Limitations of low mol. wt anticancer drugs are short tumor exposure times and toxicity to normal tissue. Methotrexate (MTX) covalently linked to human serum albumin (HSA) as a macromolecular carrier caused tumor regressions concomitant with a favorable toxicity profile in a clinical phase I trial (Hartung et aL, Clin. Cancer Res., 1999, 5, 753). We examined the uptake, intracellular degradation, metabolism and thymidylate synthase (TS) inhibition of MTX-HSA in the T-cell leukemia line CCRF-CEM and the MTX transport resistant clone CCRF-CEM/MTX. The number of MTX molecules per albumin molecule was determined by electrospray mass spectrometry. A loading ratio (LR) of approximately 1.4 mol MTX/albumin revealed intact complexes with one and two MTX molecules/albumin. In the complex with an LR of 5.7, albumin with up to 16 MTX molecules was seen. MTX-HSA was taken up by CCRF-CEM cells via endocytosis and cleaved by lysosomal enzymes. Liberated MTX was a poor substrate of folylpolyglutamate synthetase and was exported into the medium. TS was inhibited and cell survival was impaired by MTX-HSA in a time- and concentration-dependent manner. CCRF-CEM/MTX cells exhibited a growth inhibition of 30-40% after MTX-HSA treatment, but no TS inhibition. The alternative uptake route via endocytosis enables MTX-HSA to overcome transport resistance to MTX.
Background: Several clinical trials showed a reduction of both local recurrence and distant metastatic disease as well as an increase in overall survival following adjuvant radio-chemotherapy in high-risk stages of rectal cancer. The NCI and European groups currently recommend local radiation and 6 months of adjuvant chemotherapy with 5-fluorouracil (5-FU) for TNM stage II and III patients. However, modulation and schedule of application as well as optimal duration of 5-FU therapy are still subject of discussion. Experimental and clinical data show a potentiation of 5-FU cytotoxicity by folinic acid (FA), and the combination of 5-FU and FA might become standard in the treatment of colorectal carcinomas. To evaluate the effect of total duration of therapy on tumour recurrence and survival, we compare in a prospectively randomized study 6 versus 12 months of 5-FU/FA chemotherapy in patients with locally advanced or nodal-positive rectal cancer. Patients and Methods: Patients with stage II and III rectal cancer are postoperatively stratified according to tumour stage and type of operation and randomly assigned to one of two treatment arms. Patients in arm A receive a total of 12, patients in arm B a total of 6 cycles of 5-FU (450 mg/m2) and FA (100 mg/m2) on days 1-5, every 4 weeks. During the 2nd cycle local radiation up to 50.4 Gy is performed, and dose-reduced (5-FU 350 mg/m2) chemotherapy is applied weekly. The aims of the study are the registration of disease-free and overall survival as well as of toxicity. Results: Since 1993, 215 patients have been enrolled into the study. At this point, 186 patients are completely documented and available for evaluation. So far, there are no significant differences in the two treatment arms with respect to rate of local or distant relapse and of disease-free and overall survival. As reported previously, there is no increase in toxicity with the 12-month regimen. Conclusion: Our preliminary results indicate that prolonged chemotherapeutic treatment over 12 months has no relevant advantage over a 6-month protocol with 5-FU and medium-dose FA. The combination of 5-FU and medium-dose FA is well tolerated by the majority of patients and even prolonged therapy is not associated with increased toxicity.
