Surprisingly high stability of the Aβ oligomer eliminating all-d-enantiomeric peptide D3 in media simulating the route of orally administered drugs

The aggregation of the amyloid β protein (Aβ) plays an important role in the pathology of Alzheimer's disease. Previously, we have developed the all-d-enantiomeric peptide D3, which is able to eliminate neurotoxic Aβ oligomers in vitro and improve cognition in a transgenic Alzheimer's disease mouse model in vivo even after oral administration. d-Peptides are expected to be more resistant against enzymatic proteolysis compared to their l-enantiomeric equivalents, and indeed, a pharmacokinetic study with tritiated D3 revealed the oral bioavailability to be about 58%. To further investigate the underlying properties, we examined the stability of D3 in comparison to its corresponding all-l-enantiomeric mirror image l-D3 in media simulating the gastrointestinal tract, blood and liver. Potential metabolization was followed by reversed-phase high-performance liquid chromatography. In simulated gastric fluid, D3 remained almost completely stable (89%) within 24 h, while 70% of l-D3 was degraded within the same time period. Notably, in simulated intestinal fluid, D3 also remained stable (96%) for 24 h, whereas l-D3 was completely metabolized within seconds. In human plasma and human liver microsomes, l-D3 was metabolized several hundred times faster than D3. The remarkably high stability may explain the high oral bioavailability seen in previous studies allowing oral administration of the drug candidate. Thus, all-d-enantiomeric peptides may represent a promising new compound class for drug development

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ1–42 species, reduced Aβ1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein

Oral absorption enhancement of the amyloid-β oligomer eliminating compound RD2 by conjugation with folic acid

Amyloid-β (Aβ) plays a central role in the development and progression of Alzheimer’s disease (AD) with Aβ oligomers representing the most toxic species. The all-D-enantiomeric peptide RD2, which recently successfully completed clinical phase I, specifically eliminates Aβ oligomers in vitro as well as in vivo and improves cognitive deficits in various transgenic AD mouse models even after oral administration. To further enhance the oral absorption of RD2, folic acid has been conjugated to the D-peptide promoting an endocytosis-mediated uptake via a folate receptor located in the colon and rectum. Two different conjugation strategies were selected to obtain prodrugs with folic acid being cleaved after intestinal absorption releasing unmodified RD2 in order to enable RD2’s unaltered systemic efficacy. Both conjugates remained stable in simulated gastrointestinal fluids. But only one of them was suitable as prodrug as it was cleaved to RD2 in vitro in human blood plasma and liver microsomes and in vivo in mice after intravenous injection leading to a systemic release of RD2. Furthermore, the conjugate’s permeability after oral administration was strongly enhanced compared to unconjugated RD2 demonstrating the prodrug’s functionality. However, the conjugate seemed to have impaired the mice’s wellbeing shortly after oral administration

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 Alzheimer’s disease, a progressive neurodegenerative disease finally leading to dementia. Due to RD2’s 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

Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE) - and amyloid beta 1-42-induced signal transduction in glial cells

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer’s disease (AD).</p> <p>Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1–42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains.</p> <p>Results</p> <p>We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy.</p> <p>Conclusions</p> <p>The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.</p

Oral absorption enhancement of the amyloid-β oligomer eliminating compound RD2 by conjugation with folic acid

Amyloid-β (Aβ) plays a central role in the development and progression of Alzheimer's disease (AD) with Aβ oligomers representing the most toxic species. The all-d-enantiomeric peptide RD2, which recently successfully completed clinical phase I, specifically eliminates Aβ oligomers in vitro as well as in vivo and improves cognitive deficits in various transgenic AD mouse models even after oral administration. To further enhance the oral absorption of RD2, folic acid has been conjugated to the d-peptide promoting an endocytosis-mediated uptake via a folate receptor located in the intestine. Two different conjugation strategies were selected to obtain prodrugs with folic acid being cleaved after intestinal absorption releasing unmodified RD2 in order to enable RD2's unaltered systemic efficacy. Both conjugates remained stable in simulated gastrointestinal fluids. But only one of them was suitable as prodrug as it was cleaved to RD2 in vitro in human blood plasma and liver microsomes and in vivo in mice after intravenous injection leading to a systemic release of RD2. Furthermore, the conjugate's permeability in vitro and after oral administration in mice was strongly enhanced compared to unconjugated RD2 demonstrating the prodrug's functionality. However, the conjugate seemed to have impaired the mice's wellbeing shortly after oral administration possibly resulting from strain-specific hypersensitivity to folic acid. Nevertheless, we assume that the prodrug is actually non-toxic, especially in lower concentrations as verified by a cell viability test. Furthermore, lower dosages can be applied with unaltered efficacy due to its enhanced oral absorption