Efficient Synthesis and HPLC-Based Characterization for Developing Vanadium-48-Labeled Vanadyl Acetylacetonate as a Novel Cancer Radiotracer for PET Imaging.

Bis(acetylacetonato)oxidovanadium(IV) [(VO(acac)2], generally known as vanadyl acetylacetonate, has been shown to be preferentially sequestered in malignant tissue. Vanadium-48 (48V) generated with a compact medical cyclotron has been used to label VO(acac)2 as a potential radiotracer in positron emission tomography (PET) imaging for the detection of cancer, but requires lengthy synthesis. Current literature protocols for the characterization of VO(acac)2 require macroscale quantities of reactants and solvents to identify products by color and to enable crystallization that are not readily adaptable to the needs of radiotracer synthesis. We present an improved method to produce vanadium-48-labeled VO(acac)2, [48V]VO(acac)2, and characterize it using high-performance liquid chromatography (HPLC) with radiation detection in combination with UV detection. The approach is suitable for radiotracer-level quantities of material. These methods are readily applicable for production of [48V]VO(acac)2. Preliminary results of preclinical, small-animal PET studies are presented.

Preliminary investigation of 48V-labeled VO(acac)2 for cancer imaging: An initial proof-of-concept study.

In this preliminary study, a procedure for synthesizing novel PET radiotracer vanadium-48-labeled-vanadyl acetylacetonate was developed, including radioisotope production via cyclotron, separation of 48V, chelation as 48VO(acac)2, and assessment through in vitro cellular studies. We employed the beam-stop setup in a cyclotron as the target holder to irradiate titanium foils in the reaction of natTi (p,n)48V. The radioisotope production rate was 4.84 ± 0.67 µCi/µA-h. Overall radiochemical yield was 12.86 ± 0.51% with gamma-ray spectroscopy showing no detectable contaminant peaks. HPLC of 48VO(acac)2 showed a retention time (1:48) corresponding closely to that (1:50) of commercial VO(acac)2, verifying the successful synthesis of 48VO(acac)2. In vitro cellular studies demonstrated radiotracer uptake and saturation around 0.48 nM. These studies pave the way for improving methodologies and in vivo experiments, including imaging studies, in future investigations.

The vanadyl chelate bis(acetylacetonato)oxovanadium(IV) increases the fractional uptake of 2-(fluorine-18)-2-deoxy-D-glucose by cultured human breast carcinoma cells.

Detection of breast cancer by positron emission tomography (PET) imaging with 2-(fluorine-18)-2-deoxy-D-glucose (FDG) as the tracer molecule is limited in part by both tumor dimension and metabolic activity. While some types of aggressive breast cancers are associated with a high capacity for FDG uptake, more indolent breast cancers are characterized by low FDG uptake. Moreover, detection of malignant lesions in most clinical settings requires tumor dimensions ≥10 mm. Development of a method to increase the fractional uptake of FDG by cancer tissue would provide a means to detect smaller tumors. However, there is no clinically available pharmacologic reagent known to enhance the preferential uptake of FDG by cancer tissue. Because the vanadyl (VO(2+)) chelate bis(acetylacetonato)oxovanadium(IV) [VO(acac)2] is known to enhance cellular uptake of glucose, we have investigated whether VO(acac)2 facilitates enhanced uptake of FDG by cultured human breast carcinoma cells. We observed that the fractional uptake of FDG by cultured human MDA-MB-231 carcinoma cells is increased in the presence of VO(acac)2 in a dose dependent manner. Preliminary results with xenograft tumors generated in severely compromised, immunodeficient (SCID) female mice showed that VO(acac)2 treatment of mice 3-4 h prior to FDG injection enhanced FDG uptake by the malignant tissue by a factor >2.0 compared with that by normal surrounding tissue.

New vanadium-based magnetic resonance imaging probes: clinical potential for early detection of cancer.

We have developed a magnetic resonance imaging (MRI) method for improved detection of cancer with a new class of cancer-specific contrast agents, containing vanadyl (VO(2+))-chelated organic ligands, specifically bis(acetylacetonato)oxovanadium(IV) [VO(acac)(2)]. Vanadyl compounds have been found to accumulate within cells, where they interact with intracellular glycolytic enzymes. Aggressive cancers are metabolically active and highly glycolytic; an MRI contrast agent that enters cells with high glycolytic activity could provide high-resolution functional images of tumor boundaries and internal structure, which cannot be achieved by conventional contrast agents. The present work demonstrates properties of VO(acac)(2) that may give it excellent specificity for cancer detection. A high dose of VO(acac)(2) did not cause any acute or short-term adverse reactions in murine subjects. Calorimetry and spectrofluorometric methods demonstrate that VO(acac)(2) is a blood pool agent that binds to serum albumin with a dissociation constant K (d) ~ 2.5 +/- 0.7 x 10(-7) M and a binding stoichiometry n = 1.03 +/- 0.04. Owing to its prolonged blood half-life and selective leakage from hyperpermeable tumor vasculature, a low dose of VO(acac)(2) (0.15 mmol/kg) selectively enhanced in vivo magnetic resonance images of tumors, providing high-resolution images of their interior structure. The kinetics of uptake and washout are consistent with the hypothesis that VO(acac)(2) preferentially accumulates in cancer cells. Although VO(acac)(2) has a lower relaxivity than gadolinium-based MRI contrast agents, its specificity for highly glycolytic cells may lead to an innovative approach to cancer detection since it has the potential to produce MRI contrast agents that are nontoxic and highly sensitive to cancer metabolism.

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.