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68Ga-1,4,7-Triazacyclononane,1-glutaric acid-4,7-acetic acid-1,2-diaminoethane-γ-folate (P3246)

68Ga-P3246
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: September 20, 2012.

Chemical name:68Ga-1,4,7-Triazacyclononane,1-glutaric acid-4,7-acetic acid-1,2-diaminoethane-γ-folate (P3246)image 144080992 in the ncbi pubchem database
Abbreviated name:68Ga-P3246
Synonym:68Ga-NODAGA-folate
Agent category:Compound
Target:Folate receptor
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal:68Ga
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on the above structure for additional information in PubChem.

Background

[PubMed]

Folic acid (folate) is a water-soluble B vitamin (1) that is essential for methylation and DNA synthesis. The primary pathway for entry of folate into cells is through the facilitated transporter, which has a low affinity for folate (Michaelis constant (Km) = 1–5 μM). Some cells in the choroid plexus, kidney, lung, thyroid, spleen, placenta, and thymus also possess a higher-affinity receptor (dissociation constant (Kd) = 0.5 nM) that allows folate uptake via receptor-mediated endocytosis. Some human epithelial tumor cells have been found to overexpress folate receptors (2). More than 90% of human ovarian and endometrial cancers express the high-affinity folate receptor, which is absent in the corresponding normal tissues. Breast, colorectal, renal, and lung carcinomas also overexpress the high-affinity folate receptor but at lower frequencies (20%–50%). Activated macrophages, but not resting macrophages, have also been found to have the high-affinity folate receptor (3).

Several folate-based conjugates (111In-DTPA-folate, 99mTc-EC-folate, and [18F]FBA-folate) have been studied in tumor imaging (4-8). Deferoxamine (DF), a chelating agent, was conjugated to folic acid to form a mixture of two isomers, DF-α-folate and DF-γ-folate. Only the DF-γ-folate isomer was able to displace [3H]folic acid from its receptors, with a 50% inhibition concentration similar to that of folic acid (2.5 nM versus 2.4 nM) (9). Fani et al. (10) prepared a γ-folate conjugate with tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and 1,2-diaminoethane as a spacer to form P3026, which was labeled with 68Ga for positron emission tomography (PET) imaging of folate receptors in tumors. To further the quest for a 68Ga-folate conjugate for clinical application, folic acid was conjugated to 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) via 1,2-diaminoethane as a linker between the NODAGA and folic acid (P3246) (11). 68Ga-P3246 was evaluated as a PET agent for imaging folate receptor expression in a mouse tumor model. 67/68Ga-P3246 exhibited a higher tumor/blood ratio than 67/68Ga-P3026 in the same tumor model.

Synthesis

[PubMed]

Fani et al. (11) coupled P3246 (12 nmol) with 68Ga in sodium acetate buffer (pH 4.0) for 10 min at 25°C to yield 68Ga-P3246 with >92% radiochemical purity. Radiochemical yields exceeded 95% with a specific activity of ~30 MBq/nmol (0.81 mCi/nmol). 67Ga-P3246 was similarly radiolabeled with a specific activity of ~3 MBq/nmol (0.081 mCi/nmol). 68Ga-P3238 was prepared with similar specific activity as 68Ga-P3246.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The human nasopharyngeal carcinoma KB cell line folate receptors were studied with 67Ga-P3246 saturation binding studies at 4°C (11). 67Ga-P3246 showed a Kd (affinity constant) of 5.61 ± 0.96 nM, which was similar to the Kd value (4.65 ± 0.82 nM) for 67Ga-P3026 (the DOTA-folate conjugate). 67Ga-P3246 (2.5 nM) was rapidly associated (bound to the cell surface and internalized) with KB cells at 37°C, with 60% of incubation dose (ID) at 30 min and 72% ID at 4 h. Approximately 15% ID 67Ga-P3246 was internalized at 4 h. Excess folate blocked the cell-associated radioactivity to <1% ID. Approximately 76% of radioactivity was retained in the cells after 4 h incubation in fresh medium.

Animal Studies

Rodents

[PubMed]

Fani et al. (11) performed ex vivo biodistribution studies of 0.4 nmol 67/68Ga-P3246 in nude mice (n = 3–5/group) bearing KB tumor xenografts. Accumulation of 67/68Ga-P3246 in the KB tumors was 15.56 ± 3.67, 18.42 ± 0.74, 16.29 ± 4.46, and 14.32 ± 5.80% injected dose/gram (ID/g) at 1, 2, 4, and 24 h after injection, respectively. The organ with the highest accumulation at 4 h after injection was the kidney (130% ID/g), followed by the salivary gland (8.6% ID/g), pancreas (2.9% ID/g), adrenal (2.8% ID/g), heart (2.0% ID/g), muscle (1.8% ID/g), stomach (1.5% ID/g), and liver (1.1% ID/g). The accumulation in the blood was low (0.06% ID/g) at 4 h. The tumor/blood ratios were 81, 207, 254, and 391 at 1, 2, 4, and 24 h, respectively. Pretreatment with excess folate (40 nmol, 5 min before 67/68Ga-P3246 injection) reduced the radioactivity accumulation by >85% in the folate receptor-positive tumor, salivary glands, and kidneys at 4 h after injection. Pretreatment with pemetrexed (60 min before 67Ga-P3246 injection), a folate analog metabolic inhibitor, significantly reduced the kidney accumulation of 67/68Ga-P3246 by >70% at 1 h and 4 h after injection. On the other hand, little inhibition by pemetrexed was observed in the tumor and other organs. 67/68Ga-P3246 exhibited higher tumor/blood ratios than its 67/68Ga-DOTA counterpart (67/68Ga-P3026 with tumor/blood ratios of 36 at 1 h, 50 at 2 h, and 70 at 4 h).

Whole-body PET imaging scans were performed for 60 min after injection of 10 MBq (0.27 mCi) 68Ga-P3246 (0.4 nmol) in nude mice bearing KB tumor xenografts (the number of mice used was not reported) (11). The highest radioactivity levels were visualized in the KB tumor, salivary glands, and kidneys, with standard uptake values of 2.2, 1.8, and 10 at 60 min, respectively. The accumulation of 68Ga-P3246 in the kidneys was blocked by pre-injection of pemetrexed, whereas the accumulation in the tumor and salivary glands remained at the same levels. No blocking studies using excess folate were reported for the PET studies.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

1.
Stanger O. Physiology of folic acid in health and disease. Curr Drug Metab. 2002;3(2):211–23. [PubMed: 12003352]
2.
Ke C.Y., Mathias C.J., Green M.A. The folate receptor as a molecular target for tumor-selective radionuclide delivery. Nucl Med Biol. 2003;30(8):811–7. [PubMed: 14698784]
3.
Nakashima-Matsushita N., Homma T., Yu S., Matsuda T., Sunahara N., Nakamura T., Tsukano M., Ratnam M., Matsuyama T. Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis. Arthritis Rheum. 1999;42(8):1609–16. [PubMed: 10446858]
4.
Mathias C.J., Hubers D., Low P.S., Green M.A. Synthesis of [(99m)Tc]DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical. Bioconjug Chem. 2000;11(2):253–7. [PubMed: 10725102]
5.
Mathias C.J., Lewis M.R., Reichert D.E., Laforest R., Sharp T.L., Lewis J.S., Yang Z.F., Waters D.J., Snyder P.W., Low P.S., Welch M.J., Green M.A. Preparation of 66Ga- and 68Ga-labeled Ga(III)-deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals. Nucl Med Biol. 2003;30(7):725–31. [PubMed: 14499330]
6.
Mathias C.J., Wang S., Low P.S., Waters D.J., Green M.A. Receptor-mediated targeting of 67Ga-deferoxamine-folate to folate-receptor-positive human KB tumor xenografts. Nucl Med Biol. 1999;26(1):23–5. [PubMed: 10096497]
7.
Mathias C.J., Wang S., Waters D.J., Turek J.J., Low P.S., Green M.A. Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical. J Nucl Med. 1998;39(9):1579–85. [PubMed: 9744347]
8.
Bettio A., Honer M., Muller C., Bruhlmeier M., Muller U., Schibli R., Groehn V., Schubiger A.P., Ametamey S.M. Synthesis and Preclinical Evaluation of a Folic Acid Derivative Labeled with 18F for PET Imaging of Folate Receptor-Positive Tumors. J Nucl Med. 2006;47(7):1153–1160. [PubMed: 16818950]
9.
Wang S., Lee R.J., Mathias C.J., Green M.A., Low P.S. Synthesis, purification, and tumor cell uptake of 67Ga-deferoxamine--folate, a potential radiopharmaceutical for tumor imaging. Bioconjug Chem. 1996;7(1):56–62. [PubMed: 8741991]
10.
Fani M., Wang X., Nicolas G., Medina C., Raynal I., Port M., Maecke H.R. Development of new folate-based PET radiotracers: preclinical evaluation of Ga-DOTA-folate conjugates. Eur J Nucl Med Mol Imaging. 2011;38(1):108–19. [PubMed: 20799032]
11.
Fani M., Tamma M.L., Nicolas G.P., Lasri E., Medina C., Raynal I., Port M., Weber W.A., Maecke H.R. In Vivo Imaging of Folate Receptor Positive Tumor Xenografts Using Novel (68)Ga-NODAGA-Folate Conjugates. Mol Pharm. 2012;9(5):1136–45. [PubMed: 22497506]
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