Session I Transcripts
November 20th (AM) Session I: ADME
Strategic Use of Preclinical Pharmacokinetics
Studies and In Vitro ADME Models in Optimizing ADME Properties
Dhiren R. Thakker (University of North Carolina at Chapel Hill)
Question: You mention on your hepatic slide that there is this thing called intracellular
sequestration. I presume this means you suppress the compound but not transform it.
Could you explain how big an issue you think this is and how you would check for
something like that?
Thakker Response: Well, I don't think we really know how we're going to do this.
Often we are not able to relate some of the things we see in vivo to, for example, liver
perfusion studies. I think now that we have at least some handle using some real in
vitro models for hepatobiliary disposition—cellular models— we may be able to begin to
really make the connection between in vivo and in vitro observations. At least for some
of the compounds it is a real possibility that they could be sequestered pretty
extensively into organelles.
November 20th (AM) Session I: ADME
Role of Mechanistic Transport/Absorption Studies in Lead Optimization
Jerome H. Hochman (Merck and Company, Inc.)
Question: Can you tell us the origin of those cell lines that you described? The ones
for the transport studies?
Hochman Response: They came from Alfred Schinkel's lab at the Netherlands Cancer
Institute.
Question: The effect that you saw of the hydrogen-bonding sites on P-gp, do you
believe it's a direct effect with the resulting interaction of the compound with P-gp or is
that indirect because the same compound actually would also cause reduced passivity?
And I was wondering if that is reflected in having the compound slow down and P-gp
having more chance to come to life.
Hochman Response: I believe that it's a direct effect; in particular, when we put
electron-withdrawing groups adjacent to it we tend to reduce the hydrogen bond
acceptor potential without really impacting the passive permeability significantly. Also,
for the overall set of compounds, there were quite a few that had very high passive
permeability that were distinguished based on their P-gp efflux.
Question: Could you explain the difference between the 1A knockout and the 1A1B
knockout mice? Is 1B at all impacting brain distribution? Have you looked at that?
Hochman Response: With regard to brain penetration, mdr1b has no impact. In
general, I think that the results from single knockout vs. double knockout both for brain
penetration and overall P-gp parameters don't seem to show significant differences.
This suggests that the mdr1b probably isn't as active at the sites that we're concerned
about.
November 20th (AM) Session I: ADME
Metabolic Activation—Role in Toxicity and Idiosyncratic Reactions
John S. Walsh (Glaxo/SmithKline)
Question: You pointed out that there are a number of drugs with carboxylic acid that
cause toxicity due to the acyl glucuronide formation, but medicinal chemists often find
the need to maintain the presence of the carboxylic acid. Do you have any suggestions
on how one can maintain it and also have some kind of individual screens to predict the
potential of toxicity?
Walsh Response: I think some structural pharmacophores have been looked at, such
as tetrazoles, but you would not want to steer clear of carboxylic acids solely for this
reason. I think, again, if you've got a carboxylic acid drug that's forming an acyl
glucuronide, you should look to see if the acyl glucuronide is circulating, and what the
anticipated dose of the drug would be. If it's a low-dose drug, you usually have little to
worry about. The FDA is probably in reasonable agreement with this, and I think the
dose level and the circulating components are the things that they would look at.
November 20th (AM) Session I: ADME
Inhibition and Induction of Metabolic Enzymes (Cytochrome P450): Rational
Approaches to Anticipate and Minimize Drug-Drug Interactions
A. David Rodrigues (Bristol-Myers Squibb Company)
Question: Can you comment on which is better, free or total concentrations, when
using modeling?
Rodrigues Response: I would refer to the reviews in one of my slides.* I can give you
examples where you take free fraction and get a great prediction, you take total and it's
overestimated. With other drugs you take total and it's a good prediction, take free and
you underestimate completely. Prospectively, therefore, it's almost impossible. That's
why I've backed out of that and backpedaled and come up with a different strategy, as
outlined in my talk.
*Current Drug Metabolism (2003) 4:423-459; Current Drug Metabolism (2004) 5:147156.; British Journal of Clinical Pharmacology (2004) 57:473-486; AAPS PharmSci
(2002) 4:1-8.
November 20th (AM) Session I: ADME
Panel Discussion
Dhiren Thakker, Jerome Hochman, John Walsh, David Rodrigues, Lawrence Gan
Question: What has been your experience in terms of the chemists that you work with
having an understanding and an appreciation for introducing drug-like properties earlier
in drug discovery?
Gan Response: It's been very rewarding. Back in 2002, Millennium was doing mostly
in vitro screens for its discovery programs, maybe a few in vivo PK studies somewhere
in the pharmacology area. Now, we are doing a lot more in vivo PK studies to
understand the overall DMPK properties of drug molecules at much earlier phase. We
also talk about efficacy first. In the early days we'd been burned by some of discovery
molecules with poor efficacies. Project teams pushed for molecules with good ADME
profiles at the early discovery stage and settled on compounds with micromolar IC50
values, for example. In those cases, we ended up giving a whopping dose of drug in
preclinical studies, which in turn causing severe toxicities, as you can imagine. So, the
primary goal for our medicinal chemists now is to improve the efficacy of drug molecules,
we then come in at the hit to lead stage to fix the DMPK properties. Occasionally we’ve
got involved in the project at the high throughput screen stage and examined some
DMPK issues, but most of time we start the DMPK support when the project reaches
the hit to lead stage. And now it's been smooth sailing for the past 2.5 years.
Question: Lawrence, do you feel that at Millenium now you are part of the drug design
team?
Gan Response: Absolutely. We have involved colleagues from the biotransformation
group looking into molecules with the project team, not only predicting the metabolic
stability and permeability but making suggestions as to how to stabilize molecules and
how to make them more permeable and less protein bound. We do consider some in
silico models and we even perform some in vitro work to prove the hypothesis. PK
colleagues are also involved in the path forward of projects, decision-making and
selecting molecules for the efficacy studies or projecting which molecule can give good
human PK properties. So we're pretty much part of a project team.
Rodrigues Response: We respect our chemists at BMS. They actually want
leadership from the drug metabolism teams—they crave it. And on those occasions
when we don't step up and deliver good leadership, the project sometimes spirals a little
bit out of control. At least at BMS we're very good at generating leads profiling data,
SAR-type data for P450 inhibition, whatever. But if you don't support them, and mind
the SAR, then they may go off thinking the SAR is telling them something that the
difference between, say, an IC50 at 2 and 4 is significant, and you've got to keep it
realistic, so they want that. Having said that, it isn't just chemistry, it's biology, too.
Walsh Response: I'd say that probably holds for GSK. I think the DMPK groups are a
key part of the discovery teams. I'm fairly new to the discovery side myself; I've been in
development for most of the last few years. I think where we do drive things a lot is to
sort out the bad PK. This involves determining in vitro clearance coupled with
metabolite ID. I think we get a lot of interest from the chemists in being able to provide
that kind of information so as to guide the selection of more stable molecules.. And,
clearly, where we run into series with strong CYP inhibition, we find them very receptive
to suggested changes. We have a number of examples of what you would consider
minute structural changes having dramatic effects on stability or CYP inhibition so it's
difficult to predict.
Hochman Response: The chemists that I work with are a good team and they accept
us as part of the team. In doing that, the chemists are focused on making a drug and
not just a preclinical candidate and I think that's essential and is part of what drug
metabolism has to do. Part of that is education, which requires good communication
and mutual respect. From the drug metabolism point of view, we want to get a
compound with the best prediction for PK parameters and one that's going to exist in the
general population with minimal adverse effects. The chemists' focus is going to be on
potency, but they also recognize the importance of drug metabolism as far as making
the difference between the inhibitor and the drug. Likewise, from drug metabolism we
have to take into account that modifications can't necessarily be made at the expense of
potency because both sides of the formula are needed to make a good drug. To do that
requires maintaining good communication within project teams to insure that we're all
focused on the same target.
Question: Medicinal chemists can optimize the permeability based on the Caco-2 cell
permeation rates as well as the metabolism based on microsomal stability data. Are
there any examples where medicinal chemists can optimize volume of distribution and
ex solution? Lawrence showed some examples where there was a nice correlation
between volume of distribution and the levels in the brain. What are the consequences
of increasing the volume of distribution in terms of toxicology?
Gan Response: I forgot to mention that sometimes high volume of distribution can be
a problem. In fact, we have actually encountered some tissue toxicities on compounds
with high volume of distribution. There are a couple of nice papers on the prediction of
volume of distributions from Pfizer folks—two papers in J. Med. Chem. There is also a
paper in J. Pharm. Sci. talking about predicting the plasma protein binding. These
papers discuss prediction of volume of distributions based on protein binding,
lipophilicity, and log P. Again, it needs to be on a project-by-project basis. Sometimes
it can be the lipophilicity, sometimes it can simply be the pKa of molecules, which in a
way translates to lipophilicity. There are many ways we can try to increase the volume;
sometimes we need to decrease the volume of distribution by putting in more
hydrophilic groups such as amino acids.
Thakker Question: I still maintain that determining volume of distribution by in vitro
models is still somewhat beyond the realm of our capabilities because there are so
many processes involved. Lipophilicity is one, but you could actually have transporters
that are involved in efflux or influx of compounds. Uptake into the cell can also affect
the volume of distribution. So, in the absence of a clear, nice in vitro model, I wonder if
any of you use cassette dosing to just get volume of distribution data for large numbers
of compounds and use that for driving the chemistry.
Gan Response: Currently, we have no internal effort in doing the in silico prediction of
the volume of distribution—I mentioned those papers simply for your reference. Instead,
we examine volume of distribution in vivo. Cassette dosing via the IV route is one of the
strategies we used in determining the volume and clearance.
Rodrigues Response: I guess that cassette dosing went the way of the De Lorean; I
don't know, it just went out of vogue. I think it's coming back for some of the programs,
but not across the board.
Question: I have a question about using rodents for pharmacokinetic studies. You
gave an example where you had rapid clearance in rats and mice and you had fairly
good predicted human exposure. One of the challenges to do preclinical toxicology is
that you have to have exposures in two rodent species, at least. So how do you guide
project teams who see fast clearance in rodents? You don't want to go to the other side
where you create compounds that are very stable in rodents and give too long a half-life
in humans or perhaps produce reactive metabolites. What advice do you give to project
teams who are trying to achieve proper human exposure but yet to have a sufficient
exposure in rodents to get them through the toxicology studies?
Gan Response: In the tox studies you don't need more than one rodent species, and
in terms of rodents we tend to use the rat. When we see high clearances in rodent
species, it's not much of a concern. As long as you can give high doses, you can
establish the so-called MTD, in the case of oncology, or NOEL in other therapeutic
areas. We pay attention to the metabolites generated in all species. We actually go
through metabolite profiles in species like rat, monkey, dog, and mice, and compare
them to the human metabolite profile in vitro. We want to make sure that there's no
evidence of “human unique metabolites.” In other words, the metabolites we have
seen in rodent and non-rodent species qualitatively cover the human metabolites. Then,
when we change tox species, it will only be the exposure that we need to worry about.
In fact, for a high clearance drug in rodents, it probably provides more tox coverage
(quantitatively) of metabolites, as long as your parent compound reaches the desired
level. Again, all safety margins are calculated based on the parent compound level. If
NOEL level divided by the effective dose, ED50, gives you a wide safety window, you
have a drug to move forward.
Rodrigues Response: When I was at Merck and here at BMS as well, typically
male/female, differences you see are twofold, but there were some cases when we did
the reaction phenotype that it was all 3A-dependent and there were >50- to 70-fold
increases in the AUC, females vs. males. With the males we couldn't get any exposure
for the tox studies, but the females had really good exposure. So, reaction phenotyping
your molecules for the rat P450s may be very important because if your compound is
metabolized by a whole group of P450s and is smeared out over the whole reaction
phenotype, you usually get this twofold difference between genders. But if it's all one
CYP, then anything governing the expression of that CYP for a high clearance drug will
really give you dramatic gender differences to the point that, even in males, you can't do
tox studies—formulations won't allow you to get to the doses you need. So, just beware
of that.
Walsh Response: We've actually had similar types of experiences, as regards males
and females. It may also arise due to the 2C isoforms, the male-specific 2Cll or
female-specific 2C13. But in terms of the cross-species exposure, I guess we probably
do similar types of support work as everyone else, that is looking first at in vitro
clearances across species using microsomes or S9, to rank order compounds. Thus
we're going to try to anticipate how exposure might vary based on clearance
differences across species.
Question (for Rodrigues): You showed some slides showing the subtleties of SAR, of
some compounds on P450 induction and inhibition. Is there any rationale for those
relationships?
Rodrigues Response: I wish there was. On one of my backup slides I have a cartoon
of a typical S-shaped SAR hypothetical. Sometimes we're plodding along with chemists
and we're in the flat part of the SAR where they're traversing two or three orders of
magnitude in terms of potency of the target and there's no effect whatsoever on PXR or
on inhibition of P450. But they go that extra mile to get that extra low increased potency
and they've gone too far and then suddenly the PXR SAR or the P450 inhibition assay
begins and you're no longer in the flat part but in the linear part and anything they did to
the molecule gives you the sort of examples that you saw in my slides. I think that the
crystal structure of PXR is known and you could start looking at contact points in the
crystal structure and relating that to the biological activity. We're not there yet at BMS.
And could the in silico methodology predict, with any sophistication, the differences in
biology between one compound and another? We haven't put them into the in silico
models yet, so I can't tell you that. We can't predict that if you stick a nitrogen in your
compound the IC50 is going to go from .06 to 30 micromolar. That's where you have
high throughput screen biology assays to give you that information. I think where you're
going with the questions, it's that chemistry wants to know why that change happens
and where do I go from there. I would argue, once you're at 30 micromolar, you can
stop at that point; I don't know if they want to go up to 100. But that's the kind of
information they're asking for—thanks for telling us we have a problem, but what are
you going to do to fix it? They want that information from us and, unfortunately, at least
in my department, we're not yet that good at prospectively telling them, "go in this
direction." So they tend to synthesize a large number of analogs and then throw them
at the biology assays and hope that some of them have minimal PXR transactivation
and P450 inhibition.
Gan Response: In the project I discussed earlier where we had to give a lot of drug to
animals during the development phase, it was a compound that didn't show very good
efficacy in vivo. Since the project team wanted to move the project forward, we ended
up projecting a high dose for humans as well. We have to re-determine CYP inhibition
IC50 values at relatively high drug concentrations, since IC50 values for human CYPs are
normally determined at drug concentrations up to 10 micromolar.
Walsh Response: I think there's been one or two publications over the last few years
that have tended to suggest that drug failures due to induction are pretty rare, that it is
usually not a major hurdle. I don't know if it's correct or not, but I've also heard it
suggested that it could track to some degree with CYP inhibition, at least for 3A4. So
possibly as you screen out potent 3A4 inhibitors, you may be less likely to encounter
strong inducers.
Hochman Response: All the x-ray structures for the PXR have shown the binding site
to be very floppy and the orientation the drugs can have is all over the place. So,
probably any SAR that really develops is going to be based on global physical chemical
properties as opposed to conventional SAR-type of analysis. In the analysis you
showed, you primarily focus on the affinity for the PXR, in the PXR assays and
hepatocytes the extent of induction that you get varies dramatically. You can have a
compound with 1 micromolar affinity that's only going to induce 20% of what rifampin
would. How do you sort through that data? Are you just focusing on the affinity for both
modeling and decision-making or do you look at both affinity and maximal response?
Rodrigues Response: Right now we're focusing on the EC20 and EC60 primarily.
There's an Imax component and this is where a lot of confusion comes in. Do you ratio
the Imax to the EC20? We're trying to sort through that right now. What kind of
parameters do you take forward? If we have in vitro human PXR transactivation assays,
I'm reasonably optimistic that the humanized mouse may give us some bridging species
to try to predict what may happen in vivo and help us to figure out some of those
questions. Because right now we don't know what drives PXR transactivation in vivo. Is
it free fraction or total drug? What's the temporal relationship in the liver? How does
the plasma relate to it?
Question: Do you feel that the best way for an organization to approach these property
issues is to just provide the data and make the teams aware of it and through normal
chain of discussions within the teams and throughout the organization to sort out what
issues are important? Or have your organizations gone to the point of actually providing
guidance to teams or team leaders where you say, "Well, it's important to address this
issue at this point, otherwise we stop that series and we go on to a different series"? Of
the maybe 20 different properties we've heard discussed in this session, are there some
that you would say, "Stop and take a look at this and try to improve this before you go
on so you don't spend another year on this series" or should an organization just leave
this to more of an ad hoc type of normal discussion process?
Hochman Response: At Merck we tend to interact giving guidance on interpretation of
the data; in my view if we just spit out data it's really pretty useless. We have to give
interpretation to it. Another thing that came up in the discussion points today is that
there are lots of parameters that can be optimized for one drug molecule. Whether for a
given target or a given structural set we should even focus on some of these is a
question. We have limited resources, so we can't address on all of the issues. Part of
our job is to discuss with the chemists and give guidance as to what the important
issues are that we should be focusing on.
Walsh Response: At GSK we've actually moved pretty recently toward including in
vitro people in the project teams, so we are in a position to give more expert guidance
for this type of data. A certain amount of autonomy will always remain and it'll vary from
program to program in terms of how much emphasis they're going to put on this. We
may have in licenses where we have legal constraints with other companies, or there
may be only one compound in a series, so it's going to vary from case to case.
Rodrigues Response: At BMS we have pharmaceutical candidate optimization teams,
PCOTs, that interact with the discovery working groups so there's a lot of dialog. Some
folks are better than others, so we're trying to improve that process. It's always been
my firm belief that we have to get involved really early, and I want to repeat myself
because what Ron said earlier about the SAR really resonated with me. They're so
good at optimizing potency at the target that if you don't keep up with them they're going
to back you into a corner and I have never, in all the years I've been in the industry,
been able to dial out of problematic ADME space. I can't take a duck and make it into a
swan, but if I get it early on when it's still a chick and help the chemists we can help
direct in going the right direction. If you're in a deep area of ADME space where
absorption is pitiful and hERG is really bad, but they keep claiming they've got
subnanomolar potencies, I think the game's over at that point and you're going to spend
many resources in the late stages trying to optimize that molecule when you could have
done it earlier in discovery. At BMS, therefore, there is a shift toward getting ADME
data even earlier.
Gan Response: We have a senior research committee that is similar to those just
mentioned. However, many issues can be addressed at the project team level. We
have toxicology as well as in vivo and in vitro DMPK colleagues on the discovery project
team. We can gather information on the molecule quite early. Chemical series with
ADMET issues can be discussed and addressed at the compound optimization stage
and the path forward can be decided at the high level senior research committee
meeting.