The development of nuclear receptor imaging agents

Positron emission tomography (PET) is a molecular imaging technique which allows visualisation and quantification of biomarkers by administering a positron-emitting molecular probe. The steroid hormone receptors for estrogen and progesterone are over-expressed in hormone-dependent cancers of the breast and ovary. Endocrine therapies targeting estrogen receptor (ER) such as Tamoxifen, a selective estrogen receptor modulator (SERM), are among the frontline treatments for these cancers. Currently patient stratification for ER therapy is carried out using immunohistochemical (IHC) assays from biopsy samples; however, this invasive technique is unsuitable for assessing metastatic lesions (multiple and difficult biopsy sampling required). IHC assays are also prone to errors arising from discordance in methodology for assessment of results and also the effect of tumour heterogeneity. Progesterone receptor (PR) expression is regulated by ER and can therefore serve as a surrogate treatment response biomarker for endocrine therapy as PR expression reports on modulation of the ER pathway even if the receptor is saturated with tamoxifen. The use of PET for quantitative interrogation of breast tumour response to endocrine therapy has been reported with steroidal PR ligand, [¹⁸F]FFNP using an in vivo breast cancer rodent model. Correlation between PR expression and tracer uptake has been evaluated in a clinical trial.A focused library of non-steroidal PR imaging agents has been synthesised, based on the benzoxazinthione core structure of tanaproget, a well-established non-steroidal PR agonist. Novel synthetic methodology for accessing thio-carbamate Tanaproget derivatives was developed to avoid the use of Lawesson’s reagent. This thesis reports the first synthesis of a 1,2,3-triazole containing PR ligand which exhibited nanomolar potency in T47D cells.This project aimed to develop surface plasmon resonance (SPR) methodology to assess ligand-receptor binding kinetics to aid lead candidate selection for radiolabelling. The development of an assay to assess ligand binding between progesterone (used as a known PR ligand) and captured PR-ligand binding domain proved to be unsuccessful even though the receptor was able to bind anti-PR monoclonal antibody (mAb). A reduction in temperature and introducing a chaotropic agent to denature the receptor were unsuccessful attempts at getting the receptor to bind to progesterone. The dependence of ligand binding on chaperone proteins like heat shock protein-90 (HSP90) was realised and a PR-HSP90 complex receptor was captured to try and facilitate ligand binding. These receptors proved to be unsuccessful at facilitating ligand binding. Compound libraries were evaluated for potency using the T47D alkaline phosphatase assay in live cells; lead candidates were selected using this data.Radiochemical methodology was developed to label lead candidates with fluorine-18. Two lead candidates were selected from the potency data of cold compounds in the breast cancer cell line T47D; these compounds were compound 26 (EC₅₀ = 4.7 nM) and 32 (EC₅₀ = 47.6 nM). Initial steps to access compound [¹⁸F]26 were developed by radiolabelling dibromopyridine precursors as a prosthetic group; however, this radiosynthesis was not completed. The radiosynthesis of [¹⁸F]32 was achieved by one-step methodology with a fluoride incorporation of 75 – 78 % in a 15 min reaction time. Work towards developing conditions to purify [¹⁸F]32 allowed some compound to be isolated and specific activity determined (0.027 GBq/μmol). Future work will involve improving the purification method to [¹⁸F]32 in anticipation of isolating compound with higher specific activity to evaluate the compound with in vitro cell uptake studies and in vivo biodistribution in an animal model

Development of PDT/PET theranostics: synthesis and biological evaluation of an ¹⁸F-radiolabeled water soluble porphyrin

Synthesis of the first water-soluble porphyrin radiolabeled with fluorine-18 is described: a new molecular theranostic agent which integrates the therapeutic selectivity of photodynamic therapy (PDT) with the imaging efficacy of positron emission tomography (PET). Generation of the theranostic was carried out through the conjugation of a cationic water-soluble porphyrin bearing an azide functionality to a fluorine-18 radiolabeled prosthetic bearing an alkyne functionality through click conjugation, with excellent yields obtained in both cold and hot synthesis. Biological evaluation of the synthesized structures shows the first example of an 18 F-radiolabeled porphyrin retaining photocytotoxicity following radiolabeling and demonstrable conjugate uptake and potential application as a radiotracer in vivo. The promising results gained from biological evaluation demonstrate the potential of this structure as a clinically relevant theranostic agent, offering exciting possibilities for the simultaneous imaging and photodynamic treatment of tumors

Radiolabelling an 18F biologic via facile IEDDA “click” chemistry on the GE FASTLab™ platform

The use of biologics in positron emission tomography (PET) imaging is an important area of radiopharmaceutical development and new automated methods are required to facilitate their production. We report an automated radiosynthesis method to produce a radiolabelled biologic via facile inverse electron demand Diels-Alder (IEDDA) “click” chemistry on a single GE FASTLab™ cassette. We exemplified the method by producing a fluorine-18 radiolabelled interleukin-2 (IL2) radioconjugate from a trans-cyclooctene (TCO) modified IL2 precursor. The radioconjugate was produced using a fully automated radiosynthesis on a single FASTLab™ cassette in a radiochemical yield (RCY) of 19.8 ± 2.6% in 110 min (from start of synthesis); the molar activity was 132.3 ± 14.6 GBq/μmol. The in vitro uptake of [18F]TTCO-IL2 correlated with the differential receptor expression (CD25, CD122, CD132) in PC3, NK-92 and activated human PBMCs. The automated method may be adapted for the radiosynthesis of any TCO-modified protein via IEDDA chemistry

A kit-based aluminium-[18F]fluoride approach to radiolabelled microbubbles

The production of 18F-labelled microbubbles (MBs) via the aluminium-[18F]fluoride ([18F]AlF) radiolabelling method and facile inverse-electron-demand Diels-Alder (IEDDA) ‘click’ chemistry is reported. An [18F]AlF-NODA-labelled tetrazine was synthesised in excellent radiochemical yield (>95% RCY) and efficiently conjugated to a trans¬-cyclooctene (TCO) functionalised phospholipid (40-50% RCY), which was incorporated into MBs (40-50% RCY). To demonstrate the potential of producing 18F-labelled MBs for clinical studies, we also describe a kit-based approach which is amenable for use in a hospital radiopharmacy setting

Automated sulfur-[18F]fluoride exchange radiolabelling of a prostate specific membrane antigen (PSMA) targeted ligand using the GE FASTlab™ cassette-based platform

Sulfur-[18F]fluoride exchange radiochemistry is a rapid and convenient method for incorporating fluorine-18 into biologically active molecules. We report a fully automated radiolabelling procedure for the synthesis of a [18F]SO3F-bearing prostate specific membrane antigen (PSMA) targeted ligand ([18F]5) using the GE FASTLab™ cassette-based platform in a 25.0 ± 2.6% radiochemical yield (decay corrected). Uptake in vitro and in vivo correlated with PSMA expression, and the radioligand exhibited favourable biodistribution and pharmacokinetic profiles

[18F]FET-βAG-TOCA: The Design, Evaluation and Clinical Translation of a Fluorinated Octreotide

The success of Lutathera™ ([177Lu]Lu-DOTA-TATE) in the NETTER-1 clinical trial as a peptide receptor radionuclide therapy (PRRT) for somatostatin receptor expressing (SSTR) neuroendocrine tumours (NET) is likely to increase the demand for patient stratification by positron emission tomography (PET). The current gold standard of gallium-68 radiolabelled somatostatin analogues (e.g., [68Ga]Ga-DOTA-TATE) works effectively, but access is constrained by the limited availability and scalability of gallium-68 radiopharmaceutical production. The aim of this review is three-fold: firstly, we discuss the peptide library design, biological evaluation and clinical translation of [18F]fluoroethyltriazole-βAG-TOCA ([18F]FET-βAG-TOCA), our fluorine-18 radiolabelled octreotide; secondly, to exemplify the potential of the 2-[18F]fluoroethylazide prosthetic group and copper-catalysed azide-alkyne cycloaddition (CuAAC) chemistry in accessing good manufacturing practice (GMP) compatible radiopharmaceuticals; thirdly, we aim to illustrate a framework for the translation of similarly radiolabelled peptides, in which in vivo pharmacokinetics drives candidate selection, supported by robust radiochemistry methodology and a route to GMP production. It is hoped that this review will continue to inspire the development and translation of fluorine-18 radiolabelled peptides into clinical studies for the benefit of patients

The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules

The aluminium-[18F]fluoride ([18F]AlF) radiolabelling method combines the favourable decay characteristics of fluorine-18 with the convenience and familiarity of metal-based radiochemistry and has been used to parallel gallium-68 radiopharmaceutical developments. As such, the [18F]AlF method is popular and widely implemented in the development of radiopharmaceuticals for the clinic. In this review, we capture the current status of [18F]AlF-based technology and reflect upon its impact on nuclear medicine, as well as offering our perspective on what the future holds for this unique radiolabelling method

Synthesis of a benzoxazinthione derivative of tanaproget and pharmacological evaluation for PET imaging of PR expression.

BACKGROUND: The histological evaluation of estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer lesions from biopsy tissue can stratify patients to receive endocrine therapy. Furthermore, PR expression can predict response to selective estrogen receptor modulators (SERMs). Current immunohistochemical approaches to PR detection are limited by sampling error associated with biopsy and lack of standardised protocols; positron emission tomography (PET) using receptor targeted radiopharmaceuticals to provide quantitative, whole-body imaging may overcome these limitations. PR expression has been successfully imaged with PET in the clinical setting, however investigation into new radioligands with improved pharmacokinetics and metabolic stability is desirable. RESULTS: We report the synthesis of a focused library of non-steroidal PR ligands evaluated for use as PET radioligands. A lead candidate ([18F]2) with low nanomolar activity was selected and radiolabelled with a radiochemical yield of 2.29 ± 2.31% (decay-corrected), radiochemical purity (RCP) > 95% and a molar activity of 2.5 ± 1.6 GBq/μmol. Cell uptake studies showed a significant and specific accumulation of [18F]2 in T47D (PR++) breast cancer cell compared to MDA-MB-231 (PR-) control; however, in vivo evaluation was confounded by rapid defluorination of the radioligand. In vitro metabolite analysis of 2 in MLM confirmed defluorination and oxidative metabolism of the thiocarbamate to oxocarbamate moiety by mass spectrometry. CONCLUSIONS: A route to access [18F]2 was developed to allow in vitro and in vivo evaluation, albeit with low radiochemical yield and modest molar activity. [18F]2 demonstrated selective uptake in PR++ T47D cells which could be blocked in a dose dependent manner with progesterone. However, [18F]2 showed poor in vivo metabolic stability with rapid defluorination within the time frame of the imaging protocol.status: Published onlin

Synthesis of a benzoxazinthione derivative of tanaproget and pharmacological evaluation for PET imaging of PR expression

Abstract Background The histological evaluation of estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer lesions from biopsy tissue can stratify patients to receive endocrine therapy. Furthermore, PR expression can predict response to selective estrogen receptor modulators (SERMs). Current immunohistochemical approaches to PR detection are limited by sampling error associated with biopsy and lack of standardised protocols; positron emission tomography (PET) using receptor targeted radiopharmaceuticals to provide quantitative, whole-body imaging may overcome these limitations. PR expression has been successfully imaged with PET in the clinical setting, however investigation into new radioligands with improved pharmacokinetics and metabolic stability is desirable. Results We report the synthesis of a focused library of non-steroidal PR ligands evaluated for use as PET radioligands. A lead candidate ([ 18 F]2) with low nanomolar activity was selected and radiolabelled with a radiochemical yield of 2.29 ± 2.31% (decay-corrected), radiochemical purity (RCP) > 95% and a molar activity of 2.5 ± 1.6 GBq/μmol. Cell uptake studies showed a significant and specific accumulation of [ 18 F]2 in T47D (PR++) breast cancer cell compared to MDA-MB-231 (PR-) control; however, in vivo evaluation was confounded by rapid defluorination of the radioligand. In vitro metabolite analysis of 2 in MLM confirmed defluorination and oxidative metabolism of the thiocarbamate to oxocarbamate moiety by mass spectrometry. Conclusions A route to access [ 18 F]2 was developed to allow in vitro and in vivo evaluation, albeit with low radiochemical yield and modest molar activity. [ 18 F]2 demonstrated selective uptake in PR++ T47D cells which could be blocked in a dose dependent manner with progesterone. However, [ 18 F]2 showed poor in vivo metabolic stability with rapid defluorination within the time frame of the imaging protocol

Novel Non-Congeneric Derivatives of the Choline Kinase Alpha Inhibitor ICL-CCIC-0019

Choline kinase alpha (CHKA) is a promising target for the development of cancer therapeutics. We have previously reported ICL-CCIC-0019, a potent CHKA inhibitor with high cellular activity but with some unfavorable pharmacological properties. In this work, we present an active analogue of ICL-CCIC-0019 bearing a piperazine handle (CK146) to facilitate further structural elaboration of the pharmacophore and thus improve the biological profile. Two different strategies were evaluated in this study: (1) a prodrug approach whereby selective CHKA inhibition could be achieved through modulating the activity of CK146, via the incorporation of an ε-(Ac) Lys motif, cleavable by elevated levels of histone deacetylase (HDAC) and cathepsin L (CTSL) in tumour cells; (2) a prostate-specific membrane antigen (PSMA) receptor targeted delivery strategy. Prodrug (CK145) and PSMA-targeted (CK147) derivatives were successfully synthesized and evaluated in vitro. While the exploitation of CK146 in those two strategies did not deliver the expected results, important and informative structure-activity relationships were observed and have been reported