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Hi,
Does anyone have a good list of positive controls for human hepatocyte
P450 isozyme drug induction studies. I know, for example, rifampin
works on two isoforms. Would someone please be so kind as to post a
list of good, known inducers of the common P450 human isoforms,
incubated with human hepatocytes. Thanks.
Harold Boxenbaum, Ph.D.
Pharmacokinetics Consultant
Arishel Inc.
14621 Settlers Landing Way
North Potomac, MD 20878-4305
(F) 301-424-8563
harold.-a-.arishel.com
www.arishel.com
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An article published in "Drug Metabolism And Disposition" by
pharmaceutical research and manufacturers of america, has a list of
positive controls for some common P450 isoforms.The reference is:
Drug Metab Dispos. 2003 Jul;31(7):815-32.
I think this will be of some help to you.
Thank you
Ragini Vuppugalla
Graduate student
TTUHSC
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Harold,
Here's my list of inducers. I've accumulated these from the
literature and elsewhere. These are human biased (e.g. omeprazole or
rifampicin don't work in the rat);
AhR-mediated Omeprazole 100 microM (CYP1A)
PXR-mediated Rifampicin 20 microM (CYP3A, CYP2C9/19 > CYP2B6)
CAR-mediated Phenobarbital 500 microM (CYP2B6 > CYP2C9/19, CYP3A)
Other Isoniazid 100 microM (CYP2E1)
Carbamezepine and phenytoin can also be useful.
You need to keep DMSO <0.1% or the background is high.
I've not (yet) worked with PPAR, VDR, LXR, FXR etc. mediated
induction.
I hope that this helps.
Bernard
Bernard Murray, Ph.D.
Senior Research Investigator
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Bernard.Murray.aaa.abbott.com
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Bernard,
Based on Madan A et al, DMD, 31:421-431,2003, what do you think of
these:
1A2: beta-naphthoflavone
2B6: phenobarbital
2C9: phenobarbital
2C19: rifampin
2E1: isoniazid
3A4: rifampin
I realize some of these overlap your list. Anything for 2D6?
Thoughts? Please explain to us uneducated (me!), your list of the type
of mediation. Thanks. Harold.
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"I realize some of these overlap your list. Anything for 2D6?
Thoughts?
Please explain to us uneducated (me!), your list of the type of
mediation."
Harold,
The only real difference between our two lists is beta-naphthoflavone
(BNF) versus omeprazole for CYP1A. Both compounds should work fine.
I think the main reason for favouring omeprazole is that this can be
relevant in man (e.g. starting with the study by Diaz et al. in 1990)
depending upon the recipient's CYP2C19 genotype.
BNF has seen frequent use as a model CYP1A inducer in experimental
animals and has the advantage it is relatively non-toxic (unlike TCDD)
and non-mutagenic (unlike 3-methylcholanthrene). From what I understand
the only disadvantage with BNF in vivo is that it is relatively rapidly
cleared so the interval between dosing and sample collection (typically
48 hours) is more strict if you want to see the best induction. I don't
think such a situation applies with in vitro dosing.
Here's a quick overview of enzyme regulation. This is a big, and
changing, field so I'll be happy if others want to step in and clarify
or correct me.
CYP2D6 is generally considered non-inducible. This is probably
something
of an over-simplification but expression does not change markedly in
response to classic inducers.
As far as the mechanisms I hinted at are concerned;
AhR = Aryl hydrocarbon (responsiveness) Receptor (actually working as a
heterodimer with ARNT) binds ligands such as planar aromatic
hydrocarbons,
dioxin and polyhalogenated biphenyls, omeprazole, indole derivatives and
indigoids. It acts to increase transcription of members of the Ah gene
battery (e.g. CYP1A1, CYP1A2 and a variety of other, non-CYP enzymes).
CYP1A1 is pretty much excusively an extrahepatic enzyme in man at the
protein expression level so the focus in hepatocytes is more upon
CYP1A2.
PXR = Pregnane X Receptor (= SXR, Steroid and Xenobiotic Receptor) which
works as a heterodimer with RXR. PXR shows some species differences in
ligand specificity, for example rifampicin works in humans, while PCN
works
in rodents. PXR-activation leads to transcriptional activation of
CYP3A4/5/43, MDR1, CYP2C9/19 and, to a lesser extent, CYP2B6.
CAR = Constitutive Androstane Receptor (again working with RXR).
Inducers act by repression of CAR activity. The response profile can
roughly be considered CYP2B6 > CYP2C9/19 and CYP3A4/5/43. There is
overlap
in response between CAR and PXR because some ligands bind to both
receptors
and both receptors can modulate the same genes. Phenobarbital is
generally
thought a classic CAR-mediated inducer but it can also appear to act
through
PXR. This lack of selectivity is somewhat species dependent.
CYP2E1 regulation is not through mechanisms as straightforward as those
outlined above. From what I understand, the main mechanism of action of
isoniazid is post-transcriptional and is through protein stabilisation
and/or increased transcriptional efficiency. Other inducers can
supposedly
act through mRNA stabilisation. I am not aware of anything that
actually
increases the rate of transcription.
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
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Harold,
Unlike other forms of cytochrome P450 there are no known inducers of
CYP2D6
activity. It is believed that the transcription of the CYP2D6 gene is
not
inducible. The CYP2D6 gene has an inserted DNA sequence (LTR element)
probably of retroviral origin. LTR elements can have enhancer
functions.
It has been speculated that inserted LTR prevents induction of the
CYP2D6 gene by promoting antisense transcripts.
Molven A and Steen VM A retroviral element in the human CYP2D gene
cluster. Pharmacogenetics. 1996 Aug;6(4):375-7.
Charlie Fisher
I guess this might help. Though 2D6 is thought to be non inducible butBack to the Top
2D6 in brain is induced by ethanol. Regional and cellular expression
of CYP2D6 in human brain: higher levels in alcoholics. J Neurochem.
2002 Sep;82(6):1376-87.
Deepak
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