Hydroxypyridinones with enhanced iron chelating properties. Synthesis, characterization and in vivo tests of 5-hydroxy-2-(hydroxymethyl)pyridine-4(1H)-one.

The synthesis of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1) is presented, together with the evaluation of its coordination ability towards Fe(3+), studied by a combination of chemical, computational, and animal approaches. The use of complementary analytical techniques has allowed us to give evidence of the tautomeric changes of P1 as a function of pH, and to determine their influence on the coordinating ability of P1 towards Fe(3+). The pFe(3+) value 22.0 of P1-iron complexes is noticeably higher than that of deferiprone (20.6), one of the three clinical chelating agents in therapeutic use for iron overload diseases. This is due on one side to the tautomeric change to the catechol form, and on the other to the lower protonation constant of the OH group. Bio-distribution studies on mice allowed us to confirm in vivo the efficacy of P1. Furthermore the coordinating ability toward Al(3+), Cu(2+) and Zn(2+) has been studied to evaluate the possible use of P1 against a second toxic metal ion (Al(3+)), and to envisage its potential influence on the homeostatic equilibria of essential metal ions. The chelating ability of P1 toward these ions, not higher than that of the corresponding deferiprone, contributes to render P1 a more selective iron chelator.

Radiosynthesis and in vivo evaluation of a ¹8F-labelled styryl-benzoxazole derivative for ß-amyloid targeting.

The formation of ß-amyloid deposits is considered a histopathological feature of Alzheimer's disease (AD). In vivo molecular imaging by means of amyloid-avid radiotracers will allow for an early and conclusive diagnostic of AD. Herein, we describe the radiosynthesis of the radiofluorinated styryl benzoxazole derivative [¹8F]-[2-[N-methyl-N-(2'-fluoroethyl)-4'-aminostyryl]benzoxazole] ([¹8F]-1) and its pre-clinical evaluation, including metabolic and biodistribution studies in male Wistar rats. The in vivo biological evaluation of [¹8F]-1 showed that this new radiotracer has a moderate brain uptake with a slow brain washout and a poor in vivo stability.

Thermolabile liposomes: a controlled release delivery tool in diagnosis/therapy in experimental pulmonary ɶdema.

Liposomes, usually assembled from organic/synthetic lipidic compounds, are biocompatible, biodegradable, non-toxic, and do not induce immune response. Due to their structural versatility in terms of size, composition, surface charge, bilayer fluidity and ability to encapsulate drugs regardless of their solubility, liposomes enable the production of a vast number and type of formulations with potential clinical use. They can be administered through several routes of administration (e.g. i.v., i.m., oral, nasal, etc.). The use of liposomes enables the variation and control retention of drugs in biologic fluids, enhancing blood circulation and specific compartments residence. They can be tailored to target specific tissues and cells. They can play a very important role for imaging diagnosis and/or therapy. After an extensive literature review of the subject, we selected a particular area of potential clinical application: pulmonary ɶdema. This clinical entity has a variety of possible etiologies, conducing to two main types of edema: cardiogenic and non-cardiogenic. At the moment a dedicated technique for the early diagnosis/therapy of this pathology is lacking. We propose a new methodology using a specially designed GUV formulation, encapsulating chosen radiotracers labeled with 99mTc. The aim of the work has been successfully achieved in an experimental animal model of cardiogenic pulmonary oedema. Experiments using an animal model of non-cardiogenic pulmonary oedema are in course (simultaneous study with two different drugs), using the same GUV methodology. Preliminary results are very promising.

Radiolanthanide complexes with tetraazamacrocycles bearing methylphosphonate pendant arms as bone seeking agents.

AIM: Radiolanthanide complexes with ligands bearing phosphonate groups have demonstrated their usefulness as bone seeking agents. Herein, we report on the synthesis of 153Sm and 166Ho complexes with 12- to 14-membered macrocycles containing different number of methylphosphonate pendant arms and their in vitro and in vivo evaluation in order to assess the effect of the cavity size and type of appended arms on their biological behavior. METHODS: Radioactive macrocycle complexes were prepared by reaction of (153)Sm/(166)Ho nitrates with four different tetraazamacrocycles bearing methylphosphonate groups. Radiochemical behavior, in vitro stability and charge of complexes were studied by chromatography and electrophoresis. The lipophilicity, plasmatic protein binding and adsorption onto hydroxyapatite (HA) were evaluated by in vitro assays. Biodistribution was assessed in CD-1 mice. Radiolabeling efficiency depends both on radionuclide and ligand structure. All the complexes are hydrophilic with an overall negative charge and relatively low protein binding. High in vitro stability in human serum and adsorption onto HA was found for all the complexes. RESULTS: Biodistribution and in vivo stability studies have demonstrated promising biological profile for targeted radiotherapy, namely a rapid tissue clearance from most organs and rapid total excretion. Additionally, 166Ho-tritp has a high bone uptake, which led to high bone/ blood and bone/muscle ratios. CONCLUSIONS: Our results clearly demonstrate that 12- and 13-membered macrocyclic ligands led to stable complexes with biological profile adequate to radionuclide therapy. The favorable in vivo behavior highlights the interest to further investigate these or closely related complexes to be used as bone seeking agents.

Lymphatic uptake of lipid nanoparticles following endotracheal administration.

A previous publication reported the uptake into the lymphatics of pulmonary administered lipid nanoparticles (LN), after aerosolization and inhalation. In the present study LN clearance from the lungs and lymphatic uptake were further evaluated after endotracheal administration. Nanoparticles prepared with gliceryl behenate were radiolabelled by association to the lipophilic tracer D,L-hexamethylpropylene amine oxime (HMPAO) coupled with 99mTc. Labelling efficiency was 97% and stability in body fluids was demonstrated in vitro. Wistar rats were treated by endotracheal administration and lymphatic uptake was determined upon organ sampling. Endotracheally delivered LN are rapidly eliminated from rat lungs and accumulation in para-aortic, axillary and inguinal lymph nodes starts almost immediately after administration. Translocation of LN across the lung mucosa and their uptake into the lymphatics demonstrate their usefulness as potential drug carriers for lung cancer therapy, as well as for immunization purposes.

Dramatic effect of the tridentate ligand on the stability of 99mTC "3 + 1" oxo complexes bearing arylpiperazine derivatives.

Mixed-ligand model complexes of general formula [(99m)Tc(O)(kappa(3)-PNX)(kappa(1)-SPh))] [X = O (1a), S (2a)] were prepared in a one-step procedure from [(99m)TcO(4)(-)] using stannous chloride as reducing agent. Stability studies and challenge experiments with glutathione showed that complex 2a presented promising features for pursuing animal studies. The activity in the brain (% dose injected/organ) at 5 min (0.14% +/- 0.03) and 120 min (0.11% +/- 0.02) pi encouraged the synthesis of several mixed-ligand "3 + 1" oxo complexes of general formula [M(O)(kappa(3)-PNS)(kappa(1)-SL))] (M = (99m)Tc, 3a-6a, Re, 3-6), in which the tridentate ligand is the heterofunctionalized phosphine 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (PNS) and the co-ligands are different arylpiperazine derivatives (HSL1-HSL4). The (99m)Tc complexes have been characterized by comparison of their retention times in the HPLC chromatogram (gamma-detection) with the retention times of the analogous Re complexes (UV detection at 254 nm). The (99m)Tc complexes, obtained with radiochemical purity higher than 95%, after HPLC purification, are stable in saline, 0.01 M PBS (pH 7.4), rat plasma (4 h, 37 degrees C), and glutathione (10 mM solutions, 2h, 37 degrees C). Binding affinity and selectivity for 5-HT(1A) receptors (relative to the 5-HT(2A) receptor) were determined, complex 5 demonstrating the best values (IC(50) for the 5-HT(1A) 2.35 +/- 0.02 nM; competitor 5-HT(2A) 372 +/- 11 nM). Biodistribution and stability studies in mice indicated a preferred hepatobiliary excretion, a high in vivo stability, but a poor brain uptake.

Synthesis and biological evaluation of silylated mixed-ligand 99mTc complexes with the [PNS/S] donor atom set.

New oxotechnetium complexes of general formula [99mTc(O)(PNS)(S(CH2)nOSiR3)] (4-6) were synthesized by direct reduction of [99mTcO4]- with stannous chloride, in the presence of the tridentate heterofunctionalized phosphine H2PNS and of the monodentate silylated thiols [HS(CH2)nOSiR3] (n = 2, R = Ph (1); n = 3, R = Ph (2); n = 3, R = Et (3)). The mixed-ligand rhenium and technetium complexes of general formula [M(O)(PNS)(S(CH2)nOH)] (n = 2: M = 99mTc, (7), M = Re, (7a); n = 3: M = 99mTc, (8), M = Re, (8a)) were also prepared. All the 99mTc complexes were obtained with high radiochemical purity (> 95%), after purification by HPLC, and were characterized by comparison of their HPLC profiles with the ones obtained for the corresponding Re compounds. The silylated compounds 4-6 are stable in phosphate saline buffer (PBS) pH 7.4, rat plasma, human serum and whole blood, and do not bind to plasmatic proteins, and also do not challenge with glutathione. The biological behavior of [99mTc(O)(PNS)(S(CH2)nOH)] (7, 8) and [99mTc(O)(PNS)(S(CH2)nOSiR3)] (4-6) was studied. The effect of the pH on the cleavage of the O-Si bond in complexes 4-6 was also evaluated.

Synthesis, characterization and biodistribution of bisphosphonates Sm-153 complexes: correlation with molecular modeling interaction studies.

Bisphosphonates (BPs) are characterized by a P-C-P backbone structure and two phosphonic acid groups bonded to the same carbon, and are established as osteoclast-mediated bone resorption inhibitors. The nature of the groups attached to the central carbon atom are responsible in determining the potency of bisphosphonates as anti-resorption drugs. However, it is not yet clear the exact relationship between their molecular structure and pharmacologic activities. In this study, molecular geometries of pamidronate, alendronate and neridronate, differing only in the length of the aliphatic chains, were predicted by molecular mechanics and their interactions with hydroxyapatite, the main bone mineral component, were examined. We report the synthesis and radiochemical characterization of 153Sm complexes with pamidronate, alendronate and neridronate. Hydroxyapatite binding and biodistribution studies of these complexes have shown a good correlation with the theoretical molecular modeling interaction studies. So, it is possible to conclude that computational chemistry techniques are a good approach to evaluate specific interactions and may play a relevant role in determining the relative ability of BPs to mineral bone, and open new perspectives to the design of new BPs with increased pharmacological activity. These techniques could be extended to BPs as ligands to carrier radioactive metals, aiming for new bone therapeutic radiopharmaceuticals.

Synthesis and biological evaluation of two new radiolabelled estrogens: [125I](E)-3-methoxy-17alpha-iodovinylestra-1,3,5(10),6-tetraen-17beta-ol and.

The synthesis of two novel radiolabelled estrogen derivatives, [125I](E)-3-methoxy-17alpha-iodovinylestra-1,3,5(10),6-tetraen-17beta-ol (E[125I]IVDE) and [125I](Z)-3-methoxy-17alpha-iodovinylestra-1,3,5(10),6-tetraen-17beta-ol (Z[125I]IVDE), was carried out aiming to study the influence of the introduction of a C6-C7 double bond on the biological properties of the estradiol molecule. 3-Methoxyestra-1,3,5(10),6-tetraen-17-one was synthesised starting from a suitably protected estrone and subsequently converted into the 17alpha-ethynyl derivative. The radioiodinated derivatives were stereoselectively formed by radioiododestannylation of the corresponding tributylstannyl precursors. The biodistribution of the novel [125I]iodovinylestradiol derivatives was evaluated in immature female mice. Biological data indicated that the Z-isomer, owing to its higher in vivo uptake by the target tissue, has the preferable configuration for further development of similar compounds for estrogen receptor detection.

Synthesis, chelating properties towards gallium and biological evaluation of two N-substituted 3-hydroxy-4-pyridinones.

Two N-substituted 3-hydroxy-4-pyridinones (1-(3'-aminopropyl)-3-hydroxy-2-methyl-4-pyridinone (L1) and 1-(2'-carboxyethyl)-3-hydroxy-2-methyl-4-pyridinone (L2)) were prepared through one- and three-step reactions, respectively. The pKa values of the ligands and the stability constants of their Ga(III) complexes have been determined. Both the complexes are strongly coordinated to three (O,O) hydroxypyridonate moieties. There is a clear effect of the N-substituents in the lipophilic-hydrophilic balance and in the Ga(III) binding interaction; the acid derivative (L2) has lower lipophilicity but higher chelating strength than the amine derivative (L1). Both chelators are shown to interfere in the typical biological behavior of 67Ga-citrate in mice: L1 enhanced the urinary excretion leading to an increased 67Ga removal from the soft tissue, while L2 induced a lower blood clearance with a pronounced bone uptake mainly at 48 h after injection, thus suggesting that the 67Ga-L2 complex could have potential interest as a bone imaging agent.

Synthesis, characterization, and biodistribution of oxo complexes of technetium-99m with biguanide and N1-substituted ligands.

We report the synthesis, characterization, and biodistribution of 99mTc-complexes with the bidentate-N,N chelate biguanide (Big) and the N1-substituted ligands dimethyl (DMBig), phenyl (PBig), and phenethyl (PEBig). Dynamic gamma-camera studies with 99mTc-Big and 99mTc-DMSA in rabbits indicated distinct renal and urinary excretion profiles. 99mTc-Big was cleared more quickly than 99mTc-DMSA, and for the same acquisition times, the contrast in whole-body images favored 99mTc-Big. Also, the estimated radiation absorbed doses by kidneys and blood for 99mTc-DMSA were significantly higher than for 99mTc-Big. These preliminary studies show that 99mTc-Big has favourable practical and dosimetric features for renal imaging as an alternative to 99mTc-DMSA.

Human polyclonal immunoglobulin labelled with technetium-99m via NHS-MAG3: a comparison of radiochemical behavior and biological efficacy with other labelling methods.

The aim of this study was to evaluate the radiochemical behavior, biological distribution, and localization in infection sites in mice of a human polyclonal immunoglobulin (HIG) labelled with 99mTc by a novel MAG3-labelling method. The resulting [99mTc]MAG3-HIG was compared with [99mTc]HIG preparations radiolabelled directly via 2-mercaptoethanol (2-Me) or stannous ion (Sn) reduction and indirectly via 2-iminothiolane (2-Im) conjugation. All preparations showed similar UV and radioactivity HPLC profile to that of native HIG except for 2-Im-HIG, which showed aggregates. The stabilities of the label to challenge with cysteine were similar for all the preparations. By nondenaturing SDS-PAGE, all preparations other than MAG3-HIG showed evidence of lower molecular weight fragments. The tissue distribution 4 and 24 h after intravenous administration of the four preparations were compared in mice previously administered with an isolate of Staphylococcus aureus in one thigh. The pharmacokinetics varied among the different preparations. When prepared via 2-Me, Sn, and 2-Im, both blood clearance and urinary excretion were faster than that of labelled MAG3-HIG. The absolute uptake in the infected thigh at 24 h was significantly higher for HIG labelled via MAG3 and 2-Me vs. the remaining methods. The infected thigh/normal thigh radioactivity ratios were similar at both time points for labelled HIG prepared via 2-Me, 2-Im, and NHS-MAG, methods but was significantly lower at 24 h for HIG prepared via Sn. The radioactive HPLC profiles of serum at 4 and 24 h were similar to that of the radiolabelled injectates. Based on these data we conclude that each radiolabelled HIG preparation studied showed increased localization in infectious foci although [99Tc]MAG3-HIG showed superior radiochemical and biological characteristics under the conditions of this investigation.

Technetium-99m labelling of the IOR CEA 1 monoclonal antibody: evaluation of different methods.

AIM: The aim of this study was to investigate the in vivo and in vitro properties of 99mTc labelled monoclonal antibody, IOR CEA 1 when radiolabelled by different methods. METHODS: To achieve that purpose IOR CEA was directly radiolabelled via 2-mercaptoethanol (2-Me) and stannous ion (SnCl2) reduction and indirectly via the 2-iminothiolane (2-Im) conjugation. The resulting 99mTc-MoAbs were analysed for number of free sulfhydryl groups, chemical and radiochemical purity (checked by HPLC and SDS PAGE), immunoreactivity and biological distribution in mice. RESULTS: Experimental results indicated a similar radiochemical purity and immunoreactivity for direct labelling methods and a decrease of both for 2-Im method. 2-Me antibody reduction led to a high antibody fragmentation as indicated by non-denaturing SDS PAGE analysis. Nevertheless SnCl2 and 2-Im labels revealed lower in vivo stability. CONCLUSION: 99mTc-(2-Me) IOR CEA presented favorable in vitro and in vivo properties. Therefore this label was compared to 99mTc-monoclonal antibody BW 431/26. Similar characteristics were found. Clinical studies also revealed identical biodistribution profile.

Comparison of the radiochemical behaviour and biological efficacy of three 99mTc-HIG preparations.

The aim of the present study was to evaluate whether the different methodologies used for human polyclonal immunoglobulin (HIG) preparation can affect the radiochemical purity of 99mTc-HIG and its binding affinity to infection sites. Three intravenous immunoglobulin preparations, beta-propiolactone treated, hydrochloric acid/pepsin treated, and an unmodified HIG molecule were studied. The HIG preparations were analysed by size-esclusion HPLC. The UV chromatogram profiles obtained showed some percentage of polymeric and dimeric fractions in all of them. The three HIG studied were directly radiolabelled via 2-mercaptoethanol reduction. Lyophilized kits containing 1 mg of HIG and a small amount of MDP(Sn) solution were prepared and then radiolabelled by adding pertechnetate-99m. The radiolabelled products, evaluated by ITLC, showed high radiochemical purity and in vitro stability. Biodistribution studies were performed in mice with an infection in the right thigh induced by the im administration of a single isolate of S. aureus, in order to compare the ability of 99mTc-HIG to detect an infectious focus. This study suggests that any damage during immunoglobulin treatment can influence the in vivo behaviour of 99mTc-HIG.