The present invention in a first aspect relates to the use of an agent which modulates the inflammatory activation of blood platelets in the treatment of chronic or recalcitrant skin damage and in a second aspect relates to a formulation for the prevention and treatment of skin damage comprising an agent which modulates the inflammatory activation of blood platelets.

Treatment of chronic or recalcitrant skin damage is a difficult medical problem which requires a significant amount of health care resources. In the Western world the most prevalent form of chronic or recalcitrant skin damage is leg ulceration caused by venous insufficiency. Most current treatment methods are dressing based and either tend to focus on treating the symptoms of the ulcer or rely on the accepted wisdom that ulcers are inactive and require a stimulant to trigger healing. As neither approach is completely effective, the healing of chronic ulcers remains a problem for both patient and health care professional.

We have now found that the problems of chronic or recalcitrant skin damage repair may be mitigated by the use of an agent which modulates the inflammatory activation of blood platelets.

In the past it has been suggested that venous ulceration can be treated by inhibiting free radicals with

scavengers. ("Role of the leucocyte in the pathogenesis of vascular disease" Br. J. Surg. 1993, Vol 80, December 1503-1512 Bradbury et al.). This approach however suffers from the disadvantage that free radical inhibition can only be used in a later stage of the cycle leading to tissue breakdown where many processes are believed to be active. It has also been recognised that by the action of reactive oxygen metabolites, pressure wounds may have elevated levels of thromboxane receptors. ("Cellular activation of thromboxane receptors" Ann. NY Acad. Sci. 1994, 714, 270-278, Kinsella, B.T. et al. , and "Antiaggregatory activity of 8-epi-prostaglandin F _2α and other F-series prostanoids and their binding to thromboxane A ₂ prostaglandin H ₂ receptors in human platelets" J. Pharmacol. Exp. Therap. 1994 270, 1192-1196 Yin et al. It has been suggested that venous leg ulceration can be treated with aspirin. ("Randomised trial of oral aspirin for chronic venous leg ulcers" The Lancet, July 1994, 344, 164, Layton et al.) . Aspirin however does not block all the biochemical pathways to thromboxane receptor activation. We believe these targets are therefore less effective in treating and/or curing of chronic or recalcitrant skin damage.

US 4 925 873 (Friedhoff/E.R. Squibb and Sons) discloses the use of thromboxane A ₂ receptor antagonists in the treatment of skin injuries. Thromboxane A ₂ is known to play a role in ischaemic tissue damage and the use of antagonists in the treatment of sores with a non-systemic cause such as pressure sores is suggested by this reference.

We have now found that the problems of chronic or recalcitrant skin damage repair may be mitigated by the use of pharmaceutically active agents which modulate the inflammatory activation of blood platelets in blood vessels.

While not wishing to be bound by theory the present inventors believe that chronic or recalcitrant skin damage sites, far from being inactive, are highly active tissues. It is believed that cycles of ischaemia and reperfusion or peripheral venous hypertension lead to recurring microvascular damage. These damaged sites give rise to an interplay between platelet activation, leucocyte activation and endothelial damage and activation. This then causes a cascade of reactions leading to tissue breakdown and further vascular activation leading to non-healing of the damage. The present invention seeks to inhibit the vascular activation, break the cycle and allow repair of the skin damage.

Accordingly the invention provides in a first aspect the use of agent which modulates the inflammatory activation of blood platelets in the preparation of a medicament for use in the prevention and treatment of chronic lower limb ulceration. In a second aspect the invention provides a formulation for the treatment of chronic or recalcitrant skin damage comprising a thromboxane receptor antagonist in a pharmaceutically acceptable excipient.

In the context of the present invention the term chronic

lower limb ulceration includes leg ulcers, arterial ulcers, venous ulcers, diabetic ulcers, neuropathic ulcers and ulcers of mixed etiology.

Preferred agents which modulate the inflammatory activation of blood platelets for use in the present invention are thromboxane A ₂ receptor antagonists which are specific inhibitors of the actions of thromboxane A ₂ and therefore produce the effect of inhibition without causing other non-specific effects that may be undesirable. Examples of such thromboxane A ₂ receptor antagonists suitable for use herein include but are not limited to the 7-oxabicycloheptane and 7-oxabicyclo- heptene compounds disclosed in U.S. Pat. No. 4,537,981 to Snitman et al, especially (IS- [la2β(5Z) , 3β(lE,3R,4S) ,4 ] ] -7- [3- (3-hydroxy-4-phenyl-l-pentenyl) -7-oxabicyclo- [2.2.l]hept-2-yl] -5-heptenoic acid; the 7-oxabicyclo- heptane substituted amino-prostaglandin analogs disclosed in U.S. Pat. No. 4,416,896 to Nakane et al. , especially [IS- [la,2β(5Z) , 3β,4 ] ] -7- [3- [ [2- (phenylamino) carbonyllhydrazino]methyl] -7-oxabicyclo[2.2.1]hept-2yl] - 5-heptenoic acid; the 7-oxabicycloheptane substituted diamide prostaglandin analogs disclosed in U.S. Pat, No. 4,663,336 to Nakane et al. , and [IS- [l ,2β(Z) ,3β,4 ] ] -7- [3- [ [ [ [ (4-cyclohexyl-l-oxobutyl)amino]acetyl]amino] - methyl] -7-oxabicyclo[2.2.1.]hept-2-yl] -5-heptenoic acid; the phenoxyalkyl carboxylic acids disclosed in U.S. Pat, No. 4,258,058 to Witte et al. , the sulphonamidophenyl carboxylic acids disclosed in U.S. Pat. No. 4,443,477 to Witte et al. , and the arylthioalkylphenyl carboxylic acids disclosed in U.S. Pat. No. 4,752,676 especially 4-

(3- ( (4-chlorophenyl) sulfonyl)propyl)benzeneacetic acid.

Other examples of thromboxane A ₂ inhibitors suitable for use herein include, but are not limited to 3-[l-(4- chlorophenyl methyl) -5-fluoro-3-methylindol-2yl] -2,2- dimethylpropanoic acid [(L-655240 Merck-Frosst) Eur. J.

Pharmacol. 135(2):193, 17 Mar. 87], 5 (Z) -7- ( [2,4,5, -cis] - 4- (2-hydroxyphenyl) -2-trifluoromethyl-1,3-dioxan-5- yDheptenoic acid (ICI 185282, Brit. J. Pharmacol 90 (Proc. Suppl) :228 P-Abs., Mar. 87), 5 (Z) -7- [2,2-dimethyl- 4-phenyl-l,3-dioxan=cis-5-yl]heptenoic acid (ICI 159995, Brit. J. Pharmacol, 86 (Proc. Suppl) :808 P-Abs., Dec. 85) , N,N' -bis [7- (3-chlorobenzeneaminosulfonyl) -1,2,3,4- tetrahydro-isoquinolyl] disulfonylimide (SKF 88046. Pharmacologist 25(3) :116 Abs, 117 Abs, Aug. 83), [lcx(Z)- 2β,5 ] - (+)-7- [5- [[(l,l'-biphenyl)-4-yl]methoxy] -2- (4- morpholinyl) -3-oxocyclopentyl] -4-heptenoic acid (AH 23848 - Glaxo, Circulation 72(6) :1208, Dec. 85, levallorphan allyl bromide (CM 32,191, Sanofi, Life Sci. 31 (20- 21) :2261, 15 Nov. 82), (Z,2-endo-3-oxo) -7- (3-acetyl-2- bicyclo [2.2.1] heptyl-5-hepta-3Z-enoic acid, 4- phenylthiosemicarbazone (EP092 - Univ. Edinburgh, Brit, J. Pharmacol. 84(3) :595, Mar. 85) .

A particularly preferred agent which modulates the inflammatory activation of blood platelets is the compound [IS- (l ,2 , 3 ,4 ) ] -2- [3- [4 [ (pentylamino) carbonyl] -2-oxazolyl] -7-oxabicyclo [2.2.1] hept-2- yl)methyl]benzenepropanoic acid, also known as ifetroban or BMS 180,291, as disclosed in US 5 100 889, with the following chemical formula:

Ifetroban; BMS 180,291

or a pharmaceutically acceptable salt thereof such as, but not limited to, its sodium salt, potassium salt, calcium salt or magnesium salt.

Other examples of thromboxane A ₂ receptor antagonists suitable for use herein include, but are not limited to the compound referred to as SQ 30,741, [IS- [lβ,2 (5Z) , 3 ,4β] ] -7- [3- [ [ [ [(1-oxoheptyl)amino]acetyl-]amino] methyl] -7-oxabicyclo [2.2.] ] -hept-2-yl] -5-heptenoic acid and the corresponding tetrazole, disclosed in US 4,663,336 with the following formula:

SQ 30,741

the compound known as BAY u3405 (Adv. Prostaglandin Thromboxane Leukotr. Res. 20: 110, 1990) with the following formula:

BAY U3405

the compound known as ONO 3,708 (Adv. Prostaglandin Thromboxane Leukotr. Res. 17:799, 1987) with the following chemical formula:

ONO 3 , 708

the compound known as GR 32,191 (Circulation 81:142, 1990), also known as vapiprost, with the following chemical formula:

GR 32,191; Vapiprost

the compound known as R-68, 070 referred to above, also known as ridogrel (Janssen Research Laboratories), (E)-5- [ [ [ (pyridinyl) [3- (trifluoromethyl)phenyl]methylene] amino] oxy]pentanoic acid with the following chemical formula:

R-68,070; Ridogrel

the compound known as BM 13,177 (Cardiovasc. Drug Rev. 6:20, 1988), also known as SKF 95,587 or sulotroban, 4- [2- (benzenesulfamido)ethyl]phenoxyacetic with the follow¬ ing chemical formula:

BM 13 , 177 ; SKF 95 , 587 ; Sulotroban

the compound known as BM 13,505 (Drugs of the Future 13:999, 1988), also known as SKF 96,148 or daltroban, 4- [2- (4-chlorobenzenesulfonamido)ethyl] -phenylacetic acid, with the following chemical formula:

BM 13,505; SKF 96,148; Daltroban

Further examples of thromboxane A ₂ receptor antagonists suitable for use herein include, but are not limited to, sodium (E) -11- [2- (5,6-dimethyl-1-benzimidazolyl) - ethylidene] -6,11-dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate (KW-3635, Arch. Int. Pharmacodyn. Therap. 323:32, 1993), 3-carbamyl- (3 ^• -picolyl) -4- methoxylbenzamide (G 619, Pharmacol. 47:167, 1993) and its structurally related compound known as picotamide (Biochem. Biophys. Res. Commun. 100:184, 1981), 3-[l-(4- chlorophenylmethyl) -5-fluoro-3-methylindol-2-yl] -2,2-di- methylpropanoic acid (L-655,240, Eur. J. Pharmacol. 135:193, 1987) and calcium 5 (2) -1R,2S,3S,4S-7- [3- phenylsulfonylaminobicyclo[2,2]hept-2-yl-5-heptenoate hydrate (S-1452, Prostagl. Leukotr. Ess. Fatty Acids 48:343, 1993) .

Although the agent which modulates the inflammatory activation of blood platelets may be administered systemically, such as orally or parenterally in particular to humans, it is preferred that the agent which modulates the inflammatory activation of blood

platelets be administered topically so that it may be delivered through intact or damaged skin.

The topical compositions which may be employed herein will include a topical carrier which may take the form of a cream, lotion, gel, film, fibrous material, patches, hydrocolloid wound treatment agent, liquid, powder, aerosol or the like.

When the agent which modulates the inflammatory activation of blood platelets is a thromboxane A ₂ receptor antagonist, the topical formulation will comprise from about 0.001 to 10%, preferably 0.001 to 5% by weight of the antagonist. The formulation may be administered within the dosage range of about 0.1 to about lOOmg/kg, preferably about 0.2 to about 50mg/kg on a regimen in single or two to four divided daily dosages.

The systemic compositions suitable for use herein include formulations which may take the form of tablets or capsules for oral delivery, elixirs, suppositories, nasal sprays and injectable solutions. The above dosage forms will include the necessary carrier material, excipient, lubricant, buffer, antibacterial, bulking agent, or the like. With such systemic formulations, single or divided doses of from about lmg to about 2500mg, preferably from about 10 to 2000 mg up to one to four times a day in systemic dosage forms as described above for a period sufficient to facilitate healing and repair skin damage.

The compositions of the present invention may also be in the form of creams, lotions or gels and preferably comprise a gelling agent. Suitable gelling agents are natural gums such as tragacanth, carrageen, pectin, gelatin, guar gum, locust bean gum, gum karaya, collagen, agar and alginic acid; semi-synthetic materials such as sodium, calcium or other alkali metal or alkaline earth metal salt celluloses or alginates such as methyl cellulose, hydroxyethyl cellulose, hydroxy-propylmethyl cellulose, carboxymethyl cellulose and sodium carboxymethyl cellulose and the synthetic polymer Carbopol.

The following Examples represent preferred embodiments of the present invention.

Example 1

The delivery of a thromboxane receptor antagonist formulation suitable for topical administration to damaged skin was studied in vi tro as follows.

Human whole skin (epidermis plus dermis) , dermal membranes and equivalent porcine skin from the abdomen were used in in vi tro percutaneous absorption studies. 2 cm diameter skin membranes were punched out from the tissue and individually placed onto a receptor chamber, containing a small bar magnet. Formulations A or B were applied to the skin surface, a donor chamber attached and the whole cell clamped together and placed onto individually sited positions in a submerged 15 place magnetic stirrer placed in a plexiglass water bath

maintained at 32°C, skin temperature.

Formulation A: Ifetroban dissolved in distilled water to a concentration of lmg/ml. Formulation B: 3.4% Sodium carboxymethylcellulose 0.1% Pectin

15.0% Propylene Glycol 81.4% Purified Water BP 0.1% Ifetroban At timed intervals samples were removed and analysed by either HPLC or, if radiolabelled, by scintillation counting and permeation coefficients calculated.

From the coefficients it is apparent that Ifetroban permeates intact skin at an average rate, that is around 1 x 10" cm/hr. The dermal coefficients show that

Ifetroban permeates through human dermis at a reasonably rapid rate, that is around 53 x 10" cm/hr (Formulation B) and 87 x 10" cm/hr (Formulation A) . The coefficient for 6 day old porcine wounds shows a rate of 125 x 10" cm/hr (Formulation A) . Dissolution studies demonstrated that drug release from the gel formulation was almost bi- phasic with 60% of the drug being released over 50 hours, the remaining drug being released at a much slower rate. This gives the advantage of sustained release of the drug over several days.

Examples 2 and 3

A formulation for treatment of chronic or recalcitrant skin damage by oral administration comprising an agent which modulates the inflammatory activation of blood

platelets in a pharmaceutically acceptable excipient is set out below. Capsules containing 50mg of the thromboxane receptor A ₂ antagonist (Example 2) or lOOmg of the thromboxane receptor A ₂ antagonist (Example 3) were produced from the following ingredients expressed in quantity per capsule:

Formulation Example 2 Example 3

BMS 180291 52.50 105.0

Mannito1 ca. 75.30 ca 150.60

Microcrystalline Cellulose 12.00 24.00 Crospovidone 4.50 9.00

Magnesium Oxide 3.00 6.00

Colloidal Silicon Dioxide 0.45 0.90

Magnesium stearate ca 2.25 ca. 4.50

Grey Opaque Hard Gelatin Capsule 1 piece 1 piece Size #0

BMS 180291 is [IS- (lα,2 ,3α,4α) ] -2- [3- [4 [ (pentylamino) carbonyl] -2-oxazolyl] -7-oxabicyclo[2.2.l]hept-2- yl)methyl]benzenepropanoic acid produced by the method described in US 5 100 889.

Mannitol is a common sugar alcohol which was used along with microcrystalline cellulose as a diluent. Crospovidone is a synthetic cross-linked homopolymer of povidone and was used as a disintegrant. Magnesium oxide was used as an alkalising agent. Colloidal silicone dioxide was used as a glidant. Magnesium stearate acted as a lubricant.

The ingredients were mixed together by a common dry granulation process to form granules which were then used

to fill the capsules. Exam les 4 to 9

Formulations for treatment of chronic or recalcitrant skin damage by topical administration in the form of a gel comprising an agent which modulates the inflammatory activation of blood platelets in a pharmaceutically acceptable excipient are set out below.

Ingredient Example % by weight

4 5 6 7 8 9 sodium carboxymethyl 3.4 3.4 cellulose (Courose ex

Courtaulds) carboxymethylcellulose 3.4

(Blanose ex Hercules) hydroxyehthylcellulose

(Natrasol™ ex

Hercules)

Hydroxypropylmethyl cellulose (Benecel™ ex

Hercules) acrylic acid 0.8 copolymer(Carbopol 980 ex Goodrich)

Pectin 0.1

Propylene Glycol 15 15 15 15 15 15

Triethanolamine 0.8

BMS 180,291 1 1 1 1 1 1

Water 80.5 80.6 80.6 81 81 74.4

The formulation of Example 4 was prepared by dissolving pectin in water at a maximum temperature of 60°C followed by dissolving the BMS 180,291 in the pectin solution. Carboxymethyl cellulose was then dispersed in the propylene glycol and added to the pectin/BMS 180,291 solution with rapid mixing. The mixture was then stirred at a reduced rate until a homogeneous gel was formed.

Examples 5 to 8 were prepared by dissolving the BMS 180,291 in water. The carboxymethyl cellulose was dispersed in the propylene glycol and the dispersion added to the solution with rapid mixing. The mixture was then stirred at a reduced rate until a homogenous gel was formed.

Example 9 was prepared by dissolving the BMS 180,291 in water. With rapid stirring the Carbopol 980 was slowly added and at a reduced rate the propylene glycol was added and mixed until homogeneous. The triethanolamine was slowly added until full neutralisation was indicated by pH measurement.

Example 10

A formulation for treatment of chronic or recalcitrant skin damage by transdermal administration comprising an agent which modulates the inflammatory activation of blood platelets in a pharmaceutically acceptable

excipient is set out below.

Ingredient % by weight

Vistanex LMMH (a polyisobutylene ex 40

Exxon)

Carboxymethyl Cellulose (Blanose) 19.667

Pectin 19.667

Gelatin 19.667

BMS 180,291 1

The transdermal formulation was prepared by heating the Vistanex in a Brabender mixer to a temperature of approximately 120°C. On cooling to about 80°C a premixture of BMS 180,291 and carboxymethyl cellulose was added to the Vistanex. The resulting mass was extruded between silicone release papers and a polythene backing to a thickness of less than 1mm.

Example 11

A further transdermal formulation is set out below.

Ingredient % by weight

Kraton D1107 (a synthetic rubber ex 6.25

Hercules)

Butyl rubber 16.9

Vistanex LMMH 8.3

Mineral oil 7.8

Pentalyn H (a tackifier ex Hercules) 13.3

Irganox 1010 (an antioxidant ex Hercules) 0.5

Carboxymethylcellulose 15.32

Pectin 15.32

Gelatin 15.32 BMS 180,291 1

The transdermal formulation was prepared by heating the Vistanex, butyl rubber, Kraton and Irgaonox in a Brabender mixer at about 120°C until thorough mixing was achieved. As the mix cooled the Pentalyn H and mineral oil were added. At a maximum temperature of 80°C a premixture of BMS 180,291 and carboxymethylcellulose was added to the mix and the resultant mass extruded between silicone release backing and polyethylene to a thickness of less than 1mm.

Example 12

A further transdermal formulation is set out below.

Ingredient % by weight

Carboxymethyl cellulose 37.88

Pectin 16.83

Vistanex LMMH 39.69 Mineral Oil 1.35

Butylated Hydroxy Toluene 0.08

Piccolyte Resin (synthetic 3.17 resin)

BMS 180,291 1

The formulation was prepared substantially as above by mixing the Vistanex in a Brabender mixer to about 120°C and then adding the piccolyte resin with continual mixing. To this mixture butylated hydroxy toluene, mineral oil, pectin and finally BMS 180,291 premixed with carboxymethylcellulose were added. The resulting mass was extruded between silicone release paper and polythene backing to a thickness of less than lmm.

In Examples 10,11 and 12 the quantity of BMS 180,291 can be increased to up to 5%.

Example 13

A thin bioabsorbable film formulation for the delivery of a thromboxane receptor antagonist to the wound site is shown below.

Ingredient by weight Carboxymethyl cellulose 15g

Water 185ml

BMS 180,291 15mg

The film was prepared by adding BMS 180,291 to the water to form a solution to which carboxymethylcellulose was added with vigourous stirring to form a homogenous gel. The gel was then coated in a lmm layer onto MeIanex, a polyester film ex ICI and dried at 40°C in an oven for 30 minutes followed by drying at ambient temperature.

Example 14

A thin bioabsorbable film for delivery of BMS 180,291 to a wound site has the following formulation.

Ingredient Part by weight

Sodium alginate 16g

Water 184ml

BMS 180,291 15mg

The film was prepared by adding the BMS 180,291 to the water to form a solution to which was added the sodium alginate with rapid mixing until a homogeneous gel was formed. The resulting gel was coated at lmm thickness onto Melanex and dried as in Example 13.

Example 15

The performance of a thromboxane receptor antagonist formulation suitable for oral administration to study protection against ischaemic skin damage was studied in vivo in a porcine flap wound model similar to one described in "Pharmacologic Intervention of Skin Vasospasm and Ischemic Necrosis in Pigs" Journal of Cardiovascular Pharmacology 21:163-171 (1993), Pang,C H et al. Raven Press Ltd New York. An ischaemia model was used because chronic wounds only naturally occur in humans. Past study of ischaemic models has suggested that a possible mechanism of tissue injury is a thromboxane dependent activation of the inflammatory

response. This is one of the activation mechanisms that we believe is predominant in the pathophysiology of human chronic wounds.

The wound model measures the degree of tissue damage caused by surgically-applied skin ischaemia. Four

Monopedicled skin flaps measuring 10cm (length) x 4 cm (width) were raised to the level of the deep fascia on the dorsum of each young pig under anaesthesia. The flaps were returned to their original positions, held in place by sutures and dressed with non-adherent dressings and gauze swabs. The surgical procedure induced tissue ischaemia at the distal end of the flaps due to severance of some of the blood vessels supplying the area. Reduction of blood flow was confirmed by laser-Doppler flowmetry measurements along the skin flaps. Ischaemia resulted in areas of tissue damage in the flaps as indicated by zones of tissue colouration originating from the distal ends. The extent of tissue damage was observed at various time points by recording the dimensions of the damaged tissue. Ten pigs were assigned to a control group ( Group C ) and eight pigs were divided between two treatment groups ( Groups A and B ) . Group A received an oral administration (Formulation A) comprising 3mg/kg of BMS 180,291 and Group B received an oral administration (Formulation B) comprising lOmg/kg of BMS 180,291. Group C received no treatment. The doses were administered daily for 11 days between the hours of 0830 and 0930. The percentage of tissue damage was calculated from the areas of tissue colouration indicating necrotic tissue damage.

The results are presented in the following table and show that BMS 180,291 significantly protects against ischaemia-induced skin damage in this wound model.

Treatment % Tissue Damage (± SE) Control 23.74 ± 3.12

Groups A + B (Pooled*) 12.46 ± 3.48 ^♦ Data from groups A and B were pooled because there were no statistical differences between the two groups in terms of % tissue damage)

Ingredient Formualtion A Formulation B mg/kg mg/kg

BMS 180,291 3 10 water balance balance