Long Term use of Low Dose Clonazepam and More Science on the Excitatory/Inhibitory Imbalance in Schizophrenia and ASD

   

A small number of readers of this blog have followed Professor Catterall’s ideas and trialed low dose clonazepam for autism.  

This post summarizes my findings from using it long-term; it would be a good place to collect the findings of other people.

The science part of this blog is courtesy of a reader who highlighted the full-text version of a paper I mentioned.  Perhaps it was the author?

For information on Catterall’s clonazepam research, go to the “Index by Subject” tab and click on Clonazepam.

Before getting to that, I do get asked how I know, for sure, these therapies really do work for Monty, aged 12, with classic autism.  As I told Ben-Ari, the Bumetanide researcher, the best way to convince the doubting public will be to measure IQ, not autism.  If you can add 30 to 50 points to your IQ result, even the sceptics would pay attention.

I am not measuring IQ directly, but I do note things like spelling tests, math tests and handwriting.  The first pleasant surprise was actually reaching the point of sitting the same tests as the NT kids. Piano playing is another interesting proxy.

Monty’s one to one Assistant (and pal) from age 3 to 9 came to visit the other day and could not believe what his handwriting now looks like.  She had spent hundreds of hours with him practicing fine motor skills, like pencil control.  The end result was handwriting, but even then not like that of his peers. 

Cursive handwriting is now great.  Spelling tests and “quick-fire” math tests are also great.

As we now know, 20% of people diagnosed very young with quite severe autism seem to make wonderful progress.  This has happened by 5 or 6 years old, while the brain is still highly plastic.  Spontaneous accelerated development thereafter rarely seems to happen.  Monty started his Polypill therapy at the age of 9 years, in December 2012.

This is a spelling test from school, given to NT (neurotypical) ten year olds and 12 year old Monty (on paper without lines).  It is not rocket science and big brother could probably have got 20/20 in this test when he was eight years old.  But when Monty was eight years old, he was trying to break the windows of my car with his head and his handwriting did not look like this.

I have all the proof I need that modulating the excitatory/inhibitory imbalance in Monty’s autism is well worth the effort.  The effects are reversible if you stop the therapy, as should be the case.

Clonazepam

Here I am repurposing an existing drug for a different use, at a dosage so low it is highly unlikely to cause side effects.  This is mirroring the use of the same drug, at similar low doses, in mouse models of autism by Professor Catterall.

Clonazepam at “high” doses is widely used already in people with autism, to treat seizures and extreme anxiety.  

Catterall showed that the drug has a totally different effect at very low doses (less than 10% of normal), via a specific mechanism which he has identified, the positive modulation of the α _2,3  subunits of GABA_A receptors. 

GABA_A receptors are made up of five sub-units, the strict composition does indeed vary over time, just to make things even more complicated.  The most common GABA_A receptors have two αs, two βs, and one γ (α₂β₂γ). For each subunit, many subtypes exist (α_1–6, β_1–3, and γ_1–3). It is these subtypes of the subunits that Catterall showed to be key.  Clonazepam was one of the substances that he showed to be effective (in mice).

At “high” doses Clonazepam does have side effects, people build up tolerance to it and so take ever higher doses, and then they get hooked on it.

At very low doses the reverse seems to occur.  Over time you become more sensitive to it and need lower and lower doses.  This was a surprise to me.

The other surprise was that slightly above the effective “low dose” you get some anxiety and irritability.  When I first wrote about this I did wonder if this was just a coincidence, but it is not.

My chart from back then:-

Another interesting point was that some other readers found the effective dose was even less than mine.

When you read about the use of Clonazepam at regular, much higher doses, it is clear that there are wide variations in people’s sensitivity to this drug.  So much so that there is standard lab test to measure blood concentration of this drug, so that the clinician can vary the dose to achieve the desired level in blood.

Clonazepam Level (Blood) Test

It is not an expensive test and I did wonder if this could be used by clinicians to find the effective low dose in their patients with autism.

It did sound a clever idea, but then I read that even the same blood concentration of clonazepam (at high doses) can have markedly different effects in different people.  Still it is better than doing nothing and would reduce some of the guesswork with dosage.

The effective dose

In my n=1 example, the effective dose started out at 40mcg a day.  The half-life is very long and so you need three days to reach a stable level.

Other people contacted me to say that in their case 25mcg a day was effective and in one case, dosage once every two days was optimal.

In my case 40mcg, now gives the negative effects I has originally discovered at higher doses.

Currently the effective dose is 20 to 25 mcg.

This is a tiny dose, technically sub-clinical, but it really is better than giving none.  I have discontinued on several occasions.  There is cognitive loss, which is then regained when re-starting. 

The incremental cognitive effect is not as great in magnitude as I found with Bumetanide, but in people not using Bumetanide, the effect seems to be much greater.  Put more simply, Clonazepam plus Bumetanide is more beneficial than Bumetanide alone, at least in my case.

At this dose the annual cost of the therapy is one dollar/euro/pound. So it will not break the bank.

Tablets are available as 0.5mg  (giving 20 days of use) and 2mg (giving 80 days of use).  A bottle of 2mg tablets will last someone a few years.

I wish they made 0.025 mg (25 mcg) tablets.

I see no reason why, in ten to twenty years’ time, low dose clonazepam will not be a mainstream therapy for some autism; the only problem is the variability of the effective dosage.

Science

For those diehards who have made it this far, now I move from the Peter-reviewed science to the Peer-reviewed science, but from yet another Peter, Peter Penzes from Northwestern University, close by the Windy City.

Common Mechanisms of Excitatory and Inhibitory Imbalance in Schizophrenia and Autism Spectrum Disorders

Abstract: Autism Spectrum Disorders (ASD) and Schizophrenia (SCZ) are cognitive disorders with complex genetic architectures but overlapping behavioral phenotypes, which suggests common pathway perturbations. Multiple lines of evidence implicate imbalances in excitatory and inhibitory activity (E/I imbalance) as a shared pathophysiological mechanism.

Thus, understanding the molecular underpinnings of E/I imbalance may provide essential insight into the etiology of these disorders and may uncover novel targets for future drug discovery. Here, we review key genetic, physiological, neuropathological, functional, and pathway studies that suggest alterations to excitatory/inhibitory circuits are keys to ASD and SCZ pathogenesis.

This study really shows how the common genetic dysfunctions in both schizophrenia and autism come together to produce the Excitatory/Inhibitory (E/I) imbalance.  Numerous different dysfunctions result in the same imbalance, some relate to GABA and some to NMDAR, but the end result is the same.

It is a really good paper, mentioning many of the genes we have encountered in this blog, plus many of the pathways like mTOR and even PAK inhibitors.

The study does not cover any therapeutic methods to correct the E/I imbalance, but this blog has those in spades.  They relate to modulating GABA_A, GABA_B and NMDA receptors.

Low dose clonazepam is modulating GABA_{A ,} as does Bumetanide and as should Acetazolamide (Diamox).  More of that in 2016.

It’s not Autism, it’s Sotos Syndrome – and more about GABA therapies

 Peter the wild boy, with Pitt Hopkins Syndrome

I recently returned from a 25 year class reunion; of the 200 or so class members about 120 turned up. Of the 200 we know that at least 5 have a son with autism and at least one has a nephew with autism.  So I had my first ever “autism lunch” discussing all those tricky issues we are left to deal with.

What was immediately apparent was how different each child’s “autism” was and that none of them were the autism-lite variants that are now being so widely diagnosed in older children. or even adults .  Of the six, two are non-verbal, one is institutionalized, yet one talks a lot.  Three sets of parents are big ABA fans and one child did not respond to ABA.

You may be wondering about that high incidence of autism.  This was not a gathering of science boffins or mathematicians; this was at a business school.  One thing is obvious, you can correlate some autism incidence with educational level.  You can connect all sorts of measures of IQ to autism, from having a math prodigy in the family, to having professors at Ivy league type Universities, particularly in Mathematics.  It does appear to be true that the so-called clever genes are also associated with some types of autism.

I presume that if my science-only university organized such events the incidence of autism would be even higher.

On the way back home we met an acquaintance at the airport, who was telling us all about his son with Sotos Syndrome.  "It is not autism", we were informed, but then I am not quite sure what is.  When you look it up, many of the symptoms look just like autism.  In fact, it is a single gene dysfunction that leads to gigantism and various elements of autism.

This brings me to the painting above of Peter the Wild Boy; it is not me I should point out.  The above Peter was a German boy who came to live in England in the 18^th Century; he was non-verbal and is now thought to have had Pitt Hopkins Syndrome.  Like Sotos, this is another very rare single gene disorder.

We have already come across Rett Syndrome, which for some reason is treated as autism.

Fragile X is thought of as a syndrome where autism can be comorbid.

Timothy Syndrome is fortunately extremely rare, but I have already drawn on it in my own research into autism.

There are also autism related disorders involving multiple genes.

Prader–Willi syndrome  is a rare genetic disorder in which seven genes (or some subset thereof) on chromosome 15 (q 11–13) are deleted or unexpressed (chromosome 15q partial deletion) on the paternal chromosome.  If the maternally derived genetic material from the same region is affected instead, the sister Angelman Syndrome is the result.

The most frequent disorder caused by known multiple gene overexpression is Down Syndrome.  We saw in earlier post that DS is caused by the presence of all or part of a third copy of chromosome 21.  This results in over-expression of some 300 genes.

Why So Many Syndromes

Even before the days of genetic testing, these syndromes had been identified.  How could that be?  Each syndrome is marked by clear physical differences.

These physical differences where used to identify those affected.

Within autism too, sometimes there are physical differences.  Big heads, small heads, slim stature or heavy stature, advanced bone age or retarded bone age.

So many syndromes , but no therapies

Many of the rare syndromes have their own foundations funding research, mainly on the basis that if there is a known genetic dysfunction there should be matching therapy somewhere.

As of today, there are no approved therapies for any of these syndromes.

The Futility of Genetic Research?

A great deal of autism research funding goes into looking for target genes.  The idea goes that once you know which gene is the problem you can work out how to correct it.  There are numerous scientific journal dedicated to this approach.

Since no progress has been made in treating known genetic conditions leading to “autism”, is all this research effort well directed?  Some clever researchers think it is not.

All I can do is make my observations from the side lines.

What do Down Syndrome, Autism and Pitt Hopkins Syndrome all have in common?

In at least some of those affected, they have the identical excitatory-inhibitory imbalance of GABA, that can be corrected by Bumetanide.

If you did whole exome genetic testing on the responders with these three conditions you would not find a common genetic dysfunction; and yet they respond to the same therapy.

I am actually all for continued genetic research, but those involved have got to understand its limitations, as well as its potential.

More on GABA

This post returns to the theme of the dysfunctional GABA neurotransmitter because the research indicates it is present in numerous of the above-mentioned conditions. 

Table 1.  Summary of GABAA signaling alterations in neurodevelopmental disorders (click for the full Table)

·        Autism

·        Fragile X

·        Rett Syndrome

·        Down Syndrome

·        Neurofibromatosis type 1

·        Tourette syndrome

·        Schizophrenia

·        Tuberous sclerosis complex (TSC)

·        Prader-Willi syndrome

·        Angelman Syndrome

Based on feedback to me, we should add Pitt Hopkins Syndrome to the above list.

The GABA dysfunction is not the same in all the above conditions, but at least in some people, Bumetanide is effective in cases of autism, Down Syndrome and Pitt Hopkins Syndrome.  I suspect that since it works in mice with Fragile-X , it will work in at least some humans.

GABA_A has already been covered in some depth in this blog, but I am always on the lookout for more on this subject, since interventions are highly effective.  It is complicated, but for those of you using Bumetanide, Low Dose Clonazepam, Oxytocin and some even Diamox, the paper below will be of interest.

Modulation of GABAergic transmission indevelopment and neurodevelopmental disorders: investigating physiology andpathology to gain therapeutic perspectives

Regular readers will know that in autism high levels of chloride Cl⁻ inside the neuron have been shown to make GABA excitatory rather than inhibitory.  This leads to neurons firing too frequently;  this results in effects ranging from anxiety to seizures and with reduced cognitive functioning.  Therapies revolve around reducing chloride levels, this can be done by restricting the flow in ,or by increasing the flow out.  The Na⁺/K⁺/Cl⁻ cotransporter NKCC1  imports Cl⁻ into the neuron.  By blocking this transporter using Bumetanide you can achieve lower Cl⁻ within the neuron, but with this drug you also affect NKCC2, an isoform present in the kidney, which is why Bumetanide is a diuretic.  Some experimental drugs are being tested that block NKCC1 without affecting NKCC2 and better cross the blood brain barrier. 

The interesting new approach is to restore Cl⁻ balance by increasing KCC2 expression at the plasma membrane.  This means increasing the number of transporters that carry  Cl⁻  out of the neurons.

Chloride extrusion enhancers as novel therapeutics for neurological diseases

In the Modulation of GABAergic transmission paper there is no mention of acetazolamide (Diamox) which I suggested in my posts could also reduce Cl⁻, but via the AE3 exchanger.  This would explain why Diamox can reduce seizures in some people.

The paper does mention oxytocin and it does occur to me that babies born via Cesarean/Caesarean section will completely miss this surge of the oxytocin hormone.  This oxytocin surge is suggested to be key to the GABA switch, which should occur soon after birth when GABA switches from excitatory to inhibitory.  In much autism this switch never takes place.

That would suggest that perhaps all babies born via Caesarean section should perhaps receive an artificial dose of oxytocin at birth.  This might then reduce the incidence of GABA dysfunctions in later life, which would include autism and some epilepsy.

Indeed, children born by Caesarean section (CS) are 20% more likely to develop autism.

Association Between Obstetric Mode of Delivery and Autism Spectrum Disorder - :  A Population-Based Sibling Design Study

Conclusions and Relevance  This study confirms previous findings that children born by CS are approximately 20% more likely to be diagnosed as having ASD. However, the association did not persist when using sibling controls, implying that this association is due to familial confounding by genetic and/or environmental factors.

So as not to repeat the vaccine/autism scare, the researchers do not say that Caesarean section leads to more autism, rather that the kinds of people who are born by Caesarean section already had an elevated risk of autism.  This is based on analysing sibling pairs, but I do not entirely buy into that argument.  They do not want to scare people from having a procedure that can be life-saving for mother and baby.

If you look at it rationally, you can see that the oxytocin surge at birth is there for an evolutionary reason.  It is very easy to recreate it with synthetic oxytocin.

Another interesting point is in the conflict of interest statement:-

Laura Cancedda is on the Provisional Application: US 61/919,195, 2013. Modulators of Intracellular Chloride Concentration For Treating An Intellectual Disability

Regular readers will note that in this blog we have known for some time that modifying GABA_A leads to improved cognitive function.  I even suggested to Ben-Ari that IQ should be measured in their autism trials for Bumetanide.  IQ is much less subjective than measures of autism.

Conclusion

My conclusion is that while genetic testing has its place, it is more productive to look at identifying and treating the downstream dysfunctions that are shared by many individual genetic dysfunctions.

By focusing on individual genes there is a big risk of just giving up, so if you have Pitt Hopkins Syndrome, like Peter the Wild Boy, it is a single gene cause of “autism” and there is no known therapy.  Well it seems that it shares downstream consequences with many other types of autism, so it is treatable after all.

I also think more people need to consider that cognitive dysfunction (Intellectual Disability/MR) may indeed be treatable, and not just via GABA; so good luck to Laura Cancedda.

Low Dose Clonazepam for Autism - SFARI Webinar with William Catterall

Click this link  http://sfari.org/sfari-community/community-blog/webinar-series/2015/webinar-william-catterall-examines-anxiety-drugs-for-autism/

This post will be mainly of interest to the small number of people using low-dose clonazepam for autism and those considering doing so.

This therapy modifies the excitatory/inhibitory (im)balance between the GABA and Glutamate neurotransmitters.  The big advantage is that it should be very safe, is extremely inexpensive and, unlike Bumetanide, does not cause diuresis.   The disadvantage is that the effective dose is only in a narrow window, and you have to find it by trial and error.

Does it work?

It certainly does work in some children with autism.

It also appears to have an additional effect over Bumetanide alone, at least my son.

Questions remain:

·        Does it work with everyone who responds to bumetanide?

·        Does it only work in people with a Nav1.1 dysfunction?

·        Will bumetanide work in everyone who responds to Clonazepam?

One of my earlier, detailed, posts on this subject is this one:-

Channelopathies in Autism - Treating Nav1.1 with Clonazepam

Just google “clonazepam epiphany” or use the site index, for the other posts.

Professor Catterall

I have already covered the science behind low-dose Clonazepam and Professor Catterall’s trials in two mouse models.  It is quite a complex subject and in the end most people just want to know does it work in humans with autism or not.

Catterall’s research was funded by the Simons Foundation, so no surprise really that he made a Webinar for SFARI.  It covers the ground of those two papers and indicates the next steps for his research.

It is a bit lighter going than his papers, but it is a full hour of science.

Catterall plans to trial it in humans with autism, starting with those known to have sodium channel dysfunction. So he is following the same pattern he used with his mice.

The first mouse model he used was Dravet syndrome, a rare condition leading to epilepsy and autism which is caused by a sodium ion channel (Nav1.1) dysfunction.  The second experiment used a standard mouse model of autism called the BTBR mouse model, so no connection with sodium channels.

My question to Catterall was whether this therapy would only work in people with a Nav1.1 dysfunction.  He did respond via the comments on the post, but did not really answer the question.  The fact that he plans to trial his idea on humans with autism with a known sodium ion channel dysfunction, does suggest something at least.

I think that since the actual mechanism of the drug is on a sub-unit of the GABA_A receptor, sodium channels may actually be more of a coincidence, meaning that while autism Nav1.1 dysfunction may indeed indicate this therapy, it may be applicable in other autism where GABA is dysfunctional.

Bumetanide Use

The downside of bumetanide use to correct the E/I imbalance often found in autism is the diuresis and excessive loss of potassium in about 20% of people.

If you revisit the original paper suggesting an  E/I imbalance might be fundamental to many kinds of autism, you will see that this E/I imbalance is not just an ongoing issue, it is potentially an avoidable cause of disruption at key points in the brain’s development prior to maturation. In simpler terms, an E/I imbalance during development may cause the physical brain abnormalities often observed in autism.

That would suggest you should try and reverse E/I imbalance as soon as possible, well before maturation of the brain. 

One day an analog of bumetanide may be developed, that avoids the diuresis;  it is already being discussed.

Bumetanide (or low dose Clonazepam) use, even before autism has become established ?

In something like 30% of cases of classic autism there is macrocephaly (a big head), which even shows up on ultrasound scans of the pregnant mother.  A big head does not necessarily mean autism, but specific types of autism are clearly associated with big heads.

There are many other well known risk factors, like siblings with autism, siblings with other disabilities, older parents, family history (schizophrenia, bipolar, auto-immune conditions, COPD, Nobel Laureates, math prodigies) etc.

Since we also know that an indicator of this kind of E/I imbalance is that benzodiazepine drugs can show paradoxical effects (they work in reverse), it should be possible to make some kind of predictive diagnostic test.

So it would not be rocket science to identify many babies at elevated risk of autism and then treatment could be started very early and well before brain maturation.

This is rather like the Japanese researchers in the previous post suggesting that sulforaphane consumption in childhood might prevent susceptible people developing schizophrenia in adulthood.

Diamox & Bumetanide, Ion Channels Nav1.4 and Cav1.1, HypoPP, Autism and Seizures

So you’re going to spend some time at high altitude, climbing mountains or trekking, don’t forgetto pack your Diamox before you leave home, it can save your life.  She is right.

Today’s post links together subjects that have been covered previously.

It does suggest that there are multiple therapies that may be effective in the large sub-group of autism that is characterized by the neurotransmitter GABA being excitatory (E) rather than inhibitory (I).  The science was covered in the earlier very complicated post:-

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

The growing list of potential therapies is:-

·        Bumetanide (awaiting funding for Stage 3 clinical trials in humans)

·        Micro-dose Clonazepam (trials in mouse models of autism)

·        Diamox (off-label use in autism)

·        Potassium Bromide  - to be covered in a later post (in use for 150 years)

Not surprisingly, all of these drugs also have an effect on certain types of seizure.

The optimal therapy in people with this E/I imbalance will likely be a combination of some of the above.

Periodic paralysis

Periodic paralysis (Hypokalemic periodic paralysis or HypoPP) is a rare condition that causes temporary paralysis that can be reversed by taking potassium.  A similar condition is hypokalemic sensory overload, when someone becomes overwhelmed by lights or sounds, but after taking potassium all goes back to normal. Autistic sensory overload, experienced by most people with autism, can also be reduced by potassium.

Though rare, we know that HypoPP is caused by dysfunction in the ion channels Na_v1.4 and/or Ca_v1.1.

For decades one of the treatments for HypoPP has been a diuretic called Diamox/Acetazolamide.

Other treatments include raising potassium levels using supplements or potassium sparing diuretics.

Bumetanide is a diuretic, but rather than raising potassium levels, it does the opposite.  So I always thought it was odd that bumetanide would have a positive effect on HypoPP.  But the research showed a benefit.

Autism and Channelopathies

We know that autism and epilepsy are associated with various ion channel and transporter dysfunctions (channelopathies).  In a recent post I was talking about Cav1.1 to Cav1.4.

Today we are talking about Cav1.1 and Nav1.4.

We know that Nav1.1 is associated with epilepsy and some autism (Dravet syndrome).

Nav1.4 is expressed at high levels in adult skeletal muscle, at low levels in neonatal skeletal muscle, and not at all in brain

Nav1.1 expression increases during the third postnatal week and peaks at the end of the first postnatal month, after which levels decrease by about 50% in the adult.

We saw with calcium channels that a dysfunction in one of Cav1.1 to Cav1.4 can cause a dysfunction in another dysfunction in another one of Cav1.1 to Cav1.4.

We also so that in autism the change in expression of NKCC1 and KCC2 as the brain matures failed to occur and so in effect they remain immature and therefore malfunction.

So it is plausible that sodium channels may also malfunction in a similar way. 

  

Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype

Hypokalemic periodic paralysis (hypoPP) is an autosomal dominant neuromuscular disorder characterized by episodes of flaccid skeletal muscle paralysis accompanied by reduced serum potassium levels. It is caused by mutations in one of two sarcolemmal ion channel genes, CACNA1S and SCN4A1-3 that lead to dysfunction of the dihydropyridine receptor or the alpha sub-unit of the skeletal muscle voltage gated sodium channel Nav1.4. Seventy to eighty percent of cases are caused by mutations of CACNA1S and ten percent by mutations of SCN4A4. 

There are no consensus guidelines for the treatment of hypoPP. Current pharmacological agents commonly used include potassium supplements, potassium sparing diuretics and carbonic anhydrase inhibitors (acetazolamide and dichlorphenamide). Dichlorphenamide is the only therapy for hypoPP to have undergone a randomized double blind placebo controlled cross over trial. This trial showed a significant efficacy of dichlorphenamide in reducing attack frequency but the inclusion criteria were based on clinical diagnosis of hypoPP and not genetic confirmation.

  

Ca_v1.1

Ca_v1.1 also known as the calcium channel, voltage-dependent, L type, alpha 1S subunit, (CACNA1S), is a protein which in humans is encoded by the CACNA1S gene

Na_v1.4

Sodium channel protein type 4 subunit alpha is a protein that in humans is encoded by the SCN4A gene.

The Na_v1.4 voltage-gated sodium channel is encoded by the SCN4A gene. Mutations in the gene are associated with hypokalemic periodic paralysis, hyperkalemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia.

Ranolazine

Ranolazine is an antianginal and anti-ischemic drug that is used in patients with chronic angina. Ranzoline blocks Na+ currents of Nav1.4. Both muscle and neuronal Na+ channels are as sensitive to ranolazine block as their cardiac counterparts. At its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na+ channels. Ranolazine block of open Na+ channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.

Muscle channelopathies:does the predicted channel gating pore offer new treatment insights for hypokalaemic periodic paralysis?

Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis

Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K⁺ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.

Mode of action

The research does state that nobody knows why Diamox is effective in many cases of hypoPP.

My reading of the research has already taken me in a different direction.  While researching the GABA_A receptor that is dysfunctional in some autism, it occurred to me that in addition to targeting the NKCC1 receptor with bumetanide, another way of lowering chloride levels within the cells might well exist.

I suggested in an earlier post that Diamox could be used to target the AE3 exchanger.

What Diamox (acetazolamide) does is lower the pH of the blood in the following way.

Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:

hence lowering blood pH, by means of the following reaction that carbonic acid undergoes

In doing so there will be an effect on both AE3 and NDAE, below.  This will change the intracellular concentration of Cl-, and hence give a similar result to bumetanide.

This would also explain the phenomenon cited below that pH affects the excitability of the brain.

Over excitability of the brain is the cause of some of the effects seen as autism and clearly Over excitability of the brain will be the cause of some people’s seizures/epilepsy.

Not surprisingly, then one of the uses of Diamox is to avoid seizures.

Acetazolamide in the treatment of seizures.

  

Anion exchanger 3 (AE3) in autism

Anion exchange protein 3 is a membrane transport protein that in humans is encoded by the SLC4A3 gene. It exchanges chloride for bicarbonate ions.  It increases chloride concentration within the cell.  AE3 is an anion exchanger that is primarily expressed in the brain and heart

Its activity is sensitive to pH. AE3 mutations have been linked to seizures

Characterization of an Epilepsy-associated variant of the AE3 Cl-/HCO3-exchanger

Bicarbonate (HCO₃^-) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base movements in the stomach, pancreas, intestine, kidney, reproductive organs and the central nervous system.

Two types of chloride transporters are required for GABA_A receptor-mediated inhibition in C. elegans

Abstract

Chloride influx through GABA-gated Cl⁻ channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl⁻ gradient established in part by K⁺–Cl⁻ cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na⁺-driven Cl⁻–HCO₃⁻ exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K⁺–Cl⁻ cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl⁻ extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl⁻ gradient such that Cl⁻ flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl⁻ gradient. Our results show that Na⁺-driven Cl⁻–HCO₃⁻ exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

Abstract

During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (E_GABA) for GABA_A miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic E_GABA was around −30 mV. Because of the low HCO₃⁻ permeability of the GABA_A receptor, this value of E_GABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized E_GABA and reduced nerve potentials evoked by local application of a GABA_A agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO₃⁻ and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

 

Conclusion

So the science does confirm that “chloride accumulation following NKCC1 block was still clearly present”.  This means that bumetanide is likely only a partial solution.

We also see that “anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons” and “that chloride accumulation that is dependent on HCO₃⁻”.

This is a subject of some research, but it is still early days.

Bicarbonate homeostasis in excitable tissues: role of AE3 Cl−/HCO3−exchanger and carbonic anhydrase XIV interaction

  

I suggest that Diamox, via its effect on HCO₃⁻, may affect anion exchanger AE3 and further reduce chloride accumulation within cells.  This may have a further cumulative effect on GABA.

As we saw earlier, bumetanide does indeed shift GABA from excitatory to inhibitory in people who neurons remain in an immature state (like those of a typical two week old baby).  To my surprise, the use of micro-dose Clonazepam, as proposed by Professor Catterall, but in addition to Bumetanide, has a further effect on GABA’s excitatory/inhibitory imbalance.

Taken together this would highlight the possible further benefit of Diamox.

Normal blood pH is tightly regulated between 7.35 and 7.45.  I do wonder if perhaps in some people with autism, the pH of their blood is slightly elevated (alkaline), this would contribute to excitability of the brain.

Since Diamox increases the oxygen carrying capacity of the blood, I further wonder if this additional oxygen may also be beneficial in some cases.  Since some people are adamant that hypobaric oxygen therapy has beneficial (although not sustained) effects in autism, surely a better treatment would be Diamox?

Since the body is controlled via so-called feedback loops, perhaps in a small subset of people with autism who respond to extra O₂, they actually have blood pH that is higher than 7.45.  In which case measuring blood pH would be a biomarker of who would respond to hypobaric oxygen therapy.  Not surprisingly then, trials of hypobaric oxygen therapy in autism fail, because most of the trial subjects do not have elevated blood pH.

  

So there are many reasons that Diamox should be trialed in autism.  I did find one (DAN) doctor currently using it, but they do not really explain why.

Biomedical Treatment of the Young Adult with ASD