that the method comprises administering to a mammal in need thereof a prophylactic or therapeutically effective amount of an active enamel substance.

36. A method according to claim 35, characterized in that the wound is a bodily injury or a trauma associated with oral surgery that includes periodontal surgery, dental extraction or extractions, endodontic treatment, insertion of dental implants, application and use of dental prostheses.; or wherein the wound is selected from the group consisting of aseptic wounds, blunt wounds, incisional wounds, lacerated wounds, non-penetrating wounds, open wounds, penetrating wounds, puncture wounds, puncture wounds, septic wounds, infarcted and subcutaneous wounds; or wherein the wound is selected from the group consisting of ischemic ulcers, pressure ulcers, fistulas, severe bites, thermal burns and wounds at the donor site; or where the wound is an aphthous wound, a traumatic wound or a wound associated with herpes.

37. A method according to claim 36, characterized in that the active enamel substance is selected from the group consisting of enamelins, amelogenins, non-amelogenins, non-amelogenins rich in proline, amelines (tub ameloblas, sheatlin), tuftelins, and derivatives of the same and mixtures thereof.

38. A method according to claim 36, characterized in that the active enamel substance has a molecular weight of at most about 120 kDa such as, e.g., at most 100 kDa, 90 kDa, 80 kDa, 70 kDa 5 or 60 kDa as determined by SDS Page electrophoresis.

39. A method according to claim 36, characterized in that the amount of the active enamel substance applied to the wound is an amount of total protein per cm2 which corresponds to about 0.01 mg / cm2 to about 20 mg / cm2, such as about 0.1 mg / cm2 at approximately 15 mg / cm2.

40. A method for promoting repair and / or regeneration of the skin or mucosa, characterized in that the method comprises administering to a mammal in need thereof a prophylactically or therapeutically effective amount of an active enamel substance.

41. A method according to claim 40, characterized in that the active enamel substance is selected from the group consisting of enamelins, amelogenmas, non-amelogenins, non-amelogenins rich in 5 prolma, amelines (ameloblast ina, sheatlin), tuftelins, ^? fe ^^ j &g ^^ gjl ^^ and derivatives thereof and mixtures thereof.

42. A method according to claim 40, characterized in that the active enamel substance has a molecular weight of at most about 120 kDa such as, eg, at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page electrophoresis.

43. A method according to claim 40, characterized in that the amount of the active enamel substance applied to the wound is in an amount of total protein per cm2 of affected tissue surface corresponding to about 0.01 mg / cm2 to about 20 mg / cm2 , such as about 0.1 mg / cm2 to about 15 mg / cm2.

44. A method for preventing or treating an infection, characterized in that the method comprises administering to a mammal in need thereof a prophylactically or therapeutically effective amount of an active enamel substance.

45. A method according to claim 44, characterized in that the active enamel substance is selected from the group consisting of enamelins, amelogenins, non-amelogenins, non-amelogenins rich in proline, amelines (ameloblas tub, sheatlin), tuftelins, and derivatives of the same and mixtures thereof.

46. A method according to claim 44, characterized in that the active enamel substance has a molecular weight of at most about 120 kDa such as, eg, at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page electrophoresis.

47. A method according to claim 44, characterized in that the infection is a bacterial infection of the skin or a mucosal surface.

48. A method according to claim 44, characterized in that the bacterial infection is an infection of the oral cavity.

49. A method according to claim 48, characterized in that the infection is caused by bacteria causing cavities, e.g. Str ep tococcus mutans; bacteria that cause periodontal disease e.g. Actinobacillus a ct i nomycetemcom i tans, Po rphyromona s gingiva 1 i s, Prevotella intermedia, Peptos reptococcus micros, Campylobacter (Fusobacter ia, Staphylococci), B. Forsythus; bacteria that cause alveolitis, etc., e.g. Staphylococcus, Act inomyces and Bacillus; and bacteria causing periapical lesions, e.g. Spi rochetes.

50. A method for preventing or treating an inflammatory condition of the skin or mucosa, characterized in that the method comprises administering to a mammal in need thereof a prophylactically or therapeutically effective amount of an active enamel substance.

51. A method according to claim 50, characterized in that the active enamel substance is selected from the group consisting of enamelins, amelogenins, non-amelogenins, non-amelogenins rich in proline, amelines (ame lobla stina, sheatlin), tuftelins, and derivatives of them and mixtures thereof.

52. A method according to claim 50, characterized in that the active enamel substance has a molecular weight of at most about 120 kDa such as, eg, at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page electrophoresis.

53. A method according to claim 50, characterized in that the amount of the active enamel substance applied to the wound is an amount of total protein per cm2 of affected tissue surface corresponding to approximately 0.01 mg / cm2 to approximately 20 mg / cm2, such as about 0.1 mg / cm2 to about 15 mg / cm2.