Dimesna

Example 1

The present invention is based in part on the unexpected finding that the rectal administration of Mesna to mice experiencing radiation induced gastrointestinal tract injury, reduces symptoms of the condition. In the present study, 14 male C57BL/6 mice were randomly divided into two groups—treatment group and control group. The pelvic area (containing the ano-rectal part of the intestine) of each mouse was exposed to a Cs-137 source and irradiated over 2 minutes at a rate of 6 Gy/min (a 12 Gy dose). Starting from the day of irradiation and for a period of 9 days, mice in the treatment group received enemas containing Mesna at a dose of 75 mg/kg body weight. Animals in the control group received saline enemas as a control. Animals were sacrificed nine days post irradiation and gross necropsy of the anus, rectum and colon was performed and documented by high resolution color pictures. Specimens of 7 cm of the intestinal tract starting from the muco-cutaneous junction up to the colon were collected and fixed for histological evaluation in formalin, according to pathology department guidelines. FIG. 1 presents the statistical mean (black square) with the corresponding standard error (dashed line) and standard deviation (dotted line) of mucosal alteration scoring (0=no change, 3=severe change) which was employed to evaluate the efficacy of the different treatments, where the rectangles mark an area encompassing 80% of the scores for each of the groups. Surprisingly, the results showed that the mucosal alteration score was significantly reduced in animals treated with Mesna enemas.

The success of rectally administered Mesna to the treatment of radiation induced gastrointestinal tract injury may be attributed to the hydrophilic properties of Mesna, which ensure that this compound will stay in the lumen of the lower gastrointestinal tract and will not enter the gastrointestinal cells. Furthermore, lack of absorption of Mesna will not allow its conversion to dimesna, the active form of the drug for conventional treatment of cyclophosphamide and ifosfamide. Therefore Mesna is suitable and advantageous for treating and/or preventing radiation complications in the GI tract, as the present invention discloses.

Although the invention is described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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.