PSMA-specific degradable dextran for multiplexed immunotargeted siRNA therapeutics against prostate cancer.

Small interfering RNA (siRNA) is ideal for gene silencing through a sequence-specific RNA interference process. The redundancy and complexity of molecular pathways in cancer create a need for multiplexed targeting that can be achieved with multiplexed siRNA delivery. Here, we delivered multiplexed siRNA with a PSMA-targeted biocompatible dextran nanocarrier to downregulate CD46 and PD-L1 in PSMA expressing prostate cancer cells. The selected gene targets, PD-L1 and CD46, play important roles in the escape of cancer cells from immune surveillance. PSMA, abundantly expressed by prostate cancer cells, allowed the prostate cancer-specific delivery of the nanocarrier. The nanocarrier was modified with acid cleavable acetal bonds for a rapid release of siRNA. Cell imaging and flow cytometry studies confirmed the PSMA-specific delivery of CD46 and PD-L1 siRNA to high PSMA expressing PC-3 PIP cells. Immunoblot, qRT-PCR and flow cytometry methods confirmed the downregulation of CD46 and PD-L1 following treatment with multiplexed siRNA.

Dual contrast agents for fluorescence and photoacoustic imaging: evaluation in a murine model of prostate cancer.

Prostate-specific membrane antigen (PSMA) is a promising diagnostic and therapeutic target for prostate cancer (PC). Poly(amidoamine) [PAMAM] dendrimers serve as versatile scaffolds for imaging agents and drug delivery that can be tailored to different sizes and compositions depending upon the application. We have developed PSMA-targeted PAMAM dendrimers for real-time detection of PC using fluorescence (FL) and photoacoustic (PA) imaging. A generation-4, ethylenediamine core, amine-terminated dendrimer was consecutively conjugated with on average 10 lysine-glutamate-urea PSMA targeting moieties and a different number of sulfo-cyanine7.5 (Cy7.5) near-infrared dyes (2, 4, 6 and 8 denoted as conjugates II, III, IV and V, respectively). The remaining terminal primary amines were capped with butane-1,2-diol functionalities. We also prepared a conjugate composed of Cy7.5-lysine-suberic acid-lysine glutamate-urea (I) and control dendrimer conjugate (VI). Among all conjugates, IV showed superior in vivo target specificity in male NOD-SCID mice bearing isogenic PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and suitable physicochemical properties for FL and PA imaging. Such agents may prove useful in PC cancer detection and subsequent surgical guidance during excision of PSMA-expressing lesions.

A hyaluronic acid binding peptide-polymer system for treating osteoarthritis.

Hyaluronic acid (HA) is found naturally in synovial fluid and is utilized therapeutically to treat osteoarthritis (OA). Here, we employed a peptide-polymer cartilage coating platform to localize HA to the cartilage surface for the purpose of treating post traumatic osteoarthritis. The objective of this study was to increase efficacy of the peptide-polymer platform in reducing OA progression in a mouse model of post-traumatic OA without exogenous HA supplementation. The peptide-polymer is composed of an HA-binding peptide (HABP) conjugated to a heterobifunctional poly (ethylene glycol) (PEG) chain and a collagen binding peptide (COLBP). We created a library of different peptide-polymers and characterized their HA binding properties in vitro using quartz crystal microbalance (QCM-D) and isothermal calorimetry (ITC). The peptide polymers were further tested in vivo in an anterior cruciate ligament transection (ACLT) murine model of post traumatic OA. The peptide-polymer with the highest affinity to HA as tested by QCM-D (∼4-fold greater binding compared to other peptides tested) and by ITC (∼3.8-fold) was HABP2-8-arm PEG-COLBP. Biotin tagging demonstrated that HABP2-8-arm PEG-COLBP localizes to both cartilage defects and synovium. In vivo, HABP2-8-arm PEG-COLBP treatment and the clinical HA comparator Orthovisc lowered levels of inflammatory genes including IL-6, IL-1B, and MMP13 compared to saline treated animals and increased aggrecan expression in young mice. HABP2-8-arm PEG-COLBP and Orthovisc also reduced pain as measured by incapacitance and hotplate testing. Cartilage degeneration as measured by OARSI scoring was also reduced by HABP2-8-arm PEG-COLBP and Orthovisc. In aged mice, HABP2-8-arm PEG-COLBP therapeutic efficacy was similar to its efficacy in young mice, but Orthovisc was less efficacious and did not significantly improve OARSI scoring. These results demonstrate that HABP2-8-arm PEG-COLBP is effective at reducing PTOA progression.

Preclinical Comparative Study of (68)Ga-Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for Targeting PSMA.

(68)Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: (68)Ga-1, using DOTA-monoamide as the chelating agent; (68)Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA; and (68)Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). (68)Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to (68)Ga-1 at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. (68)Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation (68)Ga-2 had the most advantageous characteristics for PSMA-targeted PET imaging.

Developing imidazoles as CEST MRI pH sensors.

A series of intra-molecular hydrogen bonded imidazoles and related heterocyclic compounds were screened for their N-H chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) contrast properties. Of the compounds, imidazole-4,5-dicarboxamides (I45DCs) were found to provide the strongest contrast, with the contrast produced at a large chemical shift from water (7.8 ppm) and strongly dependent on pH. We have tested several probes based on this scaffold, and demonstrated that these probes could be applied for in vivo detection of kidney pH after intravenous administration. Copyright © 2016 John Wiley & Sons, Ltd.

Anthranilic acid analogs as diamagnetic CEST MRI contrast agents that feature an intramolecular-bond shifted hydrogen.

Diamagnetic chemical exchange saturation transfer (diaCEST) agents are a new class of imaging agents, which have unique magnetic resonance (MR) properties similar to agents used for optical imaging. Here we present a series of anthranilic acid analogs as examples of diaCEST agents that feature an exchangeable proton shifted downfield, namely, an intramolecular-bond shifted hydrogen (IM-SHY), which produces significant and tunable contrast at frequencies of 4.8-9.3 ppm from water. Five analogs of N-sulfonyl anthranilic acids are all highly soluble and produced similar CEST contrast at ~6-8 ppm. We also discovered that flufenamic acid, a commercial nonsteroidal anti-inflammatory drug, displayed CEST contrast at 4.8 ppm. For these N-H IM-SHY agents, the contrast produced was insensitive to pH, making them complementary to existing diaCEST probes. This initial IM-SHY library includes the largest reported shifts for N-H protons on small organic diaCEST agents, and should find use as multifrequency MR agents for in vivo applications.

Tuning phenols with Intra-Molecular bond Shifted HYdrogens (IM-SHY) as diaCEST MRI contrast agents.

The optimal exchange properties for chemical exchange saturation transfer (CEST) contrast agents on 3 T clinical scanners were characterized using continuous wave saturation transfer, and it was demonstrated that the exchangeable protons in phenols can be tuned to reach these criteria through proper ring substitution. Systematic modification allows the chemical shift of the exchangeable protons to be positioned between 4.8 to 12 ppm from water and enables adjustment of the proton exchange rate to maximize CEST contrast at these shifts. In particular, 44 hydrogen-bonded phenols are investigated for their potential as CEST MRI contrast agents and the stereoelectronic effects on their CEST properties are summarized. Furthermore, a pair of compounds, 2,5-dihydroxyterephthalic acid and 4,6-dihydroxyisophthalic acid, were identified which produce the highest sensitivity through incorporating two exchangeable protons per ring.

Effect of chelators on the pharmacokinetics of (99m)Tc-labeled imaging agents for the prostate-specific membrane antigen (PSMA).

Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different (99m)Tc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea-based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [(99m)Tc(CO)3](+) core (L8-L10), traditional NxSy-based chelating agents with varying charge and polarity for the (99m)Tc-oxo core (L11-L18), and a (99m)Tc-organohydrazine-labeled radioligand (L19). (99m)Tc(I)-Tricarbonyl-labeled [(99m)Tc]L8 produced the highest PSMA+ PC3 PIP to PSMA- PC3 flu tumor ratios and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of (99m)Tc-labeled radiopharmaceuticals targeting PSMA.