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

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.

Introduction

Vitamin B12 (cobalamin, Cbl) is a critical nutrient that is physiologically required to maintain cell growth and differentiation.[1−4] Cbl is involved in the biosynthesis of nucleic acids, lipids, and proteins, and its deficiency leads to a reduction in functional methionine synthase and metabolism of methylmalonic acid in humans, leading to megaloblastic anemia and/or various neurological disorders.[1−4]

Cbl gains entry into cells upon binding to transport proteins and subsequent receptor mediated transport. Cbl in blood is bound to the transport protein transcobalamin (TC) (holo-TC), which, in turn, is recognized by specific receptors such as CD320.[1−4] Upregulation of CD320 receptors has been reported in several malignancies including breast, prostate, thyroid, cervical, colorectal, and stomach cancers.[5] The important role of Cbl in cellular proliferation and the upregulation of CD320 in tumor cells has made Cbl uptake an attractive candidate for tumor imaging, mainly using single-photon emission computed tomography with 99mTechnetium- or IIIIndium-labeled Cbl.[6−13] One Cbl-positron emission tomography (PET) imaging agent, labeled with 64Cu (t1/2 ∼ 12.7 h), has also been reported.[14]

Herein, the utility of Cbl as a vector was explored for delivering the PET radionuclide 89Zr (t1/2 ∼ 3.27 days). We hypothesized that 89Zr would (1) retain the sensitivity of PET imaging and (2) provide a longer visualization window by providing a greater signal-to-noise ratio compared with prior tracers reported, allowing for an improved tumor targeting and imaging. Following radiosynthesis, we evaluated the in vitro uptake and in vivo pharmacokinetics of the CD320-positive MDA-MB-453 breast cancer in athymic nude mice.

Results

Synthesis of Cbl-Desferrioxamine (Cbl-DFO)

Cbl-desferrioxamine (Cbl-DFO) was synthesized by forming a carbamate linkage between the 5′-hydroxyl of deoxyribose moiety in Cbl and the amine group of DFO (Scheme , Figure S1).[15] Cbl was activated using 1,1′-carbonyl-di-(1,2,4-triazole) (CDT), followed by the addition of DFO, which links through the primary amine. Purification and characterization confirmed that the conjugate was of ≥97% purity (Figures S2–S4). The yield of Cbl-DFO was 40 ± 5% based on the Cbl content in the starting material. Calculated mass (m/z): 1942 [M]; observed: 972 [M + 2H]2+ and 648 [M + 3H]3+.

Scheme 1

Synthesis and Radiolabeling of Cbl-DFO

Radiolabeling of Cbl with 89Zr

Cbl-DFO was labeled with 89Zr (89Zr-Cbl) using a previously established protocol (Scheme ).[16] A radiolabeling efficiency of ∼97% was determined by instant thin-layer chromatography (iTLC, Figure S5a). The specific activity of the tracer was determined by titrating 89Zr4+ and Cbl at different mole ratios with an achieved optimum specific activity of 250 ± 20 mCi/μmol (mean ± standard deviation, n = 3).

In Vitro Stability of 89Zr-Cbl

Stability of the tracer was analyzed by incubating 89Zr-Cbl in saline and in human serum at 37 °C. Bound versus unbound radio metal was analyzed at 0, 4, 24, and 48 h after incubation using iTLC (Figure a,b). The intact tracer was located closer to the origin (40–80 mm), whereas unbound tracer was found at 100–140 mm. After 48 h of incubation, free 89Zr was <1% in both saline and serum.

Figure 1

In vitro stability of 89Zr-Cbl (a) in saline at 37 °C, (b) in human serum at 37 °C, (c) TC binding of 91Zr-Cbl and CN-Cbl, expressed as the fraction bound compared to binding without added competitor and (d) internalization of 89Zr-Cbl (0.1 μCi, 3.7 KBq, 0.4 pmol/well) with MDA-MB-453 cells at 1, 4, 24 h time points incubated at 4 and 37 °C. A competition assay was also performed at each time point using unlabeled Cbl (Cbl 40 pmol/well co-incubated with 0.1 μCi, 0.4 pmol/well of 89Zr-Cbl). The fraction of 89Zr-Cbl internalized in MDA-MB-453 cells is expressed as cpm/105. **** and ** indicate p ≤ 0.0001 and p ≤ 0.01, respectively. Data are shown as mean and standard deviation, n ≥ 3.

TC Binding Studies

Mouse TC binding of 91Zr-Cbl was studied by radiometric chase assay using 57Co-labeled Cbl employing a previously described design.[17]91Zr-Cbl was synthesized similar to 89Zr-Cbl, but with 91ZrCl4. Mouse TC binding of 91Zr-Cbl displaced 57Co-labelled Cbl in a manner comparable to that of CN-Cbl (Figure c), indicating that the modification of Zr-Cbl did not compromise binding to TC. The same results were obtained for binding to human intrinsic factor (data not shown).

In Vitro Uptake

An internalization assay was performed to test the uptake of 89Zr-Cbl on CD320 receptor cell line MDA-MB-453 at 4 and 37 °C (Figure d). The internalized fractions of 89Zr-Cbl were expressed as counts per minute (cpm) normalized to 105 cells (cpm/105 cells). Internalization of 89Zr-Cbl was higher at 37 °C versus 4 °C at all time points with 144 ± 20 versus 36 ± 12 cpm/105 cells at 1 h (p < 0.0001), 210 ± 64 versus 30 ± 9 cpm/105 cells at 4 h (p = 0.01), and 304 ± 25 versus 83 ± 15 cpm/105 cells at 24 h (p < 0.0001). Competitive assays using excess Cbl at 37 °C displayed lower binding at all time points (p < 0.01). All of the data are reported as mean ± standard deviation of four independent measurements.

PET Imaging

PET imaging was performed after the administration of ∼1 nmol/mouse (200–250 μCi, 7.4−9.3 MBq) of 89Zr-Cbl to nude mice bearing a CD320 positive MDA-MB-453 tumor (n = 3). The image showed visualization of the tumor with tracer uptake of 1.42 ± 0.48 %ID/g at 4 h p.i. with retention observed up to 48 h p.i. (Figure a). Cohorts (n = 3) that were co-injected with 200-fold excess unlabeled Cbl (Figure b) showed significantly less uptake in the tumors (0.20 ± 0.05 %ID/g) at 24 h p.i. (p ≤ 0.001). Other tissues that displayed high tracer uptake were the kidney and liver with 8.92 ± 1.45, 8.80 ± 1.06, and 8.10 ± 0.58 %ID/g for kidney and 4.27 ± 0.51, 4.48 ± 0.65, and 4.47 ± 0.69 %ID/g for liver at 4, 24, and 48 h p.i., respectively (Figure c). Tumor-to-muscle ratio (∼3:1) did not change significantly over 48 h p.i., indicating that the maximum tumor-to-background ratio was achieved at 4 h p.i. (Figure d). All percent injected dose per gram of tissue (%ID/g) values are reported as mean ± standard deviation.

Figure 2

PET images of representative mice bearing (a) MDA-MB-453 tumors imaged with 89Zr-Cbl (∼1 nmol/mouse, 9 MBq) at 4, 24, and 48 h p.i. time points, (b) MDA-MB-453 tumors imaged with 89Zr-Cbl (∼1 nmol/mouse, 9 MBq), co-injected with 200-fold excess of unradiolabeled Cbl at 4 and 24 h p.i., (c) %ID/g values for selected organs in MDA-MD-453 tumor-bearing mice, and (d) tumor-to-muscle ratios at all imaging time points. The tumor location is indicated by a white circle.

Ex Vivo Tissue Analysis

Biodistribution data obtained from tumor-bearing mice injected 0.1 nmol (25 microcuries, 0.9 MBq) of 89Zr-Cbl showed 5.11 ± 1.33, 4.16 ± 1.09, and 3.78 ± 0.77 %ID/g (mean ± standard deviation, n = 4) tumor uptake in MDA-MB-453 tumors at 4, 24, and 48 h p.i., respectively (Figure a, Table S1). The kidneys showed the highest uptake of the tracer with 94.42 ± 4.27, 103.33 ± 11.50, and 72.74 ± 8.41 %ID/g and the liver showed the second highest uptake with 20.15 ± 3.42, 16.75 ± 1.44, and 17.99 ± 2.54 %ID/g at 4, 24, and 48 h p.i., respectively. Administration of a 200-fold excess of unmodified Cbl (as CN-Cbl) together with the tracer (n = 4 mice) resulted in an approximately 100-fold decrease (0.04 ± 0.01 %ID/g) in tracer uptake in tumors at 48 h and also a decreased uptake in the kidney (1.39 ± 0.18 %ID/g) and the liver (0.08 ± 0.01 %ID/g). These results are consistent with a Cbl-specific uptake of 89Zr-Cbl (Figure b, Table S1) and all of the %ID/g values were reported as mean ± standard deviation.

Figure 3

Ex vivo tissue distribution of (a) 89Zr-Cbl in mice (n = 4) bearing MDA-MB-453 tumors at 4, 24, and 48 h p.i. and (b) a blocking study with 200-fold unmodified Cbl co-injected with 89Zr-Cbl (n = 4) and tissue collection 48 h p.i. Data are displayed as mean ± standard deviation, with 89Zr decay accounted for in the analyses.

Discussion

In this proof-of-concept study, we report the successful production of a Cbl-derived 89Zr tracer suitable for use in PET studies. Studies on nude mice bearing a human breast cancer cell tumor allow us to demonstrate the use of the tracer for PET visualization of the tumor.

In agreement with previous data,[17,18] we found that 91Zr-Cbl bound to mouse TC in a manner comparable to that of CN-Cbl (Figure c). Next, an in vitro assay was performed in the breast cancer cell line MDA-MB-453 to demonstrate a specific uptake of 89Zr-Cbl.[19] We demonstrated a greater than 4-fold uptake of 89Zr-Cbl at 37 °C versus 4 °C; blocking with 200-fold excess unlabeled Cbl had a similar reduced uptake (Figure d). These results support the idea that the targeting properties of 89Zr-Cbl in MDA-MB-453 cells rely on a Cbl-dependent internalization mechanism, likely through the CD320 receptor.

In vivo imaging with 89Zr-Cbl showed an uptake in MDA-MB-453 tumors with 1.42 ± 0.48 of %ID/g, whereas ex vivo tissue distribution studies showed a tumor uptake of 5.11 ± 1.33 %ID/g at 4 h p.i. (Figures and 3). Notable uptake was also observed in the liver and kidneys with 4.27 ± 0.51 and 8.92 ± 1.45 %ID/g at 4 h p.i., respectively (Figure c). An in vivo block using 200-fold excess of unradiolabeled Cbl showed a significantly reduced uptake (p < 0.001) of the tracer, indicating that the in vivo tumor initialization is Cbl dependent, supported in the in vitro internalization assay. Muscle-to-tumor ratio showed that the maximum tumor-to-background ratio was achieved at 4 h p.i.

To compare the uptake values with those described in the literature, the biodistribution data will be used for a more accurate comparative analysis for the rest of the discussion. Tumor uptake persisted throughout the 4–48 h imaging period without a significant change (5.11 ± 1.33 at 4 h vs 3.78 ± 0.77 at 48 h, p > 0.1), whereas blood clearance was evident between 4 and 24 h with approximately ∼80% decrease (5.28 ± 0.62 at 4 h vs 0.92 ± 0.25 %ID/g at 24 h) in the circulating 89Zr-Cbl; the final blood activity was observed to be 0.39 ± 0.06 %ID/g at 48 h p.i.

The tumor uptake achieved in our model is comparable to that of the other Cbl-based tracers reported thus far.[6−14] Ikotun et al. investigated the tumor uptake of 64Cu-labeled Cbl in pancreatic, ovarian, murine melanoma, and colorectal tumor models, with the highest %ID/g being 4.84 ± 0.32 at 6 h p.i. in the colorectal tumor models.[14] In the melanoma model, the tumor uptake was highest (3.43 ± 0.87 %ID/g) at 1 h, which decreased to 2.64 ± 0.10 %ID/g after 24 h, whereas 89Zr-Cbl had a higher accumulation and did not show a significant decrease in tumor accumulation over 4–48 h, p > 0.1.

One of the limitations of our 89Zr-Cbl tracer is the observed kidney uptake (94.42 ± 4.27 %ID/g at 4 h), a problem across all of the Cbl tracers to date. Renal processing is the most prominent route for Cbl accumulation/storage and/or reabsorption and is driven by megalin, a known TC-Cbl receptor expressed in kidney proximal tubuli.[20,21] In addition, this is the first positively charged PET tracer reported, the effect of which is unknown. An overall positive charge on the Cbl (as 89Zr-Cbl conjugate), or indeed simply the result of modification of the β-axial position, may affect the Cbl cellular processing across tissues, as has been shown previously for two forms with varying α-ligands (OH-Cbl and CN-Cbl) Cbl species, and warrants further investigation.[12,22−24]

89Zr-Cbl shows feasibility as a PET tracer to identify MDA-MB-453 tumors in vivo. The longer window for PET imaging allowed for reduced uptake in the kidneys, a problem to date in Cbl radiotracers, while still maintaining a moderate tumor uptake over 48 h p.i. This tracer is promising since, to our knowledge, tumor uptake is the highest reported to date for a B12 based PET probe.

Conclusions

We have successfully developed and evaluated the first 89Zr-labeled Cbl tracer as a viable tool for visualizing TC-mediated Cbl uptake into a CD320 positive tumor. 89Zr-Cbl displayed retained tumor uptake up to 48 h p.i., allowing for a longer imaging window. Our data paves the road for future studies to understand the kinetics of Cbl transport and to study the use as a tool for visualizing tumors capable of accumulating Cbl.

Experimental Methods

General

Reagents listed below were purchased and used without further manipulations: dimethyl sulfoxide (DMSO, 99%, Sigma), vitamin B12 (Cbl, ≥98%, Sigma), 1,1′-carbonyl-di-(1,2,4-triazole) (CDT, ≥90%, Fluka), and acetonitrile (MeCN, 99.8%, Pharmaco-Aaper). Compounds were confirmed to be >96% pure by high-performance liquid chromatography (HPLC), proton nuclear magnetic resonance (1H NMR), and/or inductively coupled plasma.

Proton nuclear magnetic resonance (1H NMR) was performed using 400 MHz Bruker spectrometer with the residual solvent peak as an internal standard. Electrospray ionization (ESI) mass spectrometry analyses were carried out on a Shimadzu LCMS-8100. Breast cancer cells (MDA-MB-453) were obtained from the American Type Culture Collection. Charcoal stripped fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma and KD medicals, respectively. Penicillin–streptomycin solution with 10 000 units of penicillin and 10 mg/mL streptomycin in 0.9% NaCl was obtained from Corning.

Analysis of the radiotracer was performed using instant thin-layer chromatography (iTLC, Eckert & Ziegler Mini Scan) with an ethylenediaminetetraacetic acid (EDTA) (50 mM) mobile phase.

Synthesis of Cbl-DFO

Cbl-DFO was synthesized through the activation of the 5′-ribose-hydroxyl group with CDT. CDT (34 mg, 0.261 mmol, 7.2 equiv) was added with cyano-Cbl (50 mg, 0.0368 mmol, 1 equiv) in anhydrous DMSO (3 mL) at 40 °C for 2 h. DFO (208 mg, 0.313 mmol, 7.4 equiv) was added to the reaction mixture and mixed overnight. Purification of Cbl-DFO was done using reversed-phase (RP)-HPLC (Agilent 1200) with a C18 column (Agilent Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm) at a flow rate of 1 mL/min. Detection was done using a UV–vis detector at 360 nm. RP-HPLC method: (A) 0.1% trifluoroacetic acid water and (B) MeCN as solvents with the following gradient: 1% B to 70% B over 15 min, (Rt = 9.4 min). Purity was ≥97% via RP-HPLC. Yield: 30–40%. 1H NMR analysis of the aromatic region: 7.178 (s, 1H), 7.016 (s, 1H), 6.426 (s, 1H), 6.218 (s, 1H), 5.989 (s, 1H). Liquid chromatography–mass spectrometry (LC–MS) analysis: expected m/z: 1942; observed: 972 [M + 2H]2+ and 648 [M + 3H]3+.

89Zr-Radiochemistry

Optimum conditions for radiolabeling of Cbl-DFO were tested by titrating with 89Zr and analyzing the incubated solution using iTLC. Optimum labeling activity was found to be 250 ± 20 mCi/μmol (9250 ± 740 MBq/micromole). Approximately 1 mCi (37 MBq) of 89Zr(C2O4)2 (3D Imaging, LLC) was diluted with 0.9% saline and the pH was adjusted to 7–7.5 by adding 1 M Na2CO3. A solution of Cbl-DFO (0.004 μmol, 10.8 μg) was added to the pH-adjusted 89Zr solution and incubated for 20 min at ambient temperature (Scheme ). Radiolabeling efficiency of >97% was determined by iTLC using silica iTLC strips and EDTA mobile phase (Figure S5a). The identity of the tracer was characterized via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis using Cbl-DFO labeled with cold Zr4+ as standard. Expected: 2030.2 [M+]; observed: 2005.2 [M+-CN– + H+]+ (Figure S5b).

Stability of 89Zr-Cbl was tested by incubating the tracer (200 μCi, 7.4 MBq, 100 μL) in saline (0.9% NaCl) and 50% (1:1 serum/saline) human serum (Sigma) at 37 °C, and the solutions were analyzed at 0, 4, 24, and 48 h intervals using iTLC (Eckert & Ziegler Mini Scan).

Mouse TC Binding to 91Zr-Cbl

Nonradioactive 91Zr-Cbl was synthesized for TC binding studies by reacting Cbl-DFO with 91ZrCl4 as described above. The conjugate was characterized by ESI-MS (data not shown). Mouse TC binding of 91Zr-Cbl was confirmed by radiometric chase assay using 57Co-labeled Cbl and compared with free Cbl (cyanocobalamin) employing a previously described protocol.[17] Mouse TC was derived as previously described.[25]

Modified internalization assay was performed. MDA-MB-453 cells were cultured in Cbl-free media (DMEM with 10% charcoal stripped FBS) and plated in six-well plates. Each well contained 200 000 cells plated and incubated overnight. To each well, 89Zr-Cbl (0.1 μCi, 3.7 KBq, 0.4 pmol of Cbl per well) was added. For the blocking experiment, unmodified Cbl was added (40 pmol per well). Plates were incubated for 1, 4, and 24 h intervals at either 37 or 4 °C. At the end of each time point, wells were serially washed with phosphate-buffered saline (1×), acid (1 mM acetic acid and 1 mM glycine), and base (1 M NaOH, 1 mL, 5 min). Each wash was collected and measured for bound activity using a γ counter (Perking Elmer 2480 WIZARD). Control wells were trypsinized and counted using a cell counter (Contessa II). Internalized activity was normalized to 105 cells.

Cell Lines and Small Animal Xenografts

All of the animal handling and manipulations were conducted in accordance with the guidelines set by WSU Animal Care and Use Committee (IACUC). For imaging and in vivo uptake experiments, female nude mice (Envigo) were kept under Cbl-deficient diet (Teklad Cbl-free custom diet, Envigo) for 3 weeks. Cells were subcutaneously implanted on the shoulder with MDA-MB-453 cancer cells (5 × 106 cells/mouse) after 2 weeks of Cbl-free diet. Cells were injected in 1:1 media/matrigel (Corning LLC) at a volume of 200 μL. The tumor volume until was calculated using the formula length × width2 × 0.52. Mice with tumors of 100–200 mm3 dimensions were used for imaging experiments.

PET Imaging Experiment

89Zr-Cbl was intravenously administered (200−250 μCi/mouse, 7.4−9.3 MBq, 0.8−1 nmol) in sterile saline in mice bearing MDA-MB-453 xenografts. PET imaging was done using a μPET scanner (Concord) at 4, 24, and 48 h p.i. time points, while the mice were anesthetized with 1–2% isoflurane. Images were reconstructed using filtered back projection algorithm. ASIPro VMTM software version 6.3.3.0 (Concord) was used to analyze the images to acquire volumes-of-interest expressed as percent injected dose per gram of tissue (%ID/g). Competitive inhibition was done by co-injecting ∼200-fold excess of unmodified Cbl (200 nmol) with the radiotracer.

Ex Vivo Distribution and Competitive Saturation

The tissue distribution of 89Zr-Cbl was studied by administering 10–25 μCi (0.37−0.93 MBq, 0.04−0.1 nmol) of the tracer on the lateral tail vain of the rodent. For the competitive saturation assay, ∼20 nmol/mouse of cold CN-Cbl was co-injected with 89Zr-Cbl. Euthanasia was performed via CO2 asphyxiation at 4, 24, and 48 h p.i.