A Rationale for Carboplatin Treatment and Abdominal Hyperthermia in Cancers Restricted to the Peritoneal Cavity1

(CANCER RESEARCH 52, 1252-1258, March 1, 1992] A Rationale for Carboplatin Treatment and Abdominal Hyperthermia in Cancers Restricted to the Peritoneal Cavity1

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 cDDP3 is one of the most effective drugs available for i.p. treatment of ovarian carcinoma (9). Trials of single agent cDDP The purpose of this study was to optimize the treatment of cancers restricted to the peritoneal cavity by combining i.p. chemotherapy with or cDDP based combination therapy Â¡.p.,in patients with abdominal hyperthermia. In vitro experiments demonstrated that the residual small volume ovarian cancer who had failed to respond uptake of carboplatin into CC531 tumor cells was increased at tempera to i.v. cDDP treatment, have demonstrated beneficial clinical tures higher than 41.5Â°Catdose levels of 5 and 50% cell kill. Carboplatin- results; 30% complete remissions after single agent cDDP DNA adduct formation and cytotoxicity, however, were already increased treatment (10) and 65% clinically free of disease after cDDP at temperatures of about 40Â°C,indicating that carboplatin-DNA adduct based combination therapy (11). We have previously demon formation and consequently cytotoxicity could be enhanced by mild strated in a rat model that drug penetration from the peritoneal hyperthermia (temperatures in the range of 39-41.5Â°C). cavity into a rat peritoneal tumor plays a key role in achieving CC531 tumor bearing rats were treated i.v. and i.p. with carboplatin better responses (12-14). Absolute concentrations of platinum (6.15 mg/kg) in combination with regional hyperthermia of the abdomen (41.5Â°Cfor 1 h). The mean temperature was 41.5 Â±0.3Â°C(SD) in the in i.p. tumor nodules were always higher after i.p. treatment peritoneal cavity and 40.5 Â±0.3Â°Cinthe esophagus. Enhanced platinum than after i.v. treatment with cDDP (15) and the effective advantage of i.p. chemotherapy with cDDP was accentuated at concentrations were found in peritoneal tumors (factor 3) and in kidney, the periphery of the tumor (14-16). liver, spleen, and lung (a factor 2 average), after the combined i.v. or i.p. carboplatin-hyperthermia treatment. Pharmacokinetic data of Â¡.p. CBDCA is a cDDP analogue with a more favorable toxic CBDCA combined with hyperthermia demonstrated an increased tumor profile and an appreciable activity against ovarian cancer (17- exposure for total and ultrafiltered platinum in plasma. The areas under 19). The occurrence of dose limiting side effects such as neph- the concentration x time curve for total platinum at 37Â°Cand 41.5Â°C rotoxicity, neurotoxicity, and vomiting associated with i.p. were 69 and 210 pM/h, respectively; for ultrafiltered platinum these cDDP treatment, has led to trials of i.p. CBDCA. In these values were 47 and 173 nM/h. This may have been due to a slower trials, slower drug elimination from the peritoneal cavity and a elimination of platinum from the blood at the higher temperature (f.,.rf for total platinum 99 and 156 min at 37 and 41.5Â°C,respectively). The lower protein binding capacity of CBDCA were demonstrated (20). In spite of these pharmacological advantages, the relative direct exposure of the tumor via the peritoneal fluid appeared to diminish, since the area under the curve for total platinum was lower at 41.5Â°C amount of CBDCA penetrating into peritoneal tumors and than at 37Â°C(576MM/hversus 1255 nM/h, respectively). tumor cells was far less than that observed for cDDP (9, 21). Our results indicate that the advantage of adding hyperthermia is In view of these results, an attempt to improve the penetration caused by an increased drug exposure of the tumor via the circulation. of CBDCA into tumor cells seemed logical. One way to achieve This was supported by the fact that platinum concentrations in peritoneal this goal might be the application of hyperthermia, since heat tumors after carboplatin treatment at elevated temperatures were similar increases cell membrane permeability to drugs (22) and mem for the i.p. and i.v. routes. brane transport of drugs (23) and alters cellular metabolism (24). In addition, overall pharmacokinetics may change, with INTRODUCTION heat affecting drug metabolism and excretion (25), all leading to increased antitumor responses (26, 27). The human peritoneal cavity is a common site of tumor The purpose of this study was to assess the effect of regional recurrence of ovarian and gastrointestinal malignancies. After heat on the efficacy of CBDCA in a rat tumor model system, initial surgery the amount of residual tumor in the peritoneal in an attempt to improve the remission rate of cancers restricted cavity affects the final outcome of the treatment (1,2). For this to the peritoneal cavity. reason, and from the recognition that tumors growing within a body cavity are less well supplied by the bloodstream, i.p. MATERIALS AND METHODS chemotherapy may improve the survival and quality of life (3- 5). Pharmacokinetic modeling has suggested that intracavitary Rats and Drugs. Male WAG/Rij rats (8-12 weeks old; 250-300 g) administration of chemotherapeutic agents by peritoneal di were obtained from the animal department of the Netherlands Cancer Institute. The animals were kept in a temperature controlled room on alysis techniques might result in a significantly greater drug a 12 h light- 12-h darkness schedule and fed standard rat chow and tap concentration in the peritoneal cavity than in plasma. This water ad libitum. CBDCA was obtained from Bristol Myers (Weesp, concentration difference offers a potential advantage in the the Netherlands). CBDCA containing vials were stored at room tem treatment of malignancies confined to the peritoneal cavity perature. (6-8). Tumor Model. The tumor used (CC531) is a well defined transplantable rat colonie adenocarcinoma (28), with a doubling time in vitro of Received 9/9/91; accepted 12/16/91. 16 h. The tumor grows s.c., i.p., and in vitro (12-15). In vitro, cells The costs of publication of this article were defrayed in part by the payment were cultured under 5% CO2 in 75-cm2 flasks (Falcon, Oxnard, CA) 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 3The abbreviations used are: cDDP, cisplatin; CBDCA, carboplatin; AUC, Society (KoningÃ«nWilhelmina Fonds). area under the concentration x time curve; PBS, phosphate-buffered saline; i.t., 2To whom all correspondence should be addressed. intratumoral. 1252

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. i.p. CHEMOTHERAPY AND REGIONAL HYPERTHERMIA with Dulbecco's modified Eagle's medium (Irvine, Scotland), contain 168 h tissues (tumor, liver, kidney, spleen, intestines, and lung) were ing 10% fetal calf serum (Gibco). Cells were subcultured after reaching collected and prepared for platinum measurements. a density of 5 x 106/175 cm2 by trypsinization and replated at a density Platinum Detection by Flameless Atomic Absorption Spectroscopy. A of 10* cells/175 cm2. model AA40 atomic absorption spectrometer with a GTA 96 graphite In Vitro Drug Uptake in Tumor Cells. In each experiment six flasks tube atomizer (with Zeeman background correction) from Varian (Vic were used for each temperature, all containing about 6 x IO6 CCS31 toria, Australia) was used for platinum analysis. Platinum concentra tumor cells. To each flask were added 20 ml of conditioned medium tions were determined in plasma, peritoneal fluid, tumor tissue, tumor with 5 i!g (a nontoxic dose) or 200 ^g of CBDCA/ ml (50% inhibitory cells, and normal tissues. Sample preparation and flameless atomic concentration). Cells (2 x IO7for each temperature) were incubated for absorption spectroscopy procedure have been described elsewhere (15). l h at 37Â°C,40'C, 41.5'C, or 43Â°C,all at 5% CO2. After incubation, CBDCA-DNA Adduct Formation. CC531 cells were cultured on glass the cells were washed with PBS, detached with trypsin, pooled, counted, slides (2.6 x 6 cm) coated with ovalbumin (100 //I 0.5% ovalbumin/ and washed twice with PBS. Finally, the cells were suspended in slide) and incubated with CBDCA (2.2 mg/ml) for 2 h at different approximately 1 ml PBS and prepared for platinum determination. temperatures (37Â°C,38.5Â°C,40Â°C,41.5'C, and 43Â°C).Cells were fixed Sensitivity of CCS31 to CBDCA. The sensitivity of CC531 cells to by successive incubations in cold (-20Â°C) 100% methanol (10 min) and CBDCA at different temperatures was tested by clonogenic assay. acetone (2 min) and air dried. CC531 cells were harvested as described before and counted. Cells in a The characteristics of the rabbit antiserum NKI-A59 against cDDP- single cell suspension were plated in 6-well tissue culture clusters modified calf thymus DNA (platinum/nucleotide ratio, 6.7 x 10~2) (Costar, Cambridge, United Kingdom) at 150 cells/well in conditioned have been described by Terheggen el al. (29). NKI-A59 (applied without medium. After 24 h of incubation at 37*C, the cells had attached to the further purification), goat anti-rabbit immunoglobulin (Campro Bene plates and 150 p\ of a CBDCA solution were added to obtain the final lux, Elst, the Netherlands) and peroxidase-(rabbit)antiperoxidase com concentrations described under "Results." The culture clusters were plex (American Qualex, La Miranda, CA) were used in 1:1800, 1:600, incubated at 37Â°C,40Â°C,41.5Â°C,or 43Â°Cfor l h and about 15 min and 1:3000 dilutions, respectively. All sera were diluted in phosphate preheating time. After incubation, cells were washed twice with PBS buffer containing 10 mM KH2PO4(pH 7.4), 140 mM NaCl, 10% normal and 3 ml of fresh medium were added. All plates were returned to the goat serum, and 0.04% Triton X-100 (BDH, Poole, England). The incubator and incubated for 7-10 days for the development of colonies. immunocytochemical procedure was carried out as described by Ter In contrast to the 24-h incubation period, cells were kept in platinum heggen et al. (30). containing medium after heat treatment for 7-10 days at 37Â°Cfor the The binding of the antibody NKI-A59 was visualized by a double continuous CBDCA incubations. Colonies were fixed with ethanol, peroxidase-antiperoxidase staining. The nuclear staining intensity of stained with crystal violet for 10 min, counted, and related to the individual nuclei was analyzed and quantified with a Knott (Munich, control. Germany) light measuring device with a beam diameter of 5 /un, which Pharmacokinetic Studies. Pharmacokinetic studies in rats were per was coupled to a Leitz Orthoplan microscope. Data were analyzed by formed by cannulation of the carotid artery. The anesthetized animals an Atari ST computer (Sunnyvale, CA) programmed with a version of were positioned with their lower part of their body in a thermostatically controlled water bath at 41.5Â°C.The temperature in the peritoneal the Histochemical Data Acquisition System [Hidacsys; Microscan, Leiden, the Netherlands (31)]. The integrated absorbance of a selected cavity of the animals steadily increased from normal body temperature (around 38Â°C)to 41.5Â°Cwithin about 30 min. CBDCA (6.15 mg/kg), area was expressed in arbitrary units. In each slide the nuclear staining density of 3 to 4 randomly selected areas, corresponding to 20-40 dissolved in saline and brought to 41.5Â°C,was injected i.p. in a volume nuclei each, was measured. of 20 ml. At different time points after i.p. administration, blood Statistics. Student's t test or the Wilcoxon test were used to study samples of about 0.5 ml were taken from the carotid artery. Clotting differences; P < 0.05 was considered to indicate significant differences. 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 RESULTS ultrafiltered through a membrane filter with a 10-kDa cutoff (Amicon, Danvers, MA) by centrifugation at 1000 x g for 10 min. Total and In Vitro Studies on CBDCA. The effect of hyperthermia on ultrafiltered platinum concentrations were determined in plasma by the cellular uptake of CBDCA was studied Â¡nCC531 cells. Cells flameless atomic absorption spectroscopy after dilution of the samples were incubated with 5 or 200 /Â¿g/mlCBDCA for l h at 37Â°C, in 0.2 M HC1/0.15 M NaCl. The CBDCA clearance from the peritoneal 40Â°C,41.5T, or 43Â°C.The uptake of CBDCA did not increase cavity was studied by sampling the peritoneal fluid at the same time significantly when the temperature was raised from 37 to points as blood. From these data the AUC, ('â€ž,.â€ž(platinumpeak 41.5"C, but at 43Â°Ca clear increase was observed (Table 1). In concentration), /m.Â»(time,peak concentration occurs), and tv,ÃŸ(thehalf- contrast, the cytotoxicity was already increased at a temperature life of platinum) were calculated. of 40Â°(Fig. 1/4). The effect of heat on the CBDCA cytotoxicity 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 after prolonged exposure to the drug was less pronounced for were positioned in a thermostatically controlled water bath at 41.5Â°C temperatures round 40Â°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 43Â°Cis the animal steadily increased from normal body temperature (around 38Â°C)to 41.5Â°Cin about 30 min. The duration of the heat treatment cell kill increased (Fig. \B), probably as a result of the higher 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 cavity (near the bladder, the spleen, and right kidney). The placement concentration cells)5(ngPt/106 CC/37-C)51.0 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 Â»ig/ml23.9 distance of 6 cm into the rectum and the temperature in the esophagus Â±0.09 Â±1.1 were monitored. The latter two were single sensor measurements. 40 0.47 Â±0.17 ND I.I Tissue Platinum Concentrations after i.p. Chemotherapy. Rats with 41.5 0.55 Â±0.19 28.3 Â±7.0 1.32.62001.01.22.5 small solid peritoneal tumors were treated i.p. or i.v. with CBDCA 43Pt 1.12 Â±0.33200 61.2 + 21Ratio (6.15 mg/kg) with or without abdominal hyperthermia. After 24 and " Mean of 3 experiments Â±SD; ND, not determined. 1253

100 100 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). i The difference in adduct specific nuclear staining intensity 0.1 between 37Â°Cand 43Â°C(Fig. 2) is statistically significant (P < 0.05). The increase in DNA-adduct formation between 37Â°C and 40Â°C,between 37Â°Cand 41.5Â°Cand between 37Â°Cand 0.01 140 210 43Â°Cwere significant (P < 0.01, P < 0.001, and P < 0.001,

CBDCA cone, (uÃ /mi) CSDCA cone, lug/ml) respectively). Fig. 1. Clonogenic assay of CC531 cells incubated with CBDCA for 1 h (A) Thermometry. During the in vivo hyperthermia experiments, and continuously (fi) at 37'C (â€¢),40'C (A), 41.5'C (T), and 43'C (â€¢).Allcurves were adjusted to the control values at 37'C, 40'C, 41.5Â°C,and 43Â°Cand were the temperatures in the esophagus, peritoneal cavity, and rectum were monitored (Fig. 4). The temperature reaches a steady state 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, after 30 min of heating. The mean temperatures over the 1-h SD. treatment for the peritoneal cavity, the rectum, and the esoph agus were 41.5 Â±0.1Â°C,41.2 Â±0.2Â°C,and 40.5 Â±0.3Â°C, 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 (41.5Â°C)(Figs. 5 and 6; Table 2). The M 5 AUC in peritoneal fluid was smaller in rats receiving abdominal i 2 hyperthermia, indicating a faster clearance of CBDCA from the ,-Ã© peritoneal cavity. During the first hours after hyperthermia treatment the platinum concentration in the peritoneal cavity declined faster at 41.5Â°Cthan at 37Â°C(Figs. 5B and 65). This is apparent from the half-lives (t

36 20 40 60 80 100

Time (min) 2.0 Fig. 4. Temperature measured in the esophagus (â€¢),rectum (T) and peritoneal cavity (â€¢)of rats plotted against heating time in a water bath of 41.5'C. Data points represent the mean Â±SD (bars) of five measurements. 1.5

Table 2 Pharmacokinetic data of i.p. CBDCA treatment in plasma and peritoneal cavity at 37'C and 41.5'C i 1.0 41.5'CParameterAUC

0.6 Pt210 Pt173 Pt69 Pt47 h)(â€ž,.,plasma (0-24 Â±89Â°170 Â±62135Â±21C23Â±2760 Â±460 (min)C^dM)tv,ÃŸ Â±14*24 Â±1716 Â±9nÂ± Â±5156 Â±5127 Â±699 0.0 Â±13*576 i70 37 38.5 40 41.5 plasma(min)(60-360 Â±32700 Â±201255 Â±14963 o 4* min)AUC Temperature! C) h/tnÃŸp.c. (0-24 Â±74*58 Â±10964 Â±80239 Â±204195 p.c.(min)'(30-360 Â±5*Free Â±10C37Total Â±35â€¢cFreeÂ±52 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 min)Total stained for CBDCA-DNA adduct formation. Each column represents the mean " Mean Â±SD; AUC expressed in ^M/h; n = 4 for 37'C and for p.c. at 41.5'C; Â±SEM (han) of at least 3 independent stained slides with 30-40 nuclei each. n = 3 for plasma at 41.5Â°C. The increase in DNA-adduct formation between 37Â°Cand 40'C, between 37Â°C 'Significant difference between treatment at 37'C and 41.5'C (P < 0.01). and 41.5'C and between 37'C and 43'C is significant (P< 0.01, P< 0.001, and c Significant difference between treatment at 37'C and 41.5'C (P < 0.05). P< 0.001, respectively), a.u., arbitrary units. d p.c., peritoneal cavity. 1254

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 41.5Â°C/37Â°Cratio, 0.01 10 15 20 25 012345 demonstrate enhanced platinum concentrations in tissues after

Time (hours) Time (hours) the combination treatment with abdominal hyperthermia (Ta Fig. S. Semilogarithmic concentration versus time plots (A) of total platinum ble 3). Further the increase in platinum concentration was (37'C) and total platinum combined with abdominal hyperthermia (41.5'C) in higher at 168 h after treatment than after 24 h, but the absolute plasma (â€¢,37'C; O, 41.5'C:') and peritoneal fluid (T, 37'C; V, 41.5'C) after i.p. platinum concentrations in nontumorous tissues after hyper administration of 6. l S mg CBDCA/kg body weight. (B) Presentation of total platinum (37'C) and total platinum combined with abdominal hyperthermia thermic treatment at 41.5Â°Cdid not substantially change be (41.5'C) in peritoneal fluid during the first 4 h. Data points represent the mean tween 24 and 168 h (the 168 h data are not shown). The tumor Â±SD (bars) of three animals (plasma) or four animals (peritoneal cavity). platinum concentrations differed slightly (factor, 1.6) 24 h after treatment at 41.5 and 37Â°C(Fig. 7). It seems, however, that the platinum clearance from the peritoneal tumor was less after treatment at 41.5Â°Cthan at 37Â°C.The difference in platinum 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

10 15 20 25 12345 hyperthermia is as good as the i.p. treatment combined with abdominal hyperthermia in terms of platinum tumor concen- Time (hours) 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 Table 3 Platinum concentrations in tissues 24 h after combination treatment with inal hyperthermia in plasma (â€¢,37'C; O, 41.5'C) and peritoneal fluid (T, 37"C; i.p. or i.v. CBDCA and abdominal hyperthermia (41.5Â°C,I h) and single i.p. or G, 41.5'C) after i.p. administration of 6.15 mg CBDCA/kg. (B) Representation i.v. treatment with CBDCA (6.15 mg/kg, normal body temperature)" 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 Pt/g37'C0.6 6.15 mg CBDCA/kg. Data are presented as the mean Â±SD (bars) of three Treatmenti.p.i.V.TissuesLiverLungSpleenIntestinesKidneyLiverLungSpleenIntestinesKidneyKg41.5'/37'C1.32.02.03.52.05.00.83.61.32.0 animals (plasma) or four animals (peritoneal cavity). Â±0.40.3 Â±0.10.6 Â±0.20.4 Â±0.10.8 the peritoneal cavity at 41.5Â°Cduring the first 2 h of treatment Â±0.10.2 Â±0.10.7 Â±0.12.1 Â±0.34.2 (/>< 0.05) (Fig. 6B), while total platinum concentrations remain Â±0.40.6 Â±0.40.7 similar at 37"C and 41.5Â°C(Fig. 5B). This relative increase of Â±0.40.3 Â±0.10.5 ultrafiltered platinum is counteracted by the faster clearance of Â±0.10.2 Â±0.10.6 CBDCA from the peritoneal cavity at 41.5 Â°Catlater times. Â±0.10.5 Â±0.20.6 The ?â€ž,,,inplasma increased simultaneously with the faster Â±0.32.7 Â±0.38.4 clearance of CBDCA from the peritoneal cavity at 41.5Â°C(Fig. Â±0.4tissue41.5'C0.7Â±1.5Ratio ' Mean of at least 3 experiments Â±SD. 5A). The AUC in plasma was also higher at 41.5"C than at 37Â°C;this can probably be explained by a slower excretion of CBDCA from the circulation at 41.5Â°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 37Â°Cto 41.5Â°C. O 0.75

The overall result appears to be an increase in tumor exposure oÃ 1 via the systemic circulation. 3 0.50 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-hyper- Â£ 0.25 thermia treatment. We therefore treated rats with peritoneal tumors i.p. or i.v. with CBDCA (6.15 mg/kg) at 37Â°Cor in combination with abdominal hyperthermia (41.5Â°Cfor 1 h). 0 0.00 24 h 168 h Rats were killed 24 and 168 h after treatment and tissues such Time (hours) as peritoneal tumors, liver, kidney, intestine, spleen, and lung 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 were collected. For i.p. administration, increased platinum con hyperthermia (41.5Â°C)for 1 h. For each time-treatment combination, the left and centrations were measured 24 h after hyperthermic treatment right bar correspond with 37Â°Cand 41.5Â°C,respectively. The results of the (41.5Â°C)in nearly all the selected tissues (Table 3; Fig. 7). treatments were the means of at least five experiments Â±SD (bars). 1255

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. i.p. CHEMOTHERAPY AND REGIONAL HYPERTHERMIA tration. More platinum, however, remains behind in the kidney cytotoxicity during the 1-h heating period will probably be lost after i.v. than after i.p. treatment at 43Â°C,8.36 Â±1.5 versus 4.2 during the following days in which CBDCA or its aquated Â±0.4 ng platinum/g tissue after 24 h and 6.5 Â±1.6 versus 3.9 products can still enter the cell and form new DNA adducts. Â±2 after 168 h for i.v. and i.p. treatment, respectively. The The fact that the cytotoxicity increased at 43Â°Cis probably latter may be reflected in increased toxicity. 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 DISCUSSION than 41.5Â°C.This is in contrast with the parent drug cDDP. An increase in temperature from 37Â°Cto 40Â°Calready strongly During the past decade efforts have been made to improve current therapy for cancers restricted to the peritoneal cavity. affected the uptake of cDDP into cells and subsequent cell One way of achieving this was to change the route of adminis survival (39). The results presented in this paper on the tration of cDDP from systemic to i.p. in patients with residual CBDCA-DNA adduct formation strongly indicate an increased small volume ovarian cancer who failed to respond to i.v. cDDP reactivity of CBDCA which might have major implications for (6,10). In spite of the fact that i.p. drug delivery is still regarded the clinic. One of the adverse differences between CBDCA and as investigational, this experimental approach provided a ben cDDP, the difference in biotransformation into aquated metab eficial effect of i.p. compared to systemic administration (8, 11, olites, may be solved by the hyperthermia approach. 14). Clinical data, however, indicated that after an initial im The pharmacokinetic data on i.p. CBDCA administration provement of the complete remission rate, most responding with and without hyperthermia show a moderate increase in patients relapsed. The latter emphasized the need to improve the AUC of total platinum in the plasma and a significant the i.p. treatment. Since drug penetration from the peritoneal decrease in the clearance of total platinum from the circulation cavity into the tumor is one of the major advantages of i.p. after treatment with hyperthermia. These changes in parame treatment, it seems logical to concentrate on this aspect in an ters apparently result in a higher exposure of all tissues, includ attempt to improve i.p. treatment. One way to increase drug ing peritoneal tumors, to CBDCA or its reactive metabolites. penetration might be a combination with hyperthermia since The pharmacokinetic data on the peritoneal fluid, however, the latter leads to altered membrane permeability (22). In a show a marked decrease in i.p. tumor exposure after hyperther previous study we demonstrated increased penetration of cDDP mia, as can be seen from the significant decrease in AUC of into a peritoneal rat tumor when the i.p. treatment was com total platinum and a significant increase in clearance of both bined with abdominal hyperthermia (32). Higher i.t. platinum total and free platinum from the peritoneal cavity. To explain concentrations were achieved and the penetration distance of this difference in exposure, one must distinguish two periods: cDDP from the periphery increased. A disadvantage of this (a) the 90-min period of heating in which the peripheral blood combination was the concomitant increase in nephrotoxicity flow increases and the central blood flow will consequently which was, however, less than for whole body hyperthermia decrease (40); (b) the period of recovery in which the blood (33). Based on these results, experiments were performed with flow returns to normal but the membrane permeability still is hyperthermic CBDCA therapy. increased (23, 41). The observed pharmacokinetics may be The in vitro experiments with low and moderately toxic explained according to this scheme. During the heating period, CBDCA doses in combination with hyperthermia resulted in a exchange between the peritoneal cavity and the circulation will significant increase of intracellular platinum only at 43Â°C.In not increase since there is a decrease in the central blood flow. contrast, the cytotoxicity of a 1-h incubation with CBDCA was Fig. 6B shows that the difference in total platinum clearance of already higher at 40Â°C;this is in accordance with the results of the peritoneal cavity becomes obvious only after about 1.5 h. The clearance of free platinum at 41.5Â°Cis slow in comparison Xu et al. (34) obtained with lower CBDCA concentrations. An with the 37Â°Ccontrol (Fig. 6Ã„).One factor involved might be explanation for this phenomenon of increased cytotoxicity at unchanged uptake levels may be found in the process of hydra- the increased formation of reactive metabolites at 41.5Â°Cwhich tion. CBDCA itself does not significantly bind to proteins or may inhibit passage through the peritoneal membrane. A sim other nucleophiles; nonenzymatic transformation gives rise to ilar phenomenon has also been demonstrated for hyperthermic strong binding to aquated metabolites (35, 36). In this way, cDDP when given i.p. to both dogs and rats; this resulted in a CBDCA binds to nucleophilic sites on DNA, causing inter- relative increase of the free platinum concentration in the strand and intrastrand cross-links (35). The extent of binding peritoneal cavity compared to normothermic cDDP treatment probably determines the cytotoxic effect (37, 38). Data from (36, 42). A change in the chemical structure of the drug might the present study demonstrate that heat induces higher also be the reason in this case. After heating, the blood flow CBDCA-DNA adduci levels. Whether this is due to activation will return to normal but CBDCA will leave the peritoneal of the hydration process or to increased permeability of the cell cavity faster because of the still present increase in membrane membrane, followed by increased intracellular platinum con permeability. The tv,ÃŸforboth total and ultrafiltered platinum centrations, is not totally clear. The data in this study certainly in the peritoneal cavity decreased if combined with heat. This do not exclude a temperature dependent activation of the hy is in contrast with data obtained with cDDP in the same model dration process. In other words, relatively more CBDCA may (39). It means that the tumor exposure to CBDCA in the have been biotransformed into aquated metabolites at higher peritoneal cavity declines which may have severe consequences temperatures. The cytotoxicity experiments support this for drug uptake by the tumor via the direct peritoneal route. hypothesis. The slower clearance of CBDCA from the plasma, however, Potentiation of CBDCA action was more effective during 1 provides a higher exposure of the tumor by the systemic circu h exposure than during the continuous exposure experiment. lation and may compensate for the loss of tumor exposure in Since biotransformation is slow, this is likely to be a limiting the peritoneal cavity. A higher systemic exposure, however, factor in the short experiments. With continuous exposure, the may also have other consequences such as an increased myelo- relative enhancement of CBDCA-DNA adduct formation and suppression. 1256

McVie. J. G. Experimental and clinical results with intraperitoneal cisplatin. The experiments on the tissue distribution of i.p. CBDCA Semin. Oncol., 12:43-46, 1985. when combined with elevated temperatures show an increased Howell. B. S., Kirmani. S., McCIay, E. F., Kim, S., Braley, P., and Plaxe, S. platinum concentration in all tissues at 24 h after treatment. Intraperitoneal cisplatin based chemotherapy for ovarian cancer. Semin. Oncol.,/Ã„.-5-10, 1991. The larger increase in platinum concentration in tumors is due 12. Los, G., Ruevekamp. M.. Bosnie, N., de Graaf, P. Wâ€žand McVie, J. G. to both the direct penetration of platinum into these abdominal Intraperitoneal tumor growth and chemotherapy in a rat model. Eur. J. tumors from the peritoneal fluid and the higher tumor exposure Cancer Clin. Oncol., 25: 1857-1866, 1989. 13. Los, G., Nagel. J. D., and McVie, J. G. Anti-tumor effect of cisplatin, by the circulation. This increase in intratumoral platinum con carboplatin, mitroxantronc, and doxorubicin on peritoneal tumor growth centration might be beneficial with respect to the antitumor after intraperitoneal and intravenous chemotherapy: a comparative study. response. At 168 h after treatment, the absolute platinum Selective Cancer Ther.. 6: 73-82, 1990. concentrations were much lower for both the 37Â°Cand 41.5Â°C 14. 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-diamminedichloropla- treated group, but the tissue ratios between both groups were tinum(II) in intraperitoneal tumors after intraperitoneal chemotherapy: a still comparable to the 24-h values. comparison with systemic chemotherapy. Cancer Res.. 49: 3380-3384,1989. 15. Los, G., Mutsaers, P. H. A., Lenglet. W. J. M., Baldew. G. S., and McVie, In order to determine the role of the increased tumor expo J. G. Platinum distribution in intraperitoneal tumors after intraperitoneal sure by the circulation, the effect of abdominal hyperthermia cisplatin treatment. Cancer Chemother. Pharmacol., 25: 389-394, 1990. combined with i.v. CBDCA administration on the biodistribu- 16. 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 tion of platinum in various tissues was studied. These experi autopsy tumor samples after treatment with platin containing regiments. In: ments showed a rise in tumor platinum concentration at both Proceedings of Sixth NCI-EORTC Symposium on New Drugs in Cancer 24 and 168 h after heat treatment compared to control (37Â°C). Chemotherapy. Abstract 220. Amsterdam, 1989. 17. Canetta, R.. Rozencwieg, M., and Carter, S. K. Carboplatin: the clinical Comparing i.p. and i.v. treatment both with abdominal hyper spectrum to date. Cancer Treat. Rev.. 12 (Suppl. A): 125, 1985. 18. Wilshaw, E. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 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.

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