Elsevier

Regulatory Peptides

Volume 144, Issues 1–3, 1 December 2007, Pages 91-100
Regulatory Peptides

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

https://doi.org/10.1016/j.regpep.2007.06.008Get rights and content

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 64Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize 64Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans.

Radiochemical purity of each analogue was > 92%. The muscle relaxivity assay revealed IC50 to be 5.3 × 10–8 M, 4.4 × 10–8 M, 8.1 × 10–8 M, 8.1 × 10–9 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 64Cu 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.

Introduction

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors known as VPAC1, VPAC2 and PAC1 have been shown to be overexpressed on human tumors [1], [2], [3], [4], [5], [6], [7]. For these receptors, two primary peptides have high affinity [8], [9]. First, the VIP, a 28-amino acid, hydrophobic peptide, isolated from porcine intestine [10]. VIP has three Lysine (Lys) residues (at positions 15, 20, and 21), two tyrosine (Tyr) residues (at positions 10 and 22), two Arginine (Arg) residues (at positions 12 and 14), an essential histidine (His) residue at the N terminus and (Asn) amidated C-terminus. All 28 amino acids are required for full biologic activity of VIP [11]. Second, the Pituitary Adenylate Cyclase Activating Polypeptide (PACAP). This 38-amino acid peptide is isolated from bovine hypothalamus [12]. PACAP was found to stimulate accumulation of intracellular and extracellular cAMP in rat anterior pituitary cells [13]. Later Gottschall et al [13] isolated a 27 amino acid peptide (PACAP27) from bovine hypothalamus which they observed to have similar properties as PACAP38. PACAP27 shares a homology of 19 amino acids with VIP28. Like VIP28, PACAP27 also has an amidated (Leu) C-terminal and His at the N-terminal.

The biological actions of VIP and PACAP are mediated by a family of three G protein-coupled receptors, which are designated as VPAC1, VPAC2 and PAC1 [14], [15], [16], [17]. These gene receptors are also detected on the cell membrane of normal intestinal and bronchial epithelial cells [5], [7], albeit receptor density has not been specified. For human tumors on the other hand, studies have revealed that VPAC1, VPAC2, and PAC1 receptors are located at the plasma membrane of the tumor cells [18]. Among the tumors on which VPAC1 receptors have been found in high density and high incidence, include cancers of breast, prostate and urinary bladder (100%), colon (96%), pancreas (65%), lung (58%), stomach (54%), and liver (49%) [1], [2], [3].

With the goal that radiolabeled VIP28 can specifically target these receptors for in vivo visualization of some of these human tumors, Virgolini et al labeled VIP28 with Iodine-123 (123I) (t1/2 13.3 h, γ = 159 keV, 90%) at tyrosine position 10 and 22 [19]. Taking into consideration, the ubiquitous availability, and its predominant world wide role in nuclear medicine, we labeled VIP28 and PACAP27 with 99mTechnetium (99mTc, t1/2 6 h, γ = 140 keV, 94%) which required modification of the peptide to covalently accommodate a group of additional amino acids to chelate 99mTc [20], [21], [22]. The use of 99mTc analogues allowed efficient imaging of human breast cancer [23], [24].

Positron Emission Tomography (PET) permits high imaging resolution leading to the visualization of small tumors (2 mm). PET imaging of tumors with 18Flourine (18F) labeled VIP has not been highly appealing [25]. For PET imaging, our goal was to label VIP and PACAP analogues with another radionuclide, namely 64Copper (64Cu). This radionuclide has a longer half life (t1/2 12.7 h, β+ = 655 keV,17.4%, β = 573 keV, 30%), is available commercially, has abundantly known chemistry and provides nearly quantitative yields so that labeled compound once prepared, can be injected without further purification.

In an attempt to develop VIP28 analogue with higher receptor affinity and better in vivo stability, Bolin et al [26], [27] have synthesized and characterized several analogues of VIP28 molecules. Based upon these data we chose two best additional VIP28 analogues, making a total of four. To enable labeling with 64Cu, each of the four peptides was further derivatized as described in the material and method section.

The purpose of this investigation was to evaluate these analogues to ascertain that their biological activity and receptor specificity was not compromised as compared to those of the VIP28 and to determine their suitability for PET imaging of human breast cancers.

Section snippets

Materials and methods

Three analogues of the VIP peptide (TP3939, TP4200 and TP3982) and one of PACAP (TP3805) all named after their molecular weight were synthesized and labeled with 64Cu or 99mTc. Their amino acid sequences are given in Table 1. We have evaluated the analogues for functional activity and receptor affinity in vitro, for blood clearance and in vivo stability in athymic nude mice bearing T47D human breast cancer cells and for their affinity for human breast cancer specimens ex vivo.

Synthesis and characterization

The amino acid sequence of each analogue is given in Table 1 together with their calculated and observed molecular weight and purity as determined by analytical HPLC.

Radiochemical purity

The radiochemical purity of each 64Cu labeled peptide as determined by HPLC was 95.43 ± 3.78%, 96.85 ± 3.97 %, 94.6 ± 5.79 % and 97.5 ± 4.02 % for Cu-TP3939, 64Cu-TP3982, 64Cu-TP4200 and 64Cu-TP3805 respectively. Retention times varied between 5.53 to 6.7 min. Fig. 1 represents a typical HPLC elution curve. The diagonal line represents

Discussion

Annually in the USA approximately 7 million breast biopsies are performed, approximately 80% of which (5.6 million) find benign pathology. The ability to noninvasively determine the benign or malignant status of suspected breast cancer mass found by mammography may minimize the number of millions of unnecessary, invasive biopsies; spare the patient from physical and psychological trauma as well as save health care dollars. Although great strides have been made for imaging breast cancer using

Acknowledgments

This work was supported by NIH grant CA-109231.

We thank Dr. Juan Pallazzo MD, and Magdalena B. Potoczek of the department of Pathology for their advice and assistance in the studies of pathological specimens. Our thanks are also due to Stephanie Schulz, PhD, Department of Pharmacology and Experimental Therapeutics for the use of the homogenizer (BioSpec products).

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