MX2011013865A - Anticalcification composition for prosthesis. - Google Patents

Abstract

The present invention refers to anticalcification compositions for human prosthesis, mainly bioprosthesis, which are characterised in that they use a single amino acid with a single free aldehyde which is used to be fixed to the glutaraldehyde (GA) and avoid the calcium fixation, thus when the amino acid is in contact with the pericardium, no free aldehyde group is free to which another substance would be fixed, such as proteins, calcium, iron, lipids, etc. In addition, these amino acid removes, from the beginning, the GA from the pericardium. The anticalcification composition of the present invention also has the advantage of being a natural, non-toxic product produced by the human body. Likewise, the anticalcification composition is manufactured in an easy manner, with a few substances, in a quick form, without risks or complications, also avoiding side effects in human prosthesis uses. Finally, the fact of storing the bioprosthesis in glycerine and not in aldehyde avoids the new fo rmations of GA in the tissue.

Description

ANTICALCIFICATION COMPOSITION FOR PROSTHESIS Field of the Invention The present invention is defined in the field of anticalcific solutions for cardiac prostheses. Particularly, the present invention refers to solutions comprising at least one amino acid and which serve to prevent calcium from being fixed in the biological tissue used in the bioprosthesis and thus preventing the cycle of calcification that leads to the deterioration of the prosthesis. and therefore of its durability and competence.

Background of the Invention Given the evidence that biological tissues end up calcifying, for several years many groups have been given the task of investigating the causes of it as well as the shorter duration of bioprostheses. These investigations have clarified several points, although in the end all the mechanisms that lead to the mentioned calcification are not fully known to satisfaction, but much progress has been made in this field. It is known, for example, that glutaraldehyde is a good tissue preparer, since it helps fix the collagen fibers of the tissues between their various layers and produces and maintains bridges between them. It is also known that it helps to make it less antigenic and maintains elasticity, in addition to sterilizing the biological tissue; but at the same time as this, it is now known that the glutaraldehyde molecules that remain in the tissues attract the calcium that ends up being fixed to the bioprostheses.

The transcendent discovery in these studies that glutaraldehyde (GA) is a two-edged weapon, since at the same time that it produces a series of beneficial effects in the tissues treated with it, produces the counterproductive phenomenon of inducing calcification, has promoted the interest by looking for other mechanisms to prepare tissues no longer with glutaraldehyde, but with others such as epoxy compounds, photo oxidation, thermal preparation, diphenylphosphorylazide, carboimic compounds, cryopreservation and acelurization.

On the other hand it is important to note that dystrophic calcification (also called "mineralization" because other substances are also added) - abnormal calcium accumulation in greater proportion and small others such as iron, lipids, gold, magnesium, manganese, etc. - no is a problem produced only by using glutaraldehyde, but it is a natural phenomenon in the human species, due to several factors, such as age, hyperparathyroidism, dyslipidemia, diabetes mellitus overweight, sedentary lifestyle, etc. - Of these, age is undoubtedly the causative to a greater degree of it, because only by aging -in this case- of calcium metabolic systems, it accumulates in practically any organ of man (brain, arteries of all sizes, hearing, eye, abdominal organs, etc. .) producing the generic disease called atherosclerosis, causing deterioration of the functions of the affected organs and when it happens in the arteries, decreases the caliber of its light and therefore of the blood supply to various tissues and organs (as an example of this, we can mention the cardiac infarction, sequela of atherosclerosis that has partially or totally obstructed some or several of the heart's own arteries, to produce the death of part of the muscle cardiac due to lack of blood supply). This same phenomenon of calcium accumulation occurs with age in the cardiac valves, especially those on the left side - aral and aortic, which receive higher pressures and therefore stress - and this calcium can be fixed in the valves, but also in the rings of those valves in their union with the heart. This phenomenon can become so important, with deformation of the leaflets in such a way that it obstructs them, prevents them from opening completely (stenosis) or closing only partially (insufficiency), a serious situation that forces the valves to be replaced. damaged by a prosthesis, as can be seen in Figure 1.

At present, calcification of the aortic valve is the most frequent cardiac disease in the world and, therefore, the replacement of this valve with a prosthesis is the most frequent of all cardiac surgeries worldwide. A few years ago this place was occupied by atherosclerotic coronary disease, but today it is occupied by adult aortic stenosis.

Therefore, being the atherosclerosis a degenerative and systemic disease, it is not strange that when a cardiac bioprosthesis is placed, there is the possibility that sooner or later it calcifies. Because of this, since the discovery of this phenomenon, a solution was found; For this, a great variety of methods have been implemented to try to reduce, delay or avoid it.

The bioprosthesis, today there is consensus, must be protected in some way against calcification, because the damage to the biological part due to this phenomenon can cause structural damage as severe as general hardening of the valves and described above, which require reoperation to the patient, as can be seen in Figure 2, where it shows a bioprosthesis with hardened, calcified valves and even rupture of one of them.

On the other hand, mechanical prostheses, not having biological components, do not calcify, but in contrast, they have other complications that make them more risky than biological ones. Being a foreign body in the human organism, it tends in some way to reject it and produces a series of inflammatory phenomena in response that end with the formation of irregular tissue in a kind of encapsulation of the prosthesis. This phenomenon is not acute, it can take months or years and of course this response is personal, because in some patients it is rapid and very intense and in others, slower and more partial. This phenomenon is called PANNUS generically and occurs in a situation similar to the reaction of a mollusc when a foreign body -like a grain of sand for example- is introduced into its habitat. The mollusk produces a secretion equal to the one that it uses to cover the interior of its valves and with it it encapsulates the foreign body. The final result is a pearl.

In the human organism, keeping the distances because the mechanisms involved to produce the pannus are much more complex than what is described for a pearl, in any case this one is produced that tries to isolate this intruder, but as it is done in an irregular way, it ends up affect the movement of the valves, in addition to deforming them, prevents their proper closing or opening and finally obstructs the light of the valves and can block them completely, and a thrombus can be added, which makes the problem even more complex, This situation is extremely serious, because when this happens, the patient dies in minutes, without any possibility of intervening.

In addition, the mechanical prosthesis suffer other very serious contingencies, since similar phenomena in the effect happen with mechanical prostheses obstructed by thrombi, a situation that does not occur in biological prostheses, in addition to the initial mechanism of rejection, in the case of bioprostheses. , is blocked precisely by glutaraldehyde.

Thus, it is clear that if the bioprostheses are not protected with some method against calcification, they will inevitably end up deteriorating and their useful life will be shorter, which would require new valve changes and the inherent risk and cost of this situation. The clinical follow-up given to these prostheses has undoubtedly shown that good results have been achieved for this serious problem when some type of protection is used. The durability of the prostheses with this treatment has improved ostensibly. In the 1990s, when the bioprostheses were only a few years old and, of course, no anti-chemical system was used, the survival of the bioprostheses was 8-10 years. By improving the designs and especially by adding some anticalcificante system, this survival is today on average between 15 and 18 years, almost twice and this undoubtedly product of the application of these systems.

The INC has also demonstrated a similar situation, since currently, (with the application of this method of more than 1200 prostheses placed in almost 10 years) none of these prostheses has suffered visible or clinical deterioration that forced to change them, which means that will be, statistically at least similar ranges, than what is shown in the world literature on the subject. In general terms, four major ideas and methods have been developed and used in clinical practice to date.

They can be included as follows: A- Removal of lipids (phospholipids, cholesterol, etc.) B- Covalent substitution C- Detergents D- GA Removal (INC investigation) The first of them is important because these normal components in the pericardium and practically in any tissue, can attract calcium from the bloodstream initiating the first stage of calcification, nucleation. The most commonly used substances for this purpose are ethanol, methanol and chloroform.

The second is also very important, since the glutaraldehyde from which the pericardium is impregnated, by a chemical reaction, can bind its free amino groups with those of circulating calcium, thus causing the initiation (nucleation) and continuation of the phenomenon of dystrophic calcification. If these possible joints are covered with other substances, in this case an anti-chemical system, in theory these can no longer be used by calcium. The most commonly used substances for this purpose are amino oleic acid, iron, aluminum and some amino acids. This mechanism is possibly the best and most effective way to avoid calcification.

The third, has a purpose similar to the previous one and the most used are sodium dodecyl sulfate, amino oleic acid and L-glutamic acid. Its effect is not as complete as the previous one.

The fourth is another mechanism to remove practically all GA from the beginning, which makes the possibility of calcium adherement minimal. To this is added that by not storing the bioprosthesis in aldehyde until its clinical use (which may last for months or years) protects it from having aldehyde on its surface and therefore less possibility of calcification).

On the other hand, there are documents such as US 7,972,376 and US 4,976,733, which describe methods to attenuate calcification in a bioprosthesis. These methods consist of treatment with a limiting agent such as an amine, an amino acid and a aminosulfonate in combination with a reducing agent such as sodium borohydride and the process of dehydrating the tissue with a glycerol ethanol solution. In addition, the tissue used in the bioprosthesis is bovine pericardial tissue. These documents protect the use of compounds that have an amino group that must act as a substituent in an aliphatic chain and subsequently must be covalently bound to the prosthesis.

Likewise, document US WO / 2003/064706 discloses an anti-chemical solution in which a coupling agent is used which promotes the formation of amide bonds between carboxyl reactive moieties and amino reactive moieties; an agent that improves coupling and a crosslinking agent. Said document describes the use of amino groups in an amount not less than 80 millimoles, in addition to other compounds.

Moreover, WO / 2006/066327 and US 20020164739 disclose a method for treating an implantable material, which consists of exposing said material in an alcohol solution and subsequently in an acid solution which must contain an amino carboxylic acid such as L-histidine, L-arginine, L-lysine, L-glutamate and L-aspartate. In addition, the use of a specific amino acid sequence which produces inhibitory activity of calcification is described.

As you can see, all attempts in the clinic are made to try to counteract glutaraldehyde, but none uses any method to remove it. Thus, currently all commercial houses that use any of the conventional methods, although they apply it in a professional and strict manner, all end up, storing their product in some aldehyde until its clinical use, which, if it had been possible to protect the tissue of a future accumulation of calcium with his system, by putting his prosthesis again for months or years in aldehyde, the amount of this that adheres to the tissue easily exceeds the level of protection he had and again many amino sites are left to join calcium and calcium. The effect achieved with the system they have used is no longer effective.

Summary of the Invention Given the above technical problem, it is an object of the present invention to provide an anticalcific solution for cardiac prostheses placed in humans, particularly bioprostheses which has the following main advantages: Removal of lipids (phospholipids, cholesterol, etc.) This is important because certain normal components in the pericardium and practically in any tissue, can attract from the bloodstream calcium initiating the first stage of calcification, nucleation. The most commonly used substances for this purpose are ethanol, methanol and chloroform.

- Covalent substitution The glutaraldehyde from which the pericardium is impregnated, by a chemical reaction, can bind its free amino groups with those of circulating calcium, thus causing the initiation (nucleation) and continuation of the phenomenon of dystrophic calcification. If these possible bonds are covered with other substances, such as the anti-chemical composition according to the present invention, these can no longer be used by calcium. The most commonly used substances for this purpose are amino oleic acid, iron, aluminum and some amino acids. This mechanism has possibly proven to be the most effective in preventing calcification.

Detergent Purpose similar to the previous one and the most commonly used are sodium dodecyl sulfate, amino oleic acid and L-glutamic acid. Its effect is not as complete as that proposed by the composition according to the present invention.

Glutaraldehyde removal Removal of the vast majority of glutaraldehyde (GA) present in the pericardium. Tissue storage The storage of the tissue, until its clinical use in 50% glycerin, prevents the tissue from becoming impregnated with GA again.

Thus, the anti-chemical composition according to the present invention is based on three essential characteristics: A- Uses a protection mechanism, with an amino acid - - simple, glycine, which by a mechanism of covalent replacement, prevents calcium from attaching to the pericardium.

B- Removes glutaraldehyde (GA) remaining tissue, as has been proven in a series of studies in vivo T in vitro and later with its practical application in more than 1,200 patients who have been placed one or more prostheses treated with this system.

C- Protects the exposure of the bioprostheses back to aldehydes and therefore they do not re-impregnate with glutaraldehyde (GA).

Description of the Figures of the Invention Figure 1 - Figure 1 shows a native calcified aortic valve.

Figure 2 - Figure 2 shows a bioprosthesis with hardened, calcified valves and even the rupture of one of them.

Figures 3a to 3c - Figures 3a to 3c show a treated pericardium. Figure 3a shows the pericardium treated only with glutaraldehyde (GA) for 14 days where the staining that produces the large amount of aldehyde is observed, when it is not treated with the solution according to the present invention. Also, Figure 3b shows a pericardium treated 7 days with glutaraldehyde (GA) and in addition with the anti-chemical solution according to the present invention, - - where it is observed how the amount of is treated. Finally Figure 3c shows a pericardium treated 24 hours with glutaraldehyde (GA) and also with the anticalcifying solution according to the present invention, where practically no glutaraldehyde (GA) exists.

Detailed description of the invention The main purpose of the anti-chemical solution according to the present invention is to remove as much as possible of the glutaraldehyde (GA) accumulated in the pericardium, since this prevents the substance that has been shown to attract circulating calcium in the blood to be able to bind to it. None of the methods previously proposed and used to date acts in this sense. Thus, the anti-chemical solution according to the present invention has, with a single substance, two positive effects of those mentioned, one, to eliminate practically all the residual glutaraldehyde (GA) in the pericardium and the second, by the method of covalent substitution, to prevent its possible union with calcium or any other component.

The solution according to the present invention uses a single amino acid, glycine, which is the simplest of all those that exist, with a single free aldehyde, which is what it uses to bind to glutaraldehyde (GA) by covalent substitution and thus avoid the fixation of calcium, therefore, does not leave a free aldehyde group so that no other substance such as calcium, proteins, iron, lipids, etc., can be fixed to it.

- - This anticalcifying solution based on glycine has the advantage of being a natural, non-toxic product of the human organism. It is one of the conditional amino acids and is a very important natural part of many tissues and organs of man.

The solution has been tested in vivo, in vitro and, more importantly, in the long-term clinic with very satisfactory results. Almost 1300 patients for almost 10 years without any of their prostheses having calcified (and requiring reoperation). This solution is also very simple, as it is a glycine preparation (0.05 molar in phosphate buffer solution for a pH of 7.4). In addition, the manufacture of the anti-chemical composition is simple, does not involve many substances, fast, without dangers or complications. that in its use in human prostheses there is no risk of undesirable side effects.

The anti-chemical solution according to the present invention is prepared in the following manner: a 0.05 molar glycine solution is prepared, with sodium monobasic phosphate approximately 2.12 g / 1 with a purity of 99.5%; sodium dibasic phosphate approximately 2.07 g / 1 with purity of 99%, sodium chloride approximately 17,532 g / 1 and glycine (biotechnological grade) and degree of purity 99.8) approximately 3.75 g per liter with a pH of 7.4.

The pericardium previously treated with glutaraldehyde (GA) is placed in a container, with sterile technique in a closed environment (laminar flow hood) in the solution of - glycine for one hour, after which it is rinsed three consecutive times in sterile saline for 5 minutes each time, trying to remove as much as possible of glutaraldehyde (GA) and stored in the preservative solution. It is only removed from this solution to manufacture the prosthesis always in a sterile environment and once it is finished it is put back in its final container with the aforementioned conservative solution (50% glycerol).

The tests to determine if the system was useful were made in the following way: In vitro experiments In vivo experiments Clinical placement In the in vitro experiments, several placement sequences of the preparation of the anticalcifying solution in bovine pericardium were made: - fresh bovine pericardium, without any preparation bovine pericardium treated with glutaraldehyde (GA) for one hour - bovine pericardium treated with glutaraldehyde (GA) for one hour plus glycine solution for one hour 14 bovine pericardium treated with glutaraldehyde (GA) for 24 hours - bovine pericardium treated with glutaraldehyde (GA) in 24 hours, plus glycine solution for one hour bovine pericardium treated with glutaraldehyde (GA) - for a week seven days - bovine pericardium treated with glutaraldehyde (GA) for one week seven days, plus glycine solution for one hour bovine pericardium treated with glutaraldehyde (GA) for 14 days - bovine pericardium treated with glutaraldehyde (GA) for 14 days, plus glycine solution.

The results showed, as seen in Figures 3a to 3c, that the longer the pericardium remains in glutaraldehyde (GA), the more amount accumulates in it and therefore, its elimination is incomplete. Conversely, when less time was left in glutaraldehyde (GA) and glycine was added, the elimination of GA was complete. All this shows that indeed the use of glycine removes practically all the glutaraldehyde (GA).

For in vivo tests, these samples were placed in guinea pigs (6 for each group) divided for each of the sample type.

Subsequently, they were sacrificed in periods of one, three and six months respectively and analysis of microscopy and calcium measurement were carried out with an atomic absorption method. These tests showed that the pericardium that had remained for 14 days was the one that had accumulated the most calcium, followed by the one that had remained for 7 days in the glutaraldehyde (GA) solution, then the 24-hour one. Of course the one who had only been 24 - - hours and that in addition it had been treated in addition with the anticalcificante solution was the one that ostensibly accumulated less calcium and at the same time the tests of microscopy with staining showed that the residual amounts of glutaraldehyde (GA) were congruent in the same sense, as can be seen in Figures 3a to 3c. The amount of normal calcium in our experimental animals without any treatment, ie fresh, was 2.83 mg / g of dry tissue.

Table 1 shows the average results of these groups: Table 1 Finally, after 5 years of these studies and verifying that the effect of decreasing the amount of calcium in the tissue treated with this anti-chemical solution was effective, it was decided to take it to the clinic and to date there are more than 1200 patients to whom one or more prostheses treated in this way have been placed and the clinical follow-up (which includes chest x-rays and echocardiography) shows no signs of calcification and some type of valvular dysfunction attributable to hardness (due to calcification) of the leaflets of said prostheses . Neither has any of the patients in this group been reoperated for structural damage to the prosthesis.

Claims (8)

EIVINDICATIONS Once the invention is described, what is considered novel and therefore its property is claimed is the following:

1. An anticalcifying composition for cardiac bioprostheses made from biological tissue, characterized in that it comprises at least one amino acid with a single free aldehyde, which serves to prevent calcium from being fixed in the biological tissue preventing the cycle of calcification that generates the deterioration of the prosthesis and therefore its durability and competence.

2. The anti-chemical composition according to claim 1, characterized in that the amino acid used is glycine.

3. The anti-chemical composition according to claim 1, characterized in that it has a glycine concentration of 0.05 molar.

4. The anti-chemical composition according to claim 1, characterized in that it also comprises sodium monobasic phosphate, dibasic sodium phosphate and sodium chloride.

5. The anti-chemical composition according to claim 1, characterized in that it has a pH of 7.4.

6. The anti-chemical composition according to claim 1, characterized in that it removes from the beginning the glutaraldehyde of the biological tissue.

7. The anticalcifying composition according to claim 1, characterized in that by being in contact with the biological tissue or by storing said biological tissue in it, it leaves no free aldehyde group so that another undesirable substance such as proteins, calcium, iron, can be fixed to it. lipids, etc.

8. The anti-chemical composition according to claim 1, characterized in that it is non-toxic and can be used in prostheses for human patients without risk of undesirable side effects. SUMMARY OF THE INVENTION The present invention relates to an anticalcifying composition for human prostheses, particularly bioprostheses, which has the advantages that it uses a single amino acid with a single free aldehyde which is used to bind to glutaraldehyde (GA) and therefore avoid fixation of Calcium, therefore, when in contact with the pericardium does not leave a free aldehyde group so that another substance can be fixed to it such as proteins, calcium, iron, lipids, etc. In addition, this amino acid removes GA from the pericardium from the beginning. The anti-chemical composition according to the present invention also has the advantage of being a natural product, non-toxic and proper to the human organism. Likewise, the anticalcifying composition is manufactured in a simple manner, it does not involve many substances, fast, without dangers or complications, besides that in its use in human prostheses there is no risk of undesirable side effects. Finally, by not storing the bioprosthesis in glycerin and not in aldehyde, the new accumulation of GA in the tissue is avoided.