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Dear Group,
I'd appreciate if you can share your invaluable experiences
on long-term term stability of metabolizing enzymes in Liver
micrsomes, S9 fractions and rCYP isozymes, procured from different
sources. Do Cytochrome P450 and the associated activities decrease
over the period of time during storage at -80 degree C, since 30-82%
decrease has been reported within 9 months of storage?
I'm wondering why the metabolic enzyme activity were so inconsistent
after 8 months even with positive controls in the similar assay
conditions and looking for a possible explanation from PharmPK group.
Thanks in advance.
Regards,
Syed Mustafa
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The following message was posted to: PharmPK
Dear Mustafa,
In my humble experience, microsomes, S9 fractions and microsomes
expressing recombinant human CYPs are stable at -80*C for at least
several years (maybe decades, manufacturers could tell us).
On the other what may cause some drop in activity is accumulation of
freeze-thaw cycles.
Assuming there was no misuse of the microsomes, I believe you might
have consider other causes of impairment of activities, such as
temperature issue during storage (do you have records of the
temperature in the deep-freezer?). Also, if microsomes have been
thawed and refrozen in aliquots: microsomal suspension should have
been carefully homogenized before aliquoting : otherwise some
aliquots could contain inconsistent amounts of microsomes (have you
checked the protein content ?)...
Best regards,
Frederic Massiere, Oroxcell, Romainville, France
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The following message was posted to: PharmPK
Dear Syed,.
The most stable CYPs source is human microsomes. As such, if you
freeze thaw
any CYP sample a few times, it would show a 30-75% decrease in activity
depending on the number of times (2-4 times is sufficient to cause
such a
drop) it has been freeze-thawed. Storage for 6 months to a year without
significant loss of activity is feasible if the sample has never been
thawed. It is advisable to store the CYPs in aliquots as soon as they
arrive.
Best wishes,
Rajeev
Rajeev Soni, Ph.D
President and COO,
PREMAS Biotech Pvt. Ltd.
Plot No. 77, Sector 4,
IMT Manesar, Gurgaon-122050,
Haryana, India
http://www.premasbiotech.com
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Dear Syed,
From my experience , we found that several CYP activities in human
microsomes are quite stable for almost ten years. However, freeze-
thaw can effect the activity of these enzymes. I did not have any
experience with human S9 but for rat S9 , the activity of CYP
enzymes seems to decrease after a few day of storage.
Wichittra
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Dear Mustafa,
There wont be much significant drop in the activity of CYP's stored
at -80 degree C upto six months unless it has been thawed. Literature
is available which suggests thers is a drop of around 30% activity
with a couple of thaws. It is better to check the activity of the
enzyme for every three months to confirm the activity. Further I
would suggest you to use all the enzyme of a vial in a single go or
atleast complete it in the second experiment i.e the more you reduce
the thawing of the enzyme the better the activity.
Hope its clear to you
Regards
Naveed Shaik
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The following message was posted to: PharmPK
Dear Wichittra,
Do you use recombinant Cyp if so from which source.
Cheers
Praveen
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Dear Praveen,
I used to use recombinant CYP such as COS expressd CYPs , Yeast
expressed CYPs. However, we do expressed these enzymes in the lab
not the comercial expressed CYPs. Anyway, my Japnaese colleaques
like to use recombinant CYPs from Gentest.
Wichittra
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The stability of the microsomal preparation also had a lot to do with
the components for cryopreservation that have been used, how they are
mixed, re-frozen and the freeze thaw cycles. There are notable
changes in individual CYP activities that are not wholesale across
the board (30-70% reduction in activity). Using some of the classical
methods with the addition of PMSF, glycerol and other "excipients"
tends to prolong activity (See Halpert, Naslund and Betner....Suicide
inactivation of rat liver cytochrome P-450 by chloramphenicol in vivo
and in vitro. Mol Pharmacol. 1983 Mar;23(2):445-52).
But the first general principles of membrane bound enzymes we learned
in biochemistry or a good martini: Don't rush things, keep them cool,
stay well mixed, but not so much as to bruise things, refreezing is
not a good idea, use them as soon as possible, fewer ice crystals
makes a better mixture.
Xenotech has done a nice job of categorizing in their FAQ, here is a
selection:
How long are microsomes stable under certain conditions?
At or below -70*C?
Based on our experience, microsomes are stable indefinitely at or
below -70*C. Refer to the manuscript by Pearce et al., 1996 for data
establishing the stability of XENOTECH's microsomes.
CAUTION: The cited study was performed by XENOTECH's personnel using
our proprietary processes, and the stability data only applies to
microsomes prepared by XENOTECH.
On ice?
Microsomes are stable on ice, 2-8*C, for several hours; however, such
exposure should be minimized, consistent with the experimental
design. Microsomes should not be left on ice, or refrigerated,
overnight. Microsomes that are thawed and maintained on ice for less
than 2 hours can be re-frozen at -80*C and reused without significant
loss of enzyme activity.
At room temperature?
Microsomes are typically thawed in a room temperature water bath. For
a 0.5-mL vial of microsomes, this procedure typically takes 2 to 5
minutes. After the microsomes are thawed, they should be immediately
placed on ice until used. Exposure to room temperature should be
minimized as much as possible depending on the experimental design
At 37*C?
Microsomes are typically incubated at 37*C for determination of
enzyme activity. Stability of microsomes at 37*C should be
ascertained based on the enzyme activity of the metabolic reaction
being studied. This is achieved by verifying that product formation
is directly proportional to incubation time at one-half and at twice
the normal/desired incubation time. XENOTECH does not recommend
incubating microsomes at 37*C for more than 2 hours. If extended
incubations are carried out, experiments should be performed to
verify that the enzyme activity is stable during the incubation
period. Microsomes that have been incubated at 37*C should not be
reused or refrozen.
How should I handle the microsomes to minimize their degradation?
Microsomes should be stored at -80*C until needed. Exposure to higher
temperatures, such as 2-8*C (on ice), room temperature, or 37*C,
should be minimized as much as possible and as allowed by the
experimental design. The important thing to remember is that the rate
of enzyme degradation is proportional to the increase in temperature
of the microsomes.
Our recommendation: thaw the microsomes quickly in a room temperature
water bath, and immediately place them on ice. Subsequently, the
microsomes may be diluted or added to incubations directly. Vortex
the microsomes as recommended below to assure homogeneity. The
diluted microsomes or incubation mixtures are kept on ice until you
are ready to start your incubations. Typically, incubations are
carried out at 37*C.
Sanjeev Thohan, PhD
Director, DMPK
Exelixis, Inc
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