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Binding induced enzyme activated methotrexate-α-peptide prodrugs for integrin targeted drug delivery

Author

Phanidhara R. Kotamraj, University of the Pacific

Date of Award

2009

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Xiaoling Li

First Committee Member

Bhaskara Jasti

Second Committee Member

David Thomas

Third Committee Member

Jay Wu

Abstract

Improving the therapeutic efficacy and quality of life of patients by reducing the side effects caused by non-specificity of cytotoxic drugs has been a challenge in cancer treatment. A hypothesis was developed where integrin binding induced conformational change in a drug conjugated to hairpin peptide with an integrin binding ligand can lead to preferential accumulation of drug and reduced collateral damage by decreased premature prodrug activation. A model drug, MTX and a tripeptide ligand, RGD, known to specifically bind tumor overexpressing α v β 3 integrin receptors, were selected to test the hypothesis. A twelve amino acid sequence that has been previously shown to preferentially adopt an anti-parallel beta hairpin conformation in aqueous environment was flanked with MTX and RGD on N and C termini respectively by solid phase peptide synthesis to form a labile link between Arg-Glu specifically cleaved by SGPE, a Streptomyces griseus derived endopeptidase. Adenoviral vector was developed using AdEasy system for β 3 cDNA transfection to overexpress integrin α v β 3 receptor. MTX-α-RGD and MTX-β-hairpin-RGD were characterized using MALDI-TOF (MTX-α-RGD, 782.6(M+H + ); MTX-β-hairpin-RGD, 2272.1(M+H + )). Cell adhesion assay using HUVEC and A549 cells that overexpress α v β 3 showed that RGD conjugated prodrugs recognize and preferentially bind to integrin α v β 3 in RGD dependent manner. In rabbit plasma, MTX-β-hairpin-RGD was found to be 3 times more stable than MTX-α-RGD. In the absence of α v β 3 binding, SGPE mediated hydrolysis rate of MTX-β-hairpin-RGD was 0.7±0.1 ng/hr, that was significantly (P<0.025) lower than that of MTX-α-RGD (1.0±0.1ng/hr), a prodrug without hairpin structure. In presence of α v β 3 over-expressing cells, significant increase (P<0.025) in hydrolysis rate of MTX-β-hairpin-RGD to 1.0±0.1 ng/hr was observed, not significantly (P=0.6) different from that of MTX-α-RGD (1.1±0.1ng/hr). In addition, there was 400% increase in the fluorescence when FRET based quenching was abolished by the binding induced unfolding. These experiments along with docking studies using molecular modeling support the binding induced unfolding. Results from this investigation suggest that drugs conjugated to peptide ligands such as RGD may reduce the dose needed to achieve therapeutic concentrations by preferential recognition and binding to overexpressed integrin markers. Secondly, reduction of premature activation of prodrugs and thus reduced collateral damage may be achieved by making the the drug release to occurs preferentially upon binding to cells expressing specific integrin markers.

Pages

247

ISBN

9781109041286

Recommended Citation

Kotamraj, Phanidhara R.. (2009). Binding induced enzyme activated methotrexate-α-peptide prodrugs for integrin targeted drug delivery. University of the Pacific, Dissertation - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/2619