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Showing posts with label Bumetanide. Show all posts
Showing posts with label Bumetanide. Show all posts

Thursday, 22 August 2019

Bumetanide 5mg for Parkinson’s Disease?



I have been asked twice about off-label therapies for Parkinson’s, both times I mentioned Bumetanide, but having rechecked the literature, there is now plenty of supporting data, enough that a clinical trial has now been put in motion in France.

Parkinson’s disease is all about a lack of dopamine and bumetanide is all about making GABA work as inhibitory. You might wonder why is Peter suggesting people to talk to their doctor about giving their elderly parents a diuretic. Well the lack of dopamine goes on to cause a GABA dysfunction, which is treatable and does improve the symptoms of Parkinson’s.

So, Bumetanide will not cure Parkinson’s, but may reduce its severity.

In the case of the last person who asked me, her mother already takes a diuretic for other reasons, so all she would have to do is to switch drugs to Bumetanide. The doctor was only too happy, when given the evidence, to switch her to Bumetanide - a rare victory for common sense. 

What caught my attention was the dosage of Bumetanide used in the published case histories and the concern about polyuria. Polyuria is too much urination. The dose used was 5mg taken all in one go and that is a lot; you would have to run to the bathroom, which might cause falls in people with poor balance.

Since we recently discovered that Azosemide has the same effect on GABA as Bumetanide, but can have a less urgent effect as a diuretic, it may be that Azosemide is a better choice for Grandma with Parkinson’s.  Incontinence can be a feature of Parkinson’s disease.  The ideal drug will be the new one being developed by Neurochloré for autism.


Standard Parkinson’s Drugs

Since most symptoms of Parkinson’s disease (PD) are caused by a lack of dopamine in the brain, many PD drugs are aimed at either temporarily replenishing dopamine or mimicking the action of dopamine. These types of drugs are called dopaminergic medications. They generally help reduce muscle rigidity, improve speed and coordination of movement and lessen tremor.

L-DOPA, the standard treatment for Parkinson’s is actually also used in some people with autism, in particular people with Angelman Syndrome, although it failed in a clinical trial.


Bumetanide for Parkinson’s?

The clinical trial for Parkinson’s will use the standard rating scale (UPDRS) that is very much centered on motor skills. There is a tiny part on memory.

Cognition is affected in Parkinson’s and this might be another area that improves with Bumetanide; but someone has to bother to measure it.

Nobody has measured the effect of Bumetanide on IQ in those with autism, even though the effect can be substantial.

                                  


Four patients suffering from idiopathic PD at the stage of motor fluctuation were included. All of them gave their written informed consent to receive open-label bumetanide. Bumetanide was progressively titrated up to 3 mg/d (once daily) received for a month. After having verified the good tolerability of the treatment, bumetanide was increased to 5 mg/d (once daily) and received for another month. Bumetanide was added to the patient's usual antiparkinsonian treatment that was maintained stable the month before and unchanged during the study. The patients were assessed before and at 1 and 2 months after the initiation of bumetanide.
At each visit, the patient was asked about any side effects having occurred since the last visit. A Unified Parkinson's Disease Rating Scale (UPDRS)19 was performed before and after 2 months of treatment in a practical OFF stage (the patients came in the afternoon, having not taken antiparkinsonian drugs for 4 hours, and confirmed to be in an OFF stage). At the end of the study, the patient was also asked to give a global impression of change compared with baseline.

Case 3

The patient was a 58-year-old man with a 21-year history of
PD. After early development of disabling motor fluctuation and dyskinesia despite an optimized drug treatment, bilateral subthalamic electrodes were implanted 16 years ago for continuous deep brain stimulation (DBS). He got an excellent control of PD motor symptoms. However, after a year of DBS treatment, he started to develop freezing of gait and dysarthria. Despite many attempts of adjusting the treatment (DBS parameters, changes in drug treatment, and physiotherapy), these symptoms remained disabling and even slowly worsened with time. Motor fluctuation and dyskinesia were well controlled by both DBS (left side: case positive, electrode 2 negative, voltage 3.5 V; right side: case positive, electrode 1 negative, voltage 3 V; for both sides: pulse width 60 microseconds, frequency 100 Hz) and drug treatment. The latter consisted of L-DOPA, 1000 mg/d (5 intakes per day); ropinirole, 2 mg/d; and amantadine, 200 mg/d. The freezing of gait was highly disabling.

At home, the patient could walk a few steps alone with a high risk of falls. Most of the time, he was wheelchair bound. After a few days of bumetanide at a dosage of 5 mg/d, the gait dramatically improved. He was able to walk almost 1000 m without any help.

The voice was unchanged. The UPDRS III in the OFF stage was hardly changed (10% improvement), and the UPDRS II in the worst state improved by 15%. The UPDRS II in the best condition was unchanged (21 to 18). The patient and the caregiver assessed the general improvement at 50%. Despite the polyuria and the fatigue, he has decided to continue the bumetanide treatment.
After a few weeks, the improvement of gait was less dramatic but still noticeable.


GABAergic inhibition in dual-transmission cholinergic and GABAergic striatal interneurons is abolished in Parkinson disease 

We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl]i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl]ilevels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson’s disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl]i levels and GABAergic inhibition.



Official Title:
A Randomized Double-blind Placebo-controlled Multicenter Proof-of-concept Trial to Assess the Efficacy and Safety of Bumetanide in Parkinson's Disease
Actual Study Start Date  :
April 26, 2019
Estimated Primary Completion Date  :
September 2020
Estimated Study Completion Date  :
August 2021



Conclusion

There is now a long list of neurological conditions that may respond to bumetanide:-

·        Autism
·        Fragile-X Syndrome
·        Down Syndrome
·        Schizophrenia
·        Huntington’s Disease
·        Parkinson’s Disease

In addition, it is obvious that some epilepsy will respond to Bumetanide. The original epilepsy drug from 150 years ago, KBr, has the same mechanism of action, lowering chloride within neurons.

Perhaps higher doses of Bumetanide need to be trialled in autism, 5mg all at once is far higher than what has been used so far in studies.




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.




Tuesday, 25 June 2019

Learning from GABAa Dysfunction in Huntington’s Disease – useful ideas for Autism therapies?



Today’s post is really for the regular readers of this blog who are interested in the GABA switch and Bumetanide. It is not light reading.  We see how advanced some Taiwanese researchers are in their understanding of GABAA dysfunctions in Huntington’s Disease.




Taipei 101, briefly the world’s tallest building


It is an excellent paper and much of it is applicable to autism. There are some omissions, but you will struggle to find a more complete paper.

They even go into the detail of altered the sub-unit expression of GABAA receptors that occurs as the disease progresses. I think that correcting sub-unit miss-expression has great potential in treating some autism.

Huntington’s is an inherited brain disorder that first manifests itself around the age of 40 and then progresses for the next 15 to 20 years.

Much autism is present prior to birth but there is a progression that occurs as the brain develops in early childhood. Some people do seem to be entirely typical at birth and only around 2 years old develop symptoms. After 5 years old you cannot really develop “autism”, just the symptoms might not get noticed till later in life.
Schizophrenia only develops in early to mid-adulthood.

It is surprising to many people that such varied disorders share some similar aspects of biology.

In terms of practical interventions, in today’s paper these include:       

·        Inhibition of NKCC1 (bumetanide)
·        Activation of KCC2 (N-Ethylmaleimide)
·        Enhancer of CKB (creatine)
·        Inhibitor of WNK/SPAK
·        Activation of extra-synaptic GABAa receptors (taurine, progesterone)
·        Activation of synaptic GABAa receptors (zolpidem, alprazolam)
·        Inhibition of GABA transport mechanism (Tiagabine)

One thing to note is that activating GABAa receptors may well have a negative effect in some people.

Sub-unit specific therapies, like very low dose clonazepam targeting α3, are not mentioned in this paper, nor is the role of GABAb on NKCC1/KCC2 expression.

We are familiar with Bumetanide as an NKCC1 blocker intervention in autism, but looking at the list there are other common autism therapies (creatine and taurine) and the female hormone progesterone. We come upon the beneficial effect of female hormones on a regular basis in this blog (estradiol, pregnenalone, progesterone …).  We even saw how a sub-SSRI dose of Prozac increases the amount of the neurosteroid 3α-hydroxy-5α-pregnan-20-one (Allo) that potently, positively, and allosterically modulates GABA action at GABAA receptors. Progesterone is converted to Allo in the body.
 
Here is the excellent paper on Huntington’s:-






                                                                                                               

An overview of the g-aminobutyric acid (GABA) signalling system. (a) GABA homeostasis is regulated by neurons and astrocytes. GABA is synthesized by GAD65/67 from glutamate in neurons, while astrocytic GABA is synthesized through MAOB. The release of GABA is mediated by membrane depolarization in neurons and Best1 in astrocytes. The reuptake of GABA is mediated through GAT1 in neurons and GAT3 in astrocytes. The metabolism of GABA is mediated by GABA-T in neurons and astrocytes. The reuptake of GABA in astrocytes is further transformed into glutamine via the TCA cycle and glutamine synthetase (GS). The glutamine is then transported to neurons and converted to glutamate for regeneration of GABA.



(b) GABAA receptors are heteropentameric complexes assembled from 19 different subunits. The compositions of different subunits determines the subcellular distributions and functional properties of the receptors. Phasic inhibition is mediated via the activation of synaptic GABAA receptors following brief exposure to a high concentration of extracellular GABA. Tonic inhibition is mediated via the activation of extrasynaptic GABAA receptors by a low concentration of ambient GABA.






c) The excitatory inhibitory response of GABA is driven by the chloride gradient across cell membranes, which can be determined via two cation–chloride cotransporters (NKCC1 and KCC2). The high expression of NKCC1 during the developmental stage maintains higher intracellular [Cl2] via chloride influx to the cell. The activation of GABAA receptors at an early developmental stage results in an outward flow of chloride and an excitatory GABAergic response. As neurons mature, the high expression of KCC2 maintains lower intracellular [Cl2] via chloride efflux out of the cell. The activation of GABAA receptors on mature neurons results in the inward flow of chloride and an inhibitory GABAergic response.



An excerpt showing data on sub-unit misexpression in different parts of the brain at different stages of the disease



5.2. Modulation of chloride homeostasis via cation – chloride cotransporters
Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e. NKCC1 or KCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal excitatory GABAA receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function of HD mice. The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice. This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143]. Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/ SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition. Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation –chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1. It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.






Figure 2. Molecular mechanism(s) underlying the abnormal GABAAergic system in HD. (a) In the normal condition, adult neurons express high KCC2 and few NKCC1 to maintain the lower intracellular chloride concentration, which results in an inward flow of chloride when GABAA receptors are activated. Astrocytes function normally for the homeostasis of glutamate, potassium and glutamate/GABA-glutamine cycle. (b) In Huntington’s disease, reduced GABAA receptor-mediated neuronal inhibition is associated with enhanced NKCC1 expression and a decreased expression in KCC2 and membrane localized GABAA receptors. The dysregulated GABAAergic system might be caused by mutant HTT, excitotoxicity, neuroinflammation or other factors. Mutant HTT in neurons alters the transcription of genes (GABAAR and KCC2) through interactions with transcriptional activators (SP1) and repressors (REST/NRSF). Mutant HTT in neurons also disrupts the intracellular trafficking of GABAARs to the cellular membrane. HD astrocytes have impaired homeostasis of extracellular potassium/glutamate (due to deficits of astrocytic Kir4.1 channel and glutamate transporters, Glt-1) and cause neuronal excitability, which might be related to the changes of KCC2, NKCC1 and GABAAR. The activity of KCC2 could be affected through its interacting proteins, such as CKB and mHTT. Neuroinflammation, which is evoked by the interaction of HD astrocyte and microglia, enhances NKCC1 expression in neurons at the transcriptional level through an NF-kB-dependent pathway. HD astrocytes also have compromised astrocytic metabolism of glutamate/GABA–glutamine cycle that contributes to lower GABA synthesis.


Notably, neuroinflammation and the GABA neurotransmitter system are reciprocally regulated in the brain (reviewed in [104,105]). Specifically, neuroinflammation induces changes in the GABA neurotransmitter system, such as reduced GABAA receptor subunit expression, while activation
of GABAA receptors likely antagonizes inflammation.

TNF-a, a proinflammatory cytokine, induces a downregulation of the surface expression of GABAARs containing a1, a2, b2/3 and g2 subunits and a decrease in inhibitory synaptic strength in a cellular model of hippocampal neuron culture [106]. The same group further demonstrated that protein phosphatase 1-dependent trafficking of GABAARs was involved in the TNF-a evoked downregulation of GABAergic neurotransmission [107]. Upregulation of TNF-a also negatively impacts the expression of GABAAR a2 subunit mRNA and thus decreases the presynaptic inhibition in the dorsal root ganglion in a rat experimental neuropathic painmodel [108]. Conversely, blockade of central GABAARs in mice by aGABAAR antagonist increased both the basal and restraint stress-induced plasma IL-6 levels [109]. Inhibition of GABAAR activation by picrotoxin increased the nuclear translocation of NF-kB in acute hippocampal slice preparations [110]. Collectively, neuroinflammation
weakens the inhibitory synaptic strength in neurons, at least partly, through the reduction of GABAARs.

The reduced expression and function of GABAARs may further increase inflammatory responses. It remains elusive whether the same mechanism occurs in the inflammatory environment in HD brains.


hyperexcitability resulting from deficiency of astrocytic Kir4.1 might have also contributed to neuronal NKCC1 upregulation and altered GABAergic signalling in HD brains.




Figure 3. Strategy to target (a) GABAAR and (b) cation–chloride cotransporters as potential therapeutic avenues. (a) The GABAergic system is influenced directly by agents that (1) target synaptic GABAAR, (2) increase tonic GABA current or interfere with synaptic GABA concentrations via a reduction of GABA reuptake (3), and (4) block GABA metabolism.

5.1. Modulating the GABAA receptor as a therapeutic target

In view of the presently discovered HD-related deficit in the GABA system, the question arises whether HD patients can benefit from drugs that stimulate the GABA system (figure 3a). HD patients suffer from motor abnormalities and
non-motor symptoms, including cognitive deficits, psychiatric symptoms, sleep disturbance, irritability, anxiety, depression and an increased incidence of seizures [74,77,116,117].
Seizures are a well-established part of juvenile HD but no more prevalent in adult-onset HD than in the general population [73,74,118]. Several pharmacological compounds can enhance inhibitory GABAergic neurotransmission by targeting GABAAR and thereby producing sedative, anxiolytic, anticonvulsant and muscle-relaxant effects. A recent study demonstrated that zolpidem, a GABAAR modulator that enhances GABA inhibition mainly via the a1-containing GABAA receptors, corrected sleep disturbance and electroencephalographic abnormalities in symptomatic HD mice (R6/2) [119]. Alprazolam, a benzodiazepine-activating GABA receptor, reversed the dysregulated circadian rhythms and improved cognitive performance of HD mice (R6/2) [120].
In addition, progesterone, a positive modulator of GABAAR, significantly reversed the behavioural impairment in a 3-nitropropionic acid (3-NP)-induced HD rat model [121]. Apart from modulating the activity of the GABAergic system by interfering directly with the receptor, pharmacological agents can also interfere with synaptic GABA concentrations. Tiagabine, a drug that specifically blocks the GABA transporter (GAT1) to increase synaptic GABA level,was found to improve motor performance and extend survival inN171-82Q and R6/2 mice [122]. It is also worth evaluating whether vigabatrin, a GABA-T inhibitor that blocksGABAcatabolismin neurons and astrocytes [123], plays a role in the compromised astrocytic glutamate–GABA–glutamine cycling [56]. Interestingly, taurine exerted GABAA agonistic and antioxidant activities in a 3-NP HD model and improved locomotor deficits and increased GABA levels [124]. However, several early studies failed to provide the expected benefits of GABA analogues in slowing disease progression in HD patients [125–127]. For example, gaboxadol, an agonist for the extrasynaptic d-containing GABAA receptor, failed to improve the decline in cognitive and motor functions of five HD patients during a short two-week trial, but it caused side effects at the maximal dose [125]. Interestingly, although treatment with muscimol (a potent agonist of GABA receptors) did not improve motor or cognitive deficits in 10HDpatients, it did ameliorate chorea in the most severely hyperkinetic patient [126]. The therapeutic failure of GABA stimulation in early clinical trials does not argue against the importance of GABAergic deficits in HD pathogenesis. The alteration of GABAergic circuits plays a primary role or is a compensatory response to excitotoxicity, and it may contribute to HD by disrupting the balance between the excitation and inhibition systems and the overall functions of neuronal circuits. Because the subunits of the GABAA receptor are brain region- or neuron subtypespecific, the choice of drugs may have distinct effects on the brain region or neuronal population targeted [128–130]. For example, the expression of GABAAR subunits is differentially altered in MSNs and other striatal interneurons in HD 54,60]. The early involvement of D2-expressing MSNs can cause chorea [131], while dysfunctional PV-expressing interneurons can cause dystonia in HD patients [132]. Specific alteration in neuronal populations and receptor subtypes during HD progression needs to be taken into consideration when treating the dysfunction of GABAergic circuitry.
Notably, striatal tonic inhibition mediated by the dcontaining GABAARs may have neuroprotective effects against excitotoxicity in the adult striatum [63]. Because the reductions in d-containing GABAARs and tonic GABA currents in D2-expressing MSNs have been observed in early HD [32,39,40,54,61], it would be of great interest to evaluate the effects of several available compounds, such as alphaxalone and ganaxolone [133], that target d-containing GABAARs, in animal models of HD.





(b) GABAAR-mediated signalling in HD neurons is depolarizing due to the high intracellular chloride concentration caused by high NKCC1 expression and low KCC2 expression. Rescuing the function of cation–chloride cotransporters can occur via (1) inhibition of NKCC1 activity using bumetanide, (2, 3) increase in KCC2 function using a KCC2 activator or CKB enhancer, and (4) inhibitors of WNK/SPAK kinases.


5.2. Modulation of chloride homeostasis via cation–chloride cotransporters

Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e.NKCC1 orKCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal   receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function ofHDmice (R6/2, Y-T Hsu,Y-GChang, Y-CLi, K-YWang, H-MChen, D-J Lee, C-HTsai, C-C Lien,YChern 2018, personal communication). The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice (Y-T Hsu, Y-G Chang, Y-C Li, K-Y Wang, H-M Chen, D-J Lee, C-H Tsai, C-C Lien, Y Chern 2018, personal communication). This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143].
Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition.
Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation–chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.




Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline–alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na+-Cl cotransporter (NCC) and Na+-K+-2Cl cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.


Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.


WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.







N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation


K+/Cl cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.


                                                                  


Tiagabine (trade name Gabitril) is n anticonvulsant medication produced by Cephalon that is used in the treatment of epilepsy. The drug is also used off-label in the treatment of anxiety disorders and panic disorder.

Tiagabine is approved by U.S. Food and Drug Administration (FDA) as an adjunctive treatment for partial seizures in individuals of age 12 and up. It may also be prescribed off-label by physicians to treat anxiety disorders and panic disorder as well as neuropathic pain (including fibromyalgia). For anxiety and neuropathic pain, tiagabine is used primarily to augment other treatments. Tiagabine may be used alongside selective serotonin reuptake inhibitorsserotonin-norepinephrine reuptake inhibitors, or benzodiazepines for anxiety, or antidepressantsgabapentin, other anticonvulsants, or opioids for neuropathic pain.[4]
Tiagabine increases the level of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, by blocking the GABA transporter 1 (GAT-1), and hence is classified as a GABA reuptake inhibitor (GRI).


Conclusion

Today’s post shows how you need to read well beyond the autism research, not to miss something useful.

Some of today’s suggested therapies for Huntington’s are likely to help some types of autism, but some will certainly have a negative effect in some people.  For example, increasing the amount of GABA in the CNS would do my son no good at all.

The emerging field of drugs that enhance KCC2 should be very beneficial to all those with autism who are bumetanide responders.

Enhancing CKB with creatine is interesting. Creatine is a muscle building supplement used by body builders and some DAN doctors. It does have interactions at high doses.