Toward New AQP4 Inhibitors: ORI-TRN-002.

Cerebral edema is a life-threatening condition that can cause permanent brain damage or death if left untreated. Existing therapies aim at mitigating the associated elevated intracranial pressure, yet they primarily alleviate pressure rather than prevent edema formation. Prophylactic anti-edema therapy necessitates novel drugs targeting edema formation. Aquaporin 4 (AQP4), an abundantly expressed water pore in mammalian glia and ependymal cells, has been proposed to be involved in cerebral edema formation. A series of novel compounds have been tested for their potential inhibitory effects on AQP4. However, selectivity, toxicity, functional inhibition, sustained therapeutic concentration, and delivery into the central nervous system are major challenges. Employing extensive density-functional theory (DFT) calculations, we identified a previously unreported thermodynamically stable tautomer of the recently identified AQP4-specific inhibitor TGN-020 (2-(nicotinamide)-1,3,4-thiadiazol). This novel form, featuring a distinct hydrogen-bonding pattern, served as a template for a COSMOsim-3D-based virtual screen of proprietary compounds from Origenis™. The screening identified ORI-TRN-002, an electronic homologue of TGN-020, demonstrating high solubility and low protein binding. Evaluating ORI-TRN-002 on AQP4-expressing Xenopus laevis oocytes using a high-resolution volume recording system revealed an IC50 of 2.9 ± 0.6 µM, establishing it as a novel AQP4 inhibitor. ORI-TRN-002 exhibits superior solubility and overcomes free fraction limitations compared to other reported AQP4 inhibitors, suggesting its potential as a promising anti-edema therapy for treating cerebral edema in the future.

Safety and Tolerability of Active Immunotherapy Targeting α-Synuclein with PD03A in Patients with Early Parkinson's Disease: A Randomized, Placebo-Controlled, Phase 1 Study.

BACKGROUND: Immunotherapies targeting α-synuclein aim to limit its extracellular spread in the brain and prevent progression of pathology in Parkinson's disease (PD). PD03A is a specific active immunotherapy (SAIT) involving immunization with a short peptide formulation. OBJECTIVE: This phase 1 study characterized the safety and tolerability of PD03A in patients with early PD. A key secondary objective was to evaluate immunological activity following immunization. METHODS: This was a phase 1 study of two different doses of PD03A versus placebo in PD patients. Patients were randomized (1:1:1) to receive four priming plus one booster vaccination of PD03A 15µg, PD03A 75µg or placebo and were followed for 52 weeks. RESULTS: Overall, 36 patients were randomized, of which 35 received five immunizations and completed the study. All patients experienced at least one adverse event. Transient local injection site reactions affected all but two patients; otherwise most AEs were considered unrelated to study treatment. A substantial IgG antibody response against PD03 was observed with a maximum titer achieved at Week-12. Differences in titers between both active groups versus placebo were statistically significant from the second immunization at Week-8 until Week-52. CONCLUSION: The safety profile and positive antibody response of PD03A supports the further development of active immunotherapeutic approaches for the treatment of PD.

A phase I study assessing the safety, tolerability, immunogenicity, and low-density lipoprotein cholesterol-lowering activity of immunotherapeutics targeting PCSK9.

PURPOSE: AT04A and AT06A are two AFFITOPE® peptide vaccine candidates being developed for the treatment of hypercholesterolemia by inducing proprotein convertase subtilisin/kexin type 9 (PCSK9)-specific antibodies. This study aimed to investigate safety, tolerability, antibody development, and reduction of low-density lipoprotein cholesterol (LDLc) following four subcutaneous immunizations. METHODS: This phase I, single-blind, randomized, placebo-controlled study was conducted in a total of 72 healthy subjects with a mean fasting LDLc level at baseline of 117.1 mg/dL (range 77-196 mg/dL). Each cohort enrolled 24 subjects to receive three priming immunizations at weeks 0, 4, and 8 and to receive a single booster immunization at week 60 of either AT04A, AT06A, or placebo. In addition to safety (primary objective), the antigenic peptide- and PCSK9-specific antibody response and the impact on LDLc were evaluated over a period of 90 weeks. RESULTS: The most common systemic treatment-related adverse events (AEs) reported were fatigue, headache, and myalgia in 75% of subjects in the AT06A group and 58% and 46% of subjects in the placebo and AT04A groups, respectively. Injection site reactions (ISR) representing 63% of all treatment-emergent adverse events (TEAEs), were transient and mostly of mild or moderate intensity and rarely severe (3%). Both active treatments triggered a robust, long-lasting antibody response towards the antigenic peptides used for immunization that optimally cross-reacted with the target epitope on PCSK9. In the AT04A group, a reduction in serum LDLc was observed with a mean peak reduction of 11.2% and 13.3% from baseline compared to placebo at week 20 and 70 respectively, and over the whole study period, the mean LDLc reduction for the AT04A group vs. placebo was -7.2% (95% CI [-10.4 to -3.9], P < 0.0001). In this group, PCSK9 target epitope titers above 50 were associated with clinically relevant LDLc reductions with an individual maximal decrease of 39%. CONCLUSIONS: Although both AT04A and AT06 were safe and immunogenic, only AT04A demonstrated significant LDLc-lowering activity, justifying further development. TRIAL REGISTRATION: EudraCT: 2015-001719-11. ClinicalTrials.gov Identifier: NCT02508896.

A Phase 1 Randomized Trial of Specific Active α-Synuclein Immunotherapies PD01A and PD03A in Multiple System Atrophy.

Multiple system atrophy (MSA) is a rare and fatal neurodegenerative disease with limited symptomatic treatment options. Aggregation of α-synuclein in oligodendrocytes is believed to be a central mechanism of the neurodegenerative process. PD01A and PD03A are 2 novel therapeutic vaccine candidates containing short peptides as antigenic moieties that are designed to induce a sustained antibody response, specifically targeting pathogenic assemblies of α-synuclein. The objectives of the current study were to evaluate primarily the safety and tolerability of PD01A and PD03A in patients with early MSA. Thirty patients (11 women) were randomized to receive 5 subcutaneous injections of either PD01A (n = 12), PD03A (n = 12), or placebo (n = 6) in this patient- and examiner-blinded, placebo-controlled, 52-week phase 1 clinical trial (ClinicalTrial.gov identifier: NCT02270489). Immunogenicity and clinical scores were assessed as secondary objectives. Twenty-nine patients reported a total of 595 treatment-emergent adverse events (mild or moderate, n = 555; severe, n = 40). Treatment-related adverse events included 190 injection-site reactions typically observed in vaccination trials with similar per-subject incidence in the treatment groups over time. Sustained IgG titers were observed in the PD01A-treated group, and 89% of treated patients developed a PD01-specific antibody response after receiving all injections. Induced antibodies displayed clear reactivity to the α-synuclein target epitope. Titers and antibody responder rate (58%) were lower in the PD03A-treated group. In conclusion, both PD01A and PD03A were safe and well tolerated. PD01A triggered a rapid and long-lasting antibody response that specifically targeted the α-synuclein epitope. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Targeting α-synuclein by PD03 AFFITOPE® and Anle138b rescues neurodegenerative pathology in a model of multiple system atrophy: clinical relevance.

BACKGROUND: Misfolded oligomeric α-synuclein plays a pivotal role in the pathogenesis of α-synucleinopathies including Parkinson's disease and multiple system atrophy, and its detection parallels activation of microglia and a loss of neurons in the substantia nigra pars compacta. Here we aimed to analyze the therapeutic efficacy of PD03, a new AFFITOPE® immunotherapy approach, either alone or in combination with Anle138b, in a PLP-α-syn mouse model. METHODS: The PLP-α-syn mice were treated with PD03 immunotherapy, Anle138b, or a combination of two. Five months after study initiation, the mice underwent behavioral testing and were sacrificed for neuropathological analysis. The treatment groups were compared to the vehicle group with regard to motor performance, nigral neuronal loss, microglial activation and α-synuclein pathology. RESULTS: The PLP-α-syn mice receiving the PD03 or Anle138b single therapy showed improvement of gait deficits and preservation of nigral dopaminergic neurons associated with the reduced α-synuclein oligomer levels and decreased microglial activation. The combined therapy with Anle138b and PD03 resulted in lower IgG binding in the brain as compared to the single immunotherapy with PD03. CONCLUSIONS: PD03 and Anle138b can selectively target oligomeric α-synuclein, resulting in attenuation of neurodegeneration in the PLP-α-syn mice. Both approaches are potential therapies that should be developed further for disease modification in α-synucleinopathies.

Safety and immunogenicity of the α-synuclein active immunotherapeutic PD01A in patients with Parkinson's disease: a randomised, single-blinded, phase 1 trial.

BACKGROUND: Robust evidence supports the role of α-synuclein pathology as a driver of neuronal dysfunction in Parkinson's disease. PD01A is a specific active immunotherapy with a short peptide formulation targeted against oligomeric α-synuclein. This phase 1 study assessed the safety and tolerability of the PD01A immunotherapeutic in patients with Parkinson's disease. METHODS: We did a first-in-human, randomised, phase 1 study of immunisations with PD01A, followed by three consecutive study extensions. Patients aged 45-65 years with a clinical diagnosis of Parkinson's disease (≤4 years since diagnosis and Hoehn and Yahr Stage 1 to 2), imaging results (dopamine transporter single photon emission CT and MRI) consistent with their Parkinson's disease diagnosis, and on stable doses of Parkinson's disease medications for at least 3 months were recruited at a single private clinic in Vienna, Austria. Patients were randomly assigned (1:1), using a computer-generated sequence with varying block size, to receive four subcutaneous immunisations with either 15 µg or 75 µg PD01A injected into the upper arms and followed up initially for 52 weeks, followed by a further 39 weeks' follow-up. Patients were then randomly assigned (1:1) again to receive the first booster immunisation at 15 µg or 75 µg and were followed up for 24 weeks. All patients received a second booster immunisation of 75 µg and were followed up for an additional 52 weeks. Patients were masked to dose allocation. Primary (safety) analyses included all treated patients. These four studies were registered with EU Clinical Trials Register, EudraCT numbers 2011-002650-31, 2013-001774-20, 2014-002489-54, and 2015-004854-16. FINDINGS: 32 patients were recruited between Feb 14, 2012, and Feb 6, 2013, and 24 were deemed eligible and randomly assigned to receive four PD01A priming immunisations. One patient had a diagnosis change to multiple system atrophy and was withdrawn and two patients withdrew consent during the studies. 21 (87%) of 24 patients received all six immunisations and completed 221-259 weeks in-study (two patients in the 15 µg dose group and one patient in the 75 µg dose group discontinued). All patients experienced at least one adverse event, but most of them were considered unrelated to study treatment (except for transient local injection site reactions, which affected all but one patient). Serial MRI assessments also ruled out inflammatory processes. Systemic treatment-related adverse events were fatigue (n=4), headache (n=3), myalgia (n=3), muscle rigidity (n=2), and tremor (n=2). The geometric group mean titre of antibodies against the immunising peptide PD01 increased from 1:46 at baseline to 1:3580 at week 12 in the 15 µg dose group, and from 1:76 to 1:2462 at week 12 in the 75 µg dose group. Antibody titres returned to baseline over 2 years, but could be rapidly reactivated after booster immunisation from week 116 onwards, reaching geometric group mean titres up to 1:20218. INTERPRETATION: Repeated administrations of PD01A were safe and well tolerated over an extended period. Specific active immunotherapy resulted in a substantial humoral immune response with target engagement. Phase 2 studies are needed to further assess the safety and efficacy of PD01A for the treatment of Parkinson's disease. FUNDING: AFFiRiS, Michael J Fox Foundation.

Effects of single and combined immunotherapy approach targeting amyloid ß protein and α-synuclein in a dementia with Lewy bodies-like model.

INTRODUCTION: Immunotherapeutic approaches targeting amyloid ß (Aß) protein and tau in Alzheimer's disease and α-synuclein (α-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting Aß and/or α-syn may be effective. METHODS: Amyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aß (AD02) or α-syn (PD-AFF1) and the combination. RESULTS: AD02 more effectively reduced Aß and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced α-syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects. DISCUSSION: Specific active immunotherapy targeting Aß and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies.

The AT04A vaccine against proprotein convertase subtilisin/kexin type 9 reduces total cholesterol, vascular inflammation, and atherosclerosis in APOE*3Leiden.CETP mice.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hypercholesterolaemia and atherosclerosis. PCSK9 binds to the low density lipoprotein receptor and enhances its degradation, which leads to the reduced clearance of low density lipoprotein cholesterol (LDLc) and a higher risk of atherosclerosis. In this study, the AT04A anti-PCSK9 vaccine was evaluated for its therapeutic potential in ameliorating or even preventing coronary heart disease in the atherogenic APOE*3Leiden.CETP mouse model. METHODS AND RESULTS: Control and AT04A vaccine-treated mice were fed western-type diet for 18 weeks. Antibody titres, plasma lipids, and inflammatory markers were monitored by ELISA, FPLC, and multiplexed immunoassay, respectively. The progression of atherosclerosis was evaluated by histological analysis of serial cross-sections from the aortic sinus. The AT04A vaccine induced high and persistent antibody levels against PCSK9, causing a significant reduction in plasma total cholesterol (-53%, P < 0.001) and LDLc compared with controls. Plasma inflammatory markers such as serum amyloid A (SAA), macrophage inflammatory protein-1ß (MIP-1ß/CCL4), macrophage-derived chemokine (MDC/CCL22), cytokine stem cell factor (SCF), and vascular endothelial growth factor A (VEGF-A) were significantly diminished in AT04A-treated mice. As a consequence, treatment with the AT04A vaccine resulted in a decrease in atherosclerotic lesion area (-64%, P = 0.004) and aortic inflammation as well as in more lesion-free aortic segments (+119%, P = 0.026), compared with control. CONCLUSIONS: AT04A vaccine induces an effective immune response against PCSK9 in APOE*3Leiden.CETP mice, leading to a significant reduction of plasma lipids, systemic and vascular inflammation, and atherosclerotic lesions in the aorta.

Peptide-based anti-PCSK9 vaccines - an approach for long-term LDLc management.

BACKGROUND: Low Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models. METHODS AND FINDING: PCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr+/- mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested. CONCLUSIONS: Peptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.