WO2001097835A1

WO2001097835A1 - Matrix protein compositions for inhibition of epithelial cell growth

Info

Publication number: WO2001097835A1
Application number: PCT/IB2001/001091
Authority: WO
Inventors: Stale Petter Lyngstadaas; Stina Gestrelius
Original assignee: Biora Bioex Ab
Priority date: 2000-06-21
Filing date: 2001-06-21
Publication date: 2001-12-27
Also published as: AU7437901A

Abstract

The present invention relates to the use of an active enamel substance for the preparation of a pharmaceutical composition for application on a medical implant or device and/or on tissue in contact with said implant or device so as to inhibit attachement, proliferation and/or growth of epithelial cells thereon, or to inhibit epithelial downgrowth along the surface of said implant or device. The preparation is utilised to bio-engineer surfaces of medical implants and devices which are in contact with epithelial tissue in such a way that growth of epithelial cells on or along the surfaces of such implants or devices is substantially inhibited. Accordingly, the present invention relates to a method of inhibiting attachment, proliferation and/or growth of epithelial cells on a medical implant or device. The invention also relates to medical implants or devices on which enamel matrix, enamel matrix derivatives and/or enamel matrix proteins have been applied.

Description

MATRIX PROTEIN COMPOSITIONS FOR INHIBITION OF EPITHELIAL CELL GROWTH

FIELD OF THE INVENTION

The present invention relates to the use of enamel matrix, enamel matrix derivatives and/or enamel matrix proteins or peptides for application on medical implants or devices. The invention also relates to medical implants or devices on which enamel matrix, enamel matrix derivatives and/or enamel matrix proteins have been applied.

BACKGROUND OF THE INVENTION

Medical implants and devices are used in numerous medical treatments as a consequence of which they are placed either permanently, intermittently or for a certain period of time so that they are in contact with or even penetrate skin or mucous membranes. Such implants may be catheters or tubes, often made of polymers, e.g. plastics, or metals, e.g. titanium or steel, including dental implants and fixations used during healing of complicated fractures of the neck, legs or arms, or for bone elongations (e.g. of legs, arms and jaws).

In the frequent cases where such implants or devices are present in the body for a longer period of time, epithelial tissue at the site where the implant or device enters the body may proliferate and grow along the surface of the implant or device. Such epithelial down- growth interferes with the function of the implanted device. The implant may fail because epithelial cells impede proper connective tissue sealing around the implant and thus increase the risk for microbial infection and/or colonisation that may trigger inflammatory responses in the surrounding tissues. Such inflammatory reactions may cause stenosis of vessels or loss of (wanted) connective tissue adherence (e.g. cervical sealing around dental implants and permanent intestinal catheters and tracheotomy catheters). Moreover, downgrowth of surface epithelium along temporary implants produces permanent fistulas (epithelium lined canals) that impair healing and wound closure after removal of the im- plants causing severe scarring, infections, or even persisting "stomas" that need radical surgery for final closing.

Enamel matrix proteins such as those present in enamel matrix are best known as pre- cursors of dental enamel. Enamel proteins and enamel matrix derivatives have previously been described in the patent literature to induce hard tissue formation (i.e. enamel formation, cf. US Patent No. 4,672,032 (Slavkin)) or binding between hard tissues (EP-B-0 337 967 and EP-B-0263086). The use of enamel matrix proteins for inhibition of epithelial cell growth on medical implants or devices has not, to the inventors' knowledge, been sug- gested previously.

SUMMARY OF THE INVENTION

In the course of research leading to the present invention, it has surprisingly been found that a composition of enamel matrix derivatives applied on tooth roots to promote tooth attachment after periodontal surgery was able to promote healing of the lesion while effectively inhibiting epithelial downgrowth. The finding has been utilised to bio-engineer surfaces of medical implants and devices which are in contact with epithelial tissue when in use in such a way that growth of epithelial cells on or along the surfaces of such im- plants or devices is substantially inhibited, thereby effectively avoiding or at least ameliorating the above-mentioned problems associated with the presence in the body of implants or devices for substantial periods of time.

Accordingly, the present invention relates to the use of a preparation of an active enamel substance for the preparation of a pharmaceutical composition for application on a medical implant or device and/or on tissue in contact with said implant or device so as to substantially inhibit attachment, proliferation and/or growth of epithelial cells thereon, or so as to substantially inhibit epithelial downgrowth along the surface of said implant or device.

In another aspect, the invention relates to a method of substantially inhibiting attachment, proliferation and/or growth of epithelial cells on a medical implant or device adapted for use in contact with tissue comprising a substantial proportion of epithelial cells, or to substantially inhibit epithelial downgrowth along the surface of said implant or device, the method comprising applying an effective amount of an active enamel substance on at least a surface thereof which, when the implant or device is in use, is in contact with tissue comprising a substantial proportion of epithelial cells.

In a further aspect, the invention relates to a medical implant or device adapted for use in contact with tissue comprising a substantial proportion of epithelial cells, the implant or device comprising an effective amount of an active enamel substance applied on at least a surface thereof which, when the implant or device is in use, is in contact with tissue comprising a substantial proportion of epithelial cells and/or on a surface of said tissue in contact with said implant or device.

In the present context, the term "implant" is intended to indicate a medical device which, when in use, lodges in the body permanently or for a longer period of time, typically for a period of more than 30 days. The term "device" is intended to indicate a medical device which is typically used for a shorter period of time such as 30 days or less. The term "at- tachment" is intended to indicate that epithelial cells adhere to the surface of the implant or device, while the terms "proliferation" and "growth" are used to indicate that the cells form colonies to cover part of the surface of the implant or device. The term "down- growth", on the other hand, is intended to indicate that the epithelial cells grow along the surface of the implant or device on neighbouring tissue which is not epithelial tissue, but which may for instance be connective tissue, so as to form a layer or lining of epithelial cells thereon which remains when the implant or device is removed.

In developing teeth, the enamel is formed by a layer of epithelial cells called ameloblasts. These, in turn, are supported by another layer of epithelial cells providing the ameloblasts with growth factors and nutrients required for their continued existence and function. The ameloblasts synthesise and secrete enamel matrix proteins which, together with mineral and water, constitute the enamel matrix which is an early developmental stage of dental enamel. Enamel matrix proteins are mainly present during the secretory stage of enamel formation. After their initial deposition, they are gradually degraded and then lost as enamel development progresses. The active enamel substance observed to be useful in the present invention is composed of a number of proteins and peptides including such degradation products. These observations are also supported by studies of guinea pig molars showing that processing of enamel proteins is linked to the reduction of the number of surrounding epithelial cells (Hamamoto and Hammarstrδm, unpublished data). Without wishing to be limited to any particular theory, it is assumed that the ability of active enamel substance to inhibit growth of epithelial cells while leaving connective tissue cells unaffected is linked to the function of enamel matrix proteins during tooth development. The action of these proteins during tooth root formation is associated with the break-up of the epithelium derived root sheet to allow mesenchymal cells to adhere to the root and produce the dental attachment apparatus. During the process of root formation the tooth erupts through the oral mucous membrane into the oral cavity without down- growth of the oral epithelium along the root. Teeth persist as lifelong biological trans- mucosal implants and are the only structure in the body that penetrates the epithelial lin- ing. Thus, some kind of mechanism must exist that restricts the epithelium from growing down along the root surface. Enamel matrix proteins are deposited onto the newly mineralised root surface prior to the formation of the tooth attachment apparatus and are present, to a limited extent, in adult teeth beneath the root cementum in a thin layer known as the granular layer of Tomes. When this layer and the overlaying cementum is removed, e.g. by root planing and scaling during treatment of periodontitis, the result is nearly always downgrowth of the oral epithelium onto the denuded root surface. Thus an intact granular layer of Tomes seems to be important for hindering epithelial downgrowth onto the tooth root and thus facilitating connective tissue attachments that can restore the integrity of the tooth attachment.

So far, no known growth factors have been detected in EMD (Gestrelius et al. 1997a). The major constituent is amelogenins, a family of hydrophobic proteins derived from a single gene by alternative splicing and controlled post secretory processing. The amelogenins are known to self-assemble into supramolecular aggregates that form an insoluble extracellular matrix (Fincham et al. 1994) with high affinity for hydroxyapaptite and colla- gens (Gestrelius et al 1997b). When applied to denuded root surfaces EMD therefore precipitates to form an extra cellular matrix layer with a hydrophobic surface with potential for supporting interactions with cells in adjacent tissues.

In epithelial cells cultured with EMD, the observed decrease in growth rate is not followed by reduction in DNA synthesis. Since DNA synthesis, and thus replication, in epithelial cells seem to be unchanged by the presence of EMD, processes outside the replication machinery could effect the observed decrease in culture growth rate. The most plausible explanation for this discrepancy between growth rate and DNA synthesis is that cell death exceeds proliferation in these cultures. A wide range of mechanisms could affect viability of cultured epithelial cells exposed to EMD, including cytotoxicity or even apoptosis.

As shown in example 2, the presence of EMD in the culture medium generated an in- crease in intracellular cAMP concentration in epithelial cells (Figure 3). The secondary messenger cAMP is a potent activator of protein kinases that again activate proteins participating in a wide array of cellular processes like growth, replication, metabolism, secretion, gene expression and apoptosis. A rise in intracellular cAMP concentration demands that an inductive signal is transduced from the outer surface of the cell membrane to the inside by an activated G-protein. The effect of cAMP signalling varies with cell type, depending on the intracellular concentration and assortment of proteins present. In epithelial cells, the increase in cAMP concurred with the onset of PDGF-AB secretion and a fall in cell proliferation. The rise of intracellular cAMP concentrations in EMD stimulated cells suggest that interactions with EMD employs cell surface structures that act as signal transducers. The nature of these structures is however unknown and so far no candidate for an "enamel matrix receptor" has been identified.

Expression of platelet-derived growth factor AB (PDGF-AB) was induced in epithelial cells when these were cultured in the presence of EMD (Figures 4). In control cultures how- ever, PDGF-AB expression was insignificant. The expression of PDGF-AB in EMD stimulated epithelial cells increased steadily over the observation period. PDGFs are potent mitogens for connective tissue cells including gingival fibroblasts and PDL cells (Bartold et al., 1992; Oates et al., 1993) and some epithelial and endothelial cells. In addition, the PDGFs are chemotactic for fibroblasts, smooth muscle cells and some white blood cells, but not for epithelium. Other activities for PDGFs include stimulation of hyaluronate and collagen synthesis and stimulation of collagenase secretion and activity. The potent activity of PDGFs as mitogens suggest that they play an important role, possibly as autocrine factors, in regulation of growth and development. Furthermore, PDGFs are important players in normal wound healing, and the applications of PDGFs have been demonstrated to accelerate the rate of healing in various types of wounds. The restrictive effect of EMD on epithelial cell growth could thus be beneficial by hindering growth of the oral epithelium into a periodontal lesion during the wound healing phase and the early regenerative process. Surprisingly, enamel matrix proteins, primarily amelogenins, which, as indicated above, are only present in developing teeth in any significant amounts (i.e. in the teeth of children of 0-12 years of age) and are not present anywhere else in the body have a profound effect on the differentiation and growth of epithelial cells located outside the normal enamel compartment and which differ from ameloblasts with respect to their function.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors surprisingly observed that when normal human dermal epithelial cells were cultured in the presence of the active enamel substance their growth was significantly inhibited (vide Example 1 below). By way of comparison, human connective tissue cells (fibroblasts) cultured under similar conditions in the presence of the active enamel substance were stimulated as to growth. Based on these results, the present inventors believe that the active enamel substance applied on a surface of a medical im- plant or device may be used for the (selective) inhibition of the proliferation and/or growth of epithelial cells when the implant or device is in contact with tissue comprising a substantial proportion of epithelial cells. Such tissue is typically skin or mucosal tissue, and the implant or device may be one which is adapted to penetrate skin or mucosal tissue.

Medical implants and devices

According to the present invention, the implant or device may be any implant or device intended for use in the human or animal body, in particular in the gastrointestinal tract, urethra, bladder, pulmonary cavity, lungs, trachea, larynx, oesophagus, joints, bone, skull, ears, sinuses, veins, arteries or abdominal cavity.

Thus, the implant or device may be a catheter which may be any conventional catheter used for insertion into a body cavity for medical or therapeutic purposes, in particular a venous, arterial, urethral or dialysis catheter. The implant or device may also be a tube such as a tube forming an integral part of an ostomy device, i.e. the part penetrating the skin and other tissues of an ostomy patient, e.g. a colostomy or trachostomy tube, or a drain. Catheters, tubes or drains may be prepared from any material conventionally used for this purpose, in particular a polymer such as plastic, expanded polyvinyl difluoroure- thane, silicone, goretex, teflon, modified collagens and other synthetic or biological polymers conventionally used in such devices or implants, and/or may optionally be coated with a suitable polymeric material. For fixation of complicated fractures, e.g. of the neck, legs or arms, or skull fractures, the implant or device may be a pin or screw conventionally used to immobilise (fix) fragments of fractured bone. Such pins or screws typically comprise a portion that penetrates the skin of the patient at or near the site of the fracture. Pins and screws for this purpose may conventionally be prepared from a metal such as titanium or steel, and may optionally be coated with a polymeric material which may typically be biodegradable or stabilised to facilitate soft tissue closure and sealing. Further- more, an implant may be an electrical conductor such as one used in, e.g., pacemakers, brain implants or biosensors.

Before application on an implant or device, the active enamel substance may be admixed with other ingredients, e.g. pharmaceutically acceptable excipients as discussed below, and coated onto the surface of the implant or device, e.g. by dipping the relevant portion of the implant or device in a solution or dispersion of the active enamel substance or by spraying a solution or dispersion of the active enamel substance onto the relevant surface of the implant or device followed, in both cases, by drying. On application, the active enamel substance is adsorbed to the surface of the implant or device and may be fixed thereon by means of conventional fixatives such as formaldehyde, glutaraldehyde or ethanol. Alternatively, the active enamel substance may be applied on the relevant surface of the implant or device by cross-linking the active enamel substance to a polymer component of the implant or device, e.g. by UV radiation or chemical treatment in a manner known per se, or by covalently binding the active enamel substance to a suitable func- tional group of a polymeric component present on the surface of the implant or device.

The amount of active enamel substance applied on the appropriate surface of the implant or device to inhibit growth of epithelial cells will normally result in an amount of total protein per cm² area of the implant or device corresponding to from about 0.005 mg/cm²to about 20 mg/cm²such as from about 0.01 tng/ctn² to about 15 mg/cm².

In accordance with the present invention, application of the active enamel substance on a surface of an implant or device for the present purpose may optionally be combined with application of other types of suitable biologically active substances, e.g. antimicrobial agents such as antibacterial or antifungal agents, or application of bacteriostatic agents or disinfectants for the prevention or treatment of microbial infections at the site where the implant or device is in contact with epithelial tissue.

Enamel matrix, enamel matrix derivatives and enamel matrix proteins

Enamel matrix is a precursor to enamel and may be obtained from any relevant natural source, i.e. a mammal in which teeth are under development. A suitable source is developing teeth from slaughtered animals such as, e.g., calves, pigs or lambs. Another source is for example fish skin.

Enamel matrix can be prepared from developing teeth as described previously (EP-B- 0 337 967 and EP-B-0 263 086). The enamel matrix is scraped off and enamel matrix derivatives are prepared, e.g. by extraction with aqueous solution such as a buffer, a dilute acid or base or a water/solvent mixture, followed by size exclusion, desalting or other purification steps, optionally followed by freeze-drying. Enzymes may be deactivated by treatment with heat or solvents, in which case the derivatives may be stored in liquid form without freeze-drying.

In the present context, enamel matrix derivatives are derivatives of enamel matrix which include one or several of enamel matrix proteins or parts of such proteins, produced naturally by alternate splicing or processing, or by either enzymatic or chemical cleavage of a natural length protein, or by synthesis of polypeptides in vitro or in vivo (recombinant DNA methods or cultivation of diploid cells). Enamel matrix protein derivatives also include enamel matrix related polypeptides or proteins. The polypeptides or proteins may be bound to a suitable biodegradable carrier molecule, such as polyamino acids or poly- saccharides, or combinations thereof. Furthermore, the term enamel matrix derivatives also encompasses synthetic analogous substances.

Proteins are biological macromolecules constituted by amino acid residues linked together by peptide bonds. Proteins, as linear polymers of amino acids, are also called polypeptides. Typically, proteins have 50-800 amino acid residues and hence have molecular weights in the range of from about 6,000 to about several hundred thousand Daltons or more. Small proteins are called peptides or oligopeptides. Enamel matrix proteins are proteins which normally are present in enamel matrix, i.e. the precursor for enamel (Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1:282-91), or proteins which can be obtained by cleavage of such proteins. In general such proteins have a molecular weight below 120,000 daltons and include amelogenins, non-amelogenins, proline-rich non-amelogenins, and tuftelins.

Examples of proteins for use according to the invention are amelogenins, proline-rich non- amelogenins, tuftelin, tuft proteins, serum proteins, salivary proteins, enamelin, and derivatives thereof, and mixtures thereof. A preparation containing an active enamel sub- stance for use according to the invention may also contain at least two of the aforementioned proteinaceous substances. A commercial product comprising amelogenins is marketed as EMDOGAIN® (Biora AB) and comprises about 30mg/ml active enamel substance in propylene-glycol- alginate (PGA).

In general, the major proteins of an enamel matrix are known as amelogenins. They constitute about 90% w/w of the matrix proteins. The remaining 10% w/w includes proline-rich non-amelogenins, tuftelin, tuft proteins, serum proteins and at least one salivary protein; however, other proteins may also be present which have been identified in association with enamel matrix. Furthermore, the various proteins may be synthesised and/or pro- cessed in several different sizes (i.e. different molecular weights). Thus, the dominating proteins in enamel matrix, amelogenins, have been found to exist in several different sizes which together form supramolecular aggregates. They are markedly hydrophobic substances which under physiologically conditions form aggregates. They may carry or be carriers for other proteins or peptides.

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

As mentioned above, the proteins, polypeptides or peptides for use according to the invention typically have a molecular weight of at the most about 120 kDa such as, e.g., at the most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS Page elec- trophoresis. As indicated above, epithelial cells associated with ameloblasts are believed to be induced to undergo apoptosis by degradation products migrating from the enamel matrix during dental enamel development. Such degradation products, which generally have a molecular weight between about 3 kDa and 25 kDa, such as between 5 kDa and 20 kDa, may be particularly effective for use according to the present invention.

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

A preparation of an active enamel substance for use according to the invention may also contain a mixture of proteins with different molecular weights.

The proteins of an enamel matrix can be divided into a high molecular weight part and a low molecular weight part, and it has been found that a well-defined fraction of enamel matrix proteins possesses valuable properties with respect to treatment of periodontal defects (i.e. periodontal wounds). This fraction contains acetic acid extractable proteins generally referred to as amelogenins and constitutes the low molecular weight part of an enamel matrix (cf. EP-B-0 337 967 and EP-B-0 263 086).

As discussed above the low molecular weight part of an enamel matrix has a suitable activity for inducing binding between hard tissues in periodontal defects. In the present context, however, the active proteins are not restricted to the low molecular weight part of an enamel matrix. At present, preferred proteins include enamel matrix proteins such as amelogenin, tuftelin, etc. with molecular weights (as measured in vitro with SDS-PAGE) below about 60,000 daltons but proteins having a molecular weight above 60,000 daltons have also promising properties as candidates for wound healing, anti-bacterial and/or anti- inflammatory agents. Accordingly, it is contemplated that the active enamel substance for use according to the invention has a molecular weight of up to about 40,000 such as, e.g. a molecular weight of between about 5,000 and about 25,000.

A method for the isolation of enamel matrix proteins involves extraction of the proteins and removal of calcium and phosphate ions from solubilized hydroxyapatite by a suitable method, e.g. gel filtration, dialysis or ultrafiltration (see e.g. Janson, J-C & 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 lyophilised protein preparation may mainly or exclusively up to 70-90% contain amelogenins with a molecular weight (MW) between 40,000 and 5,000 daltons, the 10- 30% being made up of smaller peptides, salts and residual water. The main protein bands are at 20 kDa, 12-14 kDa and around 5 kDa.

By separating the proteins, e.g. by precipitation, ion-exchange chromatography, preparative electrophoresis, gel permeation chromatography, reversed phase chromatography or affinity chromatography, the different molecular weight amelogenins can be purified.

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

As an alternative source of the enamel matrix derivatives or proteins one may also use generally applicable synthetic routes well-known for a person skilled in the art or use cultivated cells or bacteria modified by recombinant DNA-techniques (see, e.g., Sam- brook, J. et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).

Physico-chemical properties of enamel matrix, enamel matrix derivatives and enamel matrix proteins

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

At least a part of the active enamel substance may be in the form of aggregates or is capable of forming aggregates after application in vivo. The particle size of the aggregates is in a range of from about 20 nm to about 1 μm.

It is contemplated that the solubility properties of the active enamel substance are of importance in connection with the prophylactic and therapeutic activity of the substance. When a composition containing the active enamel substance is administered to e.g. a human, the proteinaceous substances will precipitate due to the pH normally prevailing under physiological conditions. This is an advantage for the present use as the active enamel substance applied on the surface of the implant or device is likely to remain as a layer thereon which will not easily be rinsed off under physiological conditions. The active enamel substances will therefore be maintained in situ on the implant or device for a prolonged period of time thereby making it unnecessary to change the implant or device frequently due to dissolution or degradation of the layer of active enamel substance. In order to enable a proteinaceous layer to be formed in situ after application it may be advantageous to incorporate a suitable buffer substance in the preparation of the active enamel substance; the purpose of such a buffer substance could be to avoid the dissolution of the active enamel substance at the application site.

Pharmaceutical compositions

For the administration to an individual (an animal or a human) the active enamel substance and/or a preparation thereof are preferably formulated into a pharmaceutical composition containing the active enamel substance and, optionally, one or more pharmaceutically acceptable excipients suitable for application onto a surface of a medical implant or device.

The compositions may be in form of a liquid composition, e.g., a solution, dispersion or suspension for application on a surface of a medical implant or device. Once applied, the composition should preferably solidify, e.g. by drying, to a solid or at least highly viscous composition which does not dissolve on storage or when the implant or device is in use. The composition is preferably applied under sterile conditions and/or sterilised after application by irradiation or exposure to ethylene oxide gas. When the composition is in the form of a liquid composition, it may also be applied shortly before the medical implant or device is to be introduced into the body. As an alternative to applying composition comprising the active enamel substance on the medical implant or device, the composition may be applied on a surface of a tissue which is in contact with the implant or device, such as a tissue comprising a substantial proportion of epithelial cells as indicated above. Furthermore, the composition may be applied on both the implant or device and on tissue in contact therewith.

The concentration of active enamel substance in the composition may suitably be in the range of 0.005-10% w/w, preferably 0.01-5% w/w, such as 0.1-3% w/w.

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

Apart from the active enamel substance, a pharmaceutical composition for use according to the invention may comprise pharmaceutically acceptable excipients.

A pharmaceutically acceptable excipient is a substance which is substantially harmless to the individual to which the composition is to be administered. Such an excipient normally fulfils the requirements given by the national health authorities. Official pharmacopoeias such as e.g. the British Pharmacopoeia, the United States of America Pharmacopoeia and The European Pharmacopoeia set standards for pharmaceutically acceptable excipients.

The choice of pharmaceutically acceptable excipient(s) in a composition for use according to the invention and the optimum concentration thereof cannot generally be predicted and must be determined on the basis of an experimental evaluation of the final composition. However, suitable excipients for the present purpose may be selected from such excipients that promote application of the composition comprising the active enamel substance on a surface of the implant or device, or that promote the adherence of the composition to the surface on application, or that prevent immediate dissolution of the composition or protract the release of the active enamel substance from the composition when the implant or device is in use. A person skilled in the art of pharmaceutical formulation can find guidance in e.g., "Remington's Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, 1990.

The pharmaceutically acceptable excipients may include solvents, suspending agents or gel-forming agents

Examples of solvents are e.g. water or alcohols.

Examples of suspending agents are e.g. celluloses and cellulose derivatives such as, e.g., carboxymethyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxy- propylmethylcellulose, carragheenan, acacia gum, arabic gum, tragacanth, and mixtures thereof.

Examples of gel bases or viscosity-increasing agents are: liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminium, zinc soaps, glycerol, propylene glycol, tragacanth, carboxyvinyl polymers, magnesium-aluminium silicates, Carbopol®, hydrophilic polymers such as, e.g. starch or cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragheenans, collagen, gelatin, pectin, chitosans, hyaluronates or alginates

The invention is further described in the following example which is not in any way intended to limit the scope of the invention as claimed.

Legends of Figures

FIGURE 1

Epithelial cells grown in cultures for 24, 48, 72, 96 and 120 hours. Cells are counted in the microscope using a fixed grid in Five different areas in each of six parallel cultures at each timepoint. Epithelial cells show a marked decrease in cell density from 48 hours when grown in the presence of EMD.

FIGURE 2

Panel showing attachment and proliferation in cell cultures. Density plot of cultured human epithelial cells show that presence of EMD decrease the growth rate of these cultures. Five different areas were counted in each of six parallel cultures at each timepoint (n=30), error bars give ±SD.

FIGURE 3 Intracellular cyclic AMP levels in cell cultures. Compared to controls both human PDL cells and human epithelium cells showed a marked increase in intracellular cAMP after growing 24 hrs in presence of EMD. n=6 at for both cell types, error bars give ±SD.

FIGURE 4 Panel showing increased autocrine growth factor secretion from cells cultured with EMD. Human epithelial cells show a rapid and strong secretion of PDGF-AB when cultures contain EMD. In contrast to the PDL cells this secretion increased throughout the experiment. PDGF-AB secretion from controls were insignificant. n=6, error bars give ±SD. Experimental Section

Example 1

Growth of human epithelial cells in the presence and absence of EMD Materials and Methods

Normal human dermal epithelial keratinocytes (NHEK (adult skin) 6168 available from BioWhittaker Inc., USA, catalog. No. CC-2501) were emloyed. The cells were grown in Modified Eagle's Medium supplemented with 10% fetal calf serum. EMD (freeze-dried enamel matrix derivative (80% w/w protein) available from Biora AB under the trademark EMDOGAIN®) was supplied by surface coating culture dishes with a 0.1% EMD solution in 0.1% HAc, and by supplementing the culture medium with 100 μg EMD per ml of medium. Epithelial cells cultured under similar conditions in the absence of EMD were used as controls. All experiments were initiated with 50,000 cells per ml of culture medium.

Results

Epithelial cells were grown in cultures for 24, 48, 72, 96 and 120 hours. Cultures were then washed with PBS and cells were counted in the microscope using a fixed grid. Five different areas were counted in each of six parallel cultures at each timepoint. As appears from Fig. 1 , epithelial cells show a marked decrease in cell density from 48 hours when grown in the presence of EMD.

Based on these results, it is concluded that epithelial cell growth is poorer in the presence of EMD.

Example 2

Materials and Methods

Cell isolation and culture conditions

Cells included in this study were all between passage 5 and 7. Human epithelial cells were HeLa cells obtained from ATTC. All cultures were established and maintained in Ea- gles MEM with 10% foetal bovine serum. EMD was added prior to commencement of cell cultures, by coating charged plastic culture dishes (Nunc^®) or Sarstedt dishes (attachment experiments only) with a 0.5mg/ml EMD solution in 0.1% HAc in PBS overnight. In addition, the medium was supplemented with 100μg EMD per millilitre. There were no 5 changes of media during the five to seven day observation period of this study. All experiments commenced with 50.000 cells per millilitre culture medium unless otherwise stated.

Cell attachment

10 To assess the cell attachment rate during the first four hours after seeding, 100.000 cells were cultured on EMD coated surfaces (Sarstedt glass dishes) for 30, 60, 120 or 240 minutes before the cultures were vigorously washed with PBS to remove all unattached cells. The washing solution was centrifuged and the numbers of unattached cells were analysed using a Bϋrker chamber. The attached cells were then removed from the sur-

15 face by trypsinization and counted in the same way for control. Uncoated dishes were used as negative control.

Cell culture densities

Cells were seeded and maintained in cultures with or without EMD for 24, 48, 72, 96 or 20 120 hours. Cultures were then carefully washed with PBS and the number of attached cells per square millimetre was calculated in the microscope using a fixed grid.

Cell metabolism

Cells were cultured for 24, 48,72, 96, 120 or 144 hours and then given a 4 hours pulse of 25 50 μCi [³⁵S]methionine (Cell culture grade, Amersham Pharmacia Biotech). The cultures were then washed with PBS and the cells were removed by trypsinization. The cells were then washed again, centrifuged and 200 μl of each cell pellet were dissolved in Univer- Sol^™ liquid scintillation cocktail (ICN Biomedicals Inc.) and counted two times 300 seconds in a Packard Tricarb scintillation counter.

30

Nucleic acid synthesis

Nucleic acid synthesis in cells cultured 24, 48,72 or 96 hrs was assessed by colorimetric analysis at 370 nm following a 4 hrs pulse with BrdU using the Boehringer Mannheim Cell Proliferation ELISA, BrdU kit (Cat. No. 1647229). During the pulse the pyrimidine analogue BrdU is incorporated in place of thymidine into the newly synthesised DNA of proliferating cells. At the end of the pulse the cells are washed, fixed and denatured and the amount of incorporated BrdU waw measured by ELISA utilising an anti-BrdU peroxidase conjugated antibody.

Cyclic-AMP detection

Cells were cultured for 24 and 120 hours in 96 well plates and then washed twice in PBS. Cells were then lysed in situ, and the released intracellular cAMP was measured by com- petitive enzymeimmunoassay (EIA) using the Amersham Pharmacia Biotech Biotrak cAMP EIA kit (RPN 225). This assay is a direct enzyme immunoassay employing a polyclonal rabbit anti-cAMP as primary antibody and a peroxidase labelled cAMP as competing agent.

Growth factor detection

TGF-β1, interleukins (IL-1α and β, II-2 and IL-6) and PDGF (AA, AB and BB) production was measured in the culture media at 8, 24, 48,72,96 or 120 hrs. TGF-β1 released into the medium was quantified by a sandwich EIA technique with the TGF-β soluble receptor type II and an enzyme-linked anti-TGF-β1 polyclonal antibody supplied in a R&D Systems Quantikine^® Human TGF-β1 Immunoassay kit (DB100). Background values from the serum-supplemented media were subtracted for each timepoint. Secretion of IL-1α and β were analysed using the Quantikine^® Human IL-1 α or β Immunoassay kit (DLA50 and DLB50 respectively). IL-2 secretion was analysed with the Quantikine^® Human IL-2 Immunoassay kit (D2050). Human IL-6 in the media was analysed using the Quantikine^®HS Human IL-6 Immunoassay kit (HS600), all from R&D Systems. These assays employ quantitative sandwich EIA using a monoclonal primary antibody specific for each interleu- kin, and a polyclonal, enzyme linked, anti-interleukin specific (IL-1α or β, 2 or 6) secondary antibody. PDGFs in media from PDL and HeLa cultures was assessed utilising the Quantikine^® human PDGF-AB and PDGF-BB Immunoassay kit, (DHD00 and DBB00) from R&D Systems. These assays are quantitative sandwich EIA using a monoclonal primary antibody specific for PDGF-AA or the PDGF-Rβ receptor as primary ligand, and a polyclonal, enzyme-linked, anti-PDGF-AA or -BB secondary antibody. Statistics

All results were plotted using the SigmaPIot for Windows Version 5.00 from SPSS Inc. No statistics were applied to the data set except for calculation of means and standard deviations (SD) which were calculated automatically by the computer programme.

Results

The experiments showed that EMD failed to improve attachment rates, and EMD coated surfaces did not differ significantly from controls. Cell density in epithelial cultures is shown in Figure 2. Presence of EMD caused cell proliferation to lag behind control cells with two full days. Unlike the negative control epithelial cells never produced confluent cultures in the presence of EMD during these experiments (five days). EMD did not cause any significant changes in metabolic rates when assessed by pulse-chase experiments with radiolabelled methionine (data not shown).

In pulse-chase experiments with the thymidine analogue bromodeoxyuridine (BrdU) a decrease in growth rate was observed in epithelial cultures (Figure 2) there were no significant changes in DNA synthesis in epithelial cells when EMD was present.

Within 24 hours after seeding on EMD, epithelial cells exhibited highly increased intracel- lular levels of cAMP when compared to controls (Figure 3). The increase in cAMP was strong and stable for at least 72 hours before starting to fade out. Significant differences between experiment and controls could however, still be observed five days after seeding.

Screening for autocrine growth factors released into the medium revealed a significant increase in only PDGF-AB in the medium from epithelial cells growing in the presence of EMD (Figure 4). Experiments failed to demonstrate autocrine IL-1α or β, IL-2, PDGF-AA or PDGF-BB production (data not shown). PDGF-AB release into the medium strongly increased throughout the experiment when EMD was present (Figure 4), whereas, in controls, PDGF-AB levels were barely detectable before 72 hours. Non of the above men- tioned growth factors could be detected in the EMD preparations, nor were any increase of these factors observed in cell free media controls incubated together with test cultures. LIST OF REFERENCES

Bartold, P.M., Narayanan, A.S. & Page, R.C. (1992) Platelet-derived growth factor reduces the inhibitory effects of lipopolysaccharide on gingival fibroblasts proliferation. 5 Journal of Periodontal Research 27, 499-505.

Fincham, A.G., Moradian-Oldak, J., Simmer, J.P., Sarte, P., Lau, E.G., Diekwisch, T. & Slavkin, H.C. (1994) Self-assembly of a recombinant amelogenin protein generates su- pramolecular structures. Journal of Structural Biology 112, 103-109. 10

Gestrelius, S., Andersson, O, Lidstrom, D., Hammarstrδm, L. & Somerman M. (1997a) In vitro studies on periodontal ligament cells and enamel matrix derivative. Journal of Clinical Periodontology 24, 685-692.

15 Gestrelius, S., Andersson, O, Johansson, A.-O, Persson, E., Brodin, A., Rydhag, L. & Hammarstrom, L. (1997b) Formulation of enamel matrix derivative for surface coating. Kinetics and cell colonization. Journal of Clinical Periodontology 24, 678-684.

Janson, J-C & Ryden, L. (Eds.), Protein purification, VCH Publishers 1989 and Harris, 20 ELV & Angal, S., Protein purification methods - A practical approach, IRL Press, Oxford 1990.

Oates, T.W., Rouse, C.A. & Cochran, D.L. (1993) Mitogenic effect of growth factors on human periodontal ligament cells in vitro. Journal of Periodontology 64, 142-148. 25

Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.

Remington's Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, 30 1990.

Sambrook, J. et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989. Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1:282-91.

Claims

1. Use of a preparation of an active enamel substance for the preparation of a pharmaceutical composition for application on a medical implant or device and/or on tissue in contact with said implant or device so as to substantially inhibit attachment, proliferation and/or growth of epithelial cells thereon, or so as to substantially inhibit epithelial down- growth along the surface of said implant or device.

2. Use according to claim 1 , wherein the preparation of active enamel substance is ap- plied on at least a surface of a medical implant or device which, when the implant or device is in use, is in contact with tissue comprising a substantial proportion of epithelial cells and/or applied on a surface of said tissue which is in contact with said implant or device.

3. Use according to claim 2, wherein said tissue comprises skin or mucosal tissue.

4. Use according to claim 2, wherein the implant or device is intended for use in the gastrointestinal tract, urethra, bladder, pulmonary cavity, lungs, trachea, larynx, oesophagus, joints, bone, skull, ears, sinuses, veins, arteries or abdominal cavity.

5. Use according to any of claims 1-4, wherein the medical implant or device is an implant or device adapted to penetrate skin or mucosal tissue.

6. Use according to any of claims 2-5, wherein the implant device is a catheter, such as a venous, arterial, urethral or dialysis catheter, a tube, such as an ostomy tube, e.g. a co- lostomy or trachostomy tube, or a drain.

7. Use according to claim 6, wherein the catheter comprises a polymeric material such as plastic, e.g. expanded polyvinyldifluorourethane, silicone, goretex, teflon or modified col- lagen.

8. Use according to any of claims 1-7, wherein the active enamel substance is enamel matrix, enamel matrix derivatives and/or enamel matrix proteins.

9. Use according to claim 8, wherein the active enamel substance is selected from the group consisting of enamelins, amelogenins, non-amelogenins, proline-rich non-amelogenins, tuftelins, and derivatives thereof and mixtures thereof.

10. Use according to claim 8, wherein the active enamel substance has a molecular weight of at the most about 120 kDa such as, e.g, at the most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS-PAGE electrophoresis.

11. Use according to any of claims 8-10, wherein the preparation of an active enamel substance contains a mixture of active enamel substances with different molecular weights.

12. Use according to claim 11 , wherein the preparation of an active enamel substance comprises at least two substances selected from the group consisting of amelogenins, proline-rich non-amelogenins, enamelins, tuftelin, tuft proteins, serum proteins, salivary proteins, and derivatives thereof.

13. Use according to any of claims 8-12, wherein the active enamel substance has a molecular weight of up to about 40,000.

14. Use according to claim 13, wherein the active enamel substance has a molecular weight of between about 5,000 and about 25,000.

15. Use according to claim 14, wherein the major part of the active enamel substance has a molecular weight of about 20 kDa.

16. Use according to any of claims 8-15, wherein at least a part of the active enamel substance is in the form of aggregates or after application in vivo is capable of forming aggregates.

17. Use according to claim 16, wherein the aggregates have a particle size of from about 20 nm to about 1 μm.

18. Use according to any of claims 8-17, wherein the protein content of the active enamel substance in the preparation is in a range of from about 0.05% w/w to 100% w/w such as, e.g., about 5-99% w/w, about 10-95% w/w, about 15-90% w/w, about 20-90% w/w, about 30-90% w/w, about 40-85% w/w, about 50-80% w/w, about 60-70% w/w, about 70-90% w/w, or about 80-90% w/w.

5 19. Use according to any of claims 1-18, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

20. Use according to claim 19, wherein the pharmaceutically acceptable excipient is a solvent or a gel-forming agent.

10

21. Use according to any of claims 1-20, wherein the pharmaceutical composition comprises about 30mg/ml active enamel substance in propylene-glycol-alginate (PGA).

22. Use of a pharmaceutical composition obtainable from a use according to claim 21 , or 15 any proteins or peptides contained therein, for application on a medical implant or device and/or on tissue in contact with said implant or device so as to substantially inhibit attachment, proliferation and/or growth of epithelial cells thereon, or so as to substantially inhibit epithelial downgrowth along the surface of the implant or device.

20 23. A method of substantially inhibiting attachment, proliferation and/or growth of epithelial cells on a medical implant or device adapted for use in contact with tissue comprising a substantial proportion of epithelial cells, or to substantially inhibit epithelial downgrowth along the surface of said implant or device, the method comprising applying an effective amount of an active enamel substance on a surface thereof which, when the implant or

25 device is in use, is in contact with tissue comprising a substantial proportion of epithelial cells and/or on a surface of said tissue in contact with said implant or device.

24. The method of claim 23, wherein said tissue comprises skin or mucosal tissue.

30 25. The method of claim 23, wherein the implant or device is intended for use in the gastrointestinal tract, urethra, bladder, pulmonary cavity, lungs, trachea, larynx, oesophagus, joints, bone, skull, ears, sinuses, veins, arteries or abdominal cavity.

26. The method of any of claims 23-25, wherein the medical implant or device is adapted 35 to penetrate skin or mucosal tissue.

27. The method of any of claims 23-26, wherein the implant or device is a catheter, such as a venous, arterial, urethral or dialysis catheter, a tube, such as an ostomy tube, e.g. a colostomy or trachostomy tube, or a drain.

28. A method according to any of claims 23-27, wherein the active enamel substance is applied in an amount of total protein per cm² area of the implant or device corresponding to from about 0.005 mg/cm² to about 20 mg/cm², such as from about 0.01 mg/cm² to about 15 mg/cm².

10

29. A medical implant or device adapted for use in contact with tissue comprising a substantial proportion of epithelial cells, the implant or device comprising an effective amount of an active enamel substance applied on at least a surface thereof which, when the implant or device is in use, is in contact with tissue comprising a substantial proportion of

15 epithelial cells.

30. An implant or device according to claim 29, which is adapted to penetrate skin or mucosal tissue.

20 31. An implant or device according to claim 29 or 30, which is a is a catheter, such as a venous, arterial, urethral or dialysis catheter, a tube, such as an ostomy tube, e.g. a colostomy or trachostomy tube, or a drain.

32. An implant or device according to any of claims 29-31 , which is intended for use in the 25 gastrointestinal tract, urethra, bladder, pulmonary cavity, lungs, trachea, larynx, oesophagus, joints, bone, skull, ears, sinuses, veins, arteries or abdominal cavity.

33. An implant or device according to any of claims 29-32, which is coated with the active enamel substance in an amount of total protein per cm² area of the implant or device cor-

30 responding to from about 0.005 mg/cm² to about 20 mg/cm², such as from about 0.01 mg/cm² to about 15 mg/cm².