INJECTABLE ANTIOXIDANT FORMULATION FOR INTRAVENOUS USE OF SODIUM ASCORBATE IN HIGH DOSAGE, N-ACETYL CYSTEINE, AND DEFEROXAMINE; METHOD OF ADMINISTRATION AND USE FOR PREVENTING INJURY DUE TO REPERFUSION; AND KIT

Abstract

The invention refers to an injectable antioxidant formulation for intravenous use comprising sodium ascorbate in high dosage as a first antioxidant agent, N-acetyl cysteine as a second oxidant agent and deferoxamine as an iron chelating agent, plus pharmaceutically acceptable excipients. And an administration method of said formulation that results in a synergic and improved effect to prevent injury due to reperfusion in an organ. In particular, to prevent injury due to early and late reperfusion in patients diagnosed with acute myocardium infarction and indication of primary coronary angioplasty. The invention further refers to the use of the formulation to prevent injury due to reperfusion in patients with acute myocardium infarction, subjected to primary coronary angioplasty.

Claims

1-11. (canceled)

12. A pharmaceutical injectable antioxidant composition to be administered intravenously, to a patient, before, during or after an angioplasty procedure, characterized in that it comprises ascorbic acid in high dosage or a pharmaceutically acceptable salt thereof as a first antioxidant agent N-acetyl cysteine as a second antioxidant agent and deferoxamine and further pharmaceutically acceptable excipients, useful for preventing injury due to reperfusion in said patients subjected to the angioplasty procedure.

13. A pharmaceutical composition according to claim 12, characterized in that the ascorbic acid is in effective amount in the range of 30 to 60 g/L of sodium ascorbate.

14. A pharmaceutical composition according to claim 13, characterized in that the ascorbic acid is in a concentration of between 160 to 320 mmol/L in an infusion oriented to achieving plasmatic concentrations of 10 mM or higher to the plasmatic concentrations prior to the angioplasty (deflation of ball).

15. A pharmaceutical composition according to claim 12, characterized in that the N-acetyl cysteine is in effective amounts in the range of 3 to 6 g/L.

16. A pharmaceutical composition according to claim 12, characterized in that the N-acetyl cysteine is in a concentration of between 18.4 to 36.8 mmol/L in an infusion.

17. A pharmaceutical composition according to claim 12, characterized in that the deferoxamine is in effective amounts in the range of 2 to 4 g/L.

18. A pharmaceutical composition according to claim 12, characterized in that the deferoxamine, is in a concentration of 3.0 and 6.1 mmol/L in an Infusion.

19. A procedure to prevent injury due to reperfusion before, during and after subjecting a patient to an angioplasty procedure, characterized in that it comprises administering the patient, a pharmaceutical composition as claimed in claim 12, by means of infusion in a concentration in the range of 10 to 20 mL/min, between 40 and 20 minutes before the angioplasty (deflation of ball), at a minimum velocity of 10 mL/min and maximum velocity of 20 mL/min, and/or administering said infusion during the first 60 minutes of the angioplasty procedure, at a minimum velocity of 10 mL/min and maximum velocity of 20 mL/min, the later optionally reduce said velocity to 3 mL/min, during the following two hours.

20. A pharmaceutical injectable antioxidant composition to be administered by means of infusion or intravenously, characterized in that it comprises sodium ascorbate in high dosage or another pharmaceutically acceptable salt thereof as a first antioxidant agent, N-acetyl cysteine as a second antioxidant agent and deferoxamine, and further pharmaceutically acceptable excipients, useful for treating injury due to reperfusion in pharmacological thrombolysis of acute myocardium infarction, reperfusion of stroke or a condition that comprises perfusing the infarction or non-infarction region of an organ.

21. A pharmaceutical kit, characterized in that it comprises: a container with visible UV filter to avoid the photo-degradation of the sodium ascorbate, containing sodium ascorbate, where the container has been sealed under an inert atmosphere in order to avoid the oxidation of the sodium ascorbate and N-acetyl cysteine, the container has further been fabricated under sterile conditions to avoid microbiological contamination, and has at least two compartments to separate the deferoxamine that is in solid state from the sodium ascorbate and the N-acetyl cysteine that are dissolved in liquid phase, and has a shape that allows easy mixing of both phases, in separate compartments, when these are combined through energetic shaking, breaking of the internal compartment or injection of a phase upon the other, and further comprises sealing means that allow an easy and fast visual evaluation of the integrity of the kit, in order to confirm that there was no previous, an unwanted mixing of the phases and also verify that mixing occurred effectively in the desired moment.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1: Illustrates the effect of the intervention in the left ventricular ejection fraction, evaluated by means of cardiac magnetic resonance on day 6 and day 84 post-primary angioplasty. FEVI: left ventricular ejection fraction; LA group: Placebo group HA group: Intervened group.

[0026] FIG. 2: Illustrates the effect of intervention on the difference in the left ventricular ejection fraction evaluated by means of cardiac magnetic resonance on day 6 and day 84 post-primary angioplasty. FEVI: left ventricular ejection fraction; LA group: Placebo group HA group: Intervened group.

[0027] FIG. 3: Effect of the interventions with both secondary components, isolated, together and in conjunction with sodium ascorbate on the recovery of heart contractibility post-reperfusion in isolated rat hearts. NaCl: “placebo” group; DFO: deferoxamine; NAC: N-acetyl cysteine; AA: sodium ascorbate. N=3 rats per group.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The object of the invention is a pharmaceutical formulation with is antioxidant and injectable, of intravenous administration, such as an infusion, which comprises sodium ascorbate in high dosage, preferably, in an amount in the range of 30 to 60 g/L, N-acetyl cysteine, preferably, in an amount in the range of 3 to 6 g/L, and deferoxamine, preferably, in an amount in the range of 2 to 4 g/L plus pharmaceutically acceptable excipients, which is useful to prevent injury due to reperfusion in patients with myocardium infarction subjected to primary angioplasty.

[0029] Another object of the invention is the preparation and administration method of said solution, where the preparation is carried out from the pure components of pharmaceutical grade, not dissolved, or in other pharmaceutical forms, injectable through intravenous administration, which can contain each component in separate form and increased concentration.

[0030] Both strategies of formulation will give through to a product with the three components mixed; a diluted solution with the tri-association.

[0031] Previous to use, the formulation can be in the tri-associated form o can be prepared immediately before commencing the infusion. The starting time of the infusion is in the range of between 40 and 20 minutes before the angioplasty, with a flow interval corresponding to a minimum velocity of 10 mL/min and maxima of 20 mL/min to maintain during the first 60 minutes of intravenous administration.

[0032] Normally, the rechanneling of the artery is produced in a standard procedure between 20 and 30 minutes in the infusion, time which is doubled or tripled for complex procedures. After the first 60 minutes, the flow of infusion is reduced to 3 mL/min, during the following two hours.

[0033] Therefore, the formulation can comprise ascorbic acid in a concentration between 160 and 320 mmol/L, in an infusion oriented to achieve plasmatic concentrations of 10 mM or higher than the plasmatic concentrations before the angioplasty (deflation of ball); N-acetyl cysteine in a concentration between 18.4 to 36.8 mmol/L in an infusion; and deferoxamine in a concentration of 3.0 to 6.1 mmol/L in an infusion.

[0034] Currently, the PREVEC formulation, massive isolated dosages of sodium ascorbate, has only been tried under very controlled conditions and with the support of professionals hired for each study, external to the clinical routine of the angioplasty procedure for diagnosis of infarction. This implies that a stable pharmaceutical form has not been developed, susceptible of incorporating the standard clinical practice for angioplasty, which allows for routine personnel of said procedure to administer the formulation in a similar way to other intravenous medicaments of common use in these interventions.

[0035] The present procedure requires that for each patient intervened the routine personnel for the angioplasty be assisted by a specially trained nurse that prepares the formulation starting from an existing very concentrated pharmaceutical formula of sodium ascorbate for intravenous use (Pascorbin) and a standard clinical flask with bi-distilled water. The nurse then must empty 375 mL of clinically bi-distilled water from the clinical flask with a graded 500 mL laboratory test tube, to fill it, using a sterile graded syringe of 50 mL attached to a hypodermic needle of calibre 16G1, with the same volume of pascorbin, corresponding to 7 and a half flasks of said product in its commercial forma (Imported specifically for the study), work that must be carried out under laminar flow conditions to maintain sterilization. This process takes several minutes and, given the chemical instability of the new solution under aerobic and lighting conditions, it must be carried out once the patient is admitted to the assistance centre, with the most haste and diligence possible in order for him to be ready upon entry of the patient to the angioplasty OR.

[0036] The current clinical practice makes it unfeasible in the present to integrate this procedure with the standard routine of angioplasty of the health systems for different reasons. The present formulation suffers from this same negative externality with the aggravating aspect of requiring a preparation with three simultaneous components, which requires a second person, a nursing technic to assist the first professional.

[0037] For this reason that for the effects of the present application the packaging of the pharmaceutical formulation in a stable form in a ready to use container is proposed to be used in primary angioplasty and adapted to the standard routine of the clinical practice.

[0038] However, to avoid photo degradation of the sodium ascorbate, the formulation is contained in a kit that comprises a container with a visible UV filter, sealed under inert atmosphere to avoid oxidation of the sodium ascorbate and N-acetyl cysteine, sterilized beforehand to avoid microbiological contamination. The container can have two compartments in order to separate two phases; the deferoxamine, in solid state or in stabilized solution through pharmaceutical technology, of the combination of sodium ascorbate and N-acetyl cysteine in solution, allowing for an easy mixture of both phases when these are combined through energetic agitation, “breaking” of one of the compartments or “injection” of un phase into another. Moreover, the container requires sealing means that allow an easy and fast visual evaluation of the integrity of the kit, to confirm that no prior unwanted mixing of the phases has occurred, which could have degraded the formulation and to verify that the desired mixing for the activation of the formula occurred in an effective manner.

[0039] For the effects of evaluating scientifically the proposed invention, two independent experimental approximations were carried out.

[0040] The attenuation of the injury due to late reperfusion was demonstrated preliminarily with the first active component in an isolated way; sodium ascorbate in massive dosage, in the PREVEC study; PHASE II clinical trial. The attenuation of the injury due to early reperfusion, was demonstrated preliminarily with the tri-association in a murine model in an isolated heart. Said model allowed to conclude that both secondary components; N-acetyl cysteine and deferoxamine have each, separately, the capacity to attenuate the injury due to early reperfusion, effect that increases synergistically when combining both components, even in absence of sodium ascorbate.

[0041] Clinical Trial in Human Patients

[0042] The main active ingredient in the present formulation of three components; sodium ascorbate in high dosage was tested isolated in a controlled PHASE II (PREVEC) clinical trial, registering favourable results in terms of capacity of attenuating the effects of injury due to late repercussion.

[0043] In a group of 50 patients subjected to a primary angioplasty due to acute infarction diagnosis, treated only with massive dosage of sodium ascorbate (equivalent dosage to the one used in the three component formulation), a recovery was observed that has never before been reported of the cardiac function in a medium term, only referable to an attenuation of the late effects of the injury due to reperfusion. The evaluation was carried out by means of cardiac magnetic resonance, technique considered the present gold standard for this kind of studies.

[0044] Methodology for Clinical Trial in Human Patients

[0045] A randomized, double blind, placebo controlled clinical trial was applied to patients diagnosed with acute myocardium infarction with indication of primary angioplasty, previously enrolled.

[0046] As inclusion criteria, was defined: Individuals of any sex older than 18 years of age; Latency of admission of up to 12 hours since presenting symptoms; First myocardium infarction; Killip 1 and 2; Capacity and willingness to sign consent form.

[0047] As exclusion criteria was established: Renal or hepathic insufficiency history; Cardiac failure history (NYHA III, IV); Cardiogenic shock; Comorbility that determines a life expectancy<6 months; Participation in another investigation; Pregnancy; Glucose 6—Phosphate Dehydrogenase deficiency.

[0048] Patients were administered an oral dose of vitamin E (800 IU), followed by a sodium ascorbate infusion (320 mmol/L). The placebo group received an inert infusion of sodium chloride solution of the same osmolarity as the sodium ascorbate infusion. Serial blood samples were taken to analyse biomarkers related to myocardium, injury, oxidative stress and inflammation in order to evaluated efficiency on injury due to early reperfusion. Cardiac magnetic resonance exams were carried out, 6 and 84 days after the angioplasty in order to determine the size of the infarction and cardiac function, in order to evaluate efficiency on injury due to early and late reperfusion.

[0049] The statistical analysis of the preliminary results, was carried out using non parametric statistics, using the median as position measurement, interquartile range as dispersion measurement and the Mann-Whitney U test to determine the statistical significance. Differences between the multiple groups was analysed by means of a Kruskal-Wallis test. For all cases, the statistical significance was assumed for P<0.05.

[0050] Results of the Clinical Trial in Human Patients

[0051] Passed three months from the moment the angioplasty took place, the cardiac function, evaluated through the left ventricular ejection fraction by means of nuclear magnetic resonance, was a 33% higher in treated patients versus placebo (p<0.05), benefit that solely referable to an attenuation of the late effects of the injury due to reperfusion (FIG. 1). The early cardiac function did not show any difference between both groups (FIG. 1).

[0052] Moreover, there was an important individual change in the left ventricular ejection experienced by each patient, between both groups, which was compared by means of the calculation of the difference between the value of this variable 84 and six days after angioplasty. The change in the left ventricular ejection was shown to be significantly higher in the treated group (p<0.05), change that also showed an improvement regarding the value of the first resonance (6 days) in comparison to the placebo group, which showed a deterioration of this variable three months after the angioplasty (FIG. 2).

[0053] In Vitro Trials in Murine Model

[0054] Laboratory trials in murine model, by means of the use of the Langendorff isolated heart model, allowed to preliminarily establish the contribution of the secondary components of the solution in isolated form, in conjunction and in tri-association with sodium ascorbate on the attenuation of lethal reperfusion by means of the evaluation of the percentage of recovery of contractibility, subsequent to the reestablishment of the blood flow, after a total ischemia.

[0055] Murine Model Methodology

[0056] Sprague-Dawley adult male rats (250 to 300 g) were anesthetized with pentobarbital (80 mg/kg IP). Heparine (100 U/kg IV) was administered and the hearts were rapidly extracted and perfused with Krebs-Henseleit solution oxygenated in a Langendorff perfusion system at 37° C. [Donoso and cols., 2014. Stimulation of NOX2 in isolated hearts reversibly sensitizes RvR2 channels to activation by cytoplasmic calcium. J Mol Cell Cardiol 68:38-46]. The ventricular pressure was measured with a latex balloon inserted, in the left ventricular and connected to a pressure transductor.

[0057] After a stabilization period (15 minutes), a temporary regional ischemia of the left ventricular was produced by means of the placement of a prolene 6-0 tie under the appearance of the first anterior descendent artery (ADA) branch. The ends of the suture were joined by means of a polyethylene tube (PE-50) to form a loop for reversible occlusion. The hearts were subjected to 30 minutes of regional ischemia followed by 60 minutes of reperfusion in the presence or absence of the solution, object of the invention.

[0058] Both antioxidant agents and deferoxamine were administered during the last 10 minutes of the regional ischemia and in all the reperfusion period, both in isolated form or in association in the indicated concentrations (mmol/L): ascorbic acid: 320, N-acetyl cysteine: 36.8; deferoxamine: 3.9, “Placebo” hearts were perfused with the same concentration of antioxidants without ischemia.

[0059] Hearts were excluded if the time between the cardiac cleavage from, the thorax and the start of the perfusion through the aorta was over two minutes (longer periods of time can produce ischemic preconditioning). Also were excluded if during the stabilization period a perfusion pressure of 60 to 70 mmHg was not achieved, if the ventricular pressure developed was lower than 65 mmHg or if the cardiac frequency could not adjust to 250 to 350 beats per minute.

[0060] Evaluation of the injury: The capacity was measured of of both components isolated, in conjunction and in tri-association with sodium ascorbate in high dosage, in order to recover the cardiac contractibility, loss associated to the injury due to reperfusion.

[0061] For the analysis of the results parametric statistic of the continuous variables was used, expressing the position in terms of arithmetic mean and dispersion based on the standard deviation. The differences between multiple groups were analysed by means of ANOVA with Bonferroni test as Post-Hoc. For all results P<0.05 was considered as statistically significant.

[0062] Murine Model Results

[0063] In an isolated heart murine model, a favourable effect was observed on the injury due to reperfusion of short term, using the 3 components of the solution of antioxidant infusion in separate and in conjunction. It can be appreciated that both secondary components N-acetyl cysteine and deferoxamine were capable of attenuating the injury due to early reperfusion, each separately, in conjunction and in tri-association with sodium ascorbate in high dosage, measured as a functional effect on the cardiac contractibility, further observing the synergic effect (FIG. 3). The recovery of the contractibility regarding the control group (Placebo) improved in 113% for deferoxamine, in 189% for N-acetyl cysteine and 302% for the combination of both compounds. This last value is equivalent to recovering the cardiac contractibility post-reperfusion in 40% versus the recovery near 10%, which is obtained in the untreated group.

[0064] The addition of sodium ascorbate in high dosage to the N-acetyl cysteine and deferoxamine group did not have effects on the attenuation of the injury due to early reperfusion, associated to these last two components, consistent observation with the evidence obtained from human patients, where the beneficial effects of this vitamin was seen in the attenuation of injury due to late reperfusion (FIG. 1 and FIG. 2).

[0065] Synergic Effect of Tri-Association

[0066] The present application is based on a temporary and spatial synergy of the association of three components, oriented specifically to attenuating both the early and late effects of the injury due to reperfusion. The synergic effect between the components of the solution, namely ascorbic acid, N-acetyl cysteine and deferoxamine, components that separately have shown effects, though beneficial, which do not reach the levels obtained with the tri-association.

[0067] Injury due to reperfusion can be classified in a simplified way in two stages; early injury and late injury [Seropian and cols., 2014. Anti-inflammatory strategies for ventricular remodelling following ST-segment elevation acute myocardial infarction. J Am Coll Cardiol.; 63: 1593-603]. The early injury comprises mainly a violent cellular death due to necrosis that prevails minutes after the reperfusion and is a direct consequence of the oxidative stress action. Late injury comprises a slow but sustained cellular death that occurs mainly as apoptosis as an indirect consequence of the oxidative stress and prevails from days to weeks after the reperfusion, along with a remodelling of the tissue that also influences in the loss of cardiac function. All the aforementioned mediated in an important way by the immune system.

[0068] The PREVEC intervention (only sodium ascorbate in massive dosage), is acting mainly on the injury level due to late reperfusion, presumably due to the fact that the levels of sodium ascorbate in its active form are enough to attenuate the oxidative stress to a level capable of inhibiting the activation of the immune system that, favours the apoptosis and remodelling. However, these levels of sodium ascorbate in its active form would not be capable of attenuating the oxidative stress to sufficient levels to reduce in the expected way the injury due to early reperfusion.

[0069] The animal model proves that when faced with the injury due to early reperfusion, both deferoxamine and N-acetyl cysteine, were capable of exercising each separately an independent effect of the sodium ascorbate, effect that showed synergy when both compounds are mixed.

[0070] The proposed global synergy for the tri-association considers: 1) a chemical spatial synergy between the secondary components, oriented to attenuating the injury due to early reperfusion, measured in an animal model and 2) a temporary synergy derived from the observation that the sodium ascorbate, as administered in human patients, attenuates the injury due to late reperfusion.

[0071] In other words, the present application proposes a tri-associated formula capable of attenuating synergistically the injury associated to injury due to reperfusion on a spatial level, considering the combined effect of N-acetyl cysteine and deferoxamine on the early injury and temporal level, by conditioning the aforementioned benefit the effect of the sodium ascorbate in high dosage on the late injury, strategy that has yet to be proposed.

[0072] Other procedures in which the following formulation can be applied are reperfusion therapy by pharmacological thrombolysis of the acute myocardium infarction, for the therapy of reperfusion for strokes, organ transplants and in general any other procedure that involves reperfusion of an infarction or non-infarction of a region of some organ.