Introduction: SOTC Analytics spent over 80 hours in Feb & Mar 2023, to conduct a deep-dive analysis of Anavex's AAIC gene poster as it relates to Parkinson's Disease. In combination with the gene poster, previous company slides, recent educational video, third-party Parkinson's pathway (KEGG & KANPHOS), and a multitude of peer-review publications over the last decade; we assess many of the pathways targeted by Blarcamesine in Alzheimer's Disease to remain relevant in Parkinson's Disease, indicating CNS-wide potential. Beyond these shared pathways (mentioned later), we identified at least four Parkinson's-specific targets, some of which are largely regulated by Blarcamesine's non-S1R binding affinities.

We assess Anavex will use the Parkinson's Disease P2 and OLE data towards Alzheimer's Disease approval considering the strong correlation between the two disorders. 

Alzheimer's & Parkinson's Disease Major Pathways

• As mentioned earlier, there are three primary pathways that are shared between Alzheimer's and Parkinson's Disease. As we have previously gone into these in great detail during our Alzheimer's deep-dive, we will only lightly touch on these throughout this article.

1. Major Pathway: Complex 1, 3 and 4 of the Electron Transport Chain and IP3R Regulation (IPR3/ATP [includes calcium])

2. Major Pathway: UPR Gene 'IRE1a', 'PERK' and 'ATF6' Activation (UPR)

3. Major Pathway: 26S Proteasome Regulation (Proteostasis)

• What's distinct between Alzheimer's and Parkinson's from a pathology & pathway perspective is greater importance of dopamine in Parkinson's Disease, as well as a few genetic mutations leading to heritability, Lewy bodies, and fairly recognized environmental susceptibility. Blarcamesine almost certainly addresses these unique Parkinson's Disease facets: 

  1. 1. 1. 1. Dopaminergic Death & Imbalances Between Dopamine & Muscarinic Activity

           2. Clears Lewy bodies & Likely Reduces Their Formation 

           3. Upregulates PARK7 - a Parkinson's-specific Gene Mutation Influencing Oxidative Stress Detection [Hereditary Causation]

           4. Upregulates TXN & TXN2 - Along with PARK7 Reduce Oxidative Stress Caused by Pesticides in Parkinson's Disease Patients

           5. Upregulates RPS27A - Part of the Ubiquitin-Proteostasis Pathway; Also Involved in Heavy Metal & Mercury Detection/Clearance

• KEY TERMS TO KNOW WHILE READING THIS REPORT

  • • A-synuclein (SNCA): A-synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release.

    • Lewy Bodies: Abnormal deposits of a-synuclein. These deposits, called Lewy bodies, affect chemicals in the brain whose changes, in turn, can lead to problems with thinking, movement, behavior, and mood. Found in the brain and gut of Parkinson's Disease and Lewy Body Dementia patients.

Visualization & Overlay of the KEGG Parkinson's Disease Map

Now that we have had a brief overview of the specific functions impacted by Blarcamesine, we can take a look at the KEGG database. The KEGG database was directly referenced by Anavex at the bottom of their AAIC 2022 gene poster. All of the individual genes shown in that poster have been displayed in the KEGG database as holding probable involvement in Parkinson's pathology. SOTC color-coded all and assigned confidence levels to 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, 95%]

• Yellow: Theorized to be upregulated by Blarcamesine in previous company graphics, relevant peer-review journals, or officer commentary [moderate confidence, 80%+]

• Purple: Probably upregulated by Blarcamesine based on multiple peer-reviewed articles featuring other S1R agonists [low confidence, 60%+]

• Blue: Calcium hubs or important functions

Perhaps even more so than in the Alzheimer's gene mapping, overlaying regulated genes unto known Parkinson's Disease pathways reveals robust upstream therapy with enticing breadth of effect. Every Parkinson's Disease pathway catalogued in the KEGG database is addressed to some extend by Blarcamesine. Let's dive a little deeper.

• Previously Covered Cross-CNS Disorder Pathways:

  • • Electron Transport Chain complexes 1, 3, and 4 as well as IP3R regulation normalizes mitochondrial calcium flow and ATP generation with lower oxidative stress and ROS creation. It is known that an increase in intracellular calcium stimulates additional secretion of a-synuclein. In regards to the electron transport chain, scientists assess complex 1 to be especially significant to Parkinson's Disease, with the other complexes also featuring lesser, but prominent dysfunction. Complex 1 impairment has been directly linked to accelerated parkinsonism. Note this fact while reading the section on PARK7 later.

    • UPR, CHOP, and Apoptosis control mechanisms are ramped into overdrive to try to suppress endoplasmic reticulum stress but usually end up destroying the cell due to irreversible & cascading damages.

    • Proteostasis (Proteasome 26S) clear misfolded and degraded proteins. Accumulation of Lewy bodies and phosphorylated a-synuclein are core components to Parkinson's pathology which can both be cleared by the Ubiquitin-Proteostasis pathway. This will be discussed in greater detail later.

• Summary:  Aggregated a-synuclein has been shown to make negative alterations in Parkinson's Disease patients by promoting neuroinflammation, and shutting off nutrient flow to the mitochondria as well as latching unto and leeching off of the mitochondrion membrane, thus taking an active roll in inhibiting ATP mechanisms. This stress prompts the collective activation of UPR genes to address the fate of the cell by reducing synthesis of proteins, increasing production of chaperones to improve folding processes, and translating deletion mechanisms to destroy existing misfolded proteins. 

As mentioned in previous SOTC articles, UPR exists to save the cell, but triggers apoptosis (cell death) if these cell survival mechanisms fail. Apoptosis isn't inherently bad; however, in CNS disorders, rampant cell death generate compounding pathological effects. CHOP is downstream of the UPR, and is a singular gene responsible for pro-survival amplification. In Parkinson's Disease, UPR is elevated and CHOP is modulated. Evidence of increased apoptosis, increased endoplasmic reticulum folding gene levels, load reduction genes, and increased proteostasis (Proteasome 26S) genes have been found in the substantia nigra of Parkinson's Disease patients. 

• Bottomline: A-synuclein disrupts nutrient flow from the endoplasmic reticulum and the mitochondria which leads to dampening of the ATP generation process and results in less cellular energy with increasing oxidative stress. Oxidative stress and alterations caused by a-synuclein result in a loop of increased protein misfolding and degradation. Proteins need to be cleared by proteasome 26S (which is also degraded, possibly due to translation error and neuroinflammation). Without the ability to effectively remove a-synuclein and other degraded proteins, stress is too great for the endoplasmic reticulum and the cell self-destructs via apoptosis.

Parkinson's Disease-centric Gene Regulation & Effects

• Dopaminergic Death & Imbalances Between Dopamine & Muscarinic Activity (Far Left on Fig. 2 Gene Map)

  • M1/M4 & D1/D2 Balance: While not immediately related to the AAIC 2022 genomic data, I wanted to take this time to elucidate the importance of muscarinic and dopaminergic receptor balance. Studied as far back as 1995, balancing muscarinic receptors - especially M1 and M4 - with dopaminergic receptors plays a core role in reducing catalepsy (prolonged muscular rigidity and immobility featuring unnatural, fixed posture). Because dopamine is depleted in Parkinson's Disease patients, researchers theorized that muscarinic antagonists would reduce muscarinic levels and resume balance (at lower levels) between the two receptors. Early research found this to be beneficial initially, but after some time found this approach elicits psychotic side effects. 

    • • M1/M4 & D1/D2 Balance Summary: This information indicates three things. Firstly, balance between dopaminergic and muscarinic activation is clearly a key piece to both motor function and psychiatric homeostasis. Secondly, reducing muscarinic function to match lowered dopamine has short term benefit but long term consequences and this approach is akin to attaching a band aid to a bigger problem. And finally, the most obvious and probably correct approach is to upregulate dopaminergic receptors (D1/D2) while maintaining muscarinic function. Functionally, Blarcamesine does enable this approach with direct binding affinity for M1 through M4 receptors and S1R activation which modulates dopaminergic receptors - D2 of which modulates locomotion.

• Clears Lewy bodies (SNCA) & Likely Reduces Their Formation (Top Middle on Fig. 2 Gene Map)

  • S1R Function: Our last Parkinson's-specific pathway not directly related to the AAIC 2022 genomic data, this pathway is straightforward and was published in 2017's Neurobiology of Aging. Simply, researchers theorized after testing S1R knockout mice that S1R inactivation was/is a primary cause of Parkinson's Disease. In 6 and 12 month old S1R knockout mice, there was age-related loss of dopaminergic neurons and decline of motor function. In addition, levels of a-synuclein oligomers and fibrillar a-synuclein were both increased. Phosphorylation of the a-synuclein was also increased. And finally, apoptosis control gene CHOP was elevated with a decline in proteasome activity (proteasome 26S). Providing Rifampicin (an antibiotic) or Salubrinal (anti-apoptotic) to the mice largely prevented these effects in later stages and decreased loss of dopaminergic neurons while improving motor control. The researchers concluded that S1R deficiency through enhanced aggregation and phosphorylation of a-synuclein causes the loss of dopaminergic neurons leading to the decline of motor coordination. In separate Parkinson's mice model experiments, treatment of PRE-084 (S1R agonist)  dramatically improved behavioral and motor function of the mice, lending to the theory that S1R agonism is having a disease modifying effect on Parkinson's disease - likely in-part through a-synuclein clearance. Preclinical trials with S1R agonists in mice have concluded that intervention is needed early or in a preventative approach, as benefit is dramatically lowered after a few weeks of onset. This does appear to be largely true in Anavex-ran human trials as well with greater effect noted earlier in disease stage. We are looking forward to the EXCELLENCE Rett Syndrome trial readout which is ran in young girls to further verify this theory.

    • • S1R Function Summary: Tying in M1/M4 & D1/D2 balancing, more and more evidence points towards proper S1R activation playing a compensatory role in neurotransmitter function. Excitedly, at least for Parkinson's Disease, S1R appears to directly affect Disease course. S1R reduction or knockout increases a-synuclein aggregates while simultaneously reducing proteasome pathways from clearing the aggregates and increasing endoplasmic reticulum stress/apoptosis pathways.

• Upregulates PARK7 - a Parkinson's-specific Gene Mutation Influencing Oxidative Stress Detection [Hereditary Causation] (Low Middle on Fig. 2 Gene Map)

  • PARK7 Upregulation: Also known as DJ-1, PARK7 is largely associated with familial (hereditary or genetic) causes of Parkinson's Disease, but does have links to sporadic Parkinson's Disease as well. PARK7 is highly expressed in cells with high-energy demands - cells with higher levels of oxidative stress and reactive oxygen species (ROS). PARK7 has many roles (seen below in Fig. 4), but perhaps most notably is its ability to detect oxidative damage, reinforce mitochondrial walls - thus preventing ROS creation/leakage, and regulating calcium channels (IP3R) between the endoplasmic reticulum and mitochondria which inhibits apoptosis activation. PARK7 is able to do this by directly interacting with complex 1 of the electron transport chain. As a whole, these roles combine to form a powerful mitochondrion regulator and stress detection/prevention tool. 

  • The nigrostriatal system is a major dopaminergic pathway connecting the substantia nigra (area dopamine is absent in Parkinson's) with other areas of the brain. Axons of the nigrostriatal system form one of the longest tracts in the brain and require additional ATP to transport the components to the distally located synaptic terminals - thus, PARK7 insurance in proper ATP function and nutrient funneling via calcium channels are of special importance. To prove it's importance, in-vitro & in-vivo testing expression levels of PARK7 in cells show that high abundance of PARK7 makes the cell resistance to oxidative stress, whereas low abundance increases the cells susceptibility. PARK7 appears to directly inhibit a-synuclein aggregates (lewy bodies), and a-synuclein fibrillation - indicating a probable transcription role in proteostasis (protein removal). A fantastic overview of PARK7 can be found here. Gene Totals: 1 total gene in PARK7, with Anavex restoring all (100%).

    • • PARK7 Upregulation Summary: PARK7 appears to modulate a wide-swath of protective functions relating to Parkinson's Disease (and other Diseases to include cancer). From ROS detection, ROS prevention, bolstering protein removal, and axonal transport functionality, PARK7 likely plays an important role in both genetic and sporadic variations of the Disease.

• Upregulates TXN & TXN2 - Along with PARK7 Reduce Oxidative Stress Caused by Pesticides in Parkinson's Disease Patients (Lower Right on Fig. 2 Gene Map)

  • TXN/TXN2 Upregulation: Thioredoxin is expressed by two genes, TXN and TXN2 (formerly known as TRX). The compound is crucial is maintaining a reduced oxygen intra-cellular environment and thus renders protection against oxidative stress. While primarily associated with Parkinson's Disease, thioredoxin was found to be one of 28 genes heavily associated with Alzheimer's Disease in a third-party 2022 gene study. In fact, it was found to be the most down-regulated gene during that study. It appears TXN and TXN2 are downstream from PARK7, indicating a detection (PARK7) and response (TXN) relationship, which may be ablated by unusually high levels of superoxide (SOD) and/or gene mutations. The PARK7 and TXN relationship is somewhat unique to the electron transport chain mentioned earlier and in past articles. While PARK7 does interact directly with complex 1 of the chain, it's ROS detection and signaling relationship with TXN reveals how ROS can be defeated whilst outside of the mitochondria vs. prevention within the mitochondria. Trials on plants have concluded that thioredoxin is able to dramatically reduce ROS specifically caused by pesticides. In addition to these more direct roles, TXN also plays a part in the transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). NF-kB has been implicated in synaptic plasticity and memory, and serves response to free radicals, bacteria, antigens, and heavy metals & pesticides (in fig 4. above). Gene Totals: 1 total gene in TXN, with Anavex restoring all (100%); 1 total gene in TXN2, with Anavex restoring all (100%).

    • • TXN/TXN2 Upregulation Summary: TXN and TXN2 are associated with ROS ablation. TXN specifically is associated with apoptosis & transcription with implications in mutation-inactivated (genetic) PARK7 to KEAP1-NRF2 signaling pathway (in fig 4. above). TXN has also been tested against ROS caused by pesticides (specifically Paraquat), with results showing improvement to the FAS-JNK signaling pathway and decreased pesticide-caused ROS. TXN2 may play a more direct role in mitochondria defense by potentiating the organelles membrane (enhancing the mitochondrial wall to prevent ROS leakage). Ultimately, some pesticides have been found to reduce dopaminergic uptake by over 50% in animals with other pesticides causing other dopamine irregularities. Meta analysis of pesticide use in humans have found that prolonged use of pesticides increase the odds of contracting Parkinson's Disease dramatically - in some cases as high as 700% increased chance, but generally 150-500% depending on the specific pesticide with higher correlation towards insecticides and herbicides (fig. 5). Considering Parkinson's Disease is largely a culmination of Alzheimer's Disease pathways with heavier inclusion of environmental and genetic causes, restoral of TXN and TXN2 is extremely exciting.

• Upregulates RPS27A - Part of the Ubiquitin-Proteostasis Pathway; Also Involved in Heavy Metal & Mercury Detection/Clearance (Top Middle on Fig. 2 Gene Map)

  • RPS27A Upregulation: The ubiquitin-proteostasis pathway is complex but easy to explain in layman terms. Essentially, ubiquitin latches unto proteins that are believed to be misfolded. Ubiquitin makes this assessment by reading the proteins shape and making sure there are no 'holes' within the protein membrane that are irregular. If the ubiquitin makes the assessment that a protein is in fact misfolded and needs to be discarded, ubiquitin latches unto the protein like a tail. The more confident the ubiquitin is that the protein needs to be discarded the larger this tail becomes. Once the tail has formed then the proteasome 26S tags and attaches to the protein. The 26S then verifies that the protein is definitively misfolded or not. If the protein is misfolded then the 26S degrades the protein, and if the protein is not misfolded then the process ends. The reason that the ubiquitin tail length matters, is that once the protein is attached to the 26S, there is a finite amount of time for the 26S to confirm misfolding. If the ubiquitin tail is longer, it gives the 26S more time to make a higher quality assessment. Ultimately, if the ubiquitin process is degraded then an abundance of misfolded proteins aren't getting tagged (tails) and the ones that do get tagged have smaller than standard tails which may not be enough time for the 26S to accomplish its mission. RPS27A is one of four main ubiquitin creating proteins and has been found to play a specific role in heavy metal detection and clearance - especially with Mercury. In a similar meta analysis as seen above (fig. 5) heavy metal exposure (i.e. copper, iron, mercury, manganese, etc.) for extended periods of time raise odds of contracting Parkinson's Disease by 200-1,000%. In bats, irregular copper exposure has decreased dopaminergic function by 40-60%. NOTE: RPS27A is monoubiquitin. Ubiquitin proteins are either mono or poly; monoubiquitin has largely been linked to chromatin regulation, protein sorting, and trafficking, whereas polyubiquitination has been associated with protein signaling and clearance through proteasomal or autophagic degradation. With that said, monoubiquitin can and does tag smaller proteins for degradation, and does so widely within humans. More information regarding RPS27A can also be found here. Gene Totals: 4 total gene in UB (Ubiquitin), with Anavex restoring 1 (25%).

    • • RPS27A Upregulation Summary: Improving RPS27As gene expression ensures continuity of our body's ability to effectively target and degrade misfolded proteins as well heavy metals (a function also addressed by NF-kB mentioned in the PARK7 portion). In addition to RPS27A, astrocytes are neuroprotective cells and protect the neuron by soaking up heavy metals. If heavy metals are allowed to run rampant, astrocytes over-accumulate the metals and become inundated. Intake of heavy metals affects astroglia homeostatic and neuroprotective cascades including glutamate/GABA-glutamine shuttle, antioxidative machinery and energy metabolism. Interestingly, Anavex had previously made mention of decreasing L-AAA in their AVATAR Rett Syndrome data release. Decreasing L-AAA is beneficial as upregulated L-AAA results in the death of astrocytes. When astrocytes die, there is less internal capability to soak up metals in homeostasis. There appear to be multiple pathways that Blarcamesine regulates metal toxicity in the neuron. More can be read regarding astrocytes and metals here.

Parkinson's Disease Genomic Study Summary

Whether due to heavy metal toxicity, pesticide/chemical exposure, S1R deficiencies, genomic mutations - or more likely, a combination of these facets and more, dopaminergic function dramatically reduces in the brains of Parkinson's Disease patients. These dopaminergic changes manifest classic motor loss with a string of potent downstream effect to include a-synuclein aggregation & lewy body abundance. 

Blarcamesine's primary effect on Parkinson's Disease (and the dementia aspects of Parkinson's Disease which are later stage and likely distinct in cause) takes root upstream by enhancing S1R expression, and prompting improvements to dopaminergic receptor function whilst balancing muscarinic receptors simultaneously. Furthermore, Blarcamesine protects against DNA damage, RNA transcription, and resuscitates aspects of the ubiquitin-proteasome process. By improving the bodies ability to identify and tag misfolded or metal-inundated proteins, the 26S proteasome (also enhanced) can clear some 80% of our brain's dysfunctional proteins - including a-synuclein & lewy bodies. 

On a more focused level, Blarcamesine is able to upregulate PARK7, TXN, and TXN2, unlocking a treasure trove of therapeutic effect by improving dopamine synthesis, reducing ROS - especially that caused by pesticides, promotes healthy autophagic function to rescue a-synuclein aggregation (in combination with S1R autophagic function), provides potent antioxidant effect, and reduces chances for apoptosis. A-synuclein nitration - which is enhanced during oxidative stress - are found within lewy bodies. 

PARK7 also communicates directly with electron transport chain complex 1, which includes enhancing the membrane wall of the mitochondria to decrease ROS creation/leakage. It is our assessment that the electron transport chain improvements noted in the Alzheimer's gene pathway report along with calcium funneling regulation (including normalizing calcium output levels) is more related to the dementia aspects of the Disease - although not exclusively. As shown in figure 3, there is immense overlap between Parkinson's and Alzheimer's Disease, but two of the most corollary facets are protein aggregation/misfolding, and oxidative stress resulting in superoxide or ROS. For the latter, the electron transport chain is at the pinnacle of importance when addressing ROS. Holes in the mitochondrial membrane, dysfunction in nutrient in-flow, and complex 1, 3, and 4 downregulation are the most prominent causes of increased ROS. Ultimately, these effects reduce ATP (energy) for the rest of the cell, nulling axonal & synaptic activity - all the while causing enhanced endoplasmic reticulum stress and ending in apoptosis (cell death).

Bottomline & Assessment: As investors prepare for Anavex's long-awaited Parkinson's Disease dementia OLE trial data, we believe it is prudent and useful to elucidate Blarcamesine's full mechanism of action, especially as we think it likely Anavex is combining the Parkinson's Disease dementia and OLE data with the Alzheimer's 2b/3 for a robust regulatory data package. With the Parkinson's Disease dementia & OLE in-hand, the company would have ~1.2 years worth of Parkinson's Disease dementia data and 9 months worth of Alzheimer's Disease data. It is feasible that Anavex will wait to submit their regulatory package until their first Alzheimer's OLE data read (hypothesized at the first 48 week mark) in order to have 3 years worth of total data between the two indications - but we see this as less likely. If that scenario holds true however, the first 48 weeks worth of Alzheimer's OLE data is expected to close out in the last week of May or the first week of June 2023. In any case, we hope to see continued improvement in both cognitive & motor domains for those with high concentrations of the drug. We also hope to see a longer time horizon result in statistically significant REM sleep improvement. We grow anxious in anticipation of the full data read for Parkinson's Disease dementia OLE and the full Alzheimer's 2b/3 topline, and are looking forward to analyzing additional genomic and biomarker data once it is available.