Bruno L. Oliveira

In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding positron emission tomographic probe.

Background—Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. Methods and Results—The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30–90 minutes, >15-fold at 240–285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic–computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator–treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator–treated animals, but enduring high thrombus activity in control rats. Conclusions—We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.

Influence of the bifunctional chelator on the pharmacokinetic properties of 99mTc(CO)3-labeled cyclic α-melanocyte stimulating hormone analog.

Aiming at the design of specific melanocortin-1 receptor (MC1R) targeted imaging probes, we report on the effect of different azolyl-ring substitution patterns (carboxylate at the 4-position and/or methyl groups at the 3,5 positions) of pyrazolyl-diamine bifunctional chelators (Pz(2)-Pz(4)) on the pharmacokinetic profile of the (99m)Tc(CO)3-labeled lactam bridge-cyclized α-melanocyte stimulating hormone derivative, βAlaNleCycMSH(hex). Three pyrazolyl-diamine-containing chelators were conjugated to βAlaNleCycMSHhex, with the resulting peptide conjugates displaying subnanomolar MC1R binding affinity. Biodistribution studies in B16F1 melanoma-bearing mice show that all radiopeptides present a good melanoma uptake. The introduction of a carboxylate group in the azolyl-ring leads to a remarkable reduction of the kidney (>89%) and liver (>91%) accumulation for (99m)Tc(CO)3-Pz(3)-βAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-βAlaNleCycMSH(hex) when compared to the radiopeptide (99m)Tc(CO)3-Pz(1)-βAlaNleCycMSH(hex), where that group is absent. The good tumor uptake and favorable tumor-to-nontarget-organs ratios of (99m)Tc(CO)3-Pz(3)-βAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-βAlaNleCycMSH(hex) highlights the potential of both compounds as melanoma imaging agents.

Re and Tc Tricarbonyl Complexes: From the Suppression of NO Biosynthesis in Macrophages to in Vivo Targeting of Inducible Nitric Oxide Synthase

The in vivo molecular imaging of nitric oxide synthase (NOS), the enzyme responsible for the catalytic oxidation of l-arginine to citrulline and nitric oxide (NO), by noninvasive modalities could provide valuable insights into NO/NOS-related diseases. Aiming at the design of innovative (⁹⁹m)Tc(I) complexes for targeting inducible NOS (iNOS) in vivo by SPECT imaging, herein we describe a set of novel (⁹⁹m)Tc(CO)₃ complexes (2-5) and the corresponding rhenium surrogates (2a-5a) containing the NOS inhibitor N(ω)-nitro-l-arginine. The latter is linked through its α-NH₂ or α-COOH group and an alkyl spacer of variable length to the metal center. The complexes 2a (propyl spacer) and 3a (hexyl spacer), in which the α-NH₂ group of the inhibitor is involved in the conjugation to the metal center, presented remarkable affinity for purified iNOS, being similar to that of the free nonconjugated inhibitor (K(i) = 3-8 μM) in the case of 3a (K(i) = 6 μM). 2a and 3a are the first examples of organometallic complexes that permeate through RAW 264.7 macrophage cell membranes, interacting specifically with the target enzyme, as confirmed by the suppression of NO biosynthesis in LPS-treated macrophages (2a, ca. 30% inhibition; 3a, ca. 50% inhibition). The (⁹⁹m)Tc(I)-complexes 2 and 3, stable both in vitro and in vivo, also presented the ability to cross cell membranes, as demonstrated by internalization studies in the same cell model. The biodistribution studies in LPS-pretreated mature female C57BL6 mice have shown that 2 presented an overall higher uptake in most tissues of the LPS-treated mice compared to the control group (30 min postinjection). This increase is significant in lung (3.98 ± 0.63 vs to 0.99 ± 0.13%ID/g), which is known to be the organ with the highest iNOS expression after LPS treatment. These results suggest that the higher uptake in that organ may be related to iNOS upregulation.

Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.

Multisite Thrombus Imaging and Fibrin Content Estimation With a Single Whole-Body PET Scan in Rats

Objective—Thrombosis is a leading cause of morbidity and mortality worldwide. Current diagnostic strategies rely on imaging modalities that are specific for distinct vascular territories, but a thrombus-specific whole-body imaging approach is still missing. Moreover, imaging techniques to assess thrombus composition are underdeveloped, although therapeutic strategies may benefit from such technology. Therefore, our goal was to test whether positron emission tomography (PET) with the fibrin-binding probe 64Cu-FBP8 allows multisite thrombus detection and fibrin content estimation. Approach and Results—Thrombosis was induced in Sprague-Dawley rats (n=32) by ferric chloride application on both carotid artery and femoral vein. 64Cu-FBP8-PET/CT imaging was performed 1, 3, or 7 days after thrombosis to detect thrombus location and to evaluate age-dependent changes in target uptake. Ex vivo biodistribution, autoradiography, and histopathology were performed to validate imaging results. Arterial and venous thrombi were localized on fused PET/CT images with high accuracy (97.6%; 95% confidence interval, 92–100). A single whole-body PET/MR imaging session was sufficient to reveal the location of both arterial and venous thrombi after 64Cu-FBP8 administration. PET imaging showed that probe uptake was greater in younger clots than in older ones for both arterial and venous thrombosis (P<0.0001). Quantitative histopathology revealed an age-dependent reduction of thrombus fibrin content (P<0.001), consistent with PET results. Biodistribution and autoradiography further confirmed the imaging findings. Conclusions—We demonstrated that 64Cu-FBP8-PET is a feasible approach for whole-body thrombus detection and that molecular imaging of fibrin can provide, noninvasively, insight into clot composition.

Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells

Abstract The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol‐containing molecules and as reagents for bioorthogonal bond‐cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine‐mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Effect of Chelate Type and Radioisotope on the Imaging Efficacy of 4 Fibrin-Specific PET Probes

Thrombus formation plays a major role in cardiovascular diseases, but noninvasive thrombus imaging is still challenging. Fibrin is a major component of both arterial and venous thrombi and represents an ideal candidate for imaging of thrombosis. Recently, we showed that 64Cu-DOTA–labeled PET probes based on fibrin-specific peptides are suitable for thrombus imaging in vivo; however, the metabolic stability of these probes was limited. Here, we describe 4 new probes using either 64Cu or aluminum fluoride (Al18F) chelated to 2 NOTA derivatives. Methods: Probes were synthesized using a known fibrin-specific peptide conjugated to either NODAGA (FBP8, FBP10) or NOTA-monoamide (FBP9, FBP11) as chelators, followed by labeling with 64Cu (FBP8 and FBP9) or Al18F (FBP10 and FBP11). PET imaging efficacy, pharmacokinetics, biodistribution, and metabolic stability were assessed in a rat model of arterial thrombosis. Results: All probes had similar nanomolar affinity (435–760 nM) for the soluble fibrin fragment DD(E). PET imaging allowed clear visualization of thrombus by all probes, with a 5-fold or higher thrombus-to-background ratio. Compared with the previous DOTA derivative, the new 64Cu probes FBP8 and FBP9 showed substantially improved metabolic stability (>85% intact in blood at 4 h after injection), resulting in high uptake at the target site (0.5–0.8 percentage injected dose per gram) that persisted over 5 h, producing increasingly greater target-to-background ratios. The thrombus uptake was 5- to 20-fold higher than the uptake in the contralateral artery, blood, muscle, lungs, bone, spleen, large intestine, and heart at 2 h after injection and 10- to 40-fold higher at 5 h. The Al18F derivatives FBP10 and FBP11 were less stable, in particular the NODAGA conjugate (FBP10, <30% intact in blood at 4 h after injection), which showed high bone uptake and low thrombus-to-background ratios that decreased over time. The high thrombus-to-contralateral ratios for all probes were confirmed by ex vivo biodistribution and autoradiography. The uptake in the liver (<0.5 percentage injected dose per gram), kidneys, and blood were similar for all tracers, and they all showed predominant renal clearance. Conclusion: FBP8, FBP9, and FBP11 showed excellent metabolic stability and high thrombus-to-background ratios and represent promising candidates for imaging of thrombosis in vivo.

Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms

AbstractSelective inhibition of the nitric oxide synthase isoforms (NOS) is a promising approach for the treatment of various disorders. However, given the high active site conservation among all NOS isoforms, the design of selective inhibitors is a challenging task. Analysis of the X-ray crystal structures of the NOS isoforms complexed with known inhibitors most often gives no clues about the structural determinants behind the selective inhibition since the inhibitors share the same binding conformation. Aimed at a better understanding of the structural factors responsible for selective inhibition of NOS isoforms we have performed MD simulations for iNOS, nNOS and eNOS complexed with Nω-NO2-L-Arg (1), and with the aminopyridine derivatives 2 and 3. The slightly better selectivity of 1 for nNOS may be assigned to the presence of extra charge–charge interactions due to its “extended” conformation. While the high affinity of 2 for iNOS can be explained by the formation of an iNOS-specific subpocket upon binding, the lack of affinity for eNOS is associated to a conformational change in Glu363. The strong van der Waals and electrostatic interactions between 3 and the active site of nNOS are most likely responsible for its higher affinity for this isoform. Owing to the elongated and narrow binding pocket of iNOS, the correct positioning of 3 over the heme group is difficult, which may account for its lower affinity toward this isoform. Brought together, our results might help to rationalize the design of selective NOS inhibitors.n FigureOverall RMSD of the protein backbone over 8 ns simulation is shown for the complexes 3:eNOSmonomer and 3:eNOSdimer

Syntheses of bifunctional 2,3-diamino propionic acid-based chelators as small and strong tripod ligands for the labelling of biomolecules with 99mTc

The labelling of targeting biomolecules requires small and hydrophilic complexes in order to not affect the binding properties of the vectors. 2,3-Diamino propionic acid (dap) is a small and strong, albeit scarcely used, tripod ligand for the fac-[(99m)Tc(CO)(3)](+) moiety. We have introduced at the alpha-carbon atom in the basic dap structure various second functionalities such as carboxylato, amino and alpha-amino acid groups via various spacers in order to yield bifunctional chelators. These dap derivatives can be coupled to targeting molecules for application in molecular imaging. Full characterizations of the bifunctional chelators, X-ray structures of intermediates and of one rhenium complex, as well as labelling studies with (99m)Tc, are presented.

Radiation Dosimetry of the Fibrin-Binding Probe 64Cu-FBP8 and Its Feasibility for PET Imaging of Deep Vein Thrombosis and Pulmonary Embolism in Rats

The diagnosis of deep venous thromboembolic disease is still challenging despite the progress of current thrombus imaging modalities and new diagnostic algorithms. We recently reported the high target uptake and thrombus imaging efficacy of the novel fibrin-specific PET probe 64Cu-FBP8. Here, we tested the feasibility of 64Cu-FBP8 PET to detect source thrombi and culprit emboli after deep vein thrombosis and pulmonary embolism (DVT-PE). To support clinical translation of 64Cu-FBP8, we performed a human dosimetry estimation using time-dependent biodistribution in rats. Methods: Sprague–Dawley rats (n = 7) underwent ferric chloride application on the femoral vein to trigger thrombosis. Pulmonary embolism was induced 30 min or 2 d after DVT by intrajugular injection of a preformed blood clot labeled with 125I-fibrinogen. PET imaging was performed to detect the clots, and SPECT was used to confirm in vivo the location of the pulmonary emboli. Ex vivo γ counting and histopathology were used to validate the imaging findings. Detailed biodistribution was performed in healthy rats (n = 30) at different time points after 64Cu-FBP8 administration to estimate human radiation dosimetry. Longitudinal whole-body PET/MR imaging (n = 2) was performed after 64Cu-FBP8 administration to further assess radioactivity clearance. Results: 64Cu-FBP8 PET imaging detected the location of lung emboli and venous thrombi after DVT-PE, revealing significant differences in uptake between target and background tissues (P < 0.001). In vivo SPECT imaging and ex vivo γ counting confirmed the location of the lung emboli. PET quantification of the venous thrombi revealed that probe uptake was greater in younger clots than in older ones, a result confirmed by ex vivo analyses (P < 0.001). Histopathology revealed an age-dependent reduction of thrombus fibrin content (P = 0.006), further supporting the imaging findings. Biodistribution and whole-body PET/MR imaging showed a rapid, primarily renal, body clearance of 64Cu-FBP8. The effective dose was 0.021 mSv/MBq for males and 0.027 mSv/MBq for females, supporting the feasibility of using 64Cu-FBP8 in human trials. Conclusion: We showed that 64Cu-FBP8 PET is a feasible approach to image DVT-PE and that radiogenic adverse health effects should not limit the clinical translation of 64Cu-FBP8.