Fully automated radiolabeling of [68Ga]Ga-EMP100 targeting c-MET for PET-CT clinical imaging.

BACKGROUND: c-MET is a transmembrane receptor involved in many biological processes and contributes to cell proliferation and migration during cancer invasion process. Its expression is measured by immunehistochemistry on tissue biopsy in clinic, although this technique has its limitations. PET-CT could allow in vivo mapping of lesions expressing c-MET, providing whole-body detection. A number of radiopharmaceuticals are under development for this purpose but are not yet in routine clinical use. EMP100 is a cyclic oligopeptide bound to a DOTA chelator, with nanomolar affinity for c-MET. The aim of this project was to develop an automated method for radiolabelling the radiopharmaceutical [68Ga]Ga-EMP100. RESULTS: The main results showed an optimal pH range between 3.25 and 3.75 for the complexation reaction and a stabilisation of the temperature at 90 °C, resulting in an almost complete incorporation of gallium-68 after 10 min of heating. In these experiments, 90 µg of EMP-100 peptide were initially used and then lower amounts (30, 50, 75 µg) were explored to determine the minimum required for sufficient synthesis yield. Radiolysis impurities were identified by radio-HPLC and ascorbic acid and ethanol were used to improve the purity of the compound. Three batches of [68Ga]Ga-EMP100 were then prepared according to the optimised parameters and all met the established specifications. Finally, the stability of [68Ga]Ga-EMP100 was assessed at room temperature over 3 h with satisfactory results in terms of appearance, pH, radiochemical purity and sterility. CONCLUSIONS: For the automated synthesis of [68Ga]Ga-EMP100, the parameters of pH, temperature, precursor peptide content and the use of adjuvants for impurity management were efficiently optimised, resulting in the production of three compliant and stable batches according to the principles of good manufacturing practice. [68Ga]Ga-EMP100 was successfully synthesised and is now available for clinical development in PET-CT imaging.

A novel fluorescent c-met targeted imaging agent for intra-operative colonic tumour mapping: Translation from the laboratory into a clinical trial.

BACKGROUND: The c-Met protein is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. METHODS: HT-29 SiRNA transfection confirmed specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in BALB/c mice, EMI-137 was injected and biodistribution analysed through in vivo fluorescent imaging. Nine patients, received a single intravenous EMI-137 bolus (0.13 mg/kg), 1-3 h before laparoscopic-assisted colon cancer surgery (NCT03360461). Tumour and LN fluorescence was assessed intraoperatively and correlated with c-Met expression in eight samples by immunohistochemistry. FINDINGS: c-Met expression HT-29 cells was silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed up to 6 h post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all participants, 4/9 showed increased tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited high c-Met protein expression. INTERPRETATION: EMI-137, binds specifically to the human c-Met protein, is safe, and with further refinement, shows potential for application in fluorescence-guided surgery.

Preclinical dosimetry models and the prediction of clinical doses of novel positron emission tomography radiotracers.

Dosimetry models using preclinical positron emission tomography (PET) data are commonly employed to predict the clinical radiological safety of novel radiotracers. However, unbiased clinical safety profiling remains difficult during the translational exercise from preclinical research to first-in-human studies for novel PET radiotracers. In this study, we assessed PET dosimetry data of six 18F-labelled radiotracers using preclinical dosimetry models, different reconstruction methods and quantified the biases of these predictions relative to measured clinical doses to ease translation of new PET radiotracers to first-in-human studies. Whole-body PET images were taken from rats over 240 min after intravenous radiotracer bolus injection. Four existing and two novel PET radiotracers were investigated: [18F]FDG, [18F]AlF-NOTA-RGDfK, [18F]AlF-NOTA-octreotide ([18F]AlF-NOTA-OC), [18F]AlF-NOTA-NOC, [18F]ENC2015 and [18F]ENC2018. Filtered-back projection (FBP) and iterative methods were used for reconstruction of PET data. Predicted and true clinical absorbed doses for [18F]FDG and [18F]AlF-NOTA-OC were then used to quantify bias of preclinical model predictions versus clinical measurements. Our results show that most dosimetry models were biased in their predicted clinical dosimetry compared to empirical values. Therefore, normalization of rat:human organ sizes and correction for reconstruction method biases are required to achieve higher precision of dosimetry estimates.

Non-invasive in vivo imaging of acute thrombosis: development of a novel factor XIIIa radiotracer.

AIMS: Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation. METHODS AND RESULTS: Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P < 0.0001). CONCLUSION: ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

Design of Bimodal Ligands of Neurotensin Receptor 1 for Positron Emission Tomography Imaging and Fluorescence-Guided Surgery of Pancreatic Cancer.

Neurotensin receptor 1 (NTSR1) is overexpressed in most human pancreatic ductal adenocarcinomas. It makes it an attractive target for the development of pancreatic cancer imaging agents. In this study, we sought to develop a bimodal positron emission tomography (PET)/fluorescent imaging agent capable of specifically targeting these receptors. Starting from the structure of a known NTSR1 agonist, a series of tracers were synthesized, radiometalated with gallium-68, and evaluated in vitro and in vivo, in mice bearing an AsPC-1 xenograft. PET imaging allowed us to identify the compound [68Ga]Ga-NODAGA-Lys(Cy5**)-AEEAc-[Me-Arg8,Tle12]-NT(7-13) as the one with the most promising biodistribution profile, characterized by high tumor uptake (2.56 ± 0.97%ID/g, 1 h post-injection) and rapid elimination from nontargeted organs, through urinary excretion. Fluorescence imaging gave similar results. On this basis, fluorescence-guided resection of tumor masses was successfully carried out on a preclinical model.

Facile Synthesis of a Next Generation Safety-Catch Acid-Labile Linker, SCAL-2, Suitable for Solid-Phase Synthesis, On-Support Display and for Post-Synthesis Tagging.

The SCAL linker, a safety catch linker, is amongst the most versatile linkers for solid phase synthesis. It was originally described in 1991 by Pátek and Lebl. Yet, its application has been hindered by the low yields of published synthetic routes. Over time, the exceptional versatility of this linker has been demonstrated in several applications of advanced solid phase synthesis of peptides and peptidomimetics. Recently, an updated synthesis of the original linker has also been presented at the 22nd American Peptide Symposium, comprising 10 steps. Herein, the design and synthesis of a next generation SCAL linker, SCAL-2, is reported. SCAL-2 features a simplified molecular architecture, which allows for a more efficient synthesis in 8 steps with superior yields. Both linkers, SCAL and SCAL-2 are compared in terms of their cleavage properties adding valuable information on how to best utilize the versatility of these linkers for solid phase synthesis.

Phenylephrine postconditioning increases myocardial injury: are alpha-1 sympathomimetic agonist cardioprotective?

OBJECTIVE: We studied effects of phenylephrine (PHE) on postischemic functional recovery and myocardial injury in an ischemia-reperfusion (I-R) experimental model. MATERIALS AND METHODS: Rat hearts were Langendorff-perfused and subjected to 30 min zero-flow ischemia (I) and 60 min reperfusion (R). During R PHE was added at doses of 1 µM (n = 10) and 50 µM (n = 12). Hearts (n = 14) subjected to 30 and 60 min of I-R served as controls. Contractile function was assessed by left ventricular developed pressure (LVDP) and the rate of increase and decrease of LVDP; apoptosis by fluorescent imaging targeting activated caspase-3, while myocardial injury by lactate dehydrogenase (LDH) released during R. Activation of kinases was measured at 5, 15, and 60 min of R using western blotting. RESULTS: PHE did not improve postischemic contractile function. PHE increased LDH release (IU/g); 102 ± 10.4 (Mean ± standard error of mean) control versus 148 ± 14.8 PHE (1), and 145.3 ± 11 PHE (50) hearts, (P < 0.05). PHE markedly increased apoptosis. Molecular analysis showed no effect of PHE on the activation of proapoptotic c-Jun N-terminal kinase signaling; a differential pattern of p38 mitogen activated protein kinase (MAPK) activation was found depending on the PHE dose used. With 1 µM PHE, p-p38/total-p38 MAPK levels at R were markedly increased, indicating its detrimental effect. With PHE 50 µM, no further changes in p38 MAPK were seen. Activation of Akt kinase was decreased implying involvement of different mechanisms in this response. CONCLUSIONS: PHE administration during reperfusion does not improve postischemic recovery due to exacerbation of myocardial necrosis and apoptosis. This finding may be of clinical and therapeutic relevance.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Biochemical characterization of a Caspase-3 far-red fluorescent probe for non-invasive optical imaging of neuronal apoptosis.

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

In vitro and ex vivo evaluation of smart infra-red fluorescent caspase-3 probes for molecular imaging of cardiovascular apoptosis.

Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo.

Postsynthetic derivatization of fluorophores with alpha-sulfo-beta-alanine dipeptide linker. Application to the preparation of water-soluble cyanine and rhodamine dyes.

A straightforward method to enhance water solubility of fluorescent organic dyes (cyanines and rhodamines) by a postsynthetic chemical derivatization of their carboxylic acid functionality with an original disulfonated heterobifunctional linker is described. Significant water solubility enhancement is achieved without compromising either the photophysical properties (especially large molar extinction coefficients and high quantum yields) or bioconjugation efficiency of the parent non-water-soluble fluorophores. The results both demonstrate the strong potential of these new compounds as fluorescent labels for a broad range of biotechnology and biomedical applications and illustrate the utility of such an easy-to-handle water-solubizing group.

Approaches to high throughput physical organic chemistry.

High throughput (HT) techniques are now extensively used for the synthesis of libraries of several thousands of compounds. More recently, HT methods began to be applied to other areas, such as physical organic chemistry. This has allowed for instance the development of tools for HT reaction assessment, HT kinetic and thermodynamic measurements, and physicochemical property profiling, using a broad set of analytical tools, ranging from mass spectrometry to image analysis based techniques. This article provides an overview of recent HT physical organic chemistry techniques. Special attention is given to the application of quantitative analytical constructs for HT monomer reactivity profiling and HT evaluation of Hammett parameters.

High-throughput physical organic chemistry--Hammett parameter evaluation.

High-throughput analysis techniques were developed to allow the rapid assessment of a range of Hammett parameters utilizing positive electrospray mass spectrometry (ESI+ -MS) as the sole quantitative tool, with the core of the approach being a so-called "analytical construct". Hammett substituent parameters were determined for a range of meta- and para-substituted anilines by high-throughput (HT) assessment of relative reaction rates for competitive amide bond formation reaction with up to five parameters determined in a single pot reaction. Sensitivity of the reaction to substituents' effects (materialized by Hammett's rho parameter) was determined in the first instance, with HT Hammett's sigma substituent parameter assessment then carried out successfully for over 30 anilines, with excellent correlation observed between the HT ESI+ -MS method of determination and literature values.

High throughput physical organic chemistry: analytical constructs for monomer reactivity profiling.

A polymer-supported analytical construct was used to quantify the reactivity of a range of monomers in the Ugi four-component condensation using positive electrospray ionization mass spectrometry (MS) as a quantitative analytical tool. The construct incorporated a bromo group to act as a peak splitter and a quaternary ammonium to act as a MS sensitizer and ionization leveler, thereby allowing direct quantitation of the cleaved adducts by MS. The relative reactivities of 10 carboxylic acids were quantified by the relative levels of product generated as determined by MS and 10 isonitriles, and 10 aldehydes were investigated in the same way. The effect of concentration variations on monomers reactivity and product profiles were rapidly determined using this approach, and the method opens up the way for studying, in a single pot, multiple reactions with a broad range of monomers under identical and self-consistent reaction conditions.