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Dear all,
I would like to know whether I could suspend the human liver
microsomes in DPBS or HBSS instead of typical 0.1M phosphate buffer.
Does any of the ions in HBSS or DPBS (Na+, K+ Cl-...) effect the CYP
activity?
Thanks
Ravi Talluri Ph.D
Sai Advantium Pharma
Pune
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Dear Ravi,
It is always better to keep buffer system as simple as possible in
metabolic stability experiment and common practice is to use phosphate
buffer with 50-100 mM ionic strength. Using complex buffer system
having more number of components like tris and MgCl2, or sulfate may
affect the enzyme kinetic parameters like Vmax and Km values. However,
if one wishes to use it he can but with proper validation.
Any additional comments are welcome.
Thanks,
Rhishikesh,
Advinus Therapeutics, Pune, India
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Dear Rishi,
I have used both tris-HCl and 100mMphosphate buffer with MgCL2; just
got confused with your answer, can you use phosphate buffer without
the presence of MgCl2, because then, will there be any effect on CYP
activity?
With regards
Sagnik
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Dear Sagnik,
Thanks for your comment.
Let me clear my point of view. While doing the experiment you can use
the various buffer systems like phosphate buffer, Tris, carbonate,
citrate, acetate but you will never get identical results using
different buffer system as the incubation conditions such as buffer
type and concentration affects the rate of reaction. For example, 1)
If one compares the glucuronidation kinetic of a compound in phosphate
and carbonate or tris buffer it may not be same 2) high ionic stregth
works better for CYP3A4 while reverse is the case with CYP2A6 which is
more active at low ionic strength and its need less to say that ionic
strength depends on compostion of buffer. That's why to make the
things simple in drug discovery it is always good to have buffer
system which models the in-vivo metabolic profile and phosphate buffer
is best in this regards and Kcl, Nacl, Mgcl2 are common variables
presents and it gives good activity to all metabolizing enzymes.
However, it does not mean that one can not use other buffer systems;
perhaps one can do that if has specific purpose but will not get
identical results in two different buffer systems, and requires
laboratory standardization.
You can refer to good research paper of Dr. Charles Crespi. The
R144C change in the CYP2C9*2 allele alters interaction of the
cytochrome P450 with NADPH:cytochrome P450 oxidoreductase.
Pharmacogenetics and Genomics. June 1997 - Volume 7 - Issue 3 and
also some other paper Human cytochrome P450 3A (CYP3A) mediated
midazolam metabolism: the effect of assay conditions and
regioselective stimulation by alpha-naphthoflavone, terfenadine and
testosterone. Pharmacogenetics. 1998 Apr;8(2):137-55.
Comments from experts are welcome.
Thanks and Regards,
Rhishikesh
Advinus Therapeutics, Pune, India.
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Friends,
I would like to add few points here though I am not hands on in this
science.
The buffer concentration definitely affect any of the chemical
reaction (extent and rate) hence an overall kinetics. So, drug
metabolism and metabolic stability reactions cannot be avoided.
depends upon the type pH, concentration in the medium, type of CPY
isoforms and chemical nature of the drug in question; it is logical to
think the effect will be diverse. Buffer generally is added for the
integrity of the pH during the reaction. But the additional effects
will be the competitive interaction with enzyme (against drug),
chelation based on the valance and ionic state of the counter ion,
alteraction of the system polarity, dielectric constant, ionic
strength and so for which has direct or indirect impact on the
activatiion energy of the biochemical reaction. I can imagine often
the memberance permeability in presence of different type and
concentration of the buffer across the hepatocytes and microsome is
unequivocally altered.
Despite these facts, the buffer constituent for the particular
metabolic stability experiment can be customized on the basis of the
spectrum of the information expected therefrom such as accuracy and
throughput which normally has inverse relation. In discovery set up
either cannot be sacrificed so they need to be properly compensated.
One can set up proper DOE and validate the difference of the result.
Suppose the factor by which the rate and extent of enzymatic reaction
kinetics in presence of minovalent (K+, Na+), divalent ion (Mg+2),
multivalent (phosphate, tris) and even their strength can be
established for the particular structural moeity under investigation
and make the SOP. But, be noted they cannot be universalized.
I appreciate the comments forth in this concern.
Amrit
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The following message was posted to: PharmPK
Besides the buffer consideration, does anyone add protein (BSA or human
plasma) to the metabolic stability incubation mixture?
Thanks,
Sean
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Dear Sean,
Occasionally we do this. We use BSA at low conc (0.1-2 mg/mL) and at high conc (40 mg/mL).
Rostam
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