The Protein Bound Fraction

[Slow_Mobius]

So lately I've been trying to get a feel for the significance of protein binding on drug efficacy. From what I understand, plasma half-lives are usually not taking the protein bound fraction into consideration, so it's basically telling you the total amount of bound and free drug in the plasma. I've covered the basics of pharmacokinetics but have no experience actually performing these experiments, so please correct me if I'm wrong.

My question is how significant the protein bound fraction is for a psychoactive substance such as benzodiazepines. Clonazepam has an elimination half life of 30-40 hrs and is 85% protein bound. Does anyone know how quickly this equilibration of protein binding occurs and how it correlates with drug effects? If it's a rapid equilibrium, then the protein bound fraction will theoretically have little to no effect on dose response since it would equilibrate at that fraction and stay there.

Another thing Im curious about is how they determine protein binding. Some drugs can significantly change in free plasma concentration at varying total plasma concentration. ie taking 100% more drug could decrease the overall amount that is protein bound and actually give you 120% more available drug (as an example of what in talking about). So does anyone know how relevant all of this is to the dose response curve? What I'm actually even more curious about is if protein binding has any significance to the reason why a benzo that has a 40 hour half life like clonazepam only had a ~12 hr duration of action.

Any clarifications, comments, articles, questions are welcome I wrote this on the fly so I'll edit later for typos/clarification

 

[serotonin2A]

The reason why duration of action is different than PK factors is usually because the EC50 for subjective/physiological responses is higher than the half-maximal plasma concentration -- hence the effects wear off before one half-life has passed.

Protein binding is usually measured by incubating the drug with protein and measuring how much the concentration drops due to binding. To do so, dialysis equlibrium experiments are performed...a drug solution is mixed with protein, and then some of the solution passes through a dialysis membrane that is permeable to the drug but not to protein. If the solution is in equilibrium across the dialysus membrane then you can to selectively measure the fraction that is unbound.

 

[Slow_Mobius]

I don't think I would agree with that being the case. Using clonazepam again as an example, I can take 0.5 mg and definitely feel it for 12 hours, and I'll maybe feel a very subtle relaxation effect for 24 hours at which point plasma concentration should be at ~0.3 mg. after 30+ hours go by I don't feel the effects, but I should have only eliminated half my dose by that point. Considering I can feel 0.125 mg doses, it seems like I should still be able to notice an effect after one half life. This is only one example, but I know other people experience and wonder about the same thing.

I'm just trying to understand what seems to be a gap between PK parameters and dose response. I really dont see how the EC50 being less than half of peak plasma concentration would explain this. The EC50 does not equate to the absence of an effect. An EC50 being above 1/2 peak plasma concentration only tells you that you'll be at less than 1/2 of your maximum response after one half life.

 

[serotonin2A]

I'm using the EC50 as a shorthand for the dose-dependence of the drug response. Sorry if that was confusing.

The situation described in your second post is much more specific than the first. The first asked why duration of action does not always match the half-life (ie, "why a benzo that has a 40 hour half life like clonazepam only had a ~12 hr duration of action."). Even though the plasma level has not declined by 50% by 12 hours, that doesn't mean that the plasma concentration is high enough to produce a detectable drug effect. When you get to the 12 h point, the plasma concentration may have declined to the point where the drug is basically inactive.

Now in your second post you have provided more detail by explaining that you think that the plasma level at one half-life should be active. You may be able to feel a 0.125 mg dose of clonazepam taken acutely, but that doesn't mean you will be able to feel the equivalent plasma concentration after your body metabolizes half of a 0.25 mg dose. There are multiple adaptations that can occur due to the presence of a drug over a short time scale (a few hours), meaning that the concentration-response curve may gradually shift to the right as time progresses. In effect, you have a one-shot tolerance. It could be a behavioral tolerance, it could be receptor phosphorylation, it could be receptor internalization, etc, but for one or more reasons the plasma concentration has dropped below the threshold necessary for an effect.

 

[Slow_Mobius]

Personally, I suspected receptor desensitization and behavioral adaptation causes the altered dose response. I was under the impression that receptor desensitization typically took an extended period of exposure, but I'm also not too familiar with this specific pathway. I might do some more reading on benzodiazepine receptors when I get the chance and address the topic again. Internalization would actually make a ton of sense.

I guess my main thought was whether protein binding is playing a role, but if it's establishing a rapid equilibrium then that's definitely not the case. And sorry for the misunderstanding, when I hear EC50 I automatically think of its definition rather than how it can be loosely used in the context of a dose response.

Just another tidbit, do you know if anyone has tried using microscale thermophoresis to measure protein binding? I know someone that just recently got the equipment, and while I'm not very familiar with the technique, it seems perfectly suited for that type of experiment. It's actually really cool!

Thanks for the replies BTW. I reread my last post I sounded like a dick! I didn't mean to come off as ungrateful : )