ES2392522A1 - COMPOSITION FOR MAINTENANCE OF CONTACT LENSES THAT RESULTS EFFECTIVELY AGAINST ACANTHAMOEBA SSP. - Google Patents

Abstract

Composition for the maintenance of contact lenses that is effective against Acanthamoeba spp, using maslinic acid as active principle and at low concentrations. It is possible to eliminate these amoebas in cystic and trophozoite form, preventing them from de-cysting or degrading corneal tissue, without causing any toxicity to the cornea.

Description

Composition for maintenance of resulting contact lenses

effective against Acanthamoeba spp.

SECTOR OF THE TECHNIQUE

The present invention relates to two sectors, on the one hand, pharmacology and on the other with the pharmaceutical sector and more specifically with contact lens maintenance solutions. An effective composition for the treatment of ocular parasites causing gender keratitis is described.

Acanthamoeba

STATE OF THE TECHNIQUE

Maslinic acid (2-alpha, 3-betadihydroxy-28-carboxyioleannan), also called crataegolic acid, is a rare acid, although it is present in more than 20 different plants. In particular, maslinic acid is abundantly found in the skin wax of olives [The Lipids of Olea-Europaea .4. Pentacyclic Triterpene Acids in Olives, Bianchi, G., Pozzi, N., Vlahov, G., Phytochemistry, 37 (1), 205-207, (1994)]. A patent developed by the University of Granada [Procedure for industrial use of 3¡3-hydroxiolean-12-en-28-oic (oleanolic) and 2a, 3¡3-dihydroxiolean-12-en-28-oic (maslinic) acids contained in the by-products of olive milling, P9601652] allows to obtain this acid industrially, from solid by-products of the industrial milling of the olive. Thus, today there is at least one affordable and virtually inexhaustible source of this acid.

The interest in the biological activity of maslinic acid is demonstrated by the large number of patents internationally. Among others, Antitumor agent US20030355201 20030131; Induction apoptosis (W02002JP13663 20021226); Antiobestic foods and drinks (W02002JP11608 20021107); External! agent for theskin and whitening agent (US20020259323 20020930); Antiobesity drugs and materials thereof

(W02002JP07709 20020730); Drugs for vascular lesion (W02002JP03189

20020329), Antitumor food or beverage (W02001JP11374 20011225) or Use

of maslinic acid as an antiparasitic against Phyllum protozoa

Apicomplexa (P200502349). In addition, the effect that this

5

product has in the extracellular proteases secreted by protozoa as

Toxoplasma gondii (De Pablos, L. M .; González, G .; Rodríguez, R .; García-

Granados, A .; Parra, A .; Osuna, A. Journal Of Natural Products, 2010 73 (5): 831-

634.).

1st

The amoebas of free life, considered by the Center for Control and Prevention

of Diseases of Atlanta (CDC, USA), within the group of protozoa

emerging, they form a group of opportunistic pathogens with high relevance,

for both human and veterinary health. In this group are:

Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia

fifteen

diploid. The free-living amoebas of the genus Acanthamoeba have a

cosmopolitan distribution and have been isolated from a wide variety of habitats.

Due to its distribution in nature, its contact with man is a fact

evident. It has been observed that up to 85% of immunocompetent populations

twenty

they possess antibodies against Acanthamoeba (Lorenzo-Morales J, Ortega Rivas A .;

Foronda P .; Abreu N .; Ballart D .; Martínez Carretero E .; Valladares B. Mol Biol

Parasitol 2005; 95 (4): 273-27; Brindley N, Matin A, Khan NA Exp Parasitol 2009,

121 (3): 254-256). The pathologies that Acanthamoeba can cause in the

man are related to the route of entry into the body and, above all,

25

with the patient's immune status. Thus, in immunosuppressed patients

they can cause encephalitis or disseminated infections while in individuals

Immunocompetent are the cause of amoebic keratitis. It should be noted that

Majority affected individuals in the case of amoebic keratitis, are

immunocompetent individuals wearing contact lenses (Marciano-Cabra! F y

30

Goat! G Clin Microbe! Rev 2003 16 (2): 273-307).

The incidence of cases of Acanthamoeba keratitis has increased in the

last years. In the US, the Center for Diseases Control and Prevention, CDC, of

   Atlanta has highlighted the recent and significant increase of this pathology in the country

(Morbidity and Mortality Weekly Report 2007). In other countries the number of cases has increased up to six times between 2006 and 2007 (Carvalho, R. (2009) Twenty years of Acanthamoeba keratitis. Cornea. Jun; 28 (5): 516-9).

The reason for this increase in cases is not very clear. However, there is evidence that multi-purpose maintenance solutions for contact lenses, which are very popular among users due to the ease of use, are not effective against Acanthamoeba [Shoff, M (2008), Variable Responses of Acanthamoeba Strains to Three Multipurpose Lens C / eaning Solutions. Optometry and Vision Science, 84 (3): 202-207]. Unfortunately, no evidence is required from manufacturers that demonstrate the efficacy of the products they market against Acanthamoeba because this opportunistic pathogen is considered a rare cause of keratitis [Saviola JF (2007) Contact lens safety and the FDA: 1976 to The present Eye Contact Lens 33: 404-409]

The pathogenesis process of Acanthamoeba begins with the adhesion of the amoeba to the host cell, followed by the secretion of extracellular proteases (mainly serine proteases) and finally phagocytosis and / or direct death of the host cell. Recently, it has been shown that Acanthamoeba extracellular serine proteases play an essential role in this process and in fact, if the genes of these proteases are inhibited, amoebas are not capable of degrading corneal tissue [Lorenzo-Morales J; Ortega Rivas A .; Foronda P .; Abreu N .; Ballart D .; Martínez Carretero E .; Valladares B. 2005 Mol Biol Parasitol 2005; 95 (4): 273-277]. In recent years, it has been shown that these proteases are related to the processes of encystment and disengagement of these amoebas [Bouyer S, Rodier MH, Guillot A, Hechard Y. Exp Parasitol. 2009 Sep; 123 (1): 90-4; Lorenzo-Morales J, Martín-Navarro CM, López-Arencibia A, Santana-Morales MA, Afonso-Lehmann RN, Maciver SK, Valladares B, Martínez-Carretero

E. Antimicrob Agents Chemother. 2010 Dec; 54 (12): 5151-5]. Therefore, and after these previous studies carried out mainly in our center, it is clear that the search for an active molecule or principle with inhibitory properties against serine proteases, could give a solution to the problem of lack of effective treatments against these pathogens .

To solve the problem of lack of treatment for Acanthamoeba keratitis, numerous active ingredients have been used. Thus, the first treatments described were non-specific antifungal agents, aminoglycosides (Neomycin), imidazoles (Cyotrimazole, Miconazole, Ketoconazole), polymyxins (Polymyxin 8) and Propamidine, which require prolonged application, usually with high toxicity and controversial stromal penetration! and cysticidal activity [Visvesvara GS. Amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment. Curr Opin lnfect Dis. 201 ODec; 23 (6): 590-4. Review.].

The first successful chemotherapy was the association of dibromopropamidine, propamidine isothionate and neomycin described by Wright in 1985 (Wright P, Warhurst D, Jones BR. Acanthamoeba keratitis successfully treated medically. Br J Ophthalmol 1985; 69: 778-82.).

Subsequently two cationic antiseptics Chlorhexidine and Polyhexamethyl Biguanide (PHMB) were shown to be effective against the different life cycle forms of Acanthamoeba, trophozoite and cyst [Silvany E, Dougherty JM, McCulley JP. Effect of contact lens preservatives on Acanthamoeba: Ophthalmology 1991; 98: 854-7 .; Kilvington S, Anthony Y, Davies DJG, Meakin BJ. Effect of contact lens disinfectants against Acanthamoeba cysts. Rev lnfect Dis 1991; 13: (s414-s415); Silvany RE, Dougherty JM, McCulley JP, Wood TS, Bowman RW, Moore MB. The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga. Ophthalmology 1990; 97: 286-90]. This treatment is important since it was possible to eliminate the cysts that remain in the corneal stroma and reactivate the disease. Therefore, the introduction of Chlorhexidine and PHMB in 1992 was a remarkable success for the clinical management of Acanthamoeba keratitis, being achieved together with Propamidine isothionate O, 1% (Brolene®), further improving its effectiveness [Radford CF, Lehmann OJ, Dart JKG Acanthamoeba keratitis: Multicenter survey in England 1992-1996. Br J Ophthal 1998; 82: 1387-92 .; Elder MJ, Dart JKG, Kilvington S, Seal DV, Hay J, Kirkness CM. Chemotherapy for Acanthamoeba keratitis. Lancet 1995; 345: 791-2].

The treatments performed topically, with the combination of 1% propamidine isothionate (Brolene®) and 0.02% PHMB and a duration of medical treatment from 2 to 11 months, managed to eradicate Acanthamoeba infection in the 100% of cases. In those cases where the lesions caused by parasitization are important, corneal transplantation is necessary [Donoso R, Mura C, and López M, Acanthamoeba keratitis treated with propamidine and polyhexamethylbiguanide. Rev. Med. Chile, Apr. 2002, vol.130, no.4, p.396-401].

Recently, it has been shown that the concentrations of biguanides (PHMB and chlorhexidine) present in maintenance solutions are not high enough to eliminate Acanthamoeba, in addition these biguanides from certain concentrations are toxic to the corneal tissue hence the The solution is not in this group of molecules [Martín-Navarro CM, Lorenzo-Morales J, Cabrera-Serra MG, Rancel F, Coronado-Aivarez NM, Piñero JE, Valladares 8 J Med Microbiol2008 57: 1399-1404].

DESCRIPTION OF THE FIGURES

Figure 1: Acanthamoeba growth inhibition curve (in% at 96h) by action of maslinic acid obtained by the method based on the oxide-reduction of Alamar Blue. The abscissa axis represents the concentration of maslinic acid in ¡.Jg / ml.

Figure 2: Determination of the effect of maslinic acid on the number of cysts in a sample. The figure shows that there is no linear relationship between the product concentration and the number of cells removed, the effect stabilizing from a concentration starting at 0.0016.

OBJECT OF THE INVENTION

The object of this invention is a composition for the maintenance of contact lenses against Acanthamoeba spp comprising maslinic acid. In particular, it is a composition comprising a buffered isotonic saline solution at pH 7.2-7.6 and an amount of maslinic acid used, between 0.0016 and 0.004%, preferably between 0.003 and 0.004%.

DESCRIPTION OF THE INVENTION

The present invention allows eliminating the presence of ocular parasites of the Acanthamoeba genus, prevents them from becoming unhinged and degrading corneal tissue, without causing any toxicity to the cornea using maslinic acid as an active ingredient and at low concentrations

Through a colorimetric test (following the Alamar 81ue assay described in Martín-Navarro CM, Lorenzo-Morales J, Cabrera-Serra MG, Rancel F, Coronado-Áivarez NM, Piñero JE, Valladares 8 J Med Microbio! 2008 57: 1399-1404 }, the IC50 and the ICgo (concentration of the active substance that inhibits the growth of 50% and 90% of the cell population) for maslinic acid against strains of

Acanthamoeba

The composition for the maintenance of contact lenses against Acanthamoeba spp, object of the invention, comprises a buffered isotonic saline solution at pH 7.2-7.6 to which maslinic acid has been incorporated in a concentration between 0.0016 and 0.004%, preferably between

0.003 and 0.004%. The use of higher amounts of masl.inic acid does not have better effects, so it cannot be considered that there is a dose-dependent relationship, as shown in Figure 2.

A substance is considered cytotoxic when it exceeds a rate greater than 35%. [Lorenzo-Morales J, Martín-Navarro CM, López-Arencibia A, Santana-Morales MA, Afonso-Lehmann RN, Maciver SK, Valladares 8, Martínez-Carretero E. Therapeutic potential of a combination of two gene-specific small interfering RNAs against clinical strains of Acanthamoeba. Antimicrob Agents Chemother. 201 O Dec; 54 (12): 5151-5].

Experiences show a cytotoxicity of 18.09 ± 0.73% at IC50 and 31.63 ± 3.22 at IC90, which indicates zero or low toxicity of this product, and makes it appropriate for the treatment of both cystic form and amoeba trophozoite , compared to previous alternatives such as biguanides, highly toxic for

the corneal epithelium

Buffer solutions, also known as buffer or buffer springs,

5 are solutions that by the addition of moderate amounts of acids or bases

strong keep the pH almost constant.

EMBODIMENTS OF THE INVENTION

10 Example 1.-Isotonic saline solution buffered to pH 7.2-7.6 with maslinic acid

at 0.004%

This solution provided an excellent response obtaining an inhibition.

of the growth of 90% of the cell population (IC90), that is, 90% of the

15 cell population is not able to divide, and on the other hand, this solution

It allows to eliminate 90% of the amebic population in less than 96 hours.

Example 2.-Isotonic saline solution buffered to pH 7.2-7.6 with 0.002% maslinic acid

20 This solution provided an excellent response obtaining a 50% growth inhibition of the cell population, that is, 50% of the cell population is not able to divide.

Example 3.- Isotonic saline solution buffered to pH 7.2-7.6 with 0.0016% maslinic acid

This solution provided an excellent response obtaining a growth inhibition of 25% of the cell population.

These proposed concentrations are valid for both the cystic and trophozoite form of these amoebas.

In this way, a solution is obtained that prevents amoebas from multiplying, penetrating into the corneal tissue or entering their resistance period, cystic form, because the drug eliminates both cysts and trophozoites, in a concentration very low, without toxicity, compared to maintenance solutions

40 known.

Claims (3)

1.-Composition for the maintenance of contact lenses that is effective against Acanthamoeba spp comprising maslinic acid in a concentration 5 between 0.0016 and 0.004%. 2.-Composition for the maintenance of contact lenses that is effective against Acanthamoeba spp comprising a buffered isotonic saline solution at pH 7.2-7.6 And an amount of maslinic acid comprised between 10 0.0016 and 0.004%. 3.-Composition for the maintenance of contact lenses that is effective against Acanthamoeba spp according to claims 1 or 2, characterized in that the amount of maslinic acid used is between 0.003 and 0.004%.