J Cancer Res Clin Oncol (1992) 118:195-200

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Cancer ~esearch Clinical 9 9 Springer-Verlag 1992

Antineoplastic activity of three ruthenium derivatives against chemically induced colorectal carcinoma in rats Matthias H. Seelig ~, Martin R. Berger ~, and Bernhard K. Keppler 2 1 Institute of Toxicology and Chemotherapy, German Cancer Research Center, Im Neuenheimer Feld 280, W-6900 Heidelberg, Federal Republic of Germany 2 Institute of Inorganic Chemistry, University of Heidelberg. Im Neuenheimer Feld 270, W-6900 Heidelberg, Federal Republic of Germany Received 9 September 1991/Accepted 4 November 1991

Summary. The antineoplastic activity of the ruthenium complexes trans-imidazolium[tetrachlorobisimidazoleruthenate(III)], HIm(RuImzC14), trans-indazolium[tetrachlorobis(1H-indazole)ruthenate(III, N2)], Hind [RuIndzC14(N2)], and trans-indazolium[tetrachlorobis(2H-indazole)ruthenate(III,N1)], HInd[RuInd2C14(N1)] was assessed in acetoxymethylmethylnitrosamineinduced autochthonous colorectal carcinomas of Sprague-Dawley rats. The model is not sensitive to clinically established antineoplastic agents, including cisplatin. An exception is the combination therapy with 5fluorouracil/leucovorin, which shows moderate activity against the tumour model. In contrast to this general trend, the new substances were all active against this tumour. HIm(RuImzCl4) was very effective at all dosages applied (7.5 mg/kg, 5.3 mg/kg, and 3.8 mg/kg), as indicated by percentage treated/control (T/C values of 23 %, 34.5%, and 44%. Toxicity was considerable as shown by a body weight change of - 3 0 % , - 1 9 % , and - 9 % . Nevertheless, the medium dose seems to be the optimum in terms of mortality (0% vs 15% in the control group), whereas at the highest dose, mortality increased as a result of substance toxicity, and at the lowest dose mortality increased through tumor growth combined with substance toxicity. HInd[RuInd2C14(N2)] showed high efficacy at the highest dosage of 13 mg/kg, reaching a T/C value of 27% combined with 0% mortality versus 15% in the control group. In equimolar dosages (10 mg/kg, 7.1 mg/kg and 5.1 mg/kg), the compound is not as active as Him(RuIm2Cl4), as indicated by T/C values of 50.2%, 45.7%, and 38.6%. HInd[RuIndzC14(N1)] was slightly but not significantly better than HInd[RuIndzC14(NZ)] at a dosage of 7.1 mg/kg and is advantageous over combination therapy with 5-fluorouracil and leucovorin (20/ 20 mg/kg) in terms of efficacy (T/C=37.6% versus 44.7%) and mortality (6% versus 33.3%). Offprint requests to: M.R. Berger, Institute of Toxicology and Chemotherapy, German Cancer Research Center, Im Neuenheimer Feld 280, W-6900 Heidelberg, FRG

Key words: Ruthenium complexes - Acetoxymethylmethylnitrosamine-induced autochthonous colorectal carcinoma - Antitumour activity, 5-fluorouracil, leucovorin

Introduction Colorectal carcinoma is the second most frequent malignancy in men and the third most frequent in women in industrialized countries with increasing rates of incidence within the last few years (Wolmark et al. 1988). Surgical tumour resection is considered the treatment of choice (Schweiger and Gall 1986) but chemotherapy is the only possibility to slow down progression of the disease in cases of metastasis and incomplete surgical treatment. This treatment mode, however, has rendered mainly disappointing results (Galeano et al. 1990). Up to now there have been only a few agents in sustained clinical use, among which 5-fluorouracil alone or in combination with leucovorin is a schedule often administered (Arbuck 1989; Abruzzese and Levin 1989). However, none of these substances has been able to show more than marginal effects so far, and this situation could be improved by new, more active drugs. We have been involved in the synthesis and preclinical development of metal complexes with antineoplastic activity. After selection of interesting compounds by several transplanted turnout models in mice (Keller et al. 1982, 1983; Keppler and Schm/ihl 1986), further evaluation has included the use of an autochthonous, chemically induced colorectal carcinoma (Berger et al. 1984; Keppler et al. 1990). This turnout model was chosen because it mimics the human situation very closely, as underlined by the following characteristics (Berger et al. 1986): tumours grow orthotopically and can be classified as adenocarcinomas with the same histology and route of metastasis formation as compared to man; they show original tumour/host interaction, relatively slow growth kinetics and are resistant to all clinically used antineoplastic agents.

196

Therefore, it can be expected that this animal tumour model implies realistic criteria that can qualify agents with respect to their antineoplastic efficacy. Several ruthenium complexes have been synthesized and tested so far and revealed high antineoplastic activity against this tumour model (Garzon et al. 1987; Berger et al. 1989; Keppler et al. 1989). Here we show the results of several experiments that were designed to determine how the antitumour potency of three ruthenium complexes depended on their ligands and their steric configuration as well as to survey whether the size of the tumour at the start of treatment would have any influence on the therapeutic efficacy of these substances (Figs. 1, 2). In addition this therapy was compared to a standard schedule consisting of 5-fluorouracil in combination with leucovorin.

re

A /~NNNH | H

CI'~" I

H

CI

B

Materials and methods

thesized (Wiessler 1975) and kindly provided by Professor Wiessler, German Cancer Research Center. 5-Fluorouracil was provided by Medac GrnbH (Hamburg) and leucovorin by Lederle Company (Wolfratshausen).

Animals and tumour induction. The animals, 315 male Sprague-Dawley-rats (Charles River Breeding, Sulzfeld, FRG), were purchased at a weight of 140-160 g and thereafter kept under conventional conditions: 2 rats per Macrolon III cage, tap water and Altromin pellets ad libitum, dark/light cycle of 12 h. Colorectal carcinomas were induced using fresh 0.2% solutions of AMMN in physiological saline: 2 mg/kg were administered intrarectally at weekly intervals over 10 weeks by means of a rectal tube, the tip of which was inserted up to the colonic flexure.

J

~

C Substances. Acetoxymethylmethylnitrosamine (AMMN) was syn-

Ruthenium complexes. Commercially available ruthenium chloride (RuCla.3HzO), which is contaminated by ruthenium in oxidation state IV, nitroso complexes and others, was purified by refluxing with ethanol and HC1 to give a solution of Ru(III), which was then reacted with indazole (Ind). At high concentrations of hydrochloric acid, the compound trans-indazolium[tetrachlorobis(2H-indazole)ruthenate(III,N1)], Hlnd[RuIndzC14(N1)], could be obtained in high purity. At lower acid concentrations trans-indazolium[tetrachlorobis(2H-indazole)ruthenate(III,N2)], HInd[RulndzCl4(N2)], inevitably contaminated with small amounts of the (N~) compound, which could not be removed, was isolated. By reaction with imidazole (Im) the corresponding complex HIm(RuIm2Cl4) was obtained (Fig. 1) (Keppler et al. 1987, 1989). The compounds were characterized by elemental analysis, which indicated that all values were within a margin of error. The characteristic infrared frequencies (cm- x) for HIm(RuImzC14) are v (RuN) 247 m, and v (Ru-CI) 333 vs. For HInd[RulndzClg(N1)] they are v (Ru-N) 222m, and v (Ru-C1) 324s (300sh). For HInd[RuInd:Clg(N2)] they are v (Ru-N) 216 s, and v (Ru-C1) 323 s (295 sh). The paramagnetic properties of ruthenium in oxidation state II1 cause the signals of the coordinated ligand to be shifted to the high field of the NMR, whereas the signals of the uncoordinated protonated cationic heterocycle appear at a normal position. The high purity of the compounds was confirmed by HPLC investigations for HIm(RuIm2Cl4) and HInd(RuInd2Clg(N1)], whereas the compound HInd[RuInd/C14(N2)] was contaminated with small amounts of the (N ~) compound, which could not be removed because of its formation from the (N 2) compound. In addition, the structure of HIm(Rulm2Clr could be resolved by X-ray analysis (Fig. 2).

CI

Cl

H

CI

H

I

e

Cl

Fig. 1. Structures of the tumour-inhibiting ruthenium compounds: A trans-imidazoliurn(tetrachlorobisimidazoleruthenate(III), Him(Rulm2C1,0: B trans-indazolium(tetrachlorobis(IH-indazole)ruthenate(III,NZ)], Hlnd[RulndzClr C trans-indazolium[tetrachlorobis(2H-indazole)ruthenate(III,N1)], Hlnd[RuIndzC14(N1)] C~

CL 3~.

C '1

C2

N 2

N 2=

~

C 3a

Ct 3

C3

Ct 2

(Rulm2CI4)- anion in HIm(Rulm2CI4) Fig.2. X-ray structure of trans-imidazolium[tretrachlorobisimidazoleruthenate(III)], HIm(RulmzCla). Selected band distances (nm): Ru-Cll 0.2350(2), Ru-Cll 0.2342(1), Ru-C13 0.2356(1), Ru-N1 0.2079(3). Selected bond angles (~ 90.2(0.1), C12-RuN l a 89.8 (0.1), C13a-Ru-N1 89.8(0.1), Cll-Ru-C13 89.9(0.1), C12Ru-C13 90.1 (0.1), C13a-Ru-Nla 90.2(0.1)

Diagnosis of tumours and their evaluation. At the beginning of week 5 after completion of the 10-week induction period the animals were anaesthesized using chloralhydrate (320 mg/kg i.p., diluted in physiological saline). Endoscopic examination of the colon was performed carefully using a paediatric bronch0scope (Olympus 3F, type 4C2, Olympus Optical, Tokyo) (Narisawa et al. 1975; Merz et al. 1981), and animals with evident tumours were randomly allo-

197 cared to treatment and control groups (Table 1). Treatment started immediately thereafter and was continued for 10 weeks except for experiment 3, where treatment started 5 weeks after endoscopic diagnosis of tumours. All animals were treated i.v. twice weekly via the tail vein, using freshly prepared Hlnd[RuInd2C14(N2)] (0.22% and 0.12%) diluted in Haemaccel (Behringwerke, Marburg, experiment 1) or in distilled water (experiments 2, 3). Hind [RuInd2C14(N1)] (0.12%) and HIm(RuIm2C14) (0.13%) were dissolved in physiological saline. The animals were sacrificed after 10 weeks of treatment; those in a moribund state were killed prematurely. They were dissected and the last 20 cm of the gut was removed, opened and weighed. The volume of each tumour was estimated by measuring three diameters according to the formula a • b x c/2. Results were analysed according to the Kruskal-Wallis test (Kruskal et al. 1952a, 1952b).

Table 1. Treatment of acetoxymethylmethylnitrosamine (AMMN)-induced colorectal carcinoma in rats with ruthenium derivatives: experimental design

Ex-

Experiment I During the experiment the mean body weight of untreated controls increased by 3% while the mean tumour volume increased about 10-fold (from 97ram 3 to 934 mm 3) and the median tumour number rose from three to nine lesions (Table 2). A spontaneous mortality of 15% was observed, which could be ascribed to the growth of tumours. Treatment with HInd[Rulnd2C14(N2)] (Table2, groups 3-6) caused a moderate decrease in body weight

Group Compound

periment 1 2 3 4 5 6 7 8 9

a Treatment was given twice weekly and started following endoscopic diagnosis of tumours b Following tumour manifestation

Table 2. Treatment of AMMN-induced colorectal carcinoma in rats with ruthenium derivatives: anticancer efficacy and toxicity

Results

Ex-

Control Control Hlnd[RulndzCla(N2)] Hlnd[Rulnd2Clg(N2)] Hlnd[Rulnd2Clg(N2)] Hlnd[Rulnd2Cl4(N2)] Hlm(Rulm2C14) Hlm(RulmzCl~) Hlm(RulmzCl~)

Weekly dosage (mg/kg)

Total dosage (mg/kg)

Route of" No. of Duration applianimals of study cation (weeks) b

-

-

-

-

-

-

2 x 13 2 x 10 2 x 7.1 2x5.1 2x7.5 2x5.3 2 x 3.8

260 200 142 102 150 106 76

i.v. i.v. i.v. i.v. i.v. i.v. i,v.

10 11 12 13 14

Control Control Hlnd[Rulnd2Clg(N 1)] 2x7.1 142 Hlnd[Rulnd2C14(N2)] 2x7.1 142 5-FU/leucovorin 2 x 20/20 400/400

15 16 17 18

Control Control Hlnd[Rulnd2C14(N1)] 2 • 7.1 Hlnd[Rulnd2C14(N2)] 2•

Group Treatment mode

periment

-

-

-

-

-

-

-

-

-

-

142 142

i.v. i.v.

T/C" (%)

(mm 3)

a Ratio of increases in mean tumour volume of treated and control groups b Percentage of initial weight at the end of therapy c Significantly different from group 2 (P < 0.05) according to the Kruskal-Wallis test a One animal with a tumour volume of 6640 mm 3 was excluded from evaluation

.10 11 12 13 14 15 16 17 18

Control 1 Control 2 Hlnd[RulndzCl4(N2)] Hlnd[RulndzC14(N2)] Hlnd[RulndzCl4(N2)] Hlnd[RulndeC14(Nz)] Hlm(Rulm2 C14) Hlm(RulmzC1,) Hlm(RulmzC14)

97 (___144) 934(_1325) 100 249 (__.228) 26.7 469 ( _ 392) 50.2 427 ( + 398) 45.7 361 (_+508) 38.6 217 (___243) ~ 23 323 (+262) 34.5 411 ( + 526) 44

Control 1 75(+51) Control 2 872 ( _ 946) Hlnd[Rulnd2C14(N1)] 328 (_+121) d Hlnd[Rulnd2Cl,(N2)] 553 (-t- 356) 5-FU/leucovorin 390 (+__176) Control 1 Control 2 Hlnd[RulndzC14(N1)] Hlnd[Rulnd2C14(NZ)]

i.v. i,v. i.p./s.c.

-

Mean tumour volume

100 37.6 63.4 44.7

371 (+290) 1163 (-t-551) 100 733 (+351) 63 881 (+__1017) 75.7

0

10 10 10 10 10 10 10 10

1 5

-

(• 1 2 3 4 5 6 7 8 9

1 5

20 5 15 15 15 15 15 15

0

20 15 7 15

10 10 10 10

1 0

4

20 15 11

14 14 14

Median tumour number(95% confidence limits) 3 (2-4) 9 (5-14) 10 (6-15)

Change Morin tality body b (%) weight (%)

3 - 9

0 15 0

8.5 (5-13) -10

7

10 (7-15)

2

7

8 (4-10)

1

13

7 (4-12) 9 (6-14) 8.5(4-10)

-30 -19 - 9

20 0 27

4 8 9 8 8

(3-8) (7-9) (4-11) (5-16) (3-10)

3.6 0.9 2.6 - 1.3

0 5 6 0 33.3

8 (5-14) 9 (7-11) 9.5 (4-14) 7 (4-14)

- 3.5 -11.6 -12.3

0 45 33.3 16.7

--

198 ( - 8%, -- 10%, --2%) at dose levels of 13 mg/kg, 10 mg/ kg and 7.1 mg/kg, except for the lowest dosage of 5.1 mg/ kg ( + 1%). The mortality in the group treated with the highest dose, 13 mg/kg, which resulted in the best antitumour activity (T/C=27%), was 0% compared to the mortality in the control group of 15%. With decreasing doses and decreasing antitumour activity, mortality increased (7%, 7%, 13%) as a result of the reduced influence on tumour growth. The therapeutic efficacy in terms of T/C values was dose-related in the highest dosages (27% and 50%) and showed a plateau effect in the lower dosages (Table 2). Treatment with equimolar dosages of HIm(RulmzC14) (7.5 mg/kg, 5.3 mg/kg and 3.8 mg/kg) resulted in a severe dose-dependent decrease in mean body weight at all dosages applied (Fig. 3) and a mortality of 20%, 0%, and 27% respectively. Tumour growth was inhibited dose-dependently, as indicated by T/C values of 23% (statistically significant, P 200 mm 3, 50-200 mm 3) increased (Fig. 4). Treatment with HInd[RuInd2C14(N~)] and Hind [RuIndaC14(N2)] was only moderately effective as shown by T/C values of 63% and 75%, respectively. There was a 12% loss in body weight in both groups. The therapeutic efficacy becomes more evident from the reduction in mortality (control 45%, Hind [RuIndzC14(N1)] 33%, HInd[RulndzC14(Na)] 17%). This means that the therapeutic efficacy of both compounds in the treatment of large tumours becomes evident in an increase in survival time rather than in a reduced turnout volume, as the reduced mortality rates lead us to expect an increased survival time. Discussion

The experiments described were designed to confirm and extend results obtained in earlier studies that had shown a high anticancer efficacy of HIm(RuImzC14) and Hind [RuIndaC14(NZ)] in AMMN-induced colorectal carcinoma (Garzon et al. 1987; Berger et al. 1989). Now the optimum dose of HIm(RuIm2CL) had to be determined. In addition, HInd[RuInd2Clg(NZ)] had previously been applied at higher doses, which had to be administered within a large volume, which is difficult to inject intravenously. Now the efficacy of lower doses of Hind [RuIndzC14CN2)] had to be evaluated. Hind [RuInd2C14(N1)] was added to this comparison to determine whether the position of the nitrogen atom within the indazole ring binding to ruthenium would influence the activity. Moreover, this complex can be synthesized and isolated in high purity, in contrast to Hind [Rulnd2C14(N2)], which still contains small amounts of the (N 1) compound that cannot be removed. HIm(Rulm2C14) was active dose-dependently, as was shown earlier. There was evidence again of the good antineoplastic activity of Hlnd[RuInd2C14(N2)] although the results are not quite as impressive as before. In terms of toxicity HIm(RuImaC14) seems less advantageous than the indazole compounds, appearing to be even more toxic than it was in the previous experiments (Garzon et al. 1987). Nevertheless, it should be pointed out that the medium dose of 5.3 mg/kg produced considerable antitumour activity (T/C = 34.5%) combined with 0% mortality as against 15% mortality in the control group. At higher doses, a better effect in terms of T/C values could be obtained, but this was combined with a high mortality of 20% and a body weight reduction of 30%. At lower doses, the antitumour activity decreased to a T/C value of 44%, and mortality increased once again to 27% owing to toxicity from ineffective treatment combined with tumour growth. Thus the dose of 5.3 mg/kg should now be considered the optimum dose in this treatment schedule against colorectal carcinomas. Hlnd[Rulnd2C14(N1)] and Hlnd[Rulnd2C14(N2)] were shown to be relatively non-toxic complexes. This is also emphasized by the fact that the standard therapy of 5-fluorouracil and leucovorin resulted in an inacceptably high mortality (33%) and only a moderate antitumour activity (T/C = 45%), while HInd[RuInd2C14(NZ)] at the

optimum dose of i3 mg/kg reached a T/C value of 27% combined with 0% mortality. Besides, this effect of the indazole compound was not as good as that obtained in earlier experiments, which resulted in T/C values between 5% and i0%. In terms of any change in tumour weight, both compounds, (N 1) and (N2), seem to be less active against large tumours (experiment 3), but the fact that both substances produced a significantly lower mortality compared to the control group is an indication that prolonged survival times can be expected. Several factors probably contribute to the fact that the results obtained in this set of experiments reveal some differences compared to former results. In contrast to the experiment with Hind [Rulnd2C14(N2)] performed earlier, the effect of lower dosages of this complex was to be evaluated in the present series, for the substance has only a low solubility and the i.v. injection volume was not to exceed 4 ml/animal of 500 g weight. Therefore, only one group with 5 animals was treated with the high dose of 13 mg/kg, which had produced the best antitumour activity in earlier experiments. This dosage was contained in a relatively large volume of a dark-violet-coloured solution. Administration of such solutions is no problem under clinical conditions but required a change in the administration route in our experiment because the tail veins of the rats became inaccessible. Intraperitoneal administration, however, is unfavourable for this type of compound as has been shown recently (Berger et al. 1990). A more suitable galenic formulation would be helpful to minimize this problem. Furthermore it must be noted that the tumour growth of the untreated controls was relatively sluggish in these experiments. The mean tumour volume increased only 11- or 12-fold (from 97mm 3 to 934mm 3 and from 75 mm 3 to 872 mm 3) while in the experiments published earlier it had increased almost 30-fold. As efficacy of therapy is determined in relation to the growth of the untreated control, a low proliferation rate in this group reduces the antineoplastic potency. It is supposed that the antineoplastic effect of ruthenium complexes is due to the more reactive Ru(II) that is generated from Ru(III) under reductive conditions in biological media (Clarke 1980). A reductive environment is found within the poorly vascularisized, necrotic centre of large tumours and therefore it was assumed that ruthenium complexes might be more effective against such large tumours. Attempts were made to imitate this situation in experiment 3, where the start of treatment was retarded for 4 weeks. However, the histological examination of the control group (group 15) could not reveal any tumour necroses at the start of therapy so that tumour masses probably were too small to show the suggested effect and the underlying hypothesis still remains to be proven. It has to be borne in mind that the tumours again showed a very slow growth kinetic: the mean tumour volume of the control group increased only 2.5-fold (Table 2, groups 15,16), and the mean tumour volume of control 2 did not differ from the control 2 groups in experiment 1 and 2, even though the tumours grew for 4 weeks longer.

200 It must be stated that HInd[RuInd2C14(NZ)] represents an antitumour agent that reduces tumour growth in this very resistant tumour model by more than 50%, at the optimum dose of this experiment by more than 70%, and in earlier experiments by more than 80%, combined with 0% mortality and tolerable toxicity in the optimum dose. One has to keep in mind that the aim of testing compounds does not include complete remission of turnouts but selection o f potent substances and that the effect of the only clinically effective combination, 5fluorouracil/leucovorin, is only moderate - about 50% reduction of tumour growth combined with an inacceptable mortality of 33% - and that all other clinically applied drugs, including cisplatin and alkylating agents, exhibit no effect in this tumor model. A suitable galenic formulation of both indazole compounds may be helpful to promote further evaluation in the clinic.

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Antineoplastic activity of three ruthenium derivatives against chemically induced colorectal carcinoma in rats.

The antineoplastic activity of the ruthenium complexes trans-imidazolium[tetracholorobisimidazole-ruthenate(III)], HIm(RuIm2Cl4), trans-indazolium-[te...
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