Dear Group,Back to the Top
The value of human hepatocytes in assessing enzyme
induction in-vitro is well known. But how about the compounds that
induce drug metabolizing enzymes by interacting with nuclear hormone
receptors (for example Rifampicin on Human pregnane X receptors), when
PXR has the role in the regulation of genes involved in Xenobiotic
metabolism, including CYP2B6, CYP2C8, CYP2C9 and MDR1. Does anyone
suggest the suitable high throughput enzyme induction assay for such
compounds? Thanks in advance.
Regards,
S Syed Mustafa,
Research Associate,
Drug Metabolism & PK,
Dr Reddy's Research Foundation,
Hyderabad-500049 INDIA
Back to the Top
Dear Mustafa
Regarding your question about CYP450 induction studies is concerned. I
would like to tell you that induction is brought about by involvement
of one of the nuclear receptor (Pregnane X receptor, which you have
mentioned), Aryl hydrocarbon X receptor, retinoic acid X receptor and
many other orphan receptors which are yet to be characterized. All
these receptors after binding to the ligand i.e. NCE (if the NCE is a
inducer) in question dimerizes with the RXR. This whole complex binds
to the promoter (DNA) upstream of the CYP450's and brings about the
increase in the transcription of the respective CYP450's, hence
induction. This is the mechanism of CYP450 induction. There are many
ways of studying CYP450 induction.
The methods (models) that are used to study CYP450 induction should be
closer to humans, since in discovery ultimate effect of any drug
interaction will affect the clinical studies and moreover there is a
wide variation of CYP450 among the various species.
Human hepatocytes are the best models for studying CYP450 induction.
Following are the various ways of studying CYP450 induction.
1. As you have already mentioned about human hepatocytes - These cells
are exposed to the NCE in question for CYP450 induction for
prerequisite times and activity of the various CYP450 is measured.
But some times it is difficult to obtain human hepatocytes - Then we
can look forward to humanoid (human like) surrogates.
2. Stable transfection of gene downstream to the promoter of nuclear
receptors in the transformed cell line of human origin (HepG2). Culture
the cells, expose it to the NCE(if it is a inducer) it brings about the
increase in the expression of the transfected gene, which can be
quatified at the level of mRNA, protein and activity. Here using
luciferase enzyme as the transfected gene gives us ample flexibility
for quantification by chemiluminescence, which as a matter of fact is
made as highthroughput screen for studying induction.
Hope I am clear to you. Any sort of queries are most welcome.
Ansar Ali Khan, M.Sc.,
Research Scientist,
Drug Metabolism and Pharmacokinetics,
Glenmark Research Centre,
Plot No.-A-607, TTC, Industrial Area,
Mahape, Navi Mumbai-400 709,
India.
Phone No: 91-22-5590 2491/92 Extn.308
Fax No.: 91-22-5590 2318
Hi all,Back to the Top
"But some times it is difficult to obtain human hepatocytes - Then we
can look forward to humanoid (human like) surrogates."
Please tell me some surrogates for human hepatocytes.
I would like also to ask you some references about design of this kind
of in vitro experiments about enzyme induction/inhibition
Thank you,
Vlase Laurian
Dept. of Pharmaceutical Technology and Biopharmaceutics
Faculty of Pharmacy
University of Medicine and Pharmacy "Iuliu Hatieganu"
13, Emil Isac
Cluj-Napoca, Cluj 3400, Romania
vlaselaur.-at-.yahoo.com
Back to the Top
Dear Ansar and Group,
No doubt enzyme induction or inhibition Properties
of NCE's plays the pivotal role in picking up a
molecule or dropping it at an early stages of drug
development.It is well demonstrated that there are
three ways to screen a NCE for induction/inhibition
properties.
1.Primary cultured Hepatocytes
2.Hepatoma cell lines such as HepG2 and Huh7 (as you
discussed)
3.CYP engineered cells
But none other than Primay cultures have shown to
produce a metabolic profile of a NCE akin to that
observed in in-vivo and various hepatoma cell lines
respond differently to external stimuli.
Different substitutes to replace human hepatocyes
because of their limited access (as you highlighted
exactly) have been explored. One such approach was to
immortalize hepatocytes using different strategies
viz., Transfection with c-myc, cH-ras, n-ras
oncogenes, transgenic animals overexpressing growth
factors or oncogenes, conditional immortalization
etc.,But none of the resulting cells had the desirable
phenotypic charecteristics to replace primary
cultures.
Because of the differences in the levels of
certain key description factors in comparison with
hepatocytes, Human hepatoma cell lines do not express
CYP genes. With this background in mind, I wonder how
effectively one can substitute hepatoma cell lines
such as HepG2 and Huh 7 for hepatocytes? Thanks in
advance.
With Regards,
S Syed Mustafa,
Research Associate,
Drug Metabolism & Pharmacokinetics,
Dr Reddy's Research Foundation,
Hyderabad-500049 INDIA
syedmustafa27.-a-.yahoo.com
Back to the Top
Dear Mustafa / Group
I do agree with you that nothing can replace the primary human
hepatocyte cultures for studying CYP450 induction. In this regard I
would like you to take a little different approach to study the drug
metablolism encompassing the drug interaction (induction). From the drug
metabolism studies we will come to know about the metabolism profile of
the NCE as well we can characterize the enzyme responsible for the NCE
metabolism. From here onwards our focus should be to study the NCE in
question as a inducer of any of the CYP450 - mechanism identification
(Here I would like to say that the surrogate model we choose to study
CYP450 induction need not resemble or possess metabolic profile of a NCE
akin to that observed in in-vivo and various hepatoma cell lines)
Why it is not necessary is because of the advent of the
Pharmacogenetics. The assays what we have spoken (previous reply)
previously is no doubt going to stay for a long time but the new
approaches are evolving very fast and they are more reliable, validated
and high throughput. I am mentioning few of them.
1. In silico screening - using computational docking of molecules
with the available crystal structure of nuclear receptors - which have
been implicated in the CYP450 induction.
2. The availability of the cloned human nuclear receptors (which
have been implicated in the CYP450 induction) can be used to do
competitive receptor binding assays with NCE in question. These assays
can be coupled with scintillation proximity assay technology in 96 well
microtitre plates.
3. Cell based reporter gene assays - Here host cell is transfected
with two DNA plasmids - an expression plasmid for receptor (human) and a
reporter plasmid. The receptor plasmid expresses the receptor, which
will interact with the NCE in question and brings about the expression
of the reporter gene - which may be encoding for luciferase or alkaline
phosphatase.
4. Finally humanized mice can be a useful tool for studying the
risk assessment in in vivo to the exposure of NCE in question. Here
transgenic mice is developed with the replacement of its endogenous
nuclear receptors with the humans - This mice reacts similar to that of
humans to the NCE in question - useful for assessing the toxicity due to
CYP450 induction with respect to drug interaction.
All these studies are very much prevalent in studying the human pregnane
X receptor (Identified and characterized in 1997, which is just 7 years
old). The advent of pharmacogenetics may bring new receptors to light in
the forth coming years. This knowledge might not only be used for
studying induction/interaction of CYP450's but also transporter proteins
like MDR's (also known to play a critical role in drug interaction) with
similar efficency.
This is just the beginning of a new era of using surrogates for humans.
Long way to go----------
References
1. Nature Reviews, April 2002, Volume 1.
Authors - Timothy M. Wilson and Steven A.Kliever
2. Endocrine Reviews 23(5);687-702.
Authors - Steven A.Kliever, Bryan Goodwin and Timothy M. Wilson
Ansar Ali Khan, M.Sc.,
Research Scientist,
Drug Metabolism and Pharmacokinetics,
Glenmark Research Centre,
Plot No.-A-607, TTC, Industrial Area,
Mahape, Navi Mumbai-400 709,
India.
Phone No: 91-22-5590 2491/92 Extn.308
Fax No.: 91-22-5590 2318
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)