An orally available Cav2.2 calcium channel inhibitor for the treatment of neuropathic pain.

BACKGROUND AND PURPOSE: Neuropathic pain affects up to 10% of the global population and is caused by an injury or a disease affecting the somatosensory, peripheral, or central nervous system. NP is characterized by chronic, severe and opioid-resistant properties. Therefore, its clinical management remains very challenging. The N-type voltage-gated calcium channel, Cav2.2, is a validated target for therapeutic intervention in chronic and neuropathic pain. The conotoxin ziconotide (Prialt®) is an FDA-approved drug that blocks Cav2.2 channel but needs to be administered intrathecally. Thus, although being principally efficient, the required application route is very much in disfavour. EXPERIMENTAL APPROACH AND KEY RESULTS: Here, we describe an orally available drug candidate, RD2, which competes with ziconotide binding to Cav2.2 at nanomolar concentrations and inhibits Cav2.2 almost completely reversible. Other voltage-gated calcium channel subtypes, like Cav1.2 and Cav3.2, were affected by RD2 only at concentrations higher than 10 µM. Data from sciatic inflammatory neuritis rat model demonstrated the in vivo proof of concept, as low-dose RD2 (5 mg·kg-1) administered orally alleviated neuropathic pain compared with vehicle controls. High-dose RD2 (50 mg·kg-1) was necessary to reduce pain sensation in acute thermal response assessed by the tail flick test. CONCLUSIONS AND IMPLICATIONS: Taken together, these results demonstrate that RD2 has antiallodynic properties. RD2 is orally available, which is the most convenient application form for patients and caregivers. The surprising and novel result from standard receptor screens opens the room for further optimization into new promising drug candidates, which address an unmet medical need.

Evaluation of the 18F-labeled analog of the therapeutic all-D-enantiomeric peptide RD2 for amyloid ß imaging.

Positron emission tomography (PET) imaging with radiotracers that bind to fibrillary amyloid ß (Aß) deposits is an important tool for the diagnosis of Alzheimer's disease (AD) and for the recruitment of patients into clinical trials. However, it has been suggested that rather than the fibrillary Aß deposits, it is smaller, soluble Aß aggregates that exert a neurotoxic effect and trigger AD pathogenesis. The aim of the current study is to develop a PET probe that is capable of detecting small aggregates and soluble Aß oligomers for improved diagnosis and therapy monitoring. An 18F-labeled radioligand was prepared based on the Aß-binding d-enantiomeric peptide RD2, which is currently being evaluated in clinical trials as a therapeutic agent to dissolve Aß oligomers. 18F-labeling was carried out using palladium-catalyzed S-arylation of RD2 with 2-[18F]fluoro-5-iodopyridine ([18F]FIPy). Specific binding of [18F]RD2-cFPy to brain material from transgenic AD (APP/PS1) mice and AD patients was demonstrated with in vitro autoradiography. In vivo uptake and biodistribution of [18F]RD2-cFPy were evaluated using PET analyses in wild-type and transgenic APP/PS1 mice. Although brain penetration and brain wash-out kinetics of the radioligand were low, this study provides proof of principle for a PET probe based on a d-enantiomeric peptide binding to soluble Aß species.

Turning chiral peptides into a racemic supraparticle to induce the self-degradation of MDM2.

INTRODUCTION: Chirality is immanent in nature, and chiral molecules can achieve their pharmacological action through chiral matching with biomolecules and molecular conformation recognition. OBJECTIVES: Clinical translation of chiral therapeutics, particularly chiral peptide molecules, has been hampered by their unsatisfactory pharmaceutical properties. METHODS: A mild and simple self-assembly strategy was developed here for the construction of peptide-derived chiral supramolecular nanomedicine with suitable pharmaceutical properties. In this proof-of-concept study, we design a D-peptide as MDM2 Self-Degradation catalysts (MSDc) to induce the self-degradation of a carcinogenic E3 Ubiquitin ligase termed MDM2. Exploiting a metal coordination between mercaptan in peptides and trivalent gold ion, chiral MSDc was self-assembled into a racemic supraparticle (MSDNc) that eliminated the consume from the T-lymphocyte/macrophage phagocytose in circulation. RESULTS: Expectedly, MSDNc down-regulated MDM2 in more action than its L-enantiomer termed CtrlMSDNc. More importantly, MSDNc preponderantly suppressed the tumor progression and synergized the tumor immunotherapy in allograft model of melanoma through p53 restoration in comparison to CtrlMSDNc. CONCLUSION: Collectively, this work not only developed a secure and efficient therapeutic agent targeting MDM2 with the potential of clinical translation, but also provided a feasible and biocompatible strategy for the construction of peptide supraparticle and expanded the application of chiral therapeutic and homo-PROTAC to peptide-derived chiral supramolecular nanomedicine.

Interaction of Therapeutic d-Peptides with Aß42 Monomers, Thermodynamics, and Binding Analysis.

The aggregation of the amyloid-ß (Aß) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. Several d-peptides have been confirmed as promising candidates to inhibit the aggregation of Aß into toxic oligomers and fibrils by specifically interacting with monomeric species. In this work, we elucidate the structural interaction and thermodynamics of binding between three d-peptides (D3, ANK6, and RD2) and Aß42 monomers by means of enhanced molecular dynamics simulations. Our study derives thermodynamic energies in good agreement with experimental values and suggests that there is an enhanced binding for D3 and ANK6, which leads to more stable complexes than for RD2. The binding of D3 to Aß42 is shown to be weakly exothermic and mainly entropically driven, whereas the complex formation between the ANK6 and RD2 with the Aß42 free monomer is weakly endothermic. In addition, the changes in the solvent-accessible surface area and the radius of gyration support that the binding between Aß42 and d-peptides is mainly driven by electrostatic and hydrophobic interactions and leads to more compact conformations.

Oral Treatment with RD2RD2 Impedes Development of Motoric Phenotype and Delays Symptom Onset in SOD1G93A Transgenic Mice.

Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.

A Novel Anti-Inflammatory d-Peptide Inhibits Disease Phenotype Progression in an ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by selective neuronal death in the brain stem and spinal cord. The cause is unknown, but an increasing amount of evidence has firmly certified that neuroinflammation plays a key role in ALS pathogenesis. Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and has been implicated as driver of disease progression. Here, we describe a treatment study demonstrating the therapeutic potential of a tandem version of the well-known all-d-peptide RD2 (RD2RD2) in a transgenic mouse model of ALS (SOD1*G93A). Mice were treated intraperitoneally for four weeks with RD2RD2 vs. placebo. SOD1*G93A mice were tested longitudinally during treatment in various behavioural and motor coordination tests. Brain and spinal cord samples were investigated immunohistochemically for gliosis and neurodegeneration. RD2RD2 treatment in SOD1*G93A mice resulted not only in a reduction of activated astrocytes and microglia in both the brain stem and lumbar spinal cord, but also in a rescue of neurons in the motor cortex. RD2RD2 treatment was able to slow progression of the disease phenotype, especially the motor deficits, to an extent that during the four weeks treatment duration, no significant progression was observed in any of the motor experiments. Based on the presented results, we conclude that RD2RD2 is a potential therapeutic candidate against ALS.

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

Amyloid-ß (Aß) plays a central role in the development and progression of Alzheimer's disease (AD) with Aß oligomers representing the most toxic species. The all-d-enantiomeric peptide RD2, which recently successfully completed clinical phase I, specifically eliminates Aß 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.

Selection of a d-Enantiomeric Peptide Specifically Binding to PHF6 for Inhibiting Tau Aggregation in Transgenic Mice.

Tauopathies refer to a group of neurodegenerative disorders caused by the accumulation of insoluble hyperphosphorylated Tau protein in the brain. The inhibition and interruption of Tau aggregation are considered important strategies to ameliorate the neurodegenerative process. Previous work has shown that hexapeptide 306VQIVYK311 (PHF6) located in the repeat domain 3 of Tau protein drives Tau aggregation and itself forms a ß-sheet structure similar to those of Tau-oligomers and neurofibrillary tangles (NFTs). In this study, a mirror image phage display technology was used to screen protease-resistant and low-immunogenic d-enantiomeric peptides for their capacity to inhibit Tau aggregation. Following the preparation of d-enantiomeric PHF6 fibrils and M13 phage peptide library biopanning, 7 sets of high specificity peptides were obtained. Through ELISA and competition inhibition assays, we chose a highly specific peptide p-NH with the sequence N-I-T-M-N-S-R-R-R-R-N-H. The molecular docking results showed that p-NH interacted with PHF6 fibrils mainly through van der Waals forces and hydrogen bonding and could inhibit PHF6 aggregation in a d-configuration and concentration-dependent manner. In vitro, p-NH prohibited the formation of PHF6 fibrils and was able to enter into mouse neuroblastoma N2a cells (N2a cells) to inhibit Tau hyperphosphorylation and aggregation. Intranasal administration of p-NH reduced NFTs and improved the cognitive ability of TauP301S transgenic mice. These findings represent a straightforward methodology to find therapeutic peptides with potential applications in tauopathies.

Toward the Mode of Action of the Clinical Stage All-d-Enantiomeric Peptide RD2 on Aß42 Aggregation.

The aggregation of amyloid-ß (Aß) into oligomers and fibrillary structures is critical for the pathogenesis of Alzheimer's disease (AD). Recently, research effort has been focused on developing novel agents that can preferentially suppress Aß oligomer mediated toxicities, for example, by directly targeting these toxic assemblies. The compound RD2 has been developed and optimized for Aß42 monomer binding and stabilization of the monomer in its native intrinsically disordered conformation. It has been demonstrated to improve and even reverse the cognitive and behavioral deficits in AD mouse models, while the detailed mechanism of action is not fully clarified. Here we focused on exploring the interaction between RD2 and Aß42 monomers and its consequences for the fibrillation of Aß42. RD2 binds to Aß42 monomers with nanomolar affinities, according to microscale thermophoresis and surface plasmon resonance measurements. Complexes between RD2 and Aß42 monomers are formed at 1:1 and other stoichiometries, as revealed by analytical ultracentrifugation. At substoichiometric levels, RD2 slows down the secondary structure conversion of Aß42 and significantly delays the fibril formation. Our research provides experimental evidence in supporting that RD2 eliminates toxic Aß assemblies by stabilizing Aß monomers in their native intrinsically disordered conformation. The study further supports the promising application of RD2 in counteracting Aß aggregation related pathologies.

Interference with Amyloid-ß Nucleation by Transient Ligand Interaction.

Amyloid-ß peptide (Aß) is an intrinsically disordered protein (IDP) associated with Alzheimer's disease. The structural flexibility and aggregation propensity of Aß pose major challenges for elucidating the interaction between Aß monomers and ligands. All-D-peptides consisting solely of D-enantiomeric amino acid residues are interesting drug candidates that combine high binding specificity with high metabolic stability. Here we characterized the interaction between the 12-residue all-D-peptide D3 and Aß42 monomers, and how the interaction influences Aß42 aggregation. We demonstrate for the first time that D3 binds to Aß42 monomers with submicromolar affinities. These two highly unstructured molecules are able to form complexes with 1:1 and other stoichiometries. Further, D3 at substoichiometric concentrations effectively slows down the ß-sheet formation and Aß42 fibrillation by modulating the nucleation process. The study provides new insights into the molecular mechanism of how D3 affects Aß assemblies and contributes to our knowledge on the interaction between two IDPs.

d-Enantiomeric RTHLVFFARK-NH2: A Potent Multifunctional Decapeptide Inhibiting Cu2+-Mediated Amyloid ß-Protein Aggregation and Remodeling Cu2+-Mediated Amyloid ß Aggregates.

Aggregation of amyloid ß-protein (Aß) into ß-sheet-rich plaques is a general feature of Alzheimer's disease (AD). Homeostasis dysregulation of Cu2+ mediates Aß to form high cytotoxic aggregates, which causes cell damage by generation of reactive oxygen species (ROS). To improve the inhibitory potency and explore the multifaceted functions of our previously designed decapeptide, RTHLVFFARK-NH2 (RK10), we have herein reformulated the decapeptide into its d-enantiomer, rk10, and the effects of chirality on Aß aggregation, Cu2+-mediated Aß aggregations, and aggregate-remodeling effects were investigated. The results revealed the following: (1) The d-enantiomer presented enhanced inhibitory potency on Aß fibrillogenesis in comparison to RK10; rk10 and RK10 increased the cell viability from 60% to 91% and 71%, respectively, at an equimolar concentration to Aß. (2) The enantiomers were chemically equivalent to Cu2+ chelation, ROS suppression and oxidative damage rescue. (3) The d-enantiomer exhibited higher performance to inhibit Cu2+-mediated Aß aggregation, and more significantly attenuated the cytotoxicity caused by Aß42-Cu2+ complex than RK10. Cell viability was rescued from 51% to 89% and 74% by coincubating with rk10 and RK10 at 50 µM, respectively. Intracellular ROS levels generated by Aß42 and Aß42-Cu2+ species were also remarkably decreased by treating with rk10. (4) The enantiomers could remodel mature Aß42-Cu2+ aggregates by Cu2+ chelation, and rk10 showed higher performance than RK10, as evidenced by the enhanced cell viability from 57% to 86% by RK10 and to 96% by rk10. The d-enantiomer also showed higher ability than RK10 on protecting the disrupted species from reaggregation. Taken together, D-chiral derivatization of the decapeptide resulted in a potent multifunctional agent in inhibiting Cu2+-mediated Aß aggregation and remodeling mature Aß-Cu2+ species. To the best of our knowledge, this is the first investigation on the chirality effect of a multifunctional peptide inhibitor on Cu2+-mediated Aß aggregation and on the remodeling effect of mature Aß-Cu2+ aggregates. The work provides new insights into the critical role of chirality in the multifaceted functions of peptide inhibitors against amyloid formation and its toxicity.

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

The aggregation of the amyloid ß protein (Aß) 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 Aß 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 24h, while 70% of l-D3 was degraded within the same time period. Notably, in simulated intestinal fluid, D3 also remained stable (96%) for 24h, 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.

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

The application of small peptides targeting amyloid beta (Aß) 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 Aß in different assembly states and eliminate toxic Aß 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.

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.

Oral treatment with the d-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer's Disease transgenic mice.

Several lines of evidence suggest that the amyloid-ß-peptide (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). Not only Aß fibrils but also small soluble Aß oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aß targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aß species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aß. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aß oligomers and thereby supports their decisive role in AD development and progression.