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Saturday, 29 March 2014

Channelopathies in Autism - Treating Nav1.1 with Clonazepan


In this post we look at another existing drug that research shows may be effective in treating core symptoms of autism.  The drug, Clonazepam, is inexpensive and is already used in larger doses to treat anxiety in autism.
 
You make have seen this Venn diagram before, it is one of those graphics I like to produce to make things easier to understand, both for you and for me.
In our quest to treat autism we first need to understand the disease as much as possible.  By far the most complex of the four main areas is the dysfunction of the ion channels and transporters in the brain, the so-called channelopathies.  Ion channels were only discovered relatively recently and science's understanding of them is still evolving.

Here is very useful layperson’s summary:- 
Autism-Linked Variations in Ion Channel Genes Increase Brain Excitability
 
"Neuronal communication guides virtually all aspects of brain development. To better understand Autism Spectrum Disorders (ASD), scientists are searching for autism-linked genes that regulate neuronal activity. Some of these genes encode ion channels, whose activation determines whether a neuron will fire a signal. Variations in ion channels influence neuronal survival, differentiation, migration, outgrowth, and synapse formation.
Ion channels are critical for shaping neuronal excitability. Neurons encode information using electrical signals derived from ion channels. At rest, each neuron has a negative charge. When a neuron receives signals from other neurons via synapses, ion channels open and the neuronal charge becomes either more positive or negative, depending on the type of ion. Once the charge of a neuron rises to a certain threshold, the neuron “fires” a signal to other neurons in a process called emitting an “action potential.”
Think of this process like the boiling of a teapot. The bottom of the teapot receives heat from the burners of the stove, much like how dendrites of a neuron receive synaptic signals. This heat boils the water in the teapot, converting it into steam, just as neurons convert synaptic signals into electrical charges. As the pressure builds, steam escapes through the spout, letting off a loud whistle. Likewise, once a neuron builds up enough positive charge, it sends a fast action potential down its axon to the next neuron.
Positive ion channels boost neuronal excitability by creating a more positive charge. However, the balance of neuronal excitability is crucial. Too much excitation leads to seizures and epilepsy, whereas too little prevents circuits from firing. Individuals with autism frequently also have epilepsy, suggesting that their brains are overexcited.
ASD-linked mutations in genes for calcium (Ca2+), sodium (Na+), and potassium (K+) ion channels enhance brain excitability, although the exact mechanisms are not well understood. Known ASD-associated mutations occur in the genes CACNA1C, CACNA1F, CACNA1G, and CACNA1H, which encode the L-type calcium channels Cav1.2 and Cav1.4 and the T-type calcium channels Cav3.1 and Cav3.2, respectively; the sodium channel genes SCN1A and SCN2A, which encode the channels Nav1.1 and Nav1.2, respectively; and the potassium channel genes KCNMA1 and KCNJ10, which encode the channels BKCa and Kir4.1, respectively.
Variations in ion channel genes are likely to affect a myriad of brain functions. Ion channels may even provide a link between genetics and the environment because environmental factors like mercury increase calcium signaling. The broad role of ion channels may help explain why ASD is so often accompanied by other neurological complications like sleep problems and epilepsy."
Catherine Croft Swanwick, Ph.D.

In this blog I have so far covered a potassium channelopathy and a chloride channelopathy.  From my own research, I already know there are more.
In today’s post we will look at some very extensive research by  Dr Catterall, who seems to be the world’s expert on a specific sodium ion channel called NaV1.1.  Catterall has shown how it is implicated in two models of autism and it can be effectively treated/reversed using existing drugs.

Dravet’s syndrome
Dravet’s syndrome is a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome are poorly understood.  It is known that a dysfunction of the gene, SCN1A,  that encodes encoding voltage-gated sodium channel NaV1.1 causes Dravet’s syndrome. 

Experiment Number One
In the first paper, Catterall used mice with a deficiency of the SCN1A gene to become his Dravets/autistic test examples.
The mice exhibited hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive.  In effect he made autistic mice.
He goes on to explain that the behavioral deficit is mediated via impairments in GABAergic neurotransmission.  He tell us that treatment with low-dose clonazepam, a positive allosteric modulator of GABAA receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model.  

Autistic-like behaviour in Scn1a+/−mice and rescue by enhanced GABA-mediated neurotransmission

"Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABAA receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravet’s syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for NaV1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome."

Experiment number two
In a recent experiment, Catterall used a standard mouse model of autism called the BTBR mouse.  This is essential a specially bred mouse that exhibits very many traits of autism.  Nobody has purposefully interfered with its SCN1A genes or NaV1.1 ion channels.

 
The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit specific—the α2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α1-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α2,3-subunit-selective positive GABAA receptor modulation may be an effective treatment

In this study, Catterall repeated his use of low-dose clonazepam to try to “cure” the autistic mouse.  He not only was able to reduce the autistic deficits, but he was able to make cognitive improvements.  In effect he made the mice less autistic and smarter.
The following excepts from his paper are quite technical and you may wish to skip past them.

Increased GABAergic Inhibitory Neurotransmission in Response to Benzodiazepines
 
"Attempts to reverse autistic-like traits by rebalancing the ratio of excitatory to inhibitory neurotransmission through pharmacological treatments that reduce excitatory neurotransmission have met with only partial success because of their limited efficacy and unwanted side effects in control groups. 

The increased GABAergic signaling after treatment with clonazepam led to a decrease in frequency of spontaneous EPSCs (Figures 1G and 1H), without change in amplitude in BTBR hippocampal slices (Figure S1D). Interestingly, the frequency of spontaneous EPSC was also decreased by clonazepam (Figure S1K), without change in amplitude (Figure S1L) in C57BL/6J slices. 

These data support the idea that low-dose clonazepam can reverse the underlying deficit in spontaneous GABAergic inhibitory neurotransmission in BTBR mice."
 

Improvement of Social Interaction by Treatment with Clonazepam



 
"To test the behavioral effects of enhancing inhibitory neurotransmission in BTBR mice, we injected low nonsedating/nonanxiolytic doses of clonazepam intraperitoneally 30 min prior to behavioral tests. In the three-chamber social interaction test, acute clonazepam treatment had no effect on social  interactions of C57BL/6J mice (Figures 2A and S2A) but increased social interactions in BTBR, with a maximal effect at 0.05 mg/kg (Figures 2B and S2B) and no sedation (Figure S2H). Measurements of the time of interaction of the test mouse with a stranger mouse versus a novel object during three-chamber tests showed that the C57BL/6J mice are unaffected by any of the test doses (Figure 2C), whereas improvement of the social deficit in BTBR mice by clonazepam is strikingly dose dependent (Figure 2D). Interestingly, the improved social interactions in BTBR mice were lost at higher doses of clonazepam (Figures 2B and 2D). Other behaviors in BTBR mice were also rescued by low-dose clonazepam. In the open-field test, a single injection of 0.05 mg/kg clonazepam significantly reduced hyperactivity, measured as the total distance moved (Figure 2E), and stereotyped circling behavior, measured as the number of 360_ rotations (Figure 2F).

In contrast, these behaviors in C57BL/6J mice were unaffected by low-dose clonazepam. These low doses of clonazepam had little effect on anxiety-like behaviors of C57BL/6J mice, such as avoidance of the center of an open field or the open arms of an elevated plus maze (Figures 2G and 2H). However, compared to C57BL/6J, BTBR mice visited the center in the open field significantly more frequently and spent more time in open arms during the elevated plus-maze test under control conditions, as if they were less anxious than C57BL/6J mice, and these indicators of abnormally low anxiety in BTBR mice were changed toward the values for C57BL/6J mice after treatment with 0.05 mg/kg clonazepam (Figures 2G and 2H) without sedation (Figure S2I).

 
Amelioration of Cognitive Deficits by Treatment with Clonazepam

 

 
 
"Cognitive problems are often associated with ASD and BTBR mice are known to have impaired fear memory. To test the effects of low dose clonazepam on cognitive deficits, we performed context dependent fear conditioning after treatment with increasing doses of clonazepam in both BTBR and C57BL/6J mice (Figures 3A and 3B). Short-term (30 min) and long-term (24 hr) memory performance in fear conditioning to the spatial context in BTBR mice were improved by treatment with 0.05 mg/kg clonazepam, but no significant effects were observed after treatment with 0.0125 mg/kg or 0.1 mg/kg clonazepam (Figures 3B and S3B). In contrast, no cognitive changes were observed in C57BL/6J mice at any dose (Figures 3A and S3A)."
 

Rescue by a2,a3-Specific Positive Allosteric Modulators of GABAA Receptors
 
"Diversity of GABA receptor function is conferred by more than 20 different subunits, and receptors with different a subunits play distinct roles in the physiological and pharmacological actions of GABA and benzodiazepines."

"These results indicate that different subtypes of GABAA receptors may have opposite roles in social behavior, with activation of GABAA receptors containing a2,3 subunits favoring and activation of GABAA receptors with a1 subunits reducing social interaction, respectively."

"Altogether, these experiments show that treatment with an a2,3-selective positive allosteric modulator of GABAA receptors is sufficient to rescue autistic-like behaviors and cognitive deficit in both a monogenic model of autism-spectrum disorder and the BTBR mouse model of idiopathic autism." 

"Subunit-selective GABAA receptor modulators may also have an important effect on cognitive behaviors."

"The bell-shaped dose-response curves observed for both L-838,417 and clonazepam may explain why high-dose benzodiazepine treatment for prevention of anxiety and seizures has not been reported to improve autistic traits in ASD patients."
 

Discussion
"Our results on mouse models of autism support the hypothesis that social and cognitive deficits in ASDs may be caused by an increased ratio of excitatory to inhibitory synaptic transmission." 

"Therapeutic approaches to treat autistic traits in animal studies or in clinical trials have primarily focused on reducing excitatory neurotransmission in glutamatergic synapses to rebalance E/I ratio in autistic brain.  However, autistic-like behaviors in ASD mouse models are only partially reversed by drugs that inhibit excitatory neurotransmission, and these drugs also have unwanted side effects on wild type mice. To overcome these drawbacks, we focused on the opposing side, the GABAergic inhibitory transmission in autistic brain. Our results highlight the potential for therapy of autistic like behaviors and cognitive deficit in ASD by low-dose treatment with subunit-selective benzodiazepines and other positive allosteric modulators of GABAA receptors. At low doses that do not induce sedative or anxiolytic effects, we found that clonazepam, clobazam, and L-838,417 all improved autistic-like behaviors and cognitive deficit in BTBR mice, supporting the hypothesis that a2,3-subunit-selective up regulation of GABAergic neurotransmission could be an effective treatment for these core features of autism."

"Consistent with this view, Astra-Zeneca and the National Institutes of Health have initiated clinical trials of the a2,3-selective positive allosteric modulator of GABAA receptors, AZD7325, for efficacy in autism."
 
Experiment number three
Experiment number three is of course to test Dr Catterall’s idea about Clonazepam on humans.  This has not yet been done, although a trial is planned with a similar drug AZD7325.
He suggests trialing a low dose of clonazepam, but it is not clear exactly how low. There is mention of 10% of the normal dose. In large doses, clonazepam is already prescribed in autism to reduce anxiety, particularly in the US.  At even larger doses, clonazepam is used to treat seizures; given about 30% of people with autism also have seizures, it would be fair to assume that some of those are also prescribed clonazepam.
The downside is that clonazepam is a benzodiazepine, and this class of drug is habit forming.  In extremely low doses, perhaps this will not be a problem.  For anxiety, plenty of people have been prescribed it for 10+ years; the problem is they cannot stop taking it.
The pharmacological property of clonazepam is modulation of the GABAA receptor; based on the mice, the effect is extremely dose dependent.  The wrong dose gives no beneficial effect.  Bumetanide, which is affecting GABA in a very similar way to make it inhibitory rather than excitory, seems to work like an on/off switch.  A low dose is ineffective, the correct dose works and a larger dose works just the same.
The optimal dose of clonazepam will be hard to find, too little does not work and neither does too much.  So for the time being it is rather trial and error.
By my calculations, a good place to start would be 0.8 Mcg (micrograms) per Kg per day and then titrate upwards gradually increasing the frequency and size of the doses.
The drug has a half-life that varies from person to person; the average is 30 hours but can vary between 18 and 50 hours.  This means that one child might need nearly 3 times as much as another, of similar weight.
To be an effective treatment the concentration of clonazepam would have to be maintained within the effective range.  This would need some clever maths, and might result in 3 unequal daily doses, and that during sleep the concentration  might be above range, and during daytime it be held in range with one or two smaller top-up doses.  
If you get the maths wrong, the drug would not work.

Conclusion
The jury is out until we see the results of experiment three, or anecdotal evidence of some home trials. One question I have is how this relates to the  NaV1.1 ion channels referred to at the beginning of this post.  We know that a defect in this ion channel will produce autism-like symptoms and that these can be reversed (in mice) using the correct type of benzodiazepine, such as clonazepam.  If we find that in a particular child with autism, clonazepam reduces their symptoms and increases cognitive performance, can we claim the route cause was a dysfunction with NaV1.1 ion channels?  It is a bit of a leap of faith, but I think it is a fair conclusion.  In which case of course, the logical next step would be to look at the underlying gene, SCN1A; that I will leave to people much cleverer than me.
The next question is whether this therapy, which is reducing excitability of the neurotransmitter GABA, is alternative or complementary to bumetanide which is, in effect, doing exactly the same thing.  For that we would need Dr Catterall to talk to Dr Ben-Ari.  
In case you are wondering if there is another connection between Dr Catterall (Clonazepam) and Dr Ben-Ari (Bumetanide), there is.  The same man is part-funding both of these research efforts – Jim Simons, via his Simons Foundation.  As a former hedge fund manager, his is very cleverly hedging his bets. 
 
 
 
 

11 comments:

  1. As the first author of these studies, I would like to answer your question "how this relates to the NaV1.1 ion channels": NaV1.1 channel is main sodium channel in GABAergic inhibitory interneurons. Mutation of Scn1a gene causes decreased GABA neurotransmission, which may cause autistic traits in Dravet's syndrome. low-dose clonazepam rescues autistic-like social behaviors in scn1a mutant mice by recovering reduced GABA neurotransmission to normal level. BTBR mice in the second study have normal Nav1.1 functions, but GABA signaling in the BTBR brain is reduced with unknown reason. low-dose clonazepam also corrected the autistic-like behaviors in BTBR mice by increasing GABA signaling. Therefore, low-dose clonazepam may work in autism population with GABA signaling dysfunction.

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    1. Thank you for your answer and taking interest in reading such a blog. Clonazepam at a very low dose does work in my son and so does Bumetanide. I did tell Ben-Ari about this and suggest they try Clonazepam in the children in their Bumetanide research program, but I doubt they will do this.

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  2. Clonazepam at a very low dose seems doe not work for my son.

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  3. Peter, thank-you for raising this. I am putting word out in the autistic community to collect some further anecdotal evidence. If you've hit a dead end trying to get researchers to test some of your hypotheses, please contact Benjine Gerber at the Channelopathy Foundation. Your blog has already been cited a number of times in the informal documentation which preceded some of their current research proposals. I will post the link to the foundation in my signature.

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    1. Thanks for the comment. Another reader did also mention the Channelopathy Foundation. At the moment I still have further to go in my own investigation and the blog is actually rather lagging behind. There is actually no shortage of existing research, there is a lack of clinicians showing any interest to read/apply it. There really are no serious autism doctors, so the research just collects dust. Since most people are frightened to self-experiment, let alone experiment on their child, there is no serious progress.

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  4. Hi Peter, I have been giving low dose clonazepam for a week now. During the first two days there was no response, but on the third day it was obvious that there was improvement regarding anxiety and sociability.
    I needed two scales: a digital, to measure the dose and bought it, and an anxiety scales in order to be objective with my findings.
    There is a teenage girl who can't afford private lessons in our neighbourhood. Her mother told my husband about her problem and my son without knowing her aggreed to help free of charge. They met twice and I had the chance to observe closely while socializing and have to say he was really good.
    I give 0,08 mg clonazepam and 1mg Bumetanide in the morning once a day.
    I've also noticed that low dose clonazepam reduces stereotypy more than Bumetanide.

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  5. Sorry, I think I've given the dose wrong. Anyway it's 0,008mg.

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    1. Out of curiosity why did you decide on 8mcg?

      Catterall used 1mcg/kg in his original study/analysis, did you try higher and ramp down?

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    2. I am still experimenting with the dose. Most of the times my dose seems good, so it's safe. If I try higher it may cause irritability or better results, I really don't know. I also don't know if it is going to work long term.
      As I am dealing with a fear conditioning issue I know I have to take a full advantage of this research which makes sense.
      Are you thinking of/ using low dose clonazepam?

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  6. Peter, Low dose Clonazepam makes my son feel much better. The problem is that I have just one packet 0,5mg which I bought it from UK. In Greece it comes only in 2mg and it would be very difficult to find the right dose.
    Do you know if I could find a lab or something to make the dose for me, or which countries around here have it available in 0,5mg?

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  7. Peter, It's ok, I've found a pharmacy with a lab to make the dose I need, either out of 0,5 or 2 mg clonazepam.

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