EA046099B1 - USE OF A BENZODIAZEPINE-BASED PRODUCT WITH ACTIVITY FOR THE PREVENTION AND TREATMENT OF DEMENTIA - Google Patents

Description

Field of technology

The present invention relates to the prevention and/or treatment of neurodegenerative diseases or diseases associated with impaired cognitive function, unwanted oxidation or pathological processes associated with aging.

Neurodegeneration is an important problem that is common to many nervous system diseases such as dementias, Alzheimer's disease (AD), Parkinson's disease (PD) and neuropathic pain (NP). These diseases are depressing, expensive to treat, and existing treatments are inadequate. Adding to the urgency of this problem is the fact that the incidence of these aging-related diseases is rapidly increasing due to ongoing demographic changes.

Progressive aging of the world population is accompanied by undesirable consequences associated with an increase in the number of neurodegenerative diseases and senile dementia. There are an estimated 46.8 million people living with dementia worldwide. It is estimated that this number will almost double every 20 years; to 74.7 million in 2030 and 131.5 million by 2050. Dementia also has a huge economic impact.

Dementia currently has an estimated global cost of $818 billion and will become a trillion-dollar disease by 2018, with a very large impact on the quality of life of both patients and their families and caregivers (Alzheimer's Disease International. World Alzheimer Report 2015. London: Alzheimer's Disease International; 2015).

From all this it follows that AD is the most widespread, with approximately 35 million people suffering from the disease, and its incidence is estimated to increase significantly over the next three decades, in line with the average age of the population (Reitz, S.; Brayne, S.; Mayeux, R. Epidemiology of Alzheimer's disease. Nat. Rev. Neurol., 2011, 7, 137-152) (Reitz, C; Mayeux, R. Alzheimer's disease: Epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem. Pharmacol. , 2014, 88, 640-651).

AD is a neurodegenerative brain disorder that causes memory loss and loss of cognitive function, progresses slowly, and is often accompanied by behavioral changes such as aggression and depression (Querfurth, H.W.; LaFerla, F.M. Alzheimer's disease. N. Engl. J. Med., 2010, 362, 329-344). In its final stage, patients are bedridden, incontinent, and dependent on care, which is very costly for families. On average, death occurs 9 years after diagnosis (Citron M. (2004). Strategies for disease modification in Alzheimer's disease. Nat Rev Neurosci. 5(9): 677-85).

The large number of people suffering from this disease, requiring 24-hour care and other services, will greatly impact medical, material and human resources (Suh Y.H. and Checler F. (2002). Amyloid precursor protein, presenilins, and alpha-15 synuclein: molecular pathogenesis and pharmacological applications in Alzheimer's disease. Pharmacol Rev. 54(3): 469-525). Amyloid precursor protein, presenilins, and alpha-15 synuclein: molecular pathogenesis and pharmacological applications in Alzheimer's disease. Pharmacol Rev. 54(3): 469-525). Thus, it is a growing problem in medicine.

AD is a prototypical cortical dementia characterized by memory loss combined with dysphasia (a language disorder in which speech production and speech understanding are impaired), dyspraxia (impaired coordination and inability to perform certain movements and gestures in the absence of motor impairment), and agnosia (inability to recognize objects, people, sounds, shapes or smells) related to the involvement of cortical association areas (Crook R. et al. (1998). A variant of Alzheimer's disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1. Nat Med. 4(4): 452-5) (Houlden H., Baker M., et al. (2000). Variant Alzheimer's disease with spastic 5 paraparesis and cotton wool plaques is caused by PS-1 mutations that lead to exceptionally high amyloid- beta concentrations. Ann Neurol. 48(5): 806-8) (Kwok J.B., Taddei K., et al. (1997) Two novel presenilin-1 mutations in early-onset Alzheimer's disease pedigrees and preliminary evidence for association of presenilin -1 mutations with a novel phenotype. Neuroreport. 8(6): 1537-42) (Verkkoniemi A., Kalimo H., et al. (2001). Variant Alzheimer disease with spastic paraparesis: neuropathological phenotype. J Neuropathol Exp Neurol. 60(5): 483-92).

Although the disease is multifactorial and heterogeneous, it shares several common characteristics, such as extensive loss of cholinergic neurons, accumulation of neurofibrillary tangles and beta-amyloid aggregates (Huang, Y.; Mucke, L. Alzheimer Mechanisms and Therapeutic Strategies. Gel/, 2012, 148 , 1204-1222).

The chemical pathology of AD shows great similarities to Parkinson's disease (PD): oxidative stress, decreased mitochondrial complex I activity, and increased free radical lipid oxidation. These similarities also include the progressive nature of the disease, proliferation of reactive microglia around dying neurons, oxidative stress and inflammation.

Despite large investments in the pharmaceutical industry, there is only

- 1 046099 several, but in fact there are no effective treatments for AD.

Today, various strategies are being followed to obtain new drugs to treat the disease, given that the currently approved drugs have been observed to provide little benefit to patients. These medications temporarily delay (at best for a year) some symptoms of the disease, but they do not prevent its development.

Even though AD was initially associated only with cholinergic deficits, other neurotransmitters such as dopamine, norepinephrine, serotonin, and glutamate have been shown to be reduced or dysregulated in AD. To date, the neurotransmitters most studied in the pathogenesis of AD are cholinergic and glutamatergic (Palmer, AM; Gershon, S. Is the neuronal basis of Alzheimer's disease cholinergic or glutamatergic? FASEB J 1990, 4, 2745-52).

Current treatment options for AD rely on inhibition of acetylcholinesterase with drugs such as donepezil, galantamine or rivastigmine, or on the ability of memantine to antagonize the glutamate receptor, NMDA (N-methyl-D-aspartate). Due to the low success rate with these drugs, new lines of research have been opened. (Bartus, RT, Dean, RL 3rd; Beer, B; Lippa, AS The cholinergic hypothesis of geriatric memory dysfunction. Science 1982, 217, 408-14) (Terry, A.V. Jr.; Buccafusco, J.J. The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: recent challenges and their implications for novel drug development. J. Pharmacol. Exp. Ther., 2003, 306, 821-827) (van Marum, R. J. Current and future therapy in Alzheimer's disease. Fundam. Clin Pharmacol., 2008, 22, 265-274).

According to the cholinergic hypothesis of AD, loss of cholinergic functions in the central nervous system (CNS) makes a significant contribution to the cognitive impairment associated with AD (Bartus, R.; Dean, R.; Beer, B.; Lippa, A. The cholinergic hypothesis of geriatric memory dysfunction. Science, 1982, 217, 408-414). Moreover, even though the relationship between cholinergic depletion, amyloidogenesis, and tau phosphorylation is complex, it appears that cholinergic depletion may increase amyloid B production and this may induce tau phosphorylation. On the other hand, the glutamatergic hypothesis of AD suggests that excitotoxic mechanisms associated with glutamate involve the NMDA receptor, leading to cell degeneration and necrosis (Bleich S, Romer K, Wiltfang J, Komhuber J. Glutamate and the glutamate receptor system: a target for drug action. Int J Geriatr Psychiatry 2003, 18,833-40). Synaptic excitation through NMDA receptors is important for storage and memory functions, but excess glutamate can cause excitotoxicity and neurodegeneration (Michaels RL, Rothman SM glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. J Neurosci 1990 1 O, 283-92).

In this scenario, treatment strategies should interfere with both systems by combining an AChe enzyme inhibitor capable of improving cholinergic tone with an NMDA receptor antagonist capable of resisting glutamate-induced neurodegeneration. However, this combination of therapies has several disadvantages. In addition to resistance to the administration of individual drugs, an additional problem for older patients suffering from AD and their caregivers is that the different pharmacokinetics of the respective drugs may have different pharmacodynamics. In practice, hospitals will not welcome combination therapy with two different ADME (Administration, Distribution, Metabolism, and Excretion) curves.

An alternative and new approach to two-drug combinations is two drugs that can act on multiple pharmacological targets, called multi-target drugs (MTDs).

The strategy of targeting two or more proteins simultaneously with a simple compound can provide superior therapeutic effects (Cavalli A, Bolognesi ML, Minarini A, Rosini M, Tumiatti V, Recanatini M, Melchiorre C. Multi-target-directed ligands to combat neurodegenerative diseases J Med Chem 2008, 51, 347-72) (Zimmerman GR, Lehar J, Keith CT. Multi-target therapeutics; when the whole is greater than the sum of the parts. Drug Discov Today 2007, 12, 34-42) (Morphy R, Ranlovic, Z. Fragments, network biology and designing multiple ligands. Drug Discov Today 2007, 12, 156-60). This may be explained by a number of potential benefits provided by the use of MTD relative to cocktails or multicomponent medications. The benefits of MTD regarding cocktails can be summarized as follows:

1) reduced variability in clinical development, given that predicting the pharmacokinetics of a single compound is much easier than for a cocktail, overcoming the problem of varying bioavailability, pharmacokinetics and metabolism;

2) pharmacodynamic safety;

3) increased efficacy due to the synergistic effect of inhibition of multiple therapeutic targets; And

4) increased safety while reducing secondary effects of consuming drug cocktails (reducing risks due to drug-drug interactions); This is especially true in drug metabolism, where competition between different drugs for the same metabolic enzyme affects their toxicity.

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Another important benefit is a simplified therapeutic regimen with improved compliance capabilities, and this is especially important for older Alzheimer's patients and their caregivers (Small, G, Dubois In A review of compliance to treatment in Alzheimer's disease: potential benefits of a transdermal patch. Curr Med Res Opin 2007, 23, 2705-13). An important aspect in this regard is that patients with Alzheimer's disease are susceptible to a wide range of medical conditions (comorbidities), which include hypertension, vascular disease and diabetes, which can often be comorbid. Thus, the problems associated with the use of multiple pharmaceutical agents in the elderly population have been recognized as critical in recent years. These problems mainly involve drug interactions, which occur more frequently in this population due to the coexistence of chronic diseases and organ failure. It cannot be assumed that two drugs that are safe on their own will also be safe when combined, especially in the elderly population. In addition, the number of drugs administered simultaneously should be reduced as much as possible, since advanced age is an unpredictable risk factor for any drug treatment (Turnheim, K. When drug therapy gets old: pharmacokinetics and pharmacodynamics in the elderly. Exp. Geront 2006, 38, 843-853). Because MTDs are particularly preferred over complex therapies due to the complexity of interactions between multifunctional drugs, comorbidities, altered pharmacodynamic sensitivities, and changes in pharmacokinetics in the elderly. Clinical use of MTD may also simplify the therapeutic regimen (Youdim, M.B., and Buccafusco, J.J. (2005) CNS Targets for multi-functional drugs in the treatment of Alzheimer's and Parkinsons diseases J. Neural Transm 112, 519-537). Compliance with prescribed medication regimens is important for effective treatment. Lack of compliance is a common problem, but it is a problem for patients with Alzheimer's disease and their caregivers (Small, G, Dubois B A review of compliance to treatment in Alzheimer's disease: potential benefits of a transdermal patch. Curr Med Res Opin 2007, 23, 2705- 13). Therefore, a simplified treatment regimen with drugs that act on multiple targets will improve treatment adherence. All of the above benefits are not available with drug cocktails.

The multi-target ligand 1 strategy is a novel approach to develop new candidates for the treatment of complex neurological diseases, especially in view of the fact that the underlying processes involved in neurodegenerative diseases are multifactorial in nature (Cavalli A, Bolognesi ML, Minarini A, Rosini M , Tumiatti V, Recanatini M, Melchiorre S. Multi-target directed ligands to combat neurodegenerative diseases J Med Chem 2008, 51, 347-72). Such a strategy is based on the concept that a single compound can act on multiple targets contributing to the neurodegenerative process proposed in Alzheimer's disease and other neurodegenerative diseases, and will thus prevent unwanted displacement between interacting pathogenic pathways. But MTDs may represent an alternative practice for the use of drug combinations. Since the bulk of the neurodegenerative mechanisms are the same for many nerve diseases, these MTDs can also be used as a drug to treat other diseases.

The global burden of ischemic stroke is almost 4 times that of hemorrhagic stroke. Existing evidence confirms that 25-30% of ischemic stroke survivors immediately or over time develop vascular cognitive impairment or vascular dementia. Dementia after stroke can include all types of cognitive impairment. As the risk of death from strokes has decreased, the number of stroke survivors with brain and cognitive impairment has increased (R.N. Kalaria, et al., Stroke injury, cognitive impairment and vascular dementia, Biochim. Biophys. Acta (2016), http: //cix.doi.Org/10.1016/j.bbadis.2016.01.015).

Dementia after stroke is considered a disease entity that defines all types of dementia occurring after stroke, in addition to whether it involves vascular, neurodegenerative, or a combination of the two. This includes complex etiologies with various combinations of large and small vessel diseases, as well as neurodegenerative pathologies (R.N. Kalaria, et al., Stroke injury, cognitive impairment and vascular dementia, Biochim. Biophys. Acta (2016), http://dx.doi .org/10.1016/j.bbadis.2016.01.015).

From the knowledge that we have regarding the ischemic injury cascade, we know that it consists of a series of complex events that are very heterogeneous (R. Brouns, P.P. De Deyn, The complexity of neurobiological processes in acute ischemic stroke, Clin. Neurol. Neurosurg. 111 (2009) 483495), developing in the minutes-days and weeks after the initial event of insufficient perfusion. Major effects include energy deprivation due to interrupted blood flow, excitotoxicity, calcium overload, oxidative stress, blood-brain barrier dysfunction, capillary damage, hemostatic activation, damage associated with inflammatory and immune responses, and cellular necrosis at the level of neurons, glia, and endothelial cells.

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Capillary breaks and breakdown of the blood-brain barrier, which can occur over many days, lead to vasogenic edema and may also cause hemorrhage. At the same time, tissues may undergo a complex range of healing and remodeling responses that include angiogenesis to limit damage and reduce sequelae. These events are discarded in the aging brain such that the parenchyma is irreversibly damaged, thereby contributing to cognitive impairment (R. N. Kalaria, et al., Stroke injury, cognitive impairment and vascular dementia, Biochim. Biophys. Acta (2016), http:// dx.doi.Org/10.1016/j.bbadis.2016.01.015).

Experimental studies have shown that cell death after ischemic insult is strongly associated with necrosis. However, recent developments have shown that much of the death of nerve cells occurs due to apoptosis, as well as mixed mechanisms (R. N. Kalaria, et al., Stroke injury, cognitive impairment and vascular dementia, Biochim. Biophys. Acta (2016).

Neuroinflammation and immunosuppression are also associated with stroke, aging, and infection. This is likely to have a devastating effect on cognitive function after strokes (B.W. McColl, S.M. Allan, N.J. Rothwell, Systemic inflammation and stroke: aetiology, pathology and targets for therapy, Biochem. Soc. Trans. 35 (2007) 1163-1165) (C Meisel, A. Meisel, Suppressing immunosuppression after stroke, N. Engl. J. Med. 365 (2011) 2134-2136) (W. Swardfager, O. A. Winer, N. Herrmann, S. Winer, K. L. Lanctot, Interleukin17 in post -stroke neurodegeneration, Neurosci. Biobehav. Rev. 37 (2013) 436-447).

Aging and neurological and psychiatric disorders cause neuronal damage and death. Among the common and relevant changes in the nervous system, we have included, but are not limited to, neuronal degeneration, ischemia, inflammation, immune responses, trauma, and cancer. As a result, neurons may die within minutes or hours, or they may survive the initial injury in a damaged state, activating neurodegeneration and ultimately leading to cell death.

Neurodegeneration in Alzheimer's disease and other neurodegenerative diseases appears to be multifactorial, such that a wide range of toxic responses including inflammation, glutamatergic neurotoxicity, increased iron and nitric oxide levels, depletion of endogenous antioxidants, decreased expression of trophic factors, dysfunction of the ubiquitin proteasome system and expression proapoptotic proteins lead to neuronal necrosis.

Given the importance of the nervous system in generating basic motor skills and sensation, there is interest in finding a therapeutic agent to protect the nervous system.

Neuroprotective action is aimed at preserving, restoring, treating or regenerating the nervous system, its cells, structure and function (Vajda et al 2002, J Clin Neurosci 9:4-8). One of the goals of neuroprotection is to prevent or minimize the impact of the initial damage to the nervous system or to prevent or minimize the consequences of endogenous or exogenous noxious processes that cause damage to axons, neurons, synapses and dendrites.

Neuroprotection is a mechanism and strategy used to protect against neuronal damage or CNS degeneration resulting from chronic neurodegenerative disease (Alzheimer's disease). The goal of neuroprotection is to limit dysfunction/death following CNS injury and attempt to preserve as much integrity as possible for cellular interactions in the brain, resulting in intact neural function.

The concept of neuroprotection has been applied to chronic brain diseases as well as acute neurological conditions, given that some of the underlying mechanisms affecting the CNS are similar to these conditions. Neurodegenerative disorders include AD, PO, Huntington's disease, and amyotrophic lateral sclerosis. Neuroprotection has been considered as the mechanism of action of the drug used to treat these conditions.

There is a wide range of neuroprotective products available or under investigation, and some products could potentially be used for more than one disease, given that many mechanisms of damage to neural tissues are similar. Products with neuroprotective effects are grouped into the following categories: free radical scavengers, antiextoxin agents, apoptosis (programmed cell death) inhibitors, anti-inflammatory agents, neurotrophic factors, metal ion chelators, ion channel modulators, and gene therapy.

Oxygen free radicals have been demonstrated to be associated with protein denaturalization, enzyme inactivation, and DNA damage, leading to lipid peroxidation of cell membranes and ultimately cell death in neurodegenerative diseases.

Research in both animal and human models shows that loss of balance among oxidant species generated by brain metabolism and antioxidant defense mechanisms causes what is called oxidative stress, where these defense systems become less effective and disrupted. This oxidative stress increases with age and is found among the main causes of AD pathogenesis (Neurobiol. Aging 2007, 28, 1009-1014), possibly related to neuronal mitochondrial dysfunctions.

We also know that foods with antioxidant properties can prevent amyloid peptide-induced apoptosis as well as changes in Ca2 homeostasis in cultured cortical neurons (Lite Sci. 2000, 66, 1879-1892). 66, 1879-1892). Therefore, there is a great need

- 4 046099 in inhibitors of acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) and with neuroprotective ability against toxic damage such as oxygenated free radicals, these compounds will be of great medical importance in the treatment of neurodegenerative diseases such as AD, PO or Huntington's.

Generally speaking, treatment strategies are often based on modulating one factor of the proposed lesion. Although we may observe that these treatments are beneficial in very limited animal models, it is less likely that they would demonstrate effectiveness in more complex human disorders with higher severity of lesions in a genetically diverse population (Faden and Stoica, 2007. Arch Neurol 64: 794-800). To a large extent, given the proposed mechanisms of neuronal death, such as oxidative stress, mitochondrial dysfunction, added proteins, apoptosis and inflammation (oudim, M.B., and Buccafusco, J.J. (2005) CNS Targets for multifunctional drugs in the treatment of Alzheimer's and Parkinsons diseases J Neural Transm 112, 519-537), they are as complex as they are varied, we would need single compounds that have multi-target effects on multiple mechanisms of injury.

The compound (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3EDR^kenzodiacepine) (JM-20), which is described in the patent document of the Republic of Cuba CU23879 by its chemical structure, may have an effect on cardiovascular, cerebrovascular and other diseases associated with the central nervous system.

Surprisingly, researchers have discovered that the compound JM-20 has a pronounced therapeutic effect on the central nervous system, preferably for the treatment of diseases such as various types of dementia.

Thus, the essence of the present invention is based on the use of JM-20 and its chemical products, which are preferably used in the treatment of diseases such as various types of dementia.

JM-20 is a benzodiazepine product, and benzodiazepines have been widely reported to induce dementia (Huber-Geismann, F, 1994) (Rosenberg, RW, 2015). (Shash, D., T. Kurth, et al. 2015) (Zhong, G., Y. Wang, et al. 2015)), however, JM-20 unexpectedly improves the condition in various types of dementia.

Although the potential utility of JM-20 has been noted in the literature, its potential as a therapeutic drug for neurodegenerative diseases such as dementia has not been established.

As a further aspect of the present invention, the compound of general formula III (JM20), its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically tested prodrugs can be administered in mixtures with at least one vehicle, diluent and/or carrier, chemically inert, non-toxic, hereinafter recognized as excipients included in the proposed drug compositions.

Drug compositions are provided for any liquid, solid or semi-solid composition, they can be administered orally, bucopharyngeal, sublingually, parenterally, for example: intramuscularly, intravenously, intradermally or subcutaneously, topically, transdermally, tracheally, bronchially, nasally, pulmonaryly, rectally or others suitable routes of administration.

The drug compositions described will contain suitable excipients for each formulation. The compositions are usually prepared using methods existing in the art. Excipients are selected according to their chosen drug form according to the route by which they will be administered.

The compound of general formula III (JM-20), its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, prodrugs for its administration to humans may be contained in pharmaceutically acceptable dosage forms, which include, but are not limited to, these dosage forms: tablets ( including sublingual, coated and chewable), hard and soft capsules (including microcapsules, nanoparticles and pellets), solutions (oral drops, syrups), parenteral solutions, transdermal patches, implants and other retard systems, ointments (creams and gels), nasal sprays, mucoadhesives, suppositories, suspensions, powders to be diluted or added to food, among other dosage forms included in the present invention.

Using known technological processes of the prior art, JM-20, its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, prodrugs, can be formulated into dosage forms adapted for their administration, mixed with excipients such as liquid, solid excipients or semi-solids consisting of organic and inorganic substances, natural or synthetic. Some of these include: solid fillers, diluents, agglutinating agents, solvents, emulsions, lubricants, disintegrants, glidants, flavors, dyes, pigments, polymers, sweeteners, plasticizers, absorption enhancers, penetration enhancers, surfactants, co-surfactants, special oils and/or buffer systems that impart physical, chemical and/or biological stability to the active compounds or their physiologically acceptable salts.

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Some excipients used in dosage forms containing a compound of general formula III or its products, without limitation to the use of other excipients, are: starches, lactase, cellulose and its products, sucrose, sorbitol, mannitol and other sugars, talc, colloidal silicon dioxide, carbonates, magnesium oxides, calcium phosphates, titanium dioxide, polyvinylpyrrolidone, povidone, gelatin, milk proteins, citrates, tartrates, alginates, dextran, ethyl cellulose, cyclodextrins, silicon elastomers, polysorbates, amylopectin, parabens, animal and vegetable oils, propylene glycol, sterilized water, mono- or polyhydric alcohols such as glycerin, magnesium stearate, calcium stearate, sodium stearyl fumarate, sodium lauryl sulfate, glycerin and polyethylene glycol waxes, among others.

Solid oral dosage forms such as tablets, microgranules, nanoparticles, pellets, reconstituted powders or capsules containing a compound of general formula III or salts thereof, enantiomeric forms and redox products of the present invention can be used for immediate or modified release.

The selected form of the medicinal product according to the present invention is a tablet containing, as its active pharmaceutical ingredient, a compound of general formula III or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, prodrugs, obtaining a mixture with microcrystalline cellulose, corn starch, crospovidone, adding dissolved polyvinylpyrrolidone and sodium lauryl sulfate to obtain granulate, which is dried in a complete fluidized bed process and mixed with magnesium stearate and talc, then the tablets are obtained using a rotary die system for their production, finally the tablets are coated with hydroxypropyl cellulose, polyethylene glycol 4000, titanium dioxide and color suspension.

By coating the tablets we achieve an attractive final appearance and avoid unpleasant taste; this is achieved by a taste masking agent such as methacrylic acid copolymer, ethylcellulose, methylhydroxypropylcellulose or other polymers. Tablets can be produced either by the wet granulation method shown above or by the direct compression method using direct compression auxiliaries and reducing the number of steps in the tableting phase, provided that low doses are used.

Tablets may be modified release and they may contain the compound of general formula III or its products in microgranules, nanoparticles or matrix systems using excipients such as: polyethylene oxide, hydroxypropylcellulose 2910, magnesium stearate, sodium chloride, red iron oxide, cellulose acetate , polyethylene glycol 3350 and opadry.

According to the present invention, drug compositions may contain pharmaceutically acceptable polymers that are permeable, biodegradable and water insoluble to control its release profile, thereby obtaining modified release (immediate, delayed or controlled) dosage forms. These polymers can be used to coat tablets, microbeads, capsules in the preparation of nanoparticles, as release matrices in pellets, tablets, granules or mixtures with other excipients included in any other dosage form specified in the present invention.

For oral administration, other suitable pharmaceutical compositions are hard capsules, soft capsules and pharmaceutical powders, the compound of general formula III or salts thereof, enantiomeric forms and physiologically acceptable oxidation-reduction products thereof can be dosed in the form of hard gelatin or cellulose capsules, for example containing inside a mixture of the active pharmaceutical ingredient with commonly used excipients in solid form, such as those described for tablets; this mixture can be obtained by dry method, wet granulation, extrusion, tableting, in the form of microcapsules or microtablets. For soft gelatin capsule doses, conventional manufacturing methods are used and may involve admixing the compound of general formula III or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, prodrugs with vegetable oils, fat or other similar media suitable for their formation .

In the case of pharmaceutical powders, they can be prepared by simply mixing the compound of general formula III (JM-20) or its salts, enantiomeric forms and physiologically acceptable prodrug products thereof with excipients, suspending agents, sweeteners, flavorings and preservatives. Although the present invention also uses atomization drying method at inlet temperatures of 100-150°C and outlet temperatures of 50-90°C to produce powders, auxiliary substances such as dextran, polyethylene glycol 4000 and sodium lauryl sulfate, among others, are used to improve solubility of the active pharmaceutical ingredient for its proper administration into the body in solutions, or adding it to foods such as juices.

For rectal administration, the compound of general formula III (JM-20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be dosed in the form of suppositories, foams or rectal solutions for microenemas, which may contain a mixture of active compounds with a base of neutral hard fat (Witespol 45) or some

- 6 046099 other similar media suitable for their composition; sorbitan monooleate, polysorbate 20, wax emulsifier, anhydrate colloidal silicone, sodium meta-bisulfite, disodium edate, methyl parahydroxyl benzoate, sodium phosphates, macrogol 300, glycerin, water, propane, isobutene and n-butane.

For liquid oral administration, the compound of general formula III (JM-20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be formulated as syrups, elixirs, concentrated drops or suspensions with a pharmaceutically acceptable vehicle such as a mixture ethanol, glycerin, propylene glycol and/or polyethylene glycol, among others, carboxymethylcellulose or other thickeners; it may contain coloring, flavoring, sweetener (sucralose, aspartame, cyclamate, stevia) and preservative (parabens, benzoates). These liquid dosages can be prepared by diluting the powdered pharmaceutical compositions with a suitable diluent prior to use.

For parenteral administration, the compound of general formula III (JM-20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be formulated as solutions for injection. These solutions may contain stabilizing, preservative and/or buffering ingredients.

In the present invention, the active pharmaceutical ingredient is in 69% ethanol solution, benzoic alcohol, propylene glycol, benzoic acid, sodium benzoate, sodium hydroxide, water for injection; other excipients can also be used, such as polyethylene glycol 400, sodium citrate and citric acid.

Solutions for parenteral administration that contain a compound of general formula 111 (JM 20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs, can also be prepared by diluting the dry pharmaceutical composition (lyophilized) with a suitable diluent before use, including the use of excipients such as mannitol, polysorbate 80, sodium chloride and others.

For subcutaneous administration, the compound of general formula III (JM 20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be dosed as implants using excipients such as silicon elastomers and anhydrate colloidal silicone, although for Other pharmaceutical polymers can be used to make pellets.

For transdermal administration, the compound of general formula III (JM 20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be formulated in the form of patches; in this case, the active pharmaceutical ingredient is contained in a support of acrylic polymer, ethanol, light liquid paraffin, isopropyl palmitate, polyethylene terephthalate, ethylene vinyl acetate solution and a silicone layer within a peel-off layer (with a nominal release rate of 15 mg/day, over a surface area of 12.75 ^cm2 ) .

Implementation examples

Preparation of the synthetic intermediate, 1,4-dihydropyridine 5-formate.

Compound II is obtained based on the transformation of I through phosphorus oxychloride, in dry dimethylformamide, a mixture of which is then added in proportions 3-9 times to dissolve I in dichloromethane or chloroform and aged at a temperature of 20-60°C for 15-20 hours (Fig. 1).

The resulting product is then subjected to basic hydrolysis, followed by extraction with chloroform or dichloromethane, purification by washing and drying.

Compound I is prepared by a multicomponent reaction in one step where 2-nitrobenzene aldehyde, Meldrum's acid, ethyl acetoacetate and ammonium acetate are mixed in equimolar quantities in acetic acid as a solvent and refluxed for 7-10 hours. The mixture is then poured into water and the resulting precipitate is purified by recrystallization in ethanol.

Preparation of compound III.

Compound IIa or IIb obtained above is reacted with orthophenylenediamine in absolute ethanol to give compound III (JM-20), which can be prepared to form various salts, depending on the conditions and reagents used.

Preparation of compound III from Iia.

To prepare compound III (JM-20), we started with an ethanol solution of compound IIa and added equimolar amounts of orthophenylenediamine, in absolute ethanol as solvent, with stirring. Equimolar amounts or larger amounts (1-2 equivalents) of triethylamine (Figure 2, Method A) were added to the reaction mixture, maintaining stirring, or sufficient amounts (1-1.5 equivalents) of sodium hydroxide were added by controlled dripping and with continuous stirring of the reaction mixture ( Fig. 2, method B).

Preparation of compound III from IIb.

To prepare compound III (JM-20), we started with compound IIb in ethanol solution and added equimolar amounts of orthophenylenediamine, in absolute ethanol as solvent, with stirring. Equimolar amounts or large (1-2 equivalents) of triethylamine (Fig. 3, method

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A) added to the reaction mixture, maintained stirring, or added sufficient amounts (1-1.5 equivalents) of sodium hydroxide by controlled dripping and with continuous stirring of the reaction mixture (Fig. 3, method B).

The structure of the isomer of compound JM20 is shown in FIG. 4. Preparation of compound III in the form of halohydrate salts.

The corresponding hydrochloride or hydrobromide salts of III (halohydrates JM-20) were prepared from IIa or IIb, respectively, with or without the use of a catalyst. When the appropriate hydrogen acid was added in suitable catalytic amounts (5-25 mol%), a catalytic process occurred which reduced the reaction time and slightly reduced the reaction yield (Fig. 5).

JM20 hydrochloride (IIIa) was suitably prepared from IIa and when suitable amounts of HCl were added, a catalytic reaction occurred. Compound JM-20 can also be prepared as hydrobromide (IIIb) from IIb (Figure 5), with bromide being a better target group than chloride, the intramolecular nucleophilic substitution reaction is facilitated, giving a fast and efficient reaction to produce JM-20 in in the form of hydrobromide.

Preparation of compound III in the form of fumarate salt.

Fumarate salt JM-20 (IIIc) is prepared from the hydrochloride or hydrobromide of III by adding monosodium fumarate to a solution of IIIa or IIIb, stirring with a magnetic stirrer for 2-5 hours (Fig. 6) and its subsequent precipitation by adding ethanol in suitable quantities.

The precipitate corresponding to fumarate IIIc is suitably filtered and washed, purified and then placed in a temperature controlled dryer under reduced pressure.

Preparation of compound III in the form of phosphate salt.

Phosphate salt JM-20 (IIId) can be prepared in a similar manner to that described above from IIa or IIb by adding equimolar amounts of phosphoric acid at the beginning of the reaction or by adding phosphoric acid to IIIa or IIIb in ethanol solution with stirring (Fig. 7 ).

Preparation of compound III in the form of sulfate salt.

JM-20 sulfate (IIIf) can be prepared from IIa or IIb or from the halohydrates IIIa or IIIb as shown in FIG. 8.

Obtaining the composition in tablet form.

Each 120.00 mg tablet contains:

Component Quantity Function JM 20 40.00 mg Active ingredient Corn starch 23.00 mg Disintegrant Polyvinylpyrrolidone K-25 4.00 mg Agglutinin Monohydrated lactase 50.50 mg Filler Magnesium stearate 1.50 mg Lubricant Colloidal silicon dioxide 1.00 mg Lubricant Ethanol class e 12.00 µl Solvent Deionized water 12.00 µl Solvent

* Evaporates during the drying process.

Brief description of the technological process.

1. Sift the active ingredient, cornstarch and lactase through a 20 mesh sieve.

2. Weigh all components of the composition according to the quantities established in the formula.

3. For the agglutinin solution, pour a mixture of water and class C ethyl alcohol into a metal container with a steam jacket, add polyvinylpyrrolidone and shake until completely dissolved.

4. Add the active ingredient, corn starch and lactase (internal phase components) into the mixer. Mix for 15 minutes.

5. Add the agglutinin solution slowly using a peristaltic pump, adjusting to the required humidity level with water and grade C ethyl alcohol (1:1) if necessary. Grind in a mill on low speed.

6. Dry the granulate in a fluidized bed. After 10 minutes, take a representative sample of the granulate, degranulate and test for its residual moisture; the specified humidity should be between 0.8 and 1.2%.

7. Mix the dry granulate with lubricants for 10 minutes.

- 8 046099

8. In a high-speed centrifuge, compress the mixture using a 6.4 mm (1/4 PBR) flat, bevel and groove punch to obtain tablets with the following parameters:

weight: 120.0 mg±10%; height: 2.6±0.10 mm;

hardness: 4.0±1 kg-s; fragility: less than 1%.

Preparation of the composition for oral drops.

Each ml (20 drops) contains:

Component Quantity Function JM-20 40.0 mg Active ingredient Propylene glycol 300.0 mg Solvent medium Kollidon 25 160.0 mg Viscosity control agent Sodium saccharinate 12.5 mg Sweetener Ponceau S, acid red 0.05 mg Dye Lemon acid 5.535 mg pH stabilizer Dehydrated sodium citrate 20.0 mg pH stabilizer Ethanol 100.0 mg Solvent medium Methylparaben 1.8 mg Preservative a.t. Propylparaben 0.2 mg Preservative a.t. Liquid strawberry flavor (soluble) 20.0 mg Flavoring Purified water (sufficient quantity) 1.0 ml Wednesday

Brief description of the technological process.

1. Measure the pH and conductivity of purified water at the time of product manufacture.

2. Pour propylene glycol into the reactor.

3. Dissolve sodium saccharin in purified water in a stainless steel auxiliary tank with an appropriate container.

4. Turn on Kollidon 25, adding it a little at a time, stirring for at least 30 minutes until it completely disappears.

5. Stir and apply heat to the preparation, maintaining the temperature at 40-50°C for 30 minutes.

6. Add the active component to the result of the first stage in small portions, maintaining constant stirring for 30 minutes.

7. Remove heat and wait until the drug reaches room temperature: 30±2°C.

8. Dissolve methylparaben and propylene glycol in grade C ethyl alcohol in a auxiliary beaker or stainless steel container with a suitable container, stirring constantly until completely dissolved.

9. Add instant liquid strawberry flavoring to the results of the previous step and mix until completely homogeneous.

10. Add the results of the previous step slowly into the reactor vessel, stirring it constantly and vigorously.

11. Dissolve citric acid and dehydrated sodium citrate in a suitable stainless steel beaker or container, stirring after each addition until completely dissolved.

12. Slowly add the results of the previous step into the reactor container, stirring it constantly and vigorously.

13. Dissolve Ponceau S, acid red in purified water in a glass or stainless steel container with a suitable container, stir until completely dissolved and administer the drug.

14. Remove the previously prepared volume of water. Stir until smooth.

15. Test that the pH is maintained at 4.0-6.0.

- 9 046099

16. Carry out final filtration; test organoleptic characteristics.

17. Bottle the final preparation in x 15 ml amber glass bottles with 15.0±1.0 ml of solution and cap properly using caps with drip reducers for oily products.

Obtaining an injection composition.

Each flask (2 ml) JM-20 contains:

Component each ml contains Quantity per dose Unit of measurement Function JM-20 5.0 mg 10.0 mg Active ingredient Cremofor ELP 527.0 mg 1054.0 mg Wednesday Hydrochloric acid 1 N sufficient quantity - - Not pH adjustable Absolute alcohol sufficient quantity 1.0 ml 2.0 ml solvent *Nitrogen sufficient amount - -

1. Check that the reactor is completely dry after sterilization; if not, wash it with dehydrated alcohol.

2. Prepare a solution of 1 N hydrochloric acid to adjust the pH.

3. Add one part Cremofor ELP and dehydrated alcohol to the reactor. Mix at 420 rpm.

4. Weigh the active ingredient and add portions of the dehydrated alcohol to the beaker containing it; disperse it with a glass stirrer and add it to the reactor; repeat this operation until all the active ingredients have been swept away and all the dehydrated alcohol has been used.

5. Maintain stirring in the reactor for 60 minutes at 420 rpm until the active component is completely dissolved.

6. Add the rest of Cremofor ELP, sweeping up the remainder with dehydrated alcohol, stirring for 10 minutes at 420 rpm.

7. Determine the pH of the solution and bring it with 1 N hydrochloric acid to 5.0-6.0.

8. Add volume of solution by adding dehydrated alcohol. Stir for 5 minutes at 420 rpm.

9. Take 10 ml of solution and send it to the laboratory to monitor the process (assessment and pH).

10. Check that the filling and nitriding systems are installed correctly.

11. Carry out an integrity check on the Sartobran P MidiCaps filter, porosity (0.45+0.2 µm) with dehydrated alcohol.

12. After completing process control, increase the reactor pressure with nitrogen (0.7-1.0 bar) to push the solution through the Sartobran P filter cartridge with a porosity of 0.45 μm+0.2 μm. Fill and seal the flasks to obtain 2.2 ml doses of solution.

Memory studies in models of dementia.

Reagents.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). JM-20 was administered in sufficient quantities for the studies offered by the Organic Synthesis Laboratory at the Department of Chemistry of the University of Havana.

For various studies, JM-20 was suspended in carboxymethylcellulose (CMC) 0.05% and administered orally. Scopolamine bromide was dissolved in 0.9% saline and administered intraperitoneally (1 mg/kg, 4 ml/kg body weight). Aluminum chloride was dissolved in normal drinking water for experimental subjects and administered orally at 500 mg/kg per dose, continuously for one month, while behavioral studies were conducted. Beta-amyloid peptides (25-35) were oligomerized and made soluble according to the manufacturer's specifications and administered by intracerebroventricular means at 100 pmol in 5 μl, into the right hemisphere of the brain.

Experimental animals. Ethical considerations.

In vivo experiments corresponding to the scopolamine and aluminum model used Wistar rats (male, 230-260 g), while in vivo experiments corresponding to the beta-amyloid model used OF-1 mice (male, 25-30 g). All animals came from the National Center for the Breeding of Animals for Laboratory Research (CENPALAB, Mayabeque, Cuba) and after their receipt they were acclimated to laboratory conditions for 7 days. They were kept at

- 10 046099 alternating cycles of 12 hours of light and darkness, at controlled temperature (22±2°C), 45-55% relative humidity, and they were given water and food ad libitum on demand. All behavioral studies were conducted between 09:00 and 17:00 under controlled conditions of light and noise levels. All animal procedures recorded in this study were performed according to criteria approved by the International Committee for the Care of Laboratory Animals and in accordance with national guidelines for animal experimentation.

Experimental design.

Protocol 1: Evaluation of the effect of JM-20 on scopolamine-induced memory loss.

We assessed the protective effect of three doses of JM-20 (2, 4 and 8 mg/kg) on the processes of acquisition and consolidation of short- and long-term memory, which was influenced by acute intraperitoneal administration of scopolamine. For this purpose, JM-20 was administered before exposure or memory acquisition and consolidation, both short-term and long-term memory, in four independent tests.

For each test, 6 experimental groups of subjects were randomly selected (n=10, per group): healthy control group (CMC and saline); JM-20 group without damage (JM-20 8 mg/kg); scopolamine group (CMC and scopolamine 1 mg/kg); groups JM-20-scopolamine (JM-20 2 mg/kg and scopolamine 1 mg/kg), (JM-20 4 mg/kg and scopolamine 1 mg/kg) and (JM-20 8 mg/kg and scopolamine 1 mg /kg).

Effects on memory were assessed by a novel object recognition test and a forced alternation maze test, using experimental designs suitable for independent studies of the acquisition and consolidation of short- and long-term memory.

At the end of the behavioral studies, we conducted various in vivo studies of brain tissue. We examined the effects of JM-20 on various markers of oxidative stress, mitochondrial function, acetylcholinesterase enzyme AChE activity levels, and histological parameters of neuronal and axonal viability in brain regions strongly involved in memory and learning.

Protocol 2: Evaluation of the effect of JM-20 on aluminum chloride-induced memory loss.

We assessed the protective effect of two doses of JM-20 (2 and 8 mg/kg) on various types of memory, which was influenced by acute intraperitoneal administration of scopolamine. For this purpose, JM-20 was administered from day 15 after the start of aluminum chloride (500 mg/kg) until the end of the behavioral studies. We assessed the effect of JM-20 and aluminum chloride on three types of memory: spatial memory according to the Morris water maze test and the T-maze test; recognizing new things through a new object recognition test; and emotional-associative memory using a passive avoidance test.

5 experimental groups were formed from randomly selected subjects (n=10, per group): healthy control group (CMC and water); group JM-20- without damage (JM-20 8 mg/kg and water); aluminum group (CMC and aluminum chloride 500 mg/kg); groups JM-20-aluminum (JM-20 2 mg/kg and aluminum 500 mg/kg) and (JM-20 8 mg/kg and aluminum 500 mg/kg).

At the end of the behavioral studies, we conducted various in vivo studies of brain tissue. We studied the effects of JM-20 on various markers of oxidative stress, mitochondrial function, and activity levels of the acetylcholinesterase enzyme AChE.

Protocol 3: evaluation of the effect of JM-20 on damage caused by beta-amyloid peptide oligomers 25-35.

We initially assessed the effect of JM-20 on the cell viability of the PC12 cell line exposed to a 1 μM concentration of amyloid beta peptide oligomers 25-35. This in vitro test assessed the effects of 3 concentrations of JM-20 (3.125, 6.25 and 12.5 µM) dissolved in dimethyl sulfoxide (DMSO). Six experimental groups were formed: untreated control group (NT); environment group (DMSO); amyloid beta group (AR 1 µM); JM-20-beta-amyloid groups (JM-20 3.125+AP 1 µM), (JM-20 6.25+AP 1 µM) and (JM-20 12.5+AP 1 µM).

Then, in vivo studies were carried out on OF-1 mice. We assessed the effect of JM-20 on memory loss caused by amyloid-beta oligomers, administered 7 days after intracerebroventricular administration of AR (100 pmL) for 10 days, orally at 10 and 30 mg/kg doses. We formed 5 experimental groups: a healthy control group (single); JM-20 group without damage (blank +JM-20 30 mg/kg); amyloid beta group (AR 100 pmol +CMC); groups JM-20beta-amyloid (JM-20 1 O mg/kg + AR 100 pmol) and (JM-20 30 mg/kg + AR 100 pmol).

Behavioral studies Y-maze. Spontaneous alternation.

The effect of JM-20 on spatial memory was assessed using a forced alternation Y-maze test (20). The labyrinth was built from plastic with three arms (55 x 20 x 20 cm) at an angle of 120° on a central platform.

The test was carried out in two sessions. During the first phase of training, we conducted a training phase in which each rat was placed on a central platform and allowed to freely explore arms (A and C) of the maze for 10 min while the third arm (B) was closed. The entrances to each arm were considered to be the entrances to the four endpoints within any arm. Sequence of entries into the sleeves

- 11 046099 was recorded on video for 10 minutes for later analysis. The assessment phase consisted of allowing rats to freely explore the arms of the Y-maze for 5 min, including the one that was previously closed. The sequence of arm entries was recorded for 5 min for subsequent analysis. This test assessed the number of entries into a new arm (B), where the percentage of entries into this arm was directly proportional to the best memory in the studied animals. The percentage of correct entries into arm B was calculated based on the proportion of alternations made out of all possible alternations, as shown in the following equation: % correct entries = (number of entries in B)/(total number of entries in the three arms) x 100. After each test, the maze cleaned with 40% ethanol solution to reduce the existence of olfactory marks.

Object recognition.

The effect of JM-20 on memory for recognizing novel objects was assessed by an object recognition test. This test was carried out in an open field for 2 consecutive days. On the first day, the animals adapted to the field; they were allowed to explore the box for 3 minutes, in two sections. On the second day, the training was conducted with learning assessment in 2 phases of 5 min each, then considered as training (E) and testing phase (P), respectively. During Stage E, rats were presented with 2 identical objects, called familiar (F) objects. After 30 min, the P phase began and rats were exposed to a familiar object F and a novel object (N). Tests were recorded for analysis of object exploration, determined by the time of exploration performed by each animal for each object. Recognition rates between objects F and N were calculated as ID=(N-F)/(N+F).

Morris Water Maze.

The Morris water maze test was performed as described by Vorhees in Williams (21) with several modifications, in a circular tank (1.52 m diameter, 0.60 m depth). We conducted 5 days of training, 4 sections per day, and on the 6th day the training was assessed. Animals had to learn to find a submerged platform, fixed in a constant position, in all sections of the training. Several foreign keys were placed and kept in a fixed position throughout the experiment. In each training session, animals were placed in the water, facing the wall of the tank, at one of 4 exit sites until they found the platform within a maximum time of 1 min. On the day of assessment, the platform was removed. We quantified escape latency (the time spent by the animal finding the platform) in learning trials, the time the animal remained in the platform quadrant during the test to assess learning, and the distance until the platform location was found.

Passive avoidance.

The passive avoidance test was conducted based on the methodology described by Kohara and co-workers (22), with some modifications. We used passive evasion equipment (UGO Basile) consisting of 2 chambers that were illuminated (30 x 30 x 30 cm) and in darkness (10 x 20 x 12 cm), connected by a guillotine-style access door. The dark chamber was equipped with an electrical circuit.

The test was carried out in 2 stages over 2 consecutive days. On the first day, each rat was placed in a lighted chamber for 10 s, then the door between the chambers was opened and the rat was allowed to move freely for a maximum of 90 s. Once the rat entered the dark chamber, the access door was closed and the rat received a 1 mA electrical charge for 5 s.

On the second day, the latency to enter the dark chamber was measured for a maximum time of 300 s.

Study of mitochondrial function. Isolation of cerebral mitochondria.

Mitochondria were isolated by differential centrifuge. Animals were killed by decapitation and their brains were immediately removed, cut into 50 ml isolation buffer containing sucrose 75 mmol/L, EGTA 1 mmol/L, mannitol 225 mmol/L, BSA 0.1% and HEPES-KOH 10 mmol/L, pH 7.2, 4°C and homogenized in Potter-Elvehjen. The suspension thus obtained was centrifuged at 2000 g for 3 minutes and the floating substance was centrifuged at 12000 g for 8 minutes. The pellet was resuspended in 10 ml of isolation buffer, also containing 20 μl of digitonin at 10%, and it was centrifuged at 12,000 g for 10 min. The mitochondrial pellet was suspended in isolation buffer without EGTA and centrifuged at 12,000 g for 10 min, the floating material was removed and gently washed with isolation buffer without EGTA. The microscale method was used to determine protein concentrations using bovine serum albumins in a standard protocol (Mirandola et al., 2010).

Mitochondria were incubated in KCl 130 mmol/L, MgCl2 1 mmol/L and HEPES-KOH, phosphate 2 mmol/L, pH 7.4. Mitochondria (1 mg protein/ml) were activated by 5 mmol/L rotenone and 2.5 μmol/L potassium succinate.

Mitochondrial membrane potential.

The mitochondrial membrane potential was determined using a POLARstar Omega fluorescent spectrophotometer (Germany) and the fluorescent marker safranin O (10 μM) was used in a POLARstar Omega fluorescent spectrophotometer (Germany) at 495/586 nm (excitation

- 12 046099 nie/emission).

Swelling of mitochondria.

The swelling of mitochondria was controlled by a decrease in the visible absorption at 540 nm of a suspension of mitochondria incubated in a standard medium, in the presence of Ca2+200 µmol/l and using a POLARstar Omega fluorescence spectrophotometer (Germany).

Obtaining reactive oxygen species.

Reactive oxygen species (ROS) were determined by spectrofluorometry using Amplex red (Molecular Probes, Oregon, Eugene) as a fluorescent marker and 1 μl/ml horseradish peroxidase at 563/587 nm (excitation/emission).

Preparing brain tissue for enzyme and redox state tests.

Rats were killed by decapitation; their brains were removed and quickly sectioned to separate 2 regions, the hippocampus and the prefrontal cortex of both hemispheres (23). These areas were homogenized in sodium phosphate buffer solution (0.1 mM, pH 7.4: NaCl 0.13 M, KCl 0.0027 M, KH2PO4 136.04 M, Na ₂ HPO ₄ 0.0016 M) in a ratio of 1:10 (p: v) and they were centrifuged at 6000 g for 20 minutes in an Eppendorf centrifuge (Germany, 5424R). Brain homogenate ratios were maintained at -80°C until used. Their protein concentrations were determined by the Lowry method (24) at POLARstar Omega (Germany) using a bovine serum albumin standard.

Acetylcholinesterase (AChE) activity.

The activity of the AChE enzyme present in the homogenate of extracted hippocampus and prefrontal cortices was measured spectrophotometrically using the method described by Ellman (25) with slight modifications (26). The test was performed in a 96-well plate with a final volume of 200 μL. The reaction medium contained 140 μl of Ellman's reagent, 50 μl of homogenate, and 10 μl of 20 mM acetylthiocol iodate (AcSCh) solution and was incubated for 1 min at 37°C. Absorbance was read at 405 nm for 20 min at 1 min intervals in a POLARstar Omega spectrophotometer (Germany). Enzyme activity was calculated and expressed as μmol hydrolyzed (AcSCh) for one minute per mg protein.

Study of parameters of redox state and oxidative damage.

Superoxide dismutase (SOD) activity.

SOD enzyme activity was determined using the pyrogallol oxidation method (27). The reagent mixture consisted of a solution of 2.8 ml of Tris O.2M-HC1 0.2 M (pH 8.2), 0.05 ml of EDTA 60 mm, 0.1 ml of homogenate and 0.05 ml of pyrogallol solution 7.37 mm. Absorbance was read at 420 nm for 1 min in a POLARstar Omega spectrophotometer (Germany). Results were expressed as units of enzyme activity per mg of protein, considering 1 unit of enzyme activity as the amount of enzyme catalyzing the conversion of 1 μmol of substrate in one minute.

Catalase activity (CAT).

The activity of CAT enzymes was determined based on the decomposition of hydrogen peroxide (H2O2) (28). The reagent mixture consisted of 0.9 ml of phosphate buffer (pH 7.0), 0.1 ml of brain tissue homogenate and 0.5 ml of H ₂ O ₂ (50 mM). Absorbance was read at 240 nm, at 10 and 70 sah, in a POLARstar Omega fluorescence spectrophotometer (Germany). The results were expressed in µmol _{of H2O2} decomposed in 1 min per mg of protein.

Reduced glutathione (GSH) levels.

GSH levels were determined following the procedure described by Ellman (29) with some modifications.

0.375 ml of phosphate buffer (50 mM, pH 8.0), 0.1 ml of homogenate and 0.025 ml of 5,5'-dithio-bis-nitrobenzoic acid (OTNB)/acetone solution (0.6 mM) were added to the tray. Absorbance was read at 412 nm in a POLARstar Omega spectrophotometer (Germany). The results were calculated using the molar extinction coefficient of the resulting chromophore (1.36 x 10 ⁴ (mol/l) ^-1 cm ^-1 . The results were expressed as nmol GSH per mg protein.

Levels of lipid peroxidation.

Levels of lipid peroxidation were determined by detecting malondialdehyde (MOD), formed by reaction with thiobarbituric acid (30). The reagent medium was formed using 350 μl of a mixture of trichloroacetic acid: thiobarbituric uric acid: chlorhydric acid, 100 μl of brain tissue homogenate and 50 μl of diterbutylated hydroxytoluene (BHT). The mixture was homogenized in a TALBOYS vibrating mixer (USA) and placed for 5 minutes in a water bath with a Grant OLS200 thermostat (USA) at 100°C. It was then centrifuged at 3000 rpm for 10 min in an Eppendorf centrifuge (Germany, 5424R). MOA, formed as a product of lipid peroxidation in brain tissue, reacts with thiobarbituric uric acid and produces a colored compound, which was determined by spectrophotometry at 535 nm on a POLARstar Omega fluorescence spectrophotometer (Germany). The MOA concentration was calculated taking into account the molar extinction coefficient of the resulting colored compound (1.56 x 10 ⁵ M ^-1 cm ^-1 ) and depending on the following equation:

C=D.Ox10 ⁴ nM 1.56;

where C represents the MOA concentration to be determined, and D.O. represents

- 13 046099 detected absorption. Results were expressed as nanomoles MOA per mg protein.

Histological examination.

Histological evaluation of lesions caused by acute intraperitoneal injection of scopolamine was carried out in areas of the hippocampus and prefrontal cortex. We used the Paxinos and Watson Atlas (31) with an anatomical description of the rat brain to identify each brain region, and they were treated with hematoxylin and eosin (HE) solutions in 4-μm-thick cortical sections, always occupying the first three sections of each region. Both hemispheres were observed using a Leica optical microscope (Microsystems, Germany). Neuronal damage was assessed in areas CA1, CA2, CA3 and the dentate gyrus of the hippocampus, and atrophy, nuclear pyknosis, dark cytoplasmic staining, or absence of neurons were considered markers of degeneration. In addition, we calculated axonal damage, specifically based on the appearance of partially or completely demyelinated axons, axonal atrophy, and expressed it as a percentage of the total number of axons present in each microscopic slide or in each region examined. By analyzing histological damage at the level of the prefrontal cortex, we calculated the average number of neurons and axons affected in three fixed regions of each cerebral hemisphere and expressed this as the percentage of neurons/axons damaged compared to the total number in each region.

Statistical analysis.

For statistical processing and analysis of the results obtained, we used the GraphPad Prism 5.0 program (GraphPad Software Inc., USA). The data were expressed as mean ±EEM (standard error of the mean), and we checked its normality and homoscedasticity.

One-way analysis of variance (ANOVA) was performed, followed by Tuckey's multiple comparison test for comparisons between different experimental groups. A p value <0.05 was considered statistically significant.

The results are shown in Fig. 9-14. Vascular dementia.

As a model of vascular dementia, animals (male Swiss albino mice) were subjected to transient occlusion of the common carotid arteries for 20 min, and cognitive impairment was assessed using the Morris water maze test.

The results are shown in Fig. 15.

Unilateral damage to the pars compacta of the substantia nigra with injection of 6hydroxydopamine.

Materials and methods. Experimental animals.

Adult male Wistar rats, weighing between 200 and 250 g at the start of the experiment, were used, coming from the National Center for Breeding Animals for Laboratory Research (CENPALAB), Havana, Cuba. Animals were maintained at the Center for Drug Research and Development (CIDEM) in plastic translucent boxes at a mean temperature of 23°C (22 ± 2°C), with food and water ad libitum and 12-hour periods of light and darkness.

Ethical considerations.

This study followed the standards set out in the code of ethics for animal experimentation. We ensured the genetic integrity of the animals by purchasing animals from recognized breeding centers. This procedure avoids the use of genetically modified animals. From a biological point of view, the importance of the genetic quality of research is that the results of the experiment can be reproduced anywhere else where they can be repeated, and from an ethical point of view it ensures the smallest number of animals that need to be used.

During the study, the animals were kept in the best living conditions. They were kept at a temperature of 23°C, thus within the commitment range established for tropical zones (22±2°C). Temperature control is important given that high temperatures cause stress in animals and temperatures above 38°C can cause death. The periods of light and darkness to which they were exposed lasted 12 h, since photoperiodicity directly and/or indirectly regulates circadian, biochemical and hormonal rhythms.

Introduction of the connection.

JM-20 was administered at 3 different doses: 10, 20 and 40 mg/kg, 24 hours after injury induction and daily for 7 days. The compound was prepared in carboxymethylcellulose (CMC) at 0.05%, intragastric cannula. There were 5 experimental groups: group I (medium/saline with ascorbic acid, n=6), group II (animals treated with 6-OHDA, n=8), group III (animals treated with 6-OHDA and co-treated with 10 mg/kg JM-20, n=8), group IV (animals treated with 6-OHDA and co-treated with 20 mg/kg JM-20, n=8) and group V (animals treated which were treated with 6-OHDA and which were co-treated with 40 mg/kg JM-20 n=8). All behavioral tests were performed on day 7.

Data processing.

Statistical analyzes were performed using the GraphPadPrism Version.5.01.2007 package. Result

- 14 046099 you showed that the mean ±SEM percent viability compared to untreated control cells. In all cases, analysis of variance was performed (OneWay ANOVA) followed by Dunnett's test (GraphPadPrism 5) to determine a significant difference with a significance level of p<0.05 (*), p<0.01 (**) and p<0.001 (** *) relative to the control used in the experiment. To compare all values obtained in the experiment with different controls and relative to each other, we performed the Tuckey test (GraphPadPrism 5) with a significance level of p<0.05 (*), p<0.01 (**) and p<0.001 (***).

Claims (2)

CLAIM 1. Application of compound formula in the treatment of dementia. 2. Use of a compound according to claim 1, characterized in that the dementia is Alzheimer's disease or vascular dementia.