Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials

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Thesis

Reference

Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials

STRANSKY-HEILKRON, Nathalie Martine

Abstract

Les fibres amyloïdes sont associées à un grand nombre de maladies dégénératives, parmi lesquelles le diabète de type 2. Le but de cette thèse de doctorat était donc de développer des agents de contraste permettant d'imager les amyloïdes pancréatiques. Dans un premier temps, la pharmacocinétique de nanoparticules a été déterminée. Les nanoparticules testées étaient composées d'un réseau de silice sur lequel étaient greffés des cycles DOTA complexant des ions gadolinium, permettant l'imagerie par résonnance magnétique, et d'un hexapeptide, ligand des fibres amyloïdes. Suite à ces études, il a été décidé de synthétiser une série de petites molécules dérivées du florbétapir et de la thioflavine. L'affinité de ces molécules pour les fibres amyloïdes pancréatiques a été déterminée. Une étude histologique sur les pancréas d'un potentiel modèle de souris a également été menée. La dernière partie de la thèse n'est pas reliée aux chapitres précédents. Elle porte sur la thermoablation des tumeurs.

STRANSKY-HEILKRON, Nathalie Martine. Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials. Thèse de doctorat : Univ. Genève, 2016, no. Sc. 4911

URN : urn:nbn:ch:unige-875973

DOI : 10.13097/archive-ouverte/unige:87597

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UNIVERSITÉ DE GENÈVE

Section des sciences pharmaceutiques FACULTÉ DES SCIENCES Professeur Éric Allémann Département de radiologie et informatique médicale FACULTÉ DE MÉDECINE

Professeur Xavier Montet

Development of Contrast Agents for Imaging Amyloids in Type 2 Diabetes –

From Chemical Synthesis to In Vivo Trials

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques

par

Nathalie Stransky-Heilkron

de Genève (GE)

Thèse n° : 4911

Genève 2016

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À mon grand-père Sam qui, outre son goût pour la bonne chère et le bon vin, m’a transmis sa soif de connaissance.

“Always look on the bright side of life.”

Monty Python (Life of Brian)

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Remerciements

Une thèse n’est pas le travail d’une seule personne. C’est un travail d’équipe. Si j’ai autant apprécié mes années de thèse, c’est en grande partie grâce à mon entourage professionnel et personnel. Je voudrais donc chaleureusement remercier les personnes qui m’ont côtoyée de près ou de loin ces dernières années. Une page ne suffirait pas à exprimer ma reconnaissance comme il se doit mais il me faut malgré tout essayer.

Je voudrais donc remercier en tout premier Éric Allémann qui m’a fait confiance non pas sur un mais sur deux sujets de thèse ! Merci de m’avoir laissé une grande liberté quant à la manière de mener mon projet, merci pour votre présence à chaque fois que j’en ai eu besoin, merci pour votre soutien dans les périodes un peu difficiles, merci surtout pour votre grande humanité et votre bienveillance.

Merci également à Xavier Montet. Chacune de nos rencontres a été enrichissante tant scientifiquement qu’humainement. Merci pour votre disponibilité.

Merci à mes jurés de thèse Chantal Csajka, Jean-Luc Coll, Julijana Kristl et Christel Marquette pour avoir accepté de lire mon manuscrit et fait le déplacement jusqu’à Genève. J’aimerais plus particulièrement remercier Christel pour son aide apportée sur la partie histologie. Je garde un très bon souvenir de mes quelques jours passés à Grenoble et de nos discussions sur différents aspects du projet.

Merci à toutes les personnes qui ont participé au projet Diamyl et plus particulièrement à Marie Plissonneau et Jonathan Pansieri, mes collègues doctorants, pour l’aide apportée à tout moment.

Un grand merci à Andrej Babič pour son aide à tous les stades de mon projet de thèse, et surtout pour la synthèse. Merci pour ta disponibilité, ta patience et tes grandes qualités didactiques.

Merci à mes autres collègues et notamment à Viktorija, Jordan, Céline et Cédric pour les discussions scientifiques et autres, les chansons Disney dans le labo, les moments de folie du jeudi après-midi, les mots fléchés, le soutien moral et tant et tant d’autres choses. Un grand merci également à Brigitte, Florence et Florence. Ma plus vive reconnaissance va aussi à Marco qui fait en sorte que tous les appareils fonctionnent correctement, ce qui n’est pas une mince affaire.

D’un point de vue plus personnel, j’aimerais remercier Daniel qui, bien qu’il ait tenté de me dissuader de me lancer dans une thèse, a été et reste d’un grand soutien. Merci de partager ma vie.

Merci à mes parents de m’avoir permis de faire des études dans d’excellentes conditions, à mes frère et sœurs pour m’aider à me rappeler des choses importantes dans la vie et à toute ma famille pour leur soutien. Je vous aime tous très fort !

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Preface

According to the World Health Organization, diabetes affected 9 % of the worldwide population in 2014. Every year, 1.5 million deaths can be directly attributed to this disease. Type 2 diabetes (T2D) accounts for more than 90 % of diabetes cases. T2D is a metabolic disease, characterized by peripheral insulin resistance and a relative lack of insulin. In these conditions, glucose homeostasis is disturbed, leading to hyperglycemia. While obesity is the main risk factor for T2D, the etiology of diabetes remains to be elucidated. It is clearly related to insulin producing cells, the pancreatic β cells, failing to meet increased insulin needs. Several complementary hypotheses coexist nowadays for T2D development, including endoplasmic reticulum and oxidative stresses leading to β cell dysfunction and death. Another feature of T2D patients is the presence of amyloids in pancreatic islets in close proximity to degenerative β cells.

Amyloids are insoluble aggregates of proteins. They form unbranched fibrils a few nanometers wide and some micrometers long. Amyloid fibrils are constituted of aggregated peptides linked through interpeptide β sheets. Several peptides are prone to spontaneously form amyloids in vivo, causing several degenerative diseases. In the case of T2D, the peptide forming pancreatic fibrils is the islet amyloid polypeptide (IAPP). While amyloids are present in several degenerative diseases among which T2D, their role in this pathology is not elucidated yet. This is partly because no longitudinal imaging of pancreatic amyloids can be performed yet. While understanding of amyloids in T2D has been mostly left aside by the scientific community, it is not the case in Alzheimer’s disease, for which several research groups have worked on developing contrast agents aimed at Aβ amyloids imaging, present in Alzheimer’s patients. These last years, several contrast agents have been approved for the diagnosis of Alzheimer’s disease. The aim of this PhD thesis was therefore to develop targeted agents aimed at imaging pancreatic amyloids.

The introduction of this thesis explains in more details what diabetes is and how it is treated, with a focus on T2D. It also reviews current research on the development of contrast agents allowing quantification of β cell mass and function.

The first chapter presents pharmacokinetic studies that were conducted on silica-based

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Preface

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these nanoparticles had a very short plasma half-life. Therefore they were considered not suitable for imaging amyloids especially due to their targeting mode. Indeed, targeting moieties consisted in hexapeptides, which had the ability to bind only at the extremities of amyloid fibrils, meaning two nanoparticles per fibril at best. It was decided to develop other targeting moieties able to bind an abundant part of amyloids, the cross β sheets.

For this objective, advantage was taken of the already developed and approved contrast agents for Alzheimer’s diagnosis that bind to cross β sheets. Two series of molecules were synthesized with the same pharmacophores as the ones found efficient to bind to Alzheimer’s amyloids. The synthesis of the first series is presented in chapter 2, while the second series, synthesized by Dr. Babič is presented in the following chapter.

The third chapter presents affinity experiments that were conducted to characterize the affinity of the newly synthesized molecules for pancreatic amyloids produced in the laboratory. Fluorescence, surface plasmon resonance, and microscale thermophoresis were used. Only fluorescence experiments gave substantial and sound results.

The fourth chapter is a histological study of a potential preclinical model for pancreatic amyloidosis.

IAPP, the peptide that forms amyloid in diabetic humans, does not aggregate in mice and rats, although its sequence is very close to that of humans. Hence, transgenic model is required. FVB/N mice expressing human IAPP under rat insulin promoter were purchased. The selected model was the only one commercially available. It was however not completely clear in the literature whether these mice exhibited pancreatic amyloids and, if it were the case, in which extent. Histological examination of the pancreas of transgenic mice in comparison to the pancreas of age-matched, control mice was thus performed.

The fifth chapter of the thesis is not related to the previous ones. Its only link is that it was my first thesis project. As a PhD thesis is rarely a bed of roses, it occurred that my thesis project had to be redirected after the first year. However, even if not related at all to the main topic presented in this manuscript, that part of the work belongs to the thesis as the other chapters since more than one year of research was dedicated to it. This is the reason why, from imaging of diabetes in the first four chapters, there is a detour to treatment of tumors by thermoablation in the fifth chapter. This last chapter is constituted of a review article that was published in the Journal of Drug Delivery Science and Technology and of an abstract of a poster presenting scientific results that were obtained in the course of my first year of thesis. This poster was presented during the 39^th Meeting of the Controlled Release Society in Quebec City, Canada.

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Preface

Thermoablation is used to treat some tumors, mostly when resection is not feasible. It consists in heating up or cooling down tissues in order to induce the death of unwanted cells. The first part of chapter 5 is a review of preclinical studies combining thermoablation with another, complementary treatment strategy. The second part of the chapter is related to ex vivo experiments that were performed using a medical device under development.

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Introduction

Type 2 diabetes:

why imaging is needed

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Introduction. Type 2 diabetes: why imaging is needed

1. Regulation of glycaemia

Glycaemia varies before and after meals. It is mainly regulated by insulin, which promotes the storage of glucose as glycogen in the liver and muscles, and as triglycerides in adipose tissues. Insulin is produced in the endocrine, pancreatic β cells. The largest part of pancreas is an exocrine gland, secreting digestive enzymes. The endocrine part of the pancreas represents 1 to 2 % of its total volume. The endocrine cells are grouped in pancreatic islets, also known as islets of Langerhans. Each islet is constituted of about 2000 endocrine cells and measures 50 to 500 μm. Pancreatic islets are mainly formed by β cells, which produce insulin and amylin (also known as islet amyloid polypeptide).

The other cells composing the islets of Langerhans are, by order of occurrence,  cells, producing glucagon,  cells, producing somatostatin, PP cells, producing pancreatic polypeptide (which must not be confused with amylin), and  cells, producing ghrelin. Pancreatic islets are highly vascularized.

The blood flow going through them is 10 to 20 % of the total pancreatic blood flow, although the islets represent less than 2 % of the total pancreas volume [1].

Insulin is a peptide hormone produced by pancreatic β cells and stored under crystalline form in granules with amylin, another peptide hormone. Insulin secretion is a complex mechanism, finely tuned by several factors. The most important one is glucose-stimulated insulin secretion (GSIS) [2].

Briefly, glucose is transported into the β cells through glucose transporters (Figure 1). Its metabolism leads to increased adenosine triphosphate (ATP) concentration, which triggers the closing of ATP- sensitive K⁺ channels. Closing of these channels leads to depolarization of the β cell membrane.

Depolarization of the cell membrane in turn induces the opening of voltage-dependent Ca²⁺

channels, triggering Ca²⁺ influx. Increase in Ca²⁺ concentration triggers the fusion of insulin granules with the β cell membrane, allowing the secretion of insulin. The concentration of insulin in blood varies even in postprandial periods. In fact, insulin secretion follows a pulsatile pattern. Two major secretory forms coexist: ultradian oscillations, which have a period of 1 to 2 hours, and rapid after-

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Introduction

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2. Dysregulation of glycaemia

Diabetes is a disease in which glucose homeostasis is disturbed, leading to hyperglycemia. Two types of diabetes are classically distinguished. Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic β cells, responsible for insulin secretion, are selectively destroyed. Type 2 diabetes (T2D) is a metabolic disease caused by peripheral insulin resistance and a relative lack of insulin.

Figure 1. Scheme of insulin release in response to glucose stimulation. Glucose is internalized through GLUT2 and metabolized, resulting in an increase in ATP/Mg-ADP ratio. Modification of the ATP/ADP ratio induces the closure of ATP-sensitive K⁺ channels. The cell membrane is thus depolarized, leading to the opening of Ca²⁺ channels, resulting in an increase in intracellular Ca²⁺

concentration. Insulin granules react to the rise of Ca²⁺ concentration by fusing with the cell membrane, releasing insulin in the surrounding environment.

According to the World Health Organization, in 2014, 9 % of the worldwide population was affected by diabetes. Every year, 1.5 million deaths are directly caused by diabetes. The consequences of diabetes include, among others, cardiovascular diseases, neuropathies, blindness, and kidney failures. T2D accounts for 90 % of the cases of diabetes worldwide.

Obesity is the main risk factor for T2D. Clinically, obesity is characterized by peripheral insulin resistance. Insulin resistance is compensated by an increase in β cell mass (BCM) [3-6], allowing higher insulin secretion [7]. After some time, β cells get exhausted and BCM decreases, leading to impaired glucose tolerance, also referred as pre-diabetes. Impaired glucose tolerance is

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Introduction

asymptomatic and is usually diagnosed by oral glucose tolerance test or elevated values of fasting plasma glucose [8]. Undiagnosed and untreated, impaired glucose tolerance evolves, inducing increasing lack of insulin, until reaching a stage at which diabetes is declared. Treatment of diabetes consists in normalizing glycaemia by reducing peripheral insulin resistance, and, if not enough, by increasing insulinemia through provoking insulin secretion or through insulin injection.

3. Treatment of T2D diabetes

Once impaired glucose tolerance is diagnosed, the first line treatment consists of decreasing the body weight through physical activity and balanced diet. If these measures are taken early enough in the course of the disease, they are sufficient to recover normal glycaemia. However, once T2D is diagnosed, the treatment is symptomatic and aims at recovering normal glycaemia.

If lifestyle modifications are not sufficient, i.e. if glycaemia does not decrease to normal values, the first pharmacological treatment prescribed is usually metformin [9, 10]. Metformin reduces peripheral resistance to insulin. If metformin alone is not enough, it is combined with one or two drugs from other antidiabetic classes. The additional molecule can be an insulin secretagogue, such as a sulfonylurea (glipizide, glibenclamide, glimepiride, gliclazide) or a glinide (repaglinide, nateglinide, mitiglinide). These two classes of molecules act by binding the sulfonylurea receptor of pancreatic β cells, inducing, after a cascade reaction, the release of insulin. It can also be a thiazolidinedione, also known as glitazone (rosiglitazone, pioglitazone). These molecules activate peroxisome proliferator-activated receptor  (PPAR), improving, among other actions, peripheral sensitivity to insulin. Dipeptidyl peptidase-4 (DPP-4) inhibitors, also known as gliptins (sitagliptin, vidagliptin, saxagliptin, etc.), are also commonly used in the treatment of T2D. They act by increasing incretin levels, i.e. glucagon-like peptide 1 (GLP-1) and gastric inhibitor polypeptide, which decrease glucagon release. Decrease in blood glucagon in turn increases insulin secretion, decreases gastric emptying, and reduces blood glucose. A GLP-1 receptor agonist (exenatide, liraglutide, etc.) can also be used. The drawback of this type of treatment is that, due to the peptide nature of the molecule, it requires parenteral administration. Insulin injection can be introduced in the treatment either as complementary to other measures or, in more advanced cases of T2D, as the treatment itself.

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Introduction

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quantification of C-peptide, reflecting insulin secretion. No direct assessment of the β cell mass or function can be realized.

4. Mechanisms of β cell dysregulation

Mechanisms for β cell dysfunction and death in diabetes have not been elucidated yet. Several hypotheses coexist nowadays. It is to note that, although obesity is the main risk factor for developing T2D, not all obese people will suffer from T2D. In most of them, higher BCM and insulin secretion will persist, compensating for insulin resistance in peripheral tissues [6]. In other patients, though, some unclear mechanisms will take place, leading to glucose intolerance and, eventually, to diabetes. These mechanisms are summarized in Figure 2. Diabetes is a multifactorial disease.

Furthermore, its etiology can differ from patient to patient. Hyperglycemia obviously plays a role in β cell toxicity. It has been shown that transient hyperglycemia induces β cell proliferation. However, in the case of prolonged hyperglycemia, proliferation stops, leaving place to apoptosis [11]. Apoptosis in response to hyperglycemic stress is strongly patient-dependent. Furthermore, hyperglycemic stress may induce inflammatory response in β cells and impair β cell secretory functions. Other markers of obesity such as dyslipidemia [12, 13] and high blood leptin [11] have also been shown to be noxious to β cells. Again, interindividual sensitivity depending on genetic factors and/or environment plays a role.

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Introduction

Figure 2. Factors influencing the onset of diabetes. Insulin resistance may lead to hyperglycemia.

Other factors modulating BCM may include dyslipidemia, leptin, and cytokines. Genetic predisposition to diabetes may include a predetermined amount of β cells and differences in the susceptibility to apoptotic signals and in the regenerative potential of the β cells. Additionally, induction of local inflammatory mediators and cell death may activate the immune system. Finally, drugs may protect or harm β cells. Adapted from Donath et al., Diabetes, 2005 [11].

Endoplasmic reticulum (ER) stress has also been pointed out as a contributor to β cell dysfunction or death. ER is the organelle in which proteins are folded before transiting to the Golgi apparatus. ER stress slows protein folding, yielding unfolded proteins. Due to the secretory nature of β cells, their endoplasmic reticulum is highly developed. They are thus more sensitive to ER stressors.

Mitochondrial dysfunction and oxidative stress have also been proposed as contributors to the

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Introduction

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reactive oxygen species, and antioxidants, leading to oxidative stress and eventually β cell dysfunction and death. Paradoxically, the treatment of diabetes with insulin secretagogues such as sulfonylureas or glinides may thus play a role in β cell failure. This hypothesis is schematized in Figure 3. On the other hand, drugs improving insulin sensitivity such as glitazones could delay the onset of diabetes [15].

Figure 3. Schematic representation on the effect of hyperinsulinemia on β cell dysfunction.

Hyperinsulinemia occurs in case of insulin resistance or of genetic condition. Hyperinsulinemia in turn induces ER and/or oxidative stress. In individuals with genetic predisposition, increased stress leads to β cell failure and eventually to diabetes. Diabetes treatment with insulin secretagogues would further promote insulin hypersecretion, leading to worsening of β cell function. Adapted from Aston-Mourney et al., Diabetologia, 2008 [14].

Islet transplantation is an option that may be realized in insulinodependant diabetic patients encountering issues to regulate their glycaemia. Due to shortage in pancreas donors and adverse effects due to the required immunosuppressive therapy, patients undergoing islet or pancreas transplantation need to be very carefully selected. They are patients who undergo frequent hypoglycemia in response to insulin treatment regimen and whose quality of life and lifespan would be improved by a graft. Usually, purified islets are injected through the portal vein into the patient’s liver [16]. Islet transplantation has the advantage of being relatively simple compared to whole pancreas transplantation. It usually results in glycaemia regulation after the graft [17]. However, both islet transplantation and whole pancreas graft present the drawback of loss of glycemic control after

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Introduction

successful transplantation [18-20]. In some patients, this failure was attributed to the development of T2D [21]. Thus, post-graft islet failure after normoglycemia was reached could have the same underlying mechanisms as the development of T2D.

5. Involvement of amyloids in diabetes

5.1. Islet amyloid polypeptide

Another, complementary hypothesis in β cell dysfunction is the involvement of islet amyloid polypeptide (IAPP). It has indeed been found in autopsies that more than 90 % of T2D patients present amyloids in pancreatic islets [22]. It is important to note that pancreatic amyloids tend to appear in aging patients who do not suffer from this condition and, furthermore, that amyloids are not present in all T2D patients. Nevertheless, T2D patients present significantly more amyloid plaques than the general population, even elder [23]. Amyloids are also found in transplanted islets after some time [24]. The extent of amyloid deposits correlates with recurrence of hyperglycemia [25].

Amyloids are a specific type of protein aggregates, forming unbranched, fibrillar structures of a few nanometers width [26]. Amyloids are characterized by the presence of interpeptide β-sheets, perpendicular to the fibril axis. This feature causes the exhibition of green birefringence when stained with Congo red and observed under polarized light. It is thought that all types of polypeptides can form amyloids, at least in vitro, but some are more prone to amyloid formation than others. The term “amyloids” originates from the beginnings of histology, when these aggregates were mistaken for starch due to blue staining in presence of iodine [27]. Amyloids are associated with a wide range of degenerative diseases such as familial amyloid polyneuropathy and Huntington’s disease. They are also involved in Alzheimer’s disease (AD) and T2D. All diseases in which amyloids are involved are grouped under the term “amyloidoses”.

In the case of T2D, amyloid plaques are mainly constituted by aggregation of IAPP, also known as amylin. IAPP is a 37 amino acid polypeptide. Its sequence is presented in Figure 4. IAPP is stored in vesicles with insulin crystals in a 1:50 molar ratio and cosecreted with this hormone. Hence,

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Introduction

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administration of IAPP in rodents reduces food intake and body weight [40]. However, no difference in food intake was seen between wild type and IAPP knock-out mice. Infusion of amylin in IAPP knock-out mice resulted in a marked anorectic effect compared with infusion in wild type mice, suggesting that the receptors and/or signaling pathways for IAPP could be up-regulated in the absence or lack of IAPP. Other studies showed a difference of weight between wild type mice and IAPP knock-out mice [39]. IAPP also suppresses glucagon release and causes vasodilatation as well as the excretion of calcium, potassium, and sodium [38]. More recently, Visa et al. assessed in vitro the long term effects of IAPP on pancreatic β cells [41]. They showed that, depending on the level of glucose to which the cells were submitted, IAPP would promote or decrease the proliferation of β cells in an autocrine fashion.

5.2. Amyloids in diabetes

IAPP is phylogenetically conserved in all mammals, proving that it has an important function [37].

However, it forms amyloids only in some species, including human, non-human primates, and cats [42]. IAPP does not form amyloids in rodents, although the peptide sequences are very close (Figure 4). Amino acids 22-27 (NFGAIL) and 23-27 (FGAIL) tend to form amyloids in vitro [43]. These amino acids are thus thought to be the ones responsible for IAPP aggregation in humans.

Human KCNTA TCATQ RLANF LVHSS NNFGA ILSST NVGSN TY

Cat KCNTA TCATQ RLANF LIRSS NNLGA ILSPT NVGSN TY Rat and mouse KCNTA TCATQ RLANF LVRSS NNLGP VLPPT NVGSN TY

Figure 4. Sequences of human, cat, rat, and mouse IAPP. All three sequences are very close from each other. However, human and cat IAPP form amyloids in vivo while that of rodents does not. The amino acids thought to be responsible for IAPP aggregation in humans are in bold.

As already mentioned, amyloid aggregates are characterized by a high content in β sheets perpendicular to fibril length. Amyloid formation, schematized in Figure 5, occurs as follows: a polypeptide misfolds in a specific way, leading to the formation of amyloidogenic intermediate [26, 37]. Two or more of these species bind through cross β sheets, forming a nucleus. Nuclei further associate, leading to the formation of soluble aggregates, also called oligomers. In turn, oligomers further assemble, forming protofibrils, and eventually mature fibrils. Protofibrils and fibrils are insoluble and very stable. Once formed, they can reside for indefinite time. The limiting step in amyloid formation is the formation of the nuclei. Once they are formed, fibril maturation occurs relatively fast.

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Introduction

Figure 5. Formation of amyloids. Under certain conditions, not fully understood yet, a peptide encounters misfolding, leading to amyloidogenic conformation. Two or more of these amyloidogenic intermediates then form soluble oligomers. They further assemble to form insoluble protofibrils, leading eventually to mature amyloid fibrils.

A correlation has been established between the extent of pancreatic amyloid deposits and diabetes severity in patients [44]. Likewise, it has been demonstrated that accumulation of amyloids in transplanted islets precedes the reoccurrence of hyperglycemia [24, 25, 45]. In studies in macaques [46, 47] and in cats [48, 49], amyloid deposition was shown to precede the appearance of hyperglycemia. Still, the role of IAPP deposits in the etiology of diabetes remains unclear. What is known is that IAPP aggregates are cytotoxic in vitro [50]. However, mature fibrils are not thought to

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Introduction

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in diabetic patients is related to the formation of pancreatic amyloids [58]. Their hypothesis was supported by the fact that nontransgenic obese mice increase their β cell mass by forming new islets of Langerhans. This is not the case of hIAPP obese mice, which fail increasing β cell mass due to higher rate of β cell apoptosis. Difference in β cell mass between transgenic and nontransgenic mice reached 80 %. Their hypothesis, supported by others [59], is that, over a certain threshold, β cells are no longer able to chaperone the folding of all IAPP. Some of the polypeptides misfold, leading to formation of toxic oligomers. This hypothesis would be in accordance with the involvement of ER stress in the development of T2D.

Other hypotheses for amyloid formation in diabetic or prediabetic patients have been proposed [60].

Glycation of IAPP in chronic hyperglycemia would make it more amyloidogenic [61]. Dysregulation of pH and Ca²⁺ concentration and in IAPP:insulin ratio in the secretory granules could cause misfolding of IAPP, resulting in the formation of amyloidogenic intermediates [62, 63]. Dyslipidemia could also play a role in amylin aggregation [64]. Additionally, impaired IAPP clearance could be involved in the formation of pancreatic amyloids [65].

Nowadays, although research aiming at understanding and at treating the underlying causes of diabetes is very intensive, a lot remains to be discovered. One of the obstacles to diabetes research is the lack of direct visualization of what happens in pancreatic islets. All characterizations can either be done in vitro, in cultured cells or islets, or ex vivo, during dissections of animal models or autopsies of diabetic patients. All markers that can be measured over the disease course, such as glycaemia, glycated hemoglobin, and C-peptide are only indirectly correlated with β cell mass and function. We currently lack a tool allowing to see what happens at the level of β cells and pancreatic islets. Finding contrast agents aiming at visualizing those would be a powerful tool. Hence, research for a contrast agent aiming at quantifying the β cells and/or assessing their function in relationship to all types of factors is a very dynamic field.

6. Imaging diabetes

Imaging of pancreatic islets would be useful to increase our knowledge of the development and evolution of diabetes. It would also be helpful to monitor the effects of current treatments in patients on the long term. Likewise, specific contrast agents would be effective tools for the development and monitoring of new treatments and for assessing β cell mass and function in prospective studies. Follow-up of pancreatic graft and islet transplantation would also benefit from the possibility of imaging the evolution of β cell mass and function over time.

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Introduction

Imaging of pancreatic islets is really challenging. As already mentioned, β cells are clustered in the pancreas in small islets, ranging from 50 μm to 500 μm in size, and representing around 2 % of the total pancreas mass. In diabetic patients, the size of the pancreatic islets is decreased due to the loss of β cell mass. Pancreatic islets are scattered all over the pancreas. The pancreas itself is deeply located in the abdomen, in close proximity to other organs (Figure 6).

Figure 6. Anatomical location of the pancreas. The pancreas is sited in the abdomen, behind the stomach. The head of the pancreas seats in the duodenum, in which it secretes pancreatic digestive fluid. It is also in close proximity to the kidneys and liver. Image modified from the Visible Human Server (http://visiblehuman.epfl.ch).

Several challenges need to be addressed for imaging pancreatic islets. First, the imaging modality should be able to detect the pancreas, and discriminate a signal coming from this organ from a signal coming from surrounding tissues. Second, the contrast agent should be islet-specific. As β cells represent a minority of the cells composing the pancreas, the probe should not be taken up significantly by other endocrine or exocrine pancreatic cells. Sweet et al. considered that, due to the small size of pancreatic islets, the accumulation of the tracer at target site should be 1000-fold higher than in the surrounding tissues [66]. For imaging transplanted islets, it should also not bind to liver tissue, as it is the site of pancreatic islet transplantation. Third, the measured signal should mirror the

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The most studied imaging techniques for pancreatic islet imaging are positron emission tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Their main characteristics are summarized in Table 1. PET and SPECT have the advantage of being very sensitive. Their drawback lies in poor spatial resolution. Ideally, they should be coupled to an imaging modality allowing precise definition of the outline of pancreas. This is of tremendous importance in long-term diabetic patients whose pancreas is atrophied and in preclinical studies with rodents, in which the pancreas is a diffuse organ, with imprecise borders. Usually, signal assessment in PET and SPECT relies on quantification of the signal in a defined volume. Hence, if the volume of the pancreas is not precisely assessed, the quantity of signal per volume can be over- or underestimated. Another disadvantage of imaging modalities requiring the use of radioisotopes is the exposure of the patient to ionizing radiations, preventing frequent imaging sessions. MRI has the advantage of providing exquisitely detailed anatomical information. Moreover, it does not require radioisotopes, allowing repeated imaging sessions. However, MRI suffers from poor sensitivity even when using contrast agents. Signal-to-noise ratio must be much higher than for PET and SPECT.

Fluorescence has also been used as preclinical imaging modality. Due to very short tissue penetration of light (1 cm to 2 cm in the near infrared region), its use is limited to small animal models or to endoscopic procedures, making it more invasive than the previously mentioned imaging modalities.

Table 1. Main characteristics of imaging modalities considered for imaging pancreatic islets.

Adapted from Frangioni, Journal of Clinical Oncology, 2008 [67].

Imaging modality Voxel dimension Contrast agents

Minimum contrast agent concentration per voxel for detection*

MRI 1 mm³ (1x1x1 mm) Gd 50 μM

SPECT 1.7 cm³(12x12x12 mm) ^99mTc, ¹¹¹In, ⁶⁷Ga 0.3 pM

PET 0.5 cm³ (8x8x8 mm) ¹⁸F 0.02 pM

* Assuming zero background

Research for finding a suitable contrast agent for imaging diabetes is reviewed in the next paragraphs. It should be noted that staining of pancreatic islets before transplantation will not be broached since this techniques aims at visualizing the position of the grafted islets in the receiver and not at assessing their function over time.

6.1. Targeting β cells with antibodies

Using antibodies as targeting moieties is very appealing due to their specificity for their target.

Several antibodies have been characterized and evaluated as β cell probes over the years.

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Introduction

Ladrière et al. attempted using antibodies directed against β cell gangliosides [68]. The antibodies were¹²⁵I-labeled, allowing their detection by radioactivity measurement (in vitro or ex vivo). The animal model was rats treated with streptozotocin (STZ). STZ is a molecule classically used in rodents to destroy pancreatic islets and make the animals artificially diabetic. Unfortunately, no difference could be detected in the radioactive content of pancreatic islets from control or diabetic rats.

Moore et al. used a ¹¹¹In-labeled β cell-targeting antibody, IC2, to assess the beta cell mass of control and STZ-induced diabetic mice [69]. Antibody uptake was much higher in control mice than in diabetic ones. The authors concluded that IC2 antibodies should be labeled with PET or MRI tracers to allow performing in vivo experiments. Unfortunately, no follow-up study could be found on this topic. IC2 antibody was shown to target sphingomyelin, a phospholipid that forms patches localized at cell surface [70]. The antibody would bind to sphingomyelin patches only in the presence of cholesterol, which stabilizes the patches. This target is fortunately also present at the surface of human β cells. Furthermore, the quantity of cholesterol-stabilized sphingomyelin patches at the surface of β cells seems to be correlated with the expression of insulin [71]. IC2 antibody seems to be a promising candidate for the selective targeting of β cells.

Ueberberg et al. generated single-chain antibodies binding selectively β cells [72]. Labeling with ¹²⁵I made quantification of the antibodies possible in vitro, successfully identifying β cells. The amount of binding correlated nicely with the glycemic status of the animals prior to sacrifice. One of these single-chain antibodies, SCA-B1, was covalently bound to ferromagnetic nanoparticles, providing MRI contrast [73]. These nanoconstructs allowed imaging single pancreatic islets in mice at high magnetic field. This strategy seems promising for studying mouse diabetes models.

Vats et al. generated a monoclonal antibody targeting transmembrane protein 27 (TMEM27) [74]. It was previously shown that this protein was present at β cell membrane and in renal tubules [75, 76].

In β cells, TMEM27 expression was correlated with that of insulin, making it a potential target to assess insulin production [76]. Using a monoclonal antibody directed against TMEM27, Vats et al.

were able to visualize insulinoma in a mouse model using a fluorescent derivative of their antibody [74]. These results seemed promising, although insulinoma is very different from native

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Introduction

16

membrane [77]. As explained earlier, depolarization of the membrane induces the opening of Ca²⁺

channels, causing an increase in intracellular Ca²⁺ concentration, which triggers the release of insulin (Figure 1). KATP channels are composed of eight subunits: four copies of Kir6.2, which compose the channel itself, and four copies of SUR1 [77, 78]. Even if SUR receptors are part of the KATP channels of cells constituting several different organs (mainly muscles), SUR1 is specific for pancreatic β cells, making it an interesting target for the development of β cell specific contrast agents. Several sulfonylureas have been studied over the years to attempt imaging β cells in vivo.

Schmitz et al. based their search for a β cell targeting contrast agent on glyburide [79]. They synthesized analogues of this sulfonylurea and labeled them with ¹⁸F. Despite the fact that these compounds bound effectively β cells in vitro, in vivo results were disappointing: no difference of uptake was observed between control and diabetic mice. The reason for this is probably that glyburide binds with similar affinity SUR1, expressed in pancreatic β cells, and SUR2, present in all types of muscles. PET is not able to discriminate signal coming from adjacent tissues. The high lipophilicity of glyburide could also contribute to these disappointing results.

Sweet et al. screened several compounds regarding their affinity for β cells compared to affinity for other pancreatic cells [66]. Glibenclamide and fluorodithizone were the compounds with the most promising specificity but the difference of affinity was not high enough to overcome the low quantity of islets compared to the whole pancreas. As previously mentioned, the authors calculated that the affinity for β cells should be at least 1000 times higher than that for surrounding environment.

Wängler et al. synthesized an ¹⁸F-labeled derivative of repaglinide [80]. Repaglinide is used as an insulin secretagogue in the treatment of diabetes. The synthesized derivative kept the ability of binding SUR1. In this study, all measurements were made in vitro and ex vivo in rats. No results of hypothetical subsequent in vivo experiments using this repaglinide derivative could be found. It is thus not known whether repaglinide can target β cells with sufficient specificity.

Kimura et al. synthesized derivatives of mitiglinide, claiming that its lower logP would prevent its accumulation in the liver [81]. The derivatives kept high affinity for SUR1. According to the hope of the authors, clearance from the liver was relatively fast. However, signal in other tissues and organs was higher than that coming from pancreas. Furthermore, within pancreas, radioactivity was also detected outside the islets of Langerhans. In conclusion, mitiglinide derivatives lacked the required specificity for pancreatic β cells.

Babic et al. synthesized several derivatives of glibenclamide [82]. The derivative with the highest affinity for SUR1 was coupled to a poly(amidoamine) (PAMAM) dendrimer. The probe showed

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Introduction

specific uptake in the endocrine pancreas. However, no in vivo imaging or assay in diabetic animals was performed. It is thus not known whether these probes would be suitable for imaging β cell mass in vivo.

Actually, selecting SUR1 as target for β cell-specific contrast agents seems compromised. SUR1 is maybe not specific enough for β cells imaging. Furthermore, sulfonylureas and glinides are very lipophilic drugs. It means that a high fraction of the injected dose binds to blood proteins. Even if the specificity for β cells were high, issues would be caused by protein binding, causing high background signal. The synthesis of derivatives keeping high affinity for SUR1 but displaying lower protein binding seems however promising.

6.3. VMAT2 targeting

Besides SUR1, vesicular monoamine transporter 2 (VMAT2) is another putative target for the specific labeling of β cells. VMAT2 is mostly known to be responsible for the storage and release of monoamines (dopamine, serotonin, etc.) in the sympathetic nervous system [83, 84] but is also expressed in pancreatic β cells [85-89]. VMAT2 binds dihydrotetrabenazine (DTBZ) and related compounds with high avidity. Several research groups have seen in radiolabeled DTBZ a potential probe for β cell imaging.

In preliminary studies using ¹¹C-DTBZ, different pancreatic uptakes were observed between normoglycemic and STZ induced diabetic rats [90]. Using the same probe, Souza et al. showed a progression in the decline of PET signal in a rat model of spontaneous T1D [91].

The drawback of this family of molecules is its high lipophilicity, leading to high uptake in the liver and making signal discrimination difficult [92]. Several derivatives of DTBZ were synthesized over the years. One of them was especially designed to shift its metabolic and excretion routes from liver to kidneys, in the hope of improving the quantification of β cell mass [93]. This DTBZ derivative maintained high affinity for VMAT2. A preliminary study in rats resulted in a much better imaging of the pancreas compared to “classical” DTBZ, as the uptake in the liver was almost completely avoided.

However, up to now, no imaging of diabetic animal models was performed using this probe. It is thus not known if better visualization of the pancreas allows quantitative assessment of β cells.

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18

Eriksson et al. chose to use a derivative of DTBZ, 9-(¹⁸F)fluoroethyl-(+)-dihydrotetrabenazine (¹⁸F-FE- (+)-DTBZ), as targeting moiety to assess β cell mass in pigs [95]. The relative uptake of ¹⁸F-FE-(+)-DTBZ in exocrine pancreas was too high to make it a suitable agent to assess β cell mass. Furthermore, pharmacokinetics showed uptake in pancreas, liver, and kidneys, the latter being the main excretion organs. Signal was also reported to accumulate in the bile duct. The too high binding of (¹⁸F)-FE-(+)- DTBZ to exocrine pancreas in comparison with endocrine pancreas and its accumulation in other organs in close proximity to pancreas made ¹⁸F-FE-(+)-DTBZ unsuitable for in vivo evaluation of β cell mass in pigs.

Using ¹⁸F-FP-(+)-DTBZ, Normandin et al. were able to detect a loss of β cells in T1D patients [96].

Furthermore, the uptake of ¹⁸F-FP-(+)-DTBZ was correlated with the amount of circulating C peptide, which is an indicator of β cell function. The same was concluded from a study from Freeby et al. [97].

Saisho et al. reported that VMAT2 was not so specific of pancreatic β cells as it is also found in some pancreatic polypeptide cells (PP cells) [87]. All studies of VMAT2 for the targeting of β cells in rodents were jeopardized in a study showing that VMAT2 is in fact absent from the β cells of rodents, whereas abundant in human and pig β cells [86]. This highlighted the fact that animal models should be carefully characterized to avoid misleading experiments.

All studies claiming that radiolabeled DTBZ could be used to image and quantify pancreatic β cells were further contradicted by a review from Blomberg et al. [98]. The authors noticed several biases in imaging studies based on radiolabeled DTBZ. First, VMAT2 is not as specific of β cells in the pancreas as it was originally thought, as it is also present in other pancreatic cell types, including PP cells. Second, the reduced volume of the pancreas in diabetic subjects was usually not taken into account by researchers when they claimed a lower uptake of DTBZ in imaging studies. PET suffers from a poor spatial resolution. Regions of interest smaller than 3 cm in diameter (which is the case of atrophic pancreas in diabetic patients) result in an underestimation of the radiotracer concentration.

Third, the authors claimed that, in long standing diabetic patients, the number of β cells should be close to zero. In all clinical studies, although much less radioactivity was observed in the pancreas of diabetic patients in comparison to controls, radioactive signal remained detectable in the pancreas.

Due to the pathology of the patients, this signal could not be attributed to β cell binding.

The use of VMAT2 as a target to perform β cell imaging remains controversial. Several studies claimed that it was specific enough to be used to assess β cell mass in patients while others asserted that VAMT2 was not the ideal target. It is to note that, to our knowledge, only DTBZ and DTBZ derivatives have been tested in human studies, due to its approved status for neuroimaging. Other

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Introduction

ligands of VMAT2 should probably be considered to assess if the targeting of VMAT2 is problematic or if it is rather DTBZ which does not show sufficient specificity for this transporter.

6.4. GLP-1R targeting

Glucagon-like peptide-1 receptor (GLP-1R) is a putative target for the development of β cell specific probes, because of its specific presence in β cells [99]. Glucagon-like peptide-1 (GLP-1) is an incretine hormone released by the intestine in response to food digestion. Its sequence is presented in Figure 7. Among other effects, GLP-1 slows gastric emptying and induces insulin release, regulating glycaemia [100]. Administration of GLP-1 in T2D patients regulates glycaemia without risk of causing hypoglycemia, which is a common side effect of insulinotropic drugs such as sulfonylureas.

Administration of GLP-1 or analogues also results in a reduction of food intake and body weight. All these characteristic make GLP-1 an appealing way of normalizing the glycaemia of T2D patients. The use of GLP-1 itself as a drug is impaired by its very short half-life (less than 2 min [101, 102]) due to rapid degradation by dimethyl-peptidyl-peptidase-IV (DPP-IV). To reach and maintain therapeutic levels, GLP-1 should be infused in a continuous way. This poor pharmacokinetic profile led to the search of GLP-1R agonists with prolonged half-life in the hope of regulating the glycaemia of T2D patients [103-105]. Exendin-4 was identified as a potent agonist of GLP-1R, with higher affinity for this receptor than GLP-1 itself. Synthetic analogues of GLP-1 have also been developed. Substitution of a single amino acid, like in liraglutide, is sufficient to escape DPP-IV metabolism, resulting in a prolonged half-life and a once daily administration. CJC-1131 is another example of analogue with prolonged half-life.

GLP-1: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

Exendin-3: HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Exendin-4: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Exendin-4(9-39): DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

Figure 7. Sequences of GLP-1, the native GLP-1R ligand, and exendins, isolated from Gila monster venom [106]. The conserved aminoacids among all peptides are in bold.

Exendin-4 (Figure 7) was isolated from Heloderma suspectum (Gila monster) venom [106]. It is an

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Introduction

20

Specific exendin-4 uptake was found in pancreas, stomach and lungs for both mice and rats.

Significant uptake in the spleen was also observed in rats. High uptake was observed in the kidneys in both species, but it was not reduced by the previous injection of cold exendin-4, showing that it was non-specific uptake. Real-time imaging of radiolabeled exendin-4 could be performed by SPECT.

Mukai et al. used radiolabeled exendin-4(9-39) with ¹²⁵I for imaging β cells in mice [109].

Measurement of the quantity of uptake was performed in vitro and ex vivo. The highest signal observed was in the lungs. High signals were also observed in the pancreas and liver. Co- administration of cold exendin-4(9-39) decreased the signal in pancreas and lungs, showing that binding in these organs was receptor-mediated. The authors further showed that signal in the pancreas was due to the probe binding pancreatic islets and not the exocrine part of pancreas.

Wang et al. also used exendin-4(9-39) but labeled with ¹⁸F on Lys27 [110]. Biodistribution study showed a high uptake of exendin-4(9-39) in kidneys and myocardium. Uptake in the pancreas was low. These differences with the previously mentioned study were attributed to the labeling site.

Lys27 seems not to be the best labeling site in exendin-4(9-39). This assessment was confirmed for exendin-4 by Jodal et al., who compared the affinity of three different exendin-4 derivatives, which differed in the binding site of 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA), a chelator [111]. The derivative with the lowest affinity for GLP-1R was the one on which DOTAGA was coupled to Lys27. The other derivatives, with DOTAGA coupled to Lys12 or Lys40, kept sufficient affinity for GLP-1R. These studies underline the fact that all modifications of a ligand should be followed by in vitro binding studies in order to check that the chemical modification of the ligand has not modified its affinity for its target. The likelihood of exendin-4(9-39) as a potential targeting moiety for in vivo imaging of pancreatic β cells is anyway dubious as it was shown that this GLP-1R antagonist has very low affinity for human β cells [112].

Reiner et al. used a near-infrared fluorescent exendin-4 analogue (E4K12-Fl) to perform intravital microscopy in mice [113]. It tagged specifically pancreatic islets both in vitro and in vivo but no biodistribution study was performed using this probe. Using another fluorescent imaging probe, E4×12-VT750, the same group could successfully image pancreatic islets intravitally using endoscopy [114]. The blood half-life of this compound was very short (85 s). Regarding biodistribution, the highest amounts were found in the kidneys and liver. The same observation was made for a ¹⁸F-labeled exendin-4 analogue (¹⁸F-E4Tz12) [115]. In this latter study, the blood half-life of the exendin-4 analogue was 6.8 min.

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Introduction

Selvaraju et al. were successful in imaging pancreatic islets using ⁶⁸Ga-labeled exendin-4 [116]. High uptake in the kidneys of rodents was observed, making quantification of the radioprobe in the pancreas difficult. Ex vivo quantification was needed to assess that pancreatic uptake in diabetic rats was lower than in euglycemic ones. In non-human primates, trapping of radiolabeled exendin-4 was observed in the renal cortex. Specific uptake was observed in the pancreas. Pursuing the study of this probe, Nalin et al. performed a study in which ⁶⁸Ga-labeled exendin-4 was injected into normoglycemic and diabetic pigs [117]. The pancreatic uptake of radiolabeled exendin-4, although competition assays had proven that it was specific, did not differ between the two groups of pigs. It suggests that GLP-1R is also present in the pancreas of diabetic pigs. Furthermore, the administration of exendin-4 induced tachycardia in pigs. This makes pigs an unsuitable model for the study of exendin-4 derivatives as imaging probes.

Exendin-4 was also labeled with ⁶⁴Cu and cyanine 5, allowing visualization by both PET and fluorescence [118]. In this study, the pancreatic uptake was negligible.

Pancreatic islets could be visualized by PET in a mouse model of islet transplantation into the liver using ¹⁸F- and ⁶⁴Cu-labeled exendin-4 [119, 120]. Signal increase in the liver was not completely proportional to the number of transplanted islet, as liver showed background. High uptake was observed in the kidneys, as previously observed, although renal uptake of ¹⁸F-labeled exendin-4 was slightly lower than that of ⁶⁴Cu-labeled exendin-4.

Kirsi et al. confirmed that ⁶⁴Cu- and ⁶⁸Ga-labeled exendin-4 were taken up by β cells in mice [121].

However, the highest uptake was once more observed in the kidneys. The pancreas could not be visualized by PET scan using these exendin-4 derivatives.

Pancreatic islets could maybe be visualized by MRI using exendin-4-conjugated superparamagnetic iron oxide nanoparticles [122]. Although most research for finding a β cell-specific imaging probe has focused on PET as an imaging modality, some groups have conducted research focusing on MRI.

Although MRI is less sensitive than PET, it has the advantages of offering high spatial resolution and not requiring the use of radioprobes. Vinet et al. conjugated exendin-4 to iron-oxide nanoparticles [123]. Wang et al. did the same [124]. In vivo, as expected, a drop of pancreatic T2

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Introduction

22

Brom et al. performed the first clinical study using ¹¹¹In-labeled exendin-4. The pancreatic uptake of diabetic patients and control subjects was compared by SPECT/CT. Pancreatic uptake was observed in both groups with high interindividual variation. No clear correlation could be established between glycemic status of the patients and pancreatic uptake of exendin-4. The radioactive signal was in average lower in diabetic patients than in healthy controls but it was not possible from the imaging session to tell if a patient was diabetic or not.

6.5. Other targets of β cells

Malaisse et al. tried to target glucose transporter 2 (GLUT2) using tritiated D-mannoheptulose or analogues [125]. The hypothesis of the authors was that β cells from T2D patients might have a defect in glucose transport, phosphorylation, or further catabolism. This defect would lead to a decrease in ATP production, which would be insufficient to induce the closing of ATP-sensitive K⁺- channels and thus not triggering the release of insulin. D-mannoheptulose is specifically taken up by GLUT2, which is expressed specifically in pancreatic β cells and hepatocytes. Targeting GLUT2 might be interesting for imaging native pancreatic islets. However, it would not allow imaging transplanted islets, because in this case the islets are localized in the liver. Malaisse et al. showed that lower radioactive signal was measured from the pancreas of STZ-diabetic rodents compared to control rodents using tritiated D-mannoheptulose [125]. However, the use of radioactive D-mannoheptulose seems to be complicated to implement for clinical use.

Observing that imaging probes targeting “classical” β cell markers did not give the expected results in terms of specificity, Sako et al. chose another target: somatostatin receptors [126]. Somatostatin receptors are highly expressed in β cells. Radiolabeled octreotide (a known ligand of somatostatin receptor) resulted in high uptake in kidneys and pancreas. Pancreatic uptake was reduced upon competition experiment with cold octreotide and also in STZ-induced diabetic rats. The major issue with targeting somatostatin receptors is that, although highly expressed in β cells, they are also present in other pancreatic endocrine cell types, such as  and  cells [127, 128]. Octreotide thus lacks β cell specificity.

Eriksson et al. considered imaging β cell through the targeting of DOPA decarboxylase (DDC) by ¹⁸F- labeled DOPA [129] or 5-hydroxy-L-¹¹C-tryptophan (¹¹C-5-HTP) [130]. The rationale behind the use of serotoninergic markers is that the endocrine pancreas shares several features with the nervous system, including dopaminergic and serotoninergic systems. These systems are absent from the exocrine part of the pancreas. DOPA has been used in previous studies to help staging insulinomas [131] and localizing the focal form of congenital β cell hyperplasia [132, 133]. Eriksson et al. showed the expression of DDC in pancreatic β cells [129]. They also evidenced that DDC expression was

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Introduction

maintained in transplanted islets. Furthermore, they showed that ¹⁸F-DOPA was taken up by pancreatic islets in a saturable and proportional way. However, it was not shown whether ¹⁸F-DOPA was specific enough for β cell imaging or if it was taken up in the exocrine pancreas or in other tissues, which could result in confusing results, preventing the assessment of β cell mass.

5-hdroxytryptophane (5-HTP) is a precursor of serotonin, metabolized by DDC. Eriksson et al. showed that, even if present in  cells, the expression of serotonin was decreased in the pancreatic islets of diabetic patients [130]. High uptake of ¹¹C-5-HTP was seen in the pancreas of rodents compared to other organs. ¹¹C-5-HTP uptake was reduced in STZ-induced diabetic rats, indicating that targeting of the serotoninergic systems might be useful in the search for imaging pancreatic islets.

Burtea et al. used peptides to target (FXYD2)a, a small ion transport regulator of the Na⁺-K⁺- ATPase [134]. This protein is specifically expressed in pneumocytes and pancreatic β cells [135]. The peptide sequence with the highest affinity for (FXYD2)a was conjugated to ultrasmall superparamagnetic iron oxide (USPIO) [134]. In vitro, the targeted USPIO were able to make as low as 156 β cells visible by MRI. In nude mice bearing (FXYD2)a positive tumors, the targeted USPIO were specifically taken up by tumor cells. No pancreatic uptake could be assessed as (FXYD2)a is not expressed in rodents.

6.6. Imaging diabetes using non-β cell specific markers

Naish et al. wanted to assess whether a difference could be seen between healthy and diabetic volunteers after intravenous glucose challenge using MRI [136]. They hypothesized that, since blood flow in the pancreas is supposed to increase with blood glucose concentration, and knowing that diabetic patients have a delayed response, a difference could probably be observed regarding the kinetic parameters between both groups. A difference was observed in the imaging of the pancreas before glucose challenge. The endocrine pancreas displayed different appearance in diabetic patients compared to controls (lower T1 in diabetic patients). The authors hypothesized that it was probably the result of compositional differences in the pancreas of diabetic patients, such as higher protein content or fat infiltration. Kinetic parameters between the two groups were the same upon glucose challenge.

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24

MRI device, Lamprianou et al. showed that individual islets could be visualized ex vivo and in vivo [138]. In these conditions, MRI had a tendency to underestimate the number and size of pancreatic islets in control mice. However, the number of islets containing β cells in STZ-induced diabetic mice was overestimated. This was due to uptake of manganese by  cells.

MRI without specific contrast agent targeting β cells would suffer from a lack of sensitivity to quantify a small change in β cell number. Contradicting this statement, in a retrospective study of manganese- enhanced MRI images, Botsikas et al. were able to differentiate T2D from normoglycemic patients [139]. The pancreatic contrast enhancement of diabetic patients upon manganese infusion was significantly lower than that of normoglycemic patients. In a later study, Meyer et al. were able to follow the course of diabetes development in mice [140]. Manganese MRI was performed some minutes after intraperitoneal administration of glucose, inducing insulin release. MRI imaging was correlated with glycaemia after glucose tolerance test. In mice subjected to high fat diet, MRI signal increased over the first weeks, corresponding to β cell compensation. With time, MRI signal decreased, corresponding to β cell exhaustion and succeeding function and/or mass decrease and diabetes. Manganese MRI seems a promising tool for the assessment of β cell mass and function.

Some issues need to be further addressed, such as manganese-related toxicity. Longitudinal prospective studies in other animal models and eventually in humans should also be performed.

Zn²⁺ is present in insulin-containing granules [141, 142] and therefore cosecreted with insulin. Taking advantage of this, Lubag et al. were able to image the increase in zinc secretion upon glucose stimulation [143]. To do so, they used a zinc-sensitive MRI T1 contrast agent: GdDOTA-diBPEN. After glucose injection, the authors were able to detect Zn²⁺-specific contrast enhancement in the pancreas. Contrast enhancement was absent from STZ-induced diabetic mice. It was increased in mice submitted to high fat diet, which induced increased β cell function to compensate for insulin resistance. Zn²⁺ sensing could be a potent tool to estimate β cell function in vivo.

7. Conclusion

Diabetes is a disease which concerns an increasing number of persons worldwide. Mainly due to lifestyle modifications, T2D is more and more prevalent. Paradoxically, T2D is a poorly understood disease. It is not known yet why some patients develop T2D and other ones not. The role of amyloids in T2D should also be elucidated.

T2D treatment consists in improving insulin sensitivity through lifestyle modification and drug treatment, and, if not sufficient, in increasing insulin secretion. Ultimately, insulin administration can

Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials

Thesis

Reference

Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials

Development of Contrast Agents for Imaging Amyloids in Type 2 Diabetes –

From Chemical Synthesis to In Vivo Trials

Remerciements

Table of contents

Preface

Introduction

Type 2 diabetes:

why imaging is needed

Introduction. Type 2 diabetes: why imaging is needed

1. Regulation of glycaemia

2. Dysregulation of glycaemia

3. Treatment of T2D diabetes

4. Mechanisms of β cell dysregulation

5. Involvement of amyloids in diabetes

5.1. Islet amyloid polypeptide

5.2. Amyloids in diabetes

6. Imaging diabetes

6.1. Targeting β cells with antibodies

6.3. VMAT2 targeting

6.4. GLP-1R targeting

6.5. Other targets of β cells

6.6. Imaging diabetes using non-β cell specific markers

7. Conclusion