Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

Clinically Translatable Hyperpolarized 13C Bicarbonate pH Imaging Method for Use in Prostate Cancer.

Solid tumors such as prostate cancer (PCa) commonly develop an acidic microenvironment with pH 6.5-7.2, owing to heterogeneous perfusion, high metabolic activity, and rapid cell proliferation. In preclinical prostate cancer models, disease progression is associated with a decrease in tumor extracellular pH, suggesting that pH imaging may reflect an imaging biomarker to detect aggressive and high-risk disease. Therefore, we developed a hyperpolarized carbon-13 MRI method to image the tumor extracellular pH (pHe) and prepared it for clinical translation for detection and risk stratification of PCa. This method relies on the rapid breakdown of hyperpolarized (HP) 1,2-glycerol carbonate (carbonyl-13C) via base-catalyzed hydrolysis to produce HP 13CO32-, which is neutralized and converted to HP H13CO3-. After injection, HP H13CO3- equilibrates with HP 13CO2 in vivo and enables the imaging of pHe. Using insights gleaned from mechanistic studies performed in the hyperpolarized state, we solved issues of polarization loss during preparation in a clinical polarizer system. We successfully customized a reaction apparatus suitable for clinical application, developed clinical standard operating procedures, and validated the radiofrequency pulse sequence and imaging data acquisition with a wide range of animal models. The results demonstrated that we can routinely produce a highly polarized and safe HP H13CO3- contrast agent suitable for human injection. Preclinical imaging studies validated the reliability and accuracy of measuring acidification in healthy kidney and prostate tumor tissue. These methods were used to support an Investigational New Drug application to the U.S. Food and Drug Administration. This methodology is now ready to be implemented in human trials, with the ultimate goal of improving the management of PCa.

Development of specialized magnetic resonance acquisition techniques for human hyperpolarized [13 C,15 N2 ]urea + [1-13 C]pyruvate simultaneous perfusion and metabolic imaging.

PURPOSE: This study aimed to develop and demonstrate the in vivo feasibility of a 3D stack-of-spiral balanced steady-state free precession(3D-bSSFP) urea sequence, interleaved with a metabolite-specific gradient echo (GRE) sequence for pyruvate and metabolic products, for improving the SNR and spatial resolution of the first hyperpolarized 13 C-MRI human study with injection of co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea. METHODS: A metabolite-specific bSSFP urea imaging sequence was designed using a urea-specific excitation pulse, optimized TR, and 3D stack-of-spiral readouts. Simulations and phantom studies were performed to validate the spectral response of the sequence. The image quality of urea data acquired by the 3D-bSSFP sequence and the 2D-GRE sequence was evaluated with 2 identical injections of co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea formula in a rat. Subsequently, the feasibility of the acquisition strategy was validated in a prostate cancer patient. RESULTS: Simulations and phantom studies demonstrated that 3D-bSSFP sequence achieved urea-only excitation, while minimally perturbing other metabolites (<1%). An animal study demonstrated that compared to GRE, bSSFP sequence provided an ∼2.5-fold improvement in SNR without perturbing urea or pyruvate kinetics, and bSSFP approach with a shorter spiral readout reduced blurring artifacts caused by J-coupling of [13 C,15 N2 ]urea. The human study demonstrated the in vivo feasibility and data quality of the acquisition strategy. CONCLUSION: The 3D-bSSFP urea sequence with a stack-of-spiral acquisition demonstrated significantly increased SNR and image quality for [13 C,15 N2 ]urea in co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea imaging studies. This work lays the foundation for future human studies to achieve high-quality and high-SNR metabolism and perfusion images.

Tumor Microenvironment Biosensors for Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopy.

Hyperpolarization (HP) of a carbon-13 molecule via dynamic nuclear polarization (DNP) involves polarization at low temperature, followed by a dissolution procedure producing a solution with highly polarized spins at room temperature. This dissolution DNP method significantly increases the signal-to-noise ratio (SNR) of nuclear magnetic resonance (NMR) over 10,000-fold and facilitates the use of magnetic resonance spectroscopy (MRS) to image not only metabolism but also the extracellular microenvironment. The extracellular tumor microenvironment (TME) closely interacts with tumor cells and stimulates their growth and metastasis. Thus, the ability to detect pathological changes in the TME is pivotal for the detection and study of cancers. This review highlights the potential use of MRS to study features of the TME-elevated export of lactate, reduced interstitial pH, imbalanced redox equilibrium, and altered metal homeostasis. The promising outcomes of both in vitro and in vivo assays suggest that DNP-MRS may be a useful technique to study aspects of the TME. With continued improvements, this tool has the potential to study the TME and provide guidance for accurate patient stratification and precise personal therapy. Graphical Abstract.

Activation by Oxidation: Ferrocene-Functionalized Ru(II)-Arene Complexes with Anticancer, Antibacterial, and Antioxidant Properties.

Organometallic Ru(II)-cymene complexes linked to ferrocene (Fc) via nitrogen heterocycles have been synthesized and studied as cytotoxic agents. These compounds are analogues of Ru(II)-arene piano-stool anticancer complexes such as RAPTA-C. The Ru center was coordinated by pyridine, imidazole, and piperidine with 0-, 1-, or 2-carbon bridges to Fc to give six bimetallic, dinuclear compounds, and the properties of these complexes were compared with their non-Fc-functionalized parent compounds. Crystal structures for five of the compounds, their Ru-cymene parent compounds, and an unusual trinuclear compound were determined. Cyclic voltammetry was used to determine the formal MIII/II potentials of each metal center of the Ru-cymene-Fc complexes, with distinct one-electron waves observed in each case. The Fc-functionalized complexes were found to exhibit good cytotoxicity against HT29 human colon adenocarcinoma cells, whereas the parent compounds were inactive. Similarly, antibacterial activity from the Ru-cymene-Fc compounds was observed against Bacillus subtilis, but not from the unfunctionalized complexes. In both cases, the IC50 values correlated quantitatively with the Fc+/0 reduction potentials. This is consistent with more facile oxidation to give ferrocenium, and subsequent generation of toxic reactive oxygen species, leading to greater cytotoxicity. The antioxidant properties of the complexes were quantified by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. EC50 values indicate that linking of the Ru and Fc centers promotes antioxidant activity.

Daylight-Mediated, Passive, and Sustained Release of the Glaucoma Drug Timolol from a Contact Lens.

Timolol, a potent inhibitor of ß-adrenergic receptors (ßARs), is a first-line drug for decreasing the intraocular pressure (IOP) of patients with glaucoma. Timolol is administered using 0.5% eye-drop solutions at >3 × 107 times the inhibitory concentration (k i) for ßARs. This high dose is wasteful and triggers off-target effects that increase medication noncompliance. Here, we introduce contact lenses that release timolol to the eye throughout the day during passive exposures to natural daylight at a more therapeutically relevant concentration (>3000 k i). Timolol is coupled to the polymer of the contact lens via a photocleavable caged cross-linker and is released exclusively to the surrounding fluid after the 400-430 nm mediated cleavage of the cross-linking group. Studies conducted in a preclinical mouse model of glaucoma show photoreleased timolol is effective as authentic timolol in reducing IOP. Our studies highlight several advantages of daylight-mediated release of timolol from lenses compared to eye-drops. First, fitted contact lenses exposed to natural daylight release sufficient timolol to sustain the inhibition of ßARs over a 10 h period. Second, the contact lenses inhibit ßARs in the eye using only 5.7% of the timolol within a single eye-drop. Third, the lenses allow the patient to passively control the amount of timolol released from the lens-for example, early morning exposure to outdoor sunlight would release enough timolol to maximally reduce the IOP, whereas subsequent periodic exposures to indoor daylight would release sufficient timolol to overcome the effects of its spontaneous dissociation from ßARs. Fourth, our lenses are disposable, designed for single day use, and manufactured at a low cost.

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization.

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

Modulation of the Aß peptide aggregation pathway by KP1019 limits Aß-associated neurotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder that is increasing worldwide due to increased life expectancy. AD is characterized by two pathological hallmarks in the brain: amyloid-ß (Aß) plaque deposits and neurofibrillary tangles. A focus of AD research has concentrated on either inhibiting Aß peptide aggregation that leads to plaque formation or breaking down pre-formed Aß peptide aggregates. An alternative approach is to modulate the Aß aggregation profile by facilitating the formation of Aß species that are off-pathway and non-toxic. Herein, we report the re-purposing of the widely studied Ru(iii) anti-cancer complex KP1019, towards regulating the aggregation profile of the Aß peptide. Using electron paramagnetic resonance (EPR) spectroscopy, we conclude that KP1019 binds to histidine residues, located at the N-terminus of the peptide, in a rapid and robust fashion. Native gels and transmission electron microscopy (TEM) analyses have provided insight into the species and structures that are generated by KP1019-Aß interactions. Finally, incubation in an in vitro human neuronal cell model has demonstrated that the formation of KP1019-Aß species rescues cell viability from Aß-associated neurotoxicity. Modulation of the Aß aggregation pathway via covalent interactions with small molecules is thus a promising AD therapeutic strategy.

Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-ß-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.