ITMO20120180A1 - SURGICAL GLUE OF SYNTHETIC ORIGIN. - Google Patents

Description

"SURGICAL GLUE OF SYNTHETIC ORIGIN".

DESCRIPTION

The present invention relates to a hemostatic adhesive composition, its medical use and a medical kit comprising this composition, called "Colla III".

The present invention also relates to a process for the preparation of said composition.

Medical applications for tissue adhesion, for both external (topical) and internal use in the human body, are available in extremely diversified forms and include conventional sutures - resorbable and non-absorbable - Staples and / or grafts (now widely used) and a series of adhesives available in various forms: gauze, patches, liquids or gels.

In any case, regardless of the product used, the goal for the medical operator remains to heal the wound in the shortest possible time and at the same time to heal the laceration, to stop bleeding, to limit the possibility of infections, to avoid patient pain and, last but not least, to obtain a good aesthetic result of the operation.

Tissue adhesives in liquid form offer the possibility to intervene quickly and with a good final aesthetic effect. These adhesives are essentially divided into two macro-groups:

a) Adhesives of natural / human origin;

b) Adhesives of synthetic origin.

Adhesives of natural origin are mainly preparations based on fibrin, a substance used since 1909 as a hemostatic agent and later, in sheet form, to stabilize surgical and traumatic bleeding. Currently, fibrin-based adhesives (generically defined as "fibrin glue") are used in microsurgery to make the edges of the wound adhere, inducing tissue adhesion without the use of sutures, to assist the surgical sutures and to favor the hemostasis. The primary function of fibrin glue, however, remains that of a hemostat rather than an adhesive. Consequently, the application of fibrin glue almost always requires the use of conventional sutures with the risk of infections, undesirable aesthetic effects and a longer duration of the surgery.

Furthermore, the so-called fibrin glue is actually a commercial product in two components to be mixed at the time of use: the first component is a concentrate of fibrinogen and factor XIII, to which fibronectin and aprotinin are often added, and the second component is consisting of thrombin. Immediately before use, the two components are brought into contact at the point of application to reproduce the final stages of the coagulation process, thus carrying out, as adhesives, both a metabolic and a mechanical action.

For these reasons, the application of the fibrin glue is laborious since double syringe devices are required and the anti-mixing product needs to be stored at temperatures around -18 ° C.

The thrombin used for these applications can derive from human or bovine blood and in the preparation process all products intended for sale must be subjected to careful sterilization and viral inactivation processes, to exclude the risk of transmission of agents such as viruses. hepatitis and HIV. In order to limit the risk of prion transmission, due to the potential for diseases such as Creut zfeldt-Jakob disease, or to immune reactions against one or more components of the glue, the production of fibrin is currently based mainly on human blood. Consequently, the costs of fibrin and its derivatives are particularly high and this factor has further limited the use of fibrin glue in general surgery, especially in cases that require the application of high volumes of product.

The glues, or tissue adhesives, of synthetic origin are mainly glues based on cyanoacrylate. Compared to adhesives of natural origin, users complain of a lower biocompatibility, although there are substantial and consolidated advantages, such as:

- greater ease of use (the compound to be applied is in fact ready to use and does not require mixing, as is the case with fibrin and thrombin, or even special applicators);

- lower cost;

absence of risk of transmission of viral agents, such as those described above.

Furthermore, cyanoacrylate-based glues have good mechanical resistance, so they can replace the suture even in the presence of moderately large surfaces, contrary to what happens for fibrin glue.

In fact, cyanoacrylate-based glues, in contact with living tissue and a humid environment, polymerize rapidly with an exothermic chemical reaction, creating a thin elastic film with good mechanical properties that guarantees solid adhesion to the fabrics. This film naturally conforms to the anatomy of the tissues on which it is affixed, is waterproof and is not affected by blood or other organic liquids. Currently, cyanoacrylate-based adhesives are used in particular for the closure of surgical wounds or traumatic lesions without the use of conventional sutures (excluding the occlusive use in embolization of cerebral vessels via catheteral injection), and mainly use cyanoacrylates which n -butyl cyanoacrylate or 2-octyl cyanoacrylate to minimize the release in the body of irritating and toxic chemical compounds, such as formaldehyde, deriving from the metabolic degradation of the components of the adhesive.

However, the glues of synthetic origin currently available do not have a haemostatic action and have a limited antibacterial action.

Furthermore, the exothermic polymerization reaction that occurs upon application can cause pain to the patient, to the point of requiring anesthetic treatment, and also trigger inflammatory processes at the application site that can interfere with the tissue repair process.

There is therefore a need for an adhesive suitable for medical use capable of combining mechanical strength, ease of application, rapidity of action and hemostatic and antibacterial action.

The main task of the present invention is to provide an adhesive composition suitable for medical use capable of forming a thin elastic film with high tensile strength, which guarantees solid adhesion to fabrics and which is easy to apply, rapid action and hemostatic and antibacterial effect, and which generates a contained exothermic reaction.

Within this aim, an object of the invention is to provide an adhesive composition with high tissue biocompatibility and polymerization speed, at the same time limiting the amount of heat produced during said reaction so as not to cause the patient further pain and tissue damage.

Another object of the present invention is to provide an adhesive composition suitable for medical use having storage stability at temperatures close to those of use, which is sterile and comfortable for the patient.

Another important object of the present invention is to provide an adhesive composition endowed with the aforementioned properties, which can be used as a glue for medical use.

Another object of the present invention is to provide a medical kit comprising an adhesive composition with the aforementioned properties. This aim, as well as these and other objects which will appear better below, are achieved by a composition comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters.

Another aspect of the present invention is the use of the composition of the present invention, comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of α carboxylic acids , β unsaturated and their esters, such as surgical glue.

Another aspect of the invention relates to a medical kit which comprises the composition of the invention, comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of acids α, β unsaturated carboxylic compounds and their esters, and a device for applying the composition. Further objects, characteristics and advantages of the invention will become clearer from the following detailed description of a preferred, but not exclusive, embodiment of the invention.

Further characteristics and advantages of the object of the present invention will become clearer with the aid of the attached drawings in which:

Figure 1: Temperature (° C) vs. time (sec) of the in vitro polymerization temperature of various "Colla III" samples.

Figure 2: FT-IR spectra relating to the variation of the peak involved in the polymerization reaction of a tissue adhesive formulation (called blend). Figure 3: Polymerization kinetics of blend and blend / surfactant (PE6200).

Figure 4: graphic representation of the histological parameters examined in the histological analysis of the reaction of a subcutaneous tissue to an adhesive material (glue III) injected in rats and evaluated at different times (7, 21, 45 and 60 days from the injection) .

Figure 5: Histological images of the adhesive material samples (in white color) injected subcutaneously into the connective tissue of the rat back and collected at different times.

In the context of the present invention, the term "comprising" also includes "including" in addition to "consisting", e.g. a composition "comprising" X may consist exclusively of X or may include other additional substances, for example X + Y.

In the context of the present invention, the percentages indicated for each component refer to the volume of the component in question with respect to the total volume of the composition.

In the context of the present invention, the term "biocompatible" indicates a material or compound that can be applied in contact with living tissue, either outside or inside the body, without causing local or systemic undesirable effects, such as reactions immune, allergic or inflammatory.

In the context of the present invention, the term "feracryl" (or "Feracrylum", register number CAS 55492-43-8) refers to the meaning commonly used in the sector to mean a mixture of iron salt (II and III) of a polyacrylic acid (2-propenoic acid) with a molecular weight greater than 100,000 daltons. Said feracryl is biodegradable and biocompatible and has haemostatic and antimicrobial properties.

Due to the different solubility characteristics of the alkyl cyanoacrylates and feracryl, it is particularly difficult to mix said compounds. For this reason, in the state of the art there are no surgical glues based on alkyl cyanoacrylates which also include said hemostatic agent. Furthermore, there are currently no synthetic-based surgical glues on the market that also include a specific agent with haemostatic action.

The inventors of the composition in question have now surprisingly found that an active principle having a haemostatic and antibacterial function, such as for example feracryl, can be incorporated in a composition based on cyanoacrylates, resulting stable and effective.

The mixing of said composition based on cyanoacrylate and said hemostatic agent can be carried out using the techniques commonly known in the state of the art, such as, for example, encapsulation, dispersion, suspension. Preferably, the hemostatic agent of the present invention is mixed with the alkyl cyanoacrylate composition of the present invention to obtain a suspension.

To obtain a correct suspension of the mixture of said composition based on cyanoacrylate and said hemostatic agent in the cyanoacrylate matrix, the following process is carried out, which is privileged but not exclusive: 1) Preparation of a powder of an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters, preferably Feracryl, where said Feracryl is originally in flakes, by grinding, preferably by means of a planetary ball mill (PM 100 CM, Retsch), where said iron salt powder comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters has a particle size lower than or equal to 45 microns. It is understood that, if said Feracryl were available directly in powder form, rather than in flakes, step 1) would be superfluous and the process for the preparation of a suspension of the mixture of said composition based on cyanoacrylates and said hemostatic agent in the matrix of cyanoacrylate would start in step 2) below.

2) Sterilization, preferably by irradiation, of the powder obtained from step 1); even more preferably, said irradiation is at 25 kGy.

3) Preparation of a "Blend" comprising at least one alkyl cyanoacrylate, wherein said mixture is preferably constituted by

- 68% of 2-octyl cyano acrylate;

- 32% of n-butyl cyanoacrylate;

(% expressed in volume on the total mixture) and sterilization of said mixture, where said sterilization preferably takes place by means of filtration, with 0.22 micron filters.

4) High pressure homogenization of

said sterilized powder obtained from step 2);

with

an aliquot equal to 10% of said mixture obtained from step 3);

where said high pressure homogenization comprises the steps of

4.i) mixing in aseptic condition of said sterilized powder obtained from step 2) with said aliquot of mixture obtained from step 3);

4.ii) high pressure homogenization of the mixture obtained according to step 4.i). Preferably, the aseptic conditions are obtained by conveying said sterilized powder obtained from step 2) and said mixture aliquot obtained from step 3) in a disposable bag (Flexboy, Sartorius).

Preferably, said high pressure homogenization takes place through recirculation for 1 hour in a high pressure homogenizer, preferably Panda Plus 200, GEA.

Thus, iron salt particles are obtained comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters, such as feracryl, with a particle size equal to or less than 0.5 micron.

5) Aseptic mixing of the homogenized product obtained from step 4) with the remaining 90% of the blend mixture obtained from step 3)

and, optionally, with

- a surfactant,

as a percentage of 1% w / v.

The final product, collected in a special preparer, is conveyed to the filling room and distributed under aseptic conditions.

The composition of the invention is particularly suitable for use as an adhesive in the medical field thanks to the presence of a hemostatic and antibacterial active principle and to the characteristics of high tensile strength and biocompatibility.

The present invention relates to a composition comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said haemostatic agent is an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters.

Preferably, in the composition of the present invention the at least one alkyl cyanoacrylate is present in an amount comprised between 50% and 99% by volume of the total composition.

Preferably, in the composition according to the invention, the alkyl cyanoacrylates are selected from the group consisting of n-butyl cyanoacrylate, 2-octyl cyanoacrylate and a mixture thereof.

More preferably, n-butyl cyanoacrylate is used in the composition of the invention. In another more preferable alternative, a mixture of n-butyl cyanoacrylate and 2-octyl cyanoacrylate is used in the composition of the invention.

Preferably, the percentage of n-butyl cyanoacrylate is in the range 25-75% by volume, and the percentage of 2-octyl cyanoacrylate is in the range 25-75% by volume.

Even more preferably, the percentages of n-butyl cyanoacrylate and 2-octyl cyanoacrylate in the mixture are 32% and 68% by volume.

The advantage deriving from the mixture of n-butyl cyanoacrylate and 2-octyl cyanoacrylate depends on the fact that it has been found that these two components offer the composition of the invention an optimal combination of physical / mechanical characteristics. In fact, 2-octyl cyanoacrylate guarantees high flexibility while n-butyl cyanoacrylate guarantees good resistance to mechanical traction of the composition. Furthermore, thanks to the mixture of nbutyl cyanoacrylate and 2-octyl cyanoacrylate, the properties of rapidity of the polymerization reaction (characteristic of n-butyl cyanoacrylate) and of flexibility (characteristic of 2-octyl cyanoacrylate) are optimized. A composition containing only n-butyl cyanoacrylate, after polymerization, would be quite fragile, especially if used on skin folds, on long incisions, or on areas subject to movement.

As mentioned, preferably in the composition of the present invention the iron salt of homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters is a mixture of iron II and III salt of the 2-propenoic acid polymer, commonly known by the generic name "feracryl"; called feracryl, it is known for its antibacterial and hemostatic characteristics. Even more preferably, the composition of the present invention comprises feracryl in an amount comprised between 0.5% and 5% by volume of the total composition. Even more preferably, feracryl is present in an amount equal to 1% by volume of the total composition.

Preferably, the composition of the present invention further comprises a non-ionic surfactant consisting of copolymer blocks in which a propylene glycol group is flanked by two polyethylene glycol groups. Even more preferably, said non-ionic surfactant is Pluronic PE6200.

Preferably, said Pluronic PE6200 is present in an amount comprised between 0.5% and 5% by volume on the total composition.

Advantageously, the presence of a non-ionic surfactant consisting of copolymer blocks in which a propylene glycol group is flanked by two polyethylene glycol groups, such as PE6200, in the composition of the present invention extremely speeds up the polymerization kinetics of the composition; said polymerization is much faster than the polymerization of a composition not comprising said non-ionic surfactant.

The composition of the invention may further comprise a biocompatible dye. The presence of a biocompatible dye allows an easy application of the composition, making it particularly suitable for surgical use. In fact, the dye makes it possible to visually ascertain where the glue has been applied and to immediately carry out any interventions to complete the application of the glue itself without having to intervene at a later time on the patient.

Preferably, the dye present in the composition can be D&C Violet n ° 2, better specified as l-hydroxy-4- [(4-methylphenyl) amino] -9; 10-anthracenedione (CAS registry number 81-48-1) .

The composition thus described according to the present invention has the characteristic of combining a series of advantageous chemical and physical properties, such as a particular tensile strength, good flexibility and speed of polymerization with the haemostatic and antibacterial properties associated with haemostatic agents such as feracryl.

In fact, while the common compounds based on cyanoacrylate have been shown to possess only bacteriostatic properties in vitro, the iron salts comprising homopolymers or copolymers of 0, β unsaturated carboxylic acids and their esters, such as feracryl, have shown a significant antibacterial and have been shown to be effective in the in vivo suppression of the following strains:

Staphylococcus aureus 209-P;

Streptococcus pyogenes 2 95;

Pneumococcus I type;

Bacillus anthracis 1312;

Corynebacterium diphtheriae P-8;

Escherichia coli 675;

Salmonella typhosa 1196;

Shigella Flexneri 2a-516;

Proteus vulgaris 5123-111;

Pseudomonas aeruginosa 165;

thus limiting the risks of infection.

Regarding the haemostatic properties mediated by feracryl, the literature data support the fact that this compound is able to act as a coagulant even in the case of patients suffering from blood coagulation disorders such as haemophilia or Werlhof disease in which it is necessary to contain the bleeding, which tends to be profuse due to pathological causes. The mechanism of action of feracryl is in fact based on the formation of water-insoluble complexes with blood proteins, mainly with albumin present at the wound.

The haemostatic and antibacterial effects, expressed in conjunction with the mechanical wound occlusion effect due to the adhesive action of the composition once polymerized on the tissue to be repaired, ensure that wound healing is rapid and prevent the onset of infections collateral, also contributing to a better final aesthetic result.

The composition according to the invention is in fact particularly suitable for use also on skin folds, on long incisions and in areas of the body subject to movements.

Furthermore, the composition of the invention has low costs, is free from the possibility of bacterial contamination, has a high tissue biocompatibility and develops a low amount of heat during polymerization. These properties make it particularly suitable for medical use and specifically for surgery, even in the case of emergency interventions and for interventions that require high volumes of product.

In particular, the composition of the invention facilitates the post-operative comfort of the patient since it is not necessary to remove the surgical glue once the wound has healed. In case of external application, the composition is not absorbed by the skin or by the underlying tissue, but it detaches spontaneously (within 1-2 weeks) following skin re-epithelization, guaranteeing a good aesthetic result. The surgical glue is also water-repellent and therefore allows patients to wash themselves without problems, as long as they do not vigorously rub the area affected by the application of the composition. In the case of internal application, on the other hand, the glue remains in place like a plant undergoing an extremely slow and negligible hydrolytic degradation process, so much so that no significant values of formaldehyde, the main product of degradation, were found in an in vivo study on rats. hydrolytic. In particular, it should be noted that cyanoacrylates with a longer alkyl chain (such as 2-octyl cyanoacrylate) degrade more slowly than those with a shorter chain (such as n-butyl cyanoacrylate), thus allowing the degradation products - in particular formaldehyde, which can cause inflammatory tissue reactions - to be metabolized more safely.

In addition, depending on the site and the type of application, the composition of the invention can be made more viscous (by inserting micronized silica into the formulation, and in particular Aerosil 200 -Evonik Degussa) thus allowing its dosage with great ease and precision, and at the same time avoiding unwanted dripping in areas surrounding the wound or contiguous to those of application, a particularly important feature for application in delicate areas such as, for example, the facial triangle.

Another aspect of the present invention relates to the use of the composition of the present invention, comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said at least one haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters, as an adhesive for medical use.

In particular, the composition of the invention can be used as an adhesive for surgical and aesthetic applications on burned tissue. Preferably, the composition is employed as an adhesive for surgical use.

Another aspect of the present invention relates to a medical kit comprising a composition of the present invention, comprising at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said at least one haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters, and a device for applying the composition.

This kit can be easily stored and transported at room temperature, considering both the excellent stability of the composition of the invention and the adoption of a dedicated packaging for this kit which includes a plastic vial as primary container and a protective aluminum bag, together with to an inert atmosphere packaging. Furthermore, given the ease of application of the composition and its speed of polymerization, this kit is easy to use even by unskilled personnel and / or outside the hospital environment and can be part of a self-medication case or a set of accessories. of first aid material.

Preferably, the medical kit comprising the composition of the invention is a disposable device. This feature allows to limit the weight and size of the kit and is made possible thanks to the chemical-physical properties of the composition of the invention.

EXAMPLES

1. IN VITRO DETERMINATION OF THE POLYMERIZATION TEMPERATURE OF CYANOACRI LICA-BASED TISSUE ADHESIVES.

An in vitro evaluation of the heat developed in the polymerization reaction of a mixture, called "Blend", consisting of:

- 68% 2-octyl cyanoacrylate; And

- 32% from n-butyl cyanoacrylate.

(% expressed in volume of the total).

To proceed with the measurement of the heat developed by the polymerization reaction of the Blend mixture, the following materials were used:

- contact microbiological plates 0 60 mm filled with Trypticase Soy Agar (TSA) nutrient medium - [Tryptic Soy Contact Agar ICR 30 mL-Manufacturer: heipa Diagnostika- pH 7.5);

- thermocouple J connected to a device for displaying the temperature;

- digital stopwatch;

- sample of cyanoacrylate glue, called Colla III, consisting of:

- 99% Blend; And

- 1% Feracryl.

For the evaluation of the heat of polymerization, the thermocouple was introduced within the thickness of the culture medium of the contact plate, in a horizontal position with respect to the surface of the medium.

The layer of soil present above the probe was approximately 2 mm thick.

At the perimeter of the probe, a slight cracking of the culture medium was observed.

The temperature detector was then activated and the gradation displayed was monitored until the temperature read by the probe was constant (TSA temperature). A drop of glue was then deposited over the area of the ground where the thermocouple was introduced, then leveling the adhesive with a plastic spatula to create a minimum thickness of glue. In this way, the initialization of the polymerization reaction derives predominantly from the humidity of the soil, rather than from the surrounding environment, and the heat of reaction at the interface between the glue and the TSA was probably greater than that developed on the adhesive / air interface, effectively allowing a change in temperature to be recorded as heat dissipation was slower.

From the moment the sample was deposited on the TSA, the temperature displayed by the temperature display was recorded for a total of 12 minutes.

Specifically, the temperature recording frequency was:

- every 10 seconds, for the first 6 minutes of the test; - every 20 seconds, for the remaining 6 minutes of testing.

The test described above was repeated 10 times. The test was carried out at room temperature in the laboratory.

Results

The results obtained are summarized in Table 1.

Table 1

In Figure 1, the summary graph of temperature (° C) vs. time (sec) obtained by tabulating the data collected in the 10 repetitions (For each test the trend line was calculated to characterize the heat developed in the polymerization reaction of glue III not only as the maximum temperature reached but also as a variation over time).

From the analysis of the summary graph of Figure 1 it was observed that:

- Within the first 100-150 seconds (about 2 minutes) from the moment of deposition of the sample on the ground, the temperature of the TSA increases by about 1.5 ° C compared to the temperature recorded before the application of the sample.

- Within the next 50 seconds from the moment in which the temperature has reached its maximum value, there is a progressive lowering of the alcohol content until the initial temperature is reached. This initial temperature is reached in a maximum of 6 minutes.

- The highest soil temperature is recorded for a maximum time of 3 minutes.

These data, although determined in vitro, are considered significant and predictive of the temperature increase that could occur in vivo following the application of the glue. In fact, the recorded temperature increase of about 1.5 ° C of the determined TSA is confirmed by a study carried out by C. Stephen et al., Carried out in 1964 entitled "Histotoxicity of Cianoacrylate Tissue Adhesive in the rat" where the polymerization temperature of 3 different cyanoacrylates, ie MethylCA, HexylCA and DecylCA was measured in vivo on anesthetized rat mesentery. Specifically, the researchers observed that the typical temperature increase of MethylCA was about 4 ° C, that of HexylCA was about 2 ° C and that of DecylCA was between 1.5 and 2 ° C.

In the light of the above data and considerations it is believed that the cyanoacrylate blend analyzed is characterized by a negligible heat of polymerization.

2. DETERMINATION OF THE POLYMERIZATION KINETICS OF THE SAMPLE Blend (2-OCA / n-BCA = 68:32) and Blend / PE6200 (100: 1).

In order to demonstrate the influence of the non-ionic surfactant Pluronic PE6200 on the polymerization kinetics of the tissue adhesive consisting of a mixture, defined as Blend, consisting of:

- 2-octyl cyanoacrylate (2-OCA); And

- normalbutyl cyanoacrylate (n-BCA);

in a 68:32 (v / v) ratio, spectrophotometric analyzes were carried out in FT-IR on:

- Blend samples; And

- Blend / PE6200 [100: 1 (v / v)].

In order to determine the polymerization kinetics of cyanoacrylate-based adhesives, Fourier transform infrared spectroscopy (FT-IR) was used, identifying and quantifying the chemical group related to the carbon-carbon double bond (C = C) monomers capable of absorbing at the specific infrared wavelength of 3128 cm-1. During the polymerization process this bond decreases in intensity, until it disappears at the end of the reaction, as can be seen from the graph in Figure 2.

Experimentally, 20 μΐ of each sample [Blend (2-OCA / n-BCA = 68:32 (v / v)) and Blend / PE6200 (100: 1) (v / v)] were placed between two microscope slides in turn suitably housed in an FT-IR spectrophotometer (Perkin-Elmer 1600 Series FTIR).

The absorbance spectra were performed in the range of wavelengths from 4000 cm <_> 1 to 2000 cm-l with a scanning period of 18 seconds, at a distance of one minute from each other for an interval time of 30 minutes for the Blend samples, at a distance of 45 seconds from each other until complete polymerization, within 3 minutes, for the Blend / PE6200 samples.

Each individual spectrum was processed by determining the subtended area between the wavelengths of 3165 cm-l and 3100 cm-l obtaining the value of the area at time t (At), compared with the value of the area subtended at time initial (AQ), obtaining the At / Ao value, hence the complementary value to the unit (1-At / AQ) which expresses the degree of polymerization in percentage terms.

Ten determinations were performed for each sample, on which the statistical mean with relative standard deviation was calculated (see Table 2 below).

Table 2

Average calculated on experimental data excluding two anomalous data (J-blend Sblend.

Results

From the graphical representation of Figure 3, which shows the degree of polymerization as a function of time, it can be seen that for the Blend sample the reaction has a sigmoidal trend with a latency phase of about 12 minutes, with a degree of polymerization of about 10%, followed by an exponential phase of up to 25 minutes, with a degree of polymerization of about 85%, and a final phase that tends to complete polymerization.

With regard to the Blend / PE62 00 sample, the graphical representation shows an extremely fast reaction in which the degree of polymerization is approximately equal to 95% after 1 minute. Polymerization is completed within 2 minutes.

3. MECHANICAL TRACTION TEST.

Mechanical tensile tests were conducted to compare the tensile strength of the following glues:

Blend [2-octyl cyanoacrylate: n-butyl cyanoacrylate: 68:32 (v / v)];

- Glue III-No PE [Blend: Feracryl in the ratio 99: 1 (v / v)];

- ITI-Con PE glue [Glue III with formula as above: PE6200 in the ratio 100: 1 (v / v)];

The mechanical tensile tests were conducted on buckskin samples (commercially available) of the size of 15mm X 60mm. The glues were distributed on a surface equal to 15mm X 10mm.

Subsequently two samples were superimposed for a length equal to 10mm.

The glue was left to cure overnight.

Subsequently, each sample was allocated in a clamp, whose grip is located 40 mm from the nearest overlapping edge, using the GADABINI model SUN1000 dynamometer and carrying out the test according to IS04587: 2003.

The tests were conducted at a constant speed of 50 mm / min.

The highest and lowest values obtained were discarded to obtain an RSD% <20.

Results

For each sample the final data are as follows (Table 3)

Table 3

The above data have been reworked with Microsoft Excel.

Table 4

Table 5

LEGEND OF ACRONYMS: SS: sum squares; df: degrees of freedom; MS: mean of squares; F: statistic of the F test; F-CRIT: critical F value.

It is noted that the differences between the means are statistically significant and that the formulation object of the present invention offers a higher mechanical performance than the state of the art (blend).

4. IN VITRO COAGULATION TEST

Hemostasis tests were performed according to ISO 10993-4: 2002 / AMDl: 2006, aimed at evaluating the following parameters:

1) Prothrombin time (PT)

The prothrombin time is used to determine the extrinsic pathway of blood clotting.

2) Activated partial thromboplast ina time (aPTT)

This test measures the clotting time of the blood intrinsically.

3) Fibrinogen

Fibrinogen is the precursor plasma protein of fibrin. Fibrinogen is essential in blood coagulation as it is transformed by thrombin (through a polymerization process) into fibrin which in turn is necessary for the formation of the hemostatic thrombus. The level / concentration of fibrinogen is inversely proportional to the clotting time.

Samples tested

The following samples were tested:

- Blend, consisting of:

2-octyl cyanoacrylate: n-butyl cyanoacrylate in the ratio 68:32 (v / v);

- Glue III-No PE, consisting of:

Blend: Feracryl in the ratio 100: 1 (v / v);

- Glue III-Con PE, consisting of

Blend: Feracryl: Pluronic PE6200 in the ratio 100: 1: 1 (v / v);

Reagents and equipment

- SEAC coaugulometer Mod. COATRON IV (DASIT);

- Thromboplastin-S (DIAGEN);

- APTT-EA (BIOPOOL);

- TriniCLOT fibrinogen (BIOTECH);

- Disposable 20 ml syringe (TERUMO);

- Sodium citrate (MERCK);

- Calcium chloride (BIOTECH).

Preparation of the blood

The human venous blood used for these tests was drawn from randomly selected patients not treated with anticoagulant therapies. The blood was then collected in silicone tubes; said silicone tubes contain a 9: 1 ratio of blood: 3.8% sodium citrate. Plasma preparation

Plasma was obtained by centrifuging the blood at 3200 rpm for 15 minutes.

Preparation of the samples to be tested

The BLEND, Glue III-NO PE, Glue III-CON PE samples were tested as cured films.

After vigorous shaking, one drop (100 μΐ) was placed on a concave slide and left for 24 hours at room temperature (RT).

The obtained film was covered with plasma for 30 minutes at RT under dynamic conditions (gently stirred).

In two tubes of Colla III-CON PE the tested material appeared completely solid; in this case 1 ml of plasma was applied to the surface of the material to be tested directly in the test tube.

The plasma was then tested as it was.

Control plasma

A negative control was performed by keeping the plasma in the same type of tubes used for the preparation of the sample to be tested, for 30 minutes at RT under dynamic conditions (gently shaken).

Basal plasma

A certain amount of untreated plasma was used as a baseline value.

Coagulation analysis

Prothrombin time (PT)

This determination was carried out with TROMBO PLASTINA-S K25050235 and the COATRON IV coagulometer.

Partial thromboplastin time (aPTT)

This determination was performed with APTT-EA S037005 and the COATRON IV coagulometer.

Fibrinogen

This determination was carried out with TriniCLOT Fibrinogen W050007a and the COATRON IV coagulometer.

Results

The results are summarized in the following Tables 6-9.

Table 6

Table 7

Table 8

Table 9

From the above data it is noted that:

i) Prothrombin time (PT):

there is no significant difference between all tested samples (Glue III-No PE; Glue III-Con PE (solid sample); Glue III-Con PE;

Blend) (<20%);

ii) Partial thromboplast ina time (aPTT):

- III-No PE glue; Glue III-Con PE (solid sample); Glue III-Con PE: there is a significant difference between the tested samples (> 20%);

Blend: there is no significant difference between the tested sample and the basal and control plasma (<20%);

iii) Fibrinogen:

Glue II I-No PE; ΙΙΙ-Con PE glue (solid); ΙΙΙ-Con PE Glue: There is a significant difference between all tested samples (> 20%) except Blend.

The cut-off value of 20% has been established as the range of acceptable values for HEMOTROL N (Standard Control for Plasma, used as an internal standard), for the three tests

- prothrombin time (PT);

- partial thromboplastin time (aPTT);

- Fibrinogen.

The experimental data above support the thesis that feracryl has a significant impact on coagulation by decreasing the time required for the process. More specifically, Feracryl "consumes" fibrin resulting in a decrease in the concentration of fibrinogen. Since the transformation of fibrinogen into fibrin is mediated by thromboplast ina which activates prothrombin into thrombin, the haemostatic effect of Feracryl is also highlighted by monitoring coagulation control parameters such as aPTT and PT.

Precisely, the 3 parameters investigated to study the action of Feracryl:

- the level of fibrinogen

- the prothrombin time (PT)

- the thromboplast time ina (aPTT) show that, in vitro, in the presence of Feracryl, the situation that occurs is the following:

- the concentration of fibrinogen decreases as it is converted into fibrin due to the sequestration of the latter by Feracryl; PT increases highlighting a sequestration of prothrombin, since thrombin (which is the activated form of prothrombin) is required to activate fibrinogen into fibrin;

- the aPTT also increases highlighting a consumption of thromboplastin, necessary to activate prothrombin in thrombin.

Feracryl therefore has an indirect haemostatic action (ie linked to the sequestration of fibrinogen) and therefore does not directly activate the coagulation factors.

The use of the excipient Pluronic PE6200 has no impact on the action of feracryl towards the coagulation process.

5. IN VIVO BIOCOMPATIBIL ITA TEST

A histological analysis of the reaction of a subcutaneous tissue to an adhesive material (Colla III) injected in rats was conducted and evaluated at 7, 21, 45 and 60 days after injection.

The adhesive material was injected subcutaneously into the backs of rats and collected at 7, 21, 45 and 60 days after injection (T7, T21, T45; T60 dpi). A histological examination was performed in order to evaluate the reaction of the subcutaneous tissue to the injected adhesive material.

Animals

The rats were sacrificed at 7, 21, 45 and 60 days after the injection. At the time of sacrifice, the outer skin layer and the underlying sub-skin (SC), including the injected adhesive material, was removed, spread on a rigid support, and fixed in a 10% formalin solution. The fixed samples were subsequently examined.

Sample preparation

Skin / SC samples comprising the injected adhesive material were cut transversely. All fixed samples were routinely processed in an automated tissue processor / sampler and were subsequently fixed in paraffin according to standard procedures.

Histopathological examination

4pm sections were stained with Hematoxylin Eosin (HE) and evaluated under the microscope. The histological evaluation of the subcutaneous tissue reaction to the injected adhesive material (including inflammation, encapsulation, and necrosis) was performed according to the criteria reported in Table 10.

Statistic analysis

A comparison between different deadlines was made with Dunn's multiple comparison test. A P value <0.05 was considered significant, a p value> 0.001 was considered very significant.

Table 10

Results

Histopathological examination

Subcutaneously injected adhesive material appears as a non-colored area.

At day T7 the surface of the adhesive material was surrounded by concentric layers mainly consisting of mature-type granulation tissues infiltrated by a moderate number of plasma cells and lymphocytes, a low number of PMNs, epithelioid macrophages and MGCs (initial foreign body reaction ). Mononuclear cell infiltrate was most severe at day T21, but subsequently progressively decreased to day T60, concomitant with a moderate foreign body reaction. Over time, mature granulation tissue gradually decreased and by day T60 was completely replaced by a moderately thick fibrous capsule. A low amount of necrotic debris was found in the adhesive-tissue zone at day T7, persistent until day T45, but absent at day T60 (see Figure 4 for graphical representation).

Representative histological images are shown in Figure 5.

The following Table II shows the average values of the histological parameters examined at different times

Table 11

<a, b>: The values in a column with different superscript letters are statistically different (* P <0.05; * P <0.01).

The following considerations can be drawn from the above data.

i) Histological examination reveals that injected glue III develops a moderate inflammatory response, initially consisting of a lymphoplasmacytic infiltrate and subsequently replaced by a foreign body reaction (chronic granulomatous inflammation). Regarding the encapsulation of glue III, the initial formation of the granulation tissue was progressively replaced by a moderately thick fibrous capsule. A small amount of necrotic filaments were detected in the interface area between the glue and the tissue at day T7, but at day T60 they were completely absent.

ii) Overall Colla III induced a moderate tissue reaction consistent with what was expected with respect to an injection of a non-degradable or poorly degradable synthetic foreign material (such as sutures or containment meshes) into the subcutaneous tissue.

iii) In conclusion, glue III is

in rat subcutaneous tissue and a similar behavior can be hypothesized rapidly in human tissue.

Claims (17)

CLAIMS 1. Composition characterized in that it comprises at least one alkyl cyanoacrylate and at least one haemostatic agent with pharmacological action, where said haemostatic agent with pharmacological action is an iron salt comprising homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters. 2. Composition according to claim 1 characterized in that said at least one alkyl cyanoacrylate is present in an amount comprised between 50% and 99% by volume of the total composition. 3. Composition according to one of the preceding claims characterized in that said at least one alkyl cyanoacrylate is n-butyl cyanoacrylate. 4. Composition according to one of claims 1 or 2 characterized in that said at least one alkyl cyanoacrylate is 2-octyl cyanoacrylate. 5. Composition according to one of claims 1 or 2 characterized in that said alkyl cyanoacrylate is a mixture of n-butyl cyanoacrylate and 2-octyl cyanoacrylate. 6. Composition according to claim 5 characterized in that the percentage of nbutyl cyanoacrylate is in the range 25-75% by volume, and the percentage of 2-octyl cyanoacrylate is in the range 25-75% by volume. 7. Composition according to claim 6 characterized in that the percentages of nbutyl cyanoacrylate and 2-octyl cyanoacrylate in the mixture are respectively 32% and 68% by volume. 8. Composition according to one of the preceding claims, characterized in that said iron salt of homopolymers or copolymers of α, β unsaturated carboxylic acids and their esters is an iron salt of homopolymers of α, β unsaturated carboxylic acids. 9. Composition according to claim 8 characterized in that said iron salt is a mixture of iron II and iron III salts of the 2-propenoic acid polymer called feracryl. 10. Composition according to claim 9 characterized in that feracryl is present in an amount comprised between 0.5% and 5% by volume on the total composition. 11. Composition according to claim 9 or 10 characterized in that feracryl is present in an amount equal to 1% by volume of the total composition. 12. Composition according to one or more of the preceding claims, characterized in that it further comprises a non-ionic surfactant consisting of copolymer blocks in which a propylene glycol group is flanked by two polyethylene glycol groups. 13. Composition according to claim 12 characterized in that said non-ionic surfactant is Pluronic PE6200. 14. Composition according to claim 13 characterized in that said Pluronic PE6200 is present in an amount comprised between 0.5% and 5% by volume on the total composition. 15. Use of the composition according to one or more of claims 1-14 as a surgical glue. 16. Medical kit comprising the composition according to one or more of claims 1-14 and a device for its application. 17. Kit according to claim 16 characterized in that it is of the disposable type.