GB2415903A

GB2415903A - Pharmaceutical preparation which promotes wound healing

Info

Publication number: GB2415903A
Application number: GB0418664A
Authority: GB
Inventors: Alicia Essler; Lorraine Nisbet
Original assignee: Ethicon Inc
Current assignee: Ethicon Inc
Priority date: 2004-07-07
Filing date: 2004-08-20
Publication date: 2006-01-11
Also published as: GB0418664D0

Abstract

A pharmaceutical product comprising one or more elastase inhibitors and one or more growth factors for simultaneous, separate or sequential use to promote wound healing. The elastase inhibitors and growth factors may be combined in a single composition or be present in separate compositions. Growth factors include EGF, PDGF, VEGF, FGF, TGF, IGF-1, tissue growth factor and keratinocyte growth factor. A particularly preferred elastase inhibitor is alpha-1-antitrypsin (AAT), but others include elafin, elastinil, secretory leukocyte proteinase inhibitor or alpha-1-macroglobin. The pharmaceutical product may be topically administered or provided in the form of a wound dressing. An example describes the protective/stabilising effect of AAT on PDGF which is said to synergistically promote wound healing.

Description

24 1 59Q3

PROMOTION OF WOUND HEALING

All documents cited herein are incorporated by reference in their entirety.

Field of the Invention

The invention relates to the combination of one or more elastase inhibitors (e.g. alpha-l antitrypsin) and one or more growth factors for simultaneous, sequential or separate administration to promote wound healing.

Background of the invention

In mammals, injury triggers an organised complex cascade of cellular and biochemical events that result in a healed wound. Wound healing is a complex dynamic process that results in the restoration of anatomic continuity and function; an ideally healed wound is one that has returned to normal anatomic structure, function and appearance.

The purpose of this invention is to facilitate the wound healing process, as delayed or compromised wound healing remains a significant unmet need.

The present invention relates to the use of elastase inhibitors (e.g. alpha-l-antitrypsin (AAT)) and growth factors for the treatment of wounds.

Alpha- I -antitrypsin (AAT), also known as alpha- I -proteinase inhibitor or serpin, is a mammalian polypeptidc having a molecular weight of approximately 54 kDa. It is a potent fluid phase inhibitor of scrine proteases, and forms a tightly bound, stoichiometric complex with clastase. It can be inactivated by cleavage within its reactive centre. For example, neutrophil collagenase (MMP8) is know to degrade and inactivate AAT.

AAT deficiency is a congenital disorder that is principally associated with liver disease in children and emphysema in young adulthood. It is thought that AAT deficiency results in loss of protection in the lung against neutrophil elastasc, resulting in breakdown of the architecture of the lung. AAT has been administered in intravenous and aerosol formats for the treatment of pulmonary emphysema.

Growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) play an important role in wound healing. These molecules act by binding to receptors on the cell surface. This binding induces conformational changes in the growth factor receptors, which in turn activates their intrinsic kinase activity and causes the phosphorylation of many intracellular proteins, incl uding phospholipasc C-g and PI-3 kinase.

Summary of the Invention

The present invention is based on the unexpected discovery that AAT stabiliscs PDGF in chronic wound fluid. The present invention provides for an increase in the rate of wound healing or the extent of wound healing through the use of one or more elastase inhibitors and one or more growth factors. Suitably, the said one or more elastase inhibitors and said one or more growth "'actors exhibit a synergistic effect in the promotion of wound healing.

A first aspect of the invention provides a pharmaceutical product comprising: (a) one or more elastasc inhibitors (e.g. AAT); and (b) one or more growth factors. The pharmaceutical product is for simultaneous, separate or sequential administration. Preferably, the pharmaceutical product further comprises a pharmaceutically acceptable carrier, diluent or excipicnt.

The pharmaceutical product may be provided in the forth of a combined preparation or a pharmaceutical composition (i.e. as an admixture of one or more elastase inhibitors and one or more growth factors).

The teem "a combined preparation" includes a "kit of parts" in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i. e.

simultaneously, separately or sequentially. The parts of the kit of parts can then, for example, be administered shnultaneously or chronologically staggered, that is at dit'ferent time points and with equal or dit'ferent tune intervals for any part of the kit of parts.

A second aspect of the invention provides the pharmaceutical product of the first aspect of the invention tor use in medicine (e.g. therapy).

A third aspect of the invention provides for the use of one or more elastase inhibitors (e.g. AAT) and one or more growth factors in the manufacture of a pharmaceutical product for the promotion of wound healing in a patient in need thereof: As mentioned above, a pharmaceutical product according to the present invention may be a pharmaceutical composition or a combined preparation.

A fourth aspect of the invention provides for the use of one or more elastase inhibitors in the manufacture of a medicament for the treatment of a wound wherein the treatment comprises the simultaneous, separate or sequential administration of one or more growth factors to the patient.

A fifth aspect of the invention provides for the use of one or more growth factors in the manufacture of a medicamcnt for the treatment of a wound wherein the treatment comprises the simultaneous, separate or sequential administration of one or more elastase inhibitors to the patient.

In a preferred embodiment the pharmaceutical product of the first aspect of the invention is provided in the forth of a wound dressing and the medicament of the third, fourth and fifth aspects of the invention is a wound dressing.

As used herein the phrase "manufacture of a medicament" includes the use of the components of the invention directly as the mcdicament in addition to their use in any stage of the preparation of such a medicament.

A sixth aspect of the invention provides a method of promoting wound healing, said method comprising administering one or more elastase inhibitors and one or more growth factors to a patient in need thereof.

The one or more elastase inhibitors and the one or more growth factors may be administered in the same composition or by separate compositions.

T he one or more elastase inhibitors and the one or more growth factors may be administered to the patient simultaneously, sequentially or separately.

A seventh aspect of the invention provides: (i) a package comprising one or more elastase inhibitors together with instructions for use in combination with one or more growth factors for the promotion of wound healing, (ii) a package comprising one or more growth factors together with instructions for use in combination with one or more elastase inhibitors for the promotion of wound healing; and (iii) a package comprising one or more elastase inhibitors and one or more growth factors together with instructions lor use in the promotion of wound healing.

An eighth aspect of the invention provides a wound dressing comprising one or more elastase inhibitors and one or more of growth factors. Suitably the wound dressing is provided in the forth of a solid substrate with one or more clastasc inhibitors and the one or more growth factors dispersed on or in the solid substrate.

As used herein, "simultaneously" is used to mean that the one or more elastase inhibitors and one or more growth factors are administered concurrently, whereas the term "in combination" is used to mean they are administered, if not simultaneously, then "sequentially" within a timeframe that they both are available to act therapeutically within the same time-frame. Thus, administration "sequentially" may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided that both the one or more elastase inhibitors and one or more growth factors are concurrently present in therapeutically effective amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction there between, and their respective half-lives.

Brief description of the figures

Figure 1: PDGF retrieval after exposure to CWF in presence of protease inhibitors. The figure shows a synergistic effect on activity when PDGF is combined with alpha I antitrypsin.

Detailed Description

Ela.vla.se inhibitors Examples of elastase inhibitors are well known in the art and include: elastinil, elafin, secretory leukocyte proteinase inhibitor alpha-l -macroglobulin, and alpha-l -antitrypsin (AAT). A particularly preferred elastase inhibitor is AAT. Thus, in the various embodiments of the invention it is preferred that the "one or more elastase inhibitors" is AAT only or AAT in combination with one or more other elastase inhibitors.

Alpha-l-antitryp.sin It is to be understood that the term "alpha-lantitrypsin" as used herein encompasses all naturally occurring polymorphs of AAT. It also encompasses functional fragments of AAT, chimeric proteins comprising AAT or functional fragments thereof, homologs obtained by analogous substitution of one or more amino acids of AAT, and species homologs. Preferably, the AAT is a product of recombinant DNA technology, and more preferably the AAT is a product of transgenic technology. For example, the gene coding for AAT can be inserted into a mammalian gene encoding a milk whey protein in such a way that the DNA sequence is expressed in the mammary gland (e.g. as described in W088/00239).

The specific examples of elastase inhibitors given above (i.e. elastinil, elafin, secretory leukocyte proteinase inhibitor and alpha-1macroglobulin) are intended to be interpretated in a similar manner, i.e. to include functional fragments etc. of the same.

T/'e one or more growth factors Examples of growth factors which may be used in the present invention include: epidermal growth factor (EGF), platelet derived growth factor (PDGF; one or more of the three subtypes may be used: AA, AB, and B), Vascular Endothelial Growth Factor (VEGF), blood-derived growth factor, fibroblast growth factor (FGF) (including basic FGF), keratinocyte growth factor transforming growth factor, tissue growth factor, TGF-alpha, TGF-beta and insulin-like growth factor (e.g. IGF- 1).

Preferably, at least PDGF and/or EGF is used.

In one embodiment, preferably one, two three, four, five or six growth factors selected from the following is used: PDGF, EGF, basic FGF, IGF, TGF-beta and VEGF.

It is to be understood that the growth factors above encompass all naturally occurring polymorphs of the growth factors. Also, functional fragments, chimeric proteins comprising one of said growth factors or a functional fragment thereof, homologs obtained by analogous substitution of one or more amino acids of the growth factor, and species homologs are encompassed. Preferably, the one or more growth factors is a product of recombinant DNA technology, and more preferably the one or more growth factors is a product of transgenic technology. For example, platelet derived growth factor may be provided in the form of a recombinant PDGF.

Additional wound healing agents Optionally, one or more other, conventional wound healing agents (e.g. peptides, proteolytic inhibitors, extracellular matrix components, fragments and peptides, steroids, cytokines, oxygen donators or vitamins) may also be used in the manufacture of the medicament, pharmaceutical compositions and combined preparations according to the invention. Such conventional wound healing agents may also be used in the method of the present invention. The inclusion of these agents may allow a synergistic effect on wound healing. Such additional wound healing agent(s) may be administered separately, simultaneously or sequentially with the one or more elastase inhibitors and/or the one or more growth factors.

Formulations A therapeutically et'fective amount of each of the combination partners (i.e. the one or more elastase inhibitors and said one or more growth factors) may be administered simultaneously, separately or sequentially and in any order. The components may be administered separately or as a fixed combination.

The pharmaceutical products, pharmaceutical compositions, combined preparations and medicaments of the invention may, for example, take the forth of solutions, suspensions, tablets, pills, capsules, salves, creams, wound dressings, sustained release formulations, or powders, and typically contain 1%-95% of active ingredient(s), preferably 2%-70%.

The said one or more elastase inhibitors and/or said one or more growth factors may be mixed with physiological tolerable and compatible diluents, excipients and pharmaceutically acceptable carriers. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents may also be present.

The tend "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaeeharides, polylaetie acids, polyglyeolie acids, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. Pharmaecutieally acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol.

Pharmaceutically acceptable salts can also be present e.g. mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

Preferably, the one or more elastase inhibitors and the one or more growth factors are provided in the form of a wound dressing. That to say, the one or more elastase inhibitors and the one or more growth factors are provided in the form of a liquid, semi-solid or solid composition for application directly to the surtaee of a wound, or the composition is applied to the surface of, or incorporated into, a solid wound contacting layer such as a wound dressing gauze or film. The wound dressing composition may be provided in the form of a fluid or a gel. The one or more elastase inhibitors and the one or more growth factors may be provided in combination with conventional pharmaceutical excipients for topical application to a wound. Suitable carriers include: Hydrogels containing cellulose derivatives, including hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose and mixtures thereof; and hydrogels containing polyacrylie acid (Carbopols). Suitable carriers also include creams/ointmcnts used for topical pharmaceutical preparations, e.g. creams based on cctomacrogol emulsifying ointment. The above carriers may include alginate (as a thickener or stimulant), preservatives such as benzyl alcohol, buffers to control pH such as disodium hydrogen phosphate/sodium dihydrogen phosphate, agents to adjust osmolarity such as sodium chloride, and stabilizers such as EDTA.

In one embodiment of the invention, the wound dressing composition may be a slow release solid composition, in which the one or more elastase inhibitors and/or one or more growth factors is dispersed in a slow release solid matrix such as a matrix of alginate, collagen, or a synthetic bioabsorbable polymer. Preferably, the wound dressing composition is sterile.

The tend "wound dressing" in this specification refers to a dressing for topical application to a wound and excludes compositions suitable for systemic administration. For example, the one or more elastase inhibitors and/or the one or more growth factors may be dispersed in or on a solid sheet of wound contacting material such as a woven or nonwoven textile material, or may be dispersed in a layer of foam such as polyurethane foam, or in a hydrogel such as a polyurethane hydrogel, a polyacrylate hydrogel, gelatin, carboxymethyl cellulose, pectin, alginate, and/or hyaluronic acid hydrogel, for example in a gel or ointment. In preferred embodiments the AAT and/or said one or more growth factors are dispersed in or on a biodegradable sheet material that provides sustained release of the active ingredients into the wound, for example a sheet of freezedried collagen, freeze-dried collagen/alginate mixtures (available under the Registered Trade Mark FIBRACOL from Johnson & Johnson Medical Limited) or freeze-dried collagen/oxidized regenerated cellulose (available under the Registered Trade Mark PROMOGRAN from Johnson & Johnson Medical Limited).

Modes of delivery A therapeutically effective amount of each of the combination partners of the combination of the invention may be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination.

The individual combination partners of the combination of the invention can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.

The one or more elastase inhibitors and said one or more growth factors may be administered by the same or different routes. Preferably one or both of said one or more elastase inhibitors and said one or more growth factors are delivered by topical administration (peripherally or directly to a wound), including but not limited to topical administration using solid supports (such as dressings and other matrices) and medicinal formulations (such as mixtures, suspensions and ointments). In one embodiment, the solid support comprises a biocompatible membrane. In another embodiment, the solid support comprises a wound dressing. In one embodiment a wash solution comprising the one or more clastasc inhibitors and/or one or more growth factors can be used locally to promote healing of the wound.

Alternative routes of administration include: intravenously, intradermal and subcutaneous administration.

1 0 Dose The effective dosage of each of the combination partners employed in the combination of the invention may vary depending on a number of factors including the particular compound or pharmaceutical composition employed, the mode of administration, the frequency of administration, the condition being treated, the severity of the condition being treated, the route of administration, the needs of a patient sub-population to be treated or the needs of the individual patient which different needs can be due to age, sex, body weight, etc. of the patients.

The ettective dose for a given situation can be determined by routine experimentation and is within the judgement of the skilled person. For example, in order to formulate a range of dosage values, cell culture assays and animal studies can be used. The dosage of such compounds preferably lies within the dose that is therapeutically effective for at least 50% of the population, and that exhibits little or no toxicity at this level.

Type of wounds The term "wound" refers broadly to injuries to the skin and subcutaneous tissue initiated in different ways (e.g., pressure sores from extended bed rest and wounds induced by trauma) and with varying characteristics. Wounds may be classified into one of four grades depending on the depth of the wound: i) Grade I: wounds limited to the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade III: wounds extending into the subcutaneous tissue; and iv) Grade IV (or full-thickness wounds): wounds wherein bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum). The term "partial thickness wound" refers to wounds that encompass Grades I- III; examples of partial thickness wounds include burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers. The term "deep wound" is meant to include both Grade III and Grade IV wounds. The present invention contemplates treating all wound types, including deep wounds and chronic wounds. The term "chronic wound" refers to a wound that has not healed within 30 days. The delay in healing may, for example, be caused by elevated levels of matrix metalloproteinases (MMP's). Typically, the wound is a chronic wound.

Preferably, it is selected from the group consisting of venous ulcers, pressure sores, diabetic ulcers and decubitis ulcers.

The patient The patient will usually be a mammal, such as a horse (e.g a racehorse), a dog (e.g. greyhound), a cow, eic. Preferred patients are humans, including children, adults and the elderly.

It will be understood that the invcution has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

EXAMPLES

Method: Chronic wound fluid (CWF) was spiked with PDGF-AA (to a final concentration of 500pg/ml) and was then incubated with AAT (1.25mg/ml) for 24 hours. This was also repeated replacing the CWF with PBS. After incubation the solutions were assayed for final PDGF-AA concentration using ELlSA.

Results: By incubation of CWF with AAT, it was possible to retrieve 100% PDGF-AA as a percentage of the positive control (PBS + PDGF-AA). After incubation of PDGF with PBS in the presence of AAT, 16% more PDGF was retrieved than that of the positive control, indicating AAT both protects PDGF-AA in CWF but that it also stabilised the growth factor in solution.

Claims

1. A pharmaceutical product comprising: (a) one or more elastase inhibitors; and (b) one or more growth factors for simultaneous, separate or sequential use to promote wound healing.

2. The pharmaceutical product according to claim I which is provided in the tong of a combined preparation.

3. The pharmaceutical product according to claim 1 which is provided in the forth of a pharmaceutical composition.

4. The use of one or more elastase inhibitors and one or more growth factors in the manufacture of a pharmaceutical product for the promotion of wound healing in a patient in need thereof.

5. The use of claim 4 wherein the pharmaceutical product is provided in the foam of a pharmaceutical composition.

6. The use of claim 4 wherein the phanmaccutical product is provided in the fonm of a combined preparation.

7. The use of AAT in the manufacture of a medicament for the treatment of a wound wherein the treatment comprises the simultaneous, separate or sequential administration of one or more elastase inhibitors and one or more growth factors to the patient.

8. The use of one or more growth factors in the manufacture of a medicament for the treatment of a wound wherein the treatment comprises the simultaneous, separate or sequential administration of one or more elastase inhibitors one or more growth factors to the patient.

9. A method of promoting wound healing, said method comprising administering one or more elastase inhibitors and one or more growth factors to a patient in need thereof.

10. The method of claim 9 wherein the one or more elastase inhibitors and the one or more growth factors are administered in the same composition.

11. The method of claim 9 wherein the one or more elastase inhibitors and the one or more growth factors are administered by separate compositions.

12. A package comprising: -1 1 (i) one or more elastase inhibitors together with instructions for use in combination with one or more growth factors for the promotion of wound healing, (ii) one or more growth factors together with instructions for use in combination with one or more elastase inhibitors for the promotion of wound healing; or (iii) one or more elastase inhibitors and one or more growth factors together with instructions for use in the promotion of wound healing.

13. The pharmaceutical product according to any one of claims I to 3, the use according to any one of claims 4 to 8, the method according to any one of claims 9 to 11 or the package of claim 12 wherein said one or more elastase inhibitors is AAT only or AAT in combination with one or more other elastase inhibitors.

14. The pharmaceutical product according to any one of claims I to 3 or 13, the use according to any one of claims 4 to 8 or 13, the method according to any one of claims 9 to I I or 13 or the package of claim 12 or 13 wherein said growth factors includes at least one, two or three growth factors selected from the group consisting of: epidermal growth factor (EGF), platelet derived growth factor (PDGF), blood-derived growth factor, Vascular Endothelial Growth Factor (VEGF), fibroblast growth factor (FGF), keratinocyte growth factor transforming growth factor, tissue growth laetor, TGF-alpha, TGF-beta and insulin-like growth factor (e.g. IGF- 1).

15. The pharmaceutical product according to any one of claims I to 3 or 13 or 14, the use according to any one of claims 4 to 8 or 13 or 14, the method according to any one of claims 9 to 11 or 13 or 14 or the package of claim 12 or 13 or 14 wherein the one or more elastase inhibitors and the one or more growth factors are simultaneously, separately or sequentially administered by topical administration.

16. The pharmaceutical product according to claim 15, the use according to claim 15, the method according to claim 16 or the package of according to claim 16 wherein the one or more elastase inhibitors and the one or more growth factors are provided in the form of a

17. The pharmaceutical product, the use, method or package according to any one of the preceding claims wherein the wound is a chronic wound.

18. A wound dressing comprising one or inore elastase inhibitors and one or more of growth factors.

19. A wound dressing according to claim 18 wherein one or more elastase inhibitors is AAT only or AAT in combination with one or more other elastase inhibitors.