Development of Multi-Functional Chelators Based on Sarcophagine Cages

A new class of multifunctionalized sarcophagine derivatives was synthesized for 64Cu chelation. The platform developed in this study could have broad applications in 64Cu-radiopharmaceuticals.


Results and Discussion
Our initial syntheses are shown in Scheme 1. The precursor Me 2 -Ba 2 Sar was synthesized as reported [23]. Further alkylation of Me 2 -Ba 2 Sar was realized using methyl 4-bromomethylbenzoate (1) in the presence of Na 2 CO 3 as base. MeOH was used as the solvent due to its good solubilization of Sar cages. Due to the low selectivity of the multiple amino groups on Me 2 -Ba 2 Sar, the reaction mixture was complicated, as indicated by HPLC. However, we could control the ratio of Me 2 -Ba 2 Sar and 1 to achieve reasonable yields for the desired products. For example, in the synthesis of Me 3 -Ba 3 Sar, we used a 1.2:1 ratio of 1 to Me 2 -Ba 2 Sar, and Me 3 -Ba 3 Sar was obtained in the yield of 32%. When 10 equivalent of 1 was used for the synthesis, Me 7 -Ba 7 Sar and Me 8 -Ba 8 Sar could be obtained in the yields of 15% and 12%, respectively. The methyl protecting groups were readily removed in 0.2 N NaOH to afford Ba 3 Sar, Ba 7 Sar, and Ba 8 Sar in almost quantitative yields. The free benzoic acids are useful for the conjugation with terminal or lysine side chain amino groups of peptides or proteins. In addition to the homo-functionalized Sar cages, it would be interesting to introduce different functional groups to the Sar cages. However, our initial test on modifying Me 2 -Ba 2 Sar with tert-butyl(4-(bromomethyl)phenyl) carbamate (2) failed to provide us the heterofunctionalized Me 2 -Ba 2 An(Boc)Sar (Scheme 1), which may be attributed to the reactivity difference between methyl 4-bromomethylbenzoate and tert-butyl (4-(bromomethyl)phenyl)carbamate. In order to obtain a heterofunctionalized sarcophagine, we synthesized the protected (An(Boc)) 2 Sar as our previous report (Scheme 1). The purified (An(Boc)) 2 Sar was then further alkylated with 1 to give Me-Ba(An(Boc)) 2 Sar with Boc and methyl protective groups on it. The overall yield for Me-Ba(An(Boc)) 2 Sar from DiAmSar was 22%.
As a proof of principle experiment, we chose Ba 3 Sar as an example to test the radiolabeling efficacy of the synthesized chelators. As shown in Figure 1, Ba 3 Sar can be efficiently labeled with 64 Cu at pH 5.5 in sodium acetate buffer after 20 min incubation at 40 °C. Even without purification, radio trace HPLC showed greater than 95% purity of 64 Cu-Ba 3 Sar. Furthermore, to broaden the application of Ba 3 Sar, we tested the 64 Cu labeling in basic conditions, potentially useful for bioligands sensitive to acids. In phosphate buffer (pH 7.4) and borate buffer (pH 8.5), the radiolabeling of Ba 3 Sar could give 71% and 81% yield after 30 min incubation at 40 °C, respectively. To estimate the highest achievable limit of the specific activity of the product, we gradually decreased the amount of Ba 3 Sar added to the reaction. When 2 µg Ba 3 Sar was loaded into 2 mCi (74 MBq) 64 Cu solution, the labeling yield was still as high as 75%. The specific activity of the radiolabeled conjugate was 500 mCi/µmol.
The in vitro stability of 64 Cu-Ba 3 Sar was evaluated after incubation in 1 × PBS and 10% mouse serum by radio HPLC (Figure 1). No significant amount of free 64 Cu was detected by radio HPLC up to 20 h post incubation. We also like to point out that less than 5% of 64 Cu was trapped on NanoSep 10 K filter, suggesting minimum activity was trapped in serum proteins. These data are consistent with the previously published stability results [9][10][11][12][13]. The cross-bridged and cage-like configuration of the Sar structure could lead to the high stability of the complex. The in vivo distribution and stability of 64 Cu-Ba 3 Sar were evaluated by static microPET scans at 5 min and 30 min after injection of 64 Cu-Ba 3 Sar via tail vain into 6-7 weeks old nude mice ( Figure 2). microPET images show that the activity is fast cleared from kidneys. At 5 min post injection, the liver uptake is 4.45 ± 0.40%ID/g, which has no significant difference compared with blood uptake (4.38 ± 0.67%ID/g). At 30 min p.i., the liver uptake (1.26 ± 0.32%ID/g) is only slightly higher than the blood (1.02 ± 0.47%ID/g) and muscle (0.83 ± 0.28%ID/g). The low liver uptake and fast clearance from body indirectly suggested the high in vivo stability of 64 Cu-Ba 3 Sar since the released free 64 Cu would be easily accumulated in liver [25]. To further investigate the localization of 64 Cu-Ba 3 Sar in normal athymic nude mice, we also performed biodistribution study at 24 h after injection. As can be seen in Figure 2C, the highest uptake was found in liver (0.56%ID/g). The low uptake in normal organs also suggests the high in vivo stability of 64 Cu-Ba 3 Sar. Overall, the circulation of 64 Cu-Ba 3 Sar is similar to our previous investigated sarcophagine derivatives. Biodistribution studies of 64 Cu-Ba 3 Sar in normal female nude mouse at 24 h after injection.

General
All chemicals obtained commercially were of analytic grade and used without further purification. The syringe filter and polyethersulfone membranes (pore size, 0.22 μm; diameter, 13 mm) were obtained from Nalge Nunc International (Rochester, NY, USA). The semi-preparative reversed-phase HPLC using a Vydac protein and peptide column (218TP510; 5 µm, 250 × 10 mm) was performed on a Dionex 680 chromatography system with a UVD 170U absorbance detector (Sunnyvale, CA, USA) and model 105S single-channel radiation detector (Carroll & Ramsey Associates, Berkeley, CA, USA). With a flow rate of 4 mL/min, the mobile phase stayed at 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% B [0.1% TFA in acetonitrile (MeCN)] at 0-2 min and was changed from 95% solvent A and 5% B at 2 min to 35% solvent A and 65% solvent B at 32 min. Analytical HPLC had the same gradient with flow rate of 1 mL/min using a Vydac protein and peptide column (218TP510; 5 µm, 250 × 4.6 mm). The UV absorbance was monitored at 218 nm and the identification of the peptides was confirmed based on the UV spectrum using a PDA detector. Copper-64 was purchased from University of Wisconsin. It was dissolved in 0.1 N HCl solution and the specific activity > 1 Ci/µmol. For all the solvents used in the labeling study, Chelex 100 resin was used to remove heavy metal.

Radiochemistry
Acidic conditions: 64 CuCl 2 (20 µL, 74 MBq in 0.1 N HCl) was diluted in 0.1 N sodium acetate (80 µL, pH 5.5) and added to Ba 3 Sar (20 µg). The reaction mixture was kept at 40 °C for 20 min. 64 Cu-labeled product was subsequently purified by analytical HPLC and the radioactive peak containing the desired product was collected. After removal of the solvent by rotary evaporation, the 64 Cu-Ba 3 Sar tracer was reconstituted in PBS (1 mL) and passed through a 0.22 µm syringe filter for in vivo animal experiments. The decay-corrected radiochemical yield (RCY) was 95%.

In Vitro Stability
20 MBq 64 Cu-Ba 3 Sar was incubated in 1× PBS (1 mL, pH 7.4) at 40 °C for 24 h. Then, the stability was measured by analytical radio HPLC with the above mentioned program.

Serum Stability of 64 Cu-Ba 3 Sar
The in vitro stability of 64 Cu-Ba 3 Sar was evaluated by incubation of 7.4 MBq (200 μCi) of 64 Cu-Ba 3 Sar with mouse serum (10%, 1 mL) at 37 °C. At 3 h and 24 h, the solution was filtered through a NanoSep 10 K centrifuge (Pall Corp., Port Washington, NY, USA) to isolate the low-molecular-weight metabolites. The NanoSep 10 K filter was washed with PBS (200 µL) two more times. The filtrates were combined and analyzed by reverse-phase HPLC using conditions identical to those used for the standard 64 Cu-Ba 3 Sar analysis.

MicroPET Imaging and Biodistribution
Animal procedures were performed according to a protocol approved by the University of Southern California Institutional Animal Care and Use Committee. For static microPET scans, the mice were injected with approximately 3.7 MBq (100 μCi) of 64 Cu-Ba 3 Sar via the tail vein (n = 3 for each group). At 5 min and 30 min post injection (p.i.), the mice were anesthetized with isoflurane (5% for induction and 2% for maintenance in 100% O 2 ) using a knock-down box. With the help of a laser beam attached to the scanner, the mice were placed in the prone position and near the center of the field of view of the scanner. The 3-min static scans were then obtained. Images were reconstructed by use of a 2-dimensional ordered-subsets expectation maximization (OSEM) algorithm. No background correction was performed. Regions of interest (ROIs; 5 pixels for coronal and transaxial slices) were drawn over the organs of interest on decay-corrected whole-body coronal images. The maximum counts per pixel per minute were obtained from the ROI and converted to counts per milliliter per minute by using a calibration constant. With the assumption of a tissue density of 1 g/mL, the ROIs were converted to counts per gram per min. Image ROI-derived %ID/g values were determined by dividing counts per gram per minute by injected dose. No attenuation correction was performed. The biodistribution study at 24 h post injection of 64 Cu-Ba 3 Sar was performed in a normal female nude mouse as reported [26].

Conclusions
Novel homo-/hetero-functionalized sarcophagine chelators for 64 Cu radiopharmaceuticals have been developed. In our initial evaluation, good in vitro and in vivo stability was observed for 64 Cu-Ba 3 Sar. These multifunctional chelators could serve as a versatile building platform for multivalent/multimodaltity imaging probe construction, which would have a broad application in both imaging and therapy related research involving copper and other radiometals.