Amyloid Imaging PET Ligands as Biomarkers for Alzheimer’s Disease, Preclinical Evaluation

Alzheimer’s disease (AD) is the most common form of dementia in the aging population. It is a complex disease that affects many brain functions and is characterized by a progressive impairment of cognitive abilities, such as memory, learning and social skills. AD was first described over hundred years ago by the German psychiatrist Dr. Alois Alzheimer, reporting of the in due course characteristic pathological changes postmortem discovered in his 56 years old patient Auguste D (Alzheimer, 1907). The disease is obviously devastating for the patients and affects everyday life for both patients and their families, but it also generates economical challenges for the heath-care system and the society as the elderly population is growing (Wimo & Winblad, 2008). Although research regarding AD is intensive worldwide and new results do get a greater understanding of the causes of the disease, the exact mechanisms and underlying cause behind AD are still unsolved. The disease progresses over decades leading to premature death. There are no diseasemodifying therapies for AD available, and the current treatment might provide symptomatic relief and slower disease progression.


Introduction
Alzheimer's disease (AD) is the most common form of dementia in the aging population. It is a complex disease that affects many brain functions and is characterized by a progressive impairment of cognitive abilities, such as memory, learning and social skills. AD was first described over hundred years ago by the German psychiatrist Dr. Alois Alzheimer, reporting of the in due course characteristic pathological changes postmortem discovered in his 56 years old patient Auguste D (Alzheimer, 1907). The disease is obviously devastating for the patients and affects everyday life for both patients and their families, but it also generates economical challenges for the heath-care system and the society as the elderly population is growing (Wimo & Winblad, 2008). Although research regarding AD is intensive worldwide and new results do get a greater understanding of the causes of the disease, the exact mechanisms and underlying cause behind AD are still unsolved. The disease progresses over decades leading to premature death. There are no diseasemodifying therapies for AD available, and the current treatment might provide symptomatic relief and slower disease progression.
The new imaging technologies based on structural and functional processes in the brain enable early diagnosis and may also differentiate between types and severity of neurodegeneration. Mild cognitive impairment (MCI) is closely related to AD and can be considered as a transitional stage between normal cognition and dementia. MCI is characterized by either isolated memory impairment or impairment in several cognitive domains, but not of sufficient severity to meet diagnostic criteria for AD (Petersen et al., 2001). Although MCI is associated with an increased risk of developing AD, about half of MCI patients progress to AD at a rate of approximately 10%-15% per year, but approximately half do not develop AD even after follow-up periods as long as 10 years (Ewers et al., 2010;Ganguli & Petersen, 2008;Ganguli et al., 2011).

Positron Emission Tomography -Current Clinical and Research Aspects 256
A clinical diagnosis of AD is assessed by several investigations including medical history and neuropsychological criteria such as the NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's disease and Related Disorders Association) (McKhann et al., 1984), but a definite diagnosis of AD can only be done postmortem. Therefore, development of efficient prevention therapies for AD would greatly benefit from a diagnosis at a prodromal stage of the disease. Positron emission tomography (PET) is considered a unique diagnostic tool that enables early detection of pathology that facilitates prediction of AD and following the progression in vivo . Highly specific and sensitive biomarkers are of great value to assess therapeutic efficacy clinically and in terms of clearance of histopathological lesions and decelerated neurodegeneration.

Pathology
AD is characterized neuropathologically by cerebral neuronal loss and the main histopathological hallmarks are intracellular neurofibrillary tangles consisting of hyperphosphorylated tau and extracellular β-amyloid (Aβ) deposits found as senile plaques in the brain parenchyma. These plaques are formed from insoluble A peptides, fibrils and oligomers, that originate through processing of the larger Amyloid Precursor Protein (APP) (Selkoe & Schenk, 2003).

Pathological protein, Aβ
The A containing plaques induce cell death and are formed during early disease progression long before the patient feels any symptoms in the prodromal phase of the disease (Braak et al., 1998;Ingelsson et al., 2004) (figure 1). Whether the formation of plaques is the major underlying event of the neurogenerative process in AD is a matter of debate. The amyloid cascade hypothesis is today one of the leading theories and suggests that Aβ depositions in the brain are the central lesion in the pathogenesis of AD (Korczyn, 2008). However, although the A plaques appear in the brain prior to the neurofibrillary tangles, it has been demonstrated that the neurofibrillary tangles are better correlated with neuronal loss and cognitive decline than to A plaques (Schönheit et al., 2004). Furthermore, plaques are present in cognitively normal elderly subjects without any signs of neurodegeneration (Hellström-Lindahl et al., 2004;Villemagne et al., 2008). Nonetheless, there is evidence that amyloid are associated with severity of dementia (Näslund et al., 2000).
Since it became possible to measure A in vivo in the brain by PET (Klunk et al., 2004), Aβ plaques has became a disease-defining pathologic marker of AD. However, the plaques might play an important role in the pathophysiological mechanisms of the disease. Various strategies to prevent aggregation, increase Aβ clearance or alter APP metabolism sought for the development of new treatment strategies of AD

Amyloid imaging
Amyloid imaging using PET has developed rapidly in recent years. With the development of the amyloid-imaging PET tracer [ 11 C]PIB (N-methyl-[ 11 C]-2-(4-methylaminophenyl)-6hydroxy-benzothiazole), a derivate of the widely used dye for plaques thioflavin-T, the possibility to study the presence of amyloid in the brains of AD patients in early stages of the disease with PET became an important tool (Archer et al., 2006;Klunk et al., 2004;Mintun et al., 2006;Nordberg, 2004). [ 11 C]PIB has a high affinity for aggregated Aβ, crosses the blood brain barrier (BBB) easily, has low toxicity and makes it possible to visualize plaque and vascular amyloid deposits in the brain. [ 11 C]PIB is today the most commonly used radioligand in the assessment of amyloid plaques in the living human brain using PET, although several new compounds are under development (see table 1) (Henriksen et al., 2008;Ono, 2009). However, the exact nature of the mechanism for the binding of [ 11 C]PIB and other amyloid ligands to Aβ in the form of β-pleated sheets is still unknown.
One of the central issues regarding the PIB-amyloid binding in the AD brain is thus to understand the mechanisms and binding properties of the ligand. It has been demonstrated that [ 11 C]PIB has high affinity for fibrillar Aβ and binds to the β-sheet structure of the Aβ fibrils . It has also been shown that [ 11 C]PIB in nanomolar concentrations does not bind to neurofibrillary tangles (Bacskai et al., 2007;Ikonomovic et al., 2008;Kadir et al., 2011;Rosen et al., 2011).

Neuroinflammation (gliosis)
The widespread cellular degeneration and neuronal loss in AD is accompanied by reactive gliosis (Kadir et al., 2011). Close to the amyloid plaques both astrocytes and microglia are clustered, probably as a result of a starting neuroinflammatory process (Akiyama et al., 2000). Activated microglia accumulate in order to remove the plaques by phagocytosis (Streit, 2004). However, the cluster of microglia produce a number of various neurotoxic substances thereby inducing inflammatory processes, possibly contributing to the neurodegeneration process (Akiyama et al., 2000;Streit, 2004). Astrocytes proliferate as a reaction to neuronal insults, and astrogliosis is thus also a phenomenon in the AD brain (Porchet et al., 2003). Moreover, astrocytes may impair the efficacy of microglia to remove the plaques (Akiyama et al., 2000).
Molecular imaging of the entities involved in neuroinflammation process that follows plaque formation is today possible, as a result of the development of a number of new different radiotracers labeling microglia or astrocytes (Akiyama et al., 2000). For the study of activated microglia, ligands labeling the peripheral benzodiazepine receptor (translocator protein 18 kDa) are used, such as PK11195 and analogues (Banati, 2002;Cagnin et al., 2002). Astrocytes, which also are involved in the neuroinflammatory process, may be studied using a tracer labeling the monoamine oxidase B (MAO-B) enzyme, such as deprenyl. However, deprenyl is rapidly metabolized in vivo, which can be diminished by using a dideuteriated deprenyl (Bergström et al., 1998;Fowler et al., 1988;Logan et al., 2000).

Chemistry
The highly conjugated fluorescent staining agents Congo red and chrysamine G (figure 2) were the first selected target molecules for the development of Aβ imaging ligands. However, those compounds were not suitable in vivo due to their low brain penetration ability (Klunk et al., 1994;Klunk et al., 1995). A number of other compounds with various chemical structures and promising Aβ binding abilities have been developed during last couple of years (Henriksen et al., 2008;Ono, 2009). Among those the most successful one so far is [ 11 C]PIB or [ 11 C]6-OH-BTA-1. All these compounds are small molecules with a central lipophilic group, a secondary or tertiary amine at one end and a polar or non-polar group at the other end. The terminal amine group and the middle lipophilic group which are the common features of all so far published molecules might have some impact on their amyloid binding properties. A list of available Aβ imaging PET ligands together with their chemical names and structures is presented in table 1.

Short names Chemical names
Structures

Short names Chemical names
Structures

Synthesis of 11 C-and 18 F-labeled Aβ binding PET ligands
The Aβ binding PET ligands have been labeled with the ideal positron emitting radionuclides 11 C or 18 F, and the terminal amino or hydroxyl group of the molecule is mainly selected as the suitable 11 C or 18 F-labeling position. In case of [ 18 F]flutemetamol, the labeling strategy was different and one of the aromatic ring of the lipophilic part of the molecule was selected for that purpose.

Synthesis of 11 C-labeled ligands
The commonly used method for the synthesis of 11 C-labeled Aβ binding PET ligands is the methylation using [ (Shimadzu et al., 2004) and [ 11 C]MeS-IMPY (Seneca et al., 2007).

Evaluation of amyloid binding PET ligands
An important part of the development of new potential tracers for visualizing amyloid with PET is the preclinical evaluation of these compounds. Several complementary in vitro and in vivo animal techniques are used for the evaluation which has to be performed before the new tracer can be given to humans. These techniques aim at finding compounds with optimal affinity for the target (Aβ), while having as low binding as possible to other targets. Moreover, the kinetics needs to be suitable for PET, i.e. the binding equilibrium should be reached in the time frame of a PET experiment. These and other important criteria for a suitable PET ligand need to be studied in the preclinical evaluation phase.
Homogenate binding assay is a standard technique for the determination of affinity and selectivity of any compound. However, many of the parameters obtained in homogenate binding studies can also be obtained using in vitro cryosection autoradiography. After the process of evaluating the binding properties of a compound to be developed into a tracer to be used in PET or SPECT, homogenate binding studies may be used for determining of the absolute density of amyloid plaques in a tissue homogenate.
Autoradiography is a commonly used method in the study of radioligands for use in PET or SPECT, as direct comparisons of in vitro autoradiography images can be made with in vivo PET images labeled with 11 C and 18 F. Autoradiography gives information on distribution, selectivity and nonspecific binding, as well as a number of kinetic parameters which all are parameters indicative of usefulness for in vivo molecular imaging. With regard to visualization of amyloid plaques in vivo, cryosection autoradiography is often used in comparison of different potential ligands, for example in structure activity relationship studies. Furthermore, the much higher resolution with in vitro autoradiography as compared with in vivo PET makes the former technique a complementary technique.
It is obvious that certain parameters can be determined solely by an in vivo administration. For example, of great importance with regard to visualization of amyloid plaques in vivo is the extent of BBB penetration. In this case, "ex vivo" autoradiography could be preferred. Here "ex vivo" is defined as administration of the radioligand in vivo followed by autoradiography in vitro, and at least some of the above mentioned parameters can be determined. On the other hand, some parameters might be very difficult, if not impossible, to determine in vivo, such as to determine if an interaction really is saturable or not. Many of the compounds developed as tracers cannot be given in high enough concentrations to determine this, due to known toxicity or lack of toxicity information. For example, in spite of the numerous clinical studies performed with [ 11 C]PIB, saturating amounts of [ 11 C]PIB cannot be given to living subjects, and from in vivo PET it is not known if all [ 11 C]PIB binding can be blocked by excess unlabeled PIB in vivo. Using in vitro autoradiography the binding was blocked totally by adding 1000-fold higher concentration with unlabeled PIB (figure 3) Svedberg et al., 2009).

In vitro binding of PIB to postmortem AD brain
Binding of [ 3 H]PIB binding to insoluble Aβ deposits in postmortem brain obtained from AD patients has been reported by several investigators. In vitro studies have shown that the Kd for [ 3 H]PIB binding to AD brain (3-4 nM) is similar to the Kd for binding synthetic Aβ1-40 and Aβ1-42 fibrils (1 nM) (Fodero-Tavoletti et al., 2007;Ikonomovic et al., 2008;Kadir et al., 2011;Klunk et al., 2005). In some AD and control brain homogenates as well as with Aβ synthetic fibrils a lower-affinity [ 3 H]PIB binding site with Kd values of 75-250 nM have been observed (Ikonomovic et al., 2008;Klunk et al., 2005). However, these low-affinity sites would not contribute significantly to in vivo binding at [ 11 C]PIB concentrations around 1 nM. No significant correlation of [ 3 H]PIB binding with soluble Aβ peptides has been observed (Ikonomovic et al., 2008;Klunk et al., 2005). The requirement that Aβ be in fibrillar form for PIB binding was verified by showing that the highly fluorescent PIB derivative 6-CN-PIB labeled both Aβ42 -and Aβ40-immunoreactive plaques whereas no labeling was detected in tissue sections pre-treated with formic acid which disrupts β-pleated sheets (Ikonomovic et al., 2008).
Although several clinical PET studies with [ 11 C]PIB have been performed Engler et al., 2006;Klunk et al., 2003;Klunk et al., 2004), in vitro evaluation studies have provided us with important new information. In agreement with others (Fodero-Tavoletti et al., 2007;Klunk et al., 2005;Rosen et al., 2011) our recent in vitro study (Svedberg et al., 2009) revealed significantly higher binding of PIB in AD brain compared to control brain when using [ 11 C]PIB autoradiography and [ 11 C]PIB radioligand assay. Moreover, we observed no specific [ 11 C]PIB binding in the cerebellum which is often used as a reference region for quantification of [ 11 C]PIB retention in PET studies. For example, the distribution of [ 11 C]PIB was investigated using autoradiography on tissue from patients that suffered from AD (Svedberg et al., 2009), which demonstrated that the binding is confined to external layers of the cerebral cortex, which due to a lower resolution is not seen in PET (figure 4). By using a combination of autoradiography and histochemical techniques Lockhart et al. (2007) showed that PIB, in addition to binding senile plaques (dense core plaques) also bound to cerebral amyloid angiopathy (CAA) and some diffuse plaques. In contrast to senile plaques where Aβ42 are much more abundant (Hellström-Lindahl et al., 2004;Näslund et al., 2000), the major amyloid peptide species in CAA is Aβ40 with less amounts of Aβ42 (Mann et al., 1996). CAA is present in 80% or more of AD patients (Jellinger, 2002) and may therefore contribute significantly to the in vivo [ 11 C]PIB signal and in vitro binding of PIB (Bacskai et al., 2007;Johnson et al., 2007;Svedberg et al., 2009). PIB binds only weakly to Aβ deposits in nonhuman species (Klunk et al., 2005;Rosen et al., 2011). Recently, Rosen et al (2011) showed that despite levels of Aβ in cortical extracts that sometimes exceeded those in AD brain, high-affinity [ 3 H]PIB in vitro binding in nonhuman primates (aged chimpanzees, rhesus macaques, and squirrel monkeys) was strikingly less than that in humans with AD. Similarly, less [ 3 H]PIB binding was detected in homogenates of transgenic PS1/APP mouse brain compared to AD brain, despite higher Aβ levels (Klunk et al., 2005). A substantial difference in the intensity of [ 11 C]PIB labeling between AD brain and APP transgenic mice has also been shown by ex vivo autoradiography (Maeda et al., 2007).

In vivo animal studies
Two major in vivo modalities are of importance in the evaluation of new PET ligands. One is organ distribution studies in normal mice or rats, which gives information on to where the compound and its metabolites distribute. Organ distribution studies are also used in calculation of dosimetry, i.e. how much of a radiolabeled compound can be given without reaching radioactive doses that may risk damages due to radiation.
A more obvious modality is the use of animal PET. In order to study binding to Aβ plaques transgenic animals are required, as plaques are not found naturally in old rats or mice (Philipson et al., 2010). Transgenic mouse models have become an important and valuable research tool in neurodegenerative disorders like AD. Attempts to model and study in detail longitudinal pathological processes in living brains and molecular mechanisms involved in the pathogenesis of the disease can provide insight into disease progression in AD patients.
Transgenic mice of different types have been developed, all with amyloid plaques in the brain (Elder et al., 2010;Lannfelt et al., 1993;Morrissette et al., 2009). In the early animal PET studies using transgenic animals no binding of [ 11 C]PIB was seen, in spite of a high amyloid load in the animal brains (Klunk et al., 2005;Toyama et al., 2005;Ye et al., 2006), The inconsistency between AD patients and transgenic mice models was for long unclear and explained by fewer binding sites or lower binding affinity of [ 11 C]PIB for Aβ plaques in transgenic mice (Klunk et al., 2005;Toyama et al., 2005) and/or that the Aβ containing plaques in the mice resembles synthetic Aβ, where low [ 11 C]PIB binding was demonstrated (Ye et al., 2006). It was demonstrated that intrinsic mouse Aβ is formed and deposited in significant amounts in the brain of an AD mouse model and is deposited together with human Aβ, and this might also explain the low efficacy of PIB binding in transgenic mice brain tissue (van Groen et al., 2006). Few years later it was demonstrated that the specific radioactivity of the tracer significantly contributed to the detection of the amyloid deposits in the mouse brain. High-level retention of [ 11 C]PIB in APP transgenic mice brain regions known to contain amyloid was obtained when high-specific activity [ 11 C]PIB was administrated (Maeda et al., 2007). It might also depend on the type of transgenic animal used, as ex vivo studies using [ 3 H]PIB and a new transgenic animal model, ARTE10, clearly showed intense labeling of the amyloid in these animals (Willuweit et al., 2009).
Senile plaques are generally enriched in Aβ 1-42 species and are additionally subjected to a wide range of post-translational and post-deposition modifications, such as N-and Cterminal truncation, oxidation, and isomerization (Lockhart, 2006). The N-terminally truncated and modified Aβ, AβN3-pyroglutamate, has been identified in AD brain (Wirths et al., 2010) and suggested to be a major contributor to PIB binding (Maeda et al., 2007). Compared to AD brain, this Aβ subtype is not so abundant in transgenic mice (Güntert et al., 2006;Kawarabayashi et al., 2001), and the levels of AβN3-pyroglutamate may therefore provide another plausible explanation for the difference in [ 11 C]PIB binding between humans and mice.

Clinical PET
[ 11 C]PIB, developed in collaboration between Uppsala University in Sweden and University of Pittsburgh School of Medicine, PA, USA, was the first tracer to detect Aβ in the brain of living patients (figure 5) (Klunk et al., 2004). The results demonstrated an increased [ 11 C]PIB retention in several brain regions known to contain Aβ in AD and none in controls (Klunk et al., 2004). The [ 11 C]PIB signal in the cortex was approximately 1.5-2 fold higher in the AD patients compared to the control subjects (Klunk et al., 2004). Interestingly enough, postmortem studies of the same patients that showed elevated [ 11 C]PIB retention in vivo during their life correlates significantly with regional in vitro measures of Aβ pathology found at autopsy (Ikonomovic et al., 2008;Kadir et al., 2011). Since the study reported by Klunk et al. (2004) (Wong et al., 2010). The results revealed an increase in [ 18 F]AV-45 retention in the cortical gray matter in the AD patients, areas that expected to have high Aβ accumulation and none in the control subjects (Wong et al., 2010) similar to previously reported for [ 11 C]PIB.

Conclusion
The development of plaques are very early events in the course of events leading to AD (Ingelsson et al., 2004;Långström et al., 2007), and imaging plaques in early stages of the disease would be of great value for the treatment. However, the "ceiling effect", i.e. that the amount of amyloid plaques reaches maximum already early in the disease (Engler et al., 2006;Nordberg, 2007), makes it difficult to follow the slow progression of the disease using in vivo imaging of amyloid plaques. The large number of compounds that are under development or are today used in in vivo PET or SPECT investigations of amyloid plaques all aim at visualizing the same entities, and will hardly overcome this problem. New ligands that visualize the formation of plaques earlier in the course of disease are therefore needed. The presently available compounds bind to plaques, but some are also binding to earlier stages in the formation of plaques, such as to fibrils and oligomers, and might therefore be of great value for finding new treatment of AD and thus also for the individual patients.
There is also great interest in finding a specific radioligand for the visualization of neurofibrillary tangles, which is another hallmark seen in AD. Some of the ligands binding to the amyloid plaques also bind to some extent to tangles, and further development of these compounds may lead to ligands also labeling tangles (see e.g Barrio et al., 2008;Okamura et al., 2005;Shoghi-Jadid et al., 2002;Åslund et al., 2009). The development of tracers labeling tangles should be complementing the tracers labeling amyloid discussed above.
It can be concluded that the use of PET in the evaluation of AD has alleviated the diagnosis of the disease considerably. The development of new tracers with even better properties and tracers labeling amyloid fibrils and neurofibrillary tangles is therefore still a highly prioritized research area.

Acknowledgment
We are grateful for valuable discussions with Prof. Bengt Långström and our colleagues at Uppsala University. This study was performed when authors OR and HH were employed at Uppsala Applied Science Lab, GE Healthcare. We are grateful to a SAMBIO funding from Vinnova, Sweden.

Positron Emission Tomography -Current Clinical and Research
Aspects This book's stated purpose is to provide a discussion of the technical basis and clinical applications of positron emission tomography (PET), as well as their recent progress in nuclear medicine. It also summarizes current literature about research and clinical science in PET. The book is divided into two broad sections: basic science and clinical science. The basic science section examines PET imaging processing, kinetic modeling, free software, and radiopharmaceuticals. The clinical science section demonstrates various clinical applications and diagnoses. The text is intended not only for scientists, but also for all clinicians seeking recent information regarding PET.