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Dear All:
We know that we can use chemical inhibitor and monoclonal antibody to do reaction phenotyping to evaluate the contribution of CYP450 isoforms on compound metabolism. Normally, when using chemical inhibitors, the test compound concentration in the incubation system should be low due to the possible competition. My question is: Is it necessary also to use low concentration for test compound when using antibody as inhibitor? What I understand is antibody is an irreversible inhibitor, so we can use much higher concentration without considering the possible competition between them for detection convenience. Am I right? Any answer is appreciated! Thanks!
Jian
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The following message was posted to: PharmPK
Hi Jian,
Using mABs, you also will see cross reactivity. What you need to do is to test them out
in titration curves to find optimal concentrations that give you the maximum inhibition
to the CYP of interest but minimum cross reactivity. In the reaction phenotyping study,
you also need to use control antibody to make sure each incubation has the same amount of
protein. Give a 10 min pre-incubation at RM before adding substrates.
The cross reactivity can be corrected using mathematical treatment, such as the one we
use (below).
We have compared mAbs, chemical inhibitors, and RAF. Now we are using chemical inhibition
as the default assay for convenience and cost.
Uttamsingh V, Lu C, Miwa G and Gan LS (2005) Relative Contributions of the five major
Cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the
proteasome inhibitor Bortezomib. Drug Metab Dispos, 33:1723-1728.
Lu C, Miwa GT, Prakash SR, Gan, LS and Balani SK (2007) A novel model for the prediction
of drug-drug interactions in humans based on in vitro CYP phenotyping. Drug Metab Dispos,
35:79-85.
Chuang
Chuang Lu, Ph.D.
Associate Director
DMPK, Drug Safety and Disposition
Millennium, The Takeda Oncology Company
35 Landsdowne Street
Cambridge, MA 02139
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Hi, Chuang:
Thank you very much for your advise. It give me many helps. But I suspect is if we can use high concentration of test compound (Substrate) such as much higher than Km in the reaction phenotyping study when mAbs used because we all know that if we use chemical inhibitors, the substrate concentration can not be high due to the possible competition.
Jian
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The following message was posted to: PharmPK
Hi Jian,
For CYP probe substrates, we use the km concentrations. For test compound, you want to use low concentration which would reflect the physiological concentration, and therefore the true fcyp. Remember the example of O-demethylation of dex mediated by CYP2D6 at physiological concentration and N-demethylation mediated by CYP3A4 at higher concentration?
We usually use 14C test compound to monitor the metabolite formation. If the compound has very low microsomal clearance, then we use RAF with higher enzyme concentrations.
For cold compounds, you can use the t1/2 method. But you need at least 40% parent disappearance at the end of the incubation to have enough window to test the effects of inhibitors. Please see out work below:
Good luck.
Chuang
Lu C, Berg C, Prakash SR, Lee FW and Balani SK (2008) Prediction of pharmacokinetic drug-drug interactions using human hepatocyte suspension in plasma and Cytochrome P450 phenotypic data. II. In Vitro-in Vivo Correlation with ketoconazole. Drug Metab Dispos, 36:1255-1260.
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Hi, Chuang:
Thanks a lot! Due to the limited condition in China, the cold compounds is my only choice. Our compounds are always very stable in human liver microsomes that even incubated for 2 h, less than 20% loss found. So the t1/2 method can not be used here. I know at this situation, to detect metabolite formation is a better choice. But if we do not have metabolite standard material, how to do the reaction phenotyping? How to calculate fmcyp?
Jian
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The following message was posted to: PharmPK
Hi Jian,
In this case, you can use the RAF approach, such as use 1 mg/mL microsomes and 100 pmol/mL Supersomes to have a meaningful clearance. The advantage of RAF is that you are not going to lose the detection window because of the chemical inhibitors or mAbs reduce clearance.
If you prefer, you can call me and let's discuss this topic in detail.
Chuang
Chuang Lu, Ph.D.
Associate Director
DMPK, Drug Safety and Disposition
Millennium, The Takeda Oncology Company
35 Landsdowne Street
Cambridge, MA 02139
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