Advances in personalized medicine to treat Autism/IDD – Rett syndrome as an example. Also, Piperine to upregulate KCC2, but what about its direct effect on GABAa receptors?

 

Source:  https://www.cell.com/neuron/pdf/S0896-6273(21)00466-9.pdf

Today’s post is drawn from a workshop I am invited to present at an autism conference in Abu Dhabi.

I decided to talk about advances in personalized medicine – no surprise there.  Since I have 2 ½ hours, I thought I will need some interesting examples to maintain the audiences interest.  One such topic is going to be Rett syndrome.

I regard Rett syndrome and all the other such syndromes in this blog as “single gene autisms” (monogenic autism).  If you apply the American DSM classification, from 2013 onwards Rett syndrome is no longer part of autism.  Hopefully there are no such purists attending in Abu Dhabi. 

Two gene therapies for Rett syndrome are currently undergoing human trials and one drug therapy has been FDA approved.  This looks very encouraging, so let’s dig a little deeper.

Rett syndrome can present with a wide range of disability ranging from mild to severe. 

Rett syndrome is the second most common cause of severe intellectual disability after Down syndrome.

Other symptoms may include:

•      Loss of speech

•      Loss of purposeful use of hands

•      Loss of mobility or gait disturbances

•      Loss of muscle tone

•      Seizures or Rett “episodes”

•      Scoliosis

•      Breathing issues

•      Sleep disturbances

•      Slowed rate of growth for head, feet and hands

Here are the new therapies: 

TSHA-102: This gene therapy, developed by Taysha Therapeutics, is a gene replacement therapy that aims to deliver a functional copy of the MECP2 gene to brain cells.  It utilizes an AAV-9 virus to carry the miniMECP2 gene product into cells for the body to produce more MeCP2 protein, which is deficient in Rett syndrome. As of February 2024, Taysha completed dosing for the first cohort (low dose) in their REVEAL Phase 1/2 adolescent and adult trial in Canada, with positive interim data on safety. They are also conducting trials in the US for both pediatric and adolescent/adult populations.

NGN-401: This gene therapy, by Neurogene Inc., employs a different approach. It uses an AAV9 vector to deliver a regulated version of the MECP2 gene called EXACT. This technology aims to control the amount of MECP2 protein produced by the gene, mitigating the risk of overproduction. NGN-401 is currently in a Phase 1/2 trial for girls with Rett syndrome aged 4 to 10 years old.

Daybue (trofinetide)

Daybue is the first and only FDA-approved treatment specifically for Rett syndrome in adults and children two years of age and older. It is not a gene therapy, but rather a medication taken orally.

The optimistic AI generated view:

Here's a breakdown of Daybue for Rett syndrome:

• Mechanism: The exact way Daybue works in Rett syndrome isn't fully understood, but it's believed to target neuroinflammation and support synaptic function.
• Dosage: The recommended dose is based on the patient's weight and is taken twice daily, morning and evening, with or without food.
• Administration: Daybue comes as an oral solution and can be taken directly or through a gastrostomy tube if swallowing is difficult.
• Efficacy: Studies have shown that Daybue can improve symptoms of Rett syndrome, including reducing scores on the Rett Syndrome Behavior Questionnaire (RSBQ) and showing improvement on the Clinical Global Impression-Improvement (CGI-I) scale.
• Side Effects: The most common side effects of Daybue are diarrhea and vomiting. Weight loss can also occur in some patients. It's important to consult with a healthcare professional for monitoring and managing any potential side effects.

Daybue is an expensive medication. Here's what we know about the cost:

• List Price: The list price of Daybue is around $21.10 per milliliter.
• Annual Cost: This translates to an estimated average annual cost of around $375,000 for patients.
• Dosage Variability: It's important to note that the dosage of Daybue is based on a patient's weight, so the annual cost can vary depending on the individual.

Insurance and Assistance Programs:

• The high cost of Daybue highlights the importance of insurance coverage. Whether insurance covers Daybue and to what extent will depend on your specific plan.
• The manufacturer, Acadia Pharmaceuticals, offers a copay program called Daybue Acadia Connect. This program may help eligible commercially insured patients pay $0 for their monthly prescription.

What are the parents' groups saying? 

Not as good as you might be expecting for $375,000 a year.

Affordable potential alternatives to Daybue/Trofinetide

Daybue/Trofinetide is the product of decades of research into a growth factor called IGF-1.

It is a complicated subject and as usual the abbreviations can be confusing.

As you will see below there already is an OTC product commercialized by one of the original researchers, Dr Jian Guan.

One Rett syndrome parent, who reads this blog, has trialed cGP and sees a benefit. You rather wonder why the Phelan-McDermid, Pitt Hopkins, Angelman and Prader-Willi parents don’t follow him and splash out 50 USD and make a trial.

 

 

Gene-therapy

Gene therapy is undoubtedly very clever and ultimately will likely be the best therapy.  It still may not be that silver bullet.

To be effective gene therapy needs to be given at a very young age, ideally as a fetal therapy prior to birth. Note that we saw that in the Rett mouse model they gave bumetanide to the pregnant mother just before birth.

Fetal therapy is not a crazy idea and much is already written about it; many pregnancies are terminated because genetic anomalies are detected prior to birth. Down syndrome is the best-known example. Fetal therapy is realistic for some disorders.

Girls with Rett syndrome are often diagnosed first with idiopathic autism and then years later with a more precise diagnosis of Rett syndrome. This is a common experience among readers of this blog.

Classic Rett syndrome 

The average age of diagnosis for this form is around 2.5 years old in the US and 5 years old in the UK.  Why do you think that is?

Research in mouse models has shown that the effect of gene therapy ranges from curative when given extremely young to more limited the later it is given.

Off-target effects

Gene therapy has the potential for off-target effects. This is a significant concern in the field and researchers are actively working on ways to minimize these risks. Here is a breakdown of what off-target effects are and why they matter:

During gene therapy, a modified gene is delivered to target cells with the aim of correcting a genetic defect.

Ideally, the modified gene integrates into the intended location in the genome.

However, there's a chance it might insert itself into unintended locations (off-target sites).

Potential Consequences of Off-Target Effects

Disrupting normal genes at off-target sites could lead to unpredictable and potentially harmful consequences. This could include triggering uncontrolled cell growth, which is a risk factor for cancer.

It can also cause unexpected side effects depending on which genes are accidentally disrupted.

Minimizing Off-Target Effects

Researchers are developing various strategies to improve the accuracy and specificity of gene therapy techniques.

This includes using more precise gene editing tools like CRISPR-Cas9 with optimized guide RNAs to reduce off-target edits.

Additionally, researchers are working on methods to detect and potentially repair any off-target modifications that might occur.

Over-expression of the target gene

Yes, there is a possibility that the replaced gene in gene therapy could overproduce the expressed protein. This can be a potential complication and researchers are working on ways to control the level of protein expression. Here's a breakdown of the concern:

• Gene Dosing: Ideally, gene therapy aims to deliver a functional copy of the gene at the right amount to compensate for the deficiency.
• Overproduction Risks: However, if the delivered gene is too active or multiple copies are inserted, it can lead to overproduction of the protein.

Consequences of Protein Overproduction:

• Overproduction of a protein can disrupt the delicate balance in the cell, potentially leading to cell dysfunction or even cell death.
• In some cases, the protein itself might have harmful effects if present in excessive amounts.

Controlling Protein Expression:

Researchers are developing several strategies to control protein expression in gene therapy:

• Promoter selection: Using promoters that have a weaker switch can help regulate protein production.
• Viral vectors: Engineering viral vectors to control the number of gene copies delivered to cells.
• Inducible systems: Developing gene therapy methods where the expression of the introduced gene can be turned on and off as needed.

The cost of gene therapy

•      Despite the high cost, gene therapy can be a cost-effective treatment for some diseases. This is because it can eliminate the need for lifelong treatment with other medications.

•      Here are some examples of the cost of currently available pediatric gene therapies:

•      Luxturna (gene therapy for Leber congenital amaurosis type 10): $425,000

•      Zolgensma (gene therapy for spinal muscular atrophy type 1): $2.1 million

•      Skysona (gene therapy for adrenoleukodystrophy): $3 million

Piperine to correct KCC2 expression in Rett syndrome?

One key feature of Rett syndrome is impaired cognition.

As regular readers are aware, there are many types of treatable intellectual disability (ID).

One type of treatable ID is caused when the GABA developmental switch fails to occur shortly after birth.  This creates an excitatory/inhibitory imbalance in neurons which impairs cognition and lowers IQ.

The faulty GABA switch is a feature of many types of autism, but far from all of them.

By using pharmaceuticals to lower chloride within neurons, you can compensate for the failure of the GABA switch.

This treatment can be achieved by:

1.     Blocking or down regulating NKCC1

2.     Up regulating KCC2

In the paper below they look at up regulating KCC2

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

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.

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.

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

Treatment with piperine (10 μM), an activator of the TRPV1 channel (51), induced a significant rise in KCC2 expression in cultured human neurons 

We already knew this was likely from earlier research from Ben Ari, see below for a reminder.  Is Piperine an interesting option for those restricted to OTC interventions?

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2-/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

Treating the mother prior to delivery with bumetanide was a partially effective therapy in the mouse model of Rett syndrome.

Piperine

Bumetanide is cheap and very possibly effective in human Rett syndrome, but it is a prescription drug.

Piperine is an OTC supplement and a compound found in black pepper. By activating the TRPV1 channel it causes an increase in expression of the KCC2 transporter that allows flow of chloride out of neurons. So piperine should lower chloride inside neurons.  Piperine can cross the blood brain barrier, so when taken orally it should have some effect on intracellular chloride.

Piperine is also a positive allosteric modulator of GABA_A receptors

This means that piperine multiplies the effect of whatever GABA is around. This means that in typical people piperine should have anti-anxiety effects.

Piperine was recently found to interact with a previously unknown  benzodiazepine-independent binding site.

Researchers are currently toying with the piperine molecule to try and separate the effect on TRPV1 from the effects on  GABA_A.  They want to create 2 new drugs.

1.     a selective TRPV1 activator

2.     a selective GABA_A modulator (PAM)

Piperine as an alternative or complement to Bumetanide?

One effect of piperine would be great to have (TRPV1 activator) but the second effect would not be helpful (positive allosteric modulator of GABAA).

The question is what is the net effect. Nobody will be able to answer that without a human trial.

I was advised long ago by one drug developer than it is best to focus on reducing flow into neurons via NKCC1, rather than increase its exit by KCC2, because nobody had yet been successful with KCC2; many have tried.  KCC2 plays a key role in neuropathic pain and that is why it has been researched.

Conclusion

We did see years ago that taking coffee with your bumetanide made sense. Coffee contains compounds that are OAT3 inhibitors and slow down the excretion of bumetanide from the body; coffee increases the effect of bumetanide. You can achieve something very similar by just increasing the dose of bumetanide.

Taking black pepper (piperine) with your bumetanide might be good, or might not be. It certainly would be easy to find out. As with Daybue/Trofinetide, the result is likely to vary from person to person. If GABA function, post- bumetanide, is still a bit excitatory amplifying GABA signaling will make autism worse. If GABA function has been shifted to inhibitory then amplifying GABA signaling will be calming.

Gene therapy will require much earlier diagnosis of single gene autisms.

“Precision medicine” therapies like Daybue/Trofinetide may not be that precise after all and large variations exist in the response, even among children with the same affected gene.

The huge expense means that for most of the world they will see no benefit from gene therapy or indeed “precision medicine.”

The low hanging fruit is to repurpose affordable existing drugs and get the benefit from their secondary effects.  This is what I term personalized medicine.

The research clearly indicates that some girls with Rett syndrome likely will benefit from Bumetanide therapy. For a young child this therapy would cost 50 US dollars/euros a year, if you pay the actual price for generics.

Why are they trialing genetic therapies for Rett instead of first doing the obvious thing and trialing cheap bumetanide? They will likely be able to sell the gene therapy for $2 million a shot.  There is little interest in trialing a $50 a year therapy.

Our new reader from Turkey, MÜCADELECI ANNE DENIZ ( = FIGHTING MOTHER DENIZ), likely does not have $2 million to spend, but seems to be on the way to creating her own personalized medicine therapy for her son. Good luck to her.

As to the cGP Max supplement, it seems to work for some and have no effect in others. Nobody has reported any side effects. It looks worth a try for Rett syndrome.  As a supplement it is not cheap, that is until you see what they charge for Daybue. 

Treating Rett syndrome, some autism and some dementia via TrkA, TrkB, BDNF, IGF-1, NGF and NDPIH. And logically why Bumetanide really should work in Rett

Source: Rett Syndrome: Crossing the Threshold to Clinical Translation

 

Today’s post is on the one hand very specific to Rett syndrome, but much is applicable to broader autism and other single gene autisms.

Today’s post did start out with the research showing Bumetanide effective in the mouse model of Rett syndrome. This ended up with figuring out why this should have been obvious based on what we already know about growth factors that are disturbed in autism and very much so in Rett.

We even know from a published human case studies that Bumetanide can benefit those with Fragile X and indeed Down syndrome, but the world takes little notice.

If Bumetanide benefits human Rett syndrome would anyone take any notice?  They really should.

To readers of this blog who have a child with Rett, the results really are important.  You can even potentially link the problem symptoms found in Rett to the biology and see how you can potentially treat multiple symptoms with the same drug.

One feature of Rett is breathing disturbances, which typically consist of alternating periods of hyperventilation and hypoventilation.

Our reader Daniel sent me a link to paper that suggest an old OTC cough medicine could be used to treat the breathing issues.

The antitussive cloperastine improves breathing abnormalities in a Rett Syndrome mouse model by blocking presynaptic GIRK channels and enhancing GABA release

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. One of the major RTT features is breathing dysfunction characterized by periodic hypo- and hyperventilation. The breathing disorders are associated with increased brainstem neuronal excitability, which can be alleviated with antagonistic agents.

Since neuronal hypoexcitability occurs in the forebrain of RTT models, it is necessary to find pharmacological agents with a relative preference to brainstem neurons. Here we show evidence for the improvement of breathing disorders of Mecp2-null mice with the brainstem-acting drug cloperastine (CPS) and its likely neuronal targets. CPS is an over-the-counter cough medicine that has an inhibitory effect on brainstem neuronal networks. In Mecp2-null mice, CPS (30 mg/kg, i.p.) decreased the occurrence of apneas/h and breath frequency variation. GIRK currents expressed in HEK cells were inhibited by CPS with IC50 1 μM. Whole-cell patch clamp recordings in locus coeruleus (LC) and dorsal tegmental nucleus (DTN) neurons revealed an overall inhibitory effect of CPS (10 μM) on neuronal firing activity. Such an effect was reversed by the GABAA receptor antagonist bicuculline (20 μM). Voltage clamp studies showed that CPS increased GABAergic sIPSCs in LC cells, which was blocked by the GABAB receptor antagonist phaclofen. Functional GABAergic connections of DTN neurons with LC cells were shown.

These results suggest that CPS improves breathing dysfunction in Mecp2-null mice by blocking GIRK channels in synaptic terminals and enhancing GABA release.

  

Cloperastine (CPS) is a central-acting antitussive working on brainstem neuronal networks The drug has several characteristics. 1) It affects the brainstem integration of multiple sensory inputs via multiple sites including K+ channels, histamine and sigma receptors. 2) Its overall effect is inhibitory, suppressing cough and reactive airway signals. 3) With a large safety margin, it has been approved as an over-the-counter medicine in several Asian and European countries.  

With the evidence that DTN cells receive GABAergic recurrent inhibition, we tested whether the inhibitory effect of CPS was caused by enhanced GABAergic transmission. Thus, we recorded the evoked firing activity of DTN cells before and during bath application of CPS in the presence of 20 μM bicuculline. Under this condition, CPS failed to decrease the excitability of DTN neurons (F(1,9) = 0.41, P > 0.05; two‐way repeated measures ANOVA) (n=9) (Fig. 8), indicating that the inhibitory effect relies on GABAA synaptic input 

 

It appeared to me that the breathing issues might be considered as another consequence of the excitatory/inhibitory (E/I) imbalance that is a core feature of much severe autism.

In the case of Rett the lack of BDNF will make any E/I imbalance worse and that by treating the E/I imbalance we will produce the inhibitory effect from GABAa receptors that is needed to ensure correct breathing.  Note that in bumetanide responsive autism there is no inhibitory effect from GABAa receptors, the effect is excitatory.

I did wonder if arrhythmia (irregular heartbeat) is present in Rett, since the breathing problems in Rett are also seen as being caused by a dysfunction in the autonomic nervous system. Arrhythmia is actually a big problem for girls with Rett syndrome.  Regular readers of this blog might then ask about Propranolol, does that help?  It turns out to have been tried and it is not so helpful.  What is effective is another drug we have come across for autism, the sodium channel blocker Phenytoin.  Phenytoin is antiepileptic drug (AED) and it works by blocking voltage gated sodium channels.

Low dose phenytoin was proposed as an autism therapy and a case study was published from Australia. In a separate case study, phenytoin was used to treat self-injury that was triggered by frontal lobe seizures.

When you treat arrhythmia in Rett girls with Phenytoin does it have an impact on their breathing problems?

If you treat the girls with Phenytoin do they still go on to develop epilepsy?

What about if you add treatment with Bumetanide to reduce symptoms of autism? 

Lots of questions looking for answers.

 

What is Rett Syndrome?

Rett syndrome was first identified in the 1950s by Dr Andreas Rett as a disorder that develops in young girls.  Only as recently as 1999 was it determined that the syndrome is caused by a mutation in the MECP2 gene on the X chromosome.  The X chromosome is very important because girls have two copies, but boys have just one.  Rett was an Austrian like many other early researchers in autism like Kanner and Asperger. Even Freud was educated in Vienna. Eugen Bleuler lived pretty close by in Switzerland and he coined the terms schizophrenia, schizoid and autism. 

Rett syndrome is a rare genetic disorder that affects brain development, resulting in severe mental and physical disability.

It is estimated to affect about 1 in 12,000 girls born each year.

Rett is a rare condition, but among these rare conditions it is quite common and so there is a lot of research going on to find treatments.  The obvious one is gene therapy to get the brain to make the missing MeCP2 protein.

Rett syndrome is thankfully rare in absolute terms, but it is one of the best known development conditions that is associated with autism symptoms.

While Rett syndrome may not officially be an ASD in the DSM-5, the link to autism remains. Many children are diagnosed as autistic before the MECP2 mutation is identified and then the diagnosis is revised to RTT/Rett. 

Fragile X  syndrome (FXS), on the other hand, is the most common inherited cause of intellectual disability (ID), as well as the most frequent single gene type of autism.

In the meantime, the logical strategy is to treat the downstream consequences of the mutated gene. Much is known about these downstream effects and there overlaps with some broader autism and indeed dementia.

One area known to be disturbed in Rett, some other autisms and dementia is growth factors inside the brain. The best known growth factors are IGF-1 (Insulin-like Growth Factor 1), BDNF (brain-derived neurotrophic factor) and my favorite NGF (Nerve growth factor).

Without wanting to get too complicated we need to note that BDNF acts via a receptor called TrkB.  You can either increase BDNF or just find something else to activate TrkB, as pointed out to me by Daniel.

For readers whose children respond to Bumetanide they are benefiting from correcting elevated levels of chloride in neurons. Too much had been entering by the transporter NKCC1 and too little exiting via KCC2.

One of the effects of having too little BDNF and hence not enough activation of TrkB is that chloride becomes elevated in neurons.  If you do not activate TrkB you do not get enough KCC2, which is what allows chloride to exit neurons.

To what extent would TrkB activation be an alternative/complement to bumetanide in broader autism?

To what extent would TrkB activation be success in treating some types of chronic pain (where KCC2 is known to be down regulated)?

Low levels of BDNF are a feature of Rett and much dementia.

So you would want to:

·        Increase BDNF

·        Activate TRKB with something else

·        Block NKCC2 to compensate for the lack of KCC2

Note that BDNF is not reduced in all types of autism, just in a sub-group.

I note that there already is solid evidence in the research:-

Restoration of motor learning in a mouse model of Rett syndrome following long-term treatment with a novel small-molecule activator of TrkB

Reduced expression of brain-derived neurotrophic factor (BDNF) and impaired activation of the BDNF receptor, tropomyosin receptor kinase B (TrkB; also known as Ntrk2), are thought to contribute significantly to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Previous studies from this and other laboratories have shown that enhancing BDNF expression and/or TrkB activation in Mecp2-deficient mouse models of RTT can ameliorate or reverse abnormal neurological phenotypes that mimic human RTT symptoms. The present study reports on the preclinical efficacy of a novel, small-molecule, non-peptide TrkB partial agonist, PTX-BD4-3, in heterozygous female Mecp2 mutant mice, a well-established RTT model that recapitulates the genetic mosaicism of the human disease. PTX-BD4-3 exhibited specificity for TrkB in cell-based assays of neurotrophin receptor activation and neuronal cell survival and in in vitro receptor binding assays. PTX-BD4-3 also activated TrkB following systemic administration to wild-type and Mecp2 mutant mice and was rapidly cleared from the brain and plasma with a half-life of ∼2 h. Chronic intermittent treatment of Mecp2 mutants with a low dose of PTX-BD4-3 (5 mg/kg, intraperitoneally, once every 3 days for 8 weeks) reversed deficits in two core RTT symptom domains – respiration and motor control – and symptom rescue was maintained for at least 24 h after the last dose. Together, these data indicate that significant clinically relevant benefit can be achieved in a mouse model of RTT with a chronic intermittent, low-dose treatment paradigm targeting the neurotrophin receptor TrkB. 

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2^−/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

    

The GABA Polarity Shift and Bumetanide Treatment: Making Sense Requires Unbiased and Undogmatic Analysis

 

GABA depolarizes and often excites immature neurons in all animal species and brain structures investigated due to a developmentally regulated reduction in intracellular chloride concentration ([Cl⁻]_i) levels. The control of [Cl⁻]_i levels is mediated by the chloride cotransporters NKCC1 and KCC2, the former usually importing chloride and the latter exporting it. The GABA polarity shift has been extensively validated in several experimental conditions using often the NKCC1 chloride importer antagonist bumetanide. In spite of an intrinsic heterogeneity, this shift is abolished in many experimental conditions associated with developmental disorders including autism, Rett syndrome, fragile X syndrome, or maternal immune activation. Using bumetanide, an EMA- and FDA-approved agent, many clinical trials have shown promising results with the expected side effects. Kaila et al. have repeatedly challenged these experimental and clinical observations. Here, we reply to the recent reviews by Kaila et al. stressing that the GABA polarity shift is solidly accepted by the scientific community as a major discovery to understand brain development and that bumetanide has shown promising effects in clinical trials.

 

Back in 2013 a case study was published showing Bumetanide worked for a boy with Fragile X syndrome. A decade later and still nobody has looked to see if it works in all Fragile X. 

Treating Fragile X syndrome with the diuretic bumetanide: a case report

https://pubmed.ncbi.nlm.nih.gov/23647528/

We report that daily administration of the diuretic NKCC1 chloride co-transporter, bumetanide, reduces the severity of autism in a 10-year-old Fragile X boy using CARS, ADOS, ABC, RDEG and RRB before and after treatment. In keeping with extensive clinical use of this diuretic, the only side effect was a small hypokalaemia. A double-blind clinical trial is warranted to test the efficacy of bumetanide in FRX.

 

What do Rett syndrome and Fragile X have in common? 

In a healthy mature neuron the level of chloride needs to be low for it to function correctly (the neurotransmitter GABA to be inhibitory).

 

Rett and Fragile X are part of a large group of conditions that feature elevated levels of chloride in neurons.

 

Elevated chloride in neurons is treatable.

 

Is Bumetanide a cure for Rett syndrome, or Fragile X?

No it is not, but it is a step in that direction because it reverses a key defect present in at least some Rett and some Fragile X.

In the mouse model of Rett, bumetanide corrected some, but not all the problems caused by the loss of function of the MECP2 gene.

 

Moving on to IGF-1

IGF-1 is a growth hormone with multiple functions throughout aging. Production of IGF-1 is stimulated by GH (growth hormone).

The lowest levels occur in infancy and old age and highest levels occur around the growth spurt before puberty.

Girls with Turner syndrome, lack their second X chromosome and this causes a lack of growth hormones and female hormones. They end up with short stature and with features of autism. Treatment is possible with GH or indeed IGF-1.

In dementia one strategy is to increase IGF-1.  This same strategy is also being applied to single gene autisms like Rett and Pitt Hopkins.

Trofinetide and NNZ-2591 are improved synthetic analogues of peptides that occur naturally in the brain and are related to IGF-1. Trofinetide is being developed to treat Rett and Fragile X syndromes, NNZ-2591 is being developed to treat Angelman, Phelan-McDermid, Pitt Hopkins and Prader-Willi syndromes.

 

NGF (nerve growth factor)

Nerve growth factor does what it says (boosting nerve growth), plus much more. NGF plays a key role in the immune system, it is produced in mast cells, and it plays a role in how pain in perceived.

NGF acts via NGF receptors, not surprisingly, but also via TrkA receptors. We saw earlier in this post that BDNF acts via TrkB receptors.

Once NGF binds to the TrkA receptor it triggers a cascade of signalling via  the Ras/MAPK pathway and the PI3K/Akt pathway.  Both pathways relate to autism and Ras itself can play a role in intellectual disability. 

These are also cancer pathways and indeed NGF seems to play a role.  Beta cells in the pancreas produce insulin and these beta cells have TrkA receptors. In type 1 diabetes these beta cells die.  Beta cells need NGF to activate their TrkA receptors to survive.

Clearly for multiple reasons you need plenty of NGF.

Lack of NGF would be one cause of dementia and that is why Rita Levi-Montalcini choose to self-treat with NGF eye drops for 30 years. Rita won a Nobel prize for discovering NGF.

In Rett syndrome we know that the level of NGF is very low in the brain.

Logical therapies for Rett would seem to include:

·        NGF itself, perhaps taken as eye drops, but tricky to administer

·        A TrkA agonist, that would mimic the effect of NGF

·        The traditional medicinal mushroom  Lion’s Mane (Hericium erinaceus) 

We should note that effect of NGF acting via TrkA is mainly in the peripheral nervous system, not the brain.

It has long been known that Lions’ Mane (Hericium erinaceus) increases NGF but it was not clear why.  This has very recently been answered.

The active chemical has been identified to be N-de phenylethyl isohericerin (NDPIH).

The opens the door to synthesizing NDPIH as drug to treat a wide range of conditions from Alzheimer’s to Rett. 

Mushrooms Magnify Memory by Boosting Nerve Growth  

Active compounds in the edible Lion’s Mane mushroom can help promote neurogenesis and enhance memory, a new study reports. Preclinical trials report the compound had a significant impact on neural growth and improved memory formation. Researchers say the compound could have clinical applications in treating and preventing neurodegenerative disorders such as Alzheimer’s disease.

Professor Frederic Meunier from the Queensland Brain Institute said the team had identified new active compounds from the mushroom, Hericium erinaceus.

“Extracts from these so-called ‘lion’s mane’ mushrooms have been used in traditional medicine in Asian countries for centuries, but we wanted to scientifically determine their potential effect on brain cells,” Professor Meunier said.

“Pre-clinical testing found the lion’s mane mushroom had a significant impact on the growth of brain cells and improving memory.

“Laboratory tests measured the neurotrophic effects of compounds isolated from Hericium erinaceus on cultured brain cells, and surprisingly we found that the active compounds promote neuron projections, extending and connecting to other neurons.

“Using super-resolution microscopy, we found the mushroom extract and its active components largely increase the size of growth cones, which are particularly important for brain cells to sense their environment and establish new connections with other neurons in the brain.” 

 

Hericerin derivatives activates a pan‐neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

 

Since the discovery of the first neurotrophin, NGF, more than 70 years ago, countless studies have demonstrated their ability to promote neurite regeneration, prevent or reverse neuronal degeneration and enhance synaptic plasticity. Neurotrophins have attracted the attention of the scientific community in the view to implement therapeutic strategies for the treatment of a number of neurological disorders. Unfortunately, their actual therapeutic applications have been limited and the potential use of their beneficial effects remain to be exploited. Neurotrophins, for example, have poor oral bioavailability, and very low stability in serum, with half-lives in the order of minutes  as well as minimal BBB permeability and restricted diffusion within brain parenchyma. In addition, their receptor signaling networks can confer undesired off-target effects such as pain, spasticity and even neurodegeneration. As a consequence, alternative strategies to increase neurotrophin levels, improve their pharmacokinetic limitations or target specific receptors have been developed. Identification of bioactive compounds derived from natural products with neurotrophic activities also provide new hope in the development of sustainable therapeutical interventions. Hericerin derivative are therefore attractive compounds for their ability to promote a pan-neurotrophic effect with converging ERK1/2 downstream signaling pathway and for their ability to promote the expression of neurotrophins. Further work will be needed to find the direct target of Hericerin capable of mediating such a potent pan-neurotrophic activity and establish whether this novel pathway can be harnessed to improve memory performance and for slowing down the cognitive decline associated with ageing and neurodegenerative diseases.

 

What this means is that there are 2 good reasons why Lion’s Mane should be helpful in Rett syndrome, both increasing BDNF and NGF.

  

Conclusion

Interestingly, one of the above papers is co-authored by a researcher from the European Brain Research Institute, founded by Rita Levi-Montalcini, the Nobel laureate who discovered NGF (Nerve growth factor). My top pick to test next in Rett syndrome would be NGF. Administration would have to follow Rita’s own example and be in the form of eye drops or follow the Lion’s Mane option, that has recently been further validated.

Rett syndrome is very well documented and many researchers are engaged in studying it.

As with broader autism, the problem is translating all the research into practical therapy today.

Clearly polytherapy will be required.

More than one type of neuronal hyperexcitability seems to be in play.

It looks like one E/I imbalance is the bumetanide responsive kind, that can be treated and will reduce autism symptoms and improve learning skills.  Then we have the hypoventilation/apnea for which Cloperastine looks a fair bet.  For the arrhythmia we have Phenytoin.  If there are still seizures after all that therapy it looks like sodium valproate is the standard treatment for Rett.

Sodium valproate is also an HDAC inhibitor and so has possibly beneficial epigenetic effects as a bonus.

I have always liked the idea of the Lion’s Mane mushrooms as a means to increase NGF (Nerve growth factor).  In today’s post we saw that it is the NDPIH from the mushrooms that acts to increase both BDNF and NGF.  You would struggle to buy NDPIH but you can buy these mushrooms. I did once buy the supplement version of these mushrooms and it was contaminated, so I think the best bet is the actual chemical or the actual mushroom.  One reader did write in once who is a big consumer of these mushrooms.

 

Lion's Mane Mushroom

Source: Igelstachelbart Nov 06

 

A Trk-B agonist that can penetrate the blood brain barrier would look a good idea.  There are some sold by the nootropic people.

7,8-dihydroxyflavone is such an agonist that showed a benefit in the mouse model.

 

7,8-dihydroxyflavone exhibits therapeutic efficacy in a mouse model of Rett syndrome

Following weaning, 7,8-DHF was administered in drinking water throughout life. Treated mutant mice lived significantly longer compared with untreated mutant littermates (80 ± 4 and 66 ± 2 days, respectively). 7,8-DHF delayed body weight loss, increased neuronal nuclei size and enhanced voluntary locomotor (running wheel) distance in Mecp2 mutant mice. In addition, administration of 7,8-DHF partially improved breathing pattern irregularities and returned tidal volumes to near wild-type levels. Thus although the specific mechanisms are not completely known, 7,8-DHF appears to reduce disease symptoms in Mecp2 mutant mice and may have potential as a therapeutic treatment for RTT patients.

Rett syndrome also features mitochondrial dysfunction and a variant of metabolic syndrome.  We have quite a resource available from broader autism, not much of it seems to have been applied in Rett.

You can see that in Rett less oxygen is available due to breathing issues and yet more oxygen is required due to “faulty” mitochondria. 

“Intensified mitochondrial O2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.”

https://www.frontiersin.org/articles/10.3389/fphys.2019.00479/full#:~:text=Rett%20syndrome%20(RTT)%2C%20an,inner%20membrane%20is%20leaking%20protons.

 

We have seen in this blog that 2 old drugs exist to increase oxygen levels in blood.  The Western world has Diamox (Acetazolamide) and the former soviet world has Mildronate/Meldonium. Mildronate also was suggested to have some wider potential benefit to mitochondria.

Rett is proposed as a neurological disorder with metabolic components, so based on what we have seen in this blog, you would think along the lines of Metformin, Pioglitazone and a lipophilic statin (Atorvastatin, Simvastatin or Lovastatin). 

The Anti-Diabetic Drug Metformin Rescues Aberrant Mitochondrial Activity and Restrains Oxidative Stress in a Female Mouse Model of Rett Syndrome

Statins improve symptoms of Rett syndrome in mice

The ultimate Rett cure will be one of the new gene therapies given to a baby before any significant progression of the disorder has occurred.

For everyone else, it looks like there is scope to develop a pretty potent individualized polytherapy, just by applying the very substantial knowledge that already exists in the research.

Good luck to Daniel and all the others seeking answers.

 

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 is more potent than bumetanide andvarious other loop diuretics to inhibit the sodium-potassium-chloride-cotransporterhuman variants hNKCC1A and hNKCC1B

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.