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HSDAVFTDNYTKLRKQ-NIe-AVKK-(3-OCH3,4-OH)-FLNSSV-GABA-L-(Dap-(BMA)2)-64Cu

64Cu-TP3939

, PhD and , PhD.

Author Information
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim
, PhD
Laboratories of Radiopharmaceutical Research and Molecular Imaging. Department of Radiology, Thomas Jefferson University, Philadelphia, PA, Corresponding Author, ude.nosreffej@rukahT.wehtaM

Created: ; Last Update: June 11, 2008.

Chemical name:HSDAVFTDNYTKLRKQ-Nle-AVKK-(3-OCH3,4OH)-FLNSSVL-GABA-(Dap-(BMA)2)-64Cu
Image TP3939Cu64.jpg
Abbreviated name:64Cu-TP3939
Synonym:64Cu-Vasoactive intestinal peptide
Agent Category:Peptide
Target:Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPAC1, VPAC2, PAC1)
Target Category:Receptor binding
Method of detection:Positron emission tomography (PET)
Source of signal/contrast:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
64Cu-TP3939. The 64Cu coordination has not been confirmed by experiment.
Click on protein, nucleotide (RefSeq), and gene for more information about VIP.

Background

[PubMed]

HSDAVFTDNYTKLRKQ-Nle-AVKK-(2-OCH3,4OH)-FLNSSV-GABA-L-(Dap-(BMA)2)-64Cu (64Cu-TP3939) is a radiolabeled molecular imaging agent developed for positron emission tomography (PET) imaging of tumors with overexpression of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors (1, 2). 64Cu is a positron emitter with a half-life (t½) of 12.7 h.

VIP and PACAP38 are two basic C-terminally amidated peptides (28 amino acids and 38 amino acids, respectively) that possess a remarkable amino acid homology (3-6). VIP was initially isolated from porcine intestine, and PACAP38 was isolated from bovine hypothalamus (7-9).(Later, another form of PACAP was isolated with sequence homologous to N-terminal 27 amino acid secued of PACAP28 and named PACAP27.). VIP (HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2) is a hydrophobic, basic peptide that contains three lysine residues (1520, and 21, ), two arginine residues (12 and 14, ), two tyrosine residues (10 and 22, ), an essential histidine residue at the N terminus, and an amidated C-terminus (10). The sequence of PACAP27 shows 68% identity with VIP (11). Like VIP, PACAP27 also has an amidated C-terminal and histidine residue at the N terminus.

VIP and PACAP are known regulators within the hypothalamus-pituitary-adrenal axis. VIP has been shown to have a broad range of activities as a neuroendocrine mediator in diverse cells and tissues (12). Similarly, PACAP has also been shown to act as a hormone, a neurohormone, a neurotransmitter, and a trophic factor in a number of tissues (11). VIP and PACAP act through common G-protein–coupled receptors (11, 12). Three main subtypes of VIP/PACAP receptors are identified as PAC1, VPAC1, and VPAC2. PACAP binds with high affinity to all three receptors, but VIP binds with high affinity to VPAC1 and VPAC2 and with low affinity to PAC1. These receptors have been shown to be overexpressed on many human tumors, including breast (100% receptor incidence), prostate (100%), ovarian (100%), pancreas (65%), lung (58%), colon (96%), stomach (54%), liver (49%), and urinary bladder (100%) carcinomas, as well as lymphomas (58%) and meningiomas (100%) (5, 13). These receptors are potential molecular targets for diagnosis, prevention, and treatment of various cancers (14-17).

123I-Labeled VIP (123I-VIP) was prepared for single-photon emission computed tomography (SPECT) of colorectal, pancreatic, and gastric carcinomas, as well as carcinoid tumors (18-20). The results were generally promising in that both primary and metastatic lesions in the liver, lung, and lymph nodes were visualized (21, 22). 99mTc-Labeled VIP (99mTc-VIP) analogs were also developed because 99mTc is more readily available for clinical applications (23-25). In the initial 99mTc radiolabeling, VIP was conjugated at the N terminal of the histidine residue with a bifunctional chelating agent for 99mTc labeling. This 99mTc-VIP analog had relatively high radiochemical impurities and a loss of biological activity because the histidine residue was required for the VIP biological activity. In an effort to improve 99mTc labeling, Pallela et al. (24) prepared TP3654 (named according to its molecular weight) using 4-aminobutyric acid as a spacer and extended the peptide to include a tetrapeptide moiety of Gly-Gly-(d)-Ala-Gly, which provided an N4 configuration for 99mTc chelation. The biological activity of 99mTc-TP3654 was similar to that of native VIP28, and the binding to the human colorectal cancer was receptor-specific with a 50% inhibition concentration (IC50) of 1.5 × 10–8 M. To prepare a PET probe for VIP imaging, Thakur et al. (25) synthesized 64Cu-TP3982 (IC50 = 8.1 × 10–8 M) for PET imaging of oncogene VPAC1 receptors overexpressed in human breast cancer cells by synthesizing a protected diaminedithiol (N2S2)-VIP for 64Cu chelation. 64Cu-TP3939, another VIP analog, was also successfully prepared for PET imaging of experimental human prostate cancer in mice (1, 2). This analog with Lys12, Nle17, (3-OH3, 4-OH) Phe22, Val26, and Thr28 was found to be more potent and more biologically stable than the native VIP28 (26, 27). It contains a carboxy terminus lysine residue separated from VIP-asparagine by a γ-aminobutyric acid spacer, and N2S2 is used as the chelating agent. Zhang et al. (1) showed that 64Cu-TP3939 is a promising molecular imaging probe for PET imaging of prostate cancer. Another study (2) from the same research group also demonstrated the potential use of PET imaging of 64Cu-TP3939 for human breast cancers.

Synthesis

[PubMed]

Zhang et al. (1, 2) described the synthesis of 64Cu-TP3939. The unlabeled peptide was obtained commercially. Briefly, TP3939 was synthesized on preloaded polyethylene glycol resin using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptide chain was assembled on the resin by repetitive removal of the Fmoc protecting group and coupling of the protected amino acid. The coupling agents were 1,3-diisopropyl carbodiimide and N-hydroxybenzotriazole. Piperidine (20%) in N,N-dimethyl formamide was used as the de-Fmoc agent. The crude peptide was precipitated from cold ether and collected by filtration. The precipitate was purified with reverse-phase high-performance liquid chromatography (HPLC). The peptide was characterized by mass spectroscopic and amino acid analyses. The observed and calculated molecular masses were 3,939.4 and 3,939 Da, respectively; and the purity of TP3939 was 96.8%. Radiolabeling of TP3939 was performed by using no-carrier-added high specific activity 64Cu in the form of 64CuCl2. Briefly, 7.4–22.2 MBq (0.2–0.6 mCi) 64CuCl2 was mixed with 20 μg (5.08 nmol based on the molecular weight of 3939) TP3939 in glycine buffer (pH 9), stannous chloride (100 μg/10 μl), and hydrochloric acid. The reaction mixture was stirred and incubated at 90°C for 45 min. The radiochemical purity determined with HPLC was 95.43 ± 3.78%.

In Vitro Studies

[PubMed]

Zhang et al. (1) performed functional assays of TP3939 based on the extent of binding to specific VIP receptors to produce a concentration-dependent decrease in the resting tension of the internal anal sphincter smooth muscle. The IC50 value of unlabeled TP3939 was determined to be 4.4 × 10–8 M. In comparison, the native VIP28 had an IC50 value of 9.1 × 10–8 M. Receptor affinity assays were conducted with 64Cu-TP3939 in androgen receptor–positive human prostate cancer (PC3) cells. The dissociation constant (Kd) of 64Cu-TP3939 was determined to be 0.77 × 10–9 M, which was the same as that of unlabeled TP3939. In comparison, 64CuCl2 showed only nonsaturable and nonspecific binding to PC3 cells. Zhang et al. (2) also reported that the Kd of 64Cu-TP3939 for estrogen receptor–positive human breast cancer T47D cells was found to be 0.33 × 10–9 M. The Kd value of native VIP28 in T47D cells was 15 × 10–9 M.

Zhang et al. (2) used sections of histologically proven human breast cancer (n = 13) and normal tissues (n = 7) for autoradiography with 64Cu-TP3939. The sections were mounted and incubated with 64Cu-TP3939 at 22°C for 90 min after washing and air drying. The sections were then analyzed with a fluorescent image analyzer. The tumor/normal radioactivity ratios were calculated by the quantitative analysis of the phosphor images. The ratios for the invasive ductal carcinoma and in situ microinvasive tumor were 3.36–10.69 and 2.6, respectively.

Animal Studies

Rodents

[PubMed]

Zhang et al. (1, 2) studied the blood clearance of 64Cu-TP3939 in normal mice (n = 3). Each mouse received 3.7–4.44 MBq (0.1–0.12 mCi) activity of 64Cu-TP3939 by intravenous administration. The blood clearance appeared to be biphasic with a t½α of ~3.1 min (70%) and a t½β of ~120 min (30%). In an in vivo stability study, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the integrity of the radiolabeled peptide in the blood. Each mouse was euthanized 2–3 min after intravenous administration of 64Cu-TP3939. SDS-PAGE analysis of the obtained blood samples showed that ~83% of the radioactivity remained as the intact 64Cu-TP3939. The remaining radioactivity was associated with proteins with a molecular weight ≥6 kDa. Negligible (<3%) transchelation of 64Cu to protein occurred when 64Cu-TP3939 was incubated with human serum albumin at 25°C.

Tissue distribution studies of 64Cu-TP3939 were performed in groups (n = 5 in each group) of nude mice bearing the subcutaneous PC3 tumor (<0.5 cm in diameter) (1). Each mouse received 0.37–0.55 MBq (0.01–0.015 mCi) 64Cu-TP3939 by intravenous administration. The tumor radioactivity levels in percent injected dose per gram (% ID/g) were 7.48 ± 3.63 and 5.78 ± 1.32 at 4 h and 24 h, respectively. The tumor/muscle ratios were 3.98 ± 1.43 and 5.17 ± 1.32 at 4 h and 24 h, respectively. The tumor/blood ratios were 1.94 ± 0.66 and 2.46 ± 0.45 at 4 h and 24 h, respectively. There was progressive elimination of the radioactivity through the kidneys, lungs, and liver; the primary route of excretion was through the feces. At 4 h, the radioactivity levels (% ID/g) in major organs were 11.00 ± 1.64 (intestine), 9.13 ± 5.63 (heart), 34.31 ± 9.76 (lungs), 19.22 ± 9.13 (spleen), 18.38 ± 9.95 (kidneys), 1.9 ± 0.5 (normal prostate gland), and 55.00 ± 12.62 (liver). At 24 h, these radioactivity levels decreased to 7.67 ± 2.48 (intestine), 4.14 ± 0.65 (heart), 19.93 ± 5.56 (lungs), 3.56 ± 0.38 (spleen), 10.06 ± 0.42 (kidneys), 2.1 ± 0.35 (normal prostate gland), and 27.58 ± 2.18 (liver). When a blocking dose of 40 μg (10.2 nmol) unlabeled TP3939 was administered 30 min before 64Cu-TP3939, the tumor radioactivity levels were significantly (P = 0.01) decreased to 1.84 ± 0.44% ID/g at 24 h showing specific binding of radiolabeled peptide t the receptors. All other tissues also had significantly (P < 0.05) decreased radioactivity levels.

Zhang et al. (1) conducted PET imaging studies in nude mice bearing the PC3 tumors. Each mouse received 3.88–4.07 MBq (0.10–0.11 mCi) 64Cu-TP3939. The PET images unequivocally delineated the xenografted prostate cancer in nude mice. Tumor/contralateral ratios determined from region-of-interest analysis were 3.4 and 4.2 at 4 h and 24 h, respectively. No ex vivo validation studies were conducted. In comparison, the corresponding ratio for 18F-FDG at 1 h was 1.66 in the same animal model.

Two TRAMP mice received ~14.8–18.5 MBq (0.4–0.5 mCi) 64Cu-TP3939 and were imaged 1 h later (1). In the TRAMP I mouse with prostate grade II hyperplasia, the hyperplasia was not detected. In the TRAMP II mouse with grade IV intraepithelial neoplasia, the lesion was clearly visualized. There was no bladder activity at 1 h. In comparison, 18F-FDG did not visualize either the prostate hyperplasia or neoplasia, but high bladder activity was present.

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.

NIH Support

NIH CA 109231.

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