USE OF 5-HYDROXY AND 5-ACYLOXY-2,3-DIHYDRO-4,6,7-TRIMETHYL-2-BENZOFURANACETIC ACIDS AND THEIR ESTERS AS AGENTS USEFUL FOR HEALING SKIN LESIONS AND STIMULANTS OF ANGIOGENESIS

Abstract: There is described the use of 5-hydroxy and 5-acyloxy-2,3-dihydro-4,6,7-trimethyl-2-benzofuranacetic acids and their esters and pharmaceutical compositions that contain them as active ingredients capable of favouring skin repair processes and stimulating angiogenesis.

Description: USE OF 5-HYDROXY AND 5-ACYLOXY-2,3-DIHYDRO-4,6,7-TRIMETHYL-2- BENZOFURANACETIC ACIDS AND THEIR ESTERS AS AGENTS USEFUL FOR HEALING SKIN LESIONS AND STIMULANTS OF ANGIOGENESIS The present invention refers to the use of 5-hydroxy and 5-acyloxy-2,3-dihydro-4,6,7- trimemyl-2-benzofuranacetic acids and their esters and pharmaceutical compositions that contain them as active ingredients capable of favouring skin repair processes and stimulating angiogenesis. These active ingredients are therefore useful in the treatment of ulcerative lesions of the skin, particularly in patients with defects in skin repair processes, such as diabetic patients. The healing of skin lesions is a complex sequence of cellular and molecular processes that include inflammation, cell migration, angiogenesis, matrix synthesis, collagen deposition and reepithelization. The healing process requires a sophisticated interaction among inflammatory cells, biochemical mediators, extracellular matrix molecules and microenvironmental cell populations affected by the lesion. Vascular endothelial growth factor (VEGF) is one of the most powerful angiogenic cytokines, which promotes all steps in the cascade process of angiogenesis; in particular, it induces degeneration of the extracellular matrix of existing vessels by proteases, causes migration and proliferation of capillary endothelial cells, and determines tube proliferation of endothelial cells. VEGF action is associated with a variety of physiological and pathological neovascular events, such as embryonic development, tumour growth and, in particular, wound repair. Experimental evidence has in fact demonstrated expression of VEGF and its receptors during wound healing processes. Impaired wound healing may be a consequence of normal aging, therapeutic intervention, or metabolic derangements such as diabetes. As a matter of fact, the healing of skin lesions is markedly delayed in diabetic patients and in experimental models of diabetes: healing impairment is characterised by delayed cellular infiltration and granulation tissue formation, reduced angiogenesis, modification of capillary permeability, decreased collagen and alteration of its metabolism. In turn, these modifications are thought to be the result of local ischemic conditions which causes an increased production of free radicals and other reactive metabolites. Today there are still no specific treatments recognised to be effective on chronic ulcers caused by tissue hypoxia secondary to an abnormal distribution of pressure and, in some cases, associated with peripheral vascular insufficiency, such as ulcers in diabetic patients' feet. A significant proportion of these patients is therefore likely to suffer gangrenous degeneration of the lesions and consequently, amputation of the limb. Another significant problem is represented by lesions associated with prolonged stasis (bed sores), thromboangiitis obliterans (Buerger's disease) or Raynaud's disease, in which similar mechanisms occur: also in this case, the commonly used treatment simply relieves the pain symptoms and blocks the development and spreading of infections. The document US 4,999,350 describes 5-hydroxy and 5-acyloxy-2,3-dihydro-4,6,7- trimethyl-2-benzofuranacetic acids and their esters, defined by the general formula (I) wherein R is hydrogen, C1-C7 acyl, bicarboxylic acid hemiacyl; Rl is hydrogen, C1-C20 alkyl, alkyl ether of the -CHR2-(CH2)n-O-(CH2)m type where R2 is a lower alkyl and n and m range from 0 to 6, alkyl diether of the -(CH2)n-0-(CH2)m-O-(CH2)p-CH3 type where n and m range from 1 to 6 and p ranges from 0 to 6, alkylamine of the CHR2- (CH2)n-NR3R4 type where n ranges from 0 to 6, R2 has the above meaning and R3 and R4 are lower alkyls, N-alkyl heterocycle of the type: wherein X is CH2, S, O, NR2 (where R2 has the above meaning), n ranges from 1 to 6, nl and n2 range from 1 to 3; and their pharmaceutically acceptable salts. These compounds, and in particular (±)-5-acetoxy-2,3-dihydro-4,6,7-trimethyl-2- benzofuranacetic acid (raxofelast) and its active metabolite (±)-2,3-dihydro-5-hydroxy- 4,6,7-trimethyl-2-benzofuranacetic acid, have antioxidizing and lipid peroxidation inhibiting properties, as described by A. Bindoli et al. in Pharmacol. Res. 1991, 24, 369- 375, and have also been found to have anti-ischemic and anti-inflammatory activities (see for example: G.M. Campo et al., Res. Commun. Chem. Pathol. Pharmacol. 1992, 76, 287-303 and S. Cuzzocrea et al, Brit. J. Pharmacol. 1999, 126, 407-414). The document EP 848,953 describes the use of the same family of compounds in the prevention and treatment of chronic complications of diabetes mellitus, particularly micro- and macroangiopathies, while PJ. Chowienczyk et al. (Diabetologia 2000, 43, 974-977) have demonstrated the action of raxofelast in restoring endothelium-dependent vasodilation in patients with non-insulin-dependent diabetes. In the present invention it was surprisingly discovered that the substances having formula (I) as defined above are capable of restoring the skin repair processes when these processes are impaired as a result of metabolic dysfunctions such as those occurring in diabetes. The compounds of formula (I) may therefore be used in therapy to speed up the wound healing process and for the treatment of ulcers in the extremities of patients affected by insulin-dependent and non-insulin-dependent diabetes mellitus. The present invention therefore refers to the systemic and topical use of a compound of formula (I) as defined above as an active ingredient for the preparation of medical products suitable for accelerating the healing of skin lesions, the treatment of diabetic foot ulcers, the treatment of bed sores and ulcers in patients suffering from thromboangiitis obliterans (Buerger's disease) or Reynaud's disease; and the prevention of gangrene in patients suffering from ulcers in their extremities. Particularly preferred is the systemic use of the compound of formula (I) wherein R is acetyl and Rl is hydrogen (raxofelast) and the topical use of the compound of formula (I) wherein R and Rl are both hydrogen for the same purposes as those mentioned previously. According to the present invention, the compounds of formula (I) can be prepared using the methods described in the literature for 2,3-dihydro-5-benzofuranol derivatives, and in particular as described in US 5,041,568. The compound of formula (I) where R is acetyl and Rl is hydrogen (raxofelast) can be prepared, for example, through cyclization in an alkaline aqueous environment of a suitable derivative of 4-(2,5-dihydroxy-3,4,6- trimethylphenyl)-(£)-2-butenoic acid followed by (9-acetylation of the resulting (±)-2,3- dihydro-5-hydroxy-4,6,7-trimethyl-2-benzofuranacetic acid. For all therapeutic uses of the present invention, the compounds in the formula (I) can be administered topically, orally, parenterally, intralesionally, transcutaneously or transmucosally, rectally or by inhalation in formulations that contain them as active ingredients at a therapeutically effective dose with non-toxic conventional pharmaceutical excipients. The term "parenteral" used herein comprises subcutaneous, intramuscular and intravenous injections. The pharmaceutical compositions for oral use include tablets and capsules in which the active ingredient is mixed with pharmaceutically acceptable, non-toxic excipients. These excipients may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating or dispersing agents, such as corn starch or alginic acid; binding agents such as starch or gelatin; lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or coated with state-of-the-art conventional techniques, to delay disintegration and absorption in the gastrointestinal tract so as to obtain a delayed and prolonged action. Aqueous suspensions generally contain the active ingredients mixed with the appropriate excipients. The excipients may be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing and wetting agents. In addition, they may contain one or more preservatives, such as ethyl or n-propyl j9-hydroxybenzoate; one or more flavouring agents; one or more natural or synthetic sweetening agents. Oily suspensions may be formulated by dispersing the active ingredient in a vegetable or mineral oil; they may contain sweetening and flavouring agents to improve the taste of the preparation. Dispersible powders and granules suitable for preparing an aqueous suspension through the addition of water contain the active ingredient in a mixture with the dispersing or wetting agent, a suspending agent and one or more preservatives. The pharmaceutical compositions suitable for the use of the present invention may also be in the form of a W/O emulsion. The oily phase may consist of a vegetable or mineral oil. The emulsifying agents may be natural rubber such as acacia or natural phosphatides, for example, lecithin or esters of natural or synthetic fatty acids. Syrups and elixirs can be formulated with sweetening agents, for example, glycerol, sorbitol or saccharose. Pharmaceutical compositions for topical use such as creams, ointments or gels may also be prepared. For example, to obtain a cream, known methods can be used to emulsify the active ingredients of the present invention with microbiologically pure water, appropriate wetting agents such as sorbitol, glycerin, sugar esters, glycols, with fatty substances such as sweet almond oil, oleic and cetyl alcohol and superior fatty esters. While, by mixing the active ingredients in the present invention with suitable gelling agents such as activated clays, silicon dioxide, mucilage or with suitable fats of vegetable or animal origin and with suitable excipients and preservatives, gels or ointments can be obtained, respectively, using known methods. Pharmaceutical compositions may also be in the form of aqueous or oily sterile injectable solutions or suspensions, formulated in accordance with known methods using non-toxic solvents or diluents suitable for parenteral use. The compounds in the present invention may also be administered rectally in the form of suppositories. These compositions may be prepared by mixing the active ingredient with a suitable non-irritant excipient which is solid at room temperature but liquid at rectal temperature. Polyethylene glycols and cocoa butter are suitable for this purpose. The therapeutically or prophylactically effective amount of a compound in the formula (I) for the use of the present invention will depend on a number of factors which include, for example, the age and weight of the patient, the severity of the specific pathology to be treated, and the route of administration. However, an effective daily quantity of the compound in the formula (I) for the treatment of lesions such as diabetic foot ulcers will generally be within the range 0.1 to 75 mg/kg of body weight, and most often in the range 1 to 25 mg/kg/day. The use of compounds in the general formula (I) according to the present invention is shown in, but not limited to, the following example: EXAMPLE Genetically diabetic female mice (db+/db+) aged 10 weeks and weighing 40-50 g were used. The animals in the control group (db+/+m) weighed 25-32 g. The plasma glucose levels in the two groups were 527 ± 25 mg/dl and 205 ± 19 mg/dl, respectively. The animals were divided into four groups of 21 animals each. The first and second groups, consisting of diabetic and healthy control mice respectively, received raxofelast intraperitoneally at a dose of 15 mg/kg for 12 days. The third group (diabetic mice) and the fourth group (control mice) were treated with vehicle (dimethylsulphoxide/NaCl 0.9% 1:1 v/v) for the same period of time. After general anesthesia and hair shaving, two deep longitudinal incisions (4 cm) were made on the dorsum of each mouse and the wound edges were closed with skin clips placed at 1-cm intervals. Seven animals for each group were then sacrificed after 3, 6 and 12 days, respectively, and the wounds were divided into three segments of 0.8 cm each. The caudal and cranial strips were used for histology, while the central one was used for biochemical and molecular analysis and for wound breaking-strength measurements. Histological Evaluation The samples were fixed in 10% buffered formalin for microscopic examination. After fixation, perpendicular sections to the anterior-posterior axis of the wound were dehydrated with ethanol and embedded in paraffin. Sections 5 μm-thick of paraffin- embedded tissues were then stained with hematoxylin/eosin and with periodic acid Schiff (PAS) and Weigert-Van Gieson's stains. Six slides (two for each stain used) were examined under the microscope by two independent pathologists unaware of the previous treatments. The measured parameters were: re-epithelization, formation of granulation tissue, angiogenesis, infiltrated inflammatory cells. The margins of the wound in each of the sections examined and normal control wounds were used as comparisons for scoring. The histological score was evaluated according to the criteria indicated in the literature for similar experimental models (Arzneimittelforschung 1996, 6, 450-455). Fig. 1 shows the time course of histological score in the four experimental groups. Breaking strength The maximum load (breaking strength) tolerated by wounds was measured blindly using a calibrated tensometer, as described in Science 1987, 237, 1333-1336. The ends of the skin strips were pulled at a constant speed (20 cm/min), and breaking strength was expressed as the mean maximum level of tensile strength before separation of the flaps of wounds. Fig. 2 shows the tensile strength values (g/mm) evaluated on the 12 th day in the wounds of the animals belonging to the four experimental groups. HydroxypiOline Test Hydroxyproline (HP) is considered a reliable indicator of the collagen content of tissues. HP concentration in the wounds was determined using the spectrophotometric method described in Arch. Biochem. Biophys. 1961, 93, 440-447. Fig. 3 shows the time course of HP levels in wound samples of the animals belonging to the four experimental groups. Determination of VEGF levels in wounds The amount of VEGF in wounds was determined by an enzyme-linked immunosorbent assay (ELISA) on the tissue homogenates after centrifugation and microfiltration with the aid of a specific kit. Fig. 4 shows the time course of VEGF levels in the wound samples of the animals belonging to the four experimental groups. The obtained results indicate that genetically diabetic mice showed delayed wound healing (as revealed by histological examination), together with decreased breaking strength and reduced collagen and VEGF wound contents. The administration of raxofelast to diabetic mice significantly improved histological parameters of the cicatrization process, increases the breaking strength and the collagen and VEGF contents.

Claims: CLAIMS 1. Use of the compound in the formula (I) wherein R is hydrogen, C1-C7 acyl, C2-C6 hemiacyl of bicarboxyhc acid; Rl is hydrogen, C1-C20 alkyl, alkyl ether of the -CHR2-(CH2)n-0-(CH2)m type where R2 is a C1-C4 lower alkyl and n and m range from 0 to 6, alkyl diether of the -(CH2)n-O- (CH2)m-0-(CH2)p-CH3 type where n and m range from 1 to 6 and p ranges from 0 to 6, alkylamine of the -CHR2-(CH2)n-NR3R4 type where n ranges from 0 to 6, R2 has the above mentioned meaning and R3 and R4 are C1-C4 lower alkyls, N-alkyl heterocycle of the type wherein X is CH2, S, O, NR2 (where R2 has the meaning above), n ranges from 1 to 6, nl and n2 range from 1 to 3; and their pharmaceutically acceptable salts, for the preparation of a medicinal product useful in therapy for healing skin lesions. 2. Use in accordance with claim 1 of the compound of formula (I) wherein R is acetyl and Rl is hydrogen. 3. Use in accordance with claim 1 of the compound of formula (I) wherein R and Rl are both hydrogen. 4. Use of the compound of formula (I) in accordance with claims 1 to 3 for the treatment of ulcers in the extremities of diabetic patients. 5. Use of the compound of formula (I) in accordance with claims 1 to 3 for the treatment of stasis ulcers. 6. Use of the compound of formula (I) in accordance with claims 1 to 3 for the treatment of ulcers in patients suffering from thromboangiitis obliterans (Buerger's disease). 7. Use of the compound of formula (I) in accordance with claims 1 to 3 for the treatment of ulcers in patients suffering from Reynaud's disease. 8. Pharmaceutical composition for use in accordance with claims 1 to 7 characterised by the fact that it contains 5 to 1500 mg/day of active ingredient together with one or more non- toxic conventional pharmaceutical excipients. 9. Composition in accordance with Claim 8 characterised by the fact that it contains, as an active ingredient, the compound of formula (I) wherein R is acetyl and Rl is hydrogen with suitable pharmaceutically acceptable excipients for systemic use. 10. Composition in accordance with Claim 8 characterised by the fact that it contains, as an active ingredient, the compound of formula (I) wherein R and Rl are both hydrogen with suitable pharmaceutically acceptable excipients for topical use.

Inventors: SQUADRITO FRANCESCO, SQUADRITO, FRANCESCO
Applicants: FOSCAMA BIOMED CHIM FARMA, SQUADRITO FRANCESCO, BIOMEDICA FOSCAMA INDUSTRIA CHIMICO-FARMACEUTICA S.P.A., SQUADRITO, FRANCESCO
Application year: 2001
Main classification: A61K31
IPC codes: A61K31, A61P9, A61P17