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Hi colleagues,
I am a green hand to working with the human liver microsomes.
Recently, I plan to do the chemical inhibition of my substrate in
human liver microsomes, and want to find which CYP supersome are
responsible for metabolism of the substrate.
I plan to choose 3 concentration levels of the chemical inhibitors (5
inhibitors for 5 CYPs) to do the inhibition, and to compare the
inhibition activity at same concentration level. And than I can get
which CYP/CYPs is/are potentially response for the metabolism. I
suppose that in 3 concentrations I can find a good concentration can
generate observed inhibition, and no make all chemical to get
inhibition activity to 100% or to 0%.
And I thank comparison must under the same concentration of the
chemical inhibitions.
But I found a paper (attachment), which used the different
concentration of the chemical inhibitor.
Please see the figure 8 (A), for example, the TEPA (for CYP2B6, 5uM)
can generated 65% remaining activity, and BEN (for CYP2C19, 1uM) is
100% remaining activity. But can it to compare them to each other at
different level? If increase the concentration of BEN to 5uM, is it
can generate higher inhibition activity than TEPA? If it can, that
means CYP2C19 is response for the metabolism. That means different
concentration can generate different conclusion.
Would some experts give me answer,
1, Is there some special principles to choose the concentrate levels
of chemical inhibitors?
2, Does the concentration level need same or not ? why?
Thank you very much,
Kaishun
[Paper not attached: Soo Kyung Bae, Shan Cao, Kyung-Ah Seo, Hyunmi
Kim, Min-Jung Kim, Ji-Hong Shon,
Kwang-Hyeon Liu, Hong-Hao Zhou, and Jae-Gook Shin 2008 Cytochrome P450
2B6 Catalyzes the Formation
of Pharmacologically Active Sibutramine (N-{1-[1-(4-
chlorophenyl)cyclobutyl]-3-methylbutyl}-N,N-dimethylamine) Metabolites
in Human Liver Microsomes, DRUG METABOLISM AND DISPOSITION,
36:1679-1688]
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The following message was posted to: PharmPK
Hi Kaishun,
It is not like that inhibiter concentration has to be same for all
inhibitors. It depends upon the type of inhibitor. You can not compare
two
different inhibiter with two different CYPs. That's why always IC50
values
will be different according to the inhibiter and type of CYP. Always you
have to look for the repeatability of IC50 value.
Thanks and Regards,
K.N.Purna chandra rao
Research Associate
PREMAS Biotech Pvt. Ltd.
Plot No. 77, Sector 4,
IMT Manesar.
Gurgaon-122050Haryana,
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Hi ,
The rationale for selecting the suitable inhibitor concentrations is
that the I/Ki ratio of the inhibitor should be > 10 (more than 80 %
Inhibition) and it would be better if you select the specific
chemical inhibitor for that enzyme.
Regards,
Raghav
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Hi Kaishun,
Each inhibitor will have different IC50 for specific CYP phenotypes .
Hence basing on the IC50's you have to choose the concentration of the
inhibitor for CYP inhibition. For some compounds, toxicity also comes
into play in determing the concentration. Recommended and preferred
CYP inhibitors are listed in FDA website with their IC50s for most of
them. You can chose your inhibitor as well as its concentration from
that.
I hope this could be helpful
Thanks
Ravi Sankar Talluri
Sai Advantium Pharma, Pune
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My recommendation for the three concentrations to be used for an
inhibition assay will be: 0.1, 1 and 10 x of /Ki of the specific
inhibitor. Ki supposed to be independent of the substrate
concentration and type of the substrate being, but in practice it does
show some variation (for example, CYP3A assays depending upon whether
you use testosterone or midazolam as a substrate).
Below is the link that you may find useful in selecting the
appropriate inhibitors and concentrations to be used for the
inhibition assays:
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm
Thanks, Kishore
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The following message was posted to: PharmPK
Dear Kishore Khan,
You wrote:
"Ki supposed to be independent of the substrate
concentration and type of the substrate being...".
I disagree with that and I'd refer you to the Cheng-Prusoff equation for
competitive inhibition:
Cheng Y. Prusoff WH. Relationship between the inhibition constant (Ki)
and the concentration of inhibitor which causes 50 per cent inhibition
(I50) of an enzymatic reaction. Biochemical Pharmacology.
22(23):3099-108, 1973 Dec 1.
I quote from the Abstract:
"The analysis described shows Ki does not equal I50 when competitive
inhibition kinetics apply; however, Ki is equal to I50 under conditions
of either noncompetitive or uncompetitive kinetics"
Best regards,
Frederik
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The following message was posted to: PharmPK
Hi Kaishun,
Things are definitely not straightforward in this subject.
To achieve identification of the CYP involved in the metabolism of
your tested compound, the inhibitor (whatever its nature, chemical or
antibody) should ideally inhibit completely one CYP isoform and leave
the others unaffected. In that respect, a single concentration should
suffice.
The main task is to identify the concentration level to use for each
inhibitor: there is considerable litterature about inhibitor
selectivities, but reported studies are often incomplete, and when you
try yourself, you may eventually come accross surprising results. One
example is ketoconazole, the well-known inhibitor of CYP3A. If keto is
used with HLM at 0.5 uM, CYP3A is >80% inhibited (not 100%, though);
however, if it is used at 5 or 10 uM, a good number of other CYPs are
also affected, especially the CYP2C isoforms. The optimal condition is
not easy to determine, and may be subject to inter-lab variability.
The ideal is to check in one's own lab the optimal conditions of use
for each inhibitor, but this represents a huge work investment to find
complete and selective suppression for up to 10 CYP target activities.
Whenever inhibitor selectivity for a given CYP target is not ideal,
you may co-incubate the test compound with several inhibitor
concentration levels to improve the conclusiveness of your data.
You may also come accross some "funny" things, such as activity
enhancement instead of inhibition (cooperativity mechanisms).
In summary, you may eventually define an optimal study design for CYP
phenotyping, but when testing your compound, conclusions will have to
be drawn with caution and include data coming from other approaches
(use of cDNA-expressed CYP enzymes, correlation of drug metabolism and
CYP activities in a range of individual microsome batches). Another
point to care about is the concentration of your compound, which
should result from preliminary studies (see Bjornsson, 2003).
Especially, enzyme saturation should be avoided, otherwise inhibition
will not be successful, leading to erroneous conclusion.
Best wishes
Frederic MASSIERE
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The following message was posted to: PharmPK
Dear Frederik
I believe you are referring to IC50 rather than Ki.
Ki is indeed supposed to be independent from substrate concentration;
on the other hand, the abstract you are quoting is true.
It should be born in mind that IC50 is not independent from substrate
concentration (and from experimental conditions).
Which is unfortunate because it is much more convenient to determine
an IC50 than a Ki...
Ki determinations can be compared from lab to lab, whereas IC50
usually cannot.
Best wishes
Frederic Massiere
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Dear Frederic,
Indeed, I should have referred to IC50 (or I50); thanks for pointing
out my mistake and apologies for the confusion.
Best regards,
Frederik
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