ES2359321B1 - POLY? HYDROPHILE MEROS AS BIOACTIVE COMPOUND RELEASE SYSTEMS IN SURGICAL APPLICATION BADS. - Google Patents

Abstract

Hydrophilic polymers as release systems for bioactive compounds in meshes for surgical application. # The present invention relates to a mesh prosthesis comprising a medical mesh impregnated with a bioactive acrylic copolymer, characterized in that the amount of bioactive acrylic copolymer is between 5 and 40% w / w with respect to the total and the bioactive acrylic copolymer comprises: between 30 and 95% w / w of one or more monomers with hydroxyl groups; between 5 and 55% w / w of one or more monomers with sulfonic groups; and between 0.01% and 50% w / w of a bioactive agent, and when using said mesh prosthesis, surgical interventions for the repair of hernia processes.

Description

Hydrophilic polymers as release systems for bioactive compounds in surgical meshes.

The present invention relates to the development of bioactive agent release systems, preferably antibiotics, based on hydrophilic polymers for use as prosthetic coatings in the form of a polymeric mesh with special interest in abdominal surgical applications. Therefore the present application falls within the field of medicine.

Prior art

The repair of blood processes is one of the most frequent surgical interventions in general surgery behind that of cataracts in the US. When major abdominal wall defects are involved: incisional hernias, (usually secondary to previous laparotomies), tumor removal, etc. The placement of a replacement and / or reinforcement biomaterial is nowadays almost completely agreed by all surgeons. The introduction for the first time of a polypropylene mesh by Uscher in 1962 was considered one of the best advances achieved in this surgery [Uscher, Hernia repair with marlex mesh. Arch Surg., 1962. 84: p. 325-8).]. The positive effects derived from the use of abdominal meshes has stimulated the search for optimal meshes with high biocompatibility and low cell adhesion. There are many meshes made of resorbable, non-absorbable synthetic materials or organic material derived from humans or pigs on the market. This wide variety of systems implies important differences in the interaction of the mesh with the microorganisms after their implantation. One of the most devastating consequences of using a prosthetic material is the appearance of an infection. In the repair of large hernias where the patient undergoes several surgical interventions, the incidence of infection can reach 10%. The microorganisms involved in these infections correspond to bacteria on the patient's skin, specifically Staphylococcus aureus (Sa) and Staphylococcus epidermidis (Se). The infection can alter the integration of the biomaterial in the tissue and therefore the healing process of the patient who may suffer another hernia and therefore another operation. The adhesion of a bacterium to the surface of a biomaterial is the determining step in the pathogenesis of the infection. Some microorganisms are capable of forming a biofilm or biofilm on the mesh that protects them from the immune system and the action of antibiotics. The bacterium is wrapped and protected, thereby achieving strong and irreversible adhesion to the surface of the implant surviving on it.

Once the mesh has been infected there is no possible treatment and the only solution is to remove the prosthesis. In the infection prevention strategy is a good surgical technique (less traumatic), minimize contamination during surgery, systemic perioperative antibiotics. The administration of prophylactic systemic antibiotics in single doses is a standard measure and has proven to be an effective measure in the surgery of various implants, including orthopedic and breast prostheses (Pittet B, Montandon D, Pittet D. Infection in breast implants. Lancet Infect Dis. 2005 Feb; 5 (2): 94-106. Review.) But not in primary mesh hernioplasty which could justify the use of the mesh as a model of local infection with antibiotic-releasing coating in addition to its simplicity. The best way to treat an implant infection is to prevent the colonization of microorganisms in the initial stages, avoiding the formation of bio fi lm, which causes them to persist in the biomaterial despite treatments. It is necessary to prevent the initial adhesion of the bacteria and one method is to modify the surface of the implant, covering it with polymers that release antibiotics and prevent this colonization. To date, this approach has not been developed experimentally or commercially.

As for the coating-forming polymers, hydrophilic acrylic systems have been widely used, specifically systems based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and / or 2-hydroxyethyl methacrylate (HEMA). AMPS is an anionic monomer, carrier of an ionized sulfonic group (strong acid) in virtually the entire pH range [Tong Z., L.X., Swelling equilibria and volume phase transition in hydrogels with strongly dissociating electrolytes. Macromolecules, 1994. 27].

On the other hand, both HEMA polymers and copolymers have wide applications in surgery and medicine, among which are: contact lenses [Rostoke M.V., L.L., Hema copolymers having high oxygen permeability., N.P.D. Corp, Editor. 1977: USA.], Healing, hemocompatible coatings, drug delivery systems, surgical prostheses, dialysis membranes, arti fi cial corneas ... etc. [López G.P., R.B.D., Rapoza R.J., Horbett T.A., Plasma deposition of ultrathin fi lms of poly (hydroxyethylmethacrylate): Surface Analysis and protein adsorption measurements. Macromolecules, 1993. 26 (13): p. 3247-3253]. This is due to its ability to form biocompatible hydrogels with excellent tolerance and good stability [Montheard J.P., C.M., Chappard D., 2hydroxyethyl methacrylate (HEMA): chemical properties and applications in the biomedical fi elds. Macromol Sci Macromol Rev, 1992. 32: p. 1-34],

Description of the invention

In view of the state of the art described, a first object or aspect of the present invention is to provide a mesh prosthesis comprising a medical mesh impregnated with a bioactive acrylic copolymer, characterized in that the amount of bioactive acrylic copolymer is between and 40% w / w (weight / weight) with respect to the total and the bioactive acrylic copolymer comprises: between 30 and 95% w / w of one or more monomers with hydroxyl groups; between 5 and 55% w / w of monomeric ovaries with sulfonic groups; and between 0.01% and 50% w / w of a bioactive agent.

These prostheses have the advantage that the polymers are reabsorbed in a few days thus avoiding possible side effects, but at the same time allowing the application and dosage in a controlled and localized way of bioactive compounds. For example, in the case that the bioactive agent is an antibiotic, they are released in a suitable way to avoid post-implant infections in the first 48 hours. In addition, the coatings of the mesh prostheses of the invention demonstrate optimum solubility and release the active ingredient in a suitable manner. If these coatings had an extreme solubility they would dissolve in a few minutes / hours, or on the contrary if they were very insoluble, they would not release the active substance.

Additionally, a second aspect of the present invention is a process for the manufacture of these mesh prostheses of the first aspect comprising at least the following steps:

i)

the bioactive acrylic copolymer is dissolved in ethanol;

ii)

the resulting solution is deposited on the medical mesh;

iii)

the solvent is evaporated, preferably at room temperature.

This procedure is known in the state of the art as "casting" (F. Lecomte J. Siepmann, M. Walther,

R.J. MacRae, R. Bodmeier, Blends of enteric and GIT-insoluble polymers used for fi lm coating: physicochemical characterization and drug releasepatterns. Journal Control release 89, 2003, 457-471) and being one of its main advantages is the simplicity of carrying it out. An important stage of the process is that the deposition of stage (ii) be as homogeneous as possible, so that it can be carried out by deposition of the solution dropwise to the total coating or by simple immersion in the ethanolic solution. Ethanol is important because of its practically zero toxicity, and its characteristics that reduce lump formation during drying compared to other solvents.

As the bioactive acrylic copolymer can be absorbed in different ways known to those skilled in the art, a third aspect is the use of a bioactive acrylic copolymer comprising between 30 and 95% w / w of one.

or several monomers with hydroxyl groups; between 5 and 55% w / w of one or more monomers with sulfonic groups; and between 0.01% and 50% w / w of a bioactive agent for impregnating medical meshes, preferably abdominal mesh.

Detailed description of the invention

A preferred embodiment of the invention are mesh prostheses characterized in that the monomer with hydroxyl groups comprises at least one hydroxyalkyl acrylate or a hydroxyalkyl methacrylate of the general formula (I):

where:

nesde0a4;

mesde0a1;

R1 is a hydrogen or a methyl radical.

2-hydroxyethyl methacrylate is the preferred hydroxyl group monomer. With respect to the monomers with preferred sulfonic groups are those of general formula (II): where:

R1 is a hydrogen or methyl radical;

R2 and R3 are the same or different from each other and represent a hydrogen or a (C1-C3) alkyl radical;

n is a value of 1-6.

The most preferred sulfonic group monomer is 2-acrylamido-2-methylpropane sulfonic.

In an even more preferred embodiment, the bioactive acrylic copolymer comprises 2-hydroxyethyl monomers and 2-acrylamido-2-methylpropane sulfonic monomers.

Bioactive agents may be of a different nature, but for biomedical applications the most important are antibiotics, antithrombogens, antiangiogenic, proangiogenic and any of their mixtures. Antibiotics are the most preferred to avoid post implantation operations.

Different antibiotics are known that may be useful for the present invention such as quinolones, glycopeptides, tetracyclines, oxazolidinones, macrolides, lycosamides, streptogramins, sulfonamides, polypeptides, penicillins, ansamycins or any of their mixtures. Glycopeptides are the preferred antibiotics. The glycopeptides include vancomycin, erythromycin, neomycin streptomycin, daptomycin, rifamycin, puromycin, lincomycin or any combination thereof, preferably vancomycin.

In another embodiment the amount of bioactive acrylic copolymer is between 15 and 30% w / w. These percentages allow the incorporation of an adequate amount of bioactive agent, correctly coating the device, without exposed areas and without blocked pores.

The bioactive acrylic copolymer shows good release profiles of the bioactive agent when this copolymer comprises between 45 and 75% w / w of one or more monomers with hydroxyl groups.

With a content of monomers with sulphonic groups in the bioactive acrylic copolymer between 10 and 35% w / w, good biocompatibility levels are obtained, and it helps the release take place during the 24 h post operation.

The bioactive agent may preferably be between 5 and 30% w / w relative to the total bioactive acrylic copolymer.

In addition other components may be in the composition to improve adhesion, release of bioactive agent or to improve the impregnation process. For example, the bioactive acrylic copolymer may additionally comprise polyethylene glycol, preferably between 10 and 30% w / w, which is a suitable range to achieve a homogeneous coating without lumping.

The medical mesh can have different natures, but preferably it is selected from the following types: Low molecular weight polypropylene monolayer; High molecular weight polypropylene mono fi lament; High molecular weight double polypropylene; Polypropylene large pore multi fi lament; Multi-lament high molecular weight polyester; Polytetra fl uoroethylene (Gore-Tex®).

Regarding the method of synthesis of mesh prostheses, preferably in step (i) the bioactive acrylic copolymer is dissolved in a proportion between 1% and 5% w / v in ethanol, obtaining suitable viscosities for the coating of the medical mesh. Viscosity is a critical parameter because if low viscosity solutions were obtained the coating could be low and, on the other hand, very high viscosities would encourage lump formation.

Normally between 5 and 20 ml of solution is deposited per gram of mesh.

The evaporation of step (iii) must be carried out in a way that the product obtained is homogeneous. This can be achieved simply by evaporating the solvent at room temperature.

In an even more preferred embodiment, the process for obtaining the bioactive acrylic copolymer comprises:

i ’)

the bioactive agent, preferably together with polyethylene glycol, is dissolved in distilled water;

ii ’)

the acrylic copolymer is added and stirred;

iii ’)

The mixture is frozen and freeze-dried.

In step (i ’) the concentration of preferably bioactive agent in the aqueous solution is between 5 mg / mL and 40 mg / mL. In step (i ’) the concentration of preferably polyethylene glycol in the aqueous solution is between 5 mg / mL and 40 mg / mL. In an even more preferred embodiment, the process for obtaining the acrylic copolymer comprises the following steps: i ”) dissolving the monomers with a radical polymerization initiator in a mixture of water: isopropanol; ii ”) deoxygenation of the reaction medium; iii ”) subject to heat treatment at a temperature between 40 and 75ºC; iv ”) isolate the reaction product by evaporation of the solvent, and purify by dissolving in water and washing by dialysis membrane; v ”) lyophilize the acrylic copolymer.

The radical polymerization initiator may be any of those known to those skilled in the art, preferably being azobis-isobutyronitrile (AIBN), and preferably being in a proportion between 0.25 and 2% w / w.

The mixture is deoxygenated and this can be carried out with a stream of nitrogen.

In step (iii ”) the heat treatment can be carried out at a temperature between 60 and 80 ° C.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

De fi nitions

The term mesh in the context of the present invention refers to a woven or interwoven polymer network of biocompatible and biostable polymers: polyolefins, polyesters, polyethers, polyurethanes or biodegradable polymers: polyhydroxyalkanoates, polylactic, polybutyrates, polyglycolics used in various surgical procedures for example abdominal, dental or orthopedic in order to isolate tissues or organs. Preferably the mesh is abdominal, among other things because of the great relevance of infections to patients.

Brief description of the fi gures

Figure 1.-ATR-FTIR standardized spectra of HA80, HA80rec and HA80recV copolymers. Transmittance vs. wavelength is represented.

Figure 2.- First derivative of the thermograms obtained by TGA for the HEMA-AMPS system.

Figure 3.-Vancomycin release curve from HA70recV, HA80recV systems.

Figure 4.-Halo inhibition at 48 h for the HA80recV system.

Figure 5.-Death curve for a) S. aureus and b) S. epidermidis.

Figure 6.-Groups and location scheme of implant site.

Figure 7.-Macroscopic photos of rabbits treated with the different systems and infected with a) S. aureus and b)

S. epidermidis.

Figure 8.-Microscopic photos of rabbits treated with the different systems and infected with a) nothing, b) S. aureus and c) S. epidermidis.

Examples

Example 1

Preparation of systems based on copolymers of 2-hydroxyethyl methacrylate-2-acrylamido-2-methylpropanesulfonic acid and vancomycin

First, HEMA / AMPS copolymers were prepared from 80/20 and 70/30% -p feed compositions (named HA80 and HA70 respectively. The reaction was carried out in solution, using water: isopropanol (50 : 50) as solvent, at 50 ° C and AIBN as initiator in concentration 1.5x10-2 M. Isopropanol was used as solvent in order to obtain copolymers of controlled molecular weight due to its low toxicity (classified by Food and Drugs Administration (FDA) as class 3) Guidance for the Industry Q3C Impurities: Residual Solvents International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) 62 FR67377 (1997) and high transfer constant [Brandrup J., IEH, Grulke EA, Polymer Handbook, ed. IEH Brandrup J., Grulke EA (ed.). 1999, New York: Wiley.] The solution was deoxygenated by bubbling nitrogen for 30 minutes.The total concentration of monomers was 0.50 M . After 24 hours of reaction, the solvent was removed from the samples, dissolved in water, washed by dialysis membrane and lyophilized.

Subsequently, the antibiotic was incorporated into the corresponding copolymer for which vancomycin (20% -p) was dissolved together with polyethylene glycol (20% -p) in distilled water and then the corresponding copolymer (HA80) was added. This mixture was left under magnetic stirring for 24 h and subsequently frozen and lyophilized. The system was named as HA80recV. The control polymer was prepared in the absence of vancomycin (HA80rec).

The structural characterization of the copolymers was carried out by infrared spectroscopy with Fourier transform by Attenuated Total Reflectance, ATR-FTIR, in a Perkin-Elmer Spectrum One device. To this end, a sample of the corresponding copolymer was deposited on the window of the equipment exerting a pressure controlled on it in order to ensure a good window-copolymer contact (Figure 1).

The thermal stability of the copolymers was evaluated using a thermobalance (TA Instruments TGAQ500). For this, the weight loss of the sample subjected to a constant heating rate of 10 ° C / min between 50 and 500 ° C was recorded under an N2 atmosphere. Each trial was performed in duplicate (Figure 2).

The degree of hydration of these copolymers was determined by immersing the sample in a phosphate buffer (0.1 M PBS) of pH = 7.4 which was prepared according to the following composition: NaCl: 0.138M; KCl: 0.0027 M.

Three samples of each of the different HEMA / AMPS copolymers, 80 / 20PEG without and with vancomycin, were immersed in the buffer solution and incubated at 37 ° C to simulate physiological conditions. The sample was extracted at different times, its surface was dried carefully with fi lter paper and weighed to a constant weight. At that time the percentage of the degree of hydration (H) was calculated by the following equation.

where PH is the weight of the swollen sample once equilibrium is reached; PS is the initial weight of the dry sample.

Hydration values are shown in table I

Example 2

Study of the in vitro release of vancomycin from prostheses coated with polymer plus antibiotic

First, prostheses coated with polymer-antibiotic of 1 cm2 in size (HA70recV; HA80recV) were used, which were immersed in 10 ml of phosphate buffer (composition detailed in the previous section) and incubated at 37 ° C. Samples of the release medium were taken during certain periods of time (Table I).

(0.5 ml) that were replaced by fresh medium (0.5 ml). TABLE II

To analyze the quantity released, Perkin Elmer's high efficiency liquid chromatography (HPLC) with peristaltic pump LC-250 and Perkin Elmer LC-95 UV-Vis detector was used as an analytical technique. The column corresponds to a C-18 μBoundapack (Waters) of 3.9 x 300 mm. The wavelength used was 215 nm. As the mobile phase, an aqueous solution Methanol / PIC A (60:40) and an elution flow of 1 ml / min were used. All samples were tested in triplicate. The antibiotic retention time was 2.95 ± 0.05 min. (mean ± S.D., n = 200). The calibration curve performed showed a correlation coefficient of 0.9957.

Example 3

Bactericidal activity of prostheses coated in liquid medium

The experimental phase in vitro in liquid medium consists in the realization of a death curve. The death curve is a microbiological method to know the concentrations in which an antibiotic kills a known bacterial strain. It is a protocol described by the NCCLS (National Committee for Clinical Laboratory Standards), so that the results are reproducible and comparable. A known concentration of antibiotic is tested in death curve studies and viable colony forming units (CFU / ml) are quantified at each time. These studies consider that a decrease of 3log − 10 CFU / ml compared to the control group (without antibiotic) indicates an adequate bactericidal response. The dose that achieves this decrease in CFU is considered bactericidal.

Three groups were made: bare polypropylene mesh (PP), which was the control group, polymer coated polypropylene mesh without antibiotic (POL) and polymer coated mesh with vancomycin (VC). Each group had three meshes. A sterility control was also used. All meshes were sterilized by ethylene oxide. The mesh that was used in the different examples herein was the low molecular weight monopropylene polypropylene (Parietene®, K991400 Sofradim, Trevoux (France)).

The death curve was performed with two microorganisms, separately, Staphylococcus epidermidis, from a sample of a patient's vascular catheter, and Staphylococcus aureus (ATCC 25923), with demonstrated sensitivity to vancomycin (MIC = 1 mg / L). At zero time a bacterial concentration of approximately 1.5 x 106 CFU / ml is achieved in tubes with 10 ml of liquid Mueller Hinton medium. For this, a bacterial suspension equivalent to a standard of 0.5 McFarland (1.5 x 108 CFU / ml) was previously prepared by nephelometry. 100 microliters of the 0.5 McFarland bacterial suspension was inoculated for each 10 cc tube. The tubes were incubated at a temperature of 37 ° C and samples were obtained at 0, 3, 6, and 24 hours for S. aureus and 0, 2, 4, 6, and 24 hours for S. epidermidis. For each time the microbiological count, previously vortexing, with the serial dilutions method in Mueller Hinton Agar, with a sample of 100 microliters, the detection limit being 10 CFU / ml.

The measurement of vancomycin levels was performed with an automated fluorescent polarization immunoassay technique (Tdx / Flx®, from Abbott laboratories), whose minimum detection threshold is 2 μg / ml.

A significant difference greater than 3log − 10 CFU / ml is seen between the naked polypropylene (PP) and vancomycin control group and the polymer and vancomycin (VC) mesh group, indicating an adequate bactericidal response in the sample of the 24 hours, for both S. aureus and S. epidermidis (Figure 3). There seems to be no difference between the control PP group and the polymer coated mesh (POL) group, so the bactericidal activity is due to vancomycin.

Example 4

Bactericidal activity of coated prostheses. Agar test

The bactericidal activity of the coated prostheses is determined by the diffusion method on plate agar Commercial blood agar plates were used and grown at 37 ° C for twenty four hours and fourteen days. As model cultures, Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis strains of a sample from a patient's vascular catheter are used. Both cultures are preserved by storing them at -80 ° C in glycerin / water mixtures (20% v / v).

We worked with three groups of plates, for each time. One without bacterial seeding, another with Staphylococcus aureus and another with Staphylococcus epidermidis. The inoculum was done by planting a colony, twenty-four hours old, for each quadrant. Each plate was divided into 4 quadrants, of which one was free and in each of the other 3 a 1 cm2 fragment of each of the meshes to be studied was placed: Polypropylene (PP) mono-lamellar reticular prosthesis, coated polypropylene polymeric (Pol) and Polypropylene with polymer and vancomycin (Vaneo). In each group, 5 plates were used for each of the study times, which were established at 24 hours and 14 days. The halo area is measured by the ImageJ morphometric program, in photos of the blood agar plates, and statistically processed with the U-Mann Whitney statistical test for the average of paired data independent of the halo of the plates with Staphylococcus aureus and Staphylococcus epidermidis

In this test the bactericidal activity of the polymer is measured by evaluating the inhibition or retardation of the development of colonies just in the areas of direct contact with the surface of the prosthesis.

In all plaques with bacterial inoculum, and until day 14, halo of inhibition can be seen in the polypropylene mesh covered with polymer with vancomycin, compared to the bare mesh or with polymer without vancomycin, in which its quadrant in the plate is Germ cover.

The mean halo for Staphylococcus aureus was 1.75 square centimeters (0.59 standard deviation) and for Staphylococcus epidermidis it was 1.84 square centimeters (0.12 standard deviation). There were no statistically significant differences in halo size for epidermidis or aureus. (Figure 4).

Example 5

In vivo experimental model: Rabbit abdominal wall defects

As an experimental animal, the white New Zealand male rabbit was used. The weight of the animals was between 3,200 and 3,500 kg at the beginning of the study.

The management of the animals was done in accordance with the current International Regulations on experimental animals (609/86 / EEC and ETS 123).

The animals were divided into 3 study groups, in total 84 rabbits were used, 12 from the control group, 36 rabbits at 14 days and 36 rabbits at 30 days. The study groups are as follows:

-

Control Group, without bacterial inoculum: The same rabbit was used for the PP and POL control subgroups, implanting each mesh to one side of the alba line.

Subgroups: PP (n = 6), Pol (n = 6), Vaneo (n = 6).

-

Infected with S. aureus (1 implant per animal):

Subgroups: PP (n = 6), Pol (n = 6), Vaneo (n = 6).

-

Infected with S. epidermidis (1 implant per animal):

Subgroups: PP (n = 6), Pol (n = 6), Vaneo (n = 6).

Prior to surgery, the animals were anesthetized with a mixture of ketamine hydrochloride, 70 mg / kg, diazepam, 1.5 mg / kg and chlorpromazine, administered intramuscularly. In some of the animals it was necessary to administer an additional dose intraperitoneally during the intervention.

After shaving and disinfection with povidone iodine of the entire anterior abdominal wall, the skin and subcutaneous cellular tissue were sectioned.

Defects were created in the anterior muscular wall of the abdomen (right and left lateral flank in the control group and right flank in the infected groups), which included the external and internal oblique, retaining the transverse muscle, the fascia transversalis and the parietal peritoneum. avoid contact of microorganisms with the visceral peritoneum. The final surface of the defect was 15 cm2, corresponding to a rectangular defect of 5 cm of longitudinal axis and 3 cm of transverse axis. This defect was repaired by one of the meshes under study (PP, Pol,

or Vaneo). The biomaterial was fixed to the edges of the defect (prosthetic interface-anchor fabric) by means of a continuous 4/0 polypropylene suture interrupted only at the corners. The skin incision was closed with a 3/0 silk. The technique can be seen in the surgical technique photos.

In the groups in which the defect bed was contaminated, it was inoculated with 0.5 ml of 108 CFU / ml suspension of Staphylococcus aureus (Sa) or Staphylococcus epidermidis (Se), before placing the biomaterial. The concentration of microorganisms was obtained by nephelometry (0.5 McFarland). The animals were sacrificed at 14 and 30 days and the abdominal wall pieces were divided into three and processed for histological examination with scanning and scanning electron microscopy, and for immunohistochemical study.

For the morphological study, samples of the prosthesis / recipient tissue interface were taken for histological study under optical microscopy.

The pieces were fixed in solution F13, included in paraffin, cut into sections of 5 μm and stained with hematoxylin-eosin or Masson's trichrome (Goldner-Gabe variety). Finally, they were observed under a Zeiss Axiophot microscope (Cari Zeiss, Oberkochen, Germany).

The immunohistochemical study was performed using a specific monoclonal antibody for Staphylococcus aureus (Abcam, ab8067, Cambridge, UK) and Staphylococcus epidermidis (Abcam, ab20942, Cambridge, UK). After rehydration of the samples in saline phosphate buffer solution (PBS) pH 7.4 and specific blocking with 3% bovine serum albumin (BSA), the samples were incubated with the primary antibody for 12 h at 4 ° C. The preparations were then washed with PBS-BSA, and incubated with a biotinylated anti-mouse antibody (IgG, Sigma, St. Louis, USA) for 1 h, washed with PBS-BSA, incubated with Streptoavidin-Alkaline Phostase (Sigma, St. Louis, USA). The marking was detected using a chromogenic substrate, Fast red. The nuclei were contrasted with hematoxylin.

The statistical analysis of the data was performed using the Graph Pad Prism 4 program. The results were expressed as mean values ± the standard deviation. The Mann Withney U test was used to compare data from different study groups. The level of statistical significance was considered with p <0.05.

There were no signs of infection or other alterations in the animals of the control group. The animals of the PP group and the POL group infected with S. aureus in the first days after the intervention presented clinical signs of infection with loss of appetite and weight loss and a mortality rate of 16.67% in the PP group. , and 8.33% in the Pol group. In the VC group there was no clinical impact of weight loss and mortality was 0%.

The macroscopic analysis of the defect area allowed to observe the presence of important abscesses, and cases of dehiscence and cutaneous necrosis in the implants of PP and Pol infected with S. aureus both at 14 and 30 days post-implant. However, vancomycin-bearing prostheses showed an appearance similar to that observed in the control group without bacterial inoculation.

In the group infected with S. epidermidis all animals survived until the end of the study. The PP and Pol group showed three cases of infection of the superficial surgical wound. A rabbit from the PP group presented cutaneous necrosis. A rabbit from the POL group developed an important seroma. The VC group demonstrated better healing of the skin wound, without infection and without abscessing.

Under optical microscopy, in the control group, the neoformed tissue was arranged concentrically around the prosthetic fi laments. It was made up of collagen fibers and inflammatory cells. In the biomaterials with polymer (Pol and Vaneo) this coating also appeared surrounded by macrophage cells and giant foreign body cells, being evident in some of the samples studied, signs of degradation (Figure 8).

In the group infected with S. aureus, the prosthetic filaments of PP and Pol appeared in most of the studied samples infiltrated by granulation tissue composed of extracellular matrix, white cells and S. aureus positive microorganisms, as evidenced by immunohistochemical dizziness ( red tide). Sometimes the marking could be seen inside macrophage cells. At 30 days, the abscesses were surrounded by compact fibrous tissue. In the Vancomycin group, the absence of these large purulent abscesses was evident compared to the other groups, with only one of the specimens remaining in infection. The rest of the samples observed in this group presented an aspect similar to that of the control group (Figure 8).

In general, the group infected with S. epidermidis did not show large differences from the control group. Immunohistochemical treatement for S. epidermidis was mild, being seen only in some samples in isolation and in most cases, in the macrophage cytoplasm (red tide, Figure 8).

Claims (29)

one.

A mesh prosthesis comprising a medical mesh impregnated with a bioactive acrylic copolymer, characterized in that the amount of bioactive acrylic copolymer is between 5 and 40% w / w relative to the total and the bioactive acrylic copolymer comprises: between 30 and a 95% w / w of one or more monomers with hydroxyl groups; between 5 and 55% w / w of monomeric ovaries with sulfonic groups; and between 0.01% and 50% w / w of a bioactive agent.

2.

The mesh prosthesis according to the preceding claim, characterized in that the monomer with hydroxyl groups comprises at least one hydroxyalkyl acrylate or a hydroxyalkyl methacrylate of the general formula (I):

where: nesde0a4; mesde0a1; R1 is a hydrogen or a methyl radical.

3.

The mesh prosthesis according to the preceding claim, characterized in that the monomer with hydroxyl groups comprises 2-hydroxyethyl methacrylate.

Four.

The mesh prosthesis according to any of the three preceding claims, characterized in that the monomer with sulfonic groups comprises monomers with the general formula (II):

where: R1 is a hydrogen or methyl radical; R2 and R3 are the same or different from each other and represent a hydrogen or a (C1-C3) alkyl radical; n is a value of 1-6.

5.

The mesh prosthesis according to the preceding claim, characterized in that the monomer with sulfonic groups comprises 2-acrylamido-2-methylpropane sulfonic.

6.

The mesh prosthesis according to the preceding claim, characterized in that it comprises 2-hydroxyethyl methacrylate monomers and 2-acrylamido-2-methylpropane sulfonic monomers.

7.

The mesh prosthesis according to any of the preceding claims, characterized in that the bioactive agent is selected from an antibiotic, an antithrombogenic, an antiangiogenic, a proangiogenic and any of its mixtures.

8.

The mesh prosthesis according to the preceding claim, characterized in that the bioactive is an antibiotic.

9.

The mesh prosthesis according to the preceding claim, characterized in that the antibiotic is a quinolone, a glycopeptide, a tetracycline, an oxazolidinone, a macrolide, a liposamide, a streptogramin sulfonamides, polypeptides, penicillins, ansamycins or any of their mixtures.

10. The mesh prosthesis according to the preceding claim, characterized in that the bioactive is a glycopeptide.

eleven.

The mesh prosthesis according to the preceding claim, characterized in that the glycopeptide is vancomycin, erythromycin, neomycin, streptomycin, daptomycin, rifamycin, puromycin, lincomycin or any combination thereof.

12.

The mesh prosthesis according to any of the preceding claims, characterized in that the amount of bioactive acrylic copolymer is between 15 and 30% w / w.

13.

The mesh prosthesis according to any of the preceding claims, characterized in that the bioactive acrylic copolymer comprises between 45 and 75% w / w of one or more monomers with hydroxyl groups.

14.

The mesh prosthesis according to any of the preceding claims, characterized in that the bioactive acrylic copolymer comprises between 10 and 35% w / w of one or more monomers with sulfonic groups.

fifteen.

The mesh prosthesis according to any of the preceding claims, characterized in that the bioactive acrylic copolymer comprises between 5-30% w / w bioactive agent.

16.

The mesh prosthesis according to any of the preceding claims, characterized in that the bioactive acrylic copolymer comprises polyethylene glycol, preferably between 10 and 30% w / w.

17.

The mesh prosthesis according to any one of the preceding claims, characterized in that the medical mesh is composed of low molecular weight mono-filament polypropylene, high molecular weight mono-filament polypropylene, high molecular weight double polypropylene polypropylene, multi-filament large pore polypropylene, high weight polyester molecular multi fi lament, polytetrafuoroethylene or any combination thereof.

18.

Procedure for obtaining the mesh prosthesis as defined in any of claims 1 to 17, characterized in that: iv) the bioactive acrylic copolymer is dissolved in ethanol; v) the resulting solution is deposited on the medical mesh, preferably drop by drop to the coating.

total or investment investment; vi) the solvent is evaporated, preferably at room temperature.

19.

The process according to the preceding claim, characterized in that in step (i) the bioactive acrylic copolymer is dissolved in a proportion between 1 and 5% w / v in ethanol.

twenty.

The method according to any of the two preceding claims, characterized in that in step (ii) between 5 and 20 ml of solution per gram of mesh is deposited.

21. The method according to any of the three preceding claims, characterized in that in the step (iii) the solvent is evaporated at room temperature.

22

The process according to any of claims 18 to 20, characterized in that the process of obtaining the bioactive acrylic copolymer comprises: i ’) the bioactive agent, preferably together with polyethylene glycol, is dissolved in distilled water;

ii ’) the acrylic copolymer is added and stirred; iii ’) the mixture is frozen and freeze-dried.

2. 3.

The process according to any of the two preceding claims, characterized in that in step (i ’) the concentration of bioactive agent in the aqueous solution is between 5 mg / mL and 40 mg / mL.

24.

The process according to any of the two preceding claims, characterized in that in step (i ’) the concentration of polyethylene glycol in the aqueous solution is between 5 mg / mL and 40 mg / mL.

25.

The process according to any of claims 18 to 24, characterized in that the process for obtaining the acrylic copolymer comprises the following steps: i ”) dissolving the monomers with a radical polymerization initiator in a mixture of water: isopropanol; ii ”) deoxygenation of the reaction medium; iii ”) subject to heat treatment at a temperature between 40 and 75ºC; iv ”) isolate the reaction product by evaporation of the solvent, and purify by dissolving in water and

dialysis membrane washing; v ”) lyophilize the acrylic copolymer.

26.

The process according to the preceding claim, characterized in that the radical polymerization initiator is azobis-isobutyronitrile (AIBN), and is in a proportion between 0.25 and 2% w / w.

27.

The process according to any of the two preceding claims, characterized in that in (ii ") the reaction mixture is deoxygenated with a stream of nitrogen.

28.

The process according to any of the three preceding claims, characterized in that in step (iii ") the heat treatment is carried out at a temperature between 60 and 80 ° C.

29.

The use of a bioactive acrylic copolymer comprising between 30 and 95% w / w of one or more monomers with hydroxyl groups; between 5 and 55% w / w of one or more monomers with sulfonic groups; and between 0.01% and 50% w / w of a bioactive agent for impregnating medical meshes.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200930975 SPAIN Date of submission of the application: 10.11.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

EP 0596615 A1 (MEDTRONIC INC) 11.05.1994, pages 2-5; example 27; claims 1.4. 1-29

TO

WO 0213871 A2 (SURMODICS INC) 21.02.2002, page 11, lines 15-26; page 13, lines 6-8; 1-29

page 16, lines 4 - page 18, line 18.

TO

WO 2008109098 A2 (BOSTON SCIENT SCIMED INC et al.) 12.09.2008, 1-29

paragraphs [0016], [0017], [0067], [0068].

TO

WO 2008024510 A2 (BOSTON SCIENT SCIMED INC et al.) 28.02.2008, 1-29

paragraphs [0005], [0012], [0067] - [0069], [0074].

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 02.03.2011

Examiner M. Bautista Sanz Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200930975 CLASSIFICATION OBJECT OF THE APPLICATION A61L27 / 34 (01.01.2006) A61L27 / 44 (01.01.2006) A61L27 / 54 (01.01.2006) A61L27 / 58 (01.01.2006) A61F2 / 00 (01.01.2006) Minimum documentation sought (classification system followed by classification symbols) A61L, A61F, C09D Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, NPL, XPESP, HCAPLUS State of the Art Report Page 2/4  WRITTEN OPINION Application number: 200930975 Date of Completion of Written Opinion: 02.03.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-29 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-29 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 200930975 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Doc.

Publication or Identification Number Pub Date

D01

EP 0596615 A1 05/11/1994

D02

WO 0213871 A2 02/21/2002

D03

WO 2008109098 A2 09/12/2008

D04

WO 2008024510 A2 02/28/2008

The object of the invention is a mesh prosthesis impregnated with a bioactive acrylic copolymer comprising one or more monomers with hydroxyl groups, one or several monomers with sulfonic groups and up to 50% of a bioactive agent. The application also includes a procedure for obtaining the mesh prosthesis and the bioactive acrylic polymer and its use to impregnate medical meshes. Document D01 discloses a medical device formed by a substrate to which an acrylic copolymer formed by copolymerization of 2-hydroxyethyl methacrylate (HEMA) and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) is grafted (example 27, claims 1 and 4) . In addition, the copolymer has an antibiotic or antimicrobial agent attached as bioactive agents that fight possible infections. Examples of bioactive agents are neomycin, vancomycin, streptomycin or erythromycin. The procedure for obtaining the medical device consists in carrying out the copolymerization in aqueous medium with Ce (IV) as initiator and subsequently incorporating the bioactive agent. See pages 2-5; example 27; claims 1, 4. Document D02 discloses a coating composition of medical devices (meshes) that allows the controlled release of a medicament (antibiotics such as vancomycin, neomycin, erythromycin, etc.). The composition is formed by a copolymer containing, in addition to other monomers, between 30 and 70% of a hydrophilic monomer (AMPS). For the preparation, the copolymer is dissolved in isopropanol, the medical device is coated by immersion and dried. The bioactive agent is subsequently incorporated. See page 11, lines 15-26; page 13, lines 6-8; page 16, lines 4-page 18, line18. Documents D03 and D04 collect surgical meshes coated with a biodegradable polymer composition containing bioactive agents such as antibiotics and their immersion manufacturing process in compositions containing the polymer and the bioactive agent. See D03: paragraphs [0016], [0017], [0067], [0068]; D04: paragraphs [0005], [0012], [0067] - [0069], [0074]. However, none of the documents cited (D01-D04), taken alone or in combination with the others, reveals or contains any suggestion that directs the person skilled in the art towards a mesh prosthesis that has between 5 and 40% of a coating of an acrylic copolymer with a bioactive agent (0.01-50%) in which the copolymer is formed between 30 and 95% of one or more monomers with hydroxyl groups and between 5 and 55% of one or several monomers with sulfonic groups as the mesh collected in the application. Therefore, it is considered that the object of claims 1 to 29 meets the requirements of novelty, inventive activity and industrial application, as set forth in Articles 6.1. and 8.1. of patent law 11/1986. State of the Art Report Page 4/4