Targeting of G-quadruplex DNA with 99mTc(I)/Re(I) Tricarbonyl Complexes Carrying Pyridostatin Derivatives.

The main goal of this work was to elucidate the potential relevance of (radio)metal chelates of 99mTc and Re targeting G-quadruplex structures for the design of new tools for cancer theranostics. 99mTc provides the complexes with the ability to perform single-photon-emission computed tomography imaging studies, while the Re complexes should act as anticancer agents upon interaction with specific G4 DNA or RNA structures present in tumor tissues. Towards this goal, we have developed isostructural 99mTc(I) and Re(I) tricarbonyl complexes anchored by a pyrazolyl-diamine (Pz) chelator carrying a pendant pyridostatin (PDS) fragment as the G4-binding motif. The interaction of the PDF-Pz-Re (8) complex with different G4-forming oligonucleotides was studied by circular dichroism, fluorescence spectroscopy and FRET-melting assays. The results showed that the Re complex retained the ability to bind and stabilize G4-structures from different DNA or RNA sequences, namely those present on the SRC proto-oncogene and telomeric RNA (TERRA sequence). PDF-Pz-Re (8) showed low to moderate cytotoxicity in PC3 and MCF-7 cancer cell lines, as typically observed for G4-binders. Biodistribution studies of the congener PDF-Pz-99mTc (12) in normal mice showed that the complex undergoes a fast blood clearance with a predominant hepatobiliary excretion, pointing also for a high in vitro stability.

DNA-Targeted Complexes of Tc and Re for Biomedical Applications.

Due to their favorable chemical features, Re and Tc complexes have been widely used for the development of new therapeutic agents and imaging probes to solve problems of biomedical relevance. This review provides an update of the most relevant research efforts towards the development of novel cancer theranostic agents using Re and Tc-based compounds interacting with specific DNA structures. This includes a variety of homometallic complexes, namely those containing M(CO)3 (M=Re, Tc) moieties, that exhibit different modes of interaction with DNA, such as covalent binding, intercalation, groove binding or G-quadruplex DNA binding. Additionally, heterometallic complexes, designed to potentiate synergistic effects of different metal centers to improve DNA-targeting, cytotoxicity and fluorescence properties, are also reviewed. Particular attention is also given to 99m Tc- and 188 Re-labeled oligonucleotides that have been widely explored to develop imaging and therapeutic radiopharmaceuticals through the in vivo hybridization with a specific complementary DNA or RNA target sequence to provide useful molecular tools in precision medicine for cancer diagnosis and treatment. Finally, the need for further improvement of DNA-targeted Re and Tc-based compounds as potential therapeutic and diagnostic agents is highlighted, and future directions are discussed.

Searching for a Paradigm Shift in Auger-Electron Cancer Therapy with Tumor-Specific Radiopeptides Targeting the Mitochondria and/or the Cell Nucleus.

Although 99mTc is not an ideal Auger electron (AE) emitter for Targeted Radionuclide Therapy (TRT) due to its relatively low Auger electron yield, it can be considered a readily available "model" radionuclide useful to validate the design of new classes of AE-emitting radioconjugates. With this in mind, we performed a detailed study of the radiobiological effects and mechanisms of cell death induced by the dual-targeted radioconjugates 99mTc-TPP-BBN and 99mTc-AO-BBN (TPP = triphenylphosphonium; AO = acridine orange; BBN = bombesin derivative) in human prostate cancer PC3 cells. 99mTc-TPP-BBN and 99mTc-AO-BBN caused a remarkably high reduction of the survival of PC3 cells when compared with the single-targeted congener 99mTc-BBN, leading to an augmented formation of γH2AX foci and micronuclei. 99mTc-TPP-BBN also caused a reduction of the mtDNA copy number, although it enhanced the ATP production by PC3 cells. These differences can be attributed to the augmented uptake of 99mTc-TPP-BBN in the mitochondria and enhanced uptake of 99mTc-AO-BBN in the nucleus, allowing the irradiation of these radiosensitive organelles with the short path-length AEs emitted by 99mTc. In particular, the results obtained for 99mTc-TPP-BBN reinforce the relevance of targeting the mitochondria to promote stronger radiobiological effects by AE-emitting radioconjugates.

Metal-Based G-Quadruplex Binders for Cancer Theranostics.

The ability of fluorescent small molecules, such as metal complexes, to selectively recognize G-quadruplex (G4) structures has opened a route to develop new probes for the visualization of these DNA structures in cells. The main goal of this review is to update the most recent research efforts towards the development of novel cancer theranostic agents using this type of metal-based probes that specifically recognize G4 structures. This encompassed a comprehensive overview of the most significant progress in the field, namely based on complexes with Cu, Pt, and Ru that are among the most studied metals to obtain this class of molecules. It is also discussed the potential interest of obtaining G4-binders with medical radiometals (e.g., 99mTc, 111In, 64Cu, 195mPt) suitable for diagnostic and/or therapeutic applications within nuclear medicine modalities, in order to enable their theranostic potential.

Synthesis and Biological Evaluation of 99mTc(I) Tricarbonyl Complexes Dual-Targeted at Tumoral Mitochondria.

For effective Auger therapy of cancer, the Auger-electron emitters must be delivered to the tumor cells in close proximity to a radiosensitive cellular target. Nuclear DNA is considered the most relevant target of Auger electrons to have augmented radiotoxic effects and significant cell death. However, there is a growing body of evidence that other targets, such as the mitochondria, could be relevant subcellular targets in Auger therapy. Thus, we developed dual-targeted 99mTc(I) tricarbonyl complexes containing a triphenylphosphonium (TPP) moiety to promote accumulation of 99mTc in the mitochondria, and a bombesin peptide to provide specificity towards the gastrin releasing peptide receptor (GRPr) overexpressed in prostate cancer cells. The designed dual-targeted complex, 99mTc-TPP-BBN, is efficiently internalized by human prostate cancer PC3 cells through a specific GRPr-mediated mechanism of uptake. Moreover, the radioconjugate provided an augmented accumulation of 99mTc in the mitochondria of the target tumor cells, most probably following its intracellular cleavage by cathepsin B. In addition, 99mTc-TPP-BBN showed an enhanced ability to reduce the survival of PC3 cells, in a dose-dependent manner.

Hospitalisation in high-risk pregnancy patients: is prenatal attachment affected?

Objective: To compare prenatal attachment in women hospitalised due to high-risk pregnancy with prenatal attachment in non-hospitalised patients. To describe the impact of social support, socio-demographic factors and the nature of the pregnancy on prenatal attachment, anxiety and depression. Study Design: An exploratory, cross-sectional and descriptive study utilising the Maternal Antenatal Attachment Scale, the Edinburgh Postnatal Depression Scale and the State-Trait Anxiety Inventory. The sample comprised 80 hospitalised and 88 non-hospitalised patients. Result: No difference in prenatal attachment was found between the two groups. The hospitalised group presented higher levels of depressive symptomatology and anxiety. Social support had a significant effect on the hospitalised group, improving attachment quality. Conclusion: Incorporation of members of the patient's support network may help to improve quality of prenatal attachment during hospitalisation. Detection and treatment of anxiety and/or depression in hospitalised patients is recommended given their impact on the mental health of mother and baby.

Lanthanide complexes with phenanthroline-based ligands: insights into cell death mechanisms obtained by microscopy techniques.

Herein we report the synthesis, characterization, and photophysical and biological evaluation of the complexes Ln(DBM)3(RPhen) (Ln = Sm, R = H; Ln = Sm, Eu, Tb, R = 5-NH2) stabilized by three ß-diketonate units (DBM) and a phenanthroline (RPhen) derivative, with the aim of contributing to the development of lanthanide-based compounds with potential application as anticancer agents. The UV-vis spectra of [Sm(DBM)3(Phen)], [Sm(DBM)3(NH2Phen)], [Eu(DBM)3(NH2Phen)] and [Tb(DBM)3(NH2Phen)] measured in DMSO and PBS showed a strong absorption band centered at ca. 350 nm in both solvents. In DMSO, all lanthanide compounds except [Sm(DBM)3(Phen)] show a ligand centered emission band at ca. 520 nm. In PBS only sharp emission peaks are detected. The complexes show similar cytotoxic effects in A2780 ovarian cancer cells, presenting IC50 values at 24 h in the range 16-27 µM. The measurement of the cellular uptake of the complexes in the A2780 cells by inductively coupled plasma mass spectrometry (ICP-MS) revealed preferential accumulation at the membrane and cytoskeleton, with the exception of [Sm(DBM)3(Phen)] that presented higher accumulation in the cytosol than in the cell membranes. All the evaluated lanthanide complexes showed low nuclear uptake, although not negligible. Spectroscopic studies on the interaction of the complexes with calf thymus DNA (ctDNA) revealed a moderate affinity with apparent binding constants in the 104 M-1 range. Complexes bind DNA not by intercalation but probably by electrostatic interactions. A morphological evaluation of the cells treated with the different complexes by electron microscopy (TEM/SEM) proved that all of them induce mitochondrial alterations, which seemed more pronounced for the NH2Phen complexes. In addition, the complex [Eu(DBM)3(NH2Phen)] presented lysosomal uptake that might explain its augmented cytotoxicity.

Unravelling the antitumoral potential of novel bis(thiosemicarbazonato) Zn(II) complexes: structural and cellular studies.

The development of pharmacologically active compounds based on bis(thiosemicarbazones) (BTSC) and on their coordination to metal centers constitutes a promising field of research. We have recently explored this class of ligands and their Cu(II) complexes for the design of cancer theranostics agents with enhanced uptake by tumoral cells. In the present work, we expand our focus to aliphatic and aromatic BTSC Zn(II) complexes bearing piperidine/morpholine pendant arms. The new complexes ZnL1-ZnL4 were characterized by a variety of analytical techniques, which included single-crystal X-ray crystallography for ZnL2 and ZnL3. Taking advantage of the fluorescent properties of the aromatic complexes, we investigated their cellular uptake kinetics and subcellular localization. Furthermore, we tried to elucidate the mechanism of action of the cytotoxic effect observed in human cancer cell line models. The results show that the aliphatic complexes (ZnL1 and ZnL2) have a symmetrical structure, while the aromatic counterparts (ZnL3 and ZnL4) have an asymmetrical nature. The cytotoxic activity was higher for the aromatic BTSC complexes, as well as the cellular uptake, evaluated by measurement of intracellular Zn accumulation. Among the most active complexes, ZnL3 presented the fastest uptake kinetics and lysosomal localization assessed by live-cell microscopy. Detailed studies of its impact on cellular production of reactive oxygen species and impairment of lysosomal membrane integrity reinforced the influence of the pendant piperidine in the biological performance of aromatic BTSC Zn(II) complexes.

Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA.

A new family of 99mTc(I)- tricarbonyl complexes and 125I-heteroaromatic compounds bearing an acridine orange (AO) DNA targeting unit was evaluated for Auger therapy. Characterization of the DNA interaction, performed with the non-radioactive Re and 127I congeners, confirmed that all compounds act as DNA intercalators. Both classes of compounds induce double strand breaks (DSB) in plasmid DNA but the extent of DNA damage is strongly dependent on the linker between the Auger emitter (99mTc or 125I) and the AO moiety. The in vitro evaluation was complemented with molecular docking studies and Monte Carlo simulations of the energy deposited at the nanometric scale, which corroborated the experimental data. Two of the tested compounds, 125I-C5 and 99mTc-C3, place the corresponding radionuclide at similar distances to DNA and produce comparable DSB yields in plasmid and cellular DNA. These results provide the first evidence that 99mTc can induce DNA damage with similar efficiency to that of 125I, when both are positioned at comparable distances to the double helix. Furthermore, the high nuclear retention of 99mTc-C3 in tumoral cells suggests that 99mTc-labelled AO derivatives are more promising for the design of Auger-emitting radiopharmaceuticals than the 125I-labelled congeners.

Biophysical characterization and antineoplastic activity of new bis(thiosemicarbazonato) Cu(II) complexes.

Aiming to explore alternative mechanisms of cellular uptake and cytotoxicity, we have studied a new family of copper(II) complexes (CuL1-CuL4) with bis(thiosemicarbazone) (BTSC) ligands containing pendant protonable cyclic amines (morpholine and piperidine). Herein, we report on the synthesis and characterization of these new complexes, as well as on their biological performance (cytotoxic activity, cellular uptake, protein and DNA binding), in comparison with the parental CuIIATSM (ATSM=diacetyl-bis(N4-methylthiosemicarbazonate) complex without pendant cyclic amines. The new compounds have been characterized by a range of analytical techniques including ESI-MS, IR spectroscopy, cyclic voltammetry, reverse-phase HPLC and X-ray spectroscopy. In vitro cytotoxicity studies revealed that the copper complexes are cytotoxic, unlike the corresponding ligands, with a similar potency to that of CuATSM. Unlike CuATSM, the new complexes were able to circumvent cisplatin cross-resistance. The presence of the protonable cyclic amines did not lead to an enhancement of the interaction of the complexes with human serum albumin or calf thymus DNA. However, CuL1-CuL4 showed a remarkably augmented cellular uptake compared with CuATSM, as proved by uptake, internalization and externalization studies that were performed using the radioactive congeners 64CuL1-64CuL4. The enhanced cellular uptake of CuL1-CuL4 indicates that this new family of CuIIBTSC complexes deserves to be further evaluated in the design of metallodrugs for cancer theranostics.

Isostructural Re(I)/(99m)Tc(I) tricarbonyl complexes for cancer theranostics.

Merging classical organic anticancer drugs with metal-based compounds in one single molecule offers the possibility of exploring new approaches for cancer theranostics, i.e. the combination of diagnostic and therapeutic modalities. For this purpose, we have synthesized and biologically evaluated a series of Re(I)/(99m)Tc(I) tricarbonyl complexes (Re1­Re4 and Tc1­Tc4, respectively) stabilized by a cysteamine-based (N,S,O) chelator and containing 2-(4'-aminophenyl)benzothiazole pharmacophores. With the exception of Re1, all the Re complexes have shown a moderate cytotoxicity in MCF7 and PC3 cancer cells (IC50 values in the 15.9­32.1 µM range after 72 h of incubation). The cytotoxic activity of the Re complexes is well correlated with cellular uptake that was quantified using the isostructural (99m)Tc congeners. There is an augmented cytotoxic effect for Re3 and Re4 (versusRe1 and Re2), and the highest cellular uptake for Tc3 and Tc4, which display a long ether-containing linker to couple the pharmacophore to the (N,S,O)-chelator framework. Moreover, fluorescence microscopy clearly confirmed the cytosolic accumulation of the most cytotoxic compound (Re3). Biodistribution studies of Tc1­Tc4 in mice confirmed that these moderately lipophilic complexes (logDo/w = 1.95­2.32) have a favorable bioavailability. Tc3 and Tc4 presented a faster excretion, as they undergo metabolic transformations, in contrast to complexes Tc1 and Tc2. In summary, our results show that benzothiazole-containing Re(I)/(99m)Tc(I) tricarbonyl complexes stabilized by cysteamine-based (N,S,O)-chelators have potential to be further applied in the design of new tools for cancer theranostics.

Bisphosphonates as radionuclide carriers for imaging or systemic therapy.

Bisphosphonates (BP's), biologically stable analogs of naturally occurring pyrophosphates, became the treatment of choice for pathologic conditions characterized by increased osteoclast-mediated bone resorption, namely Paget's disease, osteoporosis and tumor bone disease. Moreover, the clinical success of BP's is also associated with their use in (99m)Tc-based radiopharmaceuticals for bone imaging. In addition to the successful delivery of (99m)Tc (γ-emitter) to bone, BP's have also been used to deliver ß(-)-particle emitting radiometals (e.g.(153)Sm, (186/188)Re) for bone-pain palliation. The main goal of this Review is to update the most recent research efforts toward the synthesis, characterization and biological evaluation of novel BP-containing radiometal complexes and radiohalogenated compounds for diagnostic or therapeutic purposes. The structure and in vivo properties of those compounds will be discussed and compared to the clinically available ones, namely in terms of image quality and therapeutic effect. We will also mention briefly the use of BP's as carriers of multimodal nuclear and optical imaging probes.

99mTc(CO)3-labeled pamidronate and alendronate for bone imaging.

Bone scintigraphy with (99m)Technetium-methylenediphosphonate ((99m)Tc-MDP) or (99m)Technetium-hydroxymethylenediphosphonate ((99m)Tc-HMDP) presents several limitations, namely low specificity, uncertainty in the radiopharmaceutical's molecular structure and long acquisition time after injection. Aiming to find bone-seeking radiotracers based on the core fac-[(99m)Tc(CO)(3)](+) with improved chemical and biological properties, we synthesized new conjugates (pz-PAM and pz-ALN), comprising a pyrazolyl-diamine chelating unit (pz: N,N,N donor atom set) for metal stabilization and a pendant pamidronate (PAM) or alendronate (ALN) moiety for bone targeting. The reaction of the conjugates with fac-[(99m)Tc(CO)(3)](+) yielded (> 95%) the stable complexes fac-[(99m)Tc(CO)(3)(pz-PAM)](-) (2a) and fac-[(99m)Tc(CO)(3)(pz-ALN)](-) (3a), which have been characterized by comparing their HPLC gamma-traces with the UV-vis traces of the Re surrogates 2 and 3, respectively. 2a and 3a bind strongly onto hydroxyapatite. The biodistribution studies in Balb-c mice have shown that 2a and 3a presented an high bone uptake (2a 18.3 ± 0.6% I.D./g, 3a 17.3 ± 6.1% I.D./g, at 1 h post injection), similar to (99m)Tc-MDP (17.1 ± 2.4% I.D./g, at 1 h post injection), with comparable clearance from most tissues and increased total excretion (2a 66% I.D., 3a 67% I.D. and (99m)Tc-MDP 49% I.D., at 1 h post injection). The bone-to-blood (2a 86.2, 3a 74.7) and the bone-to-muscle ratios (2a 77.7, 3a 79.0) are higher than the ones found for (99m)Tc-MDP (70.9, 47.9), at 4 h post injection. Planar whole-body gamma camera images of the rats injected with the (99m)Tc(CO)(3)-labeled pamidronate (2a) and alendronate (3a) confirmed the overall adequate biological profile of the new radiotracers for bone imaging.

A new bisphosphonate-containing (99m)Tc(I) tricarbonyl complex potentially useful as bone-seeking agent: synthesis and biological evaluation.

Aiming to develop new bone-seeking radiotracers based on the organometallic core fac-[(99m)Tc(CO)(3)](+) with improved radiochemical and biological properties, we have prepared new conjugates with phosphonate pendant groups. The conjugates comprise a chelating unit for metal coordination, which corresponds to a pyrazolyl-containing backbone (pz) with a N,N,N donor-atom set, and a pendant diethyl phosphonate (pz-MPOEt), phosphonic acid (pz-MPOH) or a bisphosphonic acid (pz-BPOH) group for bone targeting. Reactions of the conjugates with the precursor [(99m)Tc(H(2)O)(3)(CO)(3)](+) yielded (mote than 95%) the single and well-defined radioactive species [(99m)Tc(CO)(3)(kappa(3)-pz-MPOEt)](+) (1a), [(99m)Tc(CO)(3)(kappa(3)-pz-MPOH](+) (2a) and [(99m)Tc(CO)(3)(kappa(3)-pz-BPOH)](+) (3a), which were characterized by reversed-phase high-performance liquid chromatography . The corresponding Re surrogates (1-3), characterized by the usual analytical techniques, including X-ray diffraction analysis in the case of 1, allowed for macroscopic identification of the radioactive conjugates. These radioactive complexes revealed high stability both in vitro (phosphate-buffered saline solution and human plasma) and in vivo, without any measurable decomposition. Biodistribution studies of the complexes in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway in the case of complex 3a. Despite presenting moderate bone uptake (3.04 +/- 0.47% injected dose per gram of organ, 4 h after injection), the high stability presented by 3a and its adequate in vivo pharmacokinetics encourages the search for new ligands with the same chelating unit and different bisphosphonic acid pendant arms.

Synthesis and structural characterization of novel Re(i) tricarbonyl complexes anchored on a phosphinoarylbenzylamine and a phosphinoaryloxazoline generated in situ.

Reduction of the amide or replacement of the hydroxyl by a bromide in 2-(diphenylphosphanyl)-N-(2-hydroxyethyl)benzamide (H2PNO) yielded the compounds 2-(diphenylphosphanyl)-N-(2-hydroxyethyl)benzylamine (H2CH2PNO, 1) and N-(2-bromoethyl)-2-(diphenylphosphanyl)benzamide (HPNBr, 2), respectively. Compound 2 is obtained in low yield and, depending on the reaction conditions, is mixed with starting material or with a product which has been identified as 2-(2-diphenylphosphinophenyl)oxazoline (PPh3oxaz, 3). Compounds 1 and 2 react with (NEt(4))(2)[ReBr(3)(CO)(3)], leading to the complexes [Re(CO)(3)(kappa(2)-H(2)CH(2)PNO)Br] (4) and [Re(CO)(3)(kappa(2)-PPh(3)oxazBr)] (5), fully characterized by (1)H and (31)P NMR spectroscopy and X-ray crystallographic analysis. Complex 5 is the first example of a Re(I) tricarbonyl anchored on a phosphorus-oxazoline ligand, which has been generated during the course of complex formation. In the unexpected and unusual complex 5, the Re atom is stabilized by a bidentate 2-(2-diphenylphosphinophenyl)oxazoline, by a bromide, and by three facially arranged carbonyl groups. In complex 4, the carbonyl groups are also facially coordinated to the metal center and the other three remaining coordination positions are occupied by a bromide and by the bidentate (P, N) ligand 2-(diphenylphosphanyl)-N-(2-hydroxyethyl)benzylamine.