WO2006079698A1 - Use of fucans for preparing a bacteriophobic and bacteriostatic material, corresponding material and applications - Google Patents

Abstract

The invention relates to the use of fucans having a molar mass ranging from 8,000 to 30,000 g/mol, preferably between 10,000 and 20,000 g/mol, for preparing a material that is bacteriophobic, bacteriostatic and inhibits the activity of the complement system as well as to a material impregnated with a solution that contains these fucans and, in particular, to a material provided in the form of an ophthalmic device such as contact lenses.

Description

USE OF FUCANS FOR THE PREPARATION OF BACTRIOPHOBIC AND BACTERIOSTATIC MATERIAL, CORRESPONDING MATERIALS AND APPLICATIONS

USE OF SELECTED FUCANES FOR PREPARATION

BACTERIOPHOBIC MATERIAL AND BACTERIOSTATIC,

CORRESPONDING MATERIAL AND APPLICATIONS

The present invention relates to the use of mass fucans

5 molar suitably selected for the preparation of a bacteriophobic material, bacteriostatic and inhibitor of complement system activity, to a material impregnated with a solution comprising such fucans, and in particular to a material in the form of an ophthalmic device.

The development of various materials with bacteriophobic and bacteriostatic properties, particularly for the manufacture of ophthalmic devices such as contact lenses, is of particular interest in order to reduce the risk of infection.

Indeed, the use of contact lenses for the correction of human vision involves an interaction between the surface of the lens and the tears, and in particular with the lachrymal proteins. These are the cause of contamination and degradation of contact lenses and are also involved in the adhesion of bacteria and inflammation of the eye. The adhesion of lachrymal proteins to the lens creates favorable conditions for inflammation and infection of the cornea. This adhesion is a function of the type of material constituting the lens. Attempts to inhibit the growth of bacteria and / or other microbes have already been made in the prior art, in particular in the European patent application EP-AI 050 314, by incorporation of silver in the contact lenses. .

Other approaches of the prior art in the preparation of carriers which possess such properties include drugs or heavy metals (International Application WO 2004/094075). However, the use of drugs can lead to the development of resistance mechanisms in bacteria and all undesirable side effects related to the use of heavy metals are not yet known. In order to reduce the risk of infection and inflammation caused by the wearing of contact lenses and consequently to provide a better visual comfort, a great deal of research has been directed towards the development of inhibitory or non-stimulating contact lenses of bacterial adhesion and proliferation.

Thus, it has already been proposed, in particular by US Pat. No. 6,514,517, to use solutions based on biocompatible acid precursors for coating materials with the aim of conferring on them bacteriopobic and inhibitory properties. microbial proliferation or even in US Pat. No. 6,592,814, contact lenses comprising an antimicrobial coating based on lactoferrin.

However, to date, the various solutions proposed are not entirely satisfactory, particularly because of the side effects of the different active ingredients used.

The inventors have therefore set themselves the goal of developing a material exhibiting bacteriopobic and bacteriostatic properties, which does not, however, exhibit the side effects heretofore generated by the materials of the prior art. The inventors have also set themselves the goal of providing materials that provide a better user comfort, in particular because of a decrease or a suppression of the inflammatory response.

On this occasion, the inventors have demonstrated that certain fucans or fractions of fucans of suitably selected molar mass, previously known for their anticoagulant activity or their properties of inactivation of the complement system, also have bacteriopobic and bacteriostatic properties which can be used to confer these properties on a material, generally biocompatible, which is impregnated.

Fucans are sulphated polysaccharides of high average molecular weight (100,000 to 800,000 g / mol), extracted from the cell wall of brown algae thalli and consist of a heterogeneous population of molecules mainly comprising L-polymers. sulphated fucose which may also contain D-xylose, D-galactose and uronic acids.

Fucans possess a variety of biological activities: they have been shown to possess anticoagulant and antithrombotic activities (Nishino T. et al., Carbohydr Res., 1992, 229, 355-362, Springer GF et al, Proc. Soc., Exp Biol Med, 1957, 94, 404-409, Church FC et al., J. Biol Chem, 1989, 264, 3618-3623; Bernardi G. and Springer GF, J. Biol. Chem., 1962, 237, 75-80; Mori H. et al., "Marine Algae in Pharmaceutical Science", Vol. 2, HA Hoppe, T. Leuring (Eds.), Walter De Gruyter, Berlin-New York, 1982), antiviral (Baba M. et al, J. AIDS, 1990, 3, 492-493), antiangiogenic (Hahnenberger R. et al., Glycoconjugate J., 1991, 8, 350-353) and anticomplementary (C. Blondin et al, 1994, Mol Immunol., 31, 247-253 ).

Moreover, further studies have shown that fucans with a molar mass of between 5,000 and 100,000 g / mol have the property of inhibiting the activation of complement and can therefore be used as anti-inflammatory agents (C. Blondin et al, Biomaterials, 1996, 17, 597-603, B. Tissot et al, BBA, 2003, 1651, 5-16).

Fucan preparations of average molar mass less than 20,000 g / mol, or even 10,000 g / mol, which also facilitates their use in a therapeutic context, have been obtained, for example, by mild acid hydrolysis or by radical degradation. fucans of high molar mass as described in particular in European Patents EP 0 403 377 and EP 0 676 207.

In the following discussion, the term "fucane" will include both high molecular weight fucans and lower molecular weight preparations (fractions) obtained therefrom. To date, it has never been envisaged or demonstrated that fucans having a molar mass of between 8,000 and 30,000 g / mol inclusive could have bacteriopobic and bacteriostatic properties. This discovery is the basis of the present invention. In addition, the inventors have now shown that the presence of these fucans in a material reduces the adhesion of bacteria on this material and their proliferation.

These new properties of fucans with a molar mass as defined above are the basis of the present invention.

In a first aspect, the invention therefore relates to the use of at least one fucan extracted from phéophycées, said fucane having a molar mass of between approximately 8 000 and 30 000 g / mol inclusive, for the preparation of a bacteriophobic and bacteriostatic material. According to a preferred embodiment of the present invention, the fucans have a molar mass of between about 10,000 and 20,000 g / mol inclusive.

The molar mass of the fucans which can be used in accordance with the invention can be determined by steric exclusion chromatography according to the techniques conventionally known for that purpose by those skilled in the art.

Preferably, the fucans used according to the invention are obtained by the implementation of a method for the controlled lysis of crude fucans as described in patent EP 0 403 377 or else by radical degradation according to the process described in the patent. EP 0 676 207.

Advantageously, a fucan containing, in addition, glucuronic acid is used. In this case, the amount of glucuronic acid, which is determined by the method described by Filiseti-Cozzi and Carpita, (Anal Biochem., 1991, 197 (1), 157-162), is between 0.5 and 20 g per 100 g of fucan. The content of sulphate groups present in the fucans used in accordance with the invention is preferably between 10 and 50 g per 100 g of fucan; the sulphate content being determined by elemental analysis of sulfur and applying the following relationship:% sulphate groups = 3.22 x S (%).

Fucans having a molar mass of between 10 000 and 20 000 g / mol, a sulphate group content of between 10 and 50 g per 100 g of fucan and containing from 0.5 to 20 g of glucuronic acid per 100 g of fucan are particularly preferred because they have a particularly remarkable bacteriophobic and bacteriostatic activity.

In a second aspect, the invention also relates to a bacteriophobic and bacteriostatic material, characterized in that it is impregnated with a solution, in a physiologically acceptable medium, of at least one fucane with a molar mass of between about 8 000 and 30,000 g / mol inclusive, preferably with a molar mass of between 10,000 and 20,000 g / mol inclusive.

In addition to its bacteriophobic and bacteriostatic properties, the fucan impregnated material according to the invention also advantageously has an activity of inhibition of the complement system because of the already known properties of said fucans. Within said solution, the fucan (s) are preferably present at a concentration of between 15 and 150 mg / ml of solution.

The physiologically acceptable medium of the fucan solution is preferably selected from water, physiological saline and buffered aqueous media compatible with cells and biological media.

The solution containing the fucan (s) and impregnating the material may further comprise one or more additional compounds, such as for example collagen (and in particular a collagen gel) or adhesive proteins such as fibronectin, vitronectin, laminin, In this case, the fucan / collagen weight ratio in said solution is preferably between 10 and 50 approximately.

According to a preferred embodiment of the invention, said material consists of a solid material based on polymers that may for example be chosen from acrylic and methacrylic polymers such as, for example, polymethyl methacrylate (PMMA), copolymers of alkylsiloxane methacrylate and methyl methacrylate, poly (hydroxyethylmethacrylate)

(PHEMA) weakly crosslinked copolymers of hydroethylmethacrylate (HEMA) and vinylpyrrolidone, copolymers HEMA / acrylamide or copolymers

HEMA / alkyl methacrylate, said copolymers being preferably in the form of hydrogels.

According to a particularly preferred embodiment of the invention, the material is an ophthalmic device, and especially a contact lens. Because of the properties evidenced by the inventors for fucans as defined above and their already known properties, the wearing of such contact lenses impregnated with fucane provides a better visual comfort.

For example, when the impregnated material is a contact lens, it then contains about 0.2 to 2 mg of fucan per lens.

Furthermore, the material according to the invention, and in particular when it is in the form of contact lenses has the additional advantage of constituting a progressive release system fucans which it is impregnated. Thus, in the particular case of contact lenses, the fucans released in the tear fluid give the latter anti-inflammatory properties and modulation of cell proliferation.

All commercially available contact lenses can thus be impregnated with the fucan solution as defined above. Among the different types of contact lenses, mention may be made in particular of lenses made of PMMA, copolymers of alkylsiloxane methacrylate and methyl methacrylate, hydrogel-type lenses prepared from poorly crosslinked PHEMA, lenses made of of HEMA / vinylpyrrolidone copolymer, of HEMA / acrylamide copolymer or of HEMA / methacrylate copolymer.

The material impregnated with fucans according to the present invention may be prepared by the implementation of any impregnation method known from the prior art. The skilled person will obviously choose the method that will be most appropriate to the nature of the material and / or its subsequent use. Among these methods, mention may be made in particular of the simple immersion of a naked material in a fucan solution as defined above. In this case, the impregnation time may for example vary between about 10 and 60 minutes. Obviously, the duration of impregnation may vary depending on the nature of the material used, the concentration of the fucan solution and their molar mass and finally depending on the temperature at which impregnation is performed.

The material thus impregnated can then be dried using any conventional drying method well known to those skilled in the art and appropriate to the type of material used such as for example a drying under air flow under sterile conditions. or drying in an oven or be stored and / or used directly.

The materials thus prepared can then be stored under appropriate conditions which do not significantly alter the properties of the material, preferably under sterile conditions. For example, in the case of contact lenses, the possible drying is carried out under sterile conditions and in particular under a laminar flow hood and the lenses can be stored in a hydrated condition in a solution of fucans, for example identical to that used to their preparation. The present invention will be better understood with the aid of the additional description which follows, which refers to examples relating to the preparation of contact lenses impregnated with fucans and to the study of the fucan desorption of this contact lens. , as well as the demonstration of the bacterophobic and bacteriostatic properties of said contact lenses.

EXAMPLE 1 PREPARATION OF IMPREGNATED CONTACT LENSES OF FUCANE

A fucan of molar mass equal to 20,000 g / mol, having a sulfate content of 27.3 g per 100 g of fucan and further containing 3.8 g of glucuronic acid per 100 g of fucan, was solubilized in a PBS buffer at a concentration of 15 mg / ml and then filtered on a MF-Millipore filtration membrane 33 mm in diameter, GS type 0.22 microns, sold by the company Millipore. The pH of the solution thus obtained was 7.2.

Acrylic type soft contact lenses with a diameter of 14.1 mm, commercially available under the reference ACUVUE Brand from Vistakon, Johnson & Johnson (Jacksonville, Florida, USA) were immersed for 20 minutes at room temperature in the fucan solution prepared as described above. The lenses were then washed twice with an aqueous solution of PBS sold by Invitrogen-Life Technologies (Cergy Pontoise, France) and having the following composition (per liter of distilled water):

- KCl: 0.2 g

- KH ₂ PO ₄ : 0.2 g

- NaCl: 8 g

- Na ₂ HPO ₄ : 1.15 g - MgCl ₂ .6 H ₂ O: in sufficient quantity (qs)

- CaCl ₂ .2 H ₂ O: qs

Unless otherwise indicated, this aqueous solution of PBS is the one that has been used in all the examples illustrating this application.

The lenses were then drained and dried under a laminar flow hood at room temperature for 16 hours. The amount of fucan was about 0.2 mg / lens. Identical lenses immersed under the same conditions in a solution of fucan at a concentration of 150 mg / ml, contained, after impregnation, of the order of 2 mg of fucan / lens.

EXAMPLE 2 Study of the Release of Fucanates from Previously Impressed Contact Lenses

The contact lenses impregnated with fucans and as prepared as indicated above in Example 1 were transferred into 1 ml of a solution of artificial tears ("tear-like fluid") consisting of human plasma diluted to 5% in an aqueous solution of phosphate buffer supplemented with lysozyme (4.7 g) and lactoferrin (1.7 g).

The study of the kinetics of fucan release from contact lenses was carried out over a period of 15 days, in 24-well plates, using the toluidine blue (BDT) assay, a colorimetric assaying and quantifying sulfate functions of a sulfated polysaccharide and as described by Smith PK et al., Anal. Biochem., 1980, 109, 466-473.

The results obtained are reported in Table I below: TABLE I

All these results show that the release of fucan increases with time to reach a plateau at the end of the tenth day (J10); the entire fucan is then released. EXAMPLE 3: BACTERIOPHOBIC PROPERTIES OF IMPREGNATED CONTACT LENSES

fucans

1) Preparation of cultures of radiolabelled bacteria Mini-cultures were prepared by incubating respectively with

30 ^° C. and at 37 ^° C. overnight, a bacterial colony of Pseudomonas aeruginosa or of Staphylococcus aureus in 1 ml of Mueller-Hinton's medium (MH) sold by the company BIO-RAD and comprising, per liter of distilled water 2 g of meat infusion, 17.5 g of casein hydrolyzate and 1.5 g of starch, 17 g of agar, 4% of NaCl, 20-25 mg of Ca ⁺⁺ and 10-12 , 5 mg of Mg ⁴⁺ . The bacterial suspension thus obtained is then centrifuged at 3500 rotations per minute (rpm) for 10 minutes. Once the supernatant has been removed, the pellet is resuspended in 1 ml of MH medium and the bacterial suspension is homogenized by vortexing.

100 μl of tritiated thymidine (1 mCi / ml) are added to 1 ml of MH broth containing 10 CFU (colony-forming unit) / ml of bacteria. The bacterial suspension is incubated for 3 hours at 30 ^° C. or 37 ^° C. so as to allow the growth of the two bacterial strains Pseudomonas aeruginosa or Staphylococcus aureus and to obtain cultures in the exponential phase of proliferation. At the end of the incubation period, the bacterial suspension is centrifuged at 3500 rpm for 10 minutes to remove unbound radioactivity. The pellet is resuspended in the aqueous solution of PBS as described in the example

1 and further containing 2% bovine serum albumin (BSA) in order to obtain bacterial concentrations of between 10 ⁶ and 10 ⁷ CFU / ml.

Bacterial concentrations were measured by counting the colony forming units and counting the incorporated radioactivity.

2) Study of the bacterophobic properties of fucan impregnated lenses

The fucan-impregnated contact lenses obtained as described in Example 1 were transferred sterilely into 24-well plates and then incubated with the solution of artificial tears as described above with the example

2 for 1 hour at room temperature, with gentle stirring. After incubation, the lenses were placed in contact with 1 ml of each bacterial suspension for 1 hour at 30 ^° C. or 37 ^° C. (depending on the bacterial strain) with gentle stirring. After washing the lenses with the aqueous solution of PBS, the lenses are transferred to counting flasks containing 10 ml of scintillation liquid. After homogenization by vortexing, the radioactivity incorporated by the bacteria adhered to the lenses is measured by the β scintillation counter type Tri carb 2100 TR sold by Packard Bioscience Company, Orsay, France).

The values of the percentages of adhesion obtained after one hour of contact of the bacteria with the fucan impregnated lens were compared with those obtained with a contact lens not impregnated with fucan (control).

The results are reported in Table II below: TABLE II

^a : Chaque valeur indiquée représente la moyenne calculée sur 60 valeurs.

^a : Each value shown represents the average calculated on 60 values.

These results show that the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa bacteria is significantly decreased on fucan-impregnated lenses compared to the same non-impregnated lenses, with an increase in the percentage inhibition of bacterial adhesion of 23 ± 4% and 31 ± 4% respectively compared to the control.

EXAMPLE 4: BACTERIOSTATIC PROPERTIES OF FUCANE IMPREGNATED CONTACT LENSES

The study of the proliferation of Staphylococcus aureus and Pseudomonas aeruginosa bacteria adhered to fucan-impregnated contact lenses consists in carrying out a first step of adhesion of the radiolabelled bacteria to tritiated thymidine on contact lenses previously incubated with the solution of tears. as described in Example 3. The lenses are washed 5 times with an aqueous solution of PBS and then transferred to tubes containing the proliferation medium MH.

The second step consists of a kinetic study of the proliferation of bacteria adhered to the surface of the lenses. For each measurement: (a) the fluid phase containing the bacteria spontaneously detached from the lenses (released bacteria) during the proliferation is recovered after 3 successive washes with the aqueous solution of PBS and then centrifuged for 3 minutes at 3500 rpm and resuspended in 1 ml aqueous solution of PBS. The number of bacteria expressed in CFU / ml is determined after dilution of the samples taken and spread on agar. To do this, 50 μl of each diluted sample (dilution factors: 10 ² , 10 ³ , 10 ⁴ , 10 ⁶ ) were plated on MH agar.

(b) the bacteria adhered to the lenses were detached with trypsin (pure trypsin at 37 ° C. with gentle shaking for 5 minutes) and then the fluid phase was recovered, centrifuged for 3 minutes at 3500 rpm and resuspended in 1 ml of aqueous solution of PBS. The number of bacteria expressed in CFU / ml was determined after dilution of the samples taken and spread on agar as above.

The proliferation of bacteria adhered to the lenses corresponds to the sum of the number of bacteria released and the number of bacteria adhered. The control (control without a lens) corresponds to the proliferation in suspension, in the absence of a lens, of a number of bacteria equal to the number of bacteria adhered to the lenses after 1 hour of incubation of the lenses with the bacterial suspensions.

Moreover, the effectiveness of detachment of bacteria adhered to the surface of the lenses was monitored by comparing the number of radiolabelled bacteria adhered to the lenses, measured before and after detachment. It appears that 92% of adhered bacteria are detached and alive.

The proliferation of bacteria was also studied on non-impregnated lenses of fucan (control lenses). In order to compare the proliferation rates of bacteria on lentils and proliferation in suspension in the absence of a lens, various proliferation parameters were taken into account: * The increase in the bacterial population (ΔLog) after 5 hours of proliferation of the bacteria, is expressed in the form Lo g CFU / ml and calculated from the following formula (1):

ΔLog = LogN ₅ - LogNo (1) in which:

N ₀ : bacterial concentration introduced initially; N ₅ : bacterial concentration obtained after 5 hours of proliferation; t K is the proliferation rate constant, expressed in number of generations per hour and calculated from the following relation (2):

K = LogNni-LogNn ₂ / 0,301 xt (generations / hour) (2) in which:

Nn ₁ is the number of bacteria at time t]; Nn ₂ is the number of bacteria at time t ₂ ; t: the time required to increase the number of bacteria from Nn ₁ to Nn ₂ ;

4 g is the doubling time, expressed in hours, g = 1 / K (hour / generation);

* S represents the stimulation of proliferation rates of bacteria compared to control (in the absence of lenses).

S = [K (lenses) / K (control)] x 100

The results are reported in Tables III and IV below: TABLE III

Proliferation of Staphylococcus aureus

Proliferation parameters

ΔLog K g S (UFC Log / lens) (generations / h) (min)

Control lens 1.23 ± 0.06 1.05 ± 0.06 57.1 ± 3 167 ± 9

Fucan lens 0.58 ± 0.03 0.62 ± 0.03 96.8 ± 5 93.7 ± 5

Control 0.55 ± 0.02 0.64 ± 0.03 93.7 ± 6 100 ± 0 (without lens) TABLE IV

Proliferation of Pseudomonas aeruginosa

Proliferation settings

ΔLog K g S (UFC Log / lens) (generations / h) (min)

Control lens 1.85 ± 0.08 1.32 ± 0.05 45.4 ± 1, 7 245 ± 16

Fucan lens 0.45 ± 0.02 0.55 ± 0.03 109.1 ± 6 103 ± 4

Control 0.42 ± 0.02 0.56 ± 0.02 107.1 ± 7 100 ± 0 (without lens)

These results show that the proliferation of bacteria adhered to the fucan impregnated lenses is lower than that obtained with the control lenses and is of the same order of magnitude as that obtained from the bacteria in suspension (control without lens).

All of these results therefore show that the impregnation of material with a solution containing fucans of molecular weight suitably selected in accordance with the invention makes it possible to confer on materials such as ophthalmic devices, bacteriopobic and bacteriostatic properties and thus of improve the comfort of use of the subjects carrying these devices.

Claims

1. Use of at least one fucan extracted from pheophyceae, said fucan having a molar mass of between 8,000 and 30,000 g / mol inclusive, for the preparation of a bacteriophobic and bacteriostatic material. 2. Use according to claim 1, characterized in that the fucan (s) have a molar mass of between 10,000 and 20,000 g / mol inclusive. 3. Use according to claim 1 or 2, characterized in that the fucan (s) further contain glucuronic acid in an amount of between 0.5 and 20 g per 100 g of fucan. 4. Use according to any one of the preceding claims, characterized in that the fucan (s) have a sulphate group content of between 10 and 50 g per 100 g of fucan. 5. Bacteriophobic and bacteriostatic material, characterized in that it is impregnated with a solution, in a physiologically acceptable medium, of at least one fucan with a molar mass of between approximately 8,000 and 30,000 g / mol inclusive, preferably between 10,000 and 20,000 g / mol. 6. Material according to claim 5, characterized in that the solution containing the fucan (s) also contains collagen in a weight ratio fucan / collagen between 10 and 50. 7. Material according to claim 5 or 6, characterized in that it consists of a solid material based on polymers chosen from acrylic and methacrylic polymers, copolymers of alkylsiloxane methacrylate and methyl methacrylate, the poly (hydroxyethyl methacrylate) weakly crosslinked, the copolymers of hydroethyl methacrylate (HEMA) and vinylpyrrolidone, the HEMA / acrylamide copolymers or the HEMA / alkyl methacrylate copolymers. 8. Material according to any one of claims 5 to 7, characterized in that it consists of an ophthalmic device. 9. Material according to claim 8, characterized in that it consists of contact lenses. 10. Contact lens according to claim 9, characterized in that they contain between 0.2 and 2 mg of fucan per lens. 11. Material according to any one of claims 5 to 10, characterized in that it constitutes a progressive release system of fucans which it is impregnated. 12. Material according to any one of claims 5 to 11, characterized in that it further exhibits an activity of inhibition of the complement system.