I have been advised by an expert in the field that it is possible toBack to the Top
utilize human microsomal preparations to produce oxidative, reductive,
hydrolyis and conjugative metabolites. One notable comment was that to
produce conjugates, the addition of agents promoting glucuronidation
and sulfation was essential. Does anyone know of a protocol, review
article, etc. outlining the procedures for this "totality" of reactions
viz.-a-viz. human microsomes? Additionally, if other preparations may
be used to better advantage (e.g., hepatocytes, liver slices, etc.),
can references, etc. be provided. Thanks. Harold
Harold Boxenbaum, Ph.D.
Arishel Inc. (Consulting)
Pharmacokinetic n Pharmaceutical n Medical n Biotechnology n
Nutraceutical
14621 Settlers Landing Way
North Potomac, MD 20878-4305
Phone: (301) 424-2806
Fax: (301) 424-8563
E-Mail: harold.aaa.arishel.com
www.arishel.com
Harold,Back to the Top
Concerning coupled P450 + glucuronidation reactions, I use the
protocol of Fisher et al. which employs alamethicin instead of
detergent for activation (and so minimises the suppression of
oxidative metabolism).
Fisher et al. Drug Metab. Dispos. (2000) 28:560-566
Of course, actual *scaling* from microsomal glucuronidation is
considerably more problematic...
I have not seen an equivalent for coupled sulphation reactions
but since STs do not need activation it may be possible to
simply add PAPS to the mix.
I hope that this helps.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Research Investigator
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Bernard.Murray.aaa.abbott.com
Harold,Back to the Top
During the preparation of microsomes, the phase 2 enzymes tend to be
predominantly on the interior of the liposome. Alamethicin can be
added to cause pores in the microsomes (I have also seen albumin addition do
disrupt the liposome)- here is an abstract from Drug metabolism and disposition
describing the use of alamethicin:
In Vitro Glucuronidation Using Human Liver Microsomes and The
Pore-Forming Peptide Alamethicin Michael B. Fisher,1 Kristina Campanale, Bradley L. Ackermann, Mark VandenBranden, and Steven A. Wrighton
The UDP-glucuronosyltransferases (UGTs) are a superfamily of
membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to
attain maximal enzyme activity. The goals of the current work were to
develop a universal method to glucuronidate xenobiotic substrates using
microsomes, and to apply this method to sequential
oxidation-glucuronidation reactions. Three assays of UGT catalytic activity
estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and
morphine-3-glucuronidation, which are relatively selective probes for
human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment
of microsomes with the pore-forming peptide alamethicin (50 ug/mg protein)
resulted in conjugation rates 2 to 3 times the rates observed with
untreated microsomes. Addition of physiological concentrations of Mg2+ to the
alamethicin-treated microsomes yielded rates that were 4 to 7 times the
rates with untreated microsomes. Optimized assay conditions were found
not to detrimentally affect cytochrome P450 activity as determined by
effects on testosterone 6-hydroxylation and 7-ethoxycoumarin deethylation.
Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of Kmapp = 0.017 mM,
Vmaxapp = 0.4 nmol/mg/min, and n = 1.8. Formation of
acetaminophen-O-glucuronide also best fit the Hill equation, with Kmapp = 4
mM, Vmaxapp = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics,
with Kmapp = 2 mM and Vmaxapp = 2.5 nmol/mg/min. Finally, alamethicin
treatment of microsomes was found to be effective in facilitating the sequential
oxidation-glucuronidation of 7-ethoxycoumarin.
Good Luck
Mike
Michael D. Cameron Ph.D.
CellzDirect, Inc.
8609 Cross Park Dr, Suite 100
Austin, TX 78754
Phone: 512-615-2286
Fax: 512-834-7767
michaelc.-a-.cellzdirect.com
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