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In Vitro and in Vivo Antitumor Activity of Methotrexate Conjugated to Human Serum Albumin in Human Cancer Cells¹

Pharmacology Department, Fujisawa-Deutschland, 81673 Munich, Germany [K. W., E. B., B. R., N. B., P. J., R. L.], and Departments of Medical Oncology [G. J. P.] and Rheumatology [G. J.], VU University Medical Center, 1081 HV Amsterdam, the Netherlands

To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [³H]MTX and [³H]MTX-HSA. However, similar uptake kinetics were measured for ¹²⁵I-HSA and ¹²⁵I-MTX-HSA (2.1 and 1.8 pmol/10⁷ cells/h when cells were treated with 10 µM ¹²⁵I-HSA and ¹²⁵I-MTX-HSA, respectively), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis. We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC₅₀, >50 µM). However, 24 h after incubation, MTX-HSA inhibited TS with an IC₅₀ of 6.9 µM. In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC₅₀ concentrations between 2 and 78 µM, respectively. MTX-HSA accumulates in vivo in the tumor tissue. Local concentrations of 18–29 µM were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice. Fourteen tumors from eight different tissues were tested. Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response. In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity.

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