Elena Schartmann

Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease

Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.

Pharmacokinetic properties of tandem d-peptides designed for treatment of Alzheimer's disease

Peptides are more and more considered for the development of drug candidates. However, they frequently exhibit severe disadvantages such as instability and unfavourable pharmacokinetic properties. Many peptides are rapidly cleared from the organism and oral bioavailabilities as well as in vivo half-lives often remain low. In contrast, some peptides consisting solely of d-enantiomeric amino acid residues were shown to combine promising therapeutic properties with high proteolytic stability and enhanced pharmacokinetic parameters. Recently, we have shown that D3 and RD2 have highly advantageous pharmacokinetic properties. Especially D3 has already proven promising properties suitable for treatment of Alzheimers disease. Here, we analyse the pharmacokinetic profiles of D3D3 and RD2D3, which are head-to-tail tandem d-peptides built of D3 and its derivative RD2. Both D3D3 and RD2D3 show proteolytic stability in mouse plasma and organ homogenates for at least 24h and in murine and human liver microsomes for 4h. Notwithstanding their high affinity to plasma proteins, both peptides are taken up into the brain following i.v. as well as i.p. administration. Although both peptides contain identical d-amino acid residues, they are arranged in a different sequence order and the peptides show differences in pharmacokinetic properties. After i.p. administration RD2D3 exhibits lower plasma clearance and higher bioavailability than D3D3. We therefore concluded that the amino acid sequence of RD2 leads to more favourable pharmacokinetic properties within the tandem peptide, which underlines the importance of particular sequence motifs, even in short peptides, for the design of further therapeutic d-peptides.

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.

Blood-brain barrier penetration of an Aβ-targeted, arginine-rich, d-enantiomeric peptide.

The application of small peptides targeting amyloid beta (Aβ) is one of many drug development strategies for the treatment of Alzheimers disease (AD). We have previously identified several peptides consisting solely of D-enantiomeric amino acid residues obtained from mirror-image phage display selection, which bind to Aβ in different assembly states and eliminate toxic Aβ aggregates. Some of these D-peptides show both diagnostic and therapeutic potential in vitro and in vivo. Here we have analysed the similarity of the arginine-rich D-peptide D3 to the arginine-rich motif (ARM) of the human immunodeficiency virus type 1 transactivator of transcription (HIV-Tat) protein, and examined its in vivo blood-brain barrier (BBB) permeability using wild type mice and transgenic mouse models of Alzheimers disease. We are able to demonstrate that D3 rapidly enters the brain where it can be found associated with amyloid plaques suggesting a direct penetration of BBB.

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 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.