Methotrexate (MTX) is one of the widely used agents in chemotherapy of oncological and hematological diseases. Low plasma half-life, toxicity for normal proliferating cells and other limitations of the drug impel scientists to search for improved forms of the MTX. Conjugation of the drug with macromolecular carriers is one of the strategy seems to be perspective in obtaining preparations with amended properties. The aim of this work was to compare two dextrans T10 and T40 as potential carriers for MTX. We hypothesized that the difference between conjugates would be evident in multiple-dose schedule due to distinct molecular weights (MW) of these macromolecules and allow us to select more prospective carrier.
Conjugates were synthesized using dextrans with MW of 10 and 40 kDa (preparations coded T10-MTX and T40-MTX, respectively). The level of substitution was 0.022 mol of MTX per 1 mol of glucose for each preparation. Female (C57Bl/6 x DBA/2)F1 mice, 12-20-week-old, weighing 21-27 g were applied. All animals were randomly divided into four groups and injected with 106 leukemia (P388) cells i.p. (day -1). Then animals were administered with appropriate agents on the 0th, 3rd and 6th days of the experiment. The mice in control and MTX treated group were administered with 0.9% saline solution and 20 mg/kg/day of free MTX, respectively. Another two groups were administered with 20 mg/kg/day of T10-MTX or T40-MTX conjugates, respectively. All doses were based on the molarity of the MTX in the conjugates. Mice body weight and survival data were collected daily. Overall toxicity in experimental groups was assessed using a number of treated mice, which died earlier the day of first death registered in control group. Two experiments with identical protocols were conducted and after obtaining comparable results were pooled and analysed together. Survival data were compared using the Cox's F test with Bonferroni correction for multiple comparisons.
There were not early deaths due to toxicity in groups treated by either of conjugates. Data on weight changes show that both tested compounds have approximately the same toxicity as free MTX when applied in multiple-dose schedule. Notably, the antileukemic effects of conjugates were not the same. Mice treated by T10-MTX compound had lower median survival time in comparison with both free MTX and T40-MTX treated groups, and these differences were statistically significant (p < 0.01 and p < 0.0001 respectively). However, T40-MTX treated mice had median survival time similar to free MTX treated mice and there was not statistically significant difference in survival between these two groups.
We explain this diversity of the properties of T10-MTX and T40-MTX conjugates by the difference in the MW of their carriers. It is known that dextran T10 is excreted unrestrictedly, while dextran T40 is not able to pass unchanged through the pores of the glomerular capillary walls due to its larger molecular size. We suggest that the T10-MTX conjugate is excreted faster than T40-MTX. Therefore, significantly lower amount of T10-MTX have time to degrade inside tumor cells in comparison with T40-MTX. The difference between two conjugates becomes well pronounced in multiple-dose schedule, when advantage of T40-MTX over T10-MTX is cumulated during the administration course. These data confirm our previously published results that MW of the carrier is the critical parameter and should be taken into an account while designing new conjugates. We are currently conducting studies in solid tumor models, to further investigate the possibility of using the conjugates as prolonged forms of the parental drug. The advantage of T40 over T10 dextran carrier is also should be confirmed in pharmacokinetics studies. |