The development of nuclear receptor imaging agents

Allott, Louis

Abstract

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.

Citation

Allott, L. (2014). The development of nuclear receptor imaging agents. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4217221

• Thesis Type: Thesis
• Deposit Date: Oct 23, 2015
• Publicly Available Date: Feb 23, 2023
• Keywords: Biological sciences
• Public URL: https://hull-repository.worktribe.com/output/4217221
• Additional Information: Department of Biological, Biomedical and Environmental Sciences, The University of Hull
• Award Date: Sep 1, 2014