⁸⁹Zr-Cobalamin PET Tracer: Synthesis, Cellular Uptake, and Use for Tumor Imaging

Abstract

Vitamin B12, or cobalamin (Cbl), is an essential nutrient. Acquisition, transport, and cellular internalization of Cbl are dependent on specific binding proteins and associated receptors. The circulating transport protein transcobalamin (TC) promotes cellular uptake via binding to specific receptors such as CD320, a receptor upregulated in several cancer cell lines. In this study, we report the successful synthesis of 89Zirconium-labeled Cbl that was derivatized with desferrioxamine (89Zr-Cbl). We document the purity of the tracer and its binding to TC compared with that of unmodified cyano-Cbl (CN-Cbl). In vitro studies employing the CD320 receptor-positive breast cancer cell line MDA-MB-453 showed a 6- to 10-fold greater uptake of 89Zr-Cbl when compared with the uptake in the presence of 200-fold excess of CN-Cbl at 37 °C. We used nude mice with MDA-MB-453 tumors to study the feasibility of employing the tracer to visualize CD320 positive tumors. In vivo positron emission tomography images displayed a clear visualization of the tumor with 1.42 ± 0.48 %ID/g uptake (n = 3) at 4 h after injection (p.i.) with the tracer retained at 48 h p.i. Ex vivo biodistribution studies using 89Zr-Cbl exhibited the highest uptake in kidney and liver at 48 h p.i. Results document the feasibility of synthesizing a Cbl-based tracer suitable for both in vivo and ex vivo studies of Cbl trafficking and with the potential to visualize tumors expressing TC receptors, such as CD320.

A vitamin B12 conjugate of exendin‐4 improves glucose tolerance without associated nausea or hypophagia in rodents

“…Further analyses supported this hypothesis, as immunohistochemical data showed colocalization of Cy5‐B12‐Ex4 with insulin in the pancreas. This suggests that B12‐Ex4 may exert its glycaemic effects via direct action at pancreatic beta cells, while CNS‐mediated effects of GLP‐1R activation such as hypophagia, nausea and malaise are minimal or absent because of the absence of penetrance of B12‐Ex4 into the brain, consistent with previously reported radio‐probe data …”

“…This report shows for the first time (see Reference for previous biochemical GLP‐1R agonism analyses) the ability of B12‐Ex4 to improve glucose tolerance in rodents without producing hypophagia, body weight loss or CTA. Supporting a recent finding showing that B12 is not substantially transported into the adult mouse brain, current immunohistochemical data suggest that the unique profile of glycaemic effects without the same hypophagic/CTA‐producing effects of Ex4 involves a direct effect of B12‐Ex4 on pancreatic beta cells, coupled with a virtual absence of CNS penetrance of the compound.…”

“…This suggests that B12-Ex4 may exert its glycaemic effects via direct action at pancreatic beta cells, while CNS-mediated effects of GLP-1R activation such as hypophagia, nausea and malaise are minimal or absent because of the absence of penetrance of B12-Ex4 into the brain, consistent with previously reported radio-probe data. 40 The current data provide novel mechanistic evidence that B12 conjugation to a GLP-1R agonist can be used as a means to retain the hypoglycaemic properties of GLP-1R agonists while greatly reducing the CNS-mediated anorexia and illness effects observed with currently approved GLP-1-based ligands. 3,8,9 These studies are far from the complete set of in vivo glycaemic analyses needed for B12-Ex4, but certainly justify the need for more comprehensive future analyses.…”

“…Collectively, this information points to a receptor‐mediated process of B12 blood‐brain barrier penetrance, but little is known of where B12 is transported in the brain, and to what extent, relative to total concentrations. It is evident that CNS uptake is considerably lower compared to other organs, especially the liver and kidney, with one recent study, using a B12‐ 89 Zr PET probe, revealing less than 0.1% injected dose per gram (ID/g) in brain in mouse models with over 5% ID/g observed in pancreas . We therefore hypothesized that a B12‐Ex4 conjugate would retain the incretin profile of a GLP‐1R agonist to improve glucose tolerance, but would prevent development of nausea/malaise through reduced, or redirected, CNS/hypothalamic penetration of the agonist.…”

Synthetic Approaches toward Vitamin B12 Conjugates

“…Vitamin B 12 was also successfully conjugated to peptide and peptide drugs,, particularly to insulin ( 28 c and 28 d ; CDI was used instead of CDT),, appetite suppressing peptide hPYY 3–36 ( 28 e ) or glucagon‐like peptide‐1 (GLP‐1) ( 28 f ) . Moreover, (CN)Cbl was functionalized with precursors that can serve as chelating agents for metals,, e. g. for manganese (conjugate 28 g ) or 89 Zr (conjugate 28 h ) complexes . Additionally, this carbamate methodology has facilitated the attachment of vitamin B 12 to nanoparticles ,,…”