Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

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

Alzheimer's disease, a multifactorial incurable disorder, is mainly characterised by progressive neurodegeneration, extracellular accumulation of amyloid-ß protein (Aß), and intracellular aggregation of hyperphosphorylated tau protein. During the last years, Aß oligomers have been claimed to be the disease causing agent. Consequently, development of compounds that are able to disrupt already existing Aß 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 Aß 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-Aß, a particular aggressive Aß species. Using the transgenic TBA2.1 mouse model, which develops pyroglutamate-Aß(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 Aß species further validates RD2 as a drug candidate for the therapeutic use in humans.

Pyroglutamate-Modified Amyloid-ß(3-42) Shows α-Helical Intermediates before Amyloid Formation.

Pyroglutamate-modified amyloid-ß (pEAß) has been described as a relevant Aß species in Alzheimer's-disease-affected brains, with pEAß (3-42) as a dominant isoform. Aß (1-40) and Aß (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAß (3-42) possibly underlying its drastically increased aggregation propensity compared to Aß (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aß species. Soluble pEAß (3-42) has an increased tendency to form ß-sheet-rich structures compared to Aß (1-42), as indicated by circular dichroism spectroscopy data. Kinetic assays monitored by thioflavin-T show drastically accelerated aggregation leading to large fibrils visualized by electron microscopy of pEAß (3-42) in contrast to Aß (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAß (3-42) in 40% TFE solution. Although the difference between pEAß (3-42) and Aß (1-42) is purely N-terminal, it has a significant impact on the chemical environment of >20% of the total amino acid residues, as revealed by their NMR chemical-shift differences. Freshly dissolved pEAß (3-42) contains two α-helical regions connected by a flexible linker, whereas the N-terminus remains unstructured. We found that these α-helices act as a transient intermediate to ß-sheet and fibril formation of pEAß (3-42).

Pharmacokinetic Properties of a Novel D-Peptide Developed to be Therapeutically Active Against Toxic ß-Amyloid Oligomers.

PURPOSE: It has been shown that amyloid ß (Aß) 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 Aß 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 (3)H-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.

The Avidity of Autoreactive Alpha-Synuclein Antibodies in Leucine-Rich Repeat Kinase 2 Mutation Carriers Is Not Altered Compared to Healthy Controls or Patients with Parkinson's Disease.

The accumulation and aggregation of alpha-synuclein (α-Syn) are pathological processes associated with Parkinson's disease, indicating that the regulation of protein is a crucial etiopathological mechanism. Interestingly, human serum and cerebrospinal fluid contain autoantibodies that recognize α-Syn. This potentially demonstrates an already existing, naturally decomposing, and protective system. Thus, quantitative or qualitative alterations, such as the modified antigen binding of so-called naturally occurring autoantibodies against α-Syn (nAbs-α-Syn), may induce disease onset and/or progression. We investigated the serum titers and binding characteristics of nAbs-α-Syn in patients suffering from sporadic Parkinson's disease (n = 38), LRRK2 mutation carriers (n = 25), and healthy controls (n = 22). METHODS: Titers of nAbs-α-Syn were assessed with ELISA and binding affinities and kinetics with SPR. Within the patient cohort, we discriminated between idiopathic and genetic (LRRK2-mutated) variants. RESULTS: ELISA experiments revealed no significant differences in nAbs-α-Syn serum titers among the three cohorts. Moreover, the α-Syn avidity of nAbs-α-Syn was also unchanged. CONCLUSIONS: Our findings indicate that nAbs-α-Syn concentrations or affinities in healthy and diseased persons do not differ, independent of mutations in LRRK2.

Effects of a Multimerized Recombinant Autoantibody Against Amyloid-ß.

Alzheimer's disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-ß. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-ß. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-ß-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-ß, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-ß, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-ß clearance and inhibition of oligomerization.

Role of Hydrophobicity and Charge of Amyloid-Beta Oligomer Eliminating d-Peptides in the Interaction with Amyloid-Beta Monomers.

Inhibition of the self-assembly process of amyloid-beta and even more the removal of already existing toxic amyloid-beta assemblies represent promising therapeutic strategies against Alzheimer's disease. To approach this aim, we selected a d-enantiomeric peptide by phage-display based on the interaction with amyloid-beta monomers. This lead compound was successfully optimized by peptide microarrays with respect to its affinity and specificity to the target resulting in d-peptides with both increased hydrophobicity and net charge. Here, we present a detailed biophysical characterization of the interactions between these optimized d-peptides and amyloid-beta monomers in comparison to the original lead compound in order to obtain a more thorough understanding of the physicochemical determinants of the interactions. Kinetics and apparent stoichiometry of complex formation were studied using surface plasmon resonance. Potential modes of binding to amyloid-beta were identified, and the influences of ionic strength on complex stability, as well as on the inhibitory effect on amyloid-beta aggregation were investigated. The results reveal a very different mode of interaction of the optimized d-peptides based on a combination of electrostatic and hydrophobic interactions as compared to the mostly electrostatically driven interaction of the lead compound. These conclusions were supported by the thermodynamic profiles of the interaction between optimized d-peptides and Aß monomers, which indicate an increase in binding entropy with respect to the lead compound.

Relevance of N-terminal residues for amyloid-ß binding to platelet integrin αIIbß3, integrin outside-in signaling and amyloid-ß fibril formation.

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-ß peptides (Aß) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aß40 binds through its RHDS sequence to integrin αIIbß3, and promotes the formation of fibrillar Aß aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aß binding to integrin αIIbß3 by using Aß11 and Aß16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aß. We observed platelet adhesion to truncated N-terminal Aß11 and Aß16 peptides that was not mediated by integrin αIIbß3. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aß fibril formation was not observed. Taken together, the RHDS motif of Aß is not sufficient for Aß binding to platelet integrin αIIbß3 and platelet mediated Aß fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aß binding to platelet integrin αIIbß3 is important to understand the role of platelets in amyloid pathology.

LPS-mediated cell surface expression of CD74 promotes the proliferation of B cells in response to MIF.

Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-α stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time- and dose-dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of IκBα phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-κB signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.

Inhibition of amyloid Aß aggregation by high pressures or specific d-enantiomeric peptides.

Pressure can shift the polymer-monomer equilibrium of Aß, increasing pressure first leads to a release of Aß-monomers, surprisingly at pressures higher than 180 MPa repolymerization is induced. By high pressure NMR spectroscopy, differences of partial molar volumes ΔV0 and compressibility factors Δß' of polymerization were determined at different temperatures. The d-enantiomeric peptides RD2 and RD2D3 bind to monomeric Aß with affinities substantially higher than those determined for fibril formation. By reducing the Aß concentration below the critical concentration for polymerization they inhibit the formation of toxic oligomers. Chemical shift perturbation allows the identification of the binding sites. The d-peptides are candidates for drugs preventing Alzheimer's disease. We show that RD2D3 has a positive effect on the cognitive behaviour of transgenic (APPSwDI) mice prone to Alzheimer's disease. The heterodimer complexes have a smaller Stokes radius than Aß alone indicating the recognition of a more compact conformation of Aß identified by high pressure NMR before.

In Vitro Potency and Preclinical Pharmacokinetic Comparison of All-D-Enantiomeric Peptides Developed for the Treatment of Alzheimer's Disease.

Diffusible amyloid-ß (Aß) oligomers are currently presumed to be the most cytotoxic Aß assembly and held responsible to trigger the pathogenesis of Alzheimer's disease (AD). Thus, Aß oligomers are a prominent target in AD drug development. Previously, we reported on our solely D-enantiomeric peptide D3 and its derivatives as AD drug candidates. Here, we compare one of the most promising D3 derivatives, ANK6, with its tandem version (tANK6), and its head-to-tail cyclized isoform (cANK6r). In vitro tests investigating the D-peptides' potencies to inhibit Aß aggregation, eliminate Aß oligomers, and reduce Aß-induced cytotoxicity revealed that all three D-peptides efficiently target Aß. Subsequent preclinical pharmacokinetic studies of the three all-D-peptides in wildtype mice showed promising blood-brain barrier permeability with cANK6r yielding the highest levels in brain. The peptides' potencies to lower Aß toxicity and their remarkable brain/plasma ratios make them promising AD drug candidates.

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 ß 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.

Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein.

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691-836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.

Aß oligomer eliminating compounds interfere successfully with pEAß(3-42) induced motor neurodegenerative phenotype in transgenic mice.

Currently, there are no causative or disease modifying treatments available for Alzheimer's disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aß 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 Aß (pEAß(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 pEAß(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 pEAß(3-42) in comparison to Aß(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 pEAß(3-42) induced neurodegeneration.

Optimization of d-Peptides for Aß Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aß Oligomers.

Amyloid-beta (Aß) oligomers are thought to be causative for the development and progression of Alzheimer's disease (AD). Starting from the Aß oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aß(1-42) to further enhance the Aß oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aß(1-42), eliminated Aß(1-42) oligomers, inhibited Aß(1-42) fibril formation, and reduced Aß(1-42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aß(1-42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aß monomers and thereby induced elimination of Aß oligomers is a suitable therapeutic strategy.

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

While amyloid-ß protein (Aß) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer's disease (AD), soluble oligomeric Aß 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 Aß42 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 Aß42. Our findings demonstrate that RD2 was significantly more efficient in Aß oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aß oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aß oligomer elimination by RD2 treatment may be also beneficial for AD patients.

Increase of Positive Net Charge and Conformational Rigidity Enhances the Efficacy of d-Enantiomeric Peptides Designed to Eliminate Cytotoxic Aß Species.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aß) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3COOH were designed: C-terminally amidated linear D3 (D3CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3CONH2. These four compounds were compared to each other according to their binding affinities to Aß(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aß(1-42) oligomer induced neurotoxicity. D3CONH2 and cD3r versions with equally increased net charge showed superior properties over D3COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aß targeting compound D3.

Optimization of the All-D Peptide D3 for Aß Oligomer Elimination.

The aggregation of amyloid-ß (Aß) is postulated to be the crucial event in Alzheimer's disease (AD). In particular, small neurotoxic Aß oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aß. The underlying hypothesis is that ligands bind monomeric Aß and stabilize these species within the various equilibria with Aß assemblies, leading ultimately to the elimination of Aß oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aß monomers with micromolar affinities; (iii) eliminate Aß oligomers; (iv) reduce Aß-induced cytotoxicity; and (v) disassemble preformed Aß aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aß monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

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 Alzheimer's 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.

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

Targeting toxic amyloid beta (Aß) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aß1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aß 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.