Search

BACKGROUND: Amyloid beta protein (Aβ) is a treatment target in Alzheimers Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen, an orally administered small molecule, binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aβ. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aβ metabolism using Stable Isotope Labeling Kinetic (SILK) analysis. METHODS: Double-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) confirmed by low CSF Aβ42/40 were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21-23 days, then underwent CSF catheter placement, intravenous infusion of 13C6-leucine, and CSF sampling for 36 h. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aβ40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aβ and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21. RESULTS: From June 2017 to December 2021, 19 participants were enrolled, randomized within dose cohorts (5 active: 3 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aβ40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs. placebo groups. CONCLUSIONS: Posiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects. TRIAL REGISTRATION: NCT02925650 on clinicaltrials.gov (registered on 10-24-2016).

ObjectiveRecent clinical trials targeting amyloid beta (Aβ) and tau in Alzheimer's disease (AD) have yet to demonstrate efficacy. Reviewing the hypotheses for AD pathogenesis and defining possible links between them may enhance insights into both upstream initiating events and downstream mechanisms, thereby promoting discovery of novel treatments. Evidence that in Down syndrome (DS), a population markedly predisposed to develop early onset AD, increased APP gene dose is necessary for both AD neuropathology and dementia points to normalization of the levels of the amyloid precursor protein (APP) and its products as a route to further define AD pathogenesis and discovering novel treatments.BackgroundAD and DS share several characteristic manifestations. DS is caused by trisomy of whole or part of chromosome 21; this chromosome contains about 233 protein-coding genes, including APP. Recent evidence points to a defining role for increased expression of the gene for APP and for its 99 amino acid C-terminal fragment (C99, also known as β-CTF) in dysregulating the endosomal/lysosomal system. The latter is critical for normal cellular function and in neurons for transmitting neurotrophic signals.New/updated hypothesisWe hypothesize that the increase in APP gene dose in DS initiates a process in which increased levels of full-length APP (fl-APP) and its products, including β-CTF and possibly Aβ peptides (Aβ42 and Aβ40), drive AD pathogenesis through an endosome-dependent mechanism(s), which compromises transport of neurotrophic signals. To test this hypothesis, we carried out studies in the Ts65Dn mouse model of DS and examined the effects of Posiphen, an orally available small molecule shown in prior studies to reduce fl-APP. In vitro, Posiphen lowered fl-APP and its C-terminal fragments, reversed Rab5 hyperactivation and early endosome enlargement, and restored retrograde transport of neurotrophin signaling. In vivo, Posiphen treatment (50 mg/kg/d, 26 days, intraperitoneal [i.p.]) of Ts65Dn mice was well tolerated and demonstrated no adverse effects in behavior. Treatment resulted in normalization of the levels of fl-APP, C-terminal fragments and small reductions in Aβ species, restoration to normal levels of Rab5 activity, reduced phosphorylated tau (p-tau), and reversed deficits in TrkB (tropomyosin receptor kinase B) activation and in the Akt (protein kinase B [PKB]), ERK (extracellular signal-regulated kinase), and CREB (cAMP response element-binding protein) signaling pathways. Remarkably, Posiphen treatment also restored the level of choline acetyltransferase protein to 2N levels. These findings support the APP gene dose hypothesis, point to the need for additional studies to explore the mechanisms by which increased APP gene expression acts to increase the risk for AD in DS, and to possible utility of treatments to normalize the levels of APP and its products for preventing AD in those with DS.Major challenges for the hypothesisImportant unanswered questions are: (1) When should one intervene in those with DS; (2) would an APP-based strategy have untoward consequences on possible adaptive changes induced by chronically increased APP gene dose; (3) do other genes present on chromosome 21, or on other chromosomes whose expression is dysregulated in DS, contribute to AD pathogenesis; and (4) can one model strategies that combine the use of an APP-based treatment with those directed at other AD phenotypes including p-tau and inflammation.Linkage to other major theoriesThe APP gene dose hypothesis interfaces with the amyloid cascade hypothesis of AD as well as with the genetic and cell biological observations that support it. Moreover, upregulation of fl-APP protein and products may drive downstream events that dysregulate tau homeostasis and inflammatory responses that contribute to propagation of AD pathogenesis.

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Posiphen's safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington's disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5'-untranslated regions (5'-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.

Background: Posiphen has been reported to decrease translation of beta amyloid precursor protein (APP) mRNA, resulting in decreased production of Aβ.1 The pharmacodynamics of Posiphen require further clarification to better assess its therapeutic potential. Method: The DISCOVER phase 1b placebo controlled RCT enrolled patients with MCI or mild AD dementia (age 55‐89, MMSE 17‐30, CDR = 0.5‐1.0, CSF Aβ42/40 < 0.131 by mass spectrometry) at five centers.2,3 Patients were randomized to sequential dose cohorts of oral Posiphen (60 mg; once (QD), twice (BID) or three times (TID) per day for 21 days) or placebo. SILK studies were performed during a confinement on day 22. Lumbar and venous catheters were placed and 13C6‐leucine infused for 9 hours with dosing of placebo or Posiphen according to assigned study arm. CSF and venous blood were collected every two hours over 36 hours. Pharmacodynamics of APP were evaluated via a recently described mathematical model of APP and Aβ kinetics.4 Result: APP production rate was calculated for a total of 14 subjects (less than study design due to COVID) by the model in Figure 1. Nonparametric analysis of group differences was not significant (p = 0.08) with n = 1‐5 per group (Figure 2A). A trend towards lower CSF Aβ40 concentrations (indicative of lower intrinsic APP production rates) prior to drug exposure was noted in the higher dose groups (Figure 2B). The calculated APP production rate was thus controlled for pre‐drug CSF Aβ40 concentrations, which showed a downward trend with increasing dose (95%CI:(‐2209.58, 147.79, p = 0.08; Figure 3) but was not significant. Conclusion: This study provides preliminary evidence that the rate of production of APP may be measured by the SILK method in Aβ lowering drug trials. Variability in APP productions rates in the population suggest that CSF Aβ concentration should be considered as part of study randomization. [ABSTRACT FROM AUTHOR]

Objective: Recent clinical trials targeting amyloid beta (Aβ) and tau in Alzheimer's disease (AD) have yet to demonstrate efficacy. Reviewing the hypotheses for AD pathogenesis and defining possible links between them may enhance insights into both upstream initiating events and downstream mechanisms, thereby promoting discovery of novel treatments. Evidence that in Down syndrome (DS), a population markedly predisposed to develop early onset AD, increased APP gene dose is necessary for both AD neuropathology and dementia points to normalization of the levels of the amyloid precursor protein (APP) and its products as a route to further define AD pathogenesis and discovering novel treatments. Background: AD and DS share several characteristic manifestations. DS is caused by trisomy of whole or part of chromosome 21; this chromosome contains about 233 protein‐coding genes, including APP. Recent evidence points to a defining role for increased expression of the gene for APP and for its 99 amino acid C‐terminal fragment (C99, also known as β‐CTF) in dysregulating the endosomal/lysosomal system. The latter is critical for normal cellular function and in neurons for transmitting neurotrophic signals. New/updated hypothesis: We hypothesize that the increase in APP gene dose in DS initiates a process in which increased levels of full‐length APP (fl‐APP) and its products, including β‐CTF and possibly Aβ peptides (Aβ42 and Aβ40), drive AD pathogenesis through an endosome‐dependent mechanism(s), which compromises transport of neurotrophic signals. To test this hypothesis, we carried out studies in the Ts65Dn mouse model of DS and examined the effects of Posiphen, an orally available small molecule shown in prior studies to reduce fl‐APP. In vitro, Posiphen lowered fl‐APP and its C‐terminal fragments, reversed Rab5 hyperactivation and early endosome enlargement, and restored retrograde transport of neurotrophin signaling. In vivo, Posiphen treatment (50 mg/kg/d, 26 days, intraperitoneal [i.p.]) of Ts65Dn mice was well tolerated and demonstrated no adverse effects in behavior. Treatment resulted in normalization of the levels of fl‐APP, C‐terminal fragments and small reductions in Aβ species, restoration to normal levels of Rab5 activity, reduced phosphorylated tau (p‐tau), and reversed deficits in TrkB (tropomyosin receptor kinase B) activation and in the Akt (protein kinase B [PKB]), ERK (extracellular signal‐regulated kinase), and CREB (cAMP response element–binding protein) signaling pathways. Remarkably, Posiphen treatment also restored the level of choline acetyltransferase protein to 2N levels. These findings support the APP gene dose hypothesis, point to the need for additional studies to explore the mechanisms by which increased APP gene expression acts to increase the risk for AD in DS, and to possible utility of treatments to normalize the levels of APP and its products for preventing AD in those with DS. Major challenges for the hypothesis: Important unanswered questions are: (1) When should one intervene in those with DS; (2) would an APP‐based strategy have untoward consequences on possible adaptive changes induced by chronically increased APP gene dose; (3) do other genes present on chromosome 21, or on other chromosomes whose expression is dysregulated in DS, contribute to AD pathogenesis; and (4) can one model strategies that combine the use of an APP‐based treatment with those directed at other AD phenotypes including p‐tau and inflammation. Linkage to other major theories: The APP gene dose hypothesis interfaces with the amyloid cascade hypothesis of AD as well as with the genetic and cell biological observations that support it. Moreover, upregulation of fl‐APP protein and products may drive downstream events that dysregulate tau homeostasis and inflammatory responses that contribute to propagation of AD pathogenesis. [ABSTRACT FROM AUTHOR]

Antibodies generated against sugar-azoproteins have been purified by affinity chromatography using elution with monosaccharide. They have high specificity for monosaccharide and they react with terminal, non-reducing sugars on the surfaces of human erythrocytes. Some of them, either native or as Fab fragments, inhibit mitogenic stimulation of lymphocytes by concanavalin A. [ABSTRACT FROM AUTHOR]

Background: Amyloid beta protein (Aβ) is a treatment target in Alzheimer's Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aβ. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aβ metabolism using Stable Isotope Labeling Kinetic (SILK) analysis., Methods: Double-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) with positive CSF biomarkers were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21-23 days, then underwent CSF catheter placement, intravenous infusion of 13 C 6 -leucine, and CSF sampling for 36 hours. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aβ40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aβ and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21., Results: From June 2017 to December 2021, 19 participants were enrolled, in dose cohorts (6 active: 2 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aβ40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs placebo groups., Conclusions: Posiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects., Trial Registration: NCT02925650 on clinicaltrials.gov., Competing Interests: Competing interests: MM is an employee of Annovis Bio, TW is an employee of C2N Diagnostics, RB is a cofounder of C2N Diagnostics

Parkinson disease (PD) is a neurodegenerative disease with motor as well as non-motor symptoms, including gastrointestinal dysfunction. In humans, these precede the motor symptoms by decades. Previously developed and characterized transgenic mice expressing the mutant human α-synuclein gene ( SNCA ) (either A53T or A30P), but not the endogenous mouse Snca , serve as models for familial PD. These animals demonstrate both robust abnormalities in enteric nervous system (ENS) function as well as synuclein-immunoreactive aggregates in ENS ganglia by 3 months of age, recapitulating early gastrointestinal abnormalities seen before the gait impairment characteristics of human and murine PD. Posiphen is a translational inhibitor of α-synuclein that targets the 5' untranslated region (UTR) of SNCA mRNA and could be a potential drug for the treatment of PD. However, its efficacy in ameliorating symptoms of PD has not yet been evaluated. Here, we used these transgenic mouse models to investigate the efficacy of Posiphen in reversing the gastrointestinal dysfunction. We show that Posiphen normalizes the colonic motility of both transgenic mouse models, although it did not affect the Whole Gut Transit Time (WGTT). Pharmacokinetics studies revealed that Posiphen is more abundant in the brain than in blood, in agreement with its lipophilicity, and the main metabolite is N 8 -NorPosiphen, a molecule with similar properties as Posiphen. The brain Posiphen levels necessary to effect optimal function were calculated and compared with efficacious brain levels from previous studies, showing that a 2-3 mM concentration of Posiphen and metabolites is sufficient for functional efficacy. Finally, 10 mg/kg Posiphen reduced α-synuclein levels in the gut of hSNCA A53T mice treated for twenty-one weeks, while 50 and 65 mg/kg Posiphen reduced α-synuclein levels in the brain of hSNCA A53T mice treated for twenty-one days. In conclusion, this is the first study showing the preclinical efficacy of Posiphen in normalizing the colonic motility in mouse models of gastrointestinal dysfunction in early PD. This result is in agreement with the ability of Posiphen to reach the nervous system, and its mechanism of action, the translational inhibition of α-synuclein expression. These significant findings support further development of Posiphen as a drug for the treatment of PD., Competing Interests: None.