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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?








39 comments:

  1. Hi Peter,

    Great blog post!

    Peter, I had identified piperine as a potential treatment based on the paper you had identified, but given its impact on absorption of other supplements, I've been concerned to add it to my current regimen (that I think has been beneficial). In your review, did you find anything that identifies the time "window" around which piperine increases the bioavailability of other ingested substances? Ideally, I would like to trial piperine, but do so without impacting the bioavailability of the other aspects of my regimen, but haven't been able to find how long after piperine ingestion does the bioavailability impact lessen.

    Thanks in advance if you have seen this somewhere. I will continue to look, but if you know this, it would be much appreciated.

    AJ

    ReplyDelete
    Replies
    1. AJ, piperine seems to have such a long half life that you cannot give it without the chance of it increasing the bioavailability of many drugs and supplements.

      Piperine was just one of the OTC products identified in the above study.

      I think you safest bet is to use Bumetanide or Azosemide.

      Delete
    2. Hi Peter,

      Thanks so much! That is the impression that I had as well with Piperine (i.e. the impact lasts too long to try to get around it).

      I agree with you Bumetanide, unfortunately it has been a challenge, even with a physician who is open to such treatments.

      Have a great day Peter!

      AJ

      Delete
  2. Hello Peter and community,

    I just saw a very interesting story / paper from the University of Toronto (U of T), in collaboration with other researchers, that I wanted to share:

    https://www.cell.com/molecular-cell/fulltext/S1097-2765(20)30006-X

    https://scitechdaily.com/link-between-autism-and-cognitive-impairment-identified-may-lead-to-new-treatments/

    I'll be digging more into the full paper, but wanted to share the above as soon as I saw it as I think it may have important implications for some.

    There is great work being done here in Canada by researchers at various institutions, such as McMaster University, U of T, Hospital for Sick Children (Sickkids), Holland Bloorview Hospital, and many others.

    Have a great day!

    AJ

    ReplyDelete
  3. Hi Peter, just to come back shortly to your previous reply about changing from Bumetanide to Azosemide - you said in your reply that bumetanide is stronger, but in the post on Azosemide you said Azosemide was? I even went a rechecked that you said that in the post, that confused me a bit. But today I am writing to ask your opinion on this: https://www.nature.com/articles/s41591-019-0608-y?fbclid=IwAR0BJGV7a3e6IZazTOFxrMZ2fsNXN3qeI2EnuTM5w6UOmpLY7_MnQDyNsC0

    ReplyDelete
    Replies
    1. tpes, 1mg of Bumetanide is far stronger than 1mg of Azosemide.

      I use 2mg of bumetanide in the morning and 60mg of Azosemide in the afternoon. I did not see any improvement when using 60mg of Azosemide twice a day, so I do not believe 60mg of Azosemide is more potent than 2mg of bumetanide.

      I use Azosemide because it causes much milder diuresis in my son's case. If I gave 2mg of Bumetanide twice a day it would be really impractical in daily life.

      mTOR is very complicated and has been covered in this blog.

      mTOR is a component of mTORC1 and mTORC2. Taken together mTORC1 and mTORC2 signaling controlling many critical aspects of human life.

      A defect in these pathways is associated with many conditions ranging from cancer to Alzheimer’s to autism.

      Over active signalling can lead to cancer.

      The autism gene TSC inhibits mTOR, so a fault in that gene means more mTOR signaling.

      The autism/cancer gene PTEN inhibits mTOR. Reduce PTEN and you increase the chance of both cancer and autism.

      mTOR affects a vital process called autophagy. Hyperactive mTOR in autism can reduce autophagy and lead to poor synaptic pruning and social behavior deficits.

      You need the "right" amount of mTOR signalling. Autism with big heads (macrocephaly) likely has too much, whereas autism with a tiny head (microcephaly) may have too tittle.

      Delete
  4. Peter, or AJ or Ling or any other sharp and helpful soul:
    can you remind me again, of the profile for a NON bumetanide responder? and what does it indicate, the dysregulation of KCC2, for someone with regular episodes of low blood sodium? still waiting on precise answers for my son's hyponatremia. another appt upcoming
    ~Tanya

    ReplyDelete
    Replies
    1. Tanya, a non-responder to Bumetanide has normal (non-elevated) levels of chloride inside neurons. KCC2 and NKCC1 are not miss-expressed, they are normal.

      I don't think it tells you anything regarding hyponatremia.

      Polydipsia (drink too much water) is common in autism.

      Excessive water drinking behavior in autism.
      https://www.ncbi.nlm.nih.gov/pubmed/10206527

      Delete
    2. Hi Tanya,

      Hope all is well!

      Tanya, did you ever have a thorough genetic test or diagnosis? I ask as this has been incredibly helpful for us. When we got our ASD diagnosis, the developmental pediatrician offered a genetic test, but I believe it was rather limited so we did the GeneDx full ASD/ID panel (~2,500 genes) which is a trio (child, mom, and dad) and it was a gamechanger for us. We would never have gotten our genetic diagnosis without such a thorough test.

      Have a great day Tanya!

      AJ

      Delete
    3. yes trying to find out that "intrinsic" factor the pubmed link you attached mentions. Unless they are referring to actual gut related Intrinsic Factor or lack of which leads to polydipsia? looking back over labs, and the last one where he was in hyponatremia (trip to ER for intense mania but docs did not mention), he had not been drinking water excessively. Neurologist consult was concerned this had been brushed off. She referred us back to a nephrologist before sedating MRI to rule out SIADH (which she doesnt think it is) - but could a GI consult help? Or am I interpreting the mention of intrinsic factor literally?
      Thanks for the help

      Delete
    4. Tanya, the paper is 20 years old from Japan. I attach an extract from the full text.

      In short, they do not really know why people with autism do this. People with autism "do weird stuff" the list is long - breath holding, hand flapping, falling into lakes/rivers etc.

      "Autism has been stated to be associated with a hypothalamic-pituitary dysfunction indicated by a blunted-plasma
      growth-hormone response following the oral administration
      of l-dopa[10], an abnormal plasma growth hormone response to insulin-induced hypoglycemia [11], and a premature or delayed response of growth hormone to clonidine
      and l-dopa [12]. The blunted growth hormone response
      exhibited by at least 30% of autistic children to a provocative challenge with l-dopa suggests an alternation of
      hypothalamic dopamine receptor sensitivity (subsensitivity)
      in autistic children [10]. The premature response of growth
      hormone to clonidine and delayed response to l-dopa suggest possible abnormalities of both dopaminergic and noradrenergic neurotransmission in subjects with autism [12].
      Furthermore, Hiratani et al. described a case of autism
      with water intoxication and the episodic release of antidiuretic hormone [13]. The thirst center is said to be located in the hypothalamus. Therefore, a possible factor causing polydipsia in autism may be a hypothalamic–pituitary dysfunction.
      In 1988, the male case described by Hiratani et al. [13],
      who was 19-years old at that time, exhibited a remarkable
      daily body weight change that was probably due to excessive water drinking. After mild water restriction and intermittent forced water restriction according to the setting of a
      body weight limit, the daily change became smaller in 1994
      [14].
      We have often observed that autistic children sometimes
      fiddle with water, or only drink from a single faucet, presumably one manifestation of the restricted interest characteristic of autism. Therefore, preservative tendencies may contribute to compulsive water drinking.
      In conclusion, in view of the present results, it is possible
      that the principal cause of polydipsia is some intrinsic factor in autism itself (e.g. a hypothalamic–pituitary dysfunction, restricted interest and activity).

      Delete
    5. Hi AJ no we never did a complete one - as my son is 20 now and when we were starting out this wasnt even on our radar. Might be worth it. I will look at their website and I am glad it was so helpful for your family. Great news
      Thanks Peter for the full excerpt. Some of the weird things they can do are worse than others. Dont want to see this again. Just correcting hyponatremia alone is dangerous.

      Delete
    6. Tanya, as I am sure you are aware polydipsia is a known cause of death in people with autism. Like many cases of people with autism drowning, these deaths are preventable, but they keep on happening.

      Delete
    7. Hi Tanya,

      Sorry to hear about these episodes. Was your son on bumetanide when hyponatremia happened? Was it associated with polydipsia in him?

      In my opinion it requires detailed diagnostics as there are several conditions which can manifest with hyponatremia and "autism" does not protect from any of them. Speaking of genetic testing, there are some rare vasopressin receptors disorders of genetic origin.

      There is one thing that came to my mind long ago with regard to some episodic symptoms in autism/PANS, including polydipsia: histamine impact on vasopressin release and brain mast cell degranulation involvement. There are some old studies on this, but even these few papers are sometimes contradictory:
      https://pubmed.ncbi.nlm.nih.gov/18184767
      “Stimulation of Brain Mast Cells by Compound 48/80, a Histamine Liberator, Evokes Renin and Vasopressin Release in Dogs”.

      Also, this is only anecdotal, but I’ve heard a sad story of a Greek girl with autism and polydipsia, who indeed died because of electrolyte imbalance due to excessive drinking. She was found to have a treatable pituitary tumor which had caused these symptoms, but it was unfortunately post-mortem diagnosis. Apparently the girl was a child of some prominent person and it was widely discussed few years ago in Greece - maybe readers from Greece can verify it. I don’t think this is relevant to your son, but in general it’s not enough to say it’s “some intrinsic factor in autism itself”.

      Good luck with your son’s next appointment!
      Agnieszka

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    8. Tanya, look up a condition called PPD (Psychogenic Polydipsia). It is particularly common in schizophrenia. In clinical trials 60% of psychiatric patients taking an ACE inhibitor decreased water consumption.

      Psychogenic polydipsia review: Etiology, differential, and treatment
      https://link.springer.com/article/10.1007/s11920-007-0025-7

      Psychogenic polydipsia: a mini review with three case-reports
      https://www.jpsychopathol.it/wp-content/uploads/2015/07/11londrillo1.pdf

      Psychogenic Polydipsia – Management Challenges
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579464/

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    9. Sorry Tanya that I wasn't around to answer in time. I see that you got a lot of help from the brain team anyway, hope you find something useful!
      Like AJ and Agnieszka, I found genetics very helpful, and each year they are able to find more diagnosese. I don't know Peter if/when you did one for Monty, but I think it's worth redoing it every 5-10 years.

      /Ling

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    10. Peter Agnieszka AJ Ling (the dream team) - thanks so much for the leads. The mast cell link, possible genetic links, also interesting about ACE inhibitors. This give me a lot to go through. I'm going to study tonight and get my questions ready for the doctor. Agnieszka - the neurologist stated exactly what you are suggesting - that we need someone who really knows hyponatremia and that it is not just to be brushed off as a behavior and an oh well watch his water (as if that hadn't occurred to us). Very tragic about the girl in Greece. My son's excessive water drinking has ebbed and flowed over years. And recently we found he was hiding cups near different sinks in the house. When he was in the craving state, he would get very anxious when you would try implement a water drinking schedule. But after the seizure and hospital stay, it was remarkable how easy it was to get him on a schedule. No anxiety whatsoever about that. However, in the last week the water craving has ramped up a bit. AJ and Ling - how hard was it to find a doctor to help work with you on the results? Did the lab provide doctor recommendations?
      Thanks again - this blog is invaluable
      ~Tanya

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    11. Tanya, this may sound obvious but often clinicians do not publish all their knowledge. If you find the current email addresses of the authors of the papers on Psychogenic Polydipsia, ask your neurologist to email them all asking for advice on your specific case. These authors are nearly always very happy to talk to other doctors, but not so keen to talk to parents.

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    12. Tanya, read this paper and you will see why an ACE inhibitor might well help.

      Thirst
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957508/

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    13. Hi Tanya,

      Hope all is well!

      Tanya, when we got the results from GeneDx, they actually provided the opportunity for us to speak with one of their genetic counsellors about the results.

      Given that there is little known about the gene we are dealing with, there wasn't much she could say but, we had a really unique coincidence. The genetic counsellor's former colleague just happened to be working on the gene at a major university, so connected me with the researcher. Having said this, even if she hadn't made the connection, I would have done so.

      In my opinion Tanya, the best thing to do is the following:

      1. Absolutely do the test. 20 is still so young. It's expensive and you may not get a hit (there's no guarantees your cause is one of the 2,500 genes) but … if you do get a hit, that will really change the course of your approach.

      2. Once you get the results, you speak to the genetic counsellor from GeneDx and they can help. If a lot is known about the gene, you'll get a lot of key info.

      3. BUT … the best researcher in the world will be you. PubMed the gene, and look for researchers working on that gene. When you find the ones who appear most active and focused on what you're looking for, send them an e-mail. That's what I have done and right now, I am in contact with 4 separate teams, 3 of which I am actively working with.

      4. You always have your friends here - you mention the gene and you have a few pubmed experts (I think Ling and I alone may be wearing out the servers at PubMed) who will dig up a ton of relevant info you. ;)

      I hope this is helpful Tanya, and best of luck if you do go down this path!

      AJ

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    14. Peter, AJ, Agnieszka:
      Peter that paper was the most helpful of anything I have found on the topic to date. It's fascinating what I have learned from it - I have a full page of notes. Some small things like cold liquids inhibiting thirst neurons more efficiently - I have noticed my son prefers room temp water - probably at least one explanation for why he downs more of it! Also learned about these receptors further down the GI tract and in stomach..And things like CCK hormone modulating thirst neuron. But the case for ACE inhibitors makes a lot of sense after reading this. I will be ready with my questions now for the next consult. Many thanks!
      AJ your explanation - so helpful. How much was the cost? I will call the lab.
      Agnieszka I forgot to answer you earlier question - No not taking bumetanide due to sulfa med allergy. in the paper Peter
      highlighted histamine is implicated as a hormone associated with modulation of thirst neurons....
      You all have made it so much easier for me. Can't thank you enough
      ~Tanya

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    15. Hi Tanya,

      It's my pleasure! As parents, we are already in a special club, and then as parents of ASD kids, we are in an even more special club, with a bond that makes us want to help each other (as we know what the others are going through)

      The standard cost for the test (which tests each parent and child using a cheek swab) was $3,500 U.S. (several years ago). I went back to them and explained that our provincial medical plan did not cover it, and negotiated it down to $2,000 US as we were paying out of pocket.

      As I said, it's expensive, but if you find out the cause via the test, it's probably the best way to spend that amount. We've spent a lot over the years, and that was the best $2,000 US I have spent by far in terms of my daughter's condition. The doors it has opened are worth 100X that amount.

      Tanya, I hope this is helpful!

      AJ (your friend in the currently frozen north) :)

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  5. Hi Peter, so you meant 1mg vs 1mg...I knew that, I meant you said its better at getting into the brain so replacing bumetanide completely would work better? I know you covered mTOR, I meant what do you think of their proposed treatment idea...since other good options have so far not popped up.

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    1. They used a single injection of Antisense oligonucleotides (ASOs) to generate an mRNA selective knockout of mTORC2. It worked in a mouse, but we do not have that option at home.

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  6. Hi Peter, Friends, and Community,

    I wanted to share a very intriguing article and relevant paper, as it may be relevant. It has to do with myelination, a subject I know Peter has delved into in the past:

    https://medicalxpress.com/news/2020-02-links-autism-specific-cell-paves.html

    https://www.nature.com/articles/s41593-019-0578-x

    What I found intriguing was that they found the myelination issues in donated brain tissues from people with ASD from a variety of causes. So this may be a common affected pathway (which I believe Peter had presciently alluded to in the past)

    A relevant quote from the article is " It appears that in many people who suffer from ASD, their OL cells are not maturing sufficiently or functioning properly"

    I'll do some digging for some potential options, and if memory serves me right, I believe Peter had noted Clemastine as a potential option for myelination disorders.

    Have a great day everyone!

    AJ (from the not so frozen north)

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    Replies
    1. AJ, the same research group published something very similar last year, which prompted this post.

      More Research to support a Trial of Clemastine in Autism and particularly in Pitt Hopkins
      https://epiphanyasd.blogspot.com/2019/10/more-research-to-support-trial-of.html

      I spend 20 cents a day on Clemastine and I think it is a wise investment.

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  7. Hello, I have an additional question on Verapamil. I remembered recently your descriptions of SIB and explosions in Monty, and we have some similar things. Recently, our daughter will hit herself or myself when she is upset. The saddest thing is when I calmly and lovingly tell her not to do something and she hits herself on the head and shouts ‘Stop it!!!’. It looks like we could give Verapamil a try. My question was: we now supplement with about 400mg potassium chloride and 1 banana and 1 cucumber a day. How do you ‘calculate’ how much you would need if you also add Verapamil?

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    1. Tpes, since you are supplementing potassium are you also giving bumetanide?

      There are multiple reasons for self injury and so there can be no single effective therapy. In our case Verapamil is extremely effective, although to a pediatrician this would look an extremely odd therapy.

      One researcher reader of this blog went into great detail to determine how much potassium you can supplement before it becomes risky. This was to determine how much of the ketone Potassium BHB it is safe to use. His conclusion was that he did not need to worry. Your 400mg plus a banana is not a lot of potassium.

      Measuring potassium levels in blood is always a good idea.

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  8. There have been a series of very interesting studies lately on photic entrainment which for a long time were relegated to the realms of quackery and pseudoscience so that no serious researcher would look into the matter. Starting around in the 1980's, there were many people looking into audio/visual entrainment using what are called "light and sound machines" for the purposes of boosting brain power or "consciousness" or "enlightenment" or any other buzzword associated with the brain at the time in a manner that did not involve burning a hole in your brain with hard psychedelics like LSD. Hobbyists took over and before you knew it the next thing you knew people were talking about using it A/V entrainment for astral projection, chakra stimulation, and just about any other topic that would scare away mainstream scientists studying the topic in an official manner.

    Fast forward to today and people still do light and sound entrainment stimulation as you can find thousands upon thousands of sessions on YouTube. I myself began experimenting with this stuff out of curiosity before I had a family about 15 years ago and eventually stopped due to simply having just about zero time and privacy with the responsibilities of kids and all that comes with it.

    Anyways, a new study came out from a research group that has been looking into the biological mechanisms of photic entrainment with respect to its use as an Alzheimer's therapy and the results if they are to be believed may be quite applicable to autism as well:

    Press Release:

    https://www.sciencedaily.com/releases/2020/02/200203141446.htm

    Paper:

    https://www.jneurosci.org/content/40/6/1211

    What the researchers found in this study was that 40hz photic stimulation (light flickering on a screen at 40 times per second) caused an upregulation of 20 cytokines (transient neuroinflammation) that stimulated microglia to get to work in cleaning out the extracellular junk that buildup naturally over time. To their amazement, they also looked at 20hz stimulation and found it downregulated neuroinflammation which may be useful in autism as there seems to be an excess of microglial activation in the many studies of autism that have looked at the matter.

    Now, you can find a lot of light and sound machines on the internet that flash LED's. Those are likely technically the best option, except that you are supposed to close your eyes and if you have a low-functioning child such as mine, then that is simply not an option.

    As for watching a screen, in order to flicker light at 40Hz, you will need a 120hz or even a 240hz monitor which are more expensive than the typical 60hz refresh rate of standard LCD monitors. Gamers use the higher refresh rate monitors because the flicker fusion threshold can be as high as 90hz in human vision so the higher refresh rate perceptually seems smoother. For the purposes of this therapy, you need a 120hz monitor because the flicker rate is 40hz which means you need one ON frame and two OFF frames or conversely two ON frames and one OFF frame to get a clean 40hz flicker. For the 20hz flicker, all you need is a 60hz monitor for obvious similar mathematical reasons. I would think the 20hz photic entrainment would be the most beneficial for autism, but unless you buy a 120hz monitor.

    You also of course will need an MP4 file (or equivalent codec that supports at least 60hz framerates) compiled with the flicker frames (YouTube will decrease the quality automatically if you upload it there) and use a media player like VLC Media Player to play the MP4 file.

    A cell phone or ipad will not work as they are just about all 30hz at best in refresh rate, which means you cannot flicker faster than 15hz. I could be wrong on some of the newer models, but a faster refresh rate means more drain on the battery which is why 30hz is still standard as best I know.

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  9. Hi,
    We have had genetic testing done through Courtagen (recently lrecently closed I believe) a long time ago but wondered if there is a preferred , most informative test you would recommend.
    Nancy

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  10. Hi Peter,
    I remember a comment you made, and then recently read again, of the need for a diagnostic flowchart leading to the relevant cluster (from the clusters of autism types), leading one to a likely drug toolkit.
    Is there such a crude flowchart in existence or in the works by anyone?
    Is it possible to make an educated guess as to the correct cluster based simply on what has had positive effects so far, enabling one to possible next steps to try?
    Nancy

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    Replies
    1. Nancy, I have not seen such a flowchart, but it remains a good idea.

      I think to be meaningful it would need to be based on data from many people. Almost all clinicians just use a handful of their pet therapies. You would need all therapies included.

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  11. Thanks, Peter. I am not well-versed in the remarkable way it seems most of those are who post to your blog. I am more trial and error over the past 3 years that I have been following. I would love to narrow the scope of my search to some degree. Wishful thinking at the moment!
    One obstacle I continue to bump up against is finding physicians who are willing to entertain these off-label uses of drugs. Does anyone know of physicians in the US who read this blog and treat autism with consideration of these therapies?
    Thanks
    Nancy

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  12. Thanks Peter.
    This next question may be more for Tyler. The BCAAs and collagen continue to be remarkably effective in reducing the intensity and the frequency of SIB and rage.
    They are not gone completely but make life more liveable for sure.
    Sleep (or lack of) continues to be an issue, and worsening this winter. My son goes right to sleep because he is exhausted from waking then continuing to be up since about anywhere from 1-3 AM nightly. And the cycle repeats... We tried the SAD light but he would not tolerate it first thing in the morning so I stopped.
    I want to refine the BCAA protocol though. I don't currently give apigenin or ECGC at all.
    I give Niagen (2 caps) plus collagen and BCAA upon waking.
    Then the BCAA and collagen around 4or 5 PM but no Niagen.
    My son (around 175 lbs) gets 5 HTP 100 mg (1 cap) at bed and again in the morning.
    Should I add in the apigenin? Possibly increase the 5 HTP? Niagen also in the afternoon?
    Thanks Tyler.
    Nancy

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    1. Nancy, I assume you have tried melatonin? Timed release is best if you can get your child to swallow caplets.

      Also, for people who crave pressure, a weighted blanket might help as well for keeping your child asleep.

      Last but not least, 5-HTP converts to serotonin and should not be given any later than 5pm or so. They serotonin production ramps down as night time approaches and what is in the blood stream gets converted to melatonin. When you wake there is a surge of serotonin that helps wake you up. The issue with sleep and serotonin can be that if you have little serotonin in your blood stream, you may have little melatonin as well which will impact sleep quality. However, if you have a bunch of serotonin floating around in your brain around 12am, you may be more easily awoken.

      Also, not sure about where you live or the windows in your house, but in winter if there is snow you can get a lot of light pollution into your house from the ambient light (street lights), so make sure you have your child's room as dark as you can get. I even bought myself a light meter a long time ago to deal with this issue.

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  13. Hi Tyler,
    My son takes 10 mg time released melatonin at bedtime. Should I make this earlier as well?
    He takes a bunch of supplements at bed (NAC, Vital Guard Supreme (from Supreme Nutrition), L-histadine, fish oil, mg citrate, cromolym sodium, and the 100 mg 5HTP). I can move the 5 HTP earlier. Should I increase it as well?
    Also, apigenin....he does not take this at all.
    His room is very dark at night. zero light getting in.
    He does not take any niagen in the afternoon. Just the BCAA/collagen. Is that right?

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  14. My son seemed to build up a tolerance to 5-HTP when we trialed it, so maybe I gave him too much per dose or it was something else. This was 5 years ago though. So I would not increase the dose of 5-HTP just move back the schedule for it to at least an hour before sundown and probably earlier since I believe the half-life was 4 hours or so. I could be wrong but as I have said it has been 5 years.

    Other than that, focus on the ambient noise and sound at night if you can before trying other stuff.

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  15. Tyler,
    Actually, I did move the 5 HTP back to a much earlier time he still woke up but nor rages. This is a huge improvement. Possibly coincidence but likely not.
    What about apigenin...Not critical?
    Thanks so much Tyler. You have been hugely helpful.
    Nancy

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  16. Apigenin does many things, but the main reason I incorporated it with BCAA's was that it was an inhibitor of indoleamine 3,5-dioxygenase (an enzyme that promotes the use of tryptophan going down the kynurenine pathway rather than the serotonin pathway) as the BCAA therapy was not intended at the time to block amino acid precursors of dopamine and serotonin, but rather to reduce the kynurenine levels in the brain as BCAA's competitively block kynurenine, thereby indirectly reducing the levels of quinolinic acid in the brain as well since quinolinic acid is a downstream metabolite of L-Kynurenine. Quinolinic acid is needed to create NAD+ which is very important (hence the use of Niagen to replace the NAD+ from blocked kynurenine), but in high levels it causes neuroinflammation as it binds to NMDA receptors with such affinity it is considered an excitotoxin.

    This was 5-6 years ago or longer and at the time I knew a lot less about dopamine and why BCAA's may be beneficial in regulating hyperdopaminergic signaling in the brain, so how BCAA's help some people with autism could actually be many different mechanisms of action.

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