WO2012013577A1 - Composition for coating medical devices containing lae and a polycationic amphoteric polymer - Google Patents

Abstract

The invention relates to antimicrobial compositions containing LAE, a polycationic or amphoteric polymer material and optionally an auxiliary material from the group consisting of cationic and amphoteric surfactants. The antimicrobial composition preferably contains polycationic material or amphoteric polymer material which is selected from the group consisting of poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PAI), poly(vinylbenzyltrimethylamine) (PVBT), polyaniline (PAN), polypyrrole and poly(pyridinium acetylene) ) for the polycationic and octylacrylamide/acrylates/butylaminoethyl methylacrylate polymer ( Amphomer®).

Description

COMPOSITION FOR COATING MEDICAL DEVICES CONTAINING LAE AND A POLYCATIONIC AMPHOTERIC POLYMER

TECHNICAL FIELD

The present invention relates to novel combinations for the treatment of medical devices. BACKGROUND ART

A large number of medical devices such as urinary catheters, endotracheal tubes, central venous catheters are used in patient care and are a leading cause of hospital-acquired infections in high risk patients. Such high-risk patients include critically ill patients admitted to intensive care units (ICU), cancer patients and patients with chronic diseases requiring long term care, such as those on total parenteral nutrition. Anti-infective devices coated with antimicrobial agents have been shown to significantly decrease the risk of device-related and hospital- acquired infections. Most nosocomial (hospital-acquired) infections are caused by contamination of medical devices. In addition, patients undergo a vast number of surgical procedures in the United States. Current data show 27 million procedures performed per year and post-surgical site infections occur in approximately 2-3 % of these cases. Whenever medical devices are used in surgical settings the risk of infection is increased.

Many medical devices could not exist without today's advanced coating

technologies. First, without antimicrobial coating, the rate of nosocomial infection would sky-rocket. Second, without proper biocompatible coatings, any device exposed to blood for more than a few minutes could lead to thrombi and third, upon contamination most of these devices form microbial biofilms, and antibiotics and antimicrobials are not very effective in preventing the formation and proliferation of such microbial films, since their activities are primarily robust against free floating bacteria, but not on biofilms. Fourth, even if the bacteria are killed at the sites of devices, this can leave toxins released from the action of the antibacterial at the surface, which might lead to toxic shock and sepsis. Many devices are coated with technologies which handle one or two of the above problems but which do not include all treatments.

Significant prior art exists on the use of antimicrobial agents in coating devices. US 2006/ 02633404A1 provides an assembly of preparations of medical devices having a porous coating comprising hydrogen peroxide for catheters, such as urinary catheters, endoscopes, laryngoscopes, tubes for feeding, tubes for drainage, guidewires, condoms, urisheets, barrier coatings, for example gloves, stents and other implants, extracorporeal blood conduits, membranes, for example for dialysis, blood filters, devices for circulatory assistance, dressings for wound care and ostomy bags. WO2007/062330682 discloses methods for coatings or impregnating surfaces with antimicrobial agents that involve coating the surface with a composition including antimicrobial agents and solvents, in curing surfaces by applying heat. W01 /95876 teaches the coating of medical devices with antibiotics and quaternary ammonium compounds to prevent bacteria from growing on the devices. European Patent Application 0590348A1 teaches the coating of venous catheters with lipodalbahepatide antibiotics for preventing development of catheter-related infections. WO2007/050565A2 relates to the coating of medical devices with bactericidal and bacteriostatic coatings to prevent infection. US Patent 5752941 discloses central venous polyurethane catheters with a thin hydrophilic coating loaded with antibiotics to prevent catheter related infection. US 2008/0020127A1 describes the coating using the bridged polycyclic compounds with quaternary ammonium moieties to prevent infection.

US2008/026080482A1 provides a composition containing polyquat to coat medical devices preferably contact lens to prevent bacterial infection. US2006/0263444A1 describes compositions containing cationic surfactants lauric arginate and antimicrobial metals such as silver and silver compounds for coating medical devices. US2007/02252220A1 discloses compositions containing cationic surfactants lauric arginate and antibiotics for coating medical devices.

DISCLOSURE OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION As indicated above the prior art does not cover multiple problems such as infection, formation of thrombi, biofilm formation proliferation and bacterial toxins left over by the antibacterial action on the devices. In addition, most antibacterial coatings disclosed in the prior art teach the use of antibiotics and metals, which tend to form resistant microorganisms which could lead to serious infections. This invention relates to a multifunctional coating of medical devices to provide control of bacterial infection, prevention of the formation of biofilms, to inhibit local thrombosis and to bind and neutralize toxins.

This is achieved through the use of broad spectrum antibacterial ethyl lauroyl arginate in combination with polycationic materials to enhance the retention and prevention of elution of LAE from such devices. Cationic surfactants are known as preservatives used in food, cosmetic and pharmaceutical industry. Cationic surfactants have turned out to be highly effective against microbial proliferation and at the same time safe for intake in humans and mammals in general. For all of this, cationic surfactants are an attractive tool in the industry.

It has been demonstrated that cationic surfactants according to formula (1 ) derived from the condensation of fatty acids and esterified dibasic amino acids are highly effective protective substances against microorganisms.

where: X^" is a counter ion derived from an organic or inorganic acid, preferably Br^", CI^" or HS0₄^", or an anion on the basis of a phenolic compound;

R-i: is a straight a Iky I chain from a saturated fatty acid or hydroxy I acid having from 8 to 14 atoms linked to the a-amino acid group via an amidic bond;

R₂: is a straight or branched a Iky I chain from 1 to 18 carbon atoms or an aromatic group;

R₃: is

— NH3

or

where n is from 0 to 4.

The organic acids which may be the source of the counter ion X^" can be citric acid, lactic acid, acetic acid, fumaric acid, maleic acid, gluconic acid, propionic acid, sorbic acid, benzoic acid, carbonic acid, glutamic acid or other amino acids, lauric acid and fatty acids such as oleic acid and linoleic acid, whereas the inorganic acids can be phosphoric acid, nitric acid and thiocyanic acid. The phenolic compound which may be the basis of the anion X^" is for instance butylated hydroxyanisole (BHA) and the related butylated hydroxytoluene, tertiary butyl hydroquinone and parabens such as methylparaben, ethylparaben, propylparaben and butylparaben. The most preferred compound of the above class of compounds is the ethyl ester of the lauramide of the arginine monohydrochloride, hereafter referred to as LAE (CAS No. 60372-77-2). This compound is now well-known for its use as an antimicrobial agent. In practical use LAE turned out to be well tolerated and to display a very low toxicity to human beings. LAE has the chemical structure of formula (2) displayed hereafter.

The compound LAE is remarkable for its activity against different micro-organisms, like bacteria, moulds and yeasts which can be present in food products (WO 03/034842) and also in cosmetic formulations and preparations (WO 03/013453, WO 03/013454 and WO 03/043593). The compound has been furthermore described for its effect on parasites in fish, such as on the larvae of Anisakis or other species (European application 07 382 004.5). Its preservative action is particularly pronounced in a combination with a polyene fungicide such as natamycin (PCT/EP2007/060598). It has furthermore been shown to be effective for killing endospores and for having an effect in virus infections (European application 08 382 025.8). The specific use for the protection of teeth against dental erosion has been described (European application 08 382 007.6).

The general preparation of the cationic surfactants is described in Spanish patent ES 512643 and international patent applications WO 96/21642, WO 01/94292 and WO 03/064669.

LAE, also known as lauric arginate, is manufactured by Laboratories Miret, S.A. (LAMIRSA, Spain). Lauric arginate is listed by the FDA (Food and Drug Administration) as being a GRAS substance (Generally Recognized As Safe) under GRN 000164. The USDA (United States Department of Agriculture) has approved its use in meat and poultry products (FSIS Directive 7120.1 ) and also as a processing aid for fresh meat and poultry products.

The metabolism of the above cationic surfactant of formula (2) in rats has been studied; these studies have shown a fast absorption and metabolisation into naturally-occurring amino acids and the fatty acid lauric acid, which are eventually excreted as carbon dioxide and urea. Toxicological studies have demonstrated that LAE is completely harmless to animals and humans. Therefore, LAE and related compounds are particularly suitable to be used in the preservation of all perishable food products. LAE and related compounds are equally suitable for use in cosmetic or medical products and in medical devices where growth of microorganisms is common. As has been remarked above, the cationic surfactants are remarkable for their inhibitory action over the proliferation of different microorganisms, such as bacteria, fungi and yeasts. The minimum inhibitory concentrations of LAE are shown in the following table 1 . Table 1

Kind Microorganism M.I.C. (ppm)

Arthrobacter oxydans ATCC 8010 64

Bacillus cereus var mycoide ATCC 1 1778 32

Bacillus subtilis ATCC 6633 16

Clostridium perfringens ATCC 77454 16

Gram + Bacteria Listeria monocytogenes ATCC 7644 10

Staphylococcus aureus ATCC 6538 32

Micrococcus luteus ATCC 9631 128

Lactobacillus delbrueckii ssp 1 act is CECT 372 16

Leuconostoc mesenteroides CETC 912 32

Alcaligenes faecalis ATCC 8750 64

Gram - Bacteria

Bordetella bronchiseptica ATCC 4617 128

It is preferred to dissolve the compound directly before use in one of the following preferred solvents of food grade: water, ethanol, propylene glycol, isopropyl alcohol, other glycols, mixtures of glycols and mixtures of glycols and water, diacetin, triacetin, glycerol, sorbitol, mannitol and xylitol. If the treatment shall be performed at a specific pH value the use of a corresponding buffer solution may be recommendable. On the other hand the compound can be easily used in its solid form or formulated with solid carriers such as salt, sugar, maltodextrine, hydrocolloids and sorbitol. For the cationic surfactants of the above formula (1 ) the antibacterial activity and the biological activity against other microorganisms such as fungi and yeasts is well documented. LAE as the preferred species of the cationic surfactants has been investigated intensively in the past. The compound is characterized through a combination of different properties.

The compound LAE has been proven to be highly suitable for the coating of surfaces. There is a large body of published literature supporting the mechanism of preventing bacterial attachment and subsequent plaque growth by the reduction of the surface energy, especially the polar component of this surface energy. The anti-attachment mechanism in the oral cavity provides a non-specific protection against bacterial adhesion. It is an advantage of this approach, that there is no risk, that the bacteria will become resistant. Measurement of the contact angle of sessile drops from liquids with a range of polarities was used to determine the polar component of the surface energy, y_s^p. In this case, y_s is reduced to 1 1 .5 dynes/cm on an LAE-coated surface, less than half of the original y_s^p of 23 dynes/cm on a bare or saliva-coated substrate (fig. 1 ). This reduction is expected to make the surface more resistant to bacterial attachment. These results indicate, that LAE was very effective at low concentration in reducing surface energy at protein coated surfaces such as mouth environment and resulting in the inhibition of bacterial attachment. Since the formation of antibiotic resistance is a major problem for the use of antibiotic and antibacterial products on devices, it was important to demonstrate that LAE was effective against known resistant organisms. A standard

antimicrobial test was run to assess this and the data showed that minimum inhibitory concentrations (MIC values) for susceptible and resistant strains were the same. For a MRSA (methicillin resistance Staphylococcus aureus) resistance strain of E. coli, the MIC of LAE was 16 tig/ml, for Staphylococcus aureus (MRSA) the MIC value was 8 ng/ml and for Acinetobacter Baumanii (MRSA) it was 16 iig/ml. This indicates, that LAE was effective against resistant strains and unlikely to suffer the deficiencies of antibiotic and metal ions such as silvers, which cause the development of resistant organisms resulting in the loss of efficacy of the devices in preventing infections. It is known in the art, that medical devices coming in contact with blood in the body will cause formation of thrombi. This could result in serious complications. Some devices have used immobilized heparin to control thrombi. However, the safety margin of heparin is very narrow and it also elutes from the surfaces rapidly. It has already been described, that LAE is highly effective in the inhibition of thrombus formation at 2.0 millimoles (Polyarkov, Ukr.Biochem.J, 2007; 79: 124-128.).

The polycationic materials used in the present invention with LAE have a plurality of positively charged groups along a polymer chain. For instance, suitable examples of such polycationic materials include, but are not limited to,

poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PAI),

poly(vinylbenzyltrimethylamine) (PVBT), polyaniline (PAN), polypyrrole and poly(pyridinium acetylene). Other cationic polymers include poly quaternium-6 and polyquaternium-7. In addition an amphoteric polymer having positive and negative charges such as Amphomer®, octylacrylamide/acrylates/butylaminoethyl methylacrylate polymer, is a preferred embodiment.

The effect of the addition of polycationic polymers according to the present invention is believed to be the enhancement of the retention of LAE on the surfaces.

A preferred combination is the combination of LAE with Amphomer^® at 0.2%. Preparations for the treatment of the medical devices are usually solutions containing LAE and the polycationic material, such as for instance poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PAI), poly(vinylbenzyltrimethylamine) (PVBT), polyaniline (PAN), polypyrrole and poly(pyridinium acetylene). The solution usually contains LAE in a concentration within the range of 0.0001 to 5% and the polycationic material in a concentration range of 0.1 to 10% . A preferred range for the concentration of LAE is 0.005 to 1 %. A preferred range for the concentration of the polycationic material is 0.005 to 1 % . It is particularly preferred to prepare a solution of LAE and the polycationic material which contains the two components in a ratio of LAE:polycationic material of 1 : 1 , 2.5 : 1 and 1 : 2.5. Preferred ratio values are 2.5: 1 and 1 :1 .

For instance for the preferred combination of LAE with Amphomer^® ,the solution shall contain the two components in a ratio of 2.5 : 1 .

Optionally an auxiliary material from the group consisting of cationic and amphoteric surfactants is present.

Suitable cationic surfactants to be added as auxiliary material may be selected from quaternary ammonium salts, amines with amide linkages, polyoxyethylene a Iky I amines, polyoxyethylene alicyclic amines, N , N , N ' , N '-tetrakis-su bstituted ethylenediamines and 2-alkyl-1 -hydroxyethyl-2-imidazoles.

Suitable amphoteric surfactants to be added as auxiliary material may be selected from glycinates, betaines and propionates or dipropionates. Suitable products are N-coco-3-aminopropionic acid or its sodium salt, N-tallow-3-iminodipropionic acid or its disodium salt, and N-carboxymethyl-N-dimethyl-N-9-octadecenyl ammonium hydroxide and N-cocoamidethyl-N-hydroxyethylglycine or its sodium salt.

The amount of the auxiliary material in the antimicrobial composition is between 0 and 10 parts by weight relative to 100 parts by weight of the antimicrobial composition, preferably between 0.1 and 5 parts by weight per 100 parts by weight of the antimicrobial composition, more preferably between 0.5 and 2.0 parts by weight per 100 parts by weight of the antimicrobial composition.

The manner of treatment of the medical devices is usually the application of a LAE solution (0.1 to 10%) for 1 to 5 minutes for killing bacteria, pathogenic fungi and viruses on hard surfaces. Medical devices which have been treated in this manner shall normally be kept in a dry and cool place. Under such circumstances the treated medical devices maintain their activity for at least 12 months. The novel and unobvious utility of this coating for multifunctional coating of medical devices will be obvious from the examples given below.

REFERENCE EXAMPLE. 1

EFFECT OF LAE ON URINARY CATHETER BIOFILMS:

A Drip-Flow Reactor (DFR) model described below was used to evaluate LAE on urinary and venous catheters:

In this experiment the effect of N"-lauroyl-L-arginine ethyl ester HCI (LAE) in the context of urinary catheter and central venous catheter biofilms was investigated. The efficacy of the compound was assessed on single species biofilms formed by Escherichia coli and Pseudomonas aeruginosa .

Single species biofilms were grown in an in-vitro model system consisting of the drip flow reactor (DFR) (fig. 2) which is designed to model a low shear

environment.

Inoculum and biofilm growth.

Single species biofilms were grown using isolates of Escherichia coli and

Pseudomonas aeruginosa. E. coli BDH 6057 and P. aeruginosa BDH 5990 were both isolated by Bozeman Deaconess Hospital from urine and are maintained at - 80°C as frozen stock cultures by the Medical Biofilms Laboratory at the CBE. The test organisms were cultured overnight from frozen stocks at 37°C in the test medium.

The DFRs were run in a 37°C incubator under aerobic conditions.

Approximately 20 minutes prior to inoculation, sterile medium was dripped-in and allowed to collect over coupons to form conditioning layer on the surface of HA- coated glass surfaces because biofilms are better retained on the slide surfaces for treatment and analysis. Each of the channel reactor was then inoculated with 1 .0 ml of an overnight culture. DFR was maintained in a horizontal position for two hours with no flow to enable bacterial attachment to the substratum. The reactor was set at 10 degree angle and sterile medium dripped through the reactor at 40 ml/hr, total 10 ml/hr per coupon. Initially the reactor was run for 3 days, this was reduced to one day. For urinary biofilm E. coli in artificial urine medium was used. For venous catheters P. aeruginosa in 10% brain heart infusion was used.

The effect of LAE on urinary catheter (E.coli ) biofilm is disclosed in the following table 2.

Table 2

Cone, of LAE ng/ml CFUIogl O, (20h post treatment)

0 4.4E10

20 1 .03E08

40 2.1 E07

50 1 .1 E07 The results indicated that LAE treatment was very effective (3 log difference) in reducing pre-formed E. coli biofilm (simulating urinary catheter contamination).

REFERENCE EXAMPLE 2:

INACTIVATION OF ENDOTOXIN PRODUCED BY E.COLI BIOFILM:

The amount of endotoxins in the above biofilms was also measured to assess whether LAE has concomitant effect on the endotoxins produced by the films. The results in the following table indicated that LAE was effective in neutralizing the toxins in addition to reducing biofilms: Table 3

CONC. LAE (ng/ml) Endotoxin(EU)

0 120000

20 60000

40 55000

50 40100 These results indicated the dual effect of LAE on disrupting biofilms and deactivating toxins produced by E.coli.

Polymixin B is a well known deactivator of endotoxins used clinically. Therefore, it was of interest to see how LAE compares with Polymixin B. Data from binding studies showed, that LAE was as effective as Polymixin B, already proven to be an inactivator of endotoxin, in deactivating endotoxins.

REFERENCE EXAMPLE 3.

EFFECT OF LAE ON VENOUS CATHETER BIOFILM:

Above flow reactor was used to assess the effect of LAE on P. aeruginosa strain 5990 biofilm, representing venous catheters biofilms. The results in the following table below indicated efficacy against those films: Table 4

CONC. LAE (,ug/ml) CFU/ML

0 7.9E03

10 3.4E03

15 3.24E03

40 4.4E02

The effect of LAE was more than 3 log difference on venous catheter biofilms. EXAMPLE 1 .

INHIBITION OF MICROBIAL BIOFILMS BY LAE.

A multi-species biofilm model (Guggenheim, J. Dental Res. 2001 ; 80, 363-370) was used to assess the effect of LAE in combinations with polycationic polymers to prevent growth and proliferation of microbial biofilms. This model simulated conditions in vivo of biofilms on protein coated surfaces in the human body.

Briefly, multi-species of bacteria and yeast were used in batch culture process, with multiple exposures to microbes and treatment. The organisms used were Streptococcus oralis, Streptococcus sobrinus, Actinomyces naeslundii, Veillonelia dispar, Fusobacterium nucleatum and Candida albicans. The substrate used for the biofilm formation consisted of hydroxyapatite (bone mineral) disc coated with saliva proteins to simulate body fluids like saliva and serum. Organisms were maintained alive by adding 100% saliva and bacteria growth medium. The sampling was done at 30 min, 16.5 hrs, 40.5 hrs and 65.5 hrs. The composition of the film was analyzed by culture technique, dead/live bacteria by Sytox stain and automated image analyses (IBES) of immunofluorescence. The solutions tested were control (vehicle), LAE combined with polycationic materials which included poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PAI),

poly(vinylbenzyltrimethylamine) (PVBT), polyaniline (PAN), polypyrrole and poly(pyridinium acetylene). The other cationic polymers included poly quaternium- 6 and polyquaternium-7. In addition, an amphoteric polymer having positive and negative charges such as octylacrylamide/acrylates/-butylaminoethyl

methylacrylate polymer (Amphomer ® ) was also tested.

The results are in the following table, counting of the microorganisms was conducted 65.5 hours after treatment. Table 5

The results indicate, that the combination of LAE + Amphomer® was effective in suppressing on a sustained basis (65.5h) the growth and proliferation of microbial biofilm on surfaces simulating human body conditions.

Similar results were obtained when Amphomer® was replaced by PAH, PAI, PVBT and PAN.

EXAMPLE 2.

METHODS OF COATING MEDICAL DEVICES WITH LAE AND POLYCATIONICS.

A combination of 1 % LAE solution with polycationics in the cone, of 0.5% either PAH, or PAI, or PVBT or PAN, was prepared in water, glycerin or propylene glycol at pH from 4 -7 for coating. The coating was accomplished via several ways. One coating process embodiment involved solely dip-coating and dip-rinsing steps. Another embodiment of coating process was by spray-coating and spray rinsing steps. However, a number of alternatives which involves various combinations of spraying and dip-coating and rinsing steps may be designed by a person ordinarily skilled in the art. One dip-coating alternative involved steps applying polycationic material to the material of the medical device by immersing said device in a solution of a polycationic material, optionally drying, followed by coating with LAE solutions. This bi-layered process can be repeated to achieve the desired film thickness of LAE on the surfaces.

Thus, the combination of LAE plus polycationics for coating medical devices provides antimicrobial activity, prevents proliferation and growth of biofilms, kills antibiotic resistant organisms, inactivates bacterial toxins and prevent thrombi when the devices are exposed to blood.

Claims

1 . Antimicrobial composition containing LAE, a polycationic or amphoteric polymer material and optionally an auxiliary material from the group consisting of cationic and amphoteric surfactants.

2. The antimicrobial composition of claim 1 , in which the polycationic material or amphoteric polymer material is selected from the group consisting of

poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PAI),

poly(vinylbenzyltrimethylamine) (PVBT), polyaniline (PAN), polypyrrole and poly(pyridinium acetylene) ) for the polycationic and Amphomer^®,

octylacrylamide/acrylates/butylaminoethyl methylacrylate polymer.

3. The antimicrobial composition of claim 1 , containing LAE in a concentration within the range of 0.0001 to 5% and the polycationic material or amphoteric material in a concentration range of 0.1 to 10%.

4. The antimicrobial composition of claim 1 , in the combination of LAE with Amphomer^® at the ratio value of 2.5: 1 .

5. Solution of the antibacterial composition of claim 1 in a suitable solvent.

6. Use of the antibacterial composition of any of claims 1 to 3 or the solution of claim 5 for the treatment of medical devices.

7. The use of claim 6, wherein said device is a urinary catheter.

8. The use of claim 6, wherein said device is a venous catheter.

9. The use of claim 6, wherein said device is a contact lense.

10. The use of claim 6, wherein said device is an endoscope.

1 1 . The use of claim 6, wherein said device is tubes for feeding and drainage.

12. The use of claim 6, wherein said devices are guide wires, stents and implants and extra corporeal blood conduits such as membranes for dialysis filters, blood filters.

13. The use of claim 6, wherein said devices wound closure covering such as staples, meshes, wound coverings filier sutures, tissue adhesives and tissue sealants and absorbable and non-absorbable hemostats.

14. The use of claim 6, wherein said devices are tracheal devices and prosthetic devices such as for hips, knees and heart valves.