One would imagine that we drug discovery and development types have a reasonable handle on how much of our latest candidate compound makes it into cells. And that we would further know how much of that is floating around freely in there, versus tied up to some protein or another. One could not be more wrong.
This is truly a major (the major?) unsolved problem in pharmacokinetics: intracellular concentrations and distribution. There are situations where we can get such data (often with a fluorescent compound or something of that sort), but those are far outnumbered by the ones where we really have only a fuzzy idea. Considering the number of drug targets that are inside the cell membrane, that’s a big gap – and we really should be saying “membranes” there, too, because once inside the cell you have the mitochondrial membrane, the nuclear membrane, and all sorts of other gates, walls, and velvet ropes that your compound might or might not pass.
This new paper (from Manchester) is a good look at the situation. As they say, understanding such things “would provide enormous clarity” which is now lacking. Unfortunately, the lack of clariy is not only due to the lack of data – we have our own misunderstandings to deal with, and the first one the paper calls out is the way that a lot of us think about compound binding (and free concentration) in the blood plasma:
A key driver of intracellular concentration in vivo must be the unbound drug present in the circulation. Unfortunately, this subject is greatly misunderstood in the scientific community. Prestige publications in high-impact international journals still carry a message that plasma protein binding determines the unbound concentration in the circulation. Anecdotal collection of publications and presentations would indicate that over one-half of the drug research scientific community has been misled.
Instead, it’s all about clearance – clearance of whatever got absorbed and escaped first-pass metabolism. Plasma protein binding is an equilibrium process, and the drain down at the bottom right of your mental picture of it is compound clearance. As the authors note, there are basically no examples of any drug-drug interaction that works through displacement of plasma protein binding. Doesn’t happen. If it really were a determining factor for unbound concentration, that wouldn’t be true.
There’s also confusion around the volume of distribution. Unconsciously (well, sometimes consciously) people assume that if a drug has a low distribution volume that it must also be reaching low concentrations inside cells, perhaps because it’s having difficulty reaching cells at all. But that doesn’t follow, either. The big influence on steady-state volume of distribution is compound binding (or, if you like, fraction of unbound compound, same thing). If your compound doesn’t bind to much, it’s going to show a high volume of distribution, since it’s going to be spread all over the aqueous compartment(s) of the body, and you can get all sorts of VoD numbers down from there as you get more or less lipophilic, charged, and so on, but these aren’t directly tied to unbound intracellular concentration.
The paper goes into several other pharmacokinetic assumptions of this sort; I definitely recommend it for clearing your head on these subjects. It then gets down to the question of intracellular concentrations, with examples from infectious disease (tuberculosis) and oncology, among others. Our data are just not all that good. As mentioned, many of the studies in the field depend on some sort of fluorescence, and tagging drug molecules with a fluorescent group profoundly changes their properties. You have cases like doxorubicin, where the drug itself has intrinsic fluorescence, but it’s pointed out that “dox” is likely a low-permeability drug to start with, and that the great majority of cellular permeability studies with it have been single-dose (with a relatively short time course) rather than multiple-dose steady-state conditions.
And getting such data without fluorescence is an even harder problem. The paper presents an analysis of published data on various tyrosine kinase inhibitors, and estimates that at best, most of them don’t have average steady-state concentrations even as high as four times the measured Ki or IC50 values, and if you assume a more physiological concentration of ATP, most of them probably have unbound plasma concentrations *below* those values. But they still work.
In the end, the paper presents two options to deal with all this uncertainty: we can try to come up with some new technology that can measure bound and unbound drug concentrations in different cell types (preferably in vivo). . .or we can just say the hell with it, assume that we need high (lipoidal) permeability to get through cell membranes, which will help to cancel out transporter effects in things like CNS, tumor cell, and bacterial penetration (and when possible, try to design away from those as well). The authors advocate the second choice! That’s largely because the technological barriers are extremely high. As they say, doing such measurements on single cells in homogeneous culture is still at or beyond the state of the art. And as soon as you move from single-cell measurements to samples that have different varieties of cells in them (that is, something approximating the real world), “the idea of technology helping to provide a global solution becomes almost fanciful“.
In the end, you’ll be trying to make compounds with the highest passive permeability you can, and avoiding the big efflux pumps like P-gP as much as possible. “But we already knew that”, is going to be the reaction, and I think that this paper is replying “Yes, you did, didn’t you? Or you should have.” The idea of measuring intracellular concentration (more particularly, unbound concentration, seems like something we would very much want, but “the more we ask the more complex it becomes“. Better, the authors say, to stick to first principles as much as you can.
I see their point! After all, those first principles are not going away, no matter how fancy our instrumentation gets. The tricky part is that these days we’re all pushing the boundaries of compound properties with exotica such as bifunctional degrader molecules, protein-protein inhibitors, drug-biomolecule conjugates and all sorts of other things. Passive permeability may be a challenge for some of those things, and the urge to know just how much of them are getting to the targets is not going to go away. But I think this paper performs a valuable service in showing just how hard a request that is, and in warning people to keep their thinking straight about pharmacokinetics in general.