Update 9: 26 Oct 2023 Early treatment with an M1 and sigma-1 receptor agonist prevents cognitive decline in a transgenic rat model displaying Alzheimer-like amyloid pathology [Article on Anavex 3-71, Review]
Bottom-line Up Front (BLUF): On 25 Oct, the results of a preventative rat study were fully reported. Results are exciting and do foreshadow multiple points investors and industry are likely to see in the forthcoming full Alzheimer's 2b/3 study paper. As expected, Anavex 3-71 was able to effectively prevent cognitive decline and preserve certain social behaviors of advanced disease-stage rats - even after a lengthy (for rats) month-long washout period. In addition, a slew of biomarkers to include amyloid, inflammation, and cholinergic receptors were assayed to confirm results. Certain percentage estimates will be provided - those colored in red are based on my visual interpretation of a chart and should be seen as an approximate value, not fact.
Study Design:
7-month-old McGill-APP rats were pooled to 7-month-old wild-type (WT, normal) rats and assigned to 4x separate cohorts:
Placebo-WT
Dosed-WT
Placebo-mutation
Dosed-mutation
The rats began dosing regimen at 7-months and continued dosing at 10 µg/kg for a 7-month period. At the end of the 7 months, and now at 14-months of age, the rats underwent clinical testing for 2 weeks before having their brains collected (RIP).
Clinical Tests:
Open Field: Used to test general activity and total locomotor skills.
New Object Location & Novel Object Recognition: One of the subtests evaluates spatial learning, which relies heavily on hippocampal activity. The other evaluates non-spatial learning of object identity, which relies on multiple brain regions.
Social Preference: Tests social engagement behaviors.
Morris Water Maze: Tests spatial learning and memory by use of hidden platforms in a maze.
Summary of Clinical Test Results:
Open Field: No differences between any groups were noted, indicating motor skills were unaffected in all groups.
New Object Location & Novel Object Recognition: Placebo-mutation rats saw a substantial worsening (37.5%) over placebo-WT. In contrast, dosed-mutation rats saw no worsening, and in fact, performed slightly better than placebo-WT, especially in male rats.
Social Preference: Placebo-mutation rats once again saw a substantial worsening (16.6%) over placebo-WT. In contrast, dosed-mutation rats saw a significant improvement over baseline (placebo-WT) of 18.8%.
Morris Water Maze: This test was over a period of 5 days. At day 3, placebo-mutation rats saw an impressive 38% worsening over placebo-WT. Meanwhile, dosed-mutation rats saw a comparable performance to baseline (placebo-WT). At day 5, there was a an extremely noticeable improvement in dosed-WT rats. To put this miraculous improvement into perspective, the dosed-WT rats appeared to complete the maze 45% faster than the placebo-WT & mutation-dosed groups, and approximately 56% faster than the placebo-mutation group.
Assessment of Clinical Test Results: Even after a 1 month washout period, Anavex 3-71 was able to prevent cognitive decline in McGill-APP mutation rats. Efficacious and significant results were garnered in working memory, spatial navigation, and reference memory tests. In addition, the drug had profound affect on the decline in social interaction. Social engagement has a protective effect against age-related cognitive decline, and social withdrawal is one of the earliest signs in Alzheimer's progression. Importantly, these combined cognitive tests show that Anavex 3-71 has marked improvement on multiple brain regions and region interaction. Finally, it is my opinion that some of the results seen lend to the possibility that completely healthy people could see improvement over baseline for both spatial learning and working memory, as well as possible minor social/behavioral benefits.
Biomarker Assays:
Amyloid-beta 42, 40, & 38: Classic amyloid biomarkers. The 42/40 ratio in particular is of great importance and has been decreased (improved) significantly in the Blarcamesine Alzheimer's 2b/3 study.
Astrocytes & Microglia: Immune cell response. Typically overabundant in Alzheimer's patients (as well as various other disease and disorders including depression and schizophrenia). Produce cytokines which provide varied inflammatory response.
Cytokine IL-1ß : Pro-inflammatory marker. Studied to a good degree and known to be associated with Alzheimer's disease.
Cytokine IL-6: Pro-inflammatory marker. Studied to a good degree and known to be associated with Alzheimer's disease.
Cytokine IL-5: Unclear role, associated with either promoting neurotrophic activities in the brain or a mitogenic factor supporting microglia growth.
Cytokine IL-10: Anti-inflammatory marker.
Cytokine IL-4: Anti-inflammatory marker.
Cytokine IL-13: Anti-inflammatory marker.
TNFa: Dual-role; pro-inflammatory but plays a role in amyloid clearance.
IFN-?: Dual-role; pro-inflammatory but decreases Aß deposits and infiltration of peripheral monocytes.
proBDNF & mBDNF: BDNF begins as proBDNF and then turns into the useful mBDNF. Conversion of proBDNF is stifled in Alzheimer's disease patients and McGill-APP mutation rats.
Cholinergic & Glutamatergic Terminals: Neurotransmitter specific terminals to carry muscarinic & glutamatergic transmissions in the neuron. Necessary for normal brain function.
A good source to find more information on some of these cytokines can be found here.
Summary of Biomarker Assay Results:
Amyloid-beta 42, 40, & 38: In placebo-mutation rats, 90% had abundant amyloid plaque in the hippocampus and 50% had abundant amyloid plaque in the cortex. In comparison, in dosed-mutation rats, only 33% had abundant amyloid plaque in the hippocampus, and 8% had amyloid plaque in the cortex. Again, this was after a 1 month washout period. Treated rats also saw less 'mature' amyloid plaques over placebo rats. And lastly, dosed rats saw significantly decreased amyloid beta 42 and 40, as well as a significantly lower 42/40 ratio than placebo rats. Overall density of plaques between placebo and dosed mutation rats was staggeringly different; in the hippocampus, dosed rats saw amyloid density reduced by 76% compared to placebo and 73% similarly in the cortex.
Astrocytes & Microglia: Dosed rats saw reduced recruitment of microglia and astrocytes toward amyloid-affected neurons in the hippocampus. The overall shape of their structure was also significantly altered, with changes to area (volume), appendage quantity, and appendage length.
Cytokine IL-1ß : Pro-inflammatory marker was significantly decreased in dosed rats. Dosed-mutation rats were completely rescued to normal levels. In fact, dosed-WT rats also saw lower levels over baseline.
Cytokine IL-6: Pro-inflammatory marker was significantly decreased in dosed rats. Dosed-mutation rats were completely rescued to normal levels. In fact, once again, dosed-WT rats also saw lower levels over baseline.
Cytokine IL-5: IL-5 was significantly increased in dosed-mutation rats. This was probably the largest response of all of the cytokines and probably warrants further investigation. IL-5 was increased by approximately 100% over baseline.
Cytokine IL-10: Anti-inflammatory marker was significantly increased in dosed rats. Both dosed-mutation and dosed-WT rats appeared to benefit.
Cytokine IL-4: Anti-inflammatory marker was significantly increased in dosed rats. Both dosed-mutation and dosed-WT rats appeared to benefit; dosed-mutation rats by approximately 90% over baseline.
Cytokine IL-13: No changes observed.
TNFa: Dosed-mutation rats saw a significant increase over baseline.
IFN-?: Dosed-mutation rats saw a significant increase over baseline.
proBDNF & mBDNF: Comparing placebo-WT and placebo-mutation, the latter saw far less proBDNF converted to mBDNF. This failure of conversion was completely rescued in dosed-mutation rats.
Cholinergic & Glutamatergic Terminals: Cholinergic terminals were reduced in quantity for placebo-mutation rats compared to placebo-WT, whereas dosed-mutation quantity was unchanged. Glutamatergic terminals was unchanged across the board.
Journal Author's Key Points: There are a number of key takeaways outlined by the authors within this paper:
3-71 prevents cognitive decline in McGill-APP rats
3-71 prevents extracellular Aß deposition in McGill-APP rats
3-71 reduces the recruitment of microglia and astrocytes toward neurons in the hippocampus
3-71 rescues microglia morphological changes and the profile of inflammatory mediators
3-71 prevented the increase in the ratio proBDNF/mBDNF
Selective M1 with no binding bleedover to M2-M5 combined with ultra low dosing likely reduces most peripheral side effects that are sometimes associated with cholinergic drugs
3-71 prevents cognitive decline, reduces amyloid pathology and neuroinflammation, and increases BDNF availability
3-71 can prevent neuroinflammation, not only by reducing the recruitment of microglia but also of astrocytes. This effect could be directly mediated by M1, as astrocytes have M1 muscarinic receptors. In addition, this anti-inflammatory effect may be explained by the activation of Sig-1Rs
Results were dramatic, even in rats that should feature later stage progression
Additional Key Points & Assumptions by SOTC:
The author spends substantial time on M1 activation. This is often publicly overlooked by Anavex, although I am certain they are aware that muscarinic and secondary bindings play a leading role in their S1R pipeline's efficacy.
3-71 did not prevent cognitive impairment by action of increasing cholinergic synapses, as the number of synapses between treated rat and non-treated rat remained the same. Significantly it has been found that S1R antiamnesic properties likely take place even in animals with complete cholinergic depletion. So, human patients who have degraded/eroded away all/most of their cholinergic sites are unlikely to see any major benefit from the M1 (or M2-M4) part of the drug. However, they do still get muted benefit via S1R. This confirms my thoughts in 2021 that the rest of the drugs binding affinities are equally as important as S1R - even though the company mostly only ever talk about S1R. This also means there is another variable that must be assessed in patients in order to see if they will be super-responders (patients still have abundant cholinergic synapses), responders (patients have some cholinergic synapses), or poor response (patients will nearly depleted cholinergic synapse). While S1R can act independent of muscarinic action, the combined effect is necessary for full response - this was alluded to a couple of times by Dr. Missling and the CSO over the last year but never described in any sort of detail.
Fascinating to me, it appears as though completely healthy rats saw benefit over baseline in a slew of performance and biomarker tests.
Final Thoughts: This paper continues to provide verification for both Blarcamesine and 3-71 as probable disease-modifying compounds by virtue of dual muscarinic-S1R modulators and their potential as prophylactic preventative medicines. Additionally, the paper serves to confirm my thoughts on secondary binding affinities of both Blarcamesine and 3-71 having direct and meaningful impact on the drug's efficacy in mostly overlapping, yet distinct indications. 3-71 benefits over Blarcamesine with extremely low dosing with a somewhat greater safety profile (not that Blarcamesine's is poor), and hyper-focus on mood/behavior disorder pathways like depression and schizophrenia. Blarcamesine on the other hand has good cognitive benefit, decent tolerability, and has notable effect on movement disorders and seizures. Blarcamesine may also see greater effect on the cardiovascular system although this remains to be seen. Moving forward, it is likely the full Blarcamesine Alzheimer's 2b/3 readout (which may include 48 week OLE data) is likely going to mirror portions of this rat study. As we know already, Blarcamesine significantly decreased brain atrophy, significantly improved amyloid 42/40 levels, and provided marked improvement over baseline in both cognitive and activities of daily living scores in a likely-sizable sub-group of patients. Upon final release, I expect the overall trial results to look something similar to the phase 2 PDD patients on page 15 of this presentation. I remain excited for Anavex's full Alzheimer's 2b/3 data readout and continue to impress upon the significance of what progress Anavex is making in the CNS precision medicine space. As described, the 2b/3 landmark study is likely to garner data identifying a clear new SOC in most patients.
