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The following message was posted to: PharmPK
With a group of NCEs, I'd like to identify which P450 enzymes are
responsible for metabolism, and also, which P450 enzymes are
inhibited inhibits. I believe there is an initial fluorescent probe
screen for both these procedures. I also believe different
fluorescent probes are used for different enzymes. Could you please
identify the commercial sources of these probes, provide a quick
synopsis how they work, as well as literature references. I heard BD
Bioscience is a good source. Thanks, Clerk Maxwell
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The following message was posted to: PharmPK
Hi Clerk,
Since you are concerned about CYP involvement, I am assuming you
know you do have CYP metabolism from results of (microsomal)
metabolic stability data for your NCEs- and therefore a suitable
analytical method to quantify how much of your compounds it is left
after such incubations?
For identification of individual CYP isozyme(s) metabolising your
NCEs, it might then be more useful to look at the stability of your
NCEs in single CYP incubations.
I know that looking at compound disappearance is not ideal, but
neither is identification of CYP isozymes involved in metabolism
purely on the basis of data gained from screening inhibition
experiments. I guess you would have to assume that any inhibition you
see is direct (not metabolite-dependent) and competitive, which is
not always the case.
CYP Inhibition screens can be done with fluorescent probes, but
preferably in single CYP preparations (recombinant cell(lines)
expressing just one CYP) as many of the fluorescent probes are not
that specific. The probes are basically converted to a fluorescent
metabolite which is quickly quantified in a plate reader.
On a given CYP isozyme plate you would assess "normal" metabolite
formation as positive control, the effect of a KNOWN inhibitor of
the CYP in question (the inhibition control, ideally with a number of
concentrations to get a good curve) on that reaction, and also the
effect of your individual NCEs on formation of the marker metabolite.
If the NCE inhibits the CYP, less metabolite will be formed than in
PCs, which can be expressed as % inhibition compared to control and
compared to your standard inhibitor.
It is a good idea to run at least 2 concs of the NCEs, eg 1 and 10uM,
or 10 and 100uM. If you see significant inhibition around 1uM NCE,
this suggests a relatively good chance of in vivo inhibition and drug
interactions.
NB: Check that your NCEs do not interfere with the fluorescent
readout, are soluble and stable under the incubation conditions and
if possible, check their recovery (non-specific and specific binding
issues).
Most suppliers (also BD, since you have already mentioned them) of
the materials also provide detailed protocols and further information/
references on their websites. For more detailed info on in vitro
Interaction studies, have a look at the latest FDA Draft guidance:
http://www.fda.gov/cber/gdlns/interactstud.pdf
Also: If you had to start from scratch establishing these tests and
the associated expertise, it MAY just be quicker and more economical
thinking about giving that work to ADME/DMPK screening providers.
Just a thought.
Hope this has been useful,
Best regards,
Constance
DMPKORE
Dr Constance Hoefer
Lannerstrasse 8
85057 Ingolstadt
www.dmpkore.com
c.hoefer.at.dmpkore.com
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Dear Maxell,
I believe these NCEs are from Lead optimization stage. In such case i
prefered to use probe substrate (Sigma) using Human liver microsomes
(BD gentest).
In order know which P450 enzymes are responsible for metabolism of NCE
(s), one has use both non-specific and specific reference inhibitor
(ref: DMD).
In case of P450 enzymes are inhibitory studies, one has to do IC50 by
using specific substrates.
Hope this helps,
Samiulla
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Hi Dr. Hoefer,
I see that you have mentioned checking for solubility of the NCE
under the incubation condition. We often face this problem. DMSO is
inhibitory to most of the recombinant CYPs. Acetonitrile is the
recommended solvent. However, some NCEs are not soluble in ACN. Also
different NCEs show solubility in different solvents. In that case we
simply run the appropriate solvent controls when we look for
inhibition. Is that acceptable?
When using fluorescent probes, we see day to day variations in the
RFU, though the percentage inhibition by standard inhibitor remains
constant. Do you have any such experience?
Regards
Tejal
Dr. Tejal Choksi
Scientist-1
Dept. ADME
Torrent Research Centre
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The following message was posted to: PharmPK
Dear Clerk,
For general considerations on those studies, you may refer to Bjornsson
et al, The conduct of in vitro and in vivo drug-drug interaction
studies: a Pharmaceutical Research and Manufacturers of America (PhRMA)
perspective. Drug Metab Dispos. 2003 Jul;31(7):815-32.
For CYP inhibition studies, BD-Gentest is indeed a good source, see
their web site.
There are also other systems based on recombinant CYPs and fluorescent
probes such as Vivid(R)) (see Trubeskoy OV, J. Biomol. Sceening 2005,
10:56-66).
Such systems are useful to screen compounds and establish a preliminary
CYP isoform inhibition profile.
When going to the preclinical stage, studies using probe CYP substrates
in human liver microsomes or hepatocytes will be more relevant.
For CYP phenotyping studies, you'll see that 3 approaches should be
combined :
1) incubation of NCE with recombinant CYPs
2) incubation with HLM (or with human hepatocytes) in the presence of
various CYP-specific inhibitors or antibodies
3) parallel incubation with a series of individual HLM batches for
correlation with CYP activities.
I believe the first approach can be used to screen out CYP isoforms that
are not involved in the first step of NCE metabolism (since recombinant
human CYPs are optimized systems). On the other hand, when NCE is
observed to be transformed by a given recombinant isoform, the next step
will be to demonstrate the significance of that reaction in vivo.
Good luck.
Frederic MASSIERE, Oroxcell.
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The following message was posted to: PharmPK
Hi Clerk,
Here is some useful literature that helps to start with the
fluorescence based assays.
1. Donato MT, etal., Fluorescence based assays for screening nine
cytochrome P450
activities in intact cells expressing individual human P450 enzymes.
Drug Metabol Dispos,
32: 699-706, 2004.
2. Ghosal A, etal., Rapid determination of enzyme activites of
recombinant human
cytochrome p450, human liver microsomes and hepatocyes. Biopharm Drug
Dispos, 24:
375-384, 2003.
However this fluorescence based screening is rapid, used for
preliminary selection of
NCEs in a large library.
Using of specific probe substrates is the most sensitive and
specific, also used to
generate most accurate Vmax, Km, and IC50 values.
I will go with 2nd method if you give me the option.
Good luck.
Sripal
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The following message was posted to: PharmPK
Hi Tejal,
Thanks for your mail and feedback!
(Aqueous) Solubility is often a problem, and the way round that you
are using is certainly acceptable providing your solvent controls
contain the same amount of solvent as the incubations.
Another problem with high DMSO content is that it affects membrane
fluidity, so it may actually alter metabolism in cell-based systems
by altering permeability e.g. through hepatocyte cell membranes....in
my experience, avoid MEOH more so than DMSO, though, in terms of
toxicity to microsomes/supersomes.
A better solution of course would be to be very stringent on the
solubility benchmark for newly synthesized compounds. Of course you
can somehow get round the solubility problem in early screening, but
it is a problem that will increase as you go through the development
process (Formulation for tox studies, Higher concentration
incubations to produce metabolites/metabolic profiles), and of course
as you move through crystallisation stages, solubility will become
worse, not better.
I have worked with a company that used to leave "solubility"
completely alone until it became time for galenic chemistry, but
changed that strategy after they got stuck on a development candidate
that remained virtually insoluble (and completely useless orally for
that reason) after spending approx 5000 manhours on Formulation....
With regards to your second question, do you mean that the
fluorescent readout varies from plate to plate for both NCE
incubations and Standard Inhibitor incubations, but the (relative)
effect of the inhibitor stays the same? (Sorry if I am being stupid!)
Have you checked your instruments' acceptable range of inherent
variation in RFU readouts? Maybe they match?
Changes in the intensity of the readout of NCE containing samples can
in theory also be due to quenching effects (not just flourescence of
the NCE), this could be checked by an analytical interference control.
Hope this has been useful,
Best regards,
Constance
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