P11.02.A PRECLINICAL STUDY TO OVERCOME METHOTREXATE-RESISTANT MOUSE LEPTOMENINGEAL CARCINOMATOSIS: INTRATHECAL METHOTREXATE AND ORAL 6-THIOGUANINE

Abstract BACKGROUND Leptomeningeal carcinomatosis (MC) is a severe complication of advanced cancer with limited therapeutic options. Methotrexate (MTX) is one of the most commonly used drugs for treating MC. However, MTX resistance is frequently observed before and during intrathecal chemotherapy. In this study, we aimed to develop a method to overcome MTX resistance in a preclinical model. MATERIAL AND METHODS MM46 transplantable ascitic breast cancer cells (O-MM46) were used, and MTX-resistant MM46 cells (R-MM46) were established through repeated intra-abdominal MTX administrations, starting at a low dose and increasing to 20 μg/mL, thrice per week. MTX resistance was confirmed by increased dihydrofolate reductase activity (O-MM46, 0.197; R-MM46, 1.253 μmol/min/mg protein) and the lack of survival benefit in MTX-treated mice bearing R-MM46 cells, in contrast to the significantly increased survival in MTX-treated mice bearing the parent O-MM46 cells. Subsequently, we assessed pyrimidine and purine metabolic activity after intra-abdominal administration of MTX (50 mg/kg) in O-MM46 and R-MM46 transplantable ascitic mammary cancer cells by measuring the levels of orotate phosphoribosyltransferase, thymidine kinase, 5-phosphoribosyl-1-pyrophosphate, and guanine phosphoribosyltransferase. Based on the observed activation of the purine salvage pathway in R-MM46 cells, we determined to use oral administration of 6-thioguanine (6-TG), a purine antagonist, as an adjuvant drug. Finally, we assessed the synergism between intrathecal MTX and oral 6-TG by adjusting their administration time in the treatment of MTX-resistant MC. RESULTS Mice with MC were divided into six groups (n = 10/group): control (sham intrathecal phosphate-buffered saline), MTX only (12.5 μg/0.05 mL), 6-TG only (0.04 mg/body), simultaneous MTX and 6-TG, MTX 2 h after 6-TG, and 6-TG 2 h after MTX. All treatment groups, except the MTX-only group, demonstrated significantly longer survival than that of the control group. Survival (mean ± standard error of the mean, days) is as follows: control, 16 ± 0.3; MTX only, 17.4 ± 0.3; 6-TG only, 21.8 ± 0.5; simultaneous MTX and 6-TG, 24.6 ± 0.5; MTX 2 h after 6-TG, 27.1 ± 0.5; and 6-TG 2 h after MTX, 28.0 ± 0.7. Notably, survival was longest in the group receiving 6-TG 2 h after MTX (simultaneous MTX and 6-TG vs. 6-TG 2 h after MTX pretreatment; p < 0.005). CONCLUSION These findings indicate that an oral chemotherapeutic protocol with 6-TG after intrathecal MTX injection may be effective for treating MTX-resistant MC. Based on these preclinical results, we applied this approach for clinical use.