Background

[PubMed]

^99mTc-CCND1-Peptide nucleic acid (PNA)-IGF1 peptide chimeras (^99mTc-WT4185) is a ^99mTc-peptide-PNA-peptide chimera that was developed as a gene expression agent for gamma planar/single photon emission computed tomography (SPECT) imaging of breast cancer (1, 2). ^99mTc-WT4185 is a radiohybridization probe for cyclin D1 (CCND1) proto-oncogene messenger RNA (mRNA) that is overexpressed in breast cancer cells.

Breast cancer is the second leading cause of cancer death in women in the United States women (3) The CCND1 oncogene exercises powerful control over the mechanisms that regulate the G₁-S transition in the mitotic cell cycle (4, 5). Excessive CCND1 expression and /or activity is common in breast cancer and some other human cancers. Evidence from gene expression profiling has suggested that alterations in CCND1 mRNA are present in the preinvasive stage of breast cancer (1, 6). Studies have shown that CCND1 mRNA is a target that can be used to distinguish precancerous and invasive cancerous changes from benign breast diseases (1, 5).

Radiolabeled antisense oligonucleotides can be used to identify and image the presence of a particular mRNA in vivo (7). Some of the major obstacles in developing a clinically useful radiolabeled antisense probe include nonspecific affinity, ribonuclease destruction of the RNA target, and the lack of a receptor-targeting ligand. Peptide nucleic acids (PNAs) are DNA/RNA mimics in which the nucleobases are attached to a pseudopeptide backbone (1, 8, 9). The achiral, uncharged, and flexible PNA peptide backbone permits more stable hybridization to DNA and RNA oligomers with improved sequence selectivity. PNAs are also more stable against nuclease and protease attack, and the uncharged backbone is less likely to react with cellular proteins. However, relatively poor cellular uptake of PNAs requires additional design strategy such as the addition of a variety of ligands or coupling to different carriers (10). Tian et al. (3) demonstrated that adding a peptide analog specific for a cell surface receptor could be an effective way to increase the cellular uptake of PNAs in vitro and in vivo. One of the approaches is targeting to the insulin-like growth factor 1 (IGF1) receptor which is frequently overexpressed in breast cancer cells. Basu and Wickstrom (11) showed that a 5- to 10-fold uptake increase in cells expressing IGF1 receptors in vitro could be achieved by solid phase synthesis of a PNA sequence linked to a cyclized D-amino acid analog of IGF1. Tian et al. (1, 3) reported successful imaging of the CCND1 cancer gene in experimental human breast cancer xenografts with the ^99mTc-WT4185 that was specific for both oncogene CCND1 and IGF1 receptor. The authors suggested that this probe could enter breast cancer cells overexpressing IGF1R, and then hybridize specifically with CCND1 mRNA.

Synthesis

[PubMed]

In PNA, the entire phosphate deoxyribose backbone of DNA/RNA is replaced by a structurally homomorphous polyamide backbone composed of N-(2-aminoethyl) glycine units. The PNA-peptide conjugates are generally prepared by fragment condensation (12). This approach requires multiple steps of preparation and purification with significant loss in yield. Basu and Wickstrom (11) used a facile PNA-peptide single-resin synthesis without intermediate conjugation steps. The peptide was first assembled on a solid phase with 9-fluorenylmethoxy carbonyl (Fmoc) coupling. The t-Boc−protected PNA monomers were coupled manually. Tian and Wickstrom (13) designed a scheme of a continuous solid-phase synthesis on a single-resin support in a single run to yield a chimera capable of radionuclide chelation for in vivo imaging of gene expression after a single purification. WT4185 is a cyclized peptide-PNA-peptide chimera, AcGlyd(Ala)GlyGlyAba-CTGGTGTTCCAT-AEEA-d(CysSerLysCys) (1, 3, 14). Briefly, the IGF1 analog d(CysSerLysCys) was assembled by Fmoc coupling. The linker Fmoc-aminoethoxyethoxyacetic acid (AEEA) and PNA monomers were then sequentially coupled to the N-terminus of the peptide resin. Finally, the chelator peptide Fmoc-Glyd(Ala)GlyGlyAba was coupled to the N-terminus of the PNA-peptide by use of a long-coupling-cycle protocol and then acetylated. The cysteines of the IGF1 analog were cyclized on the resin by iodine oxidation, the chimeras were cleaved and deprotected, then purified by reversed-phase high-performance liquid chromatography (HPLC). The overall yield of the HPLC-purified WT4185 relative to the initial solid support was 30.6%. The mass was calculated to be 4,185.0 Da and measured to be 4,187.2 Da (1, 3).

Radiolabeling of purified WT4185 was conducted by the method of ^99mTc peptide labeling method described by Pallela et al. (1, 3, 15, 16). Sodium phosphate (Na₃PO₄; 0.05 M, pH 12) and 0.1% Tween 80 were mixed with freshly eluted ^99mTc pertechnetate. This solution was mixed with the chelator-PNA-peptide in stannous chloride in hydrochloric acid (0.05 M). The mixture was incubated for 30 min at 22ºC, and the pH was adjusted to ≈7 by the addition of sodium biphosphate (NaH₂PO₄; 0.05 m; pH 4.5). The final reaction mixture was analyzed by HPLC, instant thin-layer chromatography, and denaturing gel electrophoresis. The radiochemical purity was determined to be 96% with 1.5% unlabeled ^99mTc and 2.5% ^99mTc colloids. The specific activity was 74 GBq/μmol (2 Ci/μmol). The final ^99mTc-WT4185 product was a CCND1 PNA with an IGF1 peptide loop on the C-terminus and a ^99mTc-chelator peptide on the N-terminus.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Tian et al. (1, 3) used a fluoresceinyl-PNA-peptide antisense chimera, WT4433, to study the cellular internalization of ^99mTc-WT4185. At exposure of MCF7:IGF1R estrogen receptor positive human breast cancer cells (expressing 10⁶ IGF1Rs per cell) to WT4433 at 1 μmol/liter for 8 h, significant uptake of WT4433 was observed throughout the cells. Similar results were observed at 5 μmol/liter after 4 h of exposure. No cellular uptake was found with a fluorescent peptide mismatch chimera that served as the control. ^99mTc-WT4185 was used to determine the oncogene expression levels in untreated MCF7:IGF1R cells. The CCND1 mRNA copy number was determined to be 6,089 ± 2,000 per cell (n = 12). In comparison, the PNA mismatch control probe only measured 2,388 ± 615 per cell (n = 8). The result was similar to that measured by the technique of quantitative reverse transcription-polymerase chain reaction.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH HL59769, NIH/NCI CA42960, NIH/NCI CO27175,

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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.