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The following message was posted to: PharmPK
Dear members, I am planning on performing a metabolic stability study
and determine extrapolated hepatic extraction ratio. Going through
the literature, I came up with this protocol. Could people with
experience conducting such studies please comment on the experimental
details I have or suggest better alternatives.
Thank you
Reagents:
- 20 x NADPH-generating system: NADP (20 mM), glucose-6-phosphate (100
mM), and glucose-6-phosphate dehydrogenase (20 Unit/mL).
Q1: what solvent do you to prepare these 3 components
Q2: Can you prepare stocks of these 3 components and store them? Are
they stable? Or do you prepare and premix them immediately before
every experiment?
Q3: How long should you weight before using the system to allow enough
time for NADPH generation?
Q4: Can you store and re-use (any stability issues)?
- 20x human microsomes (20 mg/ml) in 50 mM KH2PO4 pH7.4 buffer
containing 3mM MgCl2.
-20 x test compound (20 uM) in MeOH
Incubation:
1- Pipet 10 ul microsomal suspension
2- Add 179 50 mM KH2PO4 pH7.4 buffer containing 3mM MgCl2.
3- Add 1 ul test compound
4-Start reaction by adding 10 ul 20x NADPH-generating system in 96
well-plate.
5-Stop reaction with 200 ul acetonitrile .-at-. 2, 5, 10, 15, 30, 45, 60,
90, and 120 min
LC-MS analysis:
- 4 analytical standards are made in reaction mixtures terminated with
ACN before the addition of NADPH
- Centrifuge samples and standards and analyze supernatant
Calculations:
- Clint is determined per mg microsomal proteins
- Multiply by a scaling factor of 33 mg microsomalprotein/g liver
- Obtain whole liver Clint by multiplying by 1480 g average human
liver weight
- Calculate extraction ratio (E) = Clint/ Q+ Clint
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Will you be running this automated on a robot or manually. If you're
running this assay on a robotic platform, make sure that your probes
are calibrated for consistency of inter-sample variability (replicates).
-NADPH generating systems are generally prepared fresh and not stable.
-You can monitor the production of NADPH by suing a spectrophotometer
capable of running kinetic measurements in a kinetic manner (5 second
intervals over the course of 5 minutes).
-In this type of study, generally a competent NAPDH system will be
ready within 5 minutes of preincubation.
-Make sure that your organic volume does not exceed 2% of the entire
incubation volume
-A 2-3x volume additon to stop the reaction and protein precipitate
should reduce noise in the LCMS evaluation
-Will you be suing MRM to monitor disappearacen of the parent compound.
-Are you including a zero without microsomes to assess matrix effects.
-Using these inucbtions can assist you in method development for later
PK experiments also.
-Be careful with your scaling, that is not the only calcuation that is
out there, remmeber that some compounds will be resistant to oxidative
metabolism and be subject to phase II conjugation and elimination,
thus nulifying all Clint calcuations for you using microsomes.
good luck,
sanjeev
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The following message was posted to: PharmPK
Thank you for the follow up, all points are well taken. We will be
running those samples manually, we will use MRM for LC-MS analysis,
and we will make the LC-MS standards by spiking reaction mixtures pre-
terminated with ACN to take into account matrix effect on MS signal.
1- In terms of the NADPH regenerating system. Is it safe to prepare
separate stocks of the 3 components in phosphate buffer (same buffer
used in incubation), store in -80, and premix 10 minutes before
experiment (we will make sure we have competent NADPH conversion using
a spectrophotometer)
2-What are there other commonly used calculations to get an initial
estimate on hepatic extraction ratio based on microsomal data.
Thank you
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1. Storing pre-mixed NADPH generating systems can introduce high
variability in your experiments, I personally would not recommend it.
Prepare it fresh each time prior to use.
2. Look at the molecular structure and phys chem props and see what
metabolic reactions would be apparent. There are other reactions
other than CYP oxidation that are involved in the disposition of a
molecule in vivo. In vitro will not always predict (high intrinsic
clearance generally indicates high in vivo CL, but the converse is nto
always true because of alternate reaction pathways). Its nice when it
does. Running an IVIVC to show concordance is always good with any
series of new chmical entities. Learn-confirm-test. You can corerlate
in vito results with a rapid PK model in vivo to learn more about what
thedisposition of your compound appears to be and then select the
appropriate test model for the occasion, sometimes its hepatocytes,
S-9 or microsomes.... but you have to test it and see.
--
Sanjeev Thohan, PhD
SARx Consulting
SARxconsult.aaa.Gmail.com
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The following message was posted to: PharmPK
Dear sanjeev and other members,
I am working on metabolic stability assay in 96 well format and the
through put expected is about 10 compounds per day. I have to take 6
time points upto 60 mins
Has anyone used 200uL plate with 80 to 100 uL of incubation mixture?
And taken 50 uL for analysis. I mean, one individual well for each
time point instead of taking samples for all time points from one
incubation.?
If yes did you try to calculate CLint? I could calculate t1/2 but
CLint comes no where near our regular vAlues. I used 1 mg protein/ ml
and my incubation mixtrure was 80 uL. So does it mean we cannot use
this method for CLint? Why?
Any comments/suggestions are welcome.
Thanks,
Vishal
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why wold you need to take such a large sample, you can multiply sample
10 uL from a 120 uL incubation in a 200 uL well. Using a rotary
stirrer from eppendorf you can still get good mixing and thermal
control. Using differnt variantls you can run 8 compounds in 5
species using a single plate. The time course will have 0, 5, 10, 20,
30 and 60 min time points, as well as a non NADPH (cofactor not added)
incubation at 60 minutes that allows you to assess compound stability
in the microsomal mix. I sould not recommend greater than 0.33 mg/mL
for protein concentrations and 10 uM substrate. If this cannto be
done, then move to larger format tubes in a 96 well format. There are
these 500 uL tubes availbla from your local eppendorf vendor that fit
into the 96 well format.
--
Sanjeev Thohan, PhD
SARx Consulting
SARxconsult.at.Gmail.com
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