Synthesis and Preclinical Evaluation of Bifunctional Ligands for

Aug 10, 2012 - Chemistry Division, Biological and Chemical Sciences Department, Illinois Institute of Technology, Chicago, Illinois, United States. â€...
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Synthesis and Preclinical Evaluation of Bifunctional Ligands for Improved Chelation Chemistry of 90Y and 177Lu for Targeted Radioimmunotherapy Chi Soo Kang,† Xiang Sun,† Fang Jia,‡ Hyun A Song,† Yunwei Chen,† Michael Lewis,‡ and Hyun-Soon Chong*,† †

Chemistry Division, Biological and Chemical Sciences Department, Illinois Institute of Technology, Chicago, Illinois, United States Department of Veterinary Medicine and Surgery, Department of Radiology, Nuclear Science and Engineering Institute, University of Missouri-Columbia, Columbia, Missouri, United States

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S Supporting Information *

ABSTRACT: We report a practical and high-yield synthesis of a bimodal bifunctional ligand 3p-C-NETA-NCS containing the isothiocyanate group for conjugation to a tumor targeting antibody. 3p-C-NETA-NCS was conjugated to a tumortargeting antibody, trastuzumab, and the corresponding 3pC-NETA-trastuzumab conjugate was evaluated and compared to trastuzumab conjugates of the known bifunctional ligands C-DOTA, C-DTPA, and 3p-C-DEPA for radiolabeling kinetics with 90Y and 177Lu. 3p-C-NETA-trastuzumab conjugate exhibited extremely rapid complexation kinetics with 90Y and 177Lu. 90Y-3p-C-NETA-trastuzumab and 177Lu-3p-C-NETAtrastuzumab conjugates were stable in human serum for 2 weeks. A pilot biodistribution study was conducted to evaluate in vivo stability and tumor targeting of 177Lu-radiolabeled trastuzumab conjugate using nude mice bearing ZR-75-1 human breast cancer. 177 Lu-3p-C-NETA-trastuzumab conjugate displayed low radioactivity level at blood (1.6%), low organ uptake (90% at 30 min, Table 2). 3p-CDEPA-trastuzumab conjugate exhibited similar 90Y- and 177Luradiolabeling pattern to C-DOTA-trastuzumab, achieving the respective radiolabeling efficiency of ∼67% (1 h, Table 1) and >87% (30 min, Table 2) with 90Y and 177Lu. It appears that decadentate 3p-C-DEPA has too many donor groups to form a stable Y(III) or Lu(III) complex, and this may allow the formation of the Lu(III)- or Y(III)-3p-C-DEPA complex to be a reversible process. The results indicates that the tridentate pendant acyclic donors in 3p-C-NETA are essential in rapidly binding Y(III) and Lu(III) and improved complexation kinetics of NETA with the metals relative to DOTA is ascribed to cooperative and bimodal binding of acyclic and macrocyclic donors. 90 Y- or 177Lu-radiolabeled trastuzumab conjugates of 3p-CNETA, 3p-C-DEPA, and C-DOTA were further evaluated for in vitro serum stability (Supporting Information). Both 90Y-3p-C-

removed by the treatment of 8 with 4 M HCl (g) in 1,4dioxane. Reaction of 9 with thiophosgene in CHCl3/H2O provided the desired 3p-C-NETA-NCS. The synthesis of 3p-CNETA-NCS was achieved in 71% overall yield starting from the readily available compound 1. The efficient synthetic method to 7 can be readily scaled up and applied to preparation of many other bifunctional chelates. Trastuzumab is known to target HER2 (human epidermal growth factor receptor 2) overproduced in cancer cells and is a clinically available antibody for treatment of metastatic breast cancer.19,20 3p-C-NETA-NCS was conjugated with trastuzumab, and concentration of trastuzumab in the conjugate was quantified by the spectroscopic assay.16 Ligand to protein ratio (L/P = 3.1) of 3p-C-NETA-trastuzumab conjugate was determined using Lu(III)-ArsenazoIII based UV−vis spectrophotometric assay.16 Trastuzumab conjugates of other bifunctional ligands (C-DOTA, C-DTPA, and 3p-C-DEPA) were also prepared and evaluated for comparison. The purified trastuzumab conjugate (0.25 M NH4OAc, pH 5.5) was labeled with 90Y or 177Lu at room temperature (RT).18 During the reaction time (1 h), the components were analyzed using SEHPLC after challenging the reaction mixture with 10 mM DTPA, and the radiolabeling efficiency (%) was determined using both ITLC and size exclusion (SE)-HPLC (Tables 1 and 2 and Supporting Information). The radiolabeling formation 1780

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NETA-trastuzumab and 177Lu-3p-C-NETA-trastuzumab remained stable in human serum for 2 weeks as confirmed by radio-SE-HPLC analysis. However, a significant amount of the radioactivity was dissociated from 90Y-3p-C-DEPA-trastuzumab (32%, 2 days) and 177Lu-3p-C-DEPA trastuzumab (45%, 2 days). 90Y-C-DOTA-trastuzumab and 177Lu-C-DOTA-trastuzumab were quite stable releasing a minimal level of radioactivity over 2 weeks. The in vitro result suggests that 3p-C-NETA was favorably compared to C-DOTA and displayed excellent complexation kinetics and stability with 90Y and 177Lu. 1B4M-DTPA20 has been used as the chelator of 90Y in Zevalin and was reported to exhibit rapid complexation kinetics with 90Y. However, it should be noted that 90Y-1B4M-DTPA-antibody conjugate was less stable than 90Y-DOTA-antibody conjugate both in vitro and in vivo.21 The in vivo stability and tumor targeting of 177Lu-3p-CNETA-trastuzumab was evaluated by performing a pilot biodistribution study in ZR-75-1-bearing nude mice (Figure 2). The 3p-C-NETA-trastuzumab conjugate was radiolabeled

3p-C-NETA and 3p-C-DEPA, and other known bifunctional ligands, C-DOTA and C-DTPA, were evaluated for a comparative 90Y and 177Lu-radiolabeleing kinetics study. 3p-CNETA-trastuzumab conjugate instantly bound to 90Y or 177Lu with comparable radiolabeling kinetics to C-DTPA-trastuzumab conjugate, while 3p-C-DEPA- and C-DOTA-trastuzumab conjugates displayed similar and slow radiolabeling pattern in binding 90Y and 177Lu. Both 90Y-3p-C-NETA-trastuzumab and 177 Lu-3p-C-NETA-trastuzumab were stable in human serum over 2 weeks. A significant amount of the radioactivity was released from both 90Y-3p-C-DEPA-trastuzumab and 177Lu-3pC-DEPA-trastuzumab in serum over 24 h. 177Lu-3p-C-NETAtrastuzumab conjugate produced a favorable biodistribution profile and exhibited low radioactivity level in organs and high tumor uptake. The results of the in vitro and in vivo studies indicate that bimodal and cooperative binding of the acyclic and macrocyclic moieties property in 3p-C-NETA led to fast complexation kinetics and high complex stability of the octadentate chelate in binding the lanthanides Y(III) and Lu(III). The bimodal ligand 3p-C-NETA has broad applications for RIT of various cancers using 90Y and 177Lu. On the basis of the favorable in vitro complexation and in vivo biodistribution data, 3p-C-NETA-NCS radiolabeled with 90Y or 177 Lu will be further evaluated for comprehensive in vivo biodistribution, pharmacokinetics, and dosimetry using different antibodies and tumor models.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC and ITLC chromatograms for assessment of radiolabeling reaction kinetics and serum stability and in vivo biodistribution data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 2. In vivo biodistribution of 177Lu-3p-C-NETA-trastuzumab conjugate in ZR-75-1-bearing mice (% ID/g, n = 3, intravenous injection).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Phone: 312-567-3235, Fax: 312-5673494. Mailing address: 3101 S. Dearborn St, LS 182, Chemistry Division, Biological and Chemical Sciences Department, Illinois Institute of Technology, Chicago, IL, 60616.

with 177Lu at specific activity of 640 Ci/mmol and in 92.2% yield and radiochemical purity. The highest tumor uptake (10.55 ± 7.93%) of 177Lu-3p-C-NETA-trastuzumab was observed at 72 h. 177Lu-3p-C-NETA-trastuzumab exhibited the highest radioactivity level in the blood at 24 h (14.57 ± 10.63%), which was not significantly different than 72 h (12.09 ± 1.12%), but significantly decreased at 120 h (1.58 ± 1.30%). The 177Lu-3p-C-NETA-trastuzumab conjugate resulted in a very low radioactivity level in the liver (2.20 ± 0.06%) and the kidneys (1.09 ± 0.12%) at 120 h. The radioimmunoconjugate displayed low bone uptake and a higher radioactivity level in the tumor, compared to the normal organs, over the course of the study and the highest tumor-to-blood ratio (6.39) at 120 h. The biodistribution data indicate that 177Lu-3p-C-NETA remains stable in vivo and tumor targeting of 177Lu-3p-CNETA-trastuzumab can be improved by selecting an adequate HER2-positive tumor model.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the National Institutes of Health (R01CA112503 to H. S. Chong). We also acknowledge the Department of Veterans Affairs for providing resources and use of facilities at the Harry S. Truman Memorial Veterans’ Hospital in Columbia, MO.

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REFERENCES

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CONCLUSION Highly efficient and scalable synthetic route to the new bimodal bifunctional ligand 3p-C-NETA centered on the formation of N,N-bisubstituted β-amino iodide and nucleophilic ring-opening of an aziridinium ion was developed. The bimodal ligands, 1781

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