The in vivo detection of enzyme activity is a significant biomarker in tumorigenesis. Assessment of enzyme activity relative to enzyme concentration can serve as quite an accurate measurement of several disease states. Chemical Exchange Saturation Transfer (CEST) MRI is a non-invasive imaging technique that can be used to evaluate enzyme activity. Compared to other contrast agents CEST MRI agents have a slower chemical exchange rate and thus have greater specificity for detecting the intended biomarker. Chapter 1 provides an overview of the advances made in the field of molecular imaging for detection of cancer biomarkers. The molecular mechanism of each technique is explained with specific examples and advantages as well as disadvantages of each technique. Chapter 2 investigates the specific example of detection of an enzyme, γ-glutamyl transferase (GGT) in ovarian cancer tumor models using a catalyCEST MRI contrast agent. This chapter discusses the step-by step evaluation of the non-metallic contrast agent, from synthesis to evaluation of its catalytic efficiency with Michaelis Menten kinetics studies and finally in vivo GGT detection in ovarian tumor models of OVCAR-8 and OVCAR-3. Chapter 3 investigates the enzyme, Kallikrein-6 and its detection in HCT116 colon cancer tumor model. In addition to enzyme detection, enzyme inhibition using Antithrombin III inhibitor has also been explored within in vitro media and in vivo HCT116 tumor model. Chapter 4 introduces the catalyCEST agent for detection of sulfatase enzyme. This chapter discusses the synthesis of this agent and its ability to detect sulfatase in bacterial cell suspension and mammalian cell suspension. These examples portray catalyCEST MRI as a platform technology for enzyme activity detection. Finally in Chapter 5 future ideas have been proposed to improve the in vivo detection and broaden the applications of catalyCEST MRI in the field of enzyme studies.

Enzymes are important biomarkers for determining tumor growth and progression. We have developed two molecules to image enzyme response by catalyCEST MRI. This technology allows for non-invasive detection of enzymes. A background of importance of measuring enzyme activity and MRI agents developed for this purpose have been covered in Chapter 1. We have synthesized a responsive paramagnetic Chemical Exchange Saturation Transfer (CEST) agent, called Tm-DO3A-cadaverine. This contrast agents has been successfully cross-linked to the protein albumin by the enzyme transglutaminase leading to the appearance of CEST at -9.2 ppm. The enzyme catalysis has been validated by measuring chemical exchange rates. We have shown that the position of the CEST peak is influenced by the conformation of the molecule depending on the neighboring amino acids to glutamine. This is the first example to show the appearance of CEST due to formation of a covalent bond. We have also synthesized a diamagnetic CEST agent with a large chemical shift dispersion to detect cathespin B activity. Upon enzyme mediated cleavage of PheArgSal, the aryl amide CEST peak at 5.3 ppm disappears. Taking a ratio of the CEST effects from salicylic acid at 9.5 ppm and aryl amide at 5.3 ppm we can detect enzyme activity. The salicylic acid moiety also undergoes some slow response due to enzyme action, as evident by the disappearance of CEST at 9.5 ppm. However, this proof of concept study is the first example of a DIACEST agent designed to measure enzyme activity using a ratio of two CEST effects from the same substrate. The last chapter highlights suggests improvements to the catalyCEST research. The appendix shows the use of bulk magnetic susceptibility measurements by NMR to determine bio-distribution of lanthanides in ex-vivo tissue.

In ischaemic stroke a disruption of cerebral blood flow leads to impaired metabolism and the formation of an ischaemic penumbra in which tissue at risk of infarction is sought for clinical intervention. In stroke trials, therapeutic intervention has largely been based on perfusion-weighted measures, but these have not been shown to be good predictors of tissue outcome. The aim of this thesis was to develop analysis techniques for magnetic resonance imaging (MRI) of chemical exchange saturation transfer (CEST) in order to quantify metabolic signals associated with tissue fate in patients with acute ischaemic stroke. This included addressing robustness for clinical application, and developing quantitative tools that allow exploration of the in-vivo complexity. Tissue-level analyses were performed on a dataset of 12 patients who had been admitted to the John Radcliffe Hospital in Oxford with acute ischaemic stroke and recruited into a clinical imaging study. Further characterisation of signals was performed on stroke models and tissue phantoms. A comparative study of CEST analysis techniques established a model-based approach, Bloch-McConnell model analysis, as the most robust for measuring pH-weighted signals in a clinical setting. Repeatability was improved by isolating non-CEST effects which attenuate signals of interest. The Bloch-McConnell model was developed further to explore whether more biologically-precise quantification of CEST effects was both possible and necessary. The additional model complexity, whilst more reflective of tissue biology, diminished contrast that distinguishes tissue fate, implying the biology is more complex than pH alone. The same model complexity could be used reveal signal patterns associated with tissue outcome that were otherwise obscured by competing CEST processes when observed through simpler models. Improved quantification techniques were demonstrated which were sufficiently robust to be used on clinical data, but also provided insight into the different biological processes at work in ischaemic tissue in the early stages of the disease. The complex array of competing processes in pathological tissue has underscored a need for analysis tools adequate for investigating these effects in the context of human imaging. The trends herein identified at the tissue level support the use of quantitative CEST MRI analysis as a clinical metabolic imaging tool in the investigation of ischaemic stroke.