Pharmacological drug strategies in Alzheimer's Disease

Introduction. Alzheimer's disease (AD) is a neurodegenerative disorder with a poor prognosis and no cure that affects millions of people worldwide. Medicines today in clinical studies try to investigate possible therapeutic effects that progress or stop the disease. Objective. This review is necessary to create a current panorama in 2021, describing the main pathways under study to inhibit important pathways of disease progression, such as pathways cholinergic system and ROCK inhibitors, as well as new perspectives of treatment with possible combination of drugs, to decrease neuroinflammation and change the course of the disease.


INTRODUCTION
In the XXIst century, life expectancy has increased considerably, to a great extend thanks to advances in biomedicine which have decrease the mortality caused by multiple pathologies that some years ago were uncurable.
However, this increase has also been accompanied by the appearance of age-related diseases, such as Alzheimer´s disease (AD), which prevalence is rising due to the aging of the world population 1 , entailing a great social and personal burden at the health level 2 . AD is the most common form of dementia, progressive and, nowadays, uncurable that begins approximately 15-20 years before symptoms onset.
Histologically, AD is characterized by the accumulation of βamyloid (Aβ) plaques and neurofibrillary tangles (NFT) in the brain. Specifically, Aβ plaques are extraneuronal deposits composed by aggregates of various Aβ peptides derived from amyloid precursor protein (APP). By contrast, NFT are intracellular filamentous inclusions of hyperphosphorylated Tau.
AD is classified into 2 groups: familial AD (FAD) and late onset or idiopathic AD (LOAD). FAD correspond to <5% of cases 3 and normally appears between 30 and 50 years of age 4 . It has been associated to mutations mainly in three genes: APP, presenilin 1 (PSEN1) and presenilin 2 (PSEN2) 5 .
By contrast, LOAD is the most common form which affects >95% of patients with this pathology. Its aetiology remains unclear; however, environmental and genetic factors seem to be involved in its development 6 , including APOE 7 .
Throughout the years, different hypotheses have been proposed to explain the origin of the disease, of which the amyloidogenic hypothesis has been one of the most accepted, proposed approximately thirty years ago. This hypothesis suggests that the accumulation of harmful Aβ peptides in the nervous system would be the cause of AD.
These plaques are produced through APP processing in which the enzymes β-secretase and γ-secretase cleavage APP, forming Aβ deposits in the human brain 8 . Under physiological conditions, Aβ is quickly removed from the human brain by the withdrawal mechanism, ensuring a proper functioning of the system. However, in pathological situation, it is believed that there is a defect in the removal mechanism or excessive production which, culminates in an increase level of Aβ peptides, specifically those containing 42 amino acids 7,9 . These peptides have a high tendency to bond generating oligomers, protofibrils, fibrils and Aβ plaques [7][8][9] . Likewise, it is now well known that the disease correlates better with increased levels of soluble Aβ than with plaque formation, causing alterations in dendritic spines and synapses that would be responsible for the cognitive alterations that are observed at the beginning of the disease in the stage of mild cognitive impairment (MCI). In fact, its excess together with Tau accumulation impairs synaptic communications between neurons leading to neuronal death 10 .
Despite the clinical symptoms observed and the biomarkers of cerebrospinal fluid (CSF) and positron emission tomography (PET) indicated strong evidence on AD in living patients, the definitive diagnosis of AD can only be achieved by evaluating post-mortem brain tissue 7,11 .
Abnormalities found in the CSF are low levels of Aβ peptides and increased levels of tau protein 11 , not enough to conclude its diagnosis in its totality. So far, treatment for AD is restricted to the symptomatic level and there is no strategy to combat the progressive neurodegeneration caused by AD and hence its fatal outcome 12 13 .
Among these risk factors, the most strongly associated with the onset of LOAD is advancing age, cardiovascular changes and especially, the allelic variation of ApoE.
However, in recent years diet-related factors such as obesity and associated diseases including type 2 diabetes mellitus (T2DM) have been gaining ground, which accelerate aging rate by triggering the pathophysiological cascade of LOAD 13 .
Another risk factor for LOAD widely discussed in the literature which interestingly also contribute to T2DM development, is the neuroinflammation caused by microglia.
Microglia are macrophages that rest in the brain and spinal cord, and it has been classified as M2 or resting and M1 or activated.
In physiological homeostatic equilibrium conditions, the resting M2 microglial cells are responsible for the removal of Aβ from the brain. In the case of LOAD, it has been suggested that Aβ could be the responsible of a process This protection occurs by reducing the impact on pathophysiological processes that these behaviours provide.
However, none of them has been able to delay or modify the course of this devastating disease. For this reason, it is necessary to develop drugs that stop its progression, that is, the patient remains in the state of MCI and does not develop LOAD.
Therefore, the goal of this review is to analyse drug treatments to seek new solutions for the hopefully near future, minimizing the personal and social damage caused by AD.

Current drugs for the treatment of Alzheimer's Disease
Cholinergic system drugs have been shown to be an alternative treatment for AD, since studies have indicated that cholinergic neurons located in the basal forebrain are highly affected, contributing to memory and attention deficits 20,21 . Acetylcholine (Ach) is the neurotransmitter used by cholinergic neurons which has a fundamental role in cognitive and motor processes, from memory acquisition to the recovery process 22,23 . In AD brains, it has been observed that a clear reduction of Ach in the nervous system due to significant loss of neurons 24 . Therefore, the most drugs approved by the food and drug administration (FDA) have focused on increasing Ach levels in the synaptic cleft by inhibiting acetylcholinesterase (AChE) enzymes, which also has been related to Aβ and Aβ fibrils formation and growth 25 .
The use of AChE inhibitors provides a significant improvement in the functional and cognitive aspects at an initial stage of LOAD impacting patients' quality of life.
However, there is no scientific evidence that this medication delays the disease's progression 26 .
The main drugs currently used as AChE inhibitors are donepezil, galantamine and rivastigmine 27  On the other hand, the fourth drug approved by the FDA for the treatment of AD is Memantine, a non-competitive antagonist of the NMDA receptor (channel blocker) that protects neurons from glutamate excitotoxicity by preventing their apoptosis and has low toxicity. This noncompetitive antagonism will never exceed the concentration of the agonist, which in this case is glutamate or glycine, having a role in controlling the excitotoxicity of glutamate, which deal to the prevention of nerve cells death. Therefore, it provides a therapeutic power in delaying the progress of AD 33 . The dose administered is 20 mg/day and it has been shown a significant improvement in patients with cognitive impairment, but the damage caused by the disease's progress is not repaired 34   Other drug with promising results is Albutein®  Early 2021, the pharmacist Novo Nordisk published a note that the medication Semaglutide, which is already being studied for its efficacy and safety for T2DM, will start phase 3 with early AD patients. This drug is a hormone, with a metabolic function (10.25%; n=4) that stimulates insulin signaling. The aim of this strategy is to increase insulin signaling, due to it is thought to will improve the transport of glucose in the brain, reducing neurodegeneration 45 . In fact, the use of metformin (insulin synthesizer) in previous phases of the study has demonstrated a significant cognitive and memory improvements, even though it was a study with a small sample (n=20), indicating a tendency to use this drug as a treatment for AD 46 .
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ROCK inhibitors as a strategy to improve cognition in AD
The process of storing information in the brain occurs   In line with this process, it has been demonstrated that small changes in dendritic spines, such as density, number, NFTs, suggesting that these molecules and structures are strongly correlated with cognitive impairment in AD [54][55][56] .
More specifically, the selective loss of thin spines is strongly linked with impaired ability to learn in aged rhesus monkeys 57 . These alterations could be related with the MCI that is detectable in very early stages in AD patients 54 , supporting that synaptic loss is central to the progression of the pathology 58 and providing cellular evidence that remodelling the structure of dendritic spines may be a mechanism of cognitive resilience. All these data sustain that synaptic structure and synaptic activity are clearly correlated with the cognition capacity. Therefore, the cellular and molecular events that control synapses can be an early target to treat cognitive impairment in AD.
The kinases proteins (ROCKs/Rho-quinase/Rho-quinase associated) belong to the serine/threonine family of small Globally, Rho GTPases function as key intracellular switches that regulate axonal and dendritic growth together with synapsis structure and activity through actin binding proteins such as Cof-1.
Hydroxyfasudil, which is a pan-ROCK inhibitor, is being used in studies of older rats and indicates improvement in the learning and working memory of these animals. The commercial drug (Fasudil), in several different dosage protocols, is considered safe and well tolerated in humans.
Moreover, the results of the study associated with the positive clinical use of the drug corroborate that this ROCK inhibitor improves cognition and memory dysfunction in humans 70 .
Interesting research carried out in the hippocampus of an in vivo mouse model suggested that ROCK2 is the most critical isoform for dendritic spine formation and synaptic function when compared to ROCK1. Interestingly, it was found that the ROCK2 isoform is involved in both presynaptic and postsynaptic transmission, while ROCK1 is involved only in postsynaptic transmission 71 .
In addition, it is well known that NSAIDs have been studied as a potential treatment of AD to inhibit the neuroinflammatory process as selective and non-selective cyclooxygenase (COX) inhibitors. NSAIDs such as ibuprofen, indomethacin, and sulindac have also been proposed to reduce the formation of Aβ42 72 , by inactivating RhoA 73 .
Therefore, the inhibition of ROCK by some NSAIDs is not related to the inhibitory action of COX and constitutes a therapeutic target in the prevention of AD.
Aβ is also produced from ROCK2 phosphorylation in APP at some sites (T654 and S498), which highlights the importance of ROCK2 inhibition as a protective factor for AD development, as ROCK2 acts as one of the mediators in axonal degeneration, leading to apoptosis 74 .
SR3677 (ROCK2 inhibitor) reduced the action of the APP cleavage enzyme from β (BACE1) and the production of Aβ in mice. Alteration of the endocytic distribution of BACE1 and promotion of traffic from APP to lysosomes was also identified in this study. In addition, SR3677 blocked ROCK2 phosphorylation in threonine 654 (T654). These observations suggest that ROCK2 inhibition reduces levels Aβ through independent mechanisms in the rat brain 75 .
As a therapeutic strategy for age-related memory loss and AD, pharmacological inhibition of ROCK1 and ROCK2 can be a promising treatment as it acts on increasing the density of the dendritic spines favouring synaptic transmission which improves the transmission of brain information and neural plasticity. However, more research should be done in order to clearly elucidate the specific role of each isoform and its specific targets.
Challenges and future of research in drug development for AD The biggest challenge for the scientific class is to find an effective drug that reduces, slows down or regresses AD.
Advances with monotherapies are evident in clinical tests.
However, none of them has achieve its goal. In consequence, it has arisen the need to consider new strategies. One of them is the design of combinatorial therapies since it is well known that it has been effective for other diseases which share similar complexity, the fact that several pathogenic pathways or multiple targets are involved in its development.
In fact, it has been demonstrated that the combination of memantine + AchE inhibitor produced positive effects in patients with AD 77  We can conclude, AD is a complex disease where numerous pathways are involved in the neurodegenerative process. This complexity makes it necessary to address the disease by acting on different therapeutic targets such as the decrease in A levels, decrease the neuroinflammatory process, act by maintaining the stability of the dendritic spines, in addition to, acting on the mitochondria to maintain adequate levels of ATP and decrease levels of oxidative stress. Finally, it must be considered that these drugs should be administered in a state of the MCI in the disease to be effective and to be able to modify the course of the disease and delay the process of cognitive loss.