Supramolecular aggregates containing lipophilic Gd(III) complexes as contrast agents in MRI

Magnetic resonance imaging (MRI) contrast agents based on paramagnetic gadolinium complexes are widely used in biomedical research and diagnosis. Their application is intended to improve efficacy of MRI providing physiological information along with the impressive anatomical detail already obtained by images without contrast. The classical gadolinium complexes currently used for MRI contrast enhancement are all lowmolecularweightcompounds that rapidly equilibrate between the intra and extravascular spaces after intravenous administration. In order to obtain gadolinium-based agents with different pharmacokinetic properties, supramolecular aggregates such as micelles and liposomes have been recently proposed. Micelles and liposomes, obtained by the aggregation of lipophilic gadolinium complexes are here described, with the aim to correlate their structural and relaxometric properties.We report on the state of the art in the development of supramolecular aggregates obtained by self-assembly of lipophilic gadolinium complexes and aggregates in which lipophilic gadolinium complexes are assembled with surfactants. Moreover aggregates derivatized with bioactive molecules, such as peptides and antibodies, acting as target selective MRI contrast agents are described

Gastrin and cholecystokinin peptide-based radiopharmaceuticals: an in vivo and in vitro comparison

The development of suitable radioligands for targeting CCK-2 receptor expressing tumors, such as medullary thyroid carcinoma, is of great clinical interest. In the search for the best CCK-2R binding peptides, we have synthesized, evaluated and compared the CCK8 peptide (Asp-Tyr-Met-Gly-Trp-Met-Asp-PheNH(2) ) and two gastrin analogs commonly referred to as MG0 (DGlu-Glu(5)-Ala-Tyr-Gly-Trp-Met-Asp-PheNH(2) ) and MG11 (DGlu(1)-Ala-Tyr-Gly-Trp-Met-Asp-PheNH(2) ). The N-terminal portion of the three peptide sequences was derivatized by introducing the DTPAGlu or DOTA chelators to allow radiolobeling with (111) In(III) and (68) Ga(III), respectively. Saturation binding and cellular internalization experiments were performed on A431 cells overexpressing CCK2R (A431-CCK2R). All compounds showed Kd values in the nM range and were internalized with similar rates in CCK2 receptor overexpressing cells. Biodistribution experiments showed higher specific uptake of both MG0-based compounds compared to conjugates containing the CCK8 and MG11 peptide sequences. The higher retention levels of MG0-based peptides were associated with markedly elevated and undesired kidney uptake compared to the other compounds. Current indications suggest that the 5 Glu N-terminal residues while improving peptide stability and receptor-mediated tumor uptake cause unacceptably high kidney retention. Although displaying lower absolute tumor uptake values, the DOTA-coupled CCK8 peptide provided the best tumor to kidney uptake ratio and appears more suitable as lead compound for improvement of radiopharmaceutical properties

Peptide-chelating agent conjugate for selective targeting of somatostatin receptor type1: Synthesis and characterization

Previously reported results suggest that the analogue of the somatostatin des-AA 1,2,5[D-Trp 8,IAmp 9]-somatostatin (CH-275) peptide bearing chelating agents able to coordinate radioactive metals could be used for scintigraphic imaging of tumor lesions overexpressing sstr1. An efficient synthetic procedure for the preparation of the somatostatin analogue CH-275 and its conjugate DTPAGlu-Gly-CH-275, bearing the chelating agent DTPAGlu (DTPAGlu = N,N-bis[2-[bis(carboxy-ethyl)amino]ethyl]-L-glutamic acid) on the N-terminus, by solid-phase peptide synthesis and 9-flourenymethyoxycarbonyl (Fmoc) chemistry, is here reported. Rapid and efficient labeling of DTPAGlu-Gly-CH-275 was achieved by addition of IIIIn(III) to the compound. Typical yields were greater than 97% as determined by reversed phase high performance liquid chromatography (HPLC) at specific activities in the range 4-9 GBq/μmol (100-250 Ci/mmol). A preliminary biological assay of the binding ability of IIIIn-DTPAGlu-Gly-CH-275 indicates, however, that the labeled compound does not display any specific interaction with somatostatin sstrl receptors in the tested cell lines. To confirm this unexpected negative result, competition binding experiments were carried out, in which fixed tracer amounts of the 125!-labeled somatostatin-14 were incubated with the receptor-expressing cells in the presence of DTPAGlu-Gly-CH-275 or CH-275 at concentrations ranging from 10 -10 to 10 -3 M. While CH-275 shows a IC 50 of 80 nM similar to that already found in displacement experiments on CHO-Kl sstrl-transfected cells, DTPAGlu-Gly-CH-275 displays instead very low or negligible affinity towards this receptor. The NMR solution characterization indicates that the presence of DTPAGlu does not influence the conformational and chemical features of the peptide moiety, thus suggesting that the loss in binding activity should be due to steric hindrance of either the chelating agent DTPAGlu or its indium comple

The [Tc(N)(PNP)]2+ metal fragment labeled cholecystokinin-8 (CCK8) peptide for CCK-2 receptors imaging: in vitroand in vivo studies

The radiolabeling of the natural octapeptide CCK8, derivatized with a cysteine residue (Cys-Gly-CCK8), by using the metal fragment [99mTc(N)(PNP3)]2+ (PNP3 = N,N-bis(dimethoxypropylphosphinoethyl)methoxyethylamine) is reported. The [99mTc(N)(NS-Cys-Gly-CCK8)(PNP3)]+ complex was obtained according to two methods (one-step or two-step procedure) that gave the desired compound in high yield. The complex is stable in aqueous solution and in phosphate buffer. In vitro challenge experiments with an excess of cysteine and glutathione indicate that no transchelation reactions occur, confirming the high thermodynamic stability and kinetic inertness of this compound. Stability studies carried out in human and mouse serum, as well as in mouse liver homogenates, show that the radiolabeled compound remains intact for prolonged incubation at 37 degrees C. Binding properties give Kd (19.0 +/- 4.6 nmol/l) and Bmax (approximately 10(6) sites/cell) values in A431 cells overexpressing the CCK2-R. In vivo evaluation of the compound shows rapid and specific targeting to CCK2-R, a fourfold higher accumulation compared to nonreceptor expressing tumors

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I- and 131I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism

In vitro and in vivo evaluation of In-111-DTPAGlu-G-CCK8 for cholecystokinin-B receptor imaging

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging and therapy. Receptors of the cholecystokinin family have been shown to be overexpressed in different types of neuroendocrine tumors. The purposes of this study were to evaluate the cholecystokinin octapeptide amide (CCK8) peptide tagged with a diethylenetriaminepentaacetic acid derivative (DTPAGlu) and to test whether a 111In-labeled conjugate (111In-DTPAGlu-G-CCK8, a derivative containing the chelating agent DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8) is suitable for cholecystokinin-B receptor (CCKBR) imaging. Methods: CCK8 was synthesized by solidphase techniques and covalently coupled to DTPAGlu through a glycine linker at its amino terminus. The compound was labeled with 111In. The radiochemical purity and stability of the compound were assessed by chromatographic methods. NIH-3T3 and A431 cells overexpressing CCKBR were used to characterize the in vitro properties of the compound. Nude mice bearing control and CCKBR-overexpressing A431 xenografts were used as an in vivo model. Results: DTPAGlu-G-CCK8 showed rapid and efficient labeling with 111In. The radiolabeled conjugate showed specific binding to both cell lines overexpressing CCKBR. Binding was saturable, with a dissociation constant of 20 nmol/L in both cell systems. Both cell lines showed internalization of the ligand after interaction with the receptor. Biodistribution studies showed rapid localization of 111In-DTPAGlu- G-CCK8 on CCKBR-overexpressing A431 xenografts that was severalfold higher than that on control tumors at all time points tested. Unbound activity showed rapid clearance of over 80% through the kidneys by 30 min after injection. The labeled peptide conjugate was very stable in serum but showed a rapid breakdown after injection. Incubation with kidney homogenates suggested that most breakdown occurred in the kidneys, favoring the clearance of unbound activity. Conclusion: Our findings indicate that the in vitro and in vivo characteristics of 111In-DTPAGlu-G-CCK8 are favorable for CCKBR imaging, as thepeptide shows high-affinity binding to the receptor, is internalized in CCKBR-expressing cells, and shows avid uptake in CCKBR-overexpressing xenografts, with rapid clearance of unbound radioactivity through the kidneys. Furthermore, the ease of synthesis, high labeling efficiency, and chemical stability of DTPAGlu make this chelating moiety an ideal candidate for widespread use in peptide radiolabeling for nuclear medicine applications

Monitoring the correction of glycogen storage disease type 1a in a mouse model using [18F]FDG and a dedicated animal scanner

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [18F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [18F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [18F]FDG. The retention of [18F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controlsPublicad

Peptide-containing aggregates as selective nanocarriers for therapeutics

New nanocarriers are obtained by assembling two amphiphilic monomers: one containing the bioactive peptide CCK8 spaced, by a polydisperse poly(ethylene glycol), from two hydrophobic tails ((C18)2PEG2000CCK8), and the other containing a chelating agent able to give stable radiolabeled indium-111 complexes linked to the same hydrophobic moiety ((C18)2DTPAGlu). The size and shape of the supramolecular aggregates were structurally characterized by dynamic light scattering, small-angle neutron scattering, and cryogenic transmission electronic microscopy. Under the experimental conditions we investigated (pH 7.4 and molar ratio between monomers 30:70), there is the presence of high polydisperse aggregates: rod-like micelles with a radius of 40 Å and length >700 Å, open bilayer fragments with thickness 65 Å, and probably vesicles. The presence of the bioactive peptide well exposed on the external surface of the aggregate allows selective targeting of nanocarriers towards the cholecystokinin receptors overexpressed by the cancerous cells. In vitro binding assays and in vivo biodistribution studies by nuclear medicine experiments using indium-111 are reported. Moreover, preliminary data concerning the drug loading capability of the aggregates and their drug efficiency on the target cells is reported by using the cytotoxic drug doxorubicin. Incubation of receptor-positive and control cells with peptide-containing aggregates filled with doxorubicin shows significantly lower cell survival in receptor-expressing cells relative to the control, for samples incubated in the presence of doxorubicin