Modulating Neuronal Chloride via WNK



Today�s post is a little complicated, but should be relevant to parents already using bumetanide to reduce the severity of autism.



Tuning neurons via Cl-sensitive WNK

The science behind today�s post only started to evolve twenty years ago when it became understood how chloride enters and exits the neurons in your brain. Nonetheless there is now a vast amount of research and there are parts that have not yet been covered in this blog. 

A moving target
The first thing to realize is that trying to reduce the elevated level of chloride found in much autism is very much an ongoing battle. Chloride is flowing in too fast via NKCC1 and exiting too slowing via KCC2.
If you want to reduce the entry via NKCC1, or increase the exit via KCC2, either of these two strategies should lower the equilibrium level of chloride.  Most strategies in this blog target NKCC1, but in another disease (neuropathic pain) the target has been KCC2.
Whichever you target, the risk is that the body�s feedback loops come into play and undo some of your good work. This was highlighted recently in a paper by Kristopher Kahle at Yale, who looks likely to be joining this blog�s Dean�s List, which highlights the researchers who are really worth following. He is part of the new generation of higher quality researcherswho have an interest in autism.   
If all that was not complex, we have to realize that the number of these valves (cotransporters) that either let chloride enter or exit, is changing all the time.  Many factors relating to inflammation and pain affect the number of NKCC1 and KCC2 cotransporters, so in times of inflammation  you get a reduction KCC2 and/or an increase in NKCC1; hence a higher level of chloride in your neurons.
When people have a traumatic brain injury (TBI), they get an increase in NKCC1 and so an increase in neuronal chloride.  This makes the neurotransmitter GABA less inhibitory, this can lead to cognitive loss, behavioral changes and even a tendency to seizures.
In TBI not surprisingly you have elevated inflammatory signaling, such as via something called NF-?B. As pointed out by our reader AJ, when you take the supplement Astaxanthin, you reduce the expression of NKCC1 in TBI and this has been shown to be via NF-?B. So the potent antioxidant and broadly anti-inflammatory Astaxanthin is a good choice for people with elevated NF-?B.
Much is written in neuropathic pain research about KCC2 and drugs are being developed that could later be repurposed for autism (and indeed TBI). In neuropathic pain there is a lack of KCC2 expression and this is known to be linked to something called WNK1.  The WNK1 gene provides instructions for making multiple versions of the WNK1 protein. 

Mechanisms that control NKCC1 and KCC2
There are multiple mechanisms that affect the expression of NKCC1 and KCC2.  In some cases the two (NKCC1 and KCC2) are interrelated so either one is expressed or the other is expressed.  In the mature brain there should be KCC2, but little NKCC1.  

The current research by Kristopher Kahle is based on the recent discovery of a �rheostat� of chloride homeostasis, comprising the Cl- sensitive WNK-SPAK kinases and the NKCC1/KCC2 cotransporters. This rheostat provides a way to reversibly tune the strength of inhibition in neurons.
In effect this means that inhibiting WNK should make GABA more inhibitory, which is the goal for all people who have elevated chloride in their neurons.   


GABAA receptors are ligand-gated Cl- channels. GABAAR activation can elicit excitatory or inhibitory responses, depending on the intraneuronal Cl- concentration levels. Such levels are largely established by the Cl- co-transporters NKCC1 and KCC2. A progressive postnatal increase in KCC2 over NKCC1 activity drives the emergence of GABAAR-mediated synaptic inhibition, and is critical for functional brain maturation. A delay in this NKCC1/KCC2 �switch� contributes to the impairment of GABAergic inhibition observed in Rett syndrome, fragile X syndrome, and other neurodevelopmental conditions, such as epilepsy.

Kristopher Kahle and his colleagues aim to understand the mechanisms that govern these developmental changes in NKCC1/KCC2 activity. They hypothesize that an improved knowledge of these mechanisms will lead to the development of novel strategies for restoring GABAergic inhibition. The researchers propose to exploit their recent discovery of a �rheostat� of Cl- homeostasis, comprising the Cl-sensitive WNK-SPAK kinases and the NKCC1/KCC2 cotransporters1-3. This rheostat provides a phosphorylation-dependent way to reversibly tune the strength of synaptic inhibition in neurons.

The team will create genetic mouse models with inducible expression of phospho-mimetic or constitutively dephosphorylated WNK-SPAK-KCC2 pathway components. They will also develop novel WNK-SPAK kinase inhibitors that function as simultaneous NKCC1 inhibitors and KCC2 activators. These mouse models and compounds will be used to therapeutically restore GABA inhibition in the Rett syndrome MeCP2(R308/Y) mouse model. The researchers will use a combination of two-photon microscopy coupled with improved fluorescent optogenetic Cl- sensing, quantitative phosphoproteomics and patch-clamp electrophysiology to assess cellular and physiological changes in these mice.

The intracellular concentration of Cl- ([Cl-]i) in neurons is a highly regulated variable that is established and modulated by the finely tuned activity of the KCC2 cotransporter. Despite the importance of KCC2 for neurophysiology and its role in multiple neuropsychiatric diseases, our knowledge of the transporter's regulatory mechanisms is incomplete. Recent studies suggest that the phosphorylation state of KCC2 at specific residues in its cytoplasmic COOH terminus, such as Ser940 and Thr906/Thr1007, encodes discrete levels of transporter activity that elicit graded changes in neuronal Cl- extrusion to modulate the strength of synaptic inhibition via Cl--permeable GABAA receptors. In this review, we propose that the functional and physical coupling of KCC2 to Cl--sensitive kinase(s), such as the WNK1-SPAK kinase complex, constitutes a molecular �rheostat� that regulates [Cl-]i and thereby influences the functional plasticity of GABA. The rapid reversibility of (de)phosphorylation facilitates regulatory precision, and multisite phosphorylation allows for the control of KCC2 activity by different inputs via distinct or partially overlapping upstream signaling cascades that may become more or less important depending on the physiological context. While this adaptation mechanism is highly suited to maintaining homeostasis, its adjustable set points may render it vulnerable to perturbation and dysregulation. Finally, we suggest that pharmacological modulation of this kinase-KCC2 rheostat might be a particularly efficacious strategy to enhance Cl- extrusion and therapeutically restore GABA inhibition.

Dominant-negative mutation, genetic knockdown, or chemical inhibition of WNK1 in immature neurons (but not mature neurons) is sufficient to trigger a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl- extrusion secondary to a reduction of Thr906/Thr1007 inhibitory phosphorylation (). These results extended previous work by , who showed that KCC2 Thr906 phosphorylation inversely correlates with KCC2 activity in the developing mouse brain, and , who showed a phosphorylation-dependent inhibitory effect of taurine on KCC2 activity in immature neurons that was recapitulated by WNK1 overexpression in the absence of taurine. Together, these compelling data suggest that a postnatal decrease in WNK1-regulated inhibitory phosphorylation of KCC2 also contributes to increased KCC2 function (Fig. 5), and thus to the excitatory-to-inhibitory GABA shift that occurs during development. This also raises the possibility that dysfunctional phosphoregulation of these sites could be important in certain neurodevelopmental pathologies, like autism or neonatal seizures. An important issue of future investigation will be to determine how the increased levels of Cl- in immature neurons affect WNK1 kinase activity. Could taurine, a factor known to activate WNK1 in immature neurons, achieve this by decreasing the sensitivity of WNK1 to Cl-?

Recently, a few groups have developed innovative high-throughput assays to screen for compounds that modulate KCC2 activity (, ; ), and one drug shows promise as a KCC2-dependent Cl- extrusion enhancer with therapeutic effect in a model of neuropathic pain (). These early but encouraging results require validation, but they establish the validity in vivo of the concept of GABA modulation via the pharmacological targeting of CCC-dependent Cl-transport (; ; ). Could CCC phosphoregulatory mechanisms, normally employed to modulate transporter activity in response to perturbation or biological need, be harnessed to stimulate the KCCs (or inhibit NKCC1) for therapeutic benefit in disease states featuring an accumulation of intracellular Cl-?
Moreover, since the WNK kinases might also be the Cl- sensors that detect changes in intracellular Cl- (), inhibiting these molecules might prevent feedback mechanisms that would counter the effects of targeting NKCC1 or KCC2 alone.
  

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a drugable target for CNS diseases.  

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress�responsive kinase 1). The WNK family senses changes in intracellular Cl- concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na+), potassium (K+), and chloride (Cl-) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl--importing, Na+-driven CCCs or inhibit the Cl--extruding, K+-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K+ secretion to lower blood pressure while maintaining serum K+. In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl-extrusion and promote ?-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K+-sparing blood pressure�lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism. 


The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 andSTE20/SPS1-related proline-,alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection ofthePI3K pathwaythroughmTORC2 to a Ste20 proteinkinase and ion homeostasis.

Signi?cance
With no lysine [K] (WNK) protein kinases are sensitive to changes in osmotic stress. Through the downstream protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1related proline-, alanine-rich kinase, WNKs regulate a family of ion cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. We found that a major phosphoinositide 3-kinase target pathway, the mammalian target of rapamycin complex 2, also phosphorylates OSR1, coordinating with WNK1 to enhance OSR1 and ion cotransporter function.

Changes in tonicity regulate the WNK-OSR1/SPAK pathway to control ion cotransporters for volume and ion homeostasis. We ?nd that mTORC2 also contributes to enhanced OSR1 activity. Inhibiting mTORC2 does not inhibit WNK1 activity, indicating PF1 and PF2regions.

We conclude that cell homeostasis requires the multi level integration of WNK osmosensing and PI3K survival pathways.



These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl- in?ux, we propose that the targeting of WNK�SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl- extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.


WNK Inhibitors
The first orally bioavailable pan-WNK-kinase inhibitor is WNK463.

�WNK463 is an orally bioavailable pan-WNK-kinase inhibitor. In vivo: WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.�\

WNK463 is available as a research drug.

It looks like WNK2 is also very relevant, perhaps more so than WNK1, because we are interested specifically in the brain, where there is a lot of WNK2. WNK3 also looks very relevant. There is also WNK4.



Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl--dependent 86Rb+uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl-accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation.  


WNK3 KO mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared to WNK3 WT mice subjected to MCA occlusion. The neuroprotective phenotypes conferred by WNK3 KO were associated with a decrease in stimulatory hyper-phosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr203/Thr207/Thr212, as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or siRNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia.

CONCLUSION
These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury, and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection following ischemic stroke.

  

Conclusion
I think we can simplify all of this into:-

We already know that many people with autism benefit from making GABA more inhibitory.

There are currently two types of therapy:

1.     Reducing intracellular chloride

2.     Modifying GABAA a3 subunit sensitivity (low dose clonazepam from Professor Catterall)


Reducing intracellular chloride
This can be achieved by:
        Reducing the inflow via NKCC1 using bumetanide and in future years using drugs which better pass the blood brain barrier, e.g. the research drug BUM5. Consider improving the potency of the current drug bumetanide using an OAT3 inhibitor that will increase its concentration and half-life, apparently already possible with acetazolamide.

        Increasing the outflow via KCC2, possible with the research drug CLP257  

        Reducing the inflow via AE3, possible with Diamox/acetazolamide

        Substituting Br- for Cl-, using potassium bromide

        Changing the relative expression of NKCC2/KCC1

Changing the relative expression of NKCC1/KCC2
        This can be done today by treating any underlying inflammation.  Inflammation shifts the NKCC2/KCC1 balance in a way that makes GABA more excitatory, which is bad. This might be achieved by targeting IL-6, NF-?B or just treating any GI problems and allergies.  Always treat the comorbidities of autism.  

        Using WNK inhibitors it will hopefully be possible to manually tune the NKCC1/KCC2 balance, just like tuning a piano. One pan-WNK-kinase inhibitor is WNK463.

        I continue to believe that RORa could be an effective way to increase KCC2 expression and this is something that is not so hard to test.


I will be keeping a look out for further papers by Dr Kahle and be interested in any WNK-SPAK/OSR1 inhibitors he proposes.  If I was him I would start with WNK463.


There is more to the story, because naturally I want to see how estradiol relates to WNK and finally wrap up this subject. Then we will know how to treat the immature neurons often found in autism. A case of forever young.
In a following post I intend to do that; here is a sneak, but complex, preview.








Suramin, the Purinome and Autism




Purinergic signaling is one way cells communicate with each other.  It is still an emerging area of science and medicine.



The home of Cell Danger Response and
Anti-Purinergic Therapy


Purinergic signaling is an important regulatory mechanism in a wide range of inflammatory diseases. Shifting the balance between purinergic P1 and P2 signaling is an emerging therapeutic concept that aims to dampen inflammation and promote healing.  This has some similarity with shifting the balance between th1, th2 and th17 in the immune response.
Purinergic signaling plays a role in the nervous system, the immune system and the endocrine system, all implicated in autism. It is one way that microglia in the brain can be activated, which is a common feature of autism.

Robert Naviaux

Robert Naviaux, an autism researcher, believes that
the purinergic signaling complex of a cell, sometimes known as the purinome, lies behind some types of autism. He is researching the use of an old anti-parasite drug called Suramin to treat autism.  Having started on mouse models of autism he has moved on to humans and has been encouraged by his initial findings.

Naviaux promotes his idea of the Cell Danger Response (CDR) a metabolic response to a threat, which encompasses inflammation, innate immunity, oxidative stress, and the ER (Endoplasmic Reticulum) stress response.


The CDR is maintained by purinergic signaling and it seems that in some types of disease this signaling remains active. Inhibiting purigenic signaling is put forward as a therapy for some chronic disorders.
Naviaux proposes his Anti-Purinergic Therapy (APT) to correct multiple metabolic anomalies that were produced by an over- activated Cell Danger Response (CDR).  In his mouse experiments his therapy did indeed correct multiple metabolic anomalies.
When researching Anti-PurinergicTherapy (APT) and Cell Danger Response (CDR) it is hard to find anything written by anyone other than Naviaux and his team.  This is not necessarily a bad thing, but given all Naviaux�s papers it does look odd.

My conclusion is that Naviaux may well be proven correct, but for now his ideas are still outside the mainstream.

Naviaux�s initial idea seems to have been to prove that APT works in autism using an existing drug (Suramin) and then afterwards develop a new, safer drug. Over time the view has shifted towards thinking that the existing drug, suramin, is safe enough.


Suramin

Suramin has existed as a drug for a hundred years.  It is used to treat used to treat African sleeping sickness and river blindness, which are caused by parasites.

In parasites Suramin is effective by inhibiting their energy metabolism and thus killing them.

A drawback with Suramin is that it has to been injected intravenously and, as with many anti-parasitic drugs, it cannot be taken often. In people with a parasite infection there can be toxicity, but in people without such an infection, the drug is now considered safe below the level of 200 M. It reacts very little with other drugs.

Fortunately Suramin has a long half-life, usually found to be about two months, but Naviaux found it to be just two weeks in his human trial.  The longer the half-life the less often you would have to take  Suramin. I wonder if his very small initial dose has affected the half-life, which should not be the case; but there must be a reason.



Naviaux�s antipurinergic therapy research history

1.     Maternal immune activation mouse model of autism (2013)

2.     Fragile X mouse model (2014/5)

3.     Human stage 1 trial with single dose Suramin (2015/17)



Autism spectrum disorders (ASDs) are caused by both genetic and environmental factors. Mitochondria act to connect genes and environment by regulating gene-encoded metabolic networks according to changes in the chemistry of the cell and its environment. Mitochondrial ATP and other metabolites are mitokines�signaling molecules made in mitochondria�that undergo regulated release from cells to communicate cellular health and danger to neighboring cells via purinergic signaling. The role of purinergic signaling has not yet been explored in autism spectrum disorders. 
Objectives and Methods

We used the maternal immune activation (MIA) mouse model of gestational poly(IC) exposure and treatment with the non-selective purinergic antagonist suramin to test the role of purinergic signaling in C57BL/6J mice. 

Results

We found that antipurinergic therapy (APT) corrected 16 multisystem abnormalities that defined the ASD-like phenotype in this model. These included correction of the core social deficits and sensorimotor coordination abnormalities, prevention of cerebellar Purkinje cell loss, correction of the ultrastructural synaptic dysmorphology, and correction of the hypothermia, metabolic, mitochondrial, P2Y2 and P2X7 purinergic receptor expression, and ERK1/2 and CAMKII signal transduction abnormalities. 

Conclusions


Hyperpurinergia is a fundamental and treatable feature of the multisystem abnormalities in the poly(IC) mouse model of autism spectrum disorders. Antipurinergic therapy provides a new tool for refining current concepts of pathogenesis in autism and related spectrum disorders, and represents a fresh path forward for new drug development.
  


Background
This study was designed to test a new approach to drug treatment of autism spectrum disorders (ASDs) in the Fragile X (Fmr1) knockout mouse model.

Methods
We used behavioral analysis, mass spectrometry, metabolomics, electron microscopy, and western analysis to test the hypothesis that the disturbances in social behavior, novelty preference, metabolism, and synapse structure are treatable with antipurinergic therapy (APT).
Results
Weekly treatment with the purinergic antagonist suramin (20 mg/kg intraperitoneally), started at 9 weeks of age, restored normal social behavior, and improved metabolism, and brain synaptosomal structure. Abnormalities in synaptosomal glutamate, endocannabinoid, purinergic, and IP3 receptor expression, complement C1q, TDP43, and amyloid � precursor protein (APP) were corrected. Comprehensive metabolomic analysis identified 20 biochemical pathways associated with symptom improvements. Seventeen pathways were shared with human ASD, and 11 were shared with the maternal immune activation (MIA) model of ASD. These metabolic pathways were previously identified as functionally related mediators of the evolutionarily conserved cell danger response (CDR).

Conclusions

The data show that antipurinergic therapy improves the multisystem, ASD-like features of both the environmental MIA, and the genetic Fragile X models. These abnormalities appeared to be traceable to mitochondria and regulated by purinergic signaling.



Researchers at the University of California, San Diego School of Medicine have launched a clinical trial to investigate the safety and efficacy of an unprecedented drug therapy for autism.

The phase 1 clinical trial, which is recruiting 20 qualifying participants, will evaluate suramin � a century-old drug still used for African sleeping sickness � as a novel treatment for children with a diagnosis of Autism Spectrum Disorder (ASD). Previous published research by Robert K. Naviaux, MD, PhD, professor of medicine, pediatrics and pathology at UC San Diego School of Medicine, and colleagues reported that a single injection of suramin reversed symptoms of ASD in mouse models.

This trial is the first to test suramin in children with ASD.

In the trial, suramin will be given as a single dose through an intravenous line. Half of the participating children will receive suramin; half will receive a placebo (saline infusion). Behavioral and medical tests will be conducted before and after treatment, and include some blood and urine analyses.
The trial is the first clinical investigation of a novel theory, advanced by Naviaux, that posits autism may be a consequence of abnormal cell communication resulting from abnormal activation of the cell danger response.

Cells threatened or damaged by microbes, such as viruses or bacteria, or by physical forces or by chemicals, such as pollutants, react defensively, a part of the normal immune response, Naviaux said. Their membranes stiffen. Internal metabolic processes are altered � most notably mitochondria, the cells� critical �power plants� � resulting in activation of the cell danger response and reduced communications between cells.

Naviaux said the cell danger response theory does not contradict other research regarding the causes of autism. Rather, it offers another perspective and, perhaps, a new therapeutic target.

Because suramin treatment for autism is unprecedented, Naviaux emphasized it is not known whether the drug will produce any beneficial effect in humans. He noted that suramin, as currently constituted, cannot be used for more than a few months without a risk of toxicity in humans and that it is not available as an ongoing treatment. 


NEWSLETTER�The UCSD Suramin Autism Study


The 2017 Clinical Trial


I think the interviews with parents and press release from the University are actually a better read than the clinical trial and gives a different impression.



Interviews with Parents (click)



Press Release:-


Researchers Studying Century-Old Drug in Potential New Approach to Autism


Five of the 10 boys received a single, intravenous infusion of suramin, a drug originally developed in 1916 to treat trypanosomiasis (sleeping sickness) and river blindness, both caused by parasites. The other five boys received a placebo. The trial followed earlier testing in a mouse model of autism in which a single dose of suramin temporarily reversed symptoms of the neurological disorder.

Participating families also reported benefits among the children who received suramin. �We saw improvements in our son after suramin that we have never seen before,� said the parent of a 14-year-old who had not spoken a complete sentence in 12 years.

�Within an hour after the infusion, he started to make more eye contact with the doctor and nurses in the room. There was a new calmness at times, but also more emotion at other times. He started to show an interest in playing hide-and-seek with his 16-year-old brother. He started practicing making new sounds around the house. He started seeking out his dad more.
�We have tried every new treatment out there for over 10 years. Nothing has come close to all the changes in language and social interaction and new interests that we saw after suramin. We saw our son advance almost three years in development in just six weeks.�

�We had four non-verbal children in the study,� said Naviaux, �two 6-year-olds and two 14-year-olds. The six-year-old and the 14-year-old who received suramin said the first sentences of their lives about one week after the single suramin infusion. This did not happen in any of the children given the placebo.�

Additionally, Naviaux said, �that during the time the children were on suramin, benefit from all their usual therapies and enrichment programs increased dramatically. Once suramin removed the roadblocks to development, the benefit from speech therapy, occupational therapy, applied behavioral analysis and even from playing games with other children during recess at school skyrocketed. Suramin was synergistic with their other therapies.�
Naviaux and colleagues do not believe CDR is the cause of ASD, but rather a fundamental driver that combines with other factors, such as genetics or environmental toxins. And suramin, at this stage, is not the ultimate answer.

But the therapeutic benefit of suramin was temporary: Improvements in the treated boys� cognitive functions and behaviors peaked and then gradually faded after several weeks as the single dose of suramin wore off.

The primary import of the trial�s findings, said Naviaux, is that it points a way forward, that suramin should be tested in larger, more diverse cohorts of persons with ASD. (Naviaux said his research has been limited by costs; his lab is primarily supported through philanthropy.)
�This work is new and this type of clinical trial is expensive,� he said. �We did not have enough funding to do a larger study. And even with the funding we were able to raise, we had to go $500,000 in debt to complete the trial.�

But �even if suramin itself is not the best antipurinergic drug for autism, our studies have helped blaze the trail for the development of new antipurinergic drugs that might be even better,� said Naviaux. �Before our work, no one knew that purinergic signaling abnormalities were a part of autism. Now we do, and new drugs can be developed rationally and systematically.�

Levitt at USC agreed: �The suramin pilot study is too small from which to draw specific conclusions about the treatment, but there is no doubt that the pilot study reports positive outcomes for all five children who received the medication. The findings provide a strong rationale for developing a larger study that can probe functional improvements in children in greater depth.�

The potential financial cost of ASD treatment using suramin cannot yet be determined for several reasons, the study authors said. First, additional trials are required to determine the effective dosage and frequency for different types of patients. Suramin is used much differently for treating sleeping sickness, but the cost for a one month course of treatment is modest: approximately $27.

Study:-


Low-dose suramin in autism spectrum disorder: a small, phase I/II, randomized clinical trial
Objective: No drug is yet approved to treat the core symptoms of autism spectrum
disorder (ASD). Low-dose suramin was effective in the maternal immune
activation and Fragile X mouse models of ASD. The Suramin Autism Treatment-
1 (SAT-1) trial was a double-blind, placebo-controlled, translational pilot
study to examine the safety and activity of low-dose suramin in children with
ASD. Methods: Ten male subjects with ASD, ages 5�14 years, were matched by
age, IQ, and autism severity into five pairs, then randomized to receive a single,
intravenous infusion of suramin (20 mg/kg) or saline. The primary outcomes
were ADOS-2 comparison scores and Expressive One-Word Picture Vocabulary
Test (EOWPVT). Secondary outcomes were the aberrant behavior checklist,
autism treatment evaluation checklist, repetitive behavior questionnaire, and
clinical global impression questionnaire. Results: Blood levels of suramin were
12 1.5 lmol/L (mean SD) at 2 days and 1.5 0.5 lmol/L after 6 weeks.
The terminal half-life was 14.7 0.7 days. A self-limited, asymptomatic rash
was seen, but there were no serious adverse events. ADOS-2 comparison scores
improved by 1.6 0.55 points (n = 5; 95% CI = 2.3 to 0.9; Cohen�s
d = 2.9; P = 0.0028) in the suramin group and did not change in the placebo
group. EOWPVT scores did not change. Secondary outcomes also showed
improvements in language, social interaction, and decreased restricted or repetitive
behaviors. Interpretation: The safety and activity of low-dose suramin
showed promise as a novel approach to treatment of ASD in this small study.







Reviews of the trial published in 2017

Many people had great expectations from this trial.  As expected, Naviaux goes into huge detail analyzing his biological markers. 

Unfortunately the sample is just too small; only 5 people received the single dose treatment. I am sure they would have had no shortage of volunteers and the study would have had far more value with 50 people receiving the drug.

They will tell you the trial cost many hundreds of thousands of dollars.  How much more to include a few more participants?

Since all autism trials use different methods to measure the severity of autism we cannot compare the potency of its effect to say the last bumetanide trial.

Researchers should be told by the FDA/EMA to use at least one rating scale in common with other studies.

The big surprise for me was the short half-life of just 14 days. The drug is usually quoted as having a half life three times longer. 

The next stage will hopefully have more participants and compare the effect of multiple doses of increasing amount.

Please Dr Naviaux, use CARS (Childhood Autism Rating Scale), include children with epilepsy, GI problems, asthma etc.  Have a balance between early onset autism, regressive autism and of course severity of autism.

Parental reporting of improvements, while important, is hugely open to bias. All the kids that received Suramin developed a rash on their body and none of the placebo group did, so I guess the parents who saw the rash would have built up their hopes.

Nonetheless the trial did show a short term benefit from Suramin.  But is it a NAC type of benefit, or a bumetanide scale of benefit?



Reviews of Naviaux

When researching Anti-PurinergicTherapy (APT) and his Cell Danger Response (CDR) it is hard to find anything written by anyone other than Naviaux.

There is this review of his findings:-


Naviaux is clearly highly intelligent and if you read his papers it is clear he has an unusually broad knowledge of autism.  His approach of validating his ideas in multiple types of mouse model (MIA and fragile-X) and then moving on to humans, is correct.

Naviaux is also an expert in mitochondrial disease. 



Anti-purinergic Therapy and Chronic Fatigue Syndrome

One problem with neurological conditions like fibromyalgia, Chronic Fatigue Syndrome and sometimes even MS (Multiple Sclerosis) is that people do not think they are real conditions, or that sufferers exaggerate their symptoms.

Many alternative practitioners who aim to treat these conditions also treat people with autism.


Naviaux suggests that Chronic Fatigue Syndrome is an objective metabolic disorder that could also respond to antipurinergic therapy.

Naviaux may indeed be correct, but I am not sure it helps establish the credibility of his therapy for autism. 




The chemical signature that we discovered is evidence that CFS is an objective metabolic disorder that affects mitochondrial energy metabolism, immune function, GI function, the microbiome, the autonomic nervous system, neuroendocrine, and other brain functions. These 7 systems are all connected in a network that is in constant communication using the language of chemistry and metabolism.

All animals have ways of responding to changes in environmental conditions that threaten survival. We discovered that there is a remarkable uniformity to this cellular response regardless of the many triggers that can produce it. We have used the term, the cell danger response (CDR) to describe the chemical features that underlie this response. Historical changes in the seasonal availability of calories, microbial pathogens, water stress, and other environmental stresses have ensured that we all have inherited hundreds to thousands of genes that our ancestors used to survive all of these conditions.

The body responds differently to the absence of resources (eg, caloric restriction or famine) than to the presence of pathogens and toxins.  We can classify two responses: a single-step response to the absence of resources, and a two-step process in response to the presence of a threat.  Both responses are completed by a return to normal.

When resources are severely curtailed or absent, metabolism is decreased to conserve limited resources in an effort to �outlive� the famine. This is often called a caloric restriction response. On the other hand, when the cell is faced with an active viral, bacterial, or fungal attack, or certain kinds of parasitic infection, or severe physical trauma this activates the two-step response.  The first step is to acutely activate the CDR. Innate immunity and inflammation are regulated by the metabolic features of the CDR. Activation of the CDR sets in motion a powerful sequence of reactions that are tightly choreographed to fight the threat. These are tailored to defend the cell against either intracellular or extracellular pathogens, kill and remove the pathogen, circumscribe and repair the damage, remember the encounter by metabolic and immunologic memory, shut down the CDR, and to heal.

In most cases, this strategy is effective and normal metabolism is restored after a few days or weeks of illness, and recovery is complete after a few weeks or months.

However, if the CDR remains chronically active in either state, many kinds of chronic complex, chronic diseases can occur. In the case of CFS, when the CDR gets stuck, or is unable to overcome a danger, the body enters into a kind of siege metabolism that further diverts resources away from mitochondria and sequesters or jettisons key metabolites and cofactors to make them unavailable to an invading pathogen. This has the effect of further consolidating the hypometabolic state. When the hypometabolic response to threat persists for more than 6 months, it can cause CFS and lead to chronic pain and disability. Metabolomics now gives us a way to characterize this response objectively, and a way to follow the chemical response to new treatments in systematic clinical trials.



Suramin Pharmacology

Suramin has a broad effect blocking receptors both P2X and P2Y, it does not have an effect on the third type of purinergic receptors called P1.

If you believe in the idea of balancing P1 and P2 signaling, you might consider increasing the effect of the P1 receptors to counteract excessive signaling from P2.  I am not sure I agree with this because P1 agonists would make asthma worse, not better.  Unless the idea is to counter excess P2 signaling, by reducing P1 signaling. P1 antagonists (that reduce P1 signaling) include theophylline which I did suggest for other reasons might help some autism.

If you want to be an early adopter of the Dr Naviaux, you need a P2 antagonist.

Suramin is not expensive, but rarely used in developed countries.

















Conclusion 

I think that Suramin is an interesting therapy, even if not everybody is convinced at the proposed mode of action. It does help both in mouse models of autism and in a very small human trial. We now need a large trial that includes a better behavioral assessment of the result, so we can actually judge it properly.
Will it help everybody with an autism diagnosis? I doubt it, but then I do not think any single drug ever will.
The question is more are there any biomarkers for who might respond and Naviaux does mention the �fever effect�.
I think the more people consider the broader metabolic symptoms, the easier it will become to put people into sub-groups of autism and assign them effective therapies.
As with Bumetanide, which is effective in a something like 40% of autism, I expect Suramin will be partially effective and will need other therapies to be added.

A very important point is the cost of clinical trials and indeed drug approval in the US. If just the overspend on this trial was  $500,000, a trial on 10 kids with a single infusion of the trial drug, it is time to move the research to India or Eastern Europe.

North Korea will develop a ballistic missile with nuclear warheads for less money than it costs to develop a drug in the US. 

Why do you think Bumetanide is not being developed as an autism therapy in the US?  It costs too much.










Boosting Bumetanide with an OAT3 Inhibitor?



Today�s post was prompted by our reader Ling, who highlighted research suggesting another way to improve the potency of bumetanide, a drug many readers have found reduces the severity of autism.


Sometime a little extra boost is necessary


There is an ongoing debate in the literature about how poorly bumetanide crosses into the brain and whether the theoretical chloride-lowering benefit can actually take place in humans.  Well for many readers of this blog, we know the answer.

Nonetheless there are efforts underway to improve the potency of bumetanide in neurological disorders. There is a prodrug called BUM5 which has been shown to reverse types of seizure that bumetanide could not, due to much greater potency in the brain.
The French bumetanide researchers are themselves looking to develop a more potent drug.
Ling highlighted a recent paper that suggested using an old drug called Probenecid to increase the concentration of bumetanide in the brain (and plasma) threefold.
This is not a new idea, during World War Two when antibiotics were in short supply, the same drug Probenecid was used to increase the potency of antibiotics to reduce how much you needed to give patients.

Pharmacodynamics
What we want to do is increase the concentration of bumetanide in the brain and ideally increase the half-life.  Both should increase its effect.
The recent research shows that in mice Probenecid does indeed have the effect we want, but humans are not mice.
A very old study looked at the effect in humans of Probenecid on a very similar diuretic called furosemide.


Pharmacodynamic analysis of the furosemide-probenecid interaction in man

The graph above shows that probenecid had a dramatic effect on the potency of the diuretic. Consider the area under the curves lines.  The area is a proxy for the effect of the drug (but it is a log scale).  After eight hours the furosemide alone has gone to zero, whereas when probenecid is added it is as potent as furosemide was alone after 90 minutes.

The recent study highlighted by Ling:-


Bumetanide is increasingly being used for experimental treatment of brain disorders, including neonatal seizures, epilepsy, and autism, because the neuronal Na-K-Cl cotransporter NKCC1, which is inhibited by bumetanide, is implicated in the pathophysiology of such disorders. However, use of bumetanide for treatment of brain disorders is associated with problems, including poor brain penetration and systemic adverse effects such as diuresis, hypokalemic alkalosis, and hearing loss. The poor brain penetration is thought to be related to its high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, but more recently brain efflux transporters have been involved, too. Multidrug resistance protein 4 (MRP4), organic anion transporter 3 (OAT3) and organic anion transporting polypeptide 2 (OATP2) were suggested to mediate bumetanide brain efflux, but direct proof is lacking. Because MRP4, OAT3, and OATP2 can be inhibited by probenecid, we studied whether this drug alters brain levels of bumetanide in mice. Probenecid (50 mg/kg) significantly increased brain levels of bumetanide up to 3-fold; however, it also increased its plasma levels, so that the brain:plasma ratio (~0.015-0.02) was not altered. Probenecid markedly increased the plasma half-life of bumetanide, indicating reduced elimination of bumetanide most likely by inhibition of OAT-mediated transport of bumetanide in the kidney. However, the diuretic activity of bumetanide was not reduced by probenecid. In conclusion, our study demonstrates that the clinically available drug probenecid can be used to increase brain levels of bumetanide and decrease its elimination, which could have therapeutic potential in the treatment of brain disorders.


Supporting research on organic anion transporters

As is often the case, there is already a wealth of research that we can draw on and it does indeed look like an OAT3 inhibitor should modify the pharmacodynamics of bumetanide in a very helpful way. But questions do remain.


Identification of hOAT1 and hOAT3 inhibitors from drug libraries


The NIH Clinical Collection (NCC) and NIH Clinical Collection 2 (NCC2) drug libraries used for HTS consisted respectively of 446 and 281 small molecules (727 total) approved for clinical use or having a history of use in human clinical trials. The clinically tested compounds in the NCC and NCC2 libraries are highly drug-like with known safety profiles. At the indicated concentrations, 92 compounds resulted in 50 % decrease in hOAT1-mediated 6-CF transport, whereas 262 compounds resulted in 50 % decrease in hOAT3-mediated 6-CF transport (Fig. 2). All of the 92 hOAT1 inhibitors were also inhibitors for hOAT3 but with a different potency. Among the 262 inhibitors for hOAT3, 8 compounds were specific for hOAT3 (Table 1), i.e., they lacked appreciable inhibitory activity for hOAT1. For example, stiripentol inhibited hOAT3 with an IC50 of 27.6 �1.28 �M, but it barely had any effect on hOAT1 (not shown). These inhibitors for hOAT1 and hOAT3 included classes of anti-inflammatory, antiseptic/anti-infection, antineoplastic, steroid hormones, cardiovascular, antilipemic, CNS, gastrointestinal, respiratory and reproductive control drugs.

Table 1

hOAT3-specific Inhibitors

Stiripentol
Cortisol succinate
Demeclocycline
Penciclovir
Ornidazole
Benazepril
Chlorpropamide
Artesunate

Table 2

Highly potent inhibitors for hOAT1 at peak plasma concentrations

Amlexanox
Telmisartan
Mefenamic Acid
Oxaprozin
Parecoxib Na
Meclofenamic Acid
Nitazoxanide
Ketoprofen
Ketorolac Tromethamine
Diflunisal





Table 3

Highly potent inhibitors for hOAT3 at peak plasma concentrations

Mefenamic Acid
Meclofenamic Acid
Pioglitazone
Oxaprozin
Nateglinide
Amlexanox
Ketorolac Tromethamine
Diflunisal
Nitazoxanide
Irbesartan
Valsartan
Telmisartan
Balsalazide
Ethacrynic Acid



We further increased the stringency of our selection criteria by incorporation of peak unbound plasma concentration of drugs since, for drugs tightly bound to plasma proteins, the free concentration in plasma is a better estimate of the drug level interfering with OAT transport function. Further screening using the peak unbound plasma concentration yielded three inhibitors of hOAT1 (Table 4) and seven inhibitors of hOAT3 (Table 5) with potency >95% inhibition.

Table 4

Highly potent inhibitors for hOAT1 at peak unbound plasma concentrations

Compounds
IC50 in COS-7 cells (�M)
Cmax (�M)
Cmax Unbound (Cu.p) (�M)
Cu.p/IC50
Oxaprozin
0.891�0.292
50116
5.01*
5.62
Mefenamic Acid
1.085�0.124
83.0*
8.30*
7.60
Ketorolac Tromethamine
0.653�0.130
9.5017
0.10017
0.150



Table 5

Highly potent inhibitors for hOAT3 at peak unbound plasma concentrations

Compounds
IC50 in COS-7 cells (�M)
Cmax (�M)
Cmax Unbound (Cu.p) (�M)
Cu.p/IC50
Nateglinide
0.860�0.0953
18.018
0.23019
0.270
Oxaprozin
0.870�0.0704
50116
5.01*
5.76
Nitazoxanide
0.154�0.0711
31.2?
0.0300?
0.200
Valsartan
0.250�0.143
14.820
0.85021
3.47
Ethacrynic Acid
0.662�0.261
30.922
0.600?
0.910
Diflunisal
0.720�0.290
496?
0.490?
0.680
Mefenamic Acid
1.75�0.258
83.0*
8.30*
4.74


Regulatory Requirements


The FDA and EMA require that the drug interaction liability of this transporter be evaluated in vitro for drug candidates that are renally eliminated. OAT3 contributes to renal drug clearance and transporter � mediated renal drug interactions. Based on the in vitro substrate and inhibition data, decisions are made for OAT transporter�based clinical drug interaction trials, typically with probenecid.

Localization
Endogenous substrates
Substrates used experimentally
Substrate drugs
Inhibitors
Kidney, proximal tubule, basolateral membrane. Brain, choroid plexus and blood�brain barrier
prostaglandin, uric acids, bile acids; conjugated hormones
E3S, furosemide, bumetanide
NSAIDs, cefaclor, ceftizoxime
probenecid, novobiocin




APPENDIX A- Tables

Table 1. Major human transporters

Gene                  Aliases          Tissue                 Drug Substrate                  Inhibitor     

SLC22A6          OAT1       kidney,             acyclovir,                      probenecid

                                                                   adefovir,                      cefadroxil

    methotrexate,             cefamandole

    zidovudine                   cefazolin

SLC22A7          OAT2      liver, kidney    zidovudine                  

SLC22A8          OAT3     kidney, brain   cimetidine,                  probenecid

methotrexate             cefadroxil

zidovudine                  cefamandole

                                   cefazolin


Conclusion
This is a classic case where a little inexpensive experiment could be of huge value.  You just use adult volunteers to test the effect on bumetanide pharmacodynamics of a small number of OAT3 inhibitors.

There are now hundreds of kids in France who take bumetanide, meaning hundreds of parents who are probably more than willing to give up a day to sit in a clinic and give hourly blood samples, so their child might benefit.
Would this common sense approach be followed? Or would it be the case that it needs hundreds of thousands of dollars/euros to do a trial and we wait 3 years for the result?





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