Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-β Formation in a Transgenic Mouse Model of Alzheimer’s Disease: A Longitudinal in vivo Molecular Imaging Study 1

Abstract

Background:

Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer’s disease.

Objective:

We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer’s disease using positron emission tomography (PET).

Methods:

Female mice (AβPPswe/PS1A246E/TTR+/–) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[¹⁸F]florbetaben. Immunohistochemical analysis was performed at age = 14 months.

Results:

Standard uptake values relative to the cerebellum (SUVr) of [¹⁸F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [¹⁸F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months.

Conclusion:

Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions.

Keywords

Alzheimer’s disease disease-modifying drug positron emission tomography small-molecule chaperones transthyretin TTR/Aβ interaction

INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of dementia. It is the fifth leading cause of death globally, with a total of 2.4 million deaths in 2016, and the second leading cause of death among those over the age of 70. Alarmingly, these numbers are increasing and are estimated to reach 50 million dementia patients by 2050, worldwide [1]. Pathophysiologically, AD is characterized by the accumulation of amyloid-β (Aβ) aggregates [2], the occurrence of neurofibrillary tangles of hyperphosphorylated tau protein [3], and synaptic dysfunction [4]. In addition, AD progression is accompanied by neuroinflammation [5], structural cerebrovascular alterations, and deficits in cerebral glucose uptake and cerebral blood flow responses [6].

Knowledge gained on AD has enabled the development of a variety of mechanism-based therapeutic approaches, which aim at slowing down or stopping the disease progression. Most investigated treatment strategies include: 1) minimizing the amount of Aβ in the brain by inhibiting Aβ production, preventing Aβ aggregation or accelerating Aβ clearance from the brain; 2) minimizing aggregation or post-translational modifications of tau protein; and 3) targeting Apolipoprotein E (ApoE) [7]. Other neuroprotective strategies involve the use of neurotrophins and target neuroinflammation or oxidative stress. Nevertheless, despite decades of efforts, there is still no cure for AD. The outcome is especially worrying because over the last decade more than 50 drug candidates successfully passed phase II clinical trials but all failed in more advanced phases [8 –12]. Currently, there are only 132 agents in clinical trials for the treatment of AD [9] compared to more than 3,558 drugs employed in cancer trials [13]. The absence of an approved disease-modifying therapy calls for an immediate intervention, by feeding new drug candidates into the currently exhausted AD drug development pipeline of phase I clinical trials.

One possibility of alleviating AD pathophysiological stress suggests reducing levels of Aβ and its toxic species by enabling their transport out of the brain with the help of intrinsic proteins. Research in the past decade revealed that interactions of molecular chaperone proteins with toxic Aβ species minimize their harmful effects on the central nervous system (CNS) [14 –17]. Several intrinsic proteins were shown to be capable of modifying the stability/aggregation, circulation, and clearance characteristics of Aβ peptides [18]. Some examples of these proteins include Gelsolin [19], Apolipoprotein J (clusterin) [20, 21], ApoE [22], and human serum albumin [23 –25]. In fact, the AMBAR (Alzheimer Management by Albumin Replacement) program, currently in phase III clinical trials, has shown promising results in treatment of moderate AD dementia patients based on the human serum albumin/Aβ interaction [26].

Another amyloid binding protein that helps transport Aβ peptides across the blood-brain barrier (BBB) is Transthyretin (TTR) [27 –31]. This 55 kDa homotetramer [32] is present in the serum and cerebrospinal fluid (CSF) and is the main Aβ binding protein in human CSF. Although there are many reported in vitr o studies of the interaction of TTR with Aβ [33 –36], the molecular mechanisms of TTR neuroprotection have not been fully elucidated. Some researchers suggest that TTR interferes with Aβ by redirecting oligomeric nuclei into non-amyloid aggregates [37], and very recently, other authors have reported that TTR seems to inhibit both primary and secondary nucleations of Aβ peptide aggregation, reducing the toxicity of their oligomers [38]. Furthermore, there is still controversy around which TTR species is relevant for Aβ-related protection (or for the inhibition of Aβ aggregation or re-directing oligomers into inert species). While some authors showed that, in vitro, there is an inverse relation between the strength of the inhibition of Aβ aggregation and fibril formation and the stability of TTR [33], other results show that the stability of the tetrameric form of TTR plays a pivotal role in amyloidogenic properties of the protein [39], and that unstable TTR complexes bind poorly to Aβ peptide [40]. Studies on AD patients have shown that TTR has reduced ability to carry its natural stabilizer thyroxine (T₄) in blood plasma [41], and that the folded/monomeric ratio of TTR is decreased in AD patients [42]. At the preclinical level, the presence of resveratrol in AD mice led to the reduction of Aβ plaque burden and of total Aβ brain levels, and to the deceleration of TTR clearance and restoration of normal concentration levels of TTR in the brain [43] and in plasma [44], possibly due to favored TTR dimer-dimer interaction. All these results suggest a pivotal role of the tetrameric form of TTR in Aβ-related protection, but the controversy on the topic encourages further research that would give a better insight into the disease mechanism.

In a previous study, our group has shown that the TTR tetramer stabilizer iododiflunisal (IDIF) (Fig. 1), a iodinated derivative of the non-steroidal anti-inflammatory drug (NSAID) diflunisal, promotes Aβ clearance from the brain and improves animals’ cognitive functions when orally administered to AD transgenic mice (AβPPswe/PS1A246E/TTR+/–) daily for 2 months, starting just before the onset of the disease [45]. We also showed that the formation of TTR-IDIF complex enhances brain penetration of both TTR and IDIF [46]. Additionally, recent isothermal titration calorimetry studies have provided the structural basis of the chaperoning effect of IDIF on the TTR/Aβ complex formation, and have confirmed that not all TTR tetramer stabilizers behave as small-molecule chaperones (SMCs) of the TTR/Aβ interaction [47]. These results encouraged further in vivo investigation of possible therapeutic efficacy of IDIF. Furthermore, a screening process on small TTR-stabilizers recently conducted in our group showed that the drug tolcapone (Fig. 1), a good TTR stabilizer clinically used for the treatment of TTR-related amyloidosis [48, 49], has no chaperoning activity on the TTR/Aβ interaction, which consequently may serve as a good control drug to shed light on the contribution of both TTR-stabilizing and chaperoning properties on therapeutic efficacy.

Fig.1

TTR tetramer stabilizers: small-molecule compounds iododiflunisal (IDIF) and tolcapone.

Here, we present longitudinal and long term (5–14 months of age) in vivo evaluation of the therapeutic efficacy of IDIF and tolcapone in AD transgenic mice (AβPPswe/PS1A246E/TTR+/–). The treatment efficacy was determined based on the differences in the levels of Aβ between the treated and non-treated groups. Non-invasive, ultra-sensitive in vivo imaging technique, positron emission tomography (PET), using validated radiotracer [¹⁸F]florbetaben, was used as a surrogate of Aβ deposition, based on its previous success in Aβ imaging in AD patients [50] and different transgenic AD mouse models [51 –56].

METHODS

Compounds

IDIF meglumine salt was prepared as previously described [45]. In brief, to a solution of N-methyl-D-glucamine (meglumine) (1.22 g, 6.23 mmol) in water (2 mL), ethanol (0.5 mL), and IDIF (2.34 g, 4.23 mmol) were added over 15 min in small portions. The solution was stirred for 2 h, evaporated under reduced pressure and frozen. Tolcapone was isolated from the registered drug Tasmar (MEDA Pharma). In brief, the pills were triturated in the presence of ethyl acetate. The solution was filtered, and the filtrate evaporated under reduced pressure. The corresponding meglumine salt was prepared in the same way as reported for IDIF meglumine salt. Purity of all final compounds was proved to be ≥95% by means of high-performance liquid chromatography (HPLC), high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy.

Radiolabeling

[¹⁸F]Florbetaben was prepared by ¹⁸F-fluorination/hydrolysis of the N-Boc-protected precursor as previously described [57] with minor modifications. The radiosynthesis was performed using a TRACERlab FX_FN synthesis module (GE Healthcare). [¹⁸F]F^– was first trapped on a pre-conditioned Sep-Pak^® Accell Plus QMA Light cartridge (Waters, Milford, MA, USA), and then eluted with a solution of Kryptofix K_2.2.2/K₂CO₃ in a mixture of water and acetonitrile. After complete elimination of the solvent by azeotropic evaporation, a solution containing the precursor (3 mg) in dimethylsulfoxide (1 mL) was added and the mixture was heated at 165°C for 5 min. The reactor was then cooled at room temperature, 10% HCl aqueous solution was added (0.25 mL), and the mixture was heated (2.5 min, 90°C). The reaction crude was then diluted with NaOH solution (0.33 mL, 0.1 g/mL) and 3 mL of mobile phase (see below), and purified by HPLC using a Nucleosil 100-7 C18 column (Macherey-Nagel, Düren, Germany) as stationary phase and aqueous ascorbate buffer solution (20 g of ascorbic acid and 4.54 g NaOH in 2 L water, pH adjusted to 8.7 with 0.1 M NaOH; this solution diluted 1:1 with water)/acetonitrile (40/60, v/v) as the mobile phase at a flow rate of 5 mL/min. The desired fraction (retention time = 29–30 min) was collected, diluted with water (20 mL), and the radiotracer was retained on a C-18 cartridge (Sep-Pak^® Light, Waters, Milford, MA, USA) and further eluted with ethanol (1 mL) and ascorbate buffer solution (20 g of ascorbic acid +4.54 g NaOH in 2 L water, pH adjusted to 8.7 with 0.1 M NaOH; 5 mL). Filtration through a 0.22μm filter yielded the final solution, ready for injection. Chemical and radiochemical purity and molar activity were determined by HPLC using an Agilent 1200 Series system equipped with a radioactivity detector (Gabi, Raytest) and a variable wavelength detector (λ= 350 nm) connected in series. A RP-C18 column (Mediterranea Sea 18, 4.6×150 mm, 5μm particle size; Teknokroma, Spain) was used as the stationary phase and ascorbate buffer solution (20 g of ascorbic acid +4.54 g NaOH in 2 L water, pH adjusted to 8.7 with 0.1 M NaOH; this solution diluted 1:1 with water)/acetonitrile (40/60, v/v) as the mobile phase (retention time = 5.4 min).

Animals and study design

Animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes). All animal procedures were performed in accordance with the European Union Animal Directive (2010/63/EU). Experimental procedures were approved by the corresponding Ethical Committees.

The mouse model AβPPswe/PS1A246E/TTR+/– (carrying only one copy of the TTR gene), was generated as previously described [45] by crossing AβPPswe/PS1A246E transgenic mice [58] (B6/C3H background) purchased from The Jackson Laboratory with TTR-null mice (TTR–/–) (SV129 background) [59]. Mice were bred at I3S (Porto, Portugal) and a randomly selected cohort of females (n = 21) was transferred to CIC biomaGUNE (San Sebastian, Spain) at 4–5 months of age to apply treatments and conduct imaging studies. Upon arrival, mice were randomly divided into three groups: Group I, non-treated (control; n_I = 7); group II, IDIF-treated (n_II = 7); and group III, tolcapone-treated (n_III = 7) mice. The treatment was introduced in the drinking water at a concentration of 575 mg of meglumine IDIF salt and 575 mg meglumine tolcapone salt per liter (2.8 mg drug/animal/day).

The disease progression was followed using PET-[¹⁸F]florbetaben at the age of 5 (n_I = 7, n_II = 7, n_III = 7), 9 (n_I = 7, n_II = 7, n_III = 7), 11 (n_I = 7, n_II = 7, n_III = 6), and 14 months (n_I = 6, n_II = 6, n_III = 7). One of the animals (group III) was excluded from the analysis at 11-month time point due to improper injection, as deduced from the presence of the majority of the injected activity in the tail. One of the animals (group I) died at the age of 12 months and could not be submitted to the last imaging session. Another animal (group II) presented a damaged tail at the age of 14 months and administration of the tracer was not possible. All the animals were sacrificed at the age of 14 months, after being submitted to the last imaging session. The low mortality rate observed is in agreement with our previous experience with this model.

PET-CT imaging

Imaging experiments were performed using an eXplore Vista-CT small animal PET-CT system (GE Healthcare). In all cases, anesthesia was induced with 3–5% isoflurane in pure oxygen and maintained during imaging studies with 1.5–2.0% isoflurane in pure oxygen. Mice were injected intravenously (IV) with [¹⁸F]florbetaben (9.5–20.5 MBq; injected volume: 100–150μL). At each time point, a 30 min static PET image was acquired 30 min post IV injection in one bed position to assess the accumulation in the brain (energy range 400–700 keV). A CT scan was acquired immediately after PET acquisition (X-Ray energy: 40 kV, intensity: 140μA). PET images were reconstructed using filtered back projection (FBP) applying random, scatter, and attenuation corrections.

PET images were co-registered with a magnetic resonance imaging (MRI) template (M. Mirrione-T2, available in the π-MOD image processing tool) and different brain regions (the cortex, the hippocampus, and the cerebellum) were automatically delineated. The concentration of activity was determined in each region and expressed as standard uptake value (SUV). Treatment efficacy was determined based on the amount of [¹⁸F]florbetaben in different brain regions. The hippocampus (HIP) and the cortex (CTX) were chosen as brain regions of interest. The cerebellum (CB) was chosen as reference region. Aβ plaque abundance was determined as relative SUV (SUVr) of [¹⁸F]florbetaben in HIP and CTX with respect to CB.

To get representative voxel-by-voxel images showing temporal evolution of SUVr values, images obtained for one selected individual at 9, 11, and 14 months of age were divided by the image of the same animal at the age of 5 months. For voxel-by-voxel analysis, SUVr images for the individual animals within each group at the ages of 5 and 11 months were averaged. For each of the groups, the resulting averaged images at the ages of 11 and 5 months were divided on a voxel-by-voxel basis, and the histograms for the HIP and the CTX were obtained.

Immunohistochemical analysis

Aβ plaque burden was evaluated by performing free-floating immunohistochemistry assay on 30μm-thick cryostat coronal brain sections (5 slices per animal, ranging from Bregma –1.2 to –3,2), using monoclonal biotinylated Aβ(1–16) antibody (6E10) (Covance Research Products, Inc.), as previously described [60]. In brief, free-floating brain sections were washed twice in phosphate-buffered saline (PBS), and once in distilled water (dH₂O). For partial amyloid denaturation, 70% formic acid (FA) was used for 15 min at room temperature, with gentle agitation. After washing in dH₂O and then PBS, endogenous peroxidase activity was inhibited with 1% hydrogen peroxide (H₂O₂) in PBS for 20 min. Following PBS washes, sections were blocked in blocking solution (10% fetal bovine serum (FBS) and 0.5% Triton X-100) for 1 h at room temperature and then incubated with biotinylated 6E10 primary antibody overnight at 4°C, with gentle agitation. Sections were washed with PBS and incubated in Vectastain^® Elite ABC Reagent (Vector Laboratories, Inc.). After washing once more with PBS, sections were developed with diaminobenzidine (Sigma-Aldrich, Inc.), mounted on 0.1% gelatin-coated slides and dried overnight at room temperature. After dehydration, slides were coverslipped under Entellan^® (Merck & Co., Inc.) and examined using an Olympus BX50 light microscope. Plaque burden was evaluated using Image-Pro Plus software, by analyzing the immunostained area fraction in the HIP and CTX, separately, after delimitation of the respective regions in each of the five sections (slices) analyzed per animal. Aβ plaque burden is expressed as the percentage of the stained area relative to the total area of the respective brain region.

Statistical analysis

Statistical significance of differences in between time points (for each treatment) or treatment (at a single time point) was calculated using repeated measures 2-way ANOVA analysis. Differences were concluded significant for p values <0. 05: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data are presented as individual values, including mean values and min-to-max ranges. Statistical tests were performed in GraphPad Prism 7.03 (GraphPad Software, CA, USA).

RESULTS

Radiochemistry

The radiotracer [¹⁸F]florbetaben was synthesized in overall non-decay corrected radiochemical yield of 17±7%. Radiochemical purity as determined by radio-HPLC was >95% in all cases at the injection time, and no major peaks were identified in the UV chromatographic profiles, confirming sufficient chemical purity (Supplementary Figure 2 for representative chromatograms). Molar activity values at the end of the synthesis were in the range 184–534 GBq/μmol. Because each synthesis was used to image different animals consecutively, the mass dose of the radiotracer that was administered to the animals differed from the first to the last animal within one day. However, the order of scanning was randomized in order to keep the average injected dose (both in terms of amount of radioactivity and molar amount) constant along groups and ages. Complete information about the amount of radioactivity and mass dose administered to each individual animal at each time point are included in Supplementary Table 2.

PET-CT imaging

Longitudinal PET-CT imaging using [¹⁸F]florbetaben was carried out to determine the Aβ plaque burden in selected brain sub-regions in vivo. Animals submitted to different treatments were scanned at 5, 9, 11, and 14 months of age. SUVr values (determined as the ratio between SUV values in the investigated region and CB) in CTX progressively increased with age, irrespective of the treatment received by the animals (Fig. 2a). SUVr values in CTX were always below 1, indicating that the radiotracer uptake in this brain region was actually lower than the uptake in CB. Between-age difference within each group was significant only for non-treated animals (p = 0.0229 for 14 months versus 5 months), but no statistical significance in increase of Aβ load between 5 and 14 months was observed in IDIF- and tolcapone-treated treated groups (p = 0.147 and 0.056, respectively). Differences in SUVr between groups at a given age were not significant.

Fig.2

a, b) SUVr values (SUV values relative to CB) in CTX (a) and HIP (b), obtained after administration of [¹⁸F]florbetaben to control, IDIF-treated and tolcapone-treated mice at different ages. Dots represent values for individual mouse; probability values for differences of each value with respect to the same group at age = 5 months are depicted as ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, and ⁺⁺⁺⁺p < 0.0001; probability values for differences between treated groups and the control group at a given time point are depicted as **p < 0.01; (c) SUVr values (SUV values relative to CB) in HIP, obtained after administration of [¹⁸F]florbetaben to control, IDIF-treated and tolcapone-treated mice at different ages. Connected individual values correspond to the same animal.

In contrast, SUVr values determined for HIP showed significant differences between groups (Fig. 2b, c). For non-treated animals, values progressively increased from age = 5 months (0.98±0.04) to age = 11 months (1.06±0.03; p = 0.0002) and stabilized afterwards (1.07±0.03 at age = 14 months; p < 0.0001 versus SUVr at age = 5 months). For IDIF-treated animals, the trend was different. Values raised from 0.98±0.03 at age = 5 months to 1.00±0.03 at age = 11 months (non-significant increase, p = 0.455), and dramatically increased afterwards to reach a value of 1.07±0.02 at the age of 14 months (p = 0.0005 versus SUVr at age = 11 months; p < 0.0001 versus SUVr at age = 5 months). Finally, tolcapone-treated animals showed a trend that laid in between those observed for non-treated and IDIF-treated animals. For this group, values progressively increased with time, resulting in SUVr values 0.97±0.04, 1.01±0.03, 1.03±0.02, and 1.065±0.015 at 5, 9, 11, and 14 months, respectively. Noteworthy, SUVr values obtained at 11 and 14 months significantly differ from those obtained at 5 months (p = 0.0024 and <0.0001, respectively). At the age of 11 months, SUVr values obtained for IDIF-treated animals (1.00±0.03) are significantly lower than those obtained for non-treated animals (1.06±0.03; p = 0.0088) (see Fig. 3a for representative images). The differences observed in the hippocampus among groups were also evident on the images obtained by subtracting, voxel-by-voxel, the brain image at 5 months of age from the images of the same animal at 9, 11, and 14 months of age (Fig. 3b).

Fig.3

a) Representative axial (top), coronal (middle), and sagittal (bottom) PET images corresponding to a control animal at ages = 9, 11, and 14 months, and IDIF-treated mouse at the age of 11 months. PET images show SUVr values (values relative to CB) and have been co-registered with a brain mouse atlas. Volumes of interest drawn in CTX (blue), HIP (red), and CB (brown) are displayed; (b) representative axial PET images corresponding to control, IDIF-treated and tolcapone-treated animals at ages = 9, 11, and 14 months, representing, voxel-by-voxel, increased SUVr values with respect to the value at the age of 5 months for the same animal. PET images have been co-registered with a mouse brain atlas. Volumes of interest drawn in CTX (blue) and HIP (red) are displayed on each image.

Immunohistochemistry

The effect of the treatment on Aβ deposition was studied by assessing Aβ burden in all animals after the last imaging session (age = 14 months) by IHC analyses followed by quantification. IHC did not show any significant differences between treated and non-treated animals at this time point, neither in CTX (Fig. 4a) nor in HIP (Fig. 4b). In all cases, high plaque density was observed, with significant variability among individuals, as observed in the photomicrographs (Fig. 4c).

Fig.4

a, b) Aβ plaque burden in CTX and HIP of control, IDIF-treated (IDIF) and tolcapone-treated (tolcapone) mice at age = 14 months. Dots represent individual values. c) Photomicrographs illustrate immunohistochemical analysis of brain Aβ plaques using the 6E10 antibody. Brain slices (left) and selected areas for quantification (right) are shown. Slides within each group correspond to two representative animals.

DISCUSSION

TTR is an important transporter protein for the brain defense against pathophysiological stress caused by Aβ deposition, as demonstrated in AD mouse models. Overexpression of human TTR in AD mice results in protection against Aβ deposition and toxicity [29], whereas deletion of the of the endogenous TTR gene results in more severe disease [29 , 61]. Although some studies reported that mouse wild type (wt) TTR, which is more stable than human wt TTR, is less capable of preventing Aβ aggregation and oligomers toxicity [34 , 63], in vivo studies show accelerated development of the neuropathologic phenotype when the endogenous TTR gene is deleted, demonstrating that the mouse TTR has a relevant effect on the disease progression in AD mouse models [29 , 61]. This justifies the use of mouse models for the investigation of the effects of TTR stability on amyloidosis and the significance of these results for putative translation into humans.

Previous studies in AD animal models carried out by our research group have shown that oral administration of IDIF results in decreased Aβ plaque deposition and ameliorates cognitive status at early stages of the disease [45]. These results favor the hypothesis that TTR tetramer is actually the suppressor of Aβ aggregation in vivo, as suggested also by other authors [40].

To fully evaluate the potential of IDIF to act as a disease-modifying drug in AD, longitudinal therapeutic efficacy and long-term treatment effect on plaque deposition were assessed by [¹⁸F]florbetaben amyloid-PET imaging. Comparative studies were also performed with another TTR tetramer stabilizer, the drug tolcapone. This FDA-approved molecule for Parkinson’s disease is capable to penetrate the BBB [64] and is currently being repurposed for the treatment of hereditary TTR amyloidosis (ATTR) [48, 49] and CNS amyloidosis [49].

Similar to previous preclinical PET-[¹⁸F]florbetaben studies [51–53 , 55], SUVr values were used to evaluate plaque density in different brain regions. Plaque density was determined in CTX and HIP, areas that are the most affected by Aβ deposition in the animal model used for this study. SUVr values in CTX and HIP of non-treated animals progressively increased with animal age (Fig. 2a, b). Similar to previously reported studies in a different animal model [52], more profound differences in SUVr were observed in HIP than in CTX. PET images obtained at 9, 11, and 14 months (Fig. 3a) clearly showed an increase in SUVr values in HIP of non-treated animals from 9 to 11 months, while the increase in plaque load from 11 to 14 months was not apparent, according to quantification data.

As for the treatment groups, IDIF-treatment delayed Aβ plaque build-up in HIP until the age = 11 months, but did not affect plaque accumulation at the final stage of the disease. IHC analysis at the end point confirmed that there were no significant differences between IDIF-treated and non-treated animal groups (Fig. 4). The factors behind the sudden increase in the plaque load in IDIF-group at the final point of the study are unknown. The possible reasons behind these unexpected results could be a consequence of experimental flaws, physiological changes or changes in disease mechanism. The factors such as decrease in fluid intake as a consequence of the phenotype (which has been reported for other AD models [65]) or hindered mobility, which may lead to lower drug intake, could limit therapeutic efficacy. Occurrence of other disease-related processes that possibly take over the main role at more advanced stages of the disease and the inability of TTR tetramer to take its primary role in the late stages of the disease are also not excluded. The reasons behind these changes are beyond the scope of this work and will be addressed in future studies.

The positive IDIF treatment effect observed in HIP was not matched in CTX, suggesting that CTX was not affected by any of the treatments. However, CTX is a relatively large region and plaque deposition might be heterogeneous. Hence, we decided to explore local differences within this region and extended our investigation to HIP. With that aim, SUVr images for all individual animals obtained at the age of 5 and 11 months were averaged, and the ratio 11-to-5 months was calculated for each group in a voxel-by-voxel fashion. Histograms revealed quasi-perfect Gaussian distribution in the case of CTX (see Supplementary Figure 3) with almost overlapped mean values, thus confirming that no regional uptake within CTX was present. Contrarily, Gaussian curves fitted to the histograms corresponding to HIP showed increased mean values for tolcapone and control groups, which was in agreement with our quantification data. Noteworthy, the histogram for IDIF-treated group showed an asymmetric profile, suggesting that in spite of the protective effect of IDIF, Aβ plaques could be present in certain sub-regions of HIP at the age of 11 months.

One of the possible reasons behind the lack of treatment effect in CTX could be the difference in the amount of TTR in different brain regions, as reported previously [66]. Furthermore, our recent PET-imaging study shows that entrance of TTR into the brain after intravenous administration starts at the third ventricles, which suggests that TTR traffic occurs partially via the blood-cerebrospinal fluid barrier and not only through the BBB [46]. Ultimately, this led to lower and delayed TTR presence in peripheral areas of the brain [46], such as CTX, where a significant concentration of TTR could be observed only at 6 hours after administration. Assuming the same transport mechanism of IDIF- and tolcapone-stabilized TTR, this delay could be one of the reasons for insufficient influx of the complex into CTX. In turn, this would lead to ineffective Aβ clearance, resulting in the absence of differences between treated and non-treated mice.

No clear evidence of Aβ protein clearance was observed in the tolcapone-treated group. Compared to non-treated animals, tolcapone helped slow down the rate of plaque deposition in HIP, suggesting some protective effects of the treatment. Even though previous reports showed that tolcapone and entacapone inhibit Aβ fibrilization in a specific and concentration-dependent manner [67], our results suggest that, at the assayed dose, direct effect of tolcapone was not sufficient to produce significant differences between treated and non-treated animals.

Difference in effectiveness between IDIF and tolcapone suggests a different mechanism of action. In recent studies, we have demonstrated that, contrarily to IDIF, the orphan drug Tafamidis and the drug diflunisal (NSAID), both TTR tetramer stabilizers, do not show chaperoning capabilities of TTR/Aβ interaction [47]. Evidence for the lack of chaperoning effect of tolcapone in the TTR/Aβ interaction has been also observed using similar calorimetric studies (Supplementary Figure 1 and Supplementary Table 1). Hence, limited chaperoning ability in TTR/Aβ interaction could be the cause of the inability of tolcapone-TTR complex to promote Aβ clearance from the brain.

Our study presents positive and promising results; however, it has some caveats worth to be mentioned. First, animal studies were only performed in females, while a thorough analysis of therapeutic efficacy may require the use of both genders. The animal model used in this study shows gender-associated modulation of brain Aβ levels by TTR, and females present a more severe AD-like neuropathology [60]. Because of this, and also considering that women are more affected than men by AD, we decided to carry out our proof of concept studies only in females. Future studies should incorporate both males and females, in order to explore inter-gender differences in therapeutic efficacy.

The second limitation is the lack of longitudinal information regarding IHC analysis. Inclusion of extra animals to be sacrificed at 5, 9, and 11 months of age to correlate Aβ plaque burden in different brain sub-regions with imaging data would have been highly desirable to further support our in vivo results. Still, the use of a validated imaging modality and radiotracer supports the reliability of our results.

Finally, administering the drug in the drinking water is probably not optimal, as heterogeneous water intake may lead to variations in drug exposition and consequently also in therapeutic efficacy. Still, for such long-term treatments (total duration of ca. 10 months) other alternatives present severe limitations. Repeated oral gavage can affect animal health and welfare [68]. Intraperitoneal delivery has questionable accuracy [69], and repeated administration can result in a cumulative irritant effect and needle-induced damage. Refined alternatives such as using drug-loaded chows present similar limitations to the method using in our work. In all cases, the evaluation of drug-plasma levels may aid in better correlating exposure levels with pharmacodynamics measurements.

In spite of the above-mentioned limitations, our results position IDIF as a potential disease-modifying drug for AD, and encourage follow-up studies that will include investigation of both males and females, dose-response studies, evaluation of other SMCs of the TTR/Aβ interaction and translation to larger animal species.

Conclusions

In conclusion, this is the first large-scale longitudinal Aβ-PET study of cerebral amyloidosis in a transgenic AD mouse model treated with IDIF, a small-molecule compound that enhances the TTR/Aβ interaction. Our work confirms positive effects of IDIF on the delay of Aβ deposition in HIP. This study offers an insight into the possible effect of stabilization of TTR complexes on the degree of Aβ amyloidosis in the brain longitudinally. It also offers the first in vivo evidence of the importance of chaperoning ability on Aβ deposition. Although further studies are needed, these preliminary results suggest that IDIF is a valuable candidate for amelioration of Aβ aggregate-related pathological stress in the CNS. This research shows the great significance of development of small-molecule chaperones as potential disease modifying drugs for AD therapeutics and provides the basis for the design of further investigations, including dose-response studies and translation of this new disease-modifying approach to clinical trials for AD therapy.

Footnotes

ACKNOWLEDGMENTS

The work was supported by a grant from the Fundació Marató de TV3 (Neurodegenerative Diseases Call, Project Reference 20140330-31-32-33-34, projectes-financats/2013/212/). The group at CIC biomaGUNE also acknowledges The Spanish Ministry of Economy and Competitiveness for funding through Grant CTQ2017-87637-R. I. Cardoso worked under the Investigator FCT Program which is financed by national funds through the Foundation for Science and Technology (FCT, Portugal) and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2 - Promotion of Scientific Employment. G. Arsequell also acknowledges financial support from the Spanish Ministry of Economy (CTQ2016-76840-R). E.Y. Cotrina acknowledges a contract from Fundació Marató de TV3, Spain (Project ref. 20140330-31-32-33-34) and a one year contract from Ford-Fundación Apadrina la Ciencia. Part of the work was conducted under the Maria de Maeztu Units of Excellence Programme – Grant No. MDM-2017-0720. The authors thank the animal facility staff at CIC biomaGUNE for administration of the compounds; Aitor Lekuona and Víctor Salinas for assistance in cyclotron operation and automated syntheses; and Prof. Dr. Alfredo Ballesteros (University of Oviedo, Oviedo, Spain) for support on the IDIF synthesis. G. Arsequell from IQAC-CSIC acknowledges Prof. Dr Alfredo Ballesteros from Instituto Universitario de Química Organometálica “Enrique Moles” and Departamento de Química Orgánica e Inorgánica (University of Oviedo, Spain) for support on the IDIF synthesis and Dr. Rafel Prohens from Unitat de Polimorfisme i Calorimetria, Centres Científics i Tecnològic (University of Barcelona, Spain) for assistance in isothermal titration calorimetry studies.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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