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Characterization of Diagnostic Tools and Potential Treatments for Alzheimer’s Disease: PET ligands and BACE1 inhibitors
Stockholm University, Faculty of Science, Department of Neurochemistry.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands.

We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands.

Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials.

Place, publisher, year, edition, pages
Department of Neurochemistry, Stockholm University , 2016.
Keyword [en]
Alzheimer’s disease, Diagnostics, Treatment, PET ligand, BACE1
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-134894ISBN: 978-91-7649-531-5ISBN: 978-91-7649-532-2OAI: oai:DiVA.org:su-134894DiVA: diva2:1040100
Public defence
2016-12-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Submitted.

Available from: 2016-11-23 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
List of papers
1. AZD2184: a radioligand for sensitive detection of beta-amyloid deposits
Open this publication in new window or tab >>AZD2184: a radioligand for sensitive detection of beta-amyloid deposits
2009 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 108, no 5, 1177-1186 p.Article in journal (Refereed) Published
Abstract [en]

The presence of beta-amyloid plaques in brain is a hallmark of Alzheimer's disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Positron emission tomography (PET) radioligands such as Pittsburgh compound B ([(11)C]-2-(3-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol) (PIB) binds selectively to beta-amyloid and are promising new tools supporting the clinical diagnoses of AD. In addition, such methodology may be useful for evaluation of new drugs aiming at reduction of amyloid plaque load. The objective of this study is to develop a new amyloid selective PET radioligand with higher signal-to-background ratio when compared with existing amyloid PET ligands. The lead compound, AZD2184, (2-[6-(methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol) was found to have high affinity for amyloid fibrils in vitro (K(d): 8.4 +/- 1.0 nM). Two minutes after i.v. administration in rats, about 1% of the dose was in brain. In vitro autoradiography on cortical brain sections from amyloid-beta precursor protein/presenilin 1 (APP/PS1) mice and AD patients showed that while [(3)H]AZD2184 and [(3)H]PIB are mutually displaceable, [(3)H]AZD2184 displays a higher signal-to-background ratio primarily by virtue of lower background binding levels. The ratio of binding ability in prefrontal cortex (high plaque load) to subcortical white matter (background) was 4.5 for [(3)H]AZD2184 and 0.8 for [(3)H]PIB at 1 nM. In adjacent cortical sections from APP/PS1 mouse as well as from AD cortical tissue, [(3)H]AZD2184 and antibodies to human beta-amyloid labeled identical structures. In vivo administration of [(3)H]AZD2184 to APP/PS1 mice further showed that [(3)H]AZD2184 labels amyloid deposits with low non-specific background binding. Taken together, the pre-clinical profile of AZD2184 in relation to the reference ligand PIB, suggests that (11)C-labeled AZD2184 is a potential radioligand for PET-visualization of beta-amyloid deposits in the living human brain.

Keyword
beta-amyloid, Alzheimers, imaging, plaques, positron emisson tomography
National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134881 (URN)10.1111/j.1471-4159.2008.05861.x (DOI)000263044800008 ()
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
2. Characterization of AZD4694, a novel fluorinated Abeta plaque neuroimaging PET radioligand
Open this publication in new window or tab >>Characterization of AZD4694, a novel fluorinated Abeta plaque neuroimaging PET radioligand
2010 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 114, no 3, 784-794 p.Article in journal (Refereed) Published
Abstract [en]

Positron emission tomography (PET) radioligands that bind selectively to β-amyloid plaques (Aβ) are promising imaging tools aimed at supporting the diagnosis of Alzheimer’s disease and the evaluation of new drugs aiming to modify amyloid plaque load. For extended clinical use, there is a particular need for PET tracers labeled with fluorine-18, a radionuclide with 110 min half-life allowing for central synthesis followed by wide distribution. The development of fluorinated radioligands is, however, challenging because of the lipophilic nature of aromatic fluorine, rendering fluorinated ligands more prone to have high non-specific white matter binding. We have here developed the new benzofuran-derived radioligand containing fluorine, AZD4694 that shows high affinity for β-amyloid fibrils in vitro (Kd = 2.3 ± 0.3 nM). In cortical sections from human Alzheimer’s disease brain [3H]AZD4694 selectively labeled β-amyloid deposits in gray matter, whereas there was a lower level of non-displaceable binding in plaque devoid white matter. Administration of unlabeled AZD4694 to rat showed that it has a pharmacokinetic profile consistent with good PET radioligands, i.e., it quickly entered and rapidly cleared from normal rat brain tissue. Ex vivo binding data in aged Tg2576 mice after intravenous administration of [3H]AZD4694 showed selective binding to β-amyloid deposits in a reversible manner. In Tg2576 mice, plaque bound [3H]AZD4694 could still be detected 80 min after i.v. administration. Taken together, the preclinical profile of AZD4694 suggests that fluorine-18 labeled AZD4694 may have potential for PET-visualization of cerebral β-amyloid deposits in the living human brain.

National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134882 (URN)10.1111/j.1471-4159.2010.06812.x (DOI)
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
3. Altered amyloid PET ligand binding to ApoE-containing plaques in Alzheimer´s disease brain in vitro
Open this publication in new window or tab >>Altered amyloid PET ligand binding to ApoE-containing plaques in Alzheimer´s disease brain in vitro
(English)Article in journal (Refereed) Submitted
National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134893 (URN)
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
4. Discovery of AZD3839, a Potent and Selective BACE1 Inhibitor Clinical Candidate for the Treatment of Alzheimer Disease
Open this publication in new window or tab >>Discovery of AZD3839, a Potent and Selective BACE1 Inhibitor Clinical Candidate for the Treatment of Alzheimer Disease
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 49, 41245-41257 p.Article in journal (Refereed) Published
Abstract [en]

beta-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid beta peptide (A beta species. Because cerebral deposition of A beta species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, A beta and sAPP beta release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose-and time-dependent lowering of plasma, brain, and cerebrospinal fluid A beta levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of A beta in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134885 (URN)10.1074/jbc.M112.409110 (DOI)000311887600039 ()
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
5. AZ-4217: A High Potency BACE Inhibitor Displaying Acute Central Efficacy in Different In Vivo Models and Reduced Amyloid Deposition in Tg2576 Mice
Open this publication in new window or tab >>AZ-4217: A High Potency BACE Inhibitor Displaying Acute Central Efficacy in Different In Vivo Models and Reduced Amyloid Deposition in Tg2576 Mice
2013 (English)In: Journal of Neuroscience, ISSN 0270-6474, E-ISSN 1529-2401, Vol. 33, no 24, 10075-10084 p.Article in journal (Refereed) Published
Abstract [en]

A beta, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimer's disease (AD). beta-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to A beta peptides. Small molecule BACE1 inhibitors are expected to decrease A beta-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pM in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of A beta production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134887 (URN)10.1523/JNEUROSCI.1165-13.2013 (DOI)000320235300023 ()
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved
6. AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate Kinetics
Open this publication in new window or tab >>AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate Kinetics
2016 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 50, no 4, 1109-1123 p.Article in journal (Refereed) Published
Abstract [en]

A growing body of pathological, biomarker, genetic, and mechanistic data suggests that amyloid accumulation, as a result of changes in production, processing, and/or clearance of brain amyloid-beta peptide (A beta) concentrations, plays a key role in the pathogenesis of Alzheimer's disease (AD). Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid-beta protein precursor (A beta PP) to A beta peptides, with the soluble N terminal fragment of A beta PP (sA beta PP beta) as a direct product, and BACE1 inhibition is an attractive target for therapeutic intervention to reduce the production of A beta. Here, we report the in vitro and in vivo pharmacological profile of AZD3293, a potent, highly permeable, orally active, blood-brain barrier (BBB) penetrating, BACE1 inhibitor with unique slow off-rate kinetics. The in vitro potency of AZD3293 was demonstrated in several cellular models, including primary cortical neurons. In vivo in mice, guinea pigs, and dogs, AZD3293 displayed significant dose-and time-dependent reductions in plasma, cerebrospinal fluid, and brain concentrations of A beta(40), A beta(42), and sA beta PP beta. The in vitro potency of AZD3293 in mouse and guinea pig primary cortical neuronal cells was correlated to the in vivo potency expressed as free AZD3293 concentrations in mouse and guinea pig brains. In mice and dogs, the slow off-rate from BACE1 may have translated into a prolongation of the observed effect beyond the turnover rate of A beta. The preclinical data strongly support the clinical development of AZD3293, and patients with AD are currently being recruited into a combined Phase 2/3 study to test the disease-modifying properties of AZD3293.

Keyword
Alzheimer's disease, amyloid-beta, drug therapy, pharmacology, preclinical drug evaluation
National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-134890 (URN)10.3233/JAD-150834 (DOI)000371053400016 ()
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2016-11-11Bibliographically approved

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