Design, Synthesis, and Characterization of HBP-Vectorized Methotrexate Prodrug Molecule 1102-39: Evaluation of In Vitro Cytotoxicity Activity in Cell Culture Models, Preliminary In Vivo Safety and Efficacy Results in Rodents

Abstract

A novel bone-targeted prodrug, 1102-39, is discussed with the aim of enhancing the therapeutic effects of methotrexate (MTX) within bone tissues while minimizing systemic toxicity. Within the 1102-39 molecule, the central linker part forms a cleavable ester group, with MTX being also linked by a stable imine bond to the specially designed hydroxybisphosphonic (HBP) vector. Synthesized through a convergent approach starting from MTX, this prodrug advantageously modulates MTX's activity by selective esterification of its α-carboxyl group. In vitro tests revealed a 10-fold reduction in cytotoxicity compared to standard MTX, in alignment with prodrug behavior and correlated with gradual MTX release. In vivo in rodents, 1102-39 displayed preliminary encouraging antitumor effects on orthotopic osteosarcoma. Furthermore, various aspects of designing molecules for selective therapy in bone tissue based on bisphosphonate molecules as vectors for delivering active compounds to the bone are discussed. The 1102-39 molecule exhibits strong affinity for hydroxyapatite and a progressive release of MTX in aqueous environments, enhancing the safety and efficacy of bone-specific treatments and enabling sustained activity within bone and bone joints in the therapy of tumor and inflammation.

Figure 1

Synthesis scheme of the 1102–39 molecule.

Figure 2

Improved concept of the 1102–39 molecule structure.

Figure 3

Glycol hydrazide analogue of the 1102–39.

Figure 4

Key HMBC correlations proving the 1102–39 molecule structure.

Figure 5

Determination of MTX release from HA-bound 1102–39 by HPLC-UV at 308 nm. A: HPLC-UV chromatogram of methotrexate; B: HPLC-UV chromatogram of 1102–39; C: HPLC-UV chromatogram of the remaining solution after 30 min of 1102–39 fixation on HA reveals residual product in solution, confirming HA saturation; D: HPLC-UV chromatogram of methotrexate gradual liberation from 1102-39-HA (T = 288 h, DPBS); E: release of methotrexate from saturated HA with 1102–39 investigated by HPLC-UV in DBPS at two pH (6.4 and 7.4) and in conditioned medium (prepared using medium from cells that have been grown with a minimum of 80% confluency and without medium replacement for three consecutive days).

Figure 6

Stability of 1102–39 in solution in DPBS over a period of 70 h.

Figure 7

Assessment of the cytotoxicity of 1102–39 compared to MTX in viability assays conducted on established cell lines. K7M2: osteoblast from the bone of a mouse with osteosarcoma; L929: mouse fibroblasts; LM8: mouse osteosarcoma; OSRGa: rat osteosarcoma; SaOS2: human osteosarcoma; TC-71: human Ewing’s sarcoma.

Figure 8

Systemic toxicity of repeated administrations while increasing the dose of 1102–39 on the body weight of C57BL6 male mice.

Figure 9

Systemic toxicity of repeated administrations of 1102–39 on the body weight of SWISS male mice.

Figure 10

Systemic toxicity of repeated administrations of 1102–39 on the body weight of Sprague–Dawley male rats.

Figure 11

1102–39 efficacy in mice measurement of the tumor volume (mm³). A: 1102–39 treatment reduced tumor growth in the osteosarcoma model over time. Orthotopic paratibial LM8 tumors were implanted on D0 in C3H mice and IV treatment with 1102–39 or DPBS vehicle were administered every week for 3 weeks from D13. n = 10 mice per group. Mean ± SEM B: 1102–39 was potent in inhibiting terminal osteosarcoma tumor weight. Orthotopic paratibial LM8 tumors were implanted in C3H mice. Mice received IV treatment with DPBS vehicle or 1102–39 on D13, D20 and D27. n = 9 mice per group. Statistical analysis on D34: Mann–Whitney test. * p < 0.05.