99mTc-Hydrazinonicotinamide-epidermal growth factor


Leung K.

Publication Details



In vitro Rodents



Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) secreted by ectodermic cells, monocytes, kidneys and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF receptor (EGFR) is a transmembrane protein with an intracellular tyrosine kinase present on most cells. EGFR is overexpressed on many solid tumor cells, such as those of breast, non-small-cell lung, head and neck, and colon cancers (2-4). The high level of EGFR expression on cancer cells is associated with a poor prognosis (5-7). EGF has been labeled with 99mTc by various laboratories as a potential SPECT imaging agent for EGFR expression on cancer cells (8-11).

The Ras protein is a key regulator in the signaling pathway of the EGFR. Inhibition of farnesylation of Ras in cancer cells by farnesyltransferase inhibitors (FTI) led to inhibition of tumor growth, up regulation of EGFR, and decreased internalization of EGFR (12). 99mTc-HYNIC-EGF was developed for in vivo prediction of cancer cell response to FTI (8).



Hydrazinonicotinamide (HYNIC) was conjugated to human EGF to form HYNIC-EGF, which was purified by dialysis with a yield of >94% (8). A mixture of HYNIC-EGF and [99mTc]pertechnetate (74–370 MBq, 2–10 mCi) in reaction solution was incubated at room temperature for 20 min. 99mTc-HYNIC-EGF was purified by column chromatography to yield 81-95%, with a radiochemical purity of >95%. The specific activity of 99mTc-HYNIC-EGF was 1.23 Bq/nmol (33 pCi/nmol) for in vitro binding studies and 3.7–7.4 MBq/nmol (0.1–0.2 mCi/nmol) for the biodistribution studies.

In Vitro Studies: Testing in Cells and Tissues


99mTc-HYNIC-EGF was found to be stable in Ham’s F12 cell growth medium at 25ºC up to 4 h (8). LoVo human colon adenocarcinoma cells showed a Kd of 3.6 nM with 99mTc-HYNIC-EGF and 4.0 nM with [125I]EGF in saturation binding studies (8, 13). MAb88910 is a monoclonal antibody that inhibits EGF binding to EGFR. MAb88910 showed a Ki value of 1.7 nM and 1.3 nM for blocking 99mTc-HYNIC-EGF and [125I]EGF binding to LoVo cells, respectively (8, 13).

Animal Studies



Normal mice were injected with injected i.v. with 99mTc-HYNIC-EGF (0.555 MBq, 0.015 mCi, 1 μg or 0.15 nmol) (8). The liver (55.0 ± 11.2% injected dose (ID)/g at 40 min) and kidneys (31.9 ± 18.4% ID/g at 5 min) were the major organs of uptake. A small amount was taken up by the small intestine (4.3 ± 1.8% ID/g at 2 h) and large intestine (7.7 ± 1.9% ID/g at 6 h) at later time points. Stomach activity was significantly higher than blood, beginning 20 min after injection. There was also some uptake in the spleen, which is maximal at 10 min after injection. After 48 h, only 52% of the injected 99mTc-HYNIC-EGF was excreted, whereas the remaining radioactivity was in the liver, kidneys, and spleen.

99mTc-HYNIC-hEGF was taken up in LoVo tumor tissue in tumor-bearing athymic mice (8). The tumor-to-thigh ratio was maximal at 2 h (2.5 ± 0.8). The nominal value of the uptake at 2 h was 3.5 ± 1.2%ID/g. Blocking with MAb88910 resulted in a decrease of the ratio from 2.5±0.8 to 1.2±0.2 (p<0.01). Co-injection of 100 μg of unlabelled EGF decreased the ratio to 1.1 ± 0.3 (p<0.01). In both cases, the muscle uptake did not change significantly. When comparing 99mTc-HYNIC-EGF tumor uptake data to 123I-EGF values, no significant differences were seen. Both tracers showed a maximum tumor-to-thigh ratio at 2 h (123I-EGF uptake ratio at 2 h was 2.0 ± 0.3). Pretreatment of tumor-bearing mice with FTI inhibitor (R115777, 100 mg/kg) for 6-8 h before 99mTc-HYNIC-EGF injection decreased the tumor-to-muscle ratio from 2.5 to 1.0. Similar results were also obtained using planar gamma scintigraphy imaging. The control experiment using 99mTc-HSA showed that the effect of FTI inhibition was not due to an influence on vascularization (13).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available

Human Studies


No publication is currently available.


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