Radiolabeled peptide probe for tumor imaging

Radionuclide imaging is now the premier imaging method in clinical practice for its high sensitivity and tomographic capability. Current clinically available radio imaging methods mostly use positron-emission tomography (PET) and single-photon emission computed tomography (SPECT) to detect anatomic abnormalities that conventional imaging techniques typically have challenges for visualizing. Contrast agents are indispensable for radionuclide imaging, and the radionuclide is always attached to a suitable vector that achieves targeted delivery. Nowadays, peptides have attracted increasing interest in targeting vectors of contrast agents, mainly due to their high specificity for target receptors at nanomolar concentrations and low toxicity. Radiolabeled peptide probes as kinds of PET/SPECT tracers had become essential tools for clinical radionuclide diagnosis. This review mainly summarizes radiolabeled peptide probes for bioimaging, including fundamental concepts of radiolabeled peptide probe design, some typical peptide analogs radiocontrast agents for PET, SPECT, and the combination imaging.     

Study on biodistribution and imaging of radioiodinated antisense oligonucleotides in nude mice bearing human lymphoma

A 18-mer partial phosphorothioate oligonucleotide sequence was synthesized and grafted in 5′ with a tyramine group which was further radioiodinated. The Namalwa (V_H 1 family) and HL-60 cell lines were transducted with liposome-mediated ¹²⁵I-FR1-ASON. Liposome-mediated ¹³¹I-FR1-ASON and ¹³¹I labeled sense oligonucleotides were injected intratumorally into tumor-bearing BALB/c mice (6 weeks after inoculation of 10⁷ Namalwa cells). Biodistribution was monitored by sequential scintigraphy and organ radioactivity measurement at 24 hours after injection. The transduction efficiency in Namalwa cell lines reached (26.8±1.54)% that was higher than HL-60 cell lines. Antisense probe images show tracer accumulation in tumor.     

Complexation of well-controlled low-molecular weight polyelectrolytes with antisense oligonucleotides

The influences of polymer-related properties such as molecular weight, charge density, counter ion, and hydrophilic block on the complexation of polyelectrolytes and a fluorescein-labeled oligonucleotide (ON) were investigated. A series of well-defined and well-controlled 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA) polymers and block copolymers were prepared using living anionic and radical polymerization methods. Fluorescence measurement was used to reveal the effects of polymer molecular weight, charge density, and counter ion type on the complexation. PolyDMAEMA samples having double molecular weights of the chosen oligonucleotide gave the optimal complexation performance. Kinetic studies showed that high-molecular weight/high-charge density polymer samples produced very stable complexes. The fully charged polyDMAEMA displayed the strongest binding with the ON. These complexes were therefore less sensitive to the changes in the environment. PolyDMAEMA–DMSQ samples had slightly higher complexation ability than polyDMAEMA–MCQ (DMSQ: dimethylsulfate quat; MCQ: methylchloride quat). Both poly(DMAEMA-b-HEMA) and poly(DMAEMA–MCQ-b-PEG) block copolymers showed good complexation ability and steric stability [HEMA: 2-hydroxyethyl methacrylate; PEG: poly(ethylene glycol)]. PEG, but not HEMA block, enhanced the effectiveness of polyDMAEMA–MCQ binding with the ON.     

Radiolabeled small molecule protein kinase inhibitors for imaging with PET or SPECT

Imaging protein kinase expression with radiolabeled small molecule inhibitors has been actively pursued to monitor the clinical potential of targeted therapeutics and treatments as well as to determine kinase receptor density changes related to disease progression. The goal of the present review is to provide an overview of the breadth of radiolabeled small molecules that have been synthesized to target intracellular protein kinases, not only for imaging in oncology, but also for other areas of interest, particularly the central nervous system. Considerable radiotracer development has focused on imaging receptor tyrosine kinases of growth factors, protein kinases A, B and C, and glycogen synthase kinase-3?. Design considerations, structural attributes and relevant biological results are summarized.     

Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography

Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.     

Radiolabeled novel peptide for imaging somatostatin-receptor expressing tumor: synthesis and radiobiological evaluation

The aim of this study was to synthesis, a radiolabeled (^99mTc) new somatostatin-analogue 6-hydrazinopyridine-3-carboxylic-acid (HYNIC)-Asn³-octreotate (^99mTc-HYNIC-AATE), and to evaluate as a candidate for imaging somatostatin-receptor (SSTR)-positive tumors and also compare it with ^99mTc-HYNIC-Tyr³-octreotide (^99mTc-HYNIC-TOC). Synthesis was performed by Fmoc-solid-phase strategy and ^99mTc labeled by SnCl₂. Biodistribution and imaging properties of new radiopeptide were also studied in C6 tumor bearing rat. Radiolabeling was performed at high specific activities and it showed high binding-affinity for SSTR2. In biodistribution, radiopeptides have showed high and receptor-specific uptake in the SSTR2 positive organs, tumor with rapid renal excretion from non-target tissues. These results demonstrated that ^99mTc-HYNIC-AATE is a new specific radioligand for scintigraphy of somatostatin-receptor-positive tumors.     

Evaluation of ultrahigh-performance liquid chromatography columns for the analysis of unmodified and antisense oligonucleotides

Ultrahigh-performance liquid chromatography has been used for the separation and analysis of unmodified and modified antisense oligonucleotides. For this reason, we tested various columns of low particle sizes in our analysis of unmodified and phosphorothioate oligonucleotides. The influence of both the type and concentration of ion-pair reagent on the retention of the studied biomolecules was tested. The developed methods were used for separation of unmodified oligonucleotides and to determine antisense oligonucleotides in human serum samples. The results proved that octadecyl and phenyl columns are the most selective in the resolution of oligonucleotides which differ in the position of single nucleotides in the sequence. The phenyl column was selected and applied for the analysis of phosphorothioate oligonucleotides in serum samples. The calibration plots showed good linearity within the test concentration ranges. The intra-day CV of the calibration curve slopes was in the range of 1.6 to 4.2 %. The limits of detection (LODs) were in the range of 0.11–0.16 μg mL^?1, while the limit of quantification (LOQ) values were between 0.35 and 0.51 μg mL^?1. Figure

Determination of antisense phosphorothioate oligonucleotides in serum     

Efficient synthesis of antisense phosphorothioate oligonucleotides using a universal solid support

It is demonstrated that solid support containing a novel universal linker could be efficiently used to synthesize both phosphorothioate oligodeoxyribonucleotides and second-generation 2′-O-methoxyethyloligoribonucleotides with high yield and quality as judged by ion-pair-liquid chromatography-electrospray mass spectroscopy, ³¹P NMR and reversed phase HPLC. Analysis of oligonucleotides shows quality being superior to that produced with standard succinyl-linker solid supports, without contamination of materials resulting from linker or support backbone decomposition.     

Radiolabeled biomolecules for specific imaging of cancers of the breast,prostate and lungs

Despite the great strides made in its management, cancer remains a formidable disease of mankind and has continued to take one human life every 4 s of each day and night. Sophisticated screening tests for diagnosis of breast cancer (BC) or prostate cancer (PC) exist, yet histology prevails as a gold standard for which invasive biopsies must be performed. Biopsies can be morbid, cause excessive anxiety to patients, >66 % find benign pathology and are considered unnecessary costing millions of healthcare dollars annually. Here, we present a novel biomolecule, ⁶⁴Cu-TP3805, designed, synthesized and characterized in our laboratory for targeting a genomic biomarker VPAC1 that is densely expressed at the onset of many cancers including those of BC, PC and lung cancer. This article describes preclinical evaluation of ⁶⁴Cu-TP3805 and its translation in humans for early and accurate detection of cancer.     

Design,synthesis and anti-influenza virus activities of terminal modified antisense oligonucleotides

Four novel terminal modified antisense oligonucleotides (ODNs) were designed, synthesized and tested for their anti-influenza virus activity. Initial biological studies indicated that lipophilic and rimantadin emodificated Flutide exhibited more potent anti-H1N1 activity than Flutide. Among them, lipophilic modificated ODN (Flutide-I) showed the most antiviral activity. The EC₅₀ value of Flutide-I for inhibiting H1N1 induced cytopathic effect (CPE) and H1N1 RNA were respectively (0.26 ± 0.16) μM and (0.11 ± 0.03) μM. The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with the concentrations up to 20 μM.     

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.     

Radiolabeled Nanogels for Nuclear Molecular Imaging

An efficient and simple synthesis approach to form stable ⁶⁸Ga‐labeled nanogels is reported and their fundamental properties investigated. Nanogels are obtained by self‐assembly of amphiphilic statistical prepolymers derivatised with chelating groups for radiometals. The resulting nanogels exhibit a well‐defined spherical shape with a diameter of 290 ± 50 nm. The radionuclide ⁶⁸Ga is chelated in high radiochemical yields in an aqueous medium at room temperature. The phagocytosis assay demonstrates a highly increased internalization of nanogels by activated macrophages. Access to these ⁶⁸Ga‐nanogels will allow the investigation of general behavior and clearance pathways of nanogels in vivo by nuclear molecular imaging.

     

Biological efficacy of antisense oligonucleotides complementary to overlapping regions of the mRNA target

Phosphorothioate oligonucleotides complementary to target mRNA are stable in biological milieu and are capable of decreasing levels of this mRNA and the protein encoded by this mRNA (antisense knockdown). The results of our study are compared with the data published in the literature on the efficacy of three antisense 18—21-mer oligonucleotides, which are targeted to the start codon or nearby sequences of _2A-adrenoceptor mRNA, on receptor expression, and functions regulated by these receptors. The highest biological efficacy was shown by the oligonucleotide, which is complementary to the mRNA region and contains the largest number of unpaired bases in the theoretically calculated conformation corresponding to the free energy minimum. Targeting of both ends of the antisense on unpaired bases of the target also leads to the enhancement of its biological efficacy.     

Site-specific cleavage of RNA by a metal-free artificial nuclease attached to antisense oligonucleotides

RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.     

Radiolabeled phosphonates for bone metastases therapy

This study is focused on the chelating process of two phosphonates with biological activity and therapeutic potential, HEDP (1-hydroxy-ethylidene-diphosphonic acid) and TTHMP (triethylene-tetramine-hexamethylene-phosphonic acid) with therapeutic radiometals ¹⁸⁸Re (T _1/2 = 17 hrs, E _βmax = 2.12 MeV, E _γ= 155 keV) and ¹⁷⁷Lu (T _1/2 = 6.7 days, E _βmax = 490 keV, E _γ = 208 keV). The ligand structure effect on the in vitro stability and on the biological affinity of the therapeutic agents was investigated. The radiochemical purity of the labeled compounds was higher than 95%, showing a good in vitro stability, up to 48 hours. The in vivo biodistribution studies, performed in rats, show a rapid and quantitative accumulation of both labeled compounds in bones and rapid elimination via the urinary tract. The maximum values of the bone uptake were ranged from 75.14% (injected dose/g organ) for ¹⁸⁸Re-TTHMP to 94.10% for ¹⁷⁷Lu-TTHMP. The structure of the chelates determines the kinetic of bone accumulation processes of labeled phosphonates. Its influence on the biodistribution of the radiolabeled phosphonates reveals that luthetium forms more stable chelate with polyphosphonate in respect to diphosphonate. On the other hand, the less reactive rhenium coupled with HEDP shows a better in vivo behavior than Re-TTHMP.     

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).     

Radiolabeled GX1 Peptide for Tumor Angiogenesis Imaging

Early and accurate detection of primary or metastatic tumors is of great value in staging, treatment management, and prognosis. Tumor angiogenesis plays an essential role in the growth, invasion, and metastatic spread of solid cancers, and so, is a promising approach for tumor imaging. The GX1 (CGNSNPKSC) peptide was identified by phage display library and has been investigated as a marker for human cancers. This study aims to evaluate the ^99mTc-HYNIC-PEG₄-c (GX1) as a biomarker for tumor imaging. Our results showed that GX1 specifically binds to tumor cells in vitro. SKMEL28 and MDA-MB231 cells achieved total binding peak at 60 min of incubation. For B16F10 and MKN45 cells, the total and specific binding were similar during all time points, while A549 cell line showed rapid cellular total uptake of the tracer at 30 min of incubation. Biodistribution showed low non-specific uptakes and rapid renal excretion. Melanoma tumors showed enhanced GX1 uptake in animal model at 60 min, and it was significantly blocked by cold peptide. The radiotracer showed tumor specificity, especially in melanomas that are highly vascularized tumors. In this sense, it should be considered in future studies, aiming to evaluate degree of angiogenesis, progression, and invasion of tumors.