BRPI0404248B1 - S-NITROSOTIOUS INCORPORATION PROCESS IN THE STRUCTURE OF SURGICAL ADHESIVES BASED ON THE TRANSFORMATION OF FIBRINOGEN IN FIBRINE - Google Patents

Abstract

"The process of incorporating s-nitrosothiols into the structure of surgical adhesives based on the transformation of fibrinogen into fibrin." The present invention relates to four general methods and eight specific methods for incorporating nitric oxide donor s-nitrosothiols into the structure of surgical adhesives which are based on the transformation of fibrinogen into fibrin and the surgical adhesives thus obtained. More specifically, these methods provide surgical patches that can reduce the risk of inflammatory complications following tissue and organ suture. These modified patches have the potential to promote revascularization by activating angiogenesis processes, reendothelization of arteries and veins that had damaged endothelium during surgical procedures and healing by improving local blood flow. In addition, these patches have the potential to reduce thrombogenic processes, smooth muscle cell proliferation and the risk of bacterial infection at the procedure site.

Description

(54) Title: PROCESS OF INCORPORATION OF S-NITROSOTIOLS IN THE STRUCTURE OF SURGICAL ADHESIVES THAT ARE BASED ON THE TRANSFORMATION OF FIBRINOGEN IN FIBRINE (51) Int.CI .: A61L 24/10; A61K 31/04 (73) Holder (s): UNIVERSIDADE ESTADUAL DE CAMPINAS - UNICAMP. UNIVERSIDADE DE SÃO PAULO - USP (72) Inventor (s): MARCELO GANZAROLLI DE OLIVEIRA; FERNANDA IBANEZ SIMPLICIO; JOSÉ EDUARDO KRIEGER; LUÍS ALBERTO OLIVEIRA DALLAN

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PROCESS OF INCORPORATION OF S-NITROSOTIOIS IN THE STRUCTURE OF SURGICAL ADHESIVES THAT ARE BASED ON THE TRANSFORMATION OF FIBRINOGEN IN FIBRINE

FIELD OF THE INVENTION

The present invention consists of four general methods and eight specific methods for incorporating nitric oxide donor S-nitrosothioles into the structure of surgical adhesives that are based on the transformation of fibrinogen into fibrin. These methods allow obtaining surgical patches that can reduce the risk of inflammatory complications after suturing tissues and organs. These modified patches have the potential to promote revascularization by activating angiogenesis processes, re-endothelialization of arteries and veins that had the endothelium damaged during surgical procedures and healing, by improving local blood flow. In addition, these patches have the potential to reduce thrombogenic processes, the proliferation of smooth muscle cells and the risk of infection by bacteria at the procedure site.

BACKGROUND OF THE INVENTION

The discovery in 1987 that nitric oxide (NO) is the endothelium-derived relaxation factor (EDRF) ¹ ' ² and that this diatomic molecule is produced endogenously in mammalian cells by a family of NO synthases (NOSs), determined a very large increase in research into the chemical and physiological properties of NO. In addition to the constitutive NOSs (endothelial, eNOS and neuronal, nNOS) responsible for NO regulatory actions in the cardiovascular system (relaxation of smooth muscles and blood pressure control) and in the nervous system (neurotransmission modulation), induced NOS (iNOS), it can produce high concentrations of NO which, in this case, plays a fundamental role in the cellular responses of the immune system ^3,4 . Other processes mediated by the biochemical action of NO include inhibition of platelet adhesion and neutrophil aggregation ⁵ , and stimulation of angiogenesis and

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• ·· · · • · • ·

re-endothelialization of blood vessels. These NO actions are, in turn, fundamental in controlling hemostasis and immunoinflammatory processes.

As plasma and the cellular environment have several reactive species that can quickly inactivate free NO, in contrast to the relatively long life span of EDRF, NO has been considered to be stabilized by transport molecules that prolong its half life, preserving their biological functions. The best candidates for this role are the peptides containing the sulfhydryl group (R-SH). This class of thiols can be nitrosated producing Snitrosothiois (RSNOs), which in turn can release free NO in the homolytic break of the SN bond. Among them are S-nitrosocysteine, S-nitroso-N-acetylcysteine, S15 nitrosoglutathione, S-nitrosopenicillamine and Snitrosoalbumin. As RSNOs have practically all the biochemical functions of free NO, there is currently great research interest in the synthesis of RSNOs linked to other substrates and in the development of biomaterials that use these substances for the controlled and localized release of NO in biomedical applications ⁶ .

Some of the S-nitrosothiois are marketed in solid form, such as S-nitrosoglutathione (GSNO) (ICN Pharmaceutical - Costa Mesa, CA - USA; Sigma -Aldrich, St.

Louis, MO - USA; Alexis Biochemicals, San Diego - CA - USA) and S-nitroso-N-acetylpenicillamine (SNAP) (ICN Pharmaceutical Costa Mesa, CA - USA; Sigma -Aldrich, St. Louis, MO - USA; Alexis Biochemicals, San Diego - CAUSE).

The methods of synthesizing S-nitrosothiois described in the literature and in patent applications include:

A - Reaction of the thiol with sodium nitrite (NaNO ₂ ) in an acid medium (HCI) in an ice bath. The formed S-nitrosothiol is precipitated by adding a solvent with a lower polarity than that of water, such as acetone or ether. To avoid adding another solvent for the precipitation of Snitrosothiol, one can only adjust the pH of the solution to 7.4

3/22 ···· · · glutathione. Β- Patents through the addition of base (NaOH) and buffered saline (See for example: T.W. Hart, Some observations concerning the Snitroso and S-phenylsulphonyl derivatives of L-cysteine and Tetrahedron Letters, 26, 2013-2016, 1985). US, 5,593,876, 6,471,347 and

6,124,255, in which the following thios nitration methods are described:

1- Nitrosation by exposing a polypeptide to a nitric oxide donor under conditions where the donor's nitric oxide release or transfer to the polypeptide is allowed.

2- Bubbling a gas source of nitric oxide through a polypeptide solution, for the time necessary for the formation of nitrosothiol.

3- Exposure of thiois to bovine aortic endothelial cells stimulated to secrete EDRF by exposure to shear forces.

4- Exposure of thiols to nitric oxide synthase, together with a substrate and a cofactor.

5- Acidification of alkaline solutions of thiols and species containing nitrite by the addition of acid.

A class of biomaterials of great importance in surgical procedures is that of bioadhesives used to assist healing processes. The use of synthetic surgical adhesives started around then, new adhesives were synthesized,

Cyanoacrylate, Fibrin, Gelatin-Resorcin, Geltin-ResorcinFormaldehyde, Gelatin-Resorcin-Formaldehyde-Glutaraldehyde and, more recently, based on Collagen, Glutamic Acid and 30 Water-soluble Dicarbamide ^7,8 .

Fibrin glue or fibrinogen is a lyophilized concentrate of human plasma proteins, which has high levels of fibrinogen, fibronectin and coagulation factor XIII. These components are activated by concentrated thrombin 35, diluted with calcium chloride, and to enhance the action of the components,

1960; from the base

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aprotinin, ··· * · ······ • · · · · · • ··· • · • · gives »· ····· · • · · · ···· · ·· stability one agent • ···· * « antifibrinolytic, which to clot formed ⁹ . This sticker, for to be a product biological, not provokes inflammatory reactions and tissue in the

application location. It is biodegradable; it has a slow release mechanism in the tissues; it does not generate toxic degradation products and about 20% of its original mass is disintegrated by fibrinolysis within 72 hours after application. In the 1980s, the use of fibrin glue as a bioadhesive was expanded ’with the impetus of its experimental and clinical applications.

Because it is a product derived from human blood, restrictions on use are associated with the risks of transmission of viral diseases such as hepatitis and AIDS 50. Its most extensive use occurs in European countries, Canada, Japan and the United States. The main applications include: sealing the vascular suture lines ¹⁰ ; reinforcement of suture lines by staplers in the esophagus and recto-sigmoid; high-risk gastro-intestinal sutures; mechanical lung sutures ¹¹ ; fixation of partial thickness skin grafts ¹² ; reduction of perioperative hemorrhage by nebulization (spray) of the fibrin sealant * 20 over the anterior mediastinum during cardiac surgery ¹³ ;

t elimination of air leaks when applied to bronchopleural fistulas ¹⁴ ; topical applications on raw pulmonary surfaces, after decortication or dryness of the lung; elimination of cerebrospinal cerebrospinal fluid leak in dura25 mater ¹⁵ ; reduction of bleeding in the debridement of bedsores and burns ¹⁶ ; maintenance of continuity of peripheral nerves injured by trauma ¹⁷ ; eardrum surgery and microvascular anastomoses ¹⁸ .

Although the clinical applications of surgical adhesives are well established, the chemical modification of bioadhesives offers perspectives for improving the results obtained, since several adverse consequences, resulting from surgical procedures, may be associated with their use. If the surgical procedure involves vascular lesions, the inflammatory process generated by tissue trauma at the site of the procedure can cause thrombosis

5/22 ··· ··· ·· · ·· · ·· · ·· • · ···· ··· · · ··· • · ·· · · · · ··· ··· ·· ··· · * ······ ····· · ····· · • ········ · · · • ··· ·· · ·· · · «·· · ·· acute or late with serious consequences. This same process can lead to the proliferation of smooth muscle cells on the walls of injured vessels, resulting in their occlusion as part of the healing response.

No patents or information were found in the literature on the incorporation, dissolution or synthesis of

S-nitrosothiois or any other nitric oxide donor in surgical adhesives based on the transformation of fibrinogen into fibrin in general, nor in the specific surgical adhesives 10 Beriplast P® and Tissucol®.

1- de Oliveira, M.G., Rohweder, J. J. R., Shishido, S.M. and Seabra, A.B. Nitrosation System Based on the Bubbling of a Gas Mixture of Nitric Oxide and Air, for the Nitrosation of Thiols and other Substrates. Patent application BR 0100557-4 - 02/09/2001.

2- de Oliveira, M.G. and Seabra, A.B. Process of Obtaining Polinitrosated Polyesters as Nitric Oxide Polymeric Donors for Biomedical Applications. Patent application BR 0300784-7- 02/24/2003.

Therefore, the association of bioadhesives with a nitric oxide donor drug has the potential to mitigate or even avoid these consequences, since nitric oxide inhibits platelet activation and aggregation (initial stages in the formation of thrombi) and also inhibits proliferation of smooth muscle cells on the walls of the vessels, (which is responsible for the formation of neointimate layers that can lead to occlusion of the vessels). A second advantage of the association of nitric oxide donors with bioadhesives is the benefit of the bactericidal action that nitric oxide can exert, thus reducing the risks of infection after the surgical procedure.

BRIEF DESCRIPTION OF THE INVENTION The present invention consists of four general methods for the incorporation of S-nitrosothiois in the structure of surgical adhesives that are based on the transformation of fibrinogen into fibrin and eight specific methods for the incorporation of Snitrosotiois in the structure of commercial surgical adhesives

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· Ν · »·» · · · ····· · ·· · ·· ·· • · · · · · · · * · ··· · ······ ····· · · · ···· · ······· · · · • ·· · ·· · ···· · ··

Beriplast P ^L Experimental transformation is also based on fibrin. Results of surgical adhesives and Tissucol, that of the commercial characterization fibrinogen Beriplast P® and Tissucol, containing the incorporated Snitrosoglutathione, are presented as examples.

The association of surgical adhesives with a nitric oxide donor S-nitrosothiol can reduce the risk of complications resulting from the immunoinflammatory process, since the local release of NO can have the following beneficial actions: Reduction of thrombogenic processes by inhibiting adhesion and aggregation platelet.

Promotion of revascularization by activation of the angiogenic processes e.

Promotion of re-endothelialization of arteries and veins that had an endothelium damaged during surgical procedures.

Inhibition of smooth muscle cell proliferation, with mitigation of the negative consequences of vascular remodeling.

Acceleration of the healing process by improving local blood flow.

Reduction of the risk of infection by bacteria at the surgical procedure site, due to the bactericidal action that nitric oxide can exert.

An additional advantage of this method is that it prevents the rapid diffusion of S-nitrosothiois out of the adhesive structure, thus allowing the release of nitric oxide for prolonged periods. This characteristic can increase the therapeutic action of nitric oxide in adhesives.

brief description of the figures

Figure 1 shows the spectral variation observed during the agitation of a fragment of the surgical adhesive Tissucol without the incorporation of S-nitrosothiols in deionized water for 3 hours at 37 ² C. The arrow in this figure indicates the growth of the band with maximum at 280 nm .

7/22 »· · · · ·· ··· · · · · · ··« · ······ «···» · ····· · · «···« «· · * · · · · · · ··· · · ♦ · · ··

Figure 2 shows the kinetic curve corresponding to the increased band at 280 nm (Fig. 1) associated with the release of adhesive Tissucol® fibrínicos aggregates in deionized water at 37 ² C.

Figure 3 shows the spectral variation observed during the agitation of a fragment of surgical adhesive Tissucol containing GSNO in deionized water for 4 hours.

Figure 4 shows the kinetic curves based on the monitoring of the bands at 280 nm and 33 6 nm, attributed to fibrin aggregates and GSNO, respectively.

Figure 5 shows the spectral variation observed during the agitation of a fragment of surgical adhesive Tissucol containing GSNO in solution of PBS and HbO2 at 27 ² C, for 2 hours.

Figure 6 shows the kinetic curves corresponding to the decay of HbO2 concentration in the absence of adhesive (upper curve) and in the presence of fragments of the Tissucol adhesive containing embedded GSNO (lower curves). Experiments performed at temperatures of 27, 37 and 47 ° C, as shown in the figure, monitoring the absorption band of the

HbO2 at 577 nm.

Figure 7 shows the kinetic curves corresponding to the decay of HbÜ2 concentration in the absence of adhesive (upper curve) and in the presence of fragments of the Tissucol® adhesive containing embedded GSNO (lower curves). For adhesives whose prior immersion times in PBS were 1: 10 am and 1: 30 pm the masses were 0.060 g for each fragment, while for the adhesive whose prior immersion time in PBS was 3: 50 h the mass was 0.076 g. The curves were obtained by monitoring the HbÜ2 absorption band at 577 nm.

Figure 8 shows the kinetic curves corresponding to the decay of HbO2 concentration in the absence of adhesive (upper curve) and in the presence of fragments of the Beriplast P® adhesive containing embedded GSNO (lower curves). The adhesive masses used were 0.105, 0.068, 0.041 and

0.03 6g for the previous immersion times of 0, 2:30, 4 and 20 h, «·· ··· ·· · ·· · * ·» ··

0 / ΖΖ · · · ··· · ·· · ·· · ·· • ·· «· ·····« ···· «· ···· · •« ········ · · · «« ·· ·· «·· · ···· · ·· respectively. The HbO ₂ absorption band was monitored at 577 nm.

Figure 9 shows a representation of the formation of an adduct between the acetamido group of the fibrin chains of the surgical adhesives and the carbon atom attached to the terminal amino group of the S-nitrosothiol molecule, represented in this case by S-nitrosoglutathione.

DETAILED DESCRIPTION OF THE INVENTION * The present invention consists of:

A- four general methods for the incorporation of Snitrosotiois in the structure of surgical adhesives that are based on the transformation of fibrinogen into fibrin.

B- eight specific methods for the incorporation of Snitrosotiois in the structure of commercial surgical adhesives

Beriplast P® and Tissucol®, based on the transformation of fibrinogen into fibrin.

That is, the commercial surgical adhesives that can be used in this patent application, but not limited to them, are: Beriplast P® (Aventis Behring GmbH, Marburg,

Germany) and Tissucol® (Baxter AG, Vienna, Austria). The US patent numbers that describe these adhesives are: 6,648,921; 6,613,325; RE38,158; 6,562,059; 6,558,687;

6,462,018; 6,447,774; 6,440,427; 6,329,337; 6,302,898; 6,280,727; 6,268,483; 6,235,041; 6,165,488; 6,096,309; 25 6,033,401; 6,019,993; 5,962,405; 5,911,165; 5,876,451; 5,723,010; 5,631,011; 5,618,551; 5,583,114; 5,413,601;

5,318,524; 5,284,856; 5,219,328 and 5,030,215.

The methods apply to all S-nitrosothiois consisting of amino acids or polypeptides containing the S-nitrosothiol group, including S-nitrosocysteine, Snitrosohomocysteine, S-nitroso-N-acetylcysteine, Snitroso-N-acetylpenicillamine, S-nitrosoglutathione and Snitrosoalbumin and all surgical adhesives based on the transformation of fibrinogen into fibrin.

5 - METHODOLOGY

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5.1. Description of the general methods for the incorporation of Snitrosotiois in surgical adhesives based on the transformation of fibrinogen into fibrin.

These methods apply to formulations that use the following components:

Fibrinogen, fibronectin, plasminogen and one or more clotting factors.

Aprotinin solution.

Lyophilized thrombin Calcium chloride solution.

General method:

A) Add S-nitrosothiol to the bottle containing the Aprotinin solution and shake until a homogeneous solution of the two components is obtained. The S-nitrosothiol can be added in solid form, or in solution form. In each case, the concentration of S-nitrosothiol to be reached in the aprotinin vial will depend on the number of moles of the added S-nitrosothiol. This concentration can vary freely, depending on the application. As an example, Snitrosoglutathione can be added in order to reach concentrations in the range of 1.0.10 ^-3 to 1.0 mol L ¹ of

GSNO.

B) Preheat the vials containing fibrinogen, fibronectin and one or more coagulation factors and aprotinin solution for about 10 minutes in a water bath at a temperature of 37 C.

C) Add the Aprotinin solution containing S-nitrosothiol to the vial containing Fibrinogen, fibronectin and one or more clotting factors (This mixture makes up what will be called formulation 1).

D) Return formulation 1 to the water bath, at 37 ² C, for 1 minute.

E) Remove formulation 1 from the water bath and shake with slow rotating movements to avoid excessive foam formation. Then replace the bottle of

10/22 water bath for another 10-15 formulation 1 for minutes.

F) Add the calcium chloride solution to the lyophilized Thrombin (This mixture composes what we will call formulation 2).

G) Remove formulations 1 and 2 from the bath and mix them with agitation. The polymerization reaction to form the adhesive starts at this moment. Therefore, the material can be applied right after stirring.

Note: Alternatively, S-nitrosothiol can be added to lyophilized thrombin, to the calcium chloride solution or in the vial containing fibrinogen, fibronectin and one or more coagulation factors, which make up formulation 2, under stirring, so to obtain a homogeneous solution. The details of these alternative methods are described in claims 2, 3 and 4.

5.1. Description of the specific method of incorporating Snitrosotiois into the Tissucol® surgical adhesive

Remove the 5 bottles that make up the Tissucol kit, namely:

Lyophilized Tissucol (which contains: fibrinogen, fibronectin and coagulation factor XIII);

Aprotinin solution;

Lyophilized thrombin 4

- Lyophilized thrombin 500

Calcium chloride

A) Add S-nitrosothiol to the bottle containing the Aprotinin solution and shake until a homogeneous solution of the two components is obtained. The S-nitrosothiol can be added in solid form, or in solution form. In each case, the concentration of S-nitrosothiol to be reached in the aprotinin flask will depend on the number of moles of the S-nitrosothiol. This concentration can vary freely, depending on the application. As an example, Sadicionado. depending on nitrosoglutathione can be added in order to reach concentrations in the range of 1.0.10 ³ to 1.0 mol L ¹ of

GSNO.

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B) Preheat the vials containing the lyophilized Tissucol® and aprotinin solution for about 10 minutes in a water bath at a temperature of 37 ² C.

C) Add the Aprotinin solution containing the S-nitrosothiol to the Tissucol® bottle. (This mixture makes up what we call formulation 1).

D) Return formulation 1 to the water bath at 37 ² C for one minute.

E) Remove the formulation from the swirling movements of the water bath and shake slowly to avoid excessive foam formation. Then return the bottle of formulation 1 to the water bath for another 10-15 minutes.

F) Add the lyophilized calcium chloride solution to Thrombin 4 (or Thrombin 500) (This mixture makes up what we call formulation 2).

G) Remove formulations 1 and 2 from the bath and mix them with agitation. The polymerization reaction to form the adhesive starts at this moment. Therefore, the material can be applied right after stirring.

Note: Alternatively, S-nitrosothiol can be added to lyophilized thrombin, to the calcium chloride solution or in lyophilized Tissucol®, which composes formulation 2, under stirring, in order to obtain a homogeneous solution. The details of these alternative methods are described in claims 6, 7 and 8.

5.2. Description of the method of incorporating S-nitrosothiois in the surgical adhesive Beriplast P®

A) Remove the 4 vials that make up the Beriplast P® kit, namely:

Beriplast P® (containing: fibrinogen, and coagulation factor XIII);

Aprotinin solution;

Lyophilized thrombin;

Calcium chloride

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B) Add ο S-nitrosothiol to the bottle containing the Aprotinin solution and shake until a homogeneous solution of the two components is obtained. S-nitrosothiol can be added in solid form, or in solution form. In each case, the concentration of S-nitrosothiol to be reached in the aprotinin vial will depend on the number of moles of the added S-nitrosothiol. This concentration can vary freely, depending on the application. As an example, Snitrosoglutathione can be added in order to achieve concentrations in the range of 1.0.10 ³ to 1.0 mol L ' ¹ of

GSNO.

C) Add the Aprotinin solution containing S-nitrosothiol to the bottle of Beriplast P®. (This mixture makes up what we call formulation 1).

D) Add the calcium chloride solution to the lyophilized Thrombin (This mixture makes up what we call formulation 2).

E) Mix formulations 1 and 2 with stirring. The polymerization reaction to form the adhesive starts at this moment. Therefore, the material can be applied right after stirring.

Note: Alternatively, S-nitrosothiol can be added to lyophilized thrombin, to the calcium chloride solution and in the bottle of Beriplast P, which composes formulation 2, under agitation, in order to obtain a homogeneous solution. The details of these alternative methods are described in claims 10, 11 and 12.

7. EXAMPLES OF RESULTS

7.1. NO release from the incorporation of S30 nitrosoglutathione (GSNO) in the Tissucol® surgical adhesive

7.1.1. Control experiment: Tissucol® without GSNO

Fig.l shows the spectral variation obtained in the monitoring of a mass of surgical adhesive Tissucol® without GSNO, immersed in deionized water in a quartz cuvette with magnetic stirring (1000 rpm) at 37 ² C. It is observed increased band at 280 nm, attributed to an absorption of fibrin

13/22 ····· · ····· • · · · · »····· · a (probably an η-π * transition of the acetamide group (R-CONH2). This increase in absorption indicates that fibrinic aggregates are leaving the adhesive mass over time and passing into the aqueous solution (these aggregates may be the result of incomplete crosslinking during fibrin polymerization or fibrinolysis after crosslinking).

From the monitoring of the band growth at 280 nm in Fig. 1, the kinetic curve, shown in Fig.

2. This curve provides the rate at which fibrinic aggregates leave the adhesive mass into the water.

7.1.2. Tissucol® with GSNO incorporated

Fig. 3 shows the spectral variation obtained over time, of a mass of Tissucol® surgical adhesive containing GSNO, immersed in deionized water, in a quartz cuvette under magnetic stirring (1000 rpm) and temperature of 37 ² C. note that, in this case, there is a simultaneous growth of two bands (280 nm and 336 nm) over time. This result suggests that GSNO is associated with fibrin aggregates in the form of a covalent adduct.

The formation of an adduct through a covalent bond of the GSNO with the peptides that make up the fibrin reticulum is evidenced by the observation of the practically identical kinetic behavior of the two bands (Fig. 4). If the GSNO were moving to the aqueous phase by diffusion alone, the growth rate of its band at 336 nm should be greater than that of the band of fibrin aggregates, since it is a molecule of high solubility in water.

The probable mechanism for the formation of this adduct may involve a reaction of GSNO with the fibrin monomer that contains the acetamido group, mediated by the enzyme transamidase (see below).

(R)

7.2. Monitoring the release of NO from the Tissucol adhesive containing GSNO from the reaction of NO with oxyhemoglobin

Oxyhemoglobin (HbOz) is known to react with NO, constituting one of the methods for detecting and quantifying free NO ¹⁹ . Therefore, the immersion of the adhesive containing GSNO in a

14/22 • · · «· ···· · • · ·» · · · · · aqueous HbC solution> 2 provides information on the release of free NO. The equation below shows the reaction of NO with Hb02 forming methaemoglobin (metHb) and nitrate (NO3 '):

HbO ₂ + NO - »HbOONO -> metHb + N0 ₃ (1)

Since the reaction of NO with HbÜ2 is stoichiometric, there is a quantitative method for the detection of NO available. The reaction presented above can be followed by the absorption band in the UV / Vis region of both HbÜ2 (λ = 577 nm), which should decrease as a function of time, as well as metHb, which should increase with time.

7.2.1. Method

A solution of HbO2 was prepared from a solution of hemoglobin (Hb) 1.1 mmolL ^-1 in PBS (pH 7.4 solution was added sodium dithionite (30 mg), ensuring the complete reduction of Hb oxygen in this solution for approximately 40 minutes To determine the concentration of HbC> 2 formed, 5pL of this solution was added in 1500 pL of PBS and the absorbance was measured at 415 nm The concentration of HbC> 2 was calculated from the following 20 equation :

HbO2 concentration = A4i _5nm x _300/8415 where A is the absorbance read at 415 nm, the molar absorption coefficient of HbO2 at 415 nm (131 mmol L cm ¹ ). The final concentration of the HbC stock solution> 2 must be between 0.8 - 1.2 mmol L ' ¹ . This solution was kept at low temperatures and protected from light.

7.2.2. Finding the release of NO by the adhesive

Fig. 5 shows the spectral variation observed in the reaction of HbO2 with the NO released by a fragment of the surgical adhesive

Tissucol® containing GSNO, immersed in PBS buffer solution at 27 ° C. This variation proves the release of NO (Eq. 3) by the GSNO incorporated in the adhesive (Eq. 3).

GSNO - ^ · GS-SG + 2 NO (3)

7.2.3. Effect of temperature on the NO release rate of the Tissucol® adhesive

To this in order to Then bubbled (2)

8415

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7.4). Α agitation

T Tissucol® adhesive containing incorporated GSNO was divided into three pieces, with masses of approximately 0.1 g each. Each piece of adhesive was immersed in a 1 cm quartz cuvette containing a solution of HbC> 2 (8.4 x 10 ^-4 molL ' ¹ ) in PBS (pH solution was stirred with a magnetic bar at 1000 rpm for two hours in a thermostatic system at temperatures of 27, 37 and 47 ° C. During this period, spectral variations at 577 nm were monitored at 5-minute intervals corresponding to HbC consumption> 2 due to its reaction with the NO released from the adhesive . the control of this reaction for comparison was obtained by following the stability of HbO2 solution in PBS at 37 ² C in the absence of adhesive. Fig. 6 shows the kinetic curves corresponding to the decrease of the absorption band at 577 nm in relation to time, for Tissucol® samples containing GSNO at the three temperatures used, together with the control curve.

It is observed that the presence of the adhesive leads to a great increase in the speed of disappearance of HbÜ2 at the three temperatures, indicating that in the three cases the adhesive is releasing NO to the solution. It is also noted that the release speed increases significantly from 27 to 37 ° C, with total consumption of HbO2 in less than 1 hour. It should be noted here that the complete disappearance of HbÜ2 does not mean that the entire GSNO decomposed releasing NO, since the number of moles of GSNO contained in the adhesive fragment is greater than the number of moles of HbO2 present in the solution. Therefore, after consuming HbO2, GSNO is still present in the adhesive (see item below). These data only show that the speed of NO release increases significantly with temperature. In the temperature increase from 37 to 47 ° C, there was no significant increase in the reaction speed of NO released with HbO2, which indicates that a threshold of maximum speed of NO release was reached between temperatures of and 37 ° C.

Tab.

shows the first order velocity constants and Tab. 2 shows the initial reaction velocities,

16/22 »···· · ····· • · · · • · · · · · · corresponding to the curves in Fig. 7, and the initial speeds were calculated according to Eq. 4 and the constants velocities were obtained from the exponential decay adjustments of the first order of the curves, using the Origin program (Microsoft, Inc.).

V ₀ = A [HbO ₂ ] / At (4)

Where A [HbO ₂ ] is the variation in oxyhemoglobin concentration and · »

At the time variation.

. 10 Tab. 1: Pseudo l ^s order constants (k) obtained from the kinetic curves of Fig. 6. for the reaction of consumption of HbO ₂ by the NO released by the Tissucol adhesive containing GSNO

Formulation conditions and k / h ' ¹ HbO ₂ 37 ² C 8.4.10 ^-4 HbO ₂ + Tissucol 27 ² C 1.2.10 ^-4 HbO ₂ + Tissucol 37 ² C 1.5.10 ^-3 HbO ₂ + Tissucol 47 ² C 1.6.10 ^-3

Tab. 2:: Initial speeds (v _o ) obtained from the kinetic curves of Fig. 6. for the reaction of consumption of HbO ₂ by the NO released by the Tissucol® adhesive containing GSNO

Formulation conditions and v _o / molL ^-1 h ^-1 HbO ₂ 37 ² C 4.5.10 ^-8 HbO ₂ 27 ² C + Tissucol 1.2.10 ^-7 HbO ₂ 37 ² C + Tissucol 7.1.10 ^-7 HbO ₂ 47 ² C + Tissucol 6.8.10 ^-7

17/22 »·· ··· ·· · ·· · ·· · ·· • ·· ···· ··· ··· ·· ·· · · ······ ····· · ····· · ········ · · · ··· ·· · ·· · ···· · ··

7.2.4. Analysis of the residence time of GSNO in the Tissucol® adhesive immersed in phosphate buffer solution (PBS) at pH 7.4 at ° C.

In order to evaluate the time of permanence of GSNO in adhesive 5 Tissucol under conditions of immersion in a buffer solution at pH 7.4 at 37 ² C, three fragments of adhesive containing GSNO were left immersed in closed flasks containing PBS in a bath at 37 ² C. Each fragment was removed after a different total immersion time (1:10, 1:30 and 3:50 h) and dipped in „10 followed by a solution containing HbO2 also in PBS buffer and at ² C. 0 Control of this experiment was obtained with an HbO2 solution under the same conditions, in the absence of the adhesive.

Fig. 7 shows the kinetic curves corresponding to the decay of HbC> 2 in contact with the adhesive fragments during a follow-up time of 1 h, together with the control curve. It is observed that the presence of the adhesive leads to an increase in the speed of disappearance of HbO2 after the three times of previous immersion of the adhesive in PBS solution, in relation to the control. These data demonstrate that even after

3:50 h immersed in PBS solution, the adhesive still has GSNO in its structure and that this GSNO is capable of converting HbO2 into methaemoglobin. Based on this result, it can be said that the structure of the adhesive imposes an important stabilizing effect on the GSNO, allowing a release of NO into the solution over long periods under physiological conditions. This is an important result for the medical applications that this modified adhesive may have, since it is desirable that the beneficial actions of the release of NO continue during the early stages of tissue healing.

Table 3 shows the initial HbO2 consumption speeds corresponding to the curves in Fig. 8, calculated according to Eq. 4. The reaction speeds after the immersion times of 1:10 and 1:30 h are practically the same as expected. The higher reaction speed after the 3:50 h immersion time can be attributed to the greater fragment mass used in this

18/22 ·· ··· · · · ·· · ··· ····· · ····· · ····· · · · ·· · ···· case, since the It is expected that the rate of consumption of HbO2 will decrease with the increase in the time of prior immersion of the adhesive.

Tab. 3: Initial speeds (v ₀ ) obtained from the kinetic curves of Fig. 8 for the reaction of consumption of HbC> 2 by the NO released by the adhesive Tissucol containing GSNO after different times of prior immersion of the adhesive in PBS at 37 ² Ç.

Immersion formulation and time adhesive preview Vo / molL ^-1 h ^{_ ±} HbO2 without adhesive 4.5.10 ^-8 Tissucol + HbO2 - 1: 10h 1.2.10 ^-7 Tissucol + HbO2 - 1: 30h 1.3.10 ^-7 Tissucol + HbÜ2 - 3: 50h 2.1.10 ^

7.3. Analysis of the GSNO residence time in the adhesive

Beriplast P® immersed in phosphate buffer solution (PBS) at pH

7.4 at 37 ° C.

The procedure used in this case was the same as described in the previous item. Previous immersion times of 1, 2:30, 4 and 20 Η were used. Fig. 8 shows that the consumption of HbCh decreases with the increase in the time of previous immersion of the adhesive in the buffer solution, as well as in the case of the Tissucol® adhesive. In the present case, the previous immersion times were longer, extending up to 20 h. On a scale of 1 to 2 h, the Beriplast P® adhesive produced a consumption of HbC> 2 with initial speeds of 4 to 5 times greater than the Tissucol adhesive. However, after 4 h of previous immersion, the rate of consumption of HbCb obtained was the same in both cases (Table 4). After 20 h of previous immersion, the Beriplast P adhesive did not produce any increase in the decay rate of HbCh in relation to the control, indicating that after this time the entire GSNO had already decomposed during the previous immersion in the PBS buffer solution. The kinetic curves in Fig. 8 allowed the adjustment of a first order exponential decay function. In this way, the speed constants of each curve, extracted from this adjustment, are

·· · * 19/22 • • '·' ·· · presented in Tab. 5. The k values reflect the behavior expressed by _the values of v in general, and small differences between the k values (as at times 1 and 2:30 h) it has no statistical significance, since the values are affected by the differences in the masses of the adhesive fragments and also by the error of the adjustment itself.

Tab. 4: Initial velocities (v _o ) obtained from the kinetic curves of Fig. 8 for the reaction of consumption of HbO ₂ by the NO released by the Tissucol adhesive containing GSNO at different times of immersion of the adhesive in PBS T - 37 ² C .

Immersion formulation and time adhesive preview Vo / molL ¹ h ¹ HbO ₂ alone 4.5.10 ^-8 Beriplast P + HbO ₂ 0 hours 1.10 ^-b Beriplast P + HbO ₂ lh 5.7.10 ^-7 Beriplast P + HbO ₂ 2h 30 min 5.6.10 ^-7 Beriplast P + HbO ₂ 4h 2.1.10 ^-7 Beriplast P + HbO ₂ 2Oh 5.9.10 ^-8

Table 5: Velocity constants of pseudo I ^to order (k) obtained from the kinetic curves of Fig. 8. for the reaction of consumption of HbO ₂ by the NO released by the adhesive Beriplast P®. containing GSNO at different times of immersion of the adhesive in

PBS T - 37 ² C.

Immersion formulation and time adhesive preview k / h ' ¹ HbO ₂ alone 2.3.10 ^-4 Beriplast P + HbO ₂ 0 hours 2.8.10 ^-3 Beriplast P + HbO ₂ lh 5.4.10 ^-4 Beriplast P + HbO ₂ 2h 30 min 6.0.10 ^-4 Beriplast P + HbO ₂ 4h 4.4.10 ^-4 Beriplast P + HbO ₂ 2 Oh 3.9.10 ^-4

7.5. ANALYSIS OF THE POSSIBLE MECHANISM FOR THE INCORPORATION OF SNITROSOTIOIS IN THE STRUCTURE OF SURGICAL ADHESIVES

Based on the preliminary experimental results that were obtained, it can be proposed that the incorporation of the S20 / 22 «· ·····» ·· «· · · • * · ··« · ··· ·· · ·· · · · · ···· · ···· * · ·· «» · · ····· «·· ·» ······ · ·· · ·· ♦ · ·· nitrosothiols in structure of the fibrin network of the commercial adhesives Beriplast P® and Tissucol® occurs through the covalent bond between the carbon atom attached to a terminal amino group (RC-NH ₂ ) of the structure of S-nitrosothiol and the nitrogen atom of the group acetamido (R-CONH ₂ ) of the monomers of

fibrin adhesive, according the reaction chemistry represented below (Fig. 9), for the example da S- nitrosoglutathione. At the case of this example, the formation adduct

it is mediated by the enzyme transamidase and the coagulation factor XHIa (F XlIIa), present in the adhesive formulations.

The above description of the present invention has been presented for the purpose of illustration and description. In addition, the description is not intended to limit the invention to the form disclosed herein. As a result, variations and modifications compatible with the above teachings, and the skill or knowledge of the relevant technique, are within the scope of the present invention.

Accordingly, the modalities described above are intended to better explain the known methods for practicing the invention and to allow those skilled in the art to use the invention in such, or other, modalities and with various modifications necessary for the specific applications or uses of the present invention. It is the intention that the present invention includes all modifications and variations thereof, within the scope described in the report and in the appended claims.

REFERENCES

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2. Ignarro, L.J .; Buga, G.M .; Wood, K.S .; Byrns, R.E .; Chaudhuri, G., Endothelium-Derived Relaxing Factor Produced and Released from Artery and Vein is Nitric Oxide, Proc. Natl. Acaã. Know. U.S.A., 84, 9265-9269 (1987).

3. Murphy, Μ. P., Nitric Oxide and Cell Death, Biochem. Biophys. Acta., 1411, 401-414 (1999).

4. Borges, S. S. S .; Gomes, M. G., Nitric Oxide: The Molecule of

Decade. Anais Assoe. Bras. Quim., 46 (4), 242-249 (1997).

21/22 • «··· * ·» ·· • «» · »« «· · »·· · ····>

······· · · • »· · ·· · ···· · ·· *

5. Hughes, M.N. Relationships between nitric oxide, ntroxyl ion, nitrosonium cation and peroxynitrite, Biochem. Biophys. Acta, 1411, 263-272 (1999).

6. Shishido, S. M.; Seabra, A. B .; de Oliveira, M. G., Thermal and photochemical nitric oxide release from S-nitrosothiols incorporated in Pluronic F127 gel: potential uses for local and controlled nitric oxide release, Biomaterials, 24, 35433553 (2003).

7. Trott, A. T. Cyanoacrylate tissue adhesives, an advance in wound care, Jama, 277, 1559-1560 (1997).

8. Papatheofanis, F. J .; Barmada R. The principies and applications of surgical adhesives. Surg. Annu., 25, 49-81 (1993).

9. Biondo-Simões, M. L. P .; Vivi, A. A .; Fagundes, D. J., Adhesives in anastomoses of the intestinal tract. Acta Cir Bras.

8 (1), 24-27 (1993).

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C. V., A randomized trial of fibrin sealant in peripheral vascular surgery. Vox. Sang., 70, 210-212 (1996).

11. Morio, A .; Miyamoto, H .; Yamazaki, A .; Anami, Y.; Oh, S .; Izumi, H.; Hosoda, Y.; Fukuchi, Y., A case of primary lung cancer complicated with post-operative intractable pulmonary fistula, Kyobu Geka, 53 (13), 1144-1147 (2000).

12. Ronfard, V .; Rives, J. M .; Neveux, Y .; Carsin, H .; Barrandon, Y., Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix, Transplantation, 70 (11), 1588-1598 (2000).

13. Hattori, R.; Otani, H .; Omiya, H .; Tabata, S .; Nakao, Y .; Yamamura, T.; Osako, M.; Saito, Y.; Imamura, H., Fate of fibrin sealant in pericardial space, Ann Thorac Surg, 70 (6),

2132-2136 (2000).

14. Nomori, H .; Horio, H.; Suemasu, K., Mixing collagen with fibrin glue to strengthen the sealing effect for pulmonary air leakage, Ann Thorac Surg, 70 (5), 1666-1670 (2000).

22/22 ·· ··· ·· tf ·· · ·· · ······· ··· ··· ·· • · · ·· · ··· · ·· · ·· ·· · · ······ · ···· · ····· · ········ ··· ··· ·· · ·· · ···· · · «

15. Yamakami, I .; Kobayashi, E .; Ono, J.; Yamamura, A., Prevention of cerebrospinal fluid leakage and delayed loss of preserved hearing after vestibular schwannoma removal: recontruction of the internai auditory canal in the 5 suboccipital transmeatal aprroach - thecnical note, Neurol.

Med. Chir., 40 (11) 597-601 (2000).

16. Clark, R. A., Fibrin sealant in wound repair: a systematic survey of the literature, Expert Opin. Investig. Drugs, 9 (10), 2371-2392 (2000).

10 17 Jou, I. M.; Chen, W. C .; Shen, C . L .; Matsuda, H., The influence of delay and the effect of fibrin sealant on the cut surface of the peripheral nerve, Journal of Hand Surgery, 24B (6), 707-711 (1999). 18 Kipshidze, N.; Ferguson, J. J .; Keelan, Μ. H.; Sahota, 15 H. ; Komorowski, R.; Shankar, L. R.; Chawla, 0. S .; Haudenschild, C. C .; Nikolaychic, V.; Moses, J. W., Endoluminal reconstruction of the arterial wall with endothelial cell / glue matrix reduces restenosis in an atherosclerotic rabbit. J. Am. Coll. Cardiol., 36 (4), 1396- 20 1403 (2000). 19 Balcioglu, A .; Watikins, C.J .; Maher, T.J., Use of a

hemoglobin-trapping approach in the determination of nitric oxide in in vitro and in vivo systems. Neurochem. Res. 23 (5), 815-820 (1998).

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Claims (5)

1. Method for the incorporation of S-nitrosothiois of amino acids or polypeptides into the structure of surgical adhesives that are based on the transformation of fibrinogen into fibrin, characterized by having the following steps: a) Add S-nitrosothiol to the bottle containing the aprotinin solution, shake until a homogeneous solution of the two components is obtained, preferably being added, S-nitrosoglutathione, in order to reach concentrations in the range of 1.0.10 ^-3 to 1 , 0 mol L ^-1 of GSNO; b) Preheat the vials containing fibrinogen, fibronectin and one or more coagulation factors and the aprotinin solution for 10 minutes, in a water bath, at a temperature of 37 ° C; c) Add the aprotinin solution containing S-nitrosothiol to the vial containing fibrinogen, fibronectin and one or more coagulation factors, forming “formulation 1”; d) Place formulation 1 in the water bath, at 37 ° C, for 1 minute; e) Remove formulation 1 from the water bath and shake with slow rotating movements to avoid excessive foam formation and then return the bottle of formulation 1 to the water bath for another 15 minutes; f) Add the calcium chloride solution to the lyophilized thrombin, pre-heating the vial for 10 minutes in a water bath, at a temperature of 37 ° C, forming “formulation 2”; g) Remove formulations 1 and 2 from the water bath and mix them with agitation. 2. Method for incorporating S-nitrosothiois of amino acids or polypeptides into the structure of surgical adhesives that are based on the transformation of fibrinogen into fibrin, characterized by containing the following steps: a) Add S-nitrosothiol to the flask containing lyophilized thrombin, shake until a homogeneous solution of the two components is obtained, preferably being added, S-nitrosoglutathione, in order to reach concentrations in the range of 1.0.10 ^-3 to 1.0 mol L ^-1 of GSNO; b) Preheat the vials containing fibrinogen, fibronectin and one or more coagulation factors and the aprotinin solution for 10 minutes, in a water bath, at a temperature of 37 ° C; Petition 870170095130, of 12/07/2017, p. 7/12 2/3 c) Add the aprotinin solution to the vial containing fibrinogen, fibronectin and one or more coagulation factors, forming “formulation 1”; d) Place formulation 1 in the water bath, at 37 ° C, for 1 minute; e) Remove formulation 1 from the water bath and shake with slow rotating movements to avoid excessive foaming and then return the bottle of formulation 1 to the water bath for another 10-15 minutes; f) Add the calcium chloride solution to the lyophilized thrombin containing the Snitrosothiol, pre-heating the vial for 10 minutes in a water bath, at a temperature of 37 ° C, forming “formulation 2”; g) Remove formulations 1 and 2 from the bath and mix them with agitation. 3. Method for incorporating S-nitrosothiois of amino acids or polypeptides into the structure of surgical adhesives based on the transformation of fibrinogen into fibrin, characterized by containing the following steps: a) Add S-nitrosothiol to the bottle containing the calcium chloride solution, shake until a homogeneous solution of the two components is obtained, preferably adding S-nitrosoglutathione, in order to reach concentrations in the range of 1.0.10 ^{- 3} to 1.0 mol L ^-1 of GSNO; b) Preheat the vials containing fibrinogen, fibronectin and one or more clotting factors and the aprotinin solution for 10 minutes, in a water bath, at a temperature of 37 ° C; c) Add the aprotinin solution to the vial containing fibrinogen, fibronectin and one or more coagulation factors, forming “formulation 1”; d) Place formulation 1 in the water bath, at 37 ° C, for 1 minute; e) Remove formulation 1 from the water bath and shake with slow rotating movements to avoid excessive foaming and then return the bottle of formulation 1 to the water bath for another 10-15 minutes; f) Add the calcium chloride solution containing the S-nitrosothiol to the lyophilized thrombin, pre-heating the vial for 10 minutes in a water bath, at a temperature of 37 ° C, forming “formulation 2”; g) Remove formulations 1 and 2 from the bath and mix with stirring. 4. Method for the incorporation of S-nitrosothiois of amino acids or polypeptides into the structure of surgical adhesives based on the transformation of fibrinogen into fibrin, characterized by containing the following steps: Petition 870170095130, of 12/07/2017, p. 12/8 3/3 a) Add S-nitrosothiol to the bottle containing fibrinogen, fibronectin and one or more coagulation factors, shake until a homogeneous solution of the two components is obtained, preferably being added, S-nitrosoglutathione, in order to reach concentrations in the range from 1.0.10 ^-3 to 1.0 mol L ^-1 of GSNO; b) Preheat the vials containing fibrinogen, fibronectin and one or more coagulation factors and the aprotinin solution for 10 minutes, in a water bath, at a temperature of 37 ° C; c) Add the aprotinin solution to the vial containing fibrinogen, fibronectin, one or more clotting factors and GSNO, forming “formulation 1”; d) Place formulation 1 in the water bath, at 37 ° C, for 1 minute; e) Remove formulation 1 from the water bath and shake with slow rotating movements to avoid excessive foaming and then return the bottle of formulation 1 to the water bath for another 10-15 minutes; f) Add the calcium chloride solution to the lyophilized thrombin, pre-heating the vial for 10 minutes in a water bath, at a temperature of 37 ° C, forming “formulation 2”; g) Remove formulations 1 and 2 from the bath and mix them with agitation. 5. Surgical adhesives based on the transformation of fibrinogen into fibrin, characterized by incorporating its S-nitrosothiois structure of amino acids or polypeptides through the methods described in claims 1 to 4. Petition 870170095130, of 12/07/2017, p. 9/12 * · «» · «·· ·· ·» · · * * · «« φ · · · »» ♦ · • «» · · * * · · · · * · * · 4 «φ φ 1« »» * «····» ♦ »» «·» 4 «· * ·« «* · * · * +« »ΦΦ φ ΦΦ · ···· ·« 4 1/5