Dementia, a chronic and progressive disorder due to the apoptosis of neurons and worsening of cognitive function, has been posing challenges to caregivers as well as medical professionals. Dementia occurs in different forms such as vascular dementia, Lewy body dementia, Alzheimer’s disease and others.
Common symptoms are memory loss, delusions, hallucinations, violence, depression etc.
Fig. 1. Types of dementia
Source: “Guide to Understanding Dementia with Lewy Bodies” (Edited by Kenji Kosaka)
Alzheimer’s disease (AD), a neuro-degenerative disease affecting 50 million people globally, is predicted to be 152 million-strong by 2050. (1) It is the fourth leading cause of death in elderly people over the age of 65 years and the sixth leading cause of death in the US. AD is thought to begin 20 years prior to the appearance of symptoms such as memory loss, challenges in planning or solving problems, difficulty with daily activities, problem with speech, loss of ability to retrace, changes in mood and personality etc. (2)(3).
The main changes taking place in the brain of an Alzheimer’s patient include:
Fig. 2. Accumulation of amyloid plaques (Source)
AD is driven by several hypothesis such as beta amyloid hypothesis, tau hypothesis and inflammation hypothesis. The progression of outcomes from these have further led to a study of cholinergic hypothesis, dendritic hypothesis, mitochondrial cascade hypothesis and others involving oxidative stress occurring in the hippocampus, amygdale association cortices and certain subcortical nuclei of the brain. The starting point of these hypothesis is APP (Amyloid precursor protein). (4)
Overview of APP in the brain
APP belongs to the family of APLP (Amyloid precursor-like proteins) encoded genes. It plays a crucial role in brain development, neuronal plasticity, memory and neuroprotection. It is encoded by a single gene and exists in 3 isoforms, APP695, APP751 and APP770, due to differential splicing. APP695 is more predominant than other isoforms. APP751 and APP770 result from the splicing of exons encoding kunitz-like protease inhibitor domains(KPI) and OX-2 antigen domains that are lacking in APP695.
Structure
Cleavage sites
Cleavage sites for α and β secretases are in the juxtamembrane region that links E2 and TMD. γ secretases cuts in TMD.
Fig. 3. Structure of APP
APP processing
APP processing includes canonical and non-canonical pathways. Canonical pathways involve cleavage by α or β secretases, followed by γ secretase. APP, upon cleavage by α secretases and β secretases, produces APPsα and APPsβ, respectively; along with corresponding C terminal fragments (CTF), following cleavage with γ secretases. They produce Beta Amyloid (Aβ), P3 due to exoproteolytic cut at N-terminus and AICD (APP intracellular domain) due to endoproteolytic cut at E-site. The Aβ oligomers are cleared by means of proteolytic degradation by neprilysin and insulin-degrading enzymes (IDE).
Table 1 lists the different forms of enzymes that act on APP.
| Enzyme | Form |
|---|---|
| α secretases | ADAM 10 (disintegrin and metalloproteinase domain containing protein 10) |
| β secretases | BACE1 and BACE2 |
| γ secretases | PS1, PS2, NCT, PEN2, APH1 and APH2 |
Non-canonical pathway involves action of δ- secretases, η- secretases, meprin β and caspases.(5)
Fig. 4. APP-processing pathways
Pathogenic pathway
Fig. 5. Pathogenic pathway of AD (6)
The exact cause for AD is not known. Due to some unknown changes in the cleavage of APP, there is an imbalance between production and clearance of Aβ peptides, due to which there is a formation of soluble oligomers. This results in aggregation via nucleation-dependent pathways, followed by coalesce, to form fibrils that are insoluble due to beta-sheet conformation, thus resulting in senile plaques.
The Aβ42 peptides formed induce oxidative damage, promote tau hyper-phosphorylation and attract microglia that are activated. These produce pro-inflammatory cytokines, resulting in inflammation. This causes neuronal and vascular degeneration, further activating oligodendrocytes and resulting in decreased IDE function. Aβ oligomers bind to α-7nAChRS and astrocytes, which exocytose the glutamate and activate NMDAR-triggering Ca2+, leading to dysfunctional mitochondria, increased NO (nitric oxide) and ROS (reactive oxygen species). This leads to neurotoxicity and AD. (7)
There are several reasons for the imbalance in production and clearance of amyloid proteins, some of which are listed below.
New protein aggregation
Disclosure in a recent research project work reported the discovery of new protein aggregation encoded by FAM222A: a protein called aggregatin of molecular weight 47KD and consisting of 452 amino acids, which is expressed in CNS binding to amyloid deposits by interacting with Aβ, was found to be more expressed in AD patients. (8)
Alpha sheet resembling beta sheet
Current studies state that AD is not caused due to β-sheet-rich, insoluble amyloid β-peptide plaques; and plaque burden is not a cause of cognitive impairment, but due to the toxic levels of oligomers. Oligomers share a common conformational structure but not sequence-based epitope. Based on this, scientists proposed a new confirmation on α-sheets resembling β-sheets, called α-sheet hypothesis, suggesting that misfolding of proteins is the cause of cognitive disorders such as AD.(9)
Gut bacteria
Gut microbiome excrete lipopolysaccharides (LPS) that cannot enter the bidirectional communication system of the microbiota-gut-brain; but as age progresses, the gastrointestinal epithelium and the blood brain barrier are more permeable, making the LPS and amyloids engage in bidirectional communication ands resulting in AD. (10)
Mutations
It is already known that early onset of AD is due to the inheritance of mutations in APP of chromosome 21, PSEN1 of chromosome 14 and PSEN2 of chromosome 1, while late onset of AD is due to mutations in APOE gene, e4 allele of chromosome 19, in particular. Researches are focusing on identifying different mutations responsible for early onset of AD through dominantly inherited Alzheimer network (DIAN). The identification of potential biomarkers will help in predicting the development of Alzheimer's disease. (11) By using an approach called genome-wide association study (GWAS), meant for identification of novel complex disease genes, the scientists confirmed three new AD-associated loci: ADAM10, BCKDK/KAT8 and ACE. (12)
Early diagnosis of AD plays a crucial role in prevention and treatment of the disease. Diagnosis of AD is currently being carried out by brain imaging, also called neuroimaging, which involves structural imaging, functional imaging and molecular imaging.
Structural imaging is performed with magnetic resonance imaging (MRI) and computed tomography (CT), the principle being shrinkage of the brain with the progression of disease. The main drawback is that it can be misled by tumors and other pathological conditions of the brain.
Fig. 6. MRI scan of brain - Control vs. Alzheimer patients (Source)
Functional imaging involves the use of positron emission tomography (PET) to detect reduced brain activity resembling the pathological condition of AD; for example, use of fluorodeoxyglucose (FDG)-PET associated with reduced glucose in brain.
Fig. 7. PET scan image of brain - Control vs. Alzheimer patients (Source)
Molecular imaging involves use of radiotracers to represent the pathological substances involved in AD, i.e., Amyloid and tau proteins. Pittsburgh compound B (PIB) is the first radiotracer used to highlight the Amyloid plaques in the brain. Recently, Amyvid (18F-florbetapir), Vizamyl (18F-flutametamol) and Neuraceq (18F-florbetaben) have been approved by the USFDA as radiotracers.
Apart from neuroimaging, researchers are concentrating on the following for detecting AD:
Genetic risk profiling involves the identification of mutations, which involve:
Use of artificial intelligence for diagnosis
More recently, Artificial Intelligence (AI) has seen use in the early diagnosis of AD, which involves developing and training an algorithm to recognize early degradation of the brain; which is otherwise difficult to evaluate.
A deep-learning algorithm developed by Benjamin L. Franc and team at the University of California, San Francisco and the University of California, Berkeley, was trained to predict the onset of AD in the early stages.
A database of 2,109 independent Fluorine 18-Fluorodeoxyglucose (F-FDG) PET studies performed between 2005 and 2017 was used for this study.
90 % of the data was used to train the algorithm, while the remaining 10 % was used to test the algorithm.
Researchers also used other 40 (F-FDG) PET scan sets, which the algorithm was not familiar with, to test its accuracy. It was found that the algorithm was predicting AD 6.3 years prior to final diagnosis with 82 % specificity.
Although there is a growing trend of using AI in diagnosis of AD, it will take more research efforts to achieve higher accuracy (14).
Use of navigation in gaming
A game called SEA HERO QUEST, developed by Deutsche Telekom in partnership with Alzheimer's Research UK, University College London (UCL), the University of East Anglia and game developers, has the ability to detect people at risk of Alzheimer’s. It was designed for better understanding of the relation between spatial navigation and the brain.
The game has the ability to distinguish people who are genetically at risk of Alzheimer's from those who are not. People with APOE4 gene demonstrated the worst performance in spatial navigation. (15)
There are in all, 4 prescription drugs to treat AD. These include acetylcholinesterase inhibitors (AChEIs) –Rivastigmine, Galantamine, Tacrine and Donepezil and NMDA receptor antagonist–Memantine, and a combination of Donepezil + Memantine. The mode of action of AChEIs is to inhibit the action of cholinesterase to breakdown acetylcholine, a chemical messenger involved in memory. Memantine works by partially blocking NMDA receptors and prevents the overstimulation of these receptors by glutamate, which results in cell death. These drugs only delay or slow the worsening of symptoms, and moreover, their effectiveness changes from person to person. They also have side effects such as seizures, bradycardia and worsening of pulmonary problems.
The list of approved drugs is presented in the table below:
| Drug | Company | Year of approval (US) |
|---|---|---|
| Donepezil (16) | Eisai Pharmaceuticals | 1996 |
| Tacrine (17) | Parke-Davis Pharmaceuticals | 1995 |
| Galantamine (18) | Janssen Research Foundation | 2001 |
| Rivastigmine (19) | Novartis | 2000 |
| Memantine(20) | Forest Laboratories | 2003 |
Table 2. List of drugs, the originator company and the year of approval in the US.
Therapeutic approaches
The growing need for new therapeutic approaches is increasing day by day. The following flowchart depicts the different approaches for treating AD:
Fig. 8. Approaches in AD treatment(21)
Mode of action
The main approaches for treating AD include the following:
The mechanism of action involved in new approaches has been depicted in the following flowchart:
Fig. 9. Mode of Action
A recent genetic approach of using a gene variant called Christchurch mutation to delay the symptoms of early family onset of AD was discovered accidentally. In a case-study of a family with 6000 members, where everyone carrying a gene mutation called Presenilin 1 (PSEN1) E280A was affected with early AD, there was an exception found of a single woman who had both the variants and yet, did not show any symptom until her 70s. (24)
Biologics
Advances in the understanding of pathogenesis in AD to the molecular level has shifted the attention of the researchers to the use of biologics such as vaccines, recombinant proteins, peptides and other small molecules.
Immunotherapy
Aβ42 stimulates T-cells, B-cells and microglial responses. The present active immunotherapy approach includes the use of synthetic fragments of Aβ conjugated with carrier proteins. Administration of monoclonal antibodies directed against Aβ falls under passive immunotherapy. The main mode of action is plaque breakdown, preventing formation of plaques called peripheral sink and aggregation inhibitors. (25)
Vaccines
Progression of Alzheimer‘s disease could be delayed and prevented with vaccination, based on the following criteria:
The first-in-human anti Aβ vaccine AN1792 resulted in side effects such as meningoencephalitis, and the Phase 2 trial was quickly terminated. Various peptide/ protein epitope vaccines currently undergoing clinical trials are presented in table 3.
| Vaccine | Company + collaborator | Constituents | Clinical trail | |
|---|---|---|---|---|
| Targeting Aβ | ||||
| ACC-001 | Pfizer+JANSSEN Alzheimer Immunotherapy Research & Development, LLC | Aβ1-7 +diphtheria toxoid protein | Terminated at low doses Completed Phase II at high dose | |
| CAD-106 | Novartis Pharmaceuticals | Aβ1-6+QB virus like particle | Phase-III | |
| V950 | Merck Sharp & Dohme Corp | Aβ1-15 + ISCOMATRIX | Completed Phase I | |
| Targeting tau proteins | ||||
| UBITh /UB-311 | United Neuroscience Ltd | Aβ1-14+Th2 biased delivery | Completed Phase I | |
| AADvac1 | Axon Neuroscience SE | Synthetic peptide derived from amino acids 294 to 305 of the tau sequence | Phase 2 | |
Table 3. Peptide/ protein epitope vaccines currently under trials
Past trials
Although there were more than 130 entities undergoing clinical trials, not a single entity has been approved since 2003 after Menatine. Some of the failures are listed below:
Ongoing trials
A few on-going clinical trials are presented in Table 4 below.
| Drug | Phase | Company and Collaboration | Mode of action | Date of start to estimated end date |
|---|---|---|---|---|
| BIIB076 | 1 | Biogen | Anti-tau antibody | February 17, 2017 to March 3, 2020 |
| Aducanumab | 2 | Biogen and Eisai | Anti-β-amyloid antibody | September 30, 2015 to August 5, 2019 |
| AL002 | 1 | AL002 | Targets immune system receptors | November 12, 2018 to March, 2020 |
| S-equol (AUS-131) | 1/2 | Ausio Pharmaceuticals | Estrogen receptor activator | May 5, 2017 to March 30,2020 |
| COR388 >sup>(24) | 1& 2/3 | Cortexyme, Inc. | Bacterial protease inhibitor | March 28, 2019 to December 31, 2021 |
| Neflamapimod (VX-745) | 2, 2b & 3 | EIP Pharma | Inhibits p38 MAPKα | July 8, 2019 to June 2020 |
| Xanamem | 2 | Actinogen Medical | Inhibits a cortisol-producing enzyme | Mar 23, 2017 to mar 15, 2019 |
| Piromelatine | 2 | Neurim Pharmaceuticals | Binds to and activates melatonin and serotonin receptors | November 2015 to November 30, 2019 |
| Lemborexant | 2 | Eisai and Purdue Pharma | Orexin receptor antagonist | December 20, 2016 to July 26, 2018 |
| ALZT-OP1 (inhaled cromolyn and oral ibuprofen) | 3 | AZTherapies, Inc. | Anti-inflammatory | September 2015 to December 2020 |
table 4. On-going drug trials
* Biogen and Eisai, its Tokyo-based collaboration partner, announced discontinuation of clinical trials of Aducanumab, but later on, announced that high doses of the drug had shown positive results and that after discussions with FDA, they would pursue regulatory approval. At the conference on Clinical Trials on Alzheimer’s disease held in San Diego, Biogen disclosed data to explain the reason to restart the clinical trials of aducanumab. (37) (38)
China’s GV-971
Conditionally approved by China National Medical Products Administration (NMPA), Sodium oligomannate is the first new drug in the last 17 years with the potential to treat AD. Invented by a team of scientists from Geng Meiyu [Shanghai institute of Materia medica under Chinese Academy of Sciences], it is derived from a seaweed-based brown algae that is reported to treat mild to moderate AD. In the journal, Cell Research, the authors described how a sugar moiety in a sea weed can suppress certain bacteria contained in the gut that can cause AD. The mechanism was later confirmed by a clinical trial carried out by Green Valley, a Shanghai-based pharmaceutical company. The team now wishes to gain full approval from USFDA by initiating phase-III study by the spring of 2020. (33) (34)
With increase in failure of treatment options, many startups are adopting different approaches for the treatment of Alzheimer’s disease.
Alzheimer’s Association International Conference (AAIC) is the largest and most influential annual international meeting focusing on dementia. In 2019, it was more focused on life style changes, clinical trial results and new targets for treating AD.A few highlights are presented below:
Staging system
Researchers Niklas Mattsson and Oskar Hansson of Lund University presented data on a new staging system of amyloid accumulation that could help in detecting AD way before the positive identification through PET scans. They conducted a study using the longitudinal data of cerebrospinal fluid Aβ42, along with PET scans of 741 participants, under the Alzheimer’s disease Neuroimaging Initiative. They categorized the progression of AD into 4 stages based on amyloid accumulation which is presented in Table 5 below. (39)
| Stage | Rate of Progression | Amyloid accumulation |
|---|---|---|
| 0 | 15% | Low risk of developing plaques. |
| 1 | 71% | Precuneus, posterior and isthmus cingulate, insula, and medial and lateral orbitofrontal cortices. |
| 2 | 53% | Parahippocampus, medial and inferior temporal lobes, inferior parietal lobe, and superior parietal, temporal, and frontal regions |
| 3 | Precentral, postcentral, paracentral, lingual, and pericalcarine cortices |
Table 5. The 4 stages of AD progression
PET scans showed positive for AD only at later stages when the disease progressed to the next stage. The staging system will help identify people with risk of AD progression in the early stages itself, which may be useful to detect disease pathology and also identify the stage of progression. (40)
Life style changes
Healthy lifestyle reduces the risk of AD even in a population that is genetically at risk of Alzheimer’s. Five research studies based on life style interventions were presented at AAIC-19. Topics discussed include the following:
CSF – p Tau as early detector of AD
Amyloid accumulation in the brain results in the over-production of Tau proteins by neurons. These Tau proteins are modified at certain sites such as threonine 181 and serine 217, and the resultant isoforms of p-tau can be detected in CSF and plasma. This suggests that a positive blood test for presence of p-tau may indicate the presence of amyloid plaques in the brain, which can be an early detector of AD. These markers (p181, p217) can be detected before the PET scans show positive results. (42)
Update on clinical trials
Clinical data with an innovative approach towards AD was discussed.
Inhaled Insulin in mild cognitive impairment (MCI) and AD
A phase 2/3 clinical trial involved 289 participants with MCI and AD, in which the control group received 40 IU of placebo and the test group received Insulin (humulin-RU100, Eli Lilly) a day for 12 months, and an open label extension for 6 months. Two devices were used for inhaling the insulin. The first 49 participants received insulin using Kurve Technology/Device 1, whereas the remaining 240 participants used Impel NeuroPharma/Device 2, due to the varying reliability of Device 1. Outcomes were measured using Alzheimer's Disease Assessment Scale for Cognition-12 (ADASCog-12) at 15 and 18 intervals. Activities of Daily Living Scale for MCI (ADL-MCI), and Alzheimer's biomarkers in cerebrospinal fluid (CSF) were also measured. Device 1 showed good results, whereas device 2 showed no significant difference when compared with the placebo.
Porphyromonas gingivalis: Cortexyme is targeting the inhibition of toxic virulence factor Gingipains secreted by Porphyromonas gingivalis in its phase 2/3 study of COR388 (GAIN trial, called GingipAIN inhibitor for treatment of AD).(43)
Biomarkers in AD
Measuring amyloid and tau in CSF and PET scans can be expensive and invasive. A few easily available, simple and inexpensive testing technologies that are currently under development were presented at AAIC19.
Plasma amyloid levels: Scientists at the National Center for Geriatrics and Gerontology in Japan suggested that measuring plasma levels of amyloid peptides (Aβ1-42, Aβ1-40 and APP669-711) and generating a combined ratio of results in a blood biomarker has the potential to identify people who are likely to develop AD in future. The blood biomarkers, when compared with amyloid PET scans, structural MRI, FDG-PET and behavioral tests, showed significant correlation. This test will be able to detect amyloid deposits even before onset of dementia-related symptoms.
Plasma Neurofilament Light: A neurofilament light (NfL) chain is being considered as a biomarker for neurodegenerative disorders, including AD. NfL is able to distinguish between different neurodegenerative diseases based on the blood NfL levels. A cut-off point of NfL is established to differentiate among several neurodegenerative disease and health controls. (44)
A few interesting patents have been presented below:
Diagnosis
US20200080132A1 from Alisch Reid Spencer, Hogan Kirk Jeffrey and Madrid Andy titled Test for detecting Alzheimers disease deals with a method for measuring the methylation level of differentially methylated positions (DMPs) sites in B3GALT4 and/or ZADH2 using the following steps:
a) extracting genomic DNA from a blood sample of a human individual suspected of/ already having AD;
b) treating the extracted genomic DNA with bisulfite;
c) amplifying it with primers specific for B3GALT4 and a pair of primers specific for ZADH2; and
d) measuring the methylation level using methylation-specific PCR, DNA restriction enzyme analysis, microarray analysis, pyrosequencing, bisulfite genomic sequencing PCR or whole methylome sequencing.
Treatment
WO2020061150A1 from Biogen MA INC. titled 0-Glycoprotein-2-Acetamido-2-Deoxy-3-D-Glucopyranosidase inhibitors deals with pharmaceutical salts involving method of inhibiting 0-GlcNAcase to treat neurodegenerative diseases such as AD
The patent deals with a method of treating a disease such as AD characterized by hyper phosphorylation of tau in the brain, by administering an effective amount of the compound.
WO2020028290A1 from Alzheon Inc. titled Methods for treating neurodegenerative disorders deals with a method of treating a patient suffering from AD by selecting a patient with levels of 3-sulfopropaonoic acid (3-SPA) lower than a predetermine level and administering a metabolite of Tramiprosate for treatment which will inhibit the aggregation of Aβ42 into small oligomers.
Drug targeting
CN110841062A from China Pharmaceutical University titled SER34 LMTK1 and its phosphorylated protein as alzheimer drug target applications deals with LMTK1 and its Ser34 phosphorylated protein as drug targets in screening drugs for prevention and treatment of Alzheimer's disease. The drug inhibits synaptic atrophy by regulating Rab11.
Monoclonal antibodies
EP3521308A1 from Probiodrug A titled Humanized and de-immunized antibodies deals with the use of monoclonal antibodies for diagnosis and, treatment of amyloidosis. The antibodies bind to pyroglutamated amyloid beta peptide in plasma, brain, and cerebrospinal fluid and prevent the accumulation or reverse the deposition of Aβ N3pE within the brain and in various tissues in the periphery, and thus alleviate amyloidosis.
EP2994160B1 from Baxalta titled Treatment of Alzheimer's disease subpopulations with pooled immunoglobulin G deals with pooled human immunoglobulin IgG composition to treat a subject with moderately severe Alzheimer's disease or carrier of at least one APOE4 allele.
Stabilization of microtubules in the neurons
IN201811020565A from Indian Institute of Chemical Biology titled Nonapeptide of formula i, Pharmaceutical compositions and methods for preparation thereof deals with peptide-based microtubule stabilizer working on the principle of Taxol binding pocket of P-tubulin and hydrophobic region of amyloid beta. This leads to a potential nonapeptide, which strongly binds with taxol pocket of P-tubulin and serves as an excellent microtubule stabilizer, Ap aggregation inhibitor and neuroprotective agent.
The search for biomarkers is promising in diagnosis, such as, for example, the use of tyrosine phosphorylation-signaling modulation in erythrocytes. Using AI in diagnosis is definitely set to grow; and with availability of more and more data, the accuracy will indeed get better. As treatment options are getting fewer, with no new drugs on the horizon, scientists are working on techniques to catch the disease in the initial stages. The focus of AD treatment has so far been in treating the symptoms through release of chemical messengers from the cells, without addressing the underlying cause, leading to destruction of cells and progression of AD.
The current focus has also been to understand the mechanisms involved, especially the role of different proteins in the formation of plaque. Efforts are in progress to develop biologics such as monoclonal antibodies that will destroy the plaques and stop the aggregation of proteins. Influence of hormones such as insulin is gaining importance. The effect of oxidative stress and inflammatory factors; such as the influence of interleukin and glycosidase inhibition, along with signaling pathways such as amylin, insulin dependence etc.; are being studied. Scientists are also trying to understand the key proteins, such as involvement of translocator proteins and their modulation by respective agonists or antagonists to prevent or treat AD. In the near future, the direction will be towards early diagnosis and slow progression of disease.