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ISSN:1948-5956
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Journal of
Cancer Science & Therapy
The International Open Access
Journal of Cancer Science & Therapy
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Department of Cardiothoracic Surgery
New York University Langone Medical Center (USA)
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National Institute of Standards and Technology
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Jet Propulsion Laboratory (JPL)
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Digital Object Identifier: http://dx.doi.org/10.4172/1948-5956.1000003
J Canc Sci Ther Volume 1(1) : 019-024 (2009) - 019
ISSN:1948-5 956 JCST, an open access journal
Research Article OPEN ACCESS Freely available online
doi:10.4172/1948-5956.1000003
*Corresponding author: Yakisich J. Sebastian, Department of Clinical
Neuroscience, Huddinge Division of Neurology, Karolinska Institute,
Huddinge University Hospital, Sweden Hospital, S-141 86, Huddinge,
Sweden, Tel: +46 8 585 89 533; Fax: +46 8 585 83810; E-mail:
Sebastian.Yakisich@ki.se
Received November 01, 2009; Accepted November 25, 2009; Pub-
lished November 25, 2009
Citation: Avramidis D, Cruz M, Sidén Å, Tasat DR, Yakisich JS (2009)
Regrowth Concentration Zero (RC0) as Complementary Endpoint Pa-
rameter to Evaluate Compound Candidates During Preclinical Drug De-
velopment for Cancer Treatment . J Canc Sci Ther 1: 019-024.
doi:10.4172/1948-5956.1000003
Copyright: © 2009 Avramidis D, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Abstract
The screening process for potential anticancer drugs in-
volves expensive and time consuming preclinical and clini-
cal trials (CT) before a drug is approved for clinical use
(CU). At present, there is a “bottleneck” at the CT/CU
transition because many drugs that showed promising re-
sults during preclinical research did not pass clinical tri-
als. We speculated that the endpoint parameters (the in-
hibitory concentration 50 (IC
50
) or lethal concentration
100 (CL
100
)) commonly used in proliferation assays for
short-term periods (24-72 h) are not useful to predict the
antiproliferative effect in vivo, especially during clinical
trials. We propose the use of a parameter, regrowth con-
centration 0 (RC
0
), which will define the concentration
and time necessary to kill 100 % of the cells and prevent
regrowth when drug is removed. The RC
0
might introduce
a new bottleneck at the preclinical stage, “preclinical bottle-
neck”, that will select for drugs with more chances to pass
clinical trials and improve the success rate of anticancer
screening programs. Our proposal is supported by experi-
ments done with the DBTRG-05MG human glioma cell
lines exposed to short and long-term incubation with three
different DNA replication inhibitors (aphidicolin, hydrox-
yurea and etoposide) and retrospective analysis of clinical
trials for these drugs.
Regrowth Concentration Zero (RC
0
) as Complementary End-
point Parameter to Evaluate Compound Candidates During
Preclinical Drug Development for Cancer Treatment
Avramidis Dimitrios
1
, Cruz Mabel
1
, Sidén Åke
1
, Tasat Deborah Ruth
2
, Yakisich J. Sebastian
1
*
1
Department of Clinical Neuroscience, Huddinge Division of Neurology, Karolinska Institute, Huddinge University Hospital, Sweden
2
Universidad Nacional de San Martín Buenos Aires, Argentina
Keywords:
Drug screening; DNA replication; Glioma;
Aphidicolin; Hydroxyurea; Etoposide
Introduction
In general, the standard approach to evaluate novel compounds
for cancer treatment after drug synthesis or discovery is based
in preclinical testing and clinical trials (Figure 1 top). The pre-
clinical phase involves in vitro as well as in vivo research. The
entire process is very expensive and time consuming (DiMasi
et al., 2003; Emanuel et al., 2003). Commonly, in vitro experi-
ments for determining the antiproliferative effects of a poten-
tial antineoplastic drug are done either in cell free systems or in
cell lines by means of short term proliferation assays which
measure the incorporation of tritiated thymidine or BrDU into
DNA or mass cell by colorimetric methods (e.g. MTT assay).
In these assays, cell line(s) are tested against a broad range of
drug concentrations typically for 48-72 h and the results are
usually reported as a plot and the IC
50
and LC
100
(See Glossary)
are calculated by interpolation (Brown, 1997; Iljin et al., 2009).
For instance, in the NCI60 human tumor cell line anticancer
drug screen program, the GI50 (50% growth inhibition) and
LC50 (50% lethal concentration) are derived from concentration-
response curves by linear interpolation while the TGI (total
growth inhibition) is read as the x-axis intercept (Shoemaker,
2006). The main pitfall of these assays is that the short term
incubation is not enough to determine the minimum concentra-
tion of the drug that actually kill 100 % of the cells preventing
regrowth when the drug is removed from the culture. In prac-
tice, the continuous growth of the untreated cells (control) and
cells exposed to low concentration of the drug limits the assay
because of loss of linearity over time. Long term survival for
screening purposes has been assessed by the tumor colony-form-
ing assay on a moderate scale due to technical limitations (Shoe-
maker et al., 1985 ). When used, researchers often report the
IC
50
for the tested drug (Sasaki et al., 2008). After the optimal
drug concentration (e.g. IC
50
or LC
100
) is determined, the next
step involves testing the compound in animal models in vivo
where a tumour is induced by injecting cancer cells into spe-
cific organs. The drug is administered in control and experi-
mental groups and the antineoplastic effect is usually assessed
by tumor growth and/or survival rates (Kaplan-Meier plots).
Due to ethical considerations, animals are usually sacrificed after
few weeks and long term relapses are not evaluated after treat-
ment discontinuation. In case the compound shows promising
in vivo effect on tumor growth, acceptable side effects and tox-
icity, the drug is considered a good candidate to be tested in
clinical trials. These stages are associated with a significant
percentage of the total cost of the entire drug evaluation pro-
cess (DiMasi et al., 2003; Emanuel et al., 2003). There are several
examples of newly developed compounds that might be consid-
ered for clinical trials (Roth et al., 2009; Xu et al., 2009; Yakisich et
al., 2009 ) but at present it is difficult to estimate the chances that
they will be successful. Unfortunately, when promising drug
candidates are tested in clinical trials, the majority of them fail. In
most cases, the outcome has been disappointing and sometimes
Journal of Cancer Science & Therapy
- Open Access
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ISSN:1948-5 956 JCST, an open access journal
the trial terminated early due to evident failure (Robe et al., 2009).
This problem was recognized by Brown (Brown, 1997) who re-
ported that the popular NCI’s anticancer drug screening pro-
gram does not select for clinically active compounds, but no
potential solution or important changes have been implemented
to solve the problem.
Our hypothesis is that the high rate of failure when translating
preclinical drug screening into successful clinical trials is due to
the use of the IC
50
(and less common LC
100
) as guiding concen-
tration for mechanistic studies as well as goal for clinical trials.
At present, current drug development programs have a “bottle-
neck” at the “clinical trial”/”clinical use” transition, where the
success rate is low (Figure 1 Top). It is estimated that only 5% of
cancer drugs entering clinical trials reach marketing approval
(Collins and Workman, 2006). In this paper, success rate (SR) is
defined as the ratio between the number of drugs approved for
clinical use (n
CU
) and the number of drugs that enter the clinical
trial stage (n
CT
). Conceptually the SR (SR=n
CU
/n
CT
) is similar to
the “clinical approval success rate” defined as “the probability
that a compound that enters the clinical testing pipeline will
eventually be approved for marketing” (DiMasi et al., 2003). In
some cases (e.g. roscovitine as drug candidate for brain tumours),
the concentration that actually reach the target tissue is main-
tained at levels that are below the IC
50
reported in vitro and
might limit the clinical use (Yakisich et al., 2009 ). Moreover, even
if, in the target tissue, concentrations similar to the IC
50
are
reached, one can anticipate that a fraction of cells will survive
and resume cell proliferation when the drug is removed. Interest-
ingly, in a study measuring in vitro chemo sensitivity in patient-
derived cell lines by a short-term assay (24-72 h), the ID
50
(de-
fined as the dose of the drug that inhibited protein synthesis by
50 %), was used to classified between responder and non-re-
sponder patients. The relapse-free interval in patients that
showed in vitro chemo sensitivity (responders) was modestly
increased compared to patients designated as non-responder
(Thomas et al., 1985).
Increasing the number of clinical trials by simply screening
more drugs, without any additional rational guidance, will cre-
ate an unproductive “financial bottleneck” at the “preclinical/
clinical trial” transition due to the astronomical cost associated
with the clinical trials. Due to this “financial bottleneck” some
drugs that might be of clinical relevance, will probably never
be tested while other that enter the clinical trial stage will even-
tually fail at the same rate (keeping the 5 % rate success con-
stant).
In this paper we propose that the success rate and cost benefit
for drug development could be improved by introducing a
“bottleneck” during the preclinical stage (“preclinical bottle-
neck”) using the so called RC
0
(see glossary) as endpoint pa-
rameter instead of IC
50
or LC
100
(Figure 1 Bottom). The ratio-
nale of our proposal is supported by a series of experiments
using prolonged exposure to three classical DNA replication
inhibitors with different mechanism of action (aphidicolin, hy-
droxyurea and etoposide) in the human DBTRG-05MG glioma
cell line and a retrospective analysis of clinical trials with these
same drugs.
Materials and Methods
Cell lines
Stock cultures of human DBTRG-05MG glioma cell line were
obtained from the European Collection of Cell Culture
(ECACC). Cells were routinely cultured in RPMI-1640 medium
supplemented with 10 % Foetal Bovine Serum (FBS), 2 mM
glutamine, 1 % HT and 1 mM sodium pyruvate (complete me-
dia) at 37 ºC in a humidified atmosphere containing 5% CO
2
.
Drugs
Hydroxyurea, Aphidicolin and Etoposide (Sigma, Sweden)
were prepared as stock solutions (1 M in H
2
O, 2.5 mM in DMSO
and 25 mM in DMSO respectively) and stored at -20
°
C until use.
Fresh dilutions in culture media were prepared just before use.
Antiproliferative assay
DBTRG-05MG cells were plated in 96- well flat bottom plates at
5000 cells/well and were allowed to adhere overnight. Then, the
cultures were exposed to a concentration range of the three drugs
selected and control cultures where treated with the equivalent
concentration of the corresponding vehicle (DMSO or H
2
O).
After 72 h, cell growth was monitored using the CCK kit (Sigma,
Sweden) according to supplier instructions. For long-term
antiproliferative assays, drugs were maintained in culture for 2-
10 weeks. The media and drugs were changed twice a week and
were found to be sufficient to prevent cell growth (see result)
indicating that the drugs remain active during in vitro prolonged
cultures.
The presence of surviving cells during prolonged exposure
and the (re)growth after drug removal was evaluated using a
routine inverted microscope. Regrowth was defined as the abil-
Citation: Avramidis D, Cruz M, Sidén Å, Tasat DR, Yakisich JS (2009) Regrowth Concentration Zero (RC0) as Complementary
Endpoint Parameter to Evaluate Compound Candidates During Preclinical Drug Development for Cancer Treatment. J Canc Sci Ther
1: 019-024. doi:10.4172/1948-5956.1000003
Figure 1:
Top) Simplified diagram showing the common steps during drug
development from dru g discovery to clinical tri als. After discovery or
synthesis of a new potential antineoplastic drug, the compound undergoes
preclinical research where is tested in cell free systems as well as in viv o in
cell lines and animal models. Promising candidates that pass the preclinical
stage are approved for clinical trials and few reach the “clinical use” stage.
At the “clinical trial”/”drug use” transition the presence of a “bottleneck”
limits the success rate. The success rate (defined as the number of drugs
that reach the stage of clinical use divided by t he number of drugs that
enter the number of drugs clinical trial; n
CU
/n
CT
) , value between 0-1, is an
indication of the cost/benefit of the program.
B) Proposed outcome of drug development by introducing a “preclinical
bottleneck” by using the RC
0
as endpoint parameter during preclinical
research. The presence of the “preclinical bottleneck” will reduce the
number of cells entering the “clinical trial stage” and might increase the
success rate improving the cost/benefit.
Preclinical
Cell Free Systems
Discovery
or
Synthesis
Discovery
or
Synthesis
Cell lines/Animal models
Clinical trials
(CT)
“Bottleneck”
“Bottleneck”
“Preclinical Bottleneck”
Clinical Use
(CU)
SR= n/n
CU CT
SR= n/n
CU CT
J Canc Sci Ther Volume 1(1) : 019-024 (2009) - 021
ISSN:1948-5 956 JCST, an open access journal
ity of surviving cells to form a monolayer after 1-2 weeks of
incubation with drug-free media.
Results
We evaluated the short term antiproliferative effect of hydrox-
yurea (HU), aphidicolin (Aph) and etoposide (Et) on DBTRG-
05MG cells in culture. Exponentially growing cells were incu-
bated with different concentrations of each drug and cell prolif-
eration was measured at 72 h using the CCK kit assay. All three
drugs tested showed a concentration-dependent inhibitory ef-
fect reaching the maximum effect at 2.5 µM, 10 mM and 50 µM for
Aph, HU and Et respectively (Figure 2). The IC
50
was estimated
by interpolation as 0.9 µM, 0.75 mM and 0.9 µM for Aph, HU and
Et respectively.
Long-term incubation (4 weeks) with concentrations > IC
50
(Aph, 2.5 µM; HU, 10 M; Et, 5 µM), showed a subpopulation of
cells resistant to these drug concentrations. Microscopic exami-
nation revealed that a small fraction of cells remained attached
to the surface. When the drugs were removed from the culture,
the surviving cells were able to resume cell division and, they
proliferate reaching a monolayer morphologically indistinguish-
able from the original culture. To prevent cell regrowth higher
concentrations of HU (50 mM) or Et (> 25 µM) for at least one
week were required.
Dis cussion
We used the human glioma cell line DBTRG-05MG as an ex-
perimental system to retrospectively analyze the failure of sev-
eral drugs that inhibit in vitro cell proliferation of cancer cell
lines during clinical trials. Gliomas are the most common primary
brain tumours and remain poorly responsive to multimodality
therapeutic interventions, including surgery, radiotherapy, and
chemotherapy. The highly proliferative activity of glioma cells
compared to normal brain makes DNA replication an interesting
target for therapeutic purposes. In this paper, we evaluated the
outcome of DBTRG-05MG glioma cells exposed to three differ-
ent DNA replication inhibitors for short and long-term expo-
sures (Figure 2, Figure 3).
Aphidicolin: The in vitro IC
50
for aphidicolin has been deter-
mined in cell free systems as 0.5, 0.9 and 5.8 µM for polymerase
α, δ, and ε respectively (Wright et al., 1994) without affecting
dNTP pools (Sheaff et al., 1991). Due to the poor solubility and
because pharmacologically active levels had not been achieved
for aphidicolin, it was decided early (after two phase I studies)
to stop further evaluation (Beijnen et al., 1995). Therefore,
aphidicolin represents a good example of a potential anticancer
drug that reached the stage of clinical trials and its use for can-
cer treatment was stopped early based in its pharmacokinetic
properties.
Hydroxyurea: In our study, the IC
50
for DBTRG-05MG cells
was ≈ 0.5 mM (Figure 2). Pharmacological studies showed that
the level of HU after a single dose of 1200 mg can reach only 0.04
mM and 0.26 mM in cerebrospinal fluid and plasma respectively
(Gwilt et al., 2003). DBTRG-05MG cells were able to resist pro-
longed treatments (> 4 weeks) with 10 mM while higher concen-
trations (50 mM) were required to prevent regrowth. Plasma con-
centration might be enough for treatment of some types of can-
cer, but definitely not for brain tumors. Hydroxyurea alone has
been proven at early stage to be of no use for glioma treatment
and represents an example of a drug that (based only in IC
50
values) could have been stopped at the “preclinical bottleneck”
before engaging in costly and time consuming clinical trials.
Etoposide: Several large numbers of clinical trials and pharma-
cokinetic data showed that a daily oral etoposide dose of 50 mg/
m
2
produces serum concentrations >1 mg/L (~ 1.7 µM) lasting
several hours each day (Hainsworth, 1999). Potentially cyto-
toxic concentration in cerebro spinal fluid (CSF) was achieved
with doses higher than 300 mg/m
2
i.v. ( levels of 0.175 µM; range,
0.066 to 2.12) in children with acute lymphoblastic leukemia
(Relling et al., 1996 ). In our study, the IC
50
for DBTRG-05MG
cells was < 1 µM but the RC
0
was > 25 µM (Figure 3). Even
though at present etoposide alone has been of limited use in
brain tumors, our data predict that clinical trials with etoposide
will continue to fail unless intracerebral levels of > 25 µM are
reached. The use of etoposide at very high doses (800 mg/m
2
) in
combination with autologous bone marrow transplantation in-
crease the CSF levels (similar to IC
50
but not to RC
0
levels) still,
in brain tumor tissue the outcome has been disappointing
(Giannone and Wolff, 1987; Leff et al., 1988 ). Etoposide is an
example of a drug that based in IC
50
values should have been
successful for glioma treatment, but clinical trials proved to be
of very limited use (Finn et al., 1985; Fulton et al., 1996; Tirelli et
Journal of Cancer Science & Therapy
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www.omicsonline.org JCST/Vol.1 Issue 1
Figure 2:
Antiproliferative effect of short term incubation with Hydroxyurea, Aphidicolin or Etoposide. DBTRG-05MG glioma cells were incubated with
the indicated concentra tion of drugs during 72 h. Cell proliferation was measured by the CCK kit. Results are representative of three independent
experiments performed by quadruplicates.
Survival (%)
Aphidicolin (µM) Etoposide (µM)
Hydroxyurea (mM)
120
100
80
60
40
20
0
0 0 (D) 1 2,5 5 10 25 0 1 5 10 0 1 10 50 100 250
J Canc Sci Ther Volume 1(1) : 019-024 (2009) - 022
ISSN:1948-5 956 JCST, an open access journal
al., 1984). If the RC
0
have been used as guide, etoposide as single
agents would have never been attempted saving cost and time.
Instead, etoposide would have been used from the beginning in
combinational therapy with other drugs.
The three drugs we evaluated have RC
0
much higher than IC
50
and retrospectively demonstrate that clinical trials have poor
outcome when the drug concentration reach similar or just
slightly higher values than the IC
50
. In m-AMSA treated cultures
at LD
80
concentration, a fraction of stem cells survive prolonged
exposure (Sabisz and Skladanowski, 2009). m-AMSA is a
topoisomerase II inhibitor that also works as an alkylating agent,
suggesting that our study can be extrapolated to other drug
types. Thus, classical end-point proliferation parameters used
to evaluate drug effects (IC
50
, LC
100
) when used in short term
assays, are insufficient for preclinical testing since they do not
predict long term effects. This is due to the fact that a small
fraction of cells may survive prolonged exposure to similar or
slightly higher concentrations than the IC
50
. The surviving cells,
when the drug is removed from the culture will resume cell divi-
sion. At the clinical level, when the chemotherapy regime is
discontinuated, surviving cells will resume cell division leading
to relapse of the tumour. We propose the use of a new parameter,
RC
0
(Regrowth Concentration 0; defined as the lowest concen-
tration that produce 0% regrowth when the drug is removed
from the culture) as a complementary end-point proliferation
parameter. Other authors also proposed that assays that moni-
tor surviving cells should be included during drug screening
(Sabisz and Skladanowski, 2009). The main disadvantage of the
RC
0
is the length of the assay (2-4 weeks) that in turn limits the
number of drugs that can be screened. High throughput assays
such as the NCI60 (Shoemaker, 2006) or similar cell-based screen-
ing methods (Iljin et al., 2009) can in short time, select several
potential anticancer drugs (based in IC
50
values) from compound
Citation: Avramidis D, Cruz M, Sidén Å, Tasat DR, Yakisich JS (2009) Regrowth Concentration Zero (RC0) as Complementary
Endpoint Parameter to Evaluate Compound Candidates During Preclinical Drug Development for Cancer Treatment. J Canc Sci Ther
1: 019-024. doi:10.4172/1948-5956.1000003
libraries. After that, the RC
0
can further help to identify those
drugs with higher chances to succeed in clinical trials from the
initial selection.
The RC
0
provides two types of valuable information: 1) The
necessary concentration to kill 100 % of tumoral cells, 2) the
lapse of time the cells need to be exposed to a certain concentra-
tion. The time factor has been demonstrated to be important. For
instance extended-schedule oral etoposide shows more efficacy
in selected cancers (Hainsworth, 1999). Thus, preclinical in vitro
testing should include long term proliferation assays to deter-
mine the RC
0
. This can simply be done by incubating the cells
for a prolonged period of time (e.g. 2-4 weeks) after that, remove
the drugs and incubate the cells for another 1-2 weeks and deter-
mine the concentration of drug that killed 100% of the cells (No
regrowth after incubation in drug free media).
To be of clinical use, the CR
0
should fulfil two other essential
requirements: a) the drugs should be non toxic (or mild) to
normal cells for the period required to kill all tumoral cells and b)
It should be possible to reach this concentration in the target
tissue. In this context, the data obtained from phase 0 might
constitute and essential step before pursuing more advanced
research (e.g phase I). A rational use of CR
0
in combination with
pharmacokinetic and toxicological studies in healthy individu-
als (Phase 0) might lead to a more successful rate of antineoplas-
tic drugs with clinical relevance.
Since the RC
0
> LC
100
>LC
50
, higher toxicity to normal cells will
be expected, and less number of drugs will fulfil the criteria to be
approved for clinical trials. At a first glance, this will reduce the
number of clinical trials that in turn will reduce the number of
drugs that reach the market. However, by avoiding unnecessary
highly expensive clinical trials with drugs that have little or no
chances to be of clinical use, more drugs can be screened before
the “preclinical blottleneck”. Resources can be put into devel-
oping more specific drugs with RC
0
levels that can be achieved
in target tissue with tolerable toxicity. The RC
0
will eventually
move the drug development “bottleneck” from clinical trials to
preclinical stages. This displacement will eventually produce a
drop in drugs that reach clinical trials but it will select for those
with higher rates of success reducing the high expenses and
time of clinical trials.
Thus, the use of the RC
0
has the potential to increase the
successful rate and maybe, overcome the bottleneck when trans-
lating preclinical research into clinical trials. From the economi-
cal perspective, predicting which drug will successfully pass
clinical trials will have a tremendous impact in the drug industry
by lowering cost and time by stopping clinical trials of high
number of drugs. On the other hand the use of the RC
0
will help
at early stages to decide which drugs have little chance to be
useful for monotherapy but might have use in combinational
regimes.
Glossary
GI
50
: drug concentration that causes a 50% reduction in cell
number in test plates relative to control plates (equivalent to
IC
50
).
LC
100
: drug concentration that causes a 100% reduction in cell
number in test plates relative to control plates (equivalent to
Fig ure 3:
Top) Antiproliferative effect of long -term incubation with
Aphidicolin (Aph, 2.5 µM), Hydroxyurea (HU, 10 mM) or Etoposide (Et,
5 µM) on DBTRG-05MG glioma cells. Exponentially growing cells were
incubated in complete media for 2-3 days (A), exposed for 4 weeks to the
indicated drugs concentrations (B-C). During this period the media and the
drugs were changed twice a week. A clear decrease in the cell density was
observed by microscopic examination indicating extensive cell death (B)
followed by a stable low cell density (C). When the drug was removed, the
surviving cells resumed proliferation and formed a monolayer indicated as
regrowth in (D). Bottom) same as top but cells were incubated with higher
concentrations of HU (50 mM) or Et (25 µM).
A B C D
Complete Media
Proli feration Cell death
Complete Media
Proli feratio n Cell death
Survival
No Survival
Time
Cell # Cell #
Re
Growth
HU (10 mM) or Aph (2,5 µM), Et (5 µM)
HU (50 mM) or Et (> 25 µM)
Relapse
-----------------------------------------------------------------------------
-----------------------------------------------------------------------------
-----------------------------------------------------------------------------
J Canc Sci Ther Volume 1(1) : 019-024 (2009) - 023
ISSN:1948-5 956 JCST, an open access journal
IC
100
). It is usually determined by interpolation from concentra-
tion response curves.
RC
0
: drug concentration that kill 100% of cells preventing re-
growth when cells are incubated in drug free media. In contrast
to LC
100
, RC
0
is determined empirically, not by interpolation.
Disclosure of Potential Conflicts of Interest
None
Acknowledgement
This work was supported by grants from the Swedish Re-
search Council and the Karolinska Institute.
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ISSN:1948-5 956 JCST, an open access journal
Citation: Avramidis D, Cruz M, Sidén Å, Tasat DR, Yakisich JS (2009) Regrowth Concentration Zero (RC0) as Complementary
Endpoint Parameter to Evaluate Compound Candidates During Preclinical Drug Development for Cancer Treatment. J Canc Sci Ther
1: 019-024. doi:10.4172/1948-5956.1000003
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