A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen.

2[(3-Amino-3-carboxypropyl)(hydroxy)(phosphinyl)methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from nontarget organs including kidneys, but its use for imaging of PSMA is impeded by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multipresentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a (64)Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with (64)Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA(+) cell line, LNCaP, and PSMA(-) PC3 and H2009 cell lines. (64)Cu-CBT2G showed significantly higher LNCaP tumor uptake than (64)Cu-CBT1G at 1, 4, and 24 h postinjection (p.i.) (p < 0.05). In addition, tumor uptake of (64)Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50% ID/g at 1, 4, and 24 h p.i., respectively), while (64)Cu-CBT1G showed a great decrease from 1 to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA(-) tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA(+) LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (% ID/g) of (64)Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R(2) = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This may render the probe with potential application in the management of patients with prostate cancer.

Development of a locally advanced orthotopic prostate tumor model in rats for assessment of combined modality therapy.

The purpose of this study was to develop an aggressive locally advanced orthotopic prostate cancer model for assessing high-dose image-guided radiation therapy combined with biological agents. For this study, we used a modified human prostate cancer (PCa) cell line, PC3, in which we knocked down a tumor suppressor protein, DAB2IP (PC3­KD). These prostate cancer cells were implanted into the prostate of nude or Copenhagen rats using either open surgical implantation or a minimally invasive procedure under ultrasound guidance. We report that: i) these DAB2IP-deficient PCa cells form a single focus of locally advanced aggressive tumors in both nude and Copenhagen rats; ii) the resulting tumors are highly aggressive and are poorly controlled after treatment with radiation alone; iii) ultrasound-guided tumor cell implantation can be used successfully for tumor development in the rat prostate; iv) precise measurement of the tumor volume and the treatment planning for radiation therapy can be obtained from ultrasound and MRI, respectively; and v) the use of a fiducial marker for enhanced radiotherapy localization in the rat orthotopic tumor. This model recapitulates radiation-resistant prostate cancers which can be used to demonstrate and quantify therapeutic response to combined modality treatments.

Prostate cancer-specific thermo-responsive polymer-coated iron oxide nanoparticles.

Thermo-responsive poly(N-isopropylacrylamide-acrylamide-allylamine)-coated magnetic nanoparticles (PMNPs) were developed and conjugated with prostate cancer-specific R11 peptides for active targeting and imaging of prostate cancer. The stable nanoparticles with an average diameter of 100 nm and surface charge of -27.0 mV, had a lower critical solution temperature of 40 °C. Magnetic characterization showed that the nanoparticles can be recruited using a magnetic field and possess superparamagnetic behavior even after R11 conjugation. In vitro cell studies demonstrated that R11-conjugated PMNPs (R11-PMNPs) were compatible with human dermal fibroblasts and normal prostate epithelial cells to all tested concentrations up to 500 µg/ml after 24 h of incubation. Moreover, the nanoparticles were taken up by prostate cancer cells (PC3 and LNCaP) in a dose-dependent manner, which was higher in case of R11-PMNPs than PMNPs. Further, in vivo biodistribution of the nanoparticles showed significantly more R11-PMNPs accumulation in tumors than other vital organs unlike PMNPs without R11 conjugation. Moreover, R11-PMNPs decreased 30% magnetic resonance T2 signal intensity in tumors in vivo compared to 0% decrease with PMNPs. These results indicate great potential of R11-PMPs as platform technology to target and monitor prostate cancers for diagnostic and therapeutic applications.