WO2008084253A1

WO2008084253A1 - Use of a galactolipid for wound and ulcer healing

Info

Publication number: WO2008084253A1
Application number: PCT/GB2008/000107
Authority: WO
Inventors: Alvar GRÖNBERG; Britta Radeloff
Original assignee: Lipopeptide Ab
Priority date: 2007-01-12
Filing date: 2008-01-14
Publication date: 2008-07-17

Abstract

According to the invention there is provided the use of galactolipids as active ingredients in the treatment of conditions that may benefit from activation (including proliferation and/or migration) of cells, such as epithelial cells, including keratinocytes. Relevant conditions include burn wounds and ulcers.

Description

USE OF A GALACTOLIPID FOR WOUND AND ULCER HEALING

Field of the Invention

The present invention relates to a new use of known glycolipids, specifically galactolipids and materials or compositions thereof.

Background of the Invention

Traditional wound care involves removal, mechanically or enzymatically, of necrotic debris to allow formation of granulation tissue. Wounds that are heavily colonized by bacteria may require antiseptic treatment to prevent invasive infection. Numerous topical anti-microbial agents are used, such as iodine, chlorhexidine, hydrogen peroxide and silver, as well as antibiotics, but such treatments suffer from the drawback of toxic effects on the matrix and neoepidermis.

Once the wound is clean of necrotic tissue, dressings are used to promote granulation tissue formation. A large variety of such dressings are available and numerous animal studies and clinical trials have demonstrated their beneficial effect on wound healing.

However, a certain proportion of wounds remain therapy-resistant and clinicians often find a need for additional treatment.

Chronic ulcers constitute a major clinical problem and, although understanding of the physiology of wound processes has increased during recent decades, only minor therapeutic improvements have been attained.

Distinct etiologies may underlie the development of ulceration in different clinical conditions but, whatever the cause, so-called "hard-to-heal" and/or "non-healing" ulcers are characterized by an inability of the epithelium to migrate, proliferate and close the barrier defect. The most common type of chronic skin ulcer is leg ulcers, which are typically a direct result of venous insufficiency. Afflicted patients develop peripheral venous oedema with subsequent ulceration of the skin, although arterial circulation is typically unaffected. Leg and foot ulcers due to arteriosclerotic deficiencies are less common.

Additionally, skin ulcers may develop in association with immune diseases such as pyoderma gangrenosum and vasculitis. Current treatment typically comprises long-term systemic immunosuppression, but this is not always effective.

Epithelial defects and ulceration on oral, ocular, genital and gastrointestinal mucosa are common and cause significant distress. Underlying pathomechanisms are not always clear (aphtae and erosive lichen often being thought to be responsible), and present treatments are unsatisfactory.

From a mechanistic point of view, wound healing is thought to be dependent on interplay between signals triggered in the damaged epithelial cell layer, stromal cells, extracellular matrix and inflammatory cells and mediators (Hosgood, Vet. CHn. North Am. Small Anim. Pract., 36, 667-85 (2006)).

An early consequence of wounding is that epithelial cells in the wound margin migrate into the wound area (Kirfel and Herzog, Protoplasma, 223, 67-78 (2004)). This is followed by increased cell division, particularly hi the wound margin. In chronic non-healing wounds, the re-epithelialization process is inhibited for unknown reasons.

Substances that are potentially capable of reactivating the wound healing process, including epithelial cell migration, are thus attractive candidates for treatment of wounds that present a significant clinical challenge to heal. Specifically, substances that are capable of activating migration of skin keratinocyte cells may be of benefit in the treatment of "non-healing" skin ulcers, whereas stimulation of other epithelial cells may be of benefit in the treatment of ulcers at other sites such as the gastrointestinal tract, the genitalia, the respiratory tract or cornea. To this end, animal experiments have shown that endogenous polypeptides, such as epidermal cell growth factors, that are capable of stimulation of keratinocyte migration in vitro also play an important role in wound healing (Shirakata et al, J. CeIl ScL, 118, 2363-2370 (2005)).

During the past decade there has been much focus on the potential use of growth factors to accelerate wound repair. Growth factors are molecules that control cellular processes and are critical in tissue repair, having an effect on cell ^• migration, proliferation, angiogenesis and de novo synthesis of extracellular matrix. The beneficial effect of such growth factors has been suggested in a wide variety of trials (Scharffetter-Kochanelc et al, Basic Res. Cardiol, 93, 1-3 (1999)). However, at present, growth factor treatment of chronic ulcers has been largely disappointing in clinical practice (due to perceived delivery problems and rapid degradation), with becaplermin (Regranex®) the only licensed growth factor for use, preferentially in diabetic foot ulcers.

In parallel, tissue therapies have been developed using autologous and allogenic materials in bioengineered human skin equivalents. Cultured epidermal keratinocytes constitute a functioning treatment for coverage of large areas of injured skin in e.g. burn patients, but is expensive, time-consuming and requires laboratory facilities. To provide a dermal substrate, multiple strategies have been used such as acellular human cadaver and bovine collagen with or without cells. However, all methods employed have considerable disadvantages, such as the potential transmission of disease and high costs, and are hardly suited for basic wound care.

Hence there is still a real and growing need for new and/or better treatments of wounds, such as ulcers, and in particular chronic wounds.

Certain lipids have been shown to affect epithelial cell migration and wound . healing, including lysophosphatidic acid (Balazs et al, Am. J. Physiol. Regul. Integr. Comp. Physiol, 280, R466-472 (2001); and Sauer et al, J. Invest. Dermatol, 123, 840-9 (2004)) and C8-ρhosphatidic acid (Mazie et al, J. Cell ScL, 119, 1645-1654 (2006)).

Galactolipids are a group of glycolipids in which the sugar group is galactose. They are distinguished from glycosphiπgolipids in lacking nitrogen. They constitute the majority of membrane lipids in plants. Galactolipids, which comprise primarily digalactosyldiacylglycerol (DGDG) and DGDG-rich materials, have been investigated and found to be potentially useful in industrial applications such as food, cosmetics, and pharmaceutical products.

Galactolipid preparations derived from plants such as oats have been used for some time as lipid excipients in topical and peroral emulsions, and are known have excellent physical properties in this respect. WO 99/044585 and WO 99/044585 describe the use of DGDG-rich material as an emulsifier in topical formulations of the oil-in- water type. WO 95/20944 describes the use of DGDG-rich material as a bilayer-forming material in polar solvents for pharmaceutical, nutritional and cosmetic use. These lipid materials have been the subject of extensive safety and toxicological testing and are considered to be safe and well-tolerated when used by human subjects.

The biophysical properties of galactolipid/water mixtures consisting of bilayer structures, i.e. vesicles or liposomes, make galactolipids suitable carriers of amphiphilic substances for e.g. drug delivery. Such agents may be readily incorporated within the lipid bilayers.

To our knowledge, galactolipids derived from oats, and cereals dp not possess irritant properties and are not toxic. It is this biological inertness that renders them potentially useful as pharmaceutical excipients.

It has been reported that intraperitoneal injection of galactolipids in the course of abdominal surgery in rabbits reduced adhesions between intestines and the abdominal wall (Treutner et al, Journal of Surgical Research, 59, 764-771 (1995)). A galactolipid isolated from rose Mp has been described to inhibit the migration of neutrophil granulocytes (Larsen et al, J. Nat. Prod., 66, 994-5 (2003)). Monogalactosyl galactolipid has been shown to activate the protein kinase C alpha of brain oligodendrocytes and to increase regeneration of oligodendrocyte fibers (Schmidt-Schultz and Althaus, Journal of Neurochemistiy, 62, 1578-1585 (1994)).

Extracts of oats have been shown to promote healing of ex vivo human skin wound and to stimulate keratinocyte migration (Aries et al, J. Invest. Dermatol., 112, 125 (1999) and Biol. Pharm. Bull, 28, 601-6 (2005), and Boisnic et al, Int. J. Tissue React, 27, 83-9 (2005)).

International patent applications WO 2004/067025 and WO 2005/079860 describe the use of pharmaceutical formulations comprising biologically active peptides and galactolipids mixtures in in vitro models of wound healing.

Surprisingly, we have now discovered that galactolipids are potent stimulators of keratinocyte migration and that they may promote the healing of wounds, such as chronic ulcers and burn wounds.

Description of the Invention

According to a first aspect of the invention, there is provided the use of a galactolipid as an active ingredient for the manufacture of a medicament for the treatment of a condition that may benefit from activation of cells.

Thus, the invention provides the use of a galactolipid as an active ingredient for the treatment of a condition that may benefit from activation of cells.

It will be appreciated by persons skilled in the art that the galactolipid may be a substantially pure galactolipid preparation of one or more galactolipid compounds or may be a galactolipid-containing material/composition which is enriched in such galactolipid compounds. By "enriched"; in this context, we mean that the material or composition comprises at least 20% by weight of galactolipid comρound(s), for example at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.9% by weight of galactolipid.

Thus, for example, the galactolipid may be a fractionated oat oil comprising at least 20% by weight galactolipid, a commercially available galactolipid composition such as CPL-Galactolipid™ or a substantially pure l,2-diacyl-3-O- (α-D-galactopyranosyl-( 1 -6)-O-β-D-galactopyranosyl-glycerol (as discussed further below).

The galactolipid may be used directly, e.g. as the medicament, or more typically will be formulated with one or more , additional components prior to use (as described in detail below).

By the phrase "for use as an active ingredient", we mean that galactolipid materials are employed in the context of the present invention as biologically- active (e.g. pharmaceutically-active) ingredients in their own right, rather than as excipients in pharmaceutical or cosmetic formulations. In the context of the present invention, galactolipid materials may thus be packaged and presented for use as medicaments (e.g. as pharmaceuticals or as a component of medical devices). In one embodiment, the medicament is a cosmetic or cosmeceutical for use in the treatment of a condition that may benefit from the activation of cells.

The term "cells" will be well understood by those skilled in the art to include any structural and/or functional unit of a living organism (hereinafter "patient"). Cells that may be mentioned include endothelial cells and, particularly, epithelial cells. Epithelial cells include all types of such cells, such as corneal epithelial cells, gastrointestinal epithelial cells, epithelial cells of the airways and the mucosa. Epithelial cells that may be mentioned include keratinocytes.

By "activation" of cells, we mean promotion (initiation and propagation (e.g. maintenance, support and/or continuation)) of any biological process that is known to be associated with such cells, and more particularly proliferation and especially migration of such cells.

Medicaments, medical devices, pharmaceutical compositions or pharmaceutical formulations, comprising, as active ingredients, galactolipid materials are hereinafter referred to together as "the compositions of the invention".

In the context of the present invention; the term "galactolipid material" includes any lipid material in which the polar head group comprises one or more galactose units. In particular, the "galactolipid material" is enriched in such galactolipid compounds. Suitable galactolipids include DGDG and other glycolipids, such as the glycosylceramides, either natural or synthetic, in which a non-ionic carbohydrate moiety constitutes the polar head-group. This class of galactolipids possess extremely good cutaneous tolerability. DGDG (l,2-diacyl-3-O-(α-D- galactopyranosyl-(l-6)-0-β-D-galactopyranosylglycerol) is preferred in this respect. Alternatively, or in addition, the corresponding monogalactosyl galactolipid may also be used.

More preferably, the galactolipid is a purified galactolipid fraction from oats, such as CPL-Galactolipid™ (a galactolipid material manufactured by LTP Lipid

Technologies Provider AB, Sweden, now a subsidiary of DSM, The Netherlands).

This material has been employed in dermatological creams as an excipient and has been shown to be well tolerated, and to have good absorption properties (see, for example, WO 99/044585 and WO 99/044585). CPL-Galactolipid™ is stable at ambient temperature.

In accordance with the invention, galactolipids are preferably administered for systemic or, preferably, local delivery via one or more routes selected from orally, intestinally, intravenously, intramuscularly, cutaneously, subcutaneously, intracutaneously, transmucosally (e.g. sublingually or buccally), pulmonarily (e.g. tracheally or bronchially) and, preferably, topically, for example, dermally, aurally, nasally, rectally, vaginally or to another mucosal surface (such as the cornea), or via any other parenteral route, in the form of a pharmaceutical preparation comprising the galactolipid in a pharmaceutically acceptable dosage form. Preferred modes of delivery include oral, topical (in particular cutaneous) or subcutaneous.

Galactolipids may be administered alone, but will generally be administered in the form of one or more pharmaceutical or cosmetic formulations in admixture with a pharmaceutically or cosmetically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical or cosmetic practice. Such pharmaceutically or cosmetically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be commercially available or otherwise are described in the literature, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995) and the documents referred to therein, the relevant disclosures in all of which documents are hereby incorporated by reference. Otherwise, the preparation of suitable formulations may be achieved non-inventively by the skilled person using routine techniques.

Pharmaceutically or cosmetically acceptable carriers may be employed to enable the formulation of tablets, pills, capsules, powders, liquids, aerosols, gels, syrups, slurries, suspensions, creams, infusions, ointments, solutions, patches, wound dressings, or any other suitable type of formulation.

When the pharmaceutical or cosmetic formulation comprising galactolipid is in the form of a liquid, aerosol, gel, suspension, infusion, or solution, it may comprise an aqueous carrier. Such a carrier should possess physiologically- or phaπnaceutically-acceptable properties in relation to pH, ionic strength, isotonicity etc. In this respect, isotonic solutions of water and other biocompatible solvents, aqueous solutions, such as saline and glucose solutions, and hydrogel- forming materials may be employed. The aqueous carrier may further comprise a buffering agent, such as phosphate-buffered saline (PBS). Such formulations may also comprise other pharmaceutically or cosmetically acceptable excipients, such as preservatives, antioxidants, additional isotonicity agents, colouring agents and the like. In aqueous suspensions, the compositions can be combined with suspending and stabilising agents, such as non-ionic surfactants, hydrophilic polymers and the like.

Thickening agents may be employed in order to create a gel, syrup, cream, wound dressing, or ointment. In order to form a gel, a hydrogel-forming material may be employed, such as s}iithetic polymers, e.g. pqlyvinylalcohol, polyvinylpyrolidone, polyacrylic acid, polyethylene glycol, poloxamer block copolymers and the like; semi-synthetic polymers, such as cellulose ethers, including carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, methylhydroxypropylcellulose and ethylhydroxy-ethylcellulose, and the like; natural gums, such as acacia, alginate, carragenan, chitosan, pectin, starch, xanthan gum and the like.

A hydrogel may be rendered mucoadhesive by employment of materials such as hyaluronic acid and derivatives thereof, cross-linked polyacrylic acids of the carbomer and polycarbophil types, polymers that readily form gels, which are known to adhere strongly to mucous membranes.

It is also advantageous to use block copolymers of the poloxamer type,- i.e. polymers consisting of polyethylene glycol and polypropylene glycol blocks. Certain poloxamers dispersed in water are thermoreversible. At room temperature they have a low viscosity but exhibit a marked viscosity increase at elevated temperatures, resulting in a gel formation at body temperature. Thereby the contact time^' of a pharmaceutical formulation administered to the relatively warm wound may be prolonged.

Non-limiting examples of pharmaceutical or cosmetic compositions for topical administration are solutions, sprays (including aerosols), suspensions, emulsions, gels, and membranes. If desired, a bandage or a band aid or plaster can be used, or wound dressing, to which the pharmaceutical composition has been added. Alternatively, tablets, capsules, solutions or suspensions can be used for peroral administration. Other examples of materials which can be used as part of a pharmaceutically acceptable carrier in compositions (such as solid or semi-solid compositions) comprising, as active ingredients, galactolipids, include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; other membrane lipids, such as phospholipids; glycols, such as propylene glycol; polyols, such as glycerine, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate .and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate; other non-toxic compatible substances, as well as pharmaceutically-acceptable excipients, such as release agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants.

It is thus preferred that the amount of galactolipid in the formulation is less than about 75%, for example less than about 50%, such as less than about 30%, e.g. less than about 20%, e.g. less than about 10% and even less that about 5% (w/w). In one embodiment, the amount of galactolipid in the formulation is less than about 3%, for example less than about 1%, such as less than about 0.5%, e.g. less than about 0.1%, e.g. less than about 0.01% and even less that about 0.001% (w/w).

According to a further aspect of the invention, there is provided a method of activating cells in a patient, which method comprises the administration of a composition of the invention to that patient. In this respect, there is further provided a method of regeneration and/or repair of tissue, such as ^' endothelial or, preferably, epithelial tissue, in a patient, which method comprises the administration of a composition of the invention to such a patient.

Preferred conditions include wounds and epithelial damage in a general sense. Wounds that may be mentioned include open wounds (including those made by incisions, lacerations, abrasions, punctures, penetrations and, particularly, burn wounds). Wounds that are worthy of particular mention include non-healing or hard-to-heal wounds, by which we include wounds that respond poorly to conventional therapy, including ulcers.

Ulcers that may be mentioned include, particularly, skin ulcers (e.g. chronic, nonhealing skin ulcers {vide supra), such as ulcers of the leg and/or foot, including diabetic foot ulcers). Other types of ulcers that may be mentioned include Hunner's ulcers, Curling ulcers, Cushing ulcers, pressure ulcers (decubitus), crural ulcers, and, more particularly, ulcers of the oral mucosa, the tympanic membrane, the sacrum, the corneal epithelium (which may or may not result from an autoimmune disease, atopic eczema and/or an infection), the genitalia (including penile, vulvar or labial ulcers), the respiratory tract, and gastrointestinal mucosa (including oesophegal ulcers, gastric ulcers, duodenal ulcers and large and small intestinal ulcers, which may be a result of inflammatory bowel disease, such as Crohn's disease and/or ulcerative colitis).

Compositions of the invention may be employed in the treatment of ulcers caused by bacterial infection, viral infection, fungal infection, cancer (primary and secondary), venous stasis, arterial insufficiency, diabetes, rheumatoid arthritis, amyloidosis, loss of mobility and/or hypertension.

However, it will be appreciated by persons skilled in the art that the galactolipid compositions of the invention may also be employed in the treatment of acute wounds, such as minor cuts, grazes, etc. For the avoidance of doubt, in the context of the present invention, the terms "treatment", "therapy" and "therapy method" include the therapeutic, or palliative, treatment of patients in need thereof, as well as the prophylactic treatment and/or diagnosis of patients which are susceptible to a particular disease state.

"Patients" include members of any animal species, particularly reptiles, birds and mammals (especially humans).

The amount of galactolipid material in the formulation(s) will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, galactolipid materials may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a patient in the context of the present invention should be sufficient to effect a therapeutic response in the patient over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, species, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration of active ingredients may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. Suitable doses of galactolipid materials are therefore in the range of about 0.01 mg/kg of body weight to about 1,000 mg/kg of body weight. More preferred ranges are about 1 mg/kg to about 100 mg/kg on a daily basis, when given orally.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Galactolipids may be administered as active ingredients in compositions of the invention alone or may be administered in combination with other active ingredients, and in particular those that are useful in the treatment of conditions that may benefit from activation of cells (e.g. endothelial and, more particularly, epithelial cells), such as wounds and ulcers.

According to the invention, there is provided a combination product comprising:

(a) a galactolipid material as an active ingredient; and

(b) another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells, such as endothelial and, particularly, epithelial cells.

Other active ingredients that are useful in the treatment of a condition that may benefit from activation of cells (such as endothelial and, particularly, epithelial cells), for example to enhance their migration, include growth factors (including the epidermal growth factor family, platelet derived growth factors, hepatocyte growth factor, fibroblast growth factors, insulin, insulin-like growth factor I, keratinocyte growth factor, vascular endothelial growth factors, nerve growth factor, transforming growth factor beta), cytokines, such as interferon-gamma, GM-CSF, IL-I, IL-13 and IL-20, chemokines such as IL-8 and RANTES, . antimicrobial peptides and proteins (e.g. defensins, cathelicidins, lactoferrin, etc.), peptide hormones (e.g. vasoactive intestinal peptide and the like), proteases (e.g. thrombin), and biologically active non-peptide substances (e.g. lipids, eicosanoids, nucleosides, nucleotides, polyamines, short hairpin RNA, ceramide, carbohydrates, carbohydrate phosphates, amino acids, amines, and vitamins [A, B, C, D, E, K]), antimicrobial compounds, and other pharmacologically- active compounds including receptor agonists, receptor antagonists, inverse agonists, partial agonists, phosphatase inhibitors, hydrogen peroxide, antioxidants, redox active compounds, metal ions (e g zinc, silver) and protease inhibitors.

Combination products according to the invention provide for the administration of galactolipid material in conjunction with the other active ingredient, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises galactolipid material, and at least one comprises the other active ingredient, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including galactolipid material and the other active ingredient).

Thus, there is further provided:

(1) a medicament (e.g. a pharmaceutical formulation) including a galactolipid material; another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells, such as endothelial and, particularly, epithelial cells; and a pharmaceutically-acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a "combined preparation"); and

(2) a kit of parts comprising components: (a) a medicament (e.g. a pharmaceutical formulation) including a galactolipid

^• material in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and (b) a medicament (e.g. a pharmaceutical formulation) including another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells, such as endothelial and, particularly, epithelial cells, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

According to ,a further aspect of the invention, there is provided a method of making a kit of parts as defined above, which method comprises bringing component (a), as defined above, into association with a component (b), as defined above, thus rendering the two components suitable for administration in conjunction with each other.

By bringing the two components "into association with" each other, we include that components (a) and (b) of the kit of parts may be:

(i) provided as separate formulations (i.e. independently of one another), which are subsequently brought together for use in conjunction with each other in combination therapy; or (H) packaged and presented together as separate components of a "combination pack" for use in conjunction with each other in combination therapy.

Thus, there is further provided a kit of parts comprising: (I) one of components (a) and (b) as defined herein; together with (II) instructions to use that component in conjunction with the other of the two components.

The kits of parts described herein may comprise more than one formulation including an appropriate quantity/dose of a galactolipid material, and/or more than one formulation including an appropriate quantity/dose of the other active ingredient, in order to provide for repeat dosing. If more than one formulation (comprising either active compound) is present, such formulations may be the same, or may be different in terms of the dose of either compound, chemical comρosition(s) and/or physical form(s).

With respect to the kits of parts as described herein, by '%drninistration in conjunction with", we include that respective formulations comprising a galactolipid material and the other active ingredient are administered, sequentially, separately and/or simultaneously, over the course of treatment of the relevant condition.

Thus, in respect of the combination product according to the invention, the term "administration in conjunction with" includes that the two components of the combination product (galactolipid material and the other active ingredient) are administered (optionally repeatedly), either together, or sufficiently closely in time, to enable a beneficial effect for the patient, that is greater, over the course of the treatment of the relevant condition, than if either a formulation comprising a galactolipid material, or a formulation comprising the other active ingredient, are administered (optionally repeatedly) alone, in the absence of the other component, over the same course of treatment. Determination of whether a combination provides a greater beneficial effect in respect of, and over the course of treatment of, a particular condition will depend upon the condition to be treated or prevented, but may be achieved routinely by the skilled person.

Further, in the context of a kit of parts according to the invention, the term "in conjunction with" includes that one or other of the two formulations may be administered (optionally repeatedly) prior to, after, and/or at the same time as, administration with the other component. When used in this context, the terms "administered simultaneously" and "administered at the same time as" include that individual doses of galactolipid material and the other active ingredient are administered within 48 hours (e.g. 24 hours) of each other.

According to a further aspect of the invention, there is provided a composition of the invention as hereinbefore defined provided that the composition does not comprise the human cathelicidin peptide LL-37.

According to a further aspect of the invention, there is provided a composition of the invention as hereinbefore defined provided that the composition does not comprise LL-37 or a sequence containing at least 20 amino acids of the N- terminal fragment of LL-37. According to a further aspect of the invention there is provided composition of the invention as hereinbefore defined provided that the composition does not further comprise a peptide in which, in the composition, the weight ratio between the peptide and the galactolipid material is between 1 :5 and 1 :50 and the peptide is (a) an amphophilic peptide having a molecular weight below 30 kDa; and/or (b) a cationic antimicrobial peptide having a molecular weight of between 2.5 and 5 kDa (such as apolipoprotein, an apolipoprotein analogue, insulin, glucagon, erythropoietin, darbepoietin, streptokinase, somatropin, desmopressin, oxytocin, gonadorelin, nafarelin, octreotide, lanreotide, ganirelix, cetrorelix, teriparalid, salmon calcitonin, magainin 2, cecropin, histatin, LL-25, LL-26, LL-27, LL-28, LL-29, LL-30, LL-31, LL-32, LL-33, LL-34, LL-35, LL-36, LL-37 or LL-38).

According to a further aspect of the invention there is provided composition of the invention as hereinbefore defined provided that the composition does not further comprise doxycycline, such as doxycycline hydrochloride.

Wherever the word "about" is employed herein, for example in the context of doses of active ingredients, or other quantitative measures/amounts (relative or absolute), it will be appreciated that such variables are approximate and as such may vary by ± 10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the numbers specified herein.

The compositions of the invention may have the advantage that, in the treatment of the conditions mentioned hereinbefore, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compositions known in the prior art for use in the treatment of such conditions or otherwise.

The invention is illustrated by the following examples, in which: Figure 1 is a diagram showing keratinocyte migration in response to stimulation with different concentrations (%) of galactolipid.

Figure 2 is a diagram showing the closure of in vitro wounds of lung epithelial cell monolayer in response to stimulation with different concentrations of galactolipid.

Figure 3 shows the stimulatory effect of digalactosyl diglyceride on keratinocyte migration.

Example 1

Stimulation of Keratinocyte Migration

Human skin primary keratinocytes (culture passage 3-4) obtained from Karocell AB (Stockholm, Sweden) were cultured in serum free culture medium (also from Karocell AB) until reaching 70-90% confluency.

The cells were detached from the culture flask by treatment with 0.05% Trypsin/EDTA. The trypsinization was stopped by adding trypsin inhibitor and basal medium (Karocell AB) and the cells were centrifuged for 7 min at 200xg. The cells were suspended in basal medium and counted in a Bύrker chamber.

Dilutions of galactolipid (CPL-Galactolipid™; LTP AB, Stockholm, Sweden) dispersed in basal medium were prepared and 29 μL added to the lower wells of a 48-well chemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA).

Basal medium was used as negative control and 10 ng/rnL recombinant human TGF-I (Sigma-Aldrich, Stockholm, Sweden) was used as positive control.

A polycarbonate membrane with 8 μm pores was coated with 40 μg/mL collagen I (Sigma-Aldrich, Stockholm, Sweden) in distilled water for 1 h at 37⁰C, rinsed in PBS and air-dried. The membrane was added on top of the lower part of the migration chamber with the stimulating solutions. The chamber was assembled and 50 μL cell suspensions (15,000 cells) in basal medium was added to each well.

After 18 hours incubation at 37⁰C (5% CCb), the chamber was disassembled and the membrane was fixed in 70% ethanol for 30 minutes followed by 10 minutes staining in Gill's hematoxjdin. Non-migrated cells on the upper side of the membrane were removed with a cotton swab.

Cell migration was quantitated by taking 2 photographs (20x magnification) of each well area and analyzing the pictures using ImageJ software. The migrated cell number and the area of migrating cells was estimated by counting stained cell nuclei and total stained cell area (Analyze Particle mode). At least 3 replicate wells of each treatment were analyzed.

The results from one such experiment with different amounts of galactolipid are shown in Figure 1, and demonstrate that galactolipids stimulate keratinocyte migration.

Example 2 Closure of Lung Epithelial Cell Wounds in vitro

NCI-H292 cells (a transformed lung epithelial cell line from ATCC^® (LGC Promochem AB, Boras, Sweden; CRL-1848™) were cultured in RPMI supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM Na-pyruvat and 10% fetal bovine serum (Gibco, Invitrogen, Lidingδ, Sweden). Cells that were 70-90% confluent were detached by trypsinisation (0.25% trypsin with EDTA 4Na IX, Invitrogen, Gibco) for 6 minutes at 37⁰C and resuspended in complete culture medium.

The cells were counted in a Bύrker chamber and 10⁵ cells in 250 μL in complete culture medium were dispensed into the wells of a 48-well plate. When the cells had reached confluence they were serum starved for 24 h in culture medium without serum. The monolayer was wounded with the help of a piece of silicone tubing (outer diameter 4 mm) partly slipped on a 2 mm stainless nozzle to create a uniform cell- free zone in each well. Detached cells were removed by aspiration and rinsing with PBS.

Test substance diluted in serum-free culture medium were added in a total volume of 250 μL to 3-4 wells per condition. As negative control, complete culture medium and as positive control 10 ng/mL of TGF-alpha were used. The plate was incubated at 37⁰C (5% CO₂) for 48 hours with photographic documentation of the wounds at start of the incubation and every 24 hours thereafter.

Images were analyzed using the ImageJ software

(tttp://rsb.info.nm.gov/ij/index.html) freehand drawing tool to enclose the area of the wound and get its total area. The results from one such experiment with different amounts of galactolipid are shown in Figure 2, which shows that galactolipid stimulates the closure of lung epithelial cell monolayer wounds.

Example 3 Stimulation of keratinocvte migration by digalactosyl diglyceride galactolipid

Human skin primary keratinocytes (passage 3-4) obtained from Karocell AB were cultured in serum free culture medium (Karocell AB) until reaching 70-90% confiuency. The cells were detached from the culture flask by treatment with 0.05% Trypsin/EDTA. The trypsinization was stopped by adding trypsin inhibitor and basal medium (Karocell AB) and the cells were centrifuged for 7 min at 200xg. The cells were suspended in basal medium and counted in a Bύrker chamber. Dilutions of digalactosyl diglyceride galactolipid (DGDG) from whole wheat, (>95% pure, Sigma Aldrich, catalogue no. D4651, Schnelldorf, Germany) dissolved at 2 mg/ml in 50% ethanol and diluted in basal medium were prepared and 29 μl added to the lower wells of a 48-well chemotaxis chamber (Neuroprobe). Basal medium was used as negative control and 10 ng/ml recombinant human TGF-alpha (Sigma- Aldrich)- as positive control. A polycarbonate membrane with 8 μm pores was coated with 40 μg/ml collagen I (Sigma-Aldrich) in distilled water for 1 h at 37°C, rinsed in PBS and air-dried. The membrane was added on top of the lower part of the migration chamber with the stimulating solutions. The chamber was assembled and 50 μl cell suspensions (9.000 cells) in basal medium was added to each well. After 6 h of incubation at 37⁰C, 5% CO₂, the chamber was disassembled and the membrane was fixed in 70% ethanol for 30 min followed by 10 min staining in Gill's hematoxylin. Non- migrated cells on the upper side of the membrane were removed with a cotton swab. Cell migration was quantitated by taking a photograph (10x magnification) of each well area and analyzing the pictures using ImageJ software. The migrated cell number and the area of migrating cells was determined by counting stained cell nuclei and total stained cell area. At least 2 replicate wells of each treatment were analyzed. The results from one such experiment with different amounts of digalactosyl diglyceride are shown in Fig. 3. As appears therefrom, digalactosyl diglyceride stimulates keratinocyte migration.

Claims

1. The use of a galactolipid as an active ingredient for the manufacture of a medicament for the treatment of a condition that ma3' benefit from activation of cells.

2. The use of a galactolipid as an active ingredient for the treatment of a condition that may benefit from activation of cells.

3. A method of treatment of a condition that may benefit from activation of- cells, which method comprises the administration of a galactolipid to a patient in need of that treatment.

4. The use as claimed in Claim 1 or 2, or the method as claimed in Claim 3, wherein the cells are endothelial cells or epithelial cells.

5. The use or method as claimed in Claim 4, wherein the cells are epithelial cells.

6. The use or method as claimed in Claim 5 wherein the epithelial cells are keratinocytes.

7. The use or method as claimed in any one of the preceding claims, wherein the activation comprises proliferation and/or migration of cells.

8. The use or method as claimed in Claim 7, wherein the activation comprises migration.

9. The use of a galactolipid as an active ingredient for the manufacture of a medicament for the regeneration and/or repair of tissue in a patient.

10. A method for the regeneration and/or repair of tissue in a patient, which method comprises the administration of a galactolipid to a patient in need of that regeneration and/or repair.

11. The use as claimed in Claim 9, or the method as claimed in Claim 10, wherein the tissue is endothelial tissue or epithelial tissue.

12. The use or method as claimed in Claim 11, wherein the tissue is epithelial tissue.

13. A use or method as claimed in any one of the preceding claims, wherein the galactolipid is or comprises digalactosyldiacylglycerol.

14. A use or method as claimed in any one of the preceding claims, wherein the galactolipid is in the form of a material or composition which comprises at least

20% by weight of galactolipids, for example at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.9% by weight of galactolipids.

15. A use^' or method as claimed in any one of Claims 1 to 13, wherein the galactolipid is in the form of a substantially pure galactolipid preparation.

16. A use or method as claimed in any one of Claims 1 to 15, wherein the galactolipid is CPL-Galactolipid™.

17. A use or method as claimed in any one of the preceding claims, wherein the galactolipid is used or administered as a formulation or composition.

18. A use or method as claimed in Claim 17, wherein the amount of galactolipid in the formulation or composition is less than about 50% (w/w).

19. A use or method as claimed in Claim 18, wherein the amount is less than about 30% (w/w).

20. A use or method as claimed in Claim 19, wherein the amount is less than about 10%.

21. A use or method as claimed in Claim 20, wherein the amount is less than about 3%.

22. A combination product comprising:

(a) a galactolipid material; and

(b) another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells

23. A combination product as claimed in Claim 22, which comprises a pharmaceutical formulation including a galactolipid material; another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells; and a pharmaceutically-acceptable adjuvant, diluent or carrier.

24. A combination product as claimed in Claim 22, which comprises a kit of parts comprising components:

(a) a pharmaceutical formulation including a galactolipid material in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and

(b) a pharmaceutical formulation including another active ingredient that is useful in the treatment of a condition that may benefit from activation of cells, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

25. A combination product as claimed in any one of Claims 22 to 24, wherein the cells are epithelial cells.

26. A combination product as claimed in Claim 24 or Claim 25 (as dependent on Claim 24), wherein components (a) and (b) are suitable for sequential, separate and/or simultaneous use in the treatment of a condition that may benefit from activation of cells.

27. A use, method or combination product as claimed in an}' one of Claims 1 to 21 or 26 (as appropriate), wherein the condition is a wound or epithelial damage.

28. A use, method or kit as claimed in Claim 27 wherein the wound is a skin wound.

29. A use, method or kit as claimed in Claim 27 or Claim 28 wherein the wound is a burn wound.

30. A use, method or kit as claimed in any one of Claims 27 to 29 wherein the wound is a non-healing wound.

31. A use, method or kit as claimed in any one of Claims 27 to 30 wherein the wound is an ulcer.

32. A use, method or kit as claimed in Claim 31, wherein the ulcer is of the corneal epithelium.