Differential Diagnosis and Treatment in Autism – Verapamil & Curcumin for Williams Syndrome?

 

The face of Williams syndrome kids. Source: Figure 2.  GeneReviews® - NCBI Bookshelf

Continuing from the last post, today we look again at differential diagnosis and treatment, which I prefer to just call personalized medicine.

This is the subject of a conference for parents in the UK, that I agreed to draw to the attention of readers.

 

Click on the picture above to read about the upcoming event in London.

 

 

Williams syndrome

Williams syndrome: MedlinePlus Genetics (click for info)

Williams syndrome occurs when someone is missing a small piece of chromosome 7, resulting in them lacking 25 to 27 genes. Most people with Williams syndrome have not inherited the condition from a parent.

Williams syndrome can delay a child’s milestones including:

·         Learning (mild to moderate intellectual challenges)

·         Saying their first words and talking

·         Sitting and walking

Socializing is unusual – there is excessive empathy. A child will be outgoing and very friendly, but has difficulty identifying strangers. There may be attention problems, phobias, or anxiety.

Williams is another syndrome with distinct facial features that can help with diagnosis.

·         Large ears

·         Full cheeks

·         Small jaw

·         Wide mouth

·         Small teeth

·         Upturned nose

Williams syndrome is still viewed as untreatable.

In this blog we always start from the basis that all severe autism is potentially treatable.  Often some of the downstream effects of genetic mutations overlap with other types of autism and some of these effects actually are treatable.

There is a great deal in this blog about targeting both calcium channels and potassium channels to treat autism. Verapamil and Ponstan are the two drugs I have written most about.

Curcumin is an OTC therapy for autism that has been widely covered in this blog and people do regularly write to me to tell me that it is beneficial.  Just last week a reader told me that both Ponstan and Curcumin are beneficial in his specific case.

I was intrigued to read the paper from Spain below where the researchers found the combination of Verapamil + Curcumin to improve behaviors in Williams syndrome.  The mechanism was found to be by regulation of MAPK pathway and microglia overexpression.

 

Verapamil + Curcumin to treat the behavioral issues in Williams syndrome

One key takeaway is that in the model of Williams syndrome you need both verapamil (VER) and curcumin (CUR). Either intervention on its own provided no benefit – you need the combination (VERCUR). 

Co-Treatment With Verapamil and Curcumin Attenuates the Behavioral Alterations Observed in Williams–Beuren Syndrome Mice by Regulation of MAPK Pathway and Microglia Overexpression

Williams–Beuren syndrome (WBS) is a rare neurodevelopmental disorder characterized by a distinctive cognitive phenotype for which there are currently no effective treatments. We investigated the progression of behavioral deficits present in WBS complete deletion (CD) mice, after chronic treatment with curcumin, verapamil, and a combination of both. These compounds have been proven to have beneficial effects over different cognitive aspects of various murine models and, thus, may have neuroprotective effects in WBS. Treatment was administered orally dissolved in drinking water. A set of behavioral tests demonstrated the efficiency of combinatorial treatment. Some histological and molecular analyses were performed to analyze the effects of treatment and its underlying mechanism. CD mice showed an increased density of activated microglia in the motor cortex and CA1 hippocampal region, which was prevented by co-treatment. Behavioral improvement correlated with the molecular recovery of several affected pathways regarding MAPK signaling, in tight relation to the control of synaptic transmission, and inflammation. Therefore, the results show that co-treatment prevented behavioral deficits by recovering altered gene expression in the cortex of CD mice and reducing activated microglia. These findings unravel the mechanisms underlying the beneficial effects of this novel treatment on behavioral deficits observed in CD mice and suggest that the combination of curcumin and verapamil could be a potential candidate to treat the cognitive impairments in WBS patients.

Accumulated evidence has described that curcumin, the major constituent of turmeric (Curcuma longa), exerts a variety of pharmacological effects due to its antioxidant, anti-inflammatory, and neuroprotective properties. Recent studies have reported positive effects of curcumin over different cognitive aspects such as anxiety-like behaviors, memory deficits, and motor impairments of different murine models Many studies have described that its effects on the behavioral phenotype of mice models are mediated by upregulation of BDNF (brain-derived neurotrophic factor) expression BDNF has been described as a crucial molecule for neural development and plasticity processes and its mechanism of action is highly dependent on a proper maintenance of intracellular ionic homeostasis Moreover, it has also been described to prevent neuroinflammation by modulating pathways related to NRF2 and MAPK signaling.

Verapamil is a widely used medication, and its mechanism of action involves mainly the blocking of voltage-dependent calcium channels, but it has also been proven to directly bind and block voltage-gated potassium channels  and to inhibit drug efflux pump proteins like P-glycoprotein. Although it has been mainly studied for cardiovascular applications, it has also been associated with positive effects on anxiety and memory processing in murine models.

Given the properties of both compounds, we decided to explore the effects of each compound and a combinatorial treatment on the behavioral phenotype of CD mice. The results show that only the combined treatment with curcumin and verapamil improved the deficits. This improvement can be correlated with the normalization of the MAPK and inflammasome signaling pathways and with the concomitant reduction of activated microglia. 

·   The Increased Microglia Activation in Motor Cortex and Hippocampus Presented by CD Mice Is Prevented by VERCUR Co-Treatment

·    Combinatorial Treatment Prevents Hypersociability of CD Mice

·    Only VERCUR Co-Treatment Improves Motor Coordination in CD Mice

·    VERCUR Co-Treatment Prevents Gene Expression Changes in Cortex of CD Animals

·    Neuroanatomical Features of CD Mice Do Not Change After VERCUR Co-Treatment

In conclusion, we suggest that the hemizygous loss of WBSCR in the cerebral cortex of CD mice has a direct effect on the neuroinflammatory state of the brain, as well as on the expression of some genes related to synaptic signaling or extracellular matrix structure, which are crucial for a proper neural function. This may at least be partly responsible for the behavioral phenotype observed in CD animals. A treatment combining verapamil and curcumin is able to address different molecular targets and rescue some of those pathways, being a promising therapeutic approach for the cognitive phenotype of WBS patients.

  

Conclusion 

Today’s study was in a mouse model of William’s syndrome; clearly it would be more informative if the researchers had tried it on humans.  It does though raise the question as to what other treatments from idiopathic autism might be effective in this supposedly untreatable genetic condition.

The other perspective of course is to wonder what other types of autism might benefit from Verapamil plus curcumin (VERCUR). It was interesting to note that in the model neither Verapamil nor curcumin was effective by itself, they needed the combined therapy (VERCUR).

If you read the experiences that have been shared over the years in this blog you can see that some parents spend a lot of money on genetic testing, hoping to improve their child’s outcome.  It is only very rarely that you see any great success resulting.

The alternative approach is understand the commonly shared biological features of autism and try and treat those, to see whether the individual shows a benefit.  Where there is a positive response, it is a “keeper,” if there is no response, or a negative response, the therapy is dropped.  Essentially it is a process of trial and error.  Not as fancy as genetic testing, but it works.

Clearly if your child has Williams syndrome you would be well advised to look up the function of each of the 26 missing genes, to see if there are any obvious steps to take. One good tool to use is www.genecards.org.  

Old posts that refer to cucumin:

Epiphany: Curcumin (epiphanyasd.com)

 

Old posts that refer to verapamil

Epiphany: Verapamil (epiphanyasd.com)

Turmeric/Curcumin – clinically effective in humans after all? SLC6A15 Amino Acid Transporter

Turmeric powder, only in food, modified the SLC6A15 gene

I know that most readers of this blog want to treat autism with supplements and/or diet.

Many supplements and herbal medicines do show promise in the laboratory, when tests are conducted in vitro, but very often when tests are made in humans the results are much weaker, or just not present.  Turmeric/Curcumin is a perfect example; in the test tube it has a wide range of potent benefits, but due to low absorption into humans (bioavailability) it does not show such conclusive results in human studies.

One researcher a while back did send me a study that reviewed all the turmeric/curcumin trials and it concluded that curcumin has no beneficial effect in humans.

In modern medicine anecdotal evidence does not count. Some anecdotes are genuine, but some are coincidence and some are placebo. 

Mini trial of Turmeric at three UK Universities

There is a remarkably good medical program produced by the BBC in the UK, called Trust me I’m a Doctor, where the doctor presenters team up with universities to test practical medical hypotheses.

Does turmeric really help protect us from cancer?

In one study they took 100 people to assess whether turmeric has any measurable medical benefit. They teamed up with Newcastle University, Leeds University and a clever genetic researcher at University College London (UCL).

They showed that eating turmeric in your food modified a specific gene (SLC6A15) associated with certain cancers, asthma/eczema and depression.

Taking turmeric as a supplement pill or taking a placebo pill had no effect on the gene.

The researcher at UCL was measuring the epigenetic tags attached to the genes. He showed that methylation of this gene was increased by dietary turmeric. Changing the methylation of this gene will change when it turns on/off.

Anecdotally, we know that people who eat a lot of turmeric tend to have less cancer, less asthma and less eczema.

Given that this gene is also associated with depression, you might expect big eaters of turmeric to have either less, or more, depression. Probably nobody has researched this.  

SLC6 Gene Family

It is true that asthma and eczema (atopic dermatitis) are common in people with autism, but variations in the broader SLC6 family of genes are known to affect people with ADHD, Fragile X, Tourette’s and broad autism.

SLC transporters encompass approximately 350 transporters organized into 55 families. The SLC6 family is among the largest SLC families, containing 20 genes that encode a group of highly similar transporter proteins. These proteins perform transport of amino acids and amino acid derivatives into cells. 

SLC6 Transporters: Structure, Function, Regulation, Disease Association and Therapeutics

In humans, the SLC6 family of transporters defines one of the most clinically relevant protein groups with links to orthostatic intolerance, attention deficit hyperactivity disorder (ADHD), addiction, osmotic imbalance, X-linked mental retardation , Hartnup disorder, hyperekplexia, Tourette syndrome, schizophrenia, Parkinson disease (PD), autism  and mood disorders such as depression, anxiety, obsessive compulsive disorder (OCD), and post-traumatic stress disorder (PTSD).

This review will focus on the structure-function aspects of the mammalian SLC6 transporters, their regulation by both classical as well as emerging epigenetic/transgenerational mechanisms and what impact these properties may have on disease and the use of biomarkers to detect these proteins in disease states  

The functional impact of SLC6 transporter genetic variation.

Solute carrier 6 (SLC6) is a gene family of ion-coupled plasma membrane cotransporters, including transporters of neurotransmitters, amino acids, and osmolytes that mediate the movement of their substrates into cells to facilitate or regulate synaptic transmission, neurotransmitter recycling, metabolic function, and fluid homeostasis. Polymorphisms in transporter genes may influence expression and activity of transporters and contribute to behavior, traits, and disease. Determining the relationship between the monoamine transporters and complex psychiatric disorders has been a particular challenge that is being met by evolving approaches. Elucidating the functional consequences of and interactions among polymorphic sites is advancing our understanding of this relationship. Examining the influence of environmental influences, especially early-life events, has helped bridge the gap between genotype and phenotype. Refining phenotypes, through assessment of endophenotypes, specific behavioral tasks, medication response, and brain network properties has also improved detection of the impact of genetic variation on complex behavior and disease. 

Amino acids are very important and it is not just that you need them, but you need them in the right place at the right time.

It appears that one of the many effects of defective amino acid/derivative transport into cells is on behaviour.

Improving amino acid transmission is therefore a potential therapy to correct aberrant behaviour, including depression but likely much more. 

Conclusion

Modern clinical trials are often hugely expensive, but as the BBC keeps showing with its TV series, you can carry out very meaningful research without breaking the bank.

You would think that cancer researchers would now look at the modified versions of turmeric that claim higher bioavailability and see if these pills can also modify this cancer gene, since they can easily repeat the UCL laboratory analysis. I doubt this will happen any time soon.

It has long been known that turmeric is not well absorbed, but just one teaspoon a day added to food was enough to modify the gene.

Indians have a low incidence of cancer and a high consumption of turmeric. Turmeric should particularly limit breast cancer.

Source: https://vizhub.healthdata.org/gbd-compare/

The above chart, where blue is best, shows India does well, as do some other turmeric eating countries (South Asia and the Middle East). Clearly longevity and quality of healthcare also matter, so beware Africa. Europe, Russia, Argentina, Uraguay, Oz, NZ and North American might want to up their turmeric intake.

We can say that turmeric is a potential epigenetic therapy for at least one important gene (SLC6A15) and possibly more, because turmeric does not just affect methylation. It has several other better documented epigenetic properties. 

Epigenetic changes induced by curcumin and other natural compounds

Epigenetic regulation, which includes changes in DNA methylation, histone modifications, and alteration in microRNA (miRNA) expression without any change in the DNA sequence, constitutes an important mechanism by which dietary components can selectively activate or inactivate gene expression. Curcumin (diferuloylmethane), a component of the golden spice Curcuma longa, commonly known as turmeric, has recently been determined to induce epigenetic changes. This review summarizes current knowledge about the effect of curcumin on the regulation of histone deacetylases, histone acetyltransferases, DNA methyltransferase I, and miRNAs. How these changes lead to modulation of gene expression is also discussed. We also discuss other nutraceuticals which exhibit similar properties. The development of curcumin for clinical use as a regulator of epigenetic changes, however, needs further investigation to determine novel and effective chemopreventive strategies, either alone or in combination with other anticancer agents, for improving cancer treatment.

Only a few reports have so far investigated the effect of curcumin on DNA methylation. Molecular docking of the interaction between curcumin and DNMT1 suggested that curcumin covalently blocks the catalytic thiolate of DNMT1 to exert its inhibitory effect on DNA methylation. However, a more recent study showed no curcumin-dependent demethylation, which suggested that curcumin has little or no pharmacologically relevant activity as a DNMT inhibitor. To clarify these contradictions, more research is urgently needed.

Given that 5-azacitidine and decitabine, two FDA-approved hypomethylating agents for treating myelodysplastic syndrome, have a demonstrated ability to sensitize cancer cells to chemotherapeutic agents, it would be worthwhile to explore whether the hypomethylation effect of curcumin can also induce cancer cell chemosensitization. Interestingly, a phase 1 trial with curcumin administered several days before docetaxel in patients with metastatic breast cancer resulted in 5 partial remissions and stable disease in 3 of 8 patients. This unexpected high response might have resulted from the clever sequential delivery of these two agents, which capitalized on and maximized curcumin’s epigenetic activity for cancer treatment.

Docetaxel is a 20 year old chemotherapy drug produced using extracts from the leaves of the European yew tree, perhaps best taken with root (rhizome) of the Asian Curcuma Longa plant. 

The main mode of therapeutic action of docetaxel is the suppression of microtubule dynamic assembly and disassembly. It exhibits cytotoxic activity on breast, colorectal, lung, ovarian, gastric, renal and prostate cancer cells.

Under-expression (Haploinsufficiency) of ARID1B in Autism and Corpus Callosum Abnormalities

People keep telling me that my blog is too complicated; compared to the literature it really is not. If your child has a disabling condition you really should be willing to invest all the time needed to learn about it, rather than be a passive bystander.

I think you can investigate even complex sounding genetic disorders without being an expert, which is what happens in today’s post.  

Are there 20,000 types of jeans?

As readers may recall, humans only have about 20,000 genes, far less than originally was thought. Each gene provides the instructions to make one thing, usually a protein.

For the great majority of genes we have two copies, one from Mum and one from Dad. Mitochondrial genes all come from Mum.

These genes are stored on chromosomes (like recipe books).

For 22 of these recipe books you have two copies, so if one page got damaged at least you have an undamaged version from the other book.

The 23rd pair of books is special because while females have two copies, males do not. This is the X chromosome and if a male has a problem on any page in this little book, he has a big problem, while his sister has less of a problem, because she has a spare copy. The male has a Y chromosome in place of a second copy of X. 

Examples of problems on the X chromosome:-

·        The MECP2 gene is on the X chromosome and when there is one working copy and one mutated version you have Rett syndrome and you must be female. If you were male with one mutated version you cannot survive.

·        In Fragile X syndrome a problem with the FMR1 gene means not enough not enough fragile X mental retardation protein (FMRP), which is required for normal development of the connection between neurons. Females would normally have a clean spare copy of the FMR1 gene and so show much less severe symptoms that a male with Fragile X.

Problems on chromosomes 1 to 22:-

If you have a problem in the first 22 chromosomes (recipe books), boys and girls are equal. If one page got damaged you can always look up the recipe in the other book.

In case one gene got mutated but the other copy is fine, things can work out just fine, in which case it is called haplosufficiency. You get to make enough of that protein.

In some cases you really need to use that recipe a lot; that particular protein is in big demand. One copy of that gene just is not enough. This is called  haploinsufficiency.

In most cases when the gene has a problem, it just fails to produce the intended protein. In some cases it actually produces a mutated protein, which can be worse than no protein. 

Pitt Hopkins

In Pitt Hopkins Syndrome there is a problem on chromosome 18, where you find the TCF4 gene. Not enough expression of TCF4 means not enough Transcription Factor 4;  this is an example of haploinsufficiency.

Now the reason why these rare conditions are important to many other people is that they not only affect people who happened to have a random mutation and hence a severe deficit of the protein; moderately reduced transcription of this gene, for any reason, can also result in troubling symptoms.

So in the case of the Pitt Hopkins and the gene TCF4, as was pointed out to me recently, reduced expression is a feature of some MR/ID and indeed schizophrenia. 

The Intellectual Disability and Schizophrenia Associated Transcription Factor TCF4 Is Regulated by Neuronal Activity and Protein Kinase A 

Instead of just a tiny number of people with Pitt Hopkins, you can see that upregulating TCF4 expression could help a lot of people.

It appears that people with Pitt Hopkins have a “clean copy” of TCF4, so it is just a case of making it work a little harder. There are ways being researched to achieve just that.

I suspect people with schizophrenia have two “clean copies” of TCF4, but for some reason have a deficiency of the protein encoded by it.

In the above paper it was shown that Protein Kinase A (PKA) plays a key role in regulating what your TCF4 gene is producing.

We have come across PKA before in this blog and we know that in regressive autism there can be a deficit of PKA. There is also PKB and PKC. All three are very important, but complicated. 

Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A in the Frontal Cortex of Regressive Autism

Without going into all the details you can see that if someone with Pitt Hopkins has a lack of PKA, like those with regressive autism, then he will struggle to make the most of his good copy of the gene TCF4.

It all gets very complicated, but PKA is controlled by something called cAMP. In turn cAMP is controlled by PDE. PDE4 is known to be disturbed in the brains of some people with autism.

It appears that you can activate PKA with a PDE4 inhibitor. The long established Japanese asthma drug Ibudilast is such a PDE4 inhibitor. At least one reader of this blog uses Ibudilast long term.

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

PDE4 inhibitors have been explored to treat various neurological conditions like schizophrenia.

PDE4 as a target for cognition enhancement

So logically if you feed a PDE4 inhibitor to a Pitt Hopkins mouse, you might expect something good to happen. There now is such a mouse model.

I think I could keep that mouse quite busy. 

The point being you do not have to figure things out 100%, before starting to see what you have in your drug library might be truly beneficial.  

Some of the things in the drug library are actually in the kitchen cupboard, as we have already seen. 

Protein Kinase A

Protein kinase A (PKA) is something that is both complicated and important.

The effects of PKA activation vary with cell type.

PKA has always been considered important in formation of a memory.  Formation of a normal memory is highly sensitive to PKA levels; too much is bad and too little is bad.

ARID1B in Autism and Corpus Callosum Abnormalities

I don’t think anyone has set up a research foundation for agenesis of the Corpus Callosum (ACC), perhaps they should. 

There was a post on this a while back, prompted by meeting someone whose son has this condition. 

Agenesis of the Corpus Callosum

The Corpus Callosum is just a fancy name for what joins the two sides of the brain together. Agenesis of the Corpus Callosum (ACC) is what they call it when there is a complete or partial absence of the corpus callosum.

ACC is we are told a very rare condition, but clearly smaller corpus callosum variations are a key part of some autism. 

For example, in Pitt Hopkins a small corpus callosum is typical.

An estimated 7 percent of children with autism and macrocephaly (big heads) carry a PTEN mutation. This is associated with an enlarged corpus callosum. 

PTEN is an autism gene, but it is more usually thought of as a tumor suppressor, making it a cancer gene. In older people, losing PTEN appears to be often a first step to developing cancer; up to 70% of men with prostate cancer are estimated to have lost a copy of the PTEN gene at the time of diagnosis  (https://www.ncbi.nlm.nih.gov/pubmed/16079851). 

PTEN is interesting because too little can allow cancer to develop, but too much may eventually result in type 2 diabetes. So, as always, it is a balance. 

PTEN upregulation may explain the development of insulin resistance and type 2 diabetes with high dose statins. 

Evidently from the comments in this blog, regarding tumors/cancers, people with autism are likely shifted towards the direction of lacking tumor suppressing proteins. The exception would be those born very small, or with small heads. 

ARID1B gene

ARID1B is another tumor suppressing gene, like PTEN, and like PTEN it is also an autism gene.

What I found interesting was the link between ARID1B and corpus callosum anomalies. 

ARID1B mutations are the major genetic cause of corpus callosum anomalies in patients with intellectual disability  

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism inpatients with haploinsufficiency of ARID1B

Corpus callosum abnormalities are common brain malformations with a wide clinical spectrum ranging from severe intellectual disability to normal cognitive function. The etiology is expected to be genetic in as much as 30–50% of the cases, but the underlying genetic cause remains unknown in the majority of cases.

Additional functional studies including a systematic search for ARID1B target genes may show how haploinsufficiency of ARID1B predispose to CC defects and to an array of cognitive defects, including severe speech defects

Several readers of this blog have highlighted a recent study:-  

Breakthrough research suggests potential treatment for autism, intellectual disability 

We showed that cognitive and social deficits induced by an Arid1b mutation in mice are reversed by pharmacological treatment with a GABA receptor modulating drug. And, now we have a designer mouse that can be used for future studies." 

The full study:-

Arid1b haploinsufficiency disrupts cortical interneuron development and mouse behavior

Clonazepam also reversed the reduced time spent in the center and reduced moving distance displayed by Arid1b-mutant mice in the open field test (Fig. 7c,d and Supplementary Fig. 14c). However, depression measures, using the forced swim test and the tail suspension test, showed no reversible effect of clonazepam in Arid1b+/− mice compared with controls (Fig. 7e,f). Our results show that clonazepam rescues impaired recognition, social memory, and elevated anxiety in Arid1b+/− mice. 

Our mouse model effectively mirrors the behavioral characteristics of intellectual disability and ASD. Arid1b+/− and Arid1bconditional-knockout mice displayed impaired spatial learning, recognition memory, and reference memory. Open field and social behavior tests also revealed decreased social interaction in the mice. Mice with mutations in genes encoding Smarca2 and Actl6b, other subunits of the BAF complex, have severe defects in social interaction and long-term memory35. Thus, this chromatin remodeling complex may provide a cellular and molecular platform for normal intellectual and social behavior. In addition, Arid1b+/− mice showed heightened levels of anxiety- and depression-related behaviors, which are common symptoms of ASD36. 

For people with intellectual disability, the prevalence of anxiety disorders has likewise been shown to be much higher. This may be due to reduced cognitive function and increased vulnerability to environmental demands. Communication difficulties may also make it more difficult for people with cognitive disabilities to deal with anxiety or fear. ARID1B haploinsufficiency may be responsible for multiple facets of characteristic ASD behaviors. Other isoforms of Arid1b that are not affected by the Arid1b mutation could exist in the mouse line. Additionally, it is possible that the genetic background for the mouse line may impact the effect of Arid1b haploinsufficiency. Thus it is important to consider allele specificity, genetic backgrounds, and knockout strategies for comparing phenotypes of other Arid1bhaploinsufficiency models.  

GABA allosteric modulators, including clonazepam, a benzodiazepine, have been used to treat seizures and anxiety. We found that clonazepam injection rescued deficits in object and social recognition and anxiety in Arid1b+/− mice. These results suggest that treatment with a benzodiazepine could be a potential pharmacological intervention for symptoms of ASD. Furthermore, our results suggest that pharmacological manipulation of GABA signaling is a potential treatment strategy for cognitive and social dysfunctions in ASD- or intellectual disability-associated disorders due to mutations in chromatin remodeling genes.  

ACC Research Foundation

If there actually was an ACC Research Foundation, they could explore whether clonazepam was therapeutic in children who have Arid1b haploinsufficiency.

While they are at it, they might want to look into Hereditary Motor and Sensory Neuropathy with agenesis of the corpus callosum (HMSN/ACC), this is caused by mutations in the potassium-chloride co-transporter 3 (SLC12A6/KCC3) gene. This I stumbled upon a long time ago, when trying to upregulate KCC2, which causes elevated intracellular chloride in many people with autism and likely many with Down Syndrome.

KCC2 is usually associated with neuropathic pain and now we see that so is KCC3. Odd reaction to pain is a well known feature of autism. The rather ill-defined condition of fibromyalgia seems common in female relatives of those with autism and I do not think this is just a coincidence. 

The interesting thing is that the research shows you can potentially upregulate KCC3 with curcumin. 

Transit Defect of Potassium-Chloride Co-transporter 3 Is a Major Pathogenic Mechanism in Hereditary Motor and Sensory Neuropathy with Agenesis of the Corpus Callosum

HMSN/ACC is a severe and progressive neurodegenerative disease that exhibits an early onset of symptoms. Signs of HMSN/ACC, such as hypotonia and delays in motor development skills, are noticed before 1 year of age. However, the motor abilities of patients progress slowly to 4–6 years of age, and these children are able to stand and walk with some help. This is followed by a motor deterioration that generally renders affected subjects wheelchair-dependent by adolescence. 

Accordingly, we found that curcumin relieved the ER retention of dimerized R207C in mammalian cultured cells. A diet enriched in curcumin may therefore be beneficial for the relief or delay of some of the HMSN/ACC symptoms in patients bearing the R207C mutation, including the Turkish patient described in this study (as patient has not yet reached puberty).

KCC3 defects also cause the very similar Andermann syndrome also known as agenesis of corpus callosum with neuronopathy (ACCPN).

KCC3 defects are associated with epilepsy.

My question was can you have KCC3 under-expression with partial ACC, epilepsy but no peripheral neuropathy? If this was likely, then upregulating KCC3 with curcumin might help.

The gene for KCC3 is located at chromosome 15q14. Based on my “logic of associations”, if you have ACC and epilepsy you should consider KCC3 under-expression.

I did suggest to my former classmate whose son has partial ACC and epilepsy, but no neuropathy, that it might be worth trying some curcumin. Since his son is already on anti-epileptic drugs (AEDs) my suggested effect to look for was improved cognitive function.

6 months later it does indeed, apparently, improve cognitive function.  Of course this does not establish that upregulating KCC3 had anything to do with it. It is nonetheless a nice story and another parent has realized that you can change things for the better, in spite of what neurology currently says. 

The question now is can you have both ARID1B under-expression and KCC3 under-expression, in which case you would add some clonazepam, based on the latest research. At this point you should of course go and talk to your neurologist, rather than read my blog and that was my recommendation. 

Partial agenesis of the corpus callosum in a patient with juvenile myoclonic epilepsy

We describe a patient who presented at our epilepsy-monitoring unit with myoclonic jerks, and was diagnosed with juvenile myoclonic epilepsy (JME). Imaging of his brain revealed partial agenesis of the corpus callosum (ACC). We discuss the known genetic basis of both JME and ACC, as well as the role of the corpus callosum (CC) in primary generalized epilepsy. Both JME and ACC are associated with gene loci on chromosome 15q14. Structural brain abnormalities other than ACC, such as atrophy of the corpus callosum have been reported in patients with JME. ACC has been associated with seizures, suggesting an anti-epileptogenic role of the corpus callosum

Conclusion

If you have a biological diagnosis you are one big step closer to finding a therapy. Even if you have a diagnosis like partial Agenesis of the Corpus Callosum (ACC), you can go one step further and ask why. You have a 50% chance of being able to find out a specific gene that is the cause. If you know with certainty which gene is the originator of the problem, you know a lot.  I think you are then two big steps closer to a therapy.

In the case of Rett Syndrome, a really good website is run by their research foundation (Rett Syndrome Research Trust). They look like they mean business. 

https://reverserett.org/cure/

If you look at the above site you might be left wondering why the much larger and better financed autism organizations look so amateur by comparison.  The big difference is that Rett Syndrome is a biological diagnosis and autism is not. In many ways calling autism a spectrum is not helpful, as the originators of the ASD concept are beginning to realize.  The precise biological dysfunctions are what matter and lumping together hundreds of miscellaneous brain dysfunctions into a pile labelled ASD may not be so clever, in fact I would call it primitive.

A Medical Case for Curcumin? Apparently Not

One medical researcher, who reads this blog, sent me a recent article about the vast amount of research that has been carried out on curcumin, which is widely used as a supplement.

Many apparently interesting natural substances suffer from low bioavailability and the arguments put forward in the paper do apply to many other supplements.  On the other hand, there are natural substances that do have useful medical properties in humans; it is just very hard to identify which ones, without making your own research. 

Chemists warn against deceptive molecules

Inside the golden-yellow spice turmeric lurks a chemical deceiver: curcumin, a molecule that is widely touted as having medicinal activity, but which also gives false signals in drug screening tests. For years, chemists have urged caution about curcumin and other compounds that can mislead naive drug hunters.  Now, in an attempt to stem a continuing flow of muddled research, scientists have published the most comprehensive critical review yet of curcumin — concluding that there’s no evidence it has any specific therapeutic benefits, despite thousands of research papers and more than 120 clinical trials. The scientists hope that their report will prevent further wasted research and alert the unwary to the possibility that chemicals may often show up as ‘hits’ in drug screens, but be unlikely to yield a drug.

The full paper is here:-

The Essential Medicinal Chemistry of Curcumin

Curcumin is a constituent (up to ∼5%) of the traditional medicine known as turmeric. Interest in the therapeutic use of turmeric and the relative ease of isolation of curcuminoids has led to their extensive investigation. Curcumin has recently been classified as both a PAINS (panassay interference compounds) and an IMPS (invalid metabolic panaceas) candidate. The likely false activity of curcumin in vitro and in vivo has resulted in >120 clinical trials of curcuminoids against several diseases. No doubleblinded, placebo controlled clinical trial of curcumin has been successful. This manuscript reviews the essential medicinal chemistry of curcumin and provides evidence that curcumin is an unstable, reactive, nonbioavailable compound and, therefore, a highly improbable lead. On the basis of this in-depth evaluation, potential new directions for research on curcuminoids are discussed.

At first, curcumin appeared to offer great potential for the development of a therapeutic from a NP (turmeric) that is classified as a GRAS material. Unfortunately, no form of curcumin, or its closely related analogues, appears to possess the properties required for a good drug candidate (chemical stability, high water solubility, potent and selective target activity, high bioavailability, broad tissue distribution, stable metabolism, and low toxicity). The in vitro interference properties of curcumin do, however, offer many traps that can trick unprepared researchers into misinterpreting the results of their investigations.

With respect to curcumin/curcuminoids and in vivo studies and clinical trials, we believe there is rather “much ado about nothing”. Certainly, the low systemic exposure levels reported in clinical trials do not support its further investigation as a therapeutic. Circumventing the requirement for systemic circulation, curcumin might provide benefit by acting on gut microbiota. Thus far, there is limited evidence to support this hypothesis, which will also limit the utility of this delivery method. Delivery systems such as lipid vesicles, nanoparticles, and nanofibers might be able to boost the bioavailability of 1, but this could also conceivably narrow its therapeutic window and lead to off-target toxicity by aforementioned processes. Available evidence demonstrates curcumin will ultimately degrade upon release into physiologic media, no matter the delivery mechanism. Analogues of 1 might address some of the delivery challenges but would be new chemical entities and would have to proceed through expensive preclinical work to be approved for clinical trials. In our opinion, analogues of curcumin are based on a fairly weak foundation.

Conclusion

It would be wrong to conclude that natural substances and supplements are of no medicinal value. One reader of this blog with type 1 diabetes has used some of the tips in the blog to improve insulin sensitivity so far that the requirement for insulin to be injected has been reduced by 50%.  As medical readers will realise that is quite remarkable.  It was all done with antioxidants of one kind or another (alpha lipoic acid, broccoli powder and cocoa flavanols) and without side effects.

Numerous natural substances reduce cholesterol or lower blood pressure and it is very easy to measure the results and see if they really work for you; cinnamon, beetroot juice, tangeretin, the list goes on.  

One problem is that even individual compounds often have multiple medical effects and natural substances can contain 20-30 or more different compounds, so it is impossible to say with certainty why broccoli improves insulin sensitivity.

Naturally occurring compounds cannot be patented and so nobody has a financial interest to do rigorous and costly clinical trials to conclusively show beneficial effect.  Why would pharmaceutical companies want to reduce the demand for their insulin by 50%?  They are apparently not so keen on repurposing cheap existing drugs to treat autism, and neither are some researchers.

2016 To-do List

I expect many readers of this blog have a list of things to trial in 2016; I certainly do.

Monty’s older brother, codenamed Ted, did say to me recently, “I thought you said you’d be all finished with this, in a couple of years”; that was indeed the intention.  

A medicine cabinet to be proud of, but not mine

It has now been three years.  I never really intended to go so deeply into the science, and I never expected there to be so many “obvious” things un/under-investigated by researchers.

Most people diagnosed these days with “autism” are fortunate to be relatively mildly affected.  Parents of those kids likely find this blog rather shocking; how can so many pills be needed and still you want more?

Some other people also diagnosed with autism, face really big challenges, not limited to:-

  

     ·        Unable to talk
·        Unable to walk
·        Unable to eat (must use G tube)
·        Unable to be toilet trained
·        Unable to read
·        Unable to write
·        Have seizures 

So when asked by a teacher at school, if Monty, now aged 12, has severe autism I responded in the negative.  He does not tick any of the above boxes.

If you have more than “mild autism” it seems that there are likely many dysfunctions and the more you treat, the better the result.  A quest without an end.

School

Ted hates his relatives discussing his school grades and I agree with him that they are entirely his business.  We all know that typical kids vary in how smart they are and how motivated they are.  NT kids tend to get the grades they deserve.

I do break these rules with Monty, but that is because I really want to show that when a person has numerous neurological dysfunctions, as those found in classic autism, if you treat them with science (not with bleach and other nonsense), you can end up in a different, better place. 99.99999% of the world do not know this; perhaps 500 people do know.

Improving IQ will improve the person’s ability to understand and compensate for the dysfunctions that have not been treated.  

Grading academic performance at school is something we all understand and along with its limitations.  We have all been there, so let's use it.

Kids with classic autism do not get the grades they potentially deserve.  Most can be made smarter and it is easy to measure.

Before coming to my to-do list, I did receive another question about what exactly is the effect of bumetanide. 

When I collected Monty from school the other day, his assistant was proudly holding up the latest “quick fire” math test, where speed is seemingly even more important than the right answer.

So Monty, the only one with autism, came first and by a long way. 3 minutes and 35 seconds, with the runner up taking 3:56.  He got 90% correct, but that is enough to keep first place.   The previous test before Christmas he got 100%, but finished 7^th out of 16 on speed.  It must be the turkey.

The questions are very simple, since you have to be very fast; but until the age of 9, and the introduction of Bumetanide, the class teacher would never have dreamt of having Monty compete at all.  Coming a distant last in everything would be disheartening, for the teacher. Monty would not have even noticed, let alone cared.

People with Classic Autism, or what Knut termed SDA (strict definition autism), are usually hopeless academically; but with Bumetanide, it does not have to be that way. 

Many people with classic autism leave school 18 years old, still at the level of single digit addition and subtraction, or perhaps up to 20.

If you reach the academic level of Grade 2 (Year 3 in the UK system), that of a typical 7 or 8 year old, by the time you “graduate” high school, you are doing above average.

So Ted is not alone in being able to get good grades.  The PolyPill is indeed worth all the bother.

To-do list

I did have to go through by supply cupboard to see what I had not got round to testing and that I still think has some potential merit.  Some things did get thrown out.

Some old ideas are worth revisiting.

·        Biotin (high dose)

This did seem to have a marginal positive effect and is both cheap and harmless. 

·        Pregnenolone (very low dose)

This also appeared to have some positive effect and should affect GABA subunit expression. High doses have been used in a Stanford clinical trial. We saw in earlier posts that allopregnanolone possesses biphasic, U-shaped actions at the GABA_A receptor, meaning that a tiny dose can have the same effect as a large dose.

 I like low doses.  

Old ideas worth developing:-

·        Miyairi 588 bacteria, but at higher doses

This is the bacteria used as a probiotic in Japan for humans, since the 1940s.  It is also added to animal feed to avoid inflammatory disease and so produce healthier animals.

The science showed that it should be helpful to raise Butyrate levels.  It can be achieved directly via supplementation, with sodium butyrate, and indirectly by adding a butyrate-producing bacteria, such as Clostridium Butyricum or Miyari 588.

I have been using a tiny dose of Miyari 588 for months.  It achieves what it is sold for in Japan, in that it reduces gas, which is the only obvious negative side effect of Monty’s Polypill, other than diuresis.

The positive side effect of the Polypill is near perfect asthma control.  Asthma is an auto-immune/inflammatory disease, highly comorbid with autism. 

The effect of Miyari 588 is reversible because this bacteria cannot survive long in the intestines, which is why you have to take it every day.  It crowds out some of the other bacteria in the intestines, but they will soon grow back.

New ideas already in this blog:-

·        Diamox

I did suggest on several occasions that it might be possible to get a “Bumetanide plus” effect by adding Diamox.

Diamox (Acetazolamide) is another diuretic and it is a carbonic anhydrase inhibitor

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:

The mechanism of diuresis involves the proximal tubule of the kidney. The enzyme carbonic anhydrase is found here, allowing the reabsorption of bicarbonate, sodium, and chloride. By inhibiting this enzyme, these ions are excreted, along with excess water, lowering blood pressure, intracranial pressure, and intraocular pressure. By excreting bicarbonate, the blood becomes acidic, causing compensatory hyperventilation, increasing levels of oxygen and decreasing levels of carbon dioxide in the blood

This change in bicarbonate will also affect the AE3 and NDAE exchangers.

As you will see in the figure below the regulation of bicarbonate HCO3- and pH is directly connected to chloride Cl- homeostasis.  This means that via AE3 and NDAE you can affect intracellular chloride levels by change the level of HCO3-

In turns this means that Diamox (Acetazolamide) really should have an effect on the level of intracellular chloride.

This in turn suggested to me that Diamox could augment the effect that bumetanide has on NKCC1.

 In the case that Bumetanide can lower intracellular chloride, but not to the optimal level to correct the GABA dysfunction, Diamox might be able to lower chloride levels a little further so further shifting GABA to inhibitory.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317631/

Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. 

After only a couple of days of Diamox, it is pretty clear that there is indeed a “bumetanide plus” effect.  So the same changes that were noted when starting bumetanide appear again.

A promising start to 2016.

·        Ponstan

This is the NSAID that is also suggested to be useful to affect the ion channels expressed by the genes ANO 2/4/7 & KCNMA1.  We saw in this post

http://epiphanyasd.blogspot.com/2015/12/autism-treatments-proposed-by-clinical.html

where Knut highlighted that Fenamates act as CaCC inhibitors and also stimulate BKCa channel activity.  Ponstan is a Fenamate.

·        Vitamin A

This was Maja’s discovery, that in some people vitamin A will stimulate oxytocin, via upregulation of CD38.

·        Zinc

Zinc should affect GABA, particularly in immature neurons.  Zinc homeostasis is disturbed in some autism and perhaps, in some people, a small dose of zinc may actually have a positive effect.  Simple to check.

Clioquinol, the drug that shifts zinc to the “right” place, is not without risks.

·        Picamilon

Once the GABA switch has been repaired, it may be time for a little extra GABA.  GABA should not be able to cross the blood brain barrier (BBB), but in the form of Picamilion, it does cross the BBB.

·        Inositol

This it naturally produced in the body from glucose and used to be known as vitamin B8.  In some people Inositol reduces OCD and stereotypy.  Simple to check.

·        Montelukast

This is an asthma drug, considered very safe in children, that Dr Kelley (formerly of Johns Hopkins and likely the cleverest autism clinician)  uses in children with AMD, as a short term therapy, when they are sick and, very interestingly, before immunizations.  This is to avoid further mitochondrial damage.  Montelukast is a leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies.

Dr Kelley also uses Ibuprofen as a short term therapy to counter the effects of increased cytokine production.  Montelukast is more potent and has different side effects, meaning it might be a better choice than ibuprofen for some people.

Ibuprofen may be OTC, but, more than very occasional use, can cause side effects in many people.  These side effects are caused by NSAIDs also being COX-2 inhibitors, which leads to stomach and intestinal adverse reactions.

Since I have determined that in the case of autism I deal with, the surge in cytokines like IL6 causes behavioral regression, Montelukast might be a good alternative to Ibuprofen to treat some types of autism flare.  

So a new addition to the autism flare-up toolkit, I hope.

  

Ideas not yet in this blog:-

·        Curcumin

Curcumin, and particularly some of the substances within it, have been shown to have very interesting autism-relevant effects, particularly in vitro (in test tubes).  Whether taking curcumin orally, in reasonable doses, produces any of these effects in humans is a big question.  Many such substances like luteolin and resveratrol fail to meet expectations in humans, due to poor bioavailability.

There are various ways to improve the bioavailability of curcumin, so it seems worth investigating.

·        5-loxin

Frankincense has been used for 5,000 years.  More recently, two thousand years ago, three wise men did bring gifts of gold, frankincense, and myrrh.

Frankincense is an aromatic resin obtained from trees of the genus Boswellia.  Boswellia is used for inflammatory conditions like arthritis in a similar way to curcumin.

There are six boswellic acids, one is most active. This fraction is called AKBA. 5-Loxin is a boswellia supplement claiming to deliver a high standardized level of AKBA.

5-Loxin does seem to help some people with arthritis, but does it have any benefit for the pro-inflammatory aspects found in some autism?  I am not expecting much, but you never know.

  

Ideas suggested to me by others, that look interesting:-

·        Mint/Menthol

This is Natasa’s discovery and there is evidence to show that Menthol does indeed affect GABA_A receptors.

Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABA_A receptors in native PAG neurons. The development of agents that potentiate GABA_A-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABA_A-related pharmacotherapies.

  

·        NIAGEN / Nicotinamide Riboside

This was highlighted by Tyler and is another potential therapy for oxidative stress.  Not as cheap as peppermint, but definitely interesting, perhaps particularly for those with autism and mitochondrial dysfunction.

Also note that there are odd recurring links between some autism and obesity. This is not the first anti-obesity therapy that potentially has some benefit for autism.

The NAD+ Precursor Nicotinamide Riboside Enhances Oxidative Metabolism and Protects against High-Fat Diet-Induced Obesity

Summary

As NAD⁺ is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38—both NAD⁺ consumers—increases NAD⁺ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD⁺ precursor with the ability to increase NAD⁺ levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD⁺ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.

  

Nicotinamide Riboside Ameliorates Hepatic Metaflammation by Modulating NLRP3 Inflammasome in a RodentModel of Type 2 Diabetes

Low-grade chronic inflammation (metaflammation) is a major contributing factor for the onset and development of metabolic diseases, such as type 2 diabetes, obesity, and cardiovascular disease. Nicotinamide riboside (NR), which is present in milk and beer, is a functional vitamin B₃ having advantageous effects on metabolic regulation. However, the anti-inflammatory capacity of NR is unknown. This study evaluated whether NR modulates hepatic nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Male, 8-week-old KK/HlJ mice were allocated to the control or NR group. NR (100 mg/kg/day) or vehicle (phosphate-buffered saline) was administrated by an osmotic pump for 7 days. Glucose control, lipid profiles, NLRP3 inflammasome, and inflammation markers were analyzed, and structural and histological analyses were conducted. NR treatment did not affect body weight gain, food intake, and liver function. Glucose control based on the oral glucose tolerance test and levels of serum insulin and adiponectin was improved by NR treatment. Among tested lipid profiles, NR lowered the total cholesterol concentration in the liver. Histological and structural analysis by hematoxylin and eosin staining and transmission electron microscopy, respectively, showed that NR rescued the disrupted cellular integrity of the mitochondria and nucleus in the livers of obese and diabetic KK mice. In addition, NR treatment significantly improved hepatic proinflammatory markers, including tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1. These ameliorations were accompanied by significant shifts of NLRP3 inflammasome components (NLRP3, ASC, and caspase1). These results demonstrate that NR attenuates hepatic metaflammation by modulating the NLRP3 inflammasome

  

NLRP3 inflammasome and itsinhibitors: a review

  

Activation and regulation of the inflammasomes

  

An apparently crazy idea of my own, but actually serious:-

·        Propolis tincture, without the propolis

The BIO 30 Propolis from New Zealand is a (mild) PAK1 inhibitor.  One reader is convinced of its cognitive enhancing effects in autism .  I also think it had an effect, but in our case not as potent as that reader.  Now I am wondering what was it that produced this effect. 

Most propolis is made as a tincture with ethanol.  Propolis is not soluble in water.  They typically use 70% ethanol to make propolis tincture.  “Non-alcoholic” tinctures use glycol.

In the last post we saw ethanol has pronounced effects on several GABA_A receptor subunits, mainly delta but also alpha, including possibly down regulating alpha 5.

So was it the propolis, or the ethanol that has the effect?

Propolis tincture is either made with ethanol (grain alcohol) or if it is “alcohol free” they use propylene glycol.  Propylene glycol actually is a food ingredient but it is also used to de-ice aircraft in winter.  Ethylene glycol is the antifreeze in your car and you would not want to drink that.

Compared to ethanol, glycol can dissolve less propolis, 

A quick check of school chemistry reminds us that if it is an –ol , it’s an alcohol.

·        Alcohols have at least one hydroxyl group

·        Diols have two hydroxyl groups

Propylene glycol is  C₃H₈O₂  and as you can see below it has two hydroxyl groups (the – OH), so it is both a diol and an alcohol. 

So your Propolis tincture can be ethanol-free, but it cannot be alcohol-free.  Someone might point that out to the supplement makers.

It also should be noted that propylene glycol has known effects on GABA very similar to ethanol.

Propylene glycol elicits anxiolytic responses to the elevated plus-maze in male mice

  

This suggests that the users of ethanol-free BIO30 may also be seeing responses unrelated to propolis.

Propylene glycol even has an E-number, it is E1520.  It is cheap and they even sell it on Amazon.

Food grade ethanol is normally not sold to the public.

In lay terms, ethanol and alcohol are interchangeable, so one corner of the supermarket contains food grade ethanol, with some impurities.

Japanese research suggests that these impurities are much more potent than ethanol in modulating GABA receptors.  It is the fragrant compounds that accumulate over the years on wooden barrels that cause this effect.

The twenty drops of propolis suggested to me by the Japanese PAK1 researcher/doctor contained about 1ml of ethanol.  It seems that to get an effect on GABA similar to this amount of ethanol would require a much smaller amount to well-aged Japanese whiskey.

So if someone over 18 responds well to twenty drops of BIO 30 propolis, it would helpful if they could compare the effect with 1ml of Propylene glycol (E1520), 1ml of ethanol, if they find it, and with a few drops of well-aged whiskey.