In vivo structural activity and optimization studies of folate-tubulysin conjugates.

Herein we report on the potencies of 4 related folate-conjugated tubulysins constructed with either tubulysin B hydrazide (EC0305), tubulysin A hydrazide (EC0510), the N,O-acetal derivative of natural tubulysins (EC0317) or a tubulysin B ester (EC0302). Our results confirmed that EC0305 is the most favorable conjugate of the group due to its potent antitumor activity [100% cures at 1 micromol/kg, three times a week (TIW) for 2 weeks] and its favorably low toxicity profile. In contrast, the natural tubulysin B drug proved to be inactive against a human nasopharyngeal tumor model when administered at doses near to or greater than the maximum tolerated dose (MTD). When tested against more chemoresistant folate receptor expressing M109 and 4T1-cl2 tumors, EC0305 displayed superior antitumor activity over a previously disclosed folate conjugate of desacetylvinblastine monohydrazide (EC145). These studies demonstrate that EC0305 has significant antiproliferative activity against FR expressing tumors, including those which are generally more chemoresistant, and that EC0305 should be considered for development as a candidate for the treatment of advanced FR-expressing human cancers.

Preclinical antitumor activity of a novel folate-targeted dual drug conjugate.

We have designed a new type of tumor-targeted agent by tethering two different drug molecules, with distinct biological mechanisms of action, to the same ligand. This compound, named EC0225, represents the "first in class" multidrug, folate receptor (FR)-targeted agent to be disclosed. It was constructed with a single folate molecule, extended by a hydrophilic peptide-based spacer, which was in turn attached to mitomycin and Vinca alkaloid units via two separate disulfide-containing linkers. EC0225 produced potent, dose-responsive activity in vitro, and curative activity was observed against FR-positive syngeneic and xenograft tumors following the administration of well-tolerated dosing regimens. Multiple complete responses and cures were also noted when EC0225 was used to treat mice initially bearing tumors as large as 750 mm (3) in volume. Overall, EC0225's impressive preclinical activity allowed for its selection as a development candidate and for the start of Phase 1 clinical trials, which began in March of 2007, for the treatment of advanced malignancies.

Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate.

We recently developed a new group of folate-conjugated Vinca alkaloids, one of which, EC145, emerged as a candidate for clinical development. Brief treatment of nude mice bearing approximately 100 mm(3) folate receptor-positive human xenografts led to complete response (CR) in 5/5 mice and cures (i.e., remission without a relapse for >90 days post-tumor implantation) in 4/5 mice. Multiple CRs and cures were also noted when EC145 was used to treat mice initially bearing tumors as large as 750 mm(3). Likewise, complete cures (5/5) resulted following the treatment of an aggressive folate receptor-positive J6456 lymphoma model. The activity of EC145 was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, no significant antitumor activity (0/5 CR) was observed in EC145-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating the target-specific activity of EC145. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the unconjugated drug (desacetylvinblastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose levels. Subsequent dose regimen studies confirmed that EC145 given on a more frequent, qdx5 schedule resulted in the most effective antitumor response as compared with an equivalent total dose given on thrice- or single-injection-per-week schedule. Taken together, these studies show that EC145 has significant antiproliferative activity and tolerability, thus lending support to an ongoing phase 1 trial for the treatment of advanced malignancies.