Cocaine (and Methylphenidate) are DAT Inverse Agonists, not just Inhibitors

[dopamimetic]

Dopamine reuptake transporter (DAT) “inverse agonism” - A novel hypothesis to explain the enigmatic pharmacology of cocaine

The long held view is cocaine's pharmacological effects are mediated by monoamine reuptake inhibition. However, drugs with rapid brain penetration like sibutramine, bupropion, mazindol and tesofensine, which are equal to or more potent than cocaine as dopamine reuptake inhibitors, produce no discernable 78 subjective effects such as drug “highs” or euphoria in drug-experienced human volunteers. Moreover they are dysphoric and aversive when given at high doses. In vivo experiments in animals demonstrate 80 that cocaine's monoaminergic pharmacology is profoundly different from that of other prescribed 81 monoamine reuptake inhibitors, with the exception of methylphenidate. These findings led us to conclude that the highly unusual, stimulant profile of cocaine and related compounds, eg MPH, is not mediated by monoamine reuptake inhibition alone.
We describe the experimental findings which suggest cocaine serves as a negative allosteric modulator to alter the function of the dopamine reuptake transporter (DAT) and reverse its direction of transport. This results in a firing-dependent, retro-transport of dopamine into the synaptic cleft. The proposed mechanism of cocaine is, therefore, different from other small molecule negative allostereric modulators of the monoamine reuptake transporters, eg SoRI-6238, which merely reduce the rate of inward transport. Because the physiological role of DAT is to remove dopamine from the synapse and the action of cocaine is the opposite of this, we have postulated that cocaine's effect is analogous to an inverse agonist. If this hypothesis is validated then cocaine is the prototypical compound that exemplifies a new class of monoaminergic drugs; DAT “inverse agonists”.

(

I'm really unsure if bupropion should be classified as being equally or more potent than cocaine at DAT ...?)

This has been discussed before, but it's certainly a very interesting topic and we now have the full text too for those interested in it.

So this means that alpha-PVP, MDPV etc. have to be inverse agonists too, or?

One more PDF before I'm gone: Monoamine transporter and receptor interaction profiles of a new series of designer cathinones

 

[neurotic]

i think MPDV, pipradrol etc. are commonly regarded as reuptake inhibitors simply because only IC50 values were determined, but i'd expect them to behave like amphetamine and MPH (and unlike sibutramine and other not psychoactive RI's) and raise extracellular concentrations of dopamine by a significant extent, though perhaps via different mechanisms i.e. not phosphorylation of the transporter nor through the DAT cocaine site... who knows until it's been tested

 

[Nagelfar]

Someone here once said they tested actual Troparil and it was a disappointment, which made me think of this article's table caption: J Pharmacol Exp Ther. 2013 Jul; 346(1): 2–10. *click* for ref.

"Chemical structures of example cocaine-like and atypical DAT inhibitors. Whereas classic cocaine-like DAT inhibitors (A) stabilize an open-to-out transporter conformation, atypical inhibitors (B) stabilize a more inward-facing (closed-to-out) conformational state. β-CFT, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane; β-CPT, 2β-carbomethoxy-3β-phenyltropane."

perhaps meaning, if the OP subject postulation is the correct one, that the simple change from the benzoyloxy to a phenyl actually means most phenyltropanes *are not* inverse agonists like cocaine...?

 

[aced126]

So, if cocaine "reverses the role of DAT", then where does DAT get cytosolic dopamine from to pump into the cleft? Also especially considering cocaine binds to the same spot as amphetamines, it's kinda hard to believe that it is just able to " make it work in reverse". This would be hard to believe in the case of amphetamines reversing DAT via TAAR1 but it is plausible since there is a very similar endogenous molecule (phenethylamine) which regulates this anyway.

 

[Kittycat5]

Is there not free dopamine in the cytosol?

 

[SKL]

Someone here once said they tested actual Troparil and it was a disappointment, which made me think of this article's table caption: J Pharmacol Exp Ther. 2013 Jul; 346(1): 2–10. *click* for ref.

"Chemical structures of example cocaine-like and atypical DAT inhibitors. Whereas classic cocaine-like DAT inhibitors (A) stabilize an open-to-out transporter conformation, atypical inhibitors (B) stabilize a more inward-facing (closed-to-out) conformational state. β-CFT, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane; β-CPT, 2β-carbomethoxy-3β-phenyltropane."

perhaps meaning, if the OP subject postulation is the correct one, that the simple change from the benzoyloxy to a phenyl actually means most phenyltropanes *are not* inverse agonists like cocaine...?

I didn't taste troparil, but I did taste β-CFT. It was not a disappointment, but had some different qualities to it than cocaine. My trip report and discussion, perhaps relevant subjective data to the thesis here...writing the below I didn't know the information in the OP and speculated on some other reasons in it's pharmacology why it is different from cocaine but now this thread is starting to make other possibilities clearer. Pretty cool.

Overall, a worthwhile exercise. The compound is a decent utilitarian stimulant, something I would class with methylphenidate, but superior for it's transparency. It reminds me more of methylphenidate than it does of it's close cousin cocaine or with something along the lines of MDPV--it is far less physical. and also less euphoric, but it's effects are definitely pleasant and positive. I feel as though I could use it for practical purposes, to aid in concentration or as a chemical boost to confidence. The unpleasant comedown and obsessive urge to redose that are so salient with cocaine I find totally absent.
<snip>
Chemically speaking, β-CFT if fluorinated troparil, and troparil is cocaine with a phenyl group instead of a benzoyl; the removal of the ester decreases the local anesthetic activity and the sodium-channel blocking which is responsible for a lot of cocaine's cardiotoxicity and peripheral effects. Also, it is more of a pure dopamine reuptake inhibitor with less effect on the serotonin system (relative to dopamine) -- according to Lakoskii et al. (1991), β-CFT has, relative to cocaine, 1/4th the potency as a sodium channel blocker, 3x the potency on 5-HT reuptake, and 11x the potency on dopamine reuptake.

So, it is not shocking that this is a cleaner stimulant with less "gee-whiz" factor/pushy euphoria than the parent compound. It is, by and large, what it's pharmacology suggests ... a cocaine replacement it is most definitely not, but a useful stimulant it may very well be. I am not fond of methylphenidate or MDPV or indeed of most DARIs, and actually, I found β-CFT to be superior to any of the others that I have sampled.

 

[aced126]

I thought the free cytosolic DA is taken up quickly into vesicles by VMAT

 

[Kittycat5]

Sure, but there is still some. There is a theory that neurotoxicity in Parkinsons may be related to increased cytosolic DA. There is always some being released or re-entering cells, its a fluid process.

 

[aced126]

I'd still say the majority of DA is in the vesicles. Anyway this can be resolved maybe by administering reserpine (blocks VMAT2 to increase cytosolic DA concentrations but does not reverse DAT, and hence can induce depression) prior to cocaine/methylphenidate administration and seeing how much the subjective effects are heightened by. If there is an appreciable amount of cytosolic DA anyway, then prior reserpine administration would have a smaller effect than expected.

I'm still going to go with the much more likely theory in my opinion that it just blocks reuotake and extracellular concentrations will build up as the vesicles secrete the DA.

 

[Nagelfar]

So, if cocaine "reverses the role of DAT", then where does DAT get cytosolic dopamine from to pump into the cleft? Also especially considering cocaine binds to the same spot as amphetamines, it's kinda hard to believe that it is just able to " make it work in reverse". This would be hard to believe in the case of amphetamines reversing DAT via TAAR1 but it is plausible since there is a very similar endogenous molecule (phenethylamine) which regulates this anyway.

I'm imagining things on larger scale world of Newtonian physics here, but what I envision is like the same mechanism through which "siphoning" works, it pries it open just enough, and the nature of the ebb & flow, like an energy vacuum conforming to the least energy state, sucks the dopamine out.

 

[aced126]

Hmm, your mechanism seems really unselective; if dopamine can get through the way you've described, I can imagine all sorts of other cytosolic compounds getting through in that same way.

 

[Kittycat5]

I agree most is in vesicles as well but a small amount is in the cytosol. How much I am not sure but it isnt a lot. Interstingly, one of the papers on cytosolic DA and Parkinson's did exactly what you proposed. They gave methamphetamine (and I believe previously reserpine) and cocaine after to see the effects on cytosolic DA. These cells were damaged already so had higher DA concentrations in the cytosol. Interesting meth actually registered a decrease in cytosolic DA which was blocked by cocaine. They think it has to do with where the vesicles are located and the sensitivity of their detection method (sensitive to 0.1 uM).

 

[aced126]

​Link for this study?

These results seem perfectly logical to me. Methamphetamine induces transporter reversal via some mechanism (probably TAAR1 activation and subsequent phosphorylation of DAT). It would also probably initially increase cytosolic DA by releasing DA from vesicles, however this cytosolic DA is quickly pumped into the synaptic cleft by phosphorylated DAT. This would explain the cytosolic DA concentration decrease; it is all in the synaptic cleft. When cocaine is administered, this binds to phosphorylated DAT and prevents reverse transport, hereby increasing cytosolic DA concentrations (because the methamphetamine is still causing vesicular DA release, but this DA cannot get out of the nerve now due to phosphorylated DAT blockade from cocaine). The DA that does still remain in the vesicles however is eventually excreted and trapped in the synaptic cleft. It would make for an interesting study if the synaptic cleft DA concentrations could be simultaneously be monitored as well.

 

[Kittycat5]

That is exactly the authors conclusions. I will link.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677560/

 

[Nagelfar]

Hmm, your mechanism seems really unselective; if dopamine can get through the way you've described, I can imagine all sorts of other cytosolic compounds getting through in that same way.

I was figuring that this is because cocaine, as opposed to even troparil; stabilizes DAT in the "open to out" conformation (and not the closed to out); stabilized as "open" being the operative term.

​Link for this study?

These results seem perfectly logical to me. Methamphetamine induces transporter reversal via some mechanism (probably TAAR1 activation and subsequent phosphorylation of DAT). It would also probably initially increase cytosolic DA by releasing DA from vesicles, however this cytosolic DA is quickly pumped into the synaptic cleft by phosphorylated DAT. This would explain the cytosolic DA concentration decrease; it is all in the synaptic cleft. When cocaine is administered, this binds to phosphorylated DAT and prevents reverse transport, hereby increasing cytosolic DA concentrations (because the methamphetamine is still causing vesicular DA release, but this DA cannot get out of the nerve now due to phosphorylated DAT blockade from cocaine). The DA that does still remain in the vesicles however is eventually excreted and trapped in the synaptic cleft. It would make for an interesting study if the synaptic cleft DA concentrations could be simultaneously be monitored as well.

I was under the impression that the manner in which m-Amp phosphorylates DAT makes cocaine *not bind* to the re-uptake pump.(?)

 

[aced126]

If that is so then how can the above experiment results be rationalised?

 

[Nagelfar]

If that is so then how can the above experiment results be rationalised?

The immediate down-regulation due to efflux on each particular transporter not being attenuated by the existing m-amp in the system being able to migrate to other transporters not yet phosphorylated (that cocaine gets to) and keep the same amount of dopamine consistently in the synaptic cleft. That was my thinking.

 

[aced126]

Sorry if I didn't completely understand that answer but how is that supposed to increase (decrease to a lesser extent than just m-amp) cytosolic [DA]?

 

[Nagelfar]

Sorry if I didn't completely understand that answer but how is that supposed to increase (decrease to a lesser extent than just m-amp) cytosolic [DA]?

I think of it as a bell-curve sliding scale that isn't able to be maintained because the existing m-amp, at its speed of continued phosphorylation of surrounding transporters, is inhibited into going to new ones. I think we don't take into account how fast the brain reacts in its plasticity to the introduction of a substance that makes a pervasive change. A lot of the 'plateau' kept is in-fact the drug continuing to work and phosphorylate ever more transporters as it goes from cytosol to VMAT, etc.

 

[serotonin2A]

So, if cocaine "reverses the role of DAT", then where does DAT get cytosolic dopamine from to pump into the cleft? Also especially considering cocaine binds to the same spot as amphetamines, it's kinda hard to believe that it is just able to " make it work in reverse". This would be hard to believe in the case of amphetamines reversing DAT via TAAR1 but it is plausible since there is a very similar endogenous molecule (phenethylamine) which regulates this anyway.

Sorry to go back to an old post, but I don't think anyone ever answered your questions.

First, there is a cytoplasmic pool of dopamine. This is known as the newly synthesized pool. Dopamine is synthesized in the cytosol before it is taken up into vesicles.

It has also been proposed that vesicles may dock to DAT, allowing transported dopamine to be rapidly recycled to vesicles. So when cocaine reverses DAT, it might actually be able to pull dopamine out of docked vesicles. I don't think that was ever proven to occur, but it us an interesting possibility.

Second, cocaine and amphetamine DO NOT bind to the same site on DAT. Amphetamine binds to the substrate site, where dopamine binds. Cocaine binds to a secondary, allosteric site.