67Ga labeled cyclic diethylenetriamine pentaacetic acid Insulin


Chopra A.

Publication Details



In vitro Rodents



Although the main role of insulin is considered to be the regulation of glucose transport and metabolism, insulin is known to influence directly or indirectly a variety of other metabolic processes in the cell such as protein synthesis, DNA transcription, and RNA translation in that insulin receptors are found in almost all cell types (1). Because insulin plays a key role in the regulation of these important processes, it has been associated with conditions such as diabetes, obesity, heart disease, neurodegenerative diseases, and even cancer (2-5). The insulin amino acid sequence appears to be well conserved throughout the animal kingdom, and there is a difference of less than five amino acids between the human, bovine, and porcine molecules. In addition, insulin from these mammals is known to have an invariant location of three disulfide bonds (6). Insulin was initially purified mainly from bovine and porcine sources for the treatment of humans, but because of high demand insulin is now available commercially as a recombinant form expressed in either Escherichia coli or yeast cells (7). Insulin is approved by the United States Food and Drug Administration for the treatment of diabetes and is being evaluated in several diabetes-related clinical trials.

Insulin mediates its effects through the activation of a receptor tyrosine kinase that has been studied primarily in cultured cells or in isolated cell membranes (1). Radioactive iodine (123I) was used to label insulin, and the in vivo metabolism of this radiolabeled molecule has been studied in rodents and humans with the use of single-photon emission computed tomography (SPECT); however, 123I has a very short half-life (~13 h), making it of limited value in the investigation of changes in receptor densities that may occur under various pathological conditions (8-10). Positron emission tomography (PET) using insulin labeled with radioactive fluorine (18F) has been used to study insulin receptors in monkeys and under in vitro conditions in HEK-293 cells (11, 12). This labels also has a very short half-life (~110 min), therefore this limitation prohibits the use or either 123I or 18F labeled insulin in studies to investigate receptor changes over extended periods. Insulin labeled with meta-stable technetium (99mTc) was also used to study the biodistribution in rats using SPECT, but this radionuclide also has a short half-life of ~6 h (13). Jalilian et al. used radioactive gallium (67Ga), which has a half-life of >3 days, to label insulin, and these investigators used SPECT to study its biodistribution in normal rats (14).



The synthesis of 67Ga-labeled insulin was described by Jalilian et al. (14). Pharmaceutical-grade insulin was obtained from commercial sources and used without further purification. Cyclic diethylenetriamine pentaacetic acid (DTPA) was coupled to insulin as described elsewhere (15). For radiolabeling the DTPA-conjugated insulin, [67Ga]gallium chloride in 0.2 M hydrochloric acid was added to a conical vial and dried under a stream of nitrogen. The DTPA-insulin in phosphate buffer (pH 8.0) was added to the vial and mixed gently for 30 s. The resulting solution was then incubated at room temperature for 30 min, and the products were checked with instant thin-layer chromatography (ITLC) and high-performance liquid chromatography (HPLC). The Rf values for the various components on ITLC were not reported. If the purity of labeled insulin was <90%, it was purified with gel filtration on a Sephadex G-50 column. The specific activity of 67Ga-labeled insulin was reported to be 0.68–0.86 MBq/166 nmol (0.018–0.023 mCi/166 nmol). The radiochemical yield of the reaction was reported to be 99%; final purity of the product was not reported (15). The stability of 67Ga-labeled insulin was investigated in phosphate-buffered saline (PBS) containing human serum (pH 7.4) for up to 2 h at 37°C (15). No loss of radionuclide or change in 67Ga-labeled insulin was reported on exposure to human serum under these conditions as determined with ITLC or HPLC.

In Vitro Studies: Testing in Cells and Tissues


The in vitro binding activity of 67Ga-labeled insulin was studied in human white blood cells (14). Blood samples were obtained from fasting and non-fasting volunteers and centrifuged at 3,000 rpm for 5 min. The cell pellet was washed three times with PBS and gently resuspended in the same buffer. Radiolabeled insulin was added to the cell suspension, and the mixture was incubated at 37°C for up to 150 min. Cell samples were taken from the incubation mixture at different times and washed with PBS, and the bound radioactivity was determined. Blood cells from the non-fasting volunteers did not show any change in insulin binding over time. The investigators suggested that this was probably because the insulin secreted into the blood after eating competed with the labeled radiochemical. A higher binding of labeled insulin was observed in cells obtained from the fasting volunteers (14). The affinity and dissociation constants for 67Ga-labeled insulin and blocking studies using unlabeled insulin were not reported.

Animal Studies



The biodistribution of [67Ga]-labeled insulin was investigated in normal rats (14). The rats were injected with the radiolabeled compound through the tail vein, and the animals were euthanized 2–44 h after injection. The number of animals used per time point was not reported. All major organs of the animals were collected and weighed, and accumulated radioactivity was counted. SPECT imaging was performed on animals 24 and 48 h after the administration of 67Ga-labeled insulin. The radiolabel was reported to clear from the blood within 2–3 h, and the tracer accumulated primarily in the liver (>2.5% of the injected dose/gram tissue (% ID/g)), with a small fraction (<0.25% ID/g) accumulating in the spleen and kidneys. SPECT imaging revealed that radioactivity accumulated primarily in the liver, as reported earlier with 123I-labeled insulin (16). Blocking studies were not reported.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information



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