Background

[PubMed]

The ⁶⁸Ga-labeled β-aminoalanine, γ-aminohomoalanine, and ε-aminolysine conjugates of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), abbreviated as ⁶⁸Ga-1a, ⁶⁸Ga-1b, and ⁶⁸Ga-1c, respectively, were synthesized by Shetty et al. for positron emission tomography (PET) of tumors (1).

Radiolabeled amino acids represent a diverse class of tracers that target the increased amino acid transport in cancer cells (2, 3). To date, >20 distinct amino acid transporters have been identified in mammalian cells, and these transporters differ in terms of substrate specificity, tissue expression patterns, sodium and other ion dependence, pH sensitivity, and transport mechanism (4, 5). Because of increased demand for amino acids in malignant cells, some transporters have been shown to be overexpressed in different types of tumors, and the process of amino acid transport is relatively fast (1, 2, 6). These features make tumor imaging with amino acid tracers possible within 20 min. Indeed, there is growing evidence that radiolabeled amino acids have the potential to overcome some of the limitations of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) in tumor imaging, especially in the imaging of primary and recurrent brain tumors, neuroendocrine tumors, and prostate cancers (2, 3, 7). Different studies also showed that radiotracers that target different amino acid transporters exhibit different imaging properties that may provide unique biological information of tumors (1, 2, 8).

The first group of widely investigated amino acids is the analogs of phenylalanine and tyrosine (2, 3). Because of their bulky neutral side chains, these natural amino acids are the substrates of system L transporters and have been proven to be useful for tumor imaging, particularly for brain tumors. The limitation common to most of the natural amino acids is the susceptibility to in vivo metabolism, which decreases tumor specificity and complicates kinetic analysis. Because none of the natural amino acids contain fluorine or iodine, labeling with fluorine-18 or iodine-123 while retaining identical biological activity is also difficult. These shortcomings associated with natural amino acids can be partially overcome by using non-natural amino acids. Typically, non-natural amino acids are neither metabolized nor readily incorporated into protein in vivo (1-3). One group of non-natural amino acids is α,α-dialkyl amino acids, which are generated by substituting the α-carbon hydrogen of natural amino acids with a methyl group or other alkyl chains. These amino acids are primarily the substrates of system A transporters. The second group is alicyclic amino acids, which are α,α-dialkyl amino acids with side chains bonded covalently to each other to form a cyclic ring. These amino acids are the substrates of system L transporters. The third group is the non-natural proline derivatives, which exhibit different transport selectivity. One challenge in developing amino acid radiotracers is to overcome the low selectivity and the decreased recognition after radiolabeling to specific transporters (2, 3, 8). Another challenge is the low uptake of amino acid agents in tumors, which leads to less sensitivity for tumor detection than with [¹⁸F]FDG (7).

Shetty et al. synthesized a group of ⁶⁸Ga-labeled alanine and lysine derivatives of NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), and 1,4,7,10-tetraazacyclododecane-1,4,7,-triacetic acid (DO3A) (1, 8). The four bifunctional chelating agents have similar sizes, but they differ in the net charges and stability because of the different numbers of pendent carboxylate arms. The amino acids have been conjugated to one of the carboxylate arms, and the nitrogen atoms in the heterocyclic ring are presumed to coordinate with metals to form chelates. Biodistribution studies and PET imaging indicate the structure–activity relationship of the amino acid derivatives, and the selective uptakes of these compounds by different cancer tissues might provide an insight on the different modes of amino acid uptake by cancer cells (1, 8).

This chapter summarizes the data obtained with ⁶⁸Ga-labeled NOTA derivatives: ⁶⁸Ga-1a (⁶⁸Ga-NOTA-β-aminoalanine), ⁶⁸Ga-1b (⁶⁸Ga-NOTA-γ-aminohomoalanine), and ⁶⁸Ga-1c (⁶⁸Ga-NOTA-ε-aminolysine). These NOTA derivatives were comparatively analyzed with the corresponding DOTA derivatives (⁶⁸Ga-2a, ⁶⁸Ga-2b, and ⁶⁸Ga-2c, respectively) (1).

Synthesis

[PubMed]

The protected β-amino-l-alanine was synthesized from N-tert-butyl-l-serine methyl ester. Conjugation of NOTA with protected β-amino-l-alanine was performed in aqueous solution using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride as a coupling reagent to obtain the alanine conjugate. Hydrolysis of the protected esters using LiOH, followed by hydrochloric acid, resulted in the formation of compound 1a (NOTA-β-aminoalanine) as a hydrochloride salt.

The γ-amino-l-homoalanine was purchased, and the ε-aminolysine was synthesized from N^α-boc-N^ε-benzyloxycarbonyl-lysine by tert-butyl esterification and subsequent deprotection. Conjugation of γ-amino-l-homoalanine and ε-aminolysine with NOTA was performed separately with N,N'-dicyclohexylcarbodiimide as a coupling agent and pyridine as a base. Further hydrolysis and deprotection resulted in the formation of compound 1b (NOTA-γ-aminohomoalanine) and compound 1c (NOTA-ε-aminolysine), respectively. All final products were purified. The structure of Ga-complexed NOTA-aminoalanine (1a) in aqueous solution was confirmed with multinuclear magnetic resonance spectroscopy (1).

The NOTA and DOTA derivatives were labeled with ⁶⁸Ga either at room temperature or in a boiling water bath (1). The labeling efficiency was >95% for all agents, and no free ⁶⁸Ga was found after purification. The specific activities of the purified agents were in the range 1.94–9.21 GBq/μmol (52.43–248.92 mCi/µmol). Stability studies at room temperature confirmed the stability of all agents for >4 h. The chelating agents, DOTA and NOTA, were also labeled with ⁶⁸Ga as controls for the in vitro protein binding and cell uptake studies.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Shetty et al. performed protein binding studies by incubating the agents with human serum at 37°C for 10 min and 1 h, respectively (1). ⁶⁸Ga-1b showed the lowest level of protein binding (1.71% at 10 min and 1.44% at 1 h), followed by ⁶⁸Ga-1c (2.33% at 10 min and 2.64% at 1 h) and ⁶⁸Ga-1a (3.07% at 10 min and 3.77% at 1 h).

In comparison with the corresponding DOTA derivatives, all NOTA derivatives had lower levels of serum protein binding. Of these, ⁶⁸Ga-1b showed the lowest level of protein binding and ⁶⁸Ga-2a showed the highest level of binding (9.87% at 10 min and 9.81% at 1 h). Only the DOTA derivatives showed significant increases in protein binding over time (1).

Cell uptake studies after incubation of the cells with each agent showed that all three agents had significantly higher uptakes than the control compounds (⁶⁸Ga-NOTA and ⁶⁸Ga-DOTA) in both Hep3B (a human hepatoma cell line) and CT-26 (a mouse colon cancer cell line) cells (1). Of the three agents, ⁶⁸Ga-1a showed the highest uptake in both cell lines.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

References

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Shetty D., Jeong J.M., Ju C.H., Kim Y.J., Lee J.Y., Lee Y.S., Lee D.S., Chung J.K., Lee M.C. Synthesis and evaluation of macrocyclic amino acid derivatives for tumor imaging by gallium-68 positron emission tomography. Bioorg Med Chem. 2010;18(21):7338–47. [PubMed: 20926300]

2.

McConathy, J., W. Yu, N. Jarkas, W. Seo, D.M. Schuster, and M.M. Goodman, Radiohalogenated nonnatural amino acids as PET and SPECT tumor imaging agents. Med Res Rev, 2011. [PubMed: 21793016]

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Laverman P., Boerman O.C., Corstens F.H., Oyen W.J. Fluorinated amino acids for tumour imaging with positron emission tomography. Eur J Nucl Med Mol Imaging. 2002;29(5):681–90. [PubMed: 11976809]

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Ganapathy V., Thangaraju M., Prasad P.D. Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacol Ther. 2009;121(1):29–40. [PubMed: 18992769]

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Goberdhan D.C. Intracellular amino acid sensing and mTORC1-regulated growth: new ways to block an old target? Curr Opin Investig Drugs. 2010;11(12):1360–7. [PMC free article: PMC3044466] [PubMed: 21154118]

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Fuchs B.C., Bode B.P. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime? Semin Cancer Biol. 2005;15(4):254–66. [PubMed: 15916903]

7.

la Fougere C., Suchorska B., Bartenstein P., Kreth F.W., Tonn J.C. Molecular imaging of gliomas with PET: Opportunities and limitations. Neuro Oncol. 2011;13(8):806–19. [PMC free article: PMC3145468] [PubMed: 21757446]

8.

Shetty D., Jeong J.M., Ju C.H., Lee Y.S., Jeong S.Y., Choi J.Y., Yang B.Y., Lee D.S., Chung J.K., Lee M.C. Synthesis of novel 68Ga-labeled amino acid derivatives for positron emission tomography of cancer cells. Nucl Med Biol. 2010;37(8):893–902. [PubMed: 21055619]