Boosting Bismuth(III) Complexation for Targeted α-Therapy (TAT) Applications with the Mesocyclic Chelating Agent AAZTA.

Targeted α therapy (TAT) is a promising tool in the therapy of cancer. The radionuclide 213 BiIII shows favourable physical properties for this application, but the fast and stable chelation of this metal ion remains challenging. Herein, we demonstrate that the mesocyclic chelator AAZTA quickly coordinates BiIII at room temperature, leading to a robust complex. A comprehensive study of the structural, thermodynamic and kinetic properties of [Bi(AAZTA)]- is reported, along with bifunctional [Bi(AAZTA-C4-COO- )]2- and the targeted agent [Bi(AAZTA-C4-TATE)]- , which incorporates the SSR agonist Tyr3 -octreotate. An unexpected increase in the stability and kinetic inertness of the metal chelate was observed for the bifunctional derivative and was maintained for the peptide conjugate. A cyclotron-produced 205/206 Bi mixture was used as a model of 213 Bi in labelling, stability, and biodistribution experiments, allowing the efficiency of [213 Bi(AAZTA-C4-TATE)]- to be estimated. High accumulation in AR42J tumours and reduced kidney uptake were observed with respect to the macrocyclic chelate [213 Bi(DOTA-TATE)]- .

Water-Soluble [Tc(N)(PNP)] Moiety for Room-Temperature 99mTc Labeling of Sensitive Target Vectors.

The incorporation of bioactive molecules into a water-soluble [99mTc][Tc(N)(PNP)]-based mixed compound is described. The method, which exploits the chemical properties of the new [99mTc][Tc(N)(PNP3OH)]2+ synthon [PNP3OH = N,N-bis(di-hydroxymethylenphosphinoethyl)methoxyethylamine], was successfully applied to the labeling of small, medium (cysteine-functionalized biotin and c-RGDfK pentapeptide), and large molecules. Apomyoglobin was chosen as a model protein and derivatized via site-specific enzymatic reaction catalyzed by transglutaminase (TGase) with the H-Cys-Gly-Lys-Gly-OH tetrapeptide for the insertion in the protein sequence of a reactive N-terminal Cys for 99mTc chelation. Radiosyntheses were performed under physiological conditions at room temperature within 30 min. They were reproducible, highly specific, and quantitative. Heteroleptic complexes are hydrophilic and stable. Biodistributions of the selected compounds show favorable pharmacokinetics within 60 min post-injection and predominant elimination through the renal-urinary pathway. In a wider perspective, these data suggest a role of the [99mTc][Tc(N)(PNP)] technology in the labeling of temperature-sensitive biomolecules, especially targeting proteins for SPECT imaging.

Preliminary safety and imaging efficacy of the near-infrared fluorescent contrast agent DA364 during fluorescence-guided surgery in dogs with spontaneous superficial tumors.

Tumor-targeting contrast agents may facilitate resection of solid neoplasms during fluorescence-guided surgery. Preliminary safety and imaging efficacy of the near-infrared fluorescent probe DA364 were evaluated during surgical resection of spontaneous solid tumors in 24 dogs. Intra-operative imaging was performed in situ and on excised specimens to evaluate fluorescence intensities of tumor and adjacent tissues. After standard-of-care tumor resection, the wound bed was imaged again, and additional tissue was excised if residual fluorescence was detected. DA364 was well tolerated after intravenous administration. The median tumor-to-background ratio in situ for mammary tumors, mast cell tumors and sarcomas was 1.8 (range 1.2-3.9), 2.2 (range 1.0-5.6), and 4.2 (range 2.0-4.3), respectively. Qualitative intra-operative tumor identification was feasible in half of the cases. Remaining fluorescence was detected in four wound beds that contained residual disease, and in11 tumor-free wound beds, confirmed by histopathology. Overall, DA364 did not raise safety concerns and showed accumulation in different types of spontaneous tumors, showing potential to pinpoint residual disease. Larger clinical trials are necessary to select accurate dosing and imaging protocols for specific indications to evaluate the sensitivity and specificity of the agent.

Environment-specific spectral modeling: A new tool for the analysis of biological specimens.

The recent discovery of fluorescent dyes for improving pathologic tissues identification has highlighted the need of robust methods for performance validation especially in the field of fluorescence-guided surgery. Optical imaging of excised tissue samples is the reference tool to validate the association between dyes localization and the underlying histology in a controlled environment. Spectral unmixing may improve the validation process discriminating dye from endogenous signal. Here, an innovative spectral modeling approach that weights the spectral shifts associated with changes in chemical environment is described. The method is robust against spectral shift variations and its application leads to unbiased spectral weights estimates as demonstrated by numerical simulations. Finally, spectral shifts values computed pixel-wise from spectral images are used to display additional information with potential diagnostic value.

Photoacoustic imaging of integrin-overexpressing tumors using a novel ICG-based contrast agent in mice.

PhotoAcoustic Imaging (PAI) is a biomedical imaging modality currently under evaluation in preclinical and clinical settings. In this work, ICG is coupled to an integrin binding vector (ICG-RGD) to combine the good photoacoustic properties of ICG and the favourable αvß3-binding capabilities of a small RGD cyclic peptidomimetic. ICG-RGD is characterized in terms of physicochemical properties, biodistribution and imaging performance. Tumor uptake was assessed in subcutaneous xenograft mouse models of human glioblastoma (U-87MG, high αvß3 expression) and epidermoid carcinoma (A431, low αvß3 expression). ICG and ICG-RGD showed high PA signal in tumors already after 15 min post-injection. At later time points the signal of ICG rapidly decreased, while ICG-RGD showed sustained uptake in U-87MG but not in A431 tumors, likely due to the integrin-mediated retention of the probe. In conclusion, ICG-RGD is a novel targeted contrast agents for PAI with superior biodistribution, tumor uptake properties and diagnostic value compared to ICG.

Equilibrium Thermodynamics, Formation, and Dissociation Kinetics of Trivalent Iron and Gallium Complexes of Triazacyclononane-Triphosphinate (TRAP) Chelators: Unraveling the Foundations of Highly Selective Ga-68 Labeling.

In order to rationalize the influence of FeIII contamination on labeling with the 68Ga eluted from 68Ge/68Ga-generator, a detailed investigation was carried out on the equilibrium properties, formation and dissociation kinetics of GaIII- and FeIII-complexes of 1,4,7-triazacyclononane-1,4,7-tris(methylene[2-carboxyethylphosphinic acid]) (H6TRAP). The stability and protonation constants of the [Fe(TRAP)]3- complex were determined by pH-potentiometry and spectrophotometry by following the competition reaction between the TRAP ligand and benzhydroxamic acid (0.15 M NaNO3, 25°C). The formation rates of [Fe(TRAP)] and [Ga(TRAP)] complexes were determined by spectrophotometry and 31P-NMR spectroscopy in the pH range 4.5-6.5 in the presence of 5-40 fold HxTRAP(x-6) excess (x = 1 and 2, 0.15 M NaNO3, 25°C). The kinetic inertness of [Fe(TRAP)]3- and [Ga(TRAP)]3- was examined by the trans-chelation reactions with 10 to 20-fold excess of HxHBED(x-4) ligand by spectrophotometry at 25°C in 0.15 M NaCl (x = 0,1 and 2). The stability constant of [Fe(TRAP)]3- (logKFeL = 26.7) is very similar to that of [Ga(TRAP)]3- (logKGaL = 26.2). The rates of ligand exchange reaction of [Fe(TRAP)]3- and [Ga(TRAP)]3- with HxHBED(x-4) are similar. The reactions take place quite slowly via spontaneous dissociation of [M(TRAP)]3-, [M(TRAP)OH]4- and [M(TRAP)(OH)2]5- species. Dissociation half-lives (t1/2) of [Fe(TRAP)]3- and [Ga(TRAP)]3- complexes are 1.1 × 105 and 1.4 × 105 h at pH = 7.4 and 25°C. The formation reactions of [Fe(TRAP)]3- and [Ga(TRAP)]3- are also slow due to the formation of the unusually stable monoprotonated [*M(HTRAP)]2- intermediates [*logKGa(HL) = 10.4 and *logKFe(HL) = 9.9], which are much more stable than the [*Ga(HNOTA)]+ intermediate [*logKGa(HL) = 4.2]. Deprotonation and transformation of the monoprotonated [*M(HTRAP)]2- intermediates into the final complex occur via OH--assisted reactions. Rate constants (kOH) characterizing the OH--driven deprotonation and transformation of [* Ga(HTRAP)]2- and [*Fe(HTRAP)]2- intermediates are 1.4 × 105 M-1s-1 and 3.4 × 104 M-1s-1, respectively. In conclusion, the equilibrium and kinetic properties of [Fe(TRAP)] and [Ga(TRAP)] complexes are remarkably similar due to the close physico-chemical properties of FeIII and GaIII-ions. However, a slightly faster formation of [Ga(TRAP)] over [Fe(TRAP)] provides a rationale for a previously observed, selective complexation of 68GaIII in presence of excess FeIII.

Improved Efficacy of Synthesizing *MIII-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study.

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [68Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H2O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)]- (MIII= GaIII, CeIII, EuIII, YIII, and LuIII) complexes were investigated in H2O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H2O/EtOH mixtures (0-70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K1H and log K2H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)]- complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H2DOTA)]+ intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H2O (*M(HL) kH2O) and OH- (*M(HL) kOH) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL) kH2O values increase from CeIII to LuIII. However, the log KM(HL)H protonation constants, analogous to the log KH2 value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes.

AAZTA: An Ideal Chelating Agent for the Development of 44 Sc PET Imaging Agents.

Unprecedented fast and efficient complexation of ScIII was demonstrated with the chelating agent AAZTA (AAZTA=1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) under mild experimental conditions. The robustness of the 44 Sc(AAZTA)- chelate and conjugated biomolecules thereof is further shown by in vivo PET imaging in healthy and tumor mice models. The new results pave the way towards development of efficient Sc-based radiopharmaceuticals using the AAZTA chelator.

In vivo tumor targeting and biodistribution evaluation of paramagnetic solid lipid nanoparticles for magnetic resonance imaging.

The current study was performed to evaluate the in vivo efficiency of a new nano-sized contrast agent called paramagnetic Solid Lipid Nanoparticles, pSLNs, having promising relaxivity properties for Magnetic Resonance Imaging application. Good stability and stealth properties toward macrophage uptake have been demonstrated. An in vivo MRI study resulted in an improved signal enhancement in the tumor tissue particularly when folate as targeting ligand was used to decorate the nanoparticles surface. Afterward, the biodistribution of pSLNs in several organs was investigated. The accumulation of pSLNs in kidneys, femoral bones, spleen and brain was quite low while high tropism of pSLNs was found for the liver. In this regard, approaches to improve the rate of the hepatic clearance have been proposed.

Study of the binding interaction between fluorinated matrix metalloproteinase inhibitors and Human Serum Albumin.

Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [(18)F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. (19)F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with Ka in the order of 10(5) M(-1)) and fast-exchange kinetics (with Ka in the order of 10(4) M(-1)). For 2-(2-(4'-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1a) and 2-(2-(4'-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1c), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers.

Paramagnetic solid lipid nanoparticles as a novel platform for the development of molecular MRI probes.

Highly efficient magnetic resonance imaging (MRI) probes have been prepared by loading Gd(III) complexes on the surface of solid lipid nanoparticles (pSLNs). Applicability as molecular imaging probes is demonstrated by an in vitro model study with targeted pSLNs.

Optical imaging detection of microscopic mammary cancer in ErbB-2 transgenic mice through the DA364 probe binding αv ß3 integrins.

Despite spontaneous tumor growth in genetically engineered mice being one of the most recognized tools for the in vivo evaluation of novel diagnostic and therapeutic anticancer compounds, monitoring early stage lesions in live animals is a goal that has yet to be achieved. A large number of targets for the molecular imaging of various diseases have been identified and many imaging technologies, including optical techniques are emerging. One of the most commonly exploited targets in tumor imaging is αv ß3 integrin, which plays an important role in the expansion, invasiveness and metastatic capability of a number of cancers, including breast cancer. The aim of this study was to set up an optical imaging method for the early detection of autochthonous mammary cancer in female BALB/c mice transgenic for the rat-ErbB-2 oncogene (BALB-neuT). We show that DA364, a near-infrared fluorescence arginine-glycine-aspartic acid cyclic probe, was taken up by neoplastic mammary glands and that its uptake increased with cancer progression. By contrast, the nonaccumulation of DA364 in the healthy mammary glands of virgin and lactating wild-type mice suggests that the probe specifically targets breast cancers. Comparisons of optical imaging with whole-mount and histological findings showed that DA364 allows the noninvasive visualization of atypical hyperplasia and microscopic foci of in situ carcinoma 2 months before mammary lesions become detectable by palpation. Moreover, DA364 was successfully used to monitor the outcome of anticancer vaccination. Therefore, it can be considered a promising early detection tool in near-infrared noninvasive optical imaging for the early diagnosis of breast cancer.

Synthesis and preliminary evaluation in tumor bearing mice of new (18)F-labeled arylsulfone matrix metalloproteinase inhibitors as tracers for positron emission tomography.

New fluorinated, arylsulfone-based matrix metalloproteinase (MMP) inhibitors containing carboxylate as the zinc binding group were synthesized as radiotracers for positron emission tomography. Inhibitors were characterized by Ki for MMP-2 in the nanomolar range and by a fair selectivity for MMP-2/9/12/13 over MMP-1/3/14. Two of these compounds were obtained in the (18)F-radiolabeled form, with radiochemical purity and yield suitable for preliminary studies in mice xenografted with a human U-87 MG glioblastoma. Target density in xenografts was assessed by Western blot, yielding Bmax/Kd = 14. The biodistribution of the tracer was dominated by liver uptake and hepatobiliary clearance. Tumor uptake of (18)F-labeled MMP inhibitors was about 30% that of [(18)F]fluorodeoxyglucose. Accumulation of radioactivity within the tumor periphery colocalized with MMP-2 activity (evaluated by in situ zimography). However, specific tumor uptake accounted for only 18% of total uptake. The aspecific uptake was ascribed to the high binding affinity between the radiotracer and serum albumin.

MRI evaluation of the antitumor activity of paramagnetic liposomes loaded with prednisolone phosphate.

The design of long circulating liposomes co-loaded with the glucocorticoid prednisolone phosphate (PLP) and the amphiphilic paramagnetic contrast agent Gd-DOTAMA(C(18))(2) allowed the MRI-guided in vivo visualization of the delivery and biodistribution of PLP, as well as the monitoring of drug efficacy. The performance of this theranostic probe was investigated in a mouse model bearing a melanoma B16 syngeneic tumor. The release kinetics of the drug were evaluated in vitro where it displayed a peculiar behavior characterized by a fast process (completed in few hours) involving only a small portion (<5%) of the drug. Interestingly, the incorporation of the amphiphilic imaging reporter in the liposomal bilayer slightly increased the amount of the fast-release portion (<10%), thus suggesting that it could be attributed to a drug fraction embedded in the liposomal bilayer. In fact, the release of a hydrophilic imaging probe encapsulated in the inner core of the same long circulating liposomes formulated for carrying the drug, displayed different, single-step, kinetics. The in vivo monitoring of the antitumor activity of the nanomedicine revealed that the incorporation of the MRI probe into the liposome bilayer did not significantly affect the drug efficacy. The in vivo experiments also indicated a relevant and fast liposome uptake from macrophage-rich organs like spleen and liver, which reduced the tumor accumulation of the liposomes. The accumulation of the amphipatic MRI label caused the occurrence of a long-term residual T(1) contrast still detectable 1week after injection.

A new optical imaging probe targeting αVß3 integrin in glioblastoma xenografts.

α(V)ß(3) Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near-infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α(V) ß(3) integrin-overexpressing tumors. DA364's binding affinity for α(V)ß(3) integrin was first evaluated in vitro. Human α(V)ß(3) integrin-positive, U-87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α(V)ß(3) integrin on U-87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic-RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α(V)ß(3) integrin-overexpressing tumors.

Cytotoxicity of cis-platinum(II) conjugate models. The effect of chelating arms and leaving groups on cytotoxicity: a quantitative structure-activity relationship approach.

Thirteen newly synthesized or resynthesized diamine-platinum(II) complexes were characterized, and their cytotoxic activities (IC50) were tested on parental and resistant ovarian cancer cell lines. They represent models of conjugates between biologically active vectors and cytotoxic Pt(II) moieties within the "drug targeting and delivery strategy". Three drugs, routinely employed in the clinical treatment of cancer, namely, cisplatin, carboplatin, and oxaliplatin, were also included in the study as controls. The quantitative structure-activity relationship approach provides simple regression models able to predict log(1/IC50) of diamine-platinum(II) complexes on both parental and resistant ovarian cancer cell lines. The 16 complexes were characterized using 197 molecular descriptors, after which the best regression models relating a subset of these descriptors to the log(1/IC50) in the two cancer cell lines were calculated. Models with four variables proved to be endowed with very good predictive ability Q2(LMO-50%) > or = 85.6%, making it possible to discard 50% of the molecules from the test set following for cross-validation procedure. A four-variable regression model also proved effective in predicting the resistance index RI, Q2(LMO-50%) = 84.4%.