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Mary Glodowski, StarNet Teacher 2001School:
Juanita High School
District: Lake Washington
City: Kirkland, WA
Grades taught: 10-12 Principle
Investigator: Ken Thummmel, Ph.D.
Department: Pharmaceutics
Institution: University of Washington Project
Description The
Biological Basis for Interindividual Variability in Drug Metabolism
Research in Dr. Thummel's lab is focused on
understanding the role of Cytochrome P450 enzymes in drug metabolism.
This family of enzymes is responsible for metabolizing endogenous and
xenobiotic compounds, and some forms have been found to be polymorphic.
This variability plays a role in how extensively or poorly an
individual may metabolize certain compounds. The CYP3A subfamily is
the most prevalent of all CYP enzymes and is responsible for
metabolizing 50% of all drugs that are metabolized. It is expressed
primarily in the liver and intestine. Of particular focus in the
Thummel Lab are the enzymes CYP3A4 and CYP3A5. Of these two major
isoforms, only CYP3A5 has been found to be polymorphic. The CYP3A5*1
variety results in a functional protein, whereas the CYP3A5*3 variety
is prematurely truncated resulting in a nonfunctional protein. STARNet
Summer Research Project on CYP3A5
My research focused on the
role of CYP3A5 in the metabolism of Flunitrazepam. While Flunitrazepam,
often called the 'date rape' drug, has been banned from manufacture
and sale in the US, it is widely prescribed in Europe. Like all
benzodiazepines, it has a sedative, hypnotic, and anterograde amnesic
effect. Its abuse in the US has escalated, especially in the southern
states. Abuse liability includes enhancing the effects of alcohol and
marijuana, and easing heroin withdrawal symptoms and the after-effects
of crack cocaine.
Our experimental design consisted of one group
of human livers with varying amounts of CYP3A4, but no CYP3A5, and a
second group of human livers with varying amounts of both CYP3A4 and
CYP3A5. Studies were carried out in duplicate, and both groups were
determined to have the same average amount of CYP3A4 as determined by
Western Blot. The processes used in our research included incubation
of Flunitrazepam with expressed CYP3A4 and CYP3A5 and incubation of
Flunitrazepam with microsomes of the genotyped human livers. The
products of the incubations were analyzed by HPLC to separate
Flunitrazepam from its metabolites, and MS to identify compounds based
on mass.
Analysis of these results showed that there was
a statistical difference in the amount of metabolite being formed by
the human liver microsomes that contained higher levels of CYP3A5.
While it was known that other CYP enzymes played a role in the
metabolism of Flunitrazepam, studies on CYP3A5 had not been
documented.
Further Studies
The results of this research have elicited several questions that are
applicable to other drugs of abuse. It is possible that our research
can serve as a model for studying drugs of abuse based on
interindividual metabolism by CYP3A5. Some of these questions are:
- Can the CYP3A5 genotype be related to
specific drug effects as a result of its metabolism?
- Are the metabolites of the drug active?
- Do different metabolites induce different
effects? (sedation, hypnotic effect, sleep, amnesia)
- Do interindividual differences in metabolism
affect abuse potential?
Acknowledgements
Many, many thanks to the members of the Thummel Lab! Dr. Ken Thummel,
Dr. Tom Kalhorn, Yvonne Lin, Donavon McConn, Dr. Amy Dowling, Tauri
Senn, and Hitoshi Ishizuka.
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