Biology Reference
In-Depth Information
As with many purine nucleoside analogs, the enzy-
matic degradation of gemcitabine is a complex process
that can effectively reduce the concentration of active
metabolites. For example, gemcitabine is inactivated
mainly by deoxycytidine deaminase, leading to the
production of difluorodeoxyuridine (dFdU).
220
In fact,
the K
m
of 96
m
M for gemcitabine is only two-fold higher
than that for the natural substrate, deoxycytidine (K
m
¼
47
m
M) (217). Deamination of dFdCMP to 2' 2'-difluoro-
deoxyuridine monophosphate (dFdUMP) can also
occur, and this is catalyzed by the action of dCMP-
deaminase. The subsequent conversion of dFdUMP to
dFdU represents another pathway for gemcitabine inac-
tivation.
220
Since neither gemcitabine nor dFdU are
substrates for pyrimidine nucleoside phosphorylases,
both compounds are not further degraded and excreted
out of the cell.
220
The pharmacokinetic behavior of ara-C is similar to
that described for gemcitabine. A standard dose of
100200 mg/m
2
ara-C typically results in steady-state
plasma levels of 0.51
m
M of the nucleoside.
221,222
At these low concentrations, the rate-limiting factor
in ara-C uptake is transport by the hENT1 protein.
However, when high doses of ara-C are used
(2
due primarily to variations in the k
pol
values for the
various nucleotide triphosphates as the binding affinity
for the various nucleotide analogs are essentially
identical.
There is only one available crystal structure of
a human DNA polymerase bound to DNA containing
either ara-CMP or dFdCMP paired opposite a template
dG.
232
These structures for pol
l
, a specialized poly-
merase involved in DNA repair, shows that ara-CMP
and dFdCMP bind within the nascent base pair binding
pocket of the polymerase. However, there are interesting
differences in the conformations of the ribose moieties of
the nucleoside analogs that impact their pharmacolog-
ical effects. Specifically, the conformation of the ribose
moiety of ara-CTP is similar to that of the natural
substrate, dCTP, whereas the conformation of dFdCTP
is significantly different.
232
This difference provides
a structural explanation as to why pol
l
efficiently incor-
porates ara-CTP but does not incorporate dFdCTP.
232
It
will be of significant interest to determine if canonical
and other non-canonical polymerase show similar struc-
tural differences, and if these subtle differences in sugar
conformation play roles in modulating the cytotoxic
effects of ara-CTP and dFdCTP.
In addition to differences in incorporation kinetics,
gemcitabine and ara-C differ with respect to the mecha-
nism of chain termination. After incorporation into
DNA, dFdC can be elongated but only by one additional
nucleotide (
Figure 5.13
). After this, DNA synthesis is
terminated leading to an inhibition of cellular DNA
synthesis. This contrasts the action of ara-CTP which
causes DNA polymerases to terminate DNA synthesis
directly at the site of incorporation. The non-terminal
position of dFdCMP in DNA is denoted as “masked
chain termination.” This “extra” nucleotide plays an
important role in “hiding” the incorporated dFdCTP
from DNA repair enzymes as dFdCMP resistant to the
normal mechanisms of DNA repair.
233
This plays an
important pharmacological role as it prevents detection
and repair by DNA repair enzymes to induce apoptosis.
Furthermore, the 3'
3g/m
2
), plasma concentrations of the nucleoside
analog can exceed 50
m
M. At these concentrations,
simple inward diffusion rates exceed those of pump-
mediated transport.
223
Once inside the cell, ara-C is
phosphorylated by dCK and pyrimidine kinases to
theactive5'-triphosphatederivativeara-CTP
224
e
226
Conversion to the di- and tri-phosphate forms is cata-
lyzed by pyrimidine nucleoside kinases. Catabolism
of ara-C occurs from rapid deamination by cytidine
deaminase to the non-toxic metabolite arabinoside
uridine while ara-CMP is dephosphorylated by the
action of cytoplasmic 5'-nucleotidase.
227
e
Mechanism of Action
Although gemcitabine and ara-C have multiple
intracellular targets, their primary cytostatic and cyto-
toxic activities are dependent upon the inhibitory
effects on DNA synthesis.
228,229
Similar to purine
analogs such as F-ara-ATP, the triphosphate form of
gemcitabine, dFdCTP, is a substrate for various DNA
polymerases.
230,231
In vitro
DNA primer extension
assays demonstrated that dFdCTP competes with
dCTP for incorporation opposite dG in the template
DNA strand. The IC
50
values for dFdCTP are 11
m
M
and 14
m
Mforpol
a
and pol
3
, respectively. Opposite
a templating dG, the rank order of catalytic efficiencies
(k
pol
/K
d
) for the incorporation of these natural and
non-natural pyrimidine nucleotides is dCTP greater
than ara-CTP and dFdCTP which are much more effi-
cient than rCTP.
230
The differences in k
pol
/K
d
value is
5' exonuclease activity of pol
3
is
unable to excise dFdCMP from DNA whereas ara-CMP
is removed from the 3'-terminus, albeit at a ~3-fold
slower rate compared to natural nucleotides.
234
Ligation
efficiency by T4 ligase and ligase III/XRCC1 was largely
unaffected by the nucleotide analogs.
234
/
Self-Potentiating Activity of Gemcitabine
Although gemcitabine is a potent inhibitor of DNA
synthesis, its cell killing effects are not confined to
rapidly dividing cells as the nucleoside analog is equally
effective against confluent cells and cells in log-phase
growth. This represents another important difference
between gemcitabine and ara-C as this indicates the
