6-hydrazinopyridine-3-carboxylic-acid and arginyl-glycyl-aspartic-acid

HYNIC-3PRGD

Topics: Animals; Chromatography, Liquid; Drug Stability; Female; Hydrazines; Linear Models; Male; Nicotinic Acids; Oligopeptides; Peptides, Cyclic; Radiopharmaceuticals; Rats; Rats, Wistar; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Technetium

Aiming to develop a novel (99m)Tc-labeled imaging agent, for angiogenesis and tumor receptors, two peptides obtained from phage display library, namely GX1 and the heterodimer RGD-GX1, were synthesized in a cyclic conformation. They were radiolabeled with (99m)Tc, employing the HYNIC chelator, for radiochemical evaluation and biological properties.. Radiolabeling, radiochemical control, plasma protein binding, and partition coefficient were assessed for both radioconjugates. Biodistribution in healthy Balb/c mice was carried out, in order to evaluate the biological behaviour of the radiocomplexes.. The conjugates displayed a rather similar pharmacokinetic profile. They were prepared with high radiochemical purity (>96%), and both were hydrophilic (log P of -2.25 and -2.51 respectively). Preferential renal excretion was observed. Kidney uptake (42.31±5.35 %ID/g) for (99m)Tc-HYNIC-E-[c(RGDfk)-c(GX1)], 1h post-injection was about three times higher than the uptake of (99m)Tc-HYNIC-PEG4-c(GX1) (11.92±4.77%ID/g). Total blood, bone and muscle values revealed a slightly slower clearance for the RGD-GX1 radiocomplex.. The high radiochemical purity achieved, and the similar in vivo profile observed for both radioconjugates, make them potential candidates for radiopharmaceuticals for tumor imaging. Further investigations of binding affinity, and uptake of GX1 and RGD-GX1 peptides in tumor models, are warranted.

Topics: Amino Acid Sequence; Animals; Blood Proteins; Chelating Agents; Dimerization; Drug Stability; Humans; Hydrazines; Isotope Labeling; Mice; Mice, Inbred BALB C; Molecular Imaging; Neovascularization, Pathologic; Nicotinic Acids; Oligopeptides; Radiochemistry; Technetium; Tissue Distribution

In breast cancer, α(ν)β(3) and/or α(ν)β(5) integrins are overexpressed in both endothelial and tumour cells. Radiolabelled peptides based on the Arg-Gly-Asp (RGD) sequence are radiopharmaceuticals with high affinity and selectivity for these integrins. The RGD-dimer peptide (E-[c(RGDfK)]2) radiolabelled with (99m)Tc has been reported as a radiopharmaceutical with a 10-fold higher affinity for the α(ν)β(3) integrin compared with the RGD-monomer. Ethylenediamine-N,N'-diacetic acid (EDDA) is a hydrophilic molecule that may favour renal excretion when used as coligand in the (99m)Tc labelling of hydrazinonicotinamide (HYNIC) peptides and can easily be formulated in a lyophilized kit.. The aim of this study was to establish a biokinetic model for (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 prepared from lyophilized kits and evaluate its dosimetry as a tumour-imaging agent in seven healthy women and three breast cancer patients.. (99m)Tc labelling was performed by adding sodium pertechnetate solution and 0.2 mol/l phosphate buffer (pH 7.0) to a lyophilized formulation containing E-[c(RGDfK)]2, EDDA, tricine, mannitol and stannous chloride. The radiochemical purity was evaluated using reverse-phase high-performance liquid chromatography and instant thin-layer chromatography on silica gel analyses. Stability studies in human serum were carried out using size-exclusion high-performance liquid chromatography. In-vitro cell uptake was tested using breast cancer cells (MCF7, T47D and MDA-MB-231) with blocked and nonblocked receptors. Biodistribution and tumour uptake were determined in MCF7 tumour-bearing nude mice with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/PET/CT. Whole-body images from seven healthy women were acquired at 0.5, 1, 3, 6 and 24 h after (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 administration with radiochemical purities greater than 94%. Regions of interest were drawn around the source organs at each time frame. Each region of interest was converted to activity using the conjugate view counting method. The image sequence was used to extrapolate the (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 time-activity curves of each organ to adjust the biokinetic model and calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation dose estimates. In three breast cancer patients with histologically confirmed cancer, static images were obtained at 1 h in the supine position with hands placed behind the head.. (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 obtained from lyophilized kits demonstrated high stability in human serum and specific cell receptor binding. The biodistribution data from mice showed rapid blood clearance, with both renal and hepatobiliary excretion, and specific binding towards α(ν)β(3) integrins in the MCF7 tumours. In women, the blood activity showed a half-life value of 1.60 min for the fast component (T1/2α = ln2/26.01) and half-life values of 1.0 h for the first slow component (T1/2β = ln2/0.69) and 4.03 h for the second slow component (T1/2γ = ln2/0.16). Images from patients showed an average tumour/heart (blood) ratio of 3.61 ± 0.62 at 1 h. The average equivalent doses calculated for a study using 740 MBq were 4.9, 6.2, 20.7, 34.5 and 57.0 mSv for the liver, intestines, spleen, kidneys and thyroid, respectively, and the effective dose was 6.1 mSv.. All absorbed doses were comparable to those known from most of the (99m)Tc studies. (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 obtained from kit formulations showed high tumour uptake in patients with malignant lesions, making it a promising imaging radiopharmaceutical for targeting site-specific breast cancer. The results obtained in this study warrant further clinical studies to determine the specificity and sensitivity of (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Dimerization; Edetic Acid; Female; Freeze Drying; Humans; Hydrazines; Mice; Nicotinic Acids; Oligopeptides; Organotechnetium Compounds; Radiochemistry; Radiometry; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed

The aim of this study is to develop a novel MR probe containing arginine-glycine-aspartic acid (RGD) motif for imaging integrin alphanubeta3 receptor-expressed tumor.. Commercially available HYNIC-RGD conjugated with co-ligand EDDA was labeled with Gd(3+), and the mixture was isolated and purified by solid phase extract (SPE) to get the entire probe Gd-EDDA/HYNIC-RGD. Human hepatocellular carcinoma (HHCC) cell line BEL-7402 was cultured and the cells harvested and suspended in serum-free Dulbecco's modified Eagle medium (DMEM) were subcutaneously inoculated into athymic nude mice for tumor growth. In vitro cell binding assay to integrin alphanubeta3 receptor and cell viability experiments were conducted. The in vivo imaging of the three arms of xenografts were performed by MR scan with a dedicated animal coil at time points of 0, 30, 60, 90min and 24-h post-intravenous injection (p.i.). Three arms of nude mice then were sacrificed for histological examination to confirm the imaging results.. Gd-EDDA/HYNIC-RGD was successfully isolated by SPE and validity was verified on signal enhancement through in vitro and in vivo experiments. The nude mice model bearing HHCC was well established. There was approx. 30% signal enhancement on T1WI FSE images at 90min post-intravenous injection of the Gd-EDDA/HYNIC-RGD compared with baseline, and the signal to time curve is straightforward over time in the span of 0-90min p.i., while the control arms do not show this tendency.. Gd-EDDA/HYNIC-RGD has the potential to serve as an MR probe detecting integrin alphanubeta3 receptor-expressed tumor.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Contrast Media; Diethylamines; Gadolinium; Humans; Hydrazines; Image Enhancement; Integrin alphaVbeta3; Magnetic Resonance Imaging; Mice; Mice, Nude; Molecular Probe Techniques; Neovascularization, Pathologic; Nicotinic Acids; Oligopeptides; Rats; Reproducibility of Results; Sensitivity and Specificity

Radiolabeled alpha(v)beta(3)-integrin antagonists are increasingly investigated as a means of imaging angiogenesis. Several methods of labeling alpha(v)beta(3)-integrin binding peptide with (18)F have been reported recently. In the present study, we devised a straightforward means for labeling Arg-Gly-Asp (RGD) peptide with (18)F via hydrazone formation between c(RGDyK)-hydrazinonicotinic acid (HYNIC) (3) and 4-[(18)F]-fluorobenzaldehyde ([(18)F]4). The resulting reaction mixture was purified by HPLC to give 4'-[(18)F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([(18)F]5). The conjugation efficiency of 3 and 4 to form [(18)F]5 was 95.2%, and the radiochemical purity of [(18)F]5 after purification was >99%. The specific activity of [(18)F]5 estimated by radio-HPLC was 20.5 GBq/mumol (end of synthesis). Competitive binding assay of c(RGDyK) (1) and 5 was performed using [(125)I]iodo-c(RGDyK) as a radioligand, and K(i) values were found to be 2.8 and 21.7 nM, respectively. For the biodistribution study, the angiogenic mouse model was established by inducing unilateral ischemia on the left hindlimbs of ICR mice after femoral artery ablation. Seven days after inducing ischemia, [(18)F]5 was administered to the mice through the tail vein. Ischemic muscle uptake of [(18)F]5 was significantly higher than that of normal muscle (P<.01). Specific uptake was confirmed by coinjection of 1 with [(18)F]5. Here, we successfully labeled RGD peptide with (18)F via hydrazone formation between 3 and 4, resulting to [(18)F]5. [(18)F]5 was found to have high affinity for alpha(v)beta(3)-integrin and to accumulate specifically in ischemic hindlimb muscle of mice. We suggest that (18)F labeling via formation of hydrazone between HYNIC peptide and [(18)F]4 is a useful method for labeling c(RGDyK), which can be applied for imaging angiogenesis.

Topics: Animals; Fluorine Radioisotopes; Hydrazines; Hydrazones; Integrin alphaVbeta3; Ischemia; Isotope Labeling; Metabolic Clearance Rate; Mice; Muscle, Skeletal; Nicotinic Acids; Oligopeptides; Organ Specificity; Radionuclide Imaging; Radiopharmaceuticals; Tissue Distribution

There has been increasing interest in peptides containing the Arg-Gly-Asp (RGD) sequence for targeting of alpha(v)beta(3) integrins to image angiogenesis. [(18)F]Galacto-RGD has been successfully used for positron emission tomography applications in patients. Here we report on the preclinical characterization of a (99m)Tc-labeled derivative for single-photon emission computed tomography. c(RGDyK) was derivatized with HYNIC at the amino group of the lysine [c(RGDyK(HYNIC)) or HYNIC-RGD]. (99m)Tc labeling was performed using coligands (tricine and EDDA), as well as (99m)Tc(CO)(3)(H(2)O)(3). Radiolabeled peptides were characterized with regard to lipophilicity, protein binding and stability in buffer, serum and tissue homogenates. Integrin receptor activity was determined in internalization assays using alpha(v)beta(3)-receptor-positive M21 and alpha(v)beta(3)-receptor-negative M21L melanoma cells. Biodistribution was evaluated in normal and nude mice bearing M21, M21L and small cell lung tumors. HYNIC-RGD could be labeled at high specific activities using tricine, tricine-trisodium triphenylphosphine 3,3',3''-trisulfonate (TPPTS), tricine-nicotinic acid (NA) or EDDA as coligands. [(99m)Tc]EDDA/HYNIC-RGD, [(99m)Tc]tricine-TPPTS/HYNIC-RGD and [(99m)Tc]tricine-NA/HYNIC-RGD showed protein binding (<5%) considerably lower than [(99m)Tc](CO)(3)/HYNIC-RGD and [(99m)Tc]tricine/HYNIC-RGD. [(99m)Tc]EDDA/HYNIC-RGD revealed high in vitro stability accompanied by low lipophilicity with a log P value of -3.56, comparable to that of [(18)F]Galacto-RGD. In M21 cells for this compound, the highest level of specific and rapid cell uptake (1.25% mg protein(-1)) was determined. In vivo, rapid renal excretion, low blood retention, low liver and muscle uptakes and low intestinal excretion 4 h postinjection were observed. Tumor uptake values were 2.73% ID/g in M21 alpha(v)beta(3)-receptor-positive tumors versus 0.85% ID/g in receptor-negative tumors 1 h postinjection. Small cell lung tumors could be visualized using gamma camera imaging. [(99m)Tc]EDDA/HYNIC-RGD shows encouraging properties to target alpha(v)beta(3) receptors in vivo with high stability and favorable pharmacokinetics. Tumor uptake studies showed specific targeting of alpha(v)beta(3)-receptor-positive tumors with tumor-to-organ ratios comparable to those of [(18)F]Galacto-RGD.

Topics: Animals; Carcinoma, Small Cell; Cell Line, Tumor; Drug Stability; Humans; Hydrazines; Integrin alphaVbeta3; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Nicotinic Acids; Oligopeptides; Organotechnetium Compounds; Peptides, Cyclic; Protein Binding; Radionuclide Imaging; Radiopharmaceuticals; Tissue Distribution

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc.. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture.. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated.. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells.. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.

Topics: Blood Proteins; Cells, Cultured; Edetic Acid; Endothelium, Vascular; Glycine; Humans; Hydrazines; Integrins; Ligands; Metabolic Clearance Rate; Nicotinic Acids; Oligopeptides; Protein Binding; Radionuclide Imaging; Radiopharmaceuticals; Technetium; Umbilical Veins