Nancy Janzen

Catching Bubbles: Targeting Ultrasound Microbubbles Using Bioorthogonal Inverse-Electron-Demand Diels–Alder Reactions†

Ultrasound imaging remains one of the most extensively used medical imaging methods because of its high spatial and temporal sensitivity, low cost, and portability and accessibility of equipment. Contrast-enhanced ultrasound using gas-filled microbubbles (MBs) has further enhanced the utility of ultrasound and created the opportunity to employ biomolecule-targeted derivatives for molecular imaging applications. We describe here a new approach to ultrasound molecular imaging that employs the covalent and highly selective capture of functionalized MBs in vitro and in vivo through bioorthogonal inverse-electron-demand Diels–Alder reactions. While pretargeting methods for nanometer-sized materials, such as nanoparticles and liposomes, have been published recently, the work reported herein is, to our knowledge, the first example of the bioorthogonal capture of micron-sized materials and the employment of pretargeting strategies for ultrasound molecular imaging. Ultrasound contrast agents are generally comprised of an inert gas, such as a perfluorocarbon, surrounded by a lipid, synthetic polymer, or protein shell. The traditional approach to targeting MBs, which are typically 1–8 mm in diameter and therefore restricted to intravascular targets, has been to link biomolecules with a high affinity for a specific protein to the outer shell through covalent bonds (e.g., amide bonds) or strong noncovalent interactions such as biotin–streptavidin binding. These approaches, which have largely exploited antibody and peptide vectors, have demonstrated the ability to selectively localize MBs to sites of angiogenesis, inflammation, and intravascular thrombus formation. Rather than using targeting vectors to localize conjugated prosthetic groups, new strategies for creating molecular imaging probes are being exploited that employ pretargeting and bioorthogonal coupling chemistry. Here, a targeting vector is administered first, allowing time for localization and clearance from nontarget organs, followed by a fluorescent or radiolabeled coupling partner that provides a readout for the molecular signal. The inverse-electron-demandDiels–Alder reaction between tetrazines and trans-cyclooctene (TCO) is an example of a highly selective and rapid bioorthogonal coupling reaction that has been used successfully to prepare targeted nuclear and optical molecular imaging probes. A comparable strategy for localizing MBs has not been reported. Such a method could offer a way to overcome obstacles to targeting ultrasound contrast agents whose large size and ability to bind only intravascular targets where blood flow rates and shear stress are high, make it particularly challenging to achieve and maintain good contrast in a timeframe that aligns with the limited in vivo stability of MBs. To test the feasibility of capturing micron-sized bubbles, a novel tetrazine-tagged MB (MBTz) was developed, and its reactivity towards cells treated with a TCO-conjugated antivascular endothelial growth factor receptor 2 (VEGFR2) antibody evaluated (Figure 1). VEGFR2 is overexpressed on tumor cells and upon activation triggers multiple signaling pathways that contribute to angiogenesis. The choice of target also allows the use of anti-VEGFR2-tagged MBs (MBV), which were developed by Rychak, Foster, and coworkers for evaluation in preclinical models, to validate the tetrazine–TCO capture methodology. Tetrazine-functionalized bubbles were prepared using commercially available streptavidin-coated MBs (micromarker target-ready contrast agents, VisualSonics) and a biotinylated tetrazine. The biotin–tetrazine derivative 5 was synthesized from biotin in four high-yielding steps (Scheme 1). The desired product was ultimately obtained by coupling commercially available 4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride with 6-biotinamidohexanoic tetrafluorophenyl (TFP) ester (4) at room temperature. After semipreparative HPLC, compound 5 was isolated in 75% yield and the product was stable in the freezer for more than six months. The TCO-conjugated antibody (TCO–antiVEGFR2) was prepared by combining an excess (20 equiv) of commercially available (E)-cyclooct-4-enyl-2,5-dioxopyrrolidin-1-yl carbonate (TCO–NHS) with antiVEGFR2 (eBioscience) at 4 8C overnight at pH 9.0–9.5. After purification using a 30 kDa centrifugal filter (Amicon Ultra-0.5) MALDITOF MS showed an average of 2.8 TCO groups per antibody in the product. The derivatized bubbles MBTz and MBV were prepared by adding 5 or biotinylated antiVEGFR2, respectively, to freshly reconstituted streptavidin-coated MBs. Isolation of the bubbles from the biotin-containing reagents was accomplished by treating the solution with streptavidin-coated magnetic beads (New England Biolabs), which bound residual tetrazine and [*] A. Zlitni, N. Janzen, Dr. J. F. Valliant Department of Chemistry and Chemical Biology, McMaster University 1280 Main St W., Hamilton, Ont., L8S 4M1 (Canada) E-mail: valliant@mcmaster.ca

Synthesis, Characterisation, and Biodistribution of Radioiodinated C‐Hydroxy‐Carboranes

The synthesis, radiolabelling and biodistribution of iodinated C-hydroxy-nido-carborane ligands is described. Microwave heating by using NaF in aqueous ethanol was used to prepare {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate], nido-carboranol} and {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate-8-carboxylic acid], nido-salborin} in 97 and 90 % yield, respectively. Radioiodination of these nido-carboranes was completed by using both (125)I and (123)I, and the products were obtained in high radiochemical purity (>99 %) and yield (72 to 87 %). The structures of the radiolabelled products were validated through comparison to authentic standards. Biodistribution studies in BALB/c mice showed low accumulation of the labelled compounds in the liver and intestines, which are sites where labelled carboranes typically localise. The labelled cluster bearing hydroxy and carboxylic acid groups on the two carbon vertices demonstrated preferential clearance through the kidneys and low thyroid uptake. This compound had substantially reduced non-specific binding than the deshydroxy analogue making it an attractive bifunctional ligand for preparing targeted molecular imaging and therapy agents.

Triazole Appending Agent (TAAG): A New Synthon for Preparing Iodine-Based Molecular Imaging and Radiotherapy Agents.

A new prosthetic group referred to as the triazole appending agent (TAAG) was developed as a means to prepare targeted radioiodine-based molecular imaging and therapy agents. Tributyltin-TAAG and the fluorous analogue were synthesized in high yield using simple click chemistry and the products labeled in greater than 95% RCY with (123)I. A TAAG derivative of an inhibitor of prostate-specific membrane antigen was prepared and radiolabeled with (123)I in 85% yield where biodistribution studies in LNCap prostate cancer tumor models showed rapid clearance of the agent from nontarget tissues and tumor accumulation of 20% injected dose g(-1) at 1 h. The results presented demonstrate that the TAAG group promotes minimal nonspecific binding and that labeled conjugates can achieve high tumor uptake and exquisite target-to-nontarget ratios.

Isostructural Nuclear and Luminescent Probes Derived From Stabilized [2 + 1] Rhenium(I)/Technetium(I) Organometallic Complexes

A convenient method to prepare (99m)Tc analogues of a class of rhenium(I) luminophores was developed, creating isostructural pairs of nuclear and optical probes. A two-step procedure and a new one-pot procedure were used to produce a series of [2 + 1] complexes of the type [Tc(CO)3(bipy)L](+) in greater than 80% yield. The plasma stability of the reported compounds was evaluated, where the basicity of the monodentate pyridine type ligand (L) has a significant impact with half-lives ranging from 2 to 20 h. The ability to generate the radioactive complexes makes it possible to quantitate cell uptake of Re luminophores, which was demonstrated in MCF-7 breast cancer cells using (99m)Tc analogues of two Re(I)-based mitochondrial targeting dyes.

(125)I-Tetrazines and Inverse-Electron-Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies.

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels-Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with trans-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics.

Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated High-Generation Polyester Dendrimers

A fifth generation aliphatic polyester dendrimer was functionalized with vinyl groups at the periphery and a dipicolylamine Tc(I) chelate at the core. This structure was PEGylated with three different molecular weight mPEGs (mPEG160, mPEG350, and mPEG750) using thiol-ene click chemistry. The size of the resulting macromolecules was evaluated using dynamic light scattering, and it was found that the dendrimer functionalized with mPEG750 was molecularly dispersed in water, exhibiting a hydrodynamic diameter of 9.2 ± 2.1 nm. This PEGylated dendrimer was subsequently radiolabeled using [(99m)Tc(CO)3(H2O)3](+) and purified to high (>99%) radiochemical purity. Imaging studies were initially performed on healthy rats to allow comparison to previous Tc-labeled dendrimers and then on xenograft murine tumor models, which collectively showed that the dendrimers circulated in the blood for an extended period of time (up to 24 h). Furthermore, the radiolabeled dendrimer accumulated in H520 xenograft tumors, which could be visualized by single-photon emission computed tomography (SPECT). The reported PEGylated aliphatic polyester dendrimers represent a new platform for developing tumor-targeted molecular imaging probes and therapeutics.

Technetium(I) Complexes of Bathophenanthrolinedisulfonic Acid

Bathophenanthrolinedisulfonate (BPS) complexes of technetium(I) of the type [Tc(CO)3(BPS)(L)]n (L = imidazole derivatives) were synthesized and evaluated both in vitro and in vivo. [99mTc(CO)3(BPS)(MeIm)]- (MeIm = 1-methyl-1H-imidazole) was prepared in near-quantitative yield using a convenient two-step, one-pot labeling procedure. A targeted analogue capable of binding regions of calcium turnover associated with bone metabolism was also prepared. Here, a bisphosphonate was linked to the metal through an imidazole ligand to give [99mTc(CO)3(BPS)(ImAln)]2- (ImAln = an imidazole-alendronate ligand) in high yield. The technetium(I) complexes were stable in vitro, and in biodistribution studies, [99mTc(CO)3(BPS)(ImAln)]2- exhibited rapid clearance from nontarget tissues and significant accumulation in the shoulder (7.9 ± 0.2% ID/g) and knees (15.1 ± 0.9% ID/g) by 6 h, with the residence time in the skeleton reaching 24 h. A rhenium analogue, which is luminescent and has the same structure, was also prepared and used for fluorescence labeling of cells in vitro. The data reported demonstrate the potential of this class of compounds for use in creating isostructural optical and nuclear probes.

A 99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule trans-Cyclooctene Derivatives.

A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with 99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The 99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability in vitro and in vivo. A trans-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with 99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with 99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control 99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting Staphylococcus aureus infection in vitro and in vivo. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the 99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.

Preparation and Evaluation of Radiolabeled Antibody Recruiting Small Molecules That Target Prostate-Specific Membrane Antigen for Combined Radiotherapy and Immunotherapy

The feasibility of developing a single agent that can deliver radioactive iodine and also direct cellular immune function by engaging endogenous antibodies as an antibody-recruiting small molecule (ARM) was determined. A library of new prostate-specific membrane antigen (PSMA)-binding ligands that contained antibody-recruiting 2,4-dinitrophenyl (DNP) groups and iodine were synthesized and screened in vitro and in vivo. A lead compound (9b) showed high affinity for PSMA and the ability to bind anti-DNP antibodies. Biodistribution studies of the iodine-125 analogue showed 3% ID/g in LNCaP xenograft tumors at 1 h postinjection with tumor-to-blood and tumor-to-muscle ratios of 10:1 and 44:1, respectively. The radiolabeled analogue was bound and internalized by LNCaP cells, with both functions blocked using a known PSMA inhibitor. A second candidate showed high tumor uptake (>10% ID/g) but had minimal binding to anti-DNP antibodies. The compounds reported represent the first examples of small molecules developed specifically for combination immunotherapy and radiotherapy for prostate cancer.

Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.