Special Edition: Pivotal Road
This publication serves as a precursor to our forthcoming CTAD report, which will be available to all audiences on 7 Dec (CTAD donors will have first access prior to 7 Dec). The precursor focuses on upstream pathways for Alzheimer's Disease.
Introduction: SOTC Analytics spent over 95 hours in Nov 2022, to conduct a deep-dive analysis of Anavex's AAIC gene poster. In combination with the gene poster, previous company slides, recent educational video, third-party Alzheimer's pathway (KEGG & KANPHOS), and a multitude of recent peer-review publications; we assess there to be three critical pathways upregulated (normalized) directly by Blarcamesine. At least two of these three pathways are directly elucidated by in-human data revealed at AAIC 2022.
We believe this data will be immeasurably important for Blarcamesine's regulatory approval, as it hones in on upstream therapeutics provided by the drug. The level of analytic rigor conducted by Anavex, Ariana, and third-party scientific benefactors currently supersedes that of even the highest tier biopharmaceutical companies. SOTC Analytics assesses this level of data scrutinization will almost certainly propel Anavex to approval, pending a successful readout of Anavex's 2b/3 Alzheimer's trial.
This publication was written by SOTC Analytics and reviewed by the distinguished Dr. Amor Mehta, MD Neurologist, Epilepsy Specialist – President/CEO of Neurology Center for Epilepsy and Seizures, LLC. Five other constituents including investment bankers, healthcare creative directors, and other professional craftsman also contributed to this report.
Image 1: Two Separate Pathways - IP3R/ATP & UPR
Long-standing Hypothesis Playing Out
Displayed by Image 1, we see that Anavex has been focused on at least two primary pathways, IP3R/ATP, and Unfolded Protein Response (UPR); both of which have been covered to some degree in the Update Compendium on 29 Aug 2022. Along with these two pathways confirmed during the AAIC genomic findings, SOTC Analytics is tracking at least one other newly revealed critical pathway.
The last major pathway was also confirmed in the AAIC genomic findings but has not been presented by the company yet in great detail. We are alluding to the 26S proteasome (proteostasis), which is the primary mechanism for tagging and breaking down misfolded proteins. Whereas we know Blarcamesine clears misfolded proteins, it hasn't been entirely clear by which upstream mechanism the drug was accomplishing this task. We now believe this ability to be vastly better understood post-AAIC, and we will discuss the 26S proteasome later.
Together there are three major pathways - although the AAIC genomic data revealed at least partial resuscitation of a few other lesser explored pathways which won't be discussed in great detail during this report. It is possible that there are other pathways ["not only that"], but it is hard to be sure without the entire genomic listing. The three major and additional pathways are as follows:
Major Pathway: Complex 1, 3 and 4 of the Electron Transport Chain and IP3R Regulation (IPR3/ATP [includes calcium])
Major Pathway: UPR Gene 'IRE1a', 'PERK' and 'ATF6' Activation (UPR)
Major Pathway: 26S Proteasome Regulation (Proteostasis)
Other Pathway: AGE/RAGE Signal Path (Diabetic-focus)
Other Pathway: Insulin Receptor Regulation (Synaptic aid - mentioned during Anavex's first educational video)
Other Pathway: mTOR Inhibition (Autophagy)
Other Pathway: GPCR Regulation (Moderates neurotransmitter release - not proven in AAIC gene poster)
Cellular Visualization & Pathway Mapping
In order to fully grasp image 3 (below), image 2 should be examined as to understand the basic shape of the cell. Notice the endoplasmic reticulum and mitochondria share a tethering landscape known as the MAM. It is in the MAM that S1R primarily resides. Here the S1R regulates calcium channels, prompts misfolded protein degradation, reinforces cellular pro-survival genes, and more. Importantly, the MAM is the primary source of ATP (energy) transfer between the mitochondria and the rest of the cell. These core areas of the cell are surrounded by cytoplasm - proteins and other substances in liquid form.
Image 2. Brief Visualization of the Cell; the Mitochondria and it's Tether to the ER
Now that the basic cellular structure is understood, we can make sense of the KEGG database Alzheimer's mapping. The KEGG database was directly referenced by Anavex at the bottom of their AAIC gene poster. All of the individual genes shown in that poster have been displayed in the KEGG database as holding probable involvement in Alzheimer's pathology. SOTC color-coded all of the gene clusters mentioned in the AAIC gene poster which are as follows:
Red: Proven to be directly upregulated by Blarcamesine in PDD & AD patients [high confidence]
Yellow: Theorized to be upregulated by Blarcamesine in previous company graphics, relevant peer-review journals, or officer commentary [moderate confidence]
Blue: Calcium hub (notice the abundance of off-chutes and the central role played by calcium)
Purple: Probably upregulated by Blarcamesine (rationale in the 29 Aug 2022 update) [low confidence]
Image 3. KEGG Database Alzheimer's Pathways with Color Coding (high confidence, medium confidence, low confidence, interest)
Visually, it is obvious Blarcamesine is having an effect via a massive upregulation footprint in the mitochondria (top right), the 26S proteasome (center right) and the endoplasmic reticulum - especially the UPR pathway (center/center right). Other pathways influenced include the AGE-RAGE diabetic pathway, autophagic pathway, and insulin signaling pathway - none of which will be covered in great detail here (center bottom).
We will now break out the three major pathways, gene percentages upregulated in each, and their relevance to overall cellular function.
ELECTRON SUPPORT CHAIN: PATHWAY A (Located in the Mitochondria - Top Right)
Electron Transport Chain Complex 1: First step in the oxidative phosphorylation process needed to make the majority of ATP (energy) in the mitochondria - which ends up fueling the entire cell. Complex 1 is the most susceptible complex to premature electron/oxygen leakage - thus the main production site of superoxide. Some peer-reviews reveal primary dysregulation of genes within complex 1 are caused at least in part by tau. Note: Complex 2 (unlisted) is the only electron transport chain complex which does not permeate through the mitochondrial membrane and into the cell. In other words, of all five complexes, it is localized to the mitochondrial cell wall. Gene Totals: 48 total genes in complex 1, with Anavex restoring at least 26 (54%)
Electron Transport Chain Complex 3: Third step in the oxidative phosphorylation process needed to make the majority of ATP (energy) in the mitochondria - which ends up fueling the entire cell. If complex 3 is inhibited, components of the electron chain preceding complex 3 fully degrade, and those after it become oxidized. Gene Totals: 11 total genes in complex 3, with Anavex restoring at least 5 (45%)
Electron Transport Chain Complex 4: Fourth step in the oxidative phosphorylation process needed to make the majority of ATP (energy) in the mitochondria - which ends up fueling the entire cell. Complex 4 is where ATP is finally produced and is the final step in the electron transport chain. In multiple peer-reviews dating back to the 1990’s, complex 4 has been singled out as a central defect point found in Alzheimer’s patients. Some peer-reviews reveal primary dysregulation of genes within complex 4 are caused at least in part by Aβ. Note: Complex 5 is called ATP synthase, where ATP receives final catalyzation, and doesn't count as an official step in some academia. Gene Totals: 21 total genes in complex 4, with Anavex restoring at least 9 (43%)
Electron Transport Chain Summary: Readers should absolutely go back and see the 29 Aug 2022 update as it is an intensive deep dive of the electron transport chain and it's functions. However, in short, Blarcamesine has a marked effect on the primary process driving ATP in the cell. It is important to note that the electron transport chain is not the only way the cell produces energy. There are various other ways this is accomplished; the second most prominent manner is known as the TCA cycle. Whereas the electron transport chain is located along the mitochondrial membrane (lining/wall), the TCA cycle occurs within the mitochondria itself. To put in perspective the impact of the electron transport chain versus other ATP-generation methods, one should understand the amount of ATP generated by each system. It has been well-established that the electron transport chain produces 28-32 ATP per cycle, meanwhile the TCA only produces 2-3 ATP per cycle. With this in mind, it is easy to comprehend how degradation of the electron transport chain (any step) can have catastrophic outcomes for the cell including death, ATP failure, oxidative stress with ROS creation, and inflammation. Without the ability to output adequate energy, the cell will eventually begin failing in a theory known as mitochondrial cascade. Mitochondrial cascade is an Alzheimer's theory in which the primary cause of Alzheimer's is electron transport failure, ATP depletion, and cell death. At this point, SOTC Analytics assesses this theory to have the most merit as it pertains to Alzheimer's causation. The mitochondrial cascade theory leaves open the possibility for Alzheimer's to be caused by different pathways in some patients (minority). Something of personal interest regarding this is found in image 1. Anavex had previously theorized Blarcamesine's influence on ATP generation; however, the company assumed their therapeutic properties were influencing the TCA cycle specifically, not the electron transport chain. It is our opinion that the company may have not realized the magnitude of their effect on ATP until after the genomic analysis. As a final note on the subject, readers should be keenly aware that calcium regulation plays a major role in the electron transport chain; both before ATP production, and after during ATP propagation.
MITOCHONDRIAL GENES UPREGULATED NOT DIRECTLY INVOLVED IN ELECTRON SUPPORT CHAIN
ABAD: A component of cellular homeostasis within the mitochondria & ER, with exceptional abundance in the former. ABAD has cytoprotective functions and aids in stress response. When exposed to Aβ, ABAD malforms and begins promoting free radicals and oxidative stress within the mitochondria & ER (amplifying Aβ induced cellular stress). Gene Totals: 1 total gene with Anavex restoring 1 (100%)
MPTP: A pore in the mitochondria membrane which expels toxic abundances of material as well as acting as a master regulator of ATP output by maintaining ATP/thermogenesis within the mitochondria. If inhibited, it can lead to mitochondrial swelling (bloating) and cell death through apoptosis. MPTP is related to the electron support chain but not a direct step in the process. Gene Totals: 8 total genes with Anavex restoring at least 1 (13%)
IP3R (Calcium Funneling - Possible Lynchpin to Cellular Homeostasis)
Calcium is the lifeblood of the cell, exchanging nutrients in the cell's cytoplasm, ER and mitochondria. Anavex actually made the connection between S1R and the importance of calcium funneling in 2008. Specifically, the company notes regulation of IP3R via Blarcamesine and other S1R agonists. There are three types of IP3R, which act as the main receptors responsible for transferring calcium between the ER and mitochondria. There has been copious third-party research making links between calcium deficiency and ROS (oxygen species), ATP (energy) production, apoptosis (cell death), and post-transnational modification.
During a recently published test, researchers used knock out cells to cut off IP3R and observe the effect. They found that there were 45% fewer contacts (pores) between the mitochondria and ER, and that the contacts that remain were no longer as close (drifted further apart) in most cases. Interestingly, three separate peer-reviews have now mentioned that an ~50% calcium deviation is the median required to show a pronounced negative or positive effect. The knock out study proved that dysfunctional IP3R or a disruption to calcium channels massively effects homeostasis of the cell. On the flip side, there are numerous peer reviews elucidating disastrous effects when calcium is released too abundantly. We are going back to what SOTC hits on most - Blarcamesine REGULATES functions. It doesn’t only lower or raise faulty genes/processes, it brings them back to homeostasis regardless of whether the drug needs to boost or reduce processes in particular patients. In this case, calcium regulation may very well be the ultimate upstream lynchpin for most Alzheimer's patients. Without proper nutrient funneling the mitochondria fails to produce ATP. Without enough ATP the 26S proteosome (below) fails to clear proteins, and with an abundance of faulty proteins, the UPR goes into overdrive causing mass cell death via apoptosis (also below).
Did you know that when women meet menopause they begin losing the ability to retain calcium by about 2% per year? Women on average begin menopause at age 50, and most women reasonably live to at least age 70. That’s 20 years of significant calcium degradation, ~40% by age 70.
Notice in image 4 (below) how women begin experiencing Alzheimer's around age 50. There is a large spike between ages 80 and 90. I would hypothesize that at those ages, calcium loss is too abundant (in excess of 50%) to withstand normal nutrient funneling between the endoplasmic reticulum and the mitochondria. At this point, the cell begins to die (apoptosis) and dead cells/proteins begin accumulating.
This may appear to be more of a female problem initially; however, by age 65-70 men also begin losing their ability to retain calcium. Men also don’t live to longevity as frequently as women. We believe calcium to be extremely significant to Blarcamesine's MOA and Alzheimer's as a whole. Anavex also alludes to this. During their first educational video, the company mentioned calcium on at least three separate occasions. We know that neurotransmitter dysfunction, protein clumping, and inflammation are probably the key downstream causes of CNS issues, but the first catalyst - at least for most people - may very well be calcium deficiency. Sometimes the simplest answer is the right one, but in any case, we are excited to see what future genomic findings are revealed by Anavex.
26S PROTEASOME: PATHWAY B (Center Right)
26S Proteasome: The 26S proteasome is the primary mechanism for proteostasis (protein harmony), and is created by encasing a 20S subunit in 19S subunits. To put as plainly as possible, the 19S subunits 'tag' faulty misfolded proteins for deletion. The 20S subunit then processes the proteins and deletes them. The entire process is known as the ubiquitin-proteasome pathway, which has emerged as a central player in the regulation of several diverse cellular processes, affecting DNA transcription, cell cycle, inflammation, and ribosome biogenesis. At least 80% of proteins are discarded via the 26S proteasome. Gene Totals: 20 total genes in 19S, with Anavex restoring at least 2 (10%); 15 total genes in 20S, with Anavex restoring at least 10 (67%)
26S Proteasome Summary: Over the years investors have heard Dr. Missling say the phrase "misfolded protein" and how Blarcamesine acts to dispel these faulty components. Until now it wasn't truly clear how Blarcamesine was influencing this process. With the 26S proteasome being ATP-dependent, it is likely that that the protein clearing is downstream of the electron transport chain regulation. With its newfound ATP, the 26S proteasome can continue tagging and deleting toxic proteins. As this function continues, misfolded proteins are no longer able to accumulate - a key driver of neuroinflammation. The impact of Blarcamesine on the 26S proteasome, especially the 20S deletion subunit (67%!) cannot be understated.
UPR GENE ACTIVATION; CHOP: PATHWAY C (Center Right)
CHOP: I found this resource to be extremely useful during my research towards CHOP and I will quote it a few times in this section. CHOP is a transcriptional factor, which regulates the expression of many anti-apoptotic and pro-apoptotic genes. “During normal physiology, CHOP is ubiquitously expressed at very low levels. However, when pathological conditions or microbial infection-caused ER stress is overwhelming, the expression of CHOP rises sharply and apoptosis is activated, and this process can occur in a wide variety of cells. Those processes are mainly regulated by three factors, including protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring protein 1 (IRE1).” These pathways are activated after regular autophagic functions fail to correct rampant ER stress.
PERK, ATF6, and IRE1 likely sound familiar as I covered them in-depth during the 29 Aug 2022 update. Anavex has theorized IRE1’s involvement in Alzheimer’s pathology for years, but only as recent as the AAIC gene poster is this activity elucidated. Although more detail is found in the aforementioned update, the quoted article has good short descriptions of all three which are paraphrased below:
"PERK is a transmembrane protein and an important sensor that participates in the UPR by attenuating protein translation and regulating oxidative stress. Unfolded proteins in the ER stimulate PERK oligomerization and autophosphorylation, and can phosphorylate eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α promotes the transcription of ATF4, which converges on the promoters of target genes, including CHOP, GADD34, and ATF3. Research shows that PERK−/−and ATF4−/−cells and eIF2α (Ser51Ala) knock-in cells fail to induce CHOP during ER stress. The PERK/ATF4/CHOP signaling pathway is considered to play a pivotal function in inducing cell apoptosis, both in vitro and in vivo. However, research shows that CHOP may not fully induce cell death, and that CHOP and ATF4 cooperation is required for the induction of cell death."
"ATF6 is a transmembrane protein. Under ER stress, ATF6 translocates to the Golgi compartment where it is cleaved and activated. When ATF6 is activated, it translocates to the nucleus as a homo-or heterodimer and interacts with ATF/cAMP response elements and ER stress-response elements. Such complexes bind the promoters of several genes involved in UPR (such as CHOP, GRP78, XBP1) and induce target-gene transcription. Along with XBP1(s), ATF6 contributes to the augmentation of ER size and ER protein-folding capacity through target genes. ATF6 can activate the transcription of both CHOP and XBP-1, while XBP-1 can also regulate the expression of CHOP. Thus, ATF6 can cooperate with XBP-1 to activate CHOP."
"IRE1 is a transmembrane protein containing two functional domains, including an N-terminal luminal sensor domain and a C-terminal cytosolic effector (40). IRE1 contains protein kinase and endoribonuclease activities. Unfolded proteins in the ER stimulate IRE1α oligomerization and autophosphorylation, which activates the endoribonuclease activity. Upon activation, IRE1α splices the substrate precursor, XBP-1, mRNA introns to produce a mature and active XBP-1 protein. The active protein then binds the promoters of several genes involved in UPR and ERAD, and regulates gene expression (such as CHOP) to restore protein homeostasis. Thus, CHOP expression can be upregulated by XBP1(s). IRE1α can stimulate the activation of the apoptotic-signaling kinase-1 (ASK1), which then activates the downstream kinases, Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), which cause apoptosis. The P38 MAP kinase family phosphorylates Ser78 and Ser81 of CHOP, which induces cell apoptosis. Moreover, during tunicamycin-induced apoptosis, the JNK inhibitor, SP600125, could suppress CHOP upregulation and subsequent death receptor 5 (DR5) expression, indicating that JNK activation is also involved in the modulation of CHOP. JNK and p38 MAPK can also promote the phosphorylation and activation of the pro-apoptotic protein, BAX, to regulate cell apoptosis."
Gene Totals: 1 total gene in CHOP, with Anavex restoring 1 (100%).
UPR & CHOP Summary: While PERK, ATF6, and IRE1 had no direct gene expression identified in the AAIC poster, CHOP is downstream of these proteins - indicating that there is some sort of upstream effect on PERK, ATF6, and IRE1. By reducing the hyperactivity of CHOP during periods of longstanding ER stress, the road to apoptosis is negated thus saving the cell. Because apoptosis is a bit later (after calcium, mitochondrial, and ER-stress), it is possible PERK, ATF6, IRE1, and CHOP aren't being directly influenced by Blarcamesine at all. It is technically feasible that Blarcamesine's intervention during earlier stages in disease pathology is preventing the hyperactivity of CHOP to begin with. We are currently unsure of this but do think it is an interesting notion.
We present the 29 Aug 2022 assessment for the closing of the CHOP segment as we believe it summarizes our main points nicely: "Based on the recent AAIC AD/PD Gene Clustering poster and this latest UPR analysis, it is likely Blarcamesine actually enables all three UPR pathways instead of just IRE1. Additionally, and more critically, Blarcamesine restores these pathways to a state of normalcy - maintaining healthy adaptive UPR while avoiding overexpression (maladaptive) uncontrollable neuroinflammation and cell death. Based on these findings, SOTC Analytics assesses Anavex's omission of the entire AD/PD Gene list is possibly a tactic to stave off potential buyers at this stage of company development. It is our opinion that Blarcamesine has even more cross-CNS potential than previously thought and while we have no concrete evidence to support this, we believe the company may be trying to downplay (obfuscate) their IP at this time in order to preserve long term shareholder value."
"In addition to the 40 genes related to the electron support chain mentioned earlier, we observed five additional genes which are integral to healthy UPR mechanisms; MAP2K2, PSENEN, TXN, TXN2, and perhaps the most important - DDIT3. DDIT3 (aka CHOP) is a central/shared protein between all three UPR pathways (PERK, IRE1, and ATF-6). DDIT3 is involved in the regulation of genes that encode proteins involved in proliferation, differentiation and expression, and energy metabolism. As Anavex has seen DDIT3 levels restored to normalcy, this is an extremely exciting revelation. Combining electron support chain and UPR gene restoration, Anavex has proven with in-patient data how Blarcamesine reduces neuroinflammation, preserves calcium and nutrient funneling, and prevents cascading cell death which simultaneously enables healthy (adaptive) protein clearing."
Image 5. AAIC 2022 Alzheimer's & Parkinson's Disease Dementia Gene Data
CTAD Precursor Summary
On 31 July 2022, Anavex PR'd their AAIC gene poster which included the following quote, "These findings will facilitate contextualization of upcoming readout of ANAVEX®2-73 Phase 2b/3 Alzheimer’s disease clinical trial."
Listening to the Guggenheim Conference on 14 Nov 2022, it is obvious Anavex is still working to completely understand upstream effects of Blarcamesine; however, the company has given investors and researchers fantastic insights into holistic S1R MOA. This is the primary reason for this precursor report. SOTC Analytics wanted to make the most of the genomic data revealed by the company to understand what kind of indicators are being watched behind-the-scenes as well as the magnitude those indicators have on diseases.
After many years, we are finally able to narrow down specific upstream effects with quantitative and qualitative data. Pending a successful 2b/3 data release, we believe this genomic rigor will pay great dividends to the company when approaching regulatory agencies for approval.
While it cannot be 100% certain at time of writing, SOTC Analytics is confident Dr. Missling and the Anavex team is about to present overwhelmingly positive data at CTAD 2022. At last, the company's hard work and investor diligence will pay off. Please stay tuned for part two post-CTAD.