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

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

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

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 Alzheimer's 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 Alzheimer's 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

Deceleration of the neurodegenerative phenotype in pyroglutamate-Aβ accumulating transgenic mice by oral treatment with the Aβ oligomer eliminating compound RD2

Alzheimer's disease, a multifactorial incurable disorder, is mainly characterised by progressive neurodegeneration, extracellular accumulation of amyloid-β protein (Aβ), and intracellular aggregation of hyperphosphorylated tau protein. During the last years, Aβ oligomers have been claimed to be the disease causing agent. Consequently, development of compounds that are able to disrupt already existing Aβ oligomers is highly desirable. We developed d-enantiomeric peptides, consisting solely of d-enantiomeric amino acid residues, for the direct and specific elimination of toxic Aβ oligomers. The drug candidate RD2 did show high oligomer elimination efficacy in vitro and the in vivo efficacy of RD2 was demonstrated in treatment studies by enhanced cognition in transgenic mouse models of amyloidosis. Here, we report on the in vitro and in vivo efficacy of the compound towards pyroglutamate-Aβ, a particular aggressive Aβ species. Using the transgenic TBA2.1 mouse model, which develops pyroglutamate-Aβ(3–42) induced neurodegeneration, we are able to show that oral RD2 treatment resulted in a significant deceleration of the progression of the phenotype. The in vivo efficacy against this highly toxic Aβ species further validates RD2 as a drug candidate for the therapeutic use in humans

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

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

Alzheimer's disease (AD), until now, is an incurable progressive neurodegenerative disease. To target toxic amyloid β 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. cRD2D3's 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

Pharmacokinetic Properties of a Novel d-Peptide Developed to be Therapeutically Active Against Toxic β-Amyloid Oligomers

PURPOSE:It has been shown that amyloid β (Aβ) oligomers play an important role in the pathology of Alzheimer's disease (AD). D3, a peptide consisting solely of D-enantiomeric amino acid residues, was developed to specifically eliminate Aβ oligomers and is therapeutically active in transgenic AD mice. D-peptides have several advantages over L-peptides, but little is known about their pharmacokinetic potential in vivo. Here, we analysed the pharmacokinetic properties of RD2, a rationally designed and potent D3 derivative.METHODS:The pharmacokinetic analysis was performed using 3H-RD2 after administration via several routes in mice. The time dependent amount of radiolabelled RD2 was measured in plasma and several organ homogenates by liquid scintillation counting. Furthermore, binding to plasma proteins was estimated.RESULTS:RD2 penetrates into the brain, where it is thought to implement its therapeutic function. All administration routes result in a maximal brain concentration per dose (Cmax/D) of 0.06 (μg/g)/(mg/kg) with brain/plasma ratios ranging between 0.7 and 1.0. RD2 shows a small elimination constant and a long terminal half-life in plasma of more than 2 days. It also exhibits high bioavailability after i.p., s.c. or p.o. administration.CONCLUSIONS:These excellent pharmacokinetic properties confirm that RD2 is a very promising drug candidate for AD.KEYWORDS:Alzheimer’s disease; D-enantiomer; peptide; pharmacokinetics; preclinica