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Showing posts with label Down Syndrome. Show all posts
Showing posts with label Down Syndrome. Show all posts

Friday 24 April 2020

The Ketone D-BHB as a Medical Food for Heart, Kidney and Brain Disease (Alzheimer’s, some Autism …)



 Nestle’s research centre in Lausanne, Switzerland
I did write extensively about the potential to treat some autism using the ketone BHB (beta hydroxybutyrate). This can be achieved either by following a strict ketogenic diet or just by eating medical foods that contain/produce BHB.
Some readers are now big consumers of BHB supplements and anyone taking BHB should be interested in today’s paper, that I assume was paid for by Nestlé.
Nestlé make everything from baby milk formula to George Clooney’s Nespresso.  You may not be aware that they also have a business selling medical food; they have been looking at ketones to treat Alzheimer’s for some time.  This is quite similar to Mars developing Cocoa flavanols to improve heart and brain health.
Most ketone supplements are sold to help you lose weight or boost athletic performance.  The military also uses ketones in survival rations. 
We saw that you can increase the level of ketones in your body by supplementing: -
·        MCT oil (medium chain triglyceride oil, which usually contains about 60% caprylic C8 acid and 40% capric C10 acid).  This is a product already sold by Nestlé
·        Neat caprylic acid, C8
·        BHB salts (potassium, sodium, calcium etc)
·        BHB esters (also called ketone esters KE)
These products range from expensive to very expensive.
People requiring ketones as an alternative fuel to glucose, like those with Alzheimer’s need quite large amounts of the supplements.  In Alzheimer’s a glucose transporter at the blood brain barrier is restricting the flow of glucose in blood and so the brain is starved of “fuel”.  Mitochondria in the brain can be powered by both ketones and glucose, so if not enough glucose cannot get through, you have the option to increase the amount of ketones.
Babies fed with mother’s milk are on a high ketone diet.  You can safely combine both glucose and ketones as a fuel for your body.
The news from today’s paper has already been translated to a usable therapy. 
There is growing interest in the metabolism of ketones owing to their reported benefits in neurological and more recently in cardiovascular and renal diseases. As an alternative to a very high fat ketogenic diet, ketones precursors for oral intake are being developed to achieve ketosis without the need for dietary carbohydrate restriction. Here we report that an oral D-beta-hydroxybutyrate (D-BHB) supplement is rapidly absorbed and metabolized in humans and increases blood ketones to millimolar levels. At the same dose, D-BHB is significantly more ketogenic and provides fewer calories than a racemic mixture of BHB or medium chain triglyceride. In a whole body ketone positron emission tomography pilot study, we observed that after D-BHB consumption, the ketone tracer 11C-acetoacetate is rapidly metabolized, mostly by the heart and the kidneys. Beyond brain energy rescue, this opens additional opportunities for therapeutic exploration of D-BHB supplements as a “super fuel” in cardiac and chronic kidney diseases.
One of the main benefits of ketones is their ability to act as an alternative energy source to glucose or fatty acids for production of ATP by mitochondria. Caloric restriction and intermittent fasting also produce transient mild-moderate ketosis (6, 7).
While a high dose of MCT can provide a moderate increase in blood ketones (+0.5–1.0 mM), gastrointestinal intolerance and high caloric load limit their use. Second, ketone esters (KE) made of a BHB ester linked to butanediol provide one molecule of D-BHB after digestion, with the butanediol being further metabolized by the liver to D-BHB (9). KE increase blood ketones above 1 mM but are also limited at high dose by their gastric tolerability and severe bitterness (10).
Third, perhaps the most physiologic way to raise blood ketones is via the oral intake of D-BHB itself. Exogenous D-BHB is directly absorbed into the circulation, with some of it being converted to AcAc by the liver, and both ketones being distributed throughout the body. Until recently, only racemic mixtures of dextro (D) and levo (L) BHB (D+L-BHB) were available and oral human studies with them have been reported (9, 1114). As L-BHB is not metabolized significantly into energy intermediates and is slowly excreted in the urine (9, 15), D+L-BHB would be anticipated to be less ketogenic than pure D-BHB. 
Levo, Dextro and Racemic
When certain chemicals are manufactured, they usually contain an equal mixture of the left-handed and right-handed version, this is called a racemic mixture. These versions are called enantiomers.
One enantiomer is an optical stereoisomer of another enantiomer. The two molecules are mirror images of each other, which are not superimposable - much like your left and right hand.
In the case of the chemical BHB, only the right-handed version has an effect on your body.  If you take the salt potassium BHB, half of the product has no effect other than raise your level of potassium.
Zyrtec is an antihistamine made of Cetirizine, but it is a racemic mixture.  If you want pure L-Cetirizine, you would buy Xyzal not Zyrtec.
Arbaclofen/ R-baclofen is the right-handed version of baclofen
Rezular/R-verapamil is the right-handed version of verapamil.
Back to the study:
The study compared three therapies: -

D-BHB

14.1 g of pure salts of the D enantiomer of D-BHB were used. The D-BHB supplement tested was formulated as a mixture of three salts: sodium D-beta-hydroxybutyrate, magnesium (D-beta-hydroxybutyrate and calcium (D-beta-hydroxybutyrate). Each oral serving provided 12 g D-beta-hydroxybutyric acid, 0.78 g sodium, 0.42 g magnesium, and 0.88 g calcium, citrus flavouring and sweetener (Stevia), dissolved in 150 mL of drinking water.

D+L-BHB

14.5 g of an equimolar mixture of commercial D and L beta-hydroxybutyrate salt was used (KetoCaNa, KetoSports, USA). Each serving provided a mixture of 12 g D+L-Beta-hydroxybutyric acid, 1.3 g sodium, 1.2 g calcium, orange flavoring and stevia, dissolved in 150 mL of drinking water.

MCT oil

Fifteen grams of medium chain triglyceride (MCT) (60% caprylic C8 acid and 40% capric C10 acid) emulsified in 70 mL of a 5% aqueous milk protein solution.


This chart shows the concentration of ketones in your blood plasma after taking either of the three therapies.

This chart shows the concentration of just the ketone D-BHB in your blood plasma after taking either of the three therapies.
 This chart shows the concentration of the ketone ACAc in your blood plasma after taking either of the three therapies.
  

This chart shows where the ketones are going; the chart shows the distribution of the ketone “tracer” acetoacetate (AcAc) by organ after D-BHB oral intake.  The effect is greatest on the heart and kidney, but some does reach the brain.

From the dynamic brain scan, CMRAcAc and KAcAc could be determined for all main regions of the brain and compared to baseline values previously determined in healthy young adults. Overall and compared to baseline, each region demonstrated an increase in CMRAcAc and KAcAc of ~4.7 and 2.3-fold, respectively, about 1 h after taking D-BHB. This indicated that AcAc is effectively taken by the brain and by other organs particularly the heart and the kidney.
Ketone production from an exogenous dietary source has been traditionally achieved by MCT. This requires a bolus intake to saturate the liver with MCFA, producing excess acetyl-CoA which is then transformed to AcAc and BHB, which are released into systemic circulation. The Cmax achieved with MCT is usually between 300 and 600 μM, with higher values being difficult to reach due to GI side effects and liver saturation. Here we show that D-BHB, a natural and biologically active ketone isomer, raises blood ketone Cmax above 1 mM without noticeable side effects. In comparison, an equivalent dose of D+L-BHB or MCT only achieved half this ketone level, with similar Tmax at 1 h. Thus, compared to D+L-BHB, D-BHB significantly reduces the salt intake needed to achieve the same plasma ketone response.
Results from a previous study (9) comparing KE to D+L-BHB showed that at the same dose of D-BHB equivalent, the increase blood ketone iAUC had the same magnitude, suggesting that exogenous D-BHB and KE produce similar ketosis.
Note that KE means Ketone Ester and the study (9) is this one: -

On the Metabolism of Exogenous Ketones in Humans

Ketone esters are available, but horribly expensive and taste really bad.

Conclusion
In previous posts the numerous possible beneficial modes of action of BHB were outlined. The summary post is here: -

Ketone Therapy in Autism (Summary of Parts 1-6)

In practise some people with autism seem to benefit a lot, some moderately and some not at all.
Monty, aged 16 with ASD, fits in the “moderately benefits” category.  The combination of about 20ml of caprylic acid (C8) plus a scoop of Potassium BHB powder does produce more speech.
It is not a cheap or very convenient therapy, compared the others I use.
I would agree with Nestlé that the limiting factor with BHB salts is the “salt”.  As they comment in their paper 
“compared to D+L-BHB, D-BHB significantly reduces the salt intake needed to achieve the same plasma ketone response”
Giving someone with heart disease "sodium anything" is not a good idea. A potassium salt would be safer, but even then, your heart is the limiting factor on potassium use.  Calcium salts are unwise in people with autism, because it appears to be able to upset calcium ion signalling, which would also be a potential risk in heart disease.
As I mentioned to one parent who is a big time user of BHB salts, if you switch to D-BHB you can either produce twice the ketones of regular potassium BHB, with the existing potassium load, or reduce your dosage by half and keep the same effect and save some money.
I think potassium D-BHB is good choice.  If you are taking bumetanide you may no longer need a potassium supplement (K-BHB becomes your potassium supplement).
I think people with autism and genuine mitochondrial disease are highly likely to benefit from D-BHB.  These are people who show symptoms in their entire body, i.e. lack of exercise endurance. For these people, eating (or producing via diet) large amounts of ketones will increase the production of ATP in their brains and so improve cognitive function.  D-BHB undergoes a different process to glucose, as it “converted” to ATP by the process called OXPHOS
(Oxidative phosphorylation). Some people with autism lack the enzyme complexes needed to complete OXPHOS, these people who should try D-BHB.
BHB has other beneficial effects, some relating to inflammation that seem to explain its benefit in other types of autism.  The effects were investigated here.
In the brains of people with Alzheimer’s there is decreased expression of glucose transporter 1 (GLUT 1) at the blood brain barrier. This starves the brain of glucose, which is fuel for the brain. D-BHB is an alternative fuel for mitochondria that is not dependent on GLUT 1.  People with early onset Alzheimer's would seem the best ones for this therapy, that would include many people with Down Syndrome. 


Tuesday 14 May 2019

Making best use of existing NKCC1/2 Blockers in Autism






Azosemide C12H11ClN6O2S2  


Today’s post may be of interest to those already using bumetanide for autism and for those considering doing so.  It does go into the details, because they really do matter and does assume some prior knowledge from earlier posts.

There has been a very thorough new paper published by a group at Johns Hopkins:-
It does cover all the usual issues and raises some points that have not been covered yet in this blog.  One point is treating autism prenatally. This issue was studied twice in rats, and the recent study was sent to me by Dr Ben Ari.  Short term treatment during pregnancy produced a permanent benefit.

Maternal bumetanide treatment prevents the overgrowth in the VPA condition

            
Brief maternal administration of bumetanide before birth restores low neuronal intracellular chloride concentration ([Cl]i) levels, produces an excitatory-to-inhibitory shift in the action of γ-aminobutyric acid (GABA), and attenuates the severity of electrical and behavioral features of ASD (9, 10), suggesting that [Cl]i levels during birth might play an important role in the pathogenesis of ASD (7). Here, the same bumetanide treatment significantly reduced the hippocampal and neocortical volumes of P0 VPA pups, abolishing the volume increase observed during birth in the VPA condition [hippocampus: P0 VPA versus P0 VPA + BUM (P = 0.0116); neocortex: P0 VPA versus P0 VPA + BUM (P = 0.0242); KWD] (Fig. 3B). Maternal bumetanide treatment also shifted the distribution of cerebral volumes from lognormal back to normal in the population of VPA brains, restoring smaller cerebral structure volumes (Fig. 3C). It also decreased the CA3 volume to CTL level after birth, suggesting that the increased growth observed in this region could be mediated by the excitatory actions of GABA (Fig. 3D). Therefore, maternal bumetanide administration prevents the enhanced growth observed in VPA animals during birth.

One issue with Bumetanide is that it affects both:-

·        NKCC2 in your kidneys, causing diuresis
·        NKCC1 in your brain and elsewhere, which is divided into two slightly different forms NKCC1a and NKCC1b

NKCC1 is also expressed in your inner ear where it is necessary for establishing the potassium-rich endolymph that bathes part of the cochlea, an organ necessary for hearing. 

If you block NKCC1 too much you will affect hearing.

Blocking NKCC1 in children and adults is seen as safe but the paper does query what the effect on hearing might be if given prenatally as the ear is developing.

Treating Down Syndrome Prenatally

While treating autism prenatally might seem a bit unlikely, treating Down Syndrome (DS) prenatally certainly is not.  Very often DS is accurately diagnosed before birth creating a valuable treatment window.  In most countries the vast majority of DS prenatal diagnoses lead to termination, but only a small percentage of pregnancies are tested for DS. In some countries such as Ireland a significant number of DS pregnancies are not terminated, these could be treated to reduce the deficits that will otherwise inevitably follow.



The research does suggest that DS is another brain disorder that responds to bumetanide.


Back to autism and NKCC1

This should remind us that a defect in NKCC1 expression will not only cause elevated levels of chloride with in neurons, but will also affect the levels of sodium and potassium with neurons.

There are many ion channel dysfunctions (channelopathies) implicated in autism and elevated levels of sodium and potassium will affect numerous ion channels.  The paper does suggest that the benefit of bumetanide may go beyond modifying the effect of GABA, which is the beneficial mode of action put forward by Dr Ben Ari.
We have seen how hypokalemic sensory overload looks very similar to what often occurs in autism and that autistic sensory overload is reduced by taking an oral potassium supplement.

The paper also reminds us that loop diuretics like bumetanide and furosemide not only reduce inflow of chloride into neurons, but may also reduce the outflow. This is particularly known of furosemide, but also occurs with bumetanide at higher doses.
The chart below shows that the higher the concentration of bumetanide the strong its effect becomes on blocking NKCC1.


But at higher doses there will also be a counter effect of closing the NKCC2 transporter that allows chloride to leave neurons.
At some point a higher dose of bumetanide may have a detrimental effect on trying to lower chloride within neurons.

Since Dr Ben Ari’s objective is to lower chloride levels in neurons  it is important how freely these ions both enter and exit.  The net effect is what matters. (Loop diuretics block NKCC1 that lets chloride enter neurons but also block the KCC2 transporter via which they exit)

Is Bumetanide the optimal existing drug to lower chloride within neurons?  Everyone agrees that it is not, because only a tiny amount crosses into the brain. The paper gives details of the prodrugs like BUM5 that have been looked at previously in this blog; these are modified versions of bumetanide that can better slip across the blood brain barrier and then react in the brain to produce bumetanide itself.  It also highlights the recent research that suggests that Bumetanide may not be the most potent approved drug, it is quite conceivable that another old drug called Azosemide is superior.

The blood brain barrier is the problem, as is often the case.  Bumetanide has a low pH (it is acidic) which hinders its diffusion across the barrier.  Only about 1% passes through.

There is scepticism among researchers that enough bumetanide can cross into the brain to actually do any good.  This is reflected in the review paper.

The paper reminds us of the research showing how you can boost the level of bumetanide in the brain by adding Probenecid, an OAT3 inhibitor.  During World War 2 antibiotics were in short supply and so smaller doses were used, but their effect was boosted by adding Probenecid. By blocking OAT3, certain types of drug like penicillin and bumetanide are excreted at a slower rate and so the net level in blood increases.

The effect of adding Probenecid, or another less potent OAT3 inhibitors, is really no different to just increasing the dose of bumetanide.

The problem with increasing the dose of bumetanide is that via its effect on NKCC2 you cause even more diuresis, until eventually a plateau is reached.

Eventually, drugs selective for NKCC1a and/or NKCC1b will appear.

In the meantime, the prodrug BUM5 looks good. It crosses the BBB much better than bumetanide, but it still affects NKCC2 and so will cause diuresis.  But BUM5 should be better than Bumetanide + Probenecid, or a higher dose of Bumetanide.  BUM5 remains a custom-made research drug, never used in humans.

I must say that what again stands out to me is the old German drug, Azosemide.

In a study previously highlighted in this blog, we saw that Azosemide is 4 times more potent than Bumetanide at blocking NKCC1a and NKCC1b.

Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B

Azosemide is used in Japan, where recent research shows it is actually more effective than other diuretics

Azosemide, a Long-acting Loop Diuretic, is Superior to Furosemide in Prevention of Cardiovascular Death in Heart Failure Patients Without Beta-blockade 

As is often the case, Japanese medicine has taken a different course to Western medicine.

Years of safety information has already been accumulated on Azosemide.  It is not an untried research drug. It was brought to market in 1981 in Germany. It is available as Diart in Japan made by Sanwa Kagaku Kenkyusho and as a cheaper generic version by Choseido Pharmaceutical. In South Korea Azosemide is marketed as Uretin.


In any other sector other than medicine, somebody would have thought to check by now if Azosemide is better than Bumetanide.  It is not a matter of patents, Ben-Ari has patented all of the possible drugs, including Azosemide and of course Bumetanide.

So now we move on to Azosemide.



When researchers came to check the potency of the above drugs the results came as a surprise.  It turns out that the old German drug Azosemide is 4 times as potent as bumetanide.






The big question is how does it cross the blood brain barrier.


“The low brain concentrations of bumetanide obtained after systemic administration are thought to result from its high ionization (>99%) at physiological pH and its high plasma protein binding (>95%), which restrict brain entry by passive diffusion, as well as active efflux transport at the blood-brain barrier(BBB). The poor brain penetration of bumetanide is a likely explanation for its controversial efficacy in the treatment of brain diseases

“… 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 favor 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.”


Dosage equivalents of loop Diuretics


Bumetanide has very high oral bioavailablity, meaning almost all of what you swallow as a pill makes it into your bloodstream.

Furosemide and Azosemide have much lower bioavailability and so higher doses are needed to give the same effect.

Both Furosemide and Bumetanide are short acting, while Azosemide is long acting.

For a drug that needs to cross the blood brain barrier small differences might translate into profoundly different effects.

The limiting factor in all these drugs is their effect on NKCC2 that causes diuresis.

1mg of bumetanide is equivalent to 40mg of furosemide.
2mg of bumetanide is equivalent to 80mg of furosemide.

The standard dose for Azosemide in Japan, where people are smaller than in the West, is 30 mg or 60mg. 

Research suggests that the same concentration of Azosemide is 4x more potent than Bumetanide at blocking NKCC1 transporters, other factors that matter include:-

·        How much of the oral tablet ends up in the bloodstream.
·        How long does it stay in the blood stream
·        How much of the drug actually crosses the blood brain barrier
·        How does the drug bind to the NKCC1 transporters in neurons
·        How rapidly is the drug excreted from the brain
·        What effect is there on the KCC2 transporter that controls the exit of chloride ions from neurons.

All of this comes down to which is more effective in adults with autism 2mg of bumetanide or 60mg of Azosemide.

The side effects, which are mainly diuresis and loss of electrolytes will be similar, but Azosemide is a longer acting drug and so there will be differences. In fact Azosemide is claimed to be less troublesome than Bumetanide in lower potassium levels in your blood.

Conclusion  

The open question is whether generic Azosemide is “better” than generic Bumetanide for treating brain disorders in humans.

I did recently ask Dr Ben-Ari if he is aware of any data on this subject. There is none.

Many millions of dollars/euros are being spent getting Bumetanide approved for autism, so it would be a pity if Azosemide turns out to be better. (Dr Ben Ari’s company Neurochlore wants to develop a new molecule that will cross the blood brain barrier, block NKCC1 and not NKCC2 and so will not cause diuresis).

The hunch of the researchers from Hanover, Germany seems to be that the old German drug Azosemide will be better than Bumetanide.

I wonder if doctors at Johns Hopkins / Kennedy Krieger have started to prescribe bumetanide off-label to their patients with autism.  Their paper shows that they have a very comprehensive knowledge of the subject.


===========

I suggest readers consult the full version of the Johns Hopkins review paper on Bumetanide, it is peppered with links to all the relevant papers.

Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Clgradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Climporter and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN’s brain-availability and consider its actions on non-neuronal cells.

Btn Pro-Drugs and Analogs

To improve BTN accessibility to the brain, pro-drugs with lipophilic and uncharged esters, alcohol and amide analogs have been created. These pro-drugs convert to BTN after gaining access into the brain. There was a significantly higher concentration of ester prodrug, BUM5 (N,N – dimethylaminoethyl ester), in mouse brains compared to the parent BTN (10 mg/kg, IV of BTN and equimolar dose of 13 mg/kg, IV of BUM5) (Töllner et al., 2014). BUM5 stopped seizures in adult animal models where BTN failed to work (Töllner et al., 2014Erker et al., 2016). BUM5 was also less diuretic and showed better brain access when compared to the other prodrugs, BUM1 (ester prodrug), BUM7 (alcohol prodrug) and BUM10 (amide prodrug). BUM5 was reported to be more effective than BTN in altering seizure thresholds in epileptic animals post-SE and post-kindling (Töllner et al., 2014). Furthermore, BUM5 (13 mg/kg, IV) was more efficacious than BTN (10 mg/kg, IV) in promoting the anti-seizure effects of PB, in a maximal electroshock seizure model (Erker et al., 2016). Compared to BUM5 which was an efficacious adjunct to PB in the above mentioned study, BTN was not efficacious when administered as an adjunct (Erker et al., 2016). In addition to seizure thresholds, further studies need to be conducted to assess effects of BUM5 on seizure burdens, ictal events, duration and latencies.
Recently, a benzylamine derivative, bumepamine, has been investigated in pre-clinical models. Since benzylamine derivatives lack the carboxylic group of BTN, it results in lower diuretic activity (Nielsen and Feit, 1978). This prompted Brandt et al. (2018) to explore the proposed lower diuretic activity, higher lipophilicity and lower ionization rate of bumepamine at physiological pH. Since it is known that rodents metabolize BTN quicker than humans, the study used higher doses of 10 mg/kg of bumepamine similar to their previous BTN studies (Olsen, 1977Brandt et al., 2010Töllner et al., 2014). Bumepamine, while only being nominally metabolized to BTN, was more effective than BTN to support anticonvulsant effects of PB in rodent models of epilepsy. This GABAergic response, however, was not due to antagonistic actions on NKCC1; suggesting bumepamine may have an off-target effect, which remains unknown. However, the anticonvulsive effects of bumepamine, in spite of its lack of action on NKCC1, are to be noted. Additionally, in another study by the same group, it was shown that azosemide was 4-times more potent an inhibitor of NKCC1 than BTN, opening additional avenues for better BBB penetration and NKCC1-antagonizing compounds for potential neurological drug discovery (Hampel et al., 2018).

Conclusion


The beneficial effects of BTN reported in cases of autism, schizophrenia and TLE, given its poor-brain bioavailability are intriguing. The mechanisms underlying the effects of BTN, as a neuromodulator for developmental and neuropsychiatric disorders could be multifactorial due to prominent NKCC1 function at neuronal and non-neuronal sites within the CNS. Investigation of the possible off-target and systemic effects of BTN may help further this understanding with the advent of a new generation of brain-accessible BTN analogs.