Welcome. We're now going to talk about challenges for conducting quantitative in vitro in vivo extrapolation. We're going to focus on the research needs. Hopefully some of you might be able to fill those needs in your research someday. This diagram shows the kinds of research that are needed to improve this process. You can see the kinetics area is shown in the top. And the kind of things that we are looking at now in many laboratories, relate to the intestine, to the liver, and to the kidney, and to the role of other organs. So on the left in the kinetic circle we have intestinal absorption and metabolism. Currently, most laboratories use Caco-2 cells, a cell line that was developed many years ago from a tumor, a colon tumor. They put them in a cell and they grow together and then you can look at the rate of diffusion through them. And then you can use that along with information on the metabolic capacity of intestinal cells to estimate absorption and metabolism during absorption. But there's problems with the Caco-2 cell line because it actually, being a tumor derived line, is not typical of the intestinal cells anymore. And it actually has some of the wrong enzymes and doesn't have some of the right enzymes. And so there is work going on now trying to knock in or knock out particular enzymes in order to make them more relatable to the in vivo conditions. Hepatic clearance is a major area of research, there's a lot of work going on. Metabolite identification using in vitro metabolism systems that can keep cells going for long periods of time. And coupling that with metabolite identification from quantitative structure activity relationships from high resolution mass spectrometry. And from computer-assisted methods to identify metabolites. Renal excretion is a problem area. Right now, there is no way of identifying whether a chemical is likely to be subject to renal excretion or renal resorption. And so, there are people that are trying to look into that. But actually, what's needed, is more data on chemicals in the environment. In terms of whether they have evidence of renal excretion or resorption and there's not enough to actually build QSAR modeling as yet. Distribution is not too much of a problem because of the ability to predict partitioning from QSAR. But extrahepatic clearance can be very important in some cases. The kidney often is a major site of metabolism for chemicals, not as much as the liver, but enough to make it important to consider. And actually also the lung and skin are sites where metabolism takes place. And for those roots of exposure, it's important that that be understood. And so I just put this up here, you can look at this at your leisure. It's a list of the things that were identified at a meeting that put together the road map for I mean animal alternatives testing. And this reference, Basketter et al, 2012, that's a publication in ALTEX. It was one that I listed as something that you should look at. It actually talks about a number of different areas in which there is a need for research, and what is the research that is needed. Not just biokinetics, I wrote the chapter on biokinetics and there's chapters also on skin testing and chronic testing and cancer. And so I really highly recommend that article to you, and you'll find this table in there if you're looking for research areas. As I've mentioned previously, predicting metabolism is a major challenge, the major problems that we are still trying to develop. Assays where there's long-term maintenance of metabolic competence. And we are pushing the envelope, it used to be keeping them fully functional for more than two hours was a challenge. Now we're up to two weeks. And so this is important if something is very slowly metabolized, you need to wait a long time to determine its metabolism rate. And if something is going to be induced, it takes time for the enzyme to be induced. So, being able to do continuous exposures over days allows you to look for things that you can't in a period of hours. I've already mentioned the problems of chemical absorption on the tubing. Manufacturers are trying to find tubing that works better. Teflon seems to be a good choice but it's pretty difficult to work with in the laboratory, it's fairly stiff. They analytical challenges I've mentioned somewhat. It's very expensive work and often limits how much research you can do and the analytical methods take time to develop. Ultra high resolution mass spectrometry allows you to get a fairly good idea of what chemical you're working with just from its molecular mass. And so that is something that narrows down the possibilities. And so those kinds of assays, if one can afford the equipment, are a big benefit. This shows an example of the kind of, they're called bioreactors. What they mean is where you can keep hepatocytes functional and generate metabolites over a long period of time. In this case, it's accomplished by putting the hepatocytes into alginate beads. Alginate is just a semisolid medium where the cells, you can see in this picture, the cells are the white dots. The alginate beads are lighter dots. The alginate beads are the large, round, green structures. The hepatocytes are in contact with the alginate, it helps them to keep their function. The alginate beads are very small, 250 to 500 microns in diameter, in order to allow oxygen to diffuse to all the cells in the alginate. And so with this cells then you can actually generate secondary metabolites. We actually flow media through the cells and through the beads. And can recirculate it and allow the concentration of the metabolites to build up. Or we can use dialysis to transfer the metabolites to another fluid so that we can then do our metabolism studies. So this allows us to increase our sensitivity for identifying metabolites. Actually, the hepatocytes like flow anyway, it makes them happier. Silicone tubing is the one I talked about that absorbs chemicals, and this is the evidence that I would like to show you. If you look at these plots on the left is PharMed tubing which is similar to to a silicone tubing. There are many different kinds of silicon tubing. Teflon is on the right. In one of the studies we put the chemical in and then watched its concentration over time with no hepatocytes, just with tubing. And you can see that the concentration in blue goes down over time. In another one, we actually exposed the tubing to the chemical flowing in the medium passed through the tubing. And then we removed the media that had the chemical and put in fresh media without it and looked at the reappearance of the chemical into the media. So this shows the double problem. One is that the tubing absorbs the chemical. And the other is that if the chemical were metabolized, the tubing would replenish it from its stores. And so it actually makes it impossible to determine the metabolic rate of loss. In the case of Teflon you can see this doesn't occur, the blue dots stay high with there's maybe a 20% loss. And if you run the Teflon tubing with the chemical and then take chemical out then, there's very little absorbed on the tubing that comes back in to the media. So, it works much better and there are people trying to develop things that are more flexible than Teflon but still work as well. So what I've referred to a number of times but never showed you is the idea of being able to do rapid metabolite identification. In this case, there's a product called Mass-MetaSite that was developed with funding by European pharma, and is now widely used in the pharmaceutical industry for rapid identification of metabolites. And here we have something that just shows the mass peaks and the analysis that results in structures. It's a fairly complicated system but basically a computer generates synthetic mass spectra or compounds that might be predicted to be produced. For example, I showed you like OECD ToolBox. I said it over predicts the chemicals and the question is, it over predicts various metabolites that could be produced, but they're all plausible. And so actually the computer then can generate a mass spectrum for all those candidate chemicals. And then there's a process by which you can actually see what combination of those chemicals would account for the mass spectrum you have observed. And so basically that then allows to do a better job of identifying which metabolites really are produced using the data from the bioreactor and then analyzing it with the mass spec. One of the things that is being developed now using these bioreactors is what's called Human on a Plate. Where you put multiple target tissues, in this case brain, adipose, skin and liver. And in this case, so we have a liver target tissue, to see whether there's toxicity in the liver. But the metabolism is, in the first place, produced in the bioreactor. And then that, the chemical and its metabolites, are transferred by dialysis to the plate. So that you can actually be mimicking long-term exposures to the parent chemical and its in vivo metabolites and see whether those produce toxicity. This kind of system is necessary for trying to address the most difficult problem for in vitro testing which is chronic toxicity. We can't do all of our tests over a matter of hours and hope to be able to identify potential long-term toxicities of chemicals. And so a lot of work now is being devoted to making it possible to do studies over long periods of time in vitro. There is a goal and actually I'm sure you all seen examples of people developing what they call Human on a Chip. With very small flows, and with very small tissue sizes. And these can be quite impressive in terms of their biology. Lung cells that actually respirate, cardiac cells that beat like the heart. It's amazing how one can reproduce the biology, particularly it's easier on this very small scale. What's not easy on this very small scale is trying to get the chemical into the cells at concentrations that you measure. And so here the tubing can be the largest element in the assay. And getting the chemical through the tubing to the chip is the challenge. And so all those things I mentioned before are actually much harder here than they are at the macroscale. So at this point I'd like to just go back over the learning objectives to remind you of what we've attempted to share in this particular lecture. We started out by talking about the role of physiologically-based biokinetic modeling for in vitro-based chemical risk assessments. And so particularly for acute exposures, it's necessary to have this kind of modeling capability to model the absorption, distribution, metabolism and excretion of a chemical so as you can follow the time course in vivo. And compare that to the time course in vitro where the toxicity was measured. Then in the second part we talked about the key considerations for quantitative in vitro to in vivo extrapolation. We talked about the need to worry about pre-concentration, not just depend on nominal concentration, how much was added to the media. We talked about the prediction of different elements of in vivo clearance and in vivo to distribution. And how they differ from in vitro and how that has to be part of the extrapolation. We talked about in the the third block the approaches that have been applied for biokinetic modeling and to support interpretation of in vitro toxicity assays. We talked about the very simple approach that was used with ToxCast data in order to do screening for identification of the compounds. That should be followed up with further testing because of concern for a high potency or a small margin of safety. And then finally we talked about research areas that are ongoing and that need to be maintained in order to improve the ability to do relatively rapid testing. This kind of testing approach, where you consider metabolism, where you actually, it's a medium throughput process. That actually is gaining a lot of interest in companies who are interested in green chemistry. And so they want to be able to avoid chemicals that are going to have toxicity problems. And this medium throughput approach that considers metabolism allows them to downselect early in development just like pharma does. And pharma already does this kind of thing, identifying red flags and ruling out particular candidate chemicals. And the kind of work that I described in this lecture is the kind that is going to allow environmental companies to do the same thing in their chemical developments. And avoid some of the problems we've had in the past with exposures to things like dioxin and trichlorethylene. Well, thank you for listening to this lecture, and I appreciate your taking the time. I would hope someday we might even meet. I plan to continue doing this work for some time, and I think it's very important. And I hope that you will also find it interesting and important. [MUSIC]
