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Showing posts with label Rett Sydrome. Show all posts
Showing posts with label Rett Sydrome. Show all posts

Friday, 6 March 2020

Calcium Folinate (Leucovorin) and Afobazole for Autism? Good, but …


Dr Frye is embarking on a multi-million dollar trial of Calcium Folinate (Leucovorin) to improve speech in autism.  I just completed my much humbler trial of a cheap generic Calcium Folinate.

I determined it was far cheaper and simpler to make a trial, than arrange for the blood test.  The other reason is that I note in the US they are prescribing Leucovorin, even if you test negative in the test for autoantibodies.

http://iliadneuro.com/order-a-kit.html

Dr Frye thinks many people with autism have low levels of folate inside their brain due to antibodies blocking folate crossing the blood brain barrier.  He even suggests that perhaps the source of these antibodies is your gut and they are produced as a reaction to cow’s milk.

I wondered why speech would be so directly affected by folate, but speech is something that is very noticeable and measurable.

I used 30mg of calcium folinate at breakfast and 15mg in the evening.

After a few days there was very clearly more speech. On several occasions I asked Monty a question, even without facing him eye to eye, and he gave a very much longer response than usual. The response was more like what he would produce if writing with a pencil and paper.

The problem was that three times during the trial he hit me, which is not his typical behavior. Aggression is a listed side effect of high dose calcium folinate.

Excerpt from Dr Frye’s colleague, Dr Dan Rossignol:

Dan Rossignol’s  Presentation at Synchrony 2019 | November 8, 2019

Folinic acid

• The good: Improvements in expressive speech, play skills, social skills, receptive language, attention, stereotypy

• The bad: Hyperactivity, self-stimulatory behaviors, aggression


Calcium Folinate (Leucovorin) is expensive in the US, but very much cheaper in some other countries, so it would be a viable therapy for many people.

Is there a lower dosage where you get the speech benefit without getting hit? I rather doubt it. It did actually try 15mg a day, a while back and saw no effect at all.

Since we do not really know why Calcium Folinate improves speech in particular, I doubt we can say why it produces aggression.

My old post from 2016:-

Clinical Trial of Mega-dose Folinic Acid in Autism


The new trial that is planned:-

The primary objective of this study is to evaluate the cognitive and behavioral effects of liquid leucovorin calcium on young children with autism spectrum disorder (ASD) and determine whether it improves language as well as the core and associated symptoms of ASD. The investigators will enrol 80 children across two sites, between the ages of 2.5 and 5 years, with confirmed ASD and known language delays or impairments. Participation will last approximately 26 weeks from screening to end of treatment.

  
Afobazole

Afobazole is the cheap Russian OTC treatment for anxiety that works as a sigma-1R agonist.  It has an effect on NMDA receptors.

Afobazole was covered in two recent posts.

ER Stress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it -Activation of Sigma-1 Chaperone Activity by Afobazole?


Afobazole is primarily used to treat mild anxiety.  Indeed it appears that sigma-1 receptor activation ameliorates anxiety through NR2A-CREB-BDNF signalling.  NR2A is a sub-unit of NMDA receptors.



Hundreds of millions of dollars are being spent in the US to develop a safe sigma-1R agonist (Anavex 2-73). This drug is being trialed in various autisms (Rett, Fragile X and Angelman syndromes), Parkinson’s and Alzheimer’s.


Afobazole should reduce ER Stress and protein misfolding, making it an interesting potential therapy for many neurological conditions.

I did raise the issue as to whether Afobazole may affect the Excitatory-Inhibitory (E/I) imbalance that is present in bumetanide-responsive autism.

It turns out that in my trial, Afobazole was beneficial in reducing anxiety, it just takes the edge off - nothing drastic.  After several weeks I did notice a slight reduction in cognition, this was only really evident when working on maths. It was more noticeable on cessation.  If I did not teach Monty maths, all I would have noticed was the reduction in anxiety.  When I stopped Afobazole, Monty’s assistant commented how clever he was at school.

Since we are trying to keep up with typical children in academic work at mainstream school, cognitive function is the priority and so no more Afobazole.


Conclusion

I hope the millions of dollars spent on the Calcium Folinate (Leucovorin) trials produce some tangible results. Speech clearly is the area where it shows an effect, I think it has other effects that are less measurable.  It did seem to have an effect on what I would describe as “initiative”, which is completing tasks independently that otherwise you might ask for help to complete.

If you could have the benefits of Calcium Folinate (Leucovorin) without the negative effects, that would indeed be very interesting.

Perhaps giving Calcium Folinate (Leucovorin) to very young non-verbal children will give them a nudge to start speaking.  In those little children you would likely be less concerned by some aggression - they do not hit very hard.

Afobazole also has a place; anxiety is a problem in much autism and for many people a small drop in cognition, if it indeed occurs, is not such a problem.  Long term Afobazole use might produce benefits relating to reduced ER stress and less protein misfolding.

If I had a child with Rett, Fragile X or Angelman syndromes, I would definitely trial Afobazole, since the new American sigma-1R agonist (Anavex 2-73) is not yet available and I suppose will cost 100-200 times more than the Russian drug.

I think you need to find therapies free of any troubling side effects; otherwise in trying to solve one problem, you just create two new ones.





Tuesday, 28 January 2020

Piperine/Resveratrol/Sunitinib for Rett’s and indeed much Autism? Or, R-Baclofen to raise KCC2 expression in Bumetanide-responsive autism.



Piperine/Pepper             Resveratrol/Red wine          Sunitinib/Sutent
  

This post is all about lowering chloride within neurons, by increasing the expression of the transporter that lets it leave, called KCC2.


Today’s post is one I never finished writing from last year; I looked up the price of Sutent/Sunitinib and then I remembered why. It does again highlight how cancer drugs, when they become cheap generics, will provide interesting options for autism treatment. It also shows again how Rett Syndrome is getting attention from researchers.

It also highlights that really clever Americans are looking for bumetanide alternatives, in the false belief that bumetanide has troubling side effects that cannot be managed/mitigated.

The study is by some clever guys in Cambridge Massachusetts.

Another group of clever guys from MIT burned through $40 million dollars a few years ago trying to develop R-Baclofen for Fragile-X and autism.  After that Roche-funded clinical trial failed, R-Baclofen has now been resurrected and a new trial is planned, with different end points (measures of success).

Today we see why many people should indeed respond positively to R-Baclofen, but the mode of action is entirely different to the one originally targeted by the clever guys from MIT.

Tucked away in the supplementary material of today’s paper we see that R-Baclofen increases the expression of the transporter (KCC2) that takes chloride out of neurons. So, R-Baclofen is doing the same thing as Bumetanide, just to a lesser extent and in a different way.  Both lower intracellular chloride.

That means that people responsive to bumetanide should get a further boost from R baclofen, but you might need a lot of it.

Clever they may be, but these researchers do not know how to communicate their findings.  I had to dig through the supplementary tables to extract the good stuff, which is a list of what substances increase KCC2 in regular brains (Table S1) and specifically in Rett Syndrome brains (Table S2).

This blog does rather bang on about blocking/inhibiting NKCC1 that lets chloride into neurons, you can of course alternatively open up KCC2 to let the chloride flood out. This latter strategy is proposed by the MIT researchers.

What really matters is the ratio KCC2/NKCC1.  In people with bumetanide-responsive autism, which pretty clearly will include girls with Rett Syndrome, you want to increase KCC2/NKCC1. So, block/down-regulate NKCC1 and/or up-regulate KCC2.

·        NKCC1

·        KCC2


The researchers identified 14 compounds.  To be useful as drugs these compounds have to be able to cross the blood brain barrier to be of much use, many do not.

In the paper they call KCC2 expression-enhancing compounds KEECs.

We have five approved drugs to add to the list that are functionally the same to primary hit compounds. 

·        Sunitinib
·        Crenolanib
·        Indirubin Monoxiome
·        Cabozantinib
·        TWS-119


The researchers went on to test just two compounds in Rett syndrome mice; they picked piperine (from black pepper) and KW 2449 (a leukemia drug)


Even R-baclofen pops up, with a “B score” of 6.65 (needs to be >3 to increase KCC2 expression).



Abstract
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl- cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3β (GSK3β) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.





Table S1. KEECs identified from screening with WT human KCC2 reporter neurons.






Table S2. KEECs identified from screening with RTT human KCC2 reporter neurons


Note Baclofen, Quercetin, Luteolin etc

















Fig. 3. Identification of KEECs that increase KCC2 expression in human RTT neurons
B score >3 indicates compounds potentially increasing KCC2 expression

In cultured RTT neurons, treatment with KEECs KW-2449 and BIO restored the impaired KCC2 expression and rescued deficits in both GABAergic and glutamatergic neurotransmissions, as well as abnormal neuronal morphology. Previous data suggested that disrupted Cl− homeostasis in the brainstem causes abnormalities in breathing pattern (64), consistent with breathing abnormalities seen in mice carrying a conditional Mecp2 deletion in GABAergic neurons (67). The reduction in locomotion activity observed in the Mecp2 mutant mice has also been attributed to abnormalities in the GABAergic system (65). Therefore, treatment with the KEEC KW-2449 or piperine may ameliorate disease phenotypes in MeCP2 mutant mice through restoration of the impaired KCC2 expression and GABAergic inhibition.

Most KEECs that enhanced KCC2 expression in WT neurons, including KW-2449, BIO, and resveratrol, also induced a robust increase of KCC2 reporter activity in RTT neurons (Fig. 3, A and B; a complete list of hit compounds is provided in table S2). The increase in KCC2 signal induced by KEECs was higher in RTT neurons than in WT neurons,


Our results establish a causal relationship between reduced FLT3 or GSK3 signaling activity and increased KCC2 expression.

Two hit compounds, resveratrol and piperine, act on different pathways than the kinase inhibitors, activating the SIRT1 signaling pathway (50) and the TRPV1 (51), respectively

Thus, our data demonstrate that activation of the SIRT1 pathway or the TRPV1 channel enhances KCC2 expression in RTT human neurons.


The group of KEECs reported here may help to elucidate the molecular mechanisms that regulate KCC2 gene expression in neurons. A previous study conducted with a glioma cell line showed that resveratrol activates the SIRT1 pathway and reduces the expression of NRSF/REST (50), a transcription factor that suppresses KCC2 expression (52). Our results demonstrate that resveratrol increases KCC2 expression by a similar mechanism, which could contribute to the therapeutic benefit of resveratrol on a number of brain disease conditions (68, 69). We also identified a group of GSK3 pathway inhibitors as KEECs. Overactivation of the GSK3 pathway has been reported in a number of brain diseases (70). Thus, our results suggest that GSK3 pathway inhibitors could exert beneficial effects on brain function through stimulating KCC2 expression. Another major KEEC target pathway, the FLT3 kinase signaling, has been investigated as a cancer therapy target (71, 72). Although FLT3 is expressed in the brain (73), drugs that target FLT3 pathway have not been extensively studied as potential treatments for brain diseases. Our results provide the first evidence that FLT3 signaling in the brain is critical for the regulation of key neuronal genes such as KCC2. Therefore, this work lays the foundation for further research to repurpose a number of clinically approved FLT3 inhibitors as novel brain disease therapies

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron- restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders



Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl− cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression– enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration–approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3 (GSK3) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.


By screening these KCC2 reporter human neurons, we identified a number of hits KCC2 expression–enhancing compounds (KEECs) from ~900 small-molecule compounds. Identified KEECs were validated by Western blot and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments on cultured human wild-type (WT) and isogenic RTT neurons, as well as on organotypic mouse brain slices. Pharmacological and molecular biology experiments showed that identified KEECs act through inhibition of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3b (GSK3b) kinases, or activation of the sirtuin 1 (SIRT1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment of RTT neurons with KEECs rescued disease-related deficits in GABA functional switch, excitatory synapses, and neuronal morphological development. Last, injection of the identified KEEC KW-2449 or piperine into a Mecp2 mutant mice ameliorated behavioral phenotypes including breathing pauses and reduced locomotion, which represent important preclinical data, suggesting that the KEECs identified in this study may be effective in restoring impaired E/I balance in the RTT brain and provide symptomatic treatment for patients with RTT.





Fig. 2. KEEC treatment–induced enhancement of KCC2 protein and mRNA expression in cultured organotypic mouse brain slices and a hyperpolarizing EGABA shift in cultured immature neurons.

(E to G) KCC2 and NKCC1 mRNA expression induced by FLT3 inhibitors including sunitinib (n = 4), XL-184 (n = 6), crenolanib (n = 4), or a structural analog of BIO termed indirubin monoxime (n = 6). The calculated ratios of KCC2/NKCC1 mRNA expression are shown in (G). A.U., arbitrary units




Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.


In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.




The science-light version:-

Drug screen reveals potential treatments for Rett syndrome

An experimental leukemia drug and a chemical in black pepper ease breathing and movement problems in a mouse model of Rett syndrome, according to a new study.

Rett syndrome is a rare brain condition related to autism, caused by mutations in the MECP2 gene. Because the gene is located on the X chromosome, the syndrome occurs almost exclusively in girls. No drugs are available to treat Rett.
The team screened 929 compounds from three large drug libraries, including one focused on Rett therapies. They found 30 compounds that boost KCC2’s expression in the MECP2 neurons; 14 of these also increased the protein’s expression in control neurons.

The team tested two of the identified compounds in mice with mutations in MECP2: KW-2449, which is a small molecule in clinical trials for leukemia, and piperine, an herbal supplement and component of black pepper. These mice have several traits reminiscent of Rett. They are prone to seizures, breathing problems, movement difficulties and disrupted social behavior.
Injecting the mice with either drug daily for two weeks improved the animals’ mobility relative to untreated mice. The drugs also eased the mice’s breathing problems, decreasing the frequency of pauses in breathing (apnea). The findings appeared in July in Science Translational Medicine.


 

Piperine, Resveratrol and analogs thereof

Piperine and Resveratrol are commercially available supplements.

Resveratrol has been mentioned many times in this blog.  It has numerous beneficial properties, to which we can now add increasing KCC2 expression, but it is held back by its poor ability to cross the blood barrier.

The other natural substance highlighted in the study is piperine. Piperine is the substance that gets added to curcumin to increases its bioavailability and hopefully get its health benefits.

Piperine has been recently been found to be a positive allosteric modulator of GABAA receptors.

It may be that piperine has 2 different effects on GABA, or maybe it is just the same one?

The result is that people are trying to develop modified versions of piperine that could be patentable commercial drugs.

Piperine also activated TRPV1 receptors.

You might wonder what is the effect in humans of plain old piperine in bumetanide-responsive autism.

Invitro blood–brain-barrier permeability predictions for GABAA receptor modulating piperine analogs

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood–brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00–500 ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.


The alkaloid piperine, the major pungent component of black pepper (Piper nigrum L.), was recently identified as a positive allosteric γ-aminobutyric acid type A (GABAA) receptor modulator. The compound showed anxiolytic-like activity in behavioral mouse models, and was found to interact with the GABAA receptors at a binding site that was independent of the benzodiazepine binding site [1,2]. Given that the compound complied with Lipinski’s “rule of five” [1], it represented a new scaffold for the development of novel GABAA receptor modulators [1–3]. Given that piperine also activates the transient receptor potential vanilloid 1 (TRPV1) receptors [4] which are involved in pain signaling and regulation of the body temperature [5,6], structural modification of the parent compound was required to dissect GABAA and TRPV1 activating properties

For drugs acting on the central nervous system (CNS), brain penetration is required. This process is controlled by the blood-brain barrier (BBB), a tight layer of endothelial cells lining the brain capillaries that limits the passage of molecules from the blood circulation into the brain [10]. Since low BBB permeability can reduce CNS exposure [11], lead compounds should be evaluated at an early stage of the drug development process for their ability to permeate the BBB [12].

Conclusions

Piperine and five selected piperine analogs with positive GABAA receptor modulatory activity were screened in three in vitro cell-based human and animal BBB models for their ability to cross the BBB. Data from the three models differed to some extent, possibly due to protein binding of the piperine analogs. In all three models, piperine and SCT-64 displayed the highest BBB permeation potential, which could be corroborated by in silico prediction data. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human models, and moderate to high in the animal model. ER calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux. In addition to the early in vitro BBB permeability assessment of the compounds, further studies (such as PK and drug metabolism studies) are currently in progress in our laboratory. Taken together, these data will serve for selecting the most promising candidate molecule for the next cycle of medicinal chemistry optimization




Conclusion

My conclusions are a little different to the MIT researchers

“The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.”

This assumes that you cannot safely use bumetanide/azosemide, which you can.  Open your eyes and look at France, where several hundred children with autism are safely taking bumetanide.

”It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases”

We have copious evidence that elevated chloride is a feature of many conditions, not just Rett’s and an effective cheap therapy has been sitting in the pharmacy for decades.

In the clinical trial of R-Baclofen that failed, there were some positive effects on some subjects.  Were the positive effects just caused by the effect of Baclofen in increasing KCC2 expression?

Should R-Baclofen become a cheap generic, it might indeed become a useful add-on for those with bumetanide-responsive. Regular Baclofen (Lioresal) is an approved drug, but it does have some side effects, so most likely R-baclofen will have side effects in some.

Baclofen itself in modest doses has little effect on bumetanide-responsive autism.



A cheap side-effect free KCC2 enhancer would be a good drug for autism, although cheap, safe NKCC1 blockers already exist. 

I have no idea if piperine benefits bumetanide-responsive autism.  Piperine has long been used in traditional medicine.

The TRPV1 receptor also affected by piperine plays a role in pain and anxiety.

We saw in the post below that TRPV1 controls cortical microglia activation and that GABARAP modulates TRPV1 expression.

So, TRPV1 and GABAA receptors are deeply intertwined.

  

GABAa receptor trafficking, Migraine, Pain, Light Sensitivity, Autophagy, Jacobsen Syndrome,Angelman Syndrome, GABARAP, TRPV1, PX-RICS, CaMKII and CGRP ... Oh and the"fever effect"



Is Piperine going to make autism better, or worse?








Wednesday, 18 December 2019

Will Anavex for “Autisms” be worth the wait and the price, compared to Russian OTC Afobazole?





US-Russia cooperation has long been possible in Space, but not so often in Medicine. NASA reportedly pays Russia $85 million per astronaut to go the International Space Station (ISS).  The US Space Shuttle program ended in 2011, leaving a Russian Soyuz rocket the only way to the ISS.


This post comes ahead of the dietary autism post, awaited by Tanya.  It really is just a brief follow-on from the previous post. I have only just come across Anavex, which does add weight to the first post on sigma-1R.
                                                                                                               
Hundreds of millions of dollars are being spent in the US to develop a safe sigma-1R agonist (Anavex 2-73). This drug is being trialed in various autisms (Rett, Fragile X and Angelman syndromes), Parkinson’s and Alzheimer’s.

In the last post I wrote about a cheap OTC anxiety drug from Russia, called Afobazole, that appears to be a safe sigma-1R agonist.  This drug has also recently been trialed in autism and Parkinson’s - the same targets as Anavex.

I did make the point in my original sigma-1 post that I am interested in existing therapies, rather than potential ones, so I did not include Anavex, or any other research drug, in that post. Anavex is nonetheless interesting, because their research studies further support the suggestion that targeting ER stress via sigma-1 is an interesting avenue to pursue.  

ERStress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it - Activation of Sigma-1 Chaperone Activity by Afobazole?



Anavex is claiming precision medicine, but in fact sigma-1R agonists appear more like the opposite, at least in terms of who you target.  The majority of both common and rare neurological disorders look like they should benefit from reducing ER Stress (from whatever cause); it is a shared feature.  So it looks more like a shotgun approach; that is actually a good thing, if it were to drive the price down.

What is needed is an affordable, effective, mass market drug; not an ultra expensive pill just for Rett Syndrome and perhaps a different colour version for Angelman's Syndrome.

Which will prove effective - Anavex or Afobazole? Or perhaps neither.

Having already made the case for Soyuz in my earlier post, here is the case for NASA, and for those with NASA-sized budgets, courtesy of  https://www.anavex.com/





















Treatment with Anavex 2-73 was seen to improve motor skills, acoustic responses and visual acuity in a mouse model of Rett syndrome, supporting ongoing Phase 2 studies in patients.
Its use also helped to lessen abnormal movements and ease breathing in these mice, its researchers said.
Anavex 2-73 (blarcamesine) is an oral investigational therapy developed by Anavex Life Sciences that works by activating the sigma-1 receptor (S1R), a protein involved in the correct folding of other proteins.
S1R activation results in reduced toxic accumulation of misfolded proteins, as well as lesser dysfunction in mitochondria (a cell’s “powerhouse”), oxidative stress and neuroinflammation, all involved in Rett syndrome. (Oxidative stress is an imbalance between the production of free radicals — potentially harmful molecules associated with a number of diseases — and the generation of antioxidant defenses.)
Researchers at Anavex, assisted by PsychoGenics, evaluated the potential treatment’s specific effects on Rett symptoms in a validated mouse model.
They assessed motor function (balance, motor coordination, locomotion, and abnormal movements or stereotypies), sensory function (reflex responses to sound stimuli and visual clarity), and respiratory function.
Motor and sensory functions were assessed in younger mice, while visual acuity and breathing were measured in older animals.
Results showed that Anavex 2-73 significantly eased motor dysfunction, and deficits in acoustic and visual responses compared to mice given a placebo.
Anavex 2-73 also induced a significant reduction in two distinctive features of Rett syndrome found in these mice: hind-limb clasping (an abnormal posture comparable to hand stereotypies in people with Rett), and apnea (involuntary breath-holding) that is the most concerning breathing abnormality in Rett syndrome, the researchers said. These improvements were mainly dependent on treatment dose and duration.
“In conclusion, the data demonstrate that [Anavex 2-73] is effective in ameliorating multiple neurobehavioral phenotypes in [Rett] mice,” the researchers wrote. “In line with previous animal and human studies [in other neurodegenerative diseases], [Anavex 2-73] also showed a good safety profile,” they added.
These data served as a proof-of-concept for an ongoing safety and efficacy Phase 2 trial called RS-001 (NCT03758924, still enrolling) in the U.S., and for the Phase 2 AVATAR study (NCT03941444) in Australia. These trials together will evaluate Anavex 2-73 in up to 51 women with Rett syndrome.











Conclusion

It may be that Anavex is far superior to the cheap Afobazole. Like the space shuttle was far more advanced than the Soyuz. 

But what if the cheap Afobazole is quite good enough?  Like the cramped, but reliable Soyuz rocket.

Anavex/Afobazole will not cure any severe neurological condition, just improve it, so it will need to be part of a polytherapy. That means the patient will need to be able to afford multiple drugs, somehow.

Coming back to those autisms, what if your daughter has Rett Syndrome, or son has Fragile-X Syndrome ?  Wait a few years for Anavex and for someone else to pay for it? or make do with some cheap Afobazole?