Download A Rationale for Carboplatin Treatment and Abdominal Hyperthermia

(CANCER RESEARCH 52, 1252-1258, March 1, 1992]
A Rationale for Carboplatin Treatment and Abdominal Hyperthermia
Restricted to the Peritoneal Cavity1
in Cancers
Gerrit Los,2 Oskar A. G. Smals, Marianne J. H. van Vugt, Martin van der Vlist, Leo den Engelse, J. Gordon McVie,
and H. M. Pinedo
Divisions of Experimental Therapy [G. L., M. J. H. v. V., M. v. d. V., H. M. P.] and Chemical Carcinogenesis [L. d. E.J, The Netherlands Cancer Institute, 121
Plesmanlaan, 1066 CX Amsterdam, and Department of Radiotherapy, University of Amsterdam, Academic Medical Center, Amsterdam [O. A. G. S.J, The Netherlands,
and Cancer Research Campaign, London, England [J. G. M.J
ABSTRACT
The purpose of this study was to optimize the treatment of cancers
restricted to the peritoneal cavity by combining i.p. chemotherapy with
abdominal hyperthermia. In vitro experiments demonstrated that the
uptake of carboplatin into CC531 tumor cells was increased at tempera
tures higher than 41.5°Cat dose levels of 5 and 50% cell kill. CarboplatinDNA adduct formation and cytotoxicity, however, were already increased
at temperatures of about 40°C,indicating that carboplatin-DNA adduct
formation and consequently cytotoxicity could be enhanced by mild
hyperthermia (temperatures in the range of 39-41.5°C).
CC531 tumor bearing rats were treated i.v. and i.p. with carboplatin
(6.15 mg/kg) in combination with regional hyperthermia of the abdomen
(41.5°Cfor 1 h). The mean temperature was 41.5 ±0.3°C(SD) in the
peritoneal cavity and 40.5 ±0.3°Cin the esophagus. Enhanced platinum
concentrations were found in peritoneal tumors (factor 3) and in kidney,
liver, spleen, and lung (a factor 2 average), after the combined i.v. or i.p.
carboplatin-hyperthermia
treatment. Pharmacokinetic data of ¡.p.
CBDCA combined with hyperthermia demonstrated an increased tumor
exposure for total and ultrafiltered platinum in plasma. The areas under
the concentration x time curve for total platinum at 37°Cand 41.5°C
were 69 and 210 pM/h, respectively; for ultrafiltered platinum these
values were 47 and 173 nM/h. This may have been due to a slower
elimination of platinum from the blood at the higher temperature (f.,.rf
for total platinum 99 and 156 min at 37 and 41.5°C,respectively). The
direct exposure of the tumor via the peritoneal fluid appeared to diminish,
since the area under the curve for total platinum was lower at 41.5°C
than at 37°C(576 MM/hversus 1255 nM/h, respectively).
Our results indicate that the advantage of adding hyperthermia is
caused by an increased drug exposure of the tumor via the circulation.
This was supported by the fact that platinum concentrations in peritoneal
tumors after carboplatin treatment at elevated temperatures were similar
for the i.p. and i.v. routes.
INTRODUCTION
The human peritoneal cavity is a common site of tumor
recurrence of ovarian and gastrointestinal malignancies. After
initial surgery the amount of residual tumor in the peritoneal
cavity affects the final outcome of the treatment (1,2). For this
reason, and from the recognition that tumors growing within a
body cavity are less well supplied by the bloodstream, i.p.
chemotherapy may improve the survival and quality of life (35). Pharmacokinetic modeling has suggested that intracavitary
administration of chemotherapeutic agents by peritoneal di
alysis techniques might result in a significantly greater drug
concentration in the peritoneal cavity than in plasma. This
concentration difference offers a potential advantage in the
treatment of malignancies confined to the peritoneal cavity
(6-8).
Received 9/9/91; accepted 12/16/91.
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.
'This work was supported by Grant NK1 90-19 from the Dutch Cancer
Society (KoningënWilhelmina Fonds).
2To whom all correspondence should be addressed.
cDDP3 is one of the most effective drugs available for i.p.
treatment of ovarian carcinoma (9). Trials of single agent cDDP
or cDDP based combination therapy ¡.p.,in patients with
residual small volume ovarian cancer who had failed to respond
to i.v. cDDP treatment, have demonstrated beneficial clinical
results; 30% complete remissions after single agent cDDP
treatment (10) and 65% clinically free of disease after cDDP
based combination therapy (11). We have previously demon
strated in a rat model that drug penetration from the peritoneal
cavity into a rat peritoneal tumor plays a key role in achieving
better responses (12-14). Absolute concentrations of platinum
in i.p. tumor nodules were always higher after i.p. treatment
than after i.v. treatment with cDDP (15) and the effective
advantage of i.p. chemotherapy with cDDP was accentuated at
the periphery of the tumor (14-16).
CBDCA is a cDDP analogue with a more favorable toxic
profile and an appreciable activity against ovarian cancer (1719). The occurrence of dose limiting side effects such as nephrotoxicity, neurotoxicity, and vomiting associated with i.p.
cDDP treatment, has led to trials of i.p. CBDCA. In these
trials, slower drug elimination from the peritoneal cavity and a
lower protein binding capacity of CBDCA were demonstrated
(20). In spite of these pharmacological advantages, the relative
amount of CBDCA penetrating into peritoneal tumors and
tumor cells was far less than that observed for cDDP (9, 21).
In view of these results, an attempt to improve the penetration
of CBDCA into tumor cells seemed logical. One way to achieve
this goal might be the application of hyperthermia, since heat
increases cell membrane permeability to drugs (22) and mem
brane transport of drugs (23) and alters cellular metabolism
(24). In addition, overall pharmacokinetics may change, with
heat affecting drug metabolism and excretion (25), all leading
to increased antitumor responses (26, 27).
The purpose of this study was to assess the effect of regional
heat on the efficacy of CBDCA in a rat tumor model system,
in an attempt to improve the remission rate of cancers restricted
to the peritoneal cavity.
MATERIALS
AND METHODS
Rats and Drugs. Male WAG/Rij rats (8-12 weeks old; 250-300 g)
were obtained from the animal department of the Netherlands Cancer
Institute. The animals were kept in a temperature controlled room on
a 12 h light- 12-h darkness schedule and fed standard rat chow and tap
water ad libitum. CBDCA was obtained from Bristol Myers (Weesp,
the Netherlands). CBDCA containing vials were stored at room tem
perature.
Tumor Model. The tumor used (CC531) is a well defined transplantable rat colonie adenocarcinoma (28), with a doubling time in vitro of
16 h. The tumor grows s.c., i.p., and in vitro (12-15). In vitro, cells
were cultured under 5% CO2 in 75-cm2 flasks (Falcon, Oxnard, CA)
3The abbreviations used are: cDDP, cisplatin; CBDCA, carboplatin; AUC,
area under the concentration x time curve; PBS, phosphate-buffered saline; i.t.,
intratumoral.
1252
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
i.p. CHEMOTHERAPY
AND REGIONAL HYPERTHERMIA
with Dulbecco's modified Eagle's medium (Irvine, Scotland), contain
ing 10% fetal calf serum (Gibco). Cells were subcultured after reaching
a density of 5 x 106/175 cm2 by trypsinization and replated at a density
of 10* cells/175 cm2.
In Vitro Drug Uptake in Tumor Cells. In each experiment six flasks
were used for each temperature, all containing about 6 x IO6 CCS31
tumor cells. To each flask were added 20 ml of conditioned medium
with 5 i!g (a nontoxic dose) or 200 ^g of CBDCA/ ml (50% inhibitory
concentration). Cells (2 x IO7for each temperature) were incubated for
l h at 37°C,40'C, 41.5'C, or 43°C,all at 5% CO2. After incubation,
the cells were washed with PBS, detached with trypsin, pooled, counted,
and washed twice with PBS. Finally, the cells were suspended in
approximately 1 ml PBS and prepared for platinum determination.
Sensitivity of CCS31 to CBDCA. The sensitivity of CC531 cells to
CBDCA at different temperatures was tested by clonogenic assay.
CC531 cells were harvested as described before and counted. Cells in a
single cell suspension were plated in 6-well tissue culture clusters
(Costar, Cambridge, United Kingdom) at 150 cells/well in conditioned
medium. After 24 h of incubation at 37*C, the cells had attached to the
plates and 150 p\ of a CBDCA solution were added to obtain the final
concentrations described under "Results." The culture clusters were
incubated at 37°C,40°C,41.5°C,or 43°Cfor l h and about 15 min
preheating time. After incubation, cells were washed twice with PBS
and 3 ml of fresh medium were added. All plates were returned to the
incubator and incubated for 7-10 days for the development of colonies.
In contrast to the 24-h incubation period, cells were kept in platinum
containing medium after heat treatment for 7-10 days at 37°Cfor the
continuous CBDCA incubations. Colonies were fixed with ethanol,
stained with crystal violet for 10 min, counted, and related to the
control.
Pharmacokinetic Studies. Pharmacokinetic studies in rats were per
formed by cannulation of the carotid artery. The anesthetized animals
were positioned with their lower part of their body in a thermostatically
controlled water bath at 41.5°C.The temperature in the peritoneal
cavity of the animals steadily increased from normal body temperature
(around 38°C)to 41.5°Cwithin about 30 min. CBDCA (6.15 mg/kg),
dissolved in saline and brought to 41.5°C,was injected i.p. in a volume
of 20 ml. At different time points after i.p. administration, blood
samples of about 0.5 ml were taken from the carotid artery. Clotting
was prevented by injecting heparin (50 lU/ml) into the cannulas. Saline
(1 ml) was injected in the carotid artery at f = 30 min and r = 60 min
to compensate blood loss. Blood plasma was collected and 150 /iI were
ultrafiltered through a membrane filter with a 10-kDa cutoff (Amicon,
Danvers, MA) by centrifugation at 1000 x g for 10 min. Total and
ultrafiltered platinum concentrations were determined in plasma by
flameless atomic absorption spectroscopy after dilution of the samples
in 0.2 M HC1/0.15 M NaCl. The CBDCA clearance from the peritoneal
cavity was studied by sampling the peritoneal fluid at the same time
points as blood. From these data the AUC, ('„,.„
(platinum peak
concentration), /m.»
(time, peak concentration occurs), and tv,ß
(the halflife of platinum) were calculated.
Thermometry. Rats were anesthetized by giving i.m. 0.05 ml (6 mg/
kg) Rompun followed by 50 mg/kg Ketalar 12 min later. Then the rats
were positioned in a thermostatically controlled water bath at 41.5°C
168 h tissues (tumor, liver, kidney, spleen, intestines, and lung) were
collected and prepared for platinum measurements.
Platinum Detection by Flameless Atomic Absorption Spectroscopy. A
model AA40 atomic absorption spectrometer with a GTA 96 graphite
tube atomizer (with Zeeman background correction) from Varian (Vic
toria, Australia) was used for platinum analysis. Platinum concentra
tions were determined in plasma, peritoneal fluid, tumor tissue, tumor
cells, and normal tissues. Sample preparation and flameless atomic
absorption spectroscopy procedure have been described elsewhere (15).
CBDCA-DNA Adduct Formation. CC531 cells were cultured on glass
slides (2.6 x 6 cm) coated with ovalbumin (100 //I 0.5% ovalbumin/
slide) and incubated with CBDCA (2.2 mg/ml) for 2 h at different
temperatures (37°C,38.5°C,40°C,41.5'C, and 43°C).Cells were fixed
by successive incubations in cold (-20°C) 100% methanol (10 min) and
acetone (2 min) and air dried.
The characteristics of the rabbit antiserum NKI-A59 against cDDPmodified calf thymus DNA (platinum/nucleotide ratio, 6.7 x 10~2)
have been described by Terheggen el al. (29). NKI-A59 (applied without
further purification), goat anti-rabbit immunoglobulin (Campro Bene
lux, Elst, the Netherlands) and peroxidase-(rabbit)antiperoxidase com
plex (American Qualex, La Miranda, CA) were used in 1:1800, 1:600,
and 1:3000 dilutions, respectively. All sera were diluted in phosphate
buffer containing 10 mM KH2PO4(pH 7.4), 140 mM NaCl, 10% normal
goat serum, and 0.04% Triton X-100 (BDH, Poole, England). The
immunocytochemical procedure was carried out as described by Ter
heggen et al. (30).
The binding of the antibody NKI-A59 was visualized by a double
peroxidase-antiperoxidase staining. The nuclear staining intensity of
individual nuclei was analyzed and quantified with a Knott (Munich,
Germany) light measuring device with a beam diameter of 5 /un, which
was coupled to a Leitz Orthoplan microscope. Data were analyzed by
an Atari ST computer (Sunnyvale, CA) programmed with a version of
the Histochemical Data Acquisition System [Hidacsys; Microscan,
Leiden, the Netherlands (31)]. The integrated absorbance of a selected
area was expressed in arbitrary units. In each slide the nuclear staining
density of 3 to 4 randomly selected areas, corresponding to 20-40
nuclei each, was measured.
Statistics. Student's t test or the Wilcoxon test were used to study
differences; P < 0.05 was considered to indicate significant differences.
RESULTS
In Vitro Studies on CBDCA. The effect of hyperthermia on
the cellular uptake of CBDCA was studied ¡nCC531 cells. Cells
were incubated with 5 or 200 /¿g/mlCBDCA for l h at 37°C,
40°C,41.5T, or 43°C.The uptake of CBDCA did not increase
significantly when the temperature was raised from 37 to
41.5"C, but at 43°Ca clear increase was observed (Table 1). In
contrast, the cytotoxicity was already increased at a temperature
of 40°(Fig. 1/4). The effect of heat on the CBDCA cytotoxicity
after prolonged exposure to the drug was less pronounced for
temperatures round 40°C(Fig. \B). The cytotoxicity of a 1-h
and CBDCA was administered i.p. in a 0.9% NaCl solution (20 ml). In exposure is probably due to an increased activation of CBDCA
case of i.v. administration, 20 ml 0.9% NaCl were given additionally
at higher temperatures. When CBDCA was given continuously,
into the peritoneal cavity. The temperature in the peritoneal cavity of the effect of activation became less important. Only at 43°Cis
the animal steadily increased from normal body temperature (around
cell kill increased (Fig. \B), probably as a result of the higher
38°C)to 41.5°Cin about 30 min. The duration of the heat treatment
at 41.5"C was 60 min. During treatment, i.p. temperatures were mon
itored every 5 min using copper constant thermocouple probes (IT-18;
Table 1 Platinum concentration in CC53I cells after incubation with CBDCA
(5 and 200 iig/ml)" at various temperatures
diameter, 0.62 mm; Sensortek, Inc.) at three locations in the peritoneal
concentration
cavity (near the bladder, the spleen, and right kidney). The placement
CC/37-C)51.0
cells)5
(ngPt/106
of the three probes was confirmed radiographie. In addition to the
Temperature
temperatures in the peritoneal cavity, the rectal temperature at a
CC)37
jig/ml0.43
»ig/ml23.9
distance of 6 cm into the rectum and the temperature in the esophagus
±0.09
±1.1
were monitored. The latter two were single sensor measurements.
40
0.47 ±0.17
ND
I.I
41.5
Tissue Platinum Concentrations after i.p. Chemotherapy. Rats with
0.55 ±0.19
28.3 ±7.0
1.32.62001.01.22.5
43Pt
1.12 ±0.33200 61.2 + 21Ratio
small solid peritoneal tumors were treated i.p. or i.v. with CBDCA
" Mean of 3 experiments ±SD; ND, not determined.
(6.15 mg/kg) with or without abdominal hyperthermia. After 24 and
1253
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
i.p. CHEMOTHERAPY AND REGIONAL HYPERTHERMIA
intracellular platinum concentration (Table 1).
The CBDCA-DNA adduci formation data seem to confirm
the idea of increased activation (Figs. 2 and 3) because the
staining density, which is a measure for the CBDCA-DNA
adduci formation, increased. The staining density increased
linearly with concentration (Fig. 2) and temperature (Fig. 3).
The difference in adduct specific nuclear staining intensity
between 37°Cand 43°C(Fig. 2) is statistically significant (P <
0.05). The increase in DNA-adduct formation between 37°C
and 40°C,between 37°Cand 41.5°Cand between 37°Cand
43°Cwere significant (P < 0.01, P < 0.001, and P < 0.001,
100
100
*
>
i
0.1
0.01
140
210
CBDCA cone, (uà /mi)
respectively).
Thermometry. During the in vivo hyperthermia experiments,
temperatures in the esophagus, peritoneal cavity, and rectum
were monitored (Fig. 4). The temperature reaches a steady state
after 30 min of heating. The mean temperatures over the 1-h
treatment for the peritoneal cavity, the rectum, and the esoph
agus were 41.5 ±0.1°C,41.2 ±0.2°C,and 40.5 ±0.3°C,
CSDCA cone, lug/ml)
Fig. 1. Clonogenic assay of CC531 cells incubated with CBDCA for 1 h (A)
and continuously (fi) at 37'C (•),40'C (A), 41.5'C (T), and 43'C (•).All curves
were adjusted to the control values at 37'C, 40'C, 41.5°C,and 43°Cand were the
means of five wells. No colonies could be detected any more at a doses of 200
and 5 >ig/m\ at 43'C, after a 1-h and a continuous incubation, respectively. Bars,
SD.
respectively.
Pharmacokinetics of CBDCA. Pharmacokinetics of total and
free platinum in the peritoneal cavity differed between rats
treated with CBDCA alone and rats treated with CBDCA at
elevated temperatures (41.5°C)(Figs. 5 and 6; Table 2). The
M
i5 2
AUC in peritoneal fluid was smaller in rats receiving abdominal
hyperthermia, indicating a faster clearance of CBDCA from the
peritoneal cavity. During the first hours after hyperthermia
treatment the platinum concentration in the peritoneal cavity
declined faster at 41.5°Cthan at 37°C(Figs. 5B and 65). This
is apparent from the half-lives (t</,ß)
of CBDCA in the peritoneal
cavity, which were smaller for rats receiving heat (Table 2). In
contrast with the faster clearance of CBDCA from the perito
neal cavity is the relative increase in ultrafiltered platinum in
,-é
c
C
a
0.5
1.5
CBDCA
2.5
3.5
4.5
44
cone, (mg/ml)
Fig. 2. Correlation between the nuclear staining density [expressed in arbitrary
units (a.u.)] in CC531 cells and the extracelluar CBDCA concentration (0.74,
1.48, 2.22, 3.33 mg/ml) during incubation (2 h) at 37'C (•)and 43'C (•).CC531
cells were stained for CBDCA-DNA adduci formation. Each point represents the
mean of at least 3 independently stained slides with 30 to 40 nuclei each. The
best fitting curve was calculated by linear regression analysis. The correlation
coefficients were 0.97 and 0.98 for treatments at 37'C and 43'C, respectively.
0° 42
38
Both lines differed significantly (P < 0.05) as determined by Wilcoxon.
36
20
2.0
40
60
Time
(min)
80 100
Fig. 4. Temperature measured in the esophagus (•),rectum (T) and peritoneal
cavity (•)of rats plotted against heating time in a water bath of 41.5'C. Data
points represent the mean ±SD (bars) of five measurements.
1.5
i
Table 2 Pharmacokinetic data of i.p. CBDCA treatment in plasma and
peritoneal cavity at 37'C and 41.5'C
1.0
41.5'CParameterAUC
0.6
h)(„,.,
plasma (0-24
(min)C^dM)tv,ß
0.0
37
38.5
40
Temperature!
41.5
o
C)
4*
Fig. 3. Nuclear staining density in CC531 cells after incubation with CBDCA
(0.74 mg/ml) at 37'C, 38.5'C, 40'C, 41.5'C, and 43'C for 2 h. CC531 cells were
stained for CBDCA-DNA adduct formation. Each column represents the mean
±SEM (han) of at least 3 independent stained slides with 30-40 nuclei each.
The increase in DNA-adduct formation between 37°Cand 40'C, between 37°C
and 41.5'C and between 37'C and 43'C is significant (P< 0.01, P< 0.001, and
P< 0.001, respectively), a.u., arbitrary units.
Pt210
±89°170
14*24
±
±5156
13*576
±
Pt173
Pt69
±2760
±62135±21C23
1716
±
±5127
±699
32700
±
±201255
Pt47
460±
±9n±
i70
(min)(60-360
plasma
14963
±
min)AUC
74*58
±
h/tnßp.c. (0-24
10964
±
±80239 204195
±
(min)'(30-360
p.c.
±5*Free
±10C37Total ±35•cFree±52
min)Total
" Mean ±SD; AUC expressed in ^M/h; n = 4 for 37'C and for p.c. at 41.5'C;
n = 3 for plasma at 41.5°C.
'Significant difference between treatment at 37'C and 41.5'C (P < 0.01).
c Significant difference between treatment at 37'C and 41.5'C (P < 0.05).
d p.c., peritoneal cavity.
1254
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
i.p. CHEMOTHERAPY AND REGIONAL HYPERTHERMIA
However, an increase in platinum concentration in peritoneal
tumors by a factor of 3.5 was observed (Fig. 7), while the
increase in other tissues, except for the intestines, did not exceed
a factor of 2 (Table 3). Seven days (168 h) after i.p. treatment
the increase for the combined hyperthermia treatment in peri
toneal tumors was still a factor of 3 (Fig. 7).
To take advantage of the increase in the AUC of platinum in
plasma after a hyperthermic treatment, CBDCA was adminis
tered i.v.. The results, expressed as the 41.5°C/37°Cratio,
0.01
10
Time
15
20
25
012345
Time (hours)
(hours)
Fig. S. Semilogarithmic concentration versus time plots (A) of total platinum
(37'C) and total platinum combined with abdominal hyperthermia (41.5'C) in
plasma (•,37'C; O, 41.5'C:') and peritoneal fluid (T, 37'C; V, 41.5'C) after i.p.
administration of 6. l S mg CBDCA/kg body weight. (B) Presentation of total
platinum (37'C) and total platinum combined with abdominal hyperthermia
(41.5'C) in peritoneal fluid during the first 4 h. Data points represent the mean
demonstrate enhanced platinum concentrations in tissues after
the combination treatment with abdominal hyperthermia (Ta
ble 3). Further the increase in platinum concentration was
higher at 168 h after treatment than after 24 h, but the absolute
platinum concentrations in nontumorous tissues after hyper
thermic treatment at 41.5°Cdid not substantially change be
tween 24 and 168 h (the 168 h data are not shown). The tumor
platinum concentrations differed slightly (factor, 1.6) 24 h after
treatment at 41.5 and 37°C(Fig. 7). It seems, however, that the
±SD (bars) of three animals (plasma) or four animals (peritoneal cavity).
platinum clearance from the peritoneal tumor was less after
treatment at 41.5°Cthan at 37°C.The difference in platinum
10
15
Time (hours)
20
25
concentration in the tumor at 168 h was a factor 3.4 in favor
of the hyperthermic treatment.
Comparison of platinum concentrations after the hyper
thermic treatments in which CBDCA was given either i.v. or
i.p. shows comparable values for platinum concentrations in
the different tissues. In terms of tumor platinum concentra
tions, it seems that the i.v. CBDCA treatment combined with
hyperthermia is as good as the i.p. treatment combined with
abdominal hyperthermia in terms of platinum tumor concen-
12345
Time
(hours)
Fig. 6. Semilogarithmic concentration versus time plots (A) of ultrafiltered
CBDCA (free platinum) after treatment with (41.5'C) and without (37'C) abdom
inal hyperthermia in plasma (•,37'C; O, 41.5'C) and peritoneal fluid (T, 37"C;
G, 41.5'C) after i.p. administration of 6.15 mg CBDCA/kg. (B) Representation
of free platinum (37'C) and free platinum combined with abdominal hyperthermia
(41.5'C) in peritoneal fluid during the initial phase after i.p. administration of
6.15 mg CBDCA/kg. Data are presented as the mean ±SD (bars) of three
animals (plasma) or four animals (peritoneal cavity).
Table 3 Platinum concentrations in tissues 24 h after combination treatment with
i.p. or i.v. CBDCA and abdominal hyperthermia (41.5°C,I h) and single i.p. or
i.v. treatment with CBDCA (6.15 mg/kg, normal body temperature)"
Pt/g37'C0.6
41.5'/37'C1.32.02.03.52.05.00.83.61
Treatmenti.p.i.V.TissuesLiverLungSpleenIntestinesKidneyLiverLungSpleenIntestinesKidneyKg
0.40.3
±
0.20.4
±
±0.10.2
±0.12.1
±0.40.6
the peritoneal cavity at 41.5°Cduring the first 2 h of treatment
(/>< 0.05) (Fig. 6B), while total platinum concentrations remain
similar at 37"C and 41.5°C(Fig. 5B). This relative increase of
0.40.3
±
±0.10.5
±0.10.2
±0.10.6
±0.10.5
0.20.6
±
0.32.7
±
0.38.4
±
±0.4tissue41.5'C0.7
±1.5Ratio
' Mean of at least 3 experiments ±SD.
ultrafiltered platinum is counteracted by the faster clearance of
CBDCA from the peritoneal cavity at 41.5 °Cat later times.
The ?„,,,
in plasma increased simultaneously with the faster
clearance of CBDCA from the peritoneal cavity at 41.5°C(Fig.
5A). The AUC in plasma was also higher at 41.5"C than at
37°C;this can probably be explained by a slower excretion of
CBDCA from the circulation at 41.5°C(Table 2). It seems that
I-P
the exposure of the tumor via the circulation is increased while
at the same time the exposure via the peritoneal fluid is de
creased when the temperature is raised from 37°Cto 41.5°C.
The overall result appears to be an increase in tumor exposure
via the systemic circulation.
In Vivo Biodistribution of CBDCA. Pharmacokinetic data
indicated increased exposure of the tumor to CBDCA via the
blood as a possible advantage of a combined CBDCA-hyperthermia treatment. We therefore treated rats with peritoneal
tumors i.p. or i.v. with CBDCA (6.15 mg/kg) at 37°Cor in
combination with abdominal hyperthermia (41.5°Cfor 1 h).
Rats were killed 24 and 168 h after treatment and tissues such
as peritoneal tumors, liver, kidney, intestine, spleen, and lung
were collected. For i.p. administration, increased platinum con
centrations were measured 24 h after hyperthermic treatment
(41.5°C) in nearly all the selected tissues (Table 3; Fig. 7).
±0.10.6
±0.10.8
±0.10.7
0.34.2
±
0.40.7
±
O
0.75
oÃ3
0.50
£
0.25
0
0.00
1
24
h
168 h
Time (hours)
Fig. 7. Platinum concentrations in peritoneal tumors 24 and 168 h after
combination treatment with CBDCA (6.15 mg/kg) i.p. or i.v. and with or without
hyperthermia (41.5°C)for 1 h. For each time-treatment combination, the left and
right bar correspond with 37°Cand 41.5°C,respectively. The results of the
treatments were the means of at least five experiments ±SD (bars).
1255
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
i.p. CHEMOTHERAPY AND REGIONAL HYPERTHERMIA
tration. More platinum, however, remains behind in the kidney
after i.v. than after i.p. treatment at 43°C,8.36 ±1.5 versus 4.2
±0.4 ng platinum/g tissue after 24 h and 6.5 ±1.6 versus 3.9
±2 after 168 h for i.v. and i.p. treatment, respectively. The
latter may be reflected in increased toxicity.
DISCUSSION
During the past decade efforts have been made to improve
current therapy for cancers restricted to the peritoneal cavity.
One way of achieving this was to change the route of adminis
tration of cDDP from systemic to i.p. in patients with residual
small volume ovarian cancer who failed to respond to i.v. cDDP
(6,10). In spite of the fact that i.p. drug delivery is still regarded
as investigational, this experimental approach provided a ben
eficial effect of i.p. compared to systemic administration (8, 11,
14). Clinical data, however, indicated that after an initial im
provement of the complete remission rate, most responding
patients relapsed. The latter emphasized the need to improve
the i.p. treatment. Since drug penetration from the peritoneal
cavity into the tumor is one of the major advantages of i.p.
treatment, it seems logical to concentrate on this aspect in an
attempt to improve i.p. treatment. One way to increase drug
penetration might be a combination with hyperthermia since
the latter leads to altered membrane permeability (22). In a
previous study we demonstrated increased penetration of cDDP
into a peritoneal rat tumor when the i.p. treatment was com
bined with abdominal hyperthermia (32). Higher i.t. platinum
concentrations were achieved and the penetration distance of
cDDP from the periphery increased. A disadvantage of this
combination was the concomitant increase in nephrotoxicity
which was, however, less than for whole body hyperthermia
(33). Based on these results, experiments were performed with
hyperthermic CBDCA therapy.
The in vitro experiments with low and moderately toxic
CBDCA doses in combination with hyperthermia resulted in a
significant increase of intracellular platinum only at 43°C.In
contrast, the cytotoxicity of a 1-h incubation with CBDCA was
already higher at 40°C;this is in accordance with the results of
Xu et al. (34) obtained with lower CBDCA concentrations. An
explanation for this phenomenon of increased cytotoxicity at
unchanged uptake levels may be found in the process of hydration. CBDCA itself does not significantly bind to proteins or
other nucleophiles; nonenzymatic transformation gives rise to
strong binding to aquated metabolites (35, 36). In this way,
CBDCA binds to nucleophilic sites on DNA, causing interstrand and intrastrand cross-links (35). The extent of binding
probably determines the cytotoxic effect (37, 38). Data from
the present study demonstrate that heat induces higher
CBDCA-DNA adduci levels. Whether this is due to activation
of the hydration process or to increased permeability of the cell
membrane, followed by increased intracellular platinum con
centrations, is not totally clear. The data in this study certainly
do not exclude a temperature dependent activation of the hy
dration process. In other words, relatively more CBDCA may
have been biotransformed into aquated metabolites at higher
temperatures.
The cytotoxicity experiments support this
hypothesis.
Potentiation of CBDCA action was more effective during 1
h exposure than during the continuous exposure experiment.
Since biotransformation is slow, this is likely to be a limiting
factor in the short experiments. With continuous exposure, the
relative enhancement of CBDCA-DNA adduct formation and
cytotoxicity during the 1-h heating period will probably be lost
during the following days in which CBDCA or its aquated
products can still enter the cell and form new DNA adducts.
The fact that the cytotoxicity increased at 43°Cis probably
caused by a higher CBDCA uptake into the cells during the
heating period in the first hour (Table 1). The net uptake of
CBDCA appears to be affected only at temperatures higher
than 41.5°C.This is in contrast with the parent drug cDDP.
An increase in temperature from 37°Cto 40°Calready strongly
affected the uptake of cDDP into cells and subsequent cell
survival (39). The results presented in this paper on the
CBDCA-DNA adduct formation strongly indicate an increased
reactivity of CBDCA which might have major implications for
the clinic. One of the adverse differences between CBDCA and
cDDP, the difference in biotransformation into aquated metab
olites, may be solved by the hyperthermia approach.
The pharmacokinetic data on i.p. CBDCA administration
with and without hyperthermia show a moderate increase in
the AUC of total platinum in the plasma and a significant
decrease in the clearance of total platinum from the circulation
after treatment with hyperthermia. These changes in parame
ters apparently result in a higher exposure of all tissues, includ
ing peritoneal tumors, to CBDCA or its reactive metabolites.
The pharmacokinetic data on the peritoneal fluid, however,
show a marked decrease in i.p. tumor exposure after hyperther
mia, as can be seen from the significant decrease in AUC of
total platinum and a significant increase in clearance of both
total and free platinum from the peritoneal cavity. To explain
this difference in exposure, one must distinguish two periods:
(a) the 90-min period of heating in which the peripheral blood
flow increases and the central blood flow will consequently
decrease (40); (b) the period of recovery in which the blood
flow returns to normal but the membrane permeability still is
increased (23, 41). The observed pharmacokinetics may be
explained according to this scheme. During the heating period,
exchange between the peritoneal cavity and the circulation will
not increase since there is a decrease in the central blood flow.
Fig. 6B shows that the difference in total platinum clearance of
the peritoneal cavity becomes obvious only after about 1.5 h.
The clearance of free platinum at 41.5°Cis slow in comparison
with the 37°Ccontrol (Fig. 6Ä).One factor involved might be
the increased formation of reactive metabolites at 41.5°Cwhich
may inhibit passage through the peritoneal membrane. A sim
ilar phenomenon has also been demonstrated for hyperthermic
cDDP when given i.p. to both dogs and rats; this resulted in a
relative increase of the free platinum concentration in the
peritoneal cavity compared to normothermic cDDP treatment
(36, 42). A change in the chemical structure of the drug might
also be the reason in this case. After heating, the blood flow
will return to normal but CBDCA will leave the peritoneal
cavity faster because of the still present increase in membrane
permeability. The tv,ß
for both total and ultrafiltered platinum
in the peritoneal cavity decreased if combined with heat. This
is in contrast with data obtained with cDDP in the same model
(39). It means that the tumor exposure to CBDCA in the
peritoneal cavity declines which may have severe consequences
for drug uptake by the tumor via the direct peritoneal route.
The slower clearance of CBDCA from the plasma, however,
provides a higher exposure of the tumor by the systemic circu
lation and may compensate for the loss of tumor exposure in
the peritoneal cavity. A higher systemic exposure, however,
may also have other consequences such as an increased myelosuppression.
1256
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
¡.p.CHEMOTHERAPY
AND REGIONAL HYPERTHERMIA
The experiments on the tissue distribution of i.p. CBDCA
when combined with elevated temperatures show an increased
platinum concentration in all tissues at 24 h after treatment.
The larger increase in platinum concentration in tumors is due 12.
to both the direct penetration of platinum into these abdominal
tumors from the peritoneal fluid and the higher tumor exposure
13.
by the circulation. This increase in intratumoral platinum con
centration might be beneficial with respect to the antitumor
response. At 168 h after treatment, the absolute platinum
concentrations were much lower for both the 37°Cand 41.5°C 14.
treated group, but the tissue ratios between both groups were
still comparable to the 24-h values.
In order to determine the role of the increased tumor expo
sure by the circulation, the effect of abdominal hyperthermia
combined with i.v. CBDCA administration on the biodistribution of platinum in various tissues was studied. These experi
ments showed a rise in tumor platinum concentration at both
24 and 168 h after heat treatment compared to control (37°C).
Comparing i.p. and i.v. treatment both with abdominal hyper
thermia, it appears that the platinum concentrations in tumors
do not differ. This indicates that the higher tumor platinum
concentrations after i.p. treatment plus hyperthermia, in com
parison to i.p. treatment alone, are probably due to the increased
AUC in plasma. Furthermore, the platinum concentrations in
the kidney differed. It seems that 24 and 168 h after treatment
the absolute platinum concentration is higher after i.v. than
after i.p. treatment. This may have consequences for the nephrotoxicity. Thrombocytopenia on the other hand increased only
slightly when CBDCA treatment was combined with abdominal
hyperthermia (data not shown). It did not lead to a necessary
reduction in the dose.
In summary, heat seems to increase the uptake of CBDCA
into peritoneal tumors both after i.p. and i.v. treatment. The
increased CBDCA-DNA adduct formation in vitro at higher
temperatures might even suggest better tumor responses; how
ever, this is still hypothetical. The only disadvantage of i.v.
treatment over i.p. treatment in combination with heat would
be the higher platinum concentration in the kidney after i.v.
treatment which may affect renal toxicity.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Jos Hendriks
and Nel Bosnie.
29.
30.
REFERENCES
1. Aure, J. C., Hoeg, K., and Kolstad, P. Clinical and histologie studies of
ovarian carcinoma. Obstet. Gynecol., 37: 1-9, 1971.
2. Myers, C. The use of intraperitoneal chemotherapy in the treatment of
ovarian cancer. Semin. Oncol.. //: 275-284. 1984.
3. Ozols, R. Intraperitoneal chemotherapy in the management of ovarian can
cer. Semin. Oncol.. 12: 75-80. 1985.
4. Hamilton, T. C., Young, R. C., and Ozols. R. F. Experimental modelsystems
of ovarian cancer: applications to the design and evaluation of new treatment
approaches. Semin. Oncol., //: 285-298. 1984.
5. Alberts, D. S., Young, L., Mason, N., and Salmon, S. E. In vitro evaluation
of anticancer drugs against ovarian cancer at concentrations achievable by
intraperitoneal administration. Semin. Oncol., 12: 38-42, 1985.
6. Brenner, D. E. Intraperitoneal chemotherapy: a review. J. Clin. Oncol.. 4:
1135-1147, 1986.
7. Dedrick, R. L., Myers, C. E., Bungay, P. M., and DeVita, V. T. Pharmacokinetic rationale for peritoneal drug administration in the treatment of
ovarian cancer. Cancer Treat. Rep., 62:1-11, 1978.
8. Dedrick. R. L. Theoretical and experimental bases of intraperitoneal chemo
therapy. Semin. Oncol., 12 (Suppl. 4): 1-6, 1985.
9. Los, G., and McVie, J. G. Experimental and clinical status of intraperitoneal
chemotherapy. Eur. J. Cancer, 26: 755-762, 1990.
10. Ten Bokkel Huinink. W. W., Dubbelman, R., Aartsen, A.. Franklin. H.. and
McVie. J. G. Experimental and clinical results with intraperitoneal cisplatin.
Semin. Oncol., 12:43-46, 1985.
Howell. B. S., Kirmani. S., McCIay, E. F., Kim, S., Braley, P., and Plaxe, S.
Intraperitoneal cisplatin based chemotherapy for ovarian cancer. Semin.
Oncol.,/Ä.-5-10, 1991.
Los, G., Ruevekamp. M.. Bosnie, N., de Graaf, P. W„and McVie, J. G.
Intraperitoneal tumor growth and chemotherapy in a rat model. Eur. J.
Cancer Clin. Oncol., 25: 1857-1866, 1989.
Los, G., Nagel. J. D., and McVie, J. G. Anti-tumor effect of cisplatin,
carboplatin, mitroxantronc, and doxorubicin on peritoneal tumor growth
after intraperitoneal and intravenous chemotherapy: a comparative study.
Selective Cancer Ther.. 6: 73-82, 1990.
Los, G., Mutsaers, P. H. A., van der Vijgh. W. J. F.. Baldew, G. S., de Graaf,
P. W., and McVie, J. G. Direct diffusion of m-diamminedichloroplatinum(II) in intraperitoneal tumors after intraperitoneal chemotherapy: a
comparison with systemic chemotherapy. Cancer Res.. 49: 3380-3384,1989.
Los, G., Mutsaers, P. H. A., Lenglet. W. J. M., Baldew. G. S., and McVie,
J. G. Platinum distribution in intraperitoneal tumors after intraperitoneal
cisplatin treatment. Cancer Chemother. Pharmacol., 25: 389-394, 1990.
Los, G., Mutsaers, P. H. A., Dubbelman. R., van der Hoeven. C. J.. Heintz,
A. P. M., and McVie, J. G. Platinum distribution in human peritoneal
autopsy tumor samples after treatment with platin containing regiments. In:
Proceedings of Sixth NCI-EORTC Symposium on New Drugs in Cancer
Chemotherapy. Abstract 220. Amsterdam, 1989.
Canetta, R.. Rozencwieg, M., and Carter, S. K. Carboplatin: the clinical
spectrum to date. Cancer Treat. Rev.. 12 (Suppl. A): 125, 1985.
Wilshaw, E. Ovarian trial at the Royal Marsden. Cancer Treat. Rev., 12
(Suppl. A): 67, 1985.
Speyer, J. L., Beller, U., Colombo, N., Sorich, J.. Wernz, J. C., Höchster,
H., Green, M., Porges, R., Muggia. F. M., Canetta, R., and Bcckman, E. M.
Intraperitoneal carboplatin: favorable results in women with minimal residual
ovarian cancer after cisplatin therapy. J. Clin. Oncol., 8: 1335-1341, 1990.
McVie, J. G., Ten Bokkel Huinink. W.. Dubbelman. R.. Franklin, H., van
der Vijgh, W., and Klein, J. Phase I study and pharmacokinetics of intraper
itoneal carboplatin. Cancer Treat. Rev., 12: 35-41, 1985.
Los, G., Verdegaal, E. M. E., Mutsaers, P. H. A., and McVie, J. G.
Penetration of carboplatin and cisplatin into rat peritoneal tumor nodules
after intraperitoneal chemotherapy. Cancer Chemother. Pharmacol., 28:
159-165, 1991.
Arancia, G., Crateri Trovalusci, P., Mariutti. G., and Mondovi, B. Ultrastruclural changes induced by hyperthermia in Chinese hamster V79 fibroblasts.
Int. J. Hyperthermia, 5: 341-350, 1989.
Hahn. G. M., Braun, J.. and Har-Kedar, I. Thermochemotherapy: synergism
between hyperthermia (42-43'C) and Adriamycin (or bleomycin) in mam
malian cell inactivation. Proc. Nati. Acad. Sci. USA. 172: 937-940, 1975.
Hahn. G. M., and Shiu. E. C. Effect of pH and elevated temperatures on the
cytotoxicity of some therapeutic agents on Chinese hamster cells in vitro.
Cancer Res., 43: 5789-5791, 1983.
Bull, J. M. C. An update on the anticancer effects of a combination of
chemotherapy and hyperthermia. Cancer Res.. 44 (Suppl.): 4853-4856,1984.
Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res.,
39: 2264-2268. 1979.
Fisher, G. A., and Hahn, G. M. Enhancement
of r/s-platinum(II)diamminedichloride cytotoxicity by hyperthermia. Nati. Cancer Inst.
Monogr., 61: 255-257, 1982.
Marquet R. L., Westbroek. D. J.. and Jeekel. J. Interferon treatment of
transplantable rat colon adenocarcinoma: importance of tumor site. Int. J.
Cancer, 33: 689-692, 1984.
Terheggen, P. M. A. B., Floot, B. G. J., Lempers, E. L. M., van Tellingen,
' ).. Begg, A. C., and den Engelse, L. Antibodies against cisplatin-modified
DNA and cisplatin-modified (di)nucleotides. Cancer Chemother. Pharma
col.. 28: 185-191, 1991.
Terheggen. P. M. A. B.. Dijkman. R., Begg, A. C.. Bubbelman. R., Floot. B.
G. J., Hart, A. A. M., and den Engelse, L. Monitoring of interaction products
of c/'s-diamminedichloroplatinum (II) and rij-diammine(l.l-cyclobutanedi-
carboxylato)platinum (II) with DNA in cells from platinum treated cancer
patients. Cancer Res., 48: 5597-5603. 1988.
31. Scherer, E., van Benthem, J., Terheggen, P. M. A. B., Vermeulen, E.,
Winterwerp, H. H. K.. and den Engelse. L, Immunocytochemical analysis of
DNA adducts at the single cell level: anew tool for experimental carcinogenesis. chemotherapy and molecular epidemiology. In: H. Bartsch. K. Hemminki. and I. K. O'Neill (eds). Detecting Methods for DNA Damaging
Agents in Humans: Applications in Cancer Epidemiology and Prevention.
IARC Scientific Publications No. 89, pp. 286-281. Lyon. France: Interna
tional Agency for Research on Cancer, 1988.
32. Los, G., Sminia, P., Wondergem, J., Mutsaers. P. H. A.. Haveman, J., Ten
Bokkel Huinink. D.. Smals, O. A. G.. Gonzalez-Gonzales, D., and McVie,
J. G. Optimization of intraperitoneal chemotherapy with regional hyperther
mia. Eur. J. Cancer, 27:706-713, 1991.
33. Wondergem, J., Bulger, R. E., Strebel, F. R., Newman, R. A., Travis, E. L.,
Stephens, L. C., and Bull, J. M. C. Effect of c/j-diamminedichloroplatinum(II) combined with whole body hyperthermia on renal injury. Cancer
Res., 48: 440-446, 1988.
Xu, M. J., and Alberts, D. S. Potentiation of platinum analogue cytotoxicity
by hyperthermia. Cancer Chemother. Pharmacol., 21: 191-196. 1988.
Knox, R. J., Friedlos. F.. Lydall, D. A., and Roberts. J. J. Mechanism of
cytotoxicity of anticancer platinum drugs: evidence that a'j-diamminedichlo-
1257
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
i.p. CHEMOTHERAPY
AND REGIONAL HYPERTHERMIA
roplatinum(II)andai-diammine-(l,l-cyclobutanedicarboxylato)platinum(II)
differ only in the kinetics of their interaction with DNA. Cancer Res., 46:
1972-1979, 1986.
36. Los, G., Verdegaal, E., Noteborn, H. P. J. M., Ruevekamp, M., de Graeff,
A., Meesters, E. W., Ten Bokkel Huinink, D., McVie, J. G. Cellular pharmacokinetics of carboplatin and cisplatin in relation to their cytotoxic action.
D- v, DI,
â„¢¿iA4^.
I -li-i
if.1 tool
BiochPharmacol.,
JS/-JÖJ,
lyyi.
37. Reed, E., Ozols, R. F., Tarone, R., Yuspa, R., and Poirier, M. C. PlatinumDNA adducts in leukocyte DNA correlate with disease response in ovarian
cancer patients receiving platinum-based chemotherapy. Proc. Nati. Acad.
Sci. USA, 84: 5024-5028, 1987.
38. Terheggen, P. M. A. B., Begg, A. C., Emondt, J. Y., Dubbelman, R., Floot,
B. G. J., and den Engelse, L. Formation of interaction products of carboplatin
with DNA in vitro and in cancer patients. Br. J. Cancer, 63: 195-200, 1991.
39. Los, G., Sminia, P., Wondergem, J., Mutsaers, P. H. A., Haveman, J., Ten
Bokkel Huinink, D., Smals, O., Gonzalez Gonzalez, D., and McVie, J. G.
Optimization of intraperitoneal chemotherapy with regional hyperthermia
in ra's- E"r- J- Cancer, 27: 472-477, 1991.
40- Shepherd, J. T., and Webb-Peploe, M. M. Cardiac output and blood How
^'"T0".d""nSw°rkin.
hf'•'": i>- J- »^^
P Ca«&ea"d J; A- JStolwijk18.(eds.),
Physiological
andC Behavioural
Temperature
Chap.
Springfield,
IL: Charles
Thomas, Publisher,
1970. Regulation,
4, whe"atl
¿ N* Kerr? c and Gregoryi D w Heat-induced damage to
HeLa-S3 cells: correlation of viability, permeability, osmosensitivity, phasecontrast light-, scanning electron- and transmission electron-microscopical
findings. Int. J. Hyperthermia, 5: 145-162, 1989.
42. Zakris, E. L., Dewhirst, M. W., Riviere, J. E., Hoopes, P. J., Page, R. L.,
and Oleson, J. R. Pharmacokinetics and toxicity of intraperitoneal cisplatin
combined with regional hyperthermia. J. Clin. Oncol., 5: 1613-1620, 1987.
1258
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.
A Rationale for Carboplatin Treatment and Abdominal
Hyperthermia in Cancers Restricted to the Peritoneal Cavity
Gerrit Los, Oskar A. G. Smals, Marianne J. H. van Vugt, et al.
Cancer Res 1992;52:1252-1258.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/52/5/1252
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1992 American Association for Cancer Research.