18f-labeling-radiotracers-functionalized-with-silicon-fluoride

<sup>18</sup>F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor (SiFA) for Positron Emission Tomography

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the ...

University of Alberta

Our research on pathogenesis of disseminated candidiasis led to the discovery that antibodies specific for Candida albicans cell surface β-1, 2-mannotriose [β-(Man)(3)] protect mice. A 14 mer peptide Fba, which derived from the N-terminal portion of the C. albicans cytosolic/cell surface protein fructose-bisphosphate aldolase, was used as the glycan carrier and resulted in a novel synthetic glycopeptide vaccine β-(Man)(3)-Fba. By a dendritic cell-based immunization approach, this conjugate induced protective antibody responses against both the glycan and peptide parts of the vaccine. In this report, we modified the β-(Man)(3)-Fba conjugate by coupling it to tetanus toxoid (TT) in order to improve immunogenicity and allow for use of an adjuvant suitable for human use. By new immunization procedures entirely compatible with human use, the modified β-(Man)(3)-Fba-TT was administered either alone or as a mixture made with alum or monophosphoryl lipid A (MPL) adjuvants and given to mice by a subcutaneous (s.c.) route. Mice vaccinated with or, surprisingly, without adjuvant responded well by making robust antibody responses. The immunized groups showed a high degree of protection against a lethal challenge with C. albicans as evidenced by increased survival times and reduced kidney fungal burden as compared to control groups that received only adjuvant or DPBS buffer prior to challenge. To confirm that induced antibodies were protective, sera from mice immunized against the β-(Man)(3)-Fba-TT conjugate transferred protection against disseminated candidiasis to naïve mice, whereas C. albicans-absorbed immune sera did not. Similar antibody responses and protection induced by the β-(Man)(3)-Fba-TT vaccine was observed in inbred BALB/c and outbred Swiss Webster mice. We conclude that addition of TT to the glycopeptide conjugate results in a self-adjuvanting vaccine that promotes robust antibody responses without the need for additional adjuvant, which is novel and represents a major step forward in vaccine design against disseminated candidiasis.

Self-adjuvanting glycopeptide conjugate vaccine against disseminated candidiasis.

The oligosaccharides Man4 and Man5, substructures of the high-mannose glycans of HIV glycoprotein gp120, were synthesized with a terminal 1-thiomannopyranose residue. The anomeric thiol can be readily converted to an azidomethyl aglycone through reaction with dichloromethane and displacement with sodium azide. The resulting oligomannans were then conjugated to ubiquitin utilizing thiol alkylation or azide/alkyne reactive tethers of minimal length. By combining high efficiency conjugation reactions and a short tether, we sought to establish conjugation conditions that would permit high density clustering of oligomannans in conjugate vaccines that could produce antibodies able to bind gp120 and potentially neutralize virus. LC-UV-MS was used to separate, identify and quantify the ubiquitin glycoconjugates with differing degrees of oligomannan incorporation. Binding of the HIV protective monoclonal antibody 2G12 and concanavalin A to microtitre plates coated with glycoconjugates was measured by ELISA. 

Synthesis of high-mannose 1-thio glycans and their conjugation to protein.

Unorthodox (18)F-labeling strategies not employing the formation of a carbon-(18)F bond are seldom found in radiochemistry. Historically, the formation of a boron- or silicon-(18)F bond has been introduced very early on into the repertoire of labeling chemistries, but is without translation into any clinical radiotracer besides inorganic B[(18)F]F4(-) for brain tumor diagnosis. For many decades these labeling methodologies were forgotten and have just recently been revived by a handful of researchers thinking outside the box. When breaking with established paradigms such as the inability to obtain labeled compounds of high specific activity via isotopic exchange or performing radiofluorination in aqueous media, the research community often reacts skeptically. In 2005 and 2006, two novel labeling methodologies were introduced into radiochemistry for positron emission tomography (PET) tracer development: RBF3(-) labeling reported by Perrin et al. and the SiFA methodology by Schirrmacher, Jurkschat, and Waengler et al. which is based on isotopic exchange (IE). Both labeling methodologies have been complemented by other noncanonical strategies to introduce (18)F into biomolecules of diagnostic importance, thus profoundly enriching the landscape of (18)F radiolabeling. B- and Si-based labeling strategies finally revealed that IE is a viable alternative to established and traditional radiochemistry with the advantage of simplifying both the labeling effort as well as the necessary purification of the radiotracer. Hence IE will be the focus of this contribution over other noncanonical labeling methods. Peptides for tumor imaging especially lend themselves favorably toward one-step labeling via IE, but small molecules have been described as well, taking advantage of these new approaches, and have been used successfully for brain imaging. This Review gives an account of both radiochemistries centered on boron and silicon, describing the very beginnings of their basic research, the path that led to optimization of their chemistries, and the first encouraging preclinical results paving the way to their clinical use. This side by side approach will give the reader the opportunity to follow the development of a new basic discovery into a clinically applicable radiotracer including all the hurdles that have had to be overcome. 

From Unorthodox to Established: The Current Status of (18)F-Trifluoroborate- and (18)F-SiFA-Based Radiopharmaceuticals in PET Nuclear Imaging.

Over the last 20 years, intensive investigation and multiple clinical successes targeting protein kinases, mostly for cancer treatment, have identified small molecule kinase inhibitors as a prominent therapeutic class. In the course of those investigations, radiolabeled kinase inhibitors for positron emission tomography (PET) imaging have been synthesized and evaluated as diagnostic imaging probes for cancer characterization. Given that inhibitor coverage of the kinome is continuously expanding, in vivo PET imaging will likely find increasing applications for therapy monitoring and receptor density studies both in- and outside of oncological conditions. Early investigated radiolabeled inhibitors, which are mostly based on clinically approved tyrosine kinase inhibitor (TKI) isotopologues, have now entered clinical trials. Novel radioligands for cancer and PET neuroimaging originating from novel but relevant target kinases are currently being explored in preclinical studies. This article reviews the literature involving radiotracer design, radiochemistry approaches, biological tracer evaluation and nuclear imaging results of radiolabeled kinase inhibitors for PET reported between 2010 and mid-2015. Aspects regarding the usefulness of pursuing selective vs. promiscuous inhibitor scaffolds and the inherent challenges associated with intracellular enzyme imaging will be discussed. 

Recent Advances in the Development and Application of Radiolabeled Kinase Inhibitors for PET Imaging.

Tropomyosin receptor kinases (TrkA/B/C) play crucial roles in the development and maintenance of the nervous system, and aberrant expression of Trk has been implicated in neurological disorders as well as neural and non-neural neoplasms. Patent activity encompassing Trk inhibitors has grown substantially over the last 6 years, recognized by a rise in the number of pharmaceutical entrants to the field and the escalation of novel inhibitor chemotypes. Area covered: In Part I of this two part review, a biological and structural overview of Trk is provided in the context of Trk as a therapeutic target for cancer and pain, followed by the report of recent patent literature claiming small molecule inhibitors of Trk family kinases or which describe inhibitors developed for other kinase targets but include noteworthy Trk inhibition/application. The discussion of the patent literature continues in Part II of this review, which includes an in-depth view of the current clinical applications of Trk inhibitors. Expert opinion: Substantial synthetic efforts in Trk inhibitor development has propagated numerous and diverse inhibitor chemotypes, including TrkA-specific inhibitors. While many novel Trk inhibitors remain the original progeny of Trk-specific development programs, kinase inhibitors initially developed for other kinases have also been successfully repositioned for Trk.

Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 - Part I.

TrkA/B/C receptor activation supports growth, survival, and differentiation of discrete neuronal populations during development, adult life, and ageing but also plays numerous roles in human disease onset and progression. Trk-specific inhibitors have therapeutic applications in cancer and pain and thus constitute a growing area of interest in oncology and neurology. There has been substantial growth in the number of structural classes of Trk inhibitors and the number of industrial entrants to the Trk inhibitor field over the past six years. Areas covered: In Part II of this two-part review, the discussion of recent patent literature covering Trk family inhibitors is continued from Part I and clinical research with Trk inhibitors is considered. Expert opinion: Trk has been molecularly targeted for over a decade resulting in the progressive evolution of structurally diversified Trk inhibitors arising from scaffold hopping and HTS efforts. Correspondingly, there have been a growing number of clinical investigations utilizing Trk inhibitors in recent years, with a particular focus on the treatment of NTRK-fusion positive cancers and chronic pain. The observed potential of Trk inhibitors to cause adverse CNS side effects however suggests the need for a more rigorous consideration of BBB permeation capabilities during drug development.

Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 - Part II.

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in the majority of prostate cancers. The favorable positron emission tomography (PET) imaging profile of the PSMA imaging agent 2-(3-(1-carboxy-5-[(6-[F]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentane-dioic acid [F]DCFPyL in preclinical prostate cancer models and in prostate cancer patients stimulated the development and validation of other fluorine-containing PSMA inhibitors to further enhance pharmacokinetics and simplify production methods. Here, we describe the synthesis and radiopharmacological evaluation of various F-18-labeled PSMA inhibitors which were prepared through different prosthetic group chemistry strategies.

Targeting Prostate-Specific Membrane Antigen (PSMA) with F-18-Labeled Compounds: the Influence of Prosthetic Groups on Tumor Uptake and Clearance Profile.

The proto-oncogenes NTRK1/2/3 encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [C]-(R)-3 ([C]-(R)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (R)-3 is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [C]-(R)-3 readily crosses the blood-brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer's disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [C]-(R)-3 in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human.

A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human.

In this paper, we describe the use of Cu-mediated [ F]fluorodeboronation for the automated production of positron emission tomography radiotracers suitable for clinical use. Two recurrent issues with the method, low radiochemical conversion on automation and protoarene byproduct purification issues, have been successfully addressed. The new method was utilized to produce sterile injectable doses of [ F]-(±)-IPMICF17, a positron emission tomography radiotracer for tropomyosin receptor kinase B/C, using an automated synthesis module. The product was isolated in 1.9 ± 0.1% isolated radiochemical yield, excellent radiochemical purity (>99%), and high specific activity (5294 ± 1227 Ci/mmol). Quality control testing confirmed that doses were suitable for clinical use.

Automated synthesis of PET radiotracers by copper-mediated  F-fluorination of organoborons: Importance of the order of addition and competing protodeborylation.

Nanoparticles represent the most widely studied drug delivery systems targeting cancer. Polymeric nanoparticles can be easily generated through a microemulsion polymerization. Herein, the synthesis, radiolabeling, and in vivo evaluation of nanoparticles (NPs) functionalized by an organosilicon fluoride acceptor (SiFA) are reported which can be radiolabeled without further chemical modifications. Four nanoparticles in the sub-100 nm range with distinct hydrodynamic diameters of 20 nm (NP1), 33 nm (NP2), 45 nm (NP3), and 72 nm (NP4), respectively, were synthesized under size-controlled conditions. The SiFA-labeling building block acted as an initiator for the polymerization of polymer P1. The nanoparticles were radiolabeled with fluorine-18 (F) through simple isotopic exchange (IE) and analyzed in vivo in a murine mammary tumor model (EMT6). The facile F radiolabeling SiFA methodology, performed in ethanol under mild reaction conditions, gave radiochemical yields (RCYs) of 19-26% and specific activities (SA) of 0.2-0.3 GBq/mg. Based on preclinical PET analysis, the tumor uptake and clearance profiles were analyzed depending on particle size. The nanoparticle size of 33 nm showed the highest tumor accumulation of SUV 0.97 (= 4.4%ID/g) after 4 h p.i. through passive diffusion based on the Enhanced Permeability and Retention (EPR) effect. Overall, this approach exhibits a simple, robust, and reliable synthesis of F radiolabeled polymeric nanoparticles with a favorable in vivo evaluation profile. This approach represents a straightforward synthetically accessible alternative to produce radiolabeled nanoparticles without any further surface modification to attach a radioisotope.

F-Radiolabeling and In Vivo Analysis of SiFA-Derivatized Polymeric Core-Shell Nanoparticles.

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first F-labeled optimized lead suitable for in vivo imaging of Trk, [F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity.

Identification of [F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging.

Straightforward radiosynthesis protocols for F-labeled radiopharmaceuticals are an indispensable but often overlooked prerequisite to successfully perform molecular imaging studies in vivo by PET. In recent years, thanks to the expansion of the F chemical toolbox, structurally diverse and novel clinically relevant radiopharmaceuticals have been synthesized with both high efficiency and ready implementation. This article provides an overview of recent F-labeling methodologies, specifically for B-F, Si-F, Al-F, and iodine (III)-mediated radiofluorination via the spirocyclic iodonium ylide technology.

Recent Advances in F Radiochemistry: A Focus on B-F, Si-F, Al-F, and C-F Radiofluorination via Spirocyclic Iodonium Ylides.

The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer's disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted.

Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging.

The tropomyosin receptor kinase TrkA/B/C family is responsible for human neuronal growth, survival, and differentiation from early nervous system development stages onward. Downregulation of TrkA/B/C receptors characterizes numerous neurological disorders including Alzheimer's disease (AD). Abnormally expressed Trk receptors or chimeric Trk fusion proteins are also well-characterized oncogenic drivers in a variety of neurogenic and non-neurogenic human neoplasms and are currently the focus of intensive clinical research. Previously, we have described the clinical translation of a highly selective and potent carbon-11-labeled pan-Trk radioligand and the preclinical characterization of the optimized fluorine-18-labeled analogue, [F]TRACK, for in vivo Trk positron emission tomography (PET) imaging. We describe herein central nervous system selectivity assessment and first-in-human study of [F]TRACK.

Article	Year
Self-adjuvanting glycopeptide conjugate vaccine against disseminated candidiasis. PloS one\| PubMed ID: 22563378	2012
Synthesis of high-mannose 1-thio glycans and their conjugation to protein. Organic & biomolecular chemistry\| PubMed ID: 24549150	2014
From Unorthodox to Established: The Current Status of (18)F-Trifluoroborate- and (18)F-SiFA-Based Radiopharmaceuticals in PET Nuclear Imaging. Bioconjugate chemistry\| PubMed ID: 26566577	2016
Recent Advances in the Development and Application of Radiolabeled Kinase Inhibitors for PET Imaging. Molecules (Basel, Switzerland)\| PubMed ID: 26690113	2015
Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 - Part I. Expert opinion on therapeutic patents\| PubMed ID: 28270010	2017
Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 - Part II. Expert opinion on therapeutic patents\| PubMed ID: 28270021	2017
Targeting Prostate-Specific Membrane Antigen (PSMA) with F-18-Labeled Compounds: the Influence of Prosthetic Groups on Tumor Uptake and Clearance Profile. Molecular imaging and biology\| PubMed ID: 28639122	2017
A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human. Journal of medicinal chemistry\| PubMed ID: 28696690	2017
Automated synthesis of PET radiotracers by copper-mediated F-fluorination of organoborons: Importance of the order of addition and competing protodeborylation. Journal of labelled compounds & radiopharmaceuticals\| PubMed ID: 29143408	2018
F-Radiolabeling and In Vivo Analysis of SiFA-Derivatized Polymeric Core-Shell Nanoparticles. Bioconjugate chemistry\| PubMed ID: 29202229	2018
Identification of [F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging. Journal of medicinal chemistry\| PubMed ID: 29257860	2018
Recent Advances in F Radiochemistry: A Focus on B-F, Si-F, Al-F, and C-F Radiofluorination via Spirocyclic Iodonium Ylides. Journal of nuclear medicine : official publication, Society of Nuclear Medicine\| PubMed ID: 29284673	2018
Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 30609832	2019
First-in-Human Brain Imaging of [F]TRACK, a PET tracer for Tropomyosin Receptor Kinases. ACS chemical neuroscience\| PubMed ID: 31017386	2019

Justin J. Bailey

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