Increased brain exposure of an alpha-synuclein fibrillization modulator

Letter Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy Andrew G. Cairns,† Ana Vazquez-Romero,‡ Mohammad Mahdi-Moein,‡ Jörgen Ådeń ,† Charles S. Elmore,∥ Akihiro Takano,‡ Ryosuke Arakawa,‡ Andrea Varrone,‡ Fredrik Almqvist,*,† and Magnus Schou*,‡,§ †

Department of Chemistry, Umeå University, 901 87 Umeå, Sweden Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet and Stockholm County Council, SE-171 76 Stockholm, Sweden § PET Science Centre, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Karolinska Institutet, S-171 76 Stockholm, Sweden ∥ Isotope Chemistry, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden

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‡

S Supporting Information *

ABSTRACT: Previous work in our laboratories has identified a series of peptidomimetic 2-pyridone molecules as modulators of alpha-synuclein (α-syn) fibrillization in vitro. As a first step toward developing molecules from this scaffold as positron emission tomography imaging agents, we were interested in evaluating their blood-brain barrier permeability in nonhuman primates (NHP) in vivo. For this purpose, 2pyridone 12 was prepared and found to accelerate α-syn fibrillization in vitro. Acid 12, and its acetoxymethyl ester analogue 14, were then radiolabeled with 11C (t1/2 = 20.4 min) at high radiochemical purity (>99%) and high specific radioactivity (>37 GBq/μmol). Following intravenous injection of each compound in NHP, a 4-fold higher radioactivity in brain was observed for [11C]14 compared to [11C]12 (0.8 vs 0.2 SUV, respectively). [11C]14 was rapidly eliminated from plasma, with [11C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein deposits in brain. KEYWORDS: Alpha-synuclein, PET, carbon-11

P

(PET). PET is a quantitative, noninvasive molecular imaging technique that can be used in a translational fashion for a wide range of purposes. These include studies of drug molecule distribution (i.e., microdosing3), drug-target engagement studies, and studies of disease pathology.4 The latter studies relies on the development of a specific tracer molecule, which needs to fulfill a set of strict requirements.5 Though the development of a suitable imaging biomarker for α-syn aggregates represents a significant challenge, it is crucial in relation to the great unmet clinical need in PD. FN075 (Figure 1) is a ring-fused thiazolo-2-pyridone peptidomimetic notable for modulating amyloid formation,6,7 in particular accelerating the onset of α-synuclein fibrillization.8 The detailed mechanism for this acceleration is not known, but it appears to involve increased formation of the soluble

arkinson’s disease (PD) is a progressive neurodegenerative disease affecting approximately 0.1% of the population and about 1% of individuals over 60 years of age.1 PD is characterized by the presence of Lewy body inclusions in brain neurons as well as loss of dopaminergic function.2 Although Lewy bodies may be identified by histology on post mortem human brain tissue sections, it is widely recognized that novel biomarkers are required to quantify these inclusions in the brains of living patients suffering from PD. Such biomarkers would not only be highly valuable in relation to clinical research of disease pathophysiology and progression, but also provide crucial patient segmentation and end points for PD drug development. Inspired by the successful development of imaging biomarkers for Amyloid aggregates in Alzheimer’s disease (AD), we were led to investigate the development of an imaging biomarker for α-syn aggregates, the major constituent of Lewy bodies.2 In analogy with previous imaging biomarkers developed for AD, we directed our attention to positron emission tomography © XXXX American Chemical Society

Received: May 15, 2018 Accepted: June 14, 2018 Published: June 14, 2018 A

DOI: 10.1021/acschemneuro.8b00236 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Letter

ACS Chemical Neuroscience

labile in vivo,14,15 easily synthesized, and have been previously used as prodrugs for carboxylic acids in drug16 and probe molecules.17−20 Both C4′ methoxy substituted standards and the labeling precursor were synthesized by a common route. Benzylic chloro intermediate13 4 was first synthesized by ketene−imine cycloaddition,21−23 then used in an established Suzuki coupling strategy12,24 with substituted naphthyl boronic esters 7 and 8, themselves prepared using Miyaura borylation. Hydrolysis of the C4′ methoxy precursor 10 and subsequent protection with bromomethyl acetate gave the cold analogues of the acid (12) and prodrug (14) probe forms, respectively. The same sequence was performed on the C4′ O-benzyl analogue 9 and followed by hydrogenolysis to give the radiolabeling precursor (15) (Scheme 1).

Figure 1. Chemical structure of FN075 (X = H) with the peptidomimetic motif highlighted in red.

oligomer intermediates through which α-synuclein eventually develops into fibrils.8,9 Applied in vivo, FN075 has been observed to modulate the soluble α-synuclein oligomer fraction, activity profile, and lifespan of Drosophila.10 In a murine model, intranigral injection of FN075 resulted in symptoms mimicking early stages Parkinson’s disease.11 These included metabolomic profiles consistent with those observed in Parkinson’s patients, loss of motor function, and decline in the number of TH+ neurons.11 Given the evidence for FN075 interacting with α-syn and remaining incorporated in oligomeric species9 which lead to fibrils, we decided to investigate this molecule as a starting point in the search for an in vivo α-syn aggregate imaging biomarker for PET. However, because of the lipohilic nature of FN075, it was in itself considered a poor candidate for in vivo imaging of α-syn aggregates. At the onset of this project, the C3 carboxylic acid moiety of FN075 was identified as a potential risk factor for low bloodbrain barrier (BBB) permeability. Since this functionality was considered necessary for fibrilization modulation,8 a strategic decision was made to investigate the brain exposure of a tool compound from the FN075 platform and to evaluate a potential prodrug approach for delivering the molecule more efficiently to brain. The aim of this project was thus threefold: (i) to design and prepare an analogue of FN075 that contained a handle for radiolabeling, while retaining activity as an α-syn fibrillization accelerator, (ii) to radiolabel the compound, and its prodrug analogue, with the positron emitting radionuclide 11 C (t1/2 = 20.4 min), and (iii) to evaluate the brain exposure of both radioligands by PET in vivo in nonhuman primates (NHP).

Scheme 1. Synthesis of the Radiolabelling Precursor and Control Compoundsa

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RESULTS AND DISCUSSION The first challenge addressed was the design of an analogue of FN075 containing a suitable handle for facile introduction of the 11 C-label. The position of the radiolabel reflects considerations of synthetic accessibility, but also a desire to retain the acceleration effect observed with FN075. Although not strictly necessary for this work, an analogue which retains this activity can be more confidently assumed to properly mimic the in vivo behavior of FN075. Furthermore, an FN075 analogue with such a handle would be a valuable intermediate for future amyloid probe synthesis. Working from the hypothesis that retention of the hydrogen-bonding peptidomimetic component was necessary, C8 modification was considered as a possibility. However, the effects of modifying this position are variable and can lead to loss of activity. Therefore, we instead chose to investigate a C4′ modification, as variation of the pendant naphthyl is tolerated in other applications of ring fused pyridone peptidomimetics.12,13 The strategy taken for improving brain delivery was to derivatize the carboxylic acid. For this purpose, an acetoxymethyl ester moiety was utilized; these are relatively stable on the bench but

a

A list of abbreviations can be found in the Supporting Information.

Compounds 12, 14, and 15 were then tested using an in vitro fibrillization assay (Figure 2). This showed the methoxy analogue 12 to accelerate fibril formation, as indicated by the shortened lag phase, although to a lesser extent than FN075. The two acetoxymethyl ester standards 14 and 15 were inactive, as indicated by a lag phase close to that observed for α-synuclein alone. Thus, any interaction with α-synuclein in vivo following administration of the ester prodrug [11C]14 would be expected to originate from the acid [11C]12. It is important to note that the in vitro binding affinities of these B


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ethanol in propylene glycol, and sterile filtered prior to administration. With the two radioligands in hand, we next evaluated the exposure of [11C]12 and [11C]14 in the NHP brain in vivo. A total of four dynamic PET measurements were made (two with each radioligand) while keeping the head of the NHP in the field of view. Following intravenous injection of each radioligand, radioactivity was distributed homogeneously in brain, with a notably higher brain exposure observed for [11C] 14 than that observed for [11C]12 (Figure 3).

Figure 2. α-Synuclein fibril formation rates in the presence of different additives, based on ThT fluorescence. Each line shown is an average of six measurements (see the Supporting Information for individual curves). The protein CsgC is an inhibitor of fibrillization.25 Vertical traces represent the lag time until fibrillization starts.

molecules to α-synuclein and other related protein aggregates is unknown at this point. In addition, although 12 accelerates α-synuclein aggregation in vitro, the high specific radioactivity obtained with 11C leads to administration of a microdose of unlabeled compound, which is expected to be pharmacologically inactive in vivo. Encouraged by the acceleratory properties of 12, we proceeded to radiolabeling with carbon-11 (Scheme 2). For

Figure 3. Summation (3−123 min) color-coded PET-MR images showing the distribution of radioactivity in rhesus monkey brain following the consecutive intravenous injections of [11C]12 (A) and [11C]14 (B) in the same experimental session. Image intensity was corrected for injected radioactivity.

Scheme 2. Radiolabeling of [11C]12 and Acetoxymethyl Ester [11C]14

One of the advantages of PET imaging is that radioactivity concentrations can be studied in the organ of interest as a function of time. Thus, the time course for radioactivity in brain was presented in Figure 4 in the form of time−activity curves (TACs). Following intravenous injection of [11C]12, peak brain radioactivity (0.2 SUV) was observed after

this purpose, we chose 11C-methylation, which is a standard protocol for facile introduction of 11C into small druglike molecules. Gratifyingly, the acetoxymethyl ester [11C]14 was readily obtained via the reaction of phenolic precursor 15 with [11C]methyl triflate in acetone under mild alkaline conditions. [11C]14 in turn served as a convenient intermediate for the preparation of [11C]12 via saponification. Both [11C]12 and [11C]14 were obtained at high molar activity (>37 GBq/μmol) and at high radiochemical purity (>97%) within 42 min after the end of bombardment. The radioligands were formulated in an isotonic mixture between phosphate buffered saline and

Figure 4. Time−activity curves for the concentration of radioactivity in whole brain (0−123 min) following the intravenous injection of [11C]12 and [11C]14 in NHP2. C


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elimination of the ester from plasma can be viewed as both a strength and a weakness; it may limit the radioactivity concentration in brain, which in this case was on the lower end for a useful PET radioligand. On the other hand, the observed rapid elimination results in lower prodrug concentration in plasma at later time-points when quantification of the PET data is usually conducted. Lower plasma prodrug concentrations will drive washout of the ester [11C]14 from brain and increase the probability that a larger fraction of the radioactivity in brain is constituted by the acid metabolite [11C]12. This is important in relation to modeling of the data, since it is well-known that the presence of multiple radiochemical entities in brain hampers accurate quantification of the PET signal. We herein report the first in vivo imaging of a radiolabeled 2-pyridone α-synuclein fibrillization modulator. As expected at the onset of this study, the 2-pyridone 12 was unable to cross the BBB in meaningful concentrations for in vivo PET imaging and a derivatization approach was required. Although the tool compounds used in this study by no means should be considered optimal imaging biomarker candidates, we expect the identified derivatization strategy to be applicable also on other molecules from the scaffold. The focus for future development of imaging biomarkers from the 2-pyridone scaffold is to improve the physicochemical properties and to optimize their affinity and selectivity for aggregated alphasynuclein in relation to other protein aggregates.

approximately 20 min. The maximum radioactivity observed in brain after injection of the acetoxymethyl ester [11C]14 was roughly 4-fold higher (0.8 SUV), but also notably earlier (at 10 min). After reaching peak uptake, the washout from brain was slow for both radioligands, with approximately half of the radioactivity present in brain at the end of the emission measurement. Since the NHPs in this study were healthy, it is unlikely that the slow wash-out from brain is a result of specific binding to α-syn aggregates. One potential explanation for the slow washout of the acetoxymethyl ester [11C]14 from brain is that ester hydrolysis is partially taking place within the brain. Since the acid [11C]12 has limited permeability across the BBB, it could be trapped within the brain. Indeed, multiple esterases have been identified in the primate brain that would be capable of such a transformation,26 but future work is required to test this hypothesis. Alternatively, the slow washout from brain may reflect nonspecific binding to brain tissue, especially considering the lipophilic nature of 14 (clogP = 6.127). Arterial blood sampling and kinetic analysis of the PET data may help identify the most probable reason for the observed slow wash-out. The synthesis of less lipophilic fibrillization modulators from this scaffold is therefore underway in our laboratories. Nevertheless, the 4-fold increase of radioactivity delivered to brain was encouraging and supports the use of the devised prodrug approach for improving BBB permeability of radioligands derived from this scaffold. Discrete blood samples were taken during the time course of each PET measurement for the purpose of analyzing radioactive metabolites in plasma. After injection of [11C]12, the amount of unchanged radioligand in plasma decreased from approximately 96% of plasma radioactivity at 4 min to approximately 20% at 60 min (Figure 5). Based on the elution

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CONCLUSION In conclusion, a prodrug approach was developed that delivered a 2-pyridone fibrillization modulator to the nonhuman primate brain with 4-fold higher concentration of radioactivity. This approach paves the way for the future development of 2-pyridones as radioligands for in vivo imaging of α-synuclein aggregates in brain.

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METHODS

Radiochemistry. HPLC solvents were obtained from Fisher (Sweden). Unless otherwise stated, all other reagents and solvents were obtained from Sigma-Aldrich (Sweden) and used without further purification. General Procedure for Radiolabeling. No-carrier-added [11C]CH4 was produced using 16.5 MeV protons in the 14N(p,α)11C nuclear reaction on a mixture of nitrogen and hydrogen gas (10% hydrogen). [11C]CH4 was converted to [11C]CH3I by radical iodination in a gas-phase recirculation system and swept in a stream of helium through a heated glass column containing silver triflate impregnated on graphpac to produce [11C]CH3OTf. The radiolabeling agent was bubbled through a solution of phenol 15 (0.5 mg) and aqueous sodium hydroxide (0.5 M) in acetone at room temperature. After 3 min, the reaction mixture was diluted with mobile phase, purified and formulated using a computer controlled automated radiochemistry system (Scansys AS, Denmark). Semipreparative HPLC was performed using a reversed-phase C-18 column column (ACE-C18, 5 μm, 7.8 × 300 mm, Advanced Chromatography Technologies) eluted with MeCN- HCO2NH4 (0.1 mM) 70:30 v/v at 8 mL/min. The column outlet was connected to an absorbance detector (λ = 254 nm) in a series with radiation detector. Solvents were removed from the product fraction by solid phase extraction (Oasis HLB 1 cm3 cartridge, Waters). [11C]14 was formulated in a solution of ethanol (30%) in propylene glycol (3 mL) and PBS (phosphate buffered saline, pH 7.4, 10 mL) and sterilized by membrane filtration through a Millex-GV filter unit (0.22 μm) (Millipore, Billerica, MA, USA).

Figure 5. Time course for unchanged radioligand in plasma following intravenous administration of [11C]12 and [11C]14, respectively (each point represents an average of two experiments).

profile on reverse phase radio-HPLC, all of the observed radiometabolites of [11C]12 were more polar than the parent compound. Following injection of the acetoxymethyl ester [11C]14, rapid hydrolysis was observed. Approximately 40% of the measured radioactivity in plasma corresponded to the acid [11C]12 at 4 min post injection. At 60 min, approximately 10% of the parent ester was observed in plasma. The rapid D


Letter

ACS Chemical Neuroscience [11C]12 was prepared via hydrolysis of crude ester [11C]14, which was prepared according to the above method and used without HPLC and SPE purification. Following the radiolabeling reaction, a solution of sodium hydroxide (0.125 M, 1 mL) was added and the mixture was heated at 80 °C for 5 min. After cooling to room temperature, an aqueous solution of TFA (0.15 M, 1 mL) was added and the reaction mixture was purified using HPLC. Semipreparative HPLC was performed using a reversed-phase C-18 column (ACE-C18, 5 μm, 7.8 × 300 mm, Advanced Chromatography Technologies) eluted with MeCN-HCO2NH4 (0.1 mM) 40:60 v/v at 8 mL/min. [11C]12 was isolated, formulated, and sterilized in an identical fashion as that described for [11C]14 above. PET Imaging in Nonhuman Primates. The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency (Dnr 145/08, 399/08, and 386/09) and was performed according to the “Guidelines for planning, conducting and documenting experimental research” (Dnr 4820/06-600) at the Karolinska Institutet, the Guide for the Care and Use of Laboratory Animals” the AstraZeneca bioethics policy and the EU Directive 2010/63/EU. Four PET experiments were performed in three experiment sessions. Session 1: PET measurements with 70 MBq of [11C]14 in rhesus monkey 1 (6.1 kg). Session 2: PET measurement with 89 MBq of [11C]12 followed by a PET measurement with [11C]14 (125 MBq) in rhesus monkey 2 (6.5 kg). Session 3: PET measurement with 140 MBq of [11C]12 in rhesus monkey 1 (6.3 kg). A head fixation system was used to secure a fixed position of the monkey’s head throughout the PET measurements undertaken in each experimental session. In each PET experiment, the radiotracer was injected as a bolus into a sural vein during 5 s with simultaneous start of PET data acquisition. Radioactivity in the brain was measured continuously for 123 min according to a preprogrammed series of 34 frames.

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Notes

The authors declare the following competing financial interest(s): M.S. and C.E. are employees and/or shareholders at AstraZeneca.

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ACKNOWLEDGMENTS The authors thank all members of Karolinska Institutet PET Centre.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00236. Synthesis of tool compounds, protocol for fibrillization assay, and quality control data for radiolabeled

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REFERENCES

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compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +46-8 517 75598. *E-mail: [email protected]. Tel: +46-90 786 69 25. ORCID

Magnus Schou: 0000-0002-4314-2418 Author Contributions

A.G.C., A.T., R.A., A.V., F.A. and M.S. contributed to the experimental design. A.G.C., A.V.R., M.M.M., J.Å., C.S.E., R.A. performed the experiments. A.G.C., M.M.M., A.V., F.A., M.S. analyzed the data. The manuscript was written by A.G.C. and M.S. and all authors have given approval to the final version. Funding

The authors are grateful to AstraZeneca, Vinnova, and the Michael J. Fox Foundation for financial support. F.A. received funding from the Swedish Research Council, the Göran Gustafsson Foundation, the Swedish Foundation for Strategic Research, and the Kempe Foundation. E


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Increased brain exposure of an alpha-synuclein fibrillization modulator

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