Toxicity Profiles In Vivo in Mice and Antitumour Activity in Tumour-Bearing Mice of Di- and Triorganotin Compounds

The in vivo toxicity profiles in mice and the antitumour activity in tumour bearing mice were screened for four di-n-butyltin and five triorganotin carboxylates, di-n-butyltin diterebate (5), bis(phenylacetate) (6), bis(deoxycholate) (7), bis(lithocholate) (8), tri-n-butyltin terebate (9), cinnamate (10), and triphenyltin terebate (11). At their maximum tolerated dosis (MTD), no antitumour effect (T/C ~1) was observed for the compounds 5, 7, 9, 10 and 11. The compounds 6 (T/C = 0.51) and 8 (T/C = 0.42) showed clear antitumour activity after single dose administration and might therefore be of interest for further antitumour activity studies.

All compounds revealed similar in vitro chemosensitivities in two cell lines, C26-10 and C26-A, two murine undifferentiated colon carcinoma cell lines. With all compounds tested, not only cell growth was inhibited in vitro, but also cell kill was achieved. At their maximum tolerated dose (MTD), 1 and 4 were inactive in vivo against colon 26 tumours in Balb/C mice whereas compounds 2 and 3 showed slight in vivo antitumour activity. However, the cut-off level for the growth delay factor (GDF) (>1) was not reached. With the exception of 2 administered with a single dose and 3 with the 2 doses protocol, treatment with these compounds did not increase the life span of all mice. Repeated administration of compound 2 did not improve the antitumour activity compared to single dose administration.

Materials and Methods
The structures of the seven compounds screened are depicted in figure 1. Their synthesis and characterization were described earlier (Gielen, 1997) (Willem, 1997a) (Willem, 1997b).
Toxicity and in vivo screening.
The experimental procedure was described earlier (Gielen, 1996d). The test compounds were dissolved in DMSO to a concentration ranging from 50 to 100 mg/ml and diluted to 10 mg/ml in arachidis oil, which was also used for further dilutions. Occasionally the DMSO had to be acidified or made alkaline to get a clear solution. The amount of DMSO could not be increased since mice do not tolerate more than 1%. Mice were injected intra-peritoneally.
The MTD was defined as the dose resulting in 10-15% weight loss. Antitumour activity.
The murine colon tumour Colon 26 (Corbett, 1975) is maintained in our laboratory since 1983 (Peters, 1987(Peters, , 1989Van der Wilt, 1992) in Balb/c mice by s.c. transplantation in both flanks in the thoracic region of small fragments of 1-5 mm3. Several variants of the tumour have been characterized since its original characterization, all displaying different properties (summarized in Van Laar, 1996). The variant used in this work, originally described as colon 26, has been used throughout all experiments in our laboratory. When tumours had reached a volume of 50-150 mm3, treatment was started. Tumour size was determined by calliper measurement (length x width x height x 0,5) twice a week. The volume of the tumours was expressed relatively to that on the first day of treatment (day 0). Antitumour activity was evaluated by calculation of the T/C (ratio of the tumour size of the Treated mice to that of Control mice expressed in %).

Results and discussion
Dose-finding studies of compounds 5 to 11 in MTD studies Initial dose-finding studies were performed with groups of 2 mice which were treated weekly for two weeks by i.p. injection (qd7x2). Results are shown in figures 2-8 for compounds ,5 to 11. For all tested compounds, a steep dose-toxicity relation was found. In addition, for the poorly soluble compounds 8 and 11, the toxicity was unpredictable. For example, in a repeated experiment using the same dose (i.e. 20 mg/kg), a completely different toxicity compared to a former experiment was observed for these two compounds. This was probably due to the poor solubility of the compounds in DMSO resulting in a colloidal suspension in arachidis oil, resulting in non-reproducible injection of the compounds. Toxicity of compounds 5 to 11 in the antitumour activity studies Based on the results obtained in the dose-finding study, for each compound, a dose was chosen somewhat lower than the MTD dose, since experience with previous compounds demonstrated more toxicity in tumor bearing mice. The expected toxicity should be less than 10%. However, the toxicity in tumor (colon 26A) bearing Balb/C mice was unexpectedly high (figures 9 -1 1). For the compounds 6, 7, 8, 10 and 11, severe toxicities were observed already after one injection. Therefore, administration of a second injection of these compounds was not acceptable.
However, for compound 10, 3/8 mice could be treated twice. The compounds ,5 and 9 were administered according to the schedule qd7x2. For 9, this schedule was too toxic (3/5  In vivo antitumour activity Although the compounds were too toxic to administer according to the schedule used in the dosefinding study, conclusions can be drawn based on a single dose treatment of the colon26A bearing mice. The assumption was made that when even at this high dose no antitumour activity is present, it would be unlikely that lower doses at a schedule of qd7x2 would be able to produce an antitumour effect.
At their MTD, no antitumour effect was observed for the compounds 5, 7, 9, 10 and 11, i.e. the T/C (see fig. 12 to 14). Time (day) Figure 12: Antitumour activity of compounds 5 and 9 against Colon 26A in Balb/C mice The compounds 6 and 8 (see fig. 13 and 14) showed clear antitumour activity after single dose administration (for 6 and 8, T/C equals respectively 0.51 and 0.42). The dose for both compounds was too toxic, however. Because of the promising results obtained with compound 6 at a single dose of 7.5 mg/kg (fig 13), the dosis was decreased to 5 mg/kg in a second experiment (fig. 14) in order to reduce the toxicity. The reduced dose also showed a antitumour effect which was less (T/C 0.81 but showed a comparable severe toxicity.

Conclusions
The compounds 6 and 8 might be of interest for further antitumour activity studies. However, their severe toxicity makes the results difficult to interpret. An adapted formulation for administration of the compounds will be necessary for further evaluation of possible antitumour effects.