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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA070349-01A2
Application #
2411506
Study Section
Special Emphasis Panel (ZRG2-ET-2 (02))
Project Start
1997-09-01
Project End
2000-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
Wright, Joel E; Yurasek, Gregory K; Chen, Ying-Nan et al. (2003) Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity. Biochem Pharmacol 65:1427-33
Vaidya, Chitra M; Wright, Joel E; Rosowsky, Andre (2002) Synthesis and in vitro antitumor activity of new deaza analogues of the nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523). J Med Chem 45:1690-6
Forsch, Ronald A; Wright, Joel E; Rosowsky, Andre (2002) Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid. Bioorg Med Chem 10:2067-76
Wright, J E; Vaidya, C M; Chen, Y et al. (2000) Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine (PT523) and its B-ring analogues. Biochem Pharmacol 60:41-6
Rosowsky, A; Wright, J E; Vaidya, C M et al. (2000) The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta Pharmacol Ther 85:191-205
Rosowsky, A; Wright, J E; Vaidya, C M et al. (2000) Analogues of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, p-aminobenzoyl moiety, or 9,10-bridge: synthesis and in vitro antitumor activity. J Med Chem 43:1620-34
Rosowsky, A (1999) PT523 and other aminopterin analogs with a hemiphthaloyl-L-ornithine side chain: exceptionally tight-binding inhibitors of dihydrofolate reductase which are transported by the reduced folate carrier but cannot form polyglutamates. Curr Med Chem 6:329-52
Rosowsky, A; Wright, J E; Vaidya, C M et al. (1998) Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl- L-ornithine (PT523). J Med Chem 41:5310-9