WO2003041703A2

WO2003041703A2 - Use of a flavonoid for the treatment of burns

Info

Publication number: WO2003041703A2
Application number: PCT/NL2002/000742
Authority: WO
Inventors: Conradus Ghosal Gho
Original assignee: Gho Holding B.V.
Priority date: 2001-11-16
Filing date: 2002-11-15
Publication date: 2003-05-22
Also published as: WO2003041703A3

Abstract

The present invention is concerned with a new method of treating burns. More particularly the present invention relates to a method of treating a mammal suffering from a burn, which method comprises administering to said mammal a quercetin analogue in a therapeutically effective amount to prevent secondary injury of tissue that was not directly affected by the initial burn.

Description

USE OF A FLAVONOID FOR THE TREATMENT OF BURNS

TECHNICAL FIELD OF THE INVENTION

The present invention is concerned with a new method of treating burns. More particularly the present invention relates to a method of treating a mammal suffering from a burn, which method comprises administering to said mammal a flavonoid in a therapeutically effective amount to prevent secondary injury of tissue that was not directly affected by the initial burn.

BACKGROUND OF THE INVENTION

Free radical damage (oxidative stress) is a very important factor in the early progression of burn related injury. Hydroxyl radicals (OH*) are formed during the initial injury in the tissue that is directly affected by the burn. These radicals will initiate lipid peroxidation in the cell membrane through the so called Fenton reaction. As a result of the lipid peroxidation in the cell membrane, the membrane can no longer respond adequately to signals or cytokines, and apoptosis will follow. Viable tissue that is located directly adjacent to tissue that has suffered initial burn damage, will subsequently become exposed to the hydroxyl radicals from the initial thermal injury. This cascade of direct-indirect injury will cause apoptosis in tissue that was not directly affected by the initial burn. This form of secondary injury is a very detrimental aspect of burn related injuries, particularly in case of second and third degree burns.

Treatments aiming to minimise secondary injury as a result of burns are known in the art. For instance, WO 99/06056 (Gho'st Holding B.V.) describes a method of prophyiactically treating secondary injury of tissue by administering a physiologically acceptable vanadium compound, wherein the secondary injury may result from a traumatic event such as burns. US 4,466,961 (Human Oltoanyagtermeloo es Kutato Intezet) describes a pharmaceutical composition for the treatment of injuries to the skin, particularly burns. The composition is said to relieve pain and to promote healing and is locally administered. The composition comprises 5 to 20 mg of tannic material, 5 to 15 mg of a carbohydrate, 0.5 to 6 mg of an anthocyane and or a flavonone compound and/or pectin, 0.5 to 6 mg of plant wax, and 0.01 to 0.1 mg of volatile oil, balance a C₂to C₄ alcohol, to yield 100 ml of composition.

Meng et et al., "Protective effects of quercetin rutoside, and flavone against inestinal mucosal injury induced by bums in mice", Zhongguo Yaolixue Tongbao (2000), 16(6), 673- 675 report that burn-induced injury of the mouse intestinal mucosa was healed to various extents after the 3 title flavonoids were given intragastrically for 3 days.

EP-A 0 648 496 describes therapeutic compositions, comprising from 0.1 to 5 wt.% of a flavonoid glycoside and 1 to 5 wt.% of a gelling agent for use in the treatment of skin lesions and in particular dermal ulcers, wounds and burns. These therapeutic compositions are designed for topical application. The preferred flavonoid glycoside is rutin.

SUMMARY OF THE INVENTION

The inventors have unexpectedly found that flavonoids may suitably be used to minimise the detrimental secondary impact of bums on tissue that was not directly affected by the initial bum. Although we do not wish to be bound by theory, it is believed that flavonoids are capable of effectively inhibiting the Fenton-reaction that plays a crucial role in the lipid peroxidation in cell membranes as described before. It was found that in particular the group of flavonoids belonging to the quercitine (3,

3', 4', 5, 7-pentahydroxyflavone) family, can successfully be employed in the prophylactic treatment of secondary bum injury.

DETAILED DESCRIPTION OF THE INVENTION

Consequently one aspect of the invention is concerned with a method of treating of burns, wherein the method comprises administering to a mammal suffering from a bum one or more flavonoids in a therapeutically effective amount to prevent secondary injury of tissue that was not directly affected by the initial bum, wherein the one or more flavonoids are represented by the following formula:

and wherein R₅, R₇, Ry and R₄> are independently a hydrogen atom; a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl group having from 1 to 6 carbon atoms; or an acyl group with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms; and

X is a hydrogen atom; a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl group having from 1 to 6 carbon atoms; an acyl group with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms; or a mono-, di- or trisaccharide group which may or may not be substituted with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 6 carbon atoms or an acyl radical with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms.

The present method may suitably be used to treat a wide variety of bums, e.g. bums resulting from exposure to heat, as well as chemical burns and sunburns, particularly in humans. Particularly good results are obtained in case of severe burns, particularly second- or third-degree burn. Best results are obtained when the present method is used to treat mammals that suffer from third degree bums. It is noted that the present method may suitably be employed to treat bums that affect the skin and eyes, as well as internal bums, notably bums within the mouth, oesophagus, stomach, the bronchi and lungs. Most preferably the present method is employed to treat bums of the skin.

Typically the one or more flavonoids are administered in an amount of at least 0.01, preferably of at least 0.1, more preferably of at least 0.5 and most preferably of at least 1.0 mg per kg of bodyweight. Usually the administered amount will not exceed 1000 mg per kg of bodyweight, more preferably it will not exceed 200 mg per kg of bodyweight. In order to obtain particularly good results it was found advantageous to administer the one or more flavonoids in a dose which is equivalent to an intravenous dose of at least 0.01 mg per kg of bodyweight. Here by a dose is meant the amount of the one or more flavonoids that is administered within the duration of 2 hours, preferably within 1 hour. Preferably the one or more flavonoids are administered in a dose which is equivalent to an intravenous dose of between 0.02 and 1000 mg per kg of bodyweight, more preferably equivalent to an intravenous dose of between 0.03 and 200 mg per kg. The present invention encompasses a method of treatment that employs the administration of a single dose, e.g. shortly after the contraction of the bum, as well as a method that employs repeated administration of the one or more flavonoids. In case of repeated administration it is preferred to administer the one or more flavonoids at least once daily, most preferably once daily. Usually the daily dosage of the one or more flavonoids will be equivalent to an intravenous dose of at least 0.1 mg per kg of bodyweight. Normally the daily dosage will not exceed the equivalent of an intravenous dose of 1000 mg per kg bodyweight. Preferably the daily dosage of the one or more flavonoids will be equivalent to an intravenous dose of at least 0.2, more preferably of at least 0.3 and most preferably at least 0.5 mg per kg of bodyweight. In another preferred embodiment the daily dosage will be equivalent to an intravenous dosage of not more than 750 mg, more preferably of not more than 200 mg, most preferably of not more than 100 mg flavonoids per kg bodyweight. As used herein the term "daily dosage" refers to the total amount of flavonoids administered during a day.

The preferred mode of administration is parenteral. Local administration, particularly topical administration was found to be less effective than parenteral administration. Suitable methods of parenteral administration include, intravenous, subcutaneous and intramuscular administration. Most preferably the one or more flavonoids are administered intravenously.

In order to effectively minimise secondary injury as a result of primary bums, it is important that the treatment according to the present method is started as soon as possible after the bums were contracted. Especially if the treatment is started within 24 hours after the mammal contracted the bum, very good results may still be achieved. More preferably the one or more flavonoids are administered within 12 hours, most preferably within 4 hours after the mammal has contracted the bum.

The present invention encompasses the use of a variety of quercetin analogues. These quercetin analogues have in common that they are capable of acting as very effective in vivo radical scavengers. These properties are largely due to the special nature of the unsaturated heterocyclic skeleton that is characteristic of these substances. Quercetin comprises 5 hydroxyl groups wherein the hydrogen atom may suitably be substituted by other (hetero)hydrocarbyl groups without radically affecting the radical scavenging properties of the substance. Naturally, however, such modification will have an impact on solubility and metabolisation of the substance.

It was found that the solubility and stability of the flavonoids may be improved if in the aforementioned formula at least one of R₅, R , Ry and R_4' is a hydroxyl substituted alkyl group. In a preferred embodiment of the invention, R₅, R₇, R₃- and R₄> are independently a hydrogen atom; a saturated or unsaturated, linear or branched, hydroxyl substituted, alkyl group having from 1 to 6 carbon atoms; or an acyl group with a saturated or unsaturated, linear or branched, optionally substituted, alkyl radical having from 1 to 4 carbon atoms; and at least one of R₅, R₇, R₃> and R^ is an alkyl group. In particular with a view to modes administration that require a liquid medication, it was found advantageous to employ quercetin analogues that have relatively high water solubility. In order to achieve this, it is preferred that R₅, R , R₃- and Rψ are independently a hydrogen atom or an alkyl group as defined above, preferably an alkyl group having from 1 to 4 carbon atoms. Even more preferably R₅, R , R₃> and R₄> are independently a hydrogen atom or an alkyl group having from 1 to 3 carbon atoms. The alkyl group most preferably is β- hydroxy ethyl.

The solubility of the present flavonoids is also affected by X in the aforementioned formula. Preferably X is a hydrogen atom; a saturated or unsaturated, linear or branched, hydroxyl substituted, alkyl group having from 1 to 6 carbon atoms; or an acyl group with a saturated or unsaturated, linear or branched, optionally substituted, alkyl radical having from 1 to 4 carbon atoms; or a mono-, di- or trisaccharide group. In case X is an alkyl group, said alkyl group preferably is a saturated, hydroxyl substituted alkyl group having from 1-3 carbon atoms. In an especially preferred embodiment X is a hydrogen atom, an alkyl group or a mono-, di- or trisaccharide group. Most preferably X is β-hydroxy ethyl or a mono-, di- or trisaccharide group.

A naturally occurring flavonoid that is closely related to quercitin is rutoside (quercietin-3-rutinoside), also often referred to as rutin. In effect, rutoside is a quercitin analogue, wherein the hydrogen atom in the hydroxyl group in the 3 -position is replaced by a rutinose (6-O-α-L-rhamnosyl-Dglucose). In a preferred embodiment of the present invention, the one or more flavonoids are quercetin analogues wherein the hydrogen in the hydroxyl group in the 3 -position has been replaced by a mono-, di- or trisaccharide group. More preferably X is a disaccharide group. Most preferably X is rutinose.

The present method is particularly suitable for treating serious bums, i.e. bums which cause more than superficial damage to the (epithilial) tissue. The present method is particularly effective in preventing secondary injury in non-epithelial tissue, especially as a result of exposure to elevated levels of hydroxyl radicals.

Preferably the one or more flavonoids are administered in the form of a solution. In principle a wide variety of pharmaceutically acceptable solvents may be used. Preferably, however, the one or more flavonoids are administered in the form of a composition that contains less than 10 wt.%, preferably less than 5 wt.% of a C₂to C₄ alcohol. The solution that may advantageously be employed in the present method, particularly through intravenous administration, preferably contain the one or more flavonoids in a concentration of at least 1 g/1., preferably in a concentration of between 10 and 500 g/1. In a preferred embodiment of the invention the present method comprises the combined administration of the one or more flavonoids and one or more of the vanadium compounds. It was found that the co-administration of flavonoids and vanadium compounds is surprisingly effective in minimising the detrimental secondary impact of bums. It is believed that this synergistic effect is due to the fact that the flavonoids and the vanadium compounds exert their beneficial effect through clearly different mechanisms that mutually reinforce each other.

Best results are obtained when the vanadium compound is administered via a single dose, preferably intravenously, e.g. via a bolus injection, or orally. Most preferably the vanadium compound is administered intravenously. The vanadium compound is suitably administered in an amount that is equivalent to an intravenous dosage of at least 0.01 mg per kg of bodyweight. Preferably the vanadium compound is administered in an amount that is equivalent to an intravenous dose of at least 0.05, more preferably of at least 0.1 mg per kg of bodyweight. Generally the administered amount of vanadium compound does not exceed the equivalent of an intravenous dosage of 100 mg per kg of bodyweight. Even more preferably, it does not exceed the equivalent of an intravenous dosage of 50 mg per kg of bodyweight.

Suitable vanadium salts are all physiologically acceptable vanadium salts. Examples of such salts are sodium orthovanadate and vanadyl sulphate. Vanadium complexes that can be used are known, physiologically acceptable complexes. Said complexes comprise both vanadyl and vanadium complexes. Complex-forming units that can be used are, for example, maltol and kojic acid. According to the invention maltol is the preferred complex-forming unit, resulting in bis(maltolato)oxovanadium(rV) or the corresponding bis(maltolato)oxovanadate salt. Both the vanadium and vanadyl salts and complexes mentioned above and other suitable vanadium and vanadyl salts and complexes are described in US 5 583 242 and US 5 620967. In principle all salts and complexes mentioned in the said patents can be used. According to the invention the vanadium compound, salt or complex is preferably an organovanadium compound, in particular bis(maltolato)oxovanadium(rV) or the corresponding bis(maltolato)oxovanadate salt. The invention is further illustrated by means of the following examples.

EXAMPLES

Example 1

Experiments are conducted in Dutch landrace pigs (females +/- 30 kg) to determine the effect of monohydroxyethyl rutoside on secondary tissue damage resulting from bums. The Dutch landrace pig is chosen as the experimental animal because of all animal species this domestic pig has morphological and functional skin characteristics nearest to human skin and thus best fulfils the requirements of a model for human skin.

In basic architecture it resembles human skin in the relative thickness of the epidermis and dermis, the presence of epidermal ridges, a distinct dermal papillary layer and a deep layer of subdermal fat. Compared to human the elastic fibre content of porcine dermis is relatively low, but higher than in any other species. Comparison of human and porcine epidermis and its appendages also suggests common traits. Studies on the proliferation rate of porcine epidermis show parallels with those of humans. The keratinous proteins are similar. Unlike the skin of rodents, the foUicular pattern in pigs and humans is relatively sparse and arranged as single hairs or in groups of two or three follicles. Pigs do not sweat. The regulation of body temperature by the skin is more evident in humans than in the pig. In the skin of the pig no eccrine glands are found. It does have apocrine glands, but their role in thermoregulation remains debatable.

The vascular anatomy of pig skin consists of a three layered network; lower, mid-dermal and sub epidermal. The size, orientation and distribution of the vessels are strikingly similar to human skin, but it does differ from humans in that the sub epidermal network is less dense. The vascularisation of the lower region of the follicle corresponds to that in humans. The healing of deep dermal burns, which depends on this phenomenon, is also expected to be analogous. Studies on the thermal properties of porcine skin as a function of depth have been performed by measuring the tissue water content. With the use of a mathematical model the heat capacity and thermal conductivity could be calculated and the results obtained for pig skin were found to be consistent with those for human skin.

The speed of epithelialisation in pigs is dependent on several factors. In full thickness wounds the epithelialisation starts only from the wound margins. In split-thickness wounds, each viable hair follicle is an islet for the reepithelialisation. In split-thickness wounds of 2.2 x 2.2 cm, and 0.04 cm thick, in Yucatan mini pig of six months old it takes about 96 hours before complete re-epithelialisation. The SD of the mean degree of epithelialisation is ± 10%. This shows the inter individual variability. The speed of epithelialisation is depending on the age, and is considerable faster in pigs weighting 7 kg in comparison of those weighting 40 kg. There are no differences in the progression of the epithelialisation in the centre of the wound in comparison with the wound margin in split-thickness wounds.

Protocol la: In 20 pigs twelve identical deep burn wounds (7 cm wide and 7 cm long) are inflicted in each animal. The animal model used was developed by the Burn Research Institute (Beverwijk, The Netherlands). The model is accepted by the animal experimental commission of the University of Amsterdam.

Protocol lb: Twelve biopsies of 0.5 cm wide, 9 cm long and 1 cm deep are taken at regular intervals from a bumed area of each pig during a period of 2 months. Following the infliction of the bums, monohydroxyethyl rutoside is continuously administered intravenously to 12 pigs. The flavonoid is continuously administered as an aqueous solution (240 ml/day) in an amount of 100 mg per kg of bodyweight per day. To the 8 pigs in the control group no flavonoid was administered. The biopsies show that structures present in the dermis such as the deep vascular plexus, the hair-adnexes (sebaceous glands) and sweat glands are effectively protected by the monohydroxyethyl rutoside. Furthermore contractions of scars are considerably reduced and faster healing is observed.

Example 2

Example 1 was repeated with the exception that additionally the pigs received an intravenous dose of 1 mg bis(maltolato)oxovanadium(IV) per kg of body weight immediately after the infliction of the bum for three consecutive days. The results obtained showed even more pronounced reduction of scar contraction. Also the healing rate was even faster than observed in example 1.

Claims

1. Use of one or more flavonoids in the manufacture of a medicament for use in the treatment of burns, wherein the method comprises administering to a mammal suffering from a burn one or more flavonoids in a therapeutically effective amount to prevent secondary injury of tissue that was not directly affected by the initial bum, wherein the one ore more flavonoids are represented by the following formula:

and wherein R₅, R₇, R_3' and R₄> are independently a hydrogen atom; a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl group having from 1 to 6 carbon atoms; or an acyl group with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms; and X is a hydrogen atom; a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl group having from 1 to 6 carbon atoms; an acyl group with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms; or a mono-, di- or trisaccharide group which may or may not be substituted with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 6 carbon atoms or an acyl radical with a saturated or unsaturated, linear, branched or cyclic, optionally substituted, alkyl radical having from 1 to 8 carbon atoms.

2. Use according to claim 1, wherein the method comprises administering the one or more flavonoids to a mammal suffering from second- or third-degree bum.

3. Use according to claim 1 or 2, wherein the one or more flavonoids are administered in a dose which is equivalent to an intravenous dose of at least 0.01 mg/kg of bodyweight.

4. Use according to any one of claims 1-3, wherein the one or more flavonoids are administered parenterally, preferably intravenously.

5. Use according to any one of claims 1-4, wherein the one or more flavonoids are administered within 24 hours, preferably within 12 hours after the mammal has contracted the bum.

6. Use according to any of claims 1-5, wherein R₅, R , R₃> and R_4' are independently a hydrogen atom; a saturated or unsaturated, linear or branched, hydroxyl substituted, alkyl group having from 1 to 6 carbon atoms; or an acyl group with a saturated or unsaturated, linear or branched, optionally substituted, alkyl radical having from 1 to 4 carbon atoms; and at least one of R₅, R₇, R₃- and R^ is an alkyl group.

7. Use according to claim 6, wherein at least one of R₅, R₇, Ry and R^ is an alkyl group with a saturated, linear or branched, hydroxyl substituted, alkyl radical having from 1 to 3 carbon atoms.

8. Use according to claim 6 or 7, wherein R₅, R , R₃> and R_4' are independently a hydrogen atom or an alkyl group.

9. Use according to any one of claims 1-8, wherein X is a mono-, di- or trisaccharide group.

10. Use according to any one of claims 1-9, wherein the method comprises administering the one or more flavonoids in a therapeutically effective amount to prevent secondary injury in non-epithelial tissue, especially as a result of exposure to elevated levels of hydroxyl radicals.

11. Use according to any one of claims 1-10, wherein the method comprises the co- administration of one or more vanadium compounds.