Application of active enamel substance preparations

Description of the invention

The subject of the invention is the use of preparations of an active enamel substance.

The enamel matrix proteins present in the enamel matrix are well known as enamel precursors. Enamel proteins and enamel matrix derivatives have previously been described in the patent literature as being capable of inducing hard tissue formation (i.e. enamel, US Patent No. 4,672,032 (Slavkin) and hard tissue connections (EP-B-0 337 967 and EP-B-0). 263 086). According to the known art, active enamel substances have been used for the regeneration of hard (mineralized) tissues, while the present invention relates to a beneficial antibacterial and anti-inflammatory effect.

Enamel matrix, enamel matrix derivatives and enamel matrix proteins (the term "active enamel substance" hereafter includes enamel matrix, enamel matrix derivatives and enamel matrix proteins) have been found to have antibacterial and / or anti-inflammatory properties that can be used to treat both soft tissues (i.e. non-mineralized), such as collagen-containing or epithelial tissues, including skin and mucous membranes, muscles, blood and lymph vessels, nerve tissues, glands, tendons, eyes and cartilage, and hard (i.e. mineralized) tissues.

The invention relates to the use of an active enamel substance formulation for the preparation of a pharmaceutical composition for the prevention and / or treatment of infections or inflammations.

The present invention is based on the use of the anti-bacterial effect of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins in therapy and prophylaxis. Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins exhibit infection reducing properties.

As used herein, the term "infection inhibitory effect" refers to the curative or preventive effect of enamel matrix, enamel matrix derivatives, and / or enamel matrix protein on infection in the tissues of a subject.

The term "infection" refers to the invasion and proliferation or accumulation of microorganisms in body tissues that may be clinically asymptomatic or cause local cell damage due to metabolic competition, enzyme action, toxins, intracellular proliferation, or antigen-antibody responses.

According to the present invention, the infection to be prevented or treated by the manufactured medicament may be caused by microorganisms such as bacteria, viruses, yeast, fungi, protozoa and rickettsiae.

In the present context, the term "antibacterial activity" means inhibition of the growth of bacteria or their destruction. The term is not limited to any particular type of bacteria, but includes all bacteria. However, the invention is directed to i) pathogenic bacteria causing disease in mammals, including humans, and / or ii) bacteria normally present in the body of mammals and which under certain conditions can cause undesirable conditions in the body.

The invention relates to the use of an active enamel substance for the preparation of a pharmaceutical composition for the prevention or treatment of bacterial growth on body surfaces, such as the skin, mucosal surfaces or nails, or the surface of the teeth.

The term "skin" is used in a broad sense, including the epidermis layer of the skin and, in those cases where the surface of the skin is more or less damaged, also the dermis layer. In addition to the stratum corneum, the epidermal layer of the skin consists of the epithelial layer (epithelium), and the deeper layer of the connective tissue of the skin is called the dermis (dermis).

In addition to the skin, various other tissues (e.g. soft and hard tissues) can be damaged. Soft tissue injuries, including mucous membranes, and / or skin injuries are, for example, bacterial, viral, infectious, and lichenoid injuries; herpetic ulcers, aphthous (thrush) inflammations, acute necrotic gingivitis and burning mouth syndrome. Pathologies that are treatable with the compositions prepared in accordance with the present invention include anthrax, tetanus, gas gangrene, scarlet fever, beard folliculitis, erysipelas, folliculitis, impetigo, or impetigo, venereal ulcer, e.g. urethritis, cervicitis and proctitis), tropical ulcer, etc. Often the terms "wound" and "ulcer", "wound" and "ulcer" overlap and are sometimes used interchangeably. Therefore, as mentioned above, in this text the term wound includes ulcer, wound, ulceration, and the terms are used equally, unless the differences are clearly indicated.

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Healing of damage to the skin or mucous membranes takes place in several stages that lead to the repair or regeneration of the skin or mucosa. In recent years, repair and regeneration have been distinguished as two separate types of healing. Regeneration can be defined as the biological process by which the structure and function of damaged tissue is completely restored. Reparation, on the other hand, is a process during which the continuity of damaged tissue is restored with the help of other tissue that does not correspond to the structure and function of the damaged tissue. Under normal circumstances, the body has mechanisms to heal damaged skin or mucous membranes to restore the integrity of the skin or mucosa. The process of repairing even small tears or wounds can take anywhere from hours to days, even weeks. However, in ulcers, the healing process takes a long time and may take months or even years. The stages of wound healing include the processes of inflammation (usually 1-3 days), migration (usually 1-6 days), proliferation (usually 3-24 days) and maturation (usually 1-12 months).

All the aforementioned healing processes take considerable time. The speed of healing is influenced by the sterility of the wound, the general health of the individual, the presence of foreign bodies, etc. Certain pathological conditions, such as infection, maceration, dehydration, general ill health, and malnutrition can lead to chronic ulcers, such as ischemic ulcers.

Until even superficial healing, the wound is at risk of prolonged or new infection. Therefore, the sooner a wound can be healed, the sooner the risk of infection is eliminated.

Thus, any procedure which influences the acceleration of healing or positively influences the course of wound healing is very valuable.

In the present context, the term clinical healing is used to denote a situation where no tissue rupture is observed and only discrete signs of inflammation are present, such as slight redness or slight swelling of the tissue. Moreover, there are no complaints of spontaneous pain when the organ is at rest or not being touched.

Traditionally, dry or wet-to-dry dressings are the most commonly used dressings for wounds. They are gradually replaced by humid environments with the use of an occlusive dressing. In order to successfully repair or replace a damaged part of the body, the processes of wound healing, fibrosis and microbial invasion must be kept in balance. There are many methods available to reverse an infection that is hindering healing. Delayed healing or the inflammatory process may aggravate the fibrosis. In addition, it has previously been suggested that growth factors such as epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) in including acidic fibroblast growth factor (a-FGF) and basic fibroblast growth factor (b-FGF), transforming growth factor beta (TGF-B), as well as insulin-like growth factors (IGF-1 and IGF-2) are conductors of the wound healing process and are often cited as facilitating wound healing; however, they facilitate fibrosis which may interfere with effective healing. While accelerated healing reduces the risk of infection and the resulting inflammation that can lead to scar formation, therapeutic efforts to accelerate normal wound healing have been relatively ineffective. This is likely due to the fact that the repair process requires the concerted interaction of many of the factors mentioned above.

The present inventors found that in different cultures of fibroblasts (embryonic, skin, periodontal ligament, fish or birds) twice as many

TGFei in cultures stimulated with ENDOGAIN® (from BIORa AB, S-205 12 Malmó, Sweden containing 30 mg of lyophilized Enamel Matrix protein matrix (here shortened to EMD) and 1 ml of carrier solution (propylene glycol alginate), mixed before use, unless that protein and vehicle are tested separately) compared to unstimulated cultures (measured by ELISA of a culture medium sample (see Example 1 below). Growth is seen after 24 hours of cultivation, but is more pronounced in the days following (days 2 and 3). An increase in cell proliferation is also seen in the cultures stimulated with EMDOGAIN® after the second day. A similar but less pronounced increase in TGFe1 production was observed in epithelial cells. Since TGFe1 appears to play an important role in epithelialization of wound surfaces, these observations underpin the concepts of the present invention.

The present inventors have found that after the use of enamel matrix proteins and / or enamel matrix derivatives, the inflammatory reaction stage is shortened and its typical symptoms such as heat, redness, swelling and pain are less severe.

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The therapeutic and / or preventive activity of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins can of course be demonstrated by in vivo tests on humans or experimental animals (see the experimental section of this description). However, relatively simple in vitro assays, such as e.g. cell culture assays, can provide indications of the efficacy and / or activity of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins.

Preferably the composition of the use according to the invention is used when the infection is within the oral cavity, and more preferably it is for the prevention and / or treatment of oral bacterial conditions.

Preferably the composition of the use according to the invention is used when the infection is caused by cariogenic bacteria, e.g. Streptococcus mutans; periodontal disease causing bacteria, e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella Intermedia, Peptostreptococcus micros, Campylobacter (Fusobacteria, Staphylococci), B. forsythus; bacteria causing inflammation of the alveoli etc, e.g. Staphylococcus, Actinomyces and Bacillus; and bacteria that cause periapical cavities, such as Spirochetes.

Preferably the composition of the use according to the invention is used when the bacterium is present on the skin.

Preferably the composition is for the prophylaxis and / or treatment of an oral bacterial condition.

It is within the scope of the invention to use an active enamel substance formulation for the preparation of a pharmaceutical composition for the prevention and / or treatment of inflammation.

Preferably, the composition of the use according to the invention is used when inflammation occurs within the oral cavity. Preferably the composition of the use according to the invention is used when inflammation is present at the site of removal of the tissue for transplantation.

Preferably the active enamel substance is enamel matrix, enamel matrix derivatives and / or enamel matrix proteins.

Preferably the active enamel substance is selected from the group consisting of enamelins, amelogenins, proteins other than amelogenins, proteins other than amelogenins rich in proline, amelins (ameloblastin, sheatlin), tuftelins and their derivatives and mixtures thereof.

More preferably, the active enamel material has a molecular weight of at most about 120,000u, such as, e.g., 100,000u, 90,000u, 80,000u, 70,000u, or 60,000u as determined by SDS Page.

More preferably, the formulation of an active enamel substance comprises a mixture of active enamel substances of different molecular weights.

More preferably, the active enamel substance preparation consists of at least two substances selected from the group consisting of amelogenins, proline-rich proteins other than amelogenins, tuftelin, tuft proteins, serum proteins, saliva proteins, ammeline, ameloblastin, sheatlin and derivatives thereof.

Preferably, the active enamel material has a molecular weight up to about 40,000, or between 5,000 and 25,000.

Preferably the major part of the active enamel substance has a molecular weight of 20,000.

Preferably, at least a portion of the active enamel substance is aggregated or is capable of aggregating when applied in vivo, preferably with a particle size of 20 nm to 1 micrometer.

Preferably the protein content of the active enamel substance in the formulation is in the range from 0.05 wt% to 100 wt%, e.g. 5-99 wt%, 10-95 wt%, 15-90 wt%, 20-90 wt%, 30-90% by weight, 40-85% by weight, 50-80% by weight, 60-70% by weight, 70-90% by weight, or 80-90% by weight.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient which is propylene glycol alginate or hyaluronic acid or a salt or derivative thereof.

Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins can be used to treat bacterial infections with or without antibacterial agents. Gram-negative bacteria whose infections can be treated with the active enamel substance may be streptococci such as Neisseria (e.g. N. meningitis, N. gonorrhoeae) and Acinetobacter or bacilli such as Bacterioides (e.g. B. fragilis), Bordetella (e.g. B. pertussis, B. parapertussis), Brucella (e.g. B. melitentis, B. abortus Bang, B. suis), Campylobacter (e.g. C. jejuni, C. coli, C. fetus), Citrobacter, Enterobacter,

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Escherichia) e.g. E. coli), Haemophilus (e.g. H. influenzae, H. parainfluenzae), Klebsiella (e.g. K. pneumoniae), Legionella (e.g. L. pneumophila), Pasteurella (e.g. P. yersinia, P. multocida), Proteus (e.g. P.mirabilis, P. vulgaris), Pseudomonas (e.g. P. aeruginosa, P. pseudomallei, P. mallei), Salmonella (e.g. S. enteritidis, S. infantitis, S. Dublin, S typhi, S. paratyphi, S. schottmulleri, S. choleraesius, S. typhimurium, or any of 2,500 other serotypes), Serratia (e.g. S. marscences, S. liquifaciens), Shigella (e.g. S. sonnei, S. flexneri, S. desynteriae, S. boydii), Vibrio (e.g. V. cholerae, V. el tor) and Yersinia (e.g. Y. enterocolitica, Y. pseudotuberculosis, Y. pestis).

Gram-positive bacteria that can be treated with an active enamel substance are staphylococci such as Streptococcus (e.g. S. pneumoniae, S. viridans, S. faecalis, S. pyogenes), Staphylococcus (e.g. S. aureus, S. epidermidis , S. saprophyticus, S. albus), and bacilli such as Actinomyces (e.g. A. israeli), Bacillus (e.g. B. cereus, B. subtilis, B. anthracis), Clostridium (e.g. C. botulinum, C teteani, C. perfringens, C. diddicile), Corynebacterium (e.g. C. dyphtheriae), Listeria and Providencia. Other bacteria that cause infections are Propionobacterium acne and Pityosporon ovale.

Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins can also be used to treat infections caused by spirochetes such as Borrelia, Leptospira, Treponema or Pseudomonas.

The antimicrobial agent that can be used in combination with enamel matrix, enamel matrix derivatives and / or enamel matrix proteins may be an antimicrobial drug that is antibacterial by inhibiting bacterial wall synthesis, such as:

beta lactams and vancomycin, especially penicillins such as: amidocillin, ampicillin, amoxicillin, azlocillin, bacampicillin, benzathin, penicillin G., carbenicillin, cloxacillin, cyclacillin, dicloxacillin, methacillin, mesliocillin, nafcillin, oxacillin, penicillin G., and ceficillin G. cefadroxil, cefazolin, cefalexin, cephalotin, cefapyrin and cefradin, second generation drugs such as cefaclor, cefamandol, cephonicide, ceforanide, cefoxitin and cefuroxime, or third generation cephalosporins cefopresone, cefotaxime, cefotetane, ceftazidime, ceftizoxime, ceftriaxone and moxalactam; carbapenems like imipenem; or monobactams like aztreonam;

other antibacterial drugs that inhibit protein synthesis, such as chloramphenicol; other tetracyclines, especially demeclocycline, doxycycline, metacycline, minocycline, and oxytetracycline; amino glycosides such as amikacin, gentamicin kanamycin, neomycin netilmycin, paromomycin, spectinomycin, streptomycin and tobramycin;

polymyxins such as colistin, colistimatate and polymyxin B, and erythromycins and lincomycins; antibacterial drugs whose mechanism of action is based on the inhibition of nucleic acid synthesis, in particular sulfonamides such as sulfacitin, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim, quinolones, novobiocin, pyrimethamine, and rifampin.

In a particular embodiment of the invention, the infection occurs in the oral cavity and is caused by bacteria.

Preferably, the pharmaceutical composition of the use according to the invention is used when the infection is caused by cariogenic bacteria, e.g. Streptococcus mutans; periodontal disease causing bacteria, e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus micros, Campylobacter (Fusobacteria, Staphylococci), B. forsythus; bacteria causing inflammation of the alveoli etc, e.g. Staphylococcus, Actinomyces and Bacillus; and bacteria that cause periapical cavities, such as Spirochetes.

Oral bacteria are contact inhibited or otherwise combated. Examples of bacteria that cause oral conditions include:

- cariogenic bacteria, e.g. Streptococcus mutans, Lactobacillus spp.

- bacteria causing periodontal disease, e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus microns, Camphylobacter (Fusobacteria, Staphylococci), B. forsytus - bacteria causing alveolitis etc., e.g.

- bacteria causing periapical damage, such as Spirochetes and all of the above.

Many drugs are used to inhibit inflammatory symptoms, including adrenocorticosteroids, a large group of drugs including the so-called non-steroidal anti-inflammatory drugs or NSAIDs, and drugs such as immunosuppressants. Adrenocorticosteroids, and in particular glucocorticoids, when used in pharmacological doses, exert a strong anti-inflammatory effect. They inhibit in particular

The early vascular phase of the inflammatory process reduces the permeability of the vessels and thus the migration of granulocytes. Glucocorticoids also affect the late stage of inflammation and repair processes by inhibiting the proliferation of mesenchymal cells and the production of extracellular matrix macromolecules, including proteoglycans and collagen. It has been experimentally shown that glucocorticoids inhibit e.g. macrophage functions, production of humoral antibodies, cellular response and possibly the release of lysosomal enzymes.

The degree of tissue damage may depend on the antigen / antibody response in the body as well as the extent to which the products of the inflammatory reaction are absorbed in the area of ​​the injury. The accumulation of mediators of the local inflammatory reaction accelerates the process. In most cases, this process is slow, with the infiltration of the tissue with immunologically active cells and the production of granulation tissue containing inflammatory cells.

As used herein, the term "anti-inflammatory effect" means the prevention or inhibition of an inflammatory process.

The inflammation can of course be any inflammation in / on any part of the body or any inflammation that occurs within soft or hard tissues. In one embodiment of the invention, the inflammation is present within the oral cavity. Examples of inflammation in the oral cavity are: alveolitis, cheilitis, bone necrosis (after injury), fractures.

In one embodiment, the invention relates to the use of an active enamel substance preparation for the preparation of a pharmaceutical composition for the prevention or treatment of inflammation at the site of tissue removal for transplantation. Inflammation can take place within the joint cavities. An example of such inflammation is rheumatoid arthritis and conditions associated with it.

Unlike many antibiotics currently in use, enamel matrix proteins will not impair wound healing, and rapid wound healing prevents chronic inflammation from developing. Also, the reorganization of the proper tissues, as described after the application of enamel matrix derivatives to periodontal defects, is clearly advantageous due to the rapid healing of the wound without bacteria or inflammatory reactions.

The use of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins causes rapid healing of wounds after surgical incisions, presumably by creating a surface which, on contact with bacteria, inhibits their growth while enhancing fibroblast migration and collagen synthesis. When the inflammation stage is shortened, typical symptoms such as warmth, redness, swelling and pain are less severe.

Enamel matrix is ​​an enamel precursor and can be obtained from any suitable source, i.e. from mammals in which teeth develop. A convenient source is the developing teeth of slaughter animals such as calves, pigs and sheep. Another source is fish skin, for example.

The enamel matrix can be prepared from developing teeth as previously described (EPB-0 337 967 and EP-B-0 263 086). The enamel matrix is ​​scraped off and the enamel matrix is ​​derivatized, e.g. by extraction with aqueous solutions such as buffer, dilute acid or base, or a water / solvent mixture, then filtered to remove larger pieces, desalted or other purification steps followed by lyophilization. Enzymes can be inactivated by heating or using solvents, in which case the derivatives can be stored in the liquid state without lyophilization.

As used herein, enamel matrix derivatives are enamel matrix derivatives, including one or more enamel matrix proteins, or part of these proteins, produced naturally by alternative splicing or processing, or by enzymatic or chemical cleavage of the natural length of the protein, or synthesized polypeptides in vitro. or in vivo (recombinant DNA methods or diploid cell culture). Enamel matrix protein derivatives also include enamel matrix related polypeptides or proteins. The polypeptides or proteins may be bound to the appropriate biodegradable carrier molecules. Furthermore, the term "enamel matrix derivatives" also includes analogous synthetic substances.

Proteins are biological macromolecules made up of amino acid residues linked by peptide bonds. Proteins as chain polymers of amino acids are also called polypeptides. Typically proteins consist of 50-800 amino acid residues and therefore have a molecular weight of about 6,000 to several hundred thousand, or more, Daltons (1 dalton (Da) = 1 u). Small proteins are called peptides or oligopeptides.

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Enamel matrix proteins are proteins normally present in the enamel matrix, i.e. enamel precursor (Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1: 282-291), or proteins, which can be obtained by digesting these proteins. Generally, these proteins have a molecular weight below 120,000 Daltons and include amelogenins, proteins other than amelogenins, poroline-rich proteins other than amelogenins, amelins (ameloblastin, sheatlin) and tuftelins.

The preparations containing the active enamel substance used in the invention may also contain at least two of the aforementioned protein substances. A commercial product containing amelogenins and possibly other enamel matrix proteins is marketed under the name EMDOGAIN © (Biora AB).

In general, the major enamel matrix proteins are known as amelogenins. They represent about 90% by weight of the matrix proteins. The remaining 10% by weight are proline-rich proteins other than amelogenins, tuftelin, tuft proteins, serum proteins, and at least one saliva protein; however, there may be other proteins related to the enamel matrix, such as e.g. ammeline (ameloblastin, sheatlin). In addition, proteins of different sizes (i.e., different molecular weights) can be synthesized and / or processed. Thus, the dominant proteins of the enamel matrix - amelogenins - occur as molecules of different weights, together forming large-molecule aggregates. These are clearly hydrophobic substances that form aggregates under physiological conditions. They can be carried or be carriers for other proteins or peptides.

It is also contemplated to use other proteinaceous substances in the compositions for use according to the invention. Examples include proteins such as proline-rich proteins and polyproline. Other examples of substances contemplated for use in the compositions of the present invention are aggregates of such proteins, aggregates of enamel matrix derivatives and / or enamel matrix proteins, as well as metabolites of enamel matrix, enamel matrix derivatives and enamel matrix proteins. Metabolites can be of various sizes - from the size of proteins to short peptides.

As mentioned above, proteins, polypeptides or peptides to be used in the composition prepared according to the invention usually have a molecular weight of at most about 120 kDa, most often 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS electrophoresis.

Proteins to be used in the composition prepared according to the invention are normally in the form of preparations, with the active enamel substance content of the preparations being in a wide range from about 0.05% w / w. up to 100% by weight, such as about 5-99% by weight, about 10-95% by weight, about 15-90% by weight, about 20-90% by weight, about 30-90% by weight, about 40-85% by weight, about 50-80% by weight, about 60-70% by weight, about 70-90% by weight, or about 80-90% by weight.

The preparation of an active enamel substance for the preparation of a pharmaceutical composition for use according to the invention may also contain a mixture of active enamel substances with different molecular weights.

Preferably, the active enamel substance preparation consists of at least two substances selected from the group consisting of amelogenins, proline-rich proteins other than amelogenins, tuftelin, tuft proteins, serum proteins, saliva proteins, ammeline, ameloblastin, sheatlin and derivatives thereof.

Enamel matrix proteins can be classified into high molecular weight and low molecular weight, and it has been found that a well-characterized fraction of enamel matrix proteins has beneficial properties in the treatment of periodontal defects (e.g., periodontal wounds). This fraction contains proteins, generally referred to as amelogenins, extractable with acetic acid and is part of the low molecular weight enamel matrix (according to EP-B-0 337 967 and EP-B-0- 263 086).

As discussed above, the low molecular weight portion of the enamel matrix exhibits the desired activity of inducing connections between hard tissues in periodontal defects. However, as used herein, active proteins are not limited to the low molecular weight fraction of enamel matrix. Currently, priority proteins include enamel matrix proteins such as amelogenin, ammeline, tuftelin, etc. with molecular weights (determined in vitro by SDS-PAGE) below 60,000 [mu] m, but proteins having a molecular weight above 60,000 [mu] m also have promising properties as wound healing candidates, antibacterial agents and / or anti-inflammatory agents.

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In view of the foregoing, it is believed that the active enamel material for use in the present invention has a molecular weight of up to about 40,000, such as e.g.

Preferably the major part of the active enamel substance has a molecular weight of 20,000.

Also within the scope of the present invention is the use of peptides as described in WO 97/02730, i.e. peptides having at least one sequence element 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 the amino acid sequences which, in the 20 amino acid chain, show at least 80% homology with the chain of groups constituting the sequence shown in SEQ ID NO: 1 and with the amino acid sequence 1 to 103 according to SEQ ID NO: 1 and with amino acids 6 to 324 according to SEQ ID No .: 2.

By the term "sequence identity" is meant the identity of the order of matching amino acids in the identity and position of the amino acids in the peptides. A gap is considered non-identity for one or more amino acids, respectively.

Such peptides may consist of 6 to 300 amino acids, e.g. at least 20 amino acids, at least 30 amino acids, at least 90 amino acids, at least 120 amino acids, at least 150 amino acids, and at least 200 amino acids.

The method of isolating enamel matrix proteins requires the extraction of proteins and the removal of calcium and phosphorus ions from dissolved hydroxyapatites using appropriate methods, e.g. gel filtration, dialysis or ultrafiltration (see e.g. Janson, JC & Ryden, 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 may contain mainly or exclusively up to 70-90% of amelogenins with a molecular weight between 40,000 and 5,000 Daltons, the remaining 10-30% being smaller peptides, salts and residual water. The major protein bands are at the level of 20,000u, 12-14,000u, and around 5,000.

By separating proteins, e.g., by precipitation, ion exchange chromatography, reverse phase chromatography or affinity chromatography, amelogenins of different molecular weights can be purified.

The combination of amelogenins with different molecular weights can vary from a dominant component of 20 kDa to aggregates of amelogenins of different molecular weights between 40,000 and 5,000 µl to a dominant component of 5,000. those normally found in enamel matrix can be attached to amelogenin aggregates.

An alternative source of enamel matrix derivatives or proteins may be their synthesis by in vitro grown cells or by bacteria based on recombinant DNA methods (see, e.g., Sambrook, J. et al .: Molecular cloning, Cold Spring Harbor Laboratory Press, 1989).

In general, enamel matrix, enamel matrix derivatives and enamel matrix proteins are hydrophobic substances, ie less soluble in water, especially at elevated temperatures. In principle, these proteins dissolve at non-physiological pH values ​​and at lower temperatures, such as 4-20 ° C, but aggregate and precipitate at body temperature (35-37 ° C) and neutral pH.

Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins used in the preparation of the pharmaceutical composition according to the invention also include enamel active substances, at least some of the active substance being aggregated or capable of aggregating when used in vivo. Preferably, the particle size of the aggregates ranges from about 20 nm to about 1 µm.

The solubility of the enamel matrix, enamel matrix derivatives and / or enamel matrix proteins is believed to be important for the therapeutic and prophylactic activity of a substance. When a composition comprising enamel matrix, enamel matrix derivatives and / or enamel matrix proteins (hereinafter referred to as "active enamel substance" as a generic term) is administered to eg a human, the proteinaceous substances will precipitate under normal physiological pH conditions. Thus, a layer of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins is produced at the site of application, and this layer (which may also be a layer of particles in cases where aggregates are formed) is not washed out under physiological conditions. Moreover, thanks to the bioadhesive properties of the substance (see below), the precipitating layer is firmly attached to the tissue, also on

The edge between the precipitating layer and the tissue. Thus, the proteinaceous layer covers the tissue to which the enamel matrix, enamel matrix derivatives and / or enamel matrix proteins have been applied, and the active enamel substances are kept in situ for a longer time, i.e. there is no need to administer the active enamel substance too frequently. In addition, the layer formed in situ can be compared with an occlusive dressing, i.e. the created layer protects the tissue on which it was made from the environment. In the case of injured tissue, infected tissue or inflamed tissue, such a layer protects the tissue from further infection by environmental microorganisms. In addition, the protein layer may exert its effect by direct contact with the tissue or with the microorganisms present in / on / near the tissues.

In order to enable in situ formation of a protein-like layer, after administration, it may be advantageous to include a suitable buffer substance in the pharmaceutical composition of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins; the purpose of such a buffer substance may be to prevent the active enamel substance from dissolving at the application site.

Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins have been found to have bioadhesive properties, ie they have the ability to adhere to the skin surface or mucosa surfaces. These properties are most valuable for therapeutic and / or preventive management for at least the following reasons: - the active substances can be preventively and / or therapeutically held for an extended period of time at the site of application (i.e. i) the frequency of administration can be reduced, ii) the release of the active substance can be controlled and / or iii) local treatment at the application site is improved);

- the substances can themselves serve as carriers for other prophylactically or therapeutically active substances, since the enamel matrix carrier, enamel matrix derivatives and / or enamel matrix proteins can be formed as bioadhesive carriers (i.e. a new bioadhesive drug delivery system based on the bioadhesive properties of the enamel matrix , enamel matrix derivatives and / or enamel matrix proteins).

The enamel matrix is ​​an example of an extracellular protein matrix that adheres to mineral surfaces as well as protein surfaces. Proteins form insoluble macromolecular aggregates at physiological temperature and physiological pH (Fincham et al. In J. Struct. Biol. 1994 March-April; 112 (2): 103-109 and in J. Struct. Biol. 1995 July-August; 115 (1): 50-59) that are gradually degraded by proteolytic enzymes (occurring both in vivo and in vitro, provided that the proteases have not been inactivated).

The recent observation that enamel matrix is ​​produced and transiently present in root and root cementum production helps to elucidate how the use of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins facilitates periodontal tissue regeneration. However, the observation underlying the present invention that enamel matrix, enamel matrix derivatives and / or enamel matrix proteins also have beneficial anti-infective and anti-inflammatory effects is very surprising.

In many species, the remains of the enamel matrix are found in the freshly mineralized crown during the eruption of the teeth in the oral cavity. It can be argued that new teeth would be very susceptible to attack by common bacteria in the mouth if they did not have natural protection during the initial phase.

Applying insoluble enamel matrix, enamel matrix derivatives and / or enamel matrix proteins with appropriate antibacterial and anti-inflammatory properties to the wounded surface will strengthen and improve healing.

As shown in the experimental section of enamel matrix, enamel matrix derivatives and / or enamel matrix proteins or protein aggregates inhibit bacterial growth by contact inhibition, while treated cells respond to enamel matrix as a normal environment that inhibits inflammatory responses.

Enamel matrix, enamel matrix derivatives and / or enamel matrix proteins can be used in a pharmaceutical composition for both therapeutic and prophylactic purposes.

Furthermore, enamel matrix, enamel matrix derivatives, and / or enamel matrix proteins can be used together with other active drug substances, such as e.g. antibacterial, anti-inflammatory, antiviral, antifungal substances, or in combination with growth factors such as e.g. TGFB, PDGF, IGF, FGF, keratinocyte growth factor or their peptide analogs (EGF is believed to facilitate healing by increasing the migration and proliferation of epithelial cells; in addition, EGF increases the number of fibroblasts in the wound, causing increased collagen synthesis).

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Enzymes, and in particular proteases, both normally found in enamel matrix or its preparations and added, can also be used in combination with enamel matrix, enamel matrix derivatives, and / or enamel matrix proteins.

A preparation of an active enamel substance is usually formulated as a pharmaceutical composition. Such a composition comprises a protein preparation and furthermore a pharmaceutically acceptable excipient. Propylene glycol alginate or hyaluronic acid or salts or derivatives thereof are particularly preferred excipients for the pharmaceutical compositions prepared according to the invention.

Examples of suitable compositions containing the active enamel substance (s) are given below. Depending on the use of the active enamel substance (s), the composition may be a pharmaceutical composition.

For the administration to subjects (humans or animals) of enamel matrix, enamel matrix derivatives, and / or enamel matrix proteins (hereinafter also referred to as "active enamel substance") and / or their preparations, they are preferably formulated into a pharmaceutical composition containing the active ingredient. glazes and, optionally, one or more pharmaceutically acceptable excipients.

The compositions may be in the form of e.g. solid, semi-solid or liquid, such as, for example, bioabsorbable patches, dips, dressings, gel dressings, hydrocolloid dressings, membranes, sponges, flakes, bandages, patches, delivery device compositions, implants; powders, granules, capsules, chitosan beads, tablets, pills, pellets, microcapsules, microspheres, nanoparticles; sprays, aerosols, inhalation devices; gels, hydrogels, pastes, ointments, creams, soaps, suppositories, vaginal balls, toothpastes; solutions, dispersions, suspensions, emulsions, mixtures, lotions, mouthwashes, shampoos, enemas; kits consisting of two containers, the first container containing the active enamel substance optionally mixed with another therapeutically active substance and / or a pharmaceutically acceptable excipient, and the second container contains a suitable substrate to be added to the first container before use to obtain a ready-to-use material composition; and in other appropriate forms, such as implants or covers for implants, or in a suitable form for use in implantation or transplantation.

The use of an active enamel substance formulation for the preparation of a composition for application to the skin or mucous membranes is believed to be of most importance for the present invention. Thus, compositions containing the active enamel substance to be administered may be adapted for administration by any appropriate route, e.g., topically (dermally), to the mouth, cheeks, nasally, auricle, rectally or vaginally, or by administration into a body cavity such as e.g. tooth root or tooth root canal. In addition, the composition may be adapted for administration in conjunction with surgery, e.g., in combination with an incision in the body to promote the healing of internal wounds and soft tissue damage.

As mentioned above, the active enamel substance composition (s) may be useful in a surgical procedure, e.g., for topical application (e.g., to the oral cavity) in the form of a gel, film, or a dry tablet implanted subcutaneously, or as a rinse or paste treatment solution. or surface cream to prevent bacterial invasion. In connection with surgery or implantation in the area of ​​the tooth root canal, a paste can be used to seal the cavity.

Compositions may be prepared according to generally accepted pharmaceutical principles, see, for example, "Remington Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology" published by Swarbrick, J. & JC Boylan, Marcel Dekker, Inc., New York, 1988.

As mentioned above, the use of compositions containing an active enamel substance is intended for the skin and mucous membranes. Of course, other applications may also be suitable, such as, for example, dentures, bridges, implants, and application within body cavities such as the oral cavity, nasal cavity, and vaginal cavity. The mucosa is preferably selected from the mucosa of the mouth, cheeks, nose, ears and vagina.

In addition, it is very important to use it in the dental and perineal areas. Important examples are applications on periodontal (dental) pockets, on the gingiva, or on gingival wounds or on other oral wounds, or in connection with oral surgery.

It is further believed that due to the antibacterial properties of the active enamel substance described herein, it may be advantageous to apply it to the teeth or to the roots of the teeth in order to prevent caries and / or plaque. In order to support this use, it is shown in (Weinmann, JP et al .: Hereditary disturbances of enamel formation and calcification, J. Mer. Dent. Assoc. 32:

PL 204 797 B1

397-418, 1945; Sundell S, Hereditary amelogenesis imperfecta. An eoidemiological, genetic and clinical study in a Swedish child population, Swed Dent J Suppl 1986; 31: 1-38) that teeth that are abnormally developed (amelogenesis imperfecta) and consequently contain an increased amount of amelogenins are much less prone to caries.

Pharmaceutical compositions containing an active enamel substance serve as a drug delivery system. Herein, the term "drug delivery system" denotes a pharmaceutical composition (pharmaceutical formulation or dosage form) which, upon administration, presents the active substance to the human or animal body. Thus, the term "drug delivery system" includes pure pharmacological compositions such as, for example, creams, ointments, lotions, powders, tablets, etc. as well as more complex formulations, such as sprays, plasters, bandages, dressings, devices, etc.

In addition to the active enamel substance, the pharmaceutical compositions prepared according to the present invention contain pharmaceutically acceptable excipients.

A pharmaceutically acceptable excipient is a substance which is harmless to the individual to whom it is administered. Such excipient usually meets the requirements of the National Health Authority. Official pharmacopoeias, such as, for example, the British Pharmacopoeia, the United States of America Pharmacopoeia and The European Pharmacopoeia, contain standards of pharmaceutically acceptable excipients.

The choice of pharmaceutically acceptable excipient is suitable for use in pharmaceutical compositions, depending on the type of the dosage form. Examples of properly selected excipients in accordance with pharmaceutical requirements and used in various types of compositions will be given below.

An overview of suitable pharmaceutical compositions prepared according to the invention is provided below. The review is based on specific routes of administration. Preferably, however, in cases where a pharmaceutically acceptable excipient can be used in different dosage forms or in different compositions, the use of a particular pharmaceutically acceptable excipient is not limited to a particular dosage form or function.

The choice of the pharmacologically acceptable excipient (s) in the composition prepared according to the invention and its (their) optimal concentration cannot generally be predicted and must be determined on the basis of an experimental evaluation of the final composition. However, one skilled in the art may find clues, e.g. in "Remington's Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, 1990.

For application to mucous membranes or to the skin, the composition of the present invention may contain recognized pharmacologically acceptable, non-toxic carriers and excipients, including microspheres and liposomes.

Compositions for use in accordance with the invention include all kinds of solid, semi-solid and liquid ingredients. Particularly important compositions are e.g. pastes, ointments, hydrophilic ointments, creams, gels, hydrogels, solutions, emulsions, suspensions, liniment lotions, shampoos, jellies, soaps, sticks, sprays, powders, films, foams, tampons, sponges, e.g. . collagen sponges), dressings (such as e.g. absorbent dressing), drains, bandages, plasters, and transdermal delivery systems.

Pharmaceutically acceptable excipients can include solvents, buffering agents, preservatives, moisturizers, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gel forming agents, ointment bases, penetration enhancers, perfumes, and skin protectants.

Examples of solvents are e.g. water, alcohols, vegetable or marine oils (e.g. 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, sesame oil, soybean oil, sunflower oil and oil from tea seeds), mineral oils, liquid paraffin, polyethylene glycols, propylene glycols, glycerol, liquid polyalkyl siloxanes, and mixtures thereof.

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

PL 204 797 B1

Suitable examples of preservatives for use in the compositions are parabens such as methyl, ethyl propyl parahydroxybenzoate, butyl paraben, isobutyl paraben, isopropylene paraben, potassium sorbate, benzoic acid, sorbic acid, methyl benzoate, phenoxyethanol, bronopolin, bronidox, butylantocarbenzoate, MDM. iodopropynyl, EDTA, benzalkonium chloride and benzyl alcohol, or mixtures of preservatives.

Examples of moisturizers are glycerin, propylene glycol, sorbitol, lactic acid, urea, and mixtures thereof

Examples of chelators are sodium EDTA and citric acid.

Examples of antioxidants are butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, cysteine, and mixtures thereof.

Examples of emulsifying agents are naturally occurring gums, for example gum arabic or gum tragacanth; naturally occurring phosphatides, e.g. soybean lecithin; sorbitan monooleate derivatives; wool fats; lanolin; sorbitan esters; monoglycerides; fatty acids; fatty acid esters (e.g. fatty acid triglycerides); and mixtures thereof.

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

Examples of gel bases, viscosity-increasing agents or components capable of removing wound exudate are: liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminum, zinc soaps, glycerol, propylene glycol, tragacanth, carboxyvinyl polymers, magnesium aluminum silicates, Carbopol, hydrophilic polymers such as starch or cellulose derivatives such as carboxymethylcellulose and hydroxyethyl cellulose other cellulose derivatives, water-swellable hydrocolloids, carrageenans, hyaluronates (e.g. hyaluronic gel optionally containing sodium chloride) and alginates, including propylene glycol aginate.

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

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

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

Other examples of ointment bases are triethanolamine soaps, sulphonated fatty alcohols and polysorbates.

Examples of powdered mixtures are: alginate, collagen, lactose, a powder capable of forming when applied to a wound a gel (absorbs wound fluid / exudate). Typically, powders intended for use on large, open wounds must be sterile and the particles must be pulverized to the micron size.

Examples of other excipients are polymers such as carmellose, carmellose sodium, hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, pectin, xanthan gum, acacia, gelatin, carbomer, emulsifiers such as vitamin E, glycerol stearate, glycerol glycoside, glucosin hyaluronates and alginates and chitosans.

Dressings and / or bandages are also important delivery systems for the active enamel substance. When the dressing is in a dosage form, the active enamel material may be mixed with the other material / ingredients before or during the manufacturing process of the dressing, or the active enamel material may somehow coat the dressing, e.g. by dipping the dressing in a solution or dispersion of the active enamel substance or by spraying or dispersing the active enamel substance onto the dressing. Optionally, the active enamel substance can be applied in the form of a powder for the dressing. Dressings may be in the form of an absorbent dressing in exuding wounds. The dressings can also be in the form of hydrogel dressings (e.g. cross-linked polymers such as e.g. Intrasite®, which contain carboxymethylcellulose, propylene glycol or polysaccharides, disaccharides and proteins) or in the form of occlusive dressings such as alginates, chitosan, hydrophilic polyurethane membranes, collagen sheets, plates, powders, foams or sponges (e.g. polyurethane or silicone) , hydrocolloids (e.g., carboxymethylcellulose, CMC), collagen and hyaluronic acid based dressings, including a combination of the above.

PL 204 797 B1

Alginate, chitosan and hydrocolloid dressings, when applied to a wound, collect the exudative fluid. In doing so, they produce an aqueous gel on the wound surface, and this gel is believed to be beneficial to the wound healing process by keeping the wound moist.

It is also contemplated that the active enamel substance can be incorporated into a tissue glue also containing e.g. fibrinogen and thrombin, and optionally Factor XIII or another plasma coagulating factor to arrest bleeding.

Tissue adhesives can be prepared either as a premix of the active enamel substance, fibrinogen and Factor XIII, and thrombin is added only afterwards, immediately before applying the tissue adhesive to the wound. Alternatively, a pre-mixture of fibrinogen and active enamel substance, and optionally Factor XIII, may be applied to the wound prior to thrombin attachment. Under in situ conditions, thrombin converts fibrinogen to fibrin, thereby causing the coagulation phenomena normally associated with wound healing. The presence of an active enamel substance in the tissue adhesive may, as discussed above, serve to accelerate the healing process. A commercially available product suitable for incorporation of an active enamel substance is Tisseel®, a two-component fibrin sealant, manufactured by Immuno, AG, Vienna, Austria.

In the toothpaste or mouthwash, or in other preparations for application to the teeth or root of the teeth, the active enamel substance may be either dissolved in a slightly acidic base or as a dispersion in a neutral pH base. It is assumed that in use, the active enamel substance can form a protective film on the tooth surface, thereby preventing the deposition of caries-causing bacteria (see Example 4 below). In such dental care formulations, the active enamel substance may be combined with other anti-caries ingredients, particularly fluoride, and with other trace elements such as vanadium or molybdenum. It is assumed that at a neutral pH, trace elements bind to (e.g. ionic bonds) or blend into the active enamel substance, from which they release and exert an anti-caries effect when the active enamel substance is dissolved at a pH of about 5.5 or lower, e.g. due to the production of acids by caries-causing bacteria.

The above-mentioned topical compositions are most suitable for application directly to wounds, or may be suitable for application to or into the appropriate orifices of the body, e.g., the anus, vaginal urethra, ear, nose and vestibule. The compositions may simply be applied directly to the surface to be treated or otherwise conveniently administered.

Compositions that have proven suitable for topical application are those that exhibit thixotropic properties, i.e. the viscosity of the composition changes upon agitation or agitation, whereby the viscosity of the composition may be reduced during administration and the viscosity of the composition upon application to a wound increases such that remains at the injection site.

Suitable compositions according to the invention may be suspensions, emulsions or dispersions. Such compositions contain the active enamel substance in combination with a dispersing or wetting or suspending agent, as appropriate, and / or one or more preservatives and other pharmaceutically acceptable excipients. Such compositions may also be suitable for use in delivering the active enamel substance to e.g. intact or damaged mucosa of organs such as the mouth, cheeks, nose, rectum, vagina, or for administration to intact or damaged skin or wounds.

Suitable dispersing or wetting agents are, for example, naturally occurring phosphatides, e.g. lecithin, or soybean 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 hexitol or hexitol anhydride, e.g. polyoxyethylene stearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, etc.

Suitable suspending agents are, for example, naturally occurring gums such as, for example, acacia, xanthan gum, or gum tragacanth; 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, etc.

Suitable examples of preservatives for use in the compositions made according to the use of the present invention are the same as mentioned above.

PL 204 797 B1

The compositions prepared in accordance with the present invention may also be administered orally. Orally suitable compositions may be presented as a powder, or they may be solid, semi-solid, or liquid.

Compositions for oral use include solid dosage forms such as, for example, powders, granules, granules, sachets, tablets, capsules, effervescent tablets, chewable tablets, lozenges, immediate release and modified release tablets, and liquid or liquid formulations. such as solutions, suspensions, emulsions, dispersions and mixtures. Furthermore, the compositions may be in the form of powders, dispersible powders or in the form of granules for suspension in water by addition of a liquid agent, e.g. an aqueous medium. With regard to a solid dosage form for oral (or topical) use, the composition for use according to the invention usually comprises active enamel substances and, optionally, one or more pharmacologically acceptable excipients. Such excipients can 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, calcium sulfate or sodium phosphate; granulation and disintegrating agents, e.g. cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sulfate, alginates, or alginic acid and chitosans; binding agents, e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, polyvinyl cellulose, polyvinyl cellulose propylene glycol; and chitosans; lubricants, including glidants and anti-sticking agents, e.g. magnesium stearate, zinc stearate, stearic acid, silicates, hydrogenated vegetable oils, or talc.

Other pharmaceutically acceptable excipients can be dyes, fragrances, plasticizers, wetting agents, buffering agents, etc.

In those cases where the pharmaceutical composition is in the form of a solid dosage unit dosage form (e.g., tablets or capsules), the dosage unit form may be provided with one or more coatings as listed below.

In cases where the composition is in the form of a tablet, capsule, or in multiple unit compositions, the composition or the individual units or the single unit tablet or capsule may be coated, e.g. based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers (Eudragit), polyethylene glycols and / or polyvinylpyrrolidone), or enteric coated (e.g. based on methacrylic acid copolymer (Eudragit), cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate, shellac and / or ethyl cellulose).

In addition, delay materials such as, for example, glycerol monostearate or glycerol distearate, can be used.

For application to the rectal or vaginal mucosa, suitable 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 may be included, e.g., enhancers or surfactants.

For application to the nasal mucosa (as well as the oral mucosa), inhalable sprays and aerosols are suitable compositions according to the invention. A typical intranasal composition comprises the active enamel material in particulate form, optionally dispersed in a suitable carrier. The pharmaceutically acceptable carriers and auxiliaries and optionally other pharmaceutically acceptable materials present in the composition, such as diluents, enhancers, flavoring agents, preservatives, etc. are selected in accordance with accepted pharmaceutical practice in a manner understood by those skilled in the preparation of pharmaceuticals.

In the pharmaceutical compositions produced according to the invention for the skin or mucous membranes, the active enamel substance is generally present in concentrations ranging from about 0.01% to about 99.9% by weight. The amount of composition used is usually apparent

PL 204 797 B1 from the amount of total protein per cm² wound / skin / tissue, corresponding to ranging from about 0.01 mg / cm² up to about 20 mg / cm² such as from about 0.1 mg / cm² up to about 15 mg / cm².

The amount of composition used depends on the concentration of the active enamel substance in the composition and on the release rate of the active enamel substance from the composition, but is usually in the range of at most about 15-20 mg / cm2.².

In those cases where the active enamel substance is administered in the form of a liquid composition, the concentration of the active enamel substance in such composition is usually in the range of from about 0.1 to about 50 mg / ml. In some cases, higher concentrations are needed and thus concentrations of at least about 100 mg / ml can be obtained.

When the mixture is to be used in the oral cavity, the following dosages are suitable.

An experimentally made cavity area (in monkeys) within the oral cavity is usually 4 x 2 x 5-6 mm, corresponding to about 50 μl or from about 0.025 to about 0.15 mg of total protein / mm², or about 2.5-15 mg / cm². Typically up to 0.5, e.g. 0.4, 0.3, 0.2 or 0.1 ml of the composition at a concentration of about 1-40 mg / ml, e.g. 5-30 mg / ml is used.

The surfaces of cavities in the human mouth and those caused by periodontal disease are about 5-10 x 2-4 x 5-10 mm, corresponding to about 200 μl of the composition, and usually at most 0.5-1 ml, 0.2-0 , 3 ml per tooth, of a composition having a concentration of about 1-40 mg protein / ml, such as, e.g., 5-30 mg / ml. A concentration of 0.2-0.3 mg / ml corresponds to approximately 6 mg of protein per 25-100 mm² or about 0.1 mg / mm²if it is calculated for the root area only. Usually an excess volume is added to cover the entire surface. Even for multi-layer administration, only a small fraction of the above-mentioned amounts is needed.

Generally about 0.1-0.5 ml, such as 0.15-0.3 ml or about 0.25-0.35 ml of a composition containing an enamel active substance is used for volume deficiency in alveolitis (extraction holes). tooth).

The concentration of the active enamel substance in the composition is properly about 1-40 mg total protein / ml, such as e.g. 5-30 mg / ml. When 0.3-0.4 ml of such a composition is administered per wisdom tooth, this volume corresponds to approximately 0.1 mg / cm² (alveolus calculated as a cylinder with a radius of 5 mm and a height of 20 mm).

The concentration of the active enamel substance in the pharmaceutical composition depends on the type of enamel substance, its activity, the severity of the condition requiring treatment or prevention, the age and general condition of the patient. Useful methods for determining the appropriate concentration of the active enamel substance are well known to those skilled in the art and can be determined according to Good Clinical Practice (GCP) or New Drug Testing ("IND") guidelines included e.g. in International Standard ISO / DIS 14155 Clinical Investigation of medical devices, 1994, and ICH (International Cometee for Harmonization): Harmonized tripartite guideline for good clonical practice, Brokwood Medical Publications, Ltd, Surrey, UK, 1996). The skilled person using the techniques described in the available textbooks, guidelines and regulations described above as well as on the basis of his knowledge is able to establish an exact dosage regimen that can be implemented for any active enamel substance and / or select other active substances and dosing using only a routine experimental procedure .

The invention is further disclosed with reference to the accompanying drawings.

Fig. 1 shows DNA synthesis in human PDL cells stimulated by EMD or in unstimulated cells;

Figure 2 shows the production of TGF-B1 by human PDL cells stimulated by EMD and by unstimulated cells;

Fig. 3 is a schematic drawing of the flow chamber and computer system used in the flow experiment described in Examples 3 and 4 below;

Figures 4, 5 and 6 are the results of three separate experiments showing the attachment of Actinomyces viscosus to glass plates coated with EMD or acetic acid, respectively;

Figures 7, 8 and 9 are graphs showing the results of three separate experiments showing the adherence of Streptococcus mutans to glass plates coated with EMD or acetic acid, respectively;

Fig. 10A is an X-ray photograph showing postoperative damage following wisdom tooth extraction; and

Fig. 10B is an X-ray photograph showing regeneration of the periodontal ligament after EMD treatment as described in Example 12 below.

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EXPERIMENTAL PART

Materials and methods

Enamel matrix derivative, EMDOGAIN®, from BIORA AB, S-205 12, Malmo, Sweden, containing 30 mg of lyophilized enamel matrix protein (hereinafter abbreviated as EMD) and 1 ml of carrier solution (propylene glycol alginate), were mixed before use, provided that protein and vehicle were not tested separately. The weight ratio of proteins with peaks of 20, 14 and 5 kDa was 85/5/10, respectively.

A thermally treated EMD is one that has been heated for 3 hours at about 80 ° C to inactivate residual proteases.

The 20 kDa amelogenin protein and the 5 kDa rich amelogenin peptide (TRAP) 5 kDa were isolated from EMD using HPLC chromatography (TSK G-2000 SW gel, equilibrated with 30% acetonitrile in 0.9% NaCl), and purified by reverse phase chromatography (Pro-RPC, HR 5/10, Pharmacia-Upjohn, Sweden) using an acetonitrile gradient. The isolated protein / polypeptides were then added in varying amounts to the EMDOGAIN® carrier solution, unless tested separately.

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

All bacteria and yeasts were primarily isolated from patients, classified by metabolic tests and antigenically typed according to standard procedures. The species of bacteria and yeast used for the tests are listed in the table below.

Serum albumin (bovine) and type I collagen (bovine) were obtained from Sigma, St. Louis, USA.

"Brain Hart Infusion agar" agar plates (ie, containing brain and heart extract) from Difco were supplemented with human erythrocytes (100 ml per liter of agar).

Example 1

Cell proliferation and production of TGF-e1 by PDL cells treated with EMDOGAIN®

The EMD stock solution was prepared by dissolving the contents of the ampoule (containing 30 mg EMD) in 3 ml of sterile 0.1% hyaluronic acid (HAc) solution. 60 microliters of EMD stock solution was added to 6000 microliters of Dulbecco Modified Eagle's Medium containing 10% fetal calf serum and 1% penicillin-streptomycin solution. 300 μl of the mixture was added to the wells of 96-well plates (NUNC A / S, Denmark, No. catalog 167008), 1,000 human PDL cells from the human periodontal ligament (obtained from healthy human periodontal tissue from orthodontically removed premolars and cultured as described in Somerman et al., J. Dental Res. 67, 1988, p. 66-70) was added to each well and incubated for 5 days at 37 ° C in an atmosphere of 5% carbon dioxide.

PDL cells used as control were grown in Dulbecco's Modified Eagle's medium as described above but in the absence of EMD.

After incubation, a cell proliferation assay was performed by measuring 5-bromo-2-deoxyuridine (BrdU) incorporation according to the manufacturer's instructions for the assay (Boehringer Mannheim, Cat. No. 1647 229). In this procedure, BrdU is incorporated in place of thymidine in the DNA of growing cells. BrdU incorporation is detected by ELISA and the amount of BrdU measured by this assay is a measure of DNA synthesis, and consequently a measure of PDL cell proliferation.

The results shown in Fig. 1 show that in the presence of EMD, PDL cells proliferate significantly faster than PDL cells cultured without EMD.

20 µl of 1N HCl was added to 100 µl of cell culture supernatant in the wells for 10 min. in room temperature. The incubation mixture was neutralized by the addition of 20 µl of 1 N NaOH / 0.5 M HEPES. 100 µl of this mixture was added to 400 µl of a dilution buffer. 200 µl of the solution was subjected to ELISA using the Quantkines kit (Cat # DB100), available from R&D Systems, UK, according to the manufacturer's instructions.

The results presented in Fig. 2 show a significant increase in TGF-e1 production in PDL cells incubated with EMD as compared to PDL cells not incubated with EMD.

Example 2

Research on the growth of microorganisms in the presence of enamel matrix derivatives and enamel matrix proteins

The aim of these studies was to demonstrate the inhibitory effect of enamel matrix derivatives and enamel matrix proteins on the growth of microorganisms in vitro.

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The proteins used in this example were dissolved in phosphate buffered saline (PBS) adjusted to pH 5.5 with acetic acid. The microorganisms were suspended in PBS pH 6.8 to a final concentration of 0.4 corresponding to OD600.

μΐ EMDOGAIN® (30 mg EMD in 1 ml PGA) and 50 μΐ EMD, heat treated EMD, fractions A, B, C and H EMD (all 10 mg protein per 1 ml buffer) were spotted on the agar plate and allowed to dry on the surface of the plate (diameter 9 cm, plain agar for resistance testing with the necessary additives for each type of bacteria). The homogeneous suspension of microorganisms (1 ml, OD280 = 0.5) was then spread over the surface of the plate and the plates were incubated for 3 days at 35 ° C (aerobic conditions) or for 14 days under CO2 (anaerobic conditions) or other conditions. anaerobic, as required for each type of bacteria. All cultures were checked daily. Type I collagen and serum albumin (both bovine) were tested under the same culture conditions as controls.

Undiluted propylene glycol alginate (PGA-EMD vehicle), PBS buffer and hyaluronic acid (alternative vehicle for EMD) were also used as negative control.

The results are shown in Table 1. Only the enamel matrix or enamel matrix protein derivatives inhibited microbial growth. There were no traces of a diffusion zone around the protein indicating that the used EMD protein aggregated on the agar surface and that only bacteria in direct contact with the protein were inhibited. When samples were taken from the inhibition zone and grown in liquid medium (LB broth with additives), monocultures of primary bacteria were recovered, suggesting that the active proteins are not bactericidal. All control tests were negative, no non-specific mechanism influencing the results was demonstrated.

Table 1

Increase (+/-) on the surface of the test substance

<td>Strains</td><td></td><td>Type I collagen</td><td>Alginate propylene glycol</td><td>EMDOGAIN®</td><td>EMD</td><td>EMD warm</td><td>EMD fraction A</td><td>EMD fraction B</td><td>EMD fraction C.</td><td>EMD fraction H.</td><td>Hyaluronic acid</td><td>Buffer control</td>

<td>Actinobacillus actinomycetemcomitans</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td><td> +/-</td><td> +</td><td> -</td><td> +</td><td> +</td><td> +</td>

<td>Escherichia coli</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td><td> +</td><td> +</td><td> +/-</td><td> +</td><td> +</td><td> +</td>

<td>Staphylococcus aureus</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Streptococcus mutans</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Bacillus subtilis</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Candida albicans</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

EMD fraction A: mainly amelogenin ~ 26-20kDa,

EMD fraction B: proteins ~ 17-13kDa,

EMD fraction C: peptides ~ 10-5kDa,

EMD H fraction: all EMD proteins with a molecular weight above 27 kDa, + indicates normal growth of the microorganism,

- means complete inhibition of the growth of microorganisms, +/- means some inhibition of growth as compared to the negative control.

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

These results show that EMD contains proteins or peptides that aggregate on the surface can inhibit the growth of certain gram negative rods and certain gram positive cocci. On the basis of the basic behavioral characteristics of EMD proteins (ref. Jpc), since the effect is not bactericidal, a reasonable explanation for the observed phenomenon is the formation of an insoluble barrier from protein aggregates that separates the microorganisms from the growth substances they need.

PL 204 797 B1

Example 3

Effect of EMD on the degree of adhesion of Actinomyces viscosus in vitro

Admission

The effect of EMD on the initial adherence of Actinomyces viscosus, an orally abundant bacterium in plaque not commonly thought to be associated with severe periodontopathy, was investigated. Although this organism may aggregate with Porphyromonas gingivalis and may therefore have an influence on the colonization of the root surface with potential periodontal pathogens, Actinomyces spp. are found in relatively large amounts in healthy subgingival areas (these observations are confirmed by the reports of Liljemark et al., Microbiol. Immunol. 8, 1993, 5-15, who found that the percentage of Actinomyces spp. increased significantly after periodontal treatment. Haffajee et al. , J. Clin Periodont. 24, 1997, 767-776, concluded from the counts of subgingival plaque microorganisms that Actinomyces viscosus and T. denticola were relatively abundant in patients with a good response to primary periodontal therapy.

Materials

Actinomyces viscosus HG85 was provided by Dr. AJ van Winkelhoff (Department of Oral Pathology, ACTA). Emdogain was provided by BIORA (Malmo, Sweden). RBS detergent was purchased from Fluka (Fluka Chemie AG, Buchs, Switzerland).

Bacterial growth and uptake

Actinomyces viscosus was transferred from blood agar cultures to an aliquot of Schaedler broth for 24 h. at 37 ° C. This culture served to infect another Schaedler broth culture that was kept for 16 hours. Cells were harvested by centrifugation (5 min at 6,500 xg) and washed twice with demineralized water. The microorganisms were then sonicated for 20 sec. at 30W (Vibra cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to break down bacterial chains and aggregates. Sonication was performed intermittently while cooling in an ice-water bath. Cells were counted using a Burker-Turker counter. Finally, the Actinomyces viscosus was suspended in an adhesive buffer (2 mM potassium phosphate, 50 mM potassium chloride and 1 mM calcium chloride, pH 6.8)

Covering glass plates

Glass plates were thoroughly cleaned by sonication in 5% RBS detergent, rinsed copiously with tap water, rinsed in methanol and finally rinsed in distilled water. This procedure results in a water contact angle of zero degrees. The EMD was dissolved in 0.01 M acetic acid at a concentration of 7.5 mg / ml. The glass plates were divided into two halves with a Teflon marker (DAKO A / S, Glostrup, Denmark). Acetic acid (0.01 M) was applied to one side and 250 µg EMD was applied to the other. The plates were air dried in a fume hood for 4-6 hours.

Flow experiment

The flow cell and computer system used in this experiment are shown schematically in Fig. 3. Prior to each experiment, all tubes and the flow cell were filled with Adhesion Buffer, taking care that the system did not contain air bubbles. The flow rate was set at 2.5 ml / min (comparable to the average human saliva flow). The bacterial suspension circulated through the system for approximately 3-4 hours, and the cells adhered to the substrate were counted. Three independent experiments were carried out. Each experiment using 3x10⁸ cells per 250 ml of adhesion buffer. During the experiment, images were recorded every 10-15 min on 6 previously selected places on EMD-coated and control plates. The channel height of the parallel flow chamber was 0.6 mm.

Data analysis

After counting the number of adhesion cells on all recorded images, the data was converted to the number of bacteria per cm². In each experiment, the final number of microorganisms per cm²was 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 number of sticking microorganisms especially during the first 150 min of flow. After this time, the number of microorganisms attached to the EMD plateaued to 2.0x10⁶ bacteria per cm² this is about 4 times more than on the acetic acid-treated parts of the plates.

Also in experiment 2 (Fig. 5), EMD quite dramatically stimulated the adhesion of Actinomyces viscosus to the substrate. However, at the beginning of the experiment, this effect was less pronounced. Perhaps

This was due to the lower density of organisms used in the flow system. After 90 min, the number of bacteria adhering to the EMD gradually increased relative to the control, reaching a maximum of 1.4 x 10⁶ per cm², triple increase.

The third experiment (Fig. 6) showed the stimulation of the adhesion of Actinomyces viscosus to the EMD surface after just 5 min of flow. EMD induced a sharp increase in the number of adhering organisms in the first 45 min. Thereafter, the sticking progressed progressively. Sticking to the acetic acid treated surface followed a similar pattern but with less microorganisms adhering. After 200 min, the adhesion to the EMD coating was twice as high as to the acetic acid treated surface.

In all three experiments, the differences were statistically significant (p &lt;0.05).

The results indicate that the EMD used to coat the glass surface had a significant in vitro stimulating effect on the adhesion of Actinomyces viscosus. Although it is still unknown what factors are responsible for this enhanced initial adhesion effect, it is believed that organisms interact with proline residues present in abundance in the amelogenin components of commercially available protein blends. Bacterial adhesion is often determined by specific protein-peptide or lectin-carbohydrate recognition. It is known that Actinomyces viscosus with its type 1 fimbriae can bind to proline-rich proteins such as Protein Rieh Proteins (PRP's) and collagen I and III.

The specific interactions between EMDs and certain oral microorganisms can have important consequences for the composition of the oral biofilm as the ecology of the plaque can change. If such an ecological shift could be made to promote organisms unrelated to periodontal disease, the use of EMD could result in an improvement in periodontal health just through this action. This, of course, is completely independent of the other beneficial effects of EMD.

Example 4

Effect of EMD on the degree of adhesion of Streptococcus mutans in vitro

Admission

There is ample evidence of a causal role for plaque organisms in the pathogenesis of oral diseases such as periodontitis and caries. According to the recent model of supragingival plaque formation, Streptococcus spp. Are considered to be the dominant colonizers of the tooth surface. The plaque then develops by the growth of the bacteria and by the further growth of other species of bacteria. Growth occurs through bacterial-bacterial bonds or can be mediated by saliva particles. Plaque formation facilitates the production of extracellular macromolecules. Streptococcus mutans is currently considered to be one of the biofilm species that may be the focus of attention because of its association with dental caries. Even though several gram-positive bacteria (i.e. S. mutans) causes alveolar bone loss in animals derived from "sterile animals, these organisms do not contribute substantially to the development of the periodontal pocket. Nevertheless, potentially pathogenic microorganisms must be able to evade the host defense and immune mechanisms as well as initiate damage to the host's tissues.

Materials

Streptococcus mutans NS is courtesy of Dr. H van der Mei (Materia Technica, University of Groningen). EMDOGAIN was provided by BIORA (Malmo, Sweden). RBS detergent was purchased from Fluka (Fluka Chemicals AG, Buchs, Switzerland).

Bacterial growth and uptake

Streptococcus mutans were transferred from blood agar cultures to an aliquot of Todd Hewitt broth for 24 h. at 37 ° C. This culture was used to infect another Todd Hewitt broth culture that was kept for 16 hours. Cells were harvested by centrifugation (5 min at 6,500 xg) and washed twice with demineralized water. The microorganisms were then sonicated for 20 sec. at 20W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to break down bacterial chains and aggregates. Sonication was performed intermittently while cooling in an ice-water bath. Cells were counted using a Burker-Turker counter. Finally, Streptococcus mutans was suspended in the adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, 1 mM calcium chloride, pH 6.8)

Covering glass plates

Glass plates were thoroughly cleaned by sonication in 5% RBS detergent, rinsed copiously with tap water, rinsed in methanol, and finally rinsed in distilled water. This procedure

PL 204 797 B1 results in an angle of contact with water of zero degrees. The EMD was dissolved in 0.01 M acetic acid at a concentration of 7.5 mg / ml. The glass plates were divided into two halves with a Teflon marker (DAKO A / S, Glostrup, Denmark). Acetic acid (0.01 M) was applied to one side and 250 µg of EMD to the other. The plates were air dried in a fume hood for 4-6 hours.

Flow experiment

The flow cell and computer system used in this experiment are shown schematically in Fig. 3. Before each experiment, all tubes and the flow cell were filled with adhesive buffer, taking care that the system was free of air bubbles. The flow was set at 2.5 ml / min (comparable to the average human saliva flow). The bacterial suspension circulated through the system for approximately 3-4 hours, and the cells adhered to the substrate were counted. Three independent experiments were carried out. Each experiment using 3x10⁸ cells per 250 ml of adhesion buffer. During the experiment, images were recorded every 10-15 minutes in 6 previously selected places on EMD-coated plates and control plates. The channel height of the parallel flow chamber was 0.6 mm.

Data analysis

After counting the number of adhesion cells on all recorded images, the data was converted into the number of bacteria per cm². In each experiment, the final number of microorganisms per cm²was used for statistical analysis (Student's t-test for paired observations using n as the number of experiments).

Results

In each of the three experiments, EMD showed an inhibitory effect on the degree of adherence of S. mutans (Fig. 7, 8, 9; p &lt;0.05). Inhibition was 40-70% compared to the acetic acid treated control.

The first experiment (Fig. 7) showed after 10 min of flow an inhibition of the amount of S. mutans adhering to the EMD coated glass surface. After 3 hours, the adhesion was reduced to 60% of the control.

In a second experiment (Fig. 8), EMP started to inhibit the adherence of S. mutans after 1 1/2 hours of flow. The number of S. mutans adjacent to the EMD plateaued to 0.5 million per cm² after about 40 min of flow. After 3 hours the counts were around 25% from control.

The third experiment (Fig. 9) showed inhibition of S. mutans adhesion by EMD already at the beginning of the flow. As in experiment 1, inhibition reached 60% of the control value after 3 hours. flow.

The above studies show that EMD has a significant inhibitory effect on the adherence of S. mutans to a glass surface. A possible explanation for this inhibition could be the presence of hydrophobic components in the EMD mixture. One of the proteins abundantly found in this mixture is amelogenin, a protein having, in addition to an acidic hydrophilic C-terminal sequence, a hydrophobic core containing 100-300 residues rich in proline, leucine, methionine and glutamine. Saito et al., Arch. Oral Biol. 42, 1997, 539-545, found that the adherence of various S. mutans strains to immobilized hydrophobic proteins (OAIS) was inhibited. The authors attribute this effect to negative charges on the surface of microorganisms (as in the case of S. mutans). Other surface characteristics may also be related to the effect on binding to the substrate. S. mutans contains the I / II surface antigen which has an N-terminal portion particularly rich in alanine, and also contains tandem repeats. This region has a putative alpha-helical structure, adopting a bipolar spiral conformation, and may contribute to the hydrophobicity of the cell surface associated with the expression of the I / II antigen.

Example 5

Effect of EMD on the growth of certain periodontal pathogens

Prevotella intermedia and Porphyromonas gingivalis were pre-cultured for 10-14 hours at 37 ° C in thioglycol broth supplemented with 0.5 mg / L vitamin K and 5 mg / L hemin in an aerobic atmosphere produced by GasPakPlus in appropriate vessels. When the culture density reached the OD600 of 0.1-0.2 which corresponded to a cell density of 10⁶-10⁷ cfu (colony forming units) per ml, aliquots of 100 μl were taken and the bacteria were precipitated by centrifugation. The bacteria were resuspended in 100 μl of a freshly prepared mixture of human saliva and saline, and the suspensions containing 10⁵-10⁶ cells were transferred to sterile 1.5 ml Eppendorf tubes and mixed with (i) 100 μl EMD solution (3 mg EMD in 0.1 ml PGA), (ii) 100 ml PGA or (iii) 100 μl serum solution / NaCl solution as a growth control. Samples of 10 μl were taken for growth rate control at 0, 3, 6 and 24 hours. The samples were serially diluted in sterile 0.9% NaCl and 10 µΐ of the individual dilutions were plated on Schaedler agar. The breeding conditions were the same as the pre-culture. The agar plates were incubated for 3-4 days and then the cfu and cell density were calculated. All experiments were performed 6 times.

Results (given as cfu / ml as percentage of concentration at time 0)

1) Control cultures at different time points

<td></td><td> 0</td><td>3 hours</td><td>6 hours</td><td>24 hours</td>

<td>P. intermedia</td><td> 100</td><td> 160</td><td> 25</td><td> 10</td>

<td>P. gingivalis</td><td> 100</td><td> 100</td><td> 125</td><td> 150</td>

2) Cultures in the presence of PGA at different time points

<td></td><td> 0</td><td>3 hours</td><td>6 hours</td><td>24 hours</td>

<td>P. intermedia</td><td> 100</td><td> 140</td><td> 25</td><td> 10</td>

<td>P. gingivalis</td><td> 100</td><td> 75</td><td> 50</td><td> 5</td>

3) Cultures in the presence of EMD at different time points

<td></td><td> 0</td><td>3 hours</td><td>6 hours</td><td>24 hours</td>

<td>P. intermedia</td><td> 100</td><td> 40</td><td> 0</td><td> 0</td>

<td>P. gingivalis</td><td> 100</td><td> 30</td><td> 0</td><td> 0</td>

Cultures were significantly inhibited by the presence of EMD compared to controls with or without any EMD addition

Example 6

Study of the improvement of soft tissue wound healing with ENDOGAIN® after periodontal surgery

The purpose of this example is to show the effect of enamel matrix derivatives and / or enamel matrix proteins in improving healing after surgical periodontics.

Experimental damage to the marginal periodontium in more than 50 teeth of Macaque monkeys was performed with a dental drill by removing the root cement, the periodontal membrane and the edge of the alveolar bone to a cervical-apical distance of approximately 5 mm. Then, nothing was applied to the experimental lesion: nothing (control) or the enamel matrix derivatives (EMDOGAIN both as an unsuspended lyophilized powder and as a suspension) were applied. The protein concentration in the suspension was about 5-30 mg / ml and the amount applied to the lesion was about 0.1-0.2 ml.

Wound healing was assessed visually over the next 8 weeks. In the lesions where EMDOGAIN® was applied, healing was good (no redness or swelling) and insignificant plaque after 2 weeks when the sutures were removed, good healing and slight gingivitis were observed after 5 weeks and healing without complications after 8 weeks when the experiment was terminated. In contrast to control defects where there was an inflammatory reaction with gingival exposed and copious plaque after 2 weeks, with severe gingivitis and gingivitis at both 5 weeks and 8 weeks.

Example 7

Research on the influence of enamel matrix derivatives and enamel matrix proteins on the healing of wounds formed during periodontal procedures

The purpose of this example is to demonstrate the effect of enamel matrix derivatives and enamel matrix proteins on rapid wound healing in patients after periodontal surgery. Fifty-five (55) patients requiring periodontal surgery were divided into two groups - one treated with the classical Widman flap method (20 patients), and the other, following the same procedure plus EMDOGAIN® (35 patients) ( concentration was 30 mg protein / ml and about 0.3 ml was used per tooth). None of the patients received antibiotics during the procedure, but all were instructed to use aseptic fluid (chlorhexidine) to rinse the mouth daily.

PL 204 797 B1

Patients were interviewed while the sutures were removed (1-3 weeks after surgery). While 3 (= 15%) of the control patients were cases requiring antibiotic therapy, only one (= 3%) of the patients in the EMDOGAIN®-treated group required such treatment.

Example 8

Research on the influence of enamel matrix and enamel matrix proteins on wound healing after tooth extraction

The purpose of this example is to demonstrate the effect of enamel proteins / enamel matrix derivatives on wound healing following removal of the third molar. Patients aged 30 years or older, with symmetrical or incomplete impaction of the third molar, had the third molar removed according to the classic method requiring the vertical flap to be elevated to perform the necessary osteotomy and incision, while the second was removed in the same way, and its socket was filled with EMDOGAIN® before suturing. All patients received antibiotics (3 g of amoxicillin or 1 g of erythromycin) 1-2 hours before surgery and received Ibuprofen (600 mg x 3) after surgery. They were instructed to rinse the mouth with chlorhexidine (0.1%, 10 mL x 2) for 4 weeks.

The sutures were removed after 2 weeks. The healing of the EMDOGAIN® and control sites was assessed by both the patients and the dentist. In one center, 9 patients had bilateral extractions with / without EMDOGAIN®. One patient had slight irritation from the sutures on both sides, while another patient had severe pain on the control side but had no problems with the EMDOGAIN® treated side. In the second center, three out of 6 patients had pain in the control side only. Finally, in the third center, one patient had a serious complication - alveolitis - diagnosed on the control side. The EMDAGAIN® treated site healed without any problem. Another patient had slight irritation from the sutures on both sides of the removal, but only the control side was inflamed, painful, and required repeated salt irrigation and painkillers.

These clinical results indicate that applying EMDOGAIN® to the alveolar process after wisdom tooth extraction can facilitate healing and reduce frequent otherwise painful swelling.

Example 9

Study of the influence of enamel matrix derivatives and enamel matrix proteins on the healing of the so-called dry socket

The purpose of this example is to demonstrate the effect of enamel proteins / enamel matrix derivatives on the healing of alveolitis (dry socket)

After removal of the infected leftover root (radix relicta), a 70-year-old male patient complained of severe pain and swelling at the extraction socket site. Upon examination by a dentist, it turned out that a dry socket had developed, in which the initially formed clot had dissolved and the bone wall of the alveolar process was necrotic, and the adjacent bone and soft tissues were inflamed.

The patient had a history of heart failure and was treated with the Meravan anticoagulant. As a result of this condition, he had decreased peripheral blood flow. He also smoked a few cigarettes a day regularly.

Alveolitis was treated traditionally by removing dead bone and provoking new bleeding. The gums were also activated and a suture was applied to close the socket. The patient was given penicillin (apocillin 660 mg, 2 tablets in the morning and evening for 7 days) to fight the infection and was also instructed to rinse his mouth twice daily with a chlorhexidine solution. Five days after the end of antibiotic treatment, the patient examined at the dental clinic continued to complain of severe pain. The performed examination of the operating field - visual, palpation and probe - showed that the inflammation of the socket continued and that there was more necrotic bone tissue. X-ray examination showed bone damage and necrosis all the way to the apex of the root. The surgical field was cleaned once more and the damaged bone was filled with EMDOGAIN® (30 mg / ml, 0.5 ml at most) and a new suture was placed over the gingiva to cover the socket. No additional treatment was given, but the patient was asked to continue rinsing his mouth with a chlorhexidine solution. Two days later, the patient informed the clinic that the pain and swelling were gone. Clinical examination and removal of the suture one week after administration of EMDOGAIN® showed good healing without necrotic tissue or inflammation and normal gingiva without redness or swelling covering the wound area. There was no bleeding or soreness on palpation or

PL 204 797 B1 tube. There was no bad smell or oozing. The patient did not report any pain or other symptoms.

Example 10

Research on the prophylactic effect of enamel matrix derivatives and enamel matrix proteins on the formation of a dry socket

The purpose of this example is to demonstrate the preventive effect of enamel matrix derivatives and enamel matrix proteins in counteracting dry socket (alveolitis sicca).

An 82-year-old female patient suffered a longitudinal fracture of the root of tooth 44. This tooth was a pillar of the bridge between teeth 35 to 46 and was endodontically treated many years earlier. Clinically, the gums surrounding the tooth were inflamed and there was a gingival pocket along the entire length of the tooth root, all the way to the top, on the lingual side. X-ray examination showed severe local periodontitis 44.

The oral hygiene was satisfactory, but due to the condition of the heart treated with Marevan anticoagulant, gingival bleeding easily occurred during probing. Six months earlier, the patient had had her tooth 35 surgically removed due to severe periodontitis. After this operation, she suffered from long-term dry socket inflammation. She was very careful that the removal of tooth 44 did not cause a complication similar to that she had experienced before. She was informed that the combination of her age, Marevan treatment, and an infected root and gingival pocket dramatically increased the risk of postoperative complications such as alveolar inflammation, but that there was no alternative to surgical removal of the root fragments. The patient agreed to have tooth 44 removed and, as an experiment, she underwent prophylactic treatment with EMDOGAION® in order to prevent the development of a dry socket. The patient was anesthetized, incised and the cheekbone was removed to allow removal of the root fragment without loosening the bridge. After removal, the empty socket was cleaned mechanically and filled with EMDOGAIN © (30 mg / ml, 0.5 ml at most), covered with a flap and sutured. The same evening, the patient informed by telephone that there was continuing bleeding from the treatment site (treatment with Meravan was not stopped prior to the procedure), but there were no other symptoms. When the suture was removed five days later, the soft tissues of the surgical wound were completely healed. The patient did not report any other symptoms such as pain or swelling and was generally very satisfied with the treatment.

Example 11

Research on the influence of enamel matrix derivatives and enamel matrix proteins on the healing of post-traumatic complications

The purpose of this example is to demonstrate the effect of enamel proteins / enamel matrix derivatives on the healing of traumatic complications in patients.

After the accident, the patient's upper front teeth were ligated in the emergency room. The dentist diagnosed the necrosis of teeth 11 and 21, and teeth 12 and 22 had class I or II lateral incisor fractures. The marginal gingiva was severely inflamed and adhered poorly to the tooth surface. The patient complained of numbness, swelling, bad smell and taste. There was also evidence of damage to the periodontal ligament in the apical region of teeth 11 and 21. Both middle incisors were cleaned and the canals were filled with freshly mixed calcium hydroxide Ca (OH) 2.

After 4 weeks, the healing of the wound was considered unsatisfactory. Chronic inflammation developed and the teeth were considered lost. Standard treatment in such cases would be to remove all four incisors and replace them with a bridge or implants. However, the patient strongly disagreed with such a procedure and, as the final attempt to save the teeth, gingival flap surgery was performed on all four teeth (11, 12, 21, 22). Two EMDOGAIN® ampoules (60 mg in 3 ml) were used. A maximum of 0.2 ml of EMDAGAIN® (30 mg / ml) was given by syringe to each tooth prior to application of the flaps which were sutured with seven sutures. Four sutures were removed after 5 days. There was then a significant subjective and clinical improvement. The patient no longer complained of pain, the feeling of numbness was gone, and the affected area had no odor or unpleasant taste. After two weeks, the remaining sutures were removed. The gums showed no signs of inflammation and the patient had no complaints. The gum was healthy and there was no sign of inflammation; it was firmly attached to the teeth and / or the alveolar bone, it was pink in color (not clearly red as in the sites of inflammation), with normal (not swollen) interdental papillae. In addition, significant improvements were found, such as restoration of the dental ligament in the damaged parts of the teeth and the deposition of new bone tissue, as shown by X-ray examination.

PL 204 797 B1

Example 12

Healing traumatic wounds in the vicinity of teeth and nerves

A clinical case

A 39-year-old female patient experienced severe pericoronitis around the lower left wisdom tooth (38). In a state dental clinic, the tooth was partially removed, and the apical part of the mandible was left in the jaw following an iatrogenic root fracture. In pain, the patient was admitted the next day to a specialist maxillofacial surgery ward to remove a fragment of the root. Two days after the procedure, the patient came to her dentist for a checkup. She had a swollen left side with a complete mandibular nerve block. Clinical and radiographic examination revealed that drilling had severely damaged the mandibular bone, the lower apical third of the posterior root of tooth 37, and the mandibular nerve canal (see X-ray, Fig. 1A). The probe depth of the posterior pocket of tooth 37, measured with a probe, was 25 mm below the top of the posterior root apex. In order to stimulate bone and nerve healing and regeneration of the lost ligament on tooth 37, the surgical wound was opened and carefully cleaned. After removing the debris with the exposed bone, the posterior root surface, and the mandibular nerve were covered with EMDOGAIN® (30 mg / ml, excess applied; approximately 1 ml) and the wound was re-sutured with three sutures. The patient was instructed to rinse her mouth with a chlorhexidine solution (Corsodyl) twice a day for the next five days, and as a prophylactic, penicillin (Ampicillin, 66 mg x 4) was administered for 5 days.

Ten days later, the patient reported for a checkup and removal of stitches. During this time, the swelling had subsided 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 severed nerve was, at best, uncertain. In this state, the viability of tooth 37 could not be assessed due to anesthesia. Normally, root damage, as in the described case, leads to pulp necrosis and tooth syngus. Endodontic treatment is recommended to prevent such complications. However, to see if the experimental treatment could facilitate the healing of the dental ligament, the patient agreed to leave the tooth untreated for an extended period of time. She was enrolled in a monthly examination.

Two months after the above check, the patient reported local hypersensitivity in her lower left lip, a sign of nerve healing. The soft tissues in area 37-38 were completely healed with no scarring. X-ray examination also revealed new bone formation in the extraction socket. Tooth 37 and the surrounding tissues were still anesthetized.

Four months after the treatment, the anesthesia subsided and the tested tooth 37 turned out to be alive but hypersensitive by thermal and electrical tests. X-ray examination showed significant filling of the socket with bone tissue and signs of periodontal regeneration on the posterior root of tooth 37.

Five months after starting treatment with EMDOGAIN ©, the life span of tooth 37 was normal. X-ray examination showed complete regeneration of the dental ligament (Fig. 1B) and the newly formed bone of the alveolar process, with the correct drawing, filled the bone defects and the alveolus. There was no sign of a hyperplasia.

The depth of the posterior pocket of tooth 37, measured with a probe, was now 10 mm, or approximately 1 mm below the glaze cement line. After this control, the patient was considered cured and was discharged.

Comments:

Complete and rapid healing of traumatic wounds in the vicinity of teeth and nerves after the surgical removal of a wisdom tooth is rare. Usually complications as serious as those described above end up with the removal of the damaged tooth, or at least endodontic removal of the tooth pulp and root filling, ending with osteochondrosis. A ruptured nerve takes 8-12 months to heal, if it ever occurs, and often distorted sensation (paraesthesia) lasts for many years in certain regions. The quick and good quality healing of the above-mentioned case is very unusual and should be considered as a symptom of the healing abilities of EMDOGAIN®.

X-rays (X rays):

A: Patient two days after tooth 38 extraction. Noteworthy is a large posterior cavity on tooth 37 and an involvement of the mandibular canal. Also, the alveolar bone on the posterior-buccal side of tooth 37 was removed during the procedure.

B: Patient five months after surgery. The symptom of a fully functional dental ligament (hard lamina, lamina dura) in the defect at the back of the root of tooth 37 is noteworthy. There are no signs of osteochondrosis. The outline of the mandibular canal is now visible and the alveolar cavity is completely

PL 204 797 B1 filled with bone. Also noteworthy is the formation of a new alveolar bone in the posterior cheekbone around tooth 37.

Example 13

Research on the effect of enamel matrix derivatives on the healing of leg ulcers (venous ulcers)

Patient 1

The patient was a male, born in 1926, with a history of recurring clots with very bad post-thrombotic syndromes and recurrent ulceration of varicose veins. He was treated systemically with coumarin derivative anticoagulants, and the ulcers were treated topically with Crupodex (dextran monomer) BIOGAL and 3% boric acid solution. At the time of initiation of treatment with EMDOGAIN®, he had an oval-shaped venous ulcer with dimensions of 5 x 4 cm and a depth of 0.5 mm in a granulation state with very little epithelialization. The wound was decontaminated with 3% hydrogen peroxide and 500 microliters of EMDOGAIN® was dripped, then spread evenly over the entire wound surface with a sterile stick. The EMDOGAIN® was left in the air for 10 minutes and then the wound was covered with Inadine (Johnson &amp; Johnson) Rayon dressing impregnated with 10% povidone iodide ointment.

After five days, epithelialization of the distal part of the ulcer was noted and the ulcer was reduced to 1.8 x 2.2 cm, there were no side reactions (inflammation). EMDOGAIN® was not given. After 12 days, further epithelialization in the proximal part and new epithelialization in the lateral parts of the ulcer covered an area of ​​approximately 2 x 2 cm. About half of the ulcer was healed. 400 microliters of EMDOGAIN® was applied.

After 19 days, there was further epithelialization of the proximal and lateral parts of the ulcer, but not in the distal part, where the ulcer was rather deep (about 1 mm). 300 microliters of EMDOGAIN® were used. From the moment of starting treatment with EMDOGAIN®, the patient did not experience any soreness of the ulcer, in contrast to the period before treatment with EMDOGAIN®. EMDOGAIN® was then administered weekly until day 40 (200 microliters each) and the ulcer was considered fully healed after 47 days.

Patient 2

The patient was a woman, born in 1949 and had varicose veins, chronic venous insufficiency and recurrent venous ulcers. She was multi-allergic to drugs and pharmaceuticals, and she also had varicose veins. Previously, she was treated topically with Otosporin drops (polymyxin B sulfate + neomycin sulfate + hydrocortisone) and hydrocortisone compresses.

At the time of initiation of treatment with EMDOGAIN®, her varicose ulcer was 1 cm in diameter and 2 mm in depth. 300 microliters of EMDOGAIN® were administered.

After 5 days, epithelialization of the width of 2 mm was observed on the entire circumference of the wound, and there were no side effects, inflammatory reaction. No EMDOGAIN® was given. After 12 days, the wound had decreased to a diameter of 2 mm, and 100 microliters of EMDOGAIN® was administered.

After 19 days, the ulcer was still 2 mm in diameter, but the bottom was well granulated and the ulcer was not deep (0.2 mm). 100 microliters of EMDOGAIN® were administered.

The same patient had a different ulcer on the other leg, approximately 0.3 x 1 cm. 200 microliters of EMDOGAIN® were administered.

After 7 days, a new epithelialization appeared and a nice granulation of the wound bottom appeared, and the wound was reduced to 0.2 x 0.5 cm. Then 100 microliters of EMDOGAIN® was again administered for each ulcer once a week until day 40, and the ulcers were considered fully healed after 47 days. No allergic reactions to EMDOGAIN® have been observed.

Another ulcer, approximately 0.5 x 0.3 cm in size, developed on the same leg and was applied with 100 microliters of EMDOGAIN®.

Patient 3.

The patient was a female, born in 1929, had deep venous clots after rubella, and at the time of initiation of treatment with EMDOGAIN® had a very large ulcer measuring approximately 15 x 19 cm in proximal progression, which was considered a hopeless condition after many treatment attempts . 700 microliters of EMDOGAIN® was applied to an area approximately 3 cm from the top edge of the wound.

After 7 days, no epithelialization was detected, but the treated area was more translucent (more elements were visible) and contained small granulation fields, and the site was painless, and there were no signs of progression of pathology. 700 micrograms of EMDOGAIN® was applied to the same site.

PL 204 797 B1

After 4 weeks, in the distal part of the ulcer, not treated with EMDOGAIN, an infection, possibly caused by Pseudomonas, developed. 700 microliters of EMDOGAINE were administered. Infection resolved after 7 days.

Patient 4

The patient was a woman, born in 1947, with varicose veins with superficial thrombophlebitis after rubella, and at the time of initiation of EMDOGAIN® therapy, she had a leg ulcer approximately 2 x 0.8 cm with a clean but non-granulating fundus. 300 micrograms of EMDOGAIN® was administered.

After 7 days, the ulcer had decreased to about 0.7 x 0.3 cm, epithelialization appeared and granulation tissue at the bottom of the ulcer appeared. 200 microliters of EMDOGAIN® was administered, followed by 100 microliters of EMDOGAIN® once a week for five weeks. The ulcer was considered fully healed after five weeks.

Example 14

EMDOGAIN® as an adjunct to non-surgical periodontal treatment in areas with flat surfaces.

Subject

The subject of these studies was to evaluate whether the use of EMDOGAIN® could improve healing caused by non-surgical periodontitis. The effect on places with flat surfaces was particularly assessed.

Research pattern

The studies were conducted as a long-term intra-individual test of 6 months duration and included double-blind, dizzy mouth, administration of a dummy agent (placebo) and random distribution.

Objects of research of patients reported to the Periodontology Clinic, Department of Periodontology, University of Gothenburg for the treatment of moderately advanced periodontal diseases.

Inclusion criteria • At least three flat surfaces of the teeth in each of the two opposing quadrants with pockets protruding to a depth&gt; 5 mm, and at least one pair of sites with pocket depths&gt; 6 mm • The selected tooth must have viable pulp, determined by a thermal or electrical stimulus or if intended for root canal treatment, it was asymptomatic and without technical remarks.

Management (treatment)

After the baseline examination, all patients were instructed to correctly measure supragingival control plates. Tartar removal was performed and the roots were cleaned.

When bleeding from the pockets ceased, 24% EDTA gel (obtained from Biora AB, Sweden) was applied to the pockets for 2 minutes. The pockets were then gently rinsed with salt and either the test substance (EMDOGAIN®) or the control substance (PGA gel) was applied.

Assessment

The pivotal studies at weeks 1-, 2-, 3-, 8-, and 24 included the following variables:

1. Oral hygiene - presence / absence of plaque,

2. Gingival condition (gingival index, Loe 1967),

3. Pocket depth assessed with a probe,

4. The level of adhesion is assessed with a probe,

5. Bleeding after probe examination - present or absent (15 seconds),

6. Tooth sensitivity - after blowing air (yes / no),

7. Degree of discomfort - assessed at weeks 1, 2 and 3 using a 10cm visual analog scale (VAS).

PL 204 797 B1

DENTAL PLATES; mean values ​​(standard deviations)

<td></td><td>CONTROL</td><td>EMDOGAIN®</td>

<td>The starting point</td><td> 0,10 (0,30)</td><td> 0,19 (0,40)</td>

<td>1 week</td><td> 0,08 (0,27)</td><td> 0,05 (0,22)</td>

<td>2 weeks</td><td> 0,05 (0,22)</td><td> 0,02 (0,15)</td>

<td>3 weeks</td><td> 0,05 (0,22)</td><td> 0,10 (0,30)</td>

<td>6 weeks</td><td> 0,14 (0,35)</td><td> 0,19 (0,40)</td>

<td>26 weeks</td><td> 0,12 (0,33)</td><td> 0,07 (0,34)</td>

GUM INDEX; mean values ​​(sd)

<td></td><td>CONTROL</td><td>EMDOGAIN®</td>

<td>The starting point</td><td> 1,40 (0,50)</td><td> 1,40 (0,50)</td>

<td>1 week</td><td> 1,00 (0,32)</td><td> 0,87 (0,52)</td>

<td>2 weeks</td><td> 0,83 (0,44)</td><td> 0,74 (0,45)</td>

<td>3 weeks</td><td> 0,69 (0,60)</td><td> 0,60 (0,50)</td>

<td>6 weeks</td><td> 0,67 (0,53)</td><td> 0,64 (0,58)</td>

<td>26 weeks</td><td> 0,62 (0,54)</td><td> 0,62 (0,49)</td>

Bleeding after induction; %

<td></td><td>CONTROL</td><td>EMDOGAIN®</td>

<td>The starting point</td><td> 100</td><td> 100</td>

<td>1 week</td><td> 67</td><td> 44</td>

<td>2 weeks</td><td> 43</td><td> 33</td>

<td>3 weeks</td><td> 29</td><td> 26</td>

<td>6 weeks</td><td> 33</td><td> 31</td>

<td>26 weeks</td><td> 19</td><td> 24</td>

SUBJECTIVE PATIENT EVALUATION; VAS scale First week

<td></td><td>CONTROL</td><td>EMDOGAIN®</td>

<td>VAS scale</td><td></td><td></td>

<td> 0-20</td><td> 8%</td><td> 0%</td>

<td> 21-40</td><td> 15%</td><td> 8%</td>

<td> 41-60</td><td> 31%</td><td> 30%</td>

<td> 61-80</td><td> 8%</td><td> 0%</td>

<td> 81-100</td><td> 38%</td><td> 62%</td>

Third week

<td></td><td>CONTROL</td><td>EMDOGAIN®</td>

<td> 1</td><td> 2</td><td> 3</td>

<td>VAS scale</td><td></td><td></td>

<td> 0-20</td><td> 14%</td><td> 7%</td>

PL 204 797 B1 cont. table

<td> 1</td><td> 2</td><td> 3</td>

<td> 21-40</td><td> 0%</td><td> 7%</td>

<td> 41-60</td><td> 3%</td><td> 0%</td>

<td> 61-80</td><td> 21%</td><td> 22%</td>

<td> 81-100</td><td> 57%</td><td> 64%</td>

Proposal

Patients have less postoperative hassle, bleed less, less plaque, and an improvement in the gingival index. These results confirm the beneficial effect of EMDOGAIN® on wound healing.

Example 15.

A pilot study on the treatment of injuries in pigs

Introduction

Subject

The subject of this pilot study is to evaluate the healing process of dissection wounds in pigs and to evaluate the effects of EMD on these wounds.

The reason for choosing the animal species

The pig was selected as the model animal for the study because it proved to be a convenient model for assessing wound healing in humans.

Material and methods

Animals

The experiment will be performed on 4 SPF pigs (bred in Denmark, Yorkshire and Duroc). At the beginning of the acclimatization period, the weight of the animals was approximately 35 kg. An adaptation period of one week in which animals are observed daily will eliminate animals in poor condition. All observations will be recorded.

Keeping animals

The research will be carried out in an animal facility equipped with filtered air at a temperature of about 21 ° C (fluctuating up to 3 degrees), relative humidity 55% +/- 15% and air changed 10 times / hour. The room will be illuminated with 12 hours of brightness and 12 hours of darkness. The light will be from 6 am to 6 pm.

The animals will be kept separately in boxes

Litter

The litter will be "LIGNOCEL H 3/4" sawdust from Hahn &amp; Co, D 24796, Bredenbek-Kronsburg. The contamination of this litter is regularly assessed.

Diet

Commercial pig diet, "Altromin 9033" from Chr Petersen A / S, DK-4100 Ringsted will be used (approximately 800 g twice daily). The main nutrients and relevant possible contaminants are analyzed on a regular basis.

Potable water

Twice a day, animals will be given domestic drinking water. Analyzes of its contamination are carried out on a regular basis

Mutilations

The injury will be done on 1 day. The animals were anesthetized with Strensil® Vet. Janssen, Belgium (40 mg azoperone / ml, 1 ml / 10 kg) and Atropine DAK, Denmark (1 mg atropine / ml, 0.5 ml / 10 kg), given as a single intramuscular dose followed by an intravenous injection of Hypnodil® Janssen, Belgium (50 mg methomidate / ml, about 2 ml).

The dorsolateral area of ​​both sides of the back of the animals will be shaved, washed with soap and water, disinfected with 70% ethanol, which will be washed with sterile salt, and finally dried with sterile gauze.

In such a prepared field, eight layered wounds (25 x 25 x 0.4 mm) will be made, four on each side of the spine, using the ACCU dermatome (GA 630, Aesculap®). The wounds will be numbered 1 (most cephalic) to 4 (caudal) on the left side, and 5 (most cephalic) to 8 (caudal) on the right side of the animal.

PL 204 797 B1

Coagulated blood will be removed with sterile gauze. Just before surgery, approximately 8 hours after surgery, and whenever required, animals will receive an intramuscular injection of Anorphine, A / S GEA, Denmark (0.3 mg buprenorphine / ml, 0.04 ml / kg).

Post-wound dosing will be handled as follows:

<td></td><td colspan="8">Animal number</td>

<td></td><td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2"> 4</td>

<td>Location</td><td>left</td><td>rights</td><td>left</td><td>rights</td><td>left</td><td>rights</td><td>left</td><td>rights</td>

<td>headache</td><td>AND</td><td></td><td></td><td>B</td><td>AND</td><td></td><td></td><td>B</td>

<td></td><td>B</td><td>AND</td><td></td><td></td><td>B</td><td>AND</td><td></td><td></td>

<td></td><td></td><td>B</td><td>AND</td><td></td><td></td><td>B</td><td>AND</td><td></td>

<td>caudal</td><td></td><td></td><td>B</td><td>AND</td><td></td><td></td><td>B</td><td>AND</td>

A - control B - EMD

Approximately 15 minutes before administration, the EMD formulation will be prepared according to the manufacturer's instructions. EMD will be used within 2 hours of preparation. For the treatment of wounds B, the EMD will be applied as a thin layer to the wound surface. One vial of EMD will be used for 4 wounds.

Wound dressing

The wounds will be covered with a Tegaderm® dressing. The dressing will be covered with a gauze bandage attached by Fixomul®. Dressings, gauze and Fixomul will be held with a Bend-arete® mesh (Tesval, Italy). The dressing will be checked daily.

The dressings will be changed on the second day (all animals) and on the third day (animals nos. 3 and 4).

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

Wound observation

Each wound will be observed and photographed on day 2 (all animals), on day 3 (all animals) and on day 4 (animals Nos. 3 and 4). The degree of exudate and inflammation will be assessed. The appearance of the transplanted epidermis will be described in detail.

Clinical signs

Any visible signs of illness and any behavioral changes will be recorded daily. Any deviation from the norm will be described in terms of time, duration and degree of severity.

Body weight

The animals will be weighed on arrival, on the day of wounding, and at the end of the test.

Final observations

On day 3 (approximately 56 hours after wounding) animals Nos. 1 and 2 will be killed by cutting the vein and subclavian artery after stunning with a gun bullet.

On day 4 (approximately 72 hours after wounding), animals Nos. 3 and 4 will be killed by cutting the vein and subclavian artery after bullet stunning.

Tissue collection

Each wound will be cut as a whole and separated from the skeletal muscle tissue. If there is any adhesion to skeletal muscles, some of the muscles will also be included in the fixation material. Each pickup will be fixed in a buffered neutral formaldehyde 4%.

Histological procedure

After fixation of four representative samples from all wounds, they will be embedded in paraffin, cut to a thickness of 5 millimicrons and stained with hematoxylin and eosin. After staining, the sections will be viewed under a light microscope using a grid. This will allow the overall length to be measured

The wound and the length of the epithelialising surface. This ratio will be expressed as the percentage of the epithelialised wound per section. The mean values ​​for each wound will be taken and the group mean values ​​will be calculated.

Statistics

Data will be designed to obtain group mean values ​​and standard deviation. Possible values ​​deviating from the average will also be identified. Then each continuous variable will be tested for homogeneity of variance with the Bartlett test. If the variance is homogeneous, an analysis of the variance of the variables will be performed. If significant differences are found, possible intra-group differences will be assessed using Dunett's test. If the variance is heterogeneous, each variable will be tested for normality by the Shapiro-Wilk method. In the case of a normal distribution, possible differences within the groups will be identified by Student's t-test. Otherwise, possible differences within the groups will be assessed by the Kruskal-Wallis test.

If there are no significant within-group differences, subsequent group identification will be performed with the Wilcoxon Rank-Surn test.

Statistical analysis will be performed using the SAS® software (version 6.12) described in "SAS / STAT® User Guide, Version 6, 4th Edition Volume 1 + 2", 1989, SAS Institute Inc., Cary, North Carolina 27513, USA.