A colleague e-mailed me last night with an observation that he’d heard recently: “Have you noticed,” he said, “that the number we use get less and less precise, the farther away they get from the chemists?”
Thinking about it, I’d have to say that’s right, although I don’t think that we can claim any particular credit. After all, we have our feet planted in physics. Our molecular weights are based on the weights of the elementary particles, which are known. . .pretty exactly. And we’ve got a pretty good handle on molecular formulae, too, so we can go around getting mass spectra out to four decimal places and learning all kinds of things from them.
But then when these compounds get run through the primary assays, purified enzymes or the like, the numbers start getting fuzzier. Protein preps are all subtly different – ideally, they should be different in ways that make no difference, but then there’s the actual running of the assay to consider. Reproducibility varies, but no on gets worked up about a compound that shows, say, a 3 nanomolar inhibition in one assay and a six nanomolar in another. “Single digit nanomolar” is all we need to know, and it’s good odds that the next one will split the difference and come in at four or five, anyway.
But then you go to cellular assays, and things get more complicated. Cells are ridiculously more complex than enzymes, and there are so many more things that can kick around your data. Where did this batch of cells come from? How many times have they divided? What stage of their life cycles are they in, on average? What are they growing on, and in? Are they clean (no nasty mycoplasms?) Even if you’ve got all those things under control, your compounds are going to be exposed to untold numbers of other proteins now, all with potential binding sites and activities of their own. And that’s if they can even get past the cell membrane at all – many don’t, for reasons that are not always clear. No, your cellular numbers are always going to have a pretty good spread in them.
But then you go to whole animals, which have all those problems and more. Absorption from the gut and later metabolism are tricky and poorly understood processes, and they’re affected by a bewildering number of variables. Is your compound crystalline? Same way each time? What’s the particle size? How much water does that powder have in it? What are you taking the compound up in to dose it? Have the rats eaten recently? What time of day are they getting the compound? Male rats, or female? Nothing bothering them, no loud noises or change in lighting? Every single one of these things can throw your data around all over the place.
But now you’re up to clinical trials, and animal data is as orderly as a brick wall compared to human data. All those variables listed above still obtain, although you've presumably controlled for several of them by the time you're in the clinic. But that's more than made up for by the heterogeneity of your human volunteers and that of your all-too-human clinical staff. (Ask anyone who's worked up close with clinical data, and you'll hear all about it).
So we start from chemistry, where if we make a compound once we assume that we can always make it again - not always a warranted assumption, mind you, but mostly true. Then we move to in vitro assays, where you really need to have n-of-3, at least, so you can get error bars on your numbers. And we end up in human trials with hundreds (or thousands) of people taking the resulting drug, desperately hoping all the while that we'll be able to pick out an interpretable signal in all the noise. That's the business, all right.