EP1345601A2 - 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 - Google Patents

Abstract

There is described the use of 5-hydroxy and 5-acyloxy-2,3-dihydro-4,6,7-trimethyl-2-benzofuranacetic acids, including Raxofelast 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

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.