WO2010004367A1

WO2010004367A1 - Use of a mixture of superoxide dismutase and catalase for treating pruritus and alleviating its symptoms

Info

Publication number: WO2010004367A1
Application number: PCT/IB2008/052791
Authority: WO
Inventors: Silvia Cristina Chami De Diehl; Christian Diehl
Original assignee: Life Science Investments Ltd
Priority date: 2008-07-10
Filing date: 2008-07-10
Publication date: 2010-01-14
Also published as: EP2318521A1

Abstract

The invention concerns the use of a mixture of superoxide dismutase and catalase for preparing a topical composition for treating pruritus caused by external agents, or consecutive to dermatological diseases such as atopic dermatitis, psoriasis, eczema, scarring process, internal diseases or psychic disorders, or alleviating it. The inventive compositions are formulated to obtain all galenical forms conventionally used for applying a topical composition on the skin such as for example gel, milk, cream, lotion, plaster or patch, as powder to be solubilized in water or physiological serum prior to use.

Description

Description Use of a mixture of superoxide dismutase and catalase for treating pruritus and alleviating its symptoms Technical Field

[1] Pruritus is one of the most common symptoms of skin diseases, but can also be a major symptom of systemic diseases (e.g. malignancy, infection or metabolic disorders). However, despite a century of research and investigations on pruritus, there is no generally accepted therapy for the treatment of itch.

[2] Despite its common use, the word pruritus is not easy to define. A simple description is that pruritus is an unpleasant cutaneous sensation that provokes a desire to scratch, but this simple attempt to define pruritus is certainly not perfect.

[3] Pruritus can be a physiological sensation if the consecutive scratching removes a potentially agent, or pathological, if associated with skin and/or intestinal diseases, and psychic disorders, or caused by some food and drugs.

[4] Pruritus can be experienced only in the skin, because of the unique neural mechanisms it involves. Its intensity can be mild, moderate, severe and even distressing with sleep disturbances, loss of weight, discomfort, increased irritability, problems in daily activities and even stress. It can be acute or chronic, sometimes long lasting, and may affect any part of the body. It is always a diagnostic challenge to the clinician.

[5] Pruritus may be provoked both by exogenous agents or by endogenous causes or stimuli.

[6] The major pruritogens (with the receptors interacting in the genesis of pruritus) are the following: acetylcholin (nicotinergic and muscarinergic [mAChR] receptors), CGRP (CGRP receptors), CRM and POMC (CRH-Rl and CRH-R2), cytokines (Cytokine receptors), endocannabinoids (CBs - CBl and CB2), ETs (ET receptors), Endovanilloids (activation of TRPVl and sensatization of TRPVl via activation of specific receptors), histamine (histamine receptors HlR and H4R), kallikreins and proteases (PARs), kinins (bradykinin receptors BlR or B2R), leukotriene B4 (leukotriene receptors), NKA and Substance P (tachykinin receptors), NKA, BDNF, NT (specific receptors TrkA [NGF], TrkB [NT-4, BDNF], TrkC [NT-3]) and opioids (opioid receptors).

[7] The role of histamine was long overestimated, small doses of histamine can fail to produce itch, and antagonists of the histamine receptors Hl and/or H2 have often been proven to be of low or no efficacy as antipruritic drugs.

[8] Besides the various chemicals and other substances susceptible of exogenously induce pruritus, a number of pathological processes can lead to itch: inflammation, hypersensitivity, degenerative changes, malignant tumors and even psychic abnormalities.

[9] The neuroanatomical basis of pruritus is relatively well known. Itch originates in free nerve endings, near the dermo-epidermal junction and is conducted centripetally by afferent nerves entering the spinal cord via the dorsal roots. The sensitive nerves for pruritus are small, non-myelinated C fibers with a slow conduction rate. The cell bodies of these nociceptive primary neurons are located in the dorsal root ganglion. After entering into the spinal cord the primary neurons synapse secondary neurons whose axons cross to the opposite side, and then by the tractus spinothalamicus reach the laminar nuclei of the thalamus. Finally, these nuclei relay to the cerebral cortex, i.e. the sensory area in post central gyrus. Here is noted the location, nature, intensity and other quality of this sensation.

[10] Interestingly, recent work suggests that the epidermis itself, especially the ker- atinocytes which form the bulk of the epidermis, constitute the itch receptor. (Inoue et al, 2002)Keratinocytes express a range of neuropeptide mediators and receptors which appear to be involved in pruritus, including opioids, nerve growth factor (NGF), substance P and receptors including vanilloid receptors, and protease activated receptors type-2 (PAR-2). Thus, the epidermis and its associated ramifications of fine intraepidermal c-neuron filaments can be looked upon as the "itch receptor".

[11] Pruritus is caused by the release of mediators acting peripherally on receptors, cells or nerves. Some of these substances can act directly on the free nerve endings, others act indirectly through mastocytes or other cells, in particular keratinocytes.

[12] Histamine, an imidazolethylamine, was the first and most important recognized pru- ritogenic substance, but does not account for all the types of pruritus.

[13] Serotonin (5-hydroxytryptamine, 5-HT) can also cause pruritus in some circumstances.

[14] Tryptase has recently been shown to activate PAR-2 expressed on afferent C neuron terminals, stimulating the sensation of itch and also triggering the release of substance P.

[15] Substance P is a pro-inflammatory neuropeptide, produced in the dorsal ganglia and the transported to the periphery by nociceptive nerves A and C. SP-reactive fibers are localized close to mast cells, and hence release of SP from sensory afferents can stimulate mast cell secretion in vivo. SP degranulates mast cells, and therefore can release histamine from them, provoking itch.

[16] Another neuropeptide deserving interest in the pathogenesis of pruritus is probably

Calcitonin gene-related peptide (CGRP).

[17] Neurotrophins, the key prototype of which is Nerve Growth Factor (NGF) are also key molecular players in the pathogenesis of itch.

[18] Cytokines are also key molecular players in the occurrence and maintenance of pruritus.

[19] Interleukins constitute another interesting family, some members of which seem to be deeply involved in the mechanism of pruritus, namely Interleukin-1 (IL-I), In- terleukin-2 (IL-2) and Interleukin-6 (IL-6).

[20] Vessels and adhesion molecules like E-selectin appear to play a role in the occurrence and development of pruritus, at least in certain dermatological diseases.

[21] Pruritus may be elucidated by the opioid system as well: it is believed that activation of mα-opioid receptor induces while activation of ^-opioid receptors alleviates pruritus.

[22] Pharmaceutical compositions for treating and alleviating pruritus do exist. They are essentially antihistaminic Hl and H2, which are histamine antagonists, inhibiting the release of histamine or blocking Hl or H2 receptors.

[23] They are some other drugs, such as Bufexamac, a non-steroid antiinflammatory drug acting mainly on inflammation, but not primarily on pruritus.

[24] However, superoxide dismutase nor catalase were never mentioned as being able of possessing any antipruritic property. Disclosure of Invention Technical Problem

[25] Pruritus is one of the most common symptoms of skin diseases, but laso of major systemic diseases.

[26] Pruritus is always a diagnostic and a therapeutic challenge to the clinician, and despite a great amountr of research and investigation, there is still no generally accepted therapy for the treatment of itch and pruritus.

[27] Interestingly, we have discovered that a mixture of superoxide dismutase and catalase applied on the skin was able to drastically alleviate pruritus in individuals suffering it.

[28] A first clinical trial showed that among 27 patients with keloid and hypertrophic scars, 25 were complaining severe or moderate pruritus. Immediately upon application on the area displaying pruritus of a mixture of superoxide dismutase and catalase, 21 of them noted that pruritus had disappeared, while the remaining 4 felt that their pruritus, although not having totally disappeared, had been strongly alleviated.

[29] In another controlled, double -blind study, healthy voluntaries were enrolled, and experimental pruritus was provoked by heat on the forearm, after previous treatment of one forearm with a mixture of superoxide dismutase and catalase, and the second one with the excipient of this mixture. Intensity and duration of pruritus were both much lesser on the forearm treated with the mixture of superoxide dismutase and catalse, than with the excipient alone.

[30] The present invention accordingly relates to the use of a mixture of superoxide dismutase and catalase for preparing a topical composition intended for the treatment of pruritus and to alleviate it.

[31] Superoxide dismutase (SOD) is a stable enzyme of natural origin: it gives out superoxide radicals without being consumed, and it is generally soluble in water. Superoxide dismutase is either of animal or plant origin or obtained by biotechnology.

[32] Catalase is a ferriporphyrin enzyme that catalyzes the liberation of molecular oxygen from hydrogen peroxide.

[33] These enzymes are referred to as metabolic enzymes, which catalyze reactions within the cell, such as the producion of energy and detoxification. In the natural state these enzymes are frequently present simultaneously, since their mechanisms are complementary.

[34] Superoxide dismutase is protecting the cells by attacking the free superoxide radicals.

[35] There are three types of SOD: copper/zinc SOD, manganese SOD, and ferric SOD.

These enzymes protect the cells: Cu/zn SOD protects the cytoplasm, where the free radicals are produced by metabolic reactions, and Mn SOD protects the mitochondria of the cell, that contains the genetic information and acts as the site for the production of cell energy.

[36] In the formulations according to the invention, the SOD may be of any origin.

[37] SOD is naturally present in the majority of plants: it is found in extracts, apples, certain varieties of cabbage, broccolis, Brussels sprouts, tomatoes, or even cabbage or melon, which are optionnally transgenic, and also horseradish, or it may be extracted from seeds or shoots of enzyme-rich cereals, such as wheat, corn, soybean or barley.

[38] It is also produced by biotechnology: for example it may originate from a strain of

Saccharomyces cerivisiae.

[39] It is potentially extracted from bovine or human erythrocytes, produced by recombinant synthesis by microorganisms such as E.coli or yeasts, or else extracted from mammalian livers.

[40] The invention accordingly relates in one embodiment to a composition characterized in that the superoxide dismutase is obtained by biotechnology, originating for example from a natural strain of Saccharomyces cerevisiae.

[41] According to the invention the SOD may be complexed or bonded to polymers without prejudice to its enzymatic activity, for example polysaccharides.

[42] The catalase (CAT) which converts hydrogen peroxide to water and oxygen may be of any origin.

[43] The catalase may, for example, be obtained from mammalian liver extract or from microorganisms such as Aspergillus niger. [44] It may also be obtained from plant extracts or obtained by recombinant synthesis.

[45] Like the SOD, it may be bonded covalently or complexed to polymers without prejudice to its enzymatic activity, for example polysaccharides. [46] The two enzymes may be encapsulated or incorporated in polymeric microparticles, composed for example of crosslinked ionic polysaccharides and/or hydrophilic polymers. [47] These formulations of these two enzymes, alone or as a mixture, allow the enzymatic activity to be protected, for example, from the interactions with the outside environment, while promoting targeting, spreading and also pharmaceutical formulation. [48] These two enzymes may be present in a lyophilized form, powder for example, in crystalline suspension form, in ammonium sulfate suspension form or in a solution in any solvent. [49] Numerous animal or plant extracts contain these two enzymes in association: examples include tomato extract, melon extract, cabbage extract or mammalian liver extract. [50] According to the invention the SOD/CAT mixture, whether original, meaning that the extract contains the mixture of the two enzymes on extraction, or whether prepared, must allow an enzymatic activity ratio of between 14/2 and 15/5, with an enzymatic

SOD activity of between 150 and 300IU per gram.

[51] The activity of the catalase will be between approximately 30 and 80 IU per gram.

[52] The enzymatic activity of the SOD is determined by the method of Beauchamp C. and Fridovich L, Analytical Biochemistry 44, 276 (1971), modified by the method of

Spitz D, and Oberley L: An Assay for Superoxide Dismutase Activity in Mammalian

Tissue Homogenates, Annal. Biochem. 179, 8, 1989. [53] This method is an indirect method, owing to the short half-life of SOD; it allows the

SOD to be evaluated via its capacity to inhibit a flow of superoxide anion generated by the xanthine/xanthine oxydase system. [54] The enzymatic activity of the catalase is determined by the method of Clairborne A,

Catalase activity, CRC Handbook of Methods for oxygen radical research, 283-284,

1985. [55] This method is based on the decomposition of hydrogen peroxide by catalase, which is monitored using a UV spectrophotometer at 240nm. [56] The invention accordingly relates to the use of a mixture of superoxide dismutase and catalase for preparing a topical composition intended for the treatment of pruritus and to alleviate it. [57] It relates to the use described above, characterized in that the pruritus can be caused by external agents, or consecutive to dermatological diseases such as atopic dermatitis, psoriasis, eczema, scarring, internal diseases or psychic disorders. [58] It relates to the use described above, characterized in that the superoxide dismutase is extracted from apples, broccolis, Brussels sprouts, cabbage, melon, horseradish or tomato, which are optionnally transgenic.

[59] It relates to the use described above, characterized in that the superoxide dismutase is extracted from seeds or shoots of enzyme-rich cereals selected from wheat, corn, soybean and barley.

[60] It relates to the use described above, characterized in that the superoxide dismutase is obtained by biotechnology.

[61] It relates to the use described above, characterized in that the superoxide dismutase is obtained from a strain of Saccharomyces cerevisiae.

[62] It relates to the use described above, characterized in that the superoxide dismutase is extracted from bovine or human erythrocytes or extracted from mammalian livers.

[63] It relates to the use described above, characterized in that the superoxide dismutase is produced by recombinant synthesis by E. coli microorganisms or yeasts.

[64] It relates to the use described above, characterized in that the superoxide dismutase is produced by biotechnology, by a natural strain of Saccharomyces cerevisiae .

[65] It relates to the use described above, characterized in that the catalase is obtained from mammalian liver extracts or extracts of microorganisms such as Aspergillus niger.

[66] It relates to the use described above, characterized in that the catalase is obtained from plant extracts, for instance apples, broccolis, Brussels sprouts, cabbage, melon, horseradish or tomato, which are optionally transgenic.

[67] It relates to the use described above, characterized in that the catalase is obtained by recombinant synthesis.

[68] It relates to the use described above, characterized in that the catalase or the superoxide dismutase, alone or as a mixture, are bonded covalently or complexed to polymers.

[69] It relates to the use described above, characterized in that the polymers are preferably selected from polysaccharides.

[70] It relates to the use described above, characterized in that the catalase or the superoxide dismutase, alone or as a mixture, are encapsulated or incorporated in polymeric microparticles.

[71] It relates to the use described above, characterized in that the polymeric microparticles are composed of crosslinked ionic polysaccharides and/or of hydrophilic polymers.

[72] It relates to the use described above, characterized in that the catalase and the superoxide dismutase, alone or as a mixture, are in a lyophilized powder form, in crystalline suspension form, in ammonium sulfate suspension form or in solution in any solvent.

[73] It relates to the use described above, characterized in that the catalase and the superoxide dismutase are present as a mixture in a natural extract. [74] It relates to the use described above, characterized in that the natural extract is selected from melon extracts, tomato extracts, cabbage extracts or mammalian liver extracts. [75] It relates to the use described above, characterized in that the SOD/CAT mixture exhibits an enzymatic activity ratio of between 14/2 and 15/5 with a SOD enzymatic activity of between 150 and 300 IU per gram. [76] It relates to the use described above, characterized in that the catalase activity is between approximately 30 and 80 IU per gram.

[77] The invention is illustrated for example with the following formulation.

[78] Formulation examples:

[79] Creams for local external apllication to the skin

[80] Propylene glycol dipelargonate 6%

[81] Propylene Glycol 5%

[82] Glyceryl stearate and PEG stearate 3%

[83] Stearic acid 3%

[84] Xanthan Gum 2%

[85] Ethylene Glycol 1%

[86] Cetyl Palmitate 1%

[87] Petrolatum 0.5%

[88] Avocado Oil 1%

[89] Liquid paraffin 2%

[90] Triethanolamine 0.67%

[91 ] Potassium sorbate 0.2%

[92] Preservatives 0.3%

[93] SOD/CAT mixture 2%

[94] Water q.s.

[95] The SOD/CAT mixture used is a mixture extracted from a variety of Brassica napus.

[96] The enzymatic activity of the SOD is evaluated by the method described above as being approximately 280 IU per gram. [97] The enzymatic activity of the catalase by the method described above is evaluated as being 60 IU per gram. [98] In another example the SOD/CAT mixture is provided by a melon extract: the stabilized extract sold under the brand name Extramel by the company Bionov. [99] The enzymatic activity of the SOD is evaluated by the method described above as being approximately 270 IU per gram. [100] The enzymatic activity of the catalase by the method described above is evaluated as being 40 iu per gram.

[101] In another example the SOD/CAT mixture is provided by a mixture comprising SOD and catalase which are obtained fromn tomato extracts sold by the company Dirsey Corporation.

[102] The enzymatic activity of the SOD is evaluated by the method described above as being approximately 320 IU per gram.

[103] The enzymatic activity of the catalase by the method described above is being approximately 85 IU per gram.

[104] In another example the SOD/CAT mixture is provided by a mixture prepared in accordance with the final enzymatic activity required, namely 280 IU per gram for SOD and 60 iu per gram for catalase, of SOD extracted from Escherichia coli in the form of lyophilized powder (2,500 IU/mg) and from catalase extracted from Aspergillus niger in lyophilized form (170 IU/mg).

[105] The compositions according to the invention are formulated to give all of the pharmaceutical forma that are conventionnally used for the topical apllication of a composition to the skin.

[106] They are formulated, for example, in the form of a gel, milk, cream, lotion, plaster or atch, or in stick form or even as a powder to be dissolved in water or physiological serum prior to use.

[107] The compositions according to the invention may optionally contain various additives, such as suspension agents, emulsifiers, anionic, cationic, nonionic or amphoteric polmers, proteins, vitamins, surfactants, mineral oils, vegetal oils, waxes, silicone resins and/or rubbers, thickeners, acidifying or alkalifying agents, solvents, pH stablizers, UV protectors, preservatives, antibacterial agents, antifungal agents, fragrances or other adjuvants which are commonly used in cosmetology or dermatology.

Claims

[1] 1. The use of a mixture of superoxide dismutase, extracted from apples, broccolis, Brussels sprouts, tomatoes, cabbage or melon, which are optionally transgenic, or horseradish, or extracted from seeds or shoots of enzyme-rich cereals selected from wheat, corn, soybean and barley, and catalase for preparing a topical composition intended for the treatment of pruritus and to alleviate it.

2. The use according to claim 1, characterized in that the pruritus can be caused by external agents, or consecutive to dermatological diseases such as atopic dermatitis, psoriasis, eczema, scarring processes, internal diseases or psychic disorders.

3. The use according to either of claims 1 and 2, characterized in that the catalase is obtained from mammalian liver extracts or extracts of microorganisms such as Aspergillus niger.

4. The use according to one of the preceding claims, characterized in that the catalase isobtained from plant extracts.

5. The use according to either of claims 1 and 2, characterized in that the catalase is obtained by recombinant synthesis.

6. The use according to anyone of the preceding claims, characterized in that the catalase and the superoxide dismutase, alone or as a mixture, are bonded co- valently or compleed to polymers.

7. The use according to the preceding claim, characterized in that the polymers are preferably selected from polysaccharides.

8. The use according to any one of the preceding claims, characterized in that the catalase and the superoxide dismutase, alone or as a mixture, are encapsulated or incorporated in polymeric microparticles.

9. The use according to the preceding claim, characterized in that the polymeric microparticles are composed of crosslinked ionic polysaccharides and/or of hy- drophilic polymers.

10. The use according to any one of the preceding claims, characterized in that the catalase or the superoxide dismutase, alone or as a mixture, are in a lyophilized powder form, in crystalline suspension form, in ammonium sulfate suspension form or in solution in any solvent.

11. The use according to any one of the preceding claims, characterized in that the catalase and the superoxide dismutase are present as a mixture in a natural extract.

12. The use according to the preceding claim, characterized in that the natural extract is selected from melon extracts, tomato extracts, cabbage extracts or mammalian liver extracts.

13. The use according to any one of the preceding claims, characterized in that the CAT/SOD mixture exhibits an enzymatic activity ratio of between 14/2 and 15/5 with a SOD enzymatic activity of between 150 and 300 IU per gram.

14. The use according to any one of the preceding claims, characterized in that the catalase acivity is between approximately 30 and 80 IU per gram.