Piperine/Resveratrol/Sunitinib for Rett’s and indeed much Autism? Or, R-Baclofen to raise KCC2 expression in Bumetanide-responsive autism.

Piperine/Pepper             Resveratrol/Red wine          Sunitinib/Sutent

  

This post is all about lowering chloride within neurons, by increasing the expression of the transporter that lets it leave, called KCC2.

Today’s post is one I never finished writing from last year; I looked up the price of Sutent/Sunitinib and then I remembered why. It does again highlight how cancer drugs, when they become cheap generics, will provide interesting options for autism treatment. It also shows again how Rett Syndrome is getting attention from researchers.

It also highlights that really clever Americans are looking for bumetanide alternatives, in the false belief that bumetanide has troubling side effects that cannot be managed/mitigated.

The study is by some clever guys in Cambridge Massachusetts.

Another group of clever guys from MIT burned through $40 million dollars a few years ago trying to develop R-Baclofen for Fragile-X and autism.  After that Roche-funded clinical trial failed, R-Baclofen has now been resurrected and a new trial is planned, with different end points (measures of success).

Today we see why many people should indeed respond positively to R-Baclofen, but the mode of action is entirely different to the one originally targeted by the clever guys from MIT.

Tucked away in the supplementary material of today’s paper we see that R-Baclofen increases the expression of the transporter (KCC2) that takes chloride out of neurons. So, R-Baclofen is doing the same thing as Bumetanide, just to a lesser extent and in a different way.  Both lower intracellular chloride.

That means that people responsive to bumetanide should get a further boost from R baclofen, but you might need a lot of it.

Clever they may be, but these researchers do not know how to communicate their findings.  I had to dig through the supplementary tables to extract the good stuff, which is a list of what substances increase KCC2 in regular brains (Table S1) and specifically in Rett Syndrome brains (Table S2).

This blog does rather bang on about blocking/inhibiting NKCC1 that lets chloride into neurons, you can of course alternatively open up KCC2 to let the chloride flood out. This latter strategy is proposed by the MIT researchers.

What really matters is the ratio KCC2/NKCC1.  In people with bumetanide-responsive autism, which pretty clearly will include girls with Rett Syndrome, you want to increase KCC2/NKCC1. So, block/down-regulate NKCC1 and/or up-regulate KCC2.

·        NKCC1↓

·        KCC2↑

The researchers identified 14 compounds.  To be useful as drugs these compounds have to be able to cross the blood brain barrier to be of much use, many do not.

In the paper they call KCC2 expression-enhancing compounds KEECs.

We have five approved drugs to add to the list that are functionally the same to primary hit compounds. 

·        Sunitinib

·        Crenolanib

·        Indirubin Monoxiome

·        Cabozantinib

·        TWS-119


The researchers went on to test just two compounds in Rett syndrome mice; they picked piperine (from black pepper) and KW 2449 (a leukemia drug)

Even R-baclofen pops up, with a “B score” of 6.65 (needs to be >3 to increase KCC2 expression).

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

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K⁺/Cl^- cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3β (GSK3β) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.

Table S1. KEECs identified from screening with WT human KCC2 reporter neurons.

Table S2. KEECs identified from screening with RTT human KCC2 reporter neurons

Note Baclofen, Quercetin, Luteolin etc

Fig. 3. Identification of KEECs that increase KCC2 expression in human RTT neurons

B score >3 indicates compounds potentially increasing KCC2 expression

In cultured RTT neurons, treatment with KEECs KW-2449 and BIO restored the impaired KCC2 expression and rescued deficits in both GABAergic and glutamatergic neurotransmissions, as well as abnormal neuronal morphology. Previous data suggested that disrupted Cl− homeostasis in the brainstem causes abnormalities in breathing pattern (64), consistent with breathing abnormalities seen in mice carrying a conditional Mecp2 deletion in GABAergic neurons (67). The reduction in locomotion activity observed in the Mecp2 mutant mice has also been attributed to abnormalities in the GABAergic system (65). Therefore, treatment with the KEEC KW-2449 or piperine may ameliorate disease phenotypes in MeCP2 mutant mice through restoration of the impaired KCC2 expression and GABAergic inhibition.

Most KEECs that enhanced KCC2 expression in WT neurons, including KW-2449, BIO, and resveratrol, also induced a robust increase of KCC2 reporter activity in RTT neurons (Fig. 3, A and B; a complete list of hit compounds is provided in table S2). The increase in KCC2 signal induced by KEECs was higher in RTT neurons than in WT neurons,

1. 
   

Our results establish a causal relationship between reduced FLT3 or GSK3 signaling activity and increased KCC2 expression.

Two hit compounds, resveratrol and piperine, act on different pathways than the kinase inhibitors, activating the SIRT1 signaling pathway (50) and the TRPV1 (51), respectively

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

The group of KEECs reported here may help to elucidate the molecular mechanisms that regulate KCC2 gene expression in neurons. A previous study conducted with a glioma cell line showed that resveratrol activates the SIRT1 pathway and reduces the expression of NRSF/REST (50), a transcription factor that suppresses KCC2 expression (52). Our results demonstrate that resveratrol increases KCC2 expression by a similar mechanism, which could contribute to the therapeutic benefit of resveratrol on a number of brain disease conditions (68, 69). We also identified a group of GSK3 pathway inhibitors as KEECs. Overactivation of the GSK3 pathway has been reported in a number of brain diseases (70). Thus, our results suggest that GSK3 pathway inhibitors could exert beneficial effects on brain function through stimulating KCC2 expression. Another major KEEC target pathway, the FLT3 kinase signaling, has been investigated as a cancer therapy target (71, 72). Although FLT3 is expressed in the brain (73), drugs that target FLT3 pathway have not been extensively studied as potential treatments for brain diseases. Our results provide the first evidence that FLT3 signaling in the brain is critical for the regulation of key neuronal genes such as KCC2. Therefore, this work lays the foundation for further research to repurpose a number of clinically approved FLT3 inhibitors as novel brain disease therapies

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron- restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl− cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression– enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration–approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3 (GSK3) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.

By screening these KCC2 reporter human neurons, we identified a number of hits KCC2 expression–enhancing compounds (KEECs) from ~900 small-molecule compounds. Identified KEECs were validated by Western blot and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments on cultured human wild-type (WT) and isogenic RTT neurons, as well as on organotypic mouse brain slices. Pharmacological and molecular biology experiments showed that identified KEECs act through inhibition of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3b (GSK3b) kinases, or activation of the sirtuin 1 (SIRT1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment of RTT neurons with KEECs rescued disease-related deficits in GABA functional switch, excitatory synapses, and neuronal morphological development. Last, injection of the identified KEEC KW-2449 or piperine into a Mecp2 mutant mice ameliorated behavioral phenotypes including breathing pauses and reduced locomotion, which represent important preclinical data, suggesting that the KEECs identified in this study may be effective in restoring impaired E/I balance in the RTT brain and provide symptomatic treatment for patients with RTT.

Fig. 2. KEEC treatment–induced enhancement of KCC2 protein and mRNA expression in cultured organotypic mouse brain slices and a hyperpolarizing EGABA shift in cultured immature neurons.

(E to G) KCC2 and NKCC1 mRNA expression induced by FLT3 inhibitors including sunitinib (n = 4), XL-184 (n = 6), crenolanib (n = 4), or a structural analog of BIO termed indirubin monoxime (n = 6). The calculated ratios of KCC2/NKCC1 mRNA expression are shown in (G). A.U., arbitrary units

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.

The science-light version:-

Drug screen reveals potential treatments for Rett syndrome

An experimental leukemia drug and a chemical in black pepper ease breathing and movement problems in a mouse model of Rett syndrome, according to a new study.

Rett syndrome is a rare brain condition related to autism, caused by mutations in the MECP2 gene. Because the gene is located on the X chromosome, the syndrome occurs almost exclusively in girls. No drugs are available to treat Rett.

The team screened 929 compounds from three large drug libraries, including one focused on Rett therapies. They found 30 compounds that boost KCC2’s expression in the MECP2 neurons; 14 of these also increased the protein’s expression in control neurons.

The team tested two of the identified compounds in mice with mutations in MECP2: KW-2449, which is a small molecule in clinical trials for leukemia, and piperine, an herbal supplement and component of black pepper. These mice have several traits reminiscent of Rett. They are prone to seizures, breathing problems, movement difficulties and disrupted social behavior.

Injecting the mice with either drug daily for two weeks improved the animals’ mobility relative to untreated mice. The drugs also eased the mice’s breathing problems, decreasing the frequency of pauses in breathing (apnea). The findings appeared in July in Science Translational Medicine.

 

Piperine, Resveratrol and analogs thereof

Piperine and Resveratrol are commercially available supplements.

Resveratrol has been mentioned many times in this blog.  It has numerous beneficial properties, to which we can now add increasing KCC2 expression, but it is held back by its poor ability to cross the blood barrier.

The other natural substance highlighted in the study is piperine. Piperine is the substance that gets added to curcumin to increases its bioavailability and hopefully get its health benefits.

Piperine has been recently been found to be a positive allosteric modulator of GABA_A receptors.

It may be that piperine has 2 different effects on GABA, or maybe it is just the same one?

The result is that people are trying to develop modified versions of piperine that could be patentable commercial drugs.

Piperine also activated TRPV1 receptors.

You might wonder what is the effect in humans of plain old piperine in bumetanide-responsive autism.

Invitro blood–brain-barrier permeability predictions for GABAA receptor modulating piperine analogs

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABA_A) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood–brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00–500 ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.

The alkaloid piperine, the major pungent component of black pepper (Piper nigrum L.), was recently identified as a positive allosteric γ-aminobutyric acid type A (GABAA) receptor modulator. The compound showed anxiolytic-like activity in behavioral mouse models, and was found to interact with the GABAA receptors at a binding site that was independent of the benzodiazepine binding site [1,2]. Given that the compound complied with Lipinski’s “rule of five” [1], it represented a new scaffold for the development of novel GABAA receptor modulators [1–3]. Given that piperine also activates the transient receptor potential vanilloid 1 (TRPV1) receptors [4] which are involved in pain signaling and regulation of the body temperature [5,6], structural modification of the parent compound was required to dissect GABAA and TRPV1 activating properties

For drugs acting on the central nervous system (CNS), brain penetration is required. This process is controlled by the blood-brain barrier (BBB), a tight layer of endothelial cells lining the brain capillaries that limits the passage of molecules from the blood circulation into the brain [10]. Since low BBB permeability can reduce CNS exposure [11], lead compounds should be evaluated at an early stage of the drug development process for their ability to permeate the BBB [12].

Conclusions

Piperine and five selected piperine analogs with positive GABAA receptor modulatory activity were screened in three in vitro cell-based human and animal BBB models for their ability to cross the BBB. Data from the three models differed to some extent, possibly due to protein binding of the piperine analogs. In all three models, piperine and SCT-64 displayed the highest BBB permeation potential, which could be corroborated by in silico prediction data. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human models, and moderate to high in the animal model. ER calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux. In addition to the early in vitro BBB permeability assessment of the compounds, further studies (such as PK and drug metabolism studies) are currently in progress in our laboratory. Taken together, these data will serve for selecting the most promising candidate molecule for the next cycle of medicinal chemistry optimization

Conclusion

My conclusions are a little different to the MIT researchers

“The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.”

This assumes that you cannot safely use bumetanide/azosemide, which you can.  Open your eyes and look at France, where several hundred children with autism are safely taking bumetanide.

”It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases”

We have copious evidence that elevated chloride is a feature of many conditions, not just Rett’s and an effective cheap therapy has been sitting in the pharmacy for decades.

In the clinical trial of R-Baclofen that failed, there were some positive effects on some subjects.  Were the positive effects just caused by the effect of Baclofen in increasing KCC2 expression?

Should R-Baclofen become a cheap generic, it might indeed become a useful add-on for those with bumetanide-responsive. Regular Baclofen (Lioresal) is an approved drug, but it does have some side effects, so most likely R-baclofen will have side effects in some.

Baclofen itself in modest doses has little effect on bumetanide-responsive autism.

A cheap side-effect free KCC2 enhancer would be a good drug for autism, although cheap, safe NKCC1 blockers already exist. 

I have no idea if piperine benefits bumetanide-responsive autism.  Piperine has long been used in traditional medicine.

The TRPV1 receptor also affected by piperine plays a role in pain and anxiety.

We saw in the post below that TRPV1 controls cortical microglia activation and that GABARAP modulates TRPV1 expression.

So, TRPV1 and GABA_A receptors are deeply intertwined.

  

GABAa receptor trafficking, Migraine, Pain, Light Sensitivity, Autophagy, Jacobsen Syndrome,Angelman Syndrome, GABARAP, TRPV1, PX-RICS, CaMKII and CGRP ... Oh and the"fever effect"

Is Piperine going to make autism better, or worse?

Ginseng, as a GABAb Antagonist, as an "Add-on Therapy" for some Autism? Also Homotaurine and Acamprosate

Rather like negotiating with North Korea, today’s post does rather meander. It does in the end up with some interesting options for some people. 

Korea - the centre of Ginseng research

This post was prompted by research highlighted by our reader Ling, which suggested that bumetanide responders (i.e. people with high intracellular chloride) might benefit from a GABA_B antagonist. 

There has been quite a lot of coverage in this blog about agonists of GABA_B receptors, like Baclofen and Arbaclofen. Some people with an autism diagnosis do indeed seem to benefit, ranging from some with Fragile-X to others with Asperger’s. Russian-developed GABA_B agonists like Phenibut and Pantogam are widely used by adults self-treating their behavioural/emotional disturbances.

Some Aspies have commented in this blog that far from helping, Baclofen made them feel worse; perhaps the opposite therapy might help? (the Goldilocks scenario, from the previous post) 

The paper below shows how a GABA_B antagonist (the opposite of Baclofen) might benefit some with autism.

Neuronal Chloride Regulation via KCC2 Is Modulated through a GABA_B Receptor Protein Complex.

GABA_B receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABA_B receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABA_B receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABA_A receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABA_B receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel "crosstalk" between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.

SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABA_A receptors (GABA_ARs) and metabotropic GABA_B receptors (GABA_BRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABA_BR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABA_AR. We demonstrate that GABA_BR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABA_AR. Our data therefore support an additional mechanism by which GABA_BRs are able to modulate fast synaptic inhibition.

In bumetanide-responsive autism, neurons remain immature because the “GABA switch“ never flipped and so NKCC1 is overexpressed and KCC2 is underexpressed, chloride levels remain high and the neurotransmitter GABA works backwards (excitatory, rather than inhibitory).

Bumetanide partially counters the over-abundance of NKCC1 transporters that carry chloride into neurons, but is a partial solution.

The above research suggests that blocking GABA_B receptors might increase the flow of chloride ions exiting through KCC2.

All very complicated sounding, but in effect it means that a GABA_B antagonist might boost the effect of bumetanide.

Which GABA_B antagonist?

This was Ling’s question.

Saclofen is a competitive antagonist for the GABA_B receptor. This drug is an analogue of the GABA_B agonist baclofen.

Phaclofen/phosphonobaclofen, is a selective antagonist for the GABA_B receptor.

Since these “–aclofens” are not accessible we are left with a choice of homotaurine (developed to treat Alzheimer’s) or Ginsenosides from Korean/Panax ginseng.

Both homotaurine and Ginsenosides have various other effects beyond GABA_B.

Since Ling is in Scandinavia, homotaurine is an option. It seems to be banned in the US, though it is approved in Canada and sold in Europe.

Ginseng is very widely used, indeed it is the most widely consumed herbal nutritional product in the world, with sales of $400 million in 2012.

I was surprised that there actually is research in both humans and animal models using Ginseng in autism and indeed ADHD.

N-Acetyl homotaurine,  a derivative of homotaurine, is a registered drug called Acamprostate. It is used to treat alcohol dependence. It affects both NMDA and GABA receptors. Acamprostate has been shown to benefit Fragile-X, as has bumetanide. A drug that affects GABA_B will inevitably also affect NMDA receptors.

This was covered in this post from 2015.

NMDAR hypo-function causing E/I imbalance in Autism and Schizophrenia – Baclofen, Sodium benzoate and Cinnamon (again)

which highlighted this paper:

GABA_B-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction

Homotaurine 

Homotaurine has been reported as a GABA antagonist as well as a GABA agonist. In vitro studies have found that homotaurine is a GABA_A partial agonist as well as a GABA_B receptor partial agonist with low efficacy, becoming an antagonist and a displacing full agonist of GABA or baclofen at this receptor.^[15] In a study in rats, homotaurine reversed the catatonia induced by baclofen (the prototypical GABA_B agonist),^[16] and was able to produce analgesia via the GABA_B receptor, an effect that was abolished when CGP 35348, a GABA_B receptor antagonist was applied.^[17][18] 

One study suggests Homotaurine increases dopamine levels.^[19]

One study in rats showed that homotaurine suppressed ethanol-stimulated dopamine release, as well as ethanol intake and preference in rats in a way similar to the N-acetyl derivative of homotaurine, acamprosate.^[20] Acamprosate was approved by the FDA in 2004 to treat alcohol dependence.^[3]

Impact of acamprosate on behavior and brain-derived neurotrophic factor: an open-label study in youth with fragile X syndrome.

Fragile X syndrome (FXS) is an inherited form of developmental disability and a single gene cause of autism. As a disorder with increasingly understood pathophysiology, FXS is a model form of developmental disability for targeted drug development efforts. Preclinical animal model findings have focused targeted drug treatment development in FXS on an imbalance between excessive glutamate and deficient gamma-aminobutyric acid (GABA) neurotransmission.

Acamprosate was generally safe and well tolerated and was associated with a significant improvement in social behavior and a reduction in inattention/hyperactivity. The increase in BDNF that occurred with treatment may be a useful pharmacodynamic marker in future acamprosate studies. Given these findings, a double-blind, placebo-controlled study of acamprosate in youth with FXS is warranted.

Back to Ginseng

 Supplementation of Korean Red Ginseng improves behavior deviations in animal models of autism

Background

Autism spectrum disorder (ASD) is heterogeneous neurodevelopmental disorders that primarily display social and communication impairments and restricted/repetitive behaviors. ASD prevalence has increased in recent years, yet very limited therapeutic targets and treatments are available to counteract the incapacitating disorder. Korean Red Ginseng (KRG) is a popular herbal plant in South Korea known for its wide range of therapeutic effects and nutritional benefits and has recently been gaining great scientific attention, particularly for its positive effects in the central nervous system.

Objectives

Thus, in this study, we investigated the therapeutic potential of KRG in alleviating the neurobehavioral deficits found in the valproic acid (VPA)-exposed mice models of ASD.

Design

Starting at 21 days old, VPA-exposed mice were given daily oral administrations of KRG solution (100 or 200 mg/kg) until the termination of all experiments. From P28, mice behaviors were assessed in terms of social interaction capacity, locomotor activity, repetitive behaviors, short-term spatial working memory, motor coordination, and seizure susceptibility.

Results

VPA-exposed mice showed sociability and social novelty preference deficits, hyperactivity, increased repetitive behavior, impaired spatial working memory, slightly affected motor coordination, and high seizure susceptibility. Remarkably, long-term KRG treatment in both dosages normalized all the ASD-related behaviors in VPA-exposed mice, except motor coordination ability.

Conclusion

As a food and herbal supplement with various known benefits, KRG demonstrated its therapeutic potential in rescuing abnormal behaviors related to autism caused by prenatal environmental exposure to VPA.

In the trial below the dose appears very low at 250mg. In the more encouraging study in ADHD the dose was 1000mg twice a day.

First preliminary results of an observation of Panax ginseng treatment in patients with autistic disorder.

Autism is a pervasive developmental disorder, with impairments in reciprocal social interaction and verbal and nonverbal communication. There is often the need of psychopharmacological intervention in addition to psychobehavioral therapies, but benefits are limited by adverse side effects. For that reason, Panax ginseng, which is comparable with Piracetam, a substance effective in the treatment of autism, was investigated for possible improvement of autistic symptoms. There was some improvement, which suggests some benefits of Panax ginseng, at least as an add-on therapy.

Three male outpatients (age range 18.4–22.2 years; mean=21.3 years; SD =4.1 years) meeting ICD-10 criteria for autistic disorder participated in our observation. IQs ranged from 54 to 82 (68 +/− 14), which were obtained from the Wechsler Intelligence Scale. At least two child and adolescent psychiatrists independently diagnosed the subjects for autistic disorder. All subjects had no additional medical or neurological illnesses. They had been treated with either methylphenidate, or neuroleptics before entry into the study, without any positive effect (nonresponder). One patient’s language consisted of monosyllabic utterances, second patient’s language consisted of single words(10-word vocabulary),and the third patient spoke in sentences. Parents and mentors’ (i.e., the person who takes care of the patient in daily life, and supports the patient’s educational efforts) rated instruments included weekly ratings by means of the Aberrant Behavior and Symptom Checklist. Clinician ratings consisted of the Global Assessment Scale, Psychiatric Rating Scale (CPRS), and Clinical Global Improvement. Panax ginseng (oral administration of tablets containing 250-mg alcoholic Panax ginseng berry extract, pure encapsulations) was administered for 4 weeks (dosage: 250 mg daily). Patients were free of medication for at least 4 weeks before the beginning of the study. During that time, there were no changes in the symptoms of the patients. Subjects continued to receive educational and behavioral interventions, which were not altered substantially in any of the patients during their participation in the study. The means of parent and mentor ratings were averaged over the 4-week treatment period. Clinician and mentor ratings were made at the beginning of the treatment period and then weekly up to the end of the treatment. Ratings were compared by paired t-test.

RESULTS

Panax ginseng slightly improved the ratings on the ABC factors: irritability (before treatment, 13.2 +/− 5.9; after treatment, 11.3 +/− 6.2; p =.41), hyperactivity (before treatment, 20.6+/−12.4;     after,18.4+/− 9.4; p = .33), inadequate eye contact (before treatment, 8.6 +/− 5.4; after, 7.5 +/− 3.2; p .35), and inappropriate speech (before treatment, 6.1+/−2.2;after, 4.3 +/− 3.6; p = .41). The symptom checklist scores revealed a slight increase in drowsiness (before treatment, 1.6 +/− 2.2; after, 2.9+/−4.2; p =.31) and decreased activity (before treatment, 2.5 +/− 3.3; after, 4.4 +/− 3.1; p = 0.40). None of the clinician ratings showed significant improvement. This may result from different impressions of clinical visits and daily life observations of caregivers. Panax ginseng has some moderate sedative effect with effects especially on daily life, a fact that also makes it effective in the treatment of attention deficit/hyperactive disorders. None of the subjects appeared to have headaches or stomach aches, although report of such side effects was limited by the expressive language and social skills of these subjects. Medication was continued after the observation period. We did not see any significant changes in symptoms.

DISCUSSION 

Although this was a very small study (n = 3), which revealed very modest therapeutic effect of Panax ginseng in the management of autistic patients in some of the subjects (which might be due to the small sample size), it may be mentioned that its role in the management of these symptoms in patients with autistic disorder may be limited, especially because of its risk for estrogen-associated problems in females (Papapetropoulos, 07). Since there does not seem to be any significant improvement caused by Panaxginseng, its effect as an add-on therapy remains completely open and requires further investigation. Before knowing its efficacy for adults, Panax ginseng should not be recommended for treating children suffering from autism.

Ginseng for ADHD? 

Effects of Korean Red Ginseng Extract on Behavior in Children with Symptoms of Inattention and Hyperactivity/Impulsivity: A Double-Blind Randomized Placebo-Controlled Trial

Objective: There is evidence that Korean red ginseng (KRG) can reduce the production of the adrenal corticosteroids, cortisol, and dehydroepiandrosterone (DHEA), and thus may be a viable treatment for attention-deficit/hyperactivity disorder (ADHD). The present randomized double-blind placebo-controlled clinical trial tested the effect of KRG on children with ADHD symptoms.

Methods: Subjects 6–15 years, who satisfied the inclusion criteria and had ADHD symptoms, were randomized into a KRG group (n=33) or a control group (n=37). The KRG group received one pouch of KRG (1g KRG extract/pouch) twice a day, and the control group received one pouch of placebo twice a day. At the 8 week point, the primary outcomes were the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria for inattention and hyperactivity scale scores, which were measured at baseline and 8 weeks after starting treatment. Secondary outcomes were quantitative electroencephalography theta/beta ratio (QEEG TBR) (measured at baseline and week 8) and salivary cortisol and DHEA levels (measured at baseline and at 4 and 8 weeks).

Results: The baseline characteristics of the KRG and control groups were not statistically different. The mean ages of the KRG and control groups were 10.94±2.26 and 10.86±2.41, respectively. The KRG group had significantly decreased inattention/hyperactivity scores compared with the control group at week 8 (least squared means of the differences in inattention adjusted for baseline scores: −2.25 vs. −1.24, p=0.048; hyperactivity: −1.53 vs. −0.61, p=0.047). The KRG group had significantly decreased QEEG TBR compared with the control group (least squared means of the differences: −0.94 vs. −0.14, p=0.001). However, neither the KRG group nor the control group exhibited significant differences in salivary cortisol or DHEA levels at week 8 compared with the baseline levels. No serious adverse events were reported in either group.

Conclusions: These results suggest that KRG extract may be an effective and safe alternative treatment for children with inattention and hyperactivity/ impulsivity symptoms. Further studies to investigate the efficacy and safety of KRG are warranted. 

Although medications to treat psychiatric disorders for children and adolescents have been widely researched and several are on the market, natural products may also be effective in these patients while inducing fewer significant adverse effects. The present randomized controlled trial was performed to assess whether KRG, a well-known traditional medicine plant that is used particularly frequently in Eastern Asia, can improve the adrenal function and inattention/hyperactivity symptoms of chronically stressed children with ADHD symptoms. KRG extract significantly improved the inattention and hyperactivity of the subjects and had a good safety profile. However, the KRG extract did not have significant effects on cortisol or DHEA levels

Clinical Significance

To our knowledge, this is the first randomized controlled trial to investigate the efficacy and safety of Korean red ginseng extract for children with ADHD. The stimulant medications for ADHD have demonstrated not only clinical efficacy, but also significant adverse events such as poor growth, tics, and psychosis. Although KRG extract did not affect the salivary cortisol or DHEA, it significantly improved ADHD symptoms and QEEG TBR. And the safety profile of KRG extract was good. The results imply that KRG extract is a possible effective alternative medication for ADHD children.

Effect of the herbal extract combination Panax quinquefolium and Ginkgo biloba on attention-deficit hyperactivity disorder: a pilot study.

OBJECTIVE:

A combination herbal product containing American ginseng extract, Panax quinquefolium, (200 mg) and Ginkgo biloba extract (50 mg) (AD-FX; CV Technologies, Edmonton, Alta.) was tested for its ability to improve the symptoms of attention-deficit hyperactivity disorder (ADHD). 

DESIGN:

Open study. 

PATIENTS:

36 children ranging in age from 3 to 17 years who fit the diagnostic criteria for ADHD. 

INTERVENTIONS:

AD-FX capsules were taken twice a day on an empty stomach for 4 weeks. Patients were instructed not to change any other medications during the study. 

OUTCOME MEASURES:

At the beginning of the study, after 2 weeks, and then at the end of the 4-week trial, parents completed the Conners' Parent Rating Scale--revised, long version, a questionnaire that assesses a broad range of problem behaviours (and was used as an indication of ADHD symptom severity). 

RESULTS: 

After 2 weeks of treatment, the proportion of the subjects exhibiting improvement (i.e., decrease in T-score of at least 5 points) ranged from 31% for the anxious-shy attribute to 67% for the psychosomatic attribute. After 4 weeks of treatment, the proportion of subjects exhibiting improvement ranged from 44% for the social problems attribute to 74% for the Conners' ADHD index and the DSM-IV hyperactive-impulsive attribute. Five (14%) of 36 subjects reported adverse events, only 2 of which were considered related to the study medication. 

CONCLUSIONS:

These preliminary results suggest AD-FX treatment may improve symptoms of ADHD and should encourage further research on the use of ginseng and Ginkgo biloba extracts to treat ADHD symptoms.

Interactions of ginsenosides with ligand-bindings of GABA(A) and GABA(B) receptors.

Abstract

1. Total saponin fraction decreased the affinity of specific [3H]muscimol binding without changes in Bmax. Ginsenoside Rb1 Rb2, Rc, Re, Rf and Rg1 inhibited the specific [3H]muscimol binding to the high-affinity site. 2. Total saponin fraction increased the affinity of specific [3H]flunitrazepam binding. Ginsenoside Re and Rf enhanced specific [3H]flunitrazepam binding.

3. Total saponin fraction decreased the affinity of specific [35S]TBPS binding without changes in Bmax. Ginsenosides did not affect specific or non-specific [35S]TBPS binding.

4. Total saponin fraction decreased the affinity of specific [3H]baclofen binding without changes in Bmax. Ginsenoside Rc inhibited specific [3H]baclofen binding.

Emerging signals modulating potential of ginseng and its active compounds focusing on neurodegenerative diseases

very detailed paper

Also (Ling) note that there is an effect on ERbeta

A ginseng-derived oestrogen receptor beta (ERbeta) agonist, Rb1 ginsenoside, attenuates capillary morphogenesis.

 Ginseng extracts contain a variety of active ingredients and have been shown to promote or inhibit angiogenesis, depending on the presence of different ginsenosides that exert opposing effects on blood vessel growth. Leung et al. in this issue of the British Journal of Pharmacology report that Rb1, a ginsenoside that constitutes only 0.37–0.5% of ginseng extracts (depending on manufacturing and processing methods), blocks tube-like network formation by endothelial cells in vitro. At the molecular level, Rb1 binds to the oestrogen receptors and stimulates the transcription of pigment epithelium-derived factor that, in turn, inhibits matrix-driven capillary morphogenesis.

Ginseng, the root of Panax ginseng and related species, has been a key component of traditional medicine in the Far East for over a thousand years. The genus name Panax means ‘cure all' in Greek; it, thus, comes as no surprise that ginseng has been described as beneficial in many different ailments (Huang, 1999; Kiefer and Pantuso, 2003; Ng, 2006). Perhaps the most studied biological actions of ginseng extracts and constituents are those relating to its inhibitory effects on solid tumour growth (Yun, 2001). The main active ingredients in ginseng-based herbal preparations are thought to be the ginsenosides, comprising 3–6% of ginseng extracts (Huang, 1999). 

Effects and mechanisms of ginseng and ginsenosides on cognition. 

Reviewed here is the existing evidence for the effects of ginseng extracts and isolated ginsenosides relevant to cognition in humans. Clinical studies in healthy volunteers and in patients with neurological disease or deficit, evidence from preclinical models of cognition, and pharmacokinetic data are considered. Conditions under which disease modification may indirectly benefit cognition but may not translate to cognitive benefits in healthy subjects are discussed. The number of chronic studies of ginseng effects in healthy individuals is limited, and the results from acute studies are inconsistent, making overall assessment of ginseng's efficacy as a cognitive enhancer premature. However, mechanistic results are encouraging; in particular, the ginsenosides Rg3 , Rh1 , Rh2 , Rb1 , Rd, Rg2 , and Rb3 , along with the aglycones protopanaxadiol and protopanaxatriol, warrant further attention. Compound K has a promising pharmacokinetic profile and can affect neurotransmission and neuroprotection. Properly conducted trials using standardized tests in healthy individuals reflecting the target population for ginseng supplementation are required to address inconsistencies in results from acute studies. The evidence summarized here suggests ginseng has potential, but unproven, benefits on cognition.

Ginseng is the most widely consumed herbal nutritional product in the world. According to the most recent data available, ginseng had a total world export value in 2010 of over US$350 million, which was expected to rise to more than US$400 million in 2012

A survey of Korean medicine doctors’ clinical practice patterns for autism spectrum disorder: preliminary research for clinical practice guidelines

The survey had 54 respondents and 4 (8.5%) used Ginseng.

Conclusion

There is a long list of substances shown to have a benefit in some autism. Today we can add the Asian type of Ginseng and also Acamprosate (at least for Fragile-X).

It would be interesting to see the effect of Phaclofen and Saclofen which may be more selective for GABA_B receptors.

Ginseng has so many effects there is no way to know which is the one that benefited autism and ADHD in today’s highlighted posts.

We also have the problem with natural substances that there is natural variation and that supplement companies are known to cheat with ingredients. Ginseng roots are not cheap and apparently ginseng is known to get adulterated.  Drug companies are usually much more reliable.

If anyone tries out homotaurine or ginseng, let us all know the result.

Homotaurine was originally developed as an Alzheimer’s drug, but did not work well enough, its developer then tried to sell it as a supplement called Vivimind, but it was rejected by the FDA. It is sold in Canada and Europe. 

For our Aspie readers, here is a link for them:-

https://www.reddit.com/r/phenibut/comments/5ii692/homotaurine_gabab_antagonist_tolerance_reversal/