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In Vivo Antitumor Activity and in Vitro Cytotoxic Properties of Bis[1,2-

bis(diphenylphosphino)ethane]gold(I) Chloride

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In Vivo Antitumor Activity and in Vitro Cytotoxic Properties of
Bis[1,2-bis(diphenylphosphino)ethane]gold(I) Chloride
Susan J. Berners-Price, Christopher K. Mirabelli, Randall K. Johnson, et al.

Cancer Res 1986;46:5486-5493. Published online November 1, 1986.

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Copyright © 1986 American Association for Cancer Research
[CANCER RESEARCH 46, 5486-5493, November 1986]
In Vivo Antitumor Activity and in Vitro Cytotoxic Properties of
Bis[l ,2-bis(diphenylphosphino)ethane]gold(I) Chloride
Susan J. Berners-Price, Christopher K. Mirabelli,1 Randall K. Johnson, Michael R. Mattern, Francis L. McCabe,
Leo F. Faucette, Chiù-MeiSung, Shau-Ming Mong, Peter J. Sadler, and Stanley T. Crooke
Department of Chemistry, Birkbeck College, University of London, Malet Street, London, WC1E 7HX, United Kingdom [S. J. B-P., P. J. S.J, and Department of
Molecular Pharmacology, Smith Kline and French Laboratories, Swedeland, Pennsylvania 19479 [C-M. S., R. K. J., M. R. M., F. L. M., L. F. F.,
S-M. M., C. K. M., S. T. CJ
ABSTRACT
We have previously reported the cytotoxicity and antitumor activity of
bis(diphenylphosphino)ethane (DPPE) and a variety of its transition
metal complexes. During studies of the chemistry of a gold complex of
this group |( Vu( !).(' 'I'l'l )|. it was observed that this complex readily
underwent ring closure on reaction with DPPE to form the tetrahedral
complex |Ati(I>PI'K),|*. Various counterion forms (<•.#., ( 'I ) of this cation
were isolated and were found to exhibit a remarkably high stability in
solution. Evaluation of |Au(DPPE)JCl in mice bearing i.p. P388 leukemia
demonstrated that the compound produced an average of 87% increase
in life span at its maximally tolerated dose (2-3 ¿imol/kg/dayfor 5 days).
Activity was also seen in i.p. M5076 reticulum cell sarcoma (60% increase
in life span) and s.c. mammary adenocarcinoma 16/c. Modest activity
was evident in i.p. B16 melanoma and 1,1210 leukemia. A subline of
P388 leukemia resistant to cisplatin was not cross-resistant to
|Au(Dl'l'l:),|( I. In addition, combination therapy of | \u(I)PPK)..|( I and
cisplatin against i.p. P388 demonstrated an advantage over single-agent
therapy.
in vitro studies of (Au(DPPE)2)Cl showed that the compound: (a) is
o tutuvii-to tumor cell lines; (b) is only minimally inhibited in its cytotoxic
activity by the presence of serum; (c) produces DNA protein cross-links
and DNA strand breaks in cells; and (</)inhibits macromolecular synthe
sis with a preferential inhibitory effect on protein synthesis relative to
DNA and RNA synthesis. "P nuclear magnetic resonance spectroscopy
indicated that the compound is stable in the presence of serum proteins,
thiols, or disulfides and that it reacts with Cu(II) resulting in the forma
tion of a Cu(I)DPPE complex. The results of these in vivo and in vitro
experiments suggest that the contrasting pharmacological profile of
|Au( 1>1TK).|( Ãw̄ith respect to other gold(I) phosphine complexes may
be related to both the kinetic stability of the complex and its stability in
the presence of thiols.
INTRODUCTION
Evidence is emerging to show that many gold(I) phosphine
complexes are potent cytotoxic agents (1). The antiarthritic
agent, auranofin [2,3,4,6-tetra-0-acetyl-l-thio-/3-D-glucopyra-
nosato-S-triethylphosphine gold(I)] (Fig. 1), has been shown to
inhibit human lymphocyte responsiveness both in vivo and in
vitro (2, 3) and has shown in vivo antitumor activity against i.p.
P388 leukemia (4). Extensive evaluation of the in vivo antitumor
activity of auranofin in 15 tumor models revealed that the drug
was active only when administered i.p. against i.p. P388 leu
kemia (5).
In an attempt to identify a gold-containing complex with a
spectrum of in vivo antitumor activity greater than that of
auranofin, we have synthetized a variety of gold complexes and
evaluated them in vitro and in in vivo tumor systems (1). A
series of gold(I) and gold(III) complexes incorporating biden-
tate phosphine ligands were found to be reproducibly more
active than auranofin in P388 leukemia and active as well in
Received 5/23/86; revised 7/31/86; accepted 8/5/86.
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 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Birkbeck College,
University of London, Malet Street, London, WCIE 7HX, United Kingdom.
5486
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Copyright © 1986 American Association for Cancer Research
other tumor models (6).2 In addition, several of the phosphine
ligands (e.g., DPPE3) of these compounds demonstrated anti-
tumor activity but were significantly less potent than the
gold(I) and gold(III) complexes. In the series [(AuCl)2(Ph2P-
(CH2)„PPh2)],antitumor activity was maximized when n = 2
or 3. The complex with a cw-ethylene bridging alkene also was
very active, whereas the /ra/w-ethylene complex was inactive.
Since these active ligands would be able to form stable 5- or 6-
membered rings by chelation, the possibility that a ring-closed
form could be an intermediate in the metabolism of these
complexes was considered. However, gold(I) complexes gener
ally exhibit linear 2-coordinatimi, and 3- or 4-coordinate com
plexes are rare and often unattainable (7-9). They are often
unstable species. During our studies of the chemistry of
[(AuCl)2(DPPE)], we observed, unexpectedly, that this complex
readily underwent ring closure on reaction with DPPE, to form
the tetrahedral complex [Au(DPPE)2]+ (10) (Fig. 1). Complexes
containing this cation were isolated and found to exhibit a
remarkably high stability.
This report describes our evaluation of the antitumor prop
erties, cellular pharmacology, and chemical reactivity of
[Au(DPPE)2]Cl. The results of these studies are discussed in
the context of previously described data for other classes of
gold(I) phosphines in an attempt to correlate their respective
pharmacological actions with their chemical properties.
MATERIALS AND METHODS
Materials. Na[AuCU] was purchased from Johnson Matthey, Ltd.,
and DPPE was from Strem Chemicals. Thiodiglycol and glutathione
(reduced and oxidized) were purchased from Sigma and dried bovine
serum (Wellcomtrol I) was purchased from Wellcome.
NMR. Proton-decoupled 3IP NMR (|'H| "P-NMR) spectra at 24.2
MHz were measured in 10-mm tubes using a JEOL FX60 spectrometer.
When nondeuterated solvents were used, the sample was contained in
an 8 •mm tube inside a 10-mm tube containing D2O for a '!) lock.
Eighty-five % H3PO4 in D2O (85:15, v/v) was used as an external shift
reference.
Preparation of |Au(DPPE)j|Cl. Na[AuCU] 0.5H2O (0.5 g, 1.35 mmol)
was reduced by thiodiglycol (0.33 g, 2.70 mmol) in aqueous acetone
(2.5:1, 7 ml). The solution was cooled to 0-5°Cand added dropwise to
a solution of DPPE (1.07 g, 2.70 mmol) in acetone (about 30 ml). A
clear colorless solution resulted; this was stirred for 30 min; then the
solvent was concentrated to 10 ml by evaporation at room temperature.
The product was obtained as a white to cream-colored solid by addition
of 11•<
) (about 10 ml) and was recrystallized from aqueous methanol
and dried in a vacuum (1.1 g, 79%). Analysis: (CszHsoAuClOP^ C, H,
CI, P.
The complex was characterized by NMR and UV spectroscopy. The
2C. K. Mirabelli, D. T. Hill, L. F. Faucette, F. L. McCabe, G. R. Girard, B.
M. Sutton, and R. K. Johnson. In vivo antitumor activity of analogs of
bis(diphenylphosphino)ethane and their gold(I) coordination complexes, submit
ted for publication.
' The abbreviations used are: DPPE, l,2-bis(diphenylphosphine)ethane; NMR,
nuclear magnetic resonance; GSH, reduced glutathione; GSSG, oxidized gluta
thione; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; MTD, maximally
tolerated dose; ILS, increase in life span.
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I)
CHÃ--0-C-CHj
-0
S-Au-P(CzHg)s
AURANOFIN
H,C-C-0\ 0
0 \0-C-CH,
0-C-CHS
[(AuCI)2(dppe)]
AuCI AuCI
[Au(dppe)2]CI
Fig. I. Structure of gold coordination complexes described in this paper.
molar conductance of the complex in CH3CN is consistent with that of
a 1:1 electrolyte. In the 'H NMR spectrum, the phenyl protons are
slightly shielded with respect to free DPPE, and this presumably arises
as a result of ring-current effects between adjacent aromatic rings in
the tetrahedral complex. The X-ray crystal structure of the chloride
complex has recently been described by Bates and Waters (11). We
have previously reported the X-ray crystal structure of the SbF6~
complex (10), and both have identical 31P NMR chemical shifts in
CDCI3.
[Au(DPPE)2]Cl is a white solid. It is stable to light and heat and is
soluble in a range of solvents, including DMSO, methanol, ethanol,
CHC13, and acetone. Its solubility in H2O is extremely low (<1 ng/ml).
The stability of [Au(DPPE)2]Cl in solution was monitored using 3IP
NMR. No decomposition was observed in any solvent after 24 h in
solution, and the complex was stable for at least 4 days in metha-
nol:saline(l:l, v/v).
Reaction of | \u(l)l'l'K):|( I with GSH, GSSG, Bovine Serum, and
CuSO4. Aliquots of a solution of [Au(DPPE)2]Cl in DMSO were added
to solutions containing 1, 5, 10 and 50 molar equivalents of GSH in
D2O at pH 7 (meter reading) to give final gold concentrations of 10
min in D2O:DMSO (1:1, v/v). ('H) 31PNMR spectra were recorded at
l h and 8 days of reaction time.
One-mi aliquots of GSSG in 0.1 M phosphate buffer (pH 7) were
added to 23 mM solutions of [Au(DPPE)2]Cl in methanol (1 ml) giving
gold:GSSG ratios of 2:1, 1:1, and 1:5. ('H) 31P NMR spectra were
recorded after 24 h and 7 days of reaction time.
Lyophilized bovine serum was reconstituted with D2O. Fifty n\ of a
DMSO solution of [Au(DPPE)2]Cl (1.03 mg) were added with gentle
shaking to 1 ml of the serum to give a final drug concentration of 1
mM with 5% (v/v) DMSO present. The ('H) 3IP NMR spectrum was
recorded after an average reaction time of 12 h.
CuSO4-5H2O (5.0 mg, 0.02 mmol) in D2O (0.5 ml) was added to a
solution of |Au(1)1M'I ),|(1 (21.0 mg, 0.02 mmol) in methanol (1 ml).
The solution became cloudy and after stirring for 30 min a thick white
precipitate had formed. This was removed by centrifugation, washed
thoroughly with 11•(>,
and dried in a vacuum. A small amount of the
solid was digested in concentrated HNO3 and the [Au]:[Cu] ratio was take into macromolecules was measured in parallel with cells incubated
determined by atomic absorption. The remainder was dissolved in
5487
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Copyright © 1986 American Association for Cancer Research
CHLORIDE
CDC13. The ('H) 3IP NMR spectra of both the supernatant and the
precipitate were recorded.
Evaluation in i.p. P388 Leukemia. P388 leukemia cells (10") main
tained by a serial transplantation in syngeneic DBA/2 mice were
inoculated i.p. in C57BL/6 x DBA/2 F, (hereafter called B6D2F,)
mice. Twenty-four h later, if the tumor inoculum proved to be free of
bacterial contamination (as determined by 24-h incubation in thiogly-
colate broth), animals were randomized into groups of 6 and housed in
shoe box cages. [Au(DPPE)2]Cl was dissolved in 95% ethanol. Water
was added in sufficient quantity such that the desired dose was delivered
in 0.5 ml. The final concentration of ethanol was «10%.Formulation
were prepared immediately prior to injection. The drug was adminis
tered i.p. on Days 1 through 5 (i.e., treatment was initiated 24 h after
tumor inoculation). Each experiment includes 3 groups of 6 animals as
untreated controls and animals treated with a postive control, cisplatin
(Sigma Chemical Co., St. Louis, MO), at 2 dose levels.
Other in Vivo Tumor Models. Other murine tumor models were
maintained by serial transplantation in syngeneic mice and were im
planted i.p. and/or s.c. for evaluation of drug activity. Tumor models
included: M5076 reticulum cell sarcoma, B16 melanoma, and Lewis
lung carcinoma (maintained in C57BL/6 mice and evaluated in B6D2F,
mice); Madison 109 lung carcinoma and colon carcinoma 26 (main
tained and evaluated in BALB/c mice); mammary adenocarcinomas
16/c and 13/c (maintained and evaluated in C3H mice); LI210 leuke
mia and a cisplatin-resistant subline of P388 leukemia (maintained in
DBA/2 mice and evaluated in B6D2F, mice). All tumors were obtained
from the National Cancer Institute tumor bank at Frederick Cancer
Research Center, Frederick, MD.
For evaluation in the slower growing solid tumor models,
[Au(DPPE)2]Cl was administered i.p. daily for 10 days beginning 1 day
after tumor implantation. The drug was administered in each tumor
model at 5 logarithmically spaced dosage levels which encompassed the
maximally tolerated dose. Activity was assessed by inhibition of tumor
growth at the site of implant for s.c. tumors and prolongation of median
life span for i.p. tumors. Tumor volume was determined when tumors
in untreated control mice averaged about 1000 mm3 (2 to 3 weeks after
inoculation). Tumor volume was estimated by measurement of perpen
dicular diameters with vernier calipers using the equation of 0.5 x
length x (width)2.
Cell Culture Techniques. B16 melanoma cells were maintained as
monolayer cultures in minimal essential medium and P388 and 1.1210
leukemia cells were maintained in RPMI 1640 as suspension cultures.
All cell lines were grown in medium supplemented with 10% calf serum,
penicillin (100 units/ml), and streptomycin (100 /¿g/ml)in a 5% ('<>.•-
humidified incubator at 37°C.All media and calf sera were purchased
from Grand Island Biological Co., Grand Island, NY.
In Vitro Cytotoxicity Assays. Monolayer clonogenic assays were
performed using asynchronous populations of B16 cells as described
previously (5). The in vitro chemosensitivity of asynchronous popula
tions of P388 cells was determined in a soft agar colony-forming assay
in 0.17% agarose as described by Metcalfe et al. (12).
Inhibition of Macromolecular Synthesis. B16 cells were harvested and
transferred into 96-well, flat-bottomed microtiter plates at a cell con
centration of 5 x IO4 cells/well in 10(1 //I of medium. Following
overnight incubation to allow attachment of the cells, specified concen
trations of the test compound and/or 25 n\ of [3H]thymidine (80 /xCi/
ml), [3H]uridine (80 nCi/ml), or [3H]leucine (160 /zCi/ml) were added
to the appropriate wells, and the plates were returned to the incubator
for specified time periods (5 through 90 min). The medium was then
aspirated, and each well was washed once with 200 >i\of 0.15 M NaCl.
Cells were lysed with 25 n\ of 0.2 M NaOH for 20 min at room
temperature, followed by the addition of 100 *ilof 15% TCA. After 1 h
at 4"( '. the T< A precipitated material was transferred to glass micro-
fiber Illters using a multisample cell harvester and 5% TCA. The filters
were dried and placed into scintillation vials with 2 ml of Econofluor
(New England Nuclear, Boston, MA), and the incorporation of 3H
precursors into TCA-precipitable materials was measured in a Beckman
LS 9800 counter (Beckman Instruments). Inhibition of precursor up
in the absence of drug.
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I) CHLORIDE

Alkaline Elution Assay. The DNA in exponentially growing 1,1210 average of 87% ILS over 21 separate dose-response studies.
cells was radioactively labeled by incubation with [2-14C]-or [methyi-
•'H]thymidine(50 mCi/mmol and 73 Ci/mmol, respectively; New Eng This prolongation in life span at the maximally tolerated dose
land Nuclear) for 16-20 h at 37°C.14Clabeling was with 0.02 MCi/ml corresponds to a net cell kill of approximately 2 logs; i.e., at
and 3H labeling (internal standard cells) was with <0.1 »iCi/ml. the end of the 5-day course of treatment, the tumor cell burden
Unincorporated radiolabel was removed by centrifugation prior to
was reduced by 99% compared to the tumor cell burden at the
drug treatment or cell irradiation. Cells were incubated at 37°Cin drug- start of therapy.
free medium for at least 60 min before this medium was replaced with The influence of the route of administration of [Au(DPPE)2]-
37°Cmedium containing [Au(DPPE)2]Cl. Following the time period Cl on its activity against i.p. P388 leukemia was evaluated. The
allotted for drug exposure, cells were then centrifuged and washed with complex was less toxic when given i.V., s.c., or p.o. but was
phosphate-buffered saline at 0-4°C. In experiments which measured
inactive in this tumor system by these three routes of adminis
reversibility of drug effect, treated cells were centrifuged, washed, and tration (data not shown). [Au(DPPE)2]Cl was also inactive
resuspended in 37°Cdrug-free growth medium and incubated for 30
against systemic P388 leukemia (i.v. inoculated tumor) when
min. DNA single-strand breaks and DNA-protein cross-links were
the compound was administered either i.p. or i.v. (data not
measured by DNA filter elution assays as described by Kohn et al. ( 13).
Briefly, for measurement of DNA single-strand breaks 5 x IO5treated shown).
or control [l4C]thymidine-labeled cells were mixed with approximately The activity of [Au(DPPE)2]Cl against i.p. implanted M5076
equal numbers of }H-labeled cells that had been irradiated with 300 reticulum sarcoma is shown in Fig. 3. At a MTD of 1.9 ¿¿mol/
rads. The cells were deposited onto polycarbonate membrane filters (2- kg/day, [Au(DPPE)2]Cl administered i.p. daily for 10 days
¿impore diameter, Nucleopore Corp.) and lysed with 0.1 M glycine- produced 59% ILS. As in P388 leukemia, there was a dose-
0.025 M d¡M u!iuni EDTA-2% sodium dodecyl sulfate, pH 10, with or dependent prolongation of life span. [Au(DPPE)2]Cl showed
without proteinase K (0.5 mg/ml; Sigma). Elution was carried out with modest activity against B16 melanoma with an average of 39%
tetrapropylammonium hydroxide-EDTA-0.1% sodium dodecyl sulfate, ILS at the MTD in three dose-response studies (data not
pH 12.1, at 0.04 ml/min. Fractions were collected every 3 h for a total shown). [Au(DPPE)2]Cl also exhibited significant activity
of 18 h. Single-strand break frequency and percentage of break resealing
against the s.c. implanted tumor, mammary adenocarcinoma
were calculated as described previously (13). Results were expressed as
"rad equivalents" of DNA strand breakdage. The amount of "protein- 16/c, when administered either i.p. or i.v. on an intermittent
concealed" DNA strand breakage was determined in experiments in treatment schedule (Table 1). The compound was less toxic to
which duplicate drug-treated cultures were assayed, one with and the mice when given i.v., and the greater inhibition of tumor growth
other without proteinase K in the lysis solution. To measure DNA- by this route may be the result of the higher dose levels. The
protein cross-linking treated or control cells were 7-irradiated at ice compound also was evaluated in a series of other transplantable
temperature with 3000 rads and then deposited onto 2 nM polyvinyl in vivo murine tumor models. The resulting overall spectrum
chloride filters (Millipore Corp.) and lysed with a solution consisting of in vivo activity of [Au(DPPE)2]Cl is summarized in Table 2.
of 2 M NaCl, 0.2% Sarkosyl, and 0.04 M disodium EDTA, pH 10 (5
ml). DNA was eluted, following a wash with 0.04 M EDTA, pH 10,
with tetrapropylammonium hydroxide-EDTA, pH 12.1, at 0.04 ml/ 60
min. Fractions were collected as described above, and the DNA-protein
80
cross-linking frequency was calculated according to the procedure de
UJ
scribed by Kohn (13). The data were expressed in rad equivalents of 40-
DNA-protein cross-linking for comparison with the amount of DNA I 40
single-strand breakage, and the values obtained for untreated control m
t»
cells were subtracted from those obtained for the treated samples. U)
m
20
RESULTS
In Vivo Antitumor Activity. The activity of [Au(DPPE)2]Cl ¡
against ip P388 leukemia in mice is shown in Fig. 2. A MTD 048 095 191 382 7.64
of 2.9 jiinol/kg, administered i.p. daily for 5 days, produced an DOSE (>jmol/kg/doy, ip., Days I-IO)
Fig. 3. Dose-response curve for [Au(DPPE)j]CI in i.p. M5076 reticulum celi
24 sarcoma. Each point is the average of median survival times at a particular dose
in 3 experiments. Bars, SD.
3)
20 - R
801 Table 1 Activity of(Au(DPPE)JCl against mammary adenocarcinoma 16/c
implanted s.c.
UJ
16
60 â„¢
5 Optimal Mean tumor
4O o Route of dose volume* Inhibition
Compound administration" (fimol/kg) (mm3) (%) NP'
12
Experiment
1ControlCisplatin
tr ±99930
n
(ft i.p.[Au(DPPE)2]CI ±6496
m i.p.Experiment 14311±
co
o
g l 2ControlCisplatin
13 ±626097921001/246/84/70/238/8
i.p.208201187
O 036 072 14 29 57
DOSE (pmol/Kg/day, i p.Days 1-5) [Au(DPPE)2]Cl 6 430 ±291 61 0/8
Fig. 2. Activity of [Au(DPPE)2]Cl in mice bearing i.p. P388 leukemia. Tumor- 11 181 ±163 84 2/8
bearing mice were treated with drug on Days 1 and 5 with a single daily dose i.p. * All dosages were given every 4 days for 5 doses.
Each point is the average of median survival times at a particular dose in 21 *OnDay21,±SD.
experiments. Bars, SD. ' Proportion of mice without palpable tumors on Day 21.

5488
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Copyright © 1986 American Association for Cancer Research
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I) CHLORIDE

Table 2 Spectrum of activity of [Au(DPPE)JCl in transplantable murine tumors
Response of tumor to
[Au(DPPE)2]CI
Tumor model
Table 3 Combination chemotherapy of moderately advanced i.p. P3SS leukemia
cisplatin[Au(DPPE)2]Cl
with [Au(DPPE)JCl and
(%) at following cisplatin doses
Oimol/kg/day)»0510

i.p. tumors 0<mol/kg/day)°00.35

P388 leukemia
M5076 reticulum cell sarcoma
B16 melanoma 25 40 5560 95
LI210 leukemia 0.7 30 45 90
Colon carcinoma 26 1.4 3045e1.04-15
40 50 70 105
BI6 melanoma (FIO subline) 2.95.7IIS" 502.15 604.245 808.390 13516.7C

i.v. tumors " Based on median survival time of groups of 8 mice.

P388 leukemia * Administered i.p. on Days 3 through 7.
' Toxic dose.
s.c. tumors
Mammary adenocarcinoma 16/c
Mammary adenocarcinoma 13/c
ADJ-PC6 plasmacytoma ±
M5076 reticulum cell sarcoma ±
Colon carcinoma 26
Colon carcinoma 07/A
" ++, >50% ILS in i.p. tumors and >90% tumor growth inhibition in s.c.
tumor models; +, >30% ILS in i.p. tumors and >75% tumor growth inhibition
in s.c. tumor models; ±,unreproducible activity; -, <30% ILS in i.p. tumors and
<75% tumor growth inhibition in s.c. tumor models.

A. B.
5OCO \(-.\\\|
200

40i_£>>Co0
g 11i\\\\•H\\\\\
100

80
P388 PJ88
30|O

60

05«20in\-

40
20
1.25 2.5 5 10 20 038 075 1.5 3
[Au(dppe)2Ki
Cisplotin
>jmol/kg,QDx5 >imol/Kq,QDx5
Fig. 4. Dose-response curves for cisplatin (A) and [Au(DPPE)2]Cl (B) in i.p.
P388 and the i.p. cisplalin-resistant P388 tumors, till x 5, daily for 5 days.
The in vivo antitumor activity of [Au(DPPE)2]Cl was also
evaluated in a subline of P388 leukemia which is resistant to
cisplatin. As shown in Fig. 4, this subline is refractory to
cisplatin when compared to the response of the parental tumor
line. However, [Au(DPPE)2]Cl was active against both the
parental and the cisplatin-resistant line of P388 producing
similar increases in life span (85 and 77%, respectively) at the
MTD of 2.9 ixmol/kg.
The antitumor activity of the combination of [Au(DPPE)2]Cl
with cisplatin in an in vivo tumor model was assessed. P388
leukemia was used because of its sensitivity to both classes of
drug. In order to provide more of a therapeutic challenge,
treatment was initiated on Day 3 so that a moderately advanced
tumor was present. Drugs were administered i.p. concurrently,
and nontumorous mice were treated in a parallel with tumor-
bearing mice so that an accurate assessment of the toxicity of
the combinations could be made. The results are summarized
in Table 3. Cisplatin at its MTD of 8.3 /¿mol/kg/day for 5 days
5489
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Copyright © 1986 American Association for Cancer Research
\\
\\
\\
\\T0
\ai
i i
468 10
UM of [Au(dppe).]CI
Fig. 5. Cytotoxic activity of [Au(DPPE)2]CI against B16 (<J) and P388 (H)
cells as measured in the colony formation assays following a 2-h exposure to the
drug. Points, means of 3 assays; bars, SE.
produced 90% ILS. [Au(DPPE)2]Cl was less effective in these
advanced tumor-bearing mice with 45% ILS at its MTD. The
drugs could be combined at their respective maximally tolerated
doses with no lethality. There was a clear-cut advantage of this
combination over cisplatin alone, producing 135% increase in
life span at a nontoxic dose. It was also noted that low-dose
combinations, ineffective individually, were effective in pro
longing life span.
Effect of [Au(DPPE)2]Cl on Tumor Cells in Vitro. The effect
of [Au(DPPE)2]Cl on the clonogenicity of B16 and P388 cells
following a 2-h treatment in vitro is shown in Fig. 5. Both
survival curves were characterized by a shoulder; with subse
quent exponential decreases in cell survival with increases in
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I) CHLORIDE

drug concentration. The concentrations of [Au(DPPE)2]Cl re
quired to inhibit the clonogenic capacity of B16 and P388 cells
by 50% following a 2-h exposure were 2 and 6 MM,respectively.
The cytotoxic potency of [Au(DPPE)2]Cl was not markedly
decreased when cells were incubated with the compound in the
presence of fetal calf serum. The 50% inhibitory concentration
for a 2-h exposure as measured in a clonogenic assay using B16
cells was only 2-fold lower in medium not containing serum as
compared to drug exposure in medium containing 10% fetal
calf serum. This is in contrast to the cytotoxic monophosphine
gold(I) complex, auranofin (5), for which cytotoxic potency was
decreased 10-fold in the presence of serum.
Previous studies have shown that the cytotoxic effect of
auranofin against cells in culture occurs rapidly, is not the result
of direct damage to DNA or chromatin, and does not occur as
a result of preferential inhibition of DNA, RNA, and/or protein
synthesis (5, 14). To assess the nature of cell kill (i.e., acute
versus delayed) produced by [Au(DPPE)2]Cl, the ability of P388
cells to exclude trypan blue as a function of [Au(DPPE)2]Cl
treatment was evaluated and compared to clonogenicity. Fol
lowing a 2-h incubation (under experimental conditions iden
tical to those shown in Fig. 5), there was no increase in the
percentage of cells stained by trypan blue following treatment
with (Au(DPPE)2]Cl as high as 40 MM.Therefore, the concen
tration of [Au(DPPE)2]Cl required to produce acute cell death
is at least 20-fold greater than that required to inhibit the
clonogenic capacity of B16 cells.
The effects of the drug on intracellular chromatin structure
were assessed by alkaline elution. [Au(DPPE)2]Cl produced a
concentration-dependent increase in DNA single-stranded
breaks in L1210 cells treated for l h at 37°C(Fig. 6). The
ability of the cells to repair this DNA damage was assessed by
washing the cells which had been treated with the compound
for 1 hr and subsequently incubating the cells in drug-free
medium. The results indicate that there was a further increase
in the number of DNA single-strand breaks during the incuba
tion in drug-free medium. In addition to the DNA strand
breaks, [Au(DPPE)2]Cl, at low concentrations (5 MM),produced
a significant number of DNA-protein cross-links. However,
using increasing concentrations, DNA-protein cross-links de
creased and only strand breaks were evident.
The effects of [Au(DPPE)2]Cl on the incorporation of 3H-
labeled precursors into DNA, RNA, and protein by B16 mela
noma cells are shown in Fig. 7. The compound inhibited the
uptake of [3H]thymidine, [3H]uridine, and [3HJleucine into
DNA, RNA, and protein, respectively, In H16 cells in both a
concentration- and time-dependent manner. There was a some
what greater effect on protein biosynthesis than on DNA and
RNA biosynthesis by [Au(DPPE)2]Cl. For example, 15 MM
[Au(DPPE)2]Cl essentially shut down leucine incorporation
within 90 min, whereas inhibition of uridine and thymidine
incorporation was less pronounced.
Chemical Reactivity of | Am I)l>l'l ),]( 1 in Model Systems.
Previous work with auranofin and other monophosphine gold
complexes has shown that these compounds are reactive with
serum proteins, cellular proteins, glutathione, and other small
molecular weight thiols (15-18). These interactions are domi
nated by the reactivity of these gold complexes with sulfhydryl
groups contained in the respective biological ligands and these
30 r
20
o.
u
IO
30 60 90
Minutes

40

O 30
x
E 20
o.
IO

30 60 90
Minutes
S
•o
. C
s 30

20

o.
IO

20 40 60 80 100
>jM of [Au(dppe)2] CI
30 60 90
Fig. 6. DNA strand breaks U.I and DNA-protein cross-links (9) produced in
Minutes
LI210 cells treated with increasing concentrations of [Au(DPPE)2]Cl Tor l h at
37*C. In addition, cells were treated with 25 and 50 »AI concentrations of the Fig. 7. Effect of [Au(DPPE)2]Cl on DNA, RNA and protein synthesis in B16
compound for 1 h, and the cells were then washed and resuspended in drug-free melanoma cells. Cells were treated with 0 (•),S (O), IO (D), and 15 (A) ,;\i
medium and incubated at 37*C for I h and strand breaks were measured (A). concentrations of the gold complex and the incorporation of ('HJthymidine (A),
Strand breaks and DNA-protein cross-links were measured by alkaline elution ['Hhiridine (B), and [3H]leucine (Q into TCA-precipitable material was measured
(see "Materials and Methods"). as a function of the duration of drug exposure.
5490
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Copyright © 1986 American Association for Cancer Research
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I)
reactions clearly play an important role in their pharmacologi
cal activities (5, 19). To gain insight into the potential role of
sulfhydryl interactions on the in vitro and in vivo pharmacology
of [Au(DPPE)2]Cl, its reactivity in the presence of thiols, disul-
fides, and serum proteins was investigated using 31P NMR
spectroscopy. The 31P NMR resonance of [Au(DPPE)2]Cl was
unaffected by the addition of GSH after 8 days in solution.
There was no change in the chemical shift (24.1 ppm), and the
resonance remained sharp, even in the presence of a large excess
(50 mol equivalents) of GSH showing that the thiol does not
bind to the complex or displace DPPE from the gold coordi
nation sphere. The bis(triethylphosphine) gold(I) complex
[Au(PEt3)2]Cl also has been reported to be unreactive towards
thiols in model reactions (20), but it does react with disulfides
by reductively cleaving —S—S— bonds with the formation of
Et3PO as the phosphine oxidation product. [Au(DPPE)2]Cl was
found to be unreactive towards GSSG, as judged by NMR even
after 7 days of reaction time.
Incubation of 1 mM [Au(DPPE)2]Cl in bovine serum for 24
h caused no change in the 31P NMR resonances of phosphate
and phospholipids in the serum (Fig. 8). The [Au(DPPE)2]Cl
resonance at 23.9 ppm broadened slightly (AT(i= 15 Hz). The
broadening may be indicative of some binding of the
[Au(DPPE)2]+ cation to certain serum components (.e.g, fatty
acids) either by ion pairing or by hydrophobic interactions with
the phenyl rings of the complex.
A variety of structurally diverse antitumor drugs, e.g., bleo-
mycin (21), doxorubicin (22), 1,10 phenanthroline (23), and
amsacrine (24, 25), are able to interact with and/or form
coordination complexes with transition metal ions. We have
demonstrated that the in vitro cytotoxic activity and in vivo
potency of DPPE are increased in the presence of Cu(II) salts
(26, 27). In addition, in vitro and in vivo evaluation of a series
of DPPE analogues provided evidence for a relationship be
tween the ability of Cu(II) to potentiate the cytotoxic activities
of the DPPE analogues and their having in vivo antitumor
activity (26). In view of these observations, the potential for the
reaction of [Au(DPPE)2]Cl with Cu(II) was examined in vitro.
The reaction of [Au(DPPE)2]Cl with 1 mol equivalent of
CuSO4 in aqueous methanol gave a thick white precipitate. The
31P NMR spectrum of this product, when resolved in chloro
form (Fig. 9C), consisted of two broad resonances at -10.7 and
34.4 ppm. By comparison with our previous NMR studies of
[Au(dppe)2] +
30 10 -10
S/ppm
Fig. 8. ('H) "P NMR spectrum at 24.2 MHz of 1 mM [Au(DPPE)2]CI in
bovine serum after 24 h. The lyophilized serum was reconstituted in IM) before
addition of the compound. /'/.. phospholipids; Pi, inorganic phosphate.
5491
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Copyright © 1986 American Association for Cancer Research
CHLORIDE
Cudldppe
A.
I , I I , I
60 4O 20 -20
8/ppm
Fig. 9. ('H) "P NMR spectra showing the reaction of [Au(DPPE)2]CI with
CuSO4. A, 20 mM [Au(DPPE)2]CI in methanol:D2O (3:1, v/v); B, supernatant
from the reaction of [Au(DPPE)2]Cl with 1 mol equivalent of CuSO4 in metha-
nol:D2O (3:1, v/v); C, the precipitate from this reaction redissolved in CDC13.
The complex [Au(DPPE)2]2* has a 10-membered ring structure containing two
Au(I) ions.'
Cu(I)phosphine complexes (28),4 it seems likely that the major
resonance at —
10.7 ppm corresponds to a Cu(I):DPPE complex.
Indeed, atomic absorption measurements substantiated that the
precipitate contains a substantial amount of copper; the
[Au]:[Cu] ratio in the precipitate was 1:45. The 3IP NMR
spectrum of the water-soluble product(s) of the reaction of
[Au(DPPE)2]Cl with CuSO4 is shown in Fig. 9B. The single
resonance at 39.5 ppm can be assigned to the annular complex
[Au2(DPPE)2]2+ which has an identical chemical shift in
aqueous methanol.5
DISCUSSION
DPPE and a number of closely related diphosphines and gold
complexes thereof have potent cytotoxicity to tumor cells in
culture and have reproducible activity in animal tumor models
(6, 26).2 [Au(DPPE)2]Cl is unique relative to previously de
scribed gold(I) bisphosphines in that it is an unusually stable 4-
coordinate, tetrahedral complex. Tumors sensitive to
[Au(DPPE)2]Cl in vivo include P388 leukemia, M5076 reticu-
lum cell sarcoma, B16 melanoma, and the mammary adenocar-
cinoma 16/c. Activity in the latter system demonstrates that
the compound is active when administered systemically against
a solid tumor.
[Au(DPPE)2]Cl exhibits cytotoxic activity against tumor cells
in vitro at micromolar concentrations. The survival curves (Fig.
5) were characterized by a shoulder at low concentrations, and
exponential cell kill was seen with further increases in drug
concentration. This survival pattern is similar to that produced
by radiomimetic alkylating agents (29) but dissimilar to the
monophasic and exponential survival curves produced by au-
ranofin (5). The lack of immediate toxicity to cells by
[Au(DPPE)2]Cl, as measured by the ability of cells to exclude
trypan blue after l h exposure to the compound at concentra
tions determined to be cytotoxic in the clonogenic assay, sug
gests that the cytotoxic effect of [Au(DPPE)2]Cl may be medi-
4 S. J. Berners-Price and P. J. Sadler. Manuscript in preparation.
* S. J. Berners-Price and P. J. Sadler. Gold(I) complexes with bidentate tertiary
phosphine ligands: formation of annular versus tetrahedral chelated complexes,
submitted for publication.
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I)
ated by interference with the reproductive capacity of the cell.
This result is in contrast with that found for auranofin under
equivalent experimental conditions in which its cytotoxic effects
appeared to be acutely mediated (5).
Incorporation studies demonstrated that [Au(DPPE)2]Cl in
hibited the biosynthesis of DNA, RNA, and protein. However,
whereas the rates of incorporation of the precursors into DNA
and RNA were inhibited leucine incorporation into protein
appeared to be effectively terminated within 30 min of exposure
of cells to 15 fÃ-M [Au(DPPE)2]Cl. Further experiments will be
required to determine if this apparent selective inhibition of
protein synthesis by the compound is the result of an inhibition
of amino acid transport into the cells, to a more direct inhibition
of protein synthesis, or is secondary to other effects of the drug.
The observation that [Au(DPPE)2]Cl produces DNA-protein
cross-links and DNA single-strand breaks in cells suggests that
the effects observed on macromolecular synthesis may be the
result of chromai in damage which leads to inhibition of gene
replication, transcription, and translation. The relative signifi
cance of the DNA-protein cross-links and DNA single-strand
breaks in the cytotoxic effects of [Au(DPPE)2]Cl are not yet
determined; however, the DNA-protein cross-links appeared to
be the more dominant lesion in the cytotoxic concentration
range of the compound with single-strand breaks evident only
at supralethal concentrations of [Au(DPPE)2]Cl.
Gold(I) phosphine complexes generally undergo rapid ligand
exchange reactions so that the 31P NMR spectra of complexes
of type [Au(PR3)n]Cl (n = 2, 3, or 4) in the presence of excess
1'k i consist of a single resonance at ambient temperature.
Solutions usually must be cooled to below — 70°Cfor the
resolution of separate resonances for free and bound phosphine
ligands (30-32). In contrast, we have reported that
[Au(DPPE)2]+ undergoes slow exchange in the NMR time scale
with excess DPPE so that separate resonances are resolved for
free and bound DPPE in the 3IP NMR spectrum at 300°K
(10). Therefore, although 4-coordinate gold(I) complexes are
rare and generally unstable species, tetrahedral gold (I) com
plexes with bidentate chelated phosphine ligands appear to be
more inert toward phosphine exchange than 2- or 3-coordinate
gold(I) complexes containing monodentate ligands. This unu
sual chemical stability of the bis-chelated complex may contrib
ute to the contrasting pharmacological properties compared to
those of the R3PAu Y type of complexes.
These studies have shown that [Au(DPPE)2]Cl does not read
ily undergo substitution reactions with thiols in aqueous media.
This is to be expected in view of the high stability of gold-
phosphorus bonds. The high coordination number of Au(I), 4,
also may introduce considerable activation barriers to exchange.
However, complexes of type R3PAuY, where Y is a good leaving
group, for instance chlorine (20) or 7V-imide (33), readily
undergo substitution reactions with thiols in aqueous media,
and there is fast exchange between free and bound thiol on the
NMR time scale. Reactions between thiols and gold drugs in
vivo are likely to play a role in the mechanism of action of these
drugs in rheumatoid arthritis, perhaps by gold binding to sulfhy-
dryl groups in membranes (18), enzyme active sites (34), or the
metal storage protein metallothionein (35, 36). This notion is
consistent with data measured for auranofin in which the cy
totoxic activity of the drug was reduced 10-fold by the addition
of 10% fetal calf serum during the exposure of the drug to cells
in culture (5). This result is attributed to the rapid binding of
auranofin-derived gold to serum proteins. The effect of 10%
fetal calf serum on the cytotoxic potency of [Au(DPPE)2]Cl was
significantly less than that evidenced for auranofin. This result
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Copyright © 1986 American Association for Cancer Research
CHLORIDE
is consistent with our 3IP NMR studies which demonstrate that
high concentrations of [Au(DPPE)2]Cl are stable in serum,
consistent with the model reactions where no ligand exchange
with thiols or reactions with disulphide linkages were observed.
The complex is unlikely to react directly with other functional
groups on proteins such as methionine SCH3, histidine N, or
aspartic acid and glutamic acid CO2~ groups. Electrostatic
interactions may occur with negatively charged oxyanions and
hydrophobic interactions (of the van der Waals type) with ligand
side chains.
Previous studies from our laboratory have demonstrated that
DPPE has in vitro cytotoxic and in vivo antitumor activity
suggesting that the pharmacological activities of [Au(DPPE)2]-
Cl may be the consequence of the delivery of DPPE to the
appropriate biological targets (26, 27). Phosphines are strong
reducing agents (37), and the reduction of essential cellular
components or the generation of reactive radical species during
phosphine oxidation, perhaps involving redox-active metal ions
such as copper and iron, may play a role in the cytotoxic activity
of DPPE or [Au(DPPE)2]Cl. Indeed, there are data to suggest
that copper is involved in the cytotoxic and antitumor ac
tion of DPPE (26). Our investigation of the reaction of
[Au(DPPE)2]Cl with CuSO4 shows that the complex is reactive
towards Cu(II) ions, and a Cu(I)DPPE complex is one of the
products (Fig. 9). Cu(I) complexes may offer potential as anti-
cancer agents (38), and the chemical and pharmacological eval
uation of this Cu(I)-containing reaction product is currently
under way in our laboratories. In addition, our studies have
shown that [Au(DPPE)2]Cl is more stable in solution than
DPPE alone, which slowly undergoes solvent-dependent autox-
idation to the mono- and bis-phosphine oxides (1-10 days).6 In
toto, these experimental observations led us to propose that the
role of gold in the [Au(DPPE)2]Cl complex may be to protect
the ligand from oxidation in vitro and in vivo prior to its
interactions with extracellular (and/or intracellular) redox com
ponents and critical cellular target(s). This would account for
the increased in vitro and in vivo cytotoxic potency of
[Au(DPPE)2]Cl with respect to DPPE itself. While the identity
of the critical cellular target(s) is unclear, the lipophilic nature
of both DPPE and [Au(DPPE)2]Cl may selectively target these
molecules to cellular membrane organdÃ-es or allow the com
pounds access to intercellular targets.
In conclusion, the tetrahedral complex [Au(DPPE)2)Cl has
demonstrated significant and reproducible antitumor activity in
a broad spectrum of murine tumor models and potent cytotoxic
activity to tumor cells in vitro. In vitro and in vivo studies have
provided evidence which suggests that the cytotoxic mecha
nism^) of this compound are different from those of auranofin
and cisplatin. In addition, the contrasting pharmacological
profile of [Au(DPPE)2]Cl with respect to other gold(I) phos
phine complexes may be related to both the kinetic stability of
the complex and its stability in the presence of thiols.
ACKNOWLEDGMENTS
S. J. B. P. and P. J. S. are grateful to Smith Kline & French Labo
ratories and the Medical Research Council for support. We thank
Susanne Young and Shirley Peterson for their excellent secretarial
assistance in preparing this manuscript and Drs. Judith Hempel and
James McArdle for their helpful discussions.
6 S. J. Bemers-Price and P. J. Sadler, unpublished observations.
 ANTITUMOR ACTIVITY OF BIS(DPPE)GOLD(I)
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