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Showing posts with label Autism. Show all posts
Showing posts with label Autism. Show all posts

Thursday, 15 August 2019

Wandering, Water, Sense of Danger and Accidents


We were recently at the seaside in Greece, where Monty was enjoying swimming in the sea. He is now a very competent swimmer and behaves in the water just like any other confident swimmer. Together with Mum he actually rescued a Russian swimmer in distress.  Monty does not get crazy ideas to swim to islands in the distance, or anything like that. Not so far, at least. 

Water is behind a shocking number of wanderings and deaths.

In the North American media, you can see that on a very regular basis children with autism and/or ID/MR (Intellectual Disability/Mental Retardation) wander off and get lost. Very often they are found in or beside water.

In Europe you hear much less frequently about children wandering. A high-profile case recently was an Irish teenage girl with MR/ID who disappeared while on holiday at a tiny jungle resort in Malaysia.  She left behind an open ground floor window and was found 10 days later beside a stream in a ravine a mile away. 

She had holoprosencephaly, which is an umbrella term for conditions relating to when the forebrain of the embryo fails to develop into two separate hemispheres, it includes Agenesis of the Corpus Callosum (ACC) when the part of the brain that is supposed to connect the two hemispheres fails to develop. Partial ACC and the exact opposite are features appearing in some severe autism.

People with MR/ID have no sense of danger and are usually enchanted by water. Wandering is far more likely than abduction.

Another case recently was an American teenager on a cruise arranged by his residential care home, it appears that he jumped over the deck railing at night to go for a swim in the ocean.

Even a bath tub can be dangerous, a young man with autism and epilepsy was left unattended in a bath at a UK care facility. He had a seizure and drowned.

I do think much more could be done to prevent wandering and water-related accidents. Firstly, people (parents) should be made more aware of who is at risk; anyone with a low IQ and unable to travel independently is at risk.

People with ID/MR often live in a world of cartoons, where all kinds of crazy things are possible, like jumping off a cruise ship and nobody ever gets hurt.  Going to a jungle retreat, like you are living in the Jungle Book cartoon, why wouldn’t you sneak downstairs in the night and enter your private jungle world?

Just because you have never been able to wander before does not mean you never will. 

The shortened life expectancy of people with severe autism is in large part down to preventable accidents, seizures and poor basic healthcare.

I do think that treating MR/ID would be much more socially acceptable than treating autism. Understanding the danger of crossing a road, or falling into a lake is more important than being able to tie your shoe laces.  If you can improve cognition with a pill, who could possibly object to that? 

It is no surprise that we have www.Treatable-ID.org but no www.Treatable-ASD.org 

In reality you will struggle to have treating ID taken seriously, although for many people it is possible.




Tuesday, 30 July 2019

Ginseng Compound K Esters for some Epilepsy, Autism and Cancers?

Many natural products like Ginseng and Curcumin do have long known medicinal properties but suffer from extremely low bioavailability, which limits their benefit.
Ginsenosides are compounds found in the Ginseng plant. They are metabolized by the gut flora into active compounds that include Compound K.  Compound K has been shown to have a variety of pharmacological actions such as anti-inflammatory, anti-oxidant, anti-cancer and vasorelaxation.  It also has interesting effects that relate to autism and other neurological disorders.

Compound K (CK) has extremely low bioavailability (circa 5%) which limits it potential therapeutic benefit. There are expensive versions of ginseng that aim to maximize Compound K (CK) production in the gut, but they do nothing to improve how much gets from the gut into the bloodstream.

It is possible to modify Compound K by making an ester. This ester has been shown to be highly bioavailable and that means the theoretic benefits, shown in test tubes, might actually be genuinely achieved in humans.

Two types of ester have been studied, the butyl and octyl ester resulting in so-called CK-B and CK-O.






There currently is a $400 million business selling ginseng worldwide, the research and production is mainly coming from Korea and China.  There probably should be pharmaceutical production of CK-B and/or CK-O, but I would not hold your breath.

CK-O was recently proposed as a treatment for some cancers, so perhaps someone will commercialize it.

Interestingly, the standard form CK has been proposed to treat colon cancer, which does make sense since CK is produced in the gut making colon cancer a good choice. You would think that CK-O would work better.


Bcl-2 / Bax

The gene/protein Bcl-2 is relevant to both cancer and autism and has been covered previously in this blog.

A total of 25 genes make up the Bcl-2 family of proteins. Bcl-2 itself is anti-apoptotic while family member Bax is pro-apoptotic.

Apoptosis is programmed cell death.  The Bax/Bcl-2 ratio determines the apoptotic potential of a cell. Increasing the Bax/Bcl-2 ratio can be highly desirable if you have cancer, since what you want is cell death.

Bcl-2 is dysregulated in autism. Studies have shown that the expression of Bcl-2 is significantly decreased in the brain of autistic subjects. This means a reduction in a protein that blocks apoptotic cell death, i.e. this favours growth and too much growth is a bad thing.


The big head type of autism (macrocephaly) is associated with hyperactive pro-growth signalling pathways, so reduced expression of Bcl-2 is not a surprise.


CK-O for some cancer

The Compound K ester CK-O  exerts strong anti-tumour activity by suppression of anti-apoptotic protein Bcl-2 and increase of pro-apoptotic protein Bax. It increases the Bax/Bcl-2 ratio.


CK-O from some epilepsy and some autism?

There are many types of epilepsy and hundreds of types of autism.
One commonly shared feature is the imbalance between the GABA-mediated inhibition and the glutamate-mediated excitation.

CK-O looks likes it might help both conditions.

·        CK is shown to reduce the expression of NMDA receptors and to attenuate the function of the NMDA receptors in the brain.

·        GABAB receptor activation via CK can regulate KCC2 at the cell surface in a manner that reduces intracellular chloride and hence the reversal potential for GABAA receptors

·        The expression of KCC2 protein was elevated by the treatment of CK while the expression of NKCC1 protein was reversely down-regulated.

·        CK enhances the expression of GABAA receptor subunit α1 in the brain and exhibits a tendency to decrease the expression of NMDAR1 protein in the hippocampus.

                                    
Ginseng for Autism?

There is some weak evidence that Ginseng may help in some autism.

I think what is really happening is that the effect is weak rather than the evidence is weak.  Ginseng may have a weak positive effect in some autism; weak because the amount of Compound K absorbed is trivial.

If Ginseng helps, CK-O could be substantially more effective.

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



We demonstrate that GABABR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABAAR

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


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

I would not expect a dramatic effect from any commercial Ginseng product, but CK-O really could have an effect.




Although there are many research reports regarding the bioactive function of ginsenosides and ginseng, studies on the neuroprotective eect and eects on the cognitive function of compound K are limited. It is generally agreed that compound K is more bioavailable than the parent ginsenosides, including Rb1, Rb2, and Rc, and is the major contributing factor to the health benefits of ginseng. However, as most studies were conducted using disease-associated models, such as Alzheimer’s disease and ischemic stroke, the results cannot be directly translated to the healthy normal population. Furthermore, it is not clear whether compound K can cross the blood–brain barrier and exert any action on cognitive function in humans, even though the compound was reported to facilitate GABA release in the hippocampus and exhibit a protective effect against scopolamine-induced hippocampal damage in a mouse model. The possible mechanisms of action of compound K in neuroprotection and cognitive improvement include attenuation of ROS levels in neural cells through induction of antioxidant enzymes, regulation of NO, GABA, and serotonin receptors, Ca 2+ channel modulation, regulation of the MAPK pathway, and inhibition of inflammation.
Although ginseng and ginsenosides were shown to have neuroprotective and cognitive enhancing eects, further research is required to establish whether compound K is the major component of ginseng responsible for cognitive improvement in humans.

The imbalance between the GABA-mediated inhibition and the glutamate-mediated excitation is the primary pathological mechanism of epilepsy. GABAergic and glutamatergic neurotransmission have become the most important targets for controlling epilepsy. Ginsenoside compound K (GCK) is a main metabolic production of the ginsenoside Rb1, Rb2, and Rc in the intestinal microbiota. Previous studies show that GCK promoted the release of GABA from the hippocampal neurons and enhanced the activity of GABAA receptors. GCK is shown to reduce the expression of NMDAR and to attenuate the function of the NMDA receptors in the brain. The anti-seizure effects of GCK have not been reported so far. Therefore, this study aimed to investigate the effects of GCK on epilepsy and its potential mechanism. The rat model of seizure or status epilepticus (SE) was established with either Pentylenetetrazole or Lithium chloride-pilocarpine. The Racine’s scale was used to evaluate seizure activity. The levels of the amino acid neurotransmitters were detected in the pilocarpine-induced epileptic rats. The expression levels of GABAARα1, NMDAR1, KCC2, and NKCC1 protein in the hippocampus were determined via western blot or immunohistochemistry after SE. We found that GCK had deceased seizure intensity and prolonged the latency of seizures. GCK increased the contents of GABA, while the contents of glutamate remained unchanged. GCK enhanced the expression of GABAARα1 in the brain and exhibited a tendency to decrease the expression of NMDAR1 protein in the hippocampus. The expression of KCC2 protein was elevated by the treatment of GCK after SE, while the expression of NKCC1 protein was reversely down-regulated. These findings suggested that GCK exerted anti-epileptic effects by promoting the hippocampal GABA release and enhancing the GABAAR-mediated inhibitory synaptic transmission.


Absorption mechanismof ginsenoside compound K and its butyl and octyl ester prodrugs in Caco-2cells.

 

Ginsenoside compound K (CK) is a bioactive compound with poor oral bioavailability due to its high polarity, while its novel ester prodrugs, the butyl and octyl ester (CK-B and CK-O), are more lipophilic than the original drug and have an excellent bioavailability. The aim of this study was to examine the transport mechanisms of CK, CK-B, and CK-O using human Caco-2 cells. Results showed that CK had a low permeability coefficient (8.65 × 10(-7) cm/s) for apical-to-basolated (AP-BL) transport at 10-50 μM, while the transport rate for AP to BL flux of CK-B (2.97 × 10(-6) cm/s) and CK-O (2.84 × 10(-6) cm/s) was significantly greater than that of CK. Furthermore, the major transport mechanism of CK was found as passive transcellular diffusion with active efflux mediated by P-glycoprotein (P-gp). In addition, it was found that CK-B and CK-O were not the substrate of efflux transporter since the selective inhibitors (verapamil and MK-571) of efflux transporter had little effects on the transport of CK-B and CK-O in the Caco-2 cells. These results suggest that improving the lipophilicity of CK by acylation can significantly improve the transport across Caco-2 cells.

Panax ginseng C.A. Meyer, the active components of which are mainly ginsenosides, is frequently utilized as a herbal drug in traditional oriental medicine. These ginsenosides, which belong to the class of triterpene saponins, have been reported to possess various biological and pharmacological activities such as antiaging, antiinflammation and antioxidation in central nerve system, cardiovascular system and immune system. Previous studies have shown that the pharmacological actions of ginsenosides contributed to their metabolites through biotransformation by human intestinal bacteria. Compound K (CK; Figure 1) is one of the main pharmacologically active metabolites of protopanaxadiol ginsenosides (e.g., Rb1, Rb2 and Rc) and it was reported that, it was accumulated in the liver after absorption from the GI tract to the blood, and some CK was transformed into fatty acid esters which may be the active components of ginsenosides in the body. Many studies revealed that most of the ginsenosides are poorly absorbed along the human intestinal tract due to a high polarity. Odani et al. have reported that the amount of ginsenoside Rg1 absorbed via oral administration was within the range of 1.9−20.0% of the dosage in animal models. Other ginsenosides such as Rb1 and Rb2 were also slowly absorbed through digestive tract, and the oral bioavailabilities in rats were relatively low. The biological activities of drugs depend not only on their chemical structures, but also on their degree of lipophilic and membrane permeation, which could enhance their transport across the cell membrane or influence their interaction with proteins and enzymes. Recently, considerable attention has been paid to the development of ester prodrugs, which is a widely used approach to improving overall lipophicity, membrane permehave been reported to enhance its lipophilicity, bioavailability and in vivo activity. However, to date, limited information is available concerning the mechanisms of oral absorption for CK and production of ester prodrugs to enhance the oral absorption of ginsenoside CK. To increase the oral absorption of CK, esterification provides a route to obtain more lipophilic derivatives. In addition, it has been reported that acylation of cholestane glycoside increased the antitumor potency. Several acylated triterpenoid saponins isolated from the roots of Solidago virgaurea subsp. virgaurea in a low concentration also activated the metabolism of endothelial cells, which enhanced the permeability of the blood vessel walls for better adsorption of the saponins into tissues. We thus speculated that the novel ester prodrugs of CK, butyl and octyl esters (CK-B and CK-O; Figure 1), which are more lipophilic than parent compound, may have an excellent oral bioavailability. The objective of this study was to determine the transepithelial transport and absorption mechanisms of CK and its ester derivatives in the Caco-2 system. Caco-2 cell monolayers have been generally accepted as an in vitro model for prediction of drug absorption across human intestine and for mechanistic studies of intestinal drug transport since these cells show morphological and functional similarities to human small intestinal epithelial cells. In this study, both ester derivatives were utilized for transepithelial transport and absorption assays in Caco-2 monolayers compared with CK to investigate whether esterification could enhance the membrane permeability of high hydrophilic compound, thus improving the intestinal absorption of drug.
Our results are consistent with the previous reports which showed that CK had a low oral bioavailability (approximately 5%) in rats. However, as shown in our results, the low oral bioavailability of CK can be improved by esterification of CK into CK-B and CK-O.


Octyl ester ofginsenoside compound K as novel anti‐hepatoma compound: Synthesis and evaluation on murine H22 cells in vitro and in vivo


Ginsenoside compound K (M1) is the active form of major ginsenosides deglycosylated by intestinal bacteria after oral administration. However, M1 was reported to selectively accumulate in liver and transform to fatty acid esters. Ester of M1 was not excreted by bile as M1 was, which means it was accumulated in the liver longer than M1. This study reported a synthetic method of M1‐O, a mono‐octyl ester of M1, and evaluated the anticancer property against murine H22 cell both in vitro and in vivo. As a result, both M1 and M1‐O showed a dose‐dependent manner in cytotoxicity assay in vitro. At lower dose of 12.5 μm, M1‐O showed moderate detoxification. Instead, M1‐O exhibited significantly higher inhibition in H22‐bearing mice than M1. M1‐O induced murine H22 tumor cellular apoptosis in caspase‐dependent pathway given that pan‐caspase inhibitor, Z‐VAD‐FMK, could reverse the cytotoxicity induced by M1‐O. Additionally, pro‐ and anti‐apoptosis proteins, Bcl‐2 and Bax, altered and consequently induced increased expression of cleaved caspase‐3. Interestingly, cyclophosphamide regimen significantly induced atrophy of spleen and thymus, main immune organs, while M1‐O treatment greatly alleviated this atrophy. Collectively, we propose M1‐O as a candidate for live cancer treatment.

M1-O exerted strong anti-tumor activity by suppression of anti-apoptotic protein Bcl-2 and increase of pro-apoptotic protein Bax

Note: M1-O is the same think as CK-O


Ginsenosides are isolated from the Panax quinquefolius. This is a natural product triterpene saponins and steroid glycosides. Ginsenosides are the members of a dammarane family, which consists of a 4-ring and steroid-like structure. All ginsenosides have two or three hydroxyl groups in the carbon 3 and 20. Ginsenosides are converted into active metabolites like 20(S)- protopanaxadiol Rb1-Rb3, Rc, Rd, Rg3, Rh2, Rs1 (2) with help of human gut bacteria -glycosidase Eubacterium sp. A-44. Ginsenosides produced a variety of pharmacological activities such as anti-inflammatory, anti-oxidant, anti-cancer and vasorelaxation.                                                                                                                                                                       
                      

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

  

Conclusion

The ginseng compound K ester CK-O is likely to be a potent drug in humans with a range of effects, some of which do relate to autism and epilepsy.

Very often people with epilepsy are excluded from autism clinical trials.  Here is one drug where you might want to start with that very group.

CK-O will have multiple effects, meaning it is not selective, so while it may have some very good effects, there may be some negative ones.

You might think the CK-O molecule would be a good basis on which to build a modern patentable drug; a K-O (knock-out) for someone.

Natural substances with health benefits like phytoestrogens (soy etc), curcumin/turmeric, ginseng and even bee propolis either need to be eaten in large quantities or the active substance identified and synthesized. The people with neurofibromatosis (NF-1, NF-2) consuming large amounts of expensive New Zealand propolis as a PAK1 inhibitor might as well save money and buy the active ingredient itself another ester, this time caffeic acid phenethyl ester, and gives the bees a rest.






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.




Thursday, 4 July 2019

Home/Clinic based Photobiomodulation/Laser Therapy in Autism - acting on Light Sensitive Ion Channels, Mitochondria, Lymph Nodes and more




Photobiomodulation underlying mechanisms at the cellular and molecular levels. Light at 600–850 nm is absorbed by the mitochondrial electron transfer chain and leads to upregulation of the neuronal respiratory capacity. The near-infrared light at range of 900– 1100 nm is absorbed by structured water clusters formed in or on a heat/light-gated ion channels. An increase in vibrational energy of water cluster leads to perturb the protein structure and opening the channel which ultimately allows modulation of intracellular Ca2+ levels. The absorption of green light by neuronal opsin photoreceptors (OPN2-5) activates transient receptor potential channels which causes nonselective permeabilization to Ca2+ , Na+ , and Mg2+ . The cryptochromes (a class of flavoprotein blue-light signaling receptors) absorb blue light and seems to activate the transducing cellular signals via part of the optic nerve to the suprachiasmatic nucleus in the brain, which is important in regulation of the circadian clock


  
Today we return to the idea of using low power lasers to treat autism.  This follows on from the original post that reviewed a credible clinical trial that compared laser therapy with a sham red light therapy.  My conclusion was either the researchers cheated, or it really did work.   It is a pity, but experience shows us that cheating does occur in published research. I also pondered whether a cheaper LED device could give the same benefit of an expensive laser.

Low Level Laser Therapy (LLLT) for Autism – seems to work in Havana


Our reader RD has been busy at home applying the research, first using LEDs to no avail, before moving on to an expensive laser device, which does provide a benefit.  Today we dig a little deeper about what might be going on inside the brains of people treated with such devices. Click below to read RD's extensive comments and interesting links.

https://epiphanyasd.blogspot.com/2018/12/low-level-laser-therapy-lllt-for-autism.html?showComment=1560875374458#c8257042607661710259

Some autism therapies involving the use of expensive gadgets do set alarm bells ringing, but the more you look into Photobio-modulation, which is the new name of Low Level Laser Therapy (LLLT), the more credible it becomes.  There has been a great deal of recent research regarding other neurological conditions, autism only rarely gets a mention. The same therapy has been used on different parts of the body for several decades in Russia and some other countries. Where we live physiotherapists use Photobiomodulation/LLLT to treat numerous types of ache and pain.

It is still early days for Photobiomodulation and the brain. A lot depends on which parts of the brain you want to target; there are even plans for using the mouth, nose and ears as entry points to reach different parts of the brain.




Heat/light sensitive ion channels

Many human diseases are associated with ion channel dysfunctions (channelopathies).  Many people with autism have either genetic or acquired channelopathies of one kind or another.

Today our focus on light introduces us to a class of ion channels activated by heat and/or light.

We should immediately recall the so called “fever effect” in autism where in some people a rise in body temperature improved their autism, sometimes dramatically. The fever effect was replicated by one US researcher having people sit in a hot tub.

HYPERTHERMIA AND THE IMPROVEMENT OF ASD SYMPTOMS

 Five control subjects without a history of fever completed the hyperthermia condition at 102 °F, and demonstrated the safety and feasibility of the study. Ten subjects with ASD and a history of fever response were enrolled and completed the hyperthermia condition (102 °F) and control condition (98 °F) at the aquatic therapy pool. Improvement in social cognition and repetitive/restrictive behaviors were observed at the hyperthermia condition (102 °F) on parent (SRS, RBS-R) and rater (CGI-I) assessments. Pupillometry biomarker and gene expression can be correlated with clinical improvement. Side effects were minimal, and were the same as those observed in a hot tub/sauna (redness, nausea).

Discussion

We demonstrated improvement of socialization and repetitive and restricted behaviors at the hyperthermia condition (102 °F), and that we could reliably and safely increase children’s temperatures into the fever range (mean max temperature of 101.7 °F). This temperature increase was observed to cause significant and convergent improvement on clinician ratings (CGI-I) and parent ratings (SRS, RBS-R), both of which were kept blinded to the temperature of the pool. Interestingly, each child’s fever response history was correlated with the improvements observed at the elevated temperature. Those with a history of marked fever response had the most observable behavior changes. Behavior changes observed for each child were similar to those observed by parents during febrile episodes, including increased cooperation, communication and social reciprocity and decreased hyperactivity and inappropriate vocalizations. Although multiple rationales have been posited, this is the first study looking at the direct effect of temperature on ASD symptomatology.


  
TRPV1 and Autism

There has been a link suggested between TRPV1 and autism.  SHANK3 is a single gene type of autism, often used to study autism.

  
In control mice, SHANK3 tethers a protein called TRPV1 to the surface of sensory neurons, where it detects heat and chemical signals. Those signals activate TRPV1, causing calcium ions to flood into the cell, leading to a painful sensation.
Neurons from control mice show a robust influx of calcium ions in response to capsaicin, the chemical that gives chili peppers their heat. But the chemical triggers significantly less calcium flow into neurons from SHANK3 mice.
The study stokes curiosity about the connection between autism and TRPV1. This protein aids heart and lung function, and has been linked to addiction, anxiety and depression, says Camilla Bellone, assistant professor of neuroscience at the University of Geneva in Switzerland, who was not involved in the study. “It would be really interesting to see if TRPV1 dysfunction could explain other [features] associated with autism,” she says.



Pain, Rett Syndrome, MECP2 and TRPV1

It appears to be not just SHANK3 autism that has a TRPV1 connection, so does the all-female Rett Sydrome. Here the connection relates to unusual pain sensitivity in Rett Sydrome. Many people with autism have an unusual relationship with pain.


Although TRPV1 was expressed in MeCP2-positive TG neurons innervating the tongue in both wild-type and Mecp2+/- mice, a significantly smaller number of TRPV1-positive neurons were observed in the tongues of heterozygotes compared to wild-types. Together, these data suggest that the hypoalgesia observed in this mouse model is induced by the inhibition of TRPV1 expression, and this expression is dependent in part on MeCP2 signaling.
These findings suggest that tongue heat sensitivity and inflammatory hyperalgesia are dependent on TRPV1 expression in TG neurons that innervate the tongue and that this expression is regulated by MeCP2 signaling, supporting a role for MeCP2 in pain modulation. Hypoalgesia is a potentially dangerous condition that may result in more severe tissue damage from burns or other physical trauma due to a blunted pain withdrawal reflex.  Understanding how MeCP2 modulates pain might lead to therapies that improve the pain sensitivity in Rett syndrome patients, as well as treatments that might help to reduce neuropathic pain associated with other genetic or acquired conditions.



TRPV Channels in Mast Cells as a Target for Low-Level-Laser Therapy


Low-level laser irradiation in the visible as well as infrared range is applied to skin for treatment of various diseases. Here we summarize and discuss effects of laser irradiation on mast cells that leads to degranulation of the cells. This process may contribute to initial steps in the final medical effects. We suggest that activation of TRPV channels in the mast cells forms a basis for the underlying mechanisms and that released ATP and histamine may be putative mediators for therapeutic effects.



Modulation of TRPV channel gating by light-switched ligand. Putative modulation of an azo-chromophore between cis- and trans-form by light leading to activation of TRPV channel opening. As an example TRPV activation by the cis-form is cartooned.


We have shown in this review that laser irradiation in the visible and IR as well as UV range can modulate the function and expression of TRPV ion channels, and in particular TRPV1, TRPV2, and TRPV4. This may form the basis for effect of LLLT. As Ca2+-permeable ion channels, their activation may contribute to the laser-induced increase in intracellular Ca2+ that triggers degranulation and endocytotic release of ATP. Such light-induced mechanism may contribute to the basis of the medical effects of LLLT. This hypothesis still needs confirmation in animal tests and clinical trials.


Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy


Photobiomodulation (PBM) also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infra-red region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential and ATP production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.

MOLECULAR MECHANISMS OF PBM


Light sensitive ion channels

The most well-known ion channels that can be directly gated by light are the channelrhodopsins (ChRs), which are seven-transmembrane-domain proteins that can be naturally found in algae providing them with light perception. Once activated by light, these cation channels open and depolarize the membrane. They are currently being applied in neuroscientific research in the new discipline of optogenetics [35].
However, members of another broad group of ion-channels are now known to be light sensitive [36]. These channels are called "transient receptor potential" (TRP) channels as they were first discovered in a Drosophila mutant [36] and are responsible for vision in insects. There are now at least 50 different known TRP isoforms distributed amongst seven subfamilies [37], namely the TRPC (‘Canonical’) subfamily, the TRPV (‘Vanilloid’), the TRPM (‘Melastatin’), the TRPP (‘Polycystin’), the TRPML (‘Mucolipin’), the TRPA (‘Ankyrin’) and the TRPN (‘NOMPC’) subfamilies (see Figure 2). A wide range of stimuli modulate the activity of different TRP such as light, heat, cold, sound, noxious chemicals, mechanical forces, hormones, neurotransmitters, spices, and voltage. TRP are calcium channels modulated by phosphoinositides [38].

Conclusions

Low levels of red/NIR light can interact with cells, leading to changes at the molecular, cellular and tissue levels. Each tissue, however, can respond to this light-interaction differently, although it is well known that the photons, especially in the red or NIR, are predominantly absorbed in the mitochondria [132]. Therefore, it is likely that even the diverse results observed with PBM share the basic mechanism of action. What happens after the photon absorption is yet to be fully described, since many signaling pathways seem to be activated. It seems that the effects of PBM are due to an increase in the oxidative metabolism in the mitochondria [133]. Different outcomes can occur depending on the cell type, i.e. cancer cells that tend to proliferate when PBM is delivered [88]. In this review we have not discussed the response of cells and tissues to wavelengths longer than NIR, namely far IR radiation (FIR) (3 µm to 50 µm). At these wavelengths water molecules are the only credible chromophores, and the concept of structured water layers that build up on biological lipid bilayer membranes has been introduced to explain the selective absorption [134]. Nevertheless FIR therapy has significant medical benefits that are somewhat similar to those of PBM [135], and it is possible that activation of light/heat sensitive ion channels could be the missing connection between the two approaches.
As we have shown, PBM can regulate many biological processes, such as cell viability, cell proliferation and apoptosis, and these processes are dependent on molecules like protein kinase c (PKC), protein kinase B (Akt/PKB), Src tyrosine kinases and interleukin-8/1a (IL-8/1a). The effects of light on cell proliferation can be stimulatory at low fluences (which is useful in wound healing, for instance), but could be inhibitory at higher light doses (which could be useful in certain types of scar formation such as hypertrophic scars and keloids) [131].
The applications of PBM are broad. Four clinical targets, however, are the most common: shining light on injured sites to promote healing, remodeling and/or to reduce inflammation; on nerves to induce analgesia; on lymph nodes in order to reduce edema and inflammation; and on trigger points (a single one of as many as 15 points) to promote muscle relaxation and to reduce tenderness. Since it is non invasive, PBM is very useful for patients who are needle phobic or for those who cannot tolerate therapies with non-steroidal anti-inflammatory drugs [83].
The positive outcomes depend on the parameters used on the treatment. The anti-inflammatory effect of light in low intensity was reported on patients with arthritis, acrodermatitis continua, sensitive and erythematous skin, for instance [136]. With the same basic mechanism of action, which is the light absorption by mitochondrial chromophores, mainly Cox, the consequences of PBM are various, depending on the parameters used, on the signaling pathways that are activated and on the treated tissue. In order to apply PBM in clinical procedures, the clinicians should be aware of the correct parameters and the consequences for each tissue to be treated. More studies have to be performed in order to fill the gaps that still linger in the basic mechanisms underlying LLLT and PBM.


Photobiomodulation improves the frontal cognitive function of older adults.


OBJECTIVES:

The frontal lobe hypothesis of age-related cognitive decline suggests that the deterioration of the prefrontal cortical regions that occurs with aging leads to executive function deficits. Photobiomodulation (PBM) is a newly developed, noninvasive technique for enhancing brain function, which has shown promising effects on cognitive function in both animals and humans. This randomized, sham-controlled study sought to examine the effects of PBM on the frontal brain function of older adults.


METHODS/DESIGNS:

Thirty older adults without a neuropsychiatric history performed cognitive tests of frontal function (ie, the Eriksen flanker and category fluency tests) before and after a single 7.5-minute session of real or sham PBM. The PBM device consisted of three separate light-emitting diode cluster heads (633 and 870 nm), which were applied to both sides of the forehead and posterior midline, and delivered a total energy of 1349 J.


RESULTS:

Significant group (experimental, control) × time (pre-PBM, post-PBM) interactions were found for the flanker and category fluency test scores. Specifically, only the older adults who received real PBM exhibited significant improvements in their action selection, inhibition ability, and mental flexibility after vs before PBM.


CONCLUSIONS:

Our findings support that PBM may enhance the frontal brain functions of older adults in a safe and cost-effective manner.


Brain Photobiomodulation Therapy: a Narrative Review.


Brain photobiomodulation (PBM) therapy using red to near-infrared (NIR) light is an innovative treatment for a wide range of neurological and psychological conditions. Red/NIR light is able to stimulate complex IV of the mitochondrial respiratory chain (cytochrome c oxidase) and increase ATP synthesis. Moreover, light absorption by ion channels results in release of Ca2+ and leads to activation of transcription factors and gene expression. Brain PBM therapy enhances the metabolic capacity of neurons and stimulates anti-inflammatory, anti-apoptotic, and antioxidant responses, as well as neurogenesis and synaptogenesis. Its therapeutic role in disorders such as dementia and Parkinson's disease, as well as to treat stroke, brain trauma, and depression has gained increasing interest. In the transcranial PBM approach, delivering a sufficient dose to achieve optimal stimulation is challenging due to exponential attenuation of light penetration in tissue. Alternative approaches such as intracranial and intranasal light delivery methods have been suggested to overcome this limitation. This article reviews the state-of-the-art preclinical and clinical evidence regarding the efficacy of brain PBM therapy.

                                                   
Because neural tissues contain large amounts of mitochondrial CCO, application of red to NIR lights (600–850) for brain PBM therapy is highly attractive. The main problem so far has been getting enough light into the brain to accomplish the beneficial effects. In recent years, irradiation in the wavelength range between 980 and 1100 nm has been growing rapidly, and its different mechanisms of action including stimulation of ion channels and water molecules suggest it might even be combined with red/NIR. Improving cerebral metabolic function, stimulating neurogenesis and synaptogenesis, regulating neurotransmitters, and providing neuroprotection via anti-inflammatory and antioxidant biological signaling are the most important effects of brain PBM therapy (Fig. 4). The overall results from extensive preclinical and clinical studies in the brain PBM field suggest that modest levels of red and NIR light show biostimulatory effects without any thermal damage, and could improve neurobehavioral deficits associated with many brain disorders. Nevertheless, it is still not completely clear whether chronic repetition of brain PBM will be necessary for sustained clinical benefit, especially in psychological and neurodegenerative disorders. Owing to the beneficial impacts of brain PBM therapy in depression and anxiety, new trials for other psychiatric disorders such as schizophrenia autism, , bipolar, attention-deficit hyperactivity, and obsessive–compulsive disorders might well emerge in the future. Development of new techniques for effective light delivery to deeper structures of the brain is crucial, because of involvement of the limbic system and midbrain abnormalities seen in some brain disorders. In this respect, intracranial and intranasal irradiation methods, as well as the oral cavity route, even via the ear canal could be options. Although therapeutic influences of intracranial PBM therapy has been focused on PD researches, it is postulated that developing this technique also potentially effective for those conditions that are associated with limbic system dysfunctions such as anhedonia, anxiety, as well as impaired emotional processing. Preliminary evidence of benefit has been obtained in autism spectrum disorders. There is an epidemic of AD that is expected to hit the Western world as the overall population ages, and there has been a noticeable lack of any effective pharmacological therapies that have been approved for AD. Although the evidence for the effectiveness of PBM in the treatment of AD is still very preliminary, it is possible that PBM will play an even larger role in society in years to come if clinical trials now being conducted are successful. The authors conclude that clinic or home-based PBM therapy using laser or LED devices will become one of the most promising strategies for neurorehabilitation in upcoming years



Conclusion

Our reader RD is well ahead of the curve with his PBM/LLLT investigation. I do not see this kind of therapy being adopted by mainstream Western medicine, even if it did work.  It has been used in other countries for many decades by medical doctors, for all kinds of conditions, but that fact does not cut it with most Western doctors.  There are  practitioners of PBM/LLLT in Western countries, but they tend to be on the fringes of medicine, which puts PBM/LLLT clearly in the crank therapy category for most qualified Western doctors.

On the basis that we should keep an open mind about all kinds of therapies, we should consider PBM further. It is apparently safe at the power levels used. It may look a little strange, but it is non-invasive and the therapy does not take long. A single device could easily be used to treat many people, so the high price should not remain a barrier.

I was very surprised to hear that a local speech therapy company is now offering “neurofeedback therapy” using an expensive machine they have bought. I was very suspicious of a recent study carried out in Florida that was put forward to support this therapy using a commercial device, since of the 42 children in the group that had the actual therapy only 17 completed the 12 week trial and came back for the evaluation.  The trial included a similar sized group who had a sham therapy.  The likelihood of completing the trial was the same in both groups, which also looks odd.

Of the 83 subjects that completed the evaluation at the enrollment time, 34 returned for the POST evaluation after the 12 weeks of home based therapy.

If the results were so good, why did the majority of parents walk away during the trial? I was going to suggest to the speech therapist that perhaps those few thousands of euros/dollars might have been better spent on a laser, or perhaps the lottery.

For me, one big question about the laser is about how the device is used. Depending on what you believe the mode of action to be, you would have to use it in completely different ways.

If the benefit relates to improved mitochondrial function, you should really be able to measure this benefit using a PET scan that measures glucose uptake to each part of the brain. This was the method proposed by Polish researchers to show how some people benefit from a ketogenic diet to improve power/ATP output from different parts of the brain.

You would hope other researchers would try and replicate the benefit in autism, but the first group have already patented the laser idea.

Hopefully our reader RD will perfect this therapy and we await his feedback.

I did recently write about the recently discovered lymphatic system within the brain. One proposed benefit of PBM/LLLT is improved drainage of lymph. I thought that was interesting; if it was actually true then this therapy could potentially be used to prevent the onset of Alzheimer’s. We saw in that post that faulty lymph drainage may allow the accumulation of waste products (plaques etc) in aging brains and then Alzheimer's develops. Targeting the relevant lymph node with PBM/LLLT might be an alternative to the drug therapy currently being developed.

I am told that lymphatic drainage is currently "the big thing" in autism in the US, alongside anything to do with CBD (cannabis). Hopefully in the fb world of autism they have noted that in MS the problem with the brain's lymphatic system was not drainage, but the ingress of inflammatory messengers from the body into the brain, suggesting the opposite therapy.