Drugs to reduce brain AB for AD treatment | JEP - Dove Medical Press
Introduction
No definitive treatments currently reliably combat Alzheimer disease (AD). AD is a terminal and incurable neurodegenerative disease that commonly affects the geriatric population. Symptoms include a gradual irreversible loss of memory and cognitive capabilities, such as the inability to execute common daily tasks.1 Currently, no effective or reliable method exists to accurately diagnose AD before a postmortem brain autopsy is performed.2 These postmortem biopsies reveal a combination of pathologies, including brain atrophy that is correlated with both extracellular plaques comprising amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles made up of hyperphosphorylated tau proteins (tau tangles).2 These 2 proteins are hallmarks of AD and likely play a critical role in the pathological progression of the disease. Despite the increasing prevalence of AD as the most common form of dementia in the general population worldwide,3 relatively little is known about its cause.4
The prominent risk factors for AD are age, genetics, family history, and environmental factors.5 Among these factors, age is the strongest determining risk factor for developing AD as more than 33% of the general population aged 85 years and older experience AD.6 Two situations are relevant for genetic risks and family history. First, 1% to 5% of AD cases are "familial AD", in which symptoms are often displayed before the patient reaches the age of 65; in these cases, mutations have been identified in genes associated with Aβ synthesis (ie, the amyloid precursor protein [APP] and the genes for the presenilin 1 and presenilin 2 proteins that are part of the γ-secretase complex).7 For the rest of AD cases, often referred to as "late onset AD" or "sporadic AD", a family history of AD in a first-degree relative is associated with an increased risk of developing AD.8 Some of these risks are related to rare and low-penetrance missense variants in specific genes. For example, a mutation resulting in a homozygous ApoE4 genotype greatly increases the risk of developing AD, likely due to the reduced ability by the body to dispose of Aβ in the brain,9 which is suspected to result in increased Aβ plaque loads that may participate in neuronal death. Other mutations have been identified in genes, such as TREM-2, TDP-43, TOMM-40, and others. In the absence of genetic mutation, environmental factors can be implicated in both familial and sporadic AD. Such factors include a low level of education, low physical activity, unbalanced diet, smoking, and the presence of comorbid conditions, such as cardiovascular disease, hypertension, diabetes, and hepatic disease.4,5
Currently, the only standard of care for AD is acetylcholine-esterase inhibitors (AchEIs), such as donepezil, galantamine, and rivastigmine. AchEIs have been correlated with an effect that temporarily stabilizes the progression of AD.10 However, the benefit of AchEIs in controlling AD progression is unclear due to the insignificant change in conversion rate from mild cognitive impairment (MCI) to AD.10 Studies indicate that only high doses of AchEIs have proven significantly effective, but efficacy and clinical use have been questionable due to the higher frequency of adverse events associated with higher dosages and longer periods of using such medications.10 Because of the frequent adverse effects, attempts to investigate other forms of treatment are being pursued.
In the amyloidogenic pathway, Aβ is generated from APP, which is a single-pass transmembrane protein found in many tissues, via the sequential proteolysis by intramembranous proteases, namely β-secretase and the γ-secretase quaternary complex.11 However, the nonamyloidogenic pathway cleaves APP in the middle of the Aβ sequence via an α-secretase activity, then via the γ-secretase, to produce a small peptide, called p3, of unknown function. The highest levels of APP are expressed in brains with a high expression level in neurons. The predominant theory regarding AD pathogenesis is the "amyloid cascade hypothesis."12 This hypothesis proposes that the deposition of Aβ is the primary causative agent of AD pathology, with neurofibrillary tangles, cell loss, vascular damage, and dementia following as a consequence of Aβ deposition. Proposed in 1992,12 this theory continues to be a topic of debate. Effectively, clinical data show a disconnect between Aβ levels in the brain, location of accumulation, and neuronal loss associated with dementia. This issue is illustrated by the fact that most clinical trials exclusively targeting Aβ have failed to improve clinical outcomes in AD patients; these failures have prompted exploration of new therapeutic avenues.13 A recent proposed amendment to this hypothesis is that Aβ acts as a trigger for tau bundles to result in AD pathology.14 However, evidence supports Aβ's critical role, as an increased Aβ42/Aβ40 ratio in the cerebrospinal fluid (CSF) predisposes individuals to develop AD.14 Thus, in addition to the amyloid cascade hypothesis, some authors have formulated an amyloid threshold hypothesis, which proposes that, if Aβ levels are maintained below a certain level, then AD pathology may be reduced and AD symptoms never appear.15 However, other biochemical and preclinical studies have also supported that the plaques, once formed, will cause pseudo-irreversible locking of Aβ peptides despite changes in CSF Aβ concentrations.14 Alternatively, non-Aβ-centric hypotheses explain the etiology and progression of AD, which have been thoroughly summarized in a recent review.16 However, our present review focuses on anti-Aβ therapies, and thus we do not discuss other hypotheses here.
Three main methods are currently the focus of therapeutic interventions addressing the 2 Aβ-centric AD hypotheses indicated above: (i) lowering Aβ production, (ii) augmenting Aβ clearance, and (iii) preventing Aβ aggregation. Among the strategies explored are small-molecule drugs and immunotherapies to remove Aβ and downstream targets from the brain.17–20 In this article, we review current preclinical and clinical data for brain Aβ reduction using small-molecule drugs and immunotherapies that are being tested in clinical trials or are approved by the US Food and Drug Administration (FDA).
Materials and Methods
Molecular pathways involved in Aβ metabolism were reviewed via a systematic review limited to therapies in Phase II/III trials or that have already received approval by the FDA. Alzforum.com, a forum maintained by the Alzheimer's Association and dedicated to networking medications by allowing academic scientists and private companies to report initiatives and products, was a major source for finding potential therapies. From this expansive list, our selection criteria included drugs or methods that showed promise due to earlier trials or experiments with similar methodologies. Trial results were then reviewed with in-depth searches on the official websites ClinicalTrials.gov and Clinicaltrialsregister.eu, where the trials and their design were described. Supporting studies completed prior to clinical trials were reviewed in PubMed, when available. Additional scientific publications were reviewed for each drug with regard to their mechanism of action.
Small-Molecule Therapies to Reduce Aβ Loads in the Brain of AD Patients
Small molecule drugs have been heavily employed over the past 50 years as a way of modulating enzymes and receptors.21 A small-molecule therapy is often a drug that is able to pass through biological barriers (eg, blood-brain barrier [BBB], or cell membranes) easily due to a low molecular weight. It exerts its effect(s) by affecting biological molecules once inside the cell microenvironment. Most often, this type of drug is employed for targeted therapies. Because many enzymes and receptors act as "on and off switches" for cellular activity, small-molecule therapies have been used to activate or deactivate targeted cellular processes.21
With accurate chemical design, small-molecule drugs can target enzymes or receptors in a specific manner. This targeting is an efficient way to limit the effects of a drug to a single process, thus reducing the risk of a cascade effect and adverse events. Additionally, quantifying the drug dosage required to avoid toxicity is relatively straightforward because only one molecular pathway is typically affected. Importantly for AD, small molecules historically account for the vast majority of central nervous system (CNS) drugs because of their ability to pass through the BBB by passive or carrier-mediated transport.22 With the target protein Aβ being inside the brain parenchyma (ie, behind the BBB) when drugs are administered systemically, finding a method to cross the BBB is an important consideration in drug delivery paradigms. The BBB rarely restricts small molecules. Thus, those molecules can reach sites of Aβ protein production and deposition.
However, such therapeutics have drawbacks. Combination therapies are often employed because the specific design of a given small-molecule drug is made to inhibit only a single receptor or enzyme, whereas a biological system often involves several receptor types and intracellular cascades.21 If one molecular pathway is blocked, another pathway can often take over. Thus, it is difficult to anticipate whether a drug will actually modulate the whole pathobiological process of interest or only part of it, with the latter potentially being insufficient to revert to homeostasis. Additionally, localization to specific tissues is difficult to achieve. With enzymes and receptors expressed in multiple tissues, therapeutic effects may be accompanied by unintended adverse effects caused by changing the metabolism or interacting with other tissues.
Given the potential benefits and drawbacks of small molecules, the ability of small molecules to cross the BBB makes them attractive as therapeutics capable of slowing down the progression of AD; they can be used to affect Aβ metabolism or conformation. Moreover, small molecules can assist in identifying specific pathologies identified by brain imaging methodologies.23 Aβ structure can be affected by changing the protein's propensity toward its monomeric or oligomeric state. This can be achieved by stabilizing the native state of Aβ, hence preventing conformational changes. Alternatively, small-molecule inhibitors can be used to inhibit the natural aggregate-prone tendency of some peptides and proteins, such as Aβ.24–27 On the metabolic side, small-molecule drugs can alter enzymatic activities that regulate the anabolism and catabolism of Aβ. Below, we describe small-molecule drugs being tested in clinical trials that can modulate precursor proteins, cellular hyperactivity, and other metabolic dysregulations that are known to lead to increased Aβ production and agglomeration.
Oligomerization Inhibitors
Protein aggregation is critical in the formation of Aβ plaques. For folded proteins with a defined structure, small-molecule drugs have been shown to influence the folding process. However, for aggregation-prone proteins lacking a defined structure, small-molecule inhibitors have largely been limited to screening and detection applications, such as dye-binding assays (eg, florbetapir18 F-labeled tracer for positron emission tomography [PET]28). This limitation is due to an inability to create small molecules sensitive enough to individually characterize unique protein subspecies when there is a lack of defined structures in oligomers (eg, Aβ40 vs Aβ42).24 Nonetheless, new molecules are being developed to attempt to implement small-molecule therapies for abnormal protein aggregates.
If Aβ peptide monomers are prevented from forming oligomers, then fibrils and aggregates, which are suspected to be more toxic, are less likely to form.29 Rather than using an inhibitor that targets the structure of the oligomeric assembly, the small-molecule drug could target the protein binding domain(s) and thus inhibit multimerization. In such a scenario, the protein would remain in a nonfunctional and aggregate-prone state, which, in the example of AD, reduces the risk that individual peptides form neurotoxic Aβ oligomers. Several small-molecule drugs are currently in Phase III of development and show promise at inhibiting Aβ oligomerization through different mechanisms.
Alzt-Op1
ALZT-OP1 (AZTherapies, Inc.) is a combination drug therapy of cromolyn sodium and ibuprofen, also called Intal. Cromolyn is a prescription drug that suppresses cytokine release and stabilizes mast cells. It is largely used to treat asthma.30 Ibuprofen's use in AD has been widely studied in preclinical and clinical paradigms.31,32 One study performed by the Massachusetts General Hospital indicated that cromolyn both inhibited aggregation of Aβ monomers in vitro and decreased soluble Aβ levels in the brains of APP/PS1 mice receiving the drug via intraperitoneal administration.29 Ibuprofen alone was ineffective at reducing Aβ. However, when ibuprofen was used in synergy with cromolyn, then Aβ deposits were significantly reduced.33 To date, the drug has completed a Phase I trial (ClinicalTrials.gov identifier NCT04570644) using 24 healthy participants in a 2-day trial and was deemed safe. There were only mild to moderate adverse events in 3 participants while still achieving CSF concentrations sufficient to reduce Aβ production.34 A Phase III trial (NCT02547818) has been conducted to evaluate changes in clinical dementia rating (CDR) scores with recruitment completed in December 2020. The global CDR rating ranges from 0 to 3, in 0.5-point increments; the score increases as cognitive impairment worsens.35 A global CDR of 0.5 corresponds most often to mild cognitive impairment, whereas a global score of 1 most often corresponds to mild AD dementia, 2 to moderate, and 3 to severe AD dementia. Most CDRs are tallied by the sum of the boxes (SOB), which summarizes the numerical score derived from each of the 6 domains probed. The trial was an 18-month study set to enroll 600 participants aged 55 to 79 years with confirmed early AD (see published inclusion criteria for this specific trial) and comparing four groups: 1) cromolyn active plus ibuprofen active, 2) cromolyn active plus ibuprofen placebo, 3) cromolyn placebo plus ibuprofen active, and 4) cromolyn placebo plus ibuprofen placebo. To date, no results from the trial have been posted.
ALZ-801/Homotaurine
ALZ-801 (Alzheon, Inc.) is a prodrug of homotaurine, a natural amino acid found in seaweed. The drug has been previously marketed under the names tramiprosate and alzhemed. In vivo, tramiprosate is converted via a hepatic or plasma amidase to homotaurine, which increases absorption and bioavailability.36 Previous studies have shown that tramiprosate inhibits Aβ42 aggregation into toxic oligomers.37,38 Moreover, both tramiprosate and homotaurine are metabolized into 3-sulfo propanoic acid (3-SPA). 3-SPA is a naturally occurring molecule in the brain that has also been shown to inhibit Aβ42 aggregation.36 A series of Phase I randomized, placebo-controlled trials (NCT04585347 and NCT04157712) were performed with 127 healthy adult volunteers. NCT04157712 included a single ascending dose, a 14-day multiple ascending dose, and a single-dose tablet-food effect study that also lasted 14 days.36 These trials showed that drug concentrations in the brain need to reach levels 5 to 15 times higher than what is required to inhibit Aβ42 aggregation in vitro. However, the interventions also caused mild nausea and vomiting that were not dose related, which resolved after 1 week of receiving the drug.36 In December 2019, Alzheon analyzed data on the drug and found improved cortical thickness when comparing ApoE3/3 and ApoE4/4 carriers with mild AD. Currently in Phase III, the trial began in 2021 (NCT04770220) and will assess 300 ApoE4 homozygotes with early to mild AD receiving an 18-month course of drug versus placebo. The company will be monitoring CSF concentration of Aβ oligomers, a primary target of ALZ-801, as well as neurofilament light (NfL) and P-tau levels. A secondary outcome will be improvement of cortical thickness. The trial will be conducted at 85 sites in the United States, Canada, and Europe, with completion expected by May 2024.
Inhibition of Amyloid-Induced Neuronal Hyperactivation
A correlation is suspected between hyperactivation of neuronal pathways and Aβ activity. A research team at the University of Munich, Germany, identified that an impaired synaptic glutamate reuptake resulted in Aβ-induced neuronal hyperactivation when hyperactivation was observed in the APP transgenic mouse models of AD used in the study.39 The mouse models used were both male and female C57Bl/6N wild-type mice and age-matched female APP23×PS45 mice. Upon investigation, they observed that Aβ oligomers-containing brain samples from AD patients induced hyperactivity in hippocampal CA1 neurons from slices prepared from mice. Specifically, at the single-cell level, the degree of hyperactivation induced by Aβ had a positive correlation with baseline neuronal activity.39 This finding can be interpreted to mean that the greater activity rate there is in neurons, the more Aβ is present to block glutamate reuptake at the synaptic level. The approach is motivating new paradigms in clinical settings.
Levetiracetam
Although clinical trials investigating the correlation between neuronal hyperactivity and Aβ-induced AD are scarce, one small-molecule therapy has made progress. Levetiracetam is an atypical anticonvulsant that modulates synaptic vesicle glycoprotein 2A. The mechanism of action is not fully understood, but it is suspected to inhibit calcium signaling or depolarizing currents in neurons.40 A study has analyzed the effects of levetiracetam on markers of amyloidogenesis and synaptic proteins in APP knock-in mouse models. It was observed that the chronic administration of the drug lowered the levels of cortical Aβ42, decreased the levels of β-carboxyl-terminal fragment (APP-βCTF) but not full-length APP, reduced plaque burden, and restored the levels of presynaptic endocytic proteins.41 A 1-year clinical trial administering 500 to 2000 mg levetiracetam to AD patients with reported episodes of seizures (NCT01554683) showed an improved verbal fluency and attention, but it was unclear whether the improvement was a result of reduced seizures or a global cognitive benefit.42 Adverse effects included sleepiness, headache, and lack of energy. A Phase II trial (NCT04004702) began in January 2020 at the Walter Reed National Military Medical Center in Maryland to evaluate changes on the neuropsychiatric inventory scale, severity of AD, and cognitive abilities in participants with epileptiform activity on electroencephalography who were treated with levetiracetam for 1 year. No data from this study have been published to date, and the assessment of Aβ levels are not indicated in the study design. We suggest that the analysis of Aβ levels (either CSF measures or PET imaging) could be beneficial in determining whether the cognitive benefit from this drug is solely the result of seizure reduction or a global cognitive benefit derived from reduced Aβ loads.
Receptor Inhibitors
Small molecules can also be employed to bind receptors capable of halting the enzymatic process leading to Aβ production. APP is suspected to function as a cell surface receptor and has been implicated in synaptic plasticity.43 A strong association has been found between Aβ and nicotinic acetylcholine receptors (nAChRs), particularly the α7 subtype. More precisely, Aβ is capable of binding the α7-nAChRs and modulating their function.44 These nAChRs are found highly expressed in regions of cognition and memory functions, and it has been observed that the expression of α7-nAChRs is reduced in the brain of AD patients.45 Thus, a possible therapeutic approach would attempt to modulate the binding of Aβ to α7-nAChRs, because this interaction seems to exacerbate AD pathology, such as tau hyperphosphorylation (see "Simufilam" below).
Simufilam
One small-molecule drug that has been reported to affect APP synthesis is simufilam (also called PTI-125; Cassava Sciences, Inc.). Simufilam is one potential avenue to prevent Aβ accumulation. When simufilam binds to the intracellular scaffolding protein filamin that regulates the actin cytoskeleton and is involved in cell stability and motility, the protein is unable to stabilize the high-affinity interaction of extracellular soluble Aβ42 to the transmembrane α7-nAChR.46 This interaction was reported to trigger tau phosphorylation and synaptic dysfunction in several experimental systems.47,48 A study performed on triple-transgenic AD mice (3xTgAD) treated with simufilam reportedly reduced Aβ and tau deposition, lowered neuroinflammation, and restored synaptic function compared to wild-type mice.46 A Phase IIb study (NCT04079803) was performed comparing 50 mg and 100 mg simufilam twice daily for 28 days to placebo in 64 participants with mild-to-moderate AD (confirmed by CSF biomarkers), and analyzed CSF phosphorylated-tau 181, total tau, Aβ42, neurofilament light, neurogranin, and chitinase-3-like protein 1 (also called YKL-40). The company reported that all the CSF biomarkers and some plasma biomarkers improved for both doses.49 This trial is continuing as an open-label extension and is set to run until March 2023. Results on data collected thus far were met with skepticism in August 2021 by whistle blowers who complained of multiple instances of research misconduct involving clinical trial biomarker data. The FDA posted a statement of concern (docket ID# FDA-2021-P-0930) and several independent scientists corroborated the apparent data manipulation. The company denied wrongdoing, stating that figures on their poster had errors, but the underlying data analysis and conclusion were valid. Thus, additional trials will be necessary to get a better assessment of the potential of simufilam to modulate AD-associated pathologies and cognitive decline.
Metabolic Modulators
Metabolic dysfunction has also been heavily tied to the progression of AD.4,23 One theory is that metabolic dysregulation, such as in diseases like diabetes insipidus, results in an increased load of oxidative stress.50 Aβ peptides have been studied and shown to inhibit synaptic insulin sensitivity directly in cultured mouse neurons, which can be correlated with the impaired synaptic functioning in AD.51 These observations have sparked the investigation of several drugs commonly used for diabetes on AD pathologies. At present, FDA-approved metabolic altering drugs include metformin, dichloroacetic acid, and methylene blue. Below we discuss 2 of the small-molecule drugs that are most relevant to cognitive decline and AD.
Metformin
Metformin (also known as glucophage [Merck]) is a generic drug used principally in the treatment of type 2 diabetes mellitus (T2DM). Its main functions are to decrease insulin resistance and blood glucose levels.52 Insulin resistance is also found in the brain of AD patients and is a reason that AD has been referred to as "type 3 diabetes."4 Therefore, drugs that reduce insulin resistance may help with the prevention and treatment of AD. Recent studies have shown a correlation between the use of metformin and a reduction in AD-like pathologies in APP/PS1 mice, which seems to be linked to improved microglial autophagy and phagocytosis processes.53 Metformin also ameliorated cognitive impairment in a diabetic mouse model54 (diabetes induced via streptozotocin administration) and in the senescence mouse model SAMP8,55 although the improvement of cognitive functions is not consistent across AD mouse models (reviewed by Craig et al56).
Animal data have generated interest in testing metformin in clinical trials. This line of work is spearheaded by Dr. Luchsinger at Columbia University.57 A Phase II double-blind, placebo-controlled, randomized trial was started in 2008 to assess the efficacy of metformin in 80 MCI patients via the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) and selective reminding test (SRT) scales (NCT00620191). Overall, the group treated with the drug performed significantly better on the SRT scale.58 However, some toxic events limited the maximum dose tolerated by study subjects, which prompted the design of an improved multisite Phase II/III study for the use of metformin to prevent AD (NCT04098666). This ongoing 24-month dose escalation trial aims to recruit 370 amnestic MCI subjects who are overweight or obese without diabetes and not previously treated with metformin. The primary outcome measure is the free and cued SRT. It is anticipated that half of the participants will undergo amyloid and tau PET imaging at baseline and at the completion of the trial. This trial is expected to end in 2025.
Linagliptin
Linagliptin (also termed Tradjenta; Boehringer Ingelheim Pharmaceuticals, Inc.; generic drug in the United States since August 2021) is an FDA-approved small molecule drug that is used for glycemic control in T2DM. Linagliptin acts as a competitive, reversible inhibitor of circulating dipeptidyl peptidase-4 (DPP-4); thus, it slows down the catabolism and increases the levels of active glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).59 Higher levels of GLP-1 and GIP stimulate a greater release of insulin from beta cells in the pancreas, while also inhibiting the release of glucagon. Linagliptin has been studied in vitro using human neuroblastoma SH-SY5Y cells challenged with 20 μM of Aβ42 for 24 hours. The drug at 50 nM reduced Aβ42-induced cell death by ~25%.60 Furthermore, linagliptin reduced cell death at concentrations ranging from 10 to 50 μM in SK-N-MC neuroblastoma cultures in a dose-dependent manner during Aβ infusion experiments, suggesting that it is protective against Aβ-induced neurotoxicity.61 The suspected mechanism was the blockade of dipeptidyl peptidase-4, thus elevating GLP-1 levels, which leads to increased insulin release and restoration of insulin signaling. It was thought that these results and those of others showing this neuroprotective effect may be local, but it seems that linagliptin works at the neuronal level to reduce overall oxidative stress loads.62 In 3xTg-AD mice, linagliptin was shown to attenuate cognitive deficits on the Morris Water Maze and Y-maze. In addition, the drug modulated neuroinflammation and reduced both amyloid and tau pathologies.63 These data and others suggest that linagliptin has the potential to lower AD progression. Although no clinical trial testing of the drug involving AD subjects has been reported, a cognitive substudy involving 1545 subjects over the age of 50 years was derived from the larger T2DM trial, termed CARMELINA, testing the effects of 5 mg linagliptin on cardiovascular and renal microvascular outcomes in T2DM patients (NCT01897532). The cognitive tests included the Mini–Mental State Examination (MMSE), the trail making test, and the verbal fluency test administered at baseline and after 2.5 years of daily dosing. Overall, the study found no difference in cognitive decline in the drug-treated versus placebo groups, indicating that linagliptin was neither beneficial nor detrimental for cognitive abilities in T2DM patients.64 Thus, further studies are required to determine the extent of the neuroprotective effects of linagliptin in the context of AD and related dementias.
Amyloid-Beta Degradation Modulation
The body, under physiological circumstances, regulates Aβ levels via disassembly and proteolytic degradation as well as microglial phagocytosis. Although the list is not complete, a growing number of diverse peptidases and proteinases, known collectively as Aβ-degrading proteases, play a role in the metabolism of Aβ, such as neprilysin (NEP), endothelin-converting enzymes 1 and 2, insulin-degrading enzyme, and plasmin.65,66 There is anticipation that, with further research, understanding the breakdown of Aβ could lead to slowing the progression of AD or developing a possible treatment to stop the disease entirely. Here, we focus our attention on compounds shown to transition back microglial activity from a proinflammatory/neurotoxic to an anti-inflammatory/pro-phagocytic activation state.
Alzt-Op1
Previously discussed under the section on oligomerization inhibitors, ALZT-OP1 also modulates Aβ degradation in vitro in human microglial cells HMG030.29 Degradation or removal of Aβ and plaques was promoted by modulation of the inflammatory response and microglial phagocytosis both in the human cell line HMC367 and in animal experiments, such as Tg2576 mice, with a combination of ibuprofen and cromolyn or with cromolyn alone.33 BV2 murine microglial cell cultures treated with cromolyn alone or cromolyn plus ibuprofen in the range 10–1000 μM showed enhanced phagocytosis of Aβ42.33 The effect of cromolyn on Aβ was further increased when paired with drugs such as bromocriptine during in vitro screening on human-induced pluripotent stem cell differentiated into neurons.68 Whether ALZT-OP1 also regulates microglial activity and the levels and activity of Aβ-degrading enzymes in AD patients remains to be investigated, although it will likely require investigators to analyze fresh postmortem brain tissues, which can only be performed at a few investigative sites.
Sodium Oligomannate
GV-971, also known as sodium oligomannate (Shanghai Green Valley Pharmaceuticals, Inc.), is another drug with the potential to regulate Aβ degradation. Sodium oligomannate is a mixture of acidic linear oligosaccharides isolated from brown algae. Although the mechanism of action is uncertain, one proposed mechanism is that the compound breaks down Aβ aggregates (ie, disassembles plaques).69 Another proposed mechanism is that this small molecule reduces the inflammatory response exacerbated by astrocytes, thus reduces the oxidative stress applied to neurons, which results in fewer Aβ plaques being formed.70 A Phase III trial (NCT02293915) was conducted involving 818 subjects in China, which resulted in the treatment group of participants with mild-to-moderate AD having higher ADAS-Cog13 scores than the placebo group at 4, 12, 24, and 36 weeks.71 However, no biomarkers were measured in this specific trial. Nonetheless, the drug was given conditional marketing approval in China. In August 2020, the manufacturing company registered another Phase III trial (NCT04520412) that enrolled 2046 participants with mild-to-moderate AD who were not receiving any FDA-approved AD treatment. The study will be performed at multiple sites worldwide. The trial's primary outcomes are changes from baseline on the ADAS-Cog11 and Alzheimer's Disease Cooperative Study–Clinical Global Impression of Change scale total scores after 1 year of drug or placebo. This study should also test biomarker outcomes, such as blood Aβ, P-tau, NfL, inflammation, and microbiome status, plus fecal microbiome. The trial is anticipated to run through 2025.
Repurposing FDA-Approved Small-Molecule Drugs
Although small-molecule drugs are less studied than new therapies, in the past 3 decades several investigations have focused on the modulation of Aβ pathology by FDA-approved small molecules. The main classes of compounds are antidepressants acting as selective serotonin reuptake inhibitors (SSRIs) and inhibitors of acetylcholine degradation. Below, we briefly summarize the current knowledge about these small-molecule drugs with regard to Aβ metabolism.
Repurposing Antidepressants
In both young and old APP/PS1 transgenic mice, a single-dose intraperitoneal administration of 3 SSRIs (fluoxetine, desvenlafaxine, and citalopram injected individually) induced a 25% decrease in brain interstitial fluid (ISF) Aβ levels measurable 18–24 hours after administration.72,73 Additional experiments in this model have indicated that this reduction in ISF Aβ levels was not due to a change in Aβ elimination, a change in the electric activity of neurons, or a modulation of genes implicated in APP metabolism.72,74 Chronic administration of citalopram in young APP/PS1 mice for 4 months resulted in the lowering of brain Aβ and plaque burden by 50%.72 Similar results were obtained in young 3xTgAD mice administered paroxetine for 5 months.75 In middle-aged APP/PS1 mice (6 months old), daily administration of citalopram for 28 days arrested the growth of preexisting plaques and reduced the appearance of new plaques by 75%.73 Mechanistic investigations have suggested that the effects on ISF Aβ levels may be mediated by agonists of a subset of serotonin G-protein coupled receptors (5-HTR), namely, 5-HT4R, 5-HT6R, and 5-HT7R, which signal via the protein kinase A/mitogen-activated ERK kinase/extracellular signal–regulated kinase axis to regulate the APP-processing α-secretase activity at the protein level,72,74 thus driving APP toward the nonamyloidogenic pathway.
Two indirect studies by Dr. Cirrito's group have suggested that SSRIs have a role in the modulation of Aβ levels in humans. First, using a retrospective analysis paradigm, they reported that cognitively normal elderly patients taking SSRIs (N = 52) within 5 years to treat depressive symptoms displayed significantly less amyloid signals than nontreated patients (N = 134) when assessed by PET imaging with the Pittsburgh Compound B (PIB).72 A few years later, they reported the effects of an acute dose of citalopram versus placebo on CSF Aβ levels in 23 (11 females, 12 males) normal volunteers aged 18–50 years using the stable isotope labeling kinetics method developed by Dr. Bateman.73,76 The CSF was sampled hourly starting 8 hours after treatment onset and continued up to 18 hours (N = 10) or 37 hours (N = 13). Overall, the citalopram group had a 35% reduction in CSF Aβ levels compared to the placebo groups for the period of observation, whereas the clearance rate was similar between the 2 groups.73 These initial observations in humans highlight the pleiotropic effects of SSRIs, not only as serotonin modulators but also as possible inhibitors of Aβ production. Further studies are needed to better elucidate the molecular mechanisms of Aβ metabolism regulated by SSRIs.
Possible Role of Cholinesterase Inhibitors in Aβ Metabolism
As indicated in the Introduction, AChE inhibitors are the main pharmacological line of treatment for AD and relieve cognitive symptoms for several months. In the brain, acetylcholine (ACh) is hydrolyzed by two cholinesterases, AChE and butyrylcholinesterase (BuChE).77 AChE and BuChE are differentially expressed in the AD brain, with AChE often decreasing during disease progression, whereas BuChE increases. Interestingly, it was recently observed that the 3 FDA-approved AChE drugs for AD also modulate inflammation and amyloidogenesis. Here, we focus on Aβ modulation.
Donepezil is a selective AChE inhibitor.77 A recent study reported that donepezil reduced Aβ production in primary cortical cultures from rat embryos. At the molecular level, this process was dependent upon the overexpression of sorting nexin protein 33, and, similar to SSRI, an increase in α-secretase activity without increasing the expression of full-length APP.78 Whether these results can be extended to neurons from the hippocampus and other brain regions remains to be investigated.
Galantamine is also a selective reversible, competitive AChE inhibitor and allosteric nicotinic receptor modulator, but its relative hydrophilicity results in poor crossing of the BBB.77 In vitro, galantamine infused to newborn rat microglia cultures significantly increased Aβ phagocytosis, which depend upon microglial α7-nAChRs activation and calcium.79 These results were corroborated by in vivo experiments in adult rats injected with Aβ42 in the hippocampus and treated for 2 weeks with galantamine, and in 9-month-old Tg APdE980 mice that received oral treatment for 2 months. In these 2 animal models, galantamine significantly increased brain Aβ clearance.79 In 5XFAD mice, galantamine was chronically administered via addition in drinking water to 3-month-old animals, an age when plaque deposition is considerable. The treatment was stopped 2 months later, before plaque burden reached saturation levels. With this paradigm, in addition to some behavioral improvements, it was observed that plaque loads were significantly reduced in the cortex and hippocampus of treated Tg mice, irrespective of the sex, when compared to untreated controls.81 Similarly, 8 weeks of galantamine administration significantly lowered the amyloid plaque loads in 10-month-old APP/PS1 mice, which was accompanied by improved behavioral performance and reduced brain inflammation.82 Of note, the authors of these last 2 studies did not investigate the molecular mechanisms leading to the amyloid-related histopathological changes.
Rivastigmine is a pseudo-irreversible noncompetitive inhibitor of both AChE and BuChE, although it binds more efficiently to BuChE.77,83 Dr. Lahiri's group recently published a report on the modulation of Aβ metabolism by rivastigmine in several models and in the human brain.84 In a previous study, they had shown a reduction by 50% in brain Aβ40 and Aβ42 levels in 5-month-old male APP/PS1 mice treated for 3 weeks with a BuChE.85 The new study focused on rivastigmine. In vitro, in both differentiated PC12 cells and primary human embryonic brain cells, they observed an increase in byproducts of the nonamyloidogenic pathway (called sAPPα) released in the condition media, accompanied by an increase in the expression of disintegrins and metalloproteinases ADAM-9 and 10, which are suspected to act as α-secretases on APP.84 By contrast, they observed a significant decrease in byproducts of the amyloidogenic pathway, including in Aβ40 levels. These results were corroborated in hippocampal lysates of female 3xTg-AD mice (on a C57BL/6 background) administered rivastigmine intraperitoneally for 3 weeks.84 Very elegantly, they also observed an increase in sAPPα in postmortem brain tissues (Brodmann areas 21/22) of AD patients who were treated with rivastigmine compared to non-AD and AD patients who did not receive treatment with AChE/BuChE inhibitors.84 Notably, the levels of Aβ40 and Aβ42 were not affected by rivastigmine in the human brain, suggesting that the drug was not able to dissolve existing plaques. In a separate project, it was observed that donepezil and rivastigmine were also increasing the clearance of radiolabeled Aβ across the BBB and liver in rats, which could help eliminating Aβ via the sink effect.86 For the BBB, this effect was accompanied by an increase in the major Aβ transport proteins P-glycoprotein and low-density lipoprotein receptor-related protein 1.
In our opinion, although current data are promising, there is a need for more investigations in human tissues to confirm in vitro and animal data for the modulation of Aβ metabolism by AChE and BuChE inhibitors. If the pleiotropic properties of cholinesterase inhibitors are confirmed, then it will indicate that AChE and BuChE inhibitors should be used during the entire duration of AD treatment in combination with other anti-amyloid and anti-tau therapies to slow the progression of the disease more aggressively than AChE and BuChE inhibitors are capable of alone to have an impact on cognitive symptoms.
Immunotherapies Against Amyloid Beta
Aβ immunotherapy has been a source of research and interest to reduce brain plaque loads since 1999, when it was first reported that immunization of AD mouse models with Aβ may be effective in preventing and treating AD.87 The basis for Aβ immunotherapy is to use either synthetic peptides to induce active immunization in the host, or to deliver monoclonal anti-Aβ antibodies (mAbs) to provide passive immunization. Both types of immunization aim to decrease Aβ loads in the brain, which, based on the amyloid cascade hypothesis, should slow down the progression of AD.
Active immunization functions by introducing Aβ peptides as the antigen to the host, which then mounts an immune response against Aβ monomers and/or multimers that results in the production of antibodies specifically targeting Aβ.88,89 The goal of active Aβ immunotherapy is to program the patient's immune system to eliminate endogenous Aβ.90 Active immunization for AD in humans was first tested in 2002 in a study where participants were immunized with full-length Aβ42 peptides (named AN1792; Elan Pharmaceuticals) along with the immunological adjuvant QS-21 (NCT00021723). Postmortem brain samples from the participants were then neuropathologically examined and the percentage of plaque formation was assessed to determine effectiveness.89 It was found that, 6 years after immunization, study subjects who had died in the years following AN1792 administration (N = 7) had fewer brain Aβ aggregates and plaques compared to control subjects who did not receive any treatment, and they experienced slightly less cognitive decline (assessed by ADAS-Cog, MMSE, and the disability assessment for dementia scale91), but there was no effect on the progression of neurodegeneration (neuropathological assessment via hematoxylin and eosin, modified Bielschowsky silver impregnation, and immunostaining for Aβ and tau).89 Importantly, it was found that, despite high levels of serum antibodies to Aβ42, there was no clinical improvement overall after 1 year, and about 6% of participants developed meningoencephalitis, which led to early termination of the study.92 It is thought that some of the deleterious effects induced by Aβ active immunotherapies in these early studies occurred because they initiated a T-cell reaction similar to deleterious overstimulated Th-1 immune responses.93–96 Therefore, 2 common concerns for Aβ active immunotherapy treatments are that the therapy induces autoimmune inflammatory toxicity and does not address the full range of AD pathologies, and, therefore, may not consistently produce clinically beneficial results.97
Along with the use of synthetic full-length Aβ peptides, other methods of providing active Aβ immunization include the use of Aβ fragments (eg, Aβ1-10) to stimulate the production of antibodies by B cells, which then capture Aβ peptides and clear the complexes from the brain.90 Active immunotherapy has demonstrated some benefits for AD patients, and ongoing research anticipates identification of improved methods to achieve the desired benefits while mitigating the adverse effects. The current adverse effects and safety of this approach continue to be deterrents for FDA approval. Until recently, UB-311 (United Neuroscience, Inc.) was the only Aβ active immunotherapy investigated in Phase III clinical trials, with no active immunotherapies currently in Phase IV or FDA approved.
Passive immunotherapy for AD has been investigated using humanized mAbs. Passive immunotherapy is the process of injecting pre-made antibodies to provide augmented immunity to the host. These mAbs are usually administered peripherally through intravenous infusions or via subcutaneous injections and must then cross the BBB to reach the brain parenchyma. The current variations in treatment using mAbs derive from differences in selectivity for polymorphic variants, which may recognize epitopes of Aβ based either on the Aβ conformation or a specific sequence of Aβ peptides.98 A benefit to passive over active immunotherapy is that it overcomes several of the problems encountered with active immunization, including the following.90
1. The interaction between Aβ and mAbs causes a decrease in the formation of toxic Aβ aggregates inside the brain.
2. The binding between the Fc domain of the mAb and the Fc-γ receptors found on microglia results in the phagocytosis of the Aβ-mAb complex within the brain.
3. The Aβ-mAb complex activates complement-dependent cytotoxicity, which then lyses the designated target cell of the therapy.
4. mAbs interact with Aβ in the peripheral blood and form a concentration gradient that causes Aβ to flow out from the brain; this process is referred to as the "sink effect."88,90,99
This form of therapy allows for direct disassembly of Aβ in the brain. The use of mAbs is also one of the easiest ways to provide anti-Aβ antibodies without increasing the likelihood of an uncontrolled Th-1 mediated antibody response.97 Another 2 potential benefits to passive versus active immunization are (i) the possibility to target specific conformations of Aβ peptides, which could then be used to remove specific forms of Aβ such as monomers, oligomers, or fibrils; and (ii) the potential ability to rapidly clear administered antibodies in case of adverse reactions.88
Problems that have been associated with Aβ passive immunotherapy, which must be addressed in the future, include high costs; effectively crossing the BBB consistently (<0.1%);100 the risk of hemorrhage; the risk of triggering an immune response against the injected Aβ antibodies; the necessity of repeated infusions or injections over time to maintain a constant amount of therapeutic antibodies; and correctly selecting the appropriate antigens to target toxic forms of Aβ so as to not interfere with physiological Aβ functions, which play an integral role in neuroprotection, modulation of long-term potentiation, and innate immunity.88,97,101,102 Currently, 4 Aβ passive immunotherapies are in Phase III trials (donanemab, gantenerumab, lecanemab, and solanezumab) and zero are in Phase IV trials, and 1 has been conditionally FDA approved (aducanumab). Below, we provide more details about both active and passive immunotherapies tested in AD patients.
Active Immunotherapies
Ub-311
One therapy that introduces Aβ peptide as an antigen to the host to elicit an immune response is UB-311 (United Neuroscience, Inc., now Vaxxinity). UB-311 is a synthetic peptide vaccine that consists of 2 Aβ sequences covering amino acids 1–14 linked to different UBITh helper T-cell peptide epitopes,103 which are introduced with the aim of activating regulatory T-helper type 2 cells (Th-2) more than proinflammatory Th-1 cells,90 and which are packaged in a proprietary vaccine delivery system. As mentioned above, one difficulty that newer active immunotherapy vaccines must overcome is mitigating the deleterious mounting of autoimmune inflammatory responses.93–95,97 One of the most plausible reasons that earlier versions of Aβ active immunotherapies developed inflammatory processes is the fact that the Aβ peptides that were introduced as antigens included the Aβ C-terminus region, which is proposed to activate the Th-1 response.96 Current versions of Aβ active immunotherapies, including UB-311, no longer include this portion of the Aβ peptide for this reason.
UB-311 is specifically designed to avoid any cross-reactivity with similar endogenous antigens.103 Wang et al found in preclinical studies in guinea pigs (8–12 weeks of age), adult male baboons (8–10 years of age), adult male and female Cynomolgus macaques (>4 years of age), and hAPP751 transgenic mice (mice expressing a mutant human APP with both the Swedish [K670N/M671L] and London [V717I] mutations) and their littermates (14 ± 2 weeks of age) that UB-311 was able to generate N-terminal anti-Aβ antibodies against the sequence 1–10, which neutralized Aβ toxicity and promoted extracellular Aβ plaque clearance from the brain. It was also found that there were no anti-Aβ cellular responses in the hAPP751 mouse model and that acute and chronic dosing were safe and well tolerated.104 It was later confirmed in Phase I trials that the vaccine formulation appeared to be s...
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