MORϊN , A NEW INHIBITOR OF MICROBIAL HYALURONATE LYASE

This invention relates to a new inhibitor of microbial hyaluronate lyase and its use.

Microbial hyaluronate lyase is formed by invasive microbial pathogens belonging to the pneumococci, bacteroids, micrococci, peptococci, streptococci, staphylococci, Clostridia, propionic bacteria, Yersinia, enterococci, vibriones and Candida, among them a lot of species colonizing skin and mucous membranes, as well as parasites such as Leishmania, Tetrahymena and Trypanosoma. With hyaluronate lyase, they destroy the connective tissue, reduce the barrier effect of tissue boundaries, change the tissue turgor and consequently the conditions for the immigration of immune competent cells and they release proteoglycan fixed endogenic chemotactic modulators in an irregular manner. Therefore, the hyaluronate lyases are an important pathogenicity factor for many infections. As the hyaluronate lyases can remain as an active enzyme in the body even after a successful antimicrobial treatment, their pathogenetic tissue modifying and cell modulating effects on the host are not hampered. As a product of normal microbial skin flora they can also cause an accelerated skin ageing and wrinkling.

The mechanism of hyaluronate lyases principally differs from the hydrolytically acting hyaluronidases that have the effect of β-eliminases. Hyaluronidases are enzymes that hydrolytically cleave the hyaluronic acid biopolymer in combination with a hydrating effect. Therefore, it cannot be expected that the inhibitors of hyaluronidases are necessarily inhibitors of hyaluronate lyases.

In contrast to hyaluronidases, only few inhibitors of microbial hyaluronate lyases are known. An example are synthetic copolymers that are produced by the condensation of formaldehyde and Gentisic acid (H. J. Rogers, S. P. Spensley, Biochim. Biophys. Acta, 1954, 13, 293-297) and inhibit the hyaluronate lyase of Streptococcus agalactiae. Sulphated hyaluronic acid is also a good inhibitor (H. Rodig, J. H. Ozegowski, G. Peschel, P. J. Muller, ZbI. Baku 1999, 289, 835-843).

JP2000319154 indicates morin apart from other flavonoids as a phototoxic inhibitor in cosmetic compositions or food or beverages. According to JP5148125, morin is used in toothpaste for its antioxidative and stabilizing effects.

The flavonol morin belongs to the group of flavons, the yellow plant pigments. Morin or 2',3,4',5,7-pentahydroxyflavone (CAS registry number: 480-16-0) is gained from wood of different origins, for example from the wood of Morus alba (white mulberry), and is commercially available.

It is the object of the present invention to provide a new inhibitor of hyaluronate lyase and to specify its use.

In the invention, this task is solved by a flavonol according to claim 1. Advantageous embodiments are described in the following claims.

This invention is characterised by the surprising finding that with an inhibitor content of IC ₅ Q = 0.11 μM flavonol morin is a particularly effective inhibitor of microbial hyaluronate lyase and thus it considerably differs from other flavonols and similar substances, because till now and in comparison to other known hyaluronate lyase inhibitors it shows the highest inhibitory activity against the hyaluronate lyase of pathogenic streptococci (among them the Streptococcus agalactiae microorganism/

Thanks to its inhibitory effect on microbial hyaluronate lyase, morin is an ideal adjuvans to be used together with antimicrobially acting substances as a component both of pharmaceutical and cosmetic formulations and of medical products to counteract the damages to humans and animals caused by hyaluronate lyases.

The inventive application of morin has the advantage that it shows a very low toxicity with an LD ₅₀ value of 555 mg/kg. Another advantage is the fact that with IC ₅₀ = 100 μM morin has approximately 900 times the inhibitory activity of the animal-derived hyaluronidase of cattle testicle

the properties of which come close to the endogenic hyaluronidases. Thus, the application of the morin of the inventive formulations does not imply the risk of influencing endogenic hyaluronidases and does not have a toxic effect.

Therefore, morin is particularly well suitable for solving the inventive task because apart from its considerably high inhibitory activity against hyaluronate lyase and its negligible toxicity and effect on endogenic hyaluronidase, it is always available as a vegetable product.

According to this invention, morin is used in liquid formulations in concentrations between 0.0001 mM and LO mM and in solids in concentrations of at least 0.05 g/g solid.

There is a wide range for the use of morin in the manufacture of pharmaceutical formulations or medical and cosmetic products.

According to this invention, morin is used as an adjuvans together with at least one antimicrobial substance and/or an antiparasitic substance and/or an antimicrobial preservative in pharmaceutical or cosmetic formulations or in combination with the material of medical products or distributed in the latter.

A decisive reason for the use of morin as an adjuvans in disinfectants and preservatives is the fact that microorganisms survive due to the development of resistance to disinfectants and preservatives. Thanks to the blockage of the penetration-promoting effect of their hyaluronate lyase and thanks to adherent morin, the ability to penetrate into skin and mucous membrane surfaces is successfully inhibited so that the formation of colonies is considerably reduced. Preferentially, the preservatives used are the products mentioned by Wallhauβer (K:H: Wallhauβer; Praxis der Sterilisation Desinfektion - Konservierung (Practice of sterilization disinfection - preservation). 5, completely revised edition, published by Georg Thieme Verlag Stuttgart- New York) such as formalin, silver ions, ethanol, phenols, quaternary ammonium salts or iodine. Morin can also be used together with plant-

derived substances or parts of such plants or extracts that have an antimicrobial or antiparasitic effect. A very effective plant-derived antimicrobial agent is for example thymol. In humans and animals, the formulations can be used in an intracorporeal manner or they are applied onto the skin or mucous membrane. Thus, they can be introduced by injection or perfusion into tissue in the vascular system or in body cavities or they can be orally administered. Morin is used in solid formations, e.g. as a component or a coating of medical products such as plasters, wound coverings, dressings, textile formations such as orthopaedic bandages, distance tissue, sterile area formations, supporting stockings, catheters, hearing aid elements, cotton buds or hygienic products such as sanitary towels, nappies, tampons, gloves, and condoms.

Morin can be used in disinfectant solutions to be applied onto the skin or mucous membrane, for example to disinfect wounds, hands or areas. In semi-solid or viscous products, morin can be used for example in ointments, creams, lotions, medical and liquid soaps, shampoos or in lubricants. According to this invention, other liquid formulations containing morin have an effect on the mouth and pharynx areas, for example as gargles, pills and bonbons, or they can be inhaled as an aerosol so that their effect reaches up to the lungs.

Embodiment 1

Hyaluronate lyase of Streptococcus pyogenes (Hyal A) is obtained according to Gerlach et al. (Gerlach D, W. Kohler, Zentralbl Bakteriol 221, 166-172, 1972).

Hyaluronate lyase of Streptococcus equisimilis (Hyal C) and Hyaluronate lyase of Streptococcus agalactiae strain 4755 (Hyal B) are got according to Ozegowski et al (Zentralbl. Bakteriol., 249, 310-318, 1981, Zentralbl. Bakteriol 280, 497-506, 1994). Hyaluronate lyase of Streptomyces hyalurolyticus (Hyal S) and hyaluronidase of cattle testicles are purchased (Sigma Taufkirchen, Germany).

The hyaluronate lyase activity (units/ml) is determined according to Rodig et al. (Zentralbl. Bakteriol 289, 1999, 835-843).

The inhibitory activity (%) is found out in a modified method according to Hertel et al. (Arch. Pharm 339 313-8, 2006) in microtiter plates. For determining these values, the hyaluronic acid and the enzyme are dissolved in a 0.1 M acetate buffer of pH 6.0. Morin is dissolved as a solid in dimethyl sulphoxide in a concentration of 20 mM and afterwards diluted with water to the corresponding test concentration.

To determine the inhibitory activity, 50μl of a hyaluronate lyase solution containing 4 U/ml are given into each well. Then, lOμl of the inhibitor solution are added so that the final concentration of morin is between 0.0001 mM and 1.5 mM in the preparation.

After an incubation time of 10 minutes, 50μl of hyaluronic acid of 20 mg/100 ml are added and after an incubation of 30 minutes at 37°C the reaction is stopped by the addition of 200 μl of N-Cetyl-N,N,N- trimethyl ammonium bromide (2.5% w/v in 2% w/v NaOH). The residual cloudiness appearing is measured at 600 nm and the inhibitory activity is calculated according to the following equation: Inhibitory activity[%] = ((A _C ontroi-Asampie) x 100%) /(A _Cθ ntroi). (Fig. 1)

Embodiment 2

A recipe for a shampoo that fights dandruff contains 50% alkyl ether sulphate, 28 per cent, 3.0% protein hydrolyzate, 50 per cent, 3.0% diethylene glycol monolauryl ether, 1.0% pirocton olamin, 0.5% morin and water ad 100.

Embodiment 3

A peeling cream for bad skin contains 9.6% cetyl stearyl alcohol, 4% cetyl stearyl alcohol/natrium cetyl stearyl sulphate, 12% low-viscosity paraffin, 0.1% 4-hydroxybenzoic acid ethyl ester, 52.5% water, 5.0% glycerine, 1.0% morin, 0.1% 4-hydroxybenzoic acid ethyl ester, 0.3% hydroxybenzoic acid ethyl ester, 15%, polythene powder and 0.4% phenoxyethanol.

Embodiment 4

A foot powder contains 86.5% French chalk, 7.5% aluminium hydrochloride, 3% silicon dioxide, 2.0% salicylic acid and 1.0% morin.

Embodiment 5

An antiperspirant contains 97.3% isopropanol, 1.0% glycerine, 0.5% farnesol, 0.8% perfume oil, 0.2% morin and 0.2% polysorbate.

Embodiment 6 An O/W cream for the care of bad skin contains 5.0% sulphur, 5.0% middle-chain triglycerides, 19.0% lauric acid hexyl ester, 15% cetyl stearyl alcohol/sodium cetyl stearyl sulphate, 1.0% morin, 0.2% methyl p-hydroxybenzoate, 0.2% triclosan, 0.2% allantoin, 54.3% water and 0.1% perfume oil.