AmyP53, a Therapeutic Peptide Candidate for the Treatment of Alzheimer's and Parkinson's Disease: Safety, Stability, Pharmacokinetics Parameters and Nose-to Brain Delivery

Abstract

Neurodegenerative disorders are a major public health issue. Despite decades of research efforts, we are still seeking an efficient cure for these pathologies. The initial paradigm of large aggregates of amyloid proteins (amyloid plaques, Lewis bodies) as the root cause of Alzheimer's and Parkinson's diseases has been mostly dismissed. Instead, membrane-bound oligomers forming Ca²⁺-permeable amyloid pores are now considered appropriate targets for these diseases. Over the last 20 years, our group deciphered the molecular mechanisms of amyloid pore formation, which appeared to involve a common pathway for all amyloid proteins, including Aβ (Alzheimer) and α-synuclein (Parkinson). We then designed a short peptide (AmyP53), which prevents amyloid pore formation by targeting gangliosides, the plasma membrane receptors of amyloid proteins. Herein, we show that aqueous solutions of AmyP53 are remarkably stable upon storage at temperatures up to 45 °C for several months. AmyP53 appeared to be more stable in whole blood than in plasma. Pharmacokinetics studies in rats demonstrated that the peptide can rapidly and safely reach the brain after intranasal administration. The data suggest both the direct transport of AmyP53 via the olfactory bulb (and/or the trigeminal nerve) and an indirect transport via the circulation and the blood-brain barrier. In vitro experiments confirmed that AmyP53 is as active as cargo peptides in crossing the blood-brain barrier, consistent with its amino acid sequence specificities and physicochemical properties. Overall, these data open a route for the use of a nasal spray formulation of AmyP53 for the prevention and/or treatment of Alzheimer's and Parkinson's diseases in future clinical trials in humans.

Keywords: Alzheimer’s; Parkinson’s; amyloid pore; ganglioside; peptide; therapy.

Figure 1

AmyP53 blocks a mechanism of neurotoxicity shared by both Alzheimer’s and Parkinson’s diseases. AmyP53 blocks the neurotoxic cascade triggered by oligomers in the membrane of brain cells. Amyloid pores may be either preassembled in the extracellular space (soluble oligomers, in equilibrium with monomers and intermediate assemblies) or within the plasma membrane of brain cells (from monomers that bind to the membrane). Yet in both cases the formation of these amyloid pores requires gangliosides that act as specific membrane receptors. Ca²⁺ ions rush into these pores, triggering a cascade of neurotoxic events that disrupt brain activity and precipitate the disease in patients. By preventing any amyloid protein from binding to gangliosides, AmyP53 blocks the overall neurotoxicity cascade at this earliest membrane step that is common to both Alzheimer’s and Parkinson’s diseases.

Figure 2

Chemical stability of AmyP53 in aqueous solution. AmyP53 was dissolved in pure water at a concentration of 1 mM and stored for 18 months at 4 °C, 20 °C, and 45 °C. The calibration curve (upper left) is linear over the 5–100 pmol range (R² > 0.99). The minimal amount detected is 5 pmol. Representative original raw HPLC spectra at 24 h (upper right) and M18 (18-month, lower panels) are shown.

Figure 3

Biological stability of AmyP53 in aqueous solution. AmyP53 was dissolved in pure water at a concentration of 1 mM and stored for a maximal time of 18 months at 4 °C, 20 °C, and 45 °C. (A) After 18 months (M18) of storage at the indicated temperature, samples were assayed in the GM1 binding assay. Pure water was used as negative control for the Langmuir monolayer assay. (B) Comparison of AmyP53 binding to GM1 over a 18-month period at 4 °C, 20 °C, and 45 °C. Samples were analyzed at 24 h, 48 h, 72 h, 16 days, 2 months, and 18 months. The surface pressure increase induced by 8 µL of each sample added underneath a stable monolayer of ganglioside GM1 was measured at the end of the experiment. Data are expressed as mean ± SD (n = 6). The differences between AmyP53 concentrations at 4 °C, 20 °C, and 45 °C were statistically not significant (p > 0.05, Kruskal–Wallis test).

Figure 4

In vitro stability studies of AmyP53 in rat whole blood and human serum. AmyP53 was incubated at a concentration of 300 µM with rat whole blood (A) or human serum (B). At the indicated times, samples were extracted and underwent AmyP53 quantification by LC–MS. Data are expressed as mean ± SD (n = 3).

Figure 5

Pharmacokinetics studies of AmyP53 administered in rats by either the intravenous or intranasal routes. (A) Calibration curve of AmyP53 detection in brain homogenates after methanol extraction and quantification by LC–MS. The insert shows the 250–1000 ng/mL range. Data are expressed as mean ± SD (n = 4). (B) At the indicated time following intravenous (red symbols) or intranasal administration (black symbols), brain homogenates were prepared and extracted with the methanol method, and AmyP53 was quantified by LC–MS. Data are expressed as mean ± SD (n = 3). (C) Comparison of AmyP53 determinations in perfused (P) and nonperfused (NP) brain 30 min after intranasal injection. The data considered the time required for perfusion (15 min). Data are expressed as mean ± SD (n = 3). (D) AmyP53 quantified in whole blood samples after intravenous (red symbols) or intranasal administration (black symbols). Data are expressed as mean ± SD (n = 3). In panel C, the differences were not statistically significant (p > 0.05, Kruskal–Wallis test).

Figure 6

Passage of the AmyP53 peptide through the blood–brain barrier. (A) Two-compartment cell culture device. At time t0, AmyP53 is injected in the basal (donor) compartment (1), and it is progressively transported into the apical (acceptor) compartment (2). When indicated, CTX or C6 cells (3) are seeded in the basal compartment (non-contact model). The endothelial bEnd.3 cells (represented in green) (4) colonize the permeable filter (red dashed line) (5) that separates both compartments. AmyP53 molecules in the donor and acceptor compartments are respectively noted (6) and (7). (B) Detection of AmyP53 in the acceptor compartment after 1 h and 24 h of incubation. The cell models are bEnd.3 cells (a, b), bEnd.3/CTX (c), or bEnd.3/C6 (d). PBS (a) or AmyP53 (b, c, d) was added in the basal compartment. Data are expressed as mean ± SD (n = 6). (C) Simultaneous detection of AmyP53 in the donor (full squares, blue curve) and acceptor (open triangles, red curve) compartments. The data show the typical simultaneous disappearance of AmyP53 from the basal donor compartment and its progressive appearance in the apical acceptor compartment (bEnd.3/C6 model). (D) Comparison of AmyP53 transendothelial transport (black symbols) with two cargo peptides (synB3, red symbols, and synB5, blue symbols) and bovine serum albumin (BSA, green symbols) (bEnd.3/C6 model) in a typical experiment. The amino acid sequence of AmyP53 vs. syn B3 and syn B5 is shown in the left panel. Data are expressed as mean ± SD (n = 6).

Figure 7

Therapeutic efficiency of AmyP53 recovered after blood–brain-barrier transport (bEnd.3/C6 model). AmyP53 was injected in the donor compartment and recovered from the acceptor compartment after 24 h of incubation (left panel). Samples were tested in the amyloid pore assay (right panel) in presence of Aβ1-42. Control supernatants (−AmyP53) did not inhibit the Ca²⁺ flux triggered by Aβ1-42, whereas AmyP53 transported through the reconstituted blood–brain barrier (+AmyP53) was fully active. Data are expressed as mean ± SD (n = 6); *** indicates p < 0.005 (Kruskal–Wallis test).

Figure 8

Pathways of AmyP53 intranasal transport. Following intranasal administration, AmyP53 can reach the brain by direct and indirect pathways. The direct pathway may occur via the trigeminal nerve or the olfactory bulbs. The indirect pathway may involve successively blood circulation and the blood–brain barrier (BBB).