John Hey

Elucidating the Aβ42 Anti-Aggregation Mechanism of Action of Tramiprosate in Alzheimer’s Disease: Integrating Molecular Analytical Methods, Pharmacokinetic and Clinical Data

BackgroundAmyloid beta (Aβ) oligomers play a critical role in the pathogenesis of Alzheimer’s disease (AD) and represent a promising target for drug development. Tramiprosate is a small-molecule Aβ anti-aggregation agent that was evaluated in phase III clinical trials for AD but did not meet the primary efficacy endpoints; however, a pre-specified subgroup analysis revealed robust, sustained, and clinically meaningful cognitive and functional effects in patients with AD homozygous for the ε4 allele of apolipoprotein E4 (APOE4/4 homozygotes), who carry an increased risk for the disease. Therefore, to build on this important efficacy attribute and to further improve its pharmaceutical properties, we have developed a prodrug of tramiprosate ALZ-801 that is in advanced stages of clinical development. To elucidate how tramiprosate works, we investigated its molecular mechanism of action (MOA) and the translation to observed clinical outcomes.ObjectiveThe two main objectives of this research were to (1) elucidate and characterize the MOA of tramiprosate via an integrated application of three independent molecular methodologies and (2) present an integrated translational analysis that links the MOA, conformation of the target, stoichiometry, and pharmacokinetic dose exposure to the observed clinical outcome in APOE4/4 homozygote subjects.MethodWe used three molecular analytical methods—ion mobility spectrometry–mass spectrometry (IMS–MS), nuclear magnetic resonance (NMR), and molecular dynamics—to characterize the concentration-related interactions of tramiprosate versus Aβ42 monomers and the resultant conformational alterations affecting aggregation into oligomers. The molecular stoichiometry of the tramiprosate versus Aβ42 interaction was further analyzed in the context of clinical pharmacokinetic dose exposure and central nervous system Aβ42 levels (i.e., pharmacokinetic–pharmacodynamic translation in humans).ResultsWe observed a multi-ligand interaction of tramiprosate with monomeric Aβ42, which differs from the traditional 1:1 binding. This resulted in the stabilization of Aβ42 monomers and inhibition of oligomer formation and elongation, as demonstrated by IMS–MS and molecular dynamics. Using NMR spectroscopy and molecular dynamics, we also showed that tramiprosate bound to Lys16, Lys28, and Asp23, the key amino acid side chains of Aβ42 that are responsible for both conformational seed formation and neuronal toxicity. The projected molar excess of tramiprosate versus Aβ42 in humans using the dose effective in patients with AD aligned with the molecular stoichiometry of the interaction, providing a clear clinical translation of the MOA. A consistent alignment of these preclinical-to-clinical elements describes a unique example of translational medicine and supports the efficacy seen in symptomatic patients with AD. This unique “enveloping mechanism” of tramiprosate also provides a potential basis for tramiprosate dose selection for patients with homozygous AD at earlier stages of disease.ConclusionWe have identified the molecular mechanism that may account for the observed clinical efficacy of tramiprosate in patients with APOE4/4 homozygous AD. In addition, the integrated application of the molecular methodologies (i.e., IMS-MS, NMR, and thermodynamics analysis) indicates that it is feasible to modulate and control the Aβ42 conformational dynamics landscape by a small molecule, resulting in a favorable Aβ42 conformational change that leads to a clinically relevant amyloid anti-aggregation effect and inhibition of oligomer formation. This novel enveloping MOA of tramiprosate has potential utility in the development of disease-modifying therapies for AD and other neurodegenerative diseases caused by misfolded proteins.

PHASE 1 DEVELOPMENT OF ALZ-801, A NOVEL BETA AMYLOID ANTI-AGGREGATION PRODRUG OF TRAMIPROSATE WITH IMPROVED DRUG PROPERTIES, SUPPORTING BRIDGING TO THE PHASE 3 PROGRAM

Background:ALZ-801 is a novel, orally bioavailable, small-molecule prodrug of tramiprosate with improved pharmacokinetics and oral tolerability. The initial tramiprosate Phase 3 program in mild-to-moderate Alzheimer’s disease (AD) included two studies with >2,000 patients but did not show significant separation on co-primary outcomes. In these studies,w60% of patients were carriers of the e4 allele of the apolipoprotein E gene (APOE4), and w14% were homozygous for APOE4. A subgroup intention-totreat analysis of APOE4/4 patients showed that tramiprosate 150mg BID produced a significant and clinically meaningful improvement on cognition (ADAS-cog), and a positive trend on function (CDR-SB), at 65 and 78 weeks, on top of standard of care symptomatic therapy. Tramiprosate also showed a favorable safety profile: the most common AE was nausea. Safety data from Phase 3 tramiprosate studies support bridging to the ALZ801 safety database, based on bioequivalence.Methods: To advance the clinical development of ALZ-801 into Phase 3, we have completed single dose and 14-day multiple ascending dose Phase 1 bridging studies in healthy elderly volunteers to evaluate safety, tolerability and pharmacokinetics. Results: Compared with oral tramiprosate, oral ALZ-801 delivered an equivalent plasma exposure of tramiprosate with over 50% lower inter-subject variability. Oral ALZ-801 also prolonged plasma tramiprosate terminal half-life to w24 hours. Administration of ALZ-801 with food markedly reduced the incidence of GI symptoms as compared to the fasted state, while maintaining plasma tramiprosate exposure. An immediate release tablet formulation of ALZ-801 was developed, which displayed exposure and low variability similar to the loose filled capsule formulation. ALZ-801 also showed excellent dose proportionality without accumulation or decrease in plasma exposure of tramiprosate over 14 days. Conclusions: Based on the single and multiple dose pharmacokinetics of ALZ-801, the steady-state plasma exposure of the active drug tramiprosate following oral BID dosing of ALZ-801 immediate release tablet has been determined. The data indicate that 265mg BID of ALZ-801 will achieve a steady-state tramiprosate exposure that is equivalent to 150mg BID of oral tramiprosate. These bridging data will support the Phase 3 development of ALZ-801, an optimized prodrug of tramiprosate with improved GI toleration and low inter-subject pharmacokinetic variability, in APOE4/4 homozygous AD subjects.

NOVEL ENVELOPING MECHANISM OF ACTION OF ALZ-801: CONFORMATIONAL STABILIZATION OF BETA AMYLOID BY A SMALL MOLECULE RESULTS IN ANTI-OLIGOMER ACTIVITY

Background: Soluble forms of Ab are in equilibrium between the CSF and the ISF. This poster poses a new hypothesis: that Ab can be cleared from the brain with interventions where Ab is continuously removed from the CSF. All clinical trials with peripheral immunotherapy have failed to date. Also, the benefits of central immunotherapy in animal models are transient since pulsatile immune reactions lead to alternate shifts in the balance in/out from the ISF to the CSF. We expect continuous depletion of Ab in the CSF would produce a steady clearance of Ab in the ISF. Methods: Today, we can conceive several ways of removing Ab continuously from the CNS by accessing the CSF and debugging it. For instance, Ab can be targeted either by size -particularly the aggregated forms(this would be made by filtering the CSF) or by epitopes (this would be made by permanent immunotherapy on the CSF) or by a combination of both. The first pilot studies testing this strategy are currently underway.Results:This poster presents a new therapeutic hypothesis with the aim of discussing it and promoting exploratory studies. Conclusions: We conclude that continuous clearance of beta-amyloid from the CSF may represent a new therapeutic strategy in AD.