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Showing posts with label KCC2. Show all posts
Showing posts with label KCC2. Show all posts

Tuesday, 28 January 2020

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).



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,


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 GABAA 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 (GABAA) 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 GABAA 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?








Thursday, 18 July 2019

Azosemide in Autism – ça marche aussi / it works too

Rathaus/City Hall in Hanover, Germany      
Attribution: Thomas Wolf, www.foto-tw.de

The short version of this post is that the old German diuretic Azosemide delivers the same autism benefit as the popular diuretic Bumetanide, but it has a different profile of diuresis.  Azosemide may indeed be more potent at blocking NKCC1 in the brain, but this needs to be investigated/confirmed.  For some people Azosemide will be a better choice than Bumetanide.

The bulk of today’s post is really likely to be of interest only to bumetanide users and the French and German bumetanide researchers.

I did suggest recently when I published version 5 of Monty’s PolyPill, that it is getting close to the final version.  Some of the potential remaining elements have already been written about in this blog, but I have not finished evaluating them.  Azosemide falls into this category.

One theme within this blog has been to increase the “autism effect” of Bumetanide, which was the first pharmaceutical intervention going back to 2012.  I did look at modifying how the body excretes Bumetanide to increase its plasma concentration using an OAT3 inhibitor, but that is little different to just increasing the dose. There are other ways to lower chloride levels within neurons than blocking NKCC1, you can target the AE3 exchanger for example with another diuretic called Diamox, or you can just substitute bromide ions for chloride ions, using potassium bromide. Bromide is used to treat Dravet Syndrome and other hard to treat types of pediatric epilepsy.

Researchers in Germany have developed modified versions (prodrugs) of Bumetanide that better cross the blood brain barrier; one interesting example is called BUM5.  Prodrugs are out of favour because they are hard to control, meaning that they work differently in different people.

The researchers in Hanover, Germany also published data showing that an old German diuretic called Azosemide might be much more potent than bumetanide inside the brain.

This becomes even more interesting because, not-surprisingly, diuretics as drugs are produced based on their diuretic effect.  The diuresis comes from their effect on a transporter called NKCC2, but the autism effect comes from blocking the very similar transporter NKCC1 in the brain. Because Azosemide and indeed Furosemide are 40 times weaker than Bumetanide at blocking NKCC2, the pills are made as Bumetanide 1mg, but Furosemide 40mg. Azosemide is now only used in parts of Asia, where people tend to be smaller and so there are 30mg tablets (the equivalent of Bumetanide 2mg is Azosemide 60mg in smaller adults).

Then comes bio-availability, which is how much of the pill you swallow makes it into your bloodstream. Bumetanide is very well absorbed, but in the case of Azosemide it can be 20%. I was informed that you can increase this 20% by taking it with Ascorbic acid, otherwise known as vitamin C.  

In the test tube, Azosemide is 4 times more potent at blocking NKCC1 than bumetanide at the same dose.

In the test tube 60 mg of Azosemide should be very much more potent than 2mg of Bumetanide at blocking the NKCC1 transporter found in the brain.

But then we do have the blood brain barrier that seems to block 99% of bumetanide form getting through. Azosemide will also struggle to cross the blood brain barrier (BBB). The Germans think that Bumetanide is much more acidic than Azosemide and that suggests that Azosemide might be more able to cross the BBB; however the French disagree.

The conclusion of all that is to take Azosemide with orange juice.


French Researchers

You might think the French researchers at Neurochloré would have trialed Azosemide before spending millions of dollars/euros approving Bumetanide for autism.  Their patent covers all these drugs, but they would find monetizing their idea much easier with Azosemide. Bumetanide is a cheap generic drug widely available across the world. Azosemide is currently only available in some parts of Asia.

I did ask the researchers a while back if anyone had tried Azosemide for autism. The answer was no.

I think the main plan all along was to develop a more potent drug than bumetanide, without diuresis, that could be used in many neurological disorders that feature disturbed chloride levels.  The licensing of Bumetanide for autism is just an intermediate step.

There are many considerations in developing the new drug, not least what exactly is bumetanide’s mode of action. Is it the central effect of the tiny 1% that can cross the blood brain barrier? Or is it a peripheral effect?

While the German researchers think Azosemide can cross the blood brain barrier better than Bumetanide, the French do not think so.

The fact that Azosemide does have the same “autism effect” as bumetanide may help understand how it works and then this would help develop the new tailor-made drug. This is why they were interested by the news in today’s post.

I did suggest making an experiment of bumetanide and Azosemide in healthy adults to measure how much is present in spinal fluid, this is a proxy for how much is inside the brain.

In the meantime bumetanide-responders with autism have the choice of two drugs, with quite different patterns of diuresis. So for one person Bumetanide might be best, in another Azosemide and in some a combination of both drugs might be best.

Bumetanide is short-acting and causes diuresis in the first 30-90 minutes, in most people it is substantial diuresis while in some people it is minimal. Azosemide is a long-acting diuretic and the peak effect is 3 to 5 hours after taking the drug. It seems that in some people the diuretic effect is very mild and it is always delayed.
When I took Azosemide to check the effect, I did not notice any diuretic effect.  I would not have known it was a diuretic.

The higher the dose of Bumetanide/Azosemide the greater the autism benefit will be, depending on how elevated the initial chloride level was. The limiting factor is diuresis and at extreme levels ototoxicity. Very high doses of loop diuretics can damage your ears – ototoxicity.


In immature neurons you have almost exclusively NKCC1 (green above) whereas in adult neurons you have almost exclusively KCC2 (orange above), but you can be at any point in between. Also this point is not fixed in one person; external factors can shift it in either direction.

As a result the effective dose of Bumetanide/Azosemide will vary from person to person AND vary over time.

The severity of diuresis limits the dosage. This is why Azosemide clearly has a role to play at least for some people.

Here is the German paper that prompted the interest in Azosemide:-


Azosemide was the most potent NKCC1 inhibitor (IC50s 0.246 µM for hNKCC1A and 0.197 µM for NKCC1B), being about 4-times more potent than bumetanide. 

Azosemide was the most potent inhibitor of hNKCC1, inhibiting both splice variants with about the same efficacy. Azosemide lacks the carboxylic group of the 5-sulfamoylbenzoic acid derivatives (Fig. 1), demonstrating that this carboxylic group is not needed for potent inhibition of NKCC1. Clinically, Azosemide has about the same diuretic potency as furosemide, but both drugs are clearly less potent than bumetanide30, so the high potency of Azosemide to inhibit the hNKCC1 splice variants was unexpected. In contrast to the short-acting diuretic bumetanide, the long-acting Azosemide is not a carboxylic acid, so that its tissue distribution should not be restricted by a high ionization rate. However, it is highly bound to plasma proteins31, which might limit its penetration into the brain. Indeed, in a study in which the tissue distribution of Azosemide was determined 30 min following i.v. administration of 20 mg/kg in rats, brain levels were below detection limits (0.05 µg/g32).

In conclusion, the main findings of the present study on structure-activity analyses of 10 chemically diverse diuretics are that (1) none of the examined compounds were significantly more effective to inhibit NKCC1B than NKCC1A, and (2) Azosemide was more potent than any other diuretic, including bumetanide, to inhibit the two NKCC1 variants. The latter finding is particularly interesting because, in contrast to bumetanide, which is a relatively strong acid (pKa = 3.6), Azosemide is not acidic (pKa = 7.38), which should avour its tissue distribution by passive diffusion. Lipophilicity (logP) of the two drugs is in the same range (2.38 for Azosemide vs. 2.7 for bumetanide). Furthermore, Azosemide has a longer duration of action than bumetanide, which results in superior clinical efficacy26 and may be an important advantage for treatment of brain diseases with abnormal cellular chloride homeostasis.

Bumetanide in use

In 2012 I started bumetanide use at 1mg once a day and after 10 day saw a positive effect. Later I tried 0.5mg twice a day and felt the effect was much reduced.  This is not really a surprise and is highly relevant.

In the later years I increased the dose to 2mg once a day initially to combat the summertime loss of effect due to allergy (inflammation) shifting the balance of NKKC1/KCC2 further towards NKCC1.

Adding a second daily dose of 1mg produced more diuresis but no noticeable benefit. I did not try a second daily dose of 2mg because I did not want yet more diuresis.

Azosemide in use

Azosemide is a so-called long acting diuretic, whereas as Bumetanide is short acting. In practise this means there is no immediate diuresis soon after taking the drug, the diuresis comes later and can be much less. The diuretic response seems to vary widely between people.

The milder diuretic effect is attractive for the second daily dose.

After 6 years the early morning diuresis has become a normal process, but once a day is really enough. So my initial trial was Azosemide in the afternoon, while retaining bumetanide in the morning.

After a week or so there were clear signs that benefits initially enjoyed from Bumetanide have been further extended.  This is exactly as the German research suggested might occur.

After a few weeks of 2mg Bumetanide at 7am and 60mg Azosemide at 4pm I moved on to Azosemide 60mg twice a day.

Is Azosemide 60 mg more potent than Bumetanide 2mg?  It is early days, but quite possibly it is.

Bumetanide is very cheap and we have got used to the early morning diuresis, so I am less bothered with the 7am drug.

After a few years drinking a lot of water, to compensate for the diuresis of bumetanide, has become a habit. So switching from Bumetanide to Azosemide does not stop diuresis, just the urgency.

In future-users going straight to Azosemide might be a good choice.

In our case it means that a potent second daily dose is a very practical option.

Anecdotal changes include:-

Very appropriate use of bad language while driving. We live in a country with some aggressive drivers and Monty hears many people’s verbal responses to this.  Now Monty makes the comments for us.  Everyone noticed and big brother was particularly impressed.

“Car’s coming!” while extracting my car from being boxed in by three other cars in a car park, Monty noticed another car coming towards us. For the first time ever Monty has given me a loud verbal warning of danger.  He has since repeated this.  I have long wondered how a person with severe autism can ever safely drive a car, because they lack situational awareness. Many people with severe autism never learn to safely cross a road on foot.

Monty improved use of his second language. He is declining nouns and translating out loud captions and phrases he sees in cartoons.

One area I hoped would improve was at the dentist. Back in March, before the summer allergy season, we had excellent behaviour at the dentist. This gradually changed and the dentist noted this.  We are slowing repairing 2 teeth without removing the nerves and this requires visits every 7 weeks to gradually remove the decay and grow a new layer of dentine above the nerve. After Azosemide the recent anxiety disappeared and Monty’s behaviour at the dentist went back to being very cheerful and entirely cooperative.  


How to access Azosemide tablets

Thanks to our doctor reader Rene, we know that you can order Japanese drugs in specialist “international pharmacies” in Germany with a valid prescription from any European country.

So all you need is a prescription and the money.

Azosemide is available in Japan as a branded product DIART and as a cheaper generic sold as Azosemide.

The price does vary on which pharmacy you approach in Germany, one pharmacy offers these prices:-

100 Tablets   ~ 74€
           500 Tablets   ~ 286€
         1000 Tablets  ~ 524€


This is much more expensive than generic Bumetanide, but less expensive than many supplements people are buying.

If you live in North America you would have to find a different method, or take a trip to Germany.


Conclusion

Azosemide is still “under investigation”, but the prospects look good.

As with Bumetanide, it was approved as a drug a few decades ago and so there is a great deal of safety information. It is not an experimental drug; we are just looking at repurposing it for autism and other neurological conditions with elevated chloride.

Azosemide for autism is a good example of parent cooperation and self-help. Several parents have helped in this step forward for autism treatment.

More work has to be done to see how others respond and what the most effective dosage is.

I suspect that the optimal treatment will be twice a day and the lack of substantial diuresis in most people makes it more practical than Bumetanide twice a day.  Combining Bumetanide, a short acting diuretic, with Azosemide, a long acting diuretic, is also an option to explore.

The potential risk factors are the same as Bumetanide, disturbed electrolytes, dehydration and at very high doses ototoxicity. Ototoxicity is damage to your ear that can be caused by drugs that include diuretics at very large doses.

Azosemide would appear to have milder side effects than Bumetanide.