University of Groningen
Development of PET tracers for investigation of arginase-related pathways
dos Santos Clemente, Gonçalo
DOI:
10.33612/diss.143845684
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Publication date: 2020
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dos Santos Clemente, G. (2020). Development of PET tracers for investigation of arginase-related pathways. University of Groningen. https://doi.org/10.33612/diss.143845684
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Chapter 9
As arginase is considered a multifaceted enzyme important both in physiological or pathophysiological states [1], the lack of specific molecular imaging probes for the investigation of arginase-related pathways became evident [Chapter 2]. The works reported in this thesis led to the development of the first two positron emission tomography (PET) tracers specifically directed to imaging of arginase expression ([18F]FMARS and [18F]FBMARS), which were able to map arginase with highsensitivity both in inflammatory and tumorigenic processes [Chapter 4]. As the synthesis of therapeutically potent arginase inhibitors is currently a very active research topic [2], [18F]FMARS and [18F]FBMARS can also aid in assessing the target
engagement and pharmacodynamics of newly developed molecules, evaluating the efficacy of therapeutic cycles, and determining the required dose to achieve significant enzyme inhibition. Since [18F]FBMARS showed the highest
arginase-mediated uptake in PET imaging, this tracer may be used to identify poor outcomes, to select those pathologies that may respond to arginase-inhibiting or arginine deprivation therapies, and to monitor changes of arginase levels during and after treatment.
Due to species restrictions in the animal imaging licenses, and some technical constraints (e.g., need for ventilatory masks fitting hermetically to the snout, high risk of anesthesia complications [3], and lack of experience with the handling of these animals within the department), the aerosolized inhalation of [18F]FBMARS
by asthmatic guinea pigs was not achievable. However, as the preliminary tests in arginase-overexpressing tumor xenografts and in ex vivo asthmatic lung sections were promising [Chapter 4], and the use of arginase inhibitors is regarded as a potential innovative therapy for asthma [4], the inhalation of [18F]FBMARS in
treated and non-treated asthmatic guinea pigs (or other possible asthma models) should be considered in future experiments.
The promising results obtained with the original 18F-fluorinated arginase inhibitors
in this thesis can also stimulate the development of next-generation arginase imaging probes with improved radiosynthesis yields, biodistribution, and bioavailability. A persistent drawback to date is the lack of subtype-selective arginase inhibitors [5]. Achieving subtype selectivity may allow establishing an
association between Arg1 and Arg2 with disease severity, poor prognosis, or reduced therapeutic responsiveness. Therefore, finding subtype-selective arginase inhibitors remains the ultimate goal to be achieved in this field. Some other potential approaches to arginase imaging may explore the radiolabeling of anti-arginase antibodies, which can enhance the binding stability, specificity and, affinity to the enzyme, or the assembly of multivalent nanocomposites that can also be applied in theragnosis [Chapter 2]. A major challenge in the latter proposed approaches is the conjugation of the imaging agent without compromising the biological activity of the antibodies or nanocomposites.
During the initial research, leading to the synthesis of [18F]FMARS and [18F]FBMARS,
the 18F-fluorination potential of several arylBpin-containing biologically active
compounds produced by convergent multicomponent reactions (MCR) was also revealed [Chapter 3]. Future work may integrate the 18F-fluorination step in the
final stages of the MCR processes when the arylBpin labeling products are already formed. This approach would abridge the MCR and the radiolabeling procedures in a single “one-pot” phase, instead of the two distinct phases used in this thesis, and require a sole final purification process. Although it is unlikely that this combinatory approach would be successful for more advanced applications, it may potentially accelerate the access to a range of small radiolabeled molecules with diverse functional groups for preliminary screening assays.
This thesis also highlighted the influence of statins in the arginase–NO• network
[Chapter 5]. Pleiotropic effects of statins have long been reported [6], but the exact on/off-target mechanisms, the molecular determinants, and the reasons for a documented resistance or intolerance to this class of drugs by some patients are still controversial. Nevertheless, the increase of NO• levels is one of the most
regarded outcomes (parallel to the primary cholesterol-lowering effect) of the use of statins either in in vitro or in vivo experimental assays. Therefore, as with arginase inhibitors, the use of statins has been hypothesized as a potential innovative or complementary therapy for pathologies resulting from an arginase/NOS imbalance [7]. A particular interest has grown regarding the use of inhaled arginase inhibitors and statins in asthma and chronic obstructive pulmonary disease [4, 8].
Due to its high resolution and sensitivity, quantitative molecular imaging techniques relying on radiolabeled molecules can play an essential role in the development of specific assays to understand potential mechanisms of action. So, in addition to the radiofluorinated arginase inhibitors, a 18F-analog of atorvastatin
Chapter 9: Concluding remarks and future perspectives
most potent and widely used statins, being particularly associated with pleiotropic effects [9]. The access to the 18F-fluorinated atorvastatin allowed successful in vivo
pilot tests [Chapter 8] whose results may serve as a starting point for the design of further preclinical (including PET scanning times longer than 90 minutes) and clinical research to establish whether or not [18F]atorvastatin can be used, for
example, to identify patients who will be more resistant to the use of statins due to lower uptake rates or faster pharmacokinetics. Additionally, [18F]atorvastatin may
also be used to identify potential changes in HMG-CoA reductase levels related to disease or treatment.
For the abovementioned reasons, there are already plans being envisaged in the Nuclear Medicine and Molecular Imaging (NGMB) department of the University Medical Center of Groningen (UMCG) for the incubation of carotid atherosclerotic plaques from stroke patients undergoing surgery (ideally from statin resistant and non-resistant subjects) in [18F]atorvastatin (with and without pretreatment with a
standard statin). This study may help to the understanding whether [18F]atorvastatin has binding affinity to human carotid atherosclerotic plaque,
whether [18F]atorvastatin uptake can be correlated with HMG-CoA reductase
concentrations, whether [18F]atorvastatin is suitable for the identification of culprit
(leading to atherosclerosis) and non-culprit human carotid plaques, and whether are there any quantifiable HMG-CoA reductase concentration differences between culprit and non-culprit human carotid plaques.
The therapeutic impact of statins on the arginase/NOS signaling pathways remains to be elucidated. However, the work that is described in this thesis provided two main radiotracers ([18F]FBMARS and [18F]atorvastatin) and some basis to develop
further research studies. Preclinical trials with nebulized [18F]atorvastatin similar to
those previously proposed in the asthmatic guinea pig models, should be considered. Further research might also explore the potential benefits of concomitant use of statins and arginase inhibitors in arginase-overexpressing disease models. For example, this combination of drugs can be weighed against conventional treatments (β2-adrenergic agonists and corticosteroids) in asthmatic
models, with the newly developed [18F]FBMARS and [18F]atorvastatin PET probes
being useful for the pharmacokinetics and biodistribution evaluation and potential treatment follow-up.
References
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