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Prostate-Specific Membrane Antigen (PSMA)-targeted Imaging and Therapy of Prostate Cancer
Prostate cancer (PCa) is the most frequently diagnosed and second most common cause of cancer related deaths in men. Despite tremendous progress in detection of early stage disease, therapies for metastatic and advanced stages of PCa are still inadequate and compromised by severe side effects. Therefore, more effective methods for detection and treatment are highly desirable.
Among >90 known PCa biomarkers, prostate-specific membrane antigen (PSMA) is one of the most extensively studied and widely expressed biomarker that present on over 90% of PCa. PSMA is also expressed in greater abundance on prostate cancer cells than other healthy human tissues, and recent findings reveal that PSMA is also over-expressed on the tumor-associated neovasculature of most other solid malignancies, but is essentially absent from the vasculature of normal tissues. Based on this over-expression in malignant tissues, it was decided to develop a method to deliver therapeutic and imaging agents selectively to prostate cancer cells by targeting them to PSMA. Guided by in silico docking studies using a high resolution crystal structure of PSMA, we designed a high affinity PSMA-targeting ligand, 2-[3-(1, 3-dicarboxy propyl)-ureido] pentanedioic acid (DUPA), that selectively binds and enters only PSMA-expressing cells by PSMA-mediated endocytosis.1, 2 The first application of this PCa-specific ligand was to synthesize and test a 99mTc-based radioimaging agent for detection and staging of PCa. The first agent developed, DUPA-99mTc, was found to bind PSMA-positive human prostate cancer cells (LNCaP cell line) with 14 nanomolar affinity (KD ~ 14 nM).1, 2 Biodistribution studies of the radioimaging in nu/nu mice demonstrated that DUPA-99mTc localizes primarily to LNCaP cell tumor xenografts (% injected dose/ gram = 12.4 at 4 h postinjection; tumor-to-muscle ratio = 75:1). In agreement with this observation, intravenous injection of DUPA-99mTc into mice with PSMA-negative tumors (KB and A549 tumor xenografts) showed no accumulation of radioactivity in the tumors, demonstrating the in vivo specify of the PSMA-targeted radioimaging agent. Importantly, similar PSMA specificity was observed with other radionuclides such as 68Ga and 166Lu (DUPA-68Ga and DUPA-166Lu, respectively) in nude mice xenograft models.3 Based on these encouraging data, clinical trials of DUPA-99mTc have been initiated at Indiana University Medical School, Indianapolis, IN, USA.
We have also developed multiple PSMA-targeted optical imaging agents for use in defining PCa margins during surgery, detecting PCa metastases in proximal lymph nodes, and for quantitation of PCa cells in circulation (circulating tumor cells). DUPA- Fluorescein isothiocyanate (FITC, a green color dye) and DUPA-rhodamine B (a red color dye) were shown to efficiently label LNCaP cells and to internalize and traffic to intracellular endosomes.2 More importantly, DUPA-FITC was shown to be useful for detection and quantitation of PCa cells in fresh peripheral blood samples from prostate cancer patients. In a more detailed evaluation of the latter potential application of the technology, we conducted a double blind study on ~100 prostate cancer patients at different stages of progression and 17 healthy donors as controls.4 After enriching the circulating tumor cells (CTCs) in the blood samples of prostate cancer patients using Ficoll gradient, the enriched tumor cell layer was incubated with both DUPA-FITC to label PSMA+ PCa cells and anti-human CD45 Alexaflour 647 to label human peripheral blood monocytes. Flow cytometric analysis of subpopulation of cells in the FITC+/high/Alexa flour 647- quadrant with appropriate controls confirmed that DUPA-FITC was able to detect an average of ~20 CTCs/ mL blood from the prostate cancer patients whereas no fluorescently labeled cells were detected from the blood samples of the healthy donors.4 Encouraged by specificity of DUPA for PCa, we undertook to develop therapeutic conjugates of DUPA for treatment of prostate cancer.2,5 We demonstrated that DUPA is capable of mediating the targeted killing of LNCaP cells with many different therapeutic warheads.5 To this end, DUPA was linked to such cytotoxic drugs as tubulysin B, tubulysin B hydrazide, desacetylvinblastine, camptothecin, dideminin B, and verrucarin A, via hydrophilic peptide spacer containing a releasable disulfide linker. This strategy generated PCa-targeted therapeutic agents with high PCa specificity, enhanced water solubility (compared to the base drugs), and no toxicity to cells lacking PSMA. Importantly, all DUPA prodrug conjugates with the exception of DUPA-verrucarin A were demonstrated to kill PSMA-positive LNCaP cells in culture with IC50 values in the low nanomolar range (IC50 = 3 – 115 nM).5 To test the ability of DUPA to target cytotoxic drugs selectively to PCa cells in vivo, DUPA-tubulysin B hydrazide, the most potent DUPA prodrug, was injected intravenously into mice bearing subcutaneous LNCaP tumor xenografts (100 mm3). DUPA-tubulysin B hydrazide (1.0 µmol/kg, 3x/wk, 2 wk schedule) was found to eliminate established tumor xenografts in nu/nu mice within 2 weeks with no detectable weight loss.2 Blockade of tumor targeting upon administration of 100 fold excess PSMA inhibitor confirmed the specificity of each of the PSMA-targeted prodrugs.2 Moreover, pathological analysis of the tissues of mice treated with DUPA-tubulysin B hydrazide established that collateral damage to healthy cells could be eliminated by targeting the drug to PSMA.6 Interestingly, administration of 1.0 µmol/kg of nontargeted tubulysin B or tubulysin B hydrazide killed the mice within 2 days.
Last but not least, we were able to resolve the problems associated with delivery of siRNA therapeutics using DUPA as a targeting ligand.7 Despite their enormous potential as biopharmaceutical therapeutics, systemic delivery of naked siRNA to malignant sites remains a major hurdle owing to rapid enzymatic digestion in plasma, renal elimination, limited penetration across the capillary endothelium, and inefficient uptake by tissue cells. First, we demonstrated that DUPA-21-mer siRNA-5/-Cyanine 5 (DUPA-siRNA21-Cy5) efficiently labeled PSMA-positive LNCaP cells in culture in a manner that could be inhibited by excess PSMA inhibitor, suggesting that the siRNA conjugate binding was PSMA-mediated. We further established the concept by administering DUPA-siRNA21-Cy5 to mice bearing LNCaP tumor xenografts on their shoulders. The fluorescence image analysis revealed that the DUPA-siRNA21-Cy5 was localized primarily in PSMA-positive LNCaP tumors, with little or no accumulation in other tissues. Efficacy of therapeutic siRNAs is currently under investigation.
Based on the above studies, we conclude that the PSMA-specific targeting ligand, DUPA, could potentially prove useful in targeting imaging agents to PCa for the purposes of locating metastatic disease, detecting and quantitating PCa cells in circulation, monitoring response to therapy, locating positive margins during surgery, and selecting patients for subsequent DUPA-targeted chemotherapy. DUPA should also prove beneficial in treating PCa with minimal side effects.
References:
- Kularatne SA, Zhou Z, Yang J, Post CB, Low PS. Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted (99m)Tc-radioimaging agents. Mol Pharm. 2009 May-Jun;6(3):790-800.
- Kularatne SA, Wang K, Santhapuram HK, Low PS. Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand. Mol Pharm. 2009 May-Jun;6(3):780-9.
- Kularatne SA, Müller C, Low PS. Unpublished data.
- He W, Kularatne SA, Kalli KR, Prendergast FG, Amato RJ, Klee GG, Hartmann LC, Low PS. Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands. Int J Cancer. 2008 Oct 15;123(8):1968-73.
- Kularatne SA, Venkatesh C, Santhapuram HK, Wang K, Vaitilingam B, Henne WA, Low PS. Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs. J Med Chem. 2010 Nov 11;53(21):7767-77.
- Kularatne SA, van Alstine W, Low PS. Unpublished data.
- Thomas M, Kularatne SA, Qi L, Kleindl P, Leamon CP, Hansen MJ, Low PS. Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues. Ann N Y Acad Sci. 2009 Sep;1175:32-9.
Written by:
Sumith A. Kularatne, PhD and Philip S. Low, PhD as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
Sumith A. Kularatne
The Scripps Research Institute
Department of Chemistry
10550 North Torrey Pines Road, SR202
La Jolla, CA 92037
Tele: (858) 784-9303
Fax: (858) 784-9440
e-mail:
Philip S. Low
Purdue University
560 Oval Drive
Department of Chemistry
West Lafayette, IN 47907
Tele: 765-494-5273
Fax: 765-494-5272
Email:
Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs - Abstract
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