Maria Paravatou-Petsotas

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimers disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

Spacer Site Modifications for the Improvement of the in Vitro and in Vivo Binding Properties of 99mTc-N3S-X-Bombesin[2−14] Derivatives

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.

Synthesis and characterization of rhenium and technetium-99m tricarbonyl complexes bearing the 4-[3-bromophenyl]quinazoline moiety as a biomarker for EGFR-TK imaging

Aiming at the development of technetium-99m ((99m)Tc) complexes for early detection and staging of EGFR positive tumors, the tyrosine kinase inhibitor 6-amino-4-[(3-bromophenyl)amino]quinazoline was derivatized with pyridine-2-carboxaldehyde to generate the imine 6-(pyridine-2-methylimine)-4-[(3-bromophenyl)amino]quinazoline suitable for reacting with the fac-[(99m)Tc(CO)(3)](+) core as an N,N bidentate ligand. The labelling was performed in high yield (>90%) by ligand exchange reaction using fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) as precursor. The (99m)Tc complex was characterized by comparative HPLC analysis using the analogous rhenium (Re) complex as reference. The Re complex was prepared by ligand exchange reaction using the fac-[ReBr(3)(CO)(3)](2-) as precursor and was fully characterized by NMR and IR spectroscopies and elemental analysis. In vitro studies indicate that both the ligand and its Re complex inhibit the EGFR autophosphorylation (IC(50): 17+/-3.7 and 114+/-23 nM respectively) in intact A431 cells, bind the receptor in a reversible mode, and inhibit A431 cell growth (IC(50): 5.2+/-1.1 and 2.0+/-0.98 microM respectively). Biodistribution of the (99m)Tc complex in healthy animals showed a rather fast blood and soft tissue clearance between 1 and 15 min p.i. with excretion occurring mainly via the hepatobiliary system.

99mTc-labeled aminosilane-coated iron oxide nanoparticles for molecular imaging of ανβ3-mediated tumor expression and feasibility for hyperthermia treatment

HYPOTHESIS Dual-modality imaging agents, such as radiolabeled iron oxide nanoparticles (IO-NPs), are promising candidates for cancer diagnosis and therapy. We developed and evaluated aminosilane coated Fe3O4 (10±2nm) as a tumor imaging agent in nuclear medicine through 3-aminopropyltriethoxysilane (APTES) functionalization. We evaluated this multimeric system of targeted (99m)Tc-labeled nanoparticles (NPs) conjugated with a new RGD derivate (cRGDfK-Orn3-CGG), characterized as NPs-RGD as a potential thermal therapy delivery vehicle. EXPERIMENTS Transmission Electron Microscopy (TEM) and spectroscopy techniques were used to characterize the IO-NPs indicating their functionalization with peptides. Radiolabeled IO-NPs (targeted, non-targeted) were evaluated with regard to their radiochemical, radiobiological and imaging characteristics. In vivo studies were performed in normal and ανβ3-positive tumor (U87MG glioblastoma) bearing mice. We also demonstrated that this system could reach ablative temperatures in vivo. FINDINGS Both radiolabeled IO-NPs were obtained in high radiochemical yield (>98%) and proved stable in vitro. The in vivo studies for both IO-NPs have shown significant liver and spleen uptake at all examined time points in normal and U87MG glioblastoma tumor-bearing mice, due to their colloidal nature. We have confirmed through in vivo biodistribution studies that the non-targeted (99m)Tc-NPs poorly internalized in the tumor, while the targeted (99m)Tc-NPs-RGD, present 9-fold higher tumor accumulation at 1h p.i. Accumulation of both IO-NPs in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. The preliminary in vivo study of applied alternating magnetic field showed that the induced hyperthermia is feasible due to the aid of IO-NPs.

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTalpha), i.e. against either non-conjugated ProTalpha (isolated from bovine thymus) or ProTalpha conjugated to keyhole limpet hemocyanin (ProTalpha/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTalpha, while antibodies Y against ProTalpha/KLH had a better apparent titer, could better discriminate between ProTalpha and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.

Structural modifications of 99mTc-labelled bombesin-like peptides for optimizing pharmacokinetics in prostate tumor targeting

PURPOSE The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. METHODS Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2-14)-NH(2), where M: (99m)Tc or (185/187)Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)(3)-, M-BN-A; spacer: -(ornithine)(3)-, M-BN-O; have been prepared and evaluated as tumor imaging agents. RESULTS The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1h incubation) and comparable receptor binding affinity with the standard [(125)I-Tyr(4)]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic γ-camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. CONCLUSION The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Prothymosin α (ProTα) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTα immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTα or the synthetic fragments ProTα[1-28], ProTα[87-109], and ProTα[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTα[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTa in tumorigenesis.

Initial in vitro and in vivo assessment of Au@DTDTPA-RGD nanoparticles for Gd-MRI and 68Ga-PET dual modality imaging

Gadolinium chelate coated gold nanoparticles (Au@DTDTPA) can be applied as contrast agents for both in vivo X-ray and magnetic resonance imaging. In this work, our aim was to radiolabel and evaluate this gold nanoparticle with Ga-68, in order to produce a dual modality PET/MRI imaging probe. For a typical preparation of 68Ga-labeled nanoparticles, the Au@DTDTPA nanoparticles (Au@DTDTPA/Au@DTDTPA-RGD) were mixed with ammonium acetate buffer, pH 5 and 40 MBq of 68Ga eluate. The mixture was then incubated for 45 min at 65 AaC. Radiochemical purity was determined by ITLC. In vitro stability of both radiolabeled species was assessed in saline and serum. In vitro cell binding experiments were performed on integrin ανβ3 receptor-positive U87MG cancer cells. Non-specific Au@DTDTPA was used for comparison. Ex vivo biodistribution studies and in vivo PET and MRI imaging studies in U87MG tumor-bearing SCID mice followed. The Au@DTDTPA nanoparticles were labeled with Gallium-68 at high radiochemical yield (>95%) and were stable at RT, and in the presence of serum, for up to 3 h. The cell binding assay on U87MG glioma cells proved that 68Ga-cRGD-Au@DTDTPA had specific recognition for these cells. Biodistribution studies in U87MG tumor-bearing SCID mice showed that the tumor to muscle ratio increased from 1 to 2 h p.i. (3,71 ± 0.22 and 4,69 ± 0.09 respectively), showing a clear differentiation between the affected and the non-affected tissue. The acquired PET and MRI images were in accordance to the ex vivo biodistribution results. The preliminary results of this study warrant the need for further development of Au@DTDTPA nanoparticles radiolabeled with Ga-68, as possible dual-modality PET/MRI imaging agents.

Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies

A biotin derivative, namely biotin–aminocaproic acid–lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid–lysine “long-arm” was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its Nε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic “lysine–dendrimer” was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.

New labeled derivatives of the neuroprotective peptide colivelin: synthesis, characterization, and first in vitro and in vivo applications.

Colivelin (CL), first reported in 2005, is the most potent member of the humanin family of neuroprotective peptides with in vitro and in vivo rescuing action against insults associated with Alzheimers disease (AD). The objective of the present work is the design, synthesis and characterization of specific CL derivatives that can be used as molecular probes in the investigation of the unknown mechanism of CL action. Within this framework, three CL derivatives bearing suitable tags, i.e., the fluorescent moiety FITC, the streptavidin-counterpart biotinyl-group, and the (99m)Tc-radiometal chelating unit dimethylGly-Ser-Cys, were developed and subsequently applied in biological evaluation experiments. Specifically, the FITC-labeled derivative of CL was used in confocal microscopy, where specific binding at the periphery of F11 cells was observed; the biotin-labeled derivative of CL was used in an in-house developed ELISA-type assay, where specific and concentration-dependent binding with the β-amyloid peptide of AD was shown; finally, the (99m)Tc-radiolabeled derivative of CL was used in in vivo biodistribution studies in healthy Swiss Albino mice, where 0.58% of the radioactivity administered was measured in the mouse brain 2min after injection. The above first successful applications of the CL probes demonstrate their potential to contribute in the field of neuroprotective peptides.