Melanoma targeting with alpha-melanocyte stimulating hormone analogs labeled with fac-[Tc-99m(CO)(3)](+): effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized alpha-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of alpha-MSH (Ac-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of alpha-MSH analogs by comparing the pharmacokinetics profile of the (99)mTc-labeled cyclic peptide beta Ala-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2 with that of the linear analog beta Ala-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2 in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of beta Ala, and the resulting pz-peptide conjugates were reacted with the fac-[Tc-99m(CO)(3)](+) moiety. The Tc-99m(CO)(3)-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic Tc-99m(CO)(3)-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 degrees C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 +/- 0.83 and 11.31 +/- 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear Tc-99m(CO)(3)-pz-peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle(4),DPhe(7))-alpha MSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively)

In vivo and in vitro studies of [M(η6-pseudoerlotinib)2]+ sandwich complexes (M = Re, 99mTc)

The pursuit of molecular imaging for tumors has led to endeavors focused on targeting epidermal growth factor receptors (EGFR) through monoclonal antibodies or radionuclide-labelled EGF analogs with 99mTc, 111In, or 131I. In this context, various 99mTc-labeled EGFR inhibitors using quinazoline structures have been reported based on the so-called pendant approach and on two types of complexes and labelling strategies: “4 + 1” mixed ligand complexes and fac-tricarbonyl complexes. Apart from this approach, which alters lead structures by linking pharmacophores to chelator frameworks through different connectors, the integrated incorporation of topoisomerase and tyrosine kinase inhibitors into Re and 99mTc complexes has not been explored. Here we present [M(η6-inhibitor)2]+ (M = Re, 99mTc) and [Re(η6-bz)(η6-inhibitor)]+ complexes, where the core structure of an EGFR tyrosine kinase inhibitor binds directly to the metal center. These complexes exhibit potential for tumor imaging: initial biological investigations highlight the influence of one versus two bound inhibitors on the metal center

Pt(IV)/Re(I) Chitosan Conjugates as a Flexible Platform for the Transport of Therapeutic and/or Diagnostic Anticancer Agents

New chitosan derivatives modified with (3-carboxypropyl) trimethylammonium chloride (1) and coupled with (OC-6-44)-diammine(4-carboxypropanoato) dichloridoethanolatoplatinum(IV) (2), were synthesized and their preliminary biological evaluation carried out in human tumor cells. Some of these derivatives were also loaded with a chelating ligand (3) that was derived from bis(quinolin-2-ylmethyl) amine to obtain chitosan-based nanoparticles for an EPR-mediated delivery of Pt(IV) prodrugs and Re(I) tricarbonyl complexes (4), to explore a multimodal theranostic approach to cancer. The cytotoxicity of the different chitosan conjugates (C12, C123, and C1234), carrying different combinations of the Pt(IV) complex, the chelator and the Re(I) complex, was evaluated in the A2780 human ovarian cancer cell line using the MTT assay. The Pt(IV)-containing nanosystems showed low to moderate cytotoxic activity (IC50 values in the range 13.5-33.7 uM) and was comparable to that found for the free Pt(IV) complex (IC50 = 13.7 uM). Therefore, the Pt(IV)-chitosan conjugation did not enhance the cytotoxic activity of the Pt(IV) prodrug, which certainly reflects the inefficient cellular uptake of the nanoconjugates. Nevertheless, a clearer view of their potential for the delivery of anticancer agents requires further in vivo tests because the EPR effect increases extravasation and retention within the tumor tissue, not necessarily within the tumor cells

[(Cp-R)M(CO)3] (M=Re or 99mTc) Arylsulfonamide, Arylsulfamide, and Arylsulfamate Conjugates for Selective Targeting of Human Carbonic Anhydrase IX

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Tricarbonyl M(I) (M = Re, 99mTc) complexes bearing acridine fluorophores : synthesis, characterization, DNA interaction studies and nuclear targeting

© The Royal Society of Chemistry 2010New pyrazolyl-diamine ligands with acridine derivatives at the 4-position of the pyrazolyl ring were synthesized and characterized (L1 and L2). Coordination towards the fac-[M(CO)3]+ (M = Re, 99mTc) led to complexes fac-[M(CO)3(κ3-L)] (L = L1: M = Re1, Tc1; L = L2: M = Re2, Tc2). The interaction of the novel pyrazolyl-diamine ligands (L1 and L2) and rhenium(I) complexes (Re1 and Re2) with calf thymus DNA (CT-DNA) was investigated by a variety of techniques, namely UV-visible , fluorescence spectroscopy and circular and linear dichroism . Compounds L1 and Re1 have moderate affinity to CT-DNA and bind to DNA by intercalation, while L2 and Re2 have a poor affinity for CT-DNA. Moreover, LD measurements showed that L1 and Re1 act as perfect intercalators . By confocal fluorescence microscopy we found that L1 and Re1 internalize and localize in the nucleus of B16F1 murine melanoma cells . The congener Tc1 complex also targets the cell nucleus exhibiting a time-dependent cellular uptake and a fast and high nuclear internalization (67.2% of activity after 30 min). Plasmid DNA studies have shown that Tc1 converts supercoiled (sc) puc19 DNA to the open circular (oc) form.Teresa Esteves and Sofia Gama thank the FCT for a doctoral and postdoctoral research grants (SFRH/BD/29154/2006 and SFRH/BPD/29564/2006, respectively). COST Action D39 is also acknowledge. The QITMS instrument was acquired with the support of the Programa Nacional de Reequipamento Científico (Contract>REDE/1503/REM/2005-ITN) of Fundação para a Ciência e a Tecnologia and is part of RNEM - Rede Nacional de Espectrometria de Massa

The melanocortin agonist Melanotan-II reduces the orexigenic and adipogenic effects of neuropeptide Y (NPY) but does not affect the NPY-driven suppressive effects on the gonadotropic and somatotropic axes in the male rat

Neuropeptide Y (NPY) is a strong orexigenic neurotransmitter also known to modulate several neuroendocrine axes. alpha-Melanocyte-stimulating hormone (MSH) is an essential anorectic neuropeptide, acting on hypothalamic MC3/4 receptor subtypes. When given as an intracerebroventricular bolus injection, Melanotan-II (MT-II), a non selective MC receptor agonist, inhibits feeding, suppresses the NPY orexigenic action, and reduces basal insulinaemia. We evaluated the effects of a 7-day central infusion of MT-II (15 nmol/day) given either alone or in association with NPY (5 nmol/day) in male Sprague-Dawley rats. MT-II produced almost full anorexia for 1-2 days but then feeding gradually returned to normal despite continued MT-II infusion. When coinfused with NPY, MT-II also produced the same initial anorectic episode but then maintained feeding to upper normal levels, thus cancelling the hyperphagia driven by NPY. Whereas NPY infusion produced a doubling of fat pad weight, MT-II reduced adiposity by a factor of two compared to pair-fed rats, and vastly curtailed the NPY-driven increase in fat pad weight. MT-II infusion also significantly curtailed the NPY-induced rise in insulin and leptin secretions. NPY infusion significantly inhibited hypothalamic pro-opiomelanocortin mRNA expression, most likely cancelling the alpha-MSH anorectic activity. As expected from previous studies, chronic NPY infusion strongly inhibited both the gonadotropic and somatotropic axes, and coinfusion of MT-II did not reverse these NPY-driven effects, in sharp contrast with that seen for the metabolic data. MT-II infusion alone had little effect on these axes. In conclusion, chronic MT-II infusion generated a severe but transient reduction in feeding, suggesting an escape phenomenon, and clearly reduced fat pad size. When coinfused with NPY, MT-II was able to cancel most of the NPY effects on feeding, but not those on the neuroendocrine axes. It appears therefore that, as expected, NPY and alpha-MSH closely interact in the control of feeding, whereas the neural pathways by which NPY affects growth and reproduction are distinct and not sensitive to MC peptide modulation

Towards99mTc-based imaging agents with effective doxorubicin mimetics: a molecular and cellular study

Doxorubicin is a clinical benchmark drug, which is applied in the treatment of numerous cancers. Known for its accumulation in the nucleus and ability to intercalate into DNA, it targets quickly dividing i.e. hypermitotic cells. Through this mechanism, it could be an ideal structural motif for a new class of imaging agents, given that the new entity approximates the in vitro profile of the parent drug. Here we describe design, synthesis and biological activity of a small array of Doxorubicin-metalloconjugates (M = 99mTc, Re). We demonstrate that the conjugates preferably accumulate in the nuclear compartment, tightly bind to DNA and retain an appreciable cytotoxicity. Moreover, the Re conjugates effectively act as inhibitors of the human Topoisomerase II enzyme, which is the widely accepted mechanism of action of the parent drug. Since the conjugates effectively mimic the in vitro behavior of native Doxorubicin, the 99mTc compounds are prospective imaging agents

The (Cp)M(CO)3] (M=Re, 99mTc) building block for imaging agents and bioinorganic probes: Perspectives and limitations

Starting from asymmetric Thiele's acid derivatives, two different imaging probes 99mTc(CO)3(CpR)] (R=potential targeting vector) are generated simultaneously in one-pot and from one substrate. This extends the previously introduced labeling strategy of metal-mediated retro-Diels?Alder reaction with HCp-R dimers. We demonstrate that chemically active functionalities such as hydroxamic acids are not following this labeling strategy. Adopting the principle of replacing phenyl rings by Re(CO)3(Cp)] entities, potent histone deacetylase (HDAC)-inhibiting Re analogs of suberoylanlilide hydroxamic acid (SAHA; N-hydroxy-N'-phenyloctanediamide) were synthesized and characterized. Cytotoxic evaluation on different tumor cell lines revealed low IC50 values mu M] for these compounds, comparable to their purely organic congeners

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