Man, do we ever have a lot of assays in this business. Almost every drug development project has a long list of them, arranged in what we call a screening cascade. You check to make sure that your new molecule hits your protein target, then you try it on one or more living cell lines. There are assays to check its potency against related targets (some of which you may want, most of which you don’t), and assays to measure the properties of the compound itself, like how well it dissolves. Then it’s on to blood levels in animals, and finally to a disease model in some species or another.
Not all these assays are of equal importance, naturally. And not all of them do what they’re supposed to do for you. Some processes are so poorly understood that we’re willing to try all sorts of stuff to get a read on them. I would put the Caco-2 assay firmly in that category.
Caco ("cake-o")-2 cells are a human colon cancer cell line. When you grow them in a monolayer, they still remember to form an “inside” and an “outside” – the two sides of the layer act differently, and they pump compounds across from one side to the other. This sort of active transport is very widespread in living systems, and it’s very important in drug absorption and distribution, and from a practical standpoint we don’t know much about it at all. Membranes like the gut wall or the lining of the brain’s blood vessels do this sort of thing all the time, and pump out things they don’t like. Cancer cells and bacteria do it to compounds they judge to be noxious, which covers a lot of the things we try to use to kill them. Knowing how to avoid this kind of thing would be worth billions of dollars, and would give us a lot more effective drugs.
The Caco-2 cell assay is an attempt to model some of this process in a dish, so you don’t have to find out about it in a mouse (or a human). You put a test amount of your compound on one side of the layer of cells, and see how much of it gets through to the other side – then you try it in reverse, to see how much of that flow was active transport and how much was just passive leak-through diffusion. The ratio between those two amounts is supposed to give you a read on how much of a substrate your compound is for these efflux pumps, particularly a widespread one called P-glycoprotein.
I have seen examples in the literature where this assay appears to have given useful data. Unfortunately, as far as I can remember, I cannot recall ever having participated in such a project. Every time I’ve worked with Caco-2 data, it’s been a spread of numbers that didn’t correlate well with gut absorption, didn’t correlate well with brain levels, and didn’t help to prioritize anything. That may be unfair – after all, I’ve had people tell me that ‘s worked out for them – but I think that even in those cases people had to run quite a few compounds through before they believed that the assay was really telling them something. The published data on these things can turn out to be a small, shiny heap on the summit of a vast pile of compost - the unimpressive or uninterpretable attempts that never show up in any journal, anywhere.
You can think of several reasons for these difficulties, and there are surely more that none of us have thought of yet. These are colon cells, not cells from the small intestine (where the great majority of absorption takes place) or from the blood-brain barrier. They're from a carcinoma line, not a normal population (which is why they're still happily living in dishes). But that means that they’re far removed from their origins, to boot. (It’s well known that many cell lines lose some of their characteristics and abilities as you culture them. They’re not getting the stimuli they were in their native environment, and they shed functions and pathways as they’re no longer being called for). There’s also the problem that they’re human cells, but they’re often used to correlate with data from rodent models. Our major features overlap pretty well (most mouse poisons are human poisons, for example), but the fine details can be difficult to line up.
But people still run the Caco-2 assay. I think that now it’s mostly done in the hope, mostly forlorn, that this time it’ll turn out to model something crucial to this particular drug series. A representative list of compounds that have already been through the pharmacokinetic studies is tried, and the results are graphed against the blood levels. And, for the most part, the plots look like soup thrown against a wall – again. The quest to explain these things continues. . .