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Friday 6 February 2015

Tuning GABAa receptors, plus Oxytocin

Today’s post will hopefully not get too complicated.

As has been mentioned in this blog, and also at leading institutions like MIT, it does seem possible to fine-tune certain receptors in the brain that have become dysfunctional in autism.  In the case of MIT they were “tuning” a receptor called mGluR5, which they suggested was either hypo or hyper, in other words too much or too little, depending on what the underlying disease variant was.


This was done with something called an allosteric modulator, either a positive one called PAM, or a negative one called NAM.

They found that a particular glumate receptor, called mGluR5, was dysfunction in many autism-like conditions.  But the nature of the dysfunction varied, so different people would require different treatments to return the receptor performance back to normal (top dead center).   So it really becomes like tuning your car engine. 
As I have progressed in my review of the literature it becomes clear that numerous receptors are “out of tune”; so a better analogy is tuning something like a piano.

  



"Tuning" the shape (but not number) of dendritic spines also appears not to be as fanciful as it sounds.


Back to GABAA

Regular readers will know that one of the key dysfunctional receptors in autism is called GABAA.




This subject is very complicated.  In effect what appears to have happened in autism is that the neurons have not matured as they should, and so GABAA receptors continue to function in their “normal” immature state.  The concentration of chloride remains high since the NKCC1 transporter continues to exist, whereas KCC2/3 should have developed.  The result is that when the receptor is stimulated, instead of causing an inhibitory/calming effect it causes an excitatory effect.





This is fortunately treatable by inhibiting the flow of chloride into the cells, through NKCC1, using a drug called Bumetanide.

However this is not the end of the story.


At least 11 binding sites on GABAA receptors

As you can learn from Wikipedia:-


The active site of the GABAA receptor is the binding site for GABA and several drugs such as muscimol, gaboxadol, and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin, among others.

We are particularly interested in the allosteric binding sites.
The only one that is usually referred to, in any depth, is the site for benzodiazepines, but there are at least 11 different binding sites.

Abstract
gamma-Aminobutyric acid (GABA)a receptors for the inhibitory neurotransmitter GABA are likely to be found on most, if not all, neurons in the brain and spinal cord. They appear to be the most complicated of the superfamily of ligand-gated ion channels in terms of the large number of receptor subtypes and also the variety of ligands that interact with specific sites on the receptors. There appear to be at least 11 distinct sites on GABAA receptors for these ligands.




These sites include:-

·        GABA Binding Site
·        Benzodiazepine Binding Site
·        Neurosteroid Binding Site
·        Convulsant Binding Site
·        Barbiturate Binding Site
·        b Subunit Binding Site(s)


In an earlier post I highlighted the discovery by Professor Catterall, that tiny doses of a particular Benzodiazepine drug called Clonazepam had a strange effect on the GABAA receptor.

Clonazepam is a known Positive Allosteric Modulator (PAM) of the GABAA site.  In mature neurons it amplifies the calming effect when the GABA binding site is stimulated.  In mouse models of autism (we assume therefore immature neurons)   where GABA is still excitatory, the tiny dose seemed to switch it to inhibitory.

This suggests a new function, rather than a PAM, the effect was to invert the function entirely.

Now it appears that similar things may indeed also be possible at some of the other 9+ binding sites (I exclude GABA Binding Site itself)

As complicated as this subject may sound, it actually gets even more complicated since the GABA receptors are made up of sub-units.  It appears that mutations in these subunits may be a cause of some epilepsies and, I propose, some “oddities” in autism.

Recent studies have again shown that many genetic dysfunctions found in autism relate to GABA, this short article is not so recent, but gives a nice summary:-


GABA is the major inhibitory neurotransmitter in the brain. It essentially acts as a brake for brain activation. Several aspects of GABA regulation have been linked to ASD, from early brain development to adult brain function.
Variations in GABA receptor subunits have been strongly associated with ASD. GABA receptors come in two major forms: fast, “ionotropic” GABAA receptors let negatively charged chloride ions flow into the neuron, and slow, “metabotropic” GABAB receptors produce chemical messages inside the neuron. GABAA receptors, the most common form in the brain, contain five subunits that shape their properties. Genome-wide association studies have linked the GABAA receptor subunit genes GABRA4 (α4 subunit), GABRB1 (β1 subunit), and GABRB3 (β3 subunit) to autism.[1][2] In addition, deletion of a chromosomal region that contains a cluster of a variety of GABA receptor genes (region 15q11-13) causes Angelman Syndrome.[3][4]
Genes controlling the development of GABA-releasing neurons have also been associated with ASD. Autism-linked variations in the ARX and DLX family of transcription factors interfere with proper expression of GABA.[5][6][7] Absence of such GABA-releasing neurons would negatively affect early brain development as well as adult brain stability.

Notably, variations in other ASD-linked genes affect GABA signaling. New evidence shows that the gene MECP2, the mutation of which causes Rett Syndrome, is critical for normal function of GABA-releasing neurons.[8] When MECP2 expression was blocked in GABAergic neurons of mice, GABA expression and release were reduced and the mice exhibited autistic behaviors.

ASD is a complex disorder that is likely to be caused by a combination of mutations in a variety of genes. GABA receptors are a promising therapeutic target because of their important role in monitoring brain excitation. Identification and exploration of autism-linked mutations in other GABA-related genes could shed light on the pathogenesis of autism.


Over to Switzerland

At the University of Bern a small research group is looking  at the world of  GABAA receptors, here is what they say:-

“Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.” 


Here is one of their recent papers, that shows both what is known and how very much remains unknown.




Ion Conductance
The GABAA receptors are generally GABA-gated anion channels selective for Cl ions, with some permeability for bicarbonate anions (49). Exceptionally, in C. elegans, a cation-selective GABA-gated channel has been discovered (50). Excitatory neurotransmitters increase the cation conductance to depolarize the membrane, whereas inhibitory neurotransmitters increase the anion conductance to tendentially hyperpolarize the membrane. However, if the gradient for Cl ions decreases due to down-regulation of KCC2 chloride ion transporters, opening of GABAA receptors may cause an outward flux of these anions, leading to depolarization of the membrane and thereby to excitation. This phenomenon has been implicated in neuropathic pain (51). During early development (52) and in neuronal subcompartments (53), GABA similarly confers excitation. 
Although it is relatively simple to address questions at the level of individual receptor subunit isoforms, we can only speculate how many GABAA receptors are expressed in our brain and what their subunit composition is, not to mention subunit arrangement.


Conclusions
Many scientists and companies are put off by the complexity of the field of GABAA receptors, but it is exactly this complexity that offers numerous possibilities of fine-tuned pharmacological interventions.

It may be anticipated that genetic alterations of subunits of the GABAA receptor affect any of the above mentioned processes and thereby contribute to inherited human diseases. A start has been made with the analysis of point mutations that cause epilepsy






Why is all this relevant ?

We have in recent posts discovered that at least two anti-convulsants (carbamazepine and phenytoin) appear to modulate GABAA receptors in unexpected ways when given in tiny doses.

We also found out that valproate also seems to possess such qualities.  The exact mode of action of valproate is not known and perhaps it also acts a modulator of one of the many binding sites on the GABAA receptors.

We do think that valproate is working somehow via GABA.



It turns out that Carbamazepine has also been shown to potentiate GABA receptors made up of alpha1, beta2, and gamma2 subunits.

I have already established that the effect of tiny doses of Valproate is not the same as tiny doses of Clonazepam.

The next step would be to look at the effect of tiny doses of carbamazepine, phenytoin and potentially anything else that modulates those mysterious  GABAAsites.  They are clearly all there for a reason.  It seems that their role goes beyond just the allosteric modulation (amplification/reduction) of GABA’s effect.  It is likely much more subtle and they affect emotional behaviour.

Given the difficulty/impossibility of research on human brains, in the end we may need to revert to the medical world’s often used “scientific” discovery methods known as trial and error, and stumbled upon.

For the moment that will be left to Professors Sigel and Catterall and their mice, and Dr Bird, in Australia, with his human subjects.




Oxytocin and Bumetanide share the same mode of action in autism


Whilst on the subject of GABAA, I should come back to Oxytocin.



The conclusion of this Ben-Ari paper from last year is that Oxytocin and Bumetanide share the same effect in autism; they lower the level of chloride within the neurons and help switch GABA back to inhibitory.

It seems that oxytocin from the mother may be the signal to the developing brain to lower Cl levels.  Oxytocin has many other functions in the body.

Small doses of oxytocin/Syntocinon, have been shown to be effective in some people with autism.  One reader from Portugal has written on this blog how effective it has been in his young son.

Oxytocin/Syntocinon is not available everywhere, but is being reintroduced to the US.



I am wondering if in some people, who are not responders, bumetanide/oxytocin lowers the level of chloride, but not enough to show any benefit.  People using Bumetanide, which has a short half-life, comment that the effect fades through the day and that splitting the same daily dose 3 times a day is beneficial over 2 times a day.  This might suggest that combining Oxytocin with Bumetanide might give better results, by maintaining the downward pressure on chloride levels and keeping GABA more inhibitory and for longer.

In the longer term, an analog of Bumetanide is needed without the diuretic effect and with a delayed release, to maintain a constant effective level.  This is known to the researchers, but would require a big financial investment.

Larger doses of oxytocin are likely to produce effects elsewhere in the body.

If anyone tries the combination of Bumetanide + oxytocin, let me know.





Tuesday 3 February 2015

Autism & Schizophrenia - Histamine degradation via HMT (requiring SAMe) and via DAO

Today’s post is a little complicated because it links together various issues ranging from food allergies to severe headaches, brain inflammation to arthritis.

The common link here is histamine, which has been covered at length on this blog.  You may recall that the H1 histamine receptor is the one associated with hay fever, H2 is expressed in the intestines and is involved in regulating acidity levels, H3 is mainly found in the central nervous system (CNS).

The Histamine H4 receptor has been shown to be involved in mediating eosinophil shape change and mast cell chemotaxis.

Here is the full paper, for those interested in mast cells:-


In addition to all these receptors, histamine causes an increase in the pro-inflammatory cytokine IL-6.  IL-6 is elevated in autism and many other inflammatory conditions ranging from arthritis to traumatic brain injury (TBI). 

One of interesting interventions in this post is SAMe (S-Adenosyl methionine )and its precursor L-methionine.  We will see why a deficit of SAMe causes a problem when the body tries to degrade/deactivate histamine.

We will also see in a later post that the level of SAMe in the body modulates the release anti-inflammatory cytokines like IL-10 and IL-35.  Here is one link, for now.


5. Higher expression of IL-35 could be induced by higher hypomethylation status in tissues

Previous reports showed that epigenetic mechanisms, including methylation and demethylation, control T helper cell differentiation and cytokine generation [41]. As we discussed in our recent review [42], the ratio of cellular methylation donor S-adenosylmethionine (SAM) levels over S-adenosylhomocysteine (SAH) levels is an important metabolic indicator of cellular methylation status [43], [44]. A higher SAM/SAH ratio suggests a higher methylation status than normal (hypermethylation) whereas a lower SAM/SAH ratio indicates a lower methylation status than normal (hypomethylation).  A previous report showed that feeding rats with SAM, a methyl donor, inhibits the expression of TGF-βR1 and TGF-βR2 [45], suggesting that intracellular global methylation status regulates anti-inflammatory cytokine signaling.  … Cont/


Interestingly, I found that for decades SAMe  has been a mainstream drug therapy used in Italy to treat arthritis.
    

Histamine degradation

In mammals, histamine is metabolized by two major pathways: N(tau)-methylation via histamine N-methyltransferase (HMT) and oxidative deamination via diamine oxidase (DAO).

HMT and uses S-adenosyl-L-methionine (SAMe) as the methyl donor.  If SAMe is lacking HMT cannot degrade histamine.

In the brain, the neurotransmitter activity of histamine is controlled by N(tau)-methylation.  It is disputed whether diamine oxidase is found in the central nervous system.  Some sources say it is not, but other studies specifically measure DAO levels in the brain, finding them elevated in schizophrenia.

A common genetic polymorphism affects the activity levels of HMT in red blood cells.  This can be tested for.

People with low levels of DAO will not be able to degrade histamine in their body nor, it appears to me, in the brain.

People with low levels of SAMe will not be able to degrade histamine as they should, that has crossed the BBB (blood brain barrier).  Those same low levels of SAMe will have raised the inflammatory cytokines and reduced the anti-inflammatory cytokines.


Methionine metabolism


I am always very wary when I see charts like the one below.  Often they are used to justify all kinds of strange ideas.  So the following methionine description is just a cut and paste from Wikipedia.

If anything goes wrong in this metabolism, you might indeed expect strange things to happen.  The ratio of SAMe/SAH is measurable  and tends to be markedly low in people with ASD.  This why DAN doctors use vitamin B12 injections, other B vitamins and other exotic sounding “supplements”.

Metabolic biomarkers of increased oxidative stress and impairedmethylation capacity in children with autism




Methionine is an essential amino acid that must be provided by dietary intake of proteins or methyl donors (choline and betaine found in beef, eggs and some vegetables). Assimilated methionine is transformed in S-adenosyl methionine (SAM) which is a key metabolite for polyamine synthesis, e.g. spermidine, and cysteine formation (see the figure on the right). Methionine breakdown products are also recycled back into methionine by homocysteine remethylation and methylthioadenosine (MTA) conversion (see the figure on the right). Vitamins B6, B12, folic acid and choline are essential cofactors for these reactions. SAM is the substrate for methylation reactions catalyzed by DNA, RNA and protein methyltransferases.

The products of these reactions are methylated DNA, RNA or proteins and S-adenosylhomocysteine (SAH). SAH has a negative feedback on its own production as an inhibitor of methyltransferase enzymes. Therefore SAM:SAH ratio directly regulates cellular methylation, whereas levels of vitamins B6, B12, folic acid and choline regulates indirectly the methylation state via the methionine metabolism cycle.[44][45] A near ubiquitous feature of cancer is a maladaption of the methionine metabolic pathway in response to genetic or environmental conditions resulting in depletion of SAM and/or SAM-dependent methylation. Whether it is deficiency in enzymes such as methylthioadenosine phosphorylase, methionine-dependency of cancer cells, high levels of polyamine synthesis in cancer, or induction of cancer through a diet deprived of extrinsic methyl donors or enhanced in methylation inhibitors, tumor formation is strongly correlated with a decrease in levels of SAM in mice, rats and humans.[46][47]







Low levels of SAMe do seem to cause problems in some people and it is straightforward to increase it.  You can either give extra SAMe, which is expensive, or L-methionine, which is cheap.

Interestingly, L-methionine is used at Johns Hopkins to treat autism and apparently is particularly effective at increasing speech.

If L-methionine was effective it could be for reasons including:-

·        cellular methylation was dysfunction
·        histamine in the brain had been elevated
·        the level of pro/anti-inflammatory cytokines had been out of balance 

Here are some examples of the use of SAMe (methionine)




In its native form, SAMe is labile and degrades rapidly. However, several patents for stable salts of SAMe have been granted. Among them, toluenedisulfonate and 1,4-butanedisulfonate forms have been chosen for pharmaceutical development, and as a result, preclinical and clinical studies have been performed. Numerous studies over the past 2 decades have shown that SAMe is effective in the treatment of depression (46), osteoarthritis (78), and liver disease (911). Moreover, SAMe has a very favorable side-effect profile, comparable with that of placebos. Thus, SAMe offers considerable advantages as an alternative to standard medications.

Depression
Clinical studies performed as early as 1973 indicated that SAMe had antidepressant effects (38). Over the next 2 decades, the efficacy of SAMe in treating depressive disorders was confirmed in > 40 clinical trials. Several review articles that summarize these studies were published in 1988 (4), 1989 (5), 1994 (6), and 2000 (12). In a meta-analysis, Bressa (6) reviewed 25 controlled trials including a total of 791 patients. The outcome of this analysis showed that SAMe had a significantly greater response rate than did placebo and was comparable to tricyclic antidepressants. Brown et al (12) summarized the literature on the use of SAMe in depressive disorders up to the time of publication in 2000; they reported that SAMe had been studied in 16 open, uncontrolled trials (660 patients); 13 randomized, double-blind, placebo-controlled trials (537 patients); and 19 controlled trials comparing SAMe with other antidepressants (1134 patients). Significant antidepressant effects were observed in all 16 open trials. In 18 controlled trials, SAMe was as effective as was impramine, chlorimipramine, nomifensine, and minaprine. An important observation from these studies is that SAMe had far fewer side effects than did standard medications.
Neurologic disorders
Several studies indicate that a CNS methyl group deficiency may play a role in the etiology of Alzheimer disease (AD). Reduced SAMe concentrations were found in CSF (34) and in several different brain regions (51) of patients with AD. In addition, reduced phosphatidylcholine concentrations were found in postmortem brain tissue from AD patients (52), and significant changes in brain phospholipids that are dependent on SAMe metabolism were detected in vivo with 31p magnetic resonance spectroscopy in the early stages of AD (53). Deficiencies of folate and vitamin B-12 are common in the elderly (39, 40) and can lead to decreased CNS SAMe concentrations. Several studies indicate that elevated blood homocysteine concentrations, considered to be a marker for folate deficiency, vitamin B-12 deficiency, and impaired methylation, may be a risk factor for AD (5456). It is therefore important to note that preliminary studies using either SAMe (57) or alternative methyl group donors [such as betaine (58) or folate and vitamin B-12 (59, 60)] can improve measures of cognitive function. These treatments may be able to restore methyl group metabolism and normalize blood homocysteine concentrations. Reduced SAMe concentrations in CSF were also reported in patients with subacute combined degeneration of the spinal cord resulting from folate or vitamin B-12 deficiency (39) and in children with inborn errors of the methyl-transfer pathway who had demyelination (61). In these cases, treatment with methyl-group donors such as SAMe, methyltetrahydrofolate, betaine, and methionine was associated with remyelination and a clinical response (61).

Lancet. 1991 Dec 21-28;338(8782-8783):1550-4.

Association of demyelination with deficiency of cerebrospinal-fluid S-adenosylmethionine in inborn errors of methyl-transfer pathway.

We have shown that demyelination is associated with cerebrospinal-fluid S-adenosylmethionine deficiency and that restoration of S-adenosylmethionine is associated with remyelination.


Remyelination is also interesting.  Damage to the critical myelin layer has been suggested to occur with mitochondrial disease.  Most young people with autism show signs of mitochondrial disease (based on post mortem samples) but not old people with autism.

Demyelination is the loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative autoimmune diseases, including multiple sclerosis.


Liver disease
The potential benefit of SAMe in treating liver disease stems from several important aspects of SAMe metabolism. In mammals, as much as 80% of the methionine in the liver is converted into SAMe (23). Hepatic glutathione, which is dependent on methionine and SAMe metabolism, is one of the principal antioxidants involved in hepatic detoxification. Studies have shown that abnormal SAMe synthesis is associated with chronic liver disease, regardless of its etiology. Early studies indicated that patients with liver disease are unable to metabolize methionine, resulting in elevated blood concentrations (67). Subsequent studies in patients with liver disease showed that the defect resulted from decreased activity of a liver-specific isoenzyme, MAT I/III; this defect effectively blocks the conversion of methionine to SAMe (68). Several well-designed experimental studies indicated that MAT I/III is regulated by cellular concentrations of both nitric oxide and glutathione. Thus, increased nitric oxide concentrations and decreased glutathione concentrations were shown to inhibit MAT I/III via mechanisms involving increased S-nitrosylation and free radical damage to the enzyme protein (69, 70). Experimental studies and clinical trials showed that parenteral and oral SAMe administration can increase glutathione concentrations in red blood cells (71) and in hepatic tissue (72, 73) and can effectively replenish depleted glutathione pools in patients with liver disease. The literature on the clinical potential of SAMe in the treatment of liver disease (including cholestasis, hepatitis, and cirrhosis) has been the subject of several review articles (911, 74, 75).
  
Osteoarthritis
The potential benefit of SAMe in treating osteoarthritis was discovered when patients enrolled in clinical trials of SAMe for depression reported marked improvement in their osteoarthritis symptoms (76). Nine clinical trials in Europe (77) and 1 in the United States (7) with a total of > 22 000 participants have confirmed the therapeutic activity of SAMe against osteoarthritis. SAMe has effects similar to those of the nonsteroidal anti-inflammatory drugs, but its tolerability is higher.
  

Back to DAO

I think we have established the one mechanism for histamine degradation has useful pointers for those interested in autism; now it is time to look at the other one.

D-amino acid oxidase (DAAO; also DAO, OXDA, DAMOX) is an enzyme. Its function is to oxidize D-amino acids to the corresponding imino acids, producing ammonia and hydrogen peroxide.

Recently, mammalian D-amino acid oxidase has been connected to the brain D-serine metabolism and to the regulation of the glutamatergic neurotransmission. In a postmortem study, the activity of DAAO was found to be two-fold higher in schizophrenia.
DAAO is a candidate susceptibility gene and may play a role in the glutamatergic mechanisms of schizophrenia.  Risperidone and sodium benzoate are inhibitors of DAAO.


Abstract

We review the role of two susceptibility genes; G72 and DAAO in glutamate neurotransmission and the aetiology of schizophrenia. The gene product of G72 is an activator of DAAO (D-amino acid oxidase), which is the only enzyme oxidising D-serine. D-serine is an important co-agonist for the NMDA glutamate receptor and plays a role in neuronal migration and cell death. Studies of D-serine revealed lower serum levels in schizophrenia patients as compared to healthy controls. Furthermore, administration of D-serine as add-on medication reduced the symptoms of schizophrenia. The underlying mechanism of the involvement of G72 and DAAO in schizophrenia is probably based on decreased levels of D-serine and decreased NMDA receptor functioning in patients. The involvement of this gene is therefore indirect support for the glutamate dysfunction hypothesis in schizophrenia.

Abstract
D-serine has been shown to be a major endogenous coagonist of the N-methyl D-aspartate (NMDA) type of glutamate receptors. Accumulating evidence suggests that NMDA receptor hypofunction contributes to the symptomatic features of schizophrenia. d-serine degradation can be mediated by the enzyme d-amino acid oxidase (DAAO). An involvement of d-serine in the etiology of schizophrenia is suggested by the association of the disease with single nucleotide polymorphisms in the DAAO and its regulator (G72). The present study aims to further elucidate whether the DAAO activity is altered in schizophrenia. Specific DAAO activity was measured in postmortem cortex samples of bipolar disorder, major depression and schizophrenia patients, and normal controls (n=15 per group). The mean DAAO activity was two-fold higher in the schizophrenia patients group compared with the control group. There was no correlation between DAAO activity and age, age of onset, duration of disease, pH of the tissue and tissue storage time and no effect of gender, cause of death and history of alcohol and substance abuse. The group of neuroleptics users (including bipolar disorder patients) showed significantly higher D-amino acid oxidase activity. However, there was no correlation between the cumulative life-time antipsychotic usage and D-amino acid oxidase levels. In mice, either chronic exposure to antipsychotics or acute administration of the NMDA receptor blocker MK-801, did not change d-amino acid oxidase activity. These findings provide indications that D-serine availability in the nervous system may be altered in schizophrenia because of increased D-amino acid degradation by DAAO.


Abstract
We examined the association of autism spectrum disorders (ASD) with polymorphisms in the DAO and DAOA genes. The sample comprised 57 children with ASD, 47 complete trios, and 83 healthy controls in Korea. Although the transmission disequilibrium test showed no association, a population-based case-control study showed significant associations between the rs3918346 and rs3825251 SNPs of the DAO gene and boys with ASD.


DAO as a target for the treatment of schizophrenia

As noted above, both D-serine and D-alanine show some effectiveness as add-on treatment in schizophrenia, in particular for the amelioration of negative and possibly cognitive symptoms. There are also comparable approaches and data regarding glycine augmentation. Since enzymes represent viable drug targets, DAO is receiving attention as a potential alternative therapeutic means to enhance NMDAR function in schizophrenia. The fact that DAO activity appears to be increased in schizophrenia provides another reason to propose that its inhibition might be beneficial. It is also intriguing that the original antipsychotic, chlorpromazine, was shown to be a DAO inhibitor in vitro over fifty years ago,2 confirmed recently and also found to apply to risperidone; whether these observations are relevant clinically are unknown, but they do provide a precedent for the potential therapeutic benefits of selective DAO inhibitors.
To date there have been no clinical trials of DAO inhibitors in schizophrenia, but several preclinical studies which, although findings remain preliminary, show that inactivation of DAO, either in ddY/DAO- mice or after pharmacological DAO inhibition in rats and mice, produces behavioural, electrophysiological and neurochemical effects suggestive of a pro-cognitive profile (Table 4). The Table includes the three DAO inhibitors for which functional data have been published thus far: AS057278,10 CBIO,201,203 and Compound 8.202 Several other small molecule DAO inhibitors have been patented but their behavioural effects have yet to be reported.62,204

Conclusions and future directions

DAO, as the enzyme which degrades the NMDAR co-agonist D-serine, has the potential to modulate NMDAR function and to contribute to NMDAR hypofunction in schizophrenia. Both genetic and biochemical data support an involvement of DAO in the disorder, however the processes involved are difficult to interpret. This is due to the many questions left unanswered concerning the neurobiology of DAO and its physiological roles. Notably there is still much that is unclear as to its localization and activity within the brain, and its spatial and functional relationships with its substrates. In addition, D-serine and thus DAO may have roles other than NMDAR modulation, whilst other DAO substrates, especially D-alanine, may also be relevant to any involvement of DAO in schizophrenia. Similarly, although recent preclinical data hint at potential therapeutic benefits of DAO inhibitors, extensive further study is required to establish their efficacy, tolerability, and mechanism.


Many drugs act as DAO inhibitors to a limited degree, even though this is not their intended mode of action.

We have heard about Sodium benzoate and Risperidone, but there are many others.


           

Results

Chloroquine and clavulanic acid showed greatest inhibition potential on diamine oxidase (> 90%). Cimetidine and verapamil showed inhibition of about 50%.
Moderate influence on DAO was caused by isoniazid and metamizole, acetyl cysteine and amitriptyline
(>20%). Diclofenac, metoclopramide, suxamethonium and thiamine have very low inhibition potential (<20%).  Interestingly cyclophosphamide and ibuprofen displayed no effect on DAO.

Conclusion

Since even levels of about 30% inhibition may be critical, most of the observed substances, can be designated as DAO inhibitors. Other drug components than active ingredients did not affect DAO activity or its interaction with a specific drug.


Note that cimetidine (Tagamet), a histamine H2-receptor antagonist drug used in promoting the healing of active stomach and duodenal ulcers.  Verapamil is in my “Polypill” and is a potent mast cell stabilizer.   Is this link back to histamine a coincidence?  I think not.









Conclusion

The experts are yet to conclude much, but it does seem that SAMe levels are low in autism and brain DAO levels are high schizophrenia (adult onset autism).  In Korea, DAO was shown to be dysfunction in autism.

It seems that, by coincidence, Risperidone happens to be an inhibitor of DAO and this indeed accounts for some its side effects.  Risperidone has actions at several 5-HT (serotonin) receptor subtypes, Dopamine receptors, Alpha α1/2 adrenergic receptors and even H1 histamine receptors.  Risperidone seems to be drug of last resort.

There are no selective DAO inhibitors currently in use.

We did see that two old drugs Tagamet and Verapamil are potent DAO inhibitors in vitro.

This suggest to me that while sodium benzoate has been trialed “successfully” in schizophrenia, perhaps it would be worth comparing the effect of Tagamet and Verapamil.

When it comes to autism/schizophrenia, it would seem that in some people one or more of the following might be helpful:-

·        Sodium benzoate, or cinnamon a precursor
·        Tagamet the H2 antihistamine, already used by some people with mastocytosis
 ·        Verapamil, the calcium channel blocker that actually does much more
·        SAMe, or L-methionine a precursor.