Prasant K. Nanda

[64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues

Radiolabeled peptides hold promise as diagnostic/therapeutic targeting vectors for specific human cancers. We report the design and development of a targeting vector, [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, 8-Aoc = 8-aminooctanoic acid, and BBN = bombesin), having very high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRPrs are expressed on a variety of human cancers, including breast, lung, pancreatic, and prostate, making this a viable approach toward site-directed localization or therapy of these human diseases. In this study, [NOTA-X-BBN(7-14)NH2] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC50 of [NOTA-8-Aoc-BBN(7-14)NH2] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using 125I-[Tyr4]-BBN as the displacement ligand. An IC50 of 3.1 ± 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [64Cu-NOTA-X-BBN] conjugates were prepared by the reaction of 64CuCl2 with peptides in buffered aqueous solution. In vivo studies of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] in tumor-bearing PC-3 mouse models indicated very high affinity of conjugate for the GRPr. Uptake of conjugate in tumor was 3.58 ± 0.70% injected dose (ID) per g at 1 h postintravenous injection (p.i.). Minimal accumulation of radioactivity in liver tissue (1.58 ± 0.40% ID per g, 1 h p.i.) is indicative of rapid renal-urinary excretion and suggests very high in vivo kinetic stability of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] with little or no in vivo dissociation of 64Cu2+ from the NOTA chelator. Kidney accumulation at 1 h p.i. was 3.79 ± 1.09% ID per g. Molecular imaging studies in GRPr-expressing tumor models produced high-contrast, high-quality micro-positron-emission tomography images.

In vitro and in vivo analysis of [64Cu-NO2A-8-Aoc-BBN(7–14)NH2]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors

INTRODUCTION Human breast cancer, from which the T-47D cell line was derived, is known to overexpress the gastrin-releasing peptide receptor (GRPR) in some cases. Bombesin (BBN), an agonist for the GRPR, has been appended with a radionuclide capable of positron-emission tomography (PET) imaging and therapy. (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) (NO2A=1,4,7-triazacyclononane-1,4-diacetate) has produced high-quality microPET images of GRPR-positive breast cancer xenografted tumors in mice. METHODS The imaging probe was synthesized by solid-phase peptide synthesis followed by manual conjugation of the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) bifunctional chelator and radiolabeling in aqueous solution. The radiolabeled conjugate was subjected to in vitro and in vivo studies to determine its specificity for the GRPR and its pharmacokinetic profile. A T-47D tumor-bearing mouse was imaged with microPET/CT and microMRI imaging. RESULTS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) targeting vector was determined to specifically localize in GRPR-positive tissue. Accumulation was observed in the tumor in sufficient quantities to allow for identification of tumors in microPET imaging procedures. For example, uptake and retention in T-47D xenografts at 1, 4 and 24 h were determined to be 2.27+/-0.08, 1.35+/-0.14 and 0.28+/-0.07 % ID/g, respectively. CONCLUSIONS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) produced high-quality microPET images. The pharmacokinetic profile justifies investigation of this bioconjugate as a potentially useful diagnostic/therapeutic agent. Additionally, the bioconjugate would serve as a good starting point for modification and optimization of similar agents to maximize tumor uptake and minimize nontarget accumulation.

Bombesin analogues for gastrin-releasing peptide receptor imaging

OBJECTIVES The present study describes the design and development of a series of new bombesin (BBN) antagonist peptide ligands of the form [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR). METHODS [NO2A-X-D-Phe(6)-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with (64)Cu to yield [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry. RESULTS Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection. CONCLUSION The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.

Radiolabeled regulatory peptides for imaging and therapy

Purpose of reviewThe purpose of the present review is to describe new, innovative strategies of diagnosing and treating specific human cancers using a cadre of radiolabeled regulatory peptides. Recent findingsPeptide receptor-targeted radionuclide therapy is a method of site-directed radiotherapy that specifically targets human cancers expressing a cognate receptor-subtype in very high numbers. Ideally, the procedure targets only the primary or metastatic disease and is minimally invasive, with little radiation damage to normal, collateral tissues. For treatment strategies of this type to be effective, it is critical to evaluate the toxicity of the treatment protocol, the radiation dosimetry of the therapeutic regimen, and the biological profile of the radiopharmaceutical, including biodistribution and pharmacokinetics of the drug. Site-directed molecular imaging procedures via γ-scintigraphy can address many of the critical issues associated with peptide receptor-targeted radionuclide therapy and it is, therefore, necessary to describe the effective balance between the clinical benefits and risks of this treatment strategy. SummaryContinued development in the design or chemical structure of radiolabeled, biologically active peptides could do much to improve the targeting ability of these drugs, thereby creating new and innovative strategies for diagnosis or treatment of human cancers.

Radiotracers for different angiogenesis receptors in a melanoma model

Early and reliable diagnosis of melanoma, a skin tumor with a poor prognosis, is extremely important. Phage display peptide libraries are a convenient screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was to evaluate two technetium-99m tracers for angiogenesis detection in a melanoma model, using cyclic pegylated pentapeptide with RGD and NGR motifs conjugated with the bifunctional chelator mercaptoacetyltriglycine (MAG3). The conjugated peptides (10 &mgr;l of a &mgr;g/&mgr;l solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was carried out by instant thin-layer chromatography and confirmed by high-performance liquid chromatography. The partition coefficient was determined and internalization assays were performed in two melanoma cell lines (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was carried out in healthy animals at different time points and also in tumor-bearing mice, 120 min post injection. Blocking studies were also conducted by coinjection of cold peptides. The conjugates displayed a rather similar pharmacokinetic profile. They were radiolabeled with high radiochemical purity (>97%) and both were hydrophilic with preferential renal excretion. Yet, tumor uptake was higher for human than for murine melanoma cells, especially for [99mTc]-MAG3-PEG8-c(RGDyk) (7.85±2.34%injected dose/g 120 min post injection). The performance of [99mTc]-MAG3-PEG8-c(RGDyk) was better than the NGR tracer with regard to human melanoma uptake. In this sense, it should be considered for future radiotracer studies of tumor diagnosis.

Comparison of two peptide radiotracers for prostate carcinoma targeting

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95%. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32% of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP-1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

Synthesis and Selective Radiolabeling Strategies for Production of [90Y-DOTA-βala-K-64Cu-NOTA-BBN(7–14) NH2] Conjugate; A Dual Negatron/Positron Emitting Radioligand

Herein the authors introduce the novel targeting agent, [DOTA-βala-K-NOTA-BBN(7–14)NH2], and its ability to be radiolabeled with 90Y (a pure β– emitting radionuclide) and 64Cu (a β+ emitting radionuclide) under typical radiolabeling conditions. [DOTA-βala-K-NOTA-BBN(7–14)NH2] was metallated with either 64Cu/90Y or natCu/natY to form the product, [(90Y/natY)-DOTA-βala-K-(64Cu/natCu)-NOTA-BBN(7–14)NH2]. All of the new targeting probes and metallated ligands were purified by RP-HPLC, characterized by ESI-MS, and evaluated for IC50. This new, two-step labeling procedure offers the opportunity to tailor a new radioligand that contains a high-energy pure β– emitting radionuclide and a highly resolvable β+ for molecular imaging investigatons.

Molecular Imaging of Breast Cancer Tissue via Site-Directed Radiopharmaceuticals

The American Cancer Society reports that ~261,100 new cases of invasive and in situ breast cancer were diagnosed in 2010, and nearly 40,000 fatalities were attributed to this disease (American Cancer Society, 2010). Although death rates have steadily decreased since 1990, breast cancer currently ranks second in cancer deaths among women. Improvements in detection, treatment, and prevention education contribute to slow the incidence rate, and rapidly evolving nuclear medicine techniques have emerged as a formidable opponent to female breast cancer. The involvement of nuclear medicine imaging modalities in both the detection and diagnosis of breast cancer has increased in recent years (Gopalan et al., 2002). In contrast to earlier imaging methods, in which the transmission of various forms of energy through tissue is employed to generate an image, nuclear medicine imaging techniques are based on detection of the energy emitted from radioactive tracers that are injected into the body and subsequently accumulate locally in specific tissues (Nass et al., 2001). The classification of these techniques as either positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging modalities is determined by the radionuclide that is utilized to synthesize a given radiotracer. The theory behind nuclear medicine imaging techniques to detect and diagnose breast cancer is founded on preferential radiopharmaceutical uptake by cancerous cells as a result of alterations in metabolic rate, vascularity, or receptor expression which are associated with malignancy. Although both PET and SPECT are commonly employed to detect a variety of malignancies, neither imaging technique has achieved clinical acceptance as a method of imaging breast cancer due to the lack of sensitivity and specificity demonstrated by available radiotracers (Gopalan et al., 2002; Nass et al., 2001; Rosen et al., 2008). Presently, there is only one radiopharmaceutical, the SPECT imaging agent technetium-99m methoxy-isobutyl-isonitrile (99mTc-sestamibi, Miraluma®), that has received FDA approval for use as a diagnostic adjunct to mammography (Gopalan et al., 2002; Nass et al., 2001; Rosen et al., 2008). Although the mechanism governing the concentration of 99mTc-sestamibi in cancer cells is not fully understood, it may be related to the degree of cellular proliferation and vascular permeability (Nass et al., 2001). Once inside malignant cells, 99mTc-sestamibi is