Antitumor activity of platinum(II) complexes with histamine and radioiodinated histamine in a transplantable murine adenocarcinoma model

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Nuclear Medicine and Biology 35 (2008) 605 – 613 www.elsevier.com/locate/nucmedbio

Antitumor activity of platinum(II) complexes with histamine and radioiodinated histamine in a transplantable murine adenocarcinoma model Piotr Garnuszek⁎, Urszula Karczmarczyk, Michał Maurin Department of Radiopharmaceuticals, National Medicines Institute, 00-725 Warsaw, Poland Received 20 June 2007; received in revised form 3 March 2008; accepted 19 March 2008

Abstract Purpose: Antitumor activity of the dichloroplatinum(II)–histamine complexes labeled with I-125 or I-131 was investigated in a transplantable murine adenocarcinoma (MA) model. Methods: The tumor model was obtained in C3H/W female mice after subcutaneous inoculation of the tumor cells derived from the mice bearing a mammary tumor of spontaneous origin. Antitumor activities of the platinum-histamine complexes were investigated in three independent experiments, which differed in applied doses of preparations (PtCl2Hist, PtCl2[125I]Hist, PtCl2[131I]Hist, PtCl2Hist/PtCl2[125I] Hist and PtCl2Hist/PtCl2[131I]Hist), treatment schedules as well as stages of the disease progress in the animals used. Experiment 1 included long-term, multidose treatment with low single doses (treatment duration 31–32 days; 8–10 doses of ca. 0.25∙MTDPt each). Experiment 2 included short-term, multidose treatment with higher single doses (4×ca. 0.5∙MTDPt up to Day 13 of the treatment). Experiment 3 included long-term concomitant multidose treatment with higher single doses (9×0.9–0.4∙MTDPt up to Day 33). Results: The long-term treatment with the platinum-histamine preparations revealed inhibiting activity on the tumor growth and size in comparison to control groups. The most intensive and significant antitumor effects were observed for the radioactive complexes. The tumor growth delay factors (GDFs) observed in Experiment 1 were 0.4, 0.7, and 1.2 for PtCl2Hist, PtCl2Hist/PtCl2[131I]Hist, and PtCl2Hist/PtCl2 [125I]Hist, respectively. Significant (Pb.05) prolongations of median survivals (MS) were found in Experiment 2 following the treatment with higher single doses of PtCl2Hist and PtCl2His/PtCl2[125I]Hist (Ratio MStr/MScon ca. 1.4). A slightly less potent activity was observed for PtCl2Hist/PtCl2[131I]Hist, and no survival improvement was found for the groups treated mostly with the radiation (PtCl2[125I]Hist and PtCl2 [131I]Hist). The intensive and long-term concomitant scheduling of the radioactive platinum–histamine complexes labeled with I-125 and I131 (Experiment 3) resulted in a significant inhibition of the tumor growth (GDF=1.9) and survival prolongation of the tumor-bearing mice (MStr/MScon=1.5, P=.023). The treatment-related toxicity was mild. Conclusion: An enhancement of the antitumor activity due to the multidose concomitant treatment with a combination of cytotoxic/cytostatic dichloroplatinum(II)–histamine and the attached iodine radionuclides was shown in the murine model of experimental neoplasm. © 2008 Elsevier Inc. All rights reserved. Keywords: outcome

Dichloroplatinum(II)–histamine; Iodine radioisotopes/therapeutic use; Chemoradiotherapy; Experimental neoplasm; C3H mice; Treatment

1. Introduction

⁎ Corresponding author. Department of Radiopharmaceuticals, National Medicines Institute, 00-725 Warsaw, Poland. Tel.: +48 22 718 0741; fax: +48 22 718 0740. E-mail addresses: [email protected], [email protected] (P. Garnuszek). 0969-8051/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2008.03.004

The strategy of a concomitant combination of an ionizing radiation and chemotherapeutic agents has proved to be advantageous for some types of malignant tumors [1–7]. cis-Diamminedichloroplatinum(II) (Cisplatin) and other platinum-based anticancer drugs are known as excellent radiosensitizers, wherein with a combination of external irradiation or with a low-dose continuous internal radiotherapy,

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these chemotherapeutics produce significant supraadditive treatment effects toward several tumor cells [8–11]. The intensive concurrent radiochemotherapy with an external irradiation and cisplatin may produce additive or synergistic interactions, significantly improving survival over radiotherapy alone in a regionally advanced disease, though often at a price of significant toxicity [5–7]. The internal radionuclide therapy with a low irradiation dose rate that is delivered selectively to the target tissues is more often an alternative treatment option [12]. Due to the short range of β− particles, or the ultrashort path length of Auger's electrons, specific radiopharmaceuticals produce a locally efficient therapeutic dose that acts in a continuous manner. The shortrange ionizing radiation decreases the risk of normal tissue damage; thus, the toxicity of internal radionuclides therapy is much lower in comparison to external irradiation. Several radioactive analogues of cisplatin, carboplatin and iproplatin have been synthesized using platinum radioisotopes such as 195mPt (4.02 d; IT), 193mPt (4.33 d; IT) and 191 Pt (2.9 d; EC). Their utilities for the determination of pharmacokinetics of platinum-cytostatics or therapeutic efficacy of the internal radiochemotherapy have been extensively studied [8,13–17]. For example, it has been demonstrated that the radioactive 191Pt-cisplatin is a more effective drug than the nonradioactive cisplatin in retarding tumor growth on the nude mice, without addition of systemic toxic effects [14]. However, in spite of the recognized anticancer activity of cisplatin and its radioactive analogues, the structure of this chemotherapeutic agent needs some modifications in order to reduce side effects of the therapy. Furthermore, there are some obstacles in using the platinum radionuclides in the clinical practice. The most important are, for example, their limited accessibility and high production cost (191Pt) or, else, a low specific activity (195mPt).

Fig. 1. Chemical structure of the platinum(II)–histamine complexes.

Considering the growing role and benefits of the concomitant combination of an ionizing radiation and chemotherapeutic agents for combating cancer, new radioactive platinum–[*I]histamine complexes (Fig. 1) containing PtII core and a radiotherapeutic isotope (I-131–β-emitter, or I125–prolific emitter of Auger electrons) — the moieties of plausible synergistic anti-cancer potency — have been developed and evaluated in our laboratory [18–22]. In vitro experiments showed that PtCl2Histamine complex had a cytostatic and cytotoxic activity against the human mammary adenocarcinoma cell line MCF-7 and against the human colon cancer cell line COLO-205, though slightly less potent than cisplatin [19,21]. Preliminary in vivo experiments showed a marked and statistically significant survival prolongation of C57Bl6 mice bearing the transplantable colon-38 adenocarcinoma by treatment with the radioactive dichloroplatinum(II)–[125I]histamine complex [22]. The median survival time for the animals treated with the PtCl2Histamine complex was 27% longer in comparison to the control groups, whereas the almost 60% prolongation of median survival (Pb.005) was observed for the group treated concomitantly with the cold and radioactive complexes. The results of the previous in vitro and in vivo investigations [18–22] suggest a potential usefulness of platinum–[*I]histamine complexes for the radiochemotherapy of solid tumors. The present work is devoted to the examination of in vivo antitumor activity of the platinum (II)–[*I]histamine complexes in the murine model of transplantable mammary adenocarcinoma. 2. Materials and methods 2.1. Chemicals The PtCl2Histamine complex (PtHist; Mw 377 g/mol) and its radioactive derivatives labeled with iodine-125 and iodine131 were synthesized by heating the solution of potassium tetrachloroplatinate(II) with histamine or radio-iodinated histamine (i.e., 125I- or 131I-histamine), according to the developed procedure [18]. The radioactive platinum complexes were prepared and stored in stock solutions of a slightly acidified dimethylformamide (DMF). The purity and the radiochemical purity of the platinum–histamine complexes were monitored using the high-performance liquid chromatography and the paper radioelectrophoresis technique and were usually greater than 98% even after the 1-month storage at room temperature. Tested solutions of the cold and radioactive complexes were prepared immediately before use in 0.9% saline and contained the maximal DMF concentration of 15%. The specific activities of the complex used in this study were in the range of 10–30 MBq per 1 μmol of Pt. The determination of platinum contents in samples were done by the colorimetric method based on a well-known reaction between PtII and SnII, in which the orange binuclear complex is formed with a maximum absorption at λmax=401 nm (ε=13,000 M−1 cm−1).

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Table 1 Treatment schedules and doses (protocols TM.1) applied to the tumor-bearing mice in Experiment 1 Protocol no.

Treatment group

TM.1.0

Control 15% DMF in 0.9% NaCl (n=8) PtHist (n=7) PtHist/Act.I-125 (n=8) PtHist/Act.I-131 (n=5)

TM.1.1 TM.1.2 TM.1.3

Schedule of injections

Pt doses

Radioactivity doses

Single doses of the platinum complex (μmol/kg)

Total injected dose of Pt (μmol/kg)

Single doses of radionuclide (MBq/kg)

Total injected dose of radioactivity (MBq/kg)

d0, d4, d7, d11, d14, d18, d20, d25, d28, d32

–

–

–

–

d0, d4, d7, d11, d14, d18, d20, d25, d28, d32 d0, d4, d7, d11, d14, d18, d20, d25, d28, d32 d0, d3, d8, d13, d17, d21, d26, d31

10–15

135

–

–

10–15

135

17–27

211

12–15

115

50→15

258

2.2. Mice and tumor model Female C3H/W mice with transplantable mammary tumors [23] were used in the experiments. The animals were purchased from the Department of Genetics and Laboratory Animal Breeding Cancer Centre, Institute of Oncology (Warsaw, Poland). During the experiment, the mice were kept in an area maintained at the standardized temperature 22±1°C and 12-h light/dark cycle, and had access to a rodent chow and water ad libitum. The heterotopic tumor model of the transplantable murine adenocarcinoma (MA) was obtained in C3H/W female mice after subcutaneous (s.c.) inoculation of the cells suspension derived from animals bearing a spontaneously growing mammary tumor (histologically classified as mammary adenocarcinoma) [23]. The mammary tumor-bearing (MA) C3H/W mice were anaesthetized with halothane and were sacrificed by a cervical dislocation. Tumors were removed and, after washing with cold and sterile 0.9% NaCl, were sliced into 2–3-mm pieces and homogenized (1:9 w/v). A tumor cell suspension was filtered through aseptic gauze and then injected subcutaneously (150 μl) into an area of the dorsal region of a healthy mouse lightly anaesthetized with

pentobarbital sodium salt [60 mg/kg body weight (bw), intraperitoneal injection]. For the evaluation of anticancer activity of the platinum–histamine complexes, tumor-bearing C3H/MA mice obtained after the third (Experiment 1), fourteenth (Experiment 2), and sixteenth (Experiment 3) passages of mammary adenocarcinoma cells were used. They were 8–11 weeks old and weighed 18–22 g at the start of the experiments. All experiments on living animals were approved by The IVth Local Animal Ethics Committee in Warsaw and were carried out in accordance with the principles of good laboratory practice. 2.3. Assessment of maximum tolerated dose Essentially, the maximum tolerated dose (MTD) was evaluated for the nonradioactive platinum-histamine complex (doses from ca. 9 to 50 μmol/kg bw) following the single intraperitoneal (i.p.) injection of the preparation into tumor-bearing C3H/MA mice. Additionally, the reference solution of 15% DMF in 0.9% NaCl and radioactive PtCl2 [131I]Hist complex (Pt ca. 50 μmol/kg, I-131 ca. 75 MBq/kg) were also tested. The MTD was defined as the highest dose

Table 2 Treatment schedules and doses (protocols TM.2) applied to the tumor-bearing mice in Experiment 2 Protocol no.

Treatment group

TM.2.0

Control 15% DMF in 0.9% NaCl (n=11) PtHist (n=9) Act.I-125 spec. act. 15-25 MBq/μmol (n=6) PtHist/Act.I-125 (n=8) Act.I-131 Spec. act. 15–25 MBq/μmol (n=6) PtHist/Act.I-131 (n=8)

TM.2.1 TM.2.2 TM.2.3 TM.2.4 TM.2.5

Schedule of injections

Pt doses

Radioactivity doses

Single doses of the platinum complex (μmol/kg)

Total injected dose of Pt (μmol/kg)

Single doses of radionuclide (MBq/kg)

Total injected dose of radioactivity (MBq/kg)

d0, d4, d8, d13

–

–

–

–

d0, d4, d8, d13

25–35

125

–

–

d0, d4, d8, d13

9–11

40

50

200

d0, d4, d8, d13

25–35

125

50

200

d0, d4, d8, d13

8–12

40

50

200

d0, d4, d8, d13

25–35

125

50

200

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Table 3 Treatment schedules and doses (protocols TM.3) applied to the tumor-bearing mice in Experiment 3 Protocol no.

Treatment group

TM.3.0

Control 15% DMF in 0.9% NaCl (n=9) PtHist/Act.I-125/ Act.I-131 (n=6)

TM.3.1

Schedule of injections

Pt doses

Radioactivity doses

Single doses of the platinum complex (μmol/kg)

Total injected dose of Pt (μmol/kg)

Single doses of radionuclide (MBq/kg)

Total injected dose of radioactivity (MBq/kg)

d0, d3, d7, d9, d13, d16, d21, d28, d33

–

–

–

–

d0, d3, d7, d9, d13, d16, d21, d28, d33

45→20

255

I-125: 30–100

I-125: 355

I-131: 125→25

I-131: 435

determined on the basis of the bw loss of b15% within 5 days of postinjection and the absence of early death. 2.4. Treatment schedules and dosing Antitumor activities of the platinum–histamine complexes were investigated in three independent experiments, which differed in applied doses of the preparations, treatment schedules as well as stages of the disease progress in the animals used: Experiment 1: long-term multidose treatment with low single doses (treatment duration — up to 31–32 days; 8– 10 doses of ca. 0.25∙MTDPt each). Dosing and scheduling of the preparations are presented in detail in Table 1. The treatment was started 22 days after the inoculation of tumor cells (MA) into C3H mice, and cells were derived after the third passage. Experiment 2: short-term, multidose treatment with higher single doses (4× single doses of ca. 0.5∙MTDPt up to Day 13 of the treatment). Dosing and scheduling of the preparations are presented in detail in Table 2. The treatment of C3H/MA mice was initiated at Day 21 post implantation of the tumor cells (the 14th passage). Experiment 3: long-term concomitant multidose treatment with higher single doses (9× single doses of ca. 0.9–0.4∙MTDPt up to Day 33 of the treatment). Dosing and scheduling of the preparations are presented in detail in Table 3. The treatment of C3H/MA mice was initiated at Day 32 post implantation of the tumor cells (the 16th passage).

The multidose therapy was started when tumors reached ca. 0.5–1.0 cm in diameter. The animals, randomly divided in groups (min. 5, max. 11 animals per group), were treated according to the specified protocols by i.p. administration of 0.1 ml solutions containing cold PtHist complex, PtCl2 [125 I]Hist or PtCl2[131I]Hist [the radioactive complexes of low PtII contents (Act.I-125, Act.I-131, respectively)] and PtCl2Hist/PtCl2[125I]Hist, and/or PtCl2Hist/PtCl2[131 I]Hist [the radioactive complexes containing cold PtHist complex (PtHist/Act.I-125 and PtHist/Act.I-131, respectively)]. The platinum–histamine complexes were dissolved in 15% DMF in saline, and then these solutions were applied to the control groups. 2.5. Evaluation of antitumor activity Antitumor activity of the tested preparations was evaluated based on the variations of the relative tumor volumes of the treated mice and that of the control mice and/ or by the comparison of the median survival times. 2.5.1. Relative tumor volume Based on the variations of the relative tumor volumes, antitumor activity was evaluated using two methods: (i) using the GDF, defined as the mean number of tumor-doubling times (TD) gained by the treatment and (ii) calculating the T/C values — the ratio of the relative tumor volume of treated mice (RTVtr) to that of control mice (RTVcon). The tumor size was determined using an electronic caliper measurement every 2–3 days, and the tumor volume (TV)

Table 4 Applied preparations and mean variations of bw of C3H/MA female mice in the examination of the MTD Preparation

15% DMF in 0.9% NaCl PtHist PtHist PtHist PtHist PtHist/Pt[131I]Hist

No. of mice in group

Mean real dose (μmol Pt/kg)

Mean and 1S.D. of bw changes (%) 24 h

48–72 h

96–120 h

5 5 4 3 5 5

– 9.2±0.8 36.4±1.6 41.1±2.6 48.3±3.0 48.9±1.9 (73±3 MBq/kg)

4.5±4.2 3.2±1.8 0.8±2.4 −3.8±4.3 −5.1±2.1 −7.4±2.8

3.3±2.5 6.9±2.6 1.7±4.2 −0.6±2.4 −10.5±4.0 −13.4±4.7

9.2±1.4 9.3±3.8 5.6±3.9 −0.2±3.9 −1.2±5.0 0.8±3.7

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was calculated according to the following formula: TV (mm3)=0.5×length×width×height [24]. The tumor volume was expressed (%RTV) in relation to that determined on the first day of the treatment (d0). A nonlinear regression was applied for the estimation of the mean TD of the TV from an exponential curve of RTV variations for the individual groups of treated animals [25]. Growth delay factors were calculated from the expression: GDF=(TDtr− TDcon)/TDcon, where TDtr means a TD of the treated group and TDcon, of the control group. The two-way analysis of variance (ANOVA) was applied for a comparison of tumor growth variations between groups of animals during the treatment. In a significant case of overall analysis, it was followed by posttests using the Bonferroni method for pairwise comparisons. 2.5.2. Survival analysis Antitumor activity of the tested preparations was evaluated by means of the survival prolongation of tumorbearing animals as a result of the treatment received. The Kaplan–Meier method was applied for the estimation of survival curves, and a comparison of the survival curves between each treatment and control group was performed with a log-rank test (the Mantel–Haenszel test). Based on the applied methodology, the common estimates of the differences between the survival curves were calculated: the median survivals (MS) MStr/MScon ratios, and hazard ratios (HR) — the death risk in a treated group in relation to the death risk in the control group. Survival analysis and 95% confidence intervals (95% CI) for the estimated parameters were performed with the help of the GraphPad Prism computer software (Version 4.0, GraphPad Software, San Diego, CA, USA, 2003). 3. Results 3.1. Maximum tolerated dose The mean bw changes of the animals following single i.p. administration of the tested preparations are shown in Table 4. Only the dose of PtCl2Hist at ca. 50 μmol/kg (18.9 mg/kg) resulted in a significant decrease of the mean bw of ca. −10% (duration up to 72 h post i.p. administration). The administration of the radioactive Pt[131 I]Hist complex at the doses of ca. 50 μmol Pt/kg and ca. 70 MBq/kg resulted in the mean loss of the bw of 13.4±4.7%. Therefore, with respect to the observation of a

Fig. 2. Comparison of tumor growth in the tested groups of the tumorbearing C3H mice treated in Experiment 1 (mean and S.E.M. of RTV%).

significant decrease and variations of the mean bw, and considering planned experiments of the multidose treatment, the dose of ca. 50 μmol Pt[*I]Hist/kg bw has been assumed as MTD for dichloroplatinum(II)–histamine complex and its radioactive derivatives. 3.2. The treatment response 3.2.1. Treatment experiment 1 All of the three preparations, that is, PtHist, PtHist/Act. I-125 and PtHist/Act.I-131 scheduled for the tumorbearing C3H/MA mice according to the protocols of the low-dose, long-term treatment (protocols TM.1, Table 1), revealed inhibiting activity on the tumor growth and size, in comparison to the control group treated with a solution of 15% DMF (Fig. 2, Table 5). However, the most intensive and statistically significant antitumor activity was observed for the radioactive complexes and, especially, for those labeled with iodine-125 (emitter of Auger's electrons). The tumor GDFs were 0.4 and 0.7 for the group treated with PtHist and PtHist/Act.I-131, respectively, whereas for the group treated with PtHist/Act.I-125, it was 1.2. Due to a considerably long disintegration rate of the I-125 (t1/2 59.4 d), the antitumor effects of PtHist/ Act.I-125 lasted longer than that of the I-131 (t1/2 8.02 d) labeled complex (Fig. 2). Unfortunately, none of the groups of animals treated in Experiment 1 showed improvement of the median survival time in comparison to the control group.

Table 5 Summary of the antitumor activity of the tested platinum(II)–[*I]histamine complexes observed in Experiment 1 Treatment group

TD (95% CI of TD)

GDF

T/C

Days of significant T/C observed

Control PtHist PtHist/Act.I-125 PtHist/Act.I-131

7.7 d (6.5–9.5) 10.8 d (8.7–14.1) 16.9 d (13.0–24.1) 13.2 d (10.9–16.8)

– 0.4 1.2 0.7

– ca. 0.7 (Pb.01) 0.5–0.4 (Pb.001) 0.7–0.6 (Pb.01)

– 54–60 42–60 50–60

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Table 6 Summary of the survival analysis for the tumor-bearing C3H/MA mice treated with the platinum(II)-[*I]histamine complexes (Experiment 2) Treatment group

TD (95% CI of TD)

MS

Ratio MStr/MScon (95% CI of ratio)

Log-rank test (vs. control), P value

HR (95% CI of HR)

Control PtHist Act.I-125 PtHist/Act.I-125 Act.I-131 PtHist/Act.I-131

5.6 7.2 5.5 6.4 5.3 7.2

31 d 43 d 30 d 44 d 37.5 d 39 d

– 1.39 (0.99–1.79) 0.97 (0.60–1.34) 1.42 (1.05–1.79) 1.21 (0.86–1.56) 1.26 (0.87–1.65)

– P=.024 P=.774 P=.031 P=.395 P=.134

– 0.42 (0.09–0.84) 1.13 (0.36–3.90) 0.38 (0.11–0.90) 0.65 (0.22–1.81) 0.52 (0.16–1.27)

d (4.7–6.7) d (6.5–8.1) d (5.0–6.1) d (5.6–7.4) d (5.0–5.6) d (5.7–9.6)

The two-way ANOVA of the rate of tumor growth between the groups showed extremely significant overall treatment effects for all the groups treated with platinum– histamine preparations. The pairwise comparison by Bonferroni posttests revealed statistically significant differences of tumor growths beginning from Day 20 of the experiment (Day 42 of posttumor implantation). The mean RTVs in the group treated with PtHist/Act.I-125 (TM.1.2) were over two times lower in comparison to the RTVs in the control group as early as at Day 20 of the treatment (at Day 20 T/C=0.49, Pb.05; later T/C=ca. 0.4, Pb.001). Moreover, it should be emphasized that beginning from Day 28 of the experiment, variations of tumor growths between the groups treated with PtHist (TM.1.1) and PtHist/Act.I-125 were extremely significant (Pb.001), and the RTVs observed in the group treated with the 125 I-labeled complex (TM.1.2) were approximately two times lower. 3.2.2. Treatment experiment 2 Table 6 presents results of the survival analysis performed for the treatment of C3H/MA animals according to the protocols of short-term, multi-dose treatment with higher single doses (protocols TM.2, Table 2). Similar and statistically significant (Pb.05) prolongations of the median survival, as well as the HR (ca. 0.4) were found after a treatment with PtHist and PtHist/Act.I-125 preparations (TM.2.1 and TM.2.3, respectively). A slightly less potent

Fig. 3. Kaplan–Meier survival curves for the C3H/MA mice treated with platinum–[*I]histamine complexes in Experiment 2.

activity was observed for PtHist/Act.I-131 (TM.2.5), and absolutely no survival improvement in comparison to the control group (TM.2.0) was found for the animals treated with Act.I-125 and Act.I-131 preparations (TM.2.2 and TM.2.4, respectively). Fig. 3 shows the Kaplan–Meier survival curves estimated for the treated groups except that these two were treated with radioactive preparations of low content of platinum(II). In spite of the survival prolongation observed in most of the groups treated in Experiment 2, the short-term applications of the platinum–histamine complexes did not influence in general the rate of tumor growths. No statistically significant inhibition of tumor growths was observed in the treated groups in comparison to the control group (Table 6). 3.2.3. Treatment experiment 3 The intensive long-term treatment with combined scheduling of PtHist/Act.I-125 and PtHist/Act.I-131 preparations (Protocol TM.3.1, Table 3) resulted in the highest antitumor effects gained. The rate of tumor growths in the treated group was significantly lowered, and the tumor growth was occasionally completely inhibited (tumor control; Fig. 4). The overall GDF of 1.9 was achieved owing to the treatment. A significant (P=.023) prolongation of the survival time (Fig. 5), as well as the low risk of death (HR=0.32) was also gained in the treated group (Table 7). The median survival

Fig. 4. Comparison of tumor growth in the control and the treated C3H/MA mice in Experiment 3 (mean and S.E.M. of RTV%).

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overall treatment effect with the combination of PtHist and I-125 radiation (Pb.0001). Additionally, beginning from Day 28 of the experiment, the differences between RTVs observed in the animals treated with PtHist and PtHist/Act.I-125 were also extremely significant (Pb.001), and the ratios of average RTVs in the PtHist/Act.I-125 treated group was ca. 0.5 compared to that observed in the group treated with the cold complex alone (TM.1.1). On the other hand, considering results of treatments performed in Experiment 2 according to the protocol TM.2 (Table 2), it is evident that administration of the preparation containing I-125 radionuclide and a low amount of PtCl2Hist complex (Act.I-125; prot. TM.2.2) was therapeutically ineffective (Table 6). Thus, the radiotoxicity of I-125 radionuclide alone is far inadequate to produce significant antitumor effects. Therefore, it may be concluded that the significant increase of tumor GDF observed for PtHist/Act.I-125 in comparison to PtHist in the Experiment 1 (the difference between the overall GDFs ca. 0.8), might result from a synergy between chemo- and radiotoxic agents. This might be due to a radiosensitization of tumor cells [26] or inhibition of an intracellular repair mechanism arising from molecular interaction between the drug and radiation [27]. However, further studies are needed to explain the mechanism of coaction between PtCl2–histamine complex and the Auger electrons. In the case of the 131I-labeled complex (PtHist/Act.I-131; TM.1.3), a slightly worse antitumor activity was observed in comparison to the treatment with the 125I-labeled preparation (PtHist/Act.I-125; TM.1.2). Inhibiting activity of PtHist/Act. I-131, observed in Experiment 1, decreased quickly after discontinuing the dosing of the preparation (Fig. 2). Nevertheless, it is worth noting that following TM.2.4 protocol in Experiment 2, relative prolongation of mice survival treated with Act.I-131 was achieved, although statistically insignificant (Table 6). It can be assumed, therefore, that these phenomena arise due to a nonspecific radiotoxicity of I-131 radionuclide (γ 364 keV; β Emax 0.61 MeV; average 0.192 MeV), which generate a cross-fire effect that involves not only the single tumor cell that incorporated the radioactive complex but also nearby cancerous cells nested within the range of the emitted particles. Thus, spatial cooperation of I-131 radiation and in-cell deposited PtCl2Hist complex could explain a little bit higher activity of Act. I-131 preparation in comparison to Act.I-125 preparation. Our previous [19,20] as well as recent systematic biodistribution studies (data not shown) revealed that the dose of PtCl2[*I]Hist complex administered intravenously or i.p. into rodents was scattered in soft tissues, mainly in the

Fig. 5. Kaplan–Meier survival curves for the C3H/MA mice treated with the platinum–[*I]histamine complexes in Experiment 3.

was more than 50% longer compared to the control group (ratio of MStr/MScon=1.52). It is worth noting that the treatment schedule applied in the Experiment 3 did not cause serious toxicity. No toxic deaths were observed. The mean bw loss (after diminished mass of the tumor) was transient and occasionally reached only just 15% when the administered doses were near to the MTD. 4. Discussion The recent study performed in the murine heterotopic model of transplantable s.c. adenocarcinoma (C3H/MA) confirmed that the new platinum(II)–histamine complexes had in vivo anti-cancer activity inhibiting tumor growth and prolonging the median survival time of the treated animals. The degree of antitumor efficacy was dependent on the administered preparations and the treatment schedules, as well as on progression of the tumor disease in the animals employed. In Experiment 1, application of the long-term, multi-dose treatment protocols with low single doses of platinum complexes (ca. 1/4 MTD) enabled to observe an intensification of inhibiting activity on tumor growth and size in the groups of animals treated concomitantly with the cold and radioactive complexes (Table 5). Application of the complex labeled with I-125 radionuclide (PtHist/Act.I-125), the prolific Auger electrons emitter of considerable low disintegration rate (t1/2 59.4 d; EC, γ, e−; 25 Auger electrons per decay), showed marked and long-lasting antitumor effects. Statistical analysis by two-way ANOVA followed by Bonferroni post hoc test revealed extremely significant

Table 7 Summary of the antitumor effects in the tumor-bearing C3H mice treated concomitantly with PtHist and 125I/131I-labeled complexes (Experiment 3) Treatment group

TD (95% CI of TD)

GDF

MS

Log-rank test, P value

Ratio MStr/MScon (95% CI of ratio)

HR (95% CI of HR)

Control PtHist/Act.I-125/ Act.I-131

6.7 d (5.9–7.7) 19.3 d (15.7–25.1)

– 1.9

41 d 62.5 d

– P=.023

– 1.52 (1.22–1.83)

– 0.32 (0.07–0.82)

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liver, gastrointestinal tract and kidneys. In general, physiological distributions of the platinum–histamine complexes show some similarity to that observed for cisplatin and other platinum-based drugs [13,15,16]. The complex administered i.p. in the tumor-bearing mice was excreted mainly, although moderately, with urine, with an average elimination half-life of ca. 25 h. An average accumulation of the radioactive complex in a tumor tissue, observed in our studies, was at the level from ca. 1.5 to 4% injected dose per gram, depending on a specific activity of the preparation applied and the tumor model used, and usually slightly decreased with time post administration. This suggests a nonspecific mechanism of the complex accumulation in tumor, most probably due to a higher vascularization of tumor tissue and a higher tumor metabolic activity compared to a normal muscular tissue. Referring back to the results of the present study, it is obvious that the real in-cell deposition of the platinum complexes decides on the antitumor activity gained, especially in the case of PtCl2[ 125 I]Hist complex. In consequence of the intrinsic elimination rate of the platinum-histamine complexes from the tumor tissue, the mean effective dose deposited in the tumor cells is moderately low and should be continuously supplemented to achieve desirable antitumor efficacy. Considering the abovementioned speculations, preliminary, long-term intensive treatment with a combination of PtCl2Hist complex and its radioactive derivatives labeled with I-125 and I-131 radionuclides has been performed (Experiment 3). The treatment of tumor-bearing C3H/MA mice with combined scheduling of PtHist/Act.I-125 and PtHist/Act.I-131 preparations (TM.3.1, Table 3) resulted in an observation of the highest antitumor effects gained. A significant inhibition of the tumor growth and prolongation of the survival of the tumor-bearing mice was achieved (Figs. 4 and 5). It is worth noting that keeping the deposited dose at an adequately high level throughout the treatment led to an efficient inhibition of the tumor growth. Due to frequent application of higher doses of the radioactive complex, a partial response (tumor control) was also observed (Fig. 4). The tumor remission has not been achieved; however, it should be noted that even late initiation of the intensive treatment (32 days post tumor implantation) led to a significant survival benefit of the mice bearing transplantable mammary adenocarcinoma. The long-term and intensive concomitant treatment with the cold and radioactive complexes labeled with I-125 and I-131 was the most beneficial. Multifactorial cancer cell killing might result, as discussed earlier, due to synergy between the chemotoxic platinum(II)–histamine complex and a short-range Auger's electrons emitted by the attached I-125 radionuclide. However, the extreme cytocidal effects of the Auger-electron emitter are manifested only when it is incorporated into the DNA of proliferating cells or placed in close proximity to it [28,29]. It seems evident that only a part of the platinum compounds reach the cell nucleus; thus, the rest of energy carried by Auger

electrons is ineffective. Therefore, due to a higher penetration of I-131 radiation (range in tissue ca. 0.8 mm), a spatial cooperation between platinum chemotoxicity and radiation could be achieved, and this type of cooperation could be advantageous for additional influencing of the tumor mass. The main purpose of combining chemotherapy and internal radiotherapy would be not to increase the cytotoxicity of the drug but rather to improve its therapeutic index. As it was shown previously in the biodistribution studies [19,21], platinum(II)–histamine complexes, like other platinum-based anticancer drugs, accumulate substantially in liver and kidney. It might be expected that like cisplatin, these complexes could produce serious side effects. However, the present study showed that the treatment-related toxicity was generally mild to low. No toxic deaths were observed due to the treatment according to the applied protocols. The toxicity of the complexes was followed particularly within the intensive and long-term treatment (Experiment 3). In that experiment, the mean bw loss of the mice treated intensively occasionally reached only just 15%, and a transient loss of the mean bw was observed when the absorbed dose of the platinum complex was sustained for a longer time at the proximity of the MTD. In conclusion, an enhancement of antitumor activity by a concomitant combination of the two therapeutic factors, namely, cytotoxic/cytostatic activity of dichloroplatinum (II)–histamine and the attached iodine radionuclides, has been shown in vivo in the heterotopic transplantable MA model. It should be emphasized that the radioactive platinum (II)-histamine complexes fulfill essential requirements of lowered toxicity, which, considering their in vivo antitumor activity, make them promising candidates for modern multifactorial strategy of combating cancer. The results of our study justify prospective investigation of internal radiochemotherapy of solid cancers by using the developed radioactive platinum complexes. Furthermore, the study pointed out that the treatment efficacy can be enhanced substantially by increasing the deposition of the complex in tumor tissue. Therefore, an intensive investigation has been started in our laboratory to modify the structure of radioactive platinum complexes for improving tumor targeting and prolonging the tumor retention time. Acknowledgments This work has been supported by grant No. 3 P05F 004 23 (2002-2004) from the State Committee for Scientific Research (KBN; Poland) and, in part, by grant No. N405 020 31/0911 (2006-2009) from the Ministry of Science and Higher Education (Poland). References [1] Bachaud JM, Chatelut E, Canal P, Albin N, Yardeni E, David JM, et al. Radiotherapy with concomitant continuous cisplatin infusion for

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