The overall goals of this project are to conduct in vitro and in vivo studies of the biochemical and pharmacological mode of action of a class of unusually potent water-soluble nonclassical dihydrofolate reductase inhibitors whose activity does not require, and is not dependent on, polyglutamation. The prototypical member of this class is N-alpha-(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523, NSC-633713), which was first synthesized at DFCI. Biochemical and pharmacologic properties of second generation PT523 analogs being synthesized as part of a separate chemical synthesis grant will also be investigated as part of this project with the aim of determining whether any of them may be more attractive preclinical candidates than the lead compound. Also to be studied is a recently synthesized prodrug derivative of PT523 which was designed to take advantage of """"""""antibody directed enzyme prodrug therapy"""""""" (ADEPT) to decrease host toxicity and improve tumor versus host selectivity.
Specific Aim I (in vitro studies): (i) cytotoxicity of second generation PT523 analogs from the applicant's synthesis program; (ii) prevention of cytotoxicity with thymidine and/or hypoxanthine; (iii) dihydrofolate reductase inhibition; (iii) kinetics of cellular uptake; (iv) effects on cellular reduced folate pools and on inhibition and recovery of DNA synthesis; and (v) synergistic interactions of PT523 with other, non-antifolate drugs.
Specific Aim 2 (in vivo studies): (i) antitumor activity of PT523 and other second generation analogs in human xenograft tumor models; and (ii) effects on reduced folate pools and DNA synthesis inhibition/recovery in tumor versus marrow and gut.
Specific Aim 3 (resistance studies): (i) in vitro production of resistance in human tumor cell lines by PT523 via different selection protocols, and comparison of the time needed to achieve a similar level of resistance to PT523 and MTX; (ii) analysis of DHFR activity and drug uptake in PT523 resistant cells, the most likely determinants of resistance to this nonpolyglutamatable drug; and iv) pilot in vitro studies on the therapeutic potential of PT523-L-Phe as the first example of an ADEPT derivatives of PT523.
