The Experimental Study Of Folate-Targeted Penicillin G Amidase

Objective: to explore whether there're targeting effects of folate-penicillin G amidase conjugate (F-PGA) on folate receptor positive cells and solid tumors, and to investigate its pharmacokinetic parameters in vivo. These data will be helpful to establish a base for further folate-directed enzyme prodrug therapy (FDEPT).Methods: (1) F-PGA and PGA were labeled with ¹²⁵I by Iodogen method. High performance liquid chromatography (HPLC) and paper chromatography were used to determine their radiochemical purities. The stabilities of ¹²⁵I-F-PGA and ¹²⁵I-PGA in vitro were monitored by trichloroacetic acid (TCA) precipitation, and their catalytic activities were detected by p-dimethyl-aminobenzaldchyde (PDAB) method. (2) Three kinds of cell line were used in the experiment: folate receptor positive HeLa, SKOV3 cell line and folate receptor negative A549 cell line. The relation between the uptake of F-PGA or folic acid (F-A) by HeLa, SKOV3 or A549 cells and the incubation time was observed with confocal microscopy, respectively. Further more, the capabilities of HeLa, SKOV3 and A549 cells binding and internalizing ¹²⁵I-F-PGA or ¹²⁵I-PGA with the varieties of time and concentrations at 37℃or 4℃were investigated using radioligand binding assay. The dissociation constant (Kd) of F-PGA from receptors and the numbers of maximum binding sites (Bmax) were obtained by Scatchard analysis. (3) ¹²⁵I-F-PGA, ¹²⁵I-PGA, ¹²⁵I-F-PGA plus 10mg F-A or ¹²⁵I were injected intravenously into the mice bearing Hela cell tumor, respectively. To the mice bearing subcutaneous SKOV3 tumor, ¹²⁵I-F-PGA or ¹²⁵I-PGA were injected intravenously. Single photon emission computed tomography (SPECT) was performed at the various time of postinjection for examining the target ability of these tracers to the tumors in vivo. Animals were sacrificed after final imaging, and then the main organs were weighed and their radioactivities were measured. The ratios of tumor-to-muscle radioactivities and the ratios of tumor-to-nontumor were expressed as T/M value and T/NT value, respectively. (4)The...