WO2023139307A1 - Pig brain extract and its use in the treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a non-denaturing method for obtaining a Sus scrofa domestica brain extract. Furthermore, the present invention relates to the uses of said extract for the treatment of neurodegenerative diseases, particularly Alzheimer's and Parkinson's.

Description

PIG BRAIN EXTRACT AND ITS USE IN THE TREATMENT OF NEURODEGENERATIVE DISEASES

The present invention is included in the technical field of medicine, specifically in the use of neurological extracts for the prevention and/or treatment of neurodegenerative diseases. Particularly, the invention refers to a non-denaturing process for obtaining an extract from the brain of Sus scrota domestica. Furthermore, the present invention relates to the uses of said extract for the treatment of neurodegenerative diseases, particularly Alzheimer's and Parkinson's.

BACKGROUND OF THE INVENTION

The main health problems in the developed world are cardiovascular diseases (25-30%), cancer (20-25%) and central nervous system (CNS) disorders (10-15%). Within brain disorders, neurodegenerative diseases represent a priority problem in terms of morbidity and mortality. The two most prevalent neurodegenerative disorders are Alzheimer's disease (AD) and Parkinson's disease (PD).

All neurodegenerative diseases present common problems: (i) their primary cause is associated with genomic defects, epigenetic aberrations, and environmental factors that precipitate or aggravate the primary process; (ii) they develop decades before giving symptoms and when they manifest in adulthood the degree of brain damage is irreversible; (iii) they all have a highly disabling progressive course; (iv) most present in their etiopathogenesis intra or extracellular protein deposits; (v) its diagnosis is difficult due to the lack of highly reliable biomarkers; and (vi) none have curative treatment at the present time.

Alzheimer's disease is a polygenic and multifactorial disorder in which a multitude of genomic defects, epigenetic aberrations, chronic cerebrovascular damage and various metabolic factors participate, with the participation of precipitating environmental factors. AD has a prevalence of 1.8% at 65-69 years of age with an ascending progression that reaches 42.1% of the population at 95-99 years of age (with an annual incidence of 34.1 per 1,000 in those over 60 years of age), and an age-adjusted mortality rate of 24.5 per 100,000, with more than 50 million people currently affected worldwide and a projection of 70 million affected people within 20 years (GBD 2015, Neurological Disorders Collaborator Group. Global, regional, and national burden of neurological disorders during 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Neurol. 2017; 16(11):877-89 7).

In the last two decades, more than 10,000 molecules have been investigated for the treatment of AD without success. The most recent studies indicate that the therapeutic response, both in terms of safety and efficacy, depends on the pharmacogenetic profile of the patient and the chemical characteristics of the product under development. Until 2003, the only drugs available for the treatment of AD were based on the potential cholinergic dysfunction of the disease (Tacrine, Donepezil, Rivastigmine, Galantamine) and on the underlying neurotoxic process (Memantine). In 2021, the first preventive treatment for AD, the Aducanumab antibody, was approved as an anti-amyloid immunotherapy (Cacabelos R. How piausibie is an Alzheimer's disease vaccine? Expert Opin Drug Discov. 2019; 15(1):1-6).

Parkinson's disease (PD) is the second most important neurodegenerative disorder, after Alzheimer's. The age-dependent prevalence of PD varies from 41 per 100,000 at 40 years to 1,903 cases per 100,000 at 80 years, with a peak prevalence in people over 90 years of age (4,633 cases per 100,000), and a mean prevalence of 1,680 per 100,000 in people over 65 years of age (Pringsheim T, Jette N, Frolkis A, Steeves TD. The prevalence of Parkinson's disease: a systematic review and meta-analysis. Movement Disorders: Official Journal of the Movement Disorder Society. 2014; 29(13): 1583-1590).

The main risk factors for PD are genomic defects, epigenetic aberrations, toxic elements (pesticides, herbicides, drugs, environmental pollution), cerebral microtrauma, oxidative stress, neuroinflammatory reactions, neuroglial dysfunction, hypoxic conditions or low cerebral perfusion, metabolic deficit, and dysfunction of the ubiquitin-proteasome system (Cacabelos R. Parkinson's disease: From pathogenesis to pharmacogenomics Int. J. Mol. Sci., 2017; 18:551. Doi: 10.3390/ijms18030551).

All these pathogenic events contribute to abnormal protein processing with aggregation, dissemination, and intracellular deposits in dopaminergic neurons leading to progressive death (Darweesh SK, Verlinden VJ, Adams HH, Uitterlinden AG, Hofman A, Stricker BH, van Duijn CM, Koudstaal PJ, Ikram MA. Genetic risk of Parkinson's disease in the general population. Park insonism & Related Disorders. 2016; 29:54-59). In parallel, the clinical symptoms characterized by tremor at rest, rigidity, bradykinesia and postural instability, along with other satellite symptoms such as dysautonomic syndrome and emotional lability.

Current treatment for PD includes drug therapy, deep brain stimulation, and physical therapy. The standard pharmacological treatment of PD since the 1960s is with L-DOPA [(2-amino-3-(3,4-dihydroxyphenyl)propanoic acid), the precursor of dopamine, alone or with carbidopa. In recent years, other treatments aimed at enhancing dopaminergic neurotransmission have been incorporated (Müller T. Pharmacokinetics and pharmacodynamics of levodopa/carbidopa cotherapies for Parkinson's disease. Exp Opin Drug Metab Toxicol. 2020; 16(5):403-414).

All these treatments are symptomatic, lacking, in general, anti-pathogenic effect. In addition, the continued use of classic antiparkinsonian drugs loses efficacy, requiring a consequent increase in dose that, over time, induces severe adverse effects ranging from ON-OFF episodes, with stereotypies and loss of motor effect, to psychotic disorders and complex behavioral problems (Haaxma CA, Horstink MW. Zijlmans JC, Lemmens WA, Bioem BR, Borm GF. Risk of Disabling Response Flu performances and Dyskinesias for Dopamine Agonists Versus Levodopa in Parkinson's Disease. J Park Dis. 2015; 5(4):847~ 853).

The use of nutraceutical and dietary compositions comprising neurological components as adjuvants for the prevention and/or treatment of neurodegenerative diseases, or as enhancers of nervous system health, has also been described. Neurological components include, for example, glycerophospholipids such as phosphaditylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphaftidylinositol (Pl); sphingophospholipids such as sphingomyelins and ceramides; sphingoglycolipids such as gangliosides, cerebrosides, and sulfatides.

In this sense, different procedures have been described to obtain said neurological components with different degrees of purity or as a mixture thereof from biological materials such as, for example, organs of various animals, milk, nuts, cereals, or fungi.

British patent GB1256755 describes an extract of brain mitochondria, which is suitable for the treatment or prevention of brain disorders, especially those affecting the degree of consciousness and cerebral edema. The extract is obtained by means of a sequence of centrifugations at different speeds of a homogenized brain in an aqueous solution comprising mannitol and EDTA.

Russian patent application RU-A-1994012010 describes a process for extracting cerebroside from a porcine brain. Extraction is carried out with acetone, the filtrate is precipitated with ethanol, and the final product is purified by washing with acetone.

Chinese patent application CN-A-1596733 describes a process for the preparation of a porcine or bovine brain extract, which comprises crushing the brains, treating them with a combination of enzymes, hydrolysis with hydrochloric acid, neutralization and lyophilization. According to the description, said extract is suitable for improving the nutritional status of the body and regulating the functioning of the brain.

The international patent application WO-A-2010/007620 describes a porcine brain extract that includes a mixture of peptides and proteins with a molecular weight of at least 5000 Da. Its use as a protector of muscle cells and neurons against hypoxia and apoptosis is also described.

In the article Jíttiwat et al., Porcine Brain Extract Attenuates Memory Impairments Induced by Focal Cerebral Ischemia, Am. J. Applied, Sci., 2009, 6(9), 1662-1668, a porcine brain extract is described that comprises a mixture of the amino acids aspartic acid and glutamic acid, and its use as a neuroprotectant. In the article Thukham-Mee et al., Neuroprotective Effect against Alzheimer's Disease of Porcine Brain Extract, Am. J. Applied, Sci., 2012, 9(5), 700-708, the use of the same extract as a dietary supplement or adjuvant against Alzheimer's disease and other cognitive disorders due to aging is described.

Despite the solutions described in the state of the art, it is still necessary to develop new procedures, easily implementable on an industrial scale, to prepare extracts of neurological components to be used in the prevention and/or treatment of neurodegenerative diseases and as enhancers of nervous system health, particularly for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

DESCRIPTION OF THE INVENTION

The inventors have obtained, by means of a non-denaturing procedure, an extract ultrapure freeze-dried from the brain of Sus scrofa domestica, named Nosustrophin (NSTF-1). In particular, Nosustrophin is an extract rich in brain fatty acids, neuroprotective vitamins, brain neurotransmitters, neurohormones and neurotrophic factors from the juvenile brain of Sus scrofa domestica that has bioactive effects in transgenic models of Alzheimer's disease and chemically induced models of Parkinson's disease and brain neuroinflammation. Unlike the denaturing procedures of the state of the art, such as alcoholic or thermal extraction, the procedure carried out by the inventors is a non-denaturing procedure, particularly cold extraction, which makes it possible to obtain an extract that preserves all its biological properties. With the designed procedure, all the denaturing factors of the brain extract molecules associated with changes in pH, temperature and chemical alteration are eliminated.

The inventors, through microscopic and immunohistochemical analysis, cell cultures, cell viability studies, epigenetic studies, and biochemical, metabolic, and nutritional studies, have observed the bioactive effects of Nosustrophin in transgenic models of Alzheimer's disease and chemically induced models of Parkinson's disease (PD) and cerebral neuroinflammation. In Alzheimer's (AD) transgenic animals, Nosustrophin showed potent antidegenerative capacity by reducing p-amyloid protein plaques and reducing progressive lesion damage present in transgenic models. In MPTP-induced Parkinson's models, Nosustrophin reverses and prevents the neurotoxic effect induced by MPTP and restores dopaminergic activity in neurons of the substantia nigra. In LPS-induced neuroinflammation models, Nosustrophin reduces the inflammatory damage caused by LPS. Cell viability studies show that Nosustrophin potentiates neuronal survival.

All these studies confer neuroprotective and antidegenerative activity to Nosustrophin in Alzheimer's disease, in Parkinson's disease and in neuroinflammatory processes.

Thus, in a first aspect, the present invention refers to a procedure for obtaining an extract, also called in this document Nosustrophin or NSTF-1, from the brain of Sus scrofa domestica, hereinafter "the procedure or method of the invention", which comprises the following steps:

(i) subject isolated material from the brain of Sus scrofa domestica to ultrasonic disruption for a period of between 10 and 25 minutes with a frequency between 10 to 30 KHz, between 25,000 to 30,000 rpm and a temperature below 6°C,

(i¡) deep-freeze the suspension obtained in (i) at a temperature below -60°C,

(iii) lyophilize the deep-frozen suspension obtained in (ii) for a time between 3 and 5 days until reaching a humidity percentage of less than 5%,

(iv) subjecting the lyophilized extract obtained in (iii) to cold grinding at a temperature below 6°C, and

(v) filtering the ground extract obtained in (iv) until obtaining an extract comprising a particle size below 700 pm.

The term "Sus scrofa domestica brain extract", as used herein and hereinafter the "extract of the invention", refers to a substance, product, compound, moiety, obtained from cellular material, tissue or organ, particularly Sus scrofa domestica (pig) brain obtained by a non-denaturing process, particularly by freezing, ultrasonic disruption and/or lyophilization, which has been named Nosustrophin or NSTF-1 , and characterized for being a nutraceutical agent with neuroprotective and antidegenerative activity in diseases such as Alzheimer's or Parkinson's and in neuroinflammatory processes.Particularly, Nosustrophin is an extract rich in cerebral fatty acids, neuroprotective vitamins, cerebral neurotransmitters, neurohormones and neurotrophic factors of the juvenile brain of Sus scrofa domestica.

In particular, the extract is obtained from the brain of Sus scrofa domestica, common name "pig". The pig (Sus scrofa domestica), also called pig, porcine, pig, pig or pig, is a subspecies of artiodactyl mammal of the Suidae family. It is a domestic animal used for human consumption. Its scientific name is Sus scrofa ssp. domestica, Sus domesticas or Sus domestica, used interchangeably throughout the present invention.

Particularly, in the present invention the extract is obtained from the juvenile brain of Sus scrofa domestica. Thus, in a preferred embodiment of the method of the invention, the brain of Sus scrofa domestica comes from animals younger than 9 months, preferably between 6 and 9 months of age.

The extract of the invention is obtained from any material isolated from the brain of Sus scrofa domestica, particularly from the juvenile brain of Sus scrofa domestica, such as, for example, cerebral hemispheres, cerebellum and brainstem (midbrain, pons or medulla). spinal).

In a preferred embodiment of the method of the invention, the material isolated from the brain of Sus scrofa domestica is tissue from the cerebral hemispheres, especially the cortical and subcortical structures of said cerebral hemispheres, more preferably to a depth of 1 to 2 cm.

Alternatively, the method of the invention may comprise a previous step, a preprocessing or cleaning step (a'), in which the material isolated from the brain of Sus scrofa domestica is washed and cleaned from impurities and residues, such as meninges, vessels or biological remains.

Preferably, the Sus scrofa domestica brain isolate is in a saline suspension or ultrapure distilled water.

The term "saline suspension" as used herein refers to a suspension, aqueous solution of salt in water compatible with living organisms due to its characteristics of osmoticity, pH and ionic strength, preferably the saline suspension of the method of the invention is sterile saline or sodium chloride (NaCl) solution.

Thus, in another preferred embodiment of the method of the invention, the material isolated from the brain of Sus scrofa domestica is found in a suspension or saline solution (NaCl) or ultrapure distilled water.

In another more preferred embodiment, the saline suspension is an aqueous solution of sodium chloride (NaCl), preferably where the aqueous NaCl solution is at 0.9% w/v.

Preferably, in the method of the invention the concentration of Sus scrofa domestica brain isolate in suspension, preferably in the saline suspension, is in a range between 1:1 and 1:15, preferably between 1:2 and 1:10 (mass of isolate: volume of suspension, for example, Kg:L), more preferably it is a concentration of 0.5 Kg of brain tissue per liter (L) of saline suspension (1:2).

Material isolated from the brain of Sus scrofa domestica is subjected to ultrasonic disruption. The term "ultrasonic disruption" or "ultrasonication" refers to the use of cavitation, ultrasound or ultrasonic waves, preferably in an aqueous medium, to carry out the cell disruption, i.e. to pierce, use and disrupt cell walls and cell membranes, so that intracellular material and biomolecules are released into the solvent, and thus extract bioactive compounds such as vitamins, polyphenols or natural pigments.

As previously described, in the method of the invention, the ultrasonic disruption is carried out for a time of between 10 to 25 minutes with a frequency of between 10 and 30 KHz, a power of between 25,000 to 30,000 rpm and a temperature below 6°C, preferably, the ultrasonic disruption is carried out between 25,000 to 30,000 rpm in intervals of 1 to 2 minutes to avoid heating the sample.

In another preferred embodiment of the procedure of the invention, the ultrasonic disruption of stage (i) is carried out at a frequency between 15 and 25 KHz.

In another more preferred embodiment, the ultrasonic disruption of stage (i) is carried out at a frequency that is selected from: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 KHz.

In another preferred embodiment of the procedure of the invention, the ultrasonic disruption of stage (i) is carried out for a time of between 12 and 22 minutes, more preferably between 15 and 20 minutes.

In another more preferred embodiment, the ultrasonic disruption of stage (i) is carried out for 15, 16, 17, 18, 19 and 20 minutes.

Once the isolated material from the brain of Sus scrofa domestica has been subjected to ultrasonic disruption, the obtained solution is deep-frozen at a temperature below -40°C, preferably below -60°C. Thus, in another preferred embodiment of the method of the invention, the deep freezing of stage (ii) is carried out at a temperature below -60°C.

In another more preferred embodiment, the deep freezing of step (ii) is carried out at a temperature below -70°C.

In another even more preferred embodiment, the deep-freezing temperature of step (ii) is between -90°C to -70°C, more preferably the temperature is -80°C.

In another preferred embodiment of the process of the invention, the temperature of deep freezing of step (iii) is -80°C.

Next, the deep-frozen suspension obtained in (ii) is subjected to a lyophilization process for 3 and 5 days until reaching a humidity percentage of less than 5%.

The term "lyophilization" as used herein refers to the process of preservation by dehydrofreezing, freeze-drying, or dehydration, that is, the process in which the aqueous solvent (water) of a frozen suspension is sublimed directly from the solid phase to the gas phase, bypassing the liquid state.

In another more preferred embodiment of the method of the invention, the deep-frozen suspension obtained in (ii) is subjected to a lyophilization process for 3 and 5 days until reaching a moisture percentage of less than 5%, preferably less than 4%, more preferably less than 3% or 2%. In another even more preferred embodiment, the lyophilization process is carried out until the sample reaches a moisture percentage of less than 1%, preferably less than 0.5%.

Particularly, the lyophilized extract obtained in the lyophilization step (iii) is subjected to grinding, preferably cold grinding at a temperature below 6°C. It is routine practice for the person skilled in the art to know the methods and procedures for grinding freeze-dried extracts.

Finally, the lyophilized and ground extract is filtered in step (v) of the method of the invention until obtaining an extract that has a particle size below 700 pm. Thus, in another preferred embodiment of the method of the invention, the extract obtained comprises a particle size below 700 pm.

In another more preferred embodiment of the method of the invention, the particle size of the extract obtained in the method of the invention is between 500 pm and 700 pm.

Filtering methods and procedures are routine practice for one skilled in the art. Particularly, in the method of the invention, the filtering of the extract is carried out by sequential sieving in a filter gradient. Thus, in another preferred embodiment of the method of the invention, the filtration of step (v) is carried out by sequential sieving in a filter gradient. By means of the method of the invention, an extract of the brain of Sus scrofa domestica with neuroprotective, anti-inflammatory and anti-degenerative activity in diseases such as Alzheimer's or Parkinson's is obtained. Therefore, another aspect of the present invention is an extract of the brain of Sus scrofa domestica obtained by the method of the invention, described above, hereinafter the "extract obtained by the method of the invention" or "Nosustrophin" used interchangeably herein.

The method of the invention has been previously described in the present description and applies equally to this aspect of the invention, as well as to any previous preferred embodiments (alone or in combination).

Particularly, the extract obtained by the method of the invention (or Nosustrophin) comprises the chemical and nutritional composition indicated in Table 1 of the Examples. Thus, in a particular embodiment, the extract obtained by the method of the invention comprises the composition described in Table 1.

Another aspect of the present invention refers to an extract of the brain of Sus scrofa domestica with neuroprotective and antidegenerative activity, hereinafter referred to as "the extract of the invention".

The term "Sus scrofa domestica brain extract" has already been described above and applies equally to this aspect of the invention, as well as to any previous preferred embodiments (alone or in combination).

In a particular embodiment of the extract of the invention, the brain of Sus scrofa domestica comes from animals between 6 and 9 months of age.

In another particular embodiment, the extract of the invention comprises the composition described in Table 1.

As the person skilled in the art knows well, the extract of the invention or the extract obtained by the method of the invention can be included in a composition. Therefore, another aspect of the present invention relates to a composition comprising the extract of the invention, hereinafter the "composition of the invention" herein.

In another preferred embodiment of the invention, the composition of the invention is a pharmaceutical composition or a nutritional composition.

In another preferred embodiment of the invention, the composition of the invention is a pharmaceutical composition. The term "pharmaceutical composition" refers to a product, solution, composition comprising the extract of the invention in any concentration, which has at least one application in improving the physical or physiological or psychological well-being of a subject, which implies an improvement in the general health of the state or a reduction in the risk of the disease. Said pharmaceutical composition may be a medicament. The term medicament has a more limited meaning than the meaning of "pharmaceutical composition" as defined in the present invention, since the medicament necessarily implies a preventive or therapeutic effect.

In another more preferred embodiment of the composition of the invention, the pharmaceutical composition comprises a therapeutically effective amount of the extract of the invention.

In the present invention, the term "therapeutically effective amount" refers to the amount of the component of the pharmaceutical composition which, when administered to a mammal, preferably a human, is sufficient to cause the prevention and/or treatment, as defined below, of a disease or pathological condition of interest in the mammal, preferably a human. The therapeutically effective amount will vary, for example, depending on the concentration of the extract of the invention, the form of presentation; the patient's age, body weight, general health, gender and diet; the mode and timing of administration; the rate of excretion, the drug combination, or the severity of the particular disease disorder or condition. One skilled in the art can determine the therapeutically effective amount based on his or her own knowledge and that disclosure.

In another preferred embodiment of the composition of the invention, the therapeutically effective amount is between 10 to 150 mg/Kg.

In another preferred embodiment of the composition of the invention, the pharmaceutical composition is formulated for administration in liquid or solid form.

The compositions of the present invention may be administered by oral, parenteral (eg, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant) routes of administration, preferably oral administration and intraperitoneally, and also by inhalation, nasal, vaginal, rectal, sublingual, buccal or topical spray and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles suitable for each route of administration. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, dragees, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs.

In another more preferred embodiment of the composition of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle or excipient and, optionally, another active ingredient.

The term "excipient" refers to a substance that helps to absorb any of the components of the composition of the invention, stabilizes said components or helps in the preparation of the composition in the sense of giving it consistency or, if necessary, providing flavors that make it more pleasant. Thus, the excipients could have the function of holding the components together, such as, for example, starches, sugars or celluloses, a sweetening function, a coloring function, the function of protecting the medicine, such as, for example, isolating it from air and/or moisture, a loading function for a tablet, capsule or any other form of formulation, such as, for example, dibasic calcium phosphate, a disintegration function to facilitate the dissolution of the components and their absorption in the intestine, without excluding go other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as any material included in the galenic forms that is added to the active principles or their associations to allow their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, to be compatible with said components. Examples of excipients are binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, and colorants. More specific, non-limiting examples of acceptable excipients are starches, sugars, xylitol, sorbitol, calcium phosphate, spheroidal fats, talc, silica or glycerin, among others.

The term "carrier" or "vehicle" refers to a compound that facilitates the incorporation of other compounds to allow for better dosing and administration or to give consistency and shape to the composition. Therefore, the carrier is a substance that is used to dilute any of the components of the composition of the present invention to a determined volume or weight, or even without diluting said components, capable of allowing better dosage and administration or giving consistency. Preferably the carrier is pharmaceutically acceptable.

In another preferred embodiment of the invention, the composition of the invention is a nutritional or food composition. The terms "nutritional composition", "nutraceutical" or "food composition", used interchangeably in the present invention, refer to that food or composition that, regardless of whether it provides nutrients to the subject that takes it, beneficially affects one or more functions of the organism, so as to provide better health and well-being. As a consequence, said nutritional composition can be used for the prevention and/or treatment of a disease or the factor causing a disease. Therefore, the term "nutritional composition" of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food.

Therefore, in another preferred embodiment of the invention, the nutritional composition is a food or a nutritional supplement.

As the person skilled in the art knows well, any food can be a nutritional composition. In another preferred embodiment of the invention, the nutritional composition is a food that is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, cereals, or beverages.

The terms "supplement", "dietary supplement", "nutritional supplement" or "food supplement", used interchangeably in the present invention, refer to a "food ingredient" intended to supplement the diet. Examples of dietary supplements include, without limitation, vitamins, minerals, plant substances, amino acids, and food components such as enzymes and glandular extracts. They are not presented as a substitute for a conventional food or as a sole component of a meal or diet, but rather as a dietary supplement. They may be presented as solids or liquids for ingestion or for topical (local) administration (such as in an aerosol formulation).

The present invention describes a brain extract of Sus scrota domestica for the treatment of neurodegenerative diseases, particularly Alzheimer's and Parkinson's. Therefore, this extract (the extract obtained by the method of the invention, the extract of the invention or the composition of the invention) can be used as a medicine.

Thus, another aspect of the present invention refers to the extract of the invention (or the extract obtained by the method of the invention) as well as the composition of the invention, for use as a medicine.

Alternatively, another aspect of the present invention refers to the use of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for the manufacture of a medicament.

The term "drug" as used herein refers to any substance or composition used for the prevention, diagnosis, alleviation, treatment, or cure of neurodegenerative, neuroinflammatory, or neurotoxic diseases. The administration should be adjusted to a desirable dose of the extract of the invention or the composition of the invention and varies depending on the condition and weight of the subject, the severity of the disease, the form of the drug, the route and the period of administration, and can be chosen by the person skilled in the art.

A preferred embodiment refers to the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for its use in the prevention and/or treatment of a neurodegenerative disease. Alternatively, in another preferred embodiment, the present invention refers to the use of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease.

The term "treatment" (or "treating") refers to processes that involve slowing, stopping, suspending, controlling, stopping, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily imply a total elimination of all symptoms related to the disease, conditions, or disorders associated with a neurodegenerative disease. Treatment of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (eg, without deterioration of symptoms) or a delay in progression of the disorder or disease (should the cessation of progression be only transient in nature) "Treatment" of a disorder or disease may also lead to a partial response (eg, improvement of symptoms) or a complete response (eg, disappearance of symptoms) of the subject/patient suffering from the disorder or disease.

As used herein, the term "prevention" refers to the inhibition or retardation of a neurodegenerative disease.

Another more preferred embodiment refers to the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for its use in the prevention and/or treatment of a neurodegenerative disease, or to the use of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease.

The term "neurodegenerative disease" used herein refers to diseases caused by neurodegeneration (eg, the progressive loss of neuronal structure or function, including neuronal death). Many neurodegenerative diseases (including Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease) occur as a result of neurodegenerative processes. Such diseases result in progressive degeneration and/or death of neurons. Examples of neurodegenerative diseases include, without limitation, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, multiple sclerosis (MS), or amyotrophic lateral sclerosis (ALS).

Another more preferred embodiment refers to the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for its use in the prevention and/or treatment of a neurodegenerative disease or to the use of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease, where the disease is Alzheimer's (AD), Parkinson's (PD), Huntington's disease, multiple sclerosis (MS) or amyotrophic lateral sclerosis (ALS).

Another even more preferred embodiment refers to the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for its use in the prevention and/or treatment of a neurodegenerative disease or to the use of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention, for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease, wherein the disease is Alzheimer's or Parkinson's.

Another aspect of the present invention relates to a method for preventing and/or treating a neurodegenerative disease in a subject, preferably a mammal, more preferably a human being, which comprises administering to the subject an effective amount of the extract of the invention (or the extract obtained by the method of the invention) or the composition of the invention. In a preferred embodiment of the method of prevention and/or treatment of a neurodegenerative disease, wherein the disease is Alzheimer's or Parkinson's.

DESCRIPTION OF THE FIGURES

Figure 1. Appearance of the lyophilized powder of Nosustrophin.

Figure 2. Pathological regulation of Ap plaques in brains of AD mice treated with Nosustrophin. Comparative images showing brain sections of mice with AD at the cortical and hippocampal level, from 4 to 12 months of age, showing histopathology using the p-amyloid marker. Images are presented by age group and treatment. A-F; Images showing incipient plaques immunoreactive to p-amyloid in the cortical and hippocampal regions of mice (Figures 2A, 2C and 2E) treated with nosustrophin, while control sections (Figures 2B, 2D and 2F) show numerous p-amyloid plaques with a compact morphology at different stages of evolution. Figure 2G; Cross section of the brain of wild-type (WT) mice treated with nosustrophin, showing no adverse histological effects. Figure 2H: Section of the brain of transgenic mice with AD and treated with normal diet, whose cortical sections are profusely marked by p-amyloid (Ap) immunoreactive plaques of large diameter and compact central nucleus. Scale bar: 100 //m.

Figure 3. Modulation of neuroinflammation (microglia) in the brains of mice with AD treated with Nosustrophin. Comparative images showing 4- to 12-month-old AD mouse brain sections labeled with CD11b (CD11 in figure) to detect neuroinflammation (microglia). Images are presented by age group and treatment. HF; Images showing the progressive accumulation of CD11b immunoreactive cells in AD transgenic mice, as an inflammatory response, mainly in the inner neocortical and hippocampal layers (Figures 3B, 3D and 3F). In contrast, AD transgenic mice treated with nosustrophin show a density significantly fewer CD11b immunoreactive cells in early and later stages of brain development (Figures 3A, 3C and 3E). Fig 3G; Image of brain section from wild-type (WT) mice, treated with nosustrophin, and showing no adverse histological effects. Fig 3H; Section of AD mouse brain showing numerous CD11b immunoreactive inflammatory cells in affected cortical areas. Scale bar: 100 µm.

Figure 4. Comparative images showing brain regions of 4- to 12-month-old AD mice labeled with GFAP for neuroinflammation (macroglia). Images are presented by age group and treatment. A-F; Images showing the progressive accumulation of reactive dystrophic astrocytes from the dentate gyrus (hippocampus) to the outer layers of the cortex. Notable contrast of astrocytosis densities between mice treated with nosustrophin (Figures 4A, 4C and 4E) and controls (Figures 4B, 4D and 4F), in which numerous dystrophic accumulations of immunoreactive GFAP are observed. Fig 4G; Brain section of wild-type (WT) mice treated with nosustrophin, and showing no adverse histological effects. Fig 4H; Section of AD mouse brain showing numerous clusters of GFAP-immunoreactive inflammatory cells in hippocampal brain areas. Scale bar: 100 µm.

Figure 5. Pathological regulation of neurodegeneration in the hippocampus of AD models treated with nosustrophin. Comparative immunofluorescence images showing brain sections of mice with AD at the hippocampal level (DG) and CA1 region of the hippocampus (CA1), showing histopathology through the NeuN biomarkers in neuronal maturation, Cox2 and IL17 in neuroinflammation. Images are presented by age group and treatment. The images that show immunoreactive variations to the different markers in the hippocampal regions of mice treated with nosustrophin, while the control sections (treated with PBS), show a higher density of neurodegenerative labeling. Cross section of the brain of 3-9 month old wild type mice treated with nosustrophin, showing a lower density of NeuN labeling although some inflammatory labeling (Cox2/IL17) is observed although it is circumscribed to the dentate gyrus of the hippocampus, compared to PBS-treated controls. Brain sections of transgenic mice for APP (Tg-APP) aged 3-4 and 8-9 months respectively, treated with nosustrophin, and showing the same neuroprotective pattern, with higher density of NeuN labeling and lower density of inflammation than in the respective controls.

Figure 6. Pathological regulation of neurodegeneration in the substantia nigra (SN), as well as in the ventral tegmental area (VTA or Ventral Tegmental Area), of EA models treated with nosustrophin. Comparative immunofluorescence images showing brain sections of APR-transgenic (Tg-APP) mice with AD at the basal ganglia (midbrain) level, showing biomarker immunoreactivity for dopaminergic (TH) cells and neuroinflammation (COX2). Images are presented by age group and treatment. The images show immunoreactive variations to both TH and COX2 in the area of the substantia nigra (SN) of mice treated with nosustrophin, while control sections (treated with PBS) show a higher number of dopaminergic labeling. Cross sections of the brain of 3-4 month old and 8-9 month old Tg-APP transgenic mice treated with nosustrophin, showing higher density of dopaminergic cells than controls treated with PBS. The group of 3-4 month old transgenic mice for APP treated with nosustrophin shows a lower rate of neurodegeneration than the 8-9 month old group, with the same treatment.

Figure 7. Pathological regulation of Aβ plaques (amyloid β) in the cortex or cerebral cortex (Ctx) and hippocampus (CA1) of AD models treated with nosustrophin. Comparative immunofluorescence images showing brain sections of mice with AD at the cortical and hippocampal level, showing histopathology using the p-amyloid marker. Images are presented by age group and treatment. Images showing incipient plaques immunoreactive to p-amyloid in the cortical and hippocampal regions of mice treated with nosustrophin, while control sections (treated with PBS) show a greater number of p-amyloid plaques. Cross section of the brain of 3-4 month old APP transgenic (Tg-APP) mice treated with nosustrophin, showing lower density of p-amyloid plaques than controls treated with PBS. Brain section of 8-9 month old APP transgenic mice treated with nosustrophin, showing less density of large-diameter p-amyloid-immunoreactive plaques and compact central core than controls.

Figure 8. Pathological regulation of neurodegeneration in basal ganglia of PD models treated with nosustrophin. Comparative immunofluorescence images showing PD mouse brain sections at the basal ganglia (midbrain) level, showing biomarker immunoreactivity for dopaminergic (TH) cells and neuroinflammation of astroglia (GFAP) and microglia (MLH1). Images are presented by treatment. The images show immunoreactive variations to both TH and GFAP and MLH1 in the ventral tegmental area of the basal ganglia (SN) of mice treated with nosustrophin (groups A, B and C), while control sections (treated only with MPTP), show a lower number of dopaminergic labeling (group D). AC groups; Cross sections of the brain of nosustrophin-treated EP mice show improved responses to neurodegeneration when nosustrophin treatment is administered before and after the neurotoxic induction of MPTP. Group C (pre-treated with nosustrophin) shows a higher density of dopaminergic cells than group B (post-treated with nosustrophin). Controls treated only with NSTF-1 (Group E) show complete density in the analyzed region, while group D (treated only with MPTP) shows notable dopaminergic degeneration, typical of Parkinson's pathology.

Figure 9. Regulation of neurodegeneration in the basal ganglia (SN and VTA) of PD models treated with nosustrophin. Comparative immunofluorescence images showing mouse brain sections with PD at the level of the basal ganglia (midbrain), expressing immunoreactivity through the biomarker for dopaminergic (TH) cells. Images are presented by treatment. The images show TH-immunoreactive variations in the substantia nigra (SN) area of nosustrophin-treated mice (groups G and B), whereas control sections without MPTP (saline-treated, group F; nosustrophin-treated, group A) show a higher density of dopaminergic labeling. Groups G and B; Cross sections of the brain of mice with PD induced by MPTP show significant differences, since the neuroprotective effect of pre-treatment with nosustrophin (group B) is more effective than post-treatment (group G), in terms of dopaminergic density in the analyzed area of the basal ganglia.

Figure 10. Regulation of degenerative neuroinflammation in the hippocampus of PD models treated with nosustrophin. Comparative immunofluorescence images showing brain sections of mice with PD at the hippocampal (DG) level, showing histopathology by biomarkers of astroglia (GFAP) and microglia (CD11b). Images are presented by treatment. The images show immunoreactive variations to the different markers in the hippocampal regions of mice treated with nosustrophin, compared with the positive control sections (treated with saline, Group F) that show a higher density of neuroprotective labeling and with those of the negative control (treated with MPTP, Group D). Groups A, B and G; Cross section of the brain of wild-type mice treated with nosustrophin (group A), showing a clear absence of inflammatory labeling of both GFAP and CD11b, while the MPTP group with post-nosustrophin treatment (group G) shows very high levels of inflammation, higher than the group with pre-treatment (group B). The results indicate a greater anti-inflammatory effect of nosustrophin pretreatment in the presence of MPTP (group B). Figure 11. Regulation of neuroinflammation in the basal ganglia (SN and VTA) of LPS models treated with nosustrophin. Comparative immunofluorescence images showing mouse brain sections with LPS at the level of the basal ganglia (midbrain), expressing immunoreactivity through the biomarker for dopaminergic (TH) cells. Images are presented by treatment. The images show TH immunoreactive variations in the substantia nigra (SN) area of nosustrophin-treated mice (groups H and C), while control sections without MPTP (saline-treated, group F, and nosustrophin-treated group, group A), show normal density of dopaminergic labeling. Groups H and C; Cross sections of the brain of mice with neuroinflammation induced by LPS show significant differences, since the neuroinflammation protective effect of pre-treatment with nosustrophin (group C) is more effective than post-treatment (group H), in terms of dopaminergic density in the analyzed area of the basal ganglia.

Figure 12. Regulation of degenerative neuroinflammation in the hippocampus of LPS models treated with nosustrophin. Comparative immunofluorescence images showing mouse brain sections with LPS at the hippocampal (DG) level, showing histopathology by biomarkers of astroglia (GFAP) and microglia (CD11b). Images are presented by treatment. The images show immunoreactive variations to the different markers in the hippocampal regions of mice treated with nosustrophin, compared with the sections of positive control (treated with saline, Group F) that show a higher density of neuroprotective labeling and with those of the negative control (treated with LPS, Group E). Groups A, C and H; Cross section of the brain of wild-type mice treated with nosustrophin (group A), showing a clear absence of inflammatory labeling of both GFAP and CD11b, while the LPS group with post-nosustrophin treatment (group H) exhibits very high levels of inflammation, higher than the group with pre-treatment (group C). The results indicate a greater anti-inflammatory effect of the nosustrophin pre-treatment in the presence of LPS (group C).

Figure 13. Cell viability in the SH-SY5Y line. SH-SY5Y cells were treated with different concentrations of Nosustrophin (0.05-10mg/ml) for 48h. Cell viability was determined with the PrestoBlue Cell Viability kit (Thermo Fisher). Eight replicates of each condition were performed and the results are expressed as mean ± standard error. For the statistical study, a one-way ANOVA was performed followed by the Bonferroni test. *p<0.05;**p<0.01;***p<0.001. Figure 14. Cell viability in the HepG2 line. HepG2 cells were treated with different concentrations of Nosustrophin (0.01-10mg/ml) for 48h. Cell viability was determined with the PrestoBlue Cell Viability kit (Thermo Fisher). Eight replicates of each condition were performed and the results are expressed as mean ± standard error. For the statistical study, a one-way ANOVA was performed followed by the Bonferronl test. *p<0.05;**p<0.01;***p<0.001.

Figure 15. Neurotrophic effect of Nosustrophin (serum BDNF - hippocampal BDNF). TO). In the control group treated with saline, we observed an increase in the serum levels of BDNF (pg/ml) in the transgenic mice (EA model), especially in the group of older mice (EA 8-9 m). The Alzheimer model mice treated with 150 mg/kg of Nosustrophin present significantly lower levels of BDNF compared to those treated with saline solution in the two age ranges studied (EA 3-4 m / EA 8-9 m), approaching values of the group of control mice (WT). No significant differences were observed in the group of wild-type mice (WT) according to the treatment. B). In the control group treated with saline, we observed a slight increase in BDNF levels (pg/mg protein) in the hippocampus of transgenic mice (EA model) of 3-4 months of age. Alzheimer's model mice of the same age group (3-4 months) treated with Nosustrophin have significantly lower levels of hippocampal BDNF compared to those treated with saline. In general, the same trend is observed in the three groups of mice treated with Nosustrophin (WT 3-9 m; EA 3-4 m; EA 8-9 m), with higher intensity in transgenic mice. The behavior of BDNF according to the treatment is similar to that found in serum (A). A) *p<0.05 vs EA 3-4m (saline); **p<0.01 vs EA 8-9m (saline). Error bar: +/- 2 SE, B) **p<0.01 vs EA 3-4m (saline). Error bar: +/- 2 SE, 95% Cl

Figure 16. Anti-inflammatory capacity of Nosustrophin. In the group treated with saline, we observed higher levels of CRP (mg/l) in the 3-4 month old Alzheimer (AD) model mice showing a higher degree of inflammation than the control group made up of healthy wild type (WT) mice. The 3-4 month old EA mice treated with Nosustrophin present significantly lower levels compared to the same group of mice (3-4 m EA) treated with saline. The rest of the groups (WT 3-9 m; EA 8-9 m) did not present significant differences based on the treatment. The results indicate an important anti-inflammatory power of Nosustrophin in the group of young transgenic mice. **p<0.01 vs EA 3-4m (saline). Error bar: +/- 2 SE, 95% Cl Figure 17. Antioxidant power of Nosustrophin. In the group treated with saline (control group) we observed lower levels of TAS (pmol/l) (Fig 17A) in the older EA model mice (8-9 m) and of GR (U/l) (Fig 17B) in the younger group (EA 3-4 m) compared to the WT group (healthy wild type mice). Both classic antioxidation biomarkers are significantly increased in the group treated with Nosustrophin, especially in those groups that present greater oxidative stress than the control group (saline). The results show an important antioxidant power of the extract. A) ***p<0.001 vs EA 8-9m (saline). Error bar: 95% CI, B) *p<0.05 vs WT 3-9m (saline); **p<0.01 vs EA 3-4m (saline). Error bar: 95% CI

Figure 18. Renal toxicity of Nosustrophin. Serum creatinine concentration (mg/dl) in the groups of transgenic WT and EA model mice treated with saline (control group) and Nosustrophin. A decrease in creatinine levels is observed in all groups treated with Nosustrophin, being statistically significant for the group of healthy wild-type mice (WT). The results show safety of the extract at the renal level.

Figure 19. Hepatic toxicity of Nosustrophin. Serum levels of GOT and GPT liver enzymes (Ul/I) in the groups of WT and EA model mice treated with saline (control group) and Nosustrophin. In both, a decrease in activity is observed in all groups treated with Nosustrophin. The results show safety of the extract at the hepatic level. Error bars: 95% CI

EXAMPLES

Next, the invention will be illustrated by means of tests carried out by the inventors, which show the effectiveness of the product of the invention.

Nosustrophin extraction process

Nosustrophin extract (NSTF-1) is obtained from the brain of Sus scrota domestica through a non-denaturing process that includes the following steps:

(1) Physical removal of the brain from animals between the ages of 6 and 9 months.

(2) Preprocessing: Cleaning of impurities and residues (meninges, vessels, biological remains) (manual process and washing). (3) Ultrasonic disruption of brain tissue in saline (250 g brain tissue in 0.5 L saline) with a sonicator (20 KHz) for 10 to 25 minutes (keeping temperature below 6°C) for disruption of brain cell walls and their intracytoplasmic components.

(4) Deep freezing at -80°C.

(5) Industrial freeze-drying process (between 3 to 5 days).

(6) Grinding of the lyophilized extract under thermal conditions (< 6°C).

(7) Filtering of the ground extract by sequential sieving in a filter gradient until obtaining a particle size of less than 700 pm, preferably between 500 and 700 pm.

In addition, the inventors carried out a microbiological analysis and characterization of the properties of the NSTF-1 extract. Figure 1 represents the ultrapure extract of Nosustrophin (NSTF-1) obtained by the previously described procedure; a yellowish-white powder with a crystalline appearance with a particle size between 500-700 pm.

Analysis of the Composition of Nosustrophin

The chemical and nutritional analysis of the lyophilized extract of Nosustrophin (NSTF-1) reveals the composition indicated in Table 1.

Table 1. Analysis of the Composition of Nosustrophin

In addition, the extract has a moisture content of 2.8% (measured by gravimetry) and an energy value (EU) of 512 kCal/100g and 2130 kJ/100g. A. IN VITRO AND IN VIVO CELLULAR, MICROSCOPIC, IMMUNOHISTOCHEMICAL AND BEHAVIORAL STUDIES

A.1. MATERIALS AND METHODS

Treatment

Nosustrophin (NSTF-1) was integrated into the diet as pellets, adding 50% nosustrophin powder and completing with dietary feed and adding 10% purified water (milli-Q) to mix and mold, leaving the pellets to dry at room temperature (between 22 to 25°C) for 12h. The control is the intake of feed pellets without any external additive. The nutrient composition of the treatment diet is shown in Table 2. Nosustrophin was inoculated intraperitoneally (ip) to mice, once daily, at concentrations of 150 mg/kg, diluted in sterile saline (0.9% NaCI). Inoculation of vehicle only (PBS) represents the control.

Table 2. Nutritional composition of the pellet diet/treatment per 100 g.

Primary cultures of cortical neurons

Before starting the dissociation, the culture plates had to be treated with poly-lysine to improve the adhesion of the cells to their surface. The cells were obtained from Wistar rat fetuses, 17 days of gestation. For this, the rat was sacrificed by decapitation and the fetuses were extracted. They were _washed in washing buffer (150mM NaCl; _8mM Na2HPO4.2H2O; 2.7mM KCI; 1.45mM KH2PCL; 2.6mM NaHCOs _; pH=7.2) and transferred to a plate containing commercial dissection medium (L-15), where the cerebral cortices were obtained. Once the cortex is isolated, it is essential to remove the meninges before homogenizing. The clean skins were transferred to the plate containing the incubation medium (80% (v/v) MEM (Minimum Essential Medium), 10% (v/v) horse serum, 10% (v/v) fetal serum, 1.98 mM glutamine, 3.3 mM glucose and 16 mg/L gentamicin sulfate). The tissue disintegrated and mechanically homogenize. After homogenization, the number of cells obtained by the Trypon Blue exclusion method was counted in a Neubauer chamber and suspended in "incubation medium" up to a density of 2x10 ⁵ cells/cm ² . They were seeded in 12-unit multi-well Petri dishes (0=2.2cm).

During the first four days, the cells were kept in the incubation medium. On the fourth day, it was changed to a growth medium (90% (v/v) MEM, 10% (v/v) horse serum, 1.98 mM glutamine, 3.3 mM glucose and 16 mg/L gentamicin sulfate), which also contained a cytostatic agent, cytosine arabinoside (10 ^-5 M), to prevent the growth of cells that proliferate such as glia. A week after they were planted, the medium was changed again to put “growth medium” again, but this time without cytosine. With this treatment, a mostly neuronal culture was obtained, although with approximately 5% of the glial population.

Primary glial cell culture

To obtain a culture with a mainly glial population, the seeding density was 5x10 ⁴ cells/cm ² and no cytosine arabinoside was added on the fourth day after seeding. In this way, the proliferation and growth of the glia was allowed. The percentage of each cell population was: 15 ± 3% neurons, 75 + 8% astrocytes and 10 ± 2% microglia. In addition, the experiments were carried out two weeks after sowing.

Triple transgenic model of Alzheimer's (EB/3xTg-AD)

To perform the experiment, EB/3*Tg-AD (APP/Bin1/Cops5) mice were generated and used. This triple transgenic mouse model of AD, which overexpresses the Swedish mutation of APP (human amyloid precursor protein) along with BIN1 (bridge integrator 1, AMPH2) and COPS5 (COP9 constitutive photomorphogenic homolog subunit 5, Jabí), mimics the pathology of the human brain. The sequence was verified before rearing the transgenic colony. Transgenic mice were identified by PCR analysis of tail DNA.

The Cops5 cDNA was amplified by PCR using the following primers: forward, 5'-CGGAATTCATGGCGGCGTCCGGGAGCGGT-3' (SEQ ID NO: 1) and reverse: 5'-GCGTCGACTTAAGAGATGTTAATTTGATT-3' (SEQ ID NO: 2). The Bin1 cDNA was amplified by PCR using the following primers: forward, 5'-

CGGAATTCATGGCGGCGTCCGGGAGCGGT-3' (SEQ ID NO: 3) and reverse, 5'- GCGTCGACTTAAGAGATGTTAATTTGATT-3' (SEQ ID NO: 4), while the cDNA of the application was amplified by PCR using the following primers: forward, 5'-CGGAATTCATGGCGGCGTCCGGGAGCGGT-3' (SEQ ID NO: 5) and reverse, 5'-GCGTCGACTTAAGAGATGTTAATTTGATT-3' (SEQ ID NO: 6).

Mice were generated by crossing the heterozygous 1903 line of Cops5-Tg mice with the heterozygous Bin1-Tg and APPswe transgenic mice. Microinjection of cloned cDNA into the blastocyst was carried out at the central transgenic facility of the Sylvester Comprehensive Cancer Center (University of Miami) using standard techniques and strictly following animal use protocols approved by the Animal Care and Use Committee at the Torrey Pines Institute of Molecular Studies and in accordance with the guidelines of the US and Spanish National Institutes of Health.

Both the transgenic mice and the control wild mice were kept under standard animal facility conditions (temperature of 22 ± 2 °C and relative humidity of 55 ± 5%), with a 12-h light and dark cycle. Food and water were provided ad libitum. At the end of the experimental period, all the animals were sacrificed and the different samples were obtained for the various histopathological analyzes under study. All the experimental procedures with mice were adjusted to the guidelines established by the Directive of the Council of the European Communities (86/609/EEC), the EU Directive 2010/63/UE and the Royal Decree 1201/2005 of Spain for animal experimentation and were approved by the Ethics Committee of the EuroEspes Biomedical Research Center (Permission number: EE / 2017-110).

Simple transgenic model of Alzheimer's (APP)

To carry out the experiment, transgenic mice (TgAPP) for Alzheimer's disease, created at the EuroEspes Biomedical Research Center (Carrera¡, Etcheverria I, Fernandez-Novoa L, Lombardi VR, Lakshmana MK, Cacabelos R, Vigo C. A Comparative Evaluation of a Novel Vaccine in APP/PS1 Mouse Models of Alzheimer's Disease. Biomed Res In, were used. t.2015;2015:807146). These Tg-B6C3F1/J (APPswe) mice express a chimeric human/mouse amyloid precursor protein (Mo/HuAPP695swe) directed toward central nervous system (CNS) neurons, thus exhibiting Ap plaques in the hippocampus and cerebral cortex that begin to be expressed at 6 months of age (Jackson Laboratory, Bar Harbor, ME). Both the transgenic mice and the control wild mice were kept under standard animal facility conditions (temperature of 22 ± 2 °C and relative humidity of 55 ± 5%), with a 12-h light and dark cycle. Food and water were provided ad libitum. At the end of the experimental period, all the animals were sacrificed and the different samples were obtained for the various histopathological analyzes under study. All the experimental procedures with mice were adjusted to the guidelines established by the Directive of the Council of the European Communities (86/609/EEC), the EU Directive 2010/63/UE and the Royal Decree 1201/2005 of Spain for animal experimentation and were approved by the Ethics Committee of the EuroEspes Biomedical Research Center (Permission number: EE / 2019-16).

Chemically Induced Model of Parkinson's (MPTP)

For the experiments and to reproduce the neuropathology characteristics of Parkinson's disease, the neurotoxic agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPTP) was administered. Three days prior to MPTP treatment, wild-type laboratory mice (C57BL6/J 10 weeks) were administered either the treatment vehicle or an equal volume of saline intraperitoneally (ip). To chemically generate the Parkinson's model, mice were injected with MPTP (dissolved in 0.9% saline, 30 mg/kg,) for 5 consecutive days, while the control group received an equal volume of normal saline (ip). Behavioral assessments were conducted during four key periods of treatment using the OUCEM (Osaka University Computerized Electronic Maze) learning test and the Rotarod motor coordination test. Behavioral assessments were performed during two key treatment periods using the Rotarod motor coordination test. At the end of the experimental period, all the animals were sacrificed and the different samples were obtained for the various histopathological analyzes under study.

Chemically Induced Model of Neuroinflammation (LPS)

To carry out the experiment and reproduce the neuropathology characteristics of brain neuroinflammation, lipopolysaccharide (LPS) was administered. Three days prior to LPS treatment, laboratory wild type mice (C57BL6/J) were administered either the treatment vehicle or an equal volume of saline intraperitoneally (ip). To chemically generate the neuroinflammation model, mice were injected with LPS (dissolved in 0.9% saline, 0.7 mg/kg,) for 5 consecutive days, while the control group received an equal volume of normal saline (ip). During two key periods of treatment, behavioral assessments were performed using the Rotarod motor coordination test. At the end of the four weeks of experimentation, all the animals were sacrificed and the different samples were obtained for the various histopathological analyzes under study. In vivo (Animated models): Neurodegeneration (MPTP) and Neuroinflammation (LPS)

Triple transgenic model of Alzheimer's (EB/3xTg-AD)

To carry out the experiment, the different groups were experimentally conformed with this triple transgenic mouse model (Bin1/Cops5/APP), generated by means of a specific crossing strategy within the colony to obtain specimens at different stages of development. Thus, triple transgenic mice were generated and sacrificed at 4, 8, and 12 months of age, along with wild-type mice (C57BL/6) used as control groups. The EB/3*Tg-AD mice, once generated and genotyped by PCR, were randomly divided into 4 experimental groups by age, as follows:

Group treated for 14 days with nosustrophin (n=3) (1.5 grams of nosustrophin per 5g of food), 3xTgAD transgenic mice of 4 months of age and 170.3g (grams ingested of total food, feed + nosustrophin);

Group treated for 14 days with nosustrophin (n=4) 3xTgAD transgenic mice of 8 months of age and 245.7 grams ingested;

Nosustrophin-treated group (n=3), 12-month-old 3xTgAD transgenic mice and 253 grams ingested;

Group treated for 14 days with normal feed (n=3, 3xTgAD, 4 months/ n=3, 3xTgAD, 8 months/ n=3, 3xTgAD, 12 months) and 273.5 grams ingested;

Group of wild type mice treated with nosustrophin alone for half of the experimental period (n=4, 3xTgAD, 8 months of age) for 14 and 28 days, with 135.5 and 202.2 grams ingested, respectively.

Mice were housed in an acclimatized room with controlled temperature (20-21°C), humidity (40-50%), and lighting (12-h light/dark cycle) and were supplied with water ad libitum. The diet/treatment regimen was carried out for four weeks.

Simple transgenic model of Alzheimer's (A P P)

To carry out the experiment, the different groups were experimentally conformed to the simple transgenic mouse model (APPswe), and its execution was planned through a specific crossing strategy within the colony to obtain specimens at different stages of development. Thus, triple transgenic mice were generated and sacrificed at 4, 8 and 12 months of age together with wild-type mice (C57BL/6) used as control groups. The Tg-APP or EB/1 *Tg-AD mice, once generated and genotyped by PCR, were randomly divided into 4 transgenic groups and 2 experimental controls by age according to Table 3. The mice were housed in a room with controlled temperature (20-21°C), humidity (40-50%) and lighting (12-h light/dark cycle) and were supplied with water ad libitum. The treatment regimen was carried out for four weeks.

Table 3. Experimental protocol Intraperitoneal (ip) nosustrophin - tg-APP (EA) mice

Chemically Induced Model of Parkinson's (MPTP)

To carry out the experiment (nosustrophin in pellets), the different groups were experimentally conformed with the chemically induced mouse model (MPTP), and specimens were generated at the same stage of development (10 weeks), along with wild-type mice (C57BL/6) used as control groups. Mice were randomly divided into 5 experimental groups, each consisting of 8 10-week-old mice (4 MPTP mice and 4 wild-type), according to the type of treatment: Group A, ip administration of MPTP and nosutrophin pellets before and after chemically induced neurodegeneration; Group B, MPTP and nosustrophin in food after chemically induced neurodegeneration; Group C, MPTP and nosustrophin in food before chemically induced neurodegeneration; Group D, MPTP and normal food before and after chemically induced neurodegeneration; Group E, Pellets with nosustrophin (control). Mice were housed in a room with controlled temperature (20-21°C), humidity (40-50%), and lighting (12-h light/dark cycle) and were supplied with water. ad libitum. The diet/treatment regimen was carried out for ten days.

To carry out the experiment (ip-inoculated nosutrophin), the different groups were experimentally conformed with the chemically induced mouse model (MPTP), and specimens were generated at the same stage of development (10-14 weeks), along with wild-type mice (C57BL/6) used as control groups. The mice were randomly divided into 4 experimental groups, each consisting of 4 mice, according to the type of treatment: Group A, ip administration of nosustrophin; Group B, ip administration of nosustrophin (pretreatment) and ip of MPTP afterwards; Group D, IP administration of MPTP (control); Group G, ip administration of MPTP and ip nosustrophin (post-treatment) later; Group F, saline ip administration (control vehicle). Mice were housed in a room with controlled temperature (20-21'C), humidity (40-50%) and lighting (12-h light/dark cycle) and were supplied with water ad libitum. The diet/treatment regimen was carried out for three weeks.

Chemically Induced Model of Neuroinflammation (LPS)

To carry out the experiment, the different groups were conformed experimentally with the chemically induced mouse model (LPS), and specimens were generated at the same stage of development (10-14 weeks), together with wild-type mice (C57BL/6) used as control groups. The mice were randomly divided into 4 experimental groups, each consisting of 4 mice, according to the type of treatment: Group A, ip administration of nosustrophin (control); Group C, ip administration of nosustrophin (pretreatment) and ip LPS later; Group E, ip administration of LPS (control); Group F, saline ip administration (control vehicle); Group H, ip administration of LPS and ip nosustrophin (post-treatment) later. Mice were housed in a room with controlled temperature (20-21.0), humidity (40-50%) and lighting (12-h light/dark cycle) and were supplied with water ad libitum. The diet/treatment regimen was carried out for three weeks.

In vivo immunohistochemistry

Neuropathological biomarkers were analyzed using routine immunohistochemical techniques. Briefly, parallel cross sections (12-14pm) of the left half of the brain were obtained by cryostat and pre-treated with H2O2 and incubated overnight with the specific primary antibodies, as described in Table 4. Sections were successively washed in TBS-T, incubated in goat anti-rat IgG antibody (Millipore), or goat anti-mouse IgG antibody (Sigma), depending on the primary antibody, for 1 hour, and finally, they were incubated for 30 minutes using the ABC kit detection (Vectastain; Vector). Biomarker expression was revealed by incubating sections with 3,3-diaminobenzidine (Sigma) as chromogen and hydrogen peroxide as oxidant. Sections were mounted with a coverslip with Eukitt sealant (Sigma). In several adjacent sections, negative controls performed omitting primary, secondary, or tertiary antibodies showed no immunostaining. Observed neuropathological markers were digitally quantified along with behavioral test results.

Table 4. Antibodies used in immunohistological techniques.

In vivo immunofluorescence

Neuropathological biomarkers were also analyzed using indirect immunofluorescence methods. Briefly, they were pre-treated twice in 0.05 M Trizma Buffered Saline (TBS) and then incubated overnight (+4°C) with the primary antibodies in Donkey Normal Serum (Table 5). Sections were washed successively in TBS-T, incubated in mouse anti-mouse IgG (Millipore), rabbit anti-mouse IgG (Millipore) or rat anti-mouse IgG (Millipore) secondary antibodies, depending on the primary antibody, for 1 h, rewashed in TBS-T, and then incubated for 10 min with DAPI (Invitrogen). After successive washings in TBS-T, the sections were protected with a coverslip and mounted with "anti-fadincf" medium (Vectastain; Invitrogen). The expression of the biomarkers was revealed digitally by fluorescence microscopy (Leica DM6-B) activating the “confocal Thunder” function. In several adjacent sections, negative controls performed omitting primary or secondary antibodies did not show immunofluorescence.

Acquisition and treatment of images

Images were digitally visualized using fluorescence microscopy (Leica DM6-B) activating the Thunder confocal function. The brightness and contrast of the photographs were adjusted with Corel Photo-Paint (Corel 11, Ottawa, Canada) and the images were composited with Corel Draw. The immunopositive quantifications of the different histochemical and fluorescent biomarkers were performed using the Excavator 4.0 software individually in each section analyzed.

Statistic analysis

In Vitro, all the values were expressed as mean + SEM of the number of experiments indicated in each case, p<0.05 is considered significant (Newman-Keuls test). In vivo, the statistical parameters of the results obtained were analyzed with the SPSS v. 11.0 (SPSS, Inc., Chicago, IL, USA). Differences between treated groups were compared using the Kruskal-Wallis test followed by the Mann-Whitney U test for non-parametric data, while normally distributed data were tested using one-way analysis of variance. The Bonferroni correction was used to avoid false positives, while the Games-Howell was used to compare combinations of treatment groups. Data are expressed as standard error of the mean (±SEM). In the graphs, statistically significant differences are indicated below (*p<0.05 or **p<0.01).

A.2. RESULTS

Nosustrophin plays a critical role in the neuronal development of the brain of mice with Alzheimer's (AD), Parkinson's (PD) and neuroinflammation-type pathologies.

Nosustrophin inhibits progressive neuropathological deterioration in mice

The main degenerative effect of AD is the irreversible neuronal deterioration triggering processes of activation of apoptotic markers and necrosis, culminating in massive neuronal death. Therefore, in this study we have analyzed whether Nosustrophin ingested through pellets reduces the apoptotic process, keeping neuronal density stable in the regions affected by β-amyloid plaques. For this, the average density was quantified of p-amyloid plaques and adjacent neuronal structure in all groups of treated mice. The results show the anti-degenerative effect of treatment with Nosustrophin, very evident in the affected regions (cortex and hippocampus), where less affected area and better peripheral neuropathological structure are observed (Figure 2).

In the experiment with animal models of AD, treated with Nosustrophin by intraperitoneal injection, the samples were processed for the quantification of p-amyloid plaques by immunofluorescence. The neurodegenerative pathological process of AD endogenously induced by the transgenic APP gene in 3-4 month old mice resulted in 27% of p-amyloid plaques treated with PBS in contrast to only 11% plaques in the Nosustrophin-treated group, while in 8-9 month old APP mice, 35% plaques were observed in PBS-treated controls versus 19% in Nosustrophin-treated mice (Figure 7). ). Thus, in the APP mouse models of AD treated with Nosustrophin, a significant double effect has been observed, both neurotrophic and neuroprotective, resulting in a better neuronal organization of the affected areas and a lower density of p-amyloid plaques, respectively. It is worth noting the maintenance of neuronal fibers and tracts in the affected areas (cortex and hippocampus).

In addition, the effect of Nosustrophin on neuropathological inhibition was quantified through the dopaminergic biomarker tyrosine hydroxylase (TH). Animal models treated with Nosustrophin by intraperitoneal injection in AD, PD or neuroinflammation models, as well as those treated with Nosustrophin pellets, showed high rates of immunoreactivity to TH compared to controls. Thus, in 3-4 month old APP mice, a 68% dopaminergic density was observed compared to 32% in control treatment with PBS, while in 8-9 month old APP mice, the samples indicated 47% versus 24% of dopaminergic cells in controls (Figure 6). The neurodegenerative pathological process of PD induced by exogenously MPTP in mice resulted in an inhibition of dopaminergic impairment in the basal ganglia, mainly when Nosustrophin treatment is administered by ingestion before and after the neurotoxicant. When Nosustrophin treatment is administered intraperitoneally, TH-ir densities of 55% post-treatment and 78% pre-treatment are obtained by immunofluorescence, versus 36% TH-ir cell density in untreated MPTP controls (Figure 9).

In the experiment with LPS-induced neuroinflammatory models, the same pattern was also observed, in which the density of dopaminergic cells in the groups with post- treatment (58%) and pre-treatment (71%) contrast markedly with no-treatment controls (46%) (Figure 11).

Nosustrophin inhibits massive neuroinflammatory activation in models of AD, PD and Neuroinflammation

The mouse models of neurodegeneration (EA, PD and LPS) suffer a severe reactivation of astrocytes in the cerebral cortex, hippocampus and basal ganglia as a direct effect of the associated neuropathological lesions, triggering the activation of an acute neuroinflammatory process. Therefore, this study has also analyzed whether Nosustrophin inhibits the acute activation of astroglia and microglia in these areas, to improve survival and neuronal protection. For this, immunohistochemical tests were performed using specific antibodies against astroglia (GFAP), microglia (CD11b), tissue inflammation (COX-2, MLH-1) and lymphocytes (IL-17), in all experimental groups of mice.

In experiment 1, in which the effect of Nosustrophin ingested in pellets on the inflammatory index of AD animal models was studied, it was observed that the groups treated with Nosustrophin presented a significant reduction in immunoreactive labeling for the inflammatory biomarkers CD11b (Figure 3) and GFAP (Figure 4), compared to the control group without Nosustrophin. There is a correlation between the accumulation of inflammatory labeling and p-amyloid plaques, but a massive inflammatory activation is not observed as in the controls. Similar results were obtained in the experiment with animal models of AD, treated with Nosustrophin by intraperitoneal injection.

The neurodegenerative pathological process of AD endogenously induced by the transgenic APP gene in mice resulted in a notable inflammatory effect, revealed by the COX-2 and IL-17 markers, both at the cortical (CA1) and hippocampal (DG) levels in animals treated with PBS, contrasting with a less inflammatory effect by the administration of Nosutrophin, both in 3-4 month old and 8-9 month old mice. This pattern was confirmed in the region of the basal ganglia (Figure 5).

The analysis of the effect of ingesting Nosustrophin in pellets on the Parkinson's model was carried out in different groups of MPTP mice and under different treatments. Inflammatory activation indices were analyzed by immunohistochemical labeling of astroglia biomarkers (GFAP), tissue inflammation (MLH1), in all experimental groups. It was observed that the administration of Nosustrophin markedly reduces the density of astrogliosis and the activated inflammation in the treated mice (groups AC), The group treated with Nosustrophin before and after chemical induction by MPTP (group A) stands out for its low level of inflammatory labeling (Figure 8).

On the other hand, the analysis of the effect of intraperitoneal administration of Nosustrophin (Figure 10), revealed by the immunofluorescence biomarkers GFAP and CD11b, shows the same pattern, in which inflammation is significantly reduced in the groups treated with Nosustrophin, mainly when it is administered preventively (Group B).

Regarding the effect of Nosustrophin in animal models of neuroinflammation induced by LPS (Figure 12), the inflammation indices shown by the biomarkers of astroglia (GFAP) and microglia (CD11b) in the hippocampal region, indicate a significant anti-inflammatory effect of Nosustrophin, mainly when it is administered as a pre-treatment of LPS (group C). The anti-inflammatory effect is notable when comparing the groups treated with Nosustrophin (groups H and C) with the control without Nosustrophin (group E).

A.3. CONCLUSIONS

The results obtained from the preclinical experimental analysis of Nosustrophin in transgenic models of Alzheimer's (IxTgAPP and 3xTgAPP/Bin1/Cops5), the results obtained from the preclinical experimental analysis of Nosustrophin in chemically induced Parkinson's models (MPTP), as well as the results of the preclinical experimental analysis of Nosustrophin in chemically induced models of neuroinflammation (LPS), have been validated at the neurohistological level, proving to be neuro active against the effects of cognitive impairment and neuropathological degeneration. These results indicate four major effects of treatment with Nosustrophin: a) neurotrophic activity, maintaining normal levels of neurogenesis and neuronal plasticity in the affected regions (cortical and hippocampal areas, basal ganglia areas), b) anti-neuroinflammatory activity, inhibiting the massive inflammatory activation associated with each pathology, c) anti-neurodegenerative activity, reducing the development of new p-amyloid plaques, and preserving both the density of dopaminergic cells in the affected regions. sensitive areas (SN) such as motor and spatial coordination indices, and d) neuroprotective activity, inhibiting the neurodegenerative factors involved in the neuronal destruction of the affected areas, such as apoptosis, inflammation, neuropathological plaques, etc.

B. CELL FEASIBILITY STUDIES

B.1. MATERIALS AND METHODS

Cell culture

Neuroblastoma (SH-SY5Y) and hepatocarcinoma (HepG2) cell lines were used. The SH-SY5Y line has been widely used in neurodegeneration models. The HepG2 line was used as the hepatic line, to analyze the toxicity of Nosustrophin in the liver, responsible for the metabolization of the drugs. The SH-SY5Y line was grown in Roswell Park Memorial Institute (RPMI) medium supplemented with fetal calf serum (10%). The HepG2 line was grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with fetal bovine serum (10%). Cells were grown in an incubator at 37°C and 5% CO ₂ .

Cell viability analysis

10x10 ³ SH-SY6Y cells or 6x 10 ³ HepG2 cells were seeded in a 96-well plate. After 24 hours, it was treated with increasing concentrations of Nosustrophin (0.01 mg/mL-10 mg/mL) and after 48 hours, cell viability was measured with the commercial PrestoBlue Reagent kit (Invitrogen) following the instructions of the commercial company. Briefly, 90 pL of medium and 10 pL of reagent were added to each well. After a 1h incubation in the incubator, the absorbance was read at 570 nm. The absorbance reading at 600 nm was used as reference. Absorbance reading is performed with a plate reader (Epoch, BioTek Instruments).

Statistic analysis

The statistical analysis of the results is carried out with the Pism 6 program. Statistical significance was analyzed using a one-way ANOVA test, followed by the Bonferroni test. The analysis of normal distribution and the homogeneity of variances were made with the Shapiro-Wilks and Levene tests, respectively. Data are expressed as the mean ± standard error. P<0.05 (*), p<0.01(**) and p<0.001 (***).

B.2. RESULTS

First, cell viability was analyzed in the human neuroblastoma line SH- SY5Y, after treatment with increasing concentrations of Nosustrophin. Thus, it was observed that the treatment produces a significant increase in cell viability. This increase already occurs with the 0.05 mg/mL dose but reaches its maximum from the 0.5 mg/mL dose, increasing viability by approximately 50% (Figure 13).

To analyze the effects of treatment with Nosustrophin in the liver, the organ responsible for drug metabolism, cell viability assays were performed on the human HepG2 cell line. It was observed that treatment with Nosustrophin not only does not cause liver damage, but also increases cell viability in this cell model. The increase is detected from the dose of 1 mg/mL and from this concentration the increase is 25-50% (Figure 14).

On the other hand, these trials have helped to determine the appropriate dose of Nosustrophin treatment for trials in animal models (dose range: 10-150 mg/Kg). Therefore, treatment with Nosustrophin increases cell viability in cell models of neuroblastoma and hepatocellular carcinoma.

C. BIOCHEMICAL AND METABOLIC STUDIES

C.1. MATERIALS AND METHODS

The study was carried out in wild type mice and triple-EA transgenic mice (3xTgAPP/Bin1/COPS5), both between 3 to 9 months old (N=33), to which saline solution and Nosustrophin (150mg/kg) were administered.

Blood collection was performed by cardiac puncture after deep anesthesia of the mouse immediately before sacrifice. Commercial Greiner Bio-One brand tubes without anticoagulant were used. These tubes were centrifuged at 4000 rpm for 10 minutes to separate the serum from the cell layer, after allowing them to clot for 20 minutes in an upright position after extraction. The serum was stored at -80°C until the analysis of the following parameters: albumin, calcium, phosphorous, magnesium, iron, creatinine, GOT/GPT, total antioxidant capacity (TAS), glutathione reductase by UV-visible spectrophotometry, and C-reactive protein (CRP) by turbidimetric technique, all of them in the Cobas Mira Plus analyzer (Roche Diagnostics, Basel, Switzerland). Vitamins B6 and B9 were measured using commercial ELISA kits from Elabscience (USA). For the determination of malondialdehyde (MDA) the TBA method (technique colorimetric) with a kit from Elabscience (USA). The protocols recommended in the manual of techniques were followed in all cases. The reactions were read by absorbance in the EPOCH reader (Biotek Instruments, USA).

For the determination of cerebral BDNF (Brain-Derived Neurotrophic Factor), the hippocampus was chosen as it is considered the area of origin of neuronal degeneration in AD. The samples were processed according to the recommendations indicated in the technique protocol for the preparation of a tissue homogenate. The different parts of the brain were extracted from the animal and cut into small pieces and rinsed in ice-cold PBS (0.01 M, pH-7.4) to remove excess blood and avoid interference in subsequent reading. The tissue pieces used in the analysis were homogenized in PBS with a glass homogenizer on ice. For further cell disruption, the suspension was sonicated with an ultrasonic cell disrupter. The homogenates are cold centrifuged for 5 minutes at 5000 g to obtain the supernatant that was frozen at -80°C until analysis. The protein concentration was measured on the supernatant to normalize results.

The statistical parameters of the results obtained were analyzed with the SPSS v.11.0 program (SPSS, Inc., Chicago, IL, USA.). Differences between treated groups were compared using the Kruskal-Wallis test followed by the Mann-Whitney U test for non-parametric data, while normally distributed data were tested using one-way analysis of variance. Sheffé's correction was used in the analysis for between-group significance. Data are expressed as standard error of the mean (±SEM). In the graphs, statistically significant differences are indicated below (*p<0.05, **p<0.01 and ***p<0.001). Statistical significance was determined from P equal to or greater than 0.05.

C.2. RESULTS

BDNF levels increase in the transgenic model of AD (AD) in saline, proportionally with the age of the mice (3-4 months to 8-9 months). However, Nosustrophin treatment reverses BDNF levels to normal (Figure 15). In addition, Nosustrophin treatment drastically reduces CRP levels, showing a powerful anti-inflammatory effect (Figure 16). In AD, significant oxidative damage occurs at different stages of the disease. Important markers of oxiative damage and lipid peroxidation are TAS, MDA (malondialdehyde) and glutathione reductase (GR). Nosustrophin produces a significant increase in TAS and GR in EA transgenic animals (Figure 17). These data reflect a potent antioxidant effect of Nosustrophin.

Finally, treatment with Nosustrophin reduces creatinine levels, demonstrating a potential nephroprotective effect and renal safety (Figure 18) and reduces the activity of liver enzymes (GOT and GPT transaminases) in healthy and AD transgenic mice, showing hepatoprotective capacity and lack of liver toxicity (Figure 19).

Claims

1. Non-denaturing procedure for obtaining a Sus scrofa domestica brain extract comprising the following stages:

(i) submit isolated material from the brain of Sus scrofa domestica to ultrasonic disruption for a period of 10 to 25 minutes, with a frequency of 10 to 30 KHz, between 25,000 to 30,000 rpm and a temperature below 6°C,

(ii) deep-freezing the suspension obtained in (i) at a temperature below -60°C,

(iii) lyophilize the deep-frozen suspension obtained in (ii) for between 3 and 5 days until reaching a humidity percentage of less than 5%,

(iv) subjecting the lyophilized extract obtained in (iii) to cold grinding at a temperature below 6°C, and

(v) filtering the ground extract obtained in (iv) until obtaining an extract comprising a particle size below 700 pm.

2. Method according to claim 1, wherein the brain of Sus scrofa domestica comes from animals between 6 and 9 months of age.

3. Process according to claim 1 or 2, wherein the material isolated from the brain of Sus scrofa domestica is in a saline suspension.

4. Method according to claim 3, wherein the concentration of isolated material from the brain of Sus scrofa domestica in saline suspension is in a mass ratio of isolated material: volume of saline suspension between 1:2 and 1:10.

5. Method according to any one of claims 1 to 4, wherein the ultrasonic disruption of stage (i) is carried out at a frequency of 20 KHz.

6. Procedure according to any one of claims 1 to 5, wherein the ultrasonic disruption of stage (i) is carried out for a time between 15 to 20 minutes.

Process according to any one of claims 1 to 6, wherein the deep-freezing temperature of stage (ii) is -80°C.

8. Process according to any one of claims 1 to 7, wherein the extract obtained in (v) comprises a particle size between 500 and 700 pm.

Process according to any one of claims 1 to 8, wherein the filtering of step (v) is carried out by means of sequential sieving in a filter gradient.

10. Sus scrota domestica brain extract obtained by the method according to any one of claims 1 to 9.

11. Extract according to claim 10 comprising the components described in Table 1.

A composition comprising the extract according to claim 10 or 11.

The composition according to claim 12, wherein the composition is a pharmaceutical composition or a nutritional composition.

The composition according to claim 13, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient and, optionally, another active ingredient.

The composition according to any of claims 12 to 14, wherein the composition is formulated for administration in liquid or solid form.

The composition according to claim 13, wherein the nutritional composition is a food or a nutritional supplement.

The extract according to claim 10 or 11 or the pharmaceutical composition according to any one of claims 12 to 16 for use as a medicament.

The extract according to claim 10 or 11 or the pharmaceutical composition according to any one of claims 12 to 16 for use in the prevention and/or treatment of a neurodegenerative disease.

The extract or composition for use according to claim 18 wherein the neurodegenerative disease is Parkinson's or Alzheimer's.