ES2916396A1 - PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF CHARCOT-MARIE-TOOTH DISEASE - Google Patents

Abstract

Pharmaceutical composition for the treatment of Charcot-Marie-Tooth disease. The present invention discloses a pharmaceutical composition for the treatment of Charcot-Marie-Tooth disease comprising MitoQ, preferably the cause of said disease being alterations in GDAP1 and/or MFN2 and the symptoms of the disease motor deficiencies, sensory loss, deficiencies optics, diaphragmatic paralysis and/or paralysis of the vocal cords.

Description

DESCRIPTION

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF THE DISEASE

DE CHARCOT-MARIE-TOOTH

field of invention

The present invention relates to the field of biomedicine, more specifically to the treatment of mitochondrial diseases.

Background

Charcot-Marie-Tooth (CMT) disease is a neuropathy for which there are currently no effective treatments. Patients with CMT exhibit peripheral nerve degeneration, leading to muscle weakness and loss of proprioception. Loss of mitochondrial oxidative phosphorylation proteins and antioxidant response enzymes accompany nerve degeneration in skin biopsies from MTC patients.

CMT is a rare disease and the most common inherited condition of the peripheral nervous system, with an estimated general prevalence in the population of 10 to 28/100,000 inhabitants in Europe. CMT is a heterogeneous genetic disease1 that usually appears in the first two decades of life. Patients present with peripheral nerve degeneration, leading to muscle weakness and loss of proprioception and pinprick sensation, and is characterized by distal muscle weakness and atrophy. Despite efforts to characterize the disease, CMT currently lacks effective treatments or therapies.

More than 90 genes have been linked to CMT disease and related neuropathies. One of them encodes the ganglioside-induced differentiation-associated protein (GDAP1). Mutations in the GDAP1 gene cause axonal CMT with both recessive (AR-CMT2K) and dominant (CMT2K) inheritance, and CMT4A recessive demyelination. GDAP1 is a mitochondrial outer membrane (MOM) protein that is also found at both mitochondrial associated membranes (MAM) and mitochondrial membrane-lysosome contact sites. Mutations in GDAP1 have been associated with abnormal changes in the morphology and dynamics of mitochondria and with decreased Ca2+ entry via storage-operated calcium entry (SOCE), leading to failure of mitochondria. stimulation of mitochondrial respiration and inhibition of mitochondrial ATP production. The lack of GDAP1 also induces an inflammatory response in the bone marrow. spinal cord and sciatic nerve. In addition, GDAP1 participates in the defense against oxidative stress. In this context, members of the GDAP1 family have been shown to respond to and protect against stress associated with increased levels of oxidized glutathione. Likewise, GDAP1 mutations lead to defective mitochondrial complex I activity and oxidative stress. Consistent with these observations, loss of mitochondrial and antioxidant proteins has been reported in skin biopsies from CMT2 patients and in the peripheral nerves of Gdapl knock-out ( Gdapl-null) mice. Loss of mitochondrial oxidative phosphorylation proteins and antioxidant response enzymes accompany nerve degeneration in skin biopsies from MTC patients. further suggesting that mitochondrial function and oxidative stress are potential targets for treating MTC disease.

The scientific article MitoQ protects dopaminergic neurons in a 6-OHDA induced PD model by enhancing Mfn2-dependent mitochondrial fusion via activation of PGC-1a discloses the treatment with the MitoQ compound in Parkinson's, another neuropathy, however, there are no experiments in the disease CMT.

Patent EP2624832B1 discloses the medical use of specific HDAC6 enzyme inhibitors for the treatment of CMT. These inhibitors have a different chemical structure than MitoQ and can also have side effects.

The scientific article MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A discloses the use of small peptides for the treatment of a subtype of CMT caused by alterations in MFN2, as in the case of the previous patent , the use of these compounds can have secondary effects in addition to being a more expensive treatment than the one proposed by the present invention.

In the present invention, it discloses a pharmaceutical composition for the treatment of the symptoms of Charcot-Marie-Tooth disease.

Description

In the present specification the term "treatment" is to be understood as preventive use, that is, to prevent the progression of the symptoms of the disease, or therapeutic, to reverse the symptoms.

In the present memory the expression "genetic alterations" includes mutations (substitutions, deletions, duplications or inversions) of at least one base nucleotide sequence of a gene that results in a characteristic phenotype of Charcot-Marie-Tooth disease. Genetic alterations can be somatic or spontaneous and hereditary.

The terms “Gdapl-null', “Gdap1-/-, “GdapT- deficient mice ', “Gdapl knockout', and “Gdapl kd' are used interchangeably herein.

The present invention relates to a pharmaceutical composition comprising MitoQ for use in treating the symptoms of Charcot-Marie-Tooth disease in a patient.

The chemical formula for MitoQ is [10-(4,5-Dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl](triphenyl)phosphonium methanesulfonate. Additionally, MitoQ also encompasses a pharmaceutically acceptable salt, solvate, and/or ester thereof.

MitoQ is a mitochondrial targeted antioxidant consisting of a quinone moiety linked to a triphenylphosphonium by a 10-carbon alkyl chain. MitoQ has no toxic effects on treatments administered in drinking water and is marketed as a dietary supplement for humans3.

In a particular embodiment, the cause of the disease is due to genetic alterations in GDAP1 and/or MFN2.

The sequence that codes for human GDAP1 and MFN2, as well as their respective transcripts, can be found in the Ensembl database ( Human GRCh38.p13) GDAP1: ENSG00000104381 and MFN2: ENSG00000116688. This is the sequence of a particular individual in the population of human beings. Humans vary from each other in their gene sequences. These variations are very small, sometimes occurring at a frequency of about 1 to 10 nucleotides per gene. Different forms of any particular gene exist within the human population. These different forms are called allelic variants. Allelic variants often do not change the amino acid sequence of the encoded protein; such variants are called synonyms. Even if they change the encoded amino acid (not synonymous), the function of the protein is normally not affected. Such changes are developmentally or functionally neutral. When referring to human GDAP1 or human MFN2 in the present application, all allelic variants are intended to be encompassed by the term.

For the purposes of determining a genetic alteration in any of these genes, the sequences of GDAP1 and/or MFN2 determined in a sample can be compared. test sample from a first human with a given sequence obtained from a sample from a second human serving as a control, this second human may be a relative. A difference in sequence in the two tissues indicates a mutation. Alternatively, the sequence determined in a GDAP1 and/or MFN2 gene in a test sample can be compared with the sequence indicated in the previous paragraph in Ensembl. A difference between the test sample sequence and the sequence in Ensembl can be identified as a mutation. An expert in the field would know how to differentiate those genetic alterations responsible for CMT disease from other silent or innocuous alterations. Suitable body samples for testing include blood, serum, plasma, sputum, urine, feces, saliva, and cerebrospinal fluid.

In a preferred embodiment, Charcot-Marie-Tooth (CMT) neuropathy is caused by mutations in the GDAP1 gene sequence.

In yet another preferred embodiment, the subtypes of CMT disease caused by mutations in GDAP1 are: axonal recessive (AR-CMT2K), axonal dominant (CMT2K), and demyelinating recessive (CMT4A) forms.

In another preferred embodiment, Charcot-Marie-Tooth neuropathy (CMT) is caused by mutations in the MFN2 gene sequence, it is the axonal recessive form AR-CMT2A2

In yet another preferred embodiment, the subtype of CMT disease caused by mutations in MFN2 are:

In a preferred embodiment Charcot-Marie-Tooth neuropathy 2 (CMT 2) is caused by mutations in the MFN2 gene. Mutations in MFN2 cause the AR-CMT2A2 form.

Other subtypes of CMT caused by genetic alterations in the sequence of GDAP1 and/or MFN2 are also within the scope of the present invention.

In a particular embodiment, the CMT can be at least one of the subtypes, AR-CMT2K, CMT2K and CMT4A, and the preclinical model is the GDAPI-null mouse and/or cells derived from it.

Symptoms of the disease may be the physical manifestation of alterations in proteins involved in energy metabolism and redox (reduction-oxidation) proteins characteristic of CMT disease.

In a particular embodiment, the symptoms of CMT disease are motor deficiencies, sensory loss, optical deficiencies, diaphragmatic paralysis and/or vocal cord paralysis.

In another particular embodiment, the motor deficiencies comprise motor deficiencies of the lower extremities, loss of body mass in the legs and feet, high arches of the feet, bent toes (hammer toes) and/or difficulty in flexing the heel (foot drop).

In another particular embodiment, the sensory loss comprises less sensitivity or loss of sensitivity in the legs and feet. These symptoms are linked to motor deficiencies of the lower extremities.

In another particular embodiment, the optical deficiencies can be optic atrophy, giving rise to visual impairment.

The pharmaceutical composition of the invention may additionally comprise at least one or more of the following:

- a pharmaceutically acceptable excipient, and

- a pharmaceutically acceptable additive.

The pharmaceutical composition of the invention can be used as subsequent or simultaneous treatment with other therapies.

The pharmaceutical composition of the invention can be administered to an individual by topical, intranasal, rectal, intraperitoneal, oral, subcutaneous, or intramuscular routes, preferably orally.

The pharmaceutical composition indicated for oral administration can be presented in separate units, such as capsules, pills or tablets, each containing a predetermined amount of MitoQ; in granules or in granules; in solution, as a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion or powder; MitoQ can also be administered intravenously, as a preparation or puree.

The pharmaceutical composition may have any formulation selected from the group consisting of a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution, an emulsion, a syrup, an aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze drier, a suppository, and an injection.

When used orally, they can be made into, for example, tablets, lozenges, pills, oily or aqueous suspensions, dispersed powder granules, emulsions, hard or soft pastilles, syrups or elixirs.

The pharmaceutical compositions can be manufactured by a known process, for example, a general mixing, granulating, dissolving or lyophilizing process. For example, the pharmaceutical composition for oral administration can be prepared by mixing the drug with a solid carrier, granulating the mixture, adding an appropriate additive if necessary, and then formulating the mixture or the granules in the form of a tablet or a dragee

A tablet may be made by compression or molding, or optionally with one or more additional excipients and/or additives. Compact tablets may be made by compression, in a suitable free-floating MitoQ device such as sachets or granules, optionally mixed with a binder, lubricant, inert diluent, prophylactic, surface active or dispersing agent. Tablets that have been transformed, by molding them in a suitable device, into a mixture of powdered MitoQ moistened with an inert liquid diluent. The tablets may optionally be coated, scored, or made to provide slow or controlled release of MitoQ.

Pharmaceutical compositions administered orally can be manufactured by any of the known techniques for the preparation of pharmaceutical compositions and such compositions may contain one or more excipients and/or additives that contain sweetening, flavoring, coloring and preservative agents, to in order to obtain a preparation that is pleasing to the palate. Tablets containing MitoQ in mixtures with permitted non-toxic pharmaceutical excipients and that are admitted in the manufacture of tablets will be suitable.

The pharmaceutically acceptable excipient may include, in particular, a filler, for example, sugar, such as lactose, sucrose, mannitol or sorbitol, a cellulose agent and/or calcium phosphate, such as tricalcium phosphate or calcium hydrogen phosphate, and a coupling agent, for example starch pastes using corn, wheat, rice or potato starches, gelatin, tragacanth, methylcellulose and/or polyvinylpyrrolidone, and if necessary a disintegrating agent, for example the aforementioned starches, carboxymethyl starch , cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, eg sodium alginate.

Pharmaceutically acceptable additives include, in particular, a flow control agent and a lubricant, for example silicic acid, talc, stearic acid or its salt, for example. example, magnesium stearate, or calcium and/or polyethylene glycol. An appropriate gastro-resistant coating is provided to a dragee core, and, in particular, a concentrated sugar solution including gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide in an appropriate organic solvent or solvent mixture, or a stirred solution is used, or, to form the gastro-resistant coating, a solution with an appropriate cellulose agent, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, may be used.

Other pharmaceutically acceptable excipients for oral administration include encapsulating agents and dry-filled capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The dry-filled capsule may include an active ingredient in particulate form, for example, a filler, such as lactose, a coupling agent, such as starches, and/or a modifier, such as talc or magnesium stearate, and a mixture with a stabilizer if appropriate. In the soft capsule, MitoQ is dissolved or suspended in an appropriate liquid, for example, fixed oil, petrolatum oil or liquid polyethylene glycol, and a stabilizer may be added therein. The tablets may be coated or uncoated by known techniques including microencapsulation to delay disintegration and absorption from the gastrointestinal tract and thereby provide sustained action over a longer period of time. For example, retarding materials such as glycerol monostearate or glycerol distearate can be used by themselves or with a wax.

The pharmaceutical compositions of the invention can be presented as aqueous suspensions containing MitoQ in mixtures with excipients and/or appropriate additives for the preparation of aqueous suspensions. For example, they incorporate a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and dispersing or wetting agents such as natural phosphatide (for example, lecithin). , a condensation product of alkylene oxide with a fatty acid (for example, polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol, (for example, heptadecaethyleneoxycetanol), a product of condensation of ethylene oxide with a partial ester derived from a fatty acid and hexitol anhydride (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.

The pharmaceutical compositions of the invention can be presented as dispersible powders or granules allowed in the preparation of an aqueous suspension by adding water, provide the MitoQ in a mixture with an excipient and/or dispersing or wetting additive, a suspending agent or a or more preservatives.

The permitted excipients and/or dispersing or wetting additives, as well as the suspending agents, are, for example, those set forth above. Additional excipients, for example, sweeteners, flavoring or coloring agents, may also be found.

The amount of MitoQ that can be mixed with the excipients and/or additives to generate a single dosage form will vary depending on the agent employed and the specific form of administration. For example, a pharmaceutical composition with a release time designed for oral administration in humans can contain approximately 1 to 1000 mg of MitoQ enhanced with a correct and adequate amount of carrier substance that can range between 1 and 95% of MitoQ. the total compositions (w/w). The pharmaceutical composition can be prepared to provide readily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion (injection) may contain from about 3 to 500 pg (picograms) of MitoQ per milliliter of solution so that injection of an adequate volume at a rate of 30 mL/hr may occur.

Pharmaceutical compositions may be presented in unit or multi-dose containers, for example, sealed ampoules and vials, and may be stored under lyophilized conditions requiring only the addition of a sterile liquid carrier, for example, water for injection. , immediately before use. Impromptu injection solutions and suspensions are made from sterile powders, granules and tablets of the same class as those listed above. Preferred unit dose preparations are those containing a unit daily dose or sub-dose, or such a suitable fraction, of MitoQ.

The term "patient" refers to any mammal that is the recipient of the diagnosis, preferably a human. The patient may be a person presenting for routine disease evaluation or may have symptoms suggestive of CMT. The patient may also be an individual considered to be at high risk for this disease, due to a family history of this inherited disease.

The diagnosis can be made by any method known from the state of the art that is commonly used in medical practice by a physician or a specialist in this disease.

The pharmaceutical composition can be administered to the patient before the appearance of the symptoms of CMT, thus achieving the prevention or delay in the appearance of the characteristic symptoms of this neuropathy.

In one embodiment, the pharmaceutical composition can be administered throughout the patient's life, counting from the first administration, thus preventing or delaying the appearance of the characteristic symptoms of this neuropathy.

In another particular embodiment, the pharmaceutical composition can be administered with a variable administration interval, for example from 1 to 7 times per week, preferably from 2 to 6 times per week, more preferably from 3 to 5 times per week.

The effective dose of MitoQ and the pharmaceutical composition that comprises it depends on the nature of the conditions, the execution form and the pharmaceutical formulation, and will be defined by the experience of a doctor through the application of the usual investigations for an increase in the dose. The effective dose can be considered to be from 0.0001 to about 1000 mg/kg body weight per day, or from 0.01 to about 500 mg/kg body weight per day, or from 0.01 to 100 mg/kg. body weight per day, or from 0.05 to about 10 mg/kg body weight per day. For example, the daily dose that is postulated for an adult person of approximately 70 kg of body weight will be in the range of 1 mg to 1000 mg, or between 5 mg and 500 mg, and can be made by single or multiple doses, daily or No.

In a particular embodiment, the pharmaceutical composition can be used for the claimed indication by administering to an individual a dose of 0.1 mg/kg/day to 1000 mg/kg/day, specifically a dose of 1 mg/kg/day at 500 mg/kg/day, or a dose of 3 mg/kg/day to 100 mg/kg/day.

In the dose and administration interval of the pharmaceutical composition may vary depending on various factors, for example, individual conditions, such as availability and duration of the drug, an administration procedure, sex, age and weight of individuals, severity of diseases and the like. The specific route of administration and the dose can be selected by a person skilled in the art according to conditions such as age, weight, degree of disease activity and physical state. The particular conditions of the dose and administration interval can be easily determined by a person skilled in the art.

Therefore, administration of MitoQ from an early stage of diagnosis to MTC patients could help prevent the loss of mitochondrial and antioxidant proteins observed in the peripheral nerves of mildly affected MTC patients, resulting in a lower impaired motor functions.

Of note, MitoQ treatment prevented the molecular deterioration and oxidative damage observed in the sciatic nerves of Gdapl-null mice as well as the development of CMT symptoms in these mice, strongly supporting oxidative damage as an important factor that contributes to the etiology of the disease.

The invention is illustrated by the following examples which describe in detail the objects of the invention. These examples should not be considered as limiting the scope of the invention but rather as illustrative of it.

Brief description of the figures

Figure 1 MitoQ prevents the development of the phenotype associated with CMT disease in Gdapl-/- mice.

A. Graphic scheme showing the treatment of the mice. After weaning (1 month) MitoQ (0.14 mg/ml) was administered in the mice's drinking water. Motor function tests were performed at 7 and 10 months of age.

BD Wild-type mice (WT, n=9, black line bar and dots), untreated Gdap1-/- mice (KO, n=10, black line bar and squares), and Gdap1-/- mice treated with MitoQ (MITOQ, n=9, gray line bar and triangles).

B. Representative images of mice at 10 months of age. The histograms show the quantification of the change in the weight of the mice during the treatments at 7 months of age (upper panel) and 10 months of age (lower panel).

C. Representative images of 3 10-month-old mice suspended by the tail. Wild-type (WT) mice and MitoQ-treated Gdap1-/- mice show the characteristic response of attempting to escape by extending their hind limbs from their body trunk. In contrast, the hindlimbs of untreated Gdap1-/- mice remain close to the trunk in an abnormal dystonic posture. Histograms show the quantification of the angle between the right hind limb and the body axis.

D. Representative image of the hanging test with two extremities. The histograms show the quantification of the longest time during which the mice showed sustained limb tension.

The bars indicate the mean ± SEM of the n indicated at the top. **p < 0.01 when compared to WT mice and ##p < 0.01 when compared to Gdapl-' mice by Student's t-test.

Figure 2 MitoQ prevents motor deficits in Gdapl-/- mice.

AD Wild-type mice (WT, n=9, black line bar and dots), untreated Gdapl-' mice (KO, n=10, black line bar and squares), and MitoQ -treated Gdapl-' mice ( MITOQ, n=9, gray line bar and triangles).

A. Representative image of the two-leg hang test. Histograms show the quantification of the maximum time during which the mice exhibited sustained limb tension, (left panel) and the minimum grasping impulse in the two-limb hang test at 10 months of age (right panel).

B. Representative image of the grip strength test. Histograms show the quantification of the mice's force (g) over body weight (g).

C. Representative image of the four limb hang test. The histograms show the quantification of the maximum time during which the mice showed sustained limb tension. (left panel) and the minimum grasping impulse in the limb hanging test at 10 months of age (right panel).

D. Representative image of the rotarod test. The graph shows the quantification of the latency of the mice when falling (in seconds) at a speed of 4, 8, 12, 16, 20, 24, 28 and 32 rpm.

The bars indicate the mean ± SEM of the n indicated at the top. **p < 0.01 when compared to WT mice and ##p < 0.01 when compared to Gdapl-' mice by Student's t-test.

Figure 3. MitoQ prevents the metabolic and redox deficiencies observed in the sciatic nerves of GDAP1-- mice.

Sciatic nerve extracts obtained from wild-type (WT, black box, left) mice (n=9), untreated Gdapl-' mice (KO, black box, center) (n=10), and MitoQ (MITOQ, left box). grey, right) (n=9) were deposited as drops on the slides and processed. The arrays were developed with the indicated primary antibodies and the amount of protein in the extracts expressed in arbitrary units/ng of protein using the linear diagram of the C2C12 cell line as a standard. Box plots represent the 25th to 75th percentiles with the median value at the midline, and with all data plotted from minimum to maximum protein expression level values, expression levels are shown as arbitrary units, (ua). Glucose metabolism enzymes (ENO1, LDHA, PDH), TCA cycle (IDH1, MDH2), fatty acid oxidation (HADHA), OXPHOS (Oxidative Phosphorylation) (CORE2, p-F1-ATPase) are presented. , mitochondrial dynamics (MFN2, OPA1), redox homeostasis (G6PDH, GR, SOD1, Catalase, SOD2) and oxidative stress (MDA, 4HNE). * and **, indicate p < 0.05 and p < 0.001 when compared to WT mice, respectively; # and ##, p < 0.05 and p < 0.001 when compared to Gdap1-/- mice, respectively.

Figure 4. The sciatic nerve of GDAP1 -/- mice shows a marginal effect on the protein content of metabolism.

Sciatic nerve extracts derived from wild-type (WT, black box) mice (n=9), untreated Gdapl-/- mice (KO, red box) (n=10), and MitoQ-treated Gdap1-/- mice (MITOQ , yellow box) (n=9) were deposited in the form of drops on the slides and processed. Arrays were developed with the indicated primary antibodies and the amount of protein in the extracts expressed in arbitrary units/ng of protein using the linear plot of the C2C12 cell line as a standard (Figure S3). Box plots represent the 25th to 75th percentiles with the median value at the midline, and with all data plotted from the lowest to highest values of protein expression level.

Additional mitochondrial proteins from oxidative phosphorylation (NADH9, COXIV), mitochondrial dynamics (MFN1), and mitochondrial structure (HSP60) are shown. *p < 0.05 when compared to WT mice; #p < 0.05 when compared to Gdapl-- mice.

References

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2. Soldevilla, Beatriz et al. “Plasma metabolome and skinproteins in Charcot-Marie-Tooth 1A patients.'' PloS one vol. 12.6 e0178376. 2 Jun. 2017, doi:10.1371/journal.pone.0178376

3. Smith, Robin AJ et al. “Delivery of bioactive molecules to mitochondria in vivo.” Proceedings of the National Academy of Sciences of the United States of America vol. 100.9 (2003): 5407-12. doi:10.1073/pnas.0931245100

4. Barneo-Muñoz, Manuela et al. “Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.” PLoS genetics vol. 11.4 e1005115. 10 Apr. 2015, doi:10.1371/journal.pgen.1005115

5. Aartsma-Rus, Annemieke, and Maaike van Putten. “Assessing functional performance in the mdx mouse model." Journal of visualized experiments : JoVE ,8551303. 27 Mar. 2014, doi:10.3791/51303

6. Cabrera, Jorge Ruben et al. “Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection" PLoS pathogens vol. 11.1 e1004571. 22 Jan. 2015, doi:10.1371/journal.ppat.1004571

7. Santacatterina, Fulvio et al. “Different mitochondrial genetic defects exhibit the same protein signature of metabolism in skeletal muscle of PEO and MELAS patients: A role for oxidative stress." Free radical biology & medicine vol. 126 (2018): 235-248. doi:10.1016/j. freeradbiomed.2018.08.020

8. Santacatterina, Fulvio et al. "Quantitative analysis of proteins of metabolism by reverse phase protein microarrays identifies potential biomarkers of rare neuromuscular diseases." Journal of translational medicine vol. 13 65. 18 Feb.

2015, doi:10.1186/s12967-015-0424-1

9. Santacatterina, Fulvio et al. “Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis." Journal of translational medicine vol. 15,1 29. 10 Feb. 2017, doi:10.1186/s12967-017-1136- 5

Examples of realization.

The specific materials, methods and protocols used in the 3 examples of the invention are explained below. Unless otherwise stated, the conditions and The materials used in the examples of embodiment will be those that appear in the following section.

A. Materials and Methods

Cell lines. Cells were grown in a 37°C incubator with a controlled atmosphere of 10% CO ² . HCT116 and C2C12 human colorectal carcinoma cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS). To preserve cell lines, aliquots of 5 million cells were gradually frozen in 1 ml of culture medium containing 10% DMSO, dimethyl sulfoxide (14 h at -20°C, 24 h at -70°C) and then kept at liquid nitrogen). Thawing was carried out at 37°C, immediately diluting them in the culture medium.

Animals. The mice were housed in the Animal Facility with a cycle of 12 hours of light and 12 hours of darkness and temperatures of 18-23 °C with 40-60% humidity. Gdapl -deficient or Gdapl knockout ( Gdapl--) mice were previously generated and characterized and are used as study models of CMT4 disease. MitoQ (0.14 mg/ml) was administered to mice in drinking water.

Evaluation of mouse motor function

Rotarod test. Motor performance and balance were measured using an accelerating rotarod. Each mouse was subjected to the same procedure for 2 days. The first day was used to train the mice in a 2 min session, walking at 4 revolutions per minute (rpm). Mice that fell over after completing 2 min of walking within 10 min were discarded from the experiment. Test sessions were held on the second day. During the test, the speed of the rotarod was increased from 4, 8, 12, 16, 20, 24, 28 to 32 rpm. Mice spent 1 min running at each speed. Their latency was measured. Mice that fell down 6 times in 60 seconds were discarded from the experiment.

Grip strength/endurance test. The grip strength test was used to measure the maximum force a mouse could exert with its front paws by taking advantage of the animal's tendency to grip surfaces. Using a mouse each time, he was allowed to grasp the metal bars and then gently pulled back until the grip was broken. The pull was made at a constant speed and slow enough to allow the mouse to exert resistance against it. The test was repeated 5 times per mouse, with a minimum of 1 minute between each of the 5 determinations per animal.

Hanging or suspension test: two extremities. Forelimb muscle strength was measured by monitoring the ability of mice to exert sustained tension on the limbs to oppose their weight. Mice were placed on a 2 mm thick metal bar 35 cm from a padded surface and time to fall was recorded. The test ended after a suspension time of 2 min was achieved or otherwise after three sessions. Maximum hang time and minimum holding impulse (body mass, grams x hang time, hang) were calculated5.

Hanging or Suspension Test: Four Limbs . Muscle strength of all four limbs was measured by monitoring the ability of mice to exert sustained tension on the limbs to oppose their weight. Mice were placed on a wire grid 35 cm from a padded surface and time to fall was recorded. The trial ended after a 2-minute hang time was achieved, or else after three sessions. Maximum hang time and minimum grasping impulse (body mass x hang time)5 were calculated.

Protein extraction from mouse tissues. For protein extraction, tissue sections were homogenized using an OMNI Bead Ruptor 24 bead mill homogenizer (Fisher Scientific, 15515799), previously suspended the tissue section in T-PER tissue protein extraction reagent (Cat. Nope.

78510, Thermo Scientific, Ins. Madrid, Spain,) in a ratio of 1:8 (weight/volume), and frozen three more times in liquid nitrogen. Protein concentration was determined with the Bradford reagent (Cat. No. 5000001, Bio-Rad, Inc. Madrid, Spain) using Bovine Serum Albumin (BSA) as standard.

Neuronal cultures of the dorsal root ganglion. Dorsal root ganglia (DRG) were obtained from newborn (postnatal day 0-2) wild-type (WT) and Gdapl knockout ( Gdapl-/-) mice according to6. Ganglia were digested in collagenase (Worthington, USA) and trypsin (Sigma-Aldrich, St. Louis, MO, USA), dissociated by grinding, and applied in DMEM-F12 containing 50 ng/mL of NGF (Tebu-Bio, Le-Perray-en-Yvelines, France), 10% fetal bovine serum, and 5 ng/mL aphidicolin (AG Scientific, San Diego, CA, USA). DRG neurons were seeded in polylysine and coated with laminin (1 pg/mL) (Sigma-Aldrich, St. Louis, MO, USA) and used for experimentation between 2-4 DIV, (Days in vitro).

Printing and processing of reverse phase protein arrays (RPPAs). Protein extracts from tissue biopsies were diluted with T-PER ( Tissue Protein Extraction Reagent) to a final protein concentration of 2 pg/pl (w/v). Prior to printing, samples were diluted with PBS (Phosphate Buffered Saline) to a final protein concentration of 0.5 pg/pl (w/v). Serially diluted protein extracts (0-1.5 pg/pl (w/v)) derived from C2C12 mouse myoblast and HCT116 human colorectal carcinoma cells were also prepared to assess print quality and linear response. of protein recognition by the antibodies used7. Standard curves of BSA (0-2 pg/pl) and mouse IgG (immunoglobulin G) (1-30 ng/pl) were also prepared as negative and positive internal controls, respectively.

A drop of approximately 1.5 nl of each sample was added onto nitrocellulose coated glass slides (ONCYTE® SuperNOVA 8 pad - Grace Bio-Labs, 705118). Each sample was analyzed in triplicate or quintuplicate. Droplets were added using an iTWO-300P pico system printer (M2-Automation, Inc.) equipped with a 30-150 pL piezoelectric driven microdispenser (PDMD) at constant chamber humidity (RH 52%) and temperature (16 °C), and a plate temperature (10 °C).

After printing, the slides were allowed to dry and subsequently blocked with Super G blocking buffer (Cat. No. 10501, Grace Biolabs, Madrid, Spain). Slides were then incubated overnight at 4°C with the indicated dilutions of the primary antibodies listed in Table 1.

Table 1: List of primary antibodies used

After incubation, slides were washed with saline phosphate buffered solution, PBS-T and further incubated with CF™ 647-conjugated highly cross-linked goat anti-mouse or goat anti-rabbit antibodies (Sigma Aldrich, SAB4600183 - SAB4600185). ; 1:500, Madrid, Spain). Slides directly incubated with secondary antibodies and with 0.0001% Fast Green FCF (Cat. No. F7252, Sigma Aldrich, Madrid, Spain) were used to assess possible non-specific binding to unmasked mouse IgG and the total amount of protein present. in the droplets of the samples, respectively.

The microarrays were scanned using the Typhoon 9410 scanner (GE Healthcare, Inc. Madrid, Spain). The mean fluorescence intensity of the spots was measured using GenePix® Pro 7 (USA) and normalized in relation to the amount of protein contained in the sample obtained from the FCF staining pad. After quantification, the relative fluorescent intensity was converted to arbitrary units of expressed protein/ng of protein in the extract using the linear plot of the C2C128.9 cell line as a standard.

Statistic analysis. The results shown in the figures are means ± SEM (standard error of the mean). Statistical analysis was performed by Student's t-test. Statistical tests were two-tailed at the 5% level of significance. Statistical analyzes were performed using Excel Microsoft 365 and GraphPad Prism 7.

B. Examples of implementation

Example 1: Effectiveness of MitoQ in preventing the onset and development of CMT symptoms in Gdapl -deficient mice

MitoQ was administered to Gdapl-null mice from the time of weaning until 10 months of age (Figure 1A). A group of untreated wild-type and Gdap1-null mice were also included.

At seven months of age, we observed that Gdapl-null mice showed a significant increase in body weight compared to wild-type. In contrast, MitoQ prevented body weight gain in Gdapl-null mice (Fig. 1B, top histogram). Weight gain is a serious problem for CMT patients because weakened muscles make it hard for them to stay active. Consistent with this, the progressive weight gain observed in Gdapl-null mice due to visceral fat accumulation was prevented by treating such mice with MitoQ presumably because avoiding muscle weakness allowed the mice to have the same activity as the mice. control, or WT.

However, the motor coordination of the groups of mice studied, evaluated with the rotarod in mice of the 3 conditions at 7 months of age, did not reveal significant differences between them (Figure 1D).

At 10 months of age, untreated Gdapl-null mice still showed a significant increase in body weight compared to wild-type mice (FIG. 1B, photographs of mice). MitoQ treatment significantly decreased the body weight gain of Gdapl-null mice (Figure 1B lower histogram).

CMT related to genetic alterations in GDAP1 promotes weakness and wasting of the feet and hands, leading to motor deficits and loss of sensation, leading to pronounced disability in patients. Similar to that described for patients, at 10 months of age we observed an abnormal hindlimb closure reflex in Gdapl-null mice not treated with Mitoq, as assessed by the shape of the angle between the right hindlimb and the left axis. of the body, when compared to wild-type mice (Figure 1C). Notably, treatment of mice with MitoQ significantly improved the animals' hindlimb closing reflex (Figure 1C, histogram), thus, treatment of the present invention improved prevention of onset of symptoms of CMT.

The determination of the muscle strength of the forelimbs and the maximum force exerted by said limbs were evaluated by means of the hanging tests of two and four limbs (Figures 2A and 2C images of the mice) and grip strength (Figure 2B). Both maximal hang time (Figure 2A, left histogram) and minimal grasping impulse (Figure 2A, right histogram) were significantly reduced in untreated Gdapl-null mice when compared to wild-type or WT mice in the test of hung with 2 extremities. MitoQ treatment of mice significantly improved forelimb muscle strength (Figure 2A). Similar results were obtained in the grip strength test (Figure 2B), which supports the conclusion of that both MitoQ treatment prevented wasting of the forelimb muscles of Gdapl-null mice.

Four-limb muscle strength was determined by the ability of mice to oppose their weight using their limbs, using a test similar to the two-limb hang (Figure 2C). Both maximal hang time (Figure 2C, left histogram) and minimal grasping impulse (Figure 2C, right histogram) were significantly reduced in untreated Gdaplnull mice when compared to wild-type mice. Treatment of mice with MitoQ significantly improved muscle strength in all four limbs (Figure 2C), demonstrating that treatment with this compound prevents limb muscle wasting in Gdapl-null mice.

Finally, motor coordination behavior was assessed using the rotarod test (Figure 2D). Although no significant differences were observed between the 7-month-old mice from the 3 conditions, when the experiment was repeated with 10-month-old mice from the 3 conditions, it was observed that the knockdown latency was significantly reduced in the Gdapl mice. -null (Figure 2D), further reinforcing the conclusion that MitoQ acts as a “scavenger” of mitochondrial reactive oxygen species (ROS) from early life, preventing the onset of the deleterious muscle wasting phenotype of the Gdapl- mouse model. null.

Example 2: Effectiveness of MitoQ in preventing mitochondrial redox proteome degradation in Gdapl -deficient mice

It has been published that patients with CMT disease suffer a partial loss of enzymes involved in energy metabolism and ROS manipulation as a result of epidermal nerve terminal injury. Similarly, peripheral nerves from the Gdapl-null mouse model of CMT disease also show reduced enzymes of energy metabolism and antioxidant response4. Therefore, next, we explored the effect of MitoQ on the expression of certain proteins of energy metabolism and of the response to oxidative stress in tissue extracts derived from sciatic nerves, from Gdapl-null mice (Figure 3), using a high-throughput quantitative approach called reverse phase protein arrays (RPPAs)7,8. Peripheral nerves of Gdapl-null mice showed a significant loss of enzymes involved in glucose oxidation and mitochondrial respiration (LDHA, PDH, IDH1, and CORE2) and of the antioxidant response (GR, SOD1, catalase, and SOD2). compared to wild-type mice (Figure 4). According to this last observation, the modification Oxidation of cellular proteins by MDA and 4HNE was significantly increased in Gdapl-null mice (Figure 3). Other proteins did not show significant changes (Figure 3), although a decreasing trend is observed in most of them (ENO1, MDH2, HADHA, |3F1 and OPA1).

Notably, MitoQ treatment significantly prevented and/or increased the expression of a large number of energy metabolism proteins (ENO1, LDHA, PDH, IDH, CORE2, |3F1), in the antioxidant response (GR, catalase, and SOD2) and the 4HNE protein, involved in oxidative stress, in the sciatic nerves of Gdapl-null mice (Figure 3). Other proteins did not show significant changes, although in most of them a tendency to recover similar levels of WT mice is observed, or control (MDH2, CORE2, OPA1 and SOD1) (Figure 3). These results strongly support at the molecular level that alterations in energy and redox metabolism that occur in Gdapl-null mice during development can be prevented by treatment with the antioxidant MitoQ.

Quantification of mitochondrial proteins in the sciatic nerves, such as NADH9 and COXIV from the respiratory chain, further confirmed the specific deleterious effect of Gdapl gene ablation on proteins involved in mitochondrial energy production when compared to proteins involved in other mitochondrial functions (Figure 4). In addition, the positive effect of the treatment in preventing the loss of energy metabolism enzymes in Gdapl-null mice (Figure 4), specifically in NADH9 and a certain tendency in COX IV, was confirmed. These results further confirm that MitoQ prevents the development of CMT symptoms by improving energy metabolism dysfunction and redox homeostasis in peripheral nerves.

Claims (10)

Claim 1. A pharmaceutical composition comprising MitoQ for use in treating the symptoms of Charcot-Marie-Tooth disease in a patient. 2. The pharmaceutical composition according to the preceding claim, wherein the cause of the disease is due to genetic alterations in GDAP1 and/or MFN2. 3. The pharmaceutical composition according to one of the preceding claims, wherein Charcot-Marie-Tooth disease is selected from AR-CMT2K, CMT2K and CMT4A. 4. The pharmaceutical composition according to one of claims 1 or 2, wherein Charcot-Marie-Tooth disease is AR-CMT2A2. 5. The pharmaceutical composition according to one of claims 1 to 4, wherein the symptoms of Charcot-Marie-Tooth disease are motor impairments, sensory loss, optical impairments, diaphragmatic paralysis and/or vocal cord paralysis. 6. The pharmaceutical composition according to the preceding claim, wherein the motor deficiencies comprise motor deficiencies of the lower extremities, loss of body mass in the legs and feet, high arches of the feet, bent toes and/or difficulty to flex the heel. 7. The pharmaceutical composition according to one of claims 1 to 6, comprising at least one or more of the following: - a pharmaceutically acceptable excipient, and - a pharmaceutically acceptable additive. 8. The pharmaceutical composition according to one of claims 1 to 7, wherein the treatment of the patient is carried out before the appearance of the symptoms of Charcot-Marie-Tooth disease. 9. The pharmaceutical composition according to one of claims 1 to 8, wherein the treatment of the patient is carried out 1 to 7 times per week. 10. The pharmaceutical composition according to one of claims 1 to 9, wherein the administered dose is from 0.0001 to about 1000 mg/kg body weight per day.