Targeted drugs for cancer and inflammatory diseases

Sumith A Kularatne, Purdue University

Abstract

Therapies that exploit targeting ligands to deliver attached cytotoxic drugs selectively to diseased cells are currently receiving significant attention. While antibody-targeted drugs have been the first to enter the clinic, recent studies demonstrate that low molecular weight ligands can also be used to deliver attached imaging and therapeutic agents selectively to malignant cells in human cancer patients. Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in treatment of PCa, especially metastatic disease. Guided by in silico docking studies using the crystal structure of prostate-specific membrane antigen (PSMA), we have developed a PSMA-targeted ligand, namely DUPA, that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-99mTc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (KD = 14 nM). Imaging and bio-distribution studies revealed that DUPA-99mTc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (%injected dose/gram = 11.3 at 4 h post-injection; tumor-to-muscle ratio = 75:1). PSMA-targeted optical imaging agents (DUPA-FITC, DUPA-rhodamine B, DUPA-Alexaflour 647, etc.) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. The PSMA-targeted fluorophore (DUPA-FITC) is shown to selectively label circulating prostate tumor cells in fresh peripheral blood samples from PCa patients and no fluorescent cells were detected in blood samples from healthy volunteers. Further, DUPA was able to selectively deliver siRNA conjugates to PCa cells in vitro and to tumor xenograft animal model. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC50 = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy. The vitamin folic acid and folate conjugates bind to folate receptors (FR) that are over-expressed on ∼40% of human cancers (primarily FR-alpha) and mediate internalization of their attached drugs by receptor-mediated endocytosis. For these reasons, anticancer drugs have been tethered to folate via releasable disulfide linker that is susceptible to controlled drug release upon endocytosis. Based on this strategy, six folate-targeted drugs are currently in human clinical trials. Since FR is also abundant on the apical membrane of the proximal tubule of the kidney, the effect of folate-targeted drugs on kidney toxicity remains unclear. Therefore, we have developed a two-photon FRET imaging method to monitor in vivo efficiency of drug release mechanism in the kidney and tumor. The disulfide-bridged folate-FRET conjugate was found to remain intact in the blood circulation system, but drug (rhodamine) was efficiently released in the tumor via redox-mediated disulfide bond reduction. Moreover, the folate-FRET conjugate was released (within 7 - 24 h) during FR transcytosis in the kidney proximal tubule. Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes immune system to attack synovium of the joints by "mistaken identity" theory. Although the exact causes of RA remain unknown, activated macrophages are thought to play a pivotal role in its pathogenesis. Moreover, trace macrophage infiltration of the synovial tissue is characteristic of the earliest stages of RA. Since activated macrophages, but not resting macrophages or most other healthy cells, express FR-beta, we have developed FR-beta targeted PET imaging agents (folate-DOTA-68Ga-PET and Folate- 18F) and immunotherapeutic agents (folate-haptens) to early diagnosis and treatment of RA. Imaging studies revealed that folate-targeted PET imaging agents localize significantly into a rodent hind paw inflammation model suggesting that folate tracers might be equivalent to FDG-18F-PET to monitor inflammation. FR-beta targeted immunotherapeutic agents, namely folate-TNP and folate-FITC, constitute promising haptens for use in FR-targeted immunotherapy of RA.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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