Jqq$ In!. J. Cancer: 68,452-456 (1996) C 1996 Wiley-Liss, Inc. -'s;-: Publication ofthe International Union Against Cancer Publication de I'Union InternationaleConrre le Cancer TUMORAL PLATINUM CONCENTRATIONS IN PATIENTS TREATED WITH REPEATED LOW-DOSE CISPLATIN AS A RADIOSENSITIZER Jean-Leon LAG RANG^:^, Pierre-Yves BONDIAU', Eric TESSIER', Pierre CHACVEL', Nicole RENEE', and Gerard MII.ANO~.~ Marie-Christine ETIEN!W* Departments of 'Radiation Oncology and *OncopharmacoIogy, Centre Antoine-Lacassape,Nice, France. The aim of the present study was to check whether platinum (Pt) concentrations achieved in tumors with a daily low-dose schedule were close to those promoting radiosensitization. Fifteen previously untreated patients with histologically proven advanced uterine cervix tumors were studied. They received a daily irradiation30 min after a short infusion of 5 mg cisplatin for 5 consecutive days every week. A biopsy was taken from the accessible tumor mass, 4 to 6 hr after the daily injection. Blood samples were obtained once a week, just before the daily injection (HO) and 30 min after (H30). Quantificative analysis of Pt concentrationswas performedby atomic absorption spectrophotometty. Plasma and tumoral Pt concentrations exhibited a marked intersubject variability. The median tumoral Pt concentration was 1,710 mg/g. Median total and ultrafiltrable Pt concentrations in plasmataken at HO and H30 were 23 I and 360 ng/ml. and 7 and 90 ng/ml respectively. Tumoral and total plasma Pt concentrations significantly increased during the course of treatment. Present data show that tumoral Pt concentrations achieved with this CDDP schedule are in the range of tumoral Pt concentrations previously shown to promote radiosensitization(2,000 ng/g). These results suggest that this CDDP radiosensitization regimen might be started with higher CDDP doses in order to reach earlier radiosensitizing Pt tumoral concentrations. 01996 Wiley-Liss, Inc. Cisplatin (CDDP) is one of the most active chemotherapeutic agents presently available. Apart from its direct anti-tumor effect, CDDP exhibits interesting and apparently complex interactions with radiation (Kallman, 1994). Experimental data are scanty and have suggested that platinum (Pt) concentrations around 2,000 ng/ml (i.e., 10 pM) are necessary to induce radiosensitization and inhibition of DNA repair processes (Douple and Richmond, 1978; Fu et al., 1988). Additional work by Douple et al. (1991) pointed to a threshold for tissular Pt concentration allowing radiosensitization; according to these authors, this threshold value should be located around 2,000 ng/g. There is currently no general consensus for the optimal CDDP dose and optimal timing in the sequence of administration of the drug and irradiation. However, data obtained both in vitro (Lagrange et al., 1993) and in vivo (Kallman, 1994) have suggested that CDDP is more effective when given a short time before irradiation. Van Harskamp et al. (1987) have previously reported interesting therapeutic efficacy obtained with a combination of daily low-dose CDDP quickly followed by radiotherapy for inoperable, non-smallcell lung cancer. In our institute, a pilot study was previously initiated based on the same principle of daily, low-dose CDDP plus radiotherapy in patients with inoperable cervical cancer. According to this protocol, the daily CDDP dose ( 5 mg) is given 30 min before irradiation (1.8-2 Gy) for 2-5 weeks, depending on the patient's disease stage. In order to better understand this treatment strategy, we initially investigated both plasma and tumor pharmacokinetics in these patients (Milano et al., 1990). The present study concerns the analysis of tumoral Pt concentrations during the course of treatment aiming specificallyto check whether Pt concentrations achieved in tumors with this daily low-dose protocol were close to those known to promote radiosensitization. The data presented herein are complementary to those which we published prcviously (Milanoet al., 1990). PATIENTS AND METHODS Fifteen previously untreated women (mean age 58 years, range 46-76) with histologically proven advanced uterine cervix tumors were entered into this study. All patients were treated with daily irradiation (1.8-2 Gy) 30 rnin after a short infusion ( 5 min) of CDDP ( 5 mg in 50 ml 0.9% NaCI) for 5 consecutive days every week. This treatment was applied for 2 weeks to patients with stage-I1 diseasc (cumulative dose: 50 mg CDDP, 20 Gy) and for 4 to 6 weeks to patients with stage 111-IV disease (cumulative dose: 100-150 mg CDDP, 40-60 Gy). Before starting treatment, all patients had normal renal function (blood creatinine value below 150 +mol/l), a Karnofsky performance status of at least 60% and a life expectancy of at least 12 weeks. Renal function (blood electrolytes, urea and creatinine) and hematological function (RBC, WBC, platelet count) were evaluated every week during treatment. Response to treatment was evaluated at the end of the treatment period. Complete response (CR) Corresponded to the disappearance of all clinically visible or palpable lesions; partial response (PR) was defined as tumor regression of >50% and was stratified between major PR ( > 75%) and minor PR (> 50% and <75%); no response (NR) corresponded to tumor regression of s50'%,stable disease, or progressive disease. Pharmacokinetic investigations Tiirnors Every week, a clinical examination was planned to check on changes in the tumor. At this time and whenever possible a biopsy was taken from the accessible tumor mass 4 to 6 hr after the daily injection once a week on the first or second day of treatment. In total, 27 tumor biopsies were performed during the treatment: 7 in the 1st week, 6 in the 2nd week, 7 in the 3rd week. 4 in the 4th week, 2 in the 5th week and one in the 6th week. Repeated tumor sampling (2 to 4) was performed for 6 patients. In addition, for 4 patients, biopsies were performed at a certain distance after the end of treatment. Blood samples In addition to tumor biopsies, blood samples were obtained once a week on the first or second day of treatment, just before a daily injection (HO) and 30 min after the end of the injection (H30). HO and H30 samples were taken whenever possible on the same day and from the same patients. In case of practical difficulties (patient's status), only the HO sample was taken. A total of 57 blood samples were thus analyzed. Only these 2 blood samples per patient and per week were requested in order to limit the weight of pharmacological investigations for patients. 3T0whom correspondence and reprint requests should be addressed, at the Centre Antoine-Lacassagne, Oncopharmaculogy Laboratory, 33 avenuc Valombrose, 06050 Nice, France. Pax:33 04 93 81 71 31. Received: April 25,1996 and in revised form August 12, 1996. 'TUMORAL PLATINUM CONCENTRATIONS IN LOW-DOSE CISPI.ATIN TREATMENT For 8 patients it was possible to obtain both a tumoral biopsy and blood samples on the same day. 453 RESULTS Considering all available data, tumoral and plasma Pt concentrations exhibited a marked variability between subjects. Tumoral Pt concentrations were comprised between 0 Treatment of samples and 12,600 ng/g (median 1,710, mean 2,200). At HO, total Pt varied between 0 and 507 ng/ml (median 231, mean 227, Blood n = 34) and U F Pt varied between 0 and 80 ng/ml (mcdian 0, Blood samples (3 ml) obtained in ethylenediaminetetraace- mean 7.1, n = 34). At H30, total Pt varied between 120 and 579 tic acid (EDTA) tubes were immediately placed in a water ng/ml (median 360, mcan 346, n = 23) and U F Pt varied bath containing ice for transport to the laboratory (within between 10 and 160 ng/ml (mcdian 90, mean 85, n = 21). 10-15 min) and were then centrifuged at 4°C. A 500-pIaliquot Table I illustrates the variations in tumoral and plasma Pt of the resulting plasma was centrifugcd for 30 min at 2,000 g concentrations according to duration of treatment course. (4°C) in a Centrifree micropartition unit (Amicon, Denvers, Interestingly, tumoral Pt concentrations significantly increased MA). The resulting ultrafiltrate fraction was used to determine during the course of treatment and this pattern was particuultrafilterable (UF) Pt. Total Pt was measured in the whole larly pronounced between the 1st and 4th weeks of therapy. plasma fraction. Samples were stored at -20°C until analysis. Also, total Pt concentrations in plasma significantly increased during the treatment course. The accumulation in total Pt concentration was observed in plasma samples taken both at Biopsies Biopsies (10-15 mg) were taken from the accessible tumor HO and at H30 min. In contrast, U F Pt concentrations mass. Excess blood was removed and the tissue sample was mcasurcd in plasma at HO and H30 did not reveal any accumulation pattern during treatmcnt course. placed in a screw-top plastic tube that was stored at -20°C until analysis. Biopsies were then weighed (wet weight) and Tumor biopsies were obtained from 8 patients on the same digested overnight with 500 FI 50% nitric acid, then homog- day as blood samples (H30). These 8 paired samples showed enized in a glass-glass potter. The homogenate was dried no correlation (Spearman rank correlation) between tumoral under a nitrogen strcam (50°C) and the dried residue was Pt Concentration and either plasma total Pt concentration at dissolved in 200 pI H 2 0 . H30 (p = 0.23) or plasma U F Pt concentration at H30 ( p = 0.17). In 6 patients, serial tumoral biopsies were obtained. Figure 1 Pt assay Quantificative analysis of Pt in samples was performed by illustrates the intra-patient accumulation of tumoral Pt concenatomic absorption spectrophotometry (AAS) using a Pcrkin- tration during the treatment course for these 6 patients. For 4 Elmer model 3030 atomic absorption spcctrophotometer with patients, biopsies were performed at distance, between 7 and background correction by the Zeeman effect for trace analysis. 21 days after the end of treatment. Figure 2 shows the decrease Ultrafiltrates and biopsy extracts were measured without in Pt tumoral concentrations for these 4 patients. In the limited set of 8 patients available for both tumoral dilution. Before analysis, plasma was diluted 1:2 in a 0.2% H N 0 3 solution containing 0.01% Triton X-100. The injected pharmacokinetics and clinical response, tumoral Pt concentravolume was 20 ~ 1 A. standard curve (0, 162,325,650 ng/ml of tions were not significantly different between patients with NR Pt) was automatically performed with an auto-sampler using a (<SO%) or P R <75% and those with P R > 7 5 % or C R standard Pt solution. Analysis included the following steps: (Mann-Whitney test). drying at 110°C for 40 sec; ashing at 1,400"C for 20 scc and atomization at 2,650"C with a 3-scc stop flow and tube cleaning at 2,650"C for 4 scc. The limit of sensitivity (2 times the noise) DISCUSSION was 5 ng/ml for plasma and 2.5 ng/ml for U F and biopsies. A review of published data concerning Pt tumoral concentraIntcr-assay reproducibility calculated from 25 successive series of analyses was 12.2%, 9.6% and 5.5% for Pt concentrations of tions is presented in Table 11. A feature common to all studies is the marked interpatient variability in Pt tumoral levels for a 30,300 and 900 ng/ml, respectively. TABLE I - DESCRIPTION OF TUMOHAL 1'1, PLASMA TOTAI. Pt AND PIASMA U F Pt CON('ESTRATlONS ACCORDING TO TREATMENT COURSE (WEEKS) . 1 2 230 (0-3100) n=7 1830 (20-2500) n=6 3 4 5 6 Link between increase in Pt concentrations (blood, tumor) and the duration of treatment (Spearman rank correlation) HO? ~ ~~ 1710 (350-5500) n=7 5525 (920-12600) n=4 2005(410-3600) n=2 600 n=l r = 0.41 p = 0.034 UF Pt concentration in plasma' (ng/ml) Total Pt concentration in plasma' (ngiml) Tumoral PI concentrationi (ntYg) Week ~. 106 (0-210) n=9 289 (134-410) n = 11 270 (224-507) n=6 252 (10-434) n=S 311 (304-363) n=3 n=O r = 0.59 p = 0.0007 1?3IS 197 (12M18) n=7 384 (190-520) n=6 400 (321493) n=4 360 (210-579) n=3 378(363-392) n=2 45 1 n=l r = 0.49 p = 0.021 .~ tIW H302 0 (0-9) n=9 90 (10-110) n=7 85 (20-160) n=6 112 (105-140) n=3 80 (54-100) n=3 58 n = l 90 10 (0-30) n = 11 0 (0-80) n=6 0 (0-11) n=5 12(6-13) n=3 n=O NS n = l NS 'Median and extreme values, n = number of patients.-?HO means just before the daily CDDP injection; H30 means 30 min after the end of the injection. LAGKANGE ETAL. 454 I I 8 I I 8 I I I I I I 0 7 14 21 26 35 100000 c w \ -m C 10000 E 2 L .r( U m 1000 I+ a. I+ m L 0 100 E 3 F I0 42 Days o f t h e r a p y RGURE 1 - Plot of the evolution of tumoral Pt concentrations for 6 patients undergoing serial tumoral biopsies (each symbol depicts a different patient). 10000 I I I 1 I I 8 I 0 7 14 21 -I5 \ m c Y E 1 1000 c .d I+ m 100 L 0 E 3 I- 10 Days a f t e r c e s s a t i o n o f t h e r a p y F~GURE 2 - Plot of decrease in tumoral Pt concentrations for 4 patients undergoing a biopsy on the last day of treatment along with a biopsy at distance after cessation of treatment (each symbol depicts a different patient). given dose, for instance at 100 mg/m2. When differcnccs between mean values are considered, it appears that data are quite well grouped, with values varying between 1,000 and 4,000 ng/ml when determined within 48 hr after drug adminis&ration,an exception being [he study of Pujol el al. (1990) where concentrations 10 timcs higher were reported. The fact that, in this later study, tumor samples were obtained shortly after the end of cisplatin administration is not sufficient to explain this markcd difference in Pt concentration as compared to other studics. Our data concerning low daily CDDP doses concur well with those published by Stewart et al. (1995) at a dose of 10 mg/m2. Furthermore, our results obtained after 3 and 4 wceks of rcpeated daily low-dose cisplatin, giving a cumulated dose of 75-100 mg, are within thc range of TUMORAI. PLATINUM CONCENTRATIONS I N LOW-DOSE CISPI.ATIN TREATMENT 455 TABLE I1 - PLATINUM COSCENTRAIIONS I N TUMORS-A KEVIEW OF THE LITERATURE ,r;:zs Author (reference) time after Tumor localization Cisplatin dose (protocol)' cisplatin injection -. 'Troger et al. (1991) Hecquet et al. (1985) Pujol et al. (1990) Hecquet et al. (1986) Gouyette er af. (1986) 14 22 8 8 9 Esophagus Cervix (9) Head and neck (10) Breast (3) ., Lung Cervix 24 Head and neck Mattox et a/. (1983) 5 Head and neck Vennin et al. (1985) 10 Cervix Holdinget al. (1991) 27 Head and neck Stewart et al. (1995) Los et al. (1995) Lagrange et al. (present study) 20 23 Different tumors Head and neck Cervix 15 24-36 hr 48 hr 48 hr 48 hr 30 min 30 min 100 me/m2 hr (i.v.) - (i.v. . + i.a.) 24 24 hr (La.) day 21 (i.v.) day 3 (72 hr) i.a. 1 hr 50 mg/m2day 1 i.a. 6 hr 50 mg/m2day 2 (i.v. + i.a.) i.v. 1 hr i.v. 6 hr 100 rng/m?,4 patients (i.v.) 2 hr 50 mg/m', 1 patient (i.v.) 6 hr 24 hr 50 mg, 2 patients (i.v.) 2 hr 100 mg, 8 patients (i.v.) 4 hr 24 hr 48 hr 100 mgim? (i.v.) 0 hr 24 hr 41-64 hr 500 hr 700 hr 10 mg/m2 (i.v.) 1.25 hr ( 5 min to 6 hr) 150 mg/m? (La.) 24 hr 5 mg/day (i.v.) 4-6 hr week 1 week 2 week 3 week 4 Platinum concentrations mean t SD and/or range W g ) 3,750 ? 2,300 1,800 f 1,000 1,600 f 800 4,200 f 1,400 51,130 f 65,500 22,490 f 53,900 1.OOO-5.Y00 '900-5;YOO 200-500 2.760 f 2.590 1;930 -t 11250 1.270 k 350 11320 f 410 1,800 1,100-2,800) 2,700 6004,400) 1,300 1,200-1,300) 500-7,250 4,970 f 8,750 2,610 f 1,930 1.830 f 1.060 3;800 l;500 3,700 ? 1,500 2,800 (8004,300) 300 15.000 380 f 140 60040,700 680 k 1,120 1,720 k 880 2.160 f 1.800 6,140 f $230 i * 'I.V., intravenous administration; i.a., intra-arterial administration. concentrations found after a single dose of cisplatin at 100 mg/m2. The constant accumulation of tumoral Pt we found during the treatment period is in agreement with experimental data showing that cisplatin uptake is not saturable (Gately and Howell, 1993). After treatment cessation, a diminution in tumoral Pt levels was obscrvcd in samples obtained 6 to 21 days after the end of the treatment period. This is consistent with the data reported by Holding et al. (1991) and Hecquet et al. (1986, 1987). There arc few data which describe CDDP efflux from cells. We recently undertook an in v i m study to analyze more thoroughly relative influx and efflux of CDDP from human cancer cells in culture (Troger et af., 1992). We observed that C D D P can leave the cells following a 2-slope kinetic pattern with an a half-life of 1.29 hr and a p half-life of 94.4 hr. The slow release of platinum from tissues has recently been emphasized by Schicrl et al. (1995). These authors examined urinary platinum concentrations in 23 men at 150-3,022 days after C D D P treatment for testicular neoplasm. Regression analysis showed 2 phases of long-term renal platinum excretion, one occurring between 150 and 900 days of follow-up and the other beginning at 900 days after cisplatin administration. Onc can spcculate on the possible cytotoxicity exhibited by the long-term circulating Pt species. We recently reported data concerning the in vitro cytotoxic effects of a serum aliquot taken 3 weeks after cisplatin administration in a patient with renal insufficiency (Lagrange et a/., 1994). Interestingly, this serum sample induced marked in vitro cytotoxic effects on cancer cells in culture which were much greater that those which could have been anticipated considering the Pt concentration of this serum sample. Thus, the release of Pt from tissues, as stressed in the present study, may lead to Pt circulating species which can play a role in the pharmacological effects of the drug. Troger et al. (1992) have shown that the cytotoxicity of C D D P in vitro is directly related to intracellular concentrations of the drug. O n the other hand, decreased Pt accumulation develops early during the selection of resistant cclls both in vitro and in vivo (Gately and Howell, 1993). Our knowledgc concerning the hypothetical link between tumoral Pt concentrations and clinical response is limited. So far, both Bielack et al. (1989) and ourselves (Troger et al., 1991) have failed to demonstrate any relationship between tumor Pt concentration and treatment efficacy. Here again, based on a small number of cases, we reached the same conclusions. Pujol et al. (1990) found a weak correlation between simultaneous plasma and tumoral Pt concentrations. In the present study, based on a limited number of paired samples, we failed to find a relationship between these 2 parameters. Thus, the variability in plasma pharmacokinetics does not seem to account for the differences in tumor Pt concentrations. It must be emphasized that, according t o the work of Fu et al. (1988), low-dose-rate irradiation probably had no influence on the pharmacokinetics of plasma and tumor Pt. Stewart et al. (1995) recently reported on factors affecting Pt concentrations in human tumor specimens. They found, by multiple stepwise regression analysis, that the patient characteristics most closely associated with tumor Pt concentrations were tumor type, serum calcium and chloride and bilirubin levels. Some studies indicate that the radiosensitizing capability of C D D P is highly time-dependent (Lagrange et a/., 1993; Kallman, 1994). C D D P would be more effective when given a short time before irradiation. This suggests that C D D P in tissues does not remain in a radiosensitizing form (not identified) for more than a limited period of time. However, some authors have reported the attainment of tumoral Pt concentrations around 2,000 ng/g which induced radiosensitization (Carde 456 LAGRANGE E T A L and Laval, 1981; Douple et a/., 1991). Table I shows that, on average, these optimal concentrations were obtained by the 2nd week of treatment. 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