Not sure if you all saw this. For some reason, I was unable to copy the word doc so I bolded the Company's responses. After a quick perusal, this seems like a very strong response. Also, the graphs and photos would not copy...
Neurotrope Responses to
Seeking Alpha Assertions –
By D.L. Alkon, CSO (See red print)
Neurotrope's Bryostatin Is Unlikely To Produce Significant Cognitive Efficacy
Jul. 17, 2019 1:10 PM ET|
| About: Neurotrope, Inc. (NTRP)
Richelle Cutler-Strom
Long/short equity, Growth, growth at reasonable price, short-term horizon
Strom Patent Law Firm
(121 followers)
Summary
Extensive research by Neurotrope affiliated researchers indicate that Neurotrope's bryostatin 1 could improve cognition.
Bryostatin 1 activates predominantly the PKC epsilon isozyme. It also briefly activates PKC alpha, and to a much lesser extent reduces other isozymes (See Fig. 1). Importantly, all major pre-clinical efficacies,(including synaptogenesis, anti-apoptosis, anti-amyloid, anti-neufibrillary tangles, and cognitive enhancement) with Bryostatin were confirmed with PKC epsilon-specific activators (See Honpaisan et al., J. Neuroscience, 2011; Sun and Alkon, PNAS, 2009, Nelson et al, J. Biol Chem, 2009).
These isozymes have different substrates in different cells types, which when activated may have both positive and negative effects on cognition.
That is why the PKC epsilon specificity (See Fig. 1, above – BR 122 = Bryostatin) is so critical for the multimodal benefits it has for AD). These benefits, analyzed in the absence of memantine, as pre-specified in the recently published Phase II trial (J. Alzheimer’s disease, 2019) showed remarkable consistency, with up to 95% of patients who received the safe, effective Bryostatin dose showing clinically positive benefits.
The scientific rationale for the confirmatory trial is weak, indicating that bryostatin 1's negative effects prevailed in the first phase 2, which makes the confirmatory trial outcome predictable.
The effects of the safe 20 mcgm profile were strong – improvement of 7.0 SIB in the absence of memantine that has never before been observed in 100 years since the identification of Alzheimer’s disease (AD). The present confirmatory trial, all dosing recently completed, was designed to examine the efficacy of bryostatin subjects not on memantine treatment to precisely duplicate and confirm the reversal of cognitive decline already shown in the last Phase II trial (See Farlow et al., Journal of AD, 2019). This recent publication of the previous Phase II trial provides extensive mechanistic information – including references to Neurotrope’s previously published results in tens of peer-reviewed articles in top-tier journals.
The balance of bryostatin 1's effects suggests that bryostatin 1's potential is limited, reducing Neurotrope's value significantly.
The balance of Bryostatin’s effects were demonstrated with in vivo and in vitro experiments that engaged known endogenous brain enzymes. These extensive pre-clinical studies showed Bryostatin’s remarkable multi-modal efficacy: to induce synaptogenesis that reverses cognitive deficits, prevent apoptosis (neuronal death), prevent amyloid and tau deposits, induce anti-inflammation, and directly enhance cognition – even in normal animals – as well as in 3 different varieties of transgenic AD models in mice.
Bryostatin’s potential to enhance cognitive performance as currently under clinical study, therefore, has broad implications if the current trial produces positive results. This could include treating early, mild AT, preventing AD, and treating other neurodegenerative disorders such a s Multiple Sclerosis, Fragile X mental retardation, stroke, and traumatic brain injury.
Neurotrope Inc. (NASDAQ:NTRP) is currently testing bryostatin-1 (bryostatin) in patients with moderate to advanced Alzheimer's disease (AD) patients (MMSE scores of 4-15). Considering that neurodegeneration begins decades before symptoms appear, these patients are the hardest to improve. Neurotrope is currently running a confirmatory phase 2 trial of bryostatin. This article analyses the relevant research findings to indicate the probability of bryostatin's efficacy and, thus, Neurotrope's value.
An initial phase 2 result showed that the 20 ug bryostatin group improved on the Severe Impairment Battery (SIB) test compared to the placebo group (1-tailed test, p = 0.07, at week 13). This statistic was only applied to the exploratory aspect of the trial to identify a safe and effective dose.
However, as pre-specified in the Statistical Analysis plan, elimination of the patients taking memantine (almost half the 20 ug bryostatin group) from the analysis resulted in a significant treatment difference using a 2-tailed test (p = 0.035, ?4.50, at week 13). Moreover, Neurotrope does disclose all the specifics about about all of the partients including the memantine-free patient group – as readily accessed in the tables provided. Furthermore, all of the patients in all subgroups were randomly sampled with no a priori bias imposed on any sample – including the memantine-free patients. Similarly, subjects not on concurrent memantine treatment were extracted from the entire study population, with patient characteristics provided in detail. The data from the first trial can be reviewed for many listed parameters in the Journal of AD article, 2019.
Neurotrope's explanation that memantine likely interfered with bryostatin action is based on memantine blocking NMDA receptor-gated calcium entry.
Neurotrope’s explanation was not based on calcium entry but on the biochemical control exerted by PKC on the NMDA receptors. That control was suggested (cf. Farlow et. Al, JAD, 2019) to engage the NMDA receptor’s participation in the principle efficacy of the Bryotation-PKC epsilon–BDNF pathway to generate new synapses and to prevent neuronal death. (See Farlow et al., JAD, 2019)
Blocking calcium would reduce the activation of calcium responsive classic PKC isozymes (a, ß, ?) but should not reduce novel isozymes (d and ?). Bryostatin is thought to enhance long-term through PKC? activation, which requires calcium influx. (LTP is a physiological mechanism of learning and memory and reflects the strength of a synaptic connection.) Thus, memantine could reduce, through limiting calcium influx, the bryostatin-mediated PKC? activation that is required for LTP. However, memantine reportedly blocks primarily extrasynaptic glutamate receptors, and thus, memantine may not have significantly affected PKC?-mediated LTP at the synapse.
Bryostatin was shown to enhance actual associative learning and memory in a variety of mammalian species; LTP is only a model that also happens to benefit from PKC epsilon activation. Bryostatin efficacy in a range of different species consistently emphasized unique benefits for associative learning and memory – with in vivo models of learning and memory.
With this reasoning, bryostatin's efficacy in a memantine-free group is unlikely to be verified. Regardless, Neurotrope's confirmatory phase 2 trial enrolled only memantine-free patients.
It's important to keep in mind that memantine is one of only two types of drugs FDA approved for treating AD and has consistently shown to improve cognitive function and behavior in moderate to severe AD patients, at least for a short time.
The benefit of memantine / Namenda is only to slow down the cognitive decline – not to reverse the decline, i.e. improve cognition as shown with Bryostatin. No persistent benefit was found with Namenda. In the memantine-free patients who received 20ug bryostatin in the previous protocol, cognition improved - reversing the decline and persisting at least one month after all dosing had been completed – all effects not ever seen with memantine.
Memantine works by blocking NMDA receptor mediated calcium influx, thus reducing excitotoxicity from excess glutamate signaling.
The reduction of glutamate excitotoxicity has been posited, but never directly demonstrated to explain the transient Memantine benefit. This would be to reduce glutamate damage, not to increase a natural function of glutamate.
And yet, bryostatin-mediated PKC activation should increase NMDA receptor activity. Moreover, magnesium binds the NMDA receptor and increases its threshold for activation, and clinical trials indicate that magnesium treatment (MMFS-01) improves cognition. Since bryostatin should increase excitotoxicity, if efficacious, it would oppose an established theory of glutamate-mediated excitotoxicity in AD.
The rationale used by the author has not been validated, nor has it been proven effective in treating the underlying pathology of Alzheimer’s disease.
Nonetheless, there are other reasons for investors to expect that bryostatin-mediated PKC activation could improve cognition.
Bryostatin - thru PKC - regulates the NMDA receptor, not vice-versa. The NMDA receptor is important for synaptogenesis - one of the main benefits of Bryostatin. Blocking the NMDA receptor blocks synaptogenesis and thus Bryostatin synaptogenic efficacy.
First, AD is associated with high glutamate. Glutamate is recycled from synapses by glutamate transporters in astrocytes. Bryostatin mediated activation of PKC a and ? in astrocytes has the potential to increase glutamate uptake via the EAAT1 transporter and to decrease excitotoxicity, although EAAT1 is a minor glutamate transporter in the brain.
This confused thinking can be resolved by Neuroropes’ published, peer-reviewd articles (Nelson et al, JAD, 2016) and (Farow et al. JAD, 2019). AD severity is highly correlated with reduced synapses, which are necessary for learning and memory. Synapses are the contact points between two neuronal processes, the axon and dendrite spine. The loss of synapses may be related to the significant reduction of dendritic spines in the AD brain. Neurotrope's affiliated researchers report that bryostatin increases dendritic spines. Another independent group also reported that bryostatin treatment increased dendritic spines. Bryostatin's ability to increase dendritic spines may be the structural change that increases bryostatin-mediated synaptogenesis primarily through activation of PCK?. If bryostatin does increase synapses in the elderly brain, bryostatin-mediated PCK? activation would act opposite to its reported effect in the developing mouse brain.
This is comletely illogical. Brystati-PkC epsilon-BDNF replaces lost syanapses – exactly what is required in the AD brain that has lost synapses. of AD. However, administering bryostatin 3x weekly in APP/PS1 mice for 5-6 months showed no significant learning improvement, while bryostatin administered 2x weekly in Tg2576 mice for 12 weeks did improve learning performance.
The author neglects to mention that the APP/PS1 mice, in an initial study received a non-optimal dosing regimen. In all subsequent studies appropriate dosing was shown with serial electron microscopy to restore lost synapses and prevent the other pathologic hallmarks of AD.
Differences between short versus long-term administration are not surprising, given that sustained activation should downregulate PKC isozymes. Unfortunately, inventors can attest that AD mouse models have not proven to be good predictors for drug efficacy in humans. These models fail because they significantly overexpress multiple mutated human transgenes involved in the ß-amyloid processing that produces ß-amyloid neurotoxicity; it's clear that sporadic human AD is much more complex than the mouse models.
That is why Neurotrope used 4 different animal models: Normal mice, APP, Tg256, and 5XFAD AD transgenic mice. Bryostatin enhances associative learning - spatial maze memory - and associated increase in synapses in normal animals (Hongpaisan and Alkon, 2007), and prevents learning and synaptic deficits in both the Tg2576 mice and the 5X FAD mice. The author completely ignores these demonstrated Bryostatin efficacies such as enhancement of memory and synaptogenesis in two different transgenic mouse species. Enhanced cognition was also shown with the 20ug bryostatin-treated advanced AD patients (non-memantine) in the first Phase II trial.) (See Alkon, PNAS,2-007; Etecheberriaray et al., PNAS 2003; and Hongpaisan et al. J. Neursci., 2011.
In other studies, BDNF has been found to be important for controlling neurogenesis, cell survival, and learning. Neurotrope reported that mice treated semi-weekly with 20 ug bryostatin increased total brain BDNF through PKC? activation.
But there was no dose response, and BDNF upregulation was discordant with PKC? activation.
This is a completely false assertion, since Bryostatin has been shown to activate BDNF through PKC epsilon activation – in a number of Neurotrope studies.
It certainly appears that PKC? activation would be beneficial to AD patients. But it's questionable whether PKC? is truly downregulated in AD brains. A study reporting reduced PKC? in the temporal lobes from AD brains is unconvincing. Their blot meant to demonstrate lower PKC? shows uneven ß-actin loading controls, with no mention of normalization in the methods or legend.
Finally, a group reported that bryostatin activates PKC a, d, and ? in a neuroblastoma cell line and increases a-secretase APP processing to produce beneficial APPa and thus not neurotoxic ß-amyloid. However, it was recently reported that PKC d activation increases BACE1 expression, which increases the generation of ß-amyloid, although it is now questionable whether increasing ß-amyloid is counterproductive considering (1) the lack of cognitive efficacy with ß-amyloid reduction by anti-amyloid antibodies and BACE1 inhibitors and, (2) evidence that ß-amyloid's antimicrobial property is beneficial.
Neurobrastoma cell lines are not cultured human neurons, nor do they substitute for in vivo experiments AD mouse models or human patients. The multiple efficacies of the specific isozyme PKC epsilon as activated by Bryostatin was demonstrated repeatedly in Neurotrope studies. This isozyme specificity was unequivocally confirmed by Neurotrope with other PKC activators that only activated the PKC epsilon enzyme.
Bryostatin-PKC epsilon was shown by Neurotrope to degrade A Beta oligomers and amyloid plaques thru natural brain enzymes in extensive biochemical studies (J. Biol. Chem. Nelson et al.2009), Furthermore, Bryostatin-PKC epsilon blocks GSK3 Beta and thus hyperphosphorylated tau in neurofibrillary tangles - contrary to what the author claims.
Bryostatin-1
Now, let's consider what information (and lack thereof) indicates that bryostatin might not benefit the investor. First, Neurotrope has not reported bryostatin's concentration in human cerebrospinal fluid, which would confirm whether bryostatin can penetrate the human blood brain barrier (BBB), or technically, the cerebrospinal fluid barrier. BBB penetration is a significant hurdle that most drugs fail, and the absence of this measurement is concerning. Especially considering that researchers modeling kinase inhibitors predicted that bryostatin (shown above) would not penetrate the BBB, although Neurotrope and two other groups reported that bryostatin reached the brain in mice.
In numerous quantitative animal studies, Neurotrope demonstrated that i.v. Bryostatin in low concentrations penetrates the Bood-Brain-Barrier to reach Brain concentrations exactly required to optimally activate PKC epsilon to reverse cognitive deficits an to treat all major aspects of AD - without side effects - while higher dosing produced concentrations used in the oncology trials that did produce side-effects, but not anti-tumorigenesis, in the oncology patients. Only these safe low dose concentrations were effective for treating AD in both animal models and human patients. The author has completely confused low and high dose Bryostatin-PKC protocols.
Neurotrope further demonstrated the loss of PKC epsilon in AD in a double-blind study with the Harvard Brain Bank. Also, Bryostatin-PKC epsilon controls BDNF that has been universally shown to be downregulated in the AD brain, and is considered to be an important cause of AD deficits in synaptogenesis and abnormal inflammation.
Finally, it should be pointed out that CSF levels do not prove brain access. In fact, all of the failed AD trials to date showed CSF levels of candidate drugs that had no relation to drug benefit for the patients.
Second, bryostatin is known to bind and activate PKC isozymes a, ßI/II, ?, d, and ?. The isozyme type activated as well as the downstream effectors depends on the cell type and various conditions. Interestingly, PKC d and PKC ßII expressions are upregulated in the AD brain. The increased PKC expression may stem from calcium mediated activation of conventional PKCs (a, ß, ?) since calcium dysregulation is consistently reported in AD. Accordingly, three PKCa gene variants have increased activity and are associated with AD. So, while researchers initially reported that PKC isozymes were downregulated in the AD brain, this downregulation could actually result from chronic activation. Tellingly, half the phosphorylated molecules increased in AD brains are PKC phosphorylated substrates. .
Pre-clinical research (J.Biol. Chem) shows that Bryostatin activates predominantly PKC epsilon, and to a lesser extent PKC alpha. It does not activate all the other isozymes cited by this author. See the discussion in the beginning paragraph.
Third, the breakdown of the myelin that insulates axons is a significant AD pathology, and the activation of PKC in oligodendrocytes has been shown to induce demyelination.
Fourth, considering recent robust evidence that herpesviruses are involved in AD neuropathogenesis, it is prudent to try to predict bryostatin's effect on herpesvirus activity. In this regard, phorbol ester, a PKC activator with some similarity to bryostatin, induces reactivation of the herpesvirus known as human cytomegalovirus (HCMV). Reactivation is induced via PKC d induced CREB and NFkB binding. Studies of congenital and transplant HCMV infections provide insight into HCMV's discrete role in AD pathology. For instance, HCMV infection disturbs granule cell migration in the adult dentate gyrus, and granule cell generation is deficient in AD patients. Bryostatin can also upregulate cyclooxygenase 2, mediating the prostaglandin inflammatory response. Upregulation of cyclooxygenase can increase herpes simplex virus 1 reactivation. However, bryostatin has been shown to downregulate cyclooxygenase 2 expression in colorectal mucosa, so this effect might depend on the cell type.
Fifth, both herpesviruses (HHV-6 and HCMV) utilize PKC activation to downregulate the glutamate transporter EAAT2 in astrocytes, which could increase glutamate-induced excitotoxicity. Bryostatin-medicated PKC activation would further reduce EAAT2, thus negating the PKC mediated EAAT1 increase mentioned above since EAAT2 is the major glutamate transporter in the brain. Accordingly, EAAT2 expression is unfavorably reduced AD brains.
Sixth, in view of PKC-mediated herpesvirus reactivation, now consider that bryostatin can suppress the T cell response. Further, bryostatin treatment alleviated experimental multiple sclerosis by promoting Th2 lymphocytes and reducing inflammatory Th1 lymphocytes. Promoting Th2 can reduce phagocytosis and infectious immunity. Bryostatin was also found to increase neutrophil transmigration and its anti-viral response. While this sounds positive, chronic neutrophil activation can actually be neurotoxic.
This section of many irrelevant finding ignores the clearly demonstrated and extensive anti-infammatory benefits of Bryostatin in AD but also in other disease models such as Multiple Sclerosis and Parkinson’s disease
Finally, bryostatin's ability to upregulation CD22 with respect to leukemia has been established for 20 years. Regarding bryostatin's upregulation of CD22, one lab recently reported that upregulation of CD22 significantly reduced microglia phagocytosis, which would remove less debris from the brain. Reduced phagocytosis is unlikely to be beneficial in the AD brain.
In summary, it appears bryostatin could improve cognition in AD patients by increasing EAAT1-glutamate transport, dendritic spines, BDNF, and APPa. However, bryostatin could also make cognition worse by increasing demyelination and herpesvirus reactivation, and reducing EAAT2-glutamate transport, neurotoxic neutrophil activation, infectious immunity, and phagocytosis. Of course, this all depends on whether bryostatin makes it through the BBB, altogether, the problem with bryostatin is obvious: activation of multiple PKC isozymes will have different downstream effects in different cell types, and some of these effects may not be advantageous for resolving AD pathology.
Considering the balance of effects, it is unlikely that bryostatin can significantly improve cognition in patients with moderate to severe AD. With no other drugs in development, Neurotrope's value depends solely on bryostatin's success in AD. Although Neurotrope still has several other indications in preclinical research (fragile X syndrome, Niemann-Pick Type C, multiple sclerosis, and cancer), a failure in the current AD trial will most likely bring Neurotrope's business to a close.
Neurotrope licensed patents for the use of bryostatin 1 through 18 in the method of treating AD from Cognitive Research Enterprises, Inc. (CRE). Those U.S. patents expire in 2022. If trial results are favorable, CRE is almost certain to apply for a term extension, which will depend on the regulatory review period. Neurotrope has world-wide exclusive licenses from Stanford University for the synthetic bryostatin compositions and methods of synthesis. The patents expire anywhere from 2019 (composition) to possibly as late as 2027 (methods). However, infringement of methods of synthesis is hard to prove; these patents might be difficult to enforce. Other bryostatin treatment indications have much later filing and expiration dates. Neurotrope's assets as of December 31, 2018, were $26 million, and their 2018 burn rate was less than a million per month. Neurotrope has just 12.95 million outstanding shares with 150 million registered.
Bryostatin has a safety record as an anti-tumorigenic agent, but that safety record does not extend to dosing in elderly patients with compromised immunity.
Neurotrope has significant safety data from the previous AD trial, showing that treatment with 20ug bryostatin has an excellent safety profile. At the low doses used for Neurotrope’s recently published AD trial (7 doses of 20ug), safety was minimally different from placebo in subjects ranging from 55 to 85 years of age. Safety was apparent in this previous Phase II trial and there have been no drug-related serious adverse events in the current trial, as observed in the data (currently blinded). Finally, Neurotrope now has an exclusive license to synthetic Bryostatin from Stanford University that will ultimately replace the Marine form in our clinical trials.
Bryostatin's adverse effects include myalgia, phlebitis (venous inflammation at IV catheter), fever, flu-like symptoms, fatigue, weight loss, diarrhea, anaemia, transient thrombocytopenia, headache, hypotension, bradycardia, flushing, dyspnoea, photophobia, and eye pain. If bryostatin does surprise and show efficacy in phase 2 and 3 trials, FDA approval and provider benefits will be dictated by adverse side-effects, effect size, response duration, and cost. The confirmatory AD phase 2 study completion date is July 2019.
These side-effects occurred with the higher doses used in the anti-oncology trials and were mistakenly attributed to the low, entirely safe doses produced in Neurotrope’s previous Phase II trial. This author completely misunderstands the safety of the optimal 20 micgm Bryostatin dose in the recently completed Phase II trial (Farlow et al., J. Alzheimer’s Disease, 2019).
