WO2019153099A1 - Compound and procedure for optimising the transplant of vascularised solid organs and reducing the dysfunction of same - Google Patents

Abstract

The invention relates to a compound and procedure for optimising the transplant of vascularised solid organs and reducing the dysfunction of same, which comprises 400 to 1000 mg of alpha lipoic acid (ALA), preferably 600 mg of ALA, and the administration of ALA for the usual treatment of immunosuppression, for perfusion of the graft prior to the transplant and to the recipient during the first two days following the transplant in order to reduce the functional recovery time of the graft and to improve the short-term clinical parameters for patients undergoing kidney-pancreas, kidney and liver transplants.

Description

 COMPOSITE AND PROCEDURE TO OPTIMIZE THE TRANSPLANTATION OF THE VASCULARIZED SOLID ORGANS AND REDUCE THE DYSFUNCTION OF THEMSELVES

DESCRIPTIVE MEMORY

1) FIELD OF THE INVENTION

This invention relates to a "COMPOUND AND PROCEDURE FOR OPTIMIZING THE TRANSPLANTATION OF THE VASCULARIZED SOLID ORGANS AND REDUCING THE DYSFUNCTION OF THEMSELVES", especially in the stages of ablation, transplantation and post-transplantation in order to optimize the functional recovery time. of the graft and the clinical parameters of short-term patients preferably undergoing renopancreatic, renal and hepatic transplants.

2) STATE OF THE TECHNIQUE AND EXPOSURE OF PROBLEMS TO BE SOLVED

It is known in the prior art that irreversible organ damage is one of the main health challenges. One way to mitigate this problem is through performing organ transplants. While the transplant has achieved a considerable degree of success, several factors significantly limit its effectiveness. In this context, inflammatory reactions in the graft have a fundamental influence on the functioning of the organ in the short and long term. One of the main reasons for the initial inflammatory reaction is the damage caused by ischemia and reperfusion of the organ, thus conditioning the recovery time of organ function. post-transplant In the case of renal transplantation, the clinical entity called delayed graft function (DGF) is a frequent clinical situation where the organ takes time to recover the function after the transplant. In other types of transplantation, such as the liver, adverse events are also described in the immediate post-transplant stage. In this case, a syndrome called “reperfusion syndrome” (SPR) may occur, which manifests as a decompensation of the transplant recipient. Bibliographic data indicate that both reactive oxygen species and mediators of the immune response would participate in these initial adverse events. Therefore, therapeutic strategies that tend to limit the action of free radicals as well as immunomodulators, could have beneficial effects on the transplant.

Once the surgery is performed, the functioning of the organ is expected to be immediate. However, in most of the different types of vascularized solid organ transplants the possible occurrence of a phenomenon in which the graft does not work immediately due to the injury of the entire process involving ablation and transplantation is described. This phenomenon called early graft dysfunction is defined differently according to the transplanted organ, and even, for the same type of transplant, between different centers and in the literature.

Currently, the only therapeutic behavior that reduces the early adverse events of the transplant is the use of quality perfusion machines and perfusion fluids such as Wisconsin and HTK. In addition, different combinations of immunosuppressants that vary according to the type of patient and the center where the transplant is performed are also administered. This first immunosuppression is called induction therapy.

Immediate functional graft recovery is generally achieved with ideal living donors and reduced hot and cold ischemia times. Without However, these types of ideal transplants, in practice, cannot be performed routinely due to a shortage of donors. In addition, it should be borne in mind that, in order to increase the supply of organs, organ donation programs of so-called donors with extended criteria have been installed. These donors are far from ideal and have higher percentages in the appearance of the so-called early graft dysfunction. Today, there are no methods that reduce early graft dysfunction when the recipient of a transplant receives an organ from a donor with extended criteria.

Another problem in the first post-transplant hours could be the appearance of rejections. However, the problem of rejections has been partially solved with cross-match studies, HLA typing and the use of immunosuppressive treatments.

There are two types of immunosuppressive treatment that try to avoid rejection: induction treatment and maintenance treatment. Immunosuppressive induction treatment tries to avoid the phenomenon of graft rejection, and in fact it succeeds. However, this treatment is not effective to achieve a rapid functional recovery of the graft. There is currently no therapeutic strategy based on the administration of any drug or drug that manages to recover the graft's early function. As mentioned above, the strategies are based on the use of continuous perfusion machines, but these are not implemented in all transplant centers and are very expensive due to the inputs that must be replaced after each operation.

The methods used today to recover the graft's early function are based on improving preservation techniques and reducing ischemia times. This last aspect is not always possible because of what the whole organ procurement process implies. In Argentina, the preservation method used to minimize damage at the time of transfer and Organ preservation is the static hypothermic preservation whose foundation is the suppression of cellular metabolism by hypothermia at 4 ° C. However, hypothermia alone is not enough for proper preservation, so it is necessary to irrigate the organ with special solutions before, during and after storage.

Each component of preservation solutions was chosen for a purpose. First, it is necessary to avoid cellular edematization due to the inactivity of the Na / K-ATPase pump. This is achieved using preservation liquids that have a low concentration of sodium and high potassium. Ionic solutions with extracellular composition are also used. Also, preservation liquids contain impervious substances for the cell based on simple sugars, lactobionate and trisaccharides to maintain a plasma-like osmolality (310 mOsm / kg). In addition, ischemia generates tissue acidosis, for this reason it is necessary that preservation solutions have buffer substances that maintain the pH closest to the physiological pH. They also need to contain substances that increase intravascular oncotic pressure to prevent interstitial edema and capillary collapse, for which hydroxyethyl starch derivatives are used. Free radicals released during cold ischemia and reperfusion cause oxidation of cell structures and cell injury. In this situation, the natural defense against free radicals is overcome by the need for the addition of exogenous scavenger substances, such as glutathione. There are several preservation solutions, for example the University of Wisconsin (UW), EuroCollins (EC) and HTK (Histidine-Tryptophan-Ketoglutarate) solution (Table 1).

Of all of them, the most accepted solution is the UW despite being the most expensive. This solution is usually used in kidney, liver and pancreatic transplantation. The concern of transplantologists to improve the quality of donated organs has prompted in recent years the development of preservation methods better than simple cold storage [1,2]. Currently, in some countries, a cooling method called hypothermic perfusion machine (MPH) is used. This allows a longer and more effective cold preservation due to the continuous supply of oxygen, substrates for the synthesis of ATP and other metabolites, and facilitates the washing of cellular metabolism wastes. Several studies in renal transplantation have shown that MPH leads to a better initial and long-term graft function compared to static preservation in organs of donors with brain death. With this machine it has been observed that early graft dysfunction can decrease until it affects only 10% of transplanted patients. In relation to pharmacological treatments, there are numerous experimental studies focused both on inhibiting the harmful effects of ischemia and the inflammatory response associated with reperfusion.

For this purpose, drugs such as chloroquine or chlorpromazine have been administered to prevent mitochondrial dysfunctions and phospholipid degradation during ischemia. In turn, it was investigated to block the activation of neutrophils and infiltration with specific monoclonal antibodies, as well as to reduce apoptosis by blocking calcium with an antagonist. But without a doubt, and based on the knowledge about the pathophysiology of reperfusion ischemia damage, one of the main targets of action should be the production of oxydryl radicals. In this regard, the use of tocopherol to minimize the effects of reactive oxygen species was described. However, the most commonly used antioxidant in experimental and clinical models is N-acetylcysteine (NAC). The use of NAC in animal models of IRI has shown encouraging results. In one of the cases of hepatic IRI, a significant reduction in oxidative damage, increase in glutathione, decrease in lipid peroxidation, alanine aminotransferase and cellular apoptosis was shown. For other In part, in a model of renal IRI, pretreatment with atherocopherol and erdostein reduced lipid peroxidation of renal cell membranes. In turn, an increase in the survival of ischemic rats treated with a-tocopherol was observed. However, what is observed in animal models is not always efficient in humans.

Tabla 1: Composición electrolítica (en mmol/l) de las principales soluciones de preservación.

Table 1: Electrolytic composition (in mmol / l) of the main preservation solutions.

The preservation of vascularized solid organs begins in many cases prior to ablation. Once done, and in order to eliminate form elements to minimize the possibility of coagulation, the blood is replaced by a solution specifically designed to optimize the organ's tolerance to hypothermia and lack of oxygen. Given the knowledge acquired about reperfusion ischemia damage.

There are many anti-oxidant drugs available to be tested in different models of reperfusion ischemia damage. One of the most effective antioxidants and that is used in the daily clinic, is thioctic acid or alpha lipoic acid (ALA). ALA is a naturally occurring compound created in the mitochondria from octanoic acid as a precursor. It is a powerful natural antioxidant that has activity in both aqueous and lipid media.

It acts both intra and extracellularly and has two isomeric forms. Due to these properties it has a wide potential for pharmacological action. Its main biological role is as a cofactor in mitochondrial enzymes such as a-ketoglutarate dehydrogenase and pyruvate dehydrogenase. ALA also appears to be involved in the production of acetyl-CoA, through oxidative decarboxylation of pyruvate. In vivo, ALA can be reduced in dihydrolipoic acid (ADHL) which has a greater antioxidant action. Both ALA and ADHL have chelating capacity of metals (Fe ²⁺ , Cu ²⁺ and Cd ²⁺ ) and neutralized from reactive oxygen species but only ADHL is able to regenerate endogenous antioxidants (glutathione and vitamin E, C) and repair tissue damage caused by reactive oxygen species. However, not all the effects of ALA are due to its anti-oxidant activity. For example, it was observed that several of the anti-inflammatory effects of ALA have been mediated by its ability to inhibit NF-kB. In other words, ALA is not only an anti-oxidant but also a substance with anti-inflammatory activity.

The use of ALA proved to be protective in several experimental models, such as hepatic and renal reperfusion ischemia damage and acute pancreatitis. In this latest study, intraperitoneal administration of 100 mg / kg of ALA in animals that suffered 45 min of renal ischemia due to renal pedicle occlusion was able to reverse the deleterious effects of ischemia, such as increased serum creatinine, IL -1 b, IL-6 and TNF- a, among others. In rats, it was also determined that intraperitoneal administration of ALA prevented the deregulation of aquaporins and sodium transporters, which is observed in renal reperfusion and attenuated increased expression of endothelin-1, which leads to renal dysfunction. In these studies, high dose administration of ALA (100 mg / kg) was performed before ischemia and immediately before the reperfusion period.

Beneficial effects on the use of ALA in humans have also been observed. Several studies have documented a positive therapeutic effect, particularly in diseases such as diabetes, arteriosclerosis, neurodegenerative diseases, and in AIDS, among others. ALA supplements are relatively safe in doses consumed in humans. The doses of 600mg / day and 1800mg / day had no side effects for a period of 1-6 months. After oral intake, it is widely excreted by the kidney and is metabolized in the liver with a high hepatic first pass effect. In a clinical trial, ALA was observed to decrease hepatic IRI after hepatic occlusion and resection. However, the most significant therapeutic effect of ALA in humans is in diabetes-induced complications, including polyneuropathy and cataract formation. In fact, both intravenous administration of ALA and oral administration are approved for the treatment of diabetic polyneuropathy in many countries, including Argentina. In the case of renal transplantation, early graft dysfunction is called "delay graft function" or DGF (graft function delay). The term DGF is used to indicate that the patient requires dialysis sessions within the first post-transplant week.

The DGF is described as a discrepancy between the functional capacity of the graft and the physiological needs of the recipient, being a form of acute renal failure resulting in post-transplant oliguria. About 30-50% of kidney transplants suffer from DGF. However, in our country the number of patients with DGF amounts to 60-70%. With respect to the relationship between DGF and graft survival, it is known that the half-life of a kidney with DGF is 8.6 years compared to the half-life of 14.1 years of a kidney without DGF. The use of AAL would reduce the incidence of DGF.

In the case of liver transplantation, post-reperfusion syndrome (SPR) is a known cause of primary graft dysfunction. Although many of the organs that suffer from dysfunction can recover, approximately 5% fail to meet the needs of the recipient and only the urgent retransplant can save the patient. Primary graft dysfunction (DPI) is usually divided into two manifestations; early graft dysfunction (DTI) and primary non-function (NFP). Both describe different degrees of functional impairment beginning in the intraoperative period. However, the definitions are not clear in the literature. DTI refers to a malfunction of the graft within the first week post-transplant by analyzing altered laboratory results, including alanine aminotransferase, aspartate aminotransferase, prothrombin time, lactate and serum ammonium, among others [3- 9]. While the NFP has a more severe implication that is associated with a catastrophic clinical and laboratory deterioration. It is characterized by hepatic necrosis, increased serum transaminases, coagulopathy, increased lactate levels, hemodynamic instability, hypoglycemia, respiratory and renal failure. Unlike DTI, which is a condition with potential recovery, NFP can lead to graft failure, emergency retransplantation or death of the recipient. In this type of transplant, it is necessary to emphasize that liver preservation is more critical than that of the kidney, since, since an organ replacement machine is not available, it is essential that the graft functions return immediately after the transplant.

For both organs (kidney and liver) there are no therapies that prevent early graft dysfunction, except for having ideal donors, which in practice occurs in a few cases.

3) PROVIDED SOLUTION

In most transplants of vascularized solid organs there is a delay in the functional recovery of the graft. This process has been associated with the injury present in the stages of organ ablation and transplantation. An attempt has been made to reduce the occurrence of this phenomenon using better means and organ preservation systems. However, despite the use of these means, the incidence of early dysfunction is very high and has a long-term impact on organ survival.

The inventors provide a supplementary product that contains an active substance of alpha lipoic or thioctic acid (ALA) (C ₈ H- _H O ₂ S ₂ ) to the donor and / or the perfusion of the organs to be transplanted with ALA together with the administration of ALA in the first two days post-transplant allows to reduce early graft dysfunction.

In these cases, our invention does not imply modifying the usual therapies and procedures in solid organ transplantation, but rather incorporating a supplementary product to the same ALA to improve clinical parameters in the short and long term. The incorporation of ALA, either in the donor and / or in the organ to be transplanted and the transplant recipient in the first two days after transplantation, improves the graft's early function, backed by the changes observed in inflammatory mediators to local level as systemic. The procedure for administration of ALA is carried out in the following manner, whose stages are: a) Administer to the donor, particularly in the operating room prior to the surgical procedure of the transplant, an active substance of alpha lipoic acid (ALA) in amounts between 400 to 1000 mg, preferably 600 mg diluted in 100 my physiological solution that is passed for 30 minutes, together with the infusion of 1 vial of vitamin B (Bagó B1 B6 B12 or Becozym) endovenous; b) Slowly and continuously introduce to the organ to transplant a solution from the University of Wisconsin (UW), HTK (Bretschneider or Custodiol solution) or Eurocollins (EC) containing an active substance of alpha lipoic acid (ALA) in amounts between 400 at 1000 mg, preferably 600 mg diluted in 500 ml of physiological solution between 30-60 minutes prior to transplantation; and c) Incorporate into the receptor, an active substance of alpha lipoic acid (ALA) comprised in amounts between 400 to 1000 mg, preferably 600 mg diluted in 100 my physiological solution, during the surgical act, prior to implantation and one hour after surgery and in the two following days as a means of leveling the biochemical parameters.

Also another option that the inventors have is in the situation in which those donors who have difficulty perfusing the active substance of. Thioctic acid or alpha lipoic ALA, proceeds as a variant in slowly and continuously introducing this substance only to the organ and / or the receptor ) BRIEF DESCRIPTION OF THE FIGURES

• Figure 1 shows the patients with liver transplantation of the Rama Control and Rama treated with ALA groups who suffered or not having post-reperfusion (SPR).

• Figure 2 shows the relative expression in biopsies of liver transplanted patients that receives an organ perfused with ALA and whose recipient has received ALA.

• Figure 3 shows the plasma levels of SLPI and PAP in liver transplant patients. Liver transplanted patients receive perfused organs (RT Group) or not with ALA (RC Group).

• Figure 4 shows that patients with simultaneous reopancreatic transplantation who receive an organ perfused with ALA and whose recipient has received ALA have lower early graft dysfunction, greater graft and patient survival compared to untreated patients.

• Figure 5 shows the relative expression of inflammatory mediators in renal and pancreatic biopsies of renopancreatic transplanted patients treated with ALA.

• Figure 6 shows the cytokine plasma levels in renopancreatic transplanted patients measured in samples obtained in the pre-transplant (Pre-Tx) and 12 h post-transplant.

• Figure 7 shows the plasma levels of PAP (pancreatitis indicator protein) and SLPI in pre-transplant renopancreatic transplant patients, immediately after the act surgical (time 0 h post-Tx) and 12 hours after transplantation in group C, R and DR.

• Figure 8 shows the plasma levels of amylase, lipase, glucose, creatinine and urea.

• Figure 9 shows the% of DGF, the dialysis requirements and the hospitalization time of the patients receiving kidneys perfused with ALA and are treated with ALA during the first post-transplant days.

5) DETAILED DESCRIPTION OF THE INVENTION

With respect to fig. 1, this syndrome is evaluated within the operating room as persistent hypotension (TA> 30% the values of the anhepatic phase), asystole or arrhythmias that trigger hemodynamic instability, with the need for continuous infusion of vasopressors. Appearance of prolonged (more than 30 minutes) or recurrent fibrinolysis (reappearance of fibrinolysis 30 minutes after it has been resolved). The total number of patients is plotted and according to the type of donor. It can be seen that patients treated with ALA have less SPR than untreated patients. This phenomenon is much more evident when the groups are differentiated according to the type of donor, with a clear difference in the patients receiving organs from donors with extended criteria perfused with ALA vs the controls (Chi square test p = 0.032).

With respect to Fig. 2, qPCR was performed on biopsies of liver transplant patients from the RC (control) and RT (treated) group. The graph shows the relative expression of mRNA (2 ^L (- AACt)) in the biopsies of the RT patient group vs the biopsies of the RC group. Data are represented as the mean ± SD * p <0.05. Mann-Whitney test. It is observed that the levels of the transcripts of Birc2, Sestrin2, IkBa, HIF-1 a, GATA3, CCR1, IL-6 and IL-8 were similar in both groups of patients. Higher levels of SLPI were also observed in the biopsies of treated patients, although this difference was not statistically significant. However, the levels of PHD1 and 2 and REG3a / PAP transcripts were significantly lower in the treated group (RT) compared to those in the RC group, indicating that perfusion of the organ with ALA protects it from hypoxia suffered during surgical procedure.

With respect to fig. 3, plasma is obtained prior to transplantation, during reperfusion and on days 1, 2, 3 and 7 days after transplantation and a sandwich ELISA is performed. The graphs show that PAP plasma levels in the RC group were higher compared to those found in the RT group, in reperfusion and on day 1 post-transplant, being statistically significant only on the 1st post-transplant day. . In addition, plasma SLPI levels (considered an alarmine with anti-inflammatory functions) in patients in the RC group rise from the moment of vascular detachment and remain elevated during the first two days after transplantation. This profile is also observed for the group of RT patients, but the increase is much smaller; in such a way that when comparing the plasma levels of SLPI on day 2 post-transplant of the RT group vs. those of the RC group, significantly lower values are observed. These results indicate that patients treated with ALA have lower and much more stable inflammatory profiles compared to untreated patients.

This profile is also observed for the group of RT patients, but the increase is much smaller; in such a way that when comparing the plasma levels of SLPI on day 2 post-transplant of the RT group vs. those of the RC group, significantly lower values are observed. These results indicate that patients treated with ALA have lower and much more stable inflammatory profiles compared to untreated patients. With respect to fig. 4, the clinical results are shown, in the short term, obtained with patients with simultaneous reopancreatic transplantation receiving an organ perfused with ALA and whose recipient has received ALA. In the same graph, it can be seen that patients treated with ALA have a lower percentage of renal dysfunction and pancreatitis, a higher percentage of graft survival and of patients evaluated at 3 months post-transplant compared with untreated patients.

With respect to fig. 5, it can be observed that in renal biopsies of patients of the DR group (treatment of ALA in donors and recipients) showed lower levels of C3 and TNF- a and higher levels of TGF-b compared with those of control group. On the other hand, the expression of HMOX-1 did not show significant differences between the two groups (p = 0.669). When analyzing the expression in the pancreas, a high expression of both C3 (p = 0.0018) and HMOX-1 (p <0.0001) could be observed. Unlike what was observed in renal tissue, where significant differences in the relative expression of TNF- and TGF- b were seen between both groups of patients, no significant differences were observed in the pancreatic biopsies in any of the mediators mentioned. These results indicate that organs with a predominantly anti-inflammatory profile are shown in both the kidney and pancreas. The change in expression (2 ^L (- AACt)) in the biopsies of the DR patient group vs. the control biopsies are shown. Data are represented as the mean ± SD * p <0.05; ** p <0.01; *** p <0.001.

With respect to fig. 6, patients in the DR group were treated with ALA and received an organ from a cadaveric donor perfused with ALA; In both patients in group R, they were treated with ALA 1 hour prior to transplantation. Data are represented as mean ± SD. * p <0.05. Dunnett post hoc ANOVA for multiple comparisons. It can be seen that after surgery, patients who were not treated with ALA (control group) had significantly higher plasma levels of IL-8 (p <0.05), IL-6 (p <0.001) and IL-10 (p <0.0001) with respect to the values present in the pre-transplant sample. On the other hand, 12 hours after surgery, the group of patients treated with ALA and who received an organ from a donor perfused with ALA (DR group), presented plasma levels of IL-8 and IL-6 similar to those detected in the pre-transplant and, in addition, significantly lower than the levels detected in the control group. This effect of ALA, on IL-8 levels, could not be observed in patients of group R. In this same group of patients, levels of IL-10 and IL-6 showed a tendency to be lower than those of control group, but these differences were not statistically significant. With respect to the cytokines IL-12p70 and TNF- a, their plasma levels were below the detection threshold of the assay used. These results confirm that patients treated with ALA have an anti-inflammatory profile compared to the group of patients who did not receive ALA.

With respect to fig. 7, the data is represented as the mean ± SD in (A) and (B). * p <0.05. Unpaired t test compared to time 0 h post-transplant. In all groups, the plasma PAP level measured at the end of the surgery (time 0 h post-transplant) was found to be increased in relation to the levels found in the pre-transplant. However, at 12 hours after transplantation, serum PAP levels were significantly lower, compared to 0-hour levels, only for patients in the DR group (p = 0.039; Figure 15A), which suggests a lower grade. of aggression in patients treated with ALA and who received an organ from a cadaveric donor perfused with said drug. Another of the proteins analyzed in the plasma of the transplanted patients was the SLPI alarmine. An expression pattern similar to that of PAP can be observed in the Figure; that is, only patients in the DR group presented significantly lower levels of SLPI, at 12 hours after surgery, compared to the levels found at 0 hours, suggesting that both proteins, being proteins of acute phase, they would be indicating that the treatment with ALA generates an environment with a lower degree of damage of the organism during the transplant.

With respect to fig. 8, (A) levels of the first post-transplant day are shown. (B) area under the curve of the first 5 days post-transplant and from day 6 to 14. The data are represented as the mean ± SD. * p <0.05. Kruskal-Wallis test with Dunn post hoc test. It is observed that the DR group has significantly lower levels of amylase and lipase than the control group, while the recipient group only showed slightly lower levels of lipase compared to those of the control group. With this analysis, it was also observed that the patients of the DR group showed a slight tendency to have glucose, creatinine and urea levels in plasma, lower than the control group. However, when the same analysis was performed, but using the values of the clinical parameters obtained from the samples from days 6 to 14 post-transplantation, no significant differences were observed between the groups.

These results demonstrate that the use of ALA improves the early clinical parameters of function of the transplanted organs. With these results we can determine that the use of ALA did not affect graft function, nor did it prove to be a risk for graft and patient survival. Quite the contrary, some biochemical and gene parameters indicated that the administration of ALA could have some beneficial effect for the graft and for the patient, at least in the short term.

With respect to fig. 9, it shows that renal transplanted patients receiving an organ perfused with ALA have a tendency to present a lower percentage of DGF (graft function delay), but clearly showed lower dialysis requirements during the first two weeks after the transplant and they were discharged from the hospital more quickly during the period of hospitalization of the patients. These data show the benefits of ALA administration in the early post-transplant period.

6) EXAMPLES OF REALIZATION

For the development of this invention, three different types of transplantation were used: i) simultaneous renopancreatic transplantation; I) liver transplantation; and i¡¡) renal transplant. In each of them, there are particular similarities and differences that had to be taken into account given the differences in the surgical procedures of each organ. This section will initially describe the methodology used for each of the protocols, then we will describe the results and finally the conclusions will be discussed together.

S Simultaneous renopancreatic transplantation:

The study included 26 patients with simultaneous kidney-pancreas transplantation (SRP). The patients recruited in this study were 11 men and 15 women, in an age range 21 -57 years. Since both forms of intravenous and oral ALA are approved to mitigate diabetic sensitivomotor polyneuropathy in Argentina, all patients recruited for the study had such a condition.

The patients included were people between 18 and 65 years of age, recipients of SRP transplants. All patients received as induction therapy Timoglobulin (1.5 mg / kg for five days) and Solumedrol; and as maintenance therapy a triple immunosuppression (tacrolimus with levels of 10-12 ng / ml, prednisone 4 mg / day and mycophenolate sodium 1,440 mg / day). The patients were randomly divided into three groups: 1) Control: patients who were not treated with ALA; 2) R-ALA: patients who were given a dose of ALA (600 mg) immediately before the surgical procedure; and 3) DR-ALA: patients who in addition to receiving a dose of ALA (600 mg) immediately before the surgical procedure, such as the group Previously, they also received organs from cadaveric donors who were given ALA (600 mg) at the time of procurement. The objective of the administration of ALA was to reduce the unfavorable effect of the EROs that occur throughout the process known as IRI, which begins in the donor and continues in the recipient. This experimental design of comparing the DR-ALA group with the R-ALA group was aimed at unraveling the impact of EROs that occur during the different stages of the IRI process.

The treatment of the cadaveric donor, called preconditioning, was performed just before the procurement procedure, by intravenous drip (20 min) of 600 mg of ALA diluted in 250 ml of saline solution at the beginning of the ablation process, which has a total duration of approximately 90-120 minutes. The choice of dose administered to donors was based on the work of Dunschede et al and Muller et al, with humans and rodents, respectively [10,1 1]. In addition, this dose is the dose approved by our local regulatory authorities to treat diabetic neuropathy. The administration of said antioxidant is considered part of the normal organ extraction process, and therefore, no informed consent was necessary beyond that associated with organ donation. The organs were stored cold until transplantation.

Blood samples for obtaining plasma and measuring mediators are acquired at the beginning of the surgery, after blood unlocking, 12 hours after surgery and every one or two days after transplanting for at least 14 days or until patient discharge These samples served, not only for the measurement of inflammatory mediators, but also as part of the routine determination of analytes to assess renal and pancreatic function. In addition, kidney and pancreas biopsies were taken at the end of the surgery to perform a RT-qPCR study. Plasma levels of IL-8, IL-1, IL-6, IL-10, TNF- ae IL-12p70 were measured in the sample before surgery and at 12 h after transplant using the “BD ™ Cytometric Bead Array” kit (BD Biosciences, San José, CA), following the manufacturer's instructions. Both the samples and the standard curve were incubated with these reagents and then passed through a FACSCalibur flow cytometer for detection. Data analysis was carried out using FCAP Array software. Plasma SLPI levels were determined using a sandwich ELISA. For this, a human anti-SLPI mouse monoclonal antibody (1 pg / ml, R&D) was adhered to the surface of 96-well plates (high peptide binding) for 18h at 4 ° C. The plate was then blocked for 1 h at 37 ° C with 0.5% bovine serum albumin (Sigma) and 1% low-fat milk in 1 X PBS (blocking solution). Subsequently the wells were washed with 0.1% PBS / Tween 20 and the plasma and standard samples were incorporated; these were preserved for 1 h at 37 ° C. Once the incubation time was over, the second human anti-SLPI rabbit polyclonal antibody in blocking solution (1/1000) was added, leaving 1.5 h at 37 ° C. Then, the wells were washed with 0.1% PBS / Tween 20. Finally, it was incubated for 1 h with the goat anti-IgG rabbit polyclonal antibody conjugated to peroxidase (1/1500, Chemicon) in blocking solution. It was revealed with 3, 3 ', 5,5'-tetramethylbenzidine (Invitrogen), the reaction was stopped with 1 N sulfuric acid and the absorbance read at 450 nm with subtraction of 630 nm in ELISA plate reader (Rayto, China). Reg3 / PAP was determined by sandwich ELISA following the manufacturer's instructions (PAP pancreas, Dynabio, France). The kit consists of a 96-well plate with the anti-Reg3a detection antibody already attached. Seeded samples and standard curve, allowed to incubate for 3 h at room temperature. Then washings with PBS / Tween 20 0.1% were performed. After the incubation was finished, the biotinylated anti-Reg3a antibody was added for 30 min at room temperature. It was then washed and avidin peroxidase was added, incubating 15 min at room temperature.

Finally, it was washed and the substrate solution (TMB) was added, which was left 10 min, stopping the reaction with sulfuric acid. The absorbance was read in a plate reader (Rayto, China) at 450 nm with 630 nm subtraction. The Reg3a / PAP concentration was measured before and at 12 h post-transplant and expressed as nanograms per milliliter (ng / mi). The determination of transcripts of molecules in patient biopsies was performed through RT-qPCR assays. The biopsies were maintained at -80 ° C in later RNA solution (Ambrion, US) until processing. 19 biopsies were processed (1 1 controls and 8 of the DR group). Initially, mRNA purification was carried out using the RNeasy Mini Kit (QUIAGEN). With the biopsies a dry homogenate was performed using liquid nitrogen. This was resuspended in RLT Plus lysis buffer by using needles of different caliber for better degradation. A 3 min centrifugation was performed at 10,000 rpm. The supernatant was passed through a gDNA eliminator column (30 sec at 10,000 rpm). 70% ethanol was added to the eluate and 0.7 ml was transferred to the RNeasy column. After centrifugation (15 sec at 10,000 rpm), the eluate was discarded and the column was washed with RW1 buffer. RPE buffer was added to the column, and washed twice (15 sec and 2 min at 10,000 rpm). Dry centrifugation was performed to remove as much buffer as possible. Finally, 50 Di of RNA-free water was added to the column, centrifuged 1 min at 10,000 rpm and the eluate was recovered. Quantification was performed using a Nanodrop device, purity was determined by analyzing the relationship between absorbances at 260 and 280 nm. Then cDNA was obtained, using a kit "RT2 First Strancf '(QIAGEN). For this, the 50 ml obtained from RNA was centrifuged 15 sec.

The gDNA elimination mixture was prepared for each sample to which 700ng of RNA was added reaching a final volume of 10 ml. It was centrifuged and incubated at 42 ^Q C for 5 min, immediately he passed ice and allowed 1 min. The RT Cocktail was prepared (according to protocol). A mixture of gDNA elimination was added 10ml Cocktail RT, incubated for 15 min at 42 ^Q C, and the reaction was stopped by heating at 95 ^Q C for 5 min. Finally, 91 ml of distilled water were added. For the quantification of transcript levels, the kit used was that of SYBR GreenER qPCR SuperMix Universal (Invitrogen). A Master mix was prepared for each gene, with SYBR Green, ROX reference, 5 'primer, 3' primer and ultra pure water. To 22.5 ml of each master mix was added 2.5 ml of each sample. The instrument used for qPCR was a Corbett Research Rotor-Gene 6000 (QIAGEN, Valencia, CA). The cycling program used was: 5min at 50 ° C; 45 cycles of 10 seconds at 95 ° C, 15 seconds at 60 ° C and 20 seconds at 72 ° C. The specificity of the primers was checked with post-amplification agarose gel and by melting curve.

The data analysis was based on method 2 ^A (-AACt) with the normalization of the raw data in relation to the expression levels of the control gene, in this case GAPDH. The primers of each gene were designed and evaluated by our research group.

The transplanted patients were evaluated to determine the renal and pancreatic function, defining renal dysfunction as a decrease in creatinine less than 70% of baseline creatinine on the seventh day post-transplant or the need for hemodialysis during the first week. Clinical pancreatitis was defined as a clinical state of abdominal distension, abdominal pain, graft swelling and the need for rest of the pancreas with total parenteral nutrition. This diagnosis was made independently of this study by the doctors who were in charge of the patient and was recorded in the patient database. Survival of both the graft and the patient at 3 months post-transplant was also evaluated to determine the impact of these first events on the evolution of the transplant. Liver Transplant:

 It is a randomized, double-blind, placebo-controlled clinical trial to evaluate the use of a-lipoic acid in ischemia-reperfusion damage in liver transplantation. 23 patients were recruited. The inclusion criteria were that patients be over 18 years of age and receive a liver transplant regardless of their etiology; while the exclusion criteria were patients under 18 years of age or with reduced livers (related living donor, Split). Of the 23 patients enrolled, twelve were male and eleven female. The mean age of the patients was 60.9 ± 6.8 years. The etiology of cirrhosis was hepatitis C infection associated with hepatocarcinoma in 10 (44%) patients, alcohol in 5 (20%) and other causes in 7 (30%) patients.

 Regarding the characteristics of the donors: 11 of the 23 (48%) were not optimal donors and presented criteria of “donors with expanded criteria” [unemployed heart donor, patients> 65 years, serum sodium> 155 mEq / L, macro-steatosis > 30%, prolonged period of cold ischemia (> 16 hours), prolonged hot ischemia (> 90 minutes)].

The median MELD value (which is a prognostic index used to assess the severity of liver failure) of the population was 24 points and there were no differences between the two groups.

The patients were divided into two groups: Control and Treaty. Of the total patients included, 13 belonged to the treated group (treated with a-lipoic acid) and 10 to the control group. The administration of the drug or placebo was carried out as follows according to the group:

• Treated Branch: Donors, before the onset of cold ischemia, were given an infusion of ALA (600 mg in 50 ml of NaCI for 5 minutes) through the portal vein in order to administer it directly to the graft. Subsequently, once the transplant was carried out and prior to reperfusion, the ALA infusion into the recipient was administered by the portal vein. Then, the removal and reperfusion were carried out.

• Control Branch: instead of ALA, both donors and recipients received the same volume of physiological solution as the treated group.

It is conducive to clarify that all patients received the same induction and maintenance therapy. Induction treatment consisted of corticosteroids during surgery and Basiliximab in intensive therapy, repeating the dose of Basiliximab at 4 days. Maintenance therapy consisted of: steroids (meprednisone) in decreasing doses, Tacrolimus (Prograf) and Mofetil Mycophenolate.

Once the transplant was performed, special attention was paid to the development of severe post-reperfusion syndrome, which was defined according to the following criteria:

Hemodynamic instability with persistent hypotension (blood pressure> 30% values of the anhepatic phase), asystole or arrhythmias that triggered hemodynamic instability, need for continuous infusion of vasopressors. Appearance of prolonged (more than 30 minutes) or recurrent fibrinolysis (reappearance of fibrinolysis 30 minutes after it has been resolved). Total RNA was isolated from 10 biopsies (5 controls and 5 from the treated group) using the RNeasy Mini Kit (QUIAGEN), previously described. The quantification and purity thereof were determined by using the Nanodrop equipment. The cDNA retrotranscription was performed from 1 pg total RNA using the "RT2 First strancf 'kit (QIAGEN), described previously. The amplification and detection of cDNA in the quantitative real-time PCR was carried out using the GoTaq® qPCR Master Mix reagent, in a 96-well format in a Stratagene Mx 3000P device. The cycling program was: 95 ^Q C for 2 min, 40 cycles at 95 ^Q C for 15 seconds and 60 ^Q C 45 seconds. Finally, a cycle of 1 minute at 95 ^Q C, 30 seconds at 55 ^Q C and 30 seconds at 95 ^Q C. The primers used were designed and checked by the research group. The specificity was evaluated by melting curve and run on agarose gel from the result of post-qPCR amplification. The analysis of the result was carried out by method 2 ^A (-AACt), using 28S as the control gene. Reg3a / PAP and SLPI levels were determined in the same manner as for simultaneous renopancreatic transplants.

√ Kidney Transplants

 In this protocol ("Use of a-lipoic acid in renal transplantation") 22 patients were recruited. Of the 22 patients recruited, 8 belonged to the control group and 14 patients corresponded to the treated group (treatment with ALA). In the active branch (treated group) the administration of ALA was carried out through an infusion of 12 ampoules of 50 mg of ALA (600 mg) diluted in 100 my physiological solution that were passed for 30 minutes, together with the infusion of 1 Vitamin B ampoule (Bagó B1 B6 B12 or Becozym) intravenously, in the operating room prior to the surgical procedure of renal transplantation. In addition, the kidney to be transplanted was perfused with a Wisconsin solution containing 600 mg of ALA (diluted in 500 ml of physiological solution) between 30-60 minutes prior to transplantation. Patients in the control group only received 1 vial of vitamin B (Bagó B1 B6 B12 or Becozym) intravenously prior to the surgical procedure of renal transplantation. Regarding induction treatment, patients received ATG + MMF + Corticosteroids.

S Evidence of effect

As previously mentioned, transplanted patients may suffer a delay in the functioning of the organ, this being one of the complications more frequent in the post-transplant. Taking into account that this lack of functionality is directly related to the process of reperfusion ischemia damage and the reactive oxygen species being one of the main factors involved in the production of the damage, we evaluate the administration of the anti-oxidant ALA (alpha-acid lipoic acid) as a way to reduce their deleterious effect on the organ and ultimately on the short-term outcome of the transplant. For this part of the project, three protocols were implemented. In the first exploratory protocol, the effect of ALA was studied in patients who underwent renopancreatic transplants. The 2nd and 3rd protocol evaluated the effect of ALA in liver and kidney transplants, respectively.

S Renopancreatic transplants

For this study, three groups were considered: i) control group (C): those patients (n = 11) who did not receive ALA before or during the surgical procedure; I) group of treated recipients (group R): those patients (n = 8) who were given ALA one hour prior to surgery (administration was performed in the same surgery room); i¡¡) group of recipients and treated donors (DR group): in this case, ALA was administered to recipients in the same way as in the previous group, but they also received an organ from a cadaveric donor that had been perfused with ALA, 30 minutes prior to the act of ablation (n = 7). Of all these groups, plasma was obtained at different moments of the surgical act to make the biochemical determinations, as well as biopsies were obtained from the control group and from the DR group immediately after reperfusion. To avoid kidney injury, we consider it unnecessary to perform biopsies in the R group, since changes in transcript levels between the control group and the R group were unlikely, taking into account that the biopsies were taken immediately afterwards of arterial detachment. RT-qPCR was performed to determine the mRNA expression of inflammatory mediators involved in ischemia-reperfusion damage in renal and pancreatic biopsies of group C and DR. In particular, the transcript levels of C3, TNF-a, TGF-b, FIMOX-1 were analyzed. In Figure 5 it can be seen that the renal biopsies of the patients of the DR group showed lower levels of C3 and TNF-a and higher levels of TGF-b compared with those of the control group. On the other hand, the expression of FIMOX-1 did not show significant differences between both groups (p = 0.669). When analyzing the expression in the pancreas, a high expression of both C3 (p = 0.0018) and FIMOX-1 (p <0.0001) could be observed. Unlike what was observed in renal tissue, where significant differences in the relative expression of TNF- a and TGF-b were seen between both groups of patients, in pancreatic biopsies no significant differences were observed in any of the mediators mentioned (Figure 5 ).

Next, the plasma levels of the cytokines IL-12p70, TNF-a, IL-8, IL-6 and IL-10 were measured in the pre-transplant and 12 h post-transplant. Figure 6 shows that after surgery, patients who were not treated with ALA (control group) had significantly higher plasma levels of IL-8 (p <0.05), IL-6 (p <0.001) and IL-10 (p <0.0001) with respect to the values present in the pre-transplant sample. On the other hand, 12 hours after surgery, the group of patients treated with ALA and who received an organ from a donor perfused with ALA (DR group), presented plasma levels of IL-8 and IL-6 similar to those detected in the pre-transplant and, in addition, significantly lower than the levels detected in the control group. This effect of ALA, on IL-8 levels, could not be observed in patients of group R. In this same group of patients, levels of IL-10 and IL-6 showed a tendency to be lower than those of control group, but these differences were not statistically significant (Figure 6). With respect to the cytokines IL-12p70 and TNF-a, their Plasma levels were below the detection threshold of the assay used.

The surgical act represents a traumatic process, causing an increase in acute phase proteins. Among the acute phase proteins, and in the case of renopancreatic transplants, a protein called Reg3 / PAP stands out, whose expression increases more than 200 times during pancreatitis. For this reason, we decided to determine the plasma levels of this secretory protein in the plasma of patients treated with ALA. In all groups, the plasma PAP level measured at the end of the surgery (time 0 h post-transplant) was found to be increased in relation to the levels found in the pre-transplant (Figure 7A). However, at 12 hours after transplantation, PAP plasma levels were significantly lower, compared to 0-hour levels, only for patients in the DR group (p = 0.039; Figure 7A), which suggests a lower grade. of aggression in patients treated with ALA and who received an organ from a cadaveric donor perfused with said drug. Another of the proteins analyzed in the plasma of the transplanted patients was the SLPI alarmine. An expression pattern similar to that of PAP can be seen in Figure 7B; that is, only patients in the DR group had significantly lower SLPI levels, at 12 hours after surgery, compared to the levels found at 0 hours (Figure 7B). Both proteins, being acute phase proteins, would indicate that the administration of ALA generates a lower degree of damage or insult to the organism during transplantation. These results indicate that the perfusion of ALA to donors has a beneficial effect, since it transforms a graft with high inductive potential of inflammatory mediators into organs with less pro-inflammatory potential.

To determine the clinical importance of the inflammatory profile observed in the groups of patients treated with ALA, we examined biochemical parameters linked to the function of pancreatic grafts (amylase, lipase and glucose) and renal (creatinine and urea). The follow-up was carried out in a period of 14 days after surgery. In Figure 8, it is shown that the DR group had significantly lower levels of amylase and lipase than the control group, while the recipient group only showed slightly lower levels of lipase compared to those of the control group, confirming the trend found in the values of the first day. With this analysis, it was also observed that the patients of the DR group showed a slight tendency to have glucose, creatinine and urea levels in plasma, lower than the control group. However, when the same analysis was performed, but using the values of the clinical parameters obtained from the samples from days 6 to 14 post-transplantation, no significant differences were observed between the groups.

The modification observed in the gene and biochemical parameters does not necessarily imply a favorable impact on the clinic. That is why, the next step was to study the presence of delayed graft function. In these cases, it was decided to evaluate the DGF by examining the presence of renal dysfunction and pancreatitis defining renal dysfunction as a decrease of less than 70% of baseline creatinine at 7 days post-transplantation and pancreatitis as a clinical state of abdominal distention , abdominal pain, swelling of the graft and need for parenteral feeding. Using these criteria, 3 patients with early renal dysfunction were detected in the control group. In contrast, only one patient with renal dysfunction was detected within the R group and another in the DR group. When the presence of clinical pancreatitis was evaluated, 3 cases of pancreatitis were diagnosed in the control group, one in the DR group and none in the R group (Figure 4). Then, a three-month clinical follow-up was performed to evaluate more stringent clinical parameters such as graft and patient survival. Although the number of patients recruited and the time elapsed were very low, it was observed that there was a lower survival rate of the graft and of the patients in the control group compared to the treated groups (Figure 4). With these results we can determine that the use of ALA did not affect graft function, nor did it prove to be a risk for graft and patient survival. Quite the contrary, some biochemical and gene parameters indicated that the administration of ALA has some beneficial effect for the graft and for the patient, at least in the short term.

S Liver Transplants:

 The lack of toxic effects in renopancreatic transplant patients treated with ALA, together with the promising results obtained, allowed us to speculate on the possibility of using it in other types of transplants, such as liver transplantation, where the IRI has an important impact on patient evolution In these cases, unlike the renopancreatic protocol, patients were randomly divided only into two groups, a control branch (RC) and a branch of patients who received grafts that were perfused with ALA and who also received a dose of ALA by the portal vein immediately prior to vascular removal (treated branch, RT), evaluating levels of transcripts, biochemical mediators and clinical signs.

The effect of the treatment on the expression of markers linked to oxidative stress and inflammation was studied by performing RT-qPCR in liver biopsies. These were processed for the analysis of the following transcripts: Birc2, Sestrin2, IkBa, HIF-1 a, IL-8, IL-6, mTOR, Reg3a / PAP, PHD1, PHD2, GATA3, CCR1 and SLPI. Figure 2 shows that the levels of Birc2, Sestrin2, IkBa, HIF-1 a, GATA3, CCR1, IL-6 and IL-8 transcripts are similar between the RC and RT group. On the other hand, it is observed that there is an increase in SLPI, although this difference was not statistically significant, in liver biopsies from patients in the RT group compared with those in the RC group. However, the transcript levels of PHD1 and 2 and REG3a / PAP were significantly lower in the RT group. compared with those of the RC group (Figure 2), indicating that perfusion of the organ with ALA protects it from hypoxia suffered during the surgical procedure.

Based on the lower gene expression of REG3a / PAP in liver transplant patients in the RT group, we decided to analyze PAP plasma levels in both groups. As can be seen in Figure 3, as in the biopsies, the plasma PAP levels in the RC group were higher with respect to those found in the RT group, in reperfusion and on day 1 post-transplant, being Statistically significant only on the 1st post-transplant day.

As mentioned earlier, in renopancreatic transplanted patients, PAP levels correlated directly with SLPI values. Therefore, in this case plasma SLPI levels were also analyzed. In Figure 3 it can be seen that in patients of the RC group, plasma SLPI levels rise from the moment of vascular detachment and remain elevated during the first two days after transplantation. This profile is also observed for the group of RT patients, but the increase is much smaller; in such a way that when comparing the plasma levels of SLPI on day 2 post-transplant of the RT group vs. those of the RC group, significantly lower values are observed. In this way, the trend observed in biopsies (higher level of SLPI) is not evident at the plasma level; On the contrary, what has been observed in renopancreatic transplanted patients is repeated in whom lower levels of SLPI are seen in the treated group compared to the control group.

Transplanted patients in the control and treated group were clinically evaluated, paying special attention to the presence of post-reperfusion syndrome (SRP). As mentioned in the introduction, this syndrome is a latent threat related to the incorporation of the newly grafted organ into the circulation of the recipient. This syndrome manifests itself immediately after vascular detachment and is accompanied by hemodynamic manifestations, the most prominent manifestation being the sudden drop in systemic blood pressure. Other chemical and physical alterations characterized by hyperkalemia, decreased ionized serum calcium, hyperosmolarity, metabolic acidemia and hypothermia also occur.

Post-transplant follow-up showed that 6 of the 23 patients (26%) presented SRP during surgery. Of the 6 patients who presented SRP, only one belonged to the treated group, while the remaining 5 belonged to the control group (Figure 1). One of the patients in the control group and who developed SRP died during the first week after transplant. The cause of death was peritoneal carcinomatosis detected during surgery.

The protective effect of ALA administration was more evident in patients considered with extended criteria. In a longer-term follow-up (1st month), it was observed that 3 patients presented acute rejection, of which 2 belonged to the RC group and only one to the RT group. All of these patients had reperfusion syndrome. These results indicate, as in renopancreatic transplants, that the perfusion with ALA of the organs to be grafted presents a beneficial effect for the graft and patient survival. Likewise, no adverse effects attributable to the infusion of ALA were detected in this protocol.

S Kidney Transplants

In view of the good results obtained with ALA in renopancreatic and hepatic transplant patients and taking into account that DGF is one of the biggest problems in kidney transplants, it was decided to start another clinical protocol for the use of ALA in renal transplantation. However, variants to the ALA administration protocol had to be introduced, since administration of the drug to cadaveric donors was difficult. Therefore, for this protocol it was decided to administer ALA to the recipient (in the operating room prior to the surgical procedure) and perfuse ALA to the organ one hour before the transplant.

In this study, 22 patients were recruited, which were divided into a control group (n = 8) and a treated group (n = 14). From both groups, plasma was obtained at different moments of the surgical act to make the biochemical determinations, as well as the clinical status of the patients was evaluated. First, the analysis of creatinine levels was performed throughout the days of hospitalization of patients and the area under the curve (AUC) of creatinine levels (regardless of treatment) was obtained induction), but no difference could be observed between those treated with ALA and controls. Nor were significant differences in MDRD values between patients in the Control Branch and those in the Active Branch. All patients required dialysis sessions during the first week of hospitalization, therefore, it is considered that all patients suffered from DGF (Figure 9). However, when the dialysis requirements were analyzed per day of hospitalization, it was observed that the patients of the Active Branch required fewer days of dialysis compared to those of the Control Branch (p = 0.0024) (Figure 9). In addition, it was clearly observed that the hospitalization time of patients who had received ALA was statistically shorter compared to the control group.

All these results suggest that ALA could be a new therapeutic tool to avoid or reduce early complications of solid organ transplantation and that it could also have positive long-term repercussions. FOUNDATION OF USE

 The generation of ROS during transplantation, endorsed the possibility of using an antioxidant drug such as ALA in order to avoid or minimize the delay in graft function. However, the efficacy of antioxidant therapies has been questioned by a series of studies in which no beneficial effects were observed. In fact, in our renopacreatic transplant protocol, treatment of the recipient with a single dose of ALA at the time of surgery (group R) did not substantially improve most of the inflammatory or biochemical parameters analyzed within the first weeks after transplant. . Despite this, there was a tendency to improve some clinical parameters (such as pancreatitis and renal dysfunction), even treating graft recipients only. This makes us think that a better adjustment of the doses of ALA and / or the time of administration could improve the result of the transplant. In fact, preclinical animal studies have shown better results with high doses of ALA (100 mg / kg) and have shown that lower doses (10 mg / kg), as used in our study, are less effective [12] .

Of all the results obtained with ALA, it was very striking what was observed in those patients who, in addition to receiving ALA at the time of surgery, also received an organ from a donor that had been previously treated (DR group). The positive results were obtained in the inflammatory and functional parameters of the graft. In such a way that the division into three experimental groups, control group, DR group and R group made it possible to determine mainly the importance of donor treatment, indicating that an inflammatory activation phenomenon is taking place in the cadaveric donor that has an unfavorable impact on the graft. In fact, the RT-qPCR analysis of mediators in kidneys and pancreas derived from donors treated with ALA, showed that this favored the differential expression of mediators with an anti-inflammatory or less inflammatory profile. For example, low C3 expression and high TGF-b expression were detected in the kidney and in pancreas a remarkable expression of HMOX-1. In turn, a high expression of C3 in the pancreas was determined compared to organs derived from untreated donors. In this regard, it is interesting to note that it was recently proposed that the original classification of C3a be changed from a "pro-inflammatory mediator" to an "inflammatory modulator", based on the many anti-inflammatory facets found for C3a in vivo [13].

As previously mentioned, one of the factors analyzed in the biopsies of treated donors was HMOX-1. Several studies have described the beneficial effects of this antioxidant molecule in various animal models of IRI [14] \ Highly in the kidney, a high expression of HMOX-1 seems to be associated with a poor prognosis, it should be noted that in the study of inflammatory mediators in DGF patients describe an increase in the expression of said molecule, in line with the literature [14,15]. Unlike the kidney, in the pancreas, the positive regulation of HMOX-1 can prevent fibrosis by inhibiting the proliferation of pancreatic starry cells, so that the result obtained in said organ would be beneficial [16]. The overall effect observed in the biopsies of the DR group compared to the untreated group clearly indicates a beneficial effect of AAL in the donor.

Donor preconditioning to reduce IRI is not a new concept [17,18]. In an animal model, treatment with steroids or administration of the soluble ligand of P-selectin to the donor with brain death increased receptor survival compared to the untreated group [18]. In addition, in humans, steroid treatment to the cadaveric donor reduced the expression of proinflammatory cytokines [19]. In fact, in our study, the DR group had a reduced expression of IL-8 and IL-6, but not the R group. On the other hand, IL-10, considered an anti-inflammatory cytokine, showed no significant differences in any group. treaty. Importantly, the efficacy of ALA in decreasing the levels of some cytokines was already described during extracorporeal circulation [20] Without However, we believe that by administering AAL immediately before ablation, we are protecting the organ from the EROs produced during the ablation process, rather than protecting against the cytokine storm, which commonly begins earlier in the donor with brain death.

Another of the markers analyzed to evaluate the efficacy of the treatment was the protein associated with pancreatitis (PAP). This protein has been detected in post-transplant pancreatic juice [21, 22] and was described as a good serum marker for pancreatic injury [23]. In patients who were treated with ALA and in turn received grafts treated, the plasma levels of PAP 12hs post-transplant were lower than that of patients who received untreated grafts and were treated with ALA at the time of surgery. On the other hand, the same pattern could be observed for the SLPI alarm. In fact, a strong direct correlation was observed between PAP and SLPI. All these results indicate that treatment with ALA affects the levels of plasma inflammatory mediators, and this effect was more pronounced when both the donor and the recipient were treated. However, both groups of treated patients (R and DR) showed a tendency towards a decrease in the incidence of early renal dysfunction and pancreatitis.

The complexity of the multiple pathological mechanisms that cause renal dysfunction and pancreatitis could explain the lack of an ideal association between all the inflammatory markers analyzed and the clinical outcome in group R. This difference in the results according to the treatment, suggest that, preconditioning The donor with ALA plays an important protective role, but we insist that dose adjustment is likely to be necessary to observe more significant results. In turn, administration of ALA on post-transplant days may also help improve results. In general, ALA is a well tolerated drug that may have minor side effects, in our study such effects have not been detected. These results indicate that the use of an antioxidant can reduce inflammatory markers and improve some clinical parameters in human renopancreatic transplantation. In fact, this preliminary study suggests that preconditioning with ALA may be adequate to decrease inflammatory markers during the first days after transplantation, which would subsequently decrease the incidence of early renal dysfunction and graft pancreatitis.

In liver transplantation there is a high morbidity and mortality in the immediate post-transplant period, with severe complications due to IRI. In the liver transplant protocols, the patients were divided into a control group and a treated group (where the organ received twice the administration of ALA, as in the DR group of the renopancreatic protocol).

The results obtained with the use of ALA in liver transplantation also showed beneficial results for patients. Studies of mediator transcript levels in liver biopsies, obtained after reperfusion, indicated changes in the expression of some genes, such as a decrease in PHD1 and PHD2. Interestingly, these enzymes are linked to the hypoxia response.

These enzymes post-translationally modulate the activity of the HIF-1 subunit since they are responsible for hydroxylating it and thus favoring its degradation. Since the presence of oxygen is a requirement for such activity, this process is suppressed in hypoxia allowing HIF-1 to escape destruction and allow the activation of white gene transcription. Bernhardt W. et al. Determined that pretreatment of the organ with a PHD2 inhibitor improved short and long term results after allogeneic transplantation in an animal model [24]. In turn, Schneider M. et al. Observed that both knockout animals for PHD1 and those treated with a shRNA for said enzyme were shown to be protected against IRI. liver [25]. Thus, the decrease in transcript levels in biopsies would indicate protection against hypoxia generated by ischemia. On the other hand, we must emphasize that the levels of SLPI transcript in the treated group showed a tendency to be higher than in the control group. It is not described in the literature that ALA induces the expression of SLPI. However, Schneeberger et al analyzed the endogenous expression of SLPI in an animal model of heart transplantation [26]. These researchers determined that SLPI increases in IRI and the lack of expression of endogenous SLPI is associated with worse post-transplant cardiac functioning. The high expression of SLPI detected, in our studies, at the local level in patients treated with ALA would indicate greater tissue protection due to its anti-inflammatory and antiprotease activity. On the contrary, when measurements of this plasma mediator were made, it was observed that its levels were lower in the treated group compared to the control group; indicating that patients treated with ALA have a state of lower systemic inflammation, and therefore lower plasma level of SLPI alarmin [27],

Another of the transcripts that showed difference in their levels according to the treatment was Reg3a / PAP. In pancreas it is known that it increases its expression against inflammation, however, on liver there is not much described. This protein in normal liver tissue is not usually detected, but in patients with cirrhosis their serum levels are elevated [27]. In this study, it was determined that liver biopsies of patients treated with ALA had lower levels of Reg3a / PAP transcript compared to patients in the control group. For this reason we believe that being a protein that increases in inflammation, its expression is induced in transplantation, and said decrease in transcript between the treated and control group, would be demonstrating a lower level of inflammation in the liver tissue of transplanted patients. It is interesting to note that these results are also reflected in plasma levels. Patients in the control group showed an increase in PAP levels per day 1 post-transplant, unlike the patients treated with the antioxidant, which kept their values constant.

From the clinical point of view, an early complication with serious implications called post-reperfusion syndrome (SPR) can occur in liver transplantation. The incidence of this syndrome varies greatly between different studies, ranging from 12% [28,29] to 77% [30]. This variability could result from the population studied, therapeutic management, and even to the definition established for the SPR in each center. Despite this variability, it is a complication that increases perioperative mortality for which there is no effective prevention treatment [31, 32]. For this reason it is important to highlight the large difference in the incidence of SPR in our study, between the group treated versus the control group. In this way, and as in renopancreatic transplantation, the administration of ALA seems to have a beneficial effect, at least in the immediate post-transplant period.

As previously mentioned, the kidney also suffers consequences in the immediate post-transplant period characterized by the so-called DGF. Therefore, it was logical to start with another clinical trial in renal transplantation to observe the effect of ALA administration. However, for this protocol it was not possible to administer ALA to the donor, but the drug could be perfused to the organ to be transplanted in the operating room, prior to transplantation. The preliminary results so far obtained with ALA in renal transplantation, could not demonstrate a beneficial effect on the appearance of DGF, but it could be noted that the patients of the Control Branch needed more dialysis sessions, to maintain homeostasis, compared with the active branch. Which is interesting, not only for the cost of dialysis but also for the quality of life of patients. The results obtained from the study show that the use of ALA reduces inflammation mediators. These findings also reinforced the knowledge that was had about the deleterious consequences of the EROs, as one of the main mechanisms responsible for graft injury in solid organ transplantation.

Claims

1) A COMPOUND TO OPTIMIZE THE TRANSPLANTATION OF THE VASCULARIZED SOLID ORGANS AND REDUCE THE DYSFUNCTION OF THEMSELVES, especially in the stages of ablation, transplantation and post-transplantation in order to optimize the functional recovery time of the graft and the clinical parameters of the patients in the short term, preferably undergoing renopancreatic, renal and hepatic transplants characterized in that it comprises an active substance of alpha lipoic acid (ALA). 2) COMPOSITE according to claim 1, characterized in that the alpha lipoic acid (ALA) is comprised in amounts between 400 to 1000 mg. 3) COMPOSITE according to claim 2, characterized in that the alpha lipoic acid (ALA) is preferably in amounts between 500 to 600 mg. 4) COMPOSITE according to claim 3, characterized in that the alpha lipoic acid (ALA) is 600 mg. 5) PROCEDURE TO OPTIMIZE THE TRANSPLANTATION OF THE VASCULARIZED SOLID ORGANS AND REDUCE THE DYSFUNCTION OF THEMSELVES ”using the compound of the preceding claims, characterized in that it comprises the following steps: a) Administer to the previous donor or during the ablation procedure, particularly in the operating room prior to the surgical procedure of the transplant, an active substance of alpha lipoic acid (ALA) included in amounts between 400 to 1000 mg, preferably 600 mg diluted in 100 my physiological solution that are passed for 30 minutes, together with the infusion of 1 ampoule of vitamin B (Bagó B1 B6 B12 or Becozym) endovenous; b) Slowly and continuously introduce to the organ to transplant a solution from the University of Wisconsin (UW), HTK (Bretschneider or Custodiol solution) or Eurocollins (EC) containing an active substance of alpha lipoic acid (ALA) in amounts between 400 at 1000 mg, preferably 600 mg diluted in 500 ml of physiological solution between 30-60 minutes prior to transplantation; and c) Incorporate into the receptor, an active substance of alpha lipoic acid (ALA) comprised in amounts between 400 to 1000 mg, preferably 600 mg diluted in 100 my physiological solution, during the surgical act, prior to implantation and one hour after surgery and in the two following days as a means of leveling the biochemical parameters. 6) PROCEDURE TO OPTIMIZE THE TRANSPLANTATION OF THE SOLID ORGANS AS WELL AND REDUCE THE DYSFUNCTION OF THE SAME, according to clause 5, characterized in that the active substance of is slowly and continuously introduced. ALA alpha lipoic acid, only to the organ and / or to the recipient, according to stages b and / or c), preferably in those donors who have difficulty perfusing said substance.