Up to 40% of children in the “failed” phase 3 bumetanide trial were actually responders, according to AI reanalysis of the data – Treating autism in the real world

 

In some parts of the world even the words “treating autism” can still get you into trouble and some people have to go to quite extreme lengths to get their child’s developmental trajectory back on track.

I did note that in the US big changes have been made to their Interagency Autism Coordinating Committee (IACC) that coordinates all efforts within the Department of Health and Human Services (HHS) concerning autism. Now it includes some readers of this blog. Will this make a difference?

https://iacc.hhs.gov/ 

 

Over in France, the Bumetanide researchers Ben-Ari, Lemonnier and pals published their AI driven reanalysis of the “failed” phase 3 autism trial. They found that using AI they could actually predict who did actually respond; and many did. Nonetheless this large trial of all-jumbled-together kids with an autism diagnosis showed that overall bumetanide was no better than a placebo. Sounds strange to you? This is a common theme in autism trials because they do not narrow down a specific type of autism that they are trying to treat.

Over where I am, I keep getting positive reports of success. Some people are lucky and find that much of what works for my son works for theirs. There is a lot in this blog about other types of autism.

Why autism remains untreatable?

Autism is not simple to treat. Autism has no biological definition and measurement scales are all likely not fit for purpose. What would treatment success even mean?

From the perspective of severe autism with apparent ID (the old “Classic autism”) the biggest issues are to do with the slow rate of acquiring new skills. There are very well established tools to measure the skillset of such kids, such as  ABLLS (Assessment of Basic Language and Learning Skills). There are also non-verbal IQ tests. 

For young kids with classic autism you want them to add these basic skills ASAP, so that they can move on with their lives. In our case Bumetanide was the key to unlock new skill addition.

This is not what the phase 3 bumetanide trial was trying to measure.

Indeed one of the recurring comments from parents and teachers is the child has become more “present.” How do you quantify something like that?

For most children with autism in 2026, they do not have a problem with skill acquisition, they are a bit quirky, nervous, resistant to change, stim a bit, do not make friends. It is a very different condition. These issues are very real and genuinely concern some parents, but they are very different problems.

The modern cookie-cutter, protocol-driven, approach does work for most of medicine. But it will never work on an ill-defined category like autism. It actually becomes ridiculous when you look at all the varied types of autism. Even people with cerebral palsy or Down syndrome can be given an “autism” diagnosis on top, but they are completely different biological conditions.

Where to from here?

What does Ben-Ari do now?

Start again with another phase 3 trial? Paid for by who?  Will Servier come back and fund the second attempt?

In the meantime the clock keeps ticking.

I read Ben-Ari’s initial study and made my n=1 trial in 2012. My trial met its primary endpoint (Peter satisfied) and therapy started.

Academic performance went from complete basket-case to passing his high school public IGCSE exams a decade later.

Now it is 2026 and therapy still continues. No side effects,  heart ultrasound (echocardiogram) all normal.

Crazy world.

40% “disabled” at Stanford

I was surprised to read that almost 40% of undergraduates at Stanford University are claiming disability, to get extra time in exams. It does tell you a lot about the current generation of 20 year olds.

I would give them an E on their final diploma (I passed but needed Extra time). It is perfectly reasonable for a small number of clever students to need extra time, they might have a physical disability with their hands, be deaf, or blind, or dyslexic. It is perfectly reasonable to give some people extra time, but 40%?

It really is not fair on the remaining 60%. Maybe just give everyone an extra hour, those that finish early just leave early. They could get E on their results, for “I work fast and finish Early - hire me!"

What is annoying is the trivialization of the word disability.

40% of Stanford undergrads receive disability accommodations—but it’s become a college-wide phenomenon as Gen Z try to succeed in the current climate

So many people claim a disability like autism that theme parks in the US and Europe have had to roll back their privileged access schemes.

When I visited Charlotte International airport a while back and had to stand in a very long line for the passport control, I was amazed to see a never-ending procession of people appearing in wheelchairs to skip the queue. I have never seen this in Europe, but I suppose it will eventually come.

 

Back to those 40% in the Bumetanide trial.

New Analysis of the Bumetanide Phase 3 Trials: Were Responders Hidden in a “Failed” Study?

Approximately one-quarter to one-third of participants fit validated clinical profiles in which bumetanide showed statistically significant benefit on SRS-2, despite the overall trial being negative. The abstract itself says up to 40%.

Treating autism with Bumetanide: Identification of responders using Q-Finder machine learning algorithm

Bumetanide, a specific NKCC1 co-transporter inhibitor, restores deficient GABAergic inhibition implicated in various brain disorders, including Autism Spectrum Disorders (ASD). In keeping with this mechanism, nine successful phase 2 clinical trials, conducted by seven independent teams using an identical protocol, have shown significant improvements in ASD symptoms among individuals treated with Bumetanide. Despite these promising results, two large phase 3 clinical trials (over 400 children recruited in approximately 50 centers and covering age groups 2–6 and 7–17 years) failed with no significant difference between patients treated by placebo or Bumetanide. This failure may stem from the substantial heterogeneity of ASD symptom profiles across the study population, potentially diluting the overall observed treatment effect. To address this, we reanalyzed the phase 3 data using Q-Finder, a supervised machine learning algorithm, aiming to identify subgroups of patients who responded to the treatment. This analysis was based on clinical parameters collected at the baseline of trial and used the same standard endpoints and success criteria defined in the original phase 3 protocol. It enabled the identification of responder subgroups showing a statistically significant difference between placebo and Bumetanide treatment arms. We report detailed descriptions and statistical evaluations of these subgroups. The discovered responder subgroups, representing up to 40% of participants, were cross validated between the two study populations. These findings suggest that meaningful treatment responses can be uncovered within negative phase 3 trials, highlighting the limitations of a one-size-fits-all approach for heterogeneous conditions such as ASD. Machine learning appears to be a promising tool to support this precision medicine strategy.

The 2026 reanalysis published in Translational Psychiatry revisited the large Phase 3 bumetanide trials that previously failed to meet their primary endpoint.

The original Phase 3 trials included more than 400 children (ages 2–17) and found no significant overall difference between bumetanide and placebo on the primary outcome measure (CARS2).

This new study asked a different question:

Instead of “Did bumetanide work for everyone?”, could it have worked for specific subgroups that were diluted in the overall average?

To explore this, the authors used a supervised machine-learning algorithm (Q-Finder) to identify baseline clinical profiles associated with treatment response.

What They Found

The original overall result remains negative

Across the entire population:

• No significant benefit on the primary endpoint (CARS2).
• No meaningful average effect.

So the trial still officially failed.

Subgroups showing benefit were identified

When the data were stratified by symptom profiles at baseline, several subgroups showed:

• Statistically significant improvement on the SRS-2 (Social Responsiveness Scale)
• Treatment effects of roughly 12–17 points in validated groups
• Coverage of about 25–36% of participants in the largest responder profiles

Importantly, these findings were cross-validated between the younger and older trial cohorts.

A Consistent Feature of Responders

Across validated subgroups, one feature repeatedly appeared:

Mildly abnormal “adaptation to environmental changes” on CARS2

This domain reflects:

• Difficulty with transitions
• Rigidity around routines
• Stress with change

Responders were typically:

• Clearly autistic (often moderate–severe social symptoms)
• With repetitive behaviours
• But not globally or profoundly impaired across all domains

Interestingly, IQ did not emerge as a defining predictor of response.

Primary Endpoint vs Secondary Endpoint

A key nuance:

• No validated responder subgroups were found using the primary endpoint (CARS2).
• Validated subgroups were found using the secondary endpoint (SRS-2).

From a regulatory standpoint, this matters: trials are judged on their primary endpoint.

From a scientific standpoint, it suggests:

SRS-2 may have been more sensitive to the type of change bumetanide produces.

What This Means

This reanalysis does not prove bumetanide works broadly in autism.

It does suggest:

• Autism is highly heterogeneous.
• A one-size-fits-all trial design may dilute effects.
• A biologically or symptom-stratified approach may be necessary.
• Around one-quarter to one-third of participants may represent a responder subtype.

However, these findings are post hoc and exploratory.

To confirm them, a new trial would need to:

• Prospectively enroll only the identified responder phenotype.
• Use appropriate primary endpoints.
• Replicate the treatment effect.

Why This Matters for Autism Research

The study reflects a broader shift toward precision medicine:

• Rather than asking “Does this drug work for autism?”
• The better question may be:

“Which subtype of autism does it work for?”

Machine learning may help identify these subgroups, but prospective validation is essential.

The original Phase 3 trial remains negative at the population level.

This reanalysis suggests that meaningful responses may have been present in specific clinical subgroups — particularly children with:

• Mild adaptation abnormalities
• Repetitive behaviours
• Significant social impairment

Whether this represents a reproducible biological subtype remains to be tested in future trials.

Conclusion

In Rett syndrome a very expensive new drug called Trofinitide was approved, even though reports suggest it is only really effective in about 20% of these girls. I was really surprised.  It costs $300,000 to $900,00 a year depending on the girl’s weight.

It looks very odd that the large bumetanide failed, even though 25-40% were actually responders. By the way, my son’s bumetanide therapy has cost about $80 a year, for the last 13 years.

It does not fill you with great confidence.

I recently saw an article saying that “paracetamol/ acetaminophen does not, after all, increase the incidence of autism.” Well theoretically it should be harmful, by depleting glutathione, which is why it should be taken with NAC. We also know that NAC taken during pregnancy can significantly reduce the risk of miscarriage and this has been studied in a clinical trial.

N-acetyl cysteine for treatment of recurrent unexplained pregnancy loss

A controlled clinical trial studied N-acetylcysteine (NAC) in 168 pregnant women with a history of recurrent unexplained miscarriage. Women received either folic acid alone or folic acid plus NAC at 600 mg per day. In the NAC group, 52% of pregnancies continued beyond 20 weeks, compared with 27% in the control group. The take-home baby rate was 47% in the NAC group, compared with 21% in the control group. This represents more than a doubling of the live birth rate. NAC works by restoring glutathione, the cell’s main antioxidant, protecting placental and fetal tissue from oxidative stress. Oxidative stress is known to impair placental function and contribute to pregnancy loss. NAC was well tolerated, with no significant safety concerns reported. These results suggest that correcting oxidative stress can directly improve pregnancy outcomes in a defined high-risk group. This study illustrates how targeting a specific biological mechanism can dramatically change developmental outcomes.

If a professionally-managed autism trial cannot detect the 25-40% who responded to some extent, do you believe a study that effectively says nobody gets autism from pre-natal acetaminophen. Not even 1%? All you likely need to do is pair it with NAC to make the risk 0%.

For decades doctors refused to believe regressive autism existed. Once people started videoing their toddlers, it became impossible to doubt that some actually had developed speech and then lost it. Parents were not imagining it. It was just an inconvenient truth, and still is.

Advances in personalized medicine to treat Autism/IDD – Rett syndrome as an example. Also, Piperine to upregulate KCC2, but what about its direct effect on GABAa receptors?

 

Source:  https://www.cell.com/neuron/pdf/S0896-6273(21)00466-9.pdf

Today’s post is drawn from a workshop I am invited to present at an autism conference in Abu Dhabi.

I decided to talk about advances in personalized medicine – no surprise there.  Since I have 2 ½ hours, I thought I will need some interesting examples to maintain the audiences interest.  One such topic is going to be Rett syndrome.

I regard Rett syndrome and all the other such syndromes in this blog as “single gene autisms” (monogenic autism).  If you apply the American DSM classification, from 2013 onwards Rett syndrome is no longer part of autism.  Hopefully there are no such purists attending in Abu Dhabi. 

Two gene therapies for Rett syndrome are currently undergoing human trials and one drug therapy has been FDA approved.  This looks very encouraging, so let’s dig a little deeper.

Rett syndrome can present with a wide range of disability ranging from mild to severe. 

Rett syndrome is the second most common cause of severe intellectual disability after Down syndrome.

Other symptoms may include:

•      Loss of speech

•      Loss of purposeful use of hands

•      Loss of mobility or gait disturbances

•      Loss of muscle tone

•      Seizures or Rett “episodes”

•      Scoliosis

•      Breathing issues

•      Sleep disturbances

•      Slowed rate of growth for head, feet and hands

Here are the new therapies: 

TSHA-102: This gene therapy, developed by Taysha Therapeutics, is a gene replacement therapy that aims to deliver a functional copy of the MECP2 gene to brain cells.  It utilizes an AAV-9 virus to carry the miniMECP2 gene product into cells for the body to produce more MeCP2 protein, which is deficient in Rett syndrome. As of February 2024, Taysha completed dosing for the first cohort (low dose) in their REVEAL Phase 1/2 adolescent and adult trial in Canada, with positive interim data on safety. They are also conducting trials in the US for both pediatric and adolescent/adult populations.

NGN-401: This gene therapy, by Neurogene Inc., employs a different approach. It uses an AAV9 vector to deliver a regulated version of the MECP2 gene called EXACT. This technology aims to control the amount of MECP2 protein produced by the gene, mitigating the risk of overproduction. NGN-401 is currently in a Phase 1/2 trial for girls with Rett syndrome aged 4 to 10 years old.

Daybue (trofinetide)

Daybue is the first and only FDA-approved treatment specifically for Rett syndrome in adults and children two years of age and older. It is not a gene therapy, but rather a medication taken orally.

The optimistic AI generated view:

Here's a breakdown of Daybue for Rett syndrome:

• Mechanism: The exact way Daybue works in Rett syndrome isn't fully understood, but it's believed to target neuroinflammation and support synaptic function.
• Dosage: The recommended dose is based on the patient's weight and is taken twice daily, morning and evening, with or without food.
• Administration: Daybue comes as an oral solution and can be taken directly or through a gastrostomy tube if swallowing is difficult.
• Efficacy: Studies have shown that Daybue can improve symptoms of Rett syndrome, including reducing scores on the Rett Syndrome Behavior Questionnaire (RSBQ) and showing improvement on the Clinical Global Impression-Improvement (CGI-I) scale.
• Side Effects: The most common side effects of Daybue are diarrhea and vomiting. Weight loss can also occur in some patients. It's important to consult with a healthcare professional for monitoring and managing any potential side effects.

Daybue is an expensive medication. Here's what we know about the cost:

• List Price: The list price of Daybue is around $21.10 per milliliter.
• Annual Cost: This translates to an estimated average annual cost of around $375,000 for patients.
• Dosage Variability: It's important to note that the dosage of Daybue is based on a patient's weight, so the annual cost can vary depending on the individual.

Insurance and Assistance Programs:

• The high cost of Daybue highlights the importance of insurance coverage. Whether insurance covers Daybue and to what extent will depend on your specific plan.
• The manufacturer, Acadia Pharmaceuticals, offers a copay program called Daybue Acadia Connect. This program may help eligible commercially insured patients pay $0 for their monthly prescription.

What are the parents' groups saying? 

Not as good as you might be expecting for $375,000 a year.

Affordable potential alternatives to Daybue/Trofinetide

Daybue/Trofinetide is the product of decades of research into a growth factor called IGF-1.

It is a complicated subject and as usual the abbreviations can be confusing.

As you will see below there already is an OTC product commercialized by one of the original researchers, Dr Jian Guan.

One Rett syndrome parent, who reads this blog, has trialed cGP and sees a benefit. You rather wonder why the Phelan-McDermid, Pitt Hopkins, Angelman and Prader-Willi parents don’t follow him and splash out 50 USD and make a trial.

 

 

Gene-therapy

Gene therapy is undoubtedly very clever and ultimately will likely be the best therapy.  It still may not be that silver bullet.

To be effective gene therapy needs to be given at a very young age, ideally as a fetal therapy prior to birth. Note that we saw that in the Rett mouse model they gave bumetanide to the pregnant mother just before birth.

Fetal therapy is not a crazy idea and much is already written about it; many pregnancies are terminated because genetic anomalies are detected prior to birth. Down syndrome is the best-known example. Fetal therapy is realistic for some disorders.

Girls with Rett syndrome are often diagnosed first with idiopathic autism and then years later with a more precise diagnosis of Rett syndrome. This is a common experience among readers of this blog.

Classic Rett syndrome 

The average age of diagnosis for this form is around 2.5 years old in the US and 5 years old in the UK.  Why do you think that is?

Research in mouse models has shown that the effect of gene therapy ranges from curative when given extremely young to more limited the later it is given.

Off-target effects

Gene therapy has the potential for off-target effects. This is a significant concern in the field and researchers are actively working on ways to minimize these risks. Here is a breakdown of what off-target effects are and why they matter:

During gene therapy, a modified gene is delivered to target cells with the aim of correcting a genetic defect.

Ideally, the modified gene integrates into the intended location in the genome.

However, there's a chance it might insert itself into unintended locations (off-target sites).

Potential Consequences of Off-Target Effects

Disrupting normal genes at off-target sites could lead to unpredictable and potentially harmful consequences. This could include triggering uncontrolled cell growth, which is a risk factor for cancer.

It can also cause unexpected side effects depending on which genes are accidentally disrupted.

Minimizing Off-Target Effects

Researchers are developing various strategies to improve the accuracy and specificity of gene therapy techniques.

This includes using more precise gene editing tools like CRISPR-Cas9 with optimized guide RNAs to reduce off-target edits.

Additionally, researchers are working on methods to detect and potentially repair any off-target modifications that might occur.

Over-expression of the target gene

Yes, there is a possibility that the replaced gene in gene therapy could overproduce the expressed protein. This can be a potential complication and researchers are working on ways to control the level of protein expression. Here's a breakdown of the concern:

• Gene Dosing: Ideally, gene therapy aims to deliver a functional copy of the gene at the right amount to compensate for the deficiency.
• Overproduction Risks: However, if the delivered gene is too active or multiple copies are inserted, it can lead to overproduction of the protein.

Consequences of Protein Overproduction:

• Overproduction of a protein can disrupt the delicate balance in the cell, potentially leading to cell dysfunction or even cell death.
• In some cases, the protein itself might have harmful effects if present in excessive amounts.

Controlling Protein Expression:

Researchers are developing several strategies to control protein expression in gene therapy:

• Promoter selection: Using promoters that have a weaker switch can help regulate protein production.
• Viral vectors: Engineering viral vectors to control the number of gene copies delivered to cells.
• Inducible systems: Developing gene therapy methods where the expression of the introduced gene can be turned on and off as needed.

The cost of gene therapy

•      Despite the high cost, gene therapy can be a cost-effective treatment for some diseases. This is because it can eliminate the need for lifelong treatment with other medications.

•      Here are some examples of the cost of currently available pediatric gene therapies:

•      Luxturna (gene therapy for Leber congenital amaurosis type 10): $425,000

•      Zolgensma (gene therapy for spinal muscular atrophy type 1): $2.1 million

•      Skysona (gene therapy for adrenoleukodystrophy): $3 million

Piperine to correct KCC2 expression in Rett syndrome?

One key feature of Rett syndrome is impaired cognition.

As regular readers are aware, there are many types of treatable intellectual disability (ID).

One type of treatable ID is caused when the GABA developmental switch fails to occur shortly after birth.  This creates an excitatory/inhibitory imbalance in neurons which impairs cognition and lowers IQ.

The faulty GABA switch is a feature of many types of autism, but far from all of them.

By using pharmaceuticals to lower chloride within neurons, you can compensate for the failure of the GABA switch.

This treatment can be achieved by:

1.     Blocking or down regulating NKCC1

2.     Up regulating KCC2

In the paper below they look at up regulating KCC2

Pharmacological enhancement of KCC2 gene expression exerts therapeutic effects on human Rett syndrome neurons and Mecp2 mutant mice

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.

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.

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

Treatment with piperine (10 μM), an activator of the TRPV1 channel (51), induced a significant rise in KCC2 expression in cultured human neurons 

We already knew this was likely from earlier research from Ben Ari, see below for a reminder.  Is Piperine an interesting option for those restricted to OTC interventions?

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2-/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

Treating the mother prior to delivery with bumetanide was a partially effective therapy in the mouse model of Rett syndrome.

Piperine

Bumetanide is cheap and very possibly effective in human Rett syndrome, but it is a prescription drug.

Piperine is an OTC supplement and a compound found in black pepper. By activating the TRPV1 channel it causes an increase in expression of the KCC2 transporter that allows flow of chloride out of neurons. So piperine should lower chloride inside neurons.  Piperine can cross the blood brain barrier, so when taken orally it should have some effect on intracellular chloride.

Piperine is also a positive allosteric modulator of GABA_A receptors

This means that piperine multiplies the effect of whatever GABA is around. This means that in typical people piperine should have anti-anxiety effects.

Piperine was recently found to interact with a previously unknown  benzodiazepine-independent binding site.

Researchers are currently toying with the piperine molecule to try and separate the effect on TRPV1 from the effects on  GABA_A.  They want to create 2 new drugs.

1.     a selective TRPV1 activator

2.     a selective GABA_A modulator (PAM)

Piperine as an alternative or complement to Bumetanide?

One effect of piperine would be great to have (TRPV1 activator) but the second effect would not be helpful (positive allosteric modulator of GABAA).

The question is what is the net effect. Nobody will be able to answer that without a human trial.

I was advised long ago by one drug developer than it is best to focus on reducing flow into neurons via NKCC1, rather than increase its exit by KCC2, because nobody had yet been successful with KCC2; many have tried.  KCC2 plays a key role in neuropathic pain and that is why it has been researched.

Conclusion

We did see years ago that taking coffee with your bumetanide made sense. Coffee contains compounds that are OAT3 inhibitors and slow down the excretion of bumetanide from the body; coffee increases the effect of bumetanide. You can achieve something very similar by just increasing the dose of bumetanide.

Taking black pepper (piperine) with your bumetanide might be good, or might not be. It certainly would be easy to find out. As with Daybue/Trofinetide, the result is likely to vary from person to person. If GABA function, post- bumetanide, is still a bit excitatory amplifying GABA signaling will make autism worse. If GABA function has been shifted to inhibitory then amplifying GABA signaling will be calming.

Gene therapy will require much earlier diagnosis of single gene autisms.

“Precision medicine” therapies like Daybue/Trofinetide may not be that precise after all and large variations exist in the response, even among children with the same affected gene.

The huge expense means that for most of the world they will see no benefit from gene therapy or indeed “precision medicine.”

The low hanging fruit is to repurpose affordable existing drugs and get the benefit from their secondary effects.  This is what I term personalized medicine.

The research clearly indicates that some girls with Rett syndrome likely will benefit from Bumetanide therapy. For a young child this therapy would cost 50 US dollars/euros a year, if you pay the actual price for generics.

Why are they trialing genetic therapies for Rett instead of first doing the obvious thing and trialing cheap bumetanide? They will likely be able to sell the gene therapy for $2 million a shot.  There is little interest in trialing a $50 a year therapy.

Our new reader from Turkey, MÜCADELECI ANNE DENIZ ( = FIGHTING MOTHER DENIZ), likely does not have $2 million to spend, but seems to be on the way to creating her own personalized medicine therapy for her son. Good luck to her.

As to the cGP Max supplement, it seems to work for some and have no effect in others. Nobody has reported any side effects. It looks worth a try for Rett syndrome.  As a supplement it is not cheap, that is until you see what they charge for Daybue. 

Bumetanide “reverses” MR/ID in Down Syndrome

You probably know what Down Syndrome looks like, but you probably never expected the above life expectancy data.  It used to be the case that kids with this disorder were institutionalized after birth.

Source:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445685/figure/F2/

In an earlier post I suggested that some types of Mental Retardation (MR)/Intellectual Disability (ID) should be treatable.  I was thinking about RASopathies, dendritic spine morphology and the GABA E/I (Excitatory/Inhibitory) imbalance found in autism.  I even suggested to the autism researchers working on the bumetanide approval process that the simplest measure of effectiveness would be to measure IQ before and after treatment.

Recent research has shown that in Down Syndrome GABA is also excitatory.  GABA should be inhibitory, otherwise the brain cannot function properly and there will be a large risk of seizures.  In many people with autism GABA is excitatory; this reduces their cognitive function and leads to almost 80% having unusual epileptiform activity (like “pre-epilepsy”) and about 35% going on to developing seizures.

In the Down Syndrome mouse model the researchers found that Bumetanide improved cognitive function, via the shifting of GABA from excitatory to inhibitory.

Our findings demonstrate that GABA is excitatory in adult DS mice and identify a new therapeutic approach for the potential rescue of cognitive disabilities in individuals with DS.

I thought that was great news.  Perhaps other types of MR/ID are also the result of GABA E/I imbalance, they too would be treatable with Bumetanide.

The question remains, does anyone care enough to bother about these people?  Take a look at the life expectancy chart at the top of this post.  Things have got much better, but there is a long way to go.

Down Syndrome

Most people have heard about Down Syndrome (DS) and I certainly knew more about what DS looked like than what autism looked like.

People with DS look different, are short, and 99% have some degree of MR/ID.  About 10% also have autism and half will develop epilepsy.

Down syndrome is caused by having three copies of the genes on chromosome 21, rather than the usual two.  The extra genetic material present in DS results in overexpression of a portion of the 310 genes located on chromosome 21. This overexpression has been estimated at around 50%.

Some adults with DS lose the ability to use speech when they are about 30 years old.

Until 1970 children with DS used to live for just 10 years, whereas today most survive into their 50s.

About 92% of pregnancies in Europe with a diagnosis of Down Syndrome are terminated. In the United States, termination rates are around 67%.  When non-pregnant people are asked if they would have a termination if their fetus tested positive only 23–33% said yes.

Reversing excitatory GABA_AR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome

Down syndrome (DS) is the most frequent genetic cause of intellectual disability, and altered GABAergic transmission through Cl⁻-permeable GABA_A receptors (GABA_ARs) contributes considerably to learning and memory deficits in DS mouse models. However, the efficacy of GABAergic transmission has never been directly assessed in DS. Here GABA_AR signaling was found to be excitatory rather than inhibitory, and the reversal potential for GABA_AR-driven Cl⁻ currents (E_Cl) was shifted toward more positive potentials in the hippocampi of adult DS mice. Accordingly, hippocampal expression of the cation Cl⁻ cotransporter NKCC1 was increased in both trisomic mice and individuals with DS. Notably, NKCC1 inhibition by the FDA-approved drug bumetanide restored E_Cl, synaptic plasticity and hippocampus-dependent memory in adult DS mice. Our findings demonstrate that GABA is excitatory in adult DS mice and identify a new therapeutic approach for the potential rescue of cognitive disabilities in individuals with DS.

Conclusion

It would appear that excitatory GABA may be more common than anyone thought.  I wonder how many other people with MR/ID might be affected and be potential beneficiaries of this very inexpensive therapy.

I did ask one of my doctor relatives if she has any patients with DS; I said that there may be a treatment for their MR/ID.  She is not going to prescribe her patients anything off-label, so unless Ben-Ari decides he wants to help people with DS, as well as ASD, people will DS will remain untreated.  I will ask him.

Also, given the large amount of money going into the genetics of autism, perhaps it would be worth looking at those 310 genes located on chromosome 21 to see if the overexpression of one may trigger speech loss in people with regressive autism and/or might be present from birth in people with classic autism.  One of them must trigger the speech loss in that sub-group of adults with DS.

Treating autism with a diuretic: a long procedure

Several readers have asked me about the current status of Bumetanide as a treatment for autism. The process in Europe is controlled by EMA (European Medicines Agency), the equivalent of the FDA in the United States.  

Bumetanide affects the function of the GABA_A receptor.  If you click on the site index, you can refer to Bumetanide and read the research and my own son's very positive experience of using this drug since December 2012.     

Most readers in the UK and USA have difficulty getting their doctor to prescribe bumetanide, since autism is currently an off-label use.  Many readers elsewhere have been able to access this drug and are seeing its positive impact.

Dr Ben-Ari, whose research has been outlined in those earlier posts, has kindly provided this update:-

Treating autism with a diuretic: a long procedure 

We have started some time ago testing the possibility of using a diuretic to treat Autism Spectrum Disorders (ASD) relying on our promising experimental observations made in rodent. Indeed we discovered in 2 animal models of ASD (the in utero Valproate model and the Fragile X one) that cortical neurons have elevated intracellular chloride that shifts a major inhibitory mechanism to excitatory leading to perturbations of the behaviorally relevant brain oscillations (Tyzio et al Science 2014 and Eftekhari et al Science 2014). Correcting these elevated levels of chloride with a diuretic that reduces intracellular chloride ameliorated the electrical and behavioral signatures of ASD in these rodents. Relying on these and indirect observations, we had conducted with Dr Lemonnier a pilot trial followed by a phase 2 randomized double blind placebo control study on 54 children aged 3 to 11yrs old. We obtained promising results (Lemonnier et al Trans Psychiat 2012). Thus is followed now by a larger EMA approved trial with 80 children 2 to 18 yrs old that will be terminated next fall.

The procedure is long and complex as this requires many clinical controls and large sums of investments. This is however mandatory as  one cannot propose a treatment unless this has been tested and approved by the clinical authorities. Indeed, there have been many false hopes in the treatment of ASD, and one cannot give false promises without having all the elements needed that confirm that the treatment does improve the situation and has limited or no side effects.

We cannot therefore make any suggestion and promise as to the success or failure of the approach in spite of our compelling animal data and preliminary clinical observations. We follow the requirements and when and if our trials are successful , we shall pursue until we obtain an authorization to market this drug.

Sincerely

Information is available at

www.neurochlore.fr

Tuning GABAa receptors, plus Oxytocin

Today’s post will hopefully not get too complicated.

As has been mentioned in this blog, and also at leading institutions like MIT, it does seem possible to fine-tune certain receptors in the brain that have become dysfunctional in autism.  In the case of MIT they were “tuning” a receptor called mGluR5, which they suggested was either hypo or hyper, in other words too much or too little, depending on what the underlying disease variant was.

This was done with something called an allosteric modulator, either a positive one called PAM, or a negative one called NAM.

They found that a particular glumate receptor, called mGluR5, was dysfunction in many autism-like conditions.  But the nature of the dysfunction varied, so different people would require different treatments to return the receptor performance back to normal (top dead center).   So it really becomes like tuning your car engine. 

As I have progressed in my review of the literature it becomes clear that numerous receptors are “out of tune”; so a better analogy is tuning something like a piano.

  

Ketamine, Memantine, D-Cycloserin, Magnesium, Fenobam and yet more as Glutamatergic Modulators in Autism (and Fragile X)

"Tuning" the shape (but not number) of dendritic spines also appears not to be as fanciful as it sounds.

Back to GABA_A

Regular readers will know that one of the key dysfunctional receptors in autism is called GABA_A.

GABA A Receptors in Autism – How and Why to Modulate Them

This subject is very complicated.  In effect what appears to have happened in autism is that the neurons have not matured as they should, and so GABA_A receptors continue to function in their “normal” immature state.  The concentration of chloride remains high since the NKCC1 transporter continues to exist, whereas KCC2/3 should have developed.  The result is that when the receptor is stimulated, instead of causing an inhibitory/calming effect it causes an excitatory effect.

This is fortunately treatable by inhibiting the flow of chloride into the cells, through NKCC1, using a drug called Bumetanide.

However this is not the end of the story.

At least 11 binding sites on GABA_A receptors

As you can learn from Wikipedia:-

The active site of the GABA_A receptor is the binding site for GABA and several drugs such as muscimol, gaboxadol, and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin, among others.

We are particularly interested in the allosteric binding sites.

The only one that is usually referred to, in any depth, is the site for benzodiazepines, but there are at least 11 different binding sites.

GABAa receptor pharmacology.

Abstract

gamma-Aminobutyric acid (GABA)a receptors for the inhibitory neurotransmitter GABA are likely to be found on most, if not all, neurons in the brain and spinal cord. They appear to be the most complicated of the superfamily of ligand-gated ion channels in terms of the large number of receptor subtypes and also the variety of ligands that interact with specific sites on the receptors. There appear to be at least 11 distinct sites on GABAA receptors for these ligands.

Development of Subtype-Selective GABAA Receptor Compounds for the Treatment of Anxiety, Sleep Disorders and Epilepsy

These sites include:-

·        GABA Binding Site

·        Benzodiazepine Binding Site

·        Neurosteroid Binding Site

·        Convulsant Binding Site

·        Barbiturate Binding Site

·        b Subunit Binding Site(s)

In an earlier post I highlighted the discovery by Professor Catterall, that tiny doses of a particular Benzodiazepine drug called Clonazepam had a strange effect on the GABA_A receptor.

Clonazepam is a known Positive Allosteric Modulator (PAM) of the GABA_A site.  In mature neurons it amplifies the calming effect when the GABA binding site is stimulated.  In mouse models of autism (we assume therefore immature neurons)   where GABA is still excitatory, the tiny dose seemed to switch it to inhibitory.

This suggests a new function, rather than a PAM, the effect was to invert the function entirely.

Now it appears that similar things may indeed also be possible at some of the other 9+ binding sites (I exclude GABA Binding Site itself)

As complicated as this subject may sound, it actually gets even more complicated since the GABA receptors are made up of sub-units.  It appears that mutations in these subunits may be a cause of some epilepsies and, I propose, some “oddities” in autism.

Recent studies have again shown that many genetic dysfunctions found in autism relate to GABA, this short article is not so recent, but gives a nice summary:-

GABA is the major inhibitory neurotransmitter in the brain. It essentially acts as a brake for brain activation. Several aspects of GABA regulation have been linked to ASD, from early brain development to adult brain function.

Variations in GABA receptor subunits have been strongly associated with ASD. GABA receptors come in two major forms: fast, “ionotropic” GABAA receptors let negatively charged chloride ions flow into the neuron, and slow, “metabotropic” GABAB receptors produce chemical messages inside the neuron. GABAA receptors, the most common form in the brain, contain five subunits that shape their properties. Genome-wide association studies have linked the GABAA receptor subunit genes GABRA4 (α4 subunit), GABRB1 (β1 subunit), and GABRB3 (β3 subunit) to autism.[1][2] In addition, deletion of a chromosomal region that contains a cluster of a variety of GABA receptor genes (region 15q11-13) causes Angelman Syndrome.[3][4]

Genes controlling the development of GABA-releasing neurons have also been associated with ASD. Autism-linked variations in the ARX and DLX family of transcription factors interfere with proper expression of GABA.[5][6][7] Absence of such GABA-releasing neurons would negatively affect early brain development as well as adult brain stability.

Notably, variations in other ASD-linked genes affect GABA signaling. New evidence shows that the gene MECP2, the mutation of which causes Rett Syndrome, is critical for normal function of GABA-releasing neurons.[8] When MECP2 expression was blocked in GABAergic neurons of mice, GABA expression and release were reduced and the mice exhibited autistic behaviors.

ASD is a complex disorder that is likely to be caused by a combination of mutations in a variety of genes. GABA receptors are a promising therapeutic target because of their important role in monitoring brain excitation. Identification and exploration of autism-linked mutations in other GABA-related genes could shed light on the pathogenesis of autism.

Over to Switzerland

At the University of Bern a small research group is looking  at the world of  GABA_A receptors, here is what they say:-

“Many scientists and companies are put off by the complexity of the field of GABA_A receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.” 

Prof.Dr. Erwin Sigel

Here is one of their recent papers, that shows both what is known and how very much remains unknown.

Structure, Function, and Modulation of GABAA Receptors

Ion Conductance

The GABA_A receptors are generally GABA-gated anion channels selective for Cl⁻ ions, with some permeability for bicarbonate anions (49). Exceptionally, in C. elegans, a cation-selective GABA-gated channel has been discovered (50). Excitatory neurotransmitters increase the cation conductance to depolarize the membrane, whereas inhibitory neurotransmitters increase the anion conductance to tendentially hyperpolarize the membrane. However, if the gradient for Cl⁻ ions decreases due to down-regulation of KCC2 chloride ion transporters, opening of GABA_A receptors may cause an outward flux of these anions, leading to depolarization of the membrane and thereby to excitation. This phenomenon has been implicated in neuropathic pain (51). During early development (52) and in neuronal subcompartments (53), GABA similarly confers excitation. 

Although it is relatively simple to address questions at the level of individual receptor subunit isoforms, we can only speculate how many GABA_A receptors are expressed in our brain and what their subunit composition is, not to mention subunit arrangement.

Conclusions

Many scientists and companies are put off by the complexity of the field of GABA_A receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.

It may be anticipated that genetic alterations of subunits of the GABA_A receptor affect any of the above mentioned processes and thereby contribute to inherited human diseases. A start has been made with the analysis of point mutations that cause epilepsy

Mutations in GABAA receptor subunits associated with genetic epilepsies

Why is all this relevant ?

We have in recent posts discovered that at least two anti-convulsants (carbamazepine and phenytoin) appear to modulate GABAA receptors in unexpected ways when given in tiny doses.

We also found out that valproate also seems to possess such qualities.  The exact mode of action of valproate is not known and perhaps it also acts a modulator of one of the many binding sites on the GABAA receptors.

We do think that valproate is working somehow via GABA.

Modulation of the gamma-aminobutyric acid type A receptor by the antiepileptic drugs carbamazepine and phenytoin

It turns out that Carbamazepine has also been shown to potentiate GABA receptors made up of alpha1, beta2, and gamma2 subunits.

I have already established that the effect of tiny doses of Valproate is not the same as tiny doses of Clonazepam.

The next step would be to look at the effect of tiny doses of carbamazepine, phenytoin and potentially anything else that modulates those mysterious  GABA_Asites.  They are clearly all there for a reason.  It seems that their role goes beyond just the allosteric modulation (amplification/reduction) of GABA’s effect.  It is likely much more subtle and they affect emotional behaviour.

Given the difficulty/impossibility of research on human brains, in the end we may need to revert to the medical world’s often used “scientific” discovery methods known as trial and error, and stumbled upon.

For the moment that will be left to Professors Sigel and Catterall and their mice, and Dr Bird, in Australia, with his human subjects.

Oxytocin and Bumetanide share the same mode of action in autism

Whilst on the subject of GABA_A, I should come back to Oxytocin.

Oxytocin-Mediated GABA Inhibition During Delivery Attenuates Autism Pathogenesis in Rodent Offspring

The conclusion of this Ben-Ari paper from last year is that Oxytocin and Bumetanide share the same effect in autism; they lower the level of chloride within the neurons and help switch GABA back to inhibitory.

It seems that oxytocin from the mother may be the signal to the developing brain to lower Cl levels.  Oxytocin has many other functions in the body.

Small doses of oxytocin/Syntocinon, have been shown to be effective in some people with autism.  One reader from Portugal has written on this blog how effective it has been in his young son.

Oxytocin/Syntocinon is not available everywhere, but is being reintroduced to the US.

Retrophinto reintroduce Syntocinon™ Nasal Spray in the U.S. and Develop the Drug for Schizophrenia and Autism

I am wondering if in some people, who are not responders, bumetanide/oxytocin lowers the level of chloride, but not enough to show any benefit.  People using Bumetanide, which has a short half-life, comment that the effect fades through the day and that splitting the same daily dose 3 times a day is beneficial over 2 times a day.  This might suggest that combining Oxytocin with Bumetanide might give better results, by maintaining the downward pressure on chloride levels and keeping GABA more inhibitory and for longer.

In the longer term, an analog of Bumetanide is needed without the diuretic effect and with a delayed release, to maintain a constant effective level.  This is known to the researchers, but would require a big financial investment.

Larger doses of oxytocin are likely to produce effects elsewhere in the body.

If anyone tries the combination of Bumetanide + oxytocin, let me know.