WO2011144777A1 - Combination of n-acetylcysteine and lipoic acid for the treatment of a disease with axonal damage and concomitant oxidative lesions - Google Patents

Abstract

The present invention is a combination of N-acetylcysteine (NAC) and alpha lipoic acid (LA), or pharmaceutically acceptable salts thereof, for the prevention and/or treatment of a disease with axonal damage and concomitant oxidative lesions in a mammal, in particular X-linked adrenoleukodystrophy (X-ALD). The mouse model of X-ALD shows a late- onset neurological phenotype of the disease of locomotor deficit and axonal degeneration as predominant pathologies characteristic of the disease. The inventors have found that treatment of mouse models of X-ALD with these two active principles achieves reversal of oxidative stress and of the derived lesions to proteins of the spinal marrow as target organ of the disease, on the one hand, moreover however achieving prevention and reversal of axonal degeneration and furthermore correcting locomotor deficit presented by the mouse model of the disease, that is to say, the clinical symptomatology thereof.

Description

 COMBINATION OF N-ACETYL-CYSTEINE AND LIPOIC ACID FOR THE TREATMENT OF A DISEASE WITH AXONAL DAMAGE AND INJURY

CONCOMITATING OXIDATIVES

FIELD OF THE INVENTION

The present invention relates to a second medical indication of compounds known for their antioxidant activity, N-acetyl-cysteine and aifa-lipoic acid. The invention relates to the use of these compounds for the preparation of medicaments against diseases with axonal damage or axonopathy and concomitant oxidative lesions, and in particular against X-linked adrenoleukodystrophy, a demyelinating disease in the brain with axonal damage in the spinal cord and fatal outcome that There is currently no treatment, except for very specific cases of inflammatory demyelination at the onset of symptoms. The present invention is comprised in the field of drugs suitable for the treatment of said disease.

BACKGROUND OF THE INVENTION

Adrenoleukodystrophy or X-linked adrenoleukodystrophy (X-ALD) is a neurometabolic disorder characterized by adrenal gland dysfunction and extensive demyelination of the nervous system in the case of cerebral ALD, or by spinal cord axonopathy in the case of adrenomyeloneuropathy. X-ALD is the most frequent demyelinating, monogenic and inherited disease, with a minimum incidence of 1: 17,000 males. In the case of adrenomyeloneuropathy, it is a cause of motor disability in adults due to axonal degeneration of the spinal cord and peripheral nerves in the form of a chronic degenerative and disabling disease that slowly ends the patient's life. Mixed forms are possible, in which adult patients begin developing spinal cord axonopathy and end up with a rapidly demyelinating inflammatory reaction in the rapidly lethal brain.

The disease mutated gene, ABCD1, codes for an ATP-binding type transporter protein that imports very long chain fatty acids (VLCFA, chain of more than 22 carbon atoms) to the peroxisome for degradation. Its malfunction due to mutations causes the accumulation of VLCFA in tissues and plasma, which is the conspicuous characteristic of the disease.

Among the current attempts to prevent or treat the disease, the most common is based on the ingestion of the so-called "Lorenzo Oil", a mixture of glyceryl trioleate and glyceryl trierucate. This food therapy appears to reduce the levels of saturated fatty acids long chain in plasma and organs, but not in the cere.bro, ^'which explains the disappointing results of this therapy. A successful approach that is known in the art is allogeneic bone marrow transplantation when. it is performed before the onset of the disease (Shapiro, É. et al. "Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy". Lancet, 2000, Vol. 356. No. 9231, pp 713-718). However, bone marrow transplantation has many limitations, e.g. ex. It can only be done in children, when an HLA-compatible donor is available and in a narrow window of opportunity between diagnosis and the onset of the first symptoms. In addition, it has a high mortality rate (25%).

These drawbacks are also presented in lentivirus-mediated gene therapy on hematopoietic stem cells, which however have recently obtained good results in two children (Cartier, N. et al. "Hematopoietic stem cell gene therapy vvith a lentiviral vector in X-linked adrenoleukodystrophy ". Science, 2009, Vol. 326: No. 5954, pp. 818-823); but it cannot be applied to most patients, who belong to the subgroup of adrenomyeloneuropathy with spinal cord axonopathy. This majority of patients belong to the group, adult or children with inflammatory and demyelinating brain disease but who have more advanced symptoms that prevent both bone marrow transplantation and gene therapy.

Patent ES 2303441 B1 describes the use of valproic acid for the prevention or treatment of the disease. Valproic acid is a histone deacetylase inhibitor, which increases the expression of the ABCD2 gene. It is known that stable overexpression of ABCD2 (or ALDR), a homologue close to ABCD1, in the mouse model of the disease leads to normalization of the biochemical phenotype and ia prevention of neurological phenotype adrenoleukodystrophy throughout life. Thus, since ABCD2 can compensate for the absence of ABCD1 in vivo in the murine model, stimulation of ABCD2 expression in the nervous system of patients should be sufficient to prevent the onset or progression of the disease. However, its effectiveness in improving the clinical symptoms of human patients has not been proven. In a recent pilot clinical study with X-ALD patients treated with valproic acid, side effects such as tremors that further aggravate the symptoms of the disease have been detected.

Oxidative stress due to excessive production of Reactive Oxygen Species (ROS) is described as a common collateral aspect within the group of degenerative diseases, such as Alzheimer's, Parkinson's, amyotropic lateral sclerosis or Huntington's disease (Lin, MT et al. "Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases". Nature, 2006, Vol. 443. No. 7113, pp 787-795). Also in the pathogenesis of X-ALD there is oxidative stress and in fact there is oxidative damage in spinal cord proteins in mice. This oxidative damage is the result of an excess of saturated and unsaturated VLCFA, which generate free radicals, and an insufficient response to said free radicals by the body's antioxidant system (Fourcade, S, et al. "Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy ". Human Molecular Genetics, 2008, Vol. 17. No. 12, pp 1762-1773).

In this sense, Henderson (Henderson, JT et al. "Reduction of lower motor neuron degeneration in wobbler mice by N-acetyl-L-cysteine." Journal of Neuroscience, 1996, Vol. 16. No. 23, pp 7574-7582 ) has shown that N-acetylcysteine (NAC) is able to reduce muscle atrophy, motor neuron damage in Wobbler mice and also reduce the subsequent axonal atrophy that occurs, secondary to the loss of the body's fasoneuron fas. The Wobbler mouse is a model of degeneration of motor neurons, mainly of the cervical spinal cord and cranial motor nuclei. As a consequence of the disease of the neuron body, secondary axonal atrophy occurs. Axonal atrophy exists as a side effect that prevents developing a fully developed viable axon in model mice. In the X-ALD, however, the primary defect is not found in the body of the neuron but in the axon , which degenerates late once perfectly formed and in good condition for at least 16 months ^' of life in the mouse, and more than twenty years on average in patients. In addition, Henderson does not directly demonstrate the effect of NAC on oxidative damage; therefore the cause-effect relationship between oxidative damage and axonal atrophy is not discovered. Motor neuron disease associated with the Wobbler mutation, which is a spontaneous mutation in a non-identified gene, and therefore its function is unknown, and does not correspond to any human disease, is transmitted in an autosomal recessive manner. Although it does not manage to reverse atrophy, what is demonstrated by these results is that NAC is able to actively enter the brain and exert a positive effect on neurbdegeneration processes. Complementary to these results, Cui has shown that supplementary treatment with alpha-lipoic acid (LA) improves cognitive dysfunction and neurodegeneration in the hippocampus in a toxicity system exerted by D-galactose (Cui, X. et al. "Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice: protective effects of R-alpha-iipoic acid. "Journal of Neuroscience Research, 2006, Vol. 84. No. 3, pp 647-654). These results show that LA is also able to actively enter the brain and exert a positive effect, although the effect of both LA and NAC in primary axonal degeneration and in demyelinating diseases or leukodystrophies has not been demonstrated nor can it be suggested by These experiments.

There are no symptoms as such associated with oxidative stress. The. Direct consequences of oxidative stress are injuries or damage to macromolecules such as DNA, RNA or proteins. These damages can be repaired or not, depending on the specific cellular homeostatic systems. If the body fails to repair them, the malfunction of oxidized proteins or oxidized nucleic acids causes consequences in the malfunction of metabolic pathways or signaling determined in specific cell types, depending on the mechanisms that these different cell types have, to defend against damage and other primary causes of the disease. The final cellular damages are those that produce the different types of symptoms and pathology, and are autonomous and disconnected from the initial oxidative stress. Specific antioxidants or other agents effective in alleviating molecular lesions due to oxidative stress are unable to improve the pathology of the disease associated with said stress. The general lack of therapeutic effects by antioxidants in clinical trials indicates that the Oxidative stress should not be the biggest contributor to disease pathology. That is, oxidative stress would be associated with the disease without a causal relationship with its symptoms or pathology.

It is important to note that it is necessary to distinguish between oxidative stress associated with the disease, which contributes or not to the development of said disease since it can be an even beneficial reaction, and oxidative stress as true and primary cause of the pathology of the disease itself. disease. The effects associated with oxidative stress do not present a priori causality with the symptoms of the disease; the concept of "causality" is different from the concept of "association" with disease.

There are, however, very rare diseases that do cause direct oxidative stress, a circumstance impossible to predict a priori. They are also diseases not metabolically related to each other. In fact, it has not been proven that e! Oxidative stress is causative in no human pathology except in Friedreich's Ataxia, a rare disease that presents with functional defects of the frataxin protein. This protein is involved in the functioning of the iron and sulfur transporter in the mitochondria, and therefore directly involved in the homeostasis of oxidative stress. Clinical trials in patients have demonstrated in this particular case the positive effect of an antioxidant, idebenone, on the improvement of the neurogenerative symptoms of the disease (Meier, T. et a !. "Idebenone: an emerging therapy for Friedreich ataxia" . Journal of Neurology, 2009, Vol. 256. No. Suppl 1, pp 25-30; Schulz, J, B. et al. "Clinic! Experience with high-dose idebenone in Friedreich ataxia." Journal of Neurology, 2009, Vol. 256. No. Suppl 1, pp 42-45).

That is, there is no a priori any obvious relationship between the pathological consequences of oxidative stress and the pathology or causes of the disease in which they occur. In addition, not all antioxidants show efficiency in reversing lesions caused by oxidative stress itself, since the free radicals that cause it can be produced by different intracellular or extracellular mechanisms. Likewise, the various anti-oxidants also act in different ways. The positive effect of some antioxidants does not suggest its use, for this double reason, for treatment of the pathologies and symptoms of any disease associated with oxidative stress.

The application W099926657 affects the antioxidant character of NAC, which inhibits in vitro the production of cytokyries in glial cells. This publication only describes its antioxidant character. Perform the experiments cori tumor cells of glioblastoma or with cells of intestinal macrophages or with primary cultures of rat astrocytes, all in-vitro. These results do not allow to be related to any specific disease. It does not use a cellular model of X-ALD cells. They are not cells obtained from cells of X-ALD patients or from an X-ALD murine model, nor of course in treated patients. The results are based, as explained in Example 2 of the publication, on the artificial generation of cytokines through different stimuli such as LPS, a toxic derived from the bacterial wall that is used to mimic a bacterial infection. The conclusions are based on speculative reasoning about the effect of NAC on the production of cytokines, which is too broad and ambiguous because it could be applied to any disease that occurs with inflammation, that is, with production of cytokines.

The effective chemical compounds described for the treatment of diseases with axonal damage are scarce. Among them, the nearest publication of the art is the application CA2696310 A1, which describes the treatment of a murine _^ experimental autoimmune encephalitis model as a model of multiple sclerosis in humans, a common disease with demyelination and neuronal damage. The treatment is based on the administration of an antioxidant that is a substituted nitrogen heterocycle, Tempol. The antioxidant is described as effective in reducing the severity of clinical symptoms presented by this autoimmune disease model, as well as in reducing the axonal loss associated with inflammation and tissue damage caused by the immune response induced in the murine model. The axonal damage that occurs in the mouse is caused by a harmful inflammatory stimulus, the injection of reactive T cells into an SJL mouse, which is more sensitive to inflammation. The publication works with a model of acquired, non-genetic, inflammatory and demyelinating disease, with an important autoimmune component (Bischof, F. et al. "Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by se.lf-epitopes "Proceedings of the National Academy of Sciences of the United States of America, 2001, Vol. 98. No. 21, pp 12168-12173; Wekerle, H. et al. "Animal modeis". Annals of Neurology, 1994, Vol. 36., pp S47-53). Thus, animals are treated with myelin antigens (PLP, MOG) or with activated T-lymphocytes to produce an inflammatory autoimmune reaction reminiscent of the human disease multiple sclerosis. However, it has not been shown that the causes of multiple sclerosis or any other axonal or myelin disease are similar to those of the murine model used in this application, a fact that mimics the extrapolation of the results obtained to human diseases even if they occur with axonal and inflammatory myelin pathology.

The previous publication, CA2696310 A1, cites adrenoleukodystrophy as one of the diseases for which Tempol could be used for therapeutic purposes. However, the mere enumeration of diseases that occur with axonal and myelinic pathology, whether inflammatory or not, does not imply that Tempol can be applied to such diseases, since the etiopathogenesis of adrenoleukodystrophy in particular is completely different from multiple sclerosis, assuming that the model was representative of said disease. So the use of Tempol cannot be extrapolated to the treatment of adrenoleukodystrophy, and for this reason the use of any other antioxidant is also not suggested. ■ ^'

In addition, the murine models of multiple sclerosis and adrenoleukodystrophy are very different. As a monogenic disease, the X-ALD mouse model suffers the loss of function in the gene that is solely responsible for the disease also in humans, unlike multiple sclerosis and most neurodegenerative diseases of high prevalence, which are diseases multigene whose ■ pathogenesis remains mysterious to most of them. Thus, the data resulting from the model used by CA 2696310 A1 does not support extrapolation of the procedure to adrenoleukodystrophy or adrenomyeloneuropathy as possible diseases that can be treated.

Although Tempol can act as an antioxidant compound it is not mentioned that it acts as such in the model that uses CA 2696310 A1. The mechanism of action in the case of autoimmune encephalitis is purely speculative and cannot be deduced from the data that provide that other antioxidants protect from diseases related to axonal or myelin loss. Instead,. In the present invention the applicants do demonstrate that the combination of NAC + alfalipoic acid is effective in correcting oxidative damage. The combination of these two active ingredients improves the oxidative damage in the nervous system to proteins in the spinal cord of the mouse model of adrenoleukodystrophy. The inventors have managed to correlate the correction of oxidative damage with the correction of axonal lesion, and this with the clinical symptomatology developed by the murine model. ^one

The problem that arises in the technique is therefore to find a drug to improve in-vivo symptoms of X-ALD. The solution provided by the present invention is the combination of NAC and LA, which are efficient in reducing the production of ROS in human fibroblasts of adrenoleukodystrophy patients and improve the dysfunctions due to the disease. -

DESCRIPTION OF THE INVENTION

The murine model of X-ALD shows a late neurological phenotype of locomotor deficit disease and axonal degeneration as predominant and proper pathologies.

Recently, oxidative damage has been identified in the early stage of this disease, and the excess of VLCFA is responsible for the generation of ROS and oxidative protein lesions. The inventors have found that N-acetyl-cysteine (NAC) and alpha-lipoic acid (LA) as pharmaceutically acceptable antioxidants, are capable of preventing the generation of ROS dependent on VLCFA in-vitro.

The inventors have also found that the treatment of murine models of X-ALD with these two antioxidants manages to reverse oxidative stress and spinal cord protein-derived lesions as the target organ of the disease on the one hand, but also manages to prevent and correct the locomotor deficit presented by the murine model of the disease and also reverse axonal degeneration, that is, its clinical symptomatology. Axonal damage is expressed by virtue of normalization of the number of accumulations of APP (amyloid precursor protein) and alpha-synaptophysin in degenerate areas.

The inventive fact of the present invention has been to determine that oxidative stress is the cause of X-ALD disease. Until now oxidative stress as a cause of axonal damage and degeneration in murine models of human diseases had not been formally tested. An effective antioxidant treatment for axonopathic damage and derived symptomatology had not been described to date.

The invention is a combination of NAC and LA, or their pharmaceutically acceptable salts, for the prevention and / or treatment of a disease with axonal damage and concomitant oxidative lesions in a mammal. Also the clinic associated with this axonal damage. It is then a second medical use of the NAC and LA compounds, known separately for their antioxidant activity.

In the present invention it is understood that the term "combination" refers to an administration of the compounds of the invention simultaneously, sequentially or separately. In one embodiment of the invention, "combination" refers to simultaneous administration. In another embodiment, "combination" refers to the administration sequence !. In yet another embodiment of the invention, "combination" refers to administration separately. In sequential or separate administration embodiments, the delay in administration of the second component should be so that both compounds become present in the patient's body so that they are capable of acting together.

Concomitant oxidative lesions are lesions produced in the proteins of the patient's or mouse tissues, due to oxidative stress. Within the scope of the present invention they refer either to pathological lesions with external symptoms of the patient or to molecular or cellular lesions before the patient develops external symptoms of the disease. An embodiment of the invention relates to molecular or cellular lesions before the patient develops external symptoms of the disease, or "prevention" of the disease. Another embodiment of the invention relates to pathological lesions with external symptoms of the patient, or "treatment" of the disease.

The inventors have discovered that the combination of NAC and LA antioxidants prevents the production of ROS in human fibroblasts, ^" and that these antioxidants are capable of reducing oxidative stress injuries in spinal cord in double mutant Dko mice (Abcd1- / Abcd2- / -) 18 months of age This corresponds to behavioral experiments that show that they also prevent locomotor deficit in Dko mice 18 months of age.

Thus, a diet treatment enriched with alpha-lipoic acid and N-acetylcysteine normalized the clinical symptomatology of the disease similar to adrenomyeloneuropathy in Dko mice. In Figure 3 experiments are shown in A) Treadmill test, and B) and C) Bar test ("Bar-Cross").

The Treadmill test counted the number of mice that kept running on a rotating belt as a function of time. Thus, in minute 7, only 60% of the X-ALD model mice can continue, with the test, while all X-ALD mice that have received the antioxidant treatment are kept running, such as the group of controls and the group of controls that have received antioxidant treatment. X-ALD mice with treatment do not differ from controls.

In the bar test, the X-ALD mice with treatment slide much less times than the X-ALD mice that have not received treatment. No significant differences are seen between X-ALD mice with treatment and controls.

- In the bar test, X-ALD mice with antioxidant treatment take the same time to cross the bar as controls or controls with treatment, and differ significantly from X-ALD mice that have not received treatment.

The fact that NAC and LA are beneficial in adrenoleukodystrophy cannot be extrapolated to other antioxidants. The functionality of each one It is not extrapolated. The NAC works by increasing the intracellular pool of glutathione, which is decreased in X-ALD fibroblasts. Fibroblasts are sensitive to glutathione depletion caused by L-buthionine-S, R-sulfoximine (BSO) and die (Fourcade, S. et ai. "Early oxidative damage underlying neurodegeneration in. X-adrenoleukodystrophy." Human Molecular Genetics, 2008, Vol. 17. No. 12, pp 1762-1773). On the other hand, it is described that alpha-lipoic acid can neutralize different types of free radicals, in addition to regenerating reduced glutathione from oxidized glutathione (Arivazhagan, P. et al. "Effect of DL-alpha-lipoic acid on neural antioxidants in aged rats ". Pharmacological Research, 2000, Vol. 42. No. 3, pp 219 ^ 222). In addition, alpha-lipoic acid acts as a cofactor for the enzyme dehydrolipoyl dehydrogenase, which when oxidized and released from the cofactor exhibits activity, degenerates and is one of the main producers of free radicals in the mitochondria. Well, the inventors have found that this enzyme is oxidized in spinal cord extracts of X-ALD, which proposes LA as an ideal antioxidant for the treatment of X-ALD.

The fact that NAC and LA are beneficial in adrenoleukodystrophy due to its mechanism of action indicates that the treatment can be extrapolated to those diseases with similar pathology. So another embodiment is the combination of the invention in which said disease with axonal damage and concomitant oxidative lesions is selected from the group of peripheral neuropathies and spastic paraplegias (SPG); preferably a leukodystrophy and more preferably metachromatic leukodystrophies, Pelizaeus Merzbacher disease, megaloencephalic leukodystrophies or Gaucher's disease. In a very preferable embodiment of the invention, said leukodystrophy is X-linked adrenoleukodystrophy. In another embodiment, said X-linked adrenoleukodystrophy is adremieloneuropathy.

The object of protection of the present application is also the chemical composition containing the combination of the invention. Thus, a further embodiment of the invention is a pharmaceutical composition comprising the combination of NAC and LA, together with pharmaceutically acceptable excipients, for the prevention and / or treatment of a disease associated with axonal damage and concomitant oxidative lesions in a mammal. This composition is presented in any pharmaceutically viable form for administration to the mammal in need thereof. Thus, a preferred embodiment is that said pharmaceutical composition is a dosage unit, preferably a tablet. In another embodiment, the NAC is present in an acceptable amount in its oral administration, and in. Another embodiment plus LA is present in an acceptable amount in oral administration.

A very preferable embodiment is that the pharmaceutical composition of the invention be administered orally. Other embodiments of the invention are parenteral, topical, mucosal, intravenous, muscular or intramuscular administration.

The present invention extends its protection to any mammal that presents a pathology with axonal damage and concomitant oxidative lesions, due to its similar casuistry in all of them. So in one embodiment said mammal is a rodent, and in another more preferable embodiment said mammal is a human.

The combination of the invention may constitute a simple mixture with another therapeutic agent indicated for pathologies with axonal damage and concomitant oxidative lesions. So a further embodiment is a pharmaceutical composition comprising the combination of the invention, together with pharmaceutically acceptable excipients and at least one other therapeutic agent.

A very preferred embodiment of the invention is a kit that includes separate containers containing at least two pharmaceutical formulations comprising NAC and LA together or separately, together with pharmaceutically acceptable excipients, and instructions for the use of one of those components in conjunction with The other component. Preferably, it is a kit comprising NAC and LA suitable for simultaneous, separate ^'or sequential use for treatment of a disease associated with axonal loss and concomitant oxidative damage in a mammal, preferably a human disease. The most preferable embodiment of the invention is a pharmaceutical composition in the form of tablets comprising the _. combination of NAC and LA, together with pharmaceutically acceptable excipients, for the prevention and / or treatment of X-ALD in a human.

Another embodiment of the invention is a method of treating a disease with axonal damage and concomitant oxidative lesions in a mammal, preferably human, comprising administering to said mammal affected by the disease a therapeutically effective amount of the combination of NAC and LA, or its pharmaceutically acceptable salts. And another more preferable embodiment is a method of treatment of a disease with axon damage! and concomitant oxidative lesions, which comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the combination of NAC and LA to a subject, preferably human, in need thereof.

Apart from the demonstrated therapeutic function of the combination of the invention in mice, the inventors have also found that human X-ALD patients are characterized by high levels of markers of oxidative lesion in peripheral lymphocytes. Thus, these oxidative lesion markers can be used as biomarkers to monitor the antioxidant effects of specific treatments. Several human individuals with adrenomyeloneuropathy will be enrolled in the assay, from which their levels of protein oxidation in said peripheral lymphocytes will be determined before treatment with and after the combination of the invention. The doses may be adjusted individually, until satisfactory levels of oxidative damage correction in lymphocytes are achieved. These experiments can last about 12 months. Once the results are obtained, a double-blind clinical trial will begin to determine how the combination of antioxidants described relieves, prevents or reverses the clinical symptoms of the disease. For this, controls will be carried out every six months, with diagnostic tests such as magnetic resonance imaging, electromyograms and evoked potentials, in addition to clinical examination, in order to assess the evolution of the disease. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1. Level of oxidative markers present in the spinal cord.

The level of the following protein oxidative damage markers was measured: carboxymethylillisin (CML), carboxymethylisin (CEL), and malonalderidoidis (MDAL), present in spinal cord of control mice (Wt), in Abcd1- / Abcd2 - / - mice (DKO) and in DKO mice that were given the combination of the invention for 6 months (DKO + LA + NAC), from 12 to 18 months of age.

Figure 2. Assessment of the improvement in axonal pathologies.

Quantification of the accumulation of APP and synaptophysin in axonal inflammations of Wt with standard feeding (n = 8), DKO (n = 7) and DKO + LA + NAC (n = 9), at 18 months of age.

Figure 3. Locomotor deficit assessment.

The treadmill treadmill tests (A) and the bar-cross bar (B-C) were carried out in 18-month-old mice. The mice walked on the treadmill at a constant speed of 20 cm / s with a slope of 30 °. The amount of mice able to stay on the tape was measured every minute, and is represented as a percentage of mice (A). The number of errors on the bar (B) and the time taken to travel it from the beginning to the end (C) were quantified.

With the intention of showing the present invention in an illustrative manner but in no way limiting, the following examples are provided. -

PREFERRED EMBODIMENTS

Example 1: Evolution of oxidative lesions in spinal cord proteins. The generation and genotyping of the double Abcd1 / abcd2 knockout mice used as an adrenoleukodystrophy model is previously described (Lu, JF et al. "A mouse model for X-linked adrenoleukodystrophy". Proceedings of tñe National Academy of Sciences of the United States of America, 1997, Vol. 94. No. 17, pp 9366-9371; Pujol A, et al: Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a íherapeutic target for X-adrenoleukodystrophy. Hum Mol Genet 2004 Vol 1; No 13 (23); pp 2997-3006). The mice used for the experiments had a pure genetic background in strain C57BL / 6J. Alpha lipolco acid (LA) is supplemented at 0.5% w / w by mixing it with Dyets AIN-76A pellet meal (Bethlehem, PA) (Hagen, TM et al. "Feeding acetyl-L-carnitine and lipoic acid to hear rats significantly improves metabolic function while decreasing oxidative stress. "Proceedings of the National Academy of Sciences of the United States of America, 2002, Vol. 99. No. 4, pp 1870-1875). 1% N-acetylcysteine (NAC) was dissolved in water (pH 3.5). 12-month-old mice were randomly assigned to one of the following groups with a specific diet: Group I (Wild type or wild): Wlld type mice (n = 8) received a standard AIN-76A diet and water without supplementation; Group II (Abcdl- / Abcd2 -) Double knockout mice Abcdl- / Abcd2, (n = 4) received a standard AIN-76A diet and water without supplementing; Group III (Abcdf / Abcdl -) Double knockout mice (Abcdl- / Abcd2 -) (n = 4) received a diet supplemented with LA and NAC. The treatment was stopped after 6 months. After this time the animals were sacrificed and the tissues were recovered and stored at -80 ° C. All the methods used in this work were in accordance with the Guide for the Care and Use of Laboratory Animáis, published by the National Institutes of Health in the USA (Publications N1H No. 85-23, revised in 1996), and with the ethical committee of IDIBELL and the Generalitat of Catalonia.

Spinal cord samples were analyzed. The concentrations of N - (carboxymethyl) -iisin (CML), N - (carboxy-ethyl) -lisin (CEL) and N -malondialdehyde-lysine (DAL) in total proteins were measured ^; of spinal cord homogenates by gas chromatography / mass spectrometry (GC / MS), as follows:

^■ Samples were delipeded with 500 yig of protein, with chloroform / methanol (2: 1, v / v) and samples were precipitated with 10% trichloroacetic acid (final concentration) and subsequent centrifugation. Protein samples were reduced with 500 mM NaBH ₄ (final concentration) overnight in 0.2M borate buffer, pH 9.2, containing 1 drop of hexanol as an antifoam agent. The next day the proteins were precipitated with 1 ml of 20% trichloroacetic acid and subsequent centrifugation. The following isotopically labeled internal standards were added: [ ² H ₈ ] lysine (d8-Lys; CDN isotopes); [ ² H ₄ ] CML (d4-CML), [ ² H] CEL (d4-CEL) and [ ² H ₈ ] MDAL (d8-MDAL). The samples were hydrolyzed at 155 ° C for 30 min with 1 ml of 6N HCI and dried in vacuo. Derivatives Ν, Ο-trifluoroaceti! Methyl ester of the protein hydrolyzate were prepared as described above (Requena et al. Glutamic and aminoadipic semialdehydes are the main carbonyl producís of metal-catalyzed oxidation of proteins. Proc Nati Acad Sci US A. 2001 Vol 2; 98 (1) p 69-77.) GC / MS analyzes were carried out on a 6890 Hewlett-Packard 6890 gas chromatograph, equipped with a 30 m HP5MS capillary column (30m x 0.25mm x 0, 25 μητι) coupled to a Hewlett-mass detector

Packard model 5973A (Agilent, Barcelona, Spain). The injection temperature was 275 ° C; The temperature program was: 5 min at 110 ° C, then 2 ° C / min up to 150 ° C, 5 ° C / min up to 240 ° C, 25 ° C / min up to 300 ° C and finally 300 ° C for 5 min. Quantification was performed by external standardization, with standard curves made from mixtures of deuterated and non-deuterated standards. Analytes were detected by selective GC / MS monitoring. The ions used were: lysine and d8-iisin, m / z 180 and 187, respectively; CML and d4-CML, m / z 392 and 396, respectively; CEL and d4-CEL, m / z 379 and 383, respectively; and MDAL and d8-MDAL, m / z 474 and 482, respectively. The quantities of products were expressed as micromole ratios of CML, CEL or MDAL / moles of lysine. The results of the experiment are shown in Fig. 1, indicating that glycooxidation (CEL, CML) and lipooxidation (MDAL) injury markers are normalized by the effect of the antioxidants used.

Example 2. Reversal of axonal damage.

12-month-old mice were randomly assigned to one of the following groups with a specific diet. Group Ί (Wt): Wt mice (n = 8) that received only standard A1N-76A feed and water without supplementation; Group II (Abcd1- / Abcd2 /) '■ Abcdl- / Abcd2 mice (n = 7) that received only standard AIN-76A feed and water without supplementation; and Group III (Abcdl- / Abcd2- + Antx): AbcdT mice (n = 9) that were treated with a diet supplemented with LA and NAC. At 6 months, spinal cords were obtained from 18-month-old mice. Wt, Abcd1 / abcd2 - and Abcd1 / abcd2 - fed with the combination of the invention. After perfusion with 4% PFA they were embedded in paraffin and cut into serial sections of 5 µΜ transverse or longitudinal thickness. Sections were processed for immunohistochemistry with APP (Boehringer, 1: 10) and synaptophysin (Dako, monoclonal,: 500). After incubating overnight at 4 ° C with the primary antibody, peroxidase-based detection was performed with the modified streptavidin technique (DAKO LSAB2 System Peroxidase). Subsequently, the sections were dehydrated and mounted with PDX mounting solution (Fluka Biochemika, Switzerland). The specificity of the reaction with peroxidase treating the sections not exposed to the primary antibodies and the number of specific abnormal profiles in each 10 sections for each staining was counted. Five sections corresponding to the dorsal columns of the spinal cord were analyzed per animal and by staining. As Fig 2 shows, oxidative damage control prevents axonal degeneration in the spinal cord of AbcdT / Abcd2 knockout mice.

Example 3. Normalization of the clinical symptomatology of Abcdl - / abcd2- mice.

2-month-old mice were randomly assigned to one of the following groups with a specific diet: Group I (Wt): Wt mice (n = 8) that received only standard AIN-76A feed and water without supplementation; Group II {Wt; + Antx): Wt mice (n = 7) that were treated with a supplemented diet containing l_A and NAC; Group III (AbcdT / Abcdl. Abcdl- / Abcdl mice (n = 7) that received only standard AIN-76A feed and water without supplementation; and Group IV (Abcdl- / Abcd2- + Antx): AbcdT mice (n = 9) who were treated with a diet supplemented with LA and NAC.The treatment was stopped after 6 months.

The treadmill apparatus consists of a variable speed belt in terms of speed and slope. An electrified grid is placed on the back of the belt, which is used to manage discharges when mice stop running on the belt. The treadmill used (Panlab, Barcelona, Spain) consisted of a tape 50 cm long and 20 cm wide, varying in terms of speed (5 to 150 cm / s) and slope (0 ^or 25 °), enclosed in a plexiglass camera. The number of downloads received and the time in seconds that the downloads are made are measured as fitness parameters. The mice were evaluated during five trials in a single one-day session. In the first trial, the speed of the tape was 20 cm / s and the inclination of 5 ^o . In the second and third trials, the speed of the tape was 10 cm / s and the slope was increased to 10 ° and 20 ° respectively. For the fourth and fifth trials the inclination was maintained at 20 °, and the speed was increased to 20 and 30 cm / s, respectively. For the first three trials, the mice ran 1 minute. For the fourth and fifth, the time of the experiment was respectively 3 and 7 minutes. The mice were placed on the highest part of the moving belt in the opposite direction of the movement to start, so they had to run forward to avoid shocks on the hind legs. When the animals fell from the belt, the electric shocks were applied for a maximum duration of 1 second. As Figure 3b shows, significant differences were detected in the double Abcd1 / abcd2 mutants with respect to the controls, when the tape speed was 30 cm / s and the inclination of 20 °. The number of mice left on the platform was recorded every minute. In the 6th minute, only 60% of the Abcd1 / abcd2 double knockouts were able to continue running, while all wild types continued to run. Abcd1 / abcd2 mice with antioxidant treatment were able to continue the test until the end, and did not distinguish themselves from controls or controls treated with antioxidant. In addition, the number of shocks and the cumulative time during which the mice received the shocks was normalized.

The horizontal bar test is performed using a wooden bar 100 cm long and 2 cm in diameter. This bar is just wide enough for the mice to stay with their hind legs hovering at the edge of the circular bar, which implies that any false lateral passage will result in a slip. The bar is placed 50cm high on the surface of the table to prevent mice from jumping spontaneously, but not to hurt themselves if they fall.

As shown in Figure 3c, the double Abcd1 / abcd2 mutants that took a standard diet are supplemental, often showed difficulties in maintaining balance at the bar and had a greater number of falls and slips, as well as also demonstrated a longer latency at reach the platform that is at the other end of the bar. Some mice in this group showed an abnormal posture, as they hugged the bar literally with the legs in front and behind, showing signs of ataxia. The beneficial effects of antioxidants were very noticeable. The Abcd1 / abcd2 knockout mice that took an antioxidant diet, normalized the time spent crossing the bar and the number of slips, showing no differences with the controls of the wild type and wild type groups with antioxidants.

Claims

1. A combination of NAC and LA, or their pharmaceutically acceptable salts, for the prevention and / or treatment of a disease with axonal damage and concomitant oxidative lesions in a mammal.

2. A combination according to claim 1, wherein said disease with axonal damage and concomitant oxidative lesions is selected from the group of peripheral neuropathies and spastic paraplegias, SPG.

3. A combination according to claim 1, wherein said disease with axonal damage and concomitant oxidative lesions is a leukodystrophy.

4. A combination according to claim 3, wherein said leukodystrophy is selected from the group consisting of metachromatic leukodystrophies, Pelizaeus erzbacher disease, megaloencephalic leukodystrophies or Gaucher disease.

5. A combination according to claim 3, wherein said leukodystrophy is X-linked adrenoleukodystrophy.

6. A combination according to claim 5, wherein said X-linked adrenoleukodystrophy is adremieloneuropathy.

7. A pharmaceutical composition comprising the combination according to any of the preceding claims, together with pharmaceutically acceptable excipients, for the prevention and / or treatment of a disease associated with axonal damage and concomitant oxidative lesions in a mammal.

8. Pharmaceutical composition according to claim 7, in a dosage unit.

9. Pharmaceutical composition according to claim 8, wherein said dosage unit is a tablet.

10. Pharmaceutical composition according to any of claims 7 to 9, wherein the NAC is present in an acceptable amount in its oral administration.

1 1. Pharmaceutical composition according to any of claims 7 to 9, wherein LA is present in an acceptable amount in its oral administration.

12. Pharmaceutical composition according to any of claims 7 to 11, which is administered orally.

13. Pharmaceutical composition according to claim 7, which is administered parenterally !, topically, by mucosa, intravenous, muscular or intramuscular.

14. Pharmaceutical composition according to any of claims 7 to 13, wherein said mammal is a rodent.

15. Pharmaceutical composition according to any of claims 7 to 13, wherein said mammal is a human.

16. Pharmaceutical composition comprising the combination according to any one of claims 1 to 6, together with pharmaceutically acceptable excipients and at least one other therapeutic agent.

17. A kit that includes separate containers containing at least two pharmaceutical formulations comprising NAC and LA together or separately, together with pharmaceutically acceptable excipients and instructions for the use of one of those components in conjunction with the other component.

18. A kit comprising NAC and LA suitable for simultaneous, separate or sequential administration for the treatment of a disease associated with axonal loss and concomitant oxidative lesions in a human.