Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation

Abstract

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.

Fig. 1

HPLC elution profile of one of ⁶⁴Cu - TP3939, revealing 96.5% of the radioactivity is bound to a peak at 6.7 min. The UV peak is also centered around 6.7 min. The diagonal line represents % gradient as a function of time.

Fig. 2

Effect of increasing concentration of VIP₂₈ and its analogues as % relaxivity of the tissue. IC₅₀ values are presented in the inset table

Fig. 3

The cell binding data plotted as per Scatchard for ^99mTc-TP3939. Binding was performed using T47D human breast cancer cells that express VPAC1 receptors. In this case we used ^99mTc instead of ⁶⁴Cu, as the biological activity of peptide analogues is not influenced by the tracer used. The saturable plot represents receptor specific binding. The Kd (−1/slope) was estimated using the NIH Chemical Genomics Center, Assay Guidance Manual [Version 4:1; Receptor Binding Assays.htm]. The Kd values as calculated for the other analogues are given in Table. 2.

Fig. 4

Example of digital autoradiography in normal and breast cancer tissues were incubated with ⁶⁴Cu-TP4200. The images and the calculated tumor and normal PSL/mm² invariably reflect higher receptor expression in the tumor tissues. These results were consistent with those of the corresponding RT-PCR analytical data.

Fig. 5

RT-PCR of normal and human breast cancer tissues revealing distinct cycle difference between the tumor and the normal tissue. The fewer cycles are required to amplify the protein from tumor tissue as compared to the normal tissue. The density of VPAC1 receptors are more in the tumor tissue (T/N = 5 to 118) than that on the normal breast tissue.

Fig. 6

The blood clearance study of one of the analogues, TP4200. The clearance curve is biphasic, α t½ = 3.3 min and the β t½ = 150 min.

Fig. 7

Fig. 7.a. at the left is a Coomassie blue stained gel representing the protein bands and their approximate molecular weight and Fig. 7.b at right is the autoradiography representing ⁶⁴Cu radioactivity associated with the corresponding proteins. Lane 1: Invivo ⁶⁴Cu-TP3939 in mouse serum, Lane 2: ⁶⁴Cu-TP3939 incubated with HSA, exvivo, Lane 3: ⁶⁴CuCl₂ incubated with HSA exvivo and Lane 4: ⁶⁴CuCl_2.