Advances in Alzheimer’s disease’s pharmacological treatment

Alzheimer’s disease (AD) is the most common type of dementia in the elderly. Several hypotheses emerged from AD pathophysiological mechanisms. However, no neuronal protective or regenerative drug is available nowadays. Researchers still work in drug development and are finding new molecular targets to treat AD. Therefore, this study aimed to summarize main advances in AD pharmacological therapy. Clinical trials registered in the National Library of Medicine database were selected and analyzed accordingly to molecular targets, therapeutic effects, and safety profile. The most common outcome was the lack of efficacy. Only seven trials concluded that tested drugs were safe and induced any kind of therapeutic improvement. Three works showed therapeutic effects followed by toxicity. In addition to aducanumab recent FDA approval, antibodies against amyloid-β (Aβ) showed no noteworthy results. 5-HT6 antagonists, tau inhibitors and nicotinic agonists’ data were discouraging. However, anti-Aβ vaccine, BACE inhibitor and anti-neuroinflammation drugs showed promising results.


Introduction
It is estimated that 55 million people have dementia worldwide and, by 2050, this number may increase to 139 million due to population aging. In 2019, dementia global cost was estimated to be 1.3 trillion dollars and led to 1.6 million deaths (WHO, 2017;WHO, 2021;WHO, 2022). Alzheimer's disease (AD) is the most common type of dementia in the elderly and affects mainly females. It was estimated that among AD diagnosis, 44% are 75-to 84-year-old patients and 38% are 85 years or older. Thus, AD is a social and economic global burden (Hebert et al., 2013). This neurodegenerative disease is related to loss of cognitive functions caused by several pathological pathways: amyloid-β (Aβ) deposition, hyperphosphorylated tau protein, cholinergic disorder, excessive glutamatergic stimulation, oxidative stress, and neuroinflammation (Hardy and Allsop, 1991;Gomez-Isla et al., 1997;Fiala et al., 2007;Holmes et al., 2009;Tolar et al., 2020). The first case was reported by Alois Alzheimer in 1906, and, despite all improvements in understanding AD pathogenesis, nowadays, therapies only help to manage some symptoms. AD lingers without a cure or strategy to mitigate its progression (Alzheimer, 1906;Alzheimer et al., 1995;Tolar et al., 2020).
AD diagnosis continues to be mainly based on clinical evaluation of cognitive and physical examination. However, pathological changes occur years before symptoms arise, and the earlier diagnosis may be accomplished by detecting molecular biomarkers (Aβ and tau) or cortical atrophy using magnetic resonance imaging. Despite all available technology, the greatest sensitivity and efficacy is found only in postmortem cerebral autopsy (Ranasinghe et al., 2021;Vaillant-Beuchot et al., 2021;Troutwine et al., 2022).
Current treatments available include use of cholinesterase inhibitors for patients with any stage of AD and memantine for people with moderate to severe dementia. Main drugs approved are rivastigmine, galantamine, memantine and donepezil. However, they only improve quality of life when prescribed at the appropriate time (Botchway et al., 2018;Scheltens et al., 2021). Two decades after memantine approval, the United States (US) Food and Drug Administration (FDA) approved aducanumab in 2021, the first monoclonal antibody anti-Aβ and the latest AD drug approved. In addition to the entire thrill, this new drug is expensive and there is some doubt related to its benefits (Mafi et al., 2022). Therefore, this study aimed to describe main advances in AD pharmacological therapy through an analysis of latest clinical trial results registered in the US National Institutes of Health (NIH), contributing to theoretical information for drug development pipelines and future clinical practice.

Potential targets for drug design
This work analyzed 43 AD new drug clinical trials registered in the National Library of Medicine database ClinicalTrials.gov funded by the NIH with data published in PubMed between 2015 and October 2022, using the following keywords: "Alzheimer's disease" AND "drug." Molecular target, therapeutic effect, safety profile and side effects were evaluated in each study (Table 1). Only new drugs' clinical trials registered in the NIH were included. Exclusion criteria were as follows: drugs already approved by the FDA (even if it is in a new formulation or delivery system) and pharmacological strategies that aim to solve only non-cognitive or degenerative symptoms. Those works tested 27 new drugs and 23 different molecular targets. In summary, we evaluated three anti-Aβ therapeutic vaccines, five anti-Aβ antibodies, a tau aggregation inhibitor, three BACE-1 inhibitors, 2 5-HT6 receptor antagonists, 2 nicotinic receptors agonists, a muscarinic agonist, a glutaminyl cyclase inhibitor and 10 anti-neuroinflammation drugs ( Figure 1). Pathways related to those drugs are better detailed in Supplementary Data S1.

Aβ and tau protein
Main therapeutic targets studied for AD were directly or indirectly related to neurofibrillary tangles (tau protein) and senile plaques (Aβ protein). Nevertheless, only aducanumab, an antibody anti-Aβ, was approved (Tolar et al., 2020;Mafi et al., 2022). Aβ and tau proteins highlight as drug targets and are related to AD pathogenesis. Amyloid precursor protein (APP) cleavage by β-secretases (BACE-1) or γ-secretases results in insoluble Aβ protein, a hallmark of AD. Therefore, the therapeutic rationale is to disassemble and degrade amyloid plaques chemically or by recruiting microglia and activating phagocytosis to stop or undo neuronal damage triggered by those protein accumulation (Da (B) All mechanisms related to AD pathogenesis and progression are connected and were explored in these clinical trials, such as Aβ and tau aggregation; BACE-1, γ-secretase, or glutaminyl cyclase activity; neuroinflammation; excitotoxicity; 5-HT7R or 5-HT6R hyperstimulation and cholinergic impairment.
On the other hand, clinical studies of vaccines were less encouraged due to the full-length Aβ1-42 peptide (AN1792) trial safety issues. This work demonstrated decrease in Aβ plaques, benefits on some cognitive and memory measures, and reductions in cerebrospinal fluid (CSF) tau. However, meningoencephalitis was presented in approximately 6% of AN1792-treated patients due to cytotoxic T-cell response (Nicoll et al., 2003;Ferrer et al., 2004;Masliah et al., 2005). Safety is no longer an issue in recent studies, which included vanutide cridificar (ACC-001), a vaccine designed to elicit antibodies against N-terminal peptide Aβ1-7. In this work, safety and tolerability profile was acceptable, but it lacks therapeutic effect (Pasquier et al., 2016;Van Dyck et al., 2016). ABvac40, a vaccine against the C-terminal end of Aβ40, as well showed safety and tolerability, but also, most of the individuals receiving ABvac40 developed specific anti-Aβ40 antibodies (Lacosta et al., 2018).
In addition to tau central role in AD pathogenesis, clinical trials focusing on this target are rare because of toxicity and/or lack of efficacy. Tau provides microtubule stability and contributes to the regulation of intracellular trafficking by its phosphorylation. Hyperphosphorylation of tau destabilizes tau-microtubule interactions, leading to instability, transport defects along microtubules and neuronal death (Dixit et al., 2008;Iqbal et al., 2010;Vershinin et al., 2017). Methylthioninium chloride (MTC; commonly known as "methylene blue") was the first reported tau aggregation inhibitor (TAI) without disrupting normal tau-tubulin interactions (Melis et al., 2015). However, MTC had no therapeutic effect in phase 2 trial probably due to its poor tolerability and pharmacokinetic issues. MTC exists in equilibrium with its oxygen-sensitive redox couple, leucomethylthionium (LMTM) . Although LMTM combines superior pharmacological properties with TAI activity than MTC, phase 3 trial showed no benefit to AD patients and led to treatment discontinuation Wilcock et al., 2018).

BACE-1 and γ-secretases
Other important targets related to AD pathogenesis are BACE-1 and γ-secretases; once those enzyme activities were responsible for Aβ development, as described earlier in this work. However, disappointingly several inhibitors of those enzyme studies were discontinued due to futility and toxicity, including cognitive impairment. Severe toxicity was especially high in γ-secretase inhibitors, indicating that its inhibition cannot be achieved in a safe way due to its physiologic effect in the Notch pathway (Cebers et al., 2017;McDade et al., 2021;Yang et al., 2021).
PF-05212377 (SAM-760) and idalopirdine are selective antagonists of 5-HT6R with a good safety profile; however, they failed to demonstrate efficacy. Altogether, those findings suggest that 5-HT6 antagonists should not be a main target to AD therapy (Atril et al., 2018;Fullerton et al., 2018).

Cholinergic pathways
According to the cholinergic hypothesis, AD is related to the reduction of acetylcholine. Therefore, most frequent pharmacologic therapy for AD is to increase cholinergic pathways through acetylcholinesterase inhibition (IAch) (Bartus et al., 1982;Recio-Barbero et al., 2021). IAch drugs, such as rivastigmine, donepezil, tacrine and galantamine, only provide a modest and not lingering symptomatic benefit to cognitive decline (Mohammad et al., 2017;Kumar et al., 2020;Sabandal et al., 2022). Most common side effects in cholinergic drugs are gastrointestinal issues, fatigue, cramps and sinus node dysfunction (Briggs et al., 2016;Mohammad et al., 2017).
Frontiers in Pharmacology frontiersin.org 05 A novel cholinergic approach for AD is the modulation of α7 nicotinic receptors (nAChRs), important receptors in the hippocampus and prefrontal cortex for learning, memory, and executive function. Targeting only α7 nAChR, instead of all cholinergic receptors, as IAch drugs do, should reduce toxicity (Colovic et al., 2013;Yakel, 2013).

Glutamatergic pathways
Glutamatergic neurotransmission related to N-methyl-daspartate (NMDA) function in cortical and hippocampal brain regions also plays a relevant role in AD pathogenesis. Memantine is an approved medication for this target. Activation of NMDA receptor signaling pathway produces secondary messengers, such as cyclic guanosine monophosphate (cGMP). Therefore, inhibition of phosphodiesterase type 9 (PDE9), which hydrolyzes cGMP, could increase cGMP levels and enhance cognition through long-term potentiation (LTP) (Reneerkens et al., 2009;Dubois et al., 2010). BI 409306 is a PDE9 inhibitor that was promising in rodents' test, but no clinically meaningful changes were detected (Frölich et al., 2019).

Glutaminyl cyclase
Glutaminyl cyclase (QC) plays a central role in synaptotoxic Aβ oligomer formation with pro-inflammatory potential. QC is an enzyme (glutaminyl peptide cyclotransferase, EC 2.3.2.5) that converts glutamate residue at position 3 of the N-terminus of truncated Aβ to AβpE3 peptide (Coimbra et al., 2019) and is involved in several pathological disorders, especially in AD (Vijayan and Zhang, 2019). Evidence have shown that AβpE3, a modified form of Aβ, may contribute to tau hyperphosphorylation (Mandler et al., 2014;Hennekens et al., 2015;Bayer, 2022). Indeed, AβpE3 is upstream in the neurotoxic amyloid cascade triggering neurodegeneration and influencing the severity of AD pathology (Pivtoraiko et al., 2015;Moro, et al., 2018). Since QC may catalyze the generation of cerebral AβpE3, its activity is overexpressed in AD brains, showing that QC inhibitors may be a good option for the development of AD-modifying strategies. The inhibitory activity of current inhibitors is mainly triggered by zinc-binding groups that coordinate zinc ion in the active site and other common features (Coimbra et al., 2019). Moreover, the inhibition of this enzyme also reduces the formation of mature CCL2, and thus suppresses neuroinflammation (Vijayan and Zhang, 2019). Although Scheltens et al. (2018) showed cognitive improvement with PQ912, some participants discontinued the trial due to high-dose toxicity. PQ912 targeted glutaminyl cyclase enzymes in patients with mild AD. The study was promising due to the functional improvement, inhibition of deterioration of synaptic activity, and reduction of neuroinflammation in patients.

Neuroinflammation
Chronic brain inflammation is another pathological hallmark of AD. Neuroinflammation is initiated when glial cells are activated by neural environment or neuronal injury. Particularly, tumor necrosis factor-α (TNF-α) signaling plays a master role in this scenario and has been associated with neuronal excitotoxicity, synapse loss, and propagation of the inflammatory state. TNF-α signaling also exacerbates amyloidogenesis, including upregulation of BACE-1 expression (Song et al., 2021;Chen et al., 2022). Etanercept and thalidomide are TNF-α antagonists but none had therapeutic effects in clinical trials (Butchart et al., 2015;Decourt et al., 2017). Etanercept limitation is related to its pharmacokinetics; it is unable to cross the brain-blood barrier (Butchart et al., 2015). Thalidomide had poor tolerability (Decourt et al., 2017).
Some already approved drugs were also in evaluation for AD as an off-label strategy. A calcium channel blocker antihypertensive drug, nilvadipine, reduced amyloid production, increased cerebral blood flow, and has demonstrated anti-inflammatory and anti-tau activity in preclinical studies. These properties could be related to a blood pressure control or anti-amyloid mechanisms (Paris et al., 2004;Paris et al., 2014). However, clinical trial results do not suggest benefit of nilvadipine as a treatment to AD (Lawlor et al., 2018;Abdullah et al., 2020). Insulin also was evaluated due to its metabolic, mitochondrial, and protease activity, influencing clearance of Aβ peptide and phosphorylation of tau (Valla et al., 2010;Kellar and Craft, 2020). Intranasal glulisine, an insulin analog lacking potential olfactory toxicity due to zinc ingredient commonly found in insulin formulations, also failed in therapeutic effects (Rosenbloom et al., 2021). Oxidative stress is one of multiple factors contributing to AD pathogenesis. Monoamine oxidase B (MAO-B) enzymes are related to this mechanism in astrocytes due to oxidative deamination of neurotransmitters. A number of MAO-B inhibitors (MAO-Bi) have been studied for AD and Parkinson's disease, such as sembragiline a selective MAO-Bi. Sembragiline demonstrated a good safety profile and potential effect on neuropsychiatric symptoms and behaviorally impaired (Nave et al., 2017).
Natural products were also studied. A marine derivative PKC epsilon activator, namely, bryostatin, increased synaptic numbers via synaptic growth factors but showed no efficacy in trial (Farlow et al., 2019). MLC901 (NeuroAiD II) contains extracts from nine herbal components, and triggered neurogenesis and neuroproliferation in rodents and human stem cell cultures due to activating ATP-dependent potassium channels (KATP) and modulating neuroinflammation. Clinically, MLC601 as monotherapy showed better tolerability and comparable efficacy to AChEIs in patients with mild to moderate AD, vascular dementia, and mild cognitive impairment (Chen et al., 2022). Sodium oligomannate (GV-971) is a marine-derived oligosaccharide that can modulate gut microbiota, reducing neuroinflammation in the brain as observed in animal models. GV-971 demonstrated significant efficacy in improving cognition and was safe and well-tolerated (Xiao et al., 2021).
SIRT-1 showed good results in phase two clinical trials (Turner, et al., 2015;Moussa et al., 2017). SIRT-1 is a sirtuin, a deacetylase protein regulated by NAD+/NADH activated by caloric restriction, which itself decreases age-dependent cognitive decline in animal models (Turner et al., 2015;Moussa et al., 2017). Resveratrol is a potent activator of SIRT-1 and helped to maintain blood-brain barrier (BBB) integrity by reducing oxidative stress, and inhibiting of NF-κB and matrix metalloproteinase-9 (MMP9) release (Lin et al., 2010;Wang et al., 2016;Espinoza et al., 2017). In addition, resveratrol induced adaptive immune responses (Moussa et al., 2017). However, in phase 2 study, a reduction in brain volume was also found, and Turner et al. (2015) suggested lack of benefits. Moussa et al. (2017) showed that resveratrol significantly attenuated declined Aβ levels in CSF, and decreased cognitive and functional decline. The drug also reduced plasma levels of pro-inflammatory producers.

Conclusion
In addition to all technology and information in AD pathogenesis, the most common outcome of AD new drug clinical trials was the lack of efficacy. However, those results may be limited by the disease stage of patients because earlier therapy has better performance in AD. Due to aducanumab recent FDA approval, many of the studies were using antibodies against Aβ, but they showed no noteworthy results. 5-HT6 antagonists, tau inhibitors, and nicotinic agonists' data were also discouraging. However, anti-Aβ vaccine, BACE inhibitor, and antineuroinflammation drugs led to promising results with some drugs showing clinical improvements and no toxicity.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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