Modulation of Non-Targeted Activity of Folate Conjugates

Example 3

In addition, the amount of thiol activity correlates with the non-ligand-specific activity of ligand conjugates. In this example, the non-FR-specific activity of an exemplary folate conjugate (EC0531) was investigated in MDA-MD-468, H23, AN3CA, MDA-MB-231, KB, and A549 cells.

Extracellular thiol activity was determined using the DTNB method described above. Non-FR-specific activity of EC0531 was determined using a ³H-thymidine incorporation assay. Cells were seeded in 24-well tissue culture-treated plates at 1×10⁵ cells per well and allowed to attach overnight at 37° C. Serial dilutions of EC0531 were prepared in FDRPMI/HIFBS, and each well received 0.5 mL of EC0531 solution. To assess non-FR-targeted activity in FR+cells, 100 μM FA was included as a competitor along with the drug in the treatment solutions. Cells were incubated for 2 hours in the presence of drug, washed 3 times with media, and then chased in 0.5 mL of FDRPMI/HIFBS (FR+cells) or RPMI+FA/HIFBS (FR−cells) to 72 h at 37° C. Spent medium was then aspirated from the wells, and cells were incubated with 1 μCi/mL ³H-thymidine for 4 hours at 37° C. washed two times with PBS, pH 7.4, then treated with 0.4 mL 5% trichloroacetic acid per well. After 15 minutes, the trichloroacetic acid was aspirated from the wells, and cells were solubilized in 0.5 mL 0.25 N sodium hydroxide. Each sample (450 μL) was transferred to a scintillation vial containing 3 mL of Ecolite+scintillation cocktail and then counted in a liquid scintillation counter (LSC). Final results were expressed as percentage of ³H-thymidine incorporation relative to an untreated control (non-competed groups) or FA control (competed groups). Sensitivity to the base drug, tubulysin B hydrazide, was determined using the ³H-thymidine incorporation assay and the same incubation conditions as described for EC0531.

As shown in FIG. 3, the extracellular thiol activity (IC₅₀ (nM), adjusted for tubulysin B hydrazide sensitivity) of various cell lines correlates with the non-FR-specific activity of the folate conjugate EC0531 FIG. 3 demonstrates the correlation between extracellular thiol activity and non-FR-specific activity of EC0531 (data adjusted for tubulysin B hydrazide sensitivity).

Example 4

The presence of extracellular thiols can affect the non-ligand-specific activity of ligand conjugates. In this example, the non-FR-specific activity of an exemplary folate conjugate (EC0531) was investigated. The non-FR-specific activity of EC0531 was evaluated in both KB cells (cells known to be FR positive) and in A549 cells (cells known to be FR negative). FR-specific and non-FR-specific activity of EC0531 was determined by the ³H-thymidine incorporation assay using the methods described in Example 3 above. As shown in FIG. 4, the non-FR-specific activity of EC0531 is observed at concentrations of about 0.3-1 μM in both KB cells and A549 cells.

The effects of three different cell-impermeable thiol inhibitors on the non-FR-specific activity of EC0531 were evaluated in this example: 1) 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB); 2) N-(2-carboxyethyl)maleimide (NCEM), and 3) folate-maleimide. Each thiol inhibitor was separately co-administered with EC0531 (1 μM) and folic acid (100 μM), and the agents were incubated for 2 hours, followed by a 72 hour chase. The inhibition of non-FR-specific activity (i.e. cytotoxicity) was evaluated for each thiol inhibitor.

As shown in FIG. 5, DTNB, NCEM, and folate-maleimide all exhibit a dose-responsive inhibition of non-FR-specific activity in KB cells. DTNB exhibited an IC₅₀ of 4.6 μM, NCEM exhibited an IC₅₀ of 17 μM, and folate-maleimide exhibited an IC₅₀ of 3.5 μM.

However, pre-treatment of cells with thiol inhibitors prior to the administration of EC0531 did not inhibit the non-FR-specific activity. Cells were seeded in 24-well tissue culture-treated plates at 1×10⁵ cells per well and allowed to attach overnight at 37° C. To assess the effect of various inhibitors on non-targeted activity of EC0531 in the FR+KB cell line, cells were treated concurrently with DTNB (100 μM) or NCEM (10 mM) and 1 μM EC0531 plus 100 μM FA to block all FR-specific drug uptake. Cells were incubated for 2 hours in the presence of drug, competitor, and inhibitor, washed 3 times with media, and then chased in 0.5 mL of FDRPMI/HIFBS to 72 hours at 37° C. Cells were then treated as described above to determine ³H-thymidine incorporation. Final results were expressed as percentage of ³H-thymidine incorporation relative to an untreated control (non-competed groups) or FA control (competed groups). As shown in FIG. 6, pre-treatment of KB cells with DTNB or NCEM prior to administration of EC0531 and folic acid is not effective to inhibit the non-FR-specific activity in the cells.

Various thiol inhibitors were tested to evaluate their effectiveness in inhibiting the non-FR-specific activity following co-administration with EC0531. The results are shown in Table 1.