Sarah Schemmert

The Aβ oligomer eliminating D -enantiomeric peptide RD2 improves cognition without changing plaque pathology

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer’s disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the d-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aβ42. Our findings demonstrate that RD2 was significantly more efficient in Aβ oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aβ oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aβ oligomer elimination by RD2 treatment may be also beneficial for AD patients.

Large-Scale Oral Treatment Study with the Four Most Promising D3-Derivatives for the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is associated with the aggregation of the amyloid β protein (Aβ). Aβ oligomers are currently thought to be the major neurotoxic agent responsible for disease development and progression. Thus, their elimination is highly desirable for therapy development. Our therapeutic approach aims at specific and direct elimination of toxic Aβ oligomers by stabilizing Aβ monomers in an aggregation-incompetent conformation. We have proven that our lead compound “D3”, an all d-enantiomeric-peptide, specifically eliminates Aβ oligomers in vitro. In vivo, D3 enhances cognition and reduces plaque load in several transgenic AD mouse models. Here, we performed a large-scale oral proof of concept efficacy study, in which we directly compared four of the most promising D3-derivatives in transgenic mice expressing human amyloid precursor protein with Swedish and London mutations (APPSL), transgenic mice, to identify the most effective compound. RD2 and D3D3, both derived from D3 by rational design, were discovered to be the most effective derivatives in improving cognition in the Morris water maze. The performance of RD2- and D3D3-treated mice within the Morris water maze was significantly better than placebo-treated mice and, importantly, nearly as good as those of non-transgenic littermates, suggesting a complete reversal of the cognitive deficit of APPSL mice.

Comparison of blood-brain barrier penetration efficiencies between linear and cyclic all-d-enantiomeric peptides developed for the treatment of Alzheimer's disease

Abstract Alzheimers disease (AD), until now, is an incurable progressive neurodegenerative disease. To target toxic amyloid &bgr; oligomers in AD patients’ brains and to convert them into non‐toxic aggregation‐incompetent species, we designed peptides consisting solely of d‐enantiomeric amino acid residues. The original lead compound was named D3 and several D3 derivatives were designed to enhance beneficial properties. Here, we compare four d‐peptides concerning their efficiencies to pass the blood‐brain barrier (BBB). We demonstrate that the d‐peptides’ concentrations in murine brain directly correlate with concentrations in cerebrospinal fluid. The cyclic d‐enantiomeric peptide cRD2D3 is characterized by the highest efficiency to pass the BBB. For in total three cyclic peptides we show that administration of cyclic peptides resulted in up to tenfold higher peak concentrations in brain as compared to their linear equivalents which have partially been characterized before (Jiang et al., 2015; Leithold et al., 2016a). These results suggest that cyclic peptides pass the murine BBB more efficiently than their linear equivalents. cRD2D3s proteolytic stability, oral bioavailability, long duration of action and its favorable brain/plasma ratio reveal that it may become a suitable drug for long‐term AD‐treatment from a pharmacokinetic point of view. Graphical abstract Figure. No caption available.

Aβ oligomer eliminating compounds interfere successfully with pEAβ(3-42) induced motor neurodegenerative phenotype in transgenic mice

Currently, there are no causative or disease modifying treatments available for Alzheimers disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aβ oligomers in vitro, enhance cognition and reduce plaque load in AD transgenic mice. To further characterise the therapeutic potential of D3 towards N-terminally truncated and pyroglutamated Aβ (pEAβ(3-42)) we tested D3 and its head-to-tail tandem derivative D3D3 both in vitro and in vivo in the new mouse model TBA2.1. These mice produce human pEAβ(3-42) leading to a strong, early onset motor neurodegenerative phenotype. In the present study, we were able to demonstrate 1) strong binding affinity of both D3 and D3D3 to pEAβ(3-42) in comparison to Aβ(1-42) and 2) increased affinity of the tandem derivative D3D3 in comparison to D3. Subsequently we tested the therapeutic potentials of both peptides in the TBA2.1 animal model. Truly therapeutic, non-preventive treatment with D3 and D3D3 clearly slowed the progression of the neurodegenerative TBA2.1 phenotype, indicating the strong therapeutic potential of both peptides against pEAβ(3-42) induced neurodegeneration.

Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer’s Mice with Full-blown Pathology

Oligomers of the amyloid-β (Aβ) protein are suspected to be responsible for the development and progression of Alzheimer’s disease. Thus, the development of compounds that are able to eliminate already formed toxic Aβ oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aβ oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aβ pathology in old-aged transgenic Alzheimer’s Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aβ oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aβ elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aβ oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer’s disease.

Development and validation of an UHPLC-ESI-QTOF-MS method for quantification of the highly hydrophilic amyloid-β oligomer eliminating all- D -enantiomeric peptide RD2 in mouse plasma

During preclinical drug development, a method for quantification of unlabeled compounds in blood plasma samples from treatment or pharmacokinetic studies in mice is required. In the current work, a rapid, specific, sensitive and validated liquid chromatography mass-spectrometric UHPLC-ESI-QTOF-MS method was developed for the quantification of the therapeutic compound RD2 in mouse plasma. RD2 is an all-D-enantiomeric peptide developed for the treatment of Alzheimers disease, a progressive neurodegenerative disease finally leading to dementia. Due to RD2s highly hydrophilic properties, the sample preparation and the chromatographic separation and quantification were very challenging. The chromatographic separation of RD2 and its internal standard were accomplished on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size) within 6.5 min at 50 °C with a flow rate of 0.5 mL/min. Mobile phases consisted of water and acetonitrile with 1% formic acid and 0.025% heptafluorobutyric acid, respectively. Ions were generated by electrospray ionization (ESI) in the positive mode and the peptide was quantified by QTOF-MS. The developed extraction method for RD2 from mouse plasma revealed complete recovery. The linearity of the calibration curve was in the range of 5.3 ng/mL to 265 ng/mL (r2 > 0.999) with a lower limit of detection (LLOD) of 2.65 ng/mL and a lower limit of quantification (LLOQ) of 5.3 ng/mL. The intra-day and inter-day accuracy and precision of RD2 in plasma ranged from -0.54% to 2.21% and from 1.97% to 8.18%, respectively. Moreover, no matrix effects were observed and RD2 remained stable in extracted mouse plasma at different conditions. Using this validated bioanalytical method, plasma samples of unlabeled RD2 or placebo treated mice were analyzed. The herein developed UHPLC-ESI-QTOF-MS method is a suitable tool for the quantitative analysis of unlabeled RD2 in plasma samples of treated mice.

Aβ OLIGOMER ELIMINATION IMPROVES COGNITIVE AND MOTORIC IMPAIRMENT IN DIFFERENT ALZHEIMER’S DISEASE MOUSE MODELS AFTER ORAL ADMINISTRATION

Nobuo Sanjo, Hiroya Kuwahara, Tetsuya Nagata, Yoichiro Nishida, Akiko Amano, Fumiko Furukawa, Kousei Hirata, Hiroyuki Maruoka, Makoto Nakakido, Tsumoto Kohei, Yasutaka Anraku, Kazunori Kataoka, Ichio Aoki, Etsuro Matsubara, Takami Tomiyama, Takanori Yokota, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; Tokyo Medical and Dental University, Tokyo, Japan; The University of Tokyo, Tokyo, Japan; The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Innovation Center of NanoMedicine, Kawasaki, Japan; National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan; Oita University Faculty of Medicine, Oita, Japan; Osaka City University, Osaka, Japan. Contact e-mail: n-sanjo. nuro@tmd.ac.jp

CYCLIC D-ENANTIOMERIC PEPTIDE TARGETING AMYLOID-BETA IN ALZHEIMER’S DISEASE

Background: Muscarinic M1 receptor (M1R) is a promising target for CNS disorders with cholinergic deficits such as Alzheimer’s disease. We previously reported that the cooperativity (a-value) was a key to lower the risk of diarrhea by M1R positive allosteric modulators (M1PAMs). In this study, we characterized in vitro profiles of TAK-071, a novel M1R selective PAM with a low a-value and compared with that of T-662, an M1PAM with a high a-value. Methods:PAM parameters were assessed by using in vitro binding and functional analysis in Chinese hamster ovary cells stably expressing human M1R. Evaluation of mouse ileum contraction was conducted under the conditions to assess spontaneous activity or electric field stimulation (EFS)-induced activity by using the in vitro Magnus method. Electrophysiological experiments were performed by using current clamp recordings in slices of mouse medial prefrontal cortex. Results:TAK-071 had an inflection point (IP)-value of 2.7 nM, and showed an a-value of 199 for humanM1R with more than 3700-fold selectivity over other muscarinic receptor subtypes. T-662 also had potent M1PAM activity with an IP-value of 2.1 nM, and showed an a-value of 1786. In the in vitro Magnus method, T-662, but not TAK-071, strengthened spontaneous ileum contraction in a concentration-dependent manner, and augmented EFS-induced ileum contraction. Tonic activation of M1Rs is known to produce neuronal excitability through three actions; depolarizing the resting membrane potential, suppressing the after hyperpolarization that follows the spike, and revealing the afterdepolarization (ADP) that can initiate repetitive firing. Among then, T-662 induced all three actions, while TAK-071 selectively induced ADP in layer 5 pyramidal neurons. Conclusions: We searched M1PAMs with low a-value and discovered TAK-071. TAK-071 induced ADP in layer 5 pyramidal neurons, while it did not cause mouse ileum contraction in vitro. Thus, TAK-071may have a promising therapeutic potential for CNS disorders without causing diarrhea. TAK-071 is currently in clinical development (ClinicalTrials. gov, Identifier: NCT02769065). total number of participants 297

THERAPEUTIC TREATMENT WITH AN ALL-D-ENANTIOMERIC PEPTIDE IMPROVES COGNITIVE IMPAIRMENT IN OLD APPSWE/PS1 TRANSGENIC MICE

pathologic aging conditions such as dementia (e.g., Belleville, Chertkow, & Gauthier, 2007). Existing literature shows that attention can be improved with training (e.g., Sohlberg &Mateer, 2001). Backgroundmusic is an accessible and typically enjoyable tool that may exogenously facilitate attention and counteract some of the attentional declines in older adults who are healthy or in pre-clinical stages of Alzheimer’s Disease. Two particular properties of a musical piece (1) mode (i.e., major, minor, or atonal), and (2) tempo (i.e., stimulative or sedative) influence affect, arousal, and cognitive function; These properties may interact with older adults’ prioritized affective goals, ultimately enhancing or hindering cognitive performance on attention-demanding tasks (e.g., Husain, Thompson, & Schellenberg, 2002; Mather & Carstensen, 2005; Thayer, Hansen, Saus-Rose, & Johnson, 2009). Methods: Six musical pieces were selected to represent different combinations of mode and tempo. Older adults (i.e., 65 80 years-old; n 1⁄4 22) complete theMulti-Source Interference Task assessing selective attention (MSIT; Bush & Shin, 2006) at baseline and under the six counterbalanced musical conditions. In each condition, participants self-report their motivation and effort, as well as their affect and arousal on the Activation-Deactivation Adjective Checklist (Thayer, 1986).Results:MSIT performance (i.e., accuracy and reaction times) is analyzed in a repeated-measures ANCOVA, with MSIT performance and self-reported motivation and effort at baseline as covariates. Mediated regression analyses assess whether MSIT performance is mediated by arousal and/or affect. Conclusions: Results indicate which specific type of musical piece may counteract older adults’ declines in selective attention. Implications are discussed and are relevant to personal and community selections of background music to facilitate older adults’ performance on daily attention-demanding tasks.

RAISING AVAILABLE BRAIN CONCENTRATIONS OF A POTENTIAL ALZHEIMER’S DISEASE DRUG CANDIDATE BY PEPTIDE CYCLIZATION

Background:Alzheimer’s disease (AD) is a widespread progressive neurodegenerative disease but to date, no curative treatment is established. To target toxic amyloid-beta (Ab) species and to convert them into non-toxic species, we have developed and designed Denantiomeric peptides. Here, we investigated if intraperitoneal and intravenous administration of a cyclic D-enantiomeric peptide (cycP) result in increased brain and plasma concentrations compared to the equivalent linear D-peptide (linP) which has been characterized before (Leithold et al., 2016). Furthermore, cycP’s pharmacokinetic profile, proteolytic stability and plasma protein binding properties were characterized to investigate whether cycP is a potential drug candidate. Methods:To determine cycP’s concentration-time-profile in murine plasma and brain, Hlabelled peptide was administered intraperitoneally, intravenously and orally to wildtype mice (male C57BL/6N, 12 weeks). Proteolytic stability was investigated by thin layer chromatography after incubation for up to seven days in murine plasma, brain-homogenate and human liver microsomes. Plasma protein binding to human serum albumin (HSA) and a1-acid glycoprotein (AGP) was examined with TRANSIL Binding Kits. Results: Four hours after intraperitoneal or intravenous administration, cycP concentrations in the brain were up to ten times higher than linP concentrations. In plasma, cycP concentrations were up to eight times higher. Furthermore, cycP showed longer half-life in plasma than linP. Oral cycP administration resulted in a constant concentrationtime-profile. Brain concentrations of cycP after oral administration were even higher between 4 and 24 hours than those after i.p. or i.v. administration. Using thin layer chromatography we could show that cycP was not degraded within seven days. Plasma protein binding of cycP was stronger to AGP (dissociation constant [KD]: 1.2 mM, fraction unbound [fu]: 6 %) than to HSA (KD: 33 mM, fu: 5 %). Conclusions:As cycP concentrations in the brain were considerably higher than those of linP, cycP seems to pass the blood brain barrier (BBB) more efficiently than linP. This is fundamental for an Aß-targeting drug, as Aß is mainly found in AD-patients’ brains. In fact, an increased mobility to pass the BBB probably allows administration of lower doses for cycP treatment as compared to linP treatment, reducing further the risk of adverse side effects.