[CANCER RESEARCH 53, 4595-4602, October 1, 1993]

In Vitro and in Vivo Reversal of Multidrug Resistance by GF120918, an

Acridonecarboxamide Derivative
Fran~;ois Hyafil, ~ Catherine Vergely, Pierre Du Vignaud, and Thierry Grand-Perret
Laboratoires GLAXO, Centre de Recherches, 25 avenue du Quebec, 91951 Les Ulis Cedex, France

ABSTRACT models whereas plasma levels above 1 /.zM result in atrioventricular

blocks in patients (6).
N.{4- [2-(1,2,3,4- tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyi]-phe-
The design of potent M D R inhibitors devoid of other pharmaco-
nyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)
logical activities has thus become a desirable goal to test the M D R
has been selected from a chemical program aimed at identifying an opti-
mized inhibitor of muitidrug resitance (MDR). The potency of GF120918 reversal hypothesis in the clinics. In the course of a chemical program
is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and aimed at identifying potent M D R inhibitors, we have identified a
MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respec- series of acridone carboxamide derivatives (7). We describe here the
tively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 ~M and properties of one of them, GF120918 (Fig. 1).
is half maximally active at 0.02/.tM. The spectrum of drugs sensitized by In vitro, GF120918 is consistently active at concentrations around
GF120918 coincides with those having the classical MDR phenotype. In 20 nM in a series of cellular models for M D R inhibition: sensitization
CHRC5 cells, 0.01-0.1 ~M GF120918 enhances the uptake of [3H]dauno- of MDR cells to cytotoxics, increased anthracycline uptake, and in-
rubicin and blocks the effiux from preloaded cells. It is also shown that hibition of effiux. GF120918 binds to P-glycoprotein as witnessed by
GF120918 is still active several hours after being taken away from the
inhibition of [3H]azidopine photoaffinity labeling.
culture medium showing that it is not, like verapamil, effiuxed rapidly by
In two animal models of MDR, the P388/Dox leukemia and the C26
P-glycoprotein. GF120918 effectively competes with [3H]azidopine for
binding P-glycoprotein, pointing to this transport membrane protein as its colon carcinoma, a single i.v. or p.o. dose of GF120918 restores the
likely site of action. antiproliferative action of doxorubicin.
After i.v. administration to mice, GF120918 penetrates thoroughly vari-
ous organs that have a tissue level/blood level ratio above 10. It is elimi- MATERIALS AND METHODS
nated from organs and blood with a half-time of approximately 2.7 h. It is
well absorbed after p.o. administration. Cell Lines
In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or
p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose The MDR chinese hamster ovary cell line CHRC5 selected in vitro against
(5 mg/kg) of doxorubicin administered 1 h later. A statistically significant colchicine (8, 9) was provided by Dr. V. Ling. It was maintained as anchorage-
effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached dependent monolayers in a-minimum essential medium supplemented with
at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 thymidine, adenosine, 10% fetal bovine serum, 2 mM L-glutamine (Flow), 100
(10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the units/ml penicillin, and 100/xg/ml streptomycin in a humidified atmosphere of
growth of the moderately MDR C26 tumor implanted s.c. as assessed by 95% air and 5% CO2. Cells were passaged into Falcon culture flasks twice/
tumor size at day 19. GF120918 does not modify significantly the distri- week after dissociation with trypsin-EDTA.
bution or the elimination of doxorubicin in mice ruling out the possibility The human breast cancer doxorubicin-selected variant cell line (10) (MCF7/
that the antitumor effects seen might be explained by pharmacokinetic ADR) was prodived by Dr. K. Cowan. It was maintained in RMPI 1640
interactions. supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100
/xg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2. Cells
were split once a week after dissociation with trypsin-EDTA. For photoaffinity
INTRODUCTION radiolabeling experiments, expression of P-glycoprotein was boosted by 2
The M D R 2 phenotype has been well characterized since the mid- weeks of culture in the presence of increasing concentration of rhodamine 123
up to 0.2 mM (11).
1980s: cross-resistance to unrelated classes of cytotoxics (called there-
The ovarian carcinoma doxorubicin-selected cell line (12) (OV1/DXR) was
after M D R cytotoxics) is linked to the increased expression of a cell
obtained from Dr. J. Benard (Institut Gustave Roussy, Villejuif, France) and
surface p u m p n o w known as P-glycoprotein; reversal of M D R can be was cultured as MCF7/ADR.
obtained by treatment with numerous classes of lipophilic positively The C26 colon carcinoma cells line was obtained from B. Chauffert (IN-
charged drugs known as M D R inhibitors (for reviews, see Refs. 1-3). SERM U252, Dijon, France). Cells were maintained in Dulbecco's modified
Later studies have n o w substantiated the importance of the M D R Eagle's medium containing 15% fetal calf serum and split after dissociation by
phenotype in the acquired or intrinsic resistance of human tumors to gentle aspiration.
chemotherapy (4, 5) and raised hopes that addition of M D R inhibitors The P388 and P388/Dox mouse leukemia cell lines were obtained from Dr.
to cytotoxics might improve chemotherapy success rate. Clinical G. Atassi (Institute J. Bordet, Brussels, Belgium). They were maintained for up
Phase I and Phase I/II studies have been thus far disappointing, often to 30-35 passages by weekly i.p. passage in DBA/2 female mice (IFFA
CREDO, l'Arbresle, France). For in vitro sensitivity assays, ascites cells were
because of the limited tolerance of prototype M D R inhibitors by
collected and cultured for up to 30 days in Dulbecco's modified Eagle's
themselves, which precluded attainment of potentially active levels in
medium supplemented with 2 mM glutamine, 100 units/ml penicillin, 100
patients (2, 5). For example, full reversion of M D R by verapamil txg/ml streptomycin, and 10% fetal calf serum.
requires an approximate 10 p~M concentration in most cell culture The PEll7 lymphoblastoid cell line was from Dr. L. Degos (H6pital Saint
Louis, Paris, France). Other cell lines were from the American Type Culture
Received 3/19/93; accepted 7/23/93. Collection.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with Drugs
18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed. For cell culture studies GF120918 solutions were prepared as follows: a 2.5
2The abbreviationsused are: MDR, multidrug resistance; GF120918, N-{4-[2-(1, 2, 3,
4-tetrahydr•-6,7-dimeth•xy-2-is•quin••iny•)-ethy•]-pheny•}-9•••-dihydr•-5-meth•xy-9- mM solution was prepared in methanol/HCl 0.1 N (4:1). This stock solution was
oxo-4-acridine carboxamide; dox, doxorubicin;LDso, 50% lethal dose; IC5o, concentra- diluted 1:1000 in complete culture medium. We have noticed that solutions for
tion producing 50% inhibition. cell culture studies should always be prepared by diluting the stock solution in
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 MDR INHIBITOR GF120918

medium with 10% fetal calf serum containing increasing concentrations of

O
MDR inhibitors. [3H]daunorubicin (85 GBq/mmol) (New England Nuclear)
was added as a 0.05 ml solution in culture medium + serum to a final
concentration of 0.7 t~M.
After a 4-h incubation under culture condition, supernatants were thor-
oughly aspirated and the radioactive material incorporated into cells is eluted
overnight at 4~ by 0.1 ml NaOH, 0.1 M.
I "---~~ (CH2)2~ OMe
H [3H]Daunorubicin Efflux from CHRC5 Cells

Fig. 1. Structure of GF120918. Twenty-four h before the assay 105 cells/well were seeded into 24-well..
culture plates (Falcon). For the assay, cells are loaded with [3H]daunorubicin
medium containing 5% fetal calf serum or equivalent amounts of protein. in 0.5 ml culture medium deprived of glucose and containing 10 mM NaN3 and
GF120918, being hydrophobic, will adsorb to plastic or glass at low concen- 0.28 /~M [3H]daunorubicin with 5% dialysed fetal calf serum with or without
trations in the absence of added protein and dilution in protein free medium can MDR inhibitors.
lead to spurious results. After 2 h of incubation, cells were washed twice rapidly ( - 1 0 s for each
The following drugs were dissolved as GF120918: gramicidin D; colchi- wash) with efflux medium (see below) and 0.5 ml of this medium was added
cine; verapamil hydrochloride; quinidine (all from Sigma). to the well at time 0 of the kinetic. Efflux medium was normal culture medium
The following products were dissolved directly in culture medium: doxo- buffered at pH 7.8 by 10 mM NaHCO3 and 10 mM 4-(2-hydroxyethyl)-l-
rubicin chlorhydrate (Adriblastine; Farmitalia Carlo Erba, Rueil, France); piperazineethanesulfonic acid with or without MDR inhibitors.
amiodarone chlorhydrate (Cordarone, for injection; Sanofi Winthrop, Gentilly, The kinetics of efflux was assayed at 20~ in ambient air. Quantitation of
France); vincristine sulfate (Oncovin; Lilly); vinblastine sulfate (Velbe; Lilly); [3H]daunorubicin effluxed from cells was determined by harvesting the culture
mitoxantrone chlorhydrate (Novantrone; Lederle); etoposide (Vepeside; San- medium at each assay time and replacing it with fresh medium with or without
doz, Rueil, France); aclarubicine chlorhydrate (Aclacinomycine; Roger Bellon, MDR inhibitors.
Neuilly, France); vinorelbine ditartrate (Navelbine; Pierre Fabre, Boulogne, At the end of the kinetics, cells were lyzed in 0.1 M NaOH to assess
France); amsacrine (Amsidine; Parke Davis, Courbevoie); cisplastin (Lilly); remaining intracellular daunorubicin. Total incorporated [3H]daunorubicin was
Fluorouracil (Roche); methotrexate (Roger Bellon); carmustine (BICNU; Bris- calculated from the sum of [3H]daunorubicin effluxed during the assay and that
tol Myers Squibb); bleomycin (Roger Bellon); cyclosporin A (Sandimmun; left in the cells at the end. Results are expressed as the percentage of [3H]-
Sandoz). daunorubicin remaining in cells/total incorporated daunorubicin.
For photoaffinity radiolabeling experiments, GF120918 was dissolved as a
Photoaffinity Radiolabeling of P-glycoprotein with [3H]Azidopine
10 mM stock solution in dimethylsulfoxide and then diluted immediately before
use in 0.2 mM HC1. CHRC5 cell monolayers were washed twice with phosphate-buffered saline,
For in vivo studies, GF120918 is prepared as a 10 mg/ml stock solution of scraped, and centrifuged 5 min at 1500 rpm. The cell pellet was resuspended
the HCI salt in 1,2 propanediol/H20 (3/2, v/v). Immediately before the assay, in 10 mM Tris, 2 mM EDTA, 2 mM ethyleneglycol bis(/3-aminoethyl ether)-
the stock solution is diluted in H20 to the desired concentration. N,N,N',N'-tetraacetic acid (pH 7) and maintained in ice for membrane prepa-
Drug Sensitivity Assays ration. Membrane vesicles were prepared by sonicating the cell suspension
with Vibra Cell VC40 and ASI sound 4 times for 1 s at 20 kHz. Nucleus and
CHRC5 cells were seeded at a density of 104 cells/well in Falcon microtiter cell debris were eliminated by centrigugation at 1500 g 15 min and the
plates. After 24 h, the medium was removed and replaced by 0.1 ml of fresh membrane suspension was pelleted by centrifugation for 30 min at 100 000 g.
medium containing MDR inhibitors. A 0.1-ml volume of 2-fold dilution of This membrane enriched fraction was resuspended in the same buffer and
doxorubicin was added. After 72 h incubation, cell viability was assessed by stored at -80~ with 10% of glycerol.
the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Ten /xl of unlabeled cell membrane suspension (at 0.4 mg of protein/ml)
(Sigma) to a dark blue formazan product (13, 14). Briefly 20/.d of a 5 mg/ml were aliquoted in to each well in a V-shaped 96-well plates. Five /xl of
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in GF120918 were then added to each well. The plate was incubated 25 min at
phosphate-buffered saline were added to each well. After 4 h incubation at 25~ in the dark. Then, 5/zl of tritiated azidopine (1.8 TBq/mmol, Amersham)
37~ medium was aspirated and replaced by 0.1 ml dimethylsulfoxide. After (0.6 p.M in HCI 0.2 mM) were added to each well.
vigorous shaking the quantity of formazan product formed was assessed by its After 25 min of incubation at 25~ in the dark, samples were simultaneously
absorbance at 550 nm on a Dynatech MR 700. The average of duplicate wells irradiated for 2 min at 254 nm at 0~ with a thin layer chromatography-
cas used for the calculation. A LDso for doxorubicin was then derived in the designed UV lamp (Bioblock, Illkirch, France) directly in contact with the
absence or in the presence of various MDR inhibitors. To study the spectrum plate. Samples were solubilized in sodium dodecyl sulfate-polyacrylamide gel
of drugs potentialized by GF120918X, similar assays were run with a variety electrophoresis sample buffer but not heated because of described aggregation
of cytotoxics _ a fixed concentration of 50 nM GF120918. of P-glycoprotein at high temperature even in sample buffer (15). After sepa-
Drug sensitivity assays on OV1/DXR, C26, P388/Dox were carried out as ration on a 7.5% polyacrylamide gel, the gel was treated for fluorography with
for CHRC5 except for a 48 h culture between seeding and test (OV1/DXR) or Amplify (Amersham) and exposed during 3 days onto a photosensitive film
the use of round bottom plates and the introduction of centrifugation steps for (Hyperfilm MP, Amersham). The fluorography was analysed using a Camag
washing procedures for P388/Dox. thin layer chromatography Scanner II densitometer. Western blot identification
For the MCF7/ADR assay, a range of vinblastine concentration was used on of P-glycoprotein was performed using polyclonal rabbit antiserum (mdr Ab-1;
cells seeded at 2 • 104/well. Moreover, viability at the end of the assay was Oncogene Sciences) and enhanced chemiluminescence Western blot detection
assessed by a methylene blue staining technique. Briefly, at the end of 72 h kit (Amersham).
incubation, the supernatants were aspirated, and the cell monolayer was
washed with phosphate-buffered saline and fixed for 15 min with 0.1 ml Animal Models
methylene blue (Sigma) at 0.1% dissolved in distilled H20. The plates were
P388/Dox. Female C57BL/6 • DBA/2 F1 mice (IFFA CREDO) weighing
washed extensively under running water and then dried at 56~ for 15 rain. The
20-25 g were randomized by groups of 10 and acclimated for at least 4 days
dye was eluted by shaking with 0.1 ml of HC10.1 Mand absorbances were read
before the test. On day 0, P388/Dox ascites from DBA/2 mice used for
at 630 nm.
passages were pooled and adjusted to 106 cells/ml and a 0.1 ml inoculum was
Uptake of [3H]Daunorubicin by CHRC5 Cells injected i.p. to each mouse.
GF120918 was administered on day 1 in a 0.1 ml volume and injected i.v.
Four • 104 cells/well were seeded into a 96-well microtiter plate. Twenty- in the retro-orbital vein, or by gavage. One h later, a 5 mg/kg solution of
four h later, the medium was aspirated and replaced by 0.05 ml of culture doxorubicin was injected i.p. under a 0.1-ml volume.
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 MDR INHIBITORGF120918

Mortality was observed daily until day 30. Data were subjected to statistical Table 2 Doxorubicin sensitization by MDR inhibitors in multidrug-resistant Chinese
interpretation using the Kaplan-Meier method, log rank test, by Dr. R. Caillon hamster ovary cells (CHRC5)
(Medical Statistics; Glaxo, Paris, France). MDR inhibitor ECsoa 0ZM)
C26 colon carcinoma cells were resuspended at a concentration of 106 Amiodarone 2.3 __-0.6 (n=33)
cells/ml. On day 0, a 0.1 ml solution was injected s.c. to female BALB/c mice Verapamil 3
weighing 18-20 g. On day 1, 0.1 ml of a GF120918 solution was injected i.p. Quinidine 25
One h later, a 5 mg/kg solution of doxorubicin was injected i.p. under a 0.1-ml CyclosporinA 2
GF120918 0.021 __+0.005(n =33)
volume.
Tumor measurements were made with a caliper twice/week until day 16 to a Drug level which, combined with 5 /zg/ml doxorubicin, results in 50% cell kill of
CHRC5 (72 h exposure); mean +- SD.
19. Tumor volumes were calculated by the formula 1/2 • L • where L is the
larger diameter and l the smaller diameter of the tumor. Results are interpreted Table 3 Sensitization to various cytotoxics by GF120918X
according to the F test.
LDso 0xM)
Pharmaeokineties of GF120918. Female mice (C57BL/6 • DBA/2 F1)
received 10 mg/kg (i.v. route) or 20 mg/kg (p.o. route). At times 0.25, 0.5, 1, CHRC5 MCF7/ADR
2, 4, 6 h, (three) animals were sacrificed. Blood, heart, liver, lungs, brain, and Alone With GF Alone With GF
kidneys were analyzed. For each organ, GF120918 was extracted at pH 9 by Doxorubicin
diethyl ether and assayed by the following high pressure liquid chromatogra- Experiment 1 >500 0.2 15 1.5
phy technique: column PRPI (5/xm; Hamilton) 150 • 3.9 mm, mobile phase: Experiment 2 >500 0.25
borate buffer 0.05 M pH ll.5/acetonitrile (45/55 v/v), flow rate, 0.8 ml/min, Vincrisfine 250 18 9 <0.2
detection by fluorescence, excitation 270 nm, emission 418 nm. The limit of 2 <0.2
detection is 0.01 /~g GF120918/g wet tissue. Etoposide
Pharmacokinetics of Doxorubicin. Male mice (C57BL/6 • DBA/2 F1) Experiment 1 250 80 60 30
received 10 mg/kg doxorubicin by i.p. route with or without pretreatment by 10 Experiment 2 300 180 140 70
Experiment 3 200 150 150 70
mg/kg GF120918 i.v. administered 1 h before doxorubicin. Experiment 4 300 180 180 55
At every time point, 10 reference and 10 treated animals were sacrificed.
Mitoxantrone 6.5 0.55 NDa ND
Blood and the following organs were taken: heart; liver; lungs; and kidneys. Vinorelbine ND ND 2.3 0.2
For each organ doxorubicin was extracted at pH 9 by CHC13 MeOH (4:1) and 2 <0.2
Gramicidin D 40 <0.1 50 3
the amount was measured by the following high pressure liquid chromatog- Colchicine
raphy technique (16): column Novapak C18 (3/xm; Waters) 150 • 3.9 rnm; Experiment 1 30 <1 0.6 <0.05
mobile phase, forrniate buffer (4%) pH = 4.0/acetonitrile (7/3 v/v); flow rate, Experiment 2 0.9 0.06
0.8 ml/min; detection by fluorescence; excitation 485 nm; emission 550 nm. Aclarubicin
The detection limit was 0.05/xg doxombicin/g wet tissue. Experiment 1 2 0.5 ND ND
Experiment 2 1.3 0.25
Cisplastin 78 78 15 12
RESULTS 70 60 30 20
Fluorouracil NA NA 2.7 2.7
Methotrexate 650 >650 0.12 0.1
Sensitization of Various Cell Lines to MDR Cytotoxics 800 >800
Carmustine 650 650 700 700
The paradigm of M D R inhibitors is the sensitization of M D R cell Bleomycin 400 380 4 4
lines to killing by the M D R cytotoxics. This activity was studied in a ND, not done; NA, not active at maximally tested concentration.
three different cell lines: CHRC5 a chinese hamster ovary line selected
for resistance to colchicine and displaying resistance to doxorubicin as Indeed, when the sensitizing effect is measured at a fixed 8.6/~ra (5
a consequence of the M D R phenotype (8, 9); and two human cell lines ~g/ml) doxorubicin concentration, the median effective dose of
selected with doxorubicin, O V 1 / D X R derived from an ovarian carci- GF120918 for sensitization is consistently around 0.02 tXM, 100--1000-
n o m a (12) and M C F 7 / A D R derived from m a m m a r y carcinoma (10). fold more potent than prototype M D R inhibitors (Table 2). There is a
CHRC5 and O V 1 / D X R are fairly resistant to doxorubicin with very wide separation between the sensitizing effect and the intrisic
LDso above 10 /XM but are sensitized 300-fold and nearly 100-fold, toxicity of GF120918 which had an LDso of about 27/.ZM on CHRC5
respectively, w h e n coincubated with GF120918 (Table 1). On both cells.
lines, the sensitizing effect is observed at concentrations as low as To study the M C F 7 / A D R cell line, we turned to vinblastine, another
0.01 /XM and full potentiation is observed at 0.1 /XM. In contrast, the M D R cytotoxic, which gives reliable results. The potency of
sensitizing effect of prototype M D R inhibitors such as verapamil or GF120918 and of other M D R inhibitors is consistent with that ob-
amiodarone (data not shown) is observed only in the supramicromolar served on the two other cell lines (Table 1) and confirms a > 100-fold
range. increased potency of GF120918 relative to prototype inhibitors.
We also checked a number of supposedly M D R negative cell lines:
the sensitivity of parental MCF7 cells to vincristine (LDso, 1/XM) was
Table 1 Sensitization of MDR cell lines by GF120918 and verapamila unchanged by 0.5 /~M GF120918. Similarly, the sensitivity to doxo-
Cell line rubicin of the T-cell line Jurkat (LDso, 80 riM) and of the Epstein-Barr
CHR/C5 MCF7/ADR OV1/DXR virus transformed lymphoblastoid cell line P E l l 7 (LDso, 25 nM) was
not affected by GF120918. A 1.6-fold sensitization of HepG2 cells to
LD5o doxorubicin LD5ovinblastine LD5odoxorubicin
MDR inhibitor (p.M) (riM) (hi,M) doxorubicin (from 80 to 50 /XM) was observed.
None 10.2 280 17.3
Verapamil, 5 /XM 0.18 (56) 4.5 (62) 0.4 (43) Cytotoxies Sensitized by G F 1 2 0 9 1 8
Verapamil, 1 /.~M 2.9 (3.5) 50 (5.6) 1.4 (12)
GF120918, 0.5 /ZM 0.035(290) 1 (280) 0.26 (67) In order to determine the spectrum of activity of GF120918, cyto-
GFI20918, 0.1 /ZM 0.043 (240) 1 (280) 0.23 (75)
GF120918, 0.05 ~M 0.074 (140) 1.5 (190) 0.33 (52) toxicity curves on CHRC5 and M C F 7 / A D R for various cytotoxics
GF120918, 0.02 #M 0.125 (82) 5.5 (51) 0.37 (47)
GF120918, 0.01 /.ZM 0.98 (10) 120 (2.3) 3.9 (4.4) were constructed in the absence or in the presence of 50 nM GF120918
a Numbers in parentheses, sensitization factors (LDso no MDR inhibitor/LDsoMDR (Table 3). M D R cytotoxics only are sensitized by GF120918. Sensi-
inhibitor). tization factors vary according to cytotoxics: it is only 2 to 3 for
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 MDR INHIBITOR GF120918

40000-

~:1000

Fig. 2. Uptake of daunorubicin by CHRC5 cells.

Cells were cultured for 4 h with 0.7 /~M [3H]- m ,
daunorubicin in the absence (MDR inhibitor, 0) or
in the presence of various concentrations of the
following MDR inhibitors: GF120918 ((3); amio-
darone (O); verapamil (U]);quinidine (1). Mean ---
SD (n = 3).

1m

/ / . . . . . . . 1 . . . . . . . . i . . . . . . . . | . . . . . . . . i . . . . . . . . , . . . . . . . . 1
10 100 1000 10000 100000 1000000

MDR INHIBITOR(nM)

vepeside whereas it is > 100 for doxorubicin or gramicidin. In the case Characterization of P-glycoprotein and Inhibition of Azidopine
of vepeside, increasing the concentration of GF120918 to up to 1 /XM Labeling by GF120918
did not lead to a more pronounced potentiation. This reflects the fact
that a given density of P-glycoprotein on the cell membrane affords The dihydropyridine azidopine has been described as a photoaffin-
different degrees of resistance to different cytotoxics. ity ligand for calcium channels and P-glycoprotein (19-21). Photoaf-
finity labeling of cell membranes using radiolabeled azidopine re-
Uptake and Effiux of Daunorubicin vealed one major band at 170 kD in CHRC5 which is identified as
P-glycoprotein by immunoprecipitation and Western blot labeling by
Uptake of Daunorubicin. The MDR phenotype is characterized P-glycoprotein antibodies such as MDR Ab-1 (Fig. 4A) and JSB-1
by a defect in the accumulation of MDR cytotoxics relieved by MDR (data not shown). Dose-ranging experiments indicate that azidopine
inhibitors (1-3). The commercial availability of [3H]daunorubicin labeling is saturable with an apparent Kd of 0.18 ~ 4 for CHRC5
allows to set up a straighforward uptake assay to study the effects of P-glycoprotein (data not shown).
MDR inhibitors on drug uptake by MDR cells. As shown in Fig. 4B, the MDR inhibitor GF120918 can compete
GF120918 enhances up to 4-fold the uptake of [3H]daunorubicin by with azidopine and inhibit radiolabeling: IC5o is observed at 0.16 p~M.
CHRC5 cells and a near maximal effect is reached at 100 nM Similarly, the calcium channel blocker verapamil inhibits azidopine
GF120918 (Fig. 2). In contrast, well above micromolar concentrations labeling but very high concentration is required (IC5o, 45 ~M),
of amiodarone, verapamil, or quinidine are required to enhance to any whereas a cytostatic compound such as vinblastine inhibits the label-
extent the uptake of daunorubicin. ing with an IC5o of 4 p,M (not shown).
Efflux of Daunorubicin. Decreased uptake of daunorubicin in Even if allosteric regulation cannot be totally excluded, these re-
MDR cells is attributed to increased efflux mediated by P-glycopro- suits strongly suggest that GF120918 binds with a high affinity to
tein, whereas influx is not modified.
Inhibition of P-glycoprotein is achieved reversibly by incubation in
glucose-free medium containing azide (see, for example, Ref. 17).
Cells are deprived of ATP which is required for the pumping function z 1 0 0 ~
of P-glycoprotein. This results in enhanced daunorubicin uptake even
I-
in the absence of MDR inhibitors.
When cells are placed again in normal medium allowing the regen-
eration of ATP, very rapid efflux of daunorubicin follows with a <N "
half-life of 5 min (Fig. 3). The addition of MDR inhibitors in the
medium dramatically slows down the efflux of daunorubicin. Once
again GF120918 is active at 20 riM, bringing the half-life of dauno- t;
I',
rnbicin in cells above 4 h. A concentration of verapamil of 10/~M is ~z I .
W~
required for a similar effect (Fig. 3). I k
10 . . . . . i . . . . . ! . . . . . ! ,' . . . . !
Furthermore, if verapamil is present during the uptake phase but 60 120 180 240
omitted from the efflux medium, the efflux is not inhibited. Verapamil TIME OF EFFLUX (min)
has thus a short duration of action in cells probably because it is a Fig. 3. Daunorubicin efflux by CHRC5cells. A 2 h uptake incubation in glucose-free
substrate of P-glycoprotein (18). In contrast, GF120918 is still active medium, NAN3, [3H]daunorubicinprecedes the efflux kinetics in normal medium. The
in inhibiting efflux even if it is omitted in the efflux medium. This following MDR inhibitorswere added: none (• Verapamil, 10 /ZM,during both uptake
and efflux (O); Verapamil, 10/xM,only during uptake (O); GF120918, 20 nM(1) or 50 nM
indicates that GF120918 retained in cells remains in its active form for (A), during both uptake and efflux; GF120918, 20 nM (IS])or 50 nM (A), only during
several hours. uptake.
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 MDR INHIBITOR GF120918

observed in vitro: the LDso for doxorubicin was decreased from 400
molecular weight to 70 nM by GF120918.
(kD)
A Several MDR variants have been derived in various laboratories
GF120918x and we have worked on the doxorubicin-resistant variant (P388/Dox)
Front 46 69 92 200 origin (~M) derived by the NCI. The MDR character of this line is confirmed by
I I I I I I
in vitro experiments showing a 60-fold potentiation by GF120918 of
0 doxorubicin toxicity from 3500 nM to 58 nM.
0.008
Effects of GF120918 i.v. in the P388/Dox. As shown in Fig. 6A
0.06
0.4 the tumor is indeed quite insensitive to the maximally tolerated dose
2.8 of 5 mg/kg doxorubicin. Since the tumor grows i.p. and doxorubicin
2O is administered i.p., there is little rationale in treating i.p. with MDR
0 inhibitors. GF120918 is thus administered i.v., 1 h before doxorubicin
western blot injection. This allows time for distribution of the MDR in organs
including the peritoneal cavity. In these conditions, GF120918 (10

t
P-GLYCOPROTEIN
mg/kg) alone has no effect but, in combination with 5 mg/kg doxo-
rubicin, a dose-related increase in survival time is observed (Fig. 6A).
The model proved to be extremely reproducible in a series of con-
secutive experiments so that the results from these experiments could
be pooled yielding accurate and statistically significant results. A
dose-dependent sensitization by GF120918 is featured in Fig. 6B: the
B effect is minimal at 1 mg/kg; half maximal at 2.5 mg/kg; and maximal
at 5 mg/kg with a 50% increase in the mean survival time and a
o
5-10% proportion of 30-day survivors.
.z3 o 100" --/ It should be pointed out that the above regimen is not intended to
w
maximize the effect of GF120918: a 10 mg/kg bolus at t = -1 h yields
in mice a 0.14/xg/ml GF120918 blood level at the time of doxorubicin
injection (Fig. 5). An infusion regimen would certainly be more rel-
5O evant to the clinical use of GF120918 and yield better results but is not
n_
0 a practical in mice.
P
J~

IB 0 q l .................. , ....... , . . . . . .

0 "():01 0.1 1 10 9
GF120918X (pM)
Fig. 4. GF120918 inhibits P-glycoprotein labeling by [3H]azidopine. A, fluorography
of a typical experiment where membranes are exposed to various concentrations of O1
...,.
GF120918, then to 0.2/xM [3H]azidopine and irradiated. B, mean and SD of 4 independent OI
experiments. P-glycoprotein labeling is quantified by scanning the fluorography. T
d
C
O
U 0.1
~ e
CHRC5 P-glycoprotein on the same site as the dihydropyridine de-
rivative azidopine.
! i i i i i
Pharmacokinetics of GF120918 0.O1
1 2 3 4 5 6

Blood and tissue levels of GF120918 in mice were assessed after a Time(h)
single i.v. (10 mg/kg) or p.o. (20 mg/kg) administration. Fig. 5 depicts
blood levels and heart levels as representative of muscle tissue dis-
tribution. The apparent half-life of elimination from blood is estimated
to be 2.7 h. Blood levels are generally low but levels in other organs
are typically one order of magnitude higher (data not shown for liver,
kidney and lungs). This indicates a thorough distribution in most
organs except brain where levels are close to that in blood (data not
shown).
Blood and tissue levels after p.o. administration indicate a good oral
3
absorption of GF120918. d
C
0
P388 Leukemia U 0.1 e

The P388 leukemia is a classical model for anticancer drugs. The

tumor grows i.p. and induces mouse death roughly 10 days after 0.01 I I , I , t
implantation. This model is sensitive to most classical cytotoxics 0 1 2 3 4 5 6 7

administered by the i.p. route. For example, 1 administration of 2

Time(h)
mg/kg doxorubicin increased mean survival time from 9 to 14 days.
Fig. 5. Tissue distributionof GF120918 in mice.A, i.v. administration(10 mg/kg).
Cotreatment with GF120918 10 mg/kg i.v. further increased it to 17 9 heart; e, blood. B, p.o. administration(20 mg/kg). (3, heart; O, blood. Mean
days. This may be due to a low level MDR of this cell line also + SD; n = 3.
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 MDR INHIBITOR GFI20918

p.o. T r e a t m e n t . GF120918 is p.o. bioavailable in mice (Fig. 5). An 100~

effect in the P388/Dox model is thus expected and is indeed observed
in the dose-ranging study described in Fig. 7. Although no direct
comparisons are made, GF120918 seems to be 2-3 times less potent
by the p.o. than by the i.v. route. Once again, it can be stressed that the
Q
regime (GF120918 1 h before doxorubicin) is in no way optimized, o
T= 60
C26 C o l o n C a r c i n o m a

This mouse cell line has the advantage of being implanted s.c. from ".~
40-
lture ] cells and
cultured ;tnd having,
aavi: lg, as
;is many
n tany colon carcinomas,
ca] cinomas, spontaneously
sponta leot tsly a r
DR character.
MDR q'haract~,'r. In
I~ vitro sensitization
~,ensitization by
blt GF120918
(;F120918 of doxorubicin
do:r .~
cytotoxicity
toto~icity is a good ~;ood measurement
measu:'ement of the :he MDR character: 1001C0 nM 20
F120)18 enhances
GF120918 enlan(:es by l:y about
a])ou: 2-fold the
th~ sensitivity to doxorubicin,
do:~orulficin,
)m 1.1
from 1 1 to 0.5
0.:; ~xm,
~ i, showing
sh)wiJtg a~ moderate MDR M])R that may be present
I~resdmt in
many
any human
t~uman tumors.
um,)rs, os 9 ! 9

s.c. growth of the C26 tumor in syngenic mice is affected little by 10 15 20 25 30

a single
5i gle dose of doxorubicin (5 mg/kg i.p. at day 1) or by GF120918 Days
alone
~] : (10 mg/kg i.p.). In contrast, pretreatment by GF120918 1 h F 7. Oral activity of GF120918 in the P388/Dox model. A single administration of
Fig. 7.
GF120918
3Fl', )18 was administeredon day 1 followedafter 1 h by a single i.p. administrationof
55m~
mg/kg;d,doxorubicin(pool of 2 experiments). Untreated animals (0, n = 20), GF120918
20 mg/kg
~.0[] :g~alone (&, n = 20), doxorubicinalone ((3, n = 20) or associatedwith GF120918
l O O ~ I 2~.m~
mg/kg;(<(O, n = 30, P = 0.6), 6 mg/kg (D, n = 30, P = 0.002), or 20 mg/kg (A, n =
A ;0,1P = 4 10-7).
30, :

before
~ef( ~d doxorubicin consistently decreases tumor growth as assessed at
days 16 to 19 (Fig. 8).
i ~1 ~ ~ I In the
tayI~ he absence of GF120918, a 7.5 mg/kg dose of doxorubicin is not
[6
xiol efficacious
more eft than 5 mg/kg but toxicity becomes limiting (data not
shown). This is an indirect indication that the sensitizing action of
GF120918
3 F 39 is likely to be genuine MDR reversal and not pharmaco-
kinetic enhancement of doxorubicin levels.

Lga(
a c k ootf P h a r m a c o k i n e t i c Interaction

o ,, 9 m--." , 9 , ~We a~adressed in a more direct fashion the issue of modified distri-
5 10 15 20 2S 30 3uti i aand/or elimination of doxorubicin as a possible explanation for
bution
part
parl off tthe sensitizing effect. We verified on B 6 x D 2 mice (the strain
lOO fo P388/Dox experiments) the effects of a pretreatment by
ase, for
used
~F: D9 (10 mg/kg i.v.) on doxorubicin distribution and elimination
GF120918
from
ffoi tissues.
Iis Table 4 shows no apparent difference in the distribution
of
3f doxorubicin
c r in animals pretreated or not with GF120918. Elimina-
.o" riot off cdoxorubicin is very slow with an estimated half-life well above
tion
E 24
24 h. T Thus, no effect on elimination will show up in this protocol.
6 0 ~ i ~ Suc an
Such ar effect is, however, extremely unlikely since GF120918 has
i been
~ee fully
fu eliminated from blood and organs after 24 h (data not
> 40 ~t,~ ~ shown).

DISCUSSION

The first salient feature of GF120918 is its potency as an MDR

inhibitor. Activity was consistantly obtained with median effective
o dose around 20 nM on different MDR cell lines of routine, Chinese
5 10 15 20 25 30 hamster, and human origin and in a variety of cellular assays: sensi-
tization to MDR cytotoxics; enhancement of uptake; and inhibition of
effiux of doxorubicin. In all these assays, GF120918 is about 100-200
Days
times more potent than verapamil and it is also about 2 orders of
Fig. 6. Sensitization of doxorubicin antitumor effect on P388/Dox-bearing mice by
GF120918 administeredi.v. A single dose of GF120918 was administeredon day 1, 1 h magnitude more potent than other MDR inhibitors such as amiodarone
before the single 5 mg/kg i.p. dose of doxorubicin. Results of 8 pooled separate experi- or cyclosporin (Table 2).
ments are presented here. In parentheses are n = number of mice in the group and P =
probability of differencecompared to the doxorubicin alone control.A, untreated animal To demonstrate activity in animal models, other MDR inhibitors
(x, n = 79), doxorubicinalone (O, n = 79), GF120918 20 mg/kg alone (A, n = 69), or had often to be administered in multiple dose regimens (see for
doxorubicin + GF120918 20 mg/kg (D, n = 68, P < 10-18). B, dose ranging of example Refs. 22 and 23). In many cases (e.g., Ref. 23), the MDR
GF120918. Doxorubicinalone (x, n = 79) or cotreatmentwith GF120918 1 mg/kg (O,
n = 39, P = 0.0018), 2.5 mg/kg (A, n = 40, P = 7 10-~2), 5 mg/kg (lS],n = 49, P < inhibitor was administered i.p directly to the site where the tumor is
10-1s), or 10 mg/kg (0, n = 49, P < 10-18). implanted. In the case of GF120918, potency in vitro translates in
4600

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Research.
 MDR INHIBITOR GF120918

2OOO

1800,

1600
OO
1400, OO
OO Oq)
1200 OO 9
OO ~'.;o
IOO0 .~olo oo ese
go ~OO Oo
go 9 9o
8OO oOo
O09 "8
O0
oo
600- 0 ~llo e
go

400- eo oe |~

200-

Doxorubicin(mg/kg)i.p. 0 5 0 5

GF 120918(mg/kg)i.p. 0 0 1 0 1 0
Fig. 8. Effect of GF120918 on the activity of doxorubicin on mice bearing the C26 colon carcinoma. A single treatment was administered on day 1. Individual tumor sizes determined
at day 19 are indicated (O), as well as the mean • SE; P < 0.001 (doxorubicin + GF120918 versus doxorubicin alone).

Table 4 Lack of effect of GF120918 on the tissue distribution and elimination of doxorubicin in micea
Tissue
Time Treatment Heart Liver Lung Kidney Blood
15 min No pretreatment 0.93 -+ 0.18 0.09 -+ 0.01 0.52 +- 0.05 0.52 • 0.06 0.65 _ 0.10
GFI20918X 1.08 --- 0.16 0.11 • 0.01 0.76 • 0.09 0.59 • 0.09 0.35 • 0.05
1h No pretreatment 1.03 --+0.19 0.08 • 0.01 0.48 • 0.04 0.46 • 0.04 0.20 • 0.02
GF120918X 0.99 • 0.16 0.09 • 0.01 0.63 +_0.05 0.58 • 0.06 0.22 + 0.03
4h No pretreatment 0.80 • 0.08 0.07 • 0.00 0.58 • 0.04 0.38 • 0.02 0.15 • 0.01
GF120918X 0.98 • 0.13 0.10 --- 0.01 0.84 • 0.07 0.54 • 0.07 0.17 • 0.02
24 h No pretreatment 0.69 • 0.05 0.07 • 0.01 0.50 • 0.04 0.42 --- 0.03 0.17 • 0.03
GF120918X 0.91 • 0.09 0.10 __-0.01 0.61 • 0.06 0.44 • 0.04 0.17 • 0.03
'~ GF120918 (10 mg/kg; i.v.) is administered 1 h before doxorubicin (10 mg/kg; i.p.). Results are expressed as ~g doxorubicin/g wet tissue; mean --- SE (n = 10).

vivo: a single dose of M D R inhibitor i.v. or p.o. enhances the antitu- e.g.,in the case of H e p G 2 h e p a t o m a or wild type P388, it m a y be due
m o r effects of doxorubicin. This allows dose-ranging studies to be to the slight M D R character of the cell line. Indeed, sensitization by
performed. We have thus devised a very simple m o d e l that should G F 1 2 0 9 1 8 might b e c o m e a sensitive functional assay for the M D R
allow direct c o m p a r i s o n of different classes of M D R inhibitors. T h e character of cell lines.
intrinsic toxicity of verapamil (e.g., at 20 m g / k g i.v.) precluded its Importantly, Ca 2§ antagonist activity is no longer present: no re-
testing at potentially active levels in this system. laxation of rat aorta was observed up to 10 -5 M G F 1 2 0 9 1 8 (data not
B e y o n d potency, specificity of G F 1 2 0 9 1 8 as a classical M D R in- shown).
hibitor can also be stressed. Cross-linking studies d e m o n s t r a t e d its In vivo, potentiation of antitumor drugs can be expected not only
binding to P-glycoprotein and the s p e c t r u m of cellular activities de- through genuine M D R inhibition but also indirectly through pharma-
scribed above all point to P-glycoprotein inhibition as the m o s t likely cokinetic interactions increasing the tissue level of cytotoxics. Al-
m e c h a n i s m of action. However, in contrast to verapamil, G F 1 2 0 9 1 8 is t h o u g h the sensitizing effect of verapamil (26) and cyclosporin A and
probably in itself a poor substrate of P-glycoprotein so that it is not S D Z P S C 833 in animal m o d e l s can at least in part be attributed to
p u m p e d rapidly outside cells and has thus an extended duration of p h a r m a c o k i n e t i c interactions (27), such as explanation is ruled out in
action after w a s h i n g off the extracellular m e d i u m . the case of G F 1 2 0 9 1 8 s h o w i n g that the observed effects witness
G F 1 2 0 9 1 8 was obtained through a chemical p r o g r a m starting f r o m g e n u i n e in v i v o M D R inhibition.
prototype M D R inhibitors such as a m i o d a r o n e and verapamil. C h e m i - In conclusion, the potency and specificity of G F 1 2 0 9 1 8 m a k e this
cal modification w h i c h increased M D R inhibition was retained but it drug a suitable candidate for testing the clinical potential o f M D R
is important to s h o w that other nonselective effects are not also inhibition without interference f r o m other p h a r m a c o l o g i c a l activities.
retained during this i m p r o v e m e n t process. G F 1 2 0 9 1 8 is not by itself
a cytotoxic, with nonspecific toxic effects observed at concentrations
3 log above specific M D R inhibition. This is in contrast to niguldipine ACKNOWLEDGMENTS
w h i c h has both direct cytotoxic and M D R inhibition activities (24).
Special thanks to V. Revelant, A.B. Boullay, R. Tellier, S. de Villantroy, A.
A n o t h e r evidence of specificity is the lack o f sensitization of non- Costaz, and V. Beneton for skillfull technical assistance. We would also like to
M D R drugs and o f M D R - n e g a t i v e cell lines such as wild type M C F 7 thank Dr. P. Pianetti (Chemistry Department) for preparing GF120918, Dr.
and l y m p h o c y t i c cell lines. In contrast, cyclosporin derivatives are A.C. de Gouville for checking Ca 2+ antagonist activiy, Dr. J. M. Linget for
also sensitizers o f n o n - M D R cells, p r e s u m a b l y by nonspecific m e m - helping with the preparation of the manuscript, and I. Couleon for typing the
brane perturbation (25). W h e n sensitization by G F 1 2 0 9 1 8 occurs, manuscript.
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 MDR INHIBITOR GF120918

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In Vitro and in Vivo Reversal of Multidrug Resistance by
GF120918, an Acridonecarboxamide Derivative
François Hyafil, Catherine Vergely, Pierre Du Vignaud, et al.

Cancer Res 1993;53:4595-4602.

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