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Showing posts with label Leucovorin. Show all posts
Showing posts with label Leucovorin. Show all posts

Tuesday, 26 May 2020

Bumetanide for TSC-type Autism, Verapamil now for sinusitis, Lower dose Folinic Acid looks interesting for Autism in France, Roche cuts Balovaptan and Basmisanil; Stanford continue repurposing Vasopressin for Autism

 Repurposing what already exists – cheap, safe, effective and sometimes colourful


Today’s post is nice and simple.

Yet another sub-type of autism is shown in a clinical trial to respond to the cheap drug bumetanide, this time it is children diagnosed with TSC (tuberous sclerosis complex); TSC is a leading genetic cause of autism often used in research.

In France researchers repurposed Folinoral, a lower dose equivalent of Dr Frye’s, and our reader Roger’s, Leucovorin to treat autism with a positive result.  Folinoral is Calcium Folinate, but the dose was just 5mg twice a day, much less than the dose used in the US research.

The potential off-label uses for Verapamil, the old calcium channel blocker helpful in some autism, continue to grow.

Original purpose:  

Lower blood pressure by blocking L-type calcium channels

Alternative uses:

·        Treating bipolar disorder
·        Treating cluster headaches and some migraine
·        Halting the loss of insulin production in people with diabetes
·        Treating diarrhea-predominant irritable bowel syndrome (IBS-D)
·        Treating aggression/anxiety in some autism

We can now add, as our reader Lisa discovered by chance,

·        Treating chronic sinusitis

Patients with severe chronic rhinosinusitis show improvement with Verapamil treatment


"Recently, we became aware that some of the inflammation in chronic rhinosinusitis (CRS) with nasal polyps is generated by the nasal lining itself, when a particular protein pump (P-glycoprotein) is overexpressed and leads to the hyper-secretion of inflammatory cytokines," said senior author Benjamin S. Bleier, M.D., a sinus surgeon at Mass. Eye and Ear and an assistant professor of otolaryngology at Harvard Medical School. "Verapamil is a first-generation inhibitor that is well-established in blocking P-glycoprotein. In some patients with CRS with nasal polyps, we saw dramatic improvement in their symptom scores."

Roche ditching experimental autism drugs

Basmisanil which targets the alpha 5 sub-unit of GABAA receptors was originally being developed to improve cognition in Down Syndrome; those clinical trials failed. Now Roche have pulled the plug on the trials to improve cognition in Schizophrenia.
Balovaptan was Roche’s expensive bet on Vasopressin to treat autism, covered in earlier posts; it blocks the activity of the V1a vasopressin receptor.  The Balovaptan phase 3 clinical trials have also been cancelled.



Stanford still pursuing Vasopressin for autism

Stanford’s bet on Vasopressin for autism is still ongoing.  They had the much simpler idea of just putting some pharmaceutical-grade vasopressin in a nasal spray and trialling that.

Intranasal delivery of drugs to target the brain appeals to me, as do eye drops.  Your eyes are part of the central nervous system, in the case of your nose it appears that drugs are transported directly to the brain from the nasal cavity along the olfactory and trigeminal nerves. 

Mechanism of intranasal drug delivery directly to the brain


One feature of this blog is a belief that central hormonal dysfunction is a core feature of much autism.  The big problem is that you cannot easily measure hormone levels in the central nervous system (CNS) and you may get quite contradictory results measuring hormone levels in blood samples.

Plasma oxytocin and vasopressin do not predict neuropeptide concentrations in human cerebrospinal fluid.


I was encouraged to see that the Stanford vasopressin researchers measured vasopressin in samples from spinal fluid.  They found that children who went on to be diagnosed with autism has very low levels of vasopressin in their brains early in life. Making it a potential biomarker.


Autism spectrum disorder (ASD) is a brain disorder characterized by social impairments. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified. However, we recently found that cerebrospinal fluid (CSF) concentration of the “social” neuropeptide arginine vasopressin (AVP) is significantly lower in pediatric ASD cases vs. controls. As an initial step in establishing the direction of causation for this association, we capitalized upon a rare biomaterials collection of newborn CSF samples to conduct a quasi-prospective test of whether this association held before the developmental period when ASD first manifests. CSF samples had been collected in the course of medical care of 0- to 3-mo-old febrile infants (n = 913) and subsequently archived at −70 °C. We identified a subset of CSF samples from individuals later diagnosed with ASD, matched them 1:2 with appropriate controls (n = 33 total), and quantified their AVP and oxytocin (OXT) concentrations. Neonatal CSF AVP concentrations were significantly lower among ASD cases than controls and individually predicted case status, with highest precision when cases with comorbid attention-deficit/hyperactivity disorder were removed from the analysis. The associations were specific to AVP, as ASD cases and controls did not differ in neonatal CSF concentrations of the structurally related neuropeptide, OXT. These preliminary findings suggest that a neurochemical marker of ASD may be present very early in life, and if replicated in a larger, prospective study, this approach could transform how ASD is detected, both in behaviorally symptomatic children, and in infants at risk for developing it.
  
Easy to read version: -

Cerebrospinal fluid levels of a hormone called vasopressin were lower in babies who went on to develop autism than in those who did not, a study found. 

Cerebrospinal Fluid Vasopressin and Symptom Severity in Children with Autism

 








Cerebrospinal fluid (CSF) arginine vasopressin (AVP) concentration differs between children with and without autism (AUT), predicts AUT diagnosis, and predicts symptom severity. (A) CSF AVP concentration is lower in children with AUT (n = 36) compared to control children (n = 36), whereas (B) CSF oxytocin (OXT) concentration does not differ between groups. 
(C) The effect of CSF AVP concentration on predicted (line) and observed (symbols) group is plotted, corrected for the other variables in the analysis. Children with AUT plotted above, and control children plotted beneath, the dashed line (which represents 50% probability) are correctly classified. Specifically, across the range of observed CSF AVP concentrations, the likelihood of AUT increased over 1,000-fold, corresponding to nearly a 500-fold increase in risk with each 10-fold decrease in CSF AVP concentration (range odds ratio = 1,080, unit odds ratio = 494, β1 ± SE = −6.202 ± 1.898). (D) CSF AVP concentration predicts Autism Diagnostic Observation Schedule (ADOS)–Calibrated Severity Score (CSS) in male but not in female children with AUT.

I think many hormones are likely disturbed in autism and that modifying them is one potential method of treating autism.

At Stanford they have already had success by squirting vasopressin up kids’ noses:-



In a Stanford study of 30 children with autism, intranasal vasopressin improved social skills more than a placebo, suggesting that the hormone may treat core features of the disorder.



A RANDOMIZED CONTROLLED TRIAL OF INTRANASAL VASOPRESSIN TREATMENT FOR SOCIAL DEFICITS IN CHILDREN WITH AUTISM

Stanford University, Department of Comparative Medicine, Stanford Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social impairments and restricted, repetitive behaviors. Despite ASD’s prevalence, there are currently no medications that effectively treat its core features. Accumulating preclinical research suggests that arginine vasopressin (AVP), a neuropeptide involved in mammalian social functioning, may be a possible treatment for ASD. Objective: The goal of this investigation is to examine the safety and efficacy of AVP in the treatment of social deficits in children with ASD. Material and Methods: Using a double-blind, randomized, placebo-controlled, parallel design, we tested the efficacy and tolerability of 4-week intranasal AVP treatment in a sample of N=30 children with ASD aged 6-12 years. Results: AVP compared to Placebo treatment significantly enhanced social abilities in children with ASD as measured by change from baseline in the trial’s primary outcome measure, the Social Responsiveness Scale (a parent-report measure). AVP-related social improvements were likewise evident on clinician impression and child performance-based measures. AVP treatment also diminished anxiety symptoms and some restricted/repetitive behaviors. An endogenous blood AVP concentration by treatment group interaction was also observed, such that participants with the highest pre-treatment blood AVP concentrations benefitted the most from AVP (but not Placebo) treatment. AVP was well tolerated with minimal side-effects. No AVP-treated participant dropped out of the trial, and there were no differences in adverse event rates reported between the AVP and Placebo groups. Finally, no significant changes from baseline were observed in electrocardiogram, vital signs, height and weight, or clinical chemistry measurements after 4-week AVP treatment. Conclusions: These findings suggest that intranasally administered AVP is a well-tolerated and promising medication for the treatment of social impairments in children with ASD.

Using a double-blind, randomized, placebo-controlled, parallel clinical trial design, we found that the 4-week intranasal AVP treatment enhanced social abilities in children with ASD as assessed by the trial’s primary outcome measure, the SRS-2 T score. The robustness of this parent-reported social improvement score was corroborated by convergent evidence from clinician evaluation of the social communication abilities of trial participants and by performance of trial participants on laboratory tests of social cognition. These preliminary findings suggest that intranasally administered AVP may be a promising medication for treatment of core social impairments in children with ASD.


We also sought to investigate whether pretreatment neuropeptide concentrations in blood could predict AVP treatment response. We found that participants with the highest pretreatment AVP concentrations in blood benefitted the most from intranasal AVP treatment. This finding may seem counterintuitive, particularly in light of our recent studies showing that low AVP concentrations in CSF could be used to differentiate ASD cases from non-ASD control individuals (1314). One might therefore expect that it would be those children with the lowest endogenous AVP concentrations that stood to benefit the most from intranasal AVP treatment. However, being mindful of safety in this pediatric population, our pilot study used a conservative dose escalation regimen in which children were treated with fairly low doses of AVP throughout much of the trial. Assuming that blood AVP concentrations are related, in some manner, to brain AVP activity—a notion about which there is debate (142225)—it is possible that participants with lower endogenous AVP concentrations at the trial’s outset were “underdosed” in terms of drug amount or duration of treatment and, therefore, would not benefit as fully from AVP administration as those with higher endogenous AVP concentrations. This interpretation is consistent with our finding that AVP treatment enhanced simple social perceptual abilities independent of pretreatment AVP concentrations in blood, whereas it was only those AVP-treated individuals with higher pretreatment blood AVP concentrations who showed gains in complex social behaviors and a reduction in repetitive behaviors.

Pharmacological intervention

Commercially available injectable sterile AVP was used in this study. It was initially purchased from JHP Pharmaceuticals (Rochester, MI), which was subsequently acquired by Par Sterile Products (Chestnut Ridge, NY) in 2014. The placebo solution was prepared by Koshland Pharm (San Francisco, CA) and consisted of ingredients used in the active solution except for the AVP compound. A pharmacist transferred 25 ml of AVP (20 International Units (IU)/ml) or placebo solutions into standard sterile amber glass bottles with metered (0.1 ml per puff) nasal spray applicators to ensure that the AVP and placebo applicators were visually indistinguishable to the research team. These applicators were coded and given to the Stanford Health Care’s Investigational Drug Service for refrigerated storage (2°C to 8°C) and subsequent dispensing. After the first AVP dose (see below), the dose-escalation regimen at home for all participants involved administration of 4 IU twice daily (or BID) of AVP during week 1 and 8 IU BID of AVP during week 2. Participants aged 6 to 9.5 years then received 12 IU BID of AVP during weeks 3 and 4, whereas participants aged 9.6 to 12.9 years received 16 IU BID of AVP during weeks 3 and 4. A range of possible AVP doses was identified by review of the published literature; the final study doses were then determined in close consultation with the FDA.


A few years ago I did write about the hormone TRH as a potential means of improving autism.  TRH can also be squirted up your nose, although I favoured an oral TRH super-agonist called Taltirelin/Ceredist.

I also suggested that DHED, an orally active, centrally selective prodrug of estradiol, could well be a therapeutic in autism. DHED should give all the benefits of the female hormone estradiol, without any side-effects outside the CNS.  Many of the benefits are via ROR alpha.

Without having samples of spinal fluid, identifying, let alone treating, central hormonal dysfunction is rather a matter of guesswork.

Hormones are very much interrelated and perform different functions in different parts of the body, so it would be easy to get unwanted effects, as with estradiol, if taken orally.
  
Bumetanide for TSC (Tuberous Sclerosis Complex)

A small trial in children with TSC (Tuberous sclerosis complex) has shown that bumetanide improved their features of autism (social behavior, irritability and hyperactivity) but did not reduce seizures.


Conclusion

This pilot study indicates the potential efficacy of bumetanide on behavioral problems in young patients with TSC. Bumetanide improved irritable, explosive, and social behavior in the majority of patients in this sample and treatment was well tolerated.


Folinic Acid for Autism, but at a lower dose than Dr Frye

I did recently complete my trial of generic Calcium Folinate at something like Dr Frye’s Leucovorin dose.

I found that it did indeed have a positive effect on the use of expressive language.  It prompted the use of more complex sentences.

The downside was that it did also cause aggressive/violent outbursts, so I put it in my “rejected” pile of therapies.  

I was interested to see that in France a trial has been carried out using a lower dose than that proposed by Dr Frye.  Is it possible to get benefits without the side effects? 

Folinic acid improves the score of Autism in the EFFET placebo-controlled randomized trial  


Highlights 

Folinic acid treatment is well tolerated in children with Autism spectrum disorders.
Folinic acid treatment shows improvement in Autism Diagnostic Observation Schedule score.
Effect of 10 mg/d folinic acid should be confirmed by a larger a multi-center trial.
Autism spectrum disorders (ASD) are influenced by interacting maternal and environmental risk factors. High-dose folinic acid has shown improvement in verbal communication in ASD children. The EFFET randomized placebo-controlled trial (NCT02551380) aimed to evaluate the efficacy of folinic acid (FOLINORAL®) at a lower dose of 5 mg twice daily.
Nineteen children were included in the EFFET trial. The primary efficacy outcome was improvement of Autism Diagnostic Observation Schedule (ADOS) score. The secondary outcomes were the improvement in ADOS sub scores communication, social interactions, Social Responsiveness Score (SRS) and treatment safety.
The global ADOS score and social interaction and communication sub scores were significantly improved at week 12 compared to baseline in the folinic acid group (P = 0.003, P = 0.004 and P = 0.022, respectively), but not in the placebo group (P = 0.574, P = 0.780, P = 0.269, respectively). We observed a greater change of ADOS global score (−2.78 vs. −0.4 points) and (−1.78 vs. 0.20 points) in the folinic acid group, compared to the placebo group. No serious adverse events were observed.
This pilot study showed significant efficacy of folinic acid with an oral formulation that is readily available. It opens a perspective of therapeutic intervention with folinic acid but needs to be confirmed by a multi-center trial on a larger number of children.
  

Covid-19

There was concern that people with severe autism might be at increased risk during the current pandemic and indeed the death rate among people with intellectual disability/learning disability/mental retardation did double from 240 a month to 480 a month in the UK.  The real scandal though was deaths in care homes for the elderly, in countries with advanced healthcare systems, where tens of thousands of extra deaths have occurred.

In “advanced” healthcare systems like the UK, early in the epidemic, elderly people caught Covid-19 in hospital and when they returned to their care home, they infected others.  Care workers who are allowed/forced to work in multiple care homes then caught the virus in one home and transmitted it to the others.  Nobody was tested until care homes had already become breeding grounds for the virus.

In Hong Kong they report zero covid-19 deaths in care homes.  Elderly people could not return to their care home from hospital without testing negative for the virus, and procedures were in place to release elderly patients from hospital first to repurposed hotels, where they stayed until negative for the virus. Due to their grim experience with the 2003 SARS epidemic, Hong Kong already had very strict measures in place to limit infections and they even had regular rehearsals in care homes of the procedures to implement in future pandemics.

Where we live there was an outbreak in a care home and the authorities’ reaction was to arrest the boss of the care home.  I suppose that is one way to get other care homes to take matters seriously. We even had soldiers posted outside care homes to stop people entering.  In New York, Cuomo’s threat to care homes was that you might eventually lose your license to operate if you flout the rules. If most care homes are flouting the rules, they cannot all lose their licenses.

Some rich Western countries apparently implemented their much-vaunted flu pandemic procedures.  It looks like they have much to learn from other places, from Hong Kong to Greece, who did very much better.  Greece implemented a draconian lock down, very early, and has had a tiny number of cases and just 166 deaths. When Greece re-opens in July to tourists from high risk countries (UK, France, Italy, Spain etc) we will see what happens.

I do wonder why so many people are living in care homes. In Sweden, I saw on TV, one lady complaining that her fit and healthy father, capable of walking a few miles/km had caught covid-19 in his care home, was refused transfer to hospital and later died.  Why was he sent to live a care home in the first place?

Milan has an old care home called Pio Albergio Trivulzia ("Baggina"), it had over a thousand residents and media reports 200+ covid deaths.

There are horrific cases in the UK of young adults being sent to live in small mental hospitals by their parents; they subsequently deteriorate and some have even died.  Why did the parents hand their children over in the first place?  They thought they could not cope at home, but clearly some dedicated institutions have even less capacity to care. 


Conclusion

Re-purposing existing cheap drugs to treat a different medical condition makes a lot of sense, but it is not going to make the inventor or the drug firm much money.  It is not popular with drug producers.

Developing new drugs to treat any neurological condition looks great in the early stages of research and then they all seem to fade way, wasting many tens of millions of dollars.  Don’t raise your hopes.

Is intranasal vasopressin the smartest hormone to choose to modify?  It is possible today, using existing products and appears to be safe, which are the most important issues. I think there is more potential beyond this single hormone.

Treat autism and intellectual disability/mental retardation medically, so those people can live more normally, be more fulfilled and do not later need such expensive care home provision. It is a win-win strategy.









Friday, 6 March 2020

Calcium Folinate (Leucovorin) and Afobazole for Autism? Good, but …


Dr Frye is embarking on a multi-million dollar trial of Calcium Folinate (Leucovorin) to improve speech in autism.  I just completed my much humbler trial of a cheap generic Calcium Folinate.

I determined it was far cheaper and simpler to make a trial, than arrange for the blood test.  The other reason is that I note in the US they are prescribing Leucovorin, even if you test negative in the test for autoantibodies.

http://iliadneuro.com/order-a-kit.html

Dr Frye thinks many people with autism have low levels of folate inside their brain due to antibodies blocking folate crossing the blood brain barrier.  He even suggests that perhaps the source of these antibodies is your gut and they are produced as a reaction to cow’s milk.

I wondered why speech would be so directly affected by folate, but speech is something that is very noticeable and measurable.

I used 30mg of calcium folinate at breakfast and 15mg in the evening.

After a few days there was very clearly more speech. On several occasions I asked Monty a question, even without facing him eye to eye, and he gave a very much longer response than usual. The response was more like what he would produce if writing with a pencil and paper.

The problem was that three times during the trial he hit me, which is not his typical behavior. Aggression is a listed side effect of high dose calcium folinate.

Excerpt from Dr Frye’s colleague, Dr Dan Rossignol:

Dan Rossignol’s  Presentation at Synchrony 2019 | November 8, 2019

Folinic acid

• The good: Improvements in expressive speech, play skills, social skills, receptive language, attention, stereotypy

• The bad: Hyperactivity, self-stimulatory behaviors, aggression


Calcium Folinate (Leucovorin) is expensive in the US, but very much cheaper in some other countries, so it would be a viable therapy for many people.

Is there a lower dosage where you get the speech benefit without getting hit? I rather doubt it. It did actually try 15mg a day, a while back and saw no effect at all.

Since we do not really know why Calcium Folinate improves speech in particular, I doubt we can say why it produces aggression.

My old post from 2016:-

Clinical Trial of Mega-dose Folinic Acid in Autism


The new trial that is planned:-

The primary objective of this study is to evaluate the cognitive and behavioral effects of liquid leucovorin calcium on young children with autism spectrum disorder (ASD) and determine whether it improves language as well as the core and associated symptoms of ASD. The investigators will enrol 80 children across two sites, between the ages of 2.5 and 5 years, with confirmed ASD and known language delays or impairments. Participation will last approximately 26 weeks from screening to end of treatment.

  
Afobazole

Afobazole is the cheap Russian OTC treatment for anxiety that works as a sigma-1R agonist.  It has an effect on NMDA receptors.

Afobazole was covered in two recent posts.

ER Stress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it -Activation of Sigma-1 Chaperone Activity by Afobazole?


Afobazole is primarily used to treat mild anxiety.  Indeed it appears that sigma-1 receptor activation ameliorates anxiety through NR2A-CREB-BDNF signalling.  NR2A is a sub-unit of NMDA receptors.



Hundreds of millions of dollars are being spent in the US to develop a safe sigma-1R agonist (Anavex 2-73). This drug is being trialed in various autisms (Rett, Fragile X and Angelman syndromes), Parkinson’s and Alzheimer’s.


Afobazole should reduce ER Stress and protein misfolding, making it an interesting potential therapy for many neurological conditions.

I did raise the issue as to whether Afobazole may affect the Excitatory-Inhibitory (E/I) imbalance that is present in bumetanide-responsive autism.

It turns out that in my trial, Afobazole was beneficial in reducing anxiety, it just takes the edge off - nothing drastic.  After several weeks I did notice a slight reduction in cognition, this was only really evident when working on maths. It was more noticeable on cessation.  If I did not teach Monty maths, all I would have noticed was the reduction in anxiety.  When I stopped Afobazole, Monty’s assistant commented how clever he was at school.

Since we are trying to keep up with typical children in academic work at mainstream school, cognitive function is the priority and so no more Afobazole.


Conclusion

I hope the millions of dollars spent on the Calcium Folinate (Leucovorin) trials produce some tangible results. Speech clearly is the area where it shows an effect, I think it has other effects that are less measurable.  It did seem to have an effect on what I would describe as “initiative”, which is completing tasks independently that otherwise you might ask for help to complete.

If you could have the benefits of Calcium Folinate (Leucovorin) without the negative effects, that would indeed be very interesting.

Perhaps giving Calcium Folinate (Leucovorin) to very young non-verbal children will give them a nudge to start speaking.  In those little children you would likely be less concerned by some aggression - they do not hit very hard.

Afobazole also has a place; anxiety is a problem in much autism and for many people a small drop in cognition, if it indeed occurs, is not such a problem.  Long term Afobazole use might produce benefits relating to reduced ER stress and less protein misfolding.

If I had a child with Rett, Fragile X or Angelman syndromes, I would definitely trial Afobazole, since the new American sigma-1R agonist (Anavex 2-73) is not yet available and I suppose will cost 100-200 times more than the Russian drug.

I think you need to find therapies free of any troubling side effects; otherwise in trying to solve one problem, you just create two new ones.





Thursday, 8 December 2016

Nitrosative Stress, Nitric Oxide and Peroxynitrite










In this example of Brain Injury, developing oligodendrocytes are injured and killed by substances released from activated microglia, including nitric oxide and superoxide, which form peroxynitrite. Peroxynitrite has been found to kill these cells through the activation of the 12-lipoxygenase pathway for metabolizing arachidonic acid. Mitochondria may be involved in this pathway as a source of reactive oxygen species.


Much has been written in this blog about oxidative stress, which has now been extremely well researched in autism and more generally. Let’s recap oxidative stress.

The most knowledgeable researcher in this area is Abha Chauhan.  Based on her research and that of others we now know a great deal.  Recall that the body’s key antioxidant is called glutathione (GSH) and when it neutralizes a free radical GSH is converted to its oxidized form, glutathione disulfide (GSSG).  A good measure of oxidative stress is the ratio of  GSH/GSSG.


·        Autism is associated with deficits in glutathione antioxidant defence in selective regions of the brain.

·        In the cerebellum and temporal cortex from subjects with autism, GSH levels are significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG

·        There is also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism.

·        In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism

·        The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism.

·        Disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.


·        The activity of glutathione cysteine ligase (GCL), an enzyme for glutathione synthesis is impaired in autism.

·        The protein expression of its modulatory subunit GCLM was decreased in autism.

·        The activities of glutathione peroxidase (GPx) and glutathione S-transferase were decreased in autism.



For those interested, GPx is a family of enzymes that catalyze the reaction that converts GSH to GCCG.  So in order to soak up those free radicals you need both GSH and GPx.

Glutathione cysteine ligase (GCL) is a key enzyme needed to make the antioxidant GSH.  Dysregulation of GCL enzymatic function and activity is known to be involved in many human diseases, such as diabetes, Parkinson's disease, Alzheimer’s disease, COPD, HIV/AIDS, cancer and autism.  Without sufficient GCL your body cannot make enough glutathione (GSH).


I did have some conversation with Abha Chauhan a few years ago when I found that NAC (N-acetyl cysteine), a known precursor to GSH, really does have a positive behavioral impact in autism.  She is clearly very nice, but not the type to make the leap to translating her science into therapy.

As I have shown there are many other treatable aspects of oxidative stress.

The chart below is my annotated version of the original by Professor Helmut Sies, the German “Redox Pioneer”.  He has published 500 scientific papers.




Nitrosative Stress


Finally to nitrogen.

Nitrogen is the most common pure element in the earth, making up 78.1% of the entire volume of the atmosphere.  Although nitrogen is non-toxic, when released into an enclosed space it can displace oxygen, and therefore presents an asphyxiation hazard. 

Nitrogen is an anesthetic agent. Nitrous oxide (N2O) is commonly known as laughing gas.  It is used in medicine for its unaesthetic and analgesic effects

It is also used as an oxidizer in rocket propellants, and in motor racing to increase the power output of engines, like Mad Max.

In humans we are dealing with Nitric Oxide (NO) and when things go wrong with peroxynitrite and then other Reactive nitrogen species (RNS).  In simple terms Reactive nitrogen species (RNS), like Reactive oxygen species (ROS) are bad news.

Nitric Oxide (NO) itself does lots of good things in your body.  Too much may not be good, but a little more can actually do you good.  NO is a potent vasodilator.

For over 130 years, nitroglycerin has been used to treat heart conditions, such as angina and chronic heart failure.  Nitroglycerin produces nitric oxide (NO). In hospital most patients will receive nitroglycerin during and after a heart attack, people at risk of a heart attack often carry nitroglycerin with them.

If you want to lower your blood pressure or an athlete wanting to legally improve exercise endurance you can increase Nitric Oxide (NO) via diet.  One easy way is to drink beetroot juice, as is common in endurance cycling.  In people with peroxynitrite-derived radicals this may be unwise, because they may have too much NO.



Peroxynitrite

The starting point for the production of those unhelpful Reactive Nitrogen Species (RNS) is this chemical reaction



NO (nitric oxide) + O2· (superoxide) → ONOO (peroxynitrite)



NO production is affected by the enzyme nitric oxide synthase 2 (NOS2).

Superoxide production is catalyzed by NADPH oxidase.

Superoxide also produces Reactive Oxygen Species (ROS).

NADPH oxidase is implicated in many diseases including schizophrenia and autism.

NADPH oxidase 4 (Nox4) activity decreases mitochondrial function (chain complex I).

Activated microglia (as found in autism) produce both nitric oxide and superoxide and are therefore a source of peroxynitrite.




This has started to get rather complicated. So those interested in NADPH should refer to the literature.


Peroxynitrite can directly react with various biological targets and components of the cell including lipids, thiols, amino acid residues, DNA bases, and low-molecular weight antioxidants.


Additionally peroxynitrite can react with other molecules to form additional types of RNS including nitrogen dioxide (·NO2) and dinitrogen trioxide (N2O3) as well as other types of chemically reactive free radicals.



Nitric Oxide and Peroxynitrite in Health and Disease

I have referred on this blog to Abha Chauhan’s mammoth book on oxidative stress in autism on several occasions.  A work of similar quality but this time on Nitric Oxide and Peroxynitrite, is the paper below, by Hungarian Pal Pacher, who works at the US National Institute of Health’s Section on Oxidative Stress Tissue Injury.  He looks like a citation generating machine.

You could spend a long time reading this paper, but in summary peroxynitrite and its derived products have a negative effect on a very wide range of conditions including all the common neurological conditions, inflammatory diseases and again diabetes.  The answer would be peroxynitrite scavengers.



The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.


Some excerpts:-


·        The different events set in motion by the initial generation of peroxynitrite indicate that potent peroxynitrite decomposition catalysts and PARP inhibitors might represent useful therapeutic agents for debilitating chronic inflammatory diseases

·        In summary, available evidence indicates that NO plays dichotomous roles (promotion vs. suppression) in tumor initiation and progression. The activation of angiogenesis and the induction of DNA mutations represent key aspects of the procarcinogenic effects of NO. Peroxynitrite is emerging as a major NO-derived species responsible for DNA damage, mainly through guanine modifications and the inhibition of DNA repair enzymes. In chronic inflammatory states, the identification of 8-nitroguanine in tissues indicates that nitrative DNA damage consecutive to overproduction of NO and peroxynitrite may represent an essential link between inflammation and carcinogenesis.

·        In summary, the different studies listed above indicate that small amounts of NO produced by eNOS in the vasculature during the early phase of brain ischemia are essential to limit the extent of cerebral damage, whereas higher concentrations of NO, generated initially by nNOS and later by iNOS, exert essentially neurotoxic effects in the ischemic brain. Evidence that such toxicity depends, in large part, on the rapid reaction of NO with locally produced superoxide to generate peroxynitrite will be now exposed
  

·        NO is produced by all brain cells including neurons, endothelial cells, and glial cells (astrocytes, oligodendrocytes, and microglia) by Ca2+/calmodulin-dependent NOS isoforms. Physiologically NOS in neurons (nNOS, type I NOS) and endothelial cells (eNOS, type III NOS) produce nanomolar amounts of NO for short periods in response to transient increases in intracellular Ca2+, which is essential for the control of cerebral blood flow and neurotransmission and is involved in synaptic plasticity, modulation of neuroendocrine functions, memory formation, and behavioral activity (491, 890, 1229). The brain produces more NO for signal transduction than the rest of the body combined, and its synthesis is induced by excitatory stimuli. Consequently, NO plays an important role in amplifying toxicity in the CNS. Indeed, under various pathological conditions associated with inflammation (e.g., neurodegenerative disorders and cerebral ischemia), large amounts of NO are produced in the brain as a result of the induced expression of iNOS (type II NOS) in glial cells, phagocytes, and vascular cells, which can exert various deleterious roles (39, 491, 890). Thus NO may be a double-edged sword, exerting protective effects by modulating numerous physiological processes and complex immunological functions in the CNS on one hand and on the other hand mediating tissue damage (446, 491, 890). The detailed discussion of the role of NO in the pathophysiology of various neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), just mentioning a few, is the subject of numerous excellent recent overviews (77, 145, 194, 219, 491, 890, 1003, 1205, 1433) and beyond the scope of this paper. Here we cover only the role of peroxynitrite and protein nitration, which are likely responsible for most deleterious effects of NO in neurodegenerative disorders.


·        Peroxynitrite formation has been implicated in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, MS, ALS, and traumatic brain injury (reviewed in Refs. 194, 608, 1119, 1284). Nitrotyrosine immunoreactivity has been found in early stages of all of these diseases in human autopsy samples as well as in experimental animal models. Increased nitrite, nitrate, and free nitrotyrosine has been found to be present in the cerebral spinal fluid (CSF) and proposed to be useful marker of neurodegeneration (168; reviewed in Refs. 608, 1119, 1284). Once formed in the diseased brain, peroxynitrite may exert its toxic effects through multiple mechanisms, including lipid peroxidation, mitochondrial damage, protein nitration and oxidation, depletion of antioxidant reserves (especially glutathione), activation or inhibition of various signaling pathways, and DNA damage followed by the activation of the nuclear enzyme PARP (608, 1119, 1284).


·        Uric acid has proven to be a useful inhibitor of tyrosine nitration (although it is not a direct peroxynitrite scavenger) (1271) and has been shown to protect the blood-brain barrier and largely prevent the entry of inflammatory cells into the CNS (566, 567). Additionaly, it also prevented CNS injury after inflammatory cells have already migrated into the CNS (1141). Urate has also proven beneficial in reducing the morbidity associated with viral infections (710, 1141). Interestingly, in humans there is an inverse correlation between affliction with gout and MS (710, 1195). Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency (reviewed in Ref. 694). These studies have also suggested that serum uric acid levels could be used as biomarkers for monitoring disease activity in MS

  

·        Recent evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic PD and that derangements in complex I lead to α-synuclein aggregation, which contributes to the demise of dopamine neurons (293). Accumulation and aggregation of α-synuclein may further facilitate the death of dopamine neurons through impairments in protein handling and detoxification (293). As already mentioned above, both mitochondrial complex I and synuclein can be targets for peroxynitrite-induced protein nitration


·        The significance of this intricate interplay may have important ramifications not only for ALS but also for PD and AD (6, 58, 1102). Reactive astrocytes are common hallmark of neurodegeneration, and these results suggest that peroxynitrite may play an important role in promoting this phenotype as well as causing the degeneration of neurons. In ALS, the transformation of astrocytes into a reactive phenotype may explain why ALS is progressive, causing the relentless death of neighboring motor neurons. Interfering in such a cascade to stop the progressive death of motor neurons would not necessarily cure ALS but may keep it from being a death sentence.


·        There is accumulating evidence suggesting that increased oxidative stress and excessive production of NO might contribute to the development of HD by damaging neighboring neurons (reviewed in Refs. 63, 163). Accordingly, increased iNOS expression was observed in neuronal, glial, and vascular cells from brains of HD patients and mouse models of disease (206, 491). Similarly, numerous studies have demonstrated increased 3-NT formation in brain tissues (neurons, glia, and/or vasculature) of mice transgenic for the HD mutation, rats injected into the striatum with quinolinic acid (rat model of HD), and HD patients (300302, 427, 1022, 1023, 1096, 1117). Importantly, both NOS inhibitors and peroxynitrite scavengers decreased neuronal damage, improved disease progression, and also decreased brain 3-NT content in experimental models (301, 1022, 1117). These results suggest that peroxynitrite might be an important mediator of oxidative damage associated with HD.


·        The pathogenetic role of peroxynitrite in TBI is supported by evidence demonstrating increased brain 3-NT levels following TBI in experimental mouse and rat models (9294, 423, 507, 508, 898, 1171, 1360), and by the beneficial effects of NOS inhibitor and peroxynitrite scavengers in reducing neuronal injury and improving neurological recovery following injury (423, 508, 898).Collectively, multiple lines of evidence discussed above provide strong support for the important role of peroxynitrite formation and/or protein nitration in neurodegenerative disorders and suggest that the neutralization of this reactive species may offer significant therapeutic benefits in patients suffering from these devastating diseases.


·        Collectively, the evidence reviewed above support the view that peroxyntrite-induced damage plays an important role in numerous interconnected aspects of the pathogenesis of diabetes and diabetic complications. Neutralization of RNS or inhibition of downstream effector pathways including PARP activation may represent a promising strategy for the prevention or reversal of diabetic complications.

·        In conclusion, multiple lines of evidence discussed above and listed in Table 4 suggest that peroxynitrite plays an important role in various forms of cardiovascular dysfunction and injury; pharmacological neutralization of this reactive oxidant or targeting the downstream effector pathways may represent a promising strategy to treat various cardiovascular disorders.


·        In summary, circulatory shock is a leading cause of death in intensive care units. Considerable improvement in our understanding of the molecular and cellular mechanisms of shock over the past 20 years makes it now a reasonable expectation that novel, efficient mechanism-based therapies will emerge in the near future. Considerable evidence now exists that overproduction of NO and superoxide, triggering the generation of large amounts of peroxynitrite, is a central aspect of shock pathophysiology. In addition to direct cytotoxic effects such as the peroxidation of lipids, proteins, and DNA, peroxynitrite also occupies a critical position in a positive feedback loop of inflammatory injury, by (directly or indirectly, via PARP activation) activating proinflammatory signaling and by triggering the recruitment of phagocytes within injured tissues, leading to further NO, superoxide, and peroxynitrite production, which will progressively amplify the initial inflammatory reactions (see sect. VID, Fig. 14). These various observations support the view that future strategies reducing peroxynitrite or its precursors might have a considerable therapeutic impact in clinical circulatory shock.


Peroxynitrite Scavengers


We have already covered two substances in this blog that are potential Peroxynitrite Scavengers:-


Calcium Folinate

This is Roger’s magic pill to treat his Cerebral Folate Deficiency, but it may have application far beyond this, likely rare, condition, for those that tolerate it.

Tetrahydrofolic acid, or tetrahydrofolate, is a folic acid derivative. It has the potential to quench those peroxynitrite-derived radicals.




The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.


Uric Acid

Uric acid has proven to be a useful inhibitor of tyrosine nitration.  It was thought to be a scavenger of peroxynitrite, but our clever Pal from Hungary tells thatit is not a direct peroxynitrite scavenger ….Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency”.

An old paper:-



Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-γ receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.



Here we have a paper with the link to Tetrahydrobiopterin (BH4,), also known as sapropterin, covered in an old post:-




Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: Implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.


Xanthine oxidase (XO, sometimes 'XAO') is a form of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen species.[2] These enzymes catalyze the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. These enzymes play an important role in the catabolism of purines in some species, including humans.





Because xanthine oxidase is a metabolic pathway for uric acid formation, the xanthine oxidase inhibitor allopurinol is used in the treatment of gout.


Inhibition of xanthine oxidase has been proposed as a mechanism for improving cardiovascular health.  A study found that patients with chronic obstructive pulmonary disease (COPD) had a decrease in oxidative stress, including glutathione oxidation and lipid peroxidation, when xanthine oxidase was inhibited using allopurinol.


Reactive nitrogen species, such as peroxynitrite that xanthine oxidase can form, have been found to react with DNA, proteins, and cells, causing cellular damage or even toxicity. Reactive nitrogen signaling, coupled with reactive oxygen species, have been found to be a central part of myocardial and vascular function, explaining why xanthine oxidase is being researched for links to cardiovascular health.


We also should recall that abnormalities are common in autism.





So perhaps allopurinol for those with too much uric acid?  Perhaps this is a good marker for peroxynitrites ?





Conclusion

As is often the case there some contradiction in the science.  Is NO good for you or not?  Are both high and low uric acid actually indicating the same biological problem.

It looks like the research into very expensive BH4 therapy might be better directed into peroxynitrite scavengers.

I think we have found the reason why so many people with autism respond to Leucovorin (calcium folinate) and, unlike in our friend Roger, it may not be because of cerebral folate deficiency.

It looks like many other chronic conditions from diabetes to COPD to schizophrenia might also benefit from  calcium folinate.

Before I forget, in the Helmut Sies oxidative stress graphic I did highlight selenium.  The GPx enzymes contain selenium and if there is selenium deficiency the body's key antioxidant mechanism will be compromised. According to Abha Chauhan's book,  "Likewise, levels of exogenous antioxidants were also found to be reduced in autism, including vitamin C, vitamin E, and vitamin A in plasma, and zinc and selenium in erythrocytes (James et al., 2004)".  This might suggest adding a little extra selenium.

I think Allopurinol is worth a look for some autism.  Allopurinol does indeed reduce reactive nitrogen species in COPD (severe asthma), as suggested above.



“These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic "