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Experimental Therapeutics, Preclinical Pharmacology |
Department of Advanced Therapeutics, British Columbia Cancer Research Centre, Vancouver, British Columbia, V5Z 4E6 Canada [D. E. L. d. M., N. H., N. M., L. D. M.], and Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada [L. D. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism of AS3 action is believed to result from direct AS ODN-mRNA binding as well as degradation of these duplexes by RNase H both of which inhibit translation of Bcl-2 protein (10) . Bcl-2 as ODN treatment of tumor cells in vitro has been shown to down-regulate Bcl-2 protein and induce increased apoptosis susceptibility (11, 12, 13) . Early work with in vivo administration of phosphodiester ODNs was largely unsuccessful because of the rapid degradation of ODNs in the circulation (14 , 15) . The development of phosphorothioate-ODNs such as the Bcl-2 AS ODN G3139 (Genta, Inc.) has led to the clinical development of these molecules with promising early results (16) . Combining antitumor agents with Bcl-2 AS ODN presents a supplementary therapeutic strategy. Because many anticancer drugs elicit their cytotoxic activity via apoptosis, concurrent Bcl-2 AS ODN and drug treatment could enhance drug effectiveness, thus providing an attractive strategy to overcome drug resistance in cancer (2 , 17) . However, only very recently have studies examined the effects of ODN in increasing the sensitivity to chemotherapeutic agents in relevant tumor models (18, 19, 20) . The results of these reports provide promising indications that combinations of Bcl-2 AS ODN treatment with chemotherapy may provide significant improvements in antitumor activity, possibly in a synergistic fashion. Although these results are encouraging, many treatment characteristics such as tumor Bcl-2 expression during therapy, potential PK influences of the treatment combinations, and the effect of tumor characteristics on therapeutic activity have not been elucidated.
The objective of this study was to characterize the molecular and pharmacological effects of G3139 Bcl-2 AS ODN treatment alone and in combination with F-DOX in a Bcl-2-expressing model of human breast cancer grown in SCID-RAG2 mice. Studies to date have not examined potential effects of chronic ODN treatment on the PK properties of coadministered anticancer drugs. Given the high DNA binding avidity of the anticancer drug DOX, possibilities exist for DOX-ODN interactions both in tissues and the circulation. To understand the pharmacological attributes of coadministering these two agents, we examined the PK properties of DOX to better understand mechanisms involved in ODN-mediated drug sensitization. Given that F-DOX has a relatively short plasma half-life, we also encapsulated DOX in small (100 nm) DSPC/CHOL liposomes containing polyethylene glycol (PEG), in which it was predicted that liposome-mediated changes in drug PKs could lead to prolonged circulation times with reduced drug-related toxicities (21, 22, 23) . As a consequence of the circulation longevity, these liposomes can passively localize into solid tumors via the discontinuous capillaries, particularly during active tumor growth and angiogenesis (24) , thereby exposing tumor cells to higher levels of drug. We used these two DOX formulations to investigate possible influences of anticancer drug PKs and tumor delivery characteristics on therapeutic activity when combined with G3139.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ODNs.
Phosphorothioate ODNs (18-mer) with a sequence complementary for the
first six codons of the open reading frame of Bcl-2 mRNA: 5'-tct ccc
agc gtg cgc cat-3' was used as the AS ODN, G3139
to Bcl-2 and was a gift from Genta Inc. (San Diego, CA). A two-base
MM sequence, 5'-tct ccc agc atg tgc
cat-3', and a RP sequence, 5'-tac cgc gtg cga ccc tct-3', were used as
controls (Genta Inc.).
Cell Lines and Animals and Tumor Models.
The human breast cancer cell line MDA435/LCC6 was obtained from Dr. R.
Clarke (Georgetown University, Washington, DC; Ref.
25
). Cells were grown as adherent cultures and maintained
in DMEM supplemented with 10% fetal bovine serum at 37°C and
5% CO2. Cells were used in exponential growth
phase, up to a maximum of 25 in vitro passages. Female
SCID-RAG2 mice (4–6 weeks old, 18–22 g) were obtained from the
British Columbia Cancer Agency Joint Animal Facility breeding
colony and kept in aseptic environments. MDA435/LCC6 cells were
routinely maintained by serial passages of ascites, i.p. in SCID-RAG2
mice. An orthotopic tumor model of MDA435/LCC6 cells in RAG2 mice was
established by bilateral implantation of 2 x
106 in vivo passaged MDA435/LCC6 cells
into the mammary fat pad. All of the animal protocols were approved by
the British Columbia Cancer Agency Animal Welfare Committee.
Preparation of Liposomes.
SLs were composed of
DSPC:CHOL:PEG2000-DSPE (55:45:5 molar ratio). In
some experiments, [3H]CHE was included
as a nonexchangeable, nonmetabolizable lipid tracer (26)
.
Lipids were dissolved in chloroform, dried to a thin lipid film under
high vacuum, and later hydrated by vortex-mixing in 300 mM
citric acid (pH 4.0) to a final lipid concentration of 100 mg/ml. The
resultant multilamellar vesicles were then freeze-thawed in liquid
nitrogen for five cycles of 5 min each, to form large unilamellar
vesicles. The large unilamellar vesicles were then sequentially
extruded (Lipex Extruder; Lipex Biomembranes Inc., Vancouver,
British Columbia, Canada) through a series of polycarbonate filters
(Nucleopore; Pleasanton, CA) of pore sizes from 200 nm down to 100 nm
in diameter, as described previously (27)
. The mean
diameter of liposomes was approximately 100–110 nm measured using
quasielastic light-scattering (QELS Nicomp 270 Submicron Particle
sizer).
DOX was loaded into liposomes (DOX:Lipid, 0.15:1 weight ratio)
exhibiting a pH gradient (interior acidic), generated when the
liposomal external citrate buffer was titrated against 0.5
M Na2CO3
(28)
. SL-DOX was characterized for trapping efficiency
using Sephadex G-50 columns and size distribution (QELS;
Nicomp). The amount of DOX encapsulated was determined
spectrophotometrically at 
= 480 nm.
In Vitro AS Experiments.
Monolayers of MDA435/LCC6 cells in exponential growth phase were grown
in DMEM, supplemented with 10% fetal bovine serum. A 1:1 mixture of
N-N-dioleyl-N,N-dimethylammonium
chloride/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
liposomes were used as transfecting lipids. Lipid and 800
nM ODN (1.3:1, +/- charge ratio) were mixed in
serum-free media and kept on ice for 30 min to form liposome-ODN
complexes. Cells were incubated with the liposome-ODN complexes for two
4-h pulses (once a day) over two consecutive days. At the end of
incubation with ODNs, media was replaced after two washes with Hank’s
buffer. Cell lysates were prepared 48 h after the second pulse of
liposome-ODN treatment.
Western Blot Analysis of Bcl-2 Expression.
Cells or tumor tissue were treated with ice-cold lysis buffer (150
mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS,
2.5 mM EDTA, and 0.1% sodium azide), containing protease
inhibitors (Complete-Mini protease inhibitor tablets; Boehringer
Mannheim GmBH, Mannheim, Germany). After incubation for 30 min
on ice, samples were centrifuged at 14,000 rpm for 15 min and were
stored at -70°C. Protein content in the lysed extracts was
determined using a detergent-compatible Bio-Rad assay (Bio-Rad Labs,
Hercules, CA). Equal amounts of protein (20 µg/lane) were subjected
to 12.5% SDS-PAGE (Bio-Rad) for 45 min at 150 V in a glycine buffer
[1.92 M glycine and 25 mM Tris (pH 8.3)]
containing 1% SDS. Gels were transferred to nitrocellulose membranes
in a glycine transfer buffer with 20% methanol for 1 h at 70 V.
Membranes were blocked overnight at 4°C with 5% skim milk, 0.05%
sodium azide in TBS [20 mM Tris (pH 8.2) and 137
mM sodium chloride]. Mouse monoclonal antibody to
human Bcl-2 (1:3,000; DAKO, Glostrup, Denmark) and mouse
antihuman ß-actin monoclonal antibody (1:10,000; Sigma, St. Louis,
MO) were used diluted in 1% skim milk in TBS containing 0.05% Tween
20 and 0.05% sodium azide. The membranes were then incubated
for 1 h with 1:3,000 horseradish peroxidase-conjugated antimouse
IgG (Promega, Madison, WI). Proteins were detected by using an enhanced
chemiluminescence method (ECL; Amersham Pharmacia Biotech,
Buckinghamshire, England) and visualized after exposure to Kodak
film. Scanning densitometry (Molecular Dynamics, Sunnyvale, CA) was
performed to quantify band intensities by volume/area integration. The
amount of Bcl-2 protein in cells/tumor was normalized to their
ß-actin levels.
PK and Tissue Distribution.
Female RAG2 mice bearing MDA435/LCC6 tumors (0.1–0.15 g) were injected
i.p. with G3139 (5 mg/kg) on days 1–4. On day 4, 3 h after ODN
treatment, the mice were injected with a single i.v. bolus dose of 5
mg/kg of either F-DOX or SL-DOX (labeled with
[3H]CHE). After DOX dosing, mice (three/group)
were killed by CO2 asphyxiation at selected times
over 24 h. Blood was collected via cardiac puncture and placed
into EDTA-coated microtainer tubes. Plasma was isolated from whole
blood by centrifugation at 500 x g for 10 min. Major
organs (i.e., liver, spleen, lung, heart, and kidney),
muscle, and solid tumor were dissected, rinsed in PBS, dried, and
weighed in glass tubes. A 10–50% homogenate of tissues in distilled
water was prepared using a Polytron homogenizer (Kinematica,
Lucerne, Switzerland). Liposomal lipid was analyzed in plasma
and tissue homogenates using Solvable (Packard Bioscience B.V.,
Groningen, the Netherlands), a tissue solubilizer (at 50°C,
overnight) and treating with a cocktail of 200 mM
EDTA, 30% H2O2, and 10
N HCl for 1 h at room temperature. The
amount of radioactivity in samples was determined using scintillation
counting (TRI-CARB Model 1900; Packard Instrumentation, Meriden,
CT). The analysis of DOX and metabolites in tissues was determined
using a HPLC method described previously (29
, 30) .
Briefly, DOX was extracted from plasma or tissue homogenates with
acetonitrile and reconstituted in mobile phase consisting of 16
mM ammonium formate (pH 3.5):acetone:isopropanol,
75:20:5. Samples were run via isocratic elution on a Waters 2690 HPLC
with built-in autosampler (Milford, MA), C18 guard column (Waters),
Nova-Pak C18 analytical column (Waters), and Waters 474 fluorescent
detector. Concentration of drug in various organs was determined from a
previously prepared calibration curve, using background fluorescent
corrections for drug-free organs and appropriate tissue blood
correction factors.
In Vivo Antitumor Activity.
Efficacy experiments were conducted in RAG2 mice bearing MDA435/LCC6
tumors, randomly assigned into 4–6 mice/group. Treatments were
commenced on either day 3 (early treatment) or day 17 (
0.1–0.15-g
tumors) after cell inoculation. Saline (controls), Bcl-2 AS G3139 or RP
ODNs (5 or 10 mg/kg) were administered i.p. daily for five doses a week
over a 1-, 3-, or 6-week regimen. F-DOX or SL-DOX (5 or 10 mg/kg; 1, 3,
or 6 injections once a week) was administered i.v. via the tail
vein, either alone or in conjunction with ODNs. When G3139 was
administered in combination with either F-DOX or SL-DOX, the drug
treatment was staggered 3 h after ODN treatment.
Mice were observed daily and mouse body weights as well as signs of stress (e.g., lethargy, ruffed coat, ataxia, and so forth) were used to detect possible toxicities. Animals with ulcerated tumors or >25% loss of body weight were killed. Caliper measurements of tumors were converted into mean tumor weight (g) using the formula: 1/2 [length (cm)] x [width (cm)]2. An average tumor weight per mouse was calculated from the mean of the two bilateral tumors and was used to calculate the group mean tumor weight ± SE (n = 6–12 mice) from at least two independent experiments per group.
Tumor and Tissue Processing.
Mouse tissues/tumors were collected at selected times and fixed in
paraformaldehyde. Paraffin-embedded tissues were sectioned and were
subjected to gross histopathology using H&E staining. For Western
analysis, tumors were homogenized in lysis buffer using a Polytron
homogenizer (Kinematica).
Statistical Analyses.
All of the linear regression was done using Microsoft Excel (Seattle,
WA). Student’s t test was used to measure statistical
significance between two treatment groups. Multiple comparisons were
done using one-way ANOVA, and posttests that compared different
treatment means were done using Bonferroni’s test (Statistica release
4.5; StatSoft Inc., Tulsa, OK). Data were considered
significant for P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Treatments were
initiated 3 days after cell inoculation or when the tumors were
established to a size of 0.1–0.15 g (approximately day 17). G3139 was
administered using a treatment schedule of i.p. injections (1 a day)
given on days 3–7, 10–14, and 17–21. Early treatments with 5
mg/kg Bcl-2 AS G3139 significantly delayed tumor growth: Day-35 tumor
weights were 0.44 g and 0.17 g in control and AS-treated
groups, respectively (Fig. 1A
). Bcl-2 AS G3139 was
administered at a maximum of 10 mg/kg, because higher doses exceeded
the MTD in SCID RAG2 mice, which is approximately 10–15 mg/kg G3139.
Mice that were given 10 mg/kg G3139 exhibited marginal therapeutic
improvements compared with the 5-mg/kg G3139 dose; hence, for most
in vivo treatment regimens, a dose of 5 mg/kg was selected.
Administration of RP Bcl-2 ODN at 5 mg/kg as a control phosphorothioate
ODN provided no therapeutic activity (Fig. 1A
).
Additionally, none of the mice treated with ODNs displayed any signs of
toxicities, and both of the doses were well tolerated. We then
proceeded to examine the effects of G3139 on established (0.1–0.15 g)
tumors (Fig. 1B
). In this case, i.p. injections of G3139
were initiated on day 17 and continued through days 21, 23–27,
and 29–33. In contrast to the early treatment schedules, injection of
G3139 into mice bearing established tumors exerted minimal and
transient (statistically significant only on days 26–30,
P < 0.05) antitumor effects in mice despite prolonged
treatment (Fig. 1B
). Again, no signs of toxicity were noted
in the AS-treatment groups.
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. Mice implanted with established
(0.1–0.15 g) bilateral MDA435/LCC6 tumors were treated with saline,
RP-ODN, or G3139. Lysates from tumor homogenates were electrophoresed,
and membranes were probed with Bcl-2 and actin antibodies. Because
these antibodies were directed against the human proteins, this allowed
for determination of human Bcl-2 levels obtained from tumor cell
specimens without contamination from Bcl-2 derived from mouse cells.
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). Relative Bcl-2 levels were compared using scanning
densitometry after normalization to actin. No significant reduction in
Bcl-2 levels was observed in MDA435/LCC6 cells given either a 2-base
MM ODN or RP ODN treatments (Fig. 2A
, Lanes
2 and 3), and these levels were consistently maintained
at approximately 75–95% of controls (Fig. 2A
, Lane
1). In vitro exposure of MDA435/LCC6 cells to G3139 led
to a reduction in Bcl-2 protein levels by approximately 80–95% (Fig. 2A
, Lane 4) compared with saline-treated controls
(Fig. 2A
, Lane 1), which confirmed the sequence
specificity of Bcl-2 AS G3139 in down-regulating the target protein in
an in vitro culture system.
In tumor samples from mice, systemic administration of G3139 (5 mg/kg)
caused a pronounced down-regulation of Bcl-2 expression within 4 days
of initiating G3139 treatment in which Bcl-2 protein levels were
reduced to approximately 3% (Fig. 2, B–D
) of control
tumors (P < 0.001; Fig. 2C
, Lane 2
versus Lane 1). No down-regulation of Bcl-2
protein was observed with either RP ODN or F-DOX treatments (Fig. 2B
). With the AS treatment, Bcl-2 protein levels returned to
pretreatment levels within 2 days after the final ODN treatment of the
first weekly schedule (Fig. 2, C and D
). Of
particular interest was the observation that the solid tumors were
insensitive to subsequent courses of G3139 treatment. Specifically, no
detectable decrease in Bcl-2 expression was observed in the second or
third weekly course of treatment (Fig. 2, C and
D
).
Histopathology of Tumor Specimens.
To evaluate whether Bcl-2 AS (G3139) administration results in direct
tumor cell kill, we examined the histology of tumors undergoing G3139
treatment (Fig. 3)
. Experiments were
performed in parallel with the Western blot studies for tumor Bcl-2
protein levels to correlate the two features (Fig. 2, B and
C
, versus Fig. 3
). Mice with established
0.1–0.15-g MDA435/LCC6 tumors were given saline, 5 mg/kg G3139, or RP
ODN i.p. on days 17–21, 23–27, and 29–33, or F-DOX (5 mg/kg) on days
20, 27, and 34. Excised tumors were sectioned and stained with H&E.
Tumors of control mice or RP ODN-treated controls were composed of
densely packed tumor cells with varying amounts of vasculature, evenly
surrounded by epithelial and fibrous tissue (Fig. 3A
). F-DOX
treatment resulted in marginal tumor-cell kill (Fig. 3C
).
Despite the small change in tumor volume observed with Bcl-2 AS G3139
treatment (Fig. 1B
), the histology of tumors given Bcl-2
treatment revealed an increased fraction of dead cells (identified by
their amorphous shape and condensed nuclei) reflecting a large
percentage cell kill (Fig. 3, D–F
). Dead tumor cells and
areas of degenerative tissue were observed by day 20, appearing as
loosely arranged cells with the occurrence of vacuolated structures
(Fig. 3D
). By day 23, tumor cells were surrounded by areas
of dead tissue and surrounding stromal cells, creating regions (Fig. 3E
) of tumor growth that, by day 28 (Fig. 3F
) to
day 34 (not shown), developed into isolated tumor pockets among large
areas of dead tissue within the solid tumor. Areas of tumor
cells were heterogeneous throughout the tumor section (both peripheral
and central parts of tumor; Fig. 3, D–F
). In addition,
these tumor pockets were generally devoid of vasculature, although some
blood vessels were present in contiguous areas of necrotic tissue (Fig. 3F
).
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or
SL-DOX (Fig. 5)
in the presence and
absence of G3139. In these experiments, dosages were adjusted so as not
to exceed the MTD, particularly when combined with chemotherapy. A
maximum of 10 mg/kg G3139 combined with 10 mg/kg DOX could be tolerated
by SCID RAG2 mice without showing significant signs of toxicity
(i.e., 25% weight loss). Treatment regimens were initiated
either early, on day 3 after cell inoculation (Fig. 4
and 5)
, or when
tumors weighed approximately 0.1–0.15 g (day 17, Fig. 6
). Fig. 4A
presents the
in vivo efficacy results for 5 mg/kg and 10 mg/kg doses of
F-DOX to MDA435/LCC6 tumor-bearing mice compared with saline controls
in an early-treatment schedule. F-DOX administered at a dose of 5 mg/kg
i.v. caused only minor delays in tumor growth between days 25 and 40,
whereas 10 mg/kg F-DOX induced significant tumor growth suppression
beyond day 40 (Fig. 4A
).
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versus Fig. 4A
and Fig. 3A
). When G3139 and F-DOX doses were
escalated to those approaching their MTDs in RAG2 mice
(i.e., 10 mg/kg G3139 and 10 mg/kg F-DOX), an even more
pronounced delay in tumor progression was observed (Fig. 4B
). However, at these doses, mice displayed signs of
toxicity (12% transient body-weight loss).
To test for possible synergism between the G3139 and F-DOX, we compared
the mean tumor weights of G3139 or RP ODN with or without F-DOX on days
25 and 35 (Fig. 1
and Fig. 4, A and B
). On day
25, no difference in tumor weights were observed between control mice
(0.21 ± 0.03 g), RP ODN treatment (0.19 ± 0.03 g;
90% T/C), and F-DOX treatment (0.17 ± 0.03 g; 81% T/C;
P > 0.05; Fig. 1
and 4A
). G3139 (0.08 ± 0.02 g; 38% T/C) was able to suppress mouse tumor growth
compared with controls, F-DOX, or RP ODN (P < 0.05;
Fig. 1A
versus Fig. 4A
). When
administered in combination, G3139 and F-DOX resulted in day-35 tumor
weights of 0.03 g ± 0.005 (14% T/C), which were significantly
smaller than for equivalent doses of the individual treatments
(P < 0.01). Similarly, day-35 tumor weights for the
combined G3139 + F-DOX treatment group were significantly
(P < 0.01) less than those observed for either
individual treatment (Fig. 4B
versus Fig. 4A
and
1A). We then examined the antitumor effects of SL-DOX on
MDA435/LCC6 tumors when administered in the early therapeutic schedule
in RAG2 mice. Tumors were treated with SL-DOX (5 mg/kg i.v. on days 6,
13, and 20) with or without G3139 (5 mg/kg i.p. on days 3–7, 10–14,
and 17–21; Fig. 5
). SL-DOX alone exhibited antitumor effects and was
able to substantially decrease tumor growth compared with saline
controls (P < 0.01; day 25) and was superior to
equivalent doses of F-DOX (P < 0.01; day 25) using the
same treatment schedule (Fig. 4A
versus Fig. 5
).
Treatments of combined G3139 (5 g/kg) and SL-DOX (5 mg/kg) were
slightly more effective in suppressing tumor growth compared with
SL-DOX up to day 45; however, later-stage tumor growth rates were
similar (Fig. 5)
.
The results above, taken together, indicate that early-treatment
schedules of combined G3139 and SL-DOX did not represent a significant
improvement over G3139 plus F-DOX (Fig. 5
versus
Fig. 4B
). Because the pharmacology of SL-DOX is dependent on the
presence of permeable tumor blood vasculature, we then examined the
efficacy of combined G3139 and SL-DOX on established MDA435/LCC6 tumors
that exhibit an intratumoral capillary system (Fig. 3A
) that
may allow selective extravasation of SL-DOX in tumor sites. RAG2 mice
with 0.1–0.15-g MDA435/LCC6 solid tumors (day 17) were treated with 5
mg/kg Bcl-2 AS G3139 (15 doses over 3 weeks) with or without F-DOX or
SL-DOX (given on days 20, 27, and 34). In this established solid tumor
model with weekly DOX administration, there was no difference in the
tumor growth curves of mice treated with either F-DOX (day-35 tumor
weight, 0.35 ± 0.04 g) or saline controls (day-35 tumor
weight, 0.39 ± 0.05 g; Fig. 6A
), and the
antitumor activity of Bcl-2 AS G3139 (5 mg/kg) alone was marginal
(day-35 tumor weight, 0.27 ± 0.02 g; Fig. 6A
).
Combined Bcl-2 AS G3139 (5 mg/kg) and F-DOX (5 mg/kg) delayed tumor
growth and was superior to equivalent doses of either treatment given
alone (day-35 tumor weight, 0.27 ± 0.03 g; Fig. 6A
). In comparison, SL-DOX (5 mg/kg) alone (day-35 tumor
weight, 0.31 ± 0.04 g; Fig. 6B
versus
Fig. 6A
) was able to delay tumor growth, and this effect was
equivalent to that of G3139 + F-DOX (0.27 ± 0.03 g; Fig. 6A
versus Fig. 6B
). G3139 + SL-DOX (day-35
tumor weight, 0.25 ± 0.03 g) only slightly increased
antitumor activity compared with SL-DOX (day-35 tumor weight, 0.31 ± 0.04 g; Fig. 6B
) and was equivalent to the combined
G3139 + F-DOX treatment (day-35 tumor weight, 0.27 ± 0.03; Fig. 6B
versus Fig. 6A
). The antitumor effect of
combined Bcl-2 and SL-DOX treatment was enhanced when the dose of
SL-DOX was increased to 10 mg/kg (SL-DOX day-35 tumor weight, 0.21 ± 0.01 g, versus SL-DOX + G3139 day-35 tumor weight,
0.12 ± 0.01 g; Fig. 6C
), which indicated
synergistic activity. In addition to tumor growth suppression, G3139 (5
mg/kg) + SL-DOX (10 mg/kg) therapy exhibited transient tumor shrinkage
(Fig. 6)
after the first SL-DOX dose (day 20). However, with all of the
treatments, tumor growth persisted despite concurrent G3139 and/or DOX
treatment after an initial response.
PK and Tissue Distribution Studies.
To understand the mechanisms involved in AS-mediated drug
sensitization, we examined the PK properties of F-DOX and SL-DOX after
G3139 administration. Established MDA435/LCC6 tumors (0.1–0.15 g) in
RAG2 mice were treated with 5 mg/kg G3139 on days 17–20. F-DOX or
SL-DOX (5 mg/kg) was administered on day 20, 3 h after the
injection of G3139, corresponding to a similar dosing schedule used in
the efficacy studies. For comparisons, we also evaluated the PK
characteristics of F-DOX and SL-DOX in the absence of G3139 treatment.
PK profiles of F-DOX and SL-DOX in plasma and tumor with or without
G3139 are illustrated in Fig. 7
, and the
descriptive PK parameters are summarized in Table 1
. F-DOX has a very short residence time
in plasma, as manifested by its short distribution half-life
(t1/2
= 0.06 h), large volume
of distribution (Vd = 2536 ml), and small AUC (2.99
µg·h/ml; see Table 1
). Encapsulation of DOX in liposomes
significantly alters the PK of the encapsulated DOX, characterized by
its long circulation times (t1/2ß =
13 h). The retention of drug in liposomes was confirmed by
monitoring the plasma drug:lipid ratios (data not shown).
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). F-DOX in the presence of G3139 demonstrated a
marginal change in the AUC (3.2 µg·h/ml) and no change in the
distribution t1/2 (0.06 h) and displayed a
slightly lower Vd (36.5 ml), which reflected minor ODN-DOX
interactions in plasma (Table 1)
. In comparison, the plasma drug
profiles of SL-DOX in the absence and presence of G3139 showed similar
drug plasma-time curves with small changes in their descriptive PK
attributes (Fig. 7A
and Table 1
). The AUC of SL-DOX + G3139
was decreased 1.3-fold and the distribution
t1/2 decreased 2-fold, which indicated
relatively small effects on these two parameters. The observed
Vd was close to the plasma volume of mice, which indicated
limited distribution of liposomal drug into tissues, regardless of the
presence of ODN in plasma (Table 1)
.
Examination of the tumor levels of F-DOX and SL-DOX revealed very
interesting tumor-uptake profiles (Fig. 7B
). F-DOX in the
presence of G3139 treatment was found to rapidly distribute into tumors
at levels approximately 2-fold higher than in the absence of G3139
(P < 0.001). The difference in the tumor DOX uptake
properties were AS-specific and not observed with the RP ODN + F-DOX
combination (Fig. 7B
). In contrast, the G3139 effect on
mediating tumor localization of SL-DOX was smaller in comparison;
however, significantly greater levels were observed in the presence of
G3139 beyond 4 h (Fig. 7C
). Furthermore, monitoring
tumor liposomal lipid confirmed that SL-DOX drug accumulation in tumors
was attributable to tumor uptake of liposomal drug rather than drug
release from liposomes in plasma (not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Attempts to achieve therapeutic pro-apoptotic manipulations in tumor cells have focused primarily on the down-regulation of apoptosis antagonists such as Bcl-2 and Bcl-XL using AS ODNs. This is because of the difficulties associated with systemic applications of gene therapy designed to provide gain of functional changes in tumor cells (35) . In contrast, phosphorothioate or mixed chemistry-backbone ODNs are amenable to development as pharmaceutical agents and have been shown to be well tolerated when administered as systemic infusions to humans (14) . In particular, AS ODNs directed against Bcl-2 have been pursued as therapeutic agents, and one such ODN, G3139 (Genta, Inc.) has progressed to the stage of Phase II clinical testing (8) .
Numerous studies have documented the effect of Bcl-2 manipulation using gene or AS ODN therapy on the susceptibility of tumor cells to apoptotic stimuli (36) . Indications of both direct cytotoxicity and chemosensitization with Bcl-2 AS ODNs have been documented in vitro for a range of tumor types (19 , 31 , 37) . Far fewer studies on the biological effects of Bcl-2 AS ODNs in vivo have been reported. Bcl-2 AS ODNs have been shown to induce tumor growth suppression independent of additional chemotherapy in murine and human xenograft solid-tumor models (19 , 31 , 35) . However, in few reports have therapeutic effects been correlated with altered Bcl-2 expression in tumor specimens (19) . Furthermore, pharmacological analyses have not been performed when Bcl-2 AS ODN have been coadministered with anticancer agents. It is important to identify potential drug PK alterations in combined therapies that could complicate resolving the AS-specific chemosensitizing influence of Bcl-2 manipulations.
In the studies described here, we evaluated the in vivo activity of the Bcl-2 AS ODN G3139 in the MDA435/LCC6 human xenograft solid-tumor model. This model was chosen to assess how Bcl-2 manipulations may affect tumor growth/chemosensitivity in a tumor line exhibiting low inherent Bcl-2 expression, because previous preclinical studies have used high Bcl-2 protein-expressing tumors (31) , and we have demonstrated activity in the MDA435/LCC6 line in vitro.4 Also, the investigations here were designed to elucidate both the direct antitumor and chemosensitizing activity of G3139 when combined with the anticancer drug DOX by correlating Bcl-2 expression in tumor specimens and drug PK properties with therapeutic activity.
Recently, AS ODNs containing unmethylated CpG motifs within the ODN
sequence have been shown to be potent immune stimulators (38
, 39)
. To address the possibility that therapeutic activity was
attributable to AS-mRNA interaction and not to immunostimulatory
effects, we compared the efficacy of G3139 against a control Bcl-2 RP
sequence of the same codon length, containing the same nucleotides with
CpG motifs. In our studies, we demonstrated that daily i.p. G3139
treatment administered over 3 weeks was capable of achieving
significant Bcl-2 protein down-regulation in human breast xenograft
solid tumors, and this was associated with antitumor activity. A
maximum activity was obtained at a dose of 5–10 mg/kg per injection.
This effect was specific for the Bcl-2 AS G3139 sequence, and there was
no observable activity with the control Bcl-2 RP ODN. These results
confirm observations by Klasa et al. (40)
that
showed specific efficacy of this G3139 molecule and not Bcl-2 RP
control in perforin-knockout (natural killer-deficient) mice
and argued against possible immunostimulatory action, which is
dependent on functional natural-killer activity
(40)
. The degree of tumor growth suppression was
attenuated when treatment was initiated after the tumors reached a size
of 0.1–0.15 g compared with initiation before palpable tumor
formation. This is an interesting result because Western analysis of
tumor samples (0.1–0.15 g) during the course of G3139 treatment
indicated >90% Bcl-2 protein reductions during the first course of
treatment. Furthermore, the histology of these tumors revealed
significant induction of cell death, which indicated that small changes
in tumor volume could actually reflect a large percentage cell kill
(Fig. 1B
and Fig. 3, D–F
).
The fact that tumor growth suppression, Bcl-2
down-regulation, and histological evidence of cytotoxicity were
transient in the face of continued G3139 treatment is a very
interesting and potentially important observation. Several potential
mechanisms could account for the development of an apparent Bcl-2 AS
ODN-resistant tumor state. These include the selection of tumor cells
that either overexpress Bcl-2 protein after AS ODN treatment or result
from possible mutation in the mRNA message that decreases sensitivity
to the targeted ODNs. Reduced tumor ODN accumulation could
arise from poor access of ODN to tumor cells, particularly those
situated in areas devoid of vasculature (Fig. 3, D–F
).
Alternatively, the PKs and solid tumor uptake of G3139 may be altered
in later treatment courses. At this time, we are unable to resolve what
is responsible for this change in G3139 responsiveness; however, it is
clearly a feature that has significant implications for therapeutic
applications and warrants further investigation.
In addition to the evidence for direct antitumor activity of G3139 against human breast cancer solid-tumor xenografts, we obtained indications that Bcl-2 down-regulation may provide some level of chemosensitization. Synergistic effects of Bcl-2 AS G3139 combined with chemotherapy were observed only under certain conditions, i.e., Bcl-2 AS G3139 and F-DOX in the early-treatment schedule and Bcl-2 AS G3139 and SL-DOX in the larger solid tumors. For example, on day 35 in the early-treatment model, G3139-related tumor-growth inhibition was 48% relative to control tumors, whereas the tumor size was reduced by 22% in mice treated with F-DOX at 5 mg/kg in the absence of G3139. If tumors responded to these two therapies independently when they are coadministered, then one would predict a mean tumor weight of 0.19 g on day 35. This is 2.7-fold larger than the observed tumor weight of 0.07 g, which agrees with the degree of chemosensitization observed in a previous human melanoma xenograft study (19) and supports the presence of chemosensitizing activity. Similar results were obtained for SL-DOX at 10 mg/kg combined with G3139 in the treatment of 0.1–0.15-g size tumors. However, in other experiments performed here, clear evidence of chemosensitization could not be established.
The above results highlight the complexities of resolving issues of chemosensitization when both of the agents used display significant antitumor activity individually. It should be noted that a recent report documented the ability of Bcl-2 down-regulation in a Shionogi murine solid-tumor model to sensitize these tumors to mitoxantrone (35 , 37) . The observers’ identification of chemosensitization was simplified by the lack of direct cytotoxicity of Bcl-2 AS ODNs on this tumor cell line and established sound evidence for Bcl-2 AS ODN-related chemosensitization in solid tumors. Our results at this time cannot unambiguously establish whether Bcl-2 down-regulation with G3139 chemosensitizes MDA435/LCC6 tumors to DOX; however, this would seem to be possible.
We investigated the use of long-circulating formulations of SL-DOX to determine whether increasing the level and duration of tumor drug delivery may improve the effects of combining DOX with G3139-induced Bcl-2 down-regulation. Increased antitumor activity was observed with SL-DOX compared with F-DOX for established (0.1–0.15 g) tumors both in the absence and presence of G3139 coadministration. This enhanced therapy seemed related primarily to the increased activity of the SL-DOX formulation itself, although indications of G3139-induced chemosensitization were obtained for a SL-DOX dose of 10 mg/kg. Given the transient effects of Bcl-2 down-regulation observed by Western analysis, it is possible that chemosensitization effects are masked by the fact that this may occur for only the first week of therapy when Bcl-2 suppression is achieved.
No therapeutic benefit over F-DOX was observed when SL-DOX was administered using the early-treatment schedule (initiated on day-3 after tumor inoculation) either in the absence or presence of G3139. Because the pharmacology of SL-DOX is dependent on the presence of an active tumor neovasculature (22, 23, 24) , the lack of activity of SL-DOX in this early-treatment regimen is most likely related to the fact that the neovasculature through which liposomes mediate the enhanced delivery to solid tumors would not be well established shortly after tumor inoculation compared with the physiological situation of solid tumors in the size range of 0.1–0.15 (24) . This implies that simply increasing the circulation lifetime of the anticancer agent is insufficient to increase antitumor activity when combined with G3139.
One issue regarding combining AS ODN therapy with anticancer drugs for chemosensitization purposes that has not been previously addressed is the potential for PK, metabolic, or tissue distribution interactions between the ODN and coadministered chemotherapy agent. This phenomenon has created complications in interpreting the results of chemosensitization trials that use PGP inhibitors when therapeutic improvements were associated with PK alterations as well as PGP inhibition. Our results here demonstrate that DOX tumor exposure is affected by the presence of G3139 coadministration in the absence of plasma PK alterations. However, in contrast to the results from PGP modulation studies, the PK interactions between G3139 and F-DOX seem to favor selective delivery to tumor tissue. The plasma exposure of F-DOX in mice treated with G3139 was minimally reduced compared with no G3139 treatment, whereas tumor exposure to DOX increased 2- to 3-fold. The reasons for these changes are not fully understood but could be related to enhanced drug permeability of tumor tissue treated with G3139 (based on histology slides), increased tumor uptake of DOX-G3139 complexes that could form in the circulation, and increased binding of DOX to tumor cells arising from G3139-induced DNA lesions that are induced by apoptosis activation. The evidence of chemosensitizing activity by G3139 combined with SL-DOX in established tumors argues in favor of molecular-based chemosensitization because improved therapy was achieved in the absence of significant PK changes. More extensive investigations focusing on this issue will be required to establish the molecular basis and therapeutic implications of such ODN-mediated effects.
In summary, although the Bcl-2 AS ODN G3139 can cause effective reductions in tumor cell Bcl-2 protein levels that lead to increased antitumor activity, its therapeutic utility may depend on various aspects of tumor physiology in addition to the pharmacological properties of the anticancer drug(s) with which it may be combined. Such information may enhance our ability to apply therapies targeted at apoptosis regulation with conventional cytotoxic agents in a manner that will significantly improve the management of cancer with chemotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the National Cancer
Institute of Canada through funds from the Canadian Cancer Society. 
2 To whom requests for reprints should be
addressed, at Department of Advanced Therapeutics, British
Columbia Cancer Research Centre, 601 West 10th Avenue,
Vancouver, British Columbia, V5Z 4E6 Canada. Phone: (604) 877-6098;
Fax: (604) 877-6011; E-mail: lmayer{at}bccancer.bc.ca
3 The abbreviations used are: AS,
antisense; AUC, area(s) under the concentration-time curve; CHOL,
cholesterol; DOX, doxorubicin; F-DOX, free DOX; [3H]CHE,
cholesteryl-[1,2-3H-(N)]-hexadecyl ether;
DSPC, distearoylphosphatidylcholine; DSPE,
distearoylphosphatidyl-ethanolamine; HPLC, high-pressure liquid
chromatography; MM, mismatch; MTD, maximum tolerated dose; ODN,
oligonucleotide; PEG, poly(ethyleneglycol); PK, pharmacokinetic; RP,
reverse polarity; SCID, severely combined immunodeficient; SL,
sterically stabilized liposome; SL-DOX, sterically stabilized liposomal
DOX; % T/C, percent test/control; PGP, P-glycoprotein.
4 K. N. Chi, A. E. Wallis, C. H.
Lee, D. E. Lopes de Menezes, J. Sartor, V. Dragowska, and L. D. Mayer. Differentiation of cytotoxic and chemosensitizing activity of
Bcl-2 AS ODN treatment in human breast cancer cells, submitted for
publication.
Received 1/13/00; revised 4/ 6/00; accepted 4/ 7/00.
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, 
) or SL-DOX (
, •)
at a dose of 5 mg/kg was administered 3 h later, in the
presence (