EA041342B1 - APPLICATION OF A BENZODIAZEPINE-BASED PRODUCT WITH ACTIVITY FOR THE PREVENTION AND TREATMENT OF PARKINSON'S DISEASE - Google Patents

Description

Technical field

The present invention relates to the use of a benzodiazepine product with activity for the prevention and treatment of Parkinson's disease.

Neurodegeneration is an important problem that is common to many diseases of the nervous system such as dementias, Alzheimer's disease (AD), Parkinson's disease (PO) and neuropathic pain (NP). These diseases are depressing, expensive to treat, and existing treatments are not adequate. Adding to the urgency of this problem is the fact that the incidence of these diseases associated with aging is rapidly increasing due to ongoing demographic changes.

The progressive aging of the world's population is accompanied by undesirable consequences associated with an increase in the number of neurodegenerative diseases and senile dementia. An estimated 46.8 million people live 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 impact on the economy.

Today, the estimated global cost of dementia is $818 billion and by 2018 will be a trillion dollar cost disease 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 it is estimated that its frequency will increase significantly in the next three decades, on par with the average age of the population (Reitz, C.; Brayne, C.; Reitz, C.; Mayeux, R. Alzheimer's disease: Epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem. Pharmacol ., 2014, 88, 640-651).

AD is a neurodegenerative disorder of the brain that results in memory and cognitive loss, progresses slowly, 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 last stage, patients are bedridden, incontinent, and dependent on care that 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).

A large number of people suffering from this disease, requiring round-the-clock care and other services, will greatly affect 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, this is a growing problem in medicine.

AD is a prototypical cortical dementia characterized by memory loss along with dysphasia (a speech disorder in which speech production and 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 that 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 e vision 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 a strong similarity to Parkinson's disease (PD): oxidative stress, reduced mitochondrial complex I activity, increased free radical lipid oxidation. These similarities also include the progressive nature of the disease, the proliferation of reactive microglia around dying neurons, oxidative stress, and inflammation.

Despite large investments in the pharmaceutical industry, there are few, and in fact no, effective treatments for AD.

Various strategies are being followed today to develop new drugs for the treatment of a disease, provided that it has been observed that currently approved drugs

- 1 041342 provide few benefits for patients. These drugs temporarily delay (at best for a year) some of the symptoms of the disease, but they do not prevent its development.

Even though AD was initially associated only with cholinergic deficiency, other neurotransmitters such as dopamine, norepinephrine, serotonin, and glutamate have been shown to be reduced or unregulated in AD. Currently, 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).

Existing AD therapy options are based on acetylcholinesterase inhibition 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 research lines 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, the loss of cholinergic functions in the central nervous system (CNS) makes a significant contribution to the impairment of the cognitive process associated with AD (Bartus, R.; Dean, R.; Beer, B.; Lippa, A. The cholinergic hypothesis of geriatric memory dysfunction Science 1982, 217, 408-414). Furthermore, even though the relationship between cholinergic depletion, amyloidogenesis, and tau phosphorylation is complex, it appears that cholinergic decline may increase B-amyloid production and this may cause tau phosphorylation. On the other hand, the glutamatergic AD hypothesis shows 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 via NMDA receptors is important for learning 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 would interfere with both systems with a combination of an inhibitor of the AChe enzyme 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 resisting the administration of individual drugs, an additional problem for older patients suffering from AD and for their caregivers is that different pharmacokinetics of the respective drugs may have different pharmacodynamics. In practice, hospitals will not welcome combination therapy with two different ADME curves (introduction, distribution, metabolism and excretion).

An alternative and new approach to the combination of two drugs is two drugs that can act on multiple pharmacological targets, the so-called multipurpose drugs (MTD).

The strategy of targeting two or more proteins simultaneously with a single compound can provide excellent 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 can be explained by a number of potential benefits provided by the use of MTD over cocktails or multidrugs. The advantages of MTD over 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 different 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 the secondary effects of the consumption of drug cocktails (reduced 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.

Another important benefit is a simplified therapeutic regimen with improved compliance capabilities, and this is especially important for older 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). In this regard, an important aspect is that patients with Alzheimer's disease are subject 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 several pharmaceuticals in the elderly population have been recognized as critical in recent years. These problems are mainly drug interactions, which occur more often 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 at the same time should be reduced as much as possible, since old 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 combination therapies for the complexity of interactions between multifunctional drugs, comorbidities, altered pharmacodynamic sensitivities, and altered pharmacokinetics in the elderly. The clinical use of MTD may also simplify the therapeutic regimen (Youdim, M.B., and Buccafusco, JJ (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. Non-compliance is a general problem, but it is a problem for Alzheimer's patients 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 multi-target drugs will increase treatment adherence. All of the above benefits are not available for 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 C. Multi-targetdirected 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 that promote the neurodegenerative process putative in Alzheimer's disease and other neurodegenerative diseases, and that thus will prevent undesirable 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 neurological 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 suggests that 25-30% of ischemic stroke survivors develop vascular cognitive impairment or vascular dementia either immediately or over time. Post-stroke dementia can include all types of cognitive impairment. As the risk of death after stroke 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).

Post-stroke dementia is considered as a nosological entity that defines all types of dementia that occur after a stroke, whether it includes vascular, neurodegenerative, or a combination of the two. This includes complex etiology with various combinations of diseases of large and small vessels, 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 is available regarding the ischemic injury cascade, we know that it consists of a number 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 within minutes, days and weeks after the initial phenomenon of insufficient perfusion. Key events include lack of energy 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 cell necrosis at the level of neurons, glia, and endothelial cells.

Capillary damage and disruption of the blood-brain barrier, which can occur many days later, leads to vasogenic edema and can also cause hemorrhages. At the same time, tissues can undergo a complex range of healing and remodeling responses, which include angiogenesis to limit damage and reduce consequences. These phenomena are discarded in the aging brain so that the parenchyma is irreversibly damaged while contributing to cognitive impairment (R.N. Kalaria, et al., Stroke injury, cognitive impairment and vascular dementia,

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biochim. Biophys. Acta (2016), http://dx.doi.Org/10.1016/j.bbadis.2016.01.015).

Experimental studies have shown that cell death after ischemic injury is strongly associated with necrosis. However, recent developments have shown that nerve cell death is largely 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) -stroke neurodegeneration, Neurosci Biobehav Rev 37 (2013) 436-447).

Parkinson's disease (PO) is a neurodegenerative disease with symptoms of movement disorder, slow movements, stiffness, tremors at rest, and balance changes. As the disease progresses, many patients develop non-movement related symptoms, including anxiety, depression, constipation, and dementia.

These characteristics are associated with a severe decrease in striatal dopamine and a loss of dopaminergic neurons in the substantia nigra compactus (Gauthier, 1982).

Clinical signs of PO occur after dopaminergic neuron necrosis exceeds a threshold level of 70-80% and striatal nerve loss exceeds 50-60% (Agid, 1991).

Research on the mechanisms of PO development has shown that the loss of dopaminergic neurons in the compact substantia nigra is associated with a deficiency in mitochondrial complex 1 (Jenner 1998).

Although there are drugs that relieve the symptoms of Parkinson's disease, chronic use of these drugs is not effective in preventing the progression of PO and has been associated with debilitating secondary effects. Thus, of particular interest is the development of neuroprotective therapies that delay or even stop the development of degenerative processes.

Unfortunately, the development of neuroprotective therapies has been halted due to limited knowledge of the pathogenesis of degenerative neurological diseases such as PO. The etiology and pathogenesis responsible for the nervous disorders in Parkinson's disease are still unknown. Some evidence supports the theory of microglial activation and inflammatory processes are included in the cascade of events that lead to progressive neuronal degeneration (Kreutzgber, GW, 1996 Trends Neurosci, 19:312-318). Activated microglia are found near neurons during degeneration in the substantia nigra of patients with Parkinson's disease (McGeer, PL et al, 1988, Neurology, 38:1285-1291).

The course of classical treatment for PO is the administration of dopamine agonists such as levodopa (LOOPA) and carbidopa. In general, other drugs are prescribed with the express purpose of either replacing dopamine or inhibiting its degradation, such as inhibitors of catechol-O-methyltransferase and amantadine (N. L. Diaz, C. H. Waters, Expert Rev Neurother 9, 1781 (Dec 1, 2009).

Although such drugs generally relieve the patient's symptoms, they are not always effective and/or tolerated over time, with dyskinesia being a secondary effect (A. H. Schapira et al., European Journal of Neurology 16, 1090 (2009)). Another complication is the fact that daily treatment can be burdensome and reduce patient compliance. Thus, the current focus is on new drug delivery systems that can provide continuous infusion of dopamine agonists by placing minipumps (eg, enteral, subcutaneous) and for transdermal administration via dermal patches (A. H. Schapira et al., European Journal of Neurology 16, 1090 (2009)). Even with the development of such drugs and their administration systems, some patients with Parkinson's disease still do not receive sufficient symptom relief (N. L. Diaz, C. H. Waters, Expert Rev Neurother 9, 1781 (Dec 1, 2009).

Experimental models of mitochondrial diseases typically involve inhibition of enzymes involved in the electron transport chain (1). It has also been noted that many neurodegenerative diseases are associated with mitochondrial dysfunction (2-5). Defects in the 1,11 and IV complexes in the mitochondrial respiratory chain have been found in Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's diseases (6-9).

Some evidence suggests that Parkinson's disease is a disease that involves free radicals and mitochondrial dysfunction, resulting in the failure of energy productivity (10-11). Increased oxidative damage, lack of dopamine, protein nitration, iron accumulation, protein addition, and apoptosis are characteristic of Parkinson's disease (12–14).

Many epidemiological studies have shown that chronic pain is widespread, seriously affecting people's health, healthcare and social services (Torrance N, Smith BH.

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Bennett MI, Lee AJ. The epidemiology of chronic pain of predominantly neuropathic origin. Results from a general population survey. J Pain 2006; 7:281-289) (Treede RD, Jensen TS, Campbell JN, Cruccu G, Dostrovsky JO, Griffin JW, Hansson P, Hughes R, Nurmikko T, Serra J. Redefinition of neuropathic pain and a grading system for clinical use: consensus statement on clinical and research diagnostic criteria, Neurology 2008; 70:16301635). In particular, it has been estimated that chronic pain with neuropathic content affects approximately 7-8% of the general population, and therapies available for their treatment are still unsatisfactory (Torrance N, Smith BH. Bennett MI, Lee AJ. The epidemiology of chronic pain of predominantly neuropathic origin. Results from a general population survey. J Pain 2006; 7:281-289) (van Hecke O, Austin SK, Khan RA, Smith BH, Torrance N. Neuropathic pain in the general population: a systematic review of epidemiological studies. Pain 2014; 155:654-62) (Bennett MI, Rayment C, Hjermstad M, Aass N, Caraceni A, Kaasa S. Prevalence and aetiology of neuropathic pain in cancer patients: A systematic review. Pain 2012; 153 :359-365). NP is seen as a symptom of neurodegeneration, therefore, it is logical to consider neuroprotective action as a strategy to prevent its onset, fight progression, and even return the nerve damage leading to these symptoms of chronic pain (Bordet T and Pruss RM. Targeting neuroprotection as an alternative approach to preventing and treating neuropathic pain Neurotherapeutics 2009;6:648-662). Neuroprotective action includes methods that support the survival of neurons or their work in the context of pathological stress (traumatic, toxic, ischemic or metabolic events). Thus, to date, the treatment of most peripheral and central NP neuropathies (painful diabetic neuropathy, distal polyneuropathy, distal sensory, HIV-related and antiretroviral therapy, chemotherapy-induced peripheral neuropathy, postherpetic neuralgia, MS pain and post-stroke pain, etc. .) are directed to this area (Bordet T and Pruss RM. Targeting neuroprotection as an alternative approach to preventing and treating neuropathic pain. Neurotherapeutics 2009; 6:648-662) (Flatters SJL, Bennett GJ. Studies of peripheral sensory nerves in paclitaxel- induced painful peripheral neuropathy: Evidence for mitochondrial dysfunction Pain 2006; 122: 245-257) (Jaggi AS, Singh N. Mechanisms in cancer-chemotherapeutic drugs induced peripheral neuropathy. Toxicology 2012; 291 :l-9). Particular attention is paid to glutamatergic dysfunction, nitro-oxidative stress, mitochondrial dysfunction, apoptosis, trophic factors, neuroinflammation, and changes in intact fibers caused by neurodegeneration (Bordet T and Pruss RM. Targeting neuroprotection as an alternative approach to preventing and treating neuropathic pain. Neurotherapeutics 2009 ; 6:648-662) (DeLeo JA, Sorkin LS, Watkins LR, editors. Immune and glial regulation of pain. Seattle: IASP Press; 2007) (Park ES, Gao X, Chung JM, Chung K. Levels of mitochondrial reactive oxygen species increase in rat neuropathic spinal dorsal horn neurons Neuroscience Letters 2006;391:108-111). Biologists have proposed the development of new methods of pain treatment based on the participation of the electron transport chain in ATP-dependent neuropathies (Joseph EK, Levine JD. Mitochondrial electron transport in models of neuropathic and inflammatory pain. Pain 2006; 121:105-114) (Joseph EK, Levine JD. Multiple PKCεdependent mechanisms mediating mechanical hyperalgesia. Pain 201 O; 150:17-21). Mitochondrial dependence of nerve growth factor-induced mechanical hyperalgesia has also been noted in Chu C, Levine E, Gear RW, Bogen O, Levine JD. ).

According to these ideas, many immune mediators involved in the process of Wallerian degeneration (WD), which occurs when the continuity of the nerve fiber is broken, have been proposed as new targets for the treatment of NP drugs (Debovy P. Wallerian degeneration and peripheral nerve conditions for both axonal regeneration and neuropathic pain induction Annals of Anatomy 2011; 193: 267-275) 311). The role played by neuroimmune activation, microglianeural signaling, and oxidative stress has also been recognized in increased transmission after nerve injury (Berger JV, Knaepen L, Janssen SPM, Jaken RJP, Marcus MAE, Joosten EAJ, Deumens R. Cellular and molecular insights into neuropathy-induced pain hypersensitivity for mechanismbased treatment approaches. Brain Research Reviews 2011; 67: 282-310) 17-21) (Austin PJ, Moalem-Taylor G. The neuro-immune balance in neuropathic pain: Involvement of inflammatory immune cells, immune-like glial cells and cytokines Journal of Neuroimmunology 201 O; 229:26-50) (Salvemini D , Little JW, Doyle T, Neumann WL, Roles of reactive oxygen and nitrogen species in pain, Free Radical Biology &Medicine 2011;51:951-966).

Therefore, new strategies aimed at neuroprotective action and, in particular, neuroinflammation for the treatment of NP are currently at the stage of research and development (Bordet T and Pruss RM. Targeting neuroprotection as an alternative approach to preventing and treating neuropathic pain. Neurotherapeutics 2009 ; 6:648-662) (Stavniichuk R, Drel VR, Shevalye H, Maksimchyk Y, Kuchmerovska TM, Nadler JL, Obrosova IG. Baicalein alleviates diabetic peripheral neuropathy through inhibition of oxidative-nitrosative stress and p38 MAPK activation. Experimental Neurology 2011; 203:106-113).

Aging and neurological and psychiatric disorders cause neuronal damage and death. Among the frequent and relevant changes in the nervous system, we have included, among others, neuronal degeneration, ischemia, inflammation, immune responses, trauma, and cancer. As a result, neurons may die within minutes or hours, or they may sustain the initial injury in a damaged state, activating neurodegeneration and finally also leading to cell death.

Neurodegeneration in Parkinson's disease, 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, endogenous antioxidant depletion, decreased expression of trophic factors, ubiquitin dysfunction -proteasome system and expression of proapoptotic proteins lead to neuronal necrosis.

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

Neuroprotective action is aimed at maintaining, 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 neuroprotective action 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 harmful processes that cause damage to axons, neurons, synapses and dendrites.

Neuroprotective action is a mechanism and strategy used to protect against neuronal damage or degeneration of the CNS as a result of a chronic neurodegenerative disease (Alzheimer's disease, Parkinson's disease). The goal of neuroprotective action is to limit dysfunction/death after CNS injury and attempt to preserve the greatest possible integrity for cellular interactions in the brain, resulting in intact neuronal function.

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

There is a wide range of neuroprotective products available or under investigation, and some products have the potential to be used for more than one disease, given that many of the mechanisms of damage to neural tissues are similar. Products with neuroprotective effects are grouped into the following categories: free radical scavengers, anti-excitotoxic agents, inhibitors of apoptosis (programmed cell death), anti-inflammatory agents, neurotrophic factors, chelating metal ions, 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, finally, cell death in neurodegenerative diseases.

Studies in both animal and human models show that the imbalance among oxidant particles generated by brain metabolism and antioxidant defense mechanisms causes what is known as oxidative stress, when these defense systems become less effective and disrupted. This oxidative stress increases with age and has been found to be among the main causes of the pathogenesis of AD (Neurobiol. Aging 2007, 28, 1009-1014), possibly related to neuronal mitochondrial dysfunctions.

We also know that foods with antioxidant properties are able to prevent amyloid peptide-induced apoptosis as well as changes in Ca2 homeostasis in cortical neuron cultures (Lite Sci. 2000, 66, 1879-1892). 66, 1879-1892). Therefore, there is a great need for 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 disease. .

Generally speaking, treatment strategies are often based on the modulation of one factor of the proposed lesion. Although we can observe that these treatments are beneficial in very limited animal models, they are less likely to be effective in more complex human disorders with higher severity in a genetically diverse population (Faden and Stoica, 2007. Arch Neurol 64: 794-800). To a large extent, given the putative mechanisms of neuronal death such as oxidative stress, mitochondrial dysfunction, added proteins, apoptosis, and inflammation (oudim, M.B., and Buccafusco, JJ (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 different, we would need separate compounds that have multiple effects on multiple lesion mechanisms.

The compound (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1 H-pyrido[2,3-b][1,25]benzodiacepine) (JM-20), which is described in the patent document of the Republic of Cuba CU 23879 on its chemical structure, may have an effect on cardiovascular, cerebrovascular and other diseases associated with the central nervous system.

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Unexpectedly, researchers have found that the compound JM-20 has a pronounced therapeutic effect on the CNS, preferably for the treatment of diseases such as various types of dementia, Parkinson's disease and pain.

Thus, the essence of this 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, Parkinson's disease and pain.

JM-20 is a benzodiazepine product and benzodiazepines have been widely reported to induce dementias (Huber-Geismann, F, 1994) (Rosenberg, P. B., 2015). (Shash, D., T. Kurth, et al. 2015) (Zhong, G., Y. Wang, et al. 2015)), however, JM-20 surprisingly 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 in neurodegenerative diseases such as dementias, Parkinson's disease and neuropathic pain has not been established.

As an additional 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 admixture with at least one medium, diluent and/or carrier, chemically inert, non-toxic, hereinafter recognized as excipients included in the proposed compositions of medicinal products.

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

The drug compositions described will contain suitable excipients for each formulation. The compositions are usually obtained using methods existing in the art. The excipients are selected for 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, among which, among others, are these forms of release: 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 to their administration, mixed with excipients, such as auxiliary liquid, solid or semi-solid substances consisting of organic and inorganic substances, of natural or synthetic origin. Some of these include: solid fillers, diluents, agglutinants, solvents, emulsions, lubricants, disintegrants, glidants, fragrances, dyes, pigments, polymers, sweeteners, plasticizers, absorption enhancers, penetration enhancers, surfactants, auxiliary surfactants, special oils and/or buffer systems which impart physical, chemical and/or biological stability to the active compounds or their physiologically acceptable salts.

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, ethylcellulose, 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, microbeads, nanoparticles, pellets, powders to be diluted or capsules containing a compound of general formula III or its salts, enantiomeric forms and redox products according to this invention can be used for immediate or modified release.

The selected form of the drug, 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 a granulate,

- 7 041342 which is dried in a complete process in a fluidized bed and mixed with magnesium stearate and talc, then tablets are obtained using a system of rotary dies for their manufacture, finally the tablets are coated with hydroxypropyl cellulose, polyethylene glycol 4000, titanium dioxide and a coloring 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 prepared either by the wet granulation method shown above or by direct compression 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 may contain a compound of general formula III or its products in microgranules, nanoparticles or matrix systems using excipients such as polyethylene oxide, hydroxypropyl cellulose 2910, magnesium stearate, sodium chloride, iron oxide red, 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 while providing 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 its salts, enantiomeric forms and its physiologically acceptable redox products can be dosed in the form of hard gelatin or cellulose capsules, for example, containing a mixture of the active pharmaceutical ingredient with commonly used excipients in solid form, such as those described for tablets; said mixture can be obtained by dry, wet granulation, extrusion, tabletting, microcapsules or microtablets. For doses in soft gelatin capsules, conventional methods of manufacture are used and may involve mixing 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 application. education.

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

For rectal administration, a compound of general formula III (JM-20) or its salts, hydrates, crystalline forms, enantiomers, isomers, metabolites, physiologically acceptable prodrugs can be dosed as suppositories, foams or rectal microclyster solutions, which can contain a mixture of active compounds with base of neutral solid fat (Witespol 45) or some other similar media suitable for their preparation; sorbitan monooleate, polysorbate 20, wax emulsifier, colloidal silicone anhydrate, sodium meta-bisulfite, disodium edatate, methyl parahydroxyl benzoate, sodium phosphates, macrogol 300, glycerin, water, propane, isobutene and n-butane.

For liquid oral administration, a 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 other things, carboxymethyl cellulose or other thickeners; it may contain color, flavor, sweetener (sucralose, aspartame, cyclamate, stevia) and preservative (parabens, benzoates). These liquid dosages can be obtained by diluting 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 injectable solutions. These solutions may contain stabilizing, preservative and/or buffering ingredients.

For subcutaneous administration, a 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 implants using excipients such as silicon elastomers and

- 8 041342 anhydrate colloidal silicone, although other pharmaceutical polymers can be used to formulate the pellet.

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 as 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 layer of silicone inside the peel layer (at a nominal release rate of 15 mg/day, on a surface area of 12.75 cm ² ) .

Implementation examples

Preparation of Compound III

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

Obtaining compound III from IIa.

To prepare compound III (JM-20), we started with an ethanolic solution of compound Ha and added equimolar amounts of orthophenylenediamine, in absolute ethanol as solvent, with stirring. Equimolar amounts or greater (1-2 equivalents) of triethylamine (FIG. 2, method A) were added to the reaction mixture, stirring was maintained, 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).

Obtaining 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 greater (1-2 equivalents) of triethylamine (FIG. 3, method A) were added to the reaction mixture, stirring was maintained, or sufficient amounts (1-1.5 equivalents) of sodium hydroxide were added by controlled dripping and with continuous stirring of the reaction mixture ( Fig. 3, method B).

The structure of the JM20 compound isomer is shown in the drawing.

Obtaining compound III in the form of halide salts

The corresponding hydrochloride or hydrobromide salts III (JM-20 halohydrates) 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 mole %), a catalytic process occurred which reduced the reaction time and slightly reduced the reaction yield (FIG. 4).

JM20 hydrochloride (IIIa) was properly prepared from IIa, and when appropriate amounts of HCl were added, a catalytic reaction occurred. Compound JM-20 can also be prepared as hydrobromide (IIIb) from IIb (Fig. 4), with bromide characteristics being a better target group than chloride, the intramolecular nucleophilic substitution reaction is facilitated giving a fast and efficient reaction to give JM-20 in form of hydrobromide.

Preparation of compound III as fumarate salt

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

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

Preparation of Compound III as Phosphate Salt

JM-20 phosphate salt (IIId) can be prepared in a manner similar to that described above from IIa or IIb by adding equimolar amounts of phosphoric acid at the start of the reaction, or by adding phosphoric acid to IIIa or IIIb in an ethanolic solution with stirring (Fig. 6 ).

Preparation of Compound III as Sulfate Salt

JM-20 sulfate (IIIf) can be prepared from IIa or IIb or based on halohydrates IIIa or IIIb as shown in FIG. 7.

- 9 041342

Getting the composition in the form of a tablet

Each 120.00 mg tablet contains the following:______________

Component Quantity Function JM 20 40.00 mg Active ingredient Corn starch 23.00 mg Disintegrant Polyvinylpyrrolidone K-25 4.00 mg Agglutinin Monohydrate 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 drying

Brief description of the technological process.

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

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

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

4. Load the mixer with the active ingredient, cornstarch and lactase (internal phase components). Mix for 15 min.

5. Slowly add the agglutinin solution using a peristaltic pump, adjusting to the required degree of moisture with water and grade C ethyl alcohol (1:1) if necessary. Grind in a mill at low speed.

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

7. Mix dry granulate with lubricants for 10 minutes.

8. In a high-speed centrifuge, compress the mixture using a flat, beveled, and grooved die with a diameter of 6.4 mm (1/4 PBR) to obtain tablets with the following parameters:

mass: 120.0 mg ± 10%;

height: 2.6 ± 0.10 mm;

hardness: 4.0 ± 1 kg-s;

brittleness: less than 1%.

- 10 041342

Getting the composition for oral drops

Each ml (20 drops) contains the following:____________________

Component Quantity Function JM-2O 40.0 mg Active ingredient propylene glycol 300.0 mg Solvent medium Kollidon 25 160.0 mg Viscosity control aid 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 (Instant) 20.0 mg flavoring Purified water (enough) 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 the propylene glycol into the reactor.

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

4. Turn on Kollidon 25 by adding it little by little, stirring for at least 30 minutes, until it is completely gone.

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

6. Include the active ingredient in the result of the first stage in small portions, maintaining constant stirring for 30 minutes.

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

8. Dissolve the methyl paraben and propylene glycol in grade C ethanol in an auxiliary beaker or stainless steel container with a suitable capacity, stirring constantly until it is completely dissolved.

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

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

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

12. Slowly introduce the results of the previous step into the reactor vessel, stirring constantly and vigorously.

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

14. Remove the previously prepared volume with water. Mix until smooth.

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

16. Carry out final filtration; test organoleptic characteristics.

- 11 041342

17. Place the final formulation in amber glass bottles x 15 ml, with 15.0 ± 1.0 ml of solution into the bottle, cap properly using caps with drop reducers for oily products.

Getting the injection composition

Each vial (2 ml) of JM-20 contains the following: __________________________________

Component each ml contains Quantity per dose Unit of measurement Function JM-2O 5.0 mg 10.0 mg Active ingredient Cremofor ELP 527.0 mg 1054.0 mg Wednesday Hydrochloric acid 1n enough - - Not pH adjustable Absolute alcohol sufficient 1.0 ml 2.0 ml solvent *Nitrogen sufficient - -

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

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

3. Add one part of 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 to the reactor; repeat this operation until all active ingredients have been swept away and all dehydrated alcohol has been used up.

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 the residue with dehydrated alcohol, stirring it 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 min at 420 rpm.

9. Take 10 ml of the solution and send it to the laboratory for process control (assessments and pH).

10. Check the correct installation of the filling and nitriding systems.

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

12. After completing the process control, pressurize the reactor with nitrogen (0.7-1.0 bar) to push the solution through the Sartobran P filter cartridge with a porosity of 0.45+0.2 µm. Fill and seal the flasks, receiving doses of 2.2 ml of the 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 Laboratory of Organic Synthesis in the Department of Chemistry, University of Havana.

For various studies, JM-20 was suspended in carboxymethyl cellulose (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, and at the same time behavioral studies were performed. Beta-amyloid peptides (25-35) were oligomerized and made soluble according to the manufacturer's guidelines and administered by intracerebroventricular means at 100 pmol in 5 μl, to 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 Breeding Animals for Laboratory Research (CENPALAB, Mayabeque, Cuba) and after receiving them were subjected to adaptation to laboratory conditions for 7 days. They were kept at

- 12 041342 alternating cycles of 12 hours of light and dark, at a controlled temperature (22±2°C), 45-55% relative humidity, and they were given water and food freely on demand. All behavioral studies were performed from 09:00 to 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 Treatment of Laboratory Animals and according to national guidelines for animal experiments.

Experiment scheme

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

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

In each test formed 6 experimental groups of subjects selected at random (n=10, per group): healthy control group (CMC and saline); JM-20 group without damage (JM-20 8 mg/kg); the 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).

The effect on memory was assessed by the novel object recognition test and the forced alternation maze test using experimental designs suitable for independent investigation of the acquisition and retention of short-term and long-term memory.

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

Protocol 2. Evaluation of the effect of JM-20 on memory loss caused by aluminum chloride.

We evaluated the protective effect of two doses of JM-20 (2 and 8 mg/kg) on various types of memory, which was affected by acute intraperitoneal administration of scopolamine. For this purpose, JM-20 was administered from day 15 after the start of aluminum chloride administration (500 mg/kg) until the end of the behavioral studies. We evaluated the effect of JM-20 and aluminum chloride on three types of memory: spatial memory in the Morris water maze test and the T-maze test; recognition of the new by the test of recognition of new objects; and emotional-associative memory through 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- no 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 performed various in vivo studies of brain tissue. We studied the effect of JM-20 on various markers of oxidative stress, mitochondrial function, and activity levels of the AchE acetylcholinesterase enzyme.

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

Initially, we evaluated the effect of JM-20 on the viability of PC12 cells exposed to 1 μM concentration of beta-amyloid peptide 25-35 oligomers. This in vitro test evaluated the effect of 3 concentrations of JM-20 (3.125, 6.25 and 12.5 μM) dissolved in dimethyl sulfoxide (DMSO). Formed 6 experimental groups: the group of untreated control (NT); environment group (DMSO); amyloid beta group (AP 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 mice of the OF-1 line. We evaluated the effect of JM-20 on memory loss caused by beta-amyloid oligomers administered 7 days after intracerebroventricular administration of AP (100 pml) for 10 days, orally at 10 and 30 mg/kg doses. We formed 5 experimental groups: healthy control group (blank); group JM-20 without damage (blank + JM-20 30 mg/kg); amyloid beta group (AP 100 pmol+CMC); groups JM-20 beta-amyloid (JM-20 1 O mg/kg+AP 100 pmol) and (JM-20 30 mg/kg+AP 100 pmol).

Behavioral research 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 of plastic with three arms (55x20x20 cm) at an angle of 120° on a central platform.

The test was carried out in two sessions. In the first stage of training, we conducted a training phase during which each rat was placed on the central platform and allowed to freely explore the 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. Sleeve Entry Sequence

- 13 041342 was videotaped for 10 minutes for later analysis. The evaluation step was to allow the rats to freely explore the arms of the Y-maze for 5 min, including the one that was previously closed. The sequence of entries into the sleeves was recorded for 5 min for subsequent analysis. This test assessed the number of entries in the new arm (B), where the percentage of entries in this arm was directly proportional to the best memory in the studied animals. The percentage of correct entries in arm B was calculated based on the proportion of alternations drawn from all possible alternations, as shown in the following equation: % correct entries = (number of entries in B)/(total number of entries in three arms)x100. After each test, the maze was cleaned with a 40% ethanol solution to reduce the existence of olfactory marks.

Object recognition

The effect of JM-20 on memory in novel object recognition was assessed by an object recognition test. This test was performed 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 min, in two sections. On the second day, training was performed with learning evaluation in 2 stages of 5 min each, then considered as training (E) and testing phase (P), respectively. During stage E, rats were presented with 2 identical objects, referred to as familiar (F) objects. After 30 minutes, the P stage began and the rats were exposed to the familiar object F and the new object (N). The tests were recorded for the analysis of the study of objects, determined by the time of the study conducted by each animal for each object. The 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 in diameter, 0.60 m in depth). We spent 5 days of training, 4 sections per day, and on the 6th day training was evaluated. Animals had to learn to find a submerged platform, fixed in a fixed position, in all sections of the training. Several external keys were placed and kept in a fixed position during the entire experiment. At each training session, the animals were placed in the water, face to the tank wall, at one of the 4 exit sites until they found the platform for a maximum time of 1 min. On the day of evaluation, the platform was removed. We quantified the escape delay (time taken by the animal to find the platform) in the training trials, the time the animal remained in the platform quadrant during the learning test, and the distance until the platform site was found.

Passive Avoidance

The passive avoidance test was performed based on the methodology described by Kohara et al (22), with some modifications. We used passive evasion equipment (UGO Basile) consisting of 2 chambers that were lit (30x30x30cm) and dark (10x20x12cm) connected by a guillotine access door. The dark chamber was equipped with an electrical circuit.

The test was conducted in 2 stages for 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 time of 90 s. Once the rat entered the dark chamber, the access door was closed and the rat received an electrical charge of 1 mA for 5 s.

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

Study of mitochondrial function. Isolation of cerebral mitochondria

Mitochondria were isolated with a differential centrifuge. Animals were sacrificed 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 centrifuged at 12,000 g for 10 minutes. 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 washed gently with isolation buffer without EGTA. The microlevel method was used to determine the concentration of proteins using bovine serum albumin in a standard scheme (Mirandola et al., 2010).

Mitochondria were incubated in KCl 130 mmol/l, MgCl ₂ 1 mmol/l and HEPES-KOH, phosphate 2 mmol/l, pH 7.4. Mitochondria (1 mg protein/ml) were activated with 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 safranin O fluorescent marker (10 µM) was used in a POLARstar Omega fluorescent spectrophotometer (Germany) at 495/586 nm (excitation

- 14 041342 ni / issue).

Swelling of mitochondria

Mitochondrial swelling was controlled by a decrease in apparent 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 fluorescent spectrophotometer (Germany).

Production of reactive oxygen species

Reactive oxygen species (ROS) was 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 Tests

Rats were sacrificed by decapitation; their brains were removed and quickly dissected to separate 2 areas, the hippocampus and the prefrontal cortex of both hemispheres (23). These areas were homogenized in sodium phosphate buffer (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 centrifuged at 6000 g for 20 min in an Eppendorf centrifuge (Germany, 5424R). The brain homogenate ratios were kept at -80° C. until they were used up. Their protein concentration was determined by the Lowry method (24) at POLARstar Omega (Germany) using a standard of bovine serum albumin.

Acetylcholinesterase (AChE) activity

The activity of the AChE enzyme present in the extracted hippocampal and prefrontal cortex homogenate was measured spectrophotometrically using the method described by Ellman (25) with minor modifications (26). The test was carried out on 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 with an interval of 1 min in a POLARstar Omega spectrophotometer (Germany). Enzymatic activity was calculated and expressed as µmol hydrolyzed (AcSCh) for 1 min per mg of protein.

Studying the parameters of the 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 Tris 0.2 M - HCl 0.2 M (pH 8.2), 0.05 ml EDTA 60 mM, 0.1 ml homogenate and 0.05 ml solution of pyrogallol 7.37 mm. Absorbance was read at 420 nm for 1 min using a POLARstar Omega spectrophotometer (Germany). The 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 per 1 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). The absorbance was read at 240 nm, for 10 and 70 s, in a POLARstar Omega fluorescent spectrophotometer (Germany). The results were expressed in µmol H ₂ O ₂ decomposed per minute per mg of protein.

Decreased levels of glutathione (GSH).

GSH levels were set 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)/αcetone (0.6 mM) solution were added to the tray. Absorbance was read at 412 nm using a POLARstar Omega spectrophotometer (Germany). The results were calculated using the molar extinction coefficient of the obtained chromophore (1.36x104 (mol/l)' ¹ cm' ¹ . The results were expressed as nmol GSH per mg of protein.

Levels of lipid peroxidation.

Lipid peroxidation levels were determined by detection of malondialdehyde (MOA) formed by reaction with thiobarbituric acid (30). The reagent medium was formed with 350 µl of a mixture of trichloroacetic acid:thiobarbituric acid:chlorhydric acid, 100 µl of brain tissue homogenate and 50 µl of diterbutylhydroxytoluene (BHT). The mixture was homogenized in a TALBOYS vibrating mixer (USA) and placed for 5 min in a water bath with a Grant OLS200 thermostat (USA) at 100°C. Then it was centrifuged at 3000 rpm for 10 min in an Eppendorf centrifuge (Germany, 5424R). MOA, formed as a product of lipid peroxidation in the brain tissue, reacts with thiobarbituric uric acid and gives a colored compound, which was determined by spectrophotometry at 535 nm on a POLARstar Omega fluorescent spectrophotometer (Germany). The MOA concentration was calculated taking into account the molar extinction coefficient of the obtained colored compound (1.56x105 M ^-1 cm ^-1 ) and depending on the following

- 15 041342 equations:

C=DOx 10 ⁴ nM

1.56 where C is the concentration of MOA to be determined and D.O. represents the detected absorption. The results are expressed as nanomoles MOA per mg of protein.

Histological examination.

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

Statistical analysis.

For statistical processing and analysis of the obtained results, we used the GraphPad Prism 5.0 program (GraphPad Software Inc., USA). Data were expressed as mean±EEM (standard mean error) and we tested for normality and homoscedasticity. One-way analysis of variance (ANOVA) was performed, followed by Taki's test for multiple comparisons to compare between different experimental groups. A p<0.05 value was considered statistically significant.

The results are shown in FIG. 8-13.

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. 14.

Unilateral damage to the compact part of the substantia nigra by injection of 6hydroxydopamine.

Materials and methods Experimental animals

Used adult male Wistar rats, weighing 200 to 250 grams at the beginning of the experiment, coming from the National Center for Animal Breeding for Laboratory Research (CENPALAB), Havana, Cuba. Animals were housed at the Center for Research and Development of Medicines (CIDEM) in plastic translucent boxes at an average temperature of 23°C (22±2°C), with ad libitum food and water, and 12 hour periods of light and dark.

Ethical Considerations

This study complied with the standards set out in the code of ethics for animal experiments. We have guaranteed the genetic integrity of the animals by acquiring 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 lies in the fact that the results of the experiment can be reproduced in any other place 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, in the range of obligations established for tropical zones (22±2°C). Temperature control is important given that high temperatures are stressful for animals and temperatures above 38°C can lead to 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.

Connection introduction.

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 carboxymethyl cellulose (CMC) at 0.05%, intragastrically by cannula. There were 5 experimental groups: group I (media/saline with ascorbic acid, n=6), group II (animals treated with 6-OHDA, n=8), group III (animals treated with 6-OHDA, n=8),

- 16 041342 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 (6-OHDA treated animals co-treated with 40mg/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. The results 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 significant difference with significance levels p<0.05 (*), p<0.01 (**) and p<0.001 (** *) relative to the control used in the experiment. To compare all experimental values with different controls and with respect to each other, we performed Takay's test (GraphPadPrism 5) with p < 0.05 (*), p < 0.01 (**), and p < 0.001 significance levels. (***).

Methods of defeat in experimental models designed for rats

Unilateral damage to the substantia nigra by 6-hydroxydopamine injection in rats.

For the purpose of unilateral dopaminergic denervation of the striatum (right hemisphere), rats were anesthetized with chloral hydrate (0.4 g/kg of body weight, ip, Merck (Darmstadt, Germany)) and placed in a frame designed for stereotaxic surgery (Stoelting Instruments, USA), where they the right substantia nigra was injected with a physiological solution of the neurotoxin 6-OHDA-HBr (8 µg/3 µl, also containing 0.2 mg/ml ascorbic acid as an antioxidant) (Pav0n Fuentes, Nancy (2007) Efecto de la inactivacion de los receptores dopaminergicos D2 y de la manipulacion del nύcleo subtalamico sobre la conducta motora en modelos de hemiparkinsonismo en roedores Doctor en Ciencias de una Especialidad, Universidad de Ciencias Medicas de La Habana). The coordinates were calculated with Bregma as the reference point according to the Paxinos and Watson Atlas (Paxinos, G. and Watson, C., The rat brain in stereotaxic coordenates. 2nd the Academic Press. New York, 1986), referenced in the table .

Stereotaxic coordinates expressed in mm relative to Bregma ____ (excluding DV referring to the dura/surface)

Coordinates AR -4.4mm ML 1.2mm DV 7.8mm sharp streak -2.4 mm below the interaural line

When appropriate, the neurotoxin was slowly injected at a flow rate of 1 µl/min using a Manilton syringe (3 µl), remaining in place for 5 min. after completion of the injection.

IV. behavioral tests.

A. Checking the forelimbs for asymmetry.

In this test, rats were placed inside a transparent acrylic cylinder 20 cm in diameter and 30 cm high, which did not allow the animal to reach the edge. The cylindrical shape contributes to the inherent behavior of vertical wall exploration with their hind limbs when the rats were placed in a place they did not know. (Chan, H., Paur, H., Vernon, A.C., Zabarsky, V., Datla, K.P., Croucher, M.J., Dexter, D.T., 2010. Neuroprotection and functional recovery associated with decreased microglial activation following selective activation of mGluR2/3 receptors in a rodent model of Parkinson's disease ParkinsonsDis.pii, 190450). After placing the animal, the number of touches made by the animal with both front paws, right or left, is quantified up to 20 touches of the animal against the tank wall.

Animals affected by 6-OHDA on one side tend to use the less affected paw, in our case the left paw. The percentage of asymmetry presented by each animal is quantified by the following formula (Roof RL, Schielke GP, Ren X, Hall ED (2001) A comparison of long-term functional outcome after 2 middle cerebral artery occlusion models in rats. Stroke 32: 2648-2657 ).

(% ipsilateral touches) - (% touches on the other side) = (% asymmetry)

Research activity.

To assess the vertical exploratory activity of animals, we used the exploratory activity test; the animal was placed in a transparent plexiglass cage, 41x41x33 (h) cm,

- 17 041342 (UGO BASILE, MultipleActivityCage catalog #47420. Data output is controlled by the 52050-04 Data Acquisition Software Package (Windows® based). The cage is supported on a solid black plexiglass base that has 4 vertical steel bars with steel grooves, so that the horizontal/vertical detection systems are correctly attached.The sensors are IR systems capable of registering the movements of animals in forms or rather vertical examinations.The data is controlled by a computer.Animals are placed in the center of the box so that they can examine it.The box is placed in a room isolated from the researcher and environmental noise and with low lighting for 5 minutes The researcher takes the number of times the animal vertically explores the walls of the box when the light beams of the sensor in the box are interrupted (Cools, A. R., R. Brachten, D. Heeren, A. Willemen, and B. Ellenbroek, 1990. Search after neurobiological pr ofile of individual-specific features of Wistar rats. Brain Res. Bull. 24:49-69). The results are shown in FIG. 15.

Tonic pain.

We studied the effect of JM-20 (10, 20, 40 mg/kg, po) on pain behavior in a tonic pain model (5% formalin test) in rats. (Dubuisson D, Dennis SG. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine and brain-stem stimulation in rats and cats. Pain 1977;4:161-174).

The results are shown in FIG. 16A, B and 17.

neuropathic pain.

We investigated the effect of JM-20 in a permanent sciatic nerve stenosis (CCI), NP model (Bennett MI, Rayment C, Hjermstad M, Aass N, Caraceni A, Kaasa S. Prevalence and aetiology of neuropathic pain in cancer patients: A systematic review Pain 2012;153:359-365)

The results are shown in FIG. 18-22.

Carrageenan-induced peritonitis.

Subsequently, we designed various experiments in a rodent model of CA-induced peritonitis to investigate the possible anti-inflammatory activity of JM-20 under these conditions.

The results are shown in FIG. 23A-E.

Claims (1)

CLAIM Application of the formula compound A Ύ νο ₂ H ₃ CH ₂ COOC. Ύ I N / I I y—/ H H in (JM-20) in the treatment of Parkinson's disease. Scheme for obtaining Ila and IIb