Potential Suramin Alternatives - Sytrinol and Kudzu (Anti Purinergic Therapy)

necessary8

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In your ponderings you're thinking about pannexin-1 (Panx1) in terms of its relationship with P2X7.

But it was discovered in 2008/2009 by at least two different groups (Qiu et al and Ma et al) that Panx1 actually has its own receptor for external ATP (eATP) that operates independently of P2X7. External ATP is able to inhibit Panx1 via this receptor.
You know that I adressed this in the first part of my ponderings, right? I literally referenced the same two studies. I said:
So what about other inhibitory mechanisms of purinergic signaling, other than ecto-nucleotidases? The only other thing that I found, were some observations that P2X7 activation can inhibit Panx1-mediated ATP release.[source] Yes, this is the reverse of what I said at the beginning. There were two studies that reported this, one on murine cells, other on human HEK293 cells. This is a cell line cultured from embryonic kidney cells, often used in research because it's easy to modify them to do what you want, so they have different properties based on how you "bake them". In this case, Panx1 was artificially introduced to those cells through transfection.

Those observations raise two possibilities. First, that depending on the cell type, P2X7 activation might open or close Panx1 channels. The cell types in which P2X7 activation has been observed as leading to Panx1 channel opening are macrophages[source], astrocytes[source], and enteric neurons[source]. (And maybe more, I just haven't found the papers about it.) The P2X7-mediated Panx1 activation seems to be widely accepted in literature, but citations about it are often not present. It is very possible that in some cells P2X7 activation opens Panx1 and in other types closes them.
The second, more interesting possibility, is that there is some other factor, which decides if P2X7 activation in a cell opens or closes Panx1 channels. A distinction on P2X7 splice variants might be it.[source] Or it might be some yet undiscovered thing, possibly our mysterious blood factor, or something tied to it biochemically. We don't really know.
I consider the results of Qiu and Ma preliminary, because they are not on natural human cells. Qiu uses Xenopus, while Ma artificially transfects Panx1 into cells that do not have them normally. The research that shows ATP causing activation and opening of Panx1 is much higher quality, and larger. For me those two studies are indications that maybe sometimes eATP can close Panx1 channels, and we don't know what are the factors which decide if it opens or closes them.

Now, you propose, that we do know what the factor is, that it is solely the concentration. And you quote this as a source:

At low ATP concentrations, the purinergic receptors [e.g., P2X7] activate Panx1 resulting in amplified ATP release. As the ATP concentration builds up in the vicinity or within the vestibulum of the Panx1 channel, the self inhibition will limit further ATP release.
The problem here is that this sentence, in the original review that you linked, does not have a citation. It is nothing more than author's speculation. It's a very intetesting speculation, but a speculation nontheless. The Qiu and Ma studies do not prove this, because they induce Panx1 activation via electrical stimulation, not by administering low concentrations of eATP.

What would actually prove this is the case, is if someone took the same human cell type that was used in previous studies to show that eATP opens Panx1, like macrophages or astrocytes, and demonstrated that when you apply higher amounts of eATP, the Panx1 channels close. If it was done on macrophages, they would also need to apply the appropriate cytokines to ensure the macrophage stays in the M1 polarization, because a shift to M2 can occur spontaneously, and it changes how they respond to eATP.

So if you find a study like that, I will immediately admit that you're right on this, and I was wrong. But for now I'm gonna say this again - We. Do. Not. Know. why sometimes eATP makes Panx1 close. We do not even know if such an effect occurs in actual, natural human cells. The action of Panx1 in humans might be dependent on dozens of factors that we don not yet understand, and which Qiu and Ma failed to reproduce in their studies.


I gave the values/sources I'm using previously:
In the human pediatric trial study, the amount of suramin administered resulted in a blood concentration of suramin of about 12 micromolar… I'd been thinking in terms of an IC50 [for suramin at P2X7] of about 70 micromolar per this study.
Ah, yes. My bad. I missed it. Yes, it would seem that you're right here, at least in your math. But since this directly contradicts what Naviaux has said in his presentation, I think it's time we ask him directly about it, so I've sent an email to him. I'll let you know once he responds.

The one possibility I see, for you to be wrong on your conclusion, is that the initial concentration is 100uM, and only falls to 12uM after 2 days. It might be that it is the initial concentration that breaks the eATP release feedback loop, and the upregulation of P2X7 isnt enough to undo it later when the concentration falls. But we'll see what Naviaux says.


So the idea with clemastine is just to try to capture what is outwardly an apparent net effect of an up-regulation at P2X7 and see what happens.
I get that, but what you're effectively suggesting here is that the mechanism of ME/CFS involves an underactivation of P2X7, Panx1, and ATP being released to a lesser degree than in healthy people. This is not only directly opposite to what Naviaux said about ME/CFS, it is also not consistent with symptoms, and many research findings. My "ponderings" thread is basically two long essays explaining in detail, how OVERactivation of Panx1, P2X7 and INCREASED release of eATP is consistent with pretty much all the symptoms, as well as many of the key research findings. So this is why I'm extremaly skeptical of this hypothesis of yours - there is a mountain of arguments on the opposite side. I can't say for sure that you're wrong, but as it currently stands, your version seems incredibly unlikely to me.

How a biochemical result is achieved can obviously be just as important as the result itself but note that clemastine effectively up-regulates P2X7 by making it more sensitive to eATP while apparently not affecting Panx1:
See, but in the same study, they say clemastine potentiates the formation of large pores, as assesed by large molecule dye uptake. So it means that clemastine does potentiate eATP release from cells. Now, they say Panx1 is not involved here, because the potentiation of dye uptake did not change when they added carbenoxolone, which is a Panx1 inhibitor. Further, they state that carbenoxolone by itself caused dye uptake! This is the reverse of a multitude of other studies, which show carbenoxolone decreasing dye uptake. Something very weird is going on here. Now, it's interesting to note that in this study they used HEK cells, the same as the Ma et all study we mentioned earlier. To me, this is a suggestion that HEK cells are not representative of how P2X7 and Panx1 behave in natural human cells.
 
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Hip

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I went back using your formula and checked most of the interesting the drugs in the first paper, and all were way out of range.
You did some work there! It takes quite a while to do that. As you may have noticed, it takes time to find the pharmacokinetic studies for each drug (to get the Cmax value), and finding figures for the plasma protein binding for the drug.
 
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Learner1

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Spironolactone belongs to a class of medications known as potassium-sparing diuretics. Common side effects include electrolyte abnormalities, particularly high blood potassium, nausea, vomiting, headache, rashes, and a decreased desire for sex. In those with liver or kidney problems, extra care should be taken.

It is a steroid that blocks the effects of the hormones aldosterone and testosterone and has some estrogen-like effects.
Many of us are low in aldosterone and testosterone already.
 

nandixon

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You know that I adressed this in the first part of my ponderings, right? I literally referenced the same two studies. I said:
I'm not seeing where you mention Panx1 as having its own eATP binding site for inhibition purposes, which is the critical finding of both Qiu and Ma, but it's of no matter. I just wanted to make sure that everyone working on this is aware of all the relevant studies.

I consider the results of Qiu and Ma preliminary, because they are not on natural human cells. Qiu uses Xenopus, while Ma artificially transfects Panx1 into cells that do not have them normally. The research that shows ATP causing activation and opening of Panx1 is much higher quality, and larger. For me those two studies are indications that maybe sometimes eATP can close Panx1 channels, and we don't know what are the factors which decide if it opens or closes them.
There have been quite a few follow-up studies to those 2008/2009 studies, including several more by Dahl’s group (which Qiu belonged to). It's actually Dahl’s group that did the 2013 study on Brilliant Blue FCF that I mentioned found the overlap of the binding site for that molecule with the binding site for eATP on Panx1. That's the same study that sparked your interest in BB FCF as an inhibitor of Panx1 and also provided @Hip with an IC50 number for that compound.

Ah, yes. My bad. I missed it. Yes, it would seem that you're right here, at least in your math. But since this directly contradicts what Naviaux has said in his presentation, I think it's time we ask him directly about it, so I've sent an email to him. I'll let you know once he responds.
I don't think what I'm talking about contradicts Naviaux. Does Naviaux actually say anywhere that he's using suramin to inhibit P2X7 in order inhibit the release of ATP via Panx1?

He wouldn't (or shouldn't) be saying that because he presumably should be aware that suramin inhibits a number of the other purinergic (P2) receptors at an order of magnitude lower IC50 than P2X7. In other words, at the relevant concentration of suramin, several other P2 receptors are going to be almost completely inhibited by suramin while P2X7 is only inhibited to the extent of roughly 10%.

So if it's true that the gene expression of P2X7 is starting at a reduced level in the human autism patients (as in the mouse study), then by inhibiting the other P2 receptors this is going to cause a compensatory up-regulation of P2X7 in the form of increased gene expression - either because of a reduction in eATP and/or because of the reciprocity that exists among the P2 receptors.

So the up-regulation of the expression of P2X7 to normal levels would simply be part of the overall process to normalize eATP levels (under Naviaux’s theory).

And what I'm saying is that we don't know if the reason why P2X7 appears to be low in autism (at least in the mouse study) is really because of an excess of eATP. It may be low for other reasons and suramin may be capable of correcting this simply because of the inherent disparity that exists in its effect on the other P2 receptors relative to P2X7.

To put it another way, to me as a former medicinal chemist, the appearance is one of a possible serendipitous match, i.e., P2X7 happens to be low in autism (apparently) and suramin just happens to inhibit other P2 receptors an order of magnitude greater than P2X7 - thereby allowing for the selective up-regulation of P2X7.

The one possibility I see, for you to be wrong on your conclusion, is that the initial concentration is 100uM, and only falls to 12uM after 2 days. It might be that it is the initial concentration that breaks the eATP release feedback loop, and the upregulation of P2X7 isnt enough to undo it later when the concentration falls.
Yes, that's right. I got the impression though that Naviaux appeared to be looking for a minimum sustained level of suramin over some longer period of time, but I'm not sure.

See, but in the same study, they say clemastine potentiates the formation of large pores, as assesed by large molecule dye uptake. So it means that clemastine does potentiate eATP release from cells.
I thought that large pore formation in the P2X receptors themselves only allows for an influx of large (cationic) molecules into the cell, and that efflux of molecules such as ATP requires the association of Panx1. But I'll have to double-check that.
 

necessary8

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I'm not seeing where you mention Panx1 as having its own eATP binding site for inhibition purposes, which is the critical finding of both Qiu and Ma

...

There have been quite a few follow-up studies to those 2008/2009 studies, including several more by Dahl’s group (which Qiu belonged to).
This I wasn't aware of. Would you care to quote the relevant pieces? (You don't have to if it's too much of a bother, I'm still gonna read the studies myself, but I need more energy to do it)

I don't think what I'm talking about contradicts Naviaux. Does Naviaux actually say anywhere that he's using suramin to inhibit P2X7 in order inhibit the release of ATP via Panx1?
Yes he does, multiple times, in pretty much every presentation on autims or ME/CFS as well as in the Q&As. He even made a super simplified animation of suramin blocking ATP-releasing channels, for the purpose of explaining how suramin treats autism.

I thought that large pore formation in the P2X receptors themselves only allows for an influx of large (cationic) molecules into the cell, and that efflux of molecules such as ATP requires the association of Panx1. But I'll have to double-check that.
That was my impression as well. But since they are talking about YoPro uptake, that is 100% the large pore that can facilitate ATP efflux, and not the cation channel of the receptor itself (both are bidirectional btw, they only vary in size). That's why I'm saying something is very weird here.
 

dreampop

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You did some work there! It takes quite a while to do that. As you may have noticed, it takes time to find the pharmacokinetic studies for each drug (to get the Cmax value), and finding figures for the plasma protein binding for the drug.
One thing that paper says that sometimes ATP release inhibition occurs at much lower doses than current reduction. This only happens with some of the drugs, but one of them is Cossament Brilliant Blue. Dye Uptake IC50 = .1um vs Current inhibition ic50 = 3um. The .27um for BB FCF is current inhibition, and I couldn't find the dye uptake IC50.
 
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Hip

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One thing is that sometimes ATP release inhibition occurs at much lower doses than current reduction. This only happens with some of the drugs, but one of them is Cossament Brilliant Blue. Dye Uptake IC50 = .1um vs Current inhibition ic50 = 3um. The .27um for BB FCF is current inhibition, and I couldn't find the dye uptake IC50.
That's interesting. I have to admit that I don't really understand what these studies are doing when they refer to things like current inhibition and ATP release inhibition.

But if I understand you correctly, you are saying that the ATP release inhibition IC50 figures might be more appropriate, and that in the case of Coomassie Brilliant Blue, the ATP release inhibition IC50 is 30 times smaller than the current inhibition IC50 figure, and thus the same factor of 30 might apply to Brilliant Blue FCF, making its estimated ATP release inhibition IC50 around 0.009 μM.

In which case, a much lower oral dose of Brilliant Blue FCF might be adequate: instead of the 855 mg oral dose I calculated in this post for current inhibition, the Brilliant Blue FCF oral dose for ATP release inhibition would be 29 mg.
 

dreampop

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Typically, the effect of the drugs are more prominent on Panx1 mediated dye uptake or ATP release than on Panx1 channel currents. Irrespective of whether the drug blocks the channel sterically or whether the drug pushes the channel in a subconductance state such a difference is to be expected. A minor constriction of the channel by either mechanism will exclude ATP or dye from passage through the channel, while the remaining channel pore will still be larger than many selective ion channels, such as calcium, sodium or potassium channels.
Yeah, certain drugs modify the Pannexin 1 channel in a way that ATP is more inhibited relative the the channel, because ATP is one of the larger chemicals that passes through it. For example,

Artemisinin at 200 μM inhibited current by ~20% while the IC50 for inhibition of dye uptake by artemisinin was 0.14 μM. A discrepancy between IC50ies for drugs affecting Panx1 currents and Panx1 mediated dye uptake is not uncommon and probably is due to the induction of subconductance states.
This wasn't true for probenecid or glibenclamide (two drugs that are available and relative safe), their inhibtion of the dyes matched their channel inhibtion. However, the BB FCF chemically is so close to BBG (Coomassie Brilliant Blue) that it may also inhibit ATP flux at much lower levels. I don't know if it would be 30x lower, as it is for BBG. I suspect the protein binding for BB FCF will be higher than 50 though.

Also, in the denominator of your equation you have [ bioavailability x (100-%protein binding)]. If something has a bioavailability of of 80%, and 95% of it binds to protein, then only 4% would be free. But in your formula, I would get [80 x 5 = 400]. Is it meant to work like that?
 
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Hip

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Also, in the denominator of your equation you have [ bioavailability x (100-%protein binding)]. If something has a bioavailability of of 80%, and 95% of it binds to protein, then only 4% would be free. But in your formula, I would get [80 x 5 = 400]. Is it meant to work like that?
I am glad you are checking my equation, because I always worry that my brain fog will lead me to make a silly mistake.

I have two different equations that I use to calculate the human oral dose necessary to achieve a given blood plasma concentration (blood plasma concentrations are usually measured in μM or μg/ml).

The first of the two equations is the more accurate, because it utilizes data taken directly from pharmacokinetic studies of a drug (in such pharmacokinetic studies, they will give a specific oral dose of a drug or compound to humans or animals, and then measure the actual concentration of that drug in the blood plasma — that gives you an accurate result).


So my first, more accurate equation utilizes this pharmacokinetic data (there are two versions):

Oral dose (in mg) that achieves the target concentration C in the blood = (100 / (100 - P )) x C / R
this version of the equation is used when the concentration C is expressed in μM

Oral dose (in mg) that achieves the target concentration C in the blood = (100 / (100 - P)) x (1000 x C / W) / R
this version equation is used when the concentration C is expressed in μg/ml

Where:
C = EC50 or IC50 target concentration of the compound, as specified in the in vitro study
P = Percentage plasma protein binding of the compound
W = Molecular weight of the compound (in grams per mole)
R = Cmax peak blood plasma concentration (in μM) from 1 mg oral human dose (this you work out from the data given in the pharmacokinetic study).

The above equation (in its two versions) I devised myself, but I am reasonably sure it is right.



When precise pharmacokinetic data is not available, but we do know the bioavailability of a drug or compound, then I use the following less accurate equation (again there are two versions):

Dosage in milligrams = 400 x C x W / ( B x (100 - P))
this version of the equation is used when the concentration C is expressed in μM

Dosage in milligrams = 400,000 x C / ( B x (100 - P))
this version equation is used when the concentration C is expressed in μg/ml

Where:
C = EC50 or IC50 target concentration of the compound, as specified in the in vitro study
P = Percentage plasma protein binding of the compound
W = Molecular weight of the compound (in grams per mole)
B = Percentage bioavailability

The above equation (in its two versions) I again devised myself, and the rationale for this equation is given in this post. But it is not that accurate, and will provide more of a ballpark figure for the oral dose than a precise result. You get a more precise result if you have the precise pharmacokinetic data.

Basically, to create the above "ballpark figure" equation, I just assume that the human body contains 40 liters (= 40,000 ml) of "accessible" water (= the blood plus the fluids in the tissues) into which an orally taken drug or compound will dissolve. So if you take an oral dose of say 1000 mg of a drug, you can work out how much of that drug will be found in each ml of the 40 liters of water in the body.

So if for example the bioavailability was 10%, then with an oral dose of 1000 mg, you would get 100 mg into the 40 liters of water in your body, and in each ml of that water, you would have 100 / 40,000 = 0.0025 mg = 2.5 μg of the drug. So your drug concentration in the blood and body fluids is 2.5 μg/ml. Then if the plasma protein binding of the drug was say 90%, so that only 10% of the drug is free, then your free drug concentration is 10% x 2.5 = 0.25 μg/ml. So that's your final answer.

If you like, you can put these figures into my equation: Dosage in milligrams = 400,000 x C / ( B x (100 - P)), and you will see that it works out.

Note that if you insert the numerical values for C, B and P into this equation, you can then just copy and paste the equation into Google, hit enter, and Google will calculate the result.



Note though that in pharmacokinetics, there are many things that can render the above equations inaccurate. For example, with some drugs, as well as binding to proteins in the blood plasma, they can also strongly bind to the tissues of the body, and this will change the dynamics.
 
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dreampop

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I'll check this out when I can read through it all. In any case, I would not take the food dye, since it's never properly been tested in humans it can't be considered safe, and at higher doses it would have side effects not seen when used as a food coloring.