Blood-brain barrier permeable shuttle and uses thereof

Abstract

A composition comprising cyclic D,L-a-peptide according to Formula (IA) or Formula (IB) coupled to liposomes for use as a medicament; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. Related methods of synthesis are also disclosed as are alternative compositions based on metal nanoparticles.

Description

[0001] TITLE: BLOOD-BRAIN BARRIER PERMEABLE SHUTTLE AND USES THEREOF

[0002] Related applications:

[0003] This PCT application claims priority under 35 U.S.C 119(e) of US provisional application 63 / 555,102 filed on Feb 19, 2024 and having the same title and inventors as the present application; this earlier application is fully incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] Some embodiments of the invention are in the field of drug delivery across the blood brain barrier. Some embodiments of the invention are in the field of treatment for neurodegenerative diseases or other CNS (Central Nervous System) conditions such as Alzheimer's disease.

[0006] BACKGROUND OF THE INVENTION

[0007] Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common form of dementia. AD is characterized by a progressive memory loss and impairment of cognitive functions. It is estimated that 50 million people suffer from AD and related dementias, and the patient population is expected to increase to 78 million by 2030 and 139 million by 2050.

[0008] In the US, AD ranks as a leading cause of death among people over 65, ranking third after heart disease and cancer. The prevalence of AD and other dementias imposes a significant financial burden on the healthcare system.

[0009] It has long been believed that AD is associated with extracellular accumulation of senile plaques composed of amyloid-p (AP) protein and intraneuronal tangles of aberrantly phosphorylated microtubule-associated tau protein. More recently it has been suggested that that soluble low molecular weight AP oligomers (Apos) rather than insoluble amyloid plaques are the key pathogenic molecular assemblies that cause AD symptoms and disease progression.

[0010] FDA-approved monoclonal antibodies ADUCANUMAB, LECANEMAB and DONANEMAB, which are specific to Apos, have been the subject of recent clinical trials. These monoclonal antibodies target the toxic Apos and have demonstrated promising results in recent clinical trials. However, concerns over their widespread use remain.

[0011] In developing treatments for CNS disorders, the Blood Brain Barrier (BBB) presents a significant challenge in drug delivery. The BBB is a selective interface between the circulating blood and the CNS, which includes the brain and spinal cord. The primary role of the BBB is to regulate the influx and efflux of substances to maintain the brain's metabolic activity and neuronal function, and to protect the neurons from entry of toxins and pathogens. The BBB is made up of specialized endothelial cells connected by tight junctions, which seal the gaps between adjacent cells, restricting the diffusion of many substances, including ions and large molecules, across the BBB. Various ways (e.g. nanoparticles, peptides, and monoclonal antibodies) have been considered for delivery of active pharmaceutical ingredients (APIs) across the BBB.

[0012] Since Apos play a role in AD onset, their early detection is critical for improving patient screening and accurately monitoring treatment response. However Apos are unstable and appear at low concentrations which makes their detection challenging.

[0013] Available FDA-approved PET imaging agents such as AMYVID™, VIZAMYL™, NEURACEQ™ andnC-Pittsburgh B (nC-PIB) primarily target late-stage fibrils and insoluble plaques and do not effectively detect soluble Apos.

[0014] Existing probes specifically designed for detecting early Apos in vivo rely on fluorescence imaging techniques or oligomer specific antibodies detectable in PET or MRI images.

[0015] The previously characterized cyclic D,L-a-peptide CP-2 (Fig. 1A)) consisting of an even number of alternating D- and L-a-amino acids self-assembles into nanotubes that replicate the intermolecular hydrogen-bonding pattern of cross p-sheets and resemble many structural and functional features of amyloid proteins. CP-2 and its analogs interact with early soluble and low molecular weight oligomers of aggregation-prone polypeptides including AP, a-syn and tau- derived hexapeptide AcPHF6.

[0016] More specifically, previous studies suggest that CP-2 and its analogs inhibit A aggregation by interacting with and stabilizing small A oligomers (1-3 mers) and influencing the secondary -sheet conformation.

[0017] SUMMARY OF THE INVENTION

[0018] A broad aspect of the invention relates to cyclic D,L-a-peptide CP-2 (Fig. 1A) consisting of even number of alternating D- and L-a-amino acids. Without wishing to be bound by theory, it is believed that CP-2 molecules self-assemble into nanotubes that replicate the intermolecular hydrogen-bonding pattern of cross p-sheets and resemble many structural and functional features of amyloid proteins. Alternatively or additionally, it is believed that CP-2 and its analogs interact with early soluble and low molecular weight oligomers of aggregation-prone polypeptides including AP, a-syn and tau-derived hexapeptide AcPHF6.

[0019] More specifically, previous studies with related peptides suggest that CP-2 and its analogs inhibit Ap aggregation by interacting with and stabilizing small A oligomers (1-3 mers) and influencing the secondary -sheet conformation.

[0020] One aspect of some embodiments of the invention relates to use of multivalency effect to augment the interaction of CP-2 with early A species. In some exemplary embodiments of the invention, a multifunctional liposome-based platform targeting toxic Apos is provided for early diagnosis and treatment of AD. In some embodiments liposomes were conjugated with Apo specific CP-2 and labeled with Cy5 as a near-IR (NIR) fluorescent probe. Evaluation and characterization of the conjugated liposomes (LPs) in vitro and in transgenic C. elegans and mouse models of AD suggests their utility as diagnostic and therapeutic agents.

[0021] Another aspect of some embodiments of the invention relates to use of systemically delivered CP-2 as a therapeutic agent for treatment of Alzheimer's disease. In some exemplary embodiments of the invention, CP-2 is injected either intravenously or intraperitoneally and crosses BBB to bind A o and exert a therapeutic effect in the brain.

[0022] In another embodiments, CP-2 was conjugated to the metal chelator 2,2',2"-(l,4,7- triazacyclononane- l,4,7-triyl)triacetic acid (NOTA) and radiolabeled with64Cu for early diagnosis of AD by PET.

[0023] Yet another aspect of some embodiments of the invention relates to use of labeled liposomes that cross the BBB as a diagnostic tool for AD. In some embodiments the label is a fluorescent (e.g. Cy5). Alternatively or additionally , in some embodiments the label includes a radio-isotope.

[0024] Still another aspect of some embodiments of the invention relates to use of liposomes comprising l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Cholesterol; and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH) as a delivery vehicle for a therapeutic agent and / or a label to the brain. In some embodiments a molar ratio of DSPC:cholesterol: DSPE-PEG2k-COOH is 55:40:5. Alternatively or additionally, in some embodiments the therapeutic agent and / or the label are polyvalently coupled to the liposomes. In some embodiments the PEG has a molecular mass of 2Kd.

[0025] According to one aspect of some embodiments of the invention CP-2 is delivered to the brain without insulin. According to various exemplary embodiments of the invention CP-2 is coupled to a metal nanoparticle and / or to a liposome and the metal nanoparticle and / or liposome is not couped to insulin, either directly or by means of an intervening linker.

[0026] Various exemplary embodiments of the invention incorporate one, two, three, four or all five of these aspects.

[0027] It will be appreciated that the various aspects described above relate to solution of technical problems associated with early detection of Alzheimer's disease.

[0028] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solution of technical problems related to delivery of therapeutic agents to the brain across the BBB.

[0029] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solution of technical problems related to treatment of CNS disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0030] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solution of technical problems related to diagnosis of CNS disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0031] In some exemplary embodiments of the invention there is provided a composition including cyclic D,L-a-peptide according to Formula IA or Formula IB

[0032] Formula IA Formula IB coupled to liposomes for use as a medicament; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the medicament is formulated for treatment of Alzheimer's disease (AD). Alternatively or additionally, in some embodiments the medicament is formulated for intravenous injection. Alternatively or additionally, in some embodiments the liposomes include: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); cholesterol; and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE- PEG2k-COOH). Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 55:40:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 45:50:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 65:35:5. Alternatively or additionally, in some embodiments the composition is formulated as a medicament for Alzheimer's Disease (AD). Alternatively or additionally, in some embodiments the composition includes dye molecules according to Formula II coupled to the liposomes.

[0033] Alternatively or additionally, in some embodiments the dye molecules are polyvalently coupled to the liposomes.

[0034] In some exemplary embodiments of the invention there is provided a method of treatment including: administering a physiologically effective amount of a cyclic D,L-a-peptide according to Formula IA or Formula IB

[0035] Formula IA Formula IB coupled to liposomes to a subject in need thereof; wherein D and L indicate an enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the physiologically effective amount is at least 2.0 nanomoles. Alternatively or additionally, in some embodiments the administering is via intravenous injection. Alternatively or additionally, in some embodiments the liposomes include: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); cholesterol; and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH). Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE- PEG2k-COOH is 55:40:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 45:50:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 65:35:5. Alternatively or additionally, in some embodiments the method is directed towards Alzheimer's Disease (AD). Alternatively or additionally, in some embodiments the method includes a administering a label including dye molecules according to Formula II coupled to the liposomes.

[0036] Formula II

[0037] Alternatively or additionally, in some embodiments the dye molecules are polyvalently coupled to the liposomes.

[0038] In some exemplary embodiments of the invention there is provided a method of diagnosis or treatment including administering a cyclic D,L-a-peptide according to Formula IA or Formula IB coupled to metal nanoparticles to a subject in need thereof; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the metal nanoparticles comprise gold nanoparticles. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of not more than 65 nm. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of at least 45 nm. Alternatively or additionally, in some embodiments the method is directed towards diagnosis and / or treatment of Alzheimer's Disease (AD).

[0039] In some exemplary embodiments of the invention there is provided a composition including a cyclic D,L-a-peptide according to Formula IA or Formula IB

[0040] Formula IA Formula IB coupled to metal nanoparticles for use as a medicament; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the metal nanoparticles comprise gold nanoparticles. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of not more than 65 nm. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of at least 45 nm. Alternatively or additionally, in some embodiments the composition is formulated as a medicament for Alzheimer's Disease (AD).

[0041] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including: (a) a cyclic D,L-a-peptide according to Formula IA or Formula IB;

[0042]

[0043] Formula IA Formula IB

[0044] (b) a liposomal delivery vehicle; and

[0045] (c) diluents, excipients and carriers; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the pharmaceutical composition is formulated for intravenous injection. Alternatively or additionally, in some embodiments the liposomal delivery vehicle includes: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); cholesterol; and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k- COOH). Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 55:40:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-C00H is 45:50:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-C00H is 65:35:5. Alternatively or additionally, in some embodiments the pharmaceutical composition includes a label. Alternatively or additionally, in some embodiments the label comprises dye molecules according

[0046] Formula II

[0047] Alternatively or additionally, in some embodiments the label is polyvalently coupled to liposomes of the liposomal delivery vehicle. Alternatively or additionally, in some embodiments the cyclic D,L-a-peptide according to Formula IA or Formula IB is polyvalently coupled to liposomes of the liposomal delivery vehicle. In some exemplary embodiments of the invention there is provided a method of synthesis including:(a) dissolving l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH) in chloroform-methanol (4:1, v / v); (b) evaporating the chloroform-methanol and drying to form a film;(c) hydrating the film to form a suspension; and(d) co-extruding 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), cholesterol, and l,2-distearoyl-sn-glycero-3-phosphoethanolamine- PEG-carboxylic acid (DSPE-PEG2k-COOH) through 100 nm pores to produce liposomes; (e) activation of carboxylic acid groups on a surface of the liposomes with EDC / NHS; and (f) conjugation of a molecule according to Formula IA or Formula IB to the activated carboxylic acid groups.

[0048] Formula 1A Formula IB

[0049] In some embodiments a molar ratio of DSPCxholesterol:DSPE-PEG2k-COOH is 55:40:5.

[0050] Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE- PEG2k-COOH is 45:50:5. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 65:35:5. Alternatively or additionally, in some embodiments the method comprises including a DSPE-PEG2k-NH2 in the dissolving to introduce free amino groups on a surface of the liposomes. Alternatively or additionally, in some embodiments the method includes: reacting the liposomes with Cy5-NHS. Alternatively or additionally, in some embodiments a molar ratio of DSPCxholesterol:DSPE-PEG2k-COOH: DSPE- PEG2k-NH2 is 55:40:4.5:0.5.

[0051] In some exemplary embodiments of the invention there is provided a method including: (a) coating gold nanoparticles (GNPs) with citrate to produce citrate coated GNPs; (b) applying HS-PEG-COOH to a surface of the citrate coated GNPs to produce PEGylated GNPs; (c) activating carboxyl groups on the PEGylated GNPs with N-hydroxysuccinimide and l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide to produce activated GNPs; and (d) contacting a solution of CP-2 in dimethyl sulfoxide (DMSO) with the activated GNPs to produce CP-2 conjugated GNPs. In some embodiments the method includes purifying the CP-2-conjugated GNPs by centrifugation. Alternatively or additionally, in some embodiments the coating includes adding sodium citrate solution to a boiling solution of auric chloride (HAuCI4) in deionized water and cooling. Alternatively or additionally, in some embodiments the applying includes mixing the citrate coated GNPs with HS-PEG-COOH and HS-mPEG to a final PEG ratio of 2.33:1. Alternatively or additionally, in some embodiments the activating includes pre-reacting N- hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in DDW for at least 2 hours and adjusting the pH to 9.

[0052] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including:

[0053] (a) a cyclic D,L-a-peptide according to Formula IA or Formula IB;

[0054] (b) metal nanoparticles conjugated thereto; and

[0055] (c) diluents, excipients and carriers; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the metal nanoparticles comprise gold nanoparticles. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of not more than 65 nm. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of at least 45 nm. Alternatively or additionally, the pharmaceutical composition is formulated as a medicament for Alzheimer's Disease (AD). Alternatively or additionally, the pharmaceutical composition is formulated for intravenous injection. Alternatively or additionally, in some embodiments the cyclic D,L-a- peptide according to Formula IA or Formula IB is polyvalently coupled to the metal nanoparticles. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. In case of conflict, the patent specification, including definitions, will control. All materials, methods, and examples are illustrative only and are not intended to be limiting.

[0056] As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying inclusion of the stated features, integers, actions or components without precluding the addition of one or more additional features, integers, actions, components or groups thereof. This term is broader than, and includes the terms "consisting of" and "consisting essentially of" as defined by the Manual of Patent Examination Procedure of the United States Patent and Trademark Office. Thus, any recitation that an embodiment "includes" or "comprises" a feature is a specific statement that sub embodiments "consist essentially of" and / or "consist of" the recited feature.

[0057] The phrase "consisting essentially of" or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

[0058] The phrase "adapted to" as used in this specification and the accompanying claims imposes additional structural limitations on a previously recited component.

[0059] The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of architecture and / or computer science.

[0060] Percentages (%) of chemicals are V / V (volume per volume) unless otherwise indicated.

[0061] For purposes of this specification and the accompanying claims, the term "medicament" includes also diagnostic agents.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying figures. In the figures, identical and similar structures, elements or parts thereof that appear in more than one figure are generally labeled with the same or similar references in the figures in which they appear. Dimensions of components and features shown in the figures are chosen primarily for convenience and clarity of presentation and are not necessarily to scale. The attached figures are:

[0064] Fig. 1A is a diagram of the chemical structure of cyclic D,L-a-peptide (CP-2) with residues c- [Ly s1-D -Leu2-N1 e3-D -T rp4-Hi s5-D - S er6] ;

[0065] Fig. IB is a schematic representation of multifunctional liposomes prepared from DSPC, cholesterol, DSPE-PEG2k-COOH and DSPE-PEG2k-NH2 (55:40:4.5:0.5 molar ratio) with the liposome surface modified by oligomer-specific cyclic D,L-a-peptide (CP-2) and fluorescent contrast agent (Cy5);

[0066] Fig. 1C is a Cryo-EM photomicrograph of naked liposomes demonstrating monodispersed spherical particles;

[0067] Fig. ID is a Cryo-EM photomicrograph of CP-2-LPs demonstrating mono-dispersed spherical particles;

[0068] Fig. IE is a histogram of particle frequency as function of diameter in nm for naked liposomes from Cryo-EM images using ImageJ software;

[0069] Fig. IF is a histogram of particle frequency as function of diameter in nm for CP-2 conjugated LPs s from Cryo-EM images using ImageJ software;

[0070] Fig. 1G is an AFM photomicrograph of naked liposomes (LPs);

[0071] Fig. 1H is an AFM photomicrograph of CP-2 conjugated LPs (CP-2-LPs);

[0072] Fig. II is a plot of intensity as a function of size illustrating hydrodynamic size distribution of LPs and CP-2-LPs measured by DLS;

[0073] Fig. 1J is a plot of Zeta potential measurements of LPs, CP-2-LPs, and Cy5-CP-2-LPs as determined by DLS;

[0074] Fig. IK is a plot of absorbance as a function of wavelength (nm) comparing LPs, CP-2- LPs, free Cy5 and Cy5-CP-2-LPs;

[0075] Fig. IL is a plot of fluorescence intensity (a.u.) as a function of wavelength (nm) of LPs, CP-2-LPs, free Cy5 and Cy5-CP-2-LPs;

[0076] Fig. IM is a histogram of mean diameter (nm) as a function of time (days) for liposomes monitored over a period of 28 days (stored at 4-8 °C) based on DLS measurements comparing LPs and CP-2-LPs;

[0077] Fig. 2A is a plot of %aggregation as a function of time (h) for monomeric AP40 (10 pM) incubated with increasing concentrations of CP-2-LPs or CP-2 in PBS buffer (50 mM, pH 7.2) with the extent of amyloid formation monitored over time using ThT fluorescence;

[0078] Fig. 2B is a Cryo-EM micrograph of AP40 (10 pM) after 72 h incubation without CP-2- LPs;

[0079] Fig. 2C is an AFM micrograph of AP40 (10 pM) after 72 h incubation without CP-2-LPs;

[0080] Fig. 2D is a Cryo-EM micrograph of AP40 (10 pM) after 72 h incubation with CP-2-LPs (50 pM);

[0081] Fig. 2E is an AFM micrograph of AP40 (10 pM) after 72 h incubation with CP-2-LPs (50 pM);

[0082] Fig. 2F is an immuno-dot blot test illustrating the effect of CP-2-LPs and CP-2 on AP40 oligomer and fibril formation using oligomer-specific (All) and fibril-specific (OC) antibodies; AP40 (30 pM) was incubated for 0, 24, 48 or 72 h in the absence or presence of 150 pM of CP-2-LPs or CP-2, spotted onto nitrocellulose membranes, and probed with either All or OC antibodies as indicated;

[0083] Fig. 2G is bar graph of % cell viability illustrating the protective effect of CP-2-LPs and CP-2 on AP-induced toxicity in SH-SY5Y cells; AP40 (20 pM) was incubated for 48 h alone or with increasing concentrations of CP-2-LPs and CP-2 and exposed to the cells or 24 h; Cell viability was determined by the MTT assay; Naked LPs and CP-2 (200 pM) served as controls; Values are mean ± SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (ns = non-significant, *p< 0.05, ***p< 0.001);

[0084] Fig. 2H is a bar graph of % cell viability as a function of concentration for CP-2 and CP-2- LPs in human neuroblastoma SH-SY5Y cells; Cells were incubated with increasing concentrations of CP-2 or an equivalent amount of CP-2-LPs (0 - 200 pM) for 48 h; Cell viability was evaluated using the MTT assay; results are presented as a percentage of the control (untreated) cells and are expressed as the mean ± SD from two independent experiments (n = 3 for each experiment); Statistical analysis was performed using the Student's t-test, with p> 0.05 considered nonsignificant (ns).

[0085] Fig. 21 is a bar graph of % cell viability as a function of concentration for various concentrations of CP-2 and CP-2-LPs in rat PC-12 cells exposed to toxic level of AP40 (20 pM); AP40 (20 pM) was incubated for 48 h alone or with increasing concentrations (20, 100 or 200 pM) of CP-2 or CP-2-LPs and exposed to PC12 cells for 24 h; Cell viability was determined by the MTT assay; Naked LPs and CP-2 (200 pM) were used as controls; Fig. 3A is a Kaplan-Maier plot of % survival a function of time in days for C. elegans expressing human AP; CL2006 (transgenic) and CL802 (WT) worms were treated with 50 pM of CP-2 or equivalent amount of CP-2-LPs; the survival curve represents three independent studies (100 worms in each group);

[0086] Fig. 3B is a bar graph of mean lifespan (days) for CL2006 (transgenic) and CL802 (WT) worms treated with CP-2-LPs, CP-2, or nothing; values are mean ± SD from three experiments analyzed by one-way ANOVA followed by Tukey's multiple comparison test (ns=non-significant, *p<0.05, ***p<0.001);

[0087] Fig. 3C is a bar graph of number of body thrashes / min as a function of concentration (pM) of CP-2 or CP-2-LPs for transgenic CL2355 (neuronal A -expressing) and WT CL2122 worms; data are mean ± SD from three experiments (n=20) and statistical analysis was performed as described above (**p<0.01, ***p<0.001);

[0088] Fig. 3D is a bar graph of chemotaxis index (Cl) towards 0.1% benzaldehyde (in absolute Ethanol; V / V) at RT as a function of concentration (pM) of CP-2 or CP-2-LPs for transgenic CL2355 and WT CL2122 worms; Synchronized worms were fed with increasing amount of CP-2 (2-50 pM) or equivalent amount of CP-2-LPs at 16°C for 36 h and at 23°C for another 36 h; Results are mean ± SD from three independent experiments analyzed as described above (n=30; *p<0.05, ***p<0.001);

[0089] Fig. 3E is an immune-dot blot experiment developed by sequence and oligomer specific antibodies (6E10 and All, respectively) after spotting equal amounts of proteins extracted from transgenic CL2006 and WT CL802 worms treated with CP-2, CP-2-LPs or vehicle;

[0090] Fig. 3F is a western immunoblot of AP species in transgenic CL2006 and WT CL802 worms fed with CP-2, CP-2-LPs or vehicle, equal amounts of extracted proteins were loaded onto each lane and immunoblotted with an anti-A antibody (6E10); anti-a-tubulin antibody served as a loading control;

[0091] Fig. 3G is bar graph of relative band intensity (folds) for untreated transgenic CL2006 worms or the same worms treated with CP-2, CP-2-LPs based on the western blot of Fig. 3F using ImageJ;

[0092] Fig. 3H is representative in vivo fluorescence images of ~6-month-old C57BL / 6J mice 4 h post intravenous (LV.) injection with 0.1 mL of saline (Left image [animal]) or Cy5-labelled liposomes conjugated with CP-2 (Cy5-CP-2-LPs, 3.8 pM) (Right image [animal]), Images were captured using a MAESTRO II (CRi) in vivo fluorescence imaging system; Fig. 31 is a series of ex vivo fluorescence imagers showing biodistribution of Cy5-CP-2- LPs in mice; C57BL / 6J mice were perfused and 4 or 24 h post injection of vehicle or Cy5-CP-2- LPs (3.8 pM), the mice were euthanized and tissues were removed and imaged;

[0093] Fig. 3J is a bar graph of aaverage fluorescence intensity of Cy5-CP-2-LPs in different organs from three independent experiments, including the data shown in Fig. 31, providing a quantitative assessment of the distribution across different tissues;

[0094] Fig. 4A is a series of fluorescence images obtained at different time intervals (4, 24 and 72 h) from WT and 5xFAD mice brains (aged 5-6 months) after single LV. injection of 0.1 mL of Cy5-CP-2-LPs;

[0095] Fig. 4B is a plot of average fluorescence signal (X106photons / CM2 / S) as a function of time (h) for fluorescence signals obtained from brain samples from 3 independent experiments (n=3) including data from Fig 4A; Results are mean ± SD analyzed using student's unpaired t-test (n = 3 each; *p < 0.05, ***p <0.001);

[0096] Fig. 4C is a series of representative fluorescence images of 5xFAD mice brains after intravenous administration of 0.1 mL of Cy5-CP-2-LPs or control Cy5-LPs conjugated to p-alanine ethyl ester (Cy5-P-Ala-LPs);

[0097] Fig. 4D is a bar graph of average fluorescence signal (X106photons / CM2 / S) based on 3 independent experiments (n=3) including data from Fig 4C; Results are mean ± SD analyzed as described above (n = 3 each; ***p <0.0001);

[0098] Fig. 4E is a series of representative immunofluorescence images of cortex, thalamus, and hippocampus of a 5xFAD mouse brain stained with Cy5-CP-2-LPs (Left column) and sequence-specific monoclonal 6E10 antibody (second column from left); mice were injected intravenously with 0.1 mL of Cy5-CP-2-LPs, perfused after 24 h with saline and sacrificed; sections were blocked with 5% BSA, incubated with 6E10 antibody followed by incubation with Alexa Fluor 488 goat anti-mouse IgG secondary antibody, and stained with DAPI (second column from right). The three columns represent Cy5-CP-2-LPs, AP (6E10) and DAPI, respectively; all data is merged in the right column;

[0099] Fig. 4F is a plot of pixel intensity (a.u). as a function of pixel distance (a.u.) using ImageJ software for analysis of co-localization intensity profiles; Line scan graphs show the intensity obtained from the red (Cy5-CP-2-LPs) and green (6E10) fluorescence channels using the ImageJ Plot Profile tool;

[0100] Fig. 5 is a histogram of normalized absorption (a.u) as a function of wavelength (nm) for unmodified (naked) gold nanoparticles (GNPs), PEG-conjugated GNPs, and CP-2-conjugated GNPs as indicated;

[0101] Fig. 6 is a histogram of ThT fluorescence as a function of incubation time for Ap alone and A plus increasing amounts of CP-2-conjugated GNPs (4; 22 and 44 pM as indicated);

[0102] Fig. 7 is a bar graph illustrating the Effect of CP-2-GNPs on the motility of C. elegans strains GMC101 and CL2122;worms were fed increasing concentrations of CP-2-GNPs at the L4 stage for 24 hours, followed by exposure to 25°C for 24 hours. Data are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey's multiple comparison test (n=20 worms per group; ***P <0.001, ns = not significant); experiments were repeated for 3 times;

[0103] Fig. 8A is a western blot analysis of total proteins from treated and untreated transgenic GMC101 C. elegans treated with CP-2-GNPs (25 or 50 pM) or vehicle (0 pM); total proteins were analyzed using anti-A 6E10 antibody and anti-a-tubulin antibody was used for normalization; left lane is a Molecular weight (MW) marker;

[0104] Fig. 8B is a bar graph showing relative intensity (folds) of A bands at ~15 kDa for the western blot of Fig. 8A as a function of CP-2-GNP concentration in pM as quantified using IMAGEJ software;

[0105] Fig. 8C is a dot blot analysis of total proteins from treated GMC101 C. elegans worms using the All antibody, which specifically detects A oligomers;

[0106] Fig. 9 is a simplified flow diagram of a method according to some embodiments of the invention; and

[0107] Fig. 10 is a simplified flow diagram of a method according to some embodiments of the invention.

[0108] DETAILED DESCRIPTION OF EMBODIMENTS

[0109] Embodiments of the invention relate to a cyclic D,L-a-peptide according to Formula IA

[0110] Formula IA Formula IB coupled to either liposomes (LPs) or metal nanoparticles (NPs). Various exemplary embodiments of the invention relate to treatment methods, diagnostic methods, compositions for use, pharmaceutical compositions and synthesis methods.

[0111] Specifically, some embodiments of the invention can be used to treat and / or dignose neurodegenerative diseases or other CNS conditions such as Alzheimer's disease.

[0112] The principles and operation of methods, compositions for use, and pharmaceutical compositions according to exemplary embodiments of the invention may be better understood with reference to the drawings and accompanying descriptions.

[0113] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0114] Exemplary treatment method

[0115] In some exemplary embodiments of the invention there is provided a method of treatment including administering a physiologically effective amount of a cyclic D,L-a-peptide according to Formula IA or Formula IB coupled to liposomes to a subject in need thereof. In the diagrams D and L indicate an enantiomeric state of the amino acids in the cyclic peptide. The cyclic D,L-a-peptide according to Formula IA or Formula IB is referred to throughout this application as CP-2.

[0116] In some exemplary embodiments of the invention, the physiologically effective amount of CP-2 is at least 2.0 nanomoles. In experimental examples presented hereinbelow concerning CP-2 injected for diagnostic purposes, the concentration of liposomes was about 30-40 pM and 0.1 mLwas injected. For therapeutic efficacy in worms, concentration between 2 and 10 pM was found to be effective upon feeding the worms with CP-2-LPs.

[0117] According to various exemplary embodiments of the invention the minimum physiologically effective amount of CP-2 is at least 1 nanomole, at least 2 nanomoles, at least 4 nanomoles, at least 6 nanomoles, at least 8 nanomoles, at least 10 nanomoles, at least 12 nanomoles, or intermediate or greater numbers of nanomoles. Alternatively or additionally, according to various exemplary embodiments of the invention, the minimum physiologically effective amount of CP-2 does not exceed 11 nanomoles, does not exceed 9 nanomoles, does not exceed 7 nanomoles, does not exceed 5 nanomoles, does not exceed Bnanomoles, does not exceed 1 nanomoles, or intermediate or smaller numbers of nanomoles.

[0118] There is a considerable body of knowledge dealing with translation of dosages in laboratory animal (such as mice) to human dosages.

[0119] In functional terms, the physiologically effective amount of CP-2 is an amount which inhibits aggregation of AP peptide in a statistically significant manner. Methods to assay inhibition of aggregation of A peptide are presented hereinbelow.

[0120] In some exemplary embodiments of the invention, the administering of CP-2 is via intravenous injection. In other exemplary embodiments of the invention, other injection routes (e.g. intraperitoneal) are employed.

[0121] In some exemplary embodiments of the invention, the liposomes include 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC); Cholesterol; and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH). According to various exemplary embodiments of the invention a molar ratio of DSPC:cholesterol: DSPE-PEG2k-COOH is 55:40:5 or 45:50:5 or 65:35:5.

[0122] In some embodiments, the treatment method is directed towards Alzheimer's Disease (AD).

[0123] In some embodiments, the method includes administering a label comprising dye molecules according to Formula II coupled to the liposomes.

[0124] Formula II According to these embodiments, addition of the dye is for diagnosis, not treatment.

[0125] Dye molecules according to Formula II are commonly known as Cy5 and are referred to that way throughout the application.

[0126] In order to facilitate conjugation of Cy5 to the liposomes, the composition of the liposomes is changed by incorporation of DSPE-PEG(2K)-NH2. The final composition of the liposomes is: DSPC, cholesterol, DSPE-PEG2k-COOH, and DSPE-PEG2k-NH2 (55:40:4.5:0.5) molar ratio. In some exemplary embodiments of the invention, the dye molecules are polyvalently coupled to the liposomes.

[0127] Exemplary composition for use

[0128] In some exemplary embodiments of the invention there is provided a composition comprising cyclic D,L-a-peptide according to Formula IA or Formula IB

[0129] Formula IA Formula IB coupled to liposomes for use as a medicament. Again, D and L indicate enantiomeric states of the amino acids in the cyclic peptide. In some embodiments the medicament is formulated for treatment of Alzheimer's disease (AD). Additional features of the composition for use are a recited hereinabove in the context of "exemplary treatment method".

[0130] Additional exemplary medical method

[0131] In some exemplary embodiments of the invention there is provided a method of diagnosis or treatment comprising administering a cyclic D,L-a-peptide according to Formula IA or Formula IB

[0132]

[0133] Formula IA Formula IB coupled to metal nanoparticles to a subject in need thereof. Again, D and L indicate enantiomeric states of the amino acids in the cyclic peptide. In some embodiments the method is directed towards treatment and / or detection of Alzheimer's Disease (AD).

[0134] If treatment is the goal of the method, the amount of CP-2 must be physiologically effective as explained hereinabove. If diagnosis is the goal of the method, the amount of metal nanoparticles must be detectable using a relevant imaging methodology. In initial studies in mice, 25 mg / kg of gold nanoparticles was sufficient for detection.

[0135] In some exemplary embodiments of the invention, the metal nanoparticles include gold nanoparticles. In some exemplary embodiments of the invention, the gold nanoparticles have an average diameter of not more than 65 nm. Alternatively or additionally, in some embodiments the gold nanoparticles have an average diameter of at least 45 nm. Additional exemplary composition for use

[0136] In some exemplary embodiments of the invention there is provided a composition comprising a cyclic D,L-a-peptide according to Formula IA or Formula IB

[0137] Formula IA Formula IB coupled to metal nanoparticles for use as a medicament. Again D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

[0138] Again, if the medicament is therapeutic, the amount of CP-2 must be physiologically effective as explained hereinabove. Again, if the medicament is diagnostic the amount of metal nanoparticles must be detectable using a relevant imaging methodology.

[0139] Other features of the composition for use are as set forth hereinabove in the context of "Additional exemplary medical method".

[0140] Exemplary liposomal pharmaceutical composition

[0141] In some exemplary embodiments of the invention there is provided a pharmaceutical composition including:

[0142] (a) a cyclic D,L-a-peptide according to Formula IA or Formula IB;

[0143] Formula IA Formula IB

[0144] (b) a liposomal delivery vehicle; and

[0145] (c) diluents, excipients and carriers.

[0146] Again, D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

[0147] In some exemplary embodiments of the invention, the pharmaceutical composition is formulated for intravenous injection. In other exemplary embodiments of the invention, the pharmaceutical composition is formulated for other delivery routes such as intraperitoneal injection or direct application to the brain (e.g. via a port).

[0148] In some embodiments the liposomal delivery vehicle includes: l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC); Cholesterol; and l,2-distearoyl-sn-glycero-3-phosphoethanolamine- PEG-carboxylic acid (DSPE-PEG2k-COOH). According to various exemplary embodiments of the invention a molar ratio of DSPC:cholesterol: DSPE-PEG2k-COOH is 55:40:5 or 45:50:5 or 65:35:5. In some exemplary embodiments of the invention, the pharmaceutical composition includes a label. In some embodiments the label includes dye molecules according to Formula II.

[0149] Dye molecules according to Formula II are commonly known as CY5. In some embodiments the label is polyvalently coupled to liposomes of the liposomal delivery vehicle. Alternatively or additionally, in some embodiments CP-2 is polyvalently coupled to liposomes of the liposomal delivery vehicle. Those exemplary embodiments of the invention that include a label are amenable to use for diagnostic purposes.

[0150] Exemplary method of synthesis for CP-2 liposomes

[0151] Fig. 9 is a simplified flow diagram of a synthesis method, indicated generally as 900, according to some exemplary embodiments of the invention.

[0152] Depicted exemplary method 900 includes dissolving 910 l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, and l,2-distearoyl-sn-glycero-3-phosphoethanolamine- PEG-carboxylic acid (DSPE-PEG2k-C00H) in chloroform-methanol (4:1, v / v).

[0153] In the depicted embodiment, the chloroform-methanol is evaporated 920 and the lipids are dried to form a film.

[0154] Alternatively or additionally, in the depicted embodiment, the film is hydrated 930 to form a suspension.

[0155] In the depicted embodiment, method 900 includes co-extruding 940 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), cholesterol, and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-C00H) through pores to produce liposomes. In some embodiments the pores have a diameter of 100 nm.

[0156] In some exemplary embodiments of the invention, method 900 includes activation 950 of carboxylic acid groups on a surface of the liposomes with EDC / NHS.

[0157] In order to form CP-2 liposomes a molecule according to Formula IA or Formula IB is conjugated 960 to the activated carboxylic acid groups.

[0158]

[0159] Formula 1A Formula IB

[0160] According to various exemplary embodiments of the invention a molar ratio of DSPC:cholesterol:DSPE-PEG2k-COOH is 55:40:5 or 45:50:5 or 65:35:5.

[0161] In some exemplary embodiments of the invention, method 900 includes: including a DSPE-PEG2k-NH2 in dissolving 910 to introduce free amino groups on a surface of the liposomes and reacting the liposomes with Cy5-NHS. In some of these embodiments a molar ratio of DSPC:cholesterol:DSPE-PEG2k-COOH: DSPE-PEG2k-NH2 is 55:40:4.5:0.5.

[0162] Exemplary method of synthesis for CP-2 metal nanoparticles

[0163] Fig. 10 is a simplified flow diagram of a synthesis method, indicated generally as 1000, according to some exemplary embodiments of the invention.

[0164] Depicted exemplary method 1000 includes coating 1010 gold nanoparticles (GNPs) with citrate to produce citrate coated GNPs. In the depicted embodiment, method 1000 includes applying 1020 HS-PEG-COOH to a surface of the citrate coated GNPs to produce PEGylated GNPs.

[0165] In the depicted embodiment, method 1000 includes activating 1030 carboxyl groups on the PEGylated GNPs with N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide to produce activated GNPs.

[0166] Depicted exemplary method 1000 includes contacting 1040 a solution of CP-2 in dimethyl sulfoxide (DMSO) with the activated GNPs to produce CP-2 conjugated GNPs.

[0167] In some exemplary embodiments of the invention, the method includes purifying the CP-2-conjugated GNPs by centrifugation.

[0168] In some embodiments coating 1010 includes adding sodium citrate solution to a boiling solution of auric chloride (HAuCI4) in deionized water and cooling. In some exemplary embodiments of the invention, applying 1020 includes mixing the citrate coated GNPs with HS-PEG-COOH and HS-mPEG to a final PEG ratio of 2.33:1.

[0169] In some exemplary embodiments of the invention, activating 1030 includes pre-reacting N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in DDW for at least 2 hours and adjusting the pH to 9.

[0170] Additional exemplary pharmaceutical composition

[0171] In some exemplary embodiments of the invention there is provided a pharmaceutical composition comprising a cyclic D,L-a-peptide according to Formula IA or Formula IB; with metal nanoparticles conjugated thereto and diluents, excipients and carriers. Again, D and L indicate enantiomeric state of the amino acids in the cyclic peptide. In some embodiments the metal nanoparticles include gold nanoparticles. In some embodiments the gold nanoparticles have an average diameter of not more than 65 nm and / or have an average diameter of at least 45 nm. In some exemplary embodiments of the invention, the pharmaceutical composition is formulated as a medicament for Alzheimer's Disease (AD). Alternatively or additionally, in some embodiments the pharmaceutical composition is formulated for intravenous injection. In other exemplary embodiments of the invention, the pharmaceutical composition is formulated for other routes of injection such as intraperitoneal injection and direct application to the brain (e.g. via a port). Alternatively or additionally, in some embodiments the cyclic D,L-a-peptide according to Formula IA or Formula IB is polyvalently coupled to the metal nanoparticles. In some embodiments pharmaceutical compositions comprising metal nanoparticles are used as diagnostic agents.

[0172] It is expected that during the life of this patent many new liposome formulations will be developed and the scope of the invention includes all such new technologies a priori. As used herein the term "about" refers to ± 10 %.

[0173] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0174] Specifically, a variety of numerical indicators have been utilized. It should be understood that these numerical indicators could vary even further based upon a variety of engineering principles, materials, intended use and designs incorporated into the various embodiments of the invention. Additionally, components and / or actions ascribed to exemplary embodiments of the invention and depicted as a single unit may be divided into subunits. Conversely, components and / or actions ascribed to exemplary embodiments of the invention and depicted as sub-units / individual actions may be combined into a single unit / action with the described / depicted function.

[0175] Alternatively, or additionally, features used to describe a method can be used to characterize an apparatus and features used to describe an apparatus can be used to characterize a method.

[0176] It should be further understood that the individual features described hereinabove can be combined in all possible combinations and sub-combinations to produce additional embodiments of the invention. The examples given above are exemplary in nature and are not intended to limit the scope of the invention which is defined solely by the following claims.

[0177] Each recitation of an embodiment of the invention that includes a specific feature, part, component, module or process is an explicit statement that additional embodiments of the invention not including the recited feature, part, component, module or process exist.

[0178] Alternatively or additionally, various exemplary embodiments of the invention exclude any specific feature, part, component, module, process or element which is not specifically disclosed herein.

[0179] Specifically, the invention has been described in the context of Alzheimer's disease but might also be used in other CNS disorders.

[0180] All publications, references, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

[0181] The terms "include", and "have" and their conjugates as used herein mean "including but not necessarily limited to".

[0182] Additional objects, advantages, and novel features of various embodiments of the invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0183] EXAMPLES

[0184] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

[0185] The following materials and methods are used in performance of experiments described in examples hereinbelow:

[0186] Materials and Methods

[0187] A 4O was purchased from Hanhong Scientific (Shanghai, China) and stored as a lyophilized powder at -20 °C.

[0188] CP-2 Cyclic D,L-a-peptide 2 (CP-2) was synthesized using solid-phase peptide synthesis (SPPS) with the Fmoc strategy. Fmoc-L-Lys-loaded trityl chloride resin was initially swelled in N- Methyl-2-pyrrolidone (NMP) for 45 minutes. The Fmoc protecting group was removed by treatment with 20% piperidine in NMP (V / V) for 25 minutes. Each coupling step was carried out with 5 equivalents (eq.) of Fmoc-amino acids, 4.9 equivalents of hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), and 10 equivalents of N,N-Diisopropylethylamine (DIPEA), with amino acid pre-activation for 5 minutes prior to resin addition. Each coupling reaction was conducted for 45 minutes and followed by Fmoc deprotection. The amino acid sequence included Fmoc-D-Serine(tBu)-OH, Fmoc-L-Histidine(Trt)-OH, Fmoc-D-Tryptophan-OH, Fmoc-L-Norleucine-OH, and Fmoc-D-Leucine-OH.

[0189] Upon completion of the synthesis and deprotection of the final Fmoc group, the protected linear peptide was cleaved from the resin using 2% trifluoroacetic acid (TFA) in dichloromethane (V / V). The resulting solution was neutralized with 10% pyridine in methanol (V / V) and the volatiles were evaporated under reduced pressure. The cyclization of the peptide was performed in dimethylformamide (DMF) at 2 mg of crude protected peptide per 1 mL of DMF. The cyclization reaction was carried out using l-hydroxy-7-azabenzotriazole (HOAT, 1.5 eq.), O-(lH-6-chlorobenzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HCTU, 1.5 eq.), and DIPEA (3 eq.) and stirred for 72 hours at room temperature. Following cyclization, the solvent was removed, and the crude peptide was deprotected using a cleavage mixture (TFA: HZO:TIS 95:2.5:2.5; V / V) for 2-3 hours. The crude CP-2 was purified by preparative HPLC using a gradient mobile phase of acetonitrile (10%-100%) and double-distilled water (DDW) containing 0.1% TFA, over a 30-minute run. The final product showed an observed mass of 764.4 g / mol (calculated: 764.9 g / mol).

[0190] Lipid derivatives including l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG- carboxylic acid (DSPE-PEGZk-COOH) and l,2-istearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] (DSPE-PEGZk-NHz) were purchased from Nanosoft Polymers (NC, USA). l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol were purchased from TCI Chemicals.

[0191] Sulfo-Cy5 was purchased from AAPPTec (Louisville, KY, USA). All other chemicals and reagents were of analytical grade. All and OC antibodies were generously provided by Dr. Rakez Kayed (Department of Neurology, University of Texas Medical Branch), 6E10 antibody was obtained from Covance, and secondary antibodies were purchased from Merck.

[0192] Preparation of Naked Liposomes: Liposomes were prepared using a thin-film hydration method (C. Tzror-Azankot et al. ACS Nano 2021, 15, 1301-1309 and G. Kogkos et al. Biomolecules 2022, 12.) Briefly, DSPC, cholesterol and DSPE-PEGZk-COOH at a molar ratio of 55:40:5 were dissolved in chloroform-methanol (4:1, v / v) and stirred continuously for 1 h. The solvents were then evaporated using a stream of Nzgas, and the remaining lipid components were dried under high vacuum to form a thin lipid film. The film was hydrated in PBS (100 mM, pH 7.4) at 65 °C, and the resulting suspension was sonicated for 2 min in a water bath followed by vigorous mixing with a vortex. The lipid suspension was extruded through a polycarbonate membrane with a pore size of 100 nm for 20 cycles using a mini-extruder (Avanti Polar Lipids, Inc.). After extrusion, the suspension was dialyzed against PBS (100 mM, pH 7.4) for 24 h (x3, 8 h each), using a membrane with MWCO of 6000-8000 to obtain blank liposomes.

[0193] Preparation of CP-2 Conjugated Liposomes (CP-2-LPs): CP-2 was coupled through its free amine group to the carboxyl groups presented on the surface of the liposomes using 1- ethyl-3-(3- (dimethylamino)propyl)carbodiimide hydrochloride (EDC.HCI) and N- hydroxysuccinimide (NHS) (S. Senapati et al. Small 2024, e2311670). Briefly carboxyl groups of the liposomes were first activated using an excess of EDC and NHS (DSPE-PEG2k-COOH:EDC:NHS, 1:10:10 molar ratio) for 10 min at room temperature (RT) under gentle shaking. CP-2 was added to the reaction mixture at a 1:1.5 molar ratio of DSPE-PEG2k-COOH:CP-2 and the reaction was allowed to proceed for an additional 12 h. Excess of unreacted reagents was removed by dialysis against PBS (100 mM, pH 7.4) using a membrane with a molecular cutoff filter of 6-8kDa. The dialyzed solution containing CP-2-LPs was collected, and the concentration of unreacted CP-2 was determined at 280 nm using the extinction coefficient of the tryptophan residue.

[0194] Preparation of Fluorescently Labeled CP-2-LPs: Multifunctional liposomes were prepared from DSPC, cholesterol, DSPE-PEG2k-COOH and DSPE-PEG2k-NH2 at a molar ratio of 55:40:4.5:0.5, as described above. Fluorescent labeling was achieved by covalent conjugation of Cy5 with the free amine groups in the liposomes. In brief, the carboxylic groups of Cy5 were initially activated with EDC / NHS for 1 h in double-distilled water (DDW). The activated NHS-Cy5 was then added to the liposome solution at a 1:1.5 DSPE-PEG-NH2:Cy5 molar ratio and the mixture was stirred gently overnight. The free carboxylic acids of the liposomes were then activated with EDC / NHS and conjugated to CP-2, as described above. Unbound Cy5 and CP-2 were removed from the solution by dialysis in PBS (x3, 8 h each) with a 6-8 kDa cutoff filter.

[0195] Conjugation efficiency of CP-2 and Cy5 to the liposomes: The efficiency of CP-2 and Cy5 conjugation was assessed as described before (E. Suleiman et al. Pharmaceutics 2020, 12).

[0196] Briefly, following the conjugation step, non-conjugated CP-2 and Cy5 were separated from the liposomes using either dialysis through a membrane with a molecular cutoff filter of 6-8 kDa or ultracentrifugation (Beckman-Coulter Inc.) at 120,000 rpm for 30 min. The concentration of free

[0197] CP-2 and Cy5 were subsequently determined spectrophotometrically at 280 and 645 nm, utilizing the known extinction coefficient of the tryptophan residue (5690 M1cm1) and Cy5 (230000 M1cm1).

[0198] Conjugation efficacy was calculated using the equation:

[0199] (initial moles of CP-2)-(moles of unconjugated CP-2)

[0200] Conjugation efficiency= xl00

[0201] (initial moles of CP-2)

[0202] Accordingly, the conjugation efficiency of CP-2 and Cy5 with the liposome was 67% and

[0203] 63%, respectively. Synthesis of Gold Nanoparticles (GNPs)

[0204] Gold nanoparticles (GNPs) were synthesized using the Enustun and Turkevich method (Enustun, B. V.; Turkevich, J. Coagulation of Colloidal Gold. J. Am. Chem. Soc. 1963, 85, 3317- 3328). A 50% (w / v) solution of auric chloride (HAuCI4) in deionized water (HZO) was prepared and stored at 4°C. A 200 mL volume of deionized water (DDW) was heated in an oil bath while stirring in a 500 mL round-bottom flask. To this solution, 414 pL (120 mg) of HAuCI4was added, and the mixture was heated to boiling. Subsequently, 4.1 mL of 10% sodium citrate solution was introduced, causing the solution's color to change from yellow to dark purple. After 1-2 minutes of stirring, the mixture was cooled to room temperature. The citrate-coated GNPs were characterized using UV / Vis spectroscopy (wavelength range: 400-700 nm) and dynamic light scattering (DLS).

[0205] To modify the surface of the GNPs, bifunctional polyethylene glycol (PEG) derivatives were employed. The GNPs were mixed with HS-PEG-COOH (34 mg, 6.8 x 10“6mmol) and HS- mPEG (14.5 mg, 2.9 x 10“6mmol) in 1.13 mL and 0.484 mL of DDW, respectively, achieving a final PEG ratio of 2.33:1. The reaction mixture was incubated overnight, followed by purification of the conjugated GNPs by centrifugation at 18,000 RPM at 49C for 45 minutes. The resulting GNPs were characterized using DLS and zeta potential measurements.

[0206] For activation of the carboxyl groups on the PEGylated GNPs, N-hydroxysuccinimide (NHS, 43 mg, 746 pmol) and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 20 mg, 208 pmol) were dissolved in 500 pL of DDW and allowed to react for 2 hours. The pH of the solution was adjusted to 9 using a 0.01 M sodium borate buffer. CP-2 (5.4 mg, 24.3 pmol), dissolved in 291 pL of dimethyl sulfoxide (DMSO), was added to the activated GNPs and incubated for 24 hours. The CP-2-conjugated GNPs were purified by centrifugation at 14,000 RPM for 40 minutes. Unreacted CP-2 in the supernatant was quantified by high-performance liquid chromatography (HPLC) to calculate the conjugation yield.

[0207] Characterization

[0208] Cryogenic Transmission Electron Microscopy (Cryo-EM): The morphology and size of blank liposomes and CP-2-LPs were analyzed with a Tecnai G2 cryo-EM (FEI TECNAI™ G2, Hillsboro, Oregon) operating at an acceleration voltage of 120 kV. For imaging samples (3 pL) were loaded onto lacey grids, blotted, and rapidly frozen by plunging into liquid ethane at -180°C. Grids with frozen samples were stored in liquid nitrogen. Images were digitally recorded with a multiScan camera (Gatan794) at various magnifications. Atomic Force Microscopy (AFM): Samples (5 pL) were deposited on a silicon wafer substrate. After drying overnight at RT, images were acquired using a Bio FastScan scanning probe microscope (Bruker AXS) in tapping mode using a silicon probe with a spring constant of 18 N / m and a cantilever with a resonance frequency of approximately 1400 kHz.

[0209] Particle Size, Stability and Zeta Potential Measurements: The average size, polydispersity index (PDI) and ^-potential of the liposomes were determined in 10 mM PBS (pH 7.4) at RT using a ZetaSizer 3000 HS dynamic light scattering (DLS) instrument (Malvern, UK). The stability of the liposomes in PBS stored at 4-8 °C was monitored over a period of one month by assessing the hydrodynamic size.

[0210] Thioflavin T (ThT) Aggregation Assay: Anti-amyloidogenic activity was assessed by a kinetic ThT assay, as previous described (K M. Richman, et al. J. Am. Chem. Soc. 2013, 135, 3474- 3484). Briefly, monomerized solutions of AP40 (33 pM, 60 pL) in PBS (50 mM, pH 7.2) were incubated in a 96-well flat-bottom black plate with increasing concentrations of CP-2 and CP-2- LPs (20 pL of samples containing 5% DMSO). A solution of ThT (30 pM, 120 pL) in PBS was added to each well resulting in final concentrations of 10 pM for AP40 and 10, 50 and 100 pM for CP-2 or an equivalent amount of CP-2-LPs in 0.5% DMSO. The plate was covered with a transparent polyolefin film and placed in a microplate reader (Synergy Hl, BioTek, USA). Fluorescence intensity of amyloid-bound ThT samples was monitored hourly at 37°C over a period of 72 h using excitation and emission wavelengths of 430 and 492 nm, respectively. Before each reading, the plate was shaken for 2 min. The experiments were repeated three times.

[0211] For experiments with gold nanoparticles a slightly different ThT protocol was employed: Briefly, 20 pL of CP-2-GNPs (440 pM based on CP-2 content), 120 pL of PBS buffer, 60 pL of AP (67 pM), and 4 pL of ThT (1 mM) were added to each well of a black 96-well plate. The final concentrations in the wells were 20 pM for both AP and ThT, and 4-44 pM for CP-2-GNPs. The plate was sealed with clear polyolefin foil and placed in a Synergy Hl microplate reader at 37°C. The fluorescence of amyloid-bound ThT was measured hourly over a 90-hour period at excitation and emission wavelengths of 430 nm and 492 nm, respectively. Each reading was preceded by a 2-minute shaking step to ensure uniform mixing.

[0212] Antibody (All and OC) Dot-Blot Assay: Monomerized solutions of AP40 (30 pM) in PBS (50 mM, pH 7.4) were incubated at 37°C for different durations with or without 150 pM CP-2 or equivalent amount of CP-2-LPs in PBS (50 mM, pH 7.4), and stored at -80 °C until analysis. Samples (2 pL) were spotted onto nitrocellulose membranes (0.2 pm, Whatman) and dried at RT. The membranes were blocked for 1 h with 5% nonfat milk solution in Tris-buffered saline (TBS, 10 mM) containing 0.1% Tween-20 (TBST) and washed 3 times (10 min each) with TBST and incubated at 4 °C overnight with oligomer specific antibody All (at 1:5000 dilution), antiamyloid fibril antibody OC (1:5000 dilution) and anti-AP antibody 6E10 (1:1000 dilution) in 0.5% nonfat milk in TBST. The membranes were washed again thrice with TBST (10 min each) and incubated for another hour at RT with either horseradish peroxidase (HRP) conjugated antirabbit IgG (1:5000 dilution) for All and OC or HRP-conjugated anti-mouse IgG (1:10000 dilution) for 6E10 in 0.5% nonfat milk solution in TBST. The blots were washed thrice with TBST and developed using the enhanced chemiluminescence (ECL) reagent kit (Bio-rad, Israel). The chemiluminescence was measured using an I MAG EQUANT™ LAS 4000 system.

[0213] Cell Culture Studies: Neuronal-like rat pheochromocytoma PC12 cell line was cultured in low-glucose Dulbecco's modified eagle medium (DMEM) supplemented with horse serum (10%), fetal bovine serum (FBS; 5%), L-glutamine, penicillin, and streptomycin at 37 °C in a 5% CO2 incubator. Human neuroblastoma SH-SY5Y cells were grown in the same medium expect that 10% FBS was used. The effect of CP-2-LPs and CP-2 on A -induced toxicity was determined as described before. In brief, AP40 (200 pM) was aged for 48 h without or with increasing concentrations of CP-2-LPs or CP-2 in PBS containing 5% DMSO in a total volume of 50 pL. On the day of the experiment, the medium was replaced by fresh medium (90 pL) and the aged samples (10 pL) were diluted by a factor of 10 in the medium. Cells treated with 0.5% DMSO served as control (100% cell viability). After 24 h incubation, cell viability was determined by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Experiments were performed in quintuplicate and repeated three times.

[0214] C. elegans Strains and Maintenance: Transgenic C. elegans strains GMC101 [dvIslOO, expressing Api-42 and GFP]; CL2355 [dvls50, pCL45 (snb-l::Abeta l-42::3' UTR(long) + mtl- 2::GFP] and CL2006 [dvls2, pCL12(unc-54 / human Abeta peptide 1-42 minigene) + rol-6(sul006)] were used along with respective control strains CL2122 [dvlsl5, (pPD30.38) unc-54(vector) + (pCL26) mtl-2::GFP}] and CL802 [smg-l(cc546) I; rol-6(sul006) II], The C. elegans strains and Escherichia coli strain OP50 were obtained from the Caenorhabditis Genetics Center (CGC; University of Minnesota, Minneapolis, MN, USA). Animals were cultured at 16 °C on solid nematode growth medium (NGM) plates inoculated with live Escherichia coli OP50 bacteria as food. For all assays, age-synchronized nematodes were prepared by hypochlorite bleaching (0.1 M KOH, 1% NaCIO). Synchronized eggs were hatched overnight (LI) in M9 medium and cultured on fresh NGM plates.

[0215] Administration of GNPs to worms: After 24 hours (L4 stage), worms were fed dead OP50 bacteria mixed with CP-2-GNPs at concentrations ranging from 0 to 50 pM. Following 24 hours of incubation, the temperature was raised to 25°C for 24 hours to induce AP expression in GMC101 worms. Worm motility was assessed by counting thrashes (body bends) for 30 seconds. Results were expressed as mean ± standard deviation (SD) and analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. Statistical significance was considered at p < 0.001, with n = 60 worms per condition.

[0216] Lifespan Assay: The survival and lifespan of transgenic CL2006 and WT CL802 C. elegans were monitored as described previously (Habashi, M. et al., Proc. Natl. Acad. Sci. U. S. A., 2022, 119, e2210766119; Habashi, M. et al., J. Med. Chem., 2023, 66, 3058; Senapati, S. et al., Small, 2024, 10.1002 / smll.202311670, e2311670). Synchronized eggs of transgenic CL2006 and WT CL802 animals were cultured on 60 mm NGM plates at 16°C. Each treatment group consisted of 100 synchronized LI worms, which were seeded onto the plates at a density of 25 worms per plate. The worms were fed with heat inactivated OP50 solution containing the vehicle (5% DMSO), CP-2 (50 pM) or an equivalent amount of CP-2-LPs. Animals were transferred to new plates every alternate second day and their status was recorded daily. Worms with internal hatching were removed from the plates and excluded from lifespan calculations. The experiment was concluded when all worms were scored as deceased or censored. The statistical analysis of the lifespan data was performed using the Kaplan-Meier survival analysis method.

[0217] Thrashing Assay: The thrashing assay was performed as described before with minor modifications (Habashi, M. et al., Proc. Natl. Acad. Sci. U. S. A., 2022, 119, e2210766119; Habashi, M. et al., J. Med. Chem., 2023, 66, 3058; Senapati, S. et al., Small, 2024, 10.1002 / smll.202311670, e2311670). Briefly, synchronized LI worms of transgenic CL2355 and control CL2122 strains (n = 20) were cultured on NGM plates at 16 °C and fed for 36 h with heatdead OP50 solution mixed without or with increasing concentrations of CP-2 (0-50 pM) or equivalent amount of CP-2-LPs. Animals were incubated at 23 °C for 36 h and individually transferred to a 12-well plate containing 1 mL of M9 buffer. After a 2-minute settling period, the number of thrashes for each animal was counted for 1 min. Body thrash was recorded as bending the worm's body from one side to the other and back to the original position. Each experiment was repeated three times.

[0218] Chemotaxis Assay: The chemotaxis assay was performed following a previously described protocol with some modifications (Y. Wu et al. J. Neurosci. 2006, 26, 13102-13113). Briefly, synchronized transgenic C. elegans strains CL2355 and the WT control strain CL2122 were cultured on NGM plates at 16 °C and fed with heat-dead OP50 solution containing the vehicle (5% DMSO), CP-2 (0-50 pM) or an equivalent amount of CP-2-LPs for 36 h. The animals were incubated at 23 °C for an additional 36 h, collected, and washed three times with M9 buffer. To perform the assay, odorants were prepared by spotting sodium azide (1 pL, 0.25 M, Sigma-Aldrich) and benzaldehyde (0.1% V / V, 1 pL, Sigma-Aldrich) in absolute ethanol as "attractant" spots on opposite corners of a 100 mm agar plate. Absolute ethanol (1 pL) and sodium azide (lpL) were spotted on the two other corners as controls. A suspension of worms (5 pL, ~30 worms) was immediately applied to the center of each agar plate. The plates were incubated at 23 °C for 1 h, and the number of worms in each quadrant was scored. The chemotaxis index (Cl) was calculated by dividing the difference between the number of worms in both attractant and both control quadrants by the total number of scored worms.

[0219] Immunoblot Assay: The effect of each treatment on the oligomeric state of AP42 in worms was investigated as described (J. S. Sangha et al. PLoS One 2012, 7, e43990). Treated worms were collected, washed with M9 buffer, rapidly frozen in liquid nitrogen, and stored at - 80 °C until analysis. Protein samples were extracted using a 2x volume of lysis buffer solution (2% SDS, 10% glycerol, 4% p-mercaptoethanol and protease inhibitor cocktail in 62 mM Tris-HCI, pH 6.8) and boiled at 101 °C for 5 min. The protein content was quantified using the Bradford reagent (Bio-Rad) or by measuring the absorbance at 280 nm using a NanoDrop spectrophotometer. Equal amounts of protein samples were spotted in duplicate on a nitrocellulose membrane (0.2 pm) and blocked with 5% BSA in PBS buffer (pH 7.5) containing 0.1% (v / v) Tween 20 (PBST). The blots were incubated overnight at 5 °C with either 6E10 monoclonal (1:1000 dilution) or All (1:5000) antibody in PBST with 5% BSA. After washing four times with PBST, the blots were incubated at RT with HRP-conjugated anti-mouse IgG (1:10000 in 0.5% BSA in PBST) or anti-rabbit IgG (1:5000 in 0.5% BSA in PBST) for 1 h. The membranes were washed five times with PBST and developed with an ECL reagent kit (Bio-Rad). Western blot analysis

[0220] The AP species in treated worms were also identified by Western blot analysis. Equal amounts of protein lysate were heated at 101 °C for 3 min with sample buffer containing 5% - mercaptoethanol, loaded on a 15% Tricine-SDS-PAGE gel and run at 100 V for 1 h. The protein bands were transferred to a nitrocellulose membrane using transfer buffer containing 10% methanol at 75 V for 1 h. The blots were blocked with 5% BSA in TBS containing 0.01% (v / v) Tween 20 for 1 h at RT, incubated with the 6E10 antibody, and developed as above. Identical blots were reacted with an anti-a-tubulin antibody and developed. The mean density of A reactive bands was analyzed using ImageJ (National Institutes of Health, USA).

[0221] Mice: Transgenic 5xFAD (Jackson Laboratory) mice were bred with non-transgenic background C57BL / 6J (Jackson Laboratory) and maintained at the Bar-Han University animal facility. The mice were kept in a controlled environment with a 12 / 12 h light / dark cycle and provided with ad libitum access to food and water. Housing, breeding and all animal experiments were conducted in compliance with the Bar-Han University Animal Care and Use Committee.

[0222] Biodistribution Study in Wild Type Mice: Five-month-old wild type female C57BL / 6J mice (n=6, each group) were injected intravenously through the tail vein with 0.1 mL of Cy5- labeled CP-2-LPs (Cy5-CP-2-LPs) in PBS containing 3.8 pM of Cy5 and 28 mM of total lipids. Mice treated with vehicle were used as control. At different time intervals post injection, the mice were euthanized and perfused intracardially with PBS (100 mM, pH 7.4). The major organs including brain, kidney, heart, liver, spleen and blood were collected and imaged using a Maestro II fluorescence imaging system (Cri, Inc., MA, USA). For quantitative analysis, the average fluorescence signal (measured in xlo6photonsxcnT2xs_1) was determined for each specific area.

[0223] BBB Permeability and APO Targeting Studies: Five- to six-month-old female transgenic 5xFAD mice and WT C57BL / 6J mice (n=6, each group) were injected intravenously with O.lmL of Cy5-CP-2-LPs in PBS under isoflurane anesthesia. At different time points (4, 24 and 72 h) post injections, the mice were euthanized and perfused with 4% PFA solution. Brains were collected and imaged using the Maestro II fluorescence imaging system, as described previously. Immunofluorescent staining was performed on PFA fixed 40 pm thick sections from 5xFAD mouse brains. Free-floating sections were washed with 0.1 M PBS (3x10 min) and blocked with 2% BSA for 1 h. The sections were incubated with the primary 6E10 mouse monoclonal antibody (1: 1000) in 2% BSA in 0.1 M PBS with 0.3% Triton X (PBST) overnight at 4°C. Sections were washed (3x10 min with 0.1 M PBS) and incubated with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG antibody (1:1000, ThermoFisher) in 2% BSA in PBST for 1 h at RT. Sections were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride; 1:500, MP Biomedicals) and washed for 10 min in 0.1% PBST. The stained sections were mounted on super frost glass slides, embedded with ImmunoMount (Sigma-Aldrich), cover-slipped and stored in dark for microscopic imaging.

[0224] Statistical Analysis: Data were analyzed by one-way ANOVA followed by post-hoc analysis where appropriate or by two-tailed unpaired Student's t-test as indicated in the respective figure legends.

[0225] EXAMPLE 1:

[0226] Preparation and Characterization of Multifunctional Liposomes

[0227] In order to establish a way to deliver CP-2across the blood brain barrier cyclic D,L-a- peptide, CP-2 was synthesized using a solid-phase peptide synthesis method (M. Richman et al. J. Am. Chem. Soc. 2013, 135, 3474-3484) purified to homogeneity by RP-HPLC, and characterized by mass spectroscopy. Fig. IB outlines the preparation of multifunctional liposomes conjugated to the fluorescent molecule Cy5 and oligomer-specific CP-2 (hereinafter Cy5-CP-2-LPs). Liposomes were prepared as described hereinabove under "Preparation of Naked Liposomes". Liposome-conjugated CP-2 (CP-2-LPs) were prepared as described hereinabove under "Preparation of CP-2 Conjugated Liposomes (CP-2-LPs)".

[0228] For preparation of multifunctional liposomes containing both CP-2 and Cy5 NIR fluorescent probe, the same liposome composition was used but a small amount of DSPE-PEGzk-NF was added to introduce free amino groups on the liposome surface. The generated liposomes were first reacted with Cy5-NHS and then the free carboxylic acids were activated with NHS / EDC and coupled with CP-2. The conjugation efficiency of CP-2 and Cy5 with the liposome was about 67% and 63%, respectively.

[0229] Cryo-EM, AFM and DLS were used to analyze morphology, hydrodynamic size and potential of the particles.

[0230] Cryo-EM studies confirmed the presence of well-dispersed spherical liposomes with average size of 109±3 nm and 113±5 nm for the blank liposomes and CP-2-LPs, respectively (Fig. 1C, Fig. ID, Fig. IE, and Fig. IF).

[0231] Fig. 1C is a Cryo-EM photomicrograph of naked liposomes demonstrating monodispersed spherical particles. Fig. ID is a Cryo-EM photomicrograph of CP-2-LPs demonstrating mono-dispersed spherical particles.

[0232] Fig IE is a histogram of particle frequency as function of diameter in nm for naked liposomes from Cryo-EM images using ImageJ software.

[0233] Fig. IF is a histogram of particle frequency as function of diameter in nm for CP-2 conjugated LPs s from Cryo-EM images using ImageJ software. As expected, the average particle size is slightly larger than for naked LPs.

[0234] AFM images (Fig. 1G and Fig. 1H) further confirmed the spherical morphology of both the naked liposomes and CP-2-LPs.

[0235] Fig. 1G is an AFM photomicrograph of naked liposomes (LPs).

[0236] Fig. 1H is an AFM photomicrograph of CP-2 conjugated LPs (CP-2-LPs);

[0237] Fig. II is a plot of intensity as a function of size illustrating hydrodynamic size distribution of LPs and CP-2-LPs measured by DLS with average size of 114±3 and 115±2 nm, respectively. Consistent with the Cryo-EM and AFM studies, DLS measurements demonstrated an average hydrodynamic size of 118±4 and 123±2 nm for the naked liposomes and CP-2-LPs with a PDI of 0.14 and 0.17, respectively (Figure II).

[0238] Fig. 1J is a plot of Zeta potential measurements of LPs, CP-2-LPs, and Cy5-CP-2-LPs as determined by DLS. Experiments were carried out in triplicate and values are mean ± SD. The blank liposomes had a potential of -18.3±1.1 mV, indicating a negative charge due to the presence of free -COOH groups from DSPE-PEG2k-COOH (Figure IF). Upon conjugation of carboxylic acids with CP-2 to generate CP-2-LPs, the potential increased to -7.6±0.64 mV. The potential of Cy5-CP-2-LPs was more negative (-13.510.73 mV) confirming successful incorporation of the negatively charged Cy5.

[0239] Fig. IK is a plot of absorbance as a function of wavelength (nm) comparing LPs, CP-2 - LPs, free Cy5 and Cy5-CP-2-LPs.

[0240] Fig. IL is a plot of fluorescence intensity (a.u.) as a function of wavelength (nm) of LPs, CP-2-LPs, free Cy5 and Cy5-CP-2-LPs.

[0241] Conjugation of Cy5 to the particles was further validated with visible and fluorescence spectroscopy, which showed similar absorption and fluorescence spectra for free Cy5 and Cy5- CP-2-LPs (Fig. IK and Fig. IL).

[0242] Fig. IM is a histogram of mean diameter (nm) as a function of time (days) for liposomes monitored over a period of 28 days (stored at 4-8 °C) based on DLS measurements comparing LPs and CP-2-LPs. DLS was used to assess the stability of the liposomes over a period of ~1 month. Up to 14 days of incubation at 4-8 °C, no significant change in particle size was apparent indicating a high stability with no evidence of particle fusion or aggregation.

[0243] However, minor changes in particle size occurred after 21 days of storage suggesting some instability during prolonged incubation or storage.

[0244] Results presented in Fig. 1C, Fig. ID, Fig. IE, Fig. IF, Fig. 1G, Fig. 1H, Fig. II, Fig. 1J, Fig. IK, Fig. IL, and Fig. IM confirm that it is possible to prepare liposomes that are multivalent with respect to CP-2 and / or Cy5. These CP-2-LPs and Cy5-CP-2-LPs provided a basis for further study.

[0245] EXAMPLE 2:

[0246] Effect of CP-2- LPs on A 6 in cells

[0247] In order to study the effect of CP-2-LPs on AP, a series of experiments was conducted in tissue culture cells.

[0248] Fig. 2A is a plot of % aggregation as a function of time (h) for monomeric A 40 (10 pM) incubated with increasing concentrations of CP-2-LPs or CP-2 in PBS buffer (50 mM, pH 7.2) with the extent of amyloid formation monitored over time using ThT fluorescence;

[0249] Initially, a ThT fluorescence assay was used to monitor A aggregation in the absence and presence of the liposomes. ThT binds specifically to the cross-p-sheets of amyloid fibrils to generate distinctive fluorescence signals. Results presented in Fig. 2A demonstrate that Fibrillization of AP40 follows a sigmoidal amyloid kinetic with a lag phase (~ 20 h), an elongation phase, and a plateau phase. These results are in line with expectations for disease progression associate with AP aggregation into fibrils.

[0250] Fig. 2A shows that CP-2-LPs dose dependently reduced the aggregation of AP40 even at the lowest concentration tested. Aggregation of AP was decreased by 76% and almost completely with AP:CP-2-LPs ratios of 1:1 and 1:5, respectively.

[0251] In control experiments CP-2 (not conjugated to liposomes) reduced the aggregation of AP40 only by 35 and 60% at the same ratios. One possible explanation for the observed difference is that the enhanced efficacy of CP-2-LPs in inhibiting AP aggregation (compared to free CP-2) is a multivalency effect.

[0252] The ThT results also demonstrate that naked liposomes had no significant effect on AP aggregation. Without being bound by theory, the lack of an impact of naked liposomes on AP aggregation in this study is likely attributable to differences in lipid composition and surface characteristics, including phase behavior. Fig. 2B is a Cryo-EM micrograph of AP40 (10 pM) after 72 h incubation without CP-2-

[0253] LPs.

[0254] Fig. 2C is an AFM micrograph of AP40 (10 pM) after 72 h incubation without CP-2- LPs.

[0255] Fig. 2D is a Cryo-EM micrograph of AP40 (10 pM) after 72 h incubation with CP-2- LPs (50 pM).

[0256] Fig. 2E is an AFM micrograph of AP40 (10 pM) after 72 h incubation with CP-2- LPs (50 pM).

[0257] Results presented in Fig. 2B; Fig. 2C; Fig. 2D and Fig. 2E employ Cryo-EM and AFM to examine the morphology of AP in absence or presence of CP-2-LPs. AP forms long fibrillar structures after 72 h of incubation. Consistent with the ThT results, significantly fewer fibrils were formed when AP was treated with a 5-fold excess of CP-2-LPs.

[0258] Fig. 2F is an immuno-dot blot test illustrating the effect of CP-2-LPs and CP-2 on AP40 oligomer and fibril formation using oligomer-specific (All) and fibril-specific (OC) antibodies; AP40 (30 pM) was incubated for 0, 24, 48 or 72 h in the absence or presence of 150 pM of CP- 2-LPs or CP-2, spotted onto nitrocellulose membranes, and probed with either All or OC antibodies as indicated.

[0259] The time course effect of CP-2-LPs on AP oligomer and fibril formation in Fig. 2F employed conformation-specific antibodies All and OC which recognize respectively soluble oligomers and fibrils. The reactivity of AP to both All and OC antibodies increased steadily over time, indicating conversion of monomeric AP to oligomers and fibrils. Soluble toxic oligomers and fibril content both decreased more effectively upon incubation of AP40 with five-fold excess of CP-2-LPs compared with a similar excess of CP-2. In control experiments naked liposomes had no effect on the generation of AP oligomers or fibrils.

[0260] These results suggest that CP-2-LPs have the potential to retard formation of AP oligomers and fibrils which should inhibit progression of AD. In addition, the influence of CP-2- LPs appears to be greater than the sum of the effect of free CP-2 and free LPs.

[0261] Fig. 2G is bar graph of % cell viability illustrating the protective effect of CP-2-LPs and CP-2 on AP-induced toxicity in SH-SY5Y cells. AP40 (20 pM) was incubated for 48 h alone or with increasing concentrations of CP-2-LPs and CP-2 and exposed to the cells for 24 h. Cell viability was determined by the MTT assay. Naked LPs and CP-2 (200 pM) served as controls. Values are mean ± SD and were analyzed by one-way ANOVA followed by Tukey's multiple comparison test (ns = non-significant, *p< 0.05, ***p< 0.001).

[0262] Fig. 2H is a bar graph of % cell viability as a function of concentration for CP-2 and CP-2-LPs in human neuroblastoma SH-SY5Y cells; Cells were incubated with increasing concentrations of CP-2 or an equivalent amount of CP-2-LPs (0 - 200 pM) for 48 h. Cell viability was evaluated using the MTT assay. Results are presented as a percentage of the control (untreated) cells and are expressed as the mean ± SD from two independent experiments (n = 3 for each experiment). Statistical analysis was performed using the Student's t-test, with p> 0.05 considered nonsignificant (ns).

[0263] Since Apos are believed to play a crucial role in the pathogenesis of AD the demonstrated ability of CP-2-conjugated liposomes to modulate A -induced neurotoxicity was examined using SH-SY5Y human neuroblastoma (Fig. 2G and Fig. 2H) and neuronal-like rat pheochromocytoma PC12 cells (Fig. 21).

[0264] Initially, the toxicity profile of CP-2-LPs was assessed by exposing SH-SY5Y cells to increasing concentrations of CP-2-LPs and evaluating the cells' metabolic activity through the well-established MTT assay. Unconjugated CP-2 was used as the control. As expected naked LPs and CP-2 produced no significant reduction in cell viability in the MTT assay, even at the highest concentration of CP-2-LPs tested (200 pM). This suggests that neither CP-2 nor CP-2-LPs exert toxicity on the cells (Fig. 2G and Fig. 2H).

[0265] Fig. 21 is a bar graph of % cell viability as a function of concentration for various concentrations of CP-2 and CP-2-LPs in rat PC-12 cells exposed to toxic level of AP40 (20 pM). AP40 (20 pM) was incubated for 48 h alone or with increasing concentrations (20, 100 or 200 pM) of CP-2 or CP-2-LPs and exposed to PC12 cells for 24 h. Cell viability was determined by the MTT assay. Naked LPs and CP-2 (200 pM) were used as controls.

[0266] PC12 cells are a rat adrenal pheochromocytoma cell line that are largely used in the field since they are "neuronal like". Although PC12 cells do not fully replicate human neurons, their use in viability assays provides a cost-effective, reproducible, and biologically relevant system for initial screening of neurotoxic effects and neuroprotective strategies. SH-SY5Y is a better model to study AD.

[0267] These results suggest that CP-2-LPs dose-dependently decrease Ap toxicity, without exerting any toxicity of their own. The immunoblotting results of Fig. 2F show that Ap requires an incubation period of at least 24 h to generate A os and fibrils. Accordingly, A 40 was incubated for 24 h with and without increasing concentrations of CP-2-LPs and CP-2 and incubated with the cells for an additional 48 h. Cell viability was then assessed by the MTT assay. Incubation of SH-SY5Y and PC12 cells with pre-incubated Ap40 decreased cell viability to "'60%. However, at a 1:5 Ap:CP-2 equivalent ratio, CP-2 and CP-2-LPs increased cell viability to 75% and 92%, respectively. Notably, only CP-2-LPs demonstrated significant neuroprotective activity (78%) at a 1:1 ratio. Similar results were obtained when PC12 cells were incubated with Ap40 and CP-2-LPs (Fig. 21).

[0268] Results of the experiments presented in this example demonstrate that the non-toxic CP-2-LPs retard aggregation and fibril formation of Ap40 and reduced its toxicity in relevant cell types and suggest that CP-2-LPs have the potential to retard AD progression.

[0269] EXAMPLE 3:

[0270] Evaluation of CP-2-LPs in Transgenic Worm Models and in WT Mice

[0271] In order to confirm the cell culture results of Example 2, additional experiments were conducted in accepted transgenic models of AD in C. elegans and in wild type (WT) mice.

[0272] The therapeutic effect of CP-2-LPs was evaluated in two in vivo C. elegans models expressing human A 42. These transgenic C. elegans models are widely utilized to study AD and other age-related neurodegenerative diseases due to their age-dependent and human-like physiological changes observed at the tissue, cellular and molecular levels.

[0273] Briefly, the C. elegans strain CL2006 constitutively expresses human A 42 in the bodywall muscles. Aggregation of A 42 leads to progressive paralysis and premature death. The untreated CL2006 worms display an average mean lifespan of 12.8 days, while control WT CL802 worms have a significantly longer lifespan of 14.6 days.

[0274] As shown in Fig. 3A and Fig. 3B, treatment of CL2006 worms with 50 pM of CP-2 increased the mean lifespan to 13.9 days (p<0.05). Treatment of the transgenic worms with an equivalent amount of CP-2-LPs significantly further extended the lifespan to that of WT worms (14.6 days; p<0.001) and mitigated the adverse effects of over-expressed AP (Fig. 3A and Fig. 3B). These findings indicate that CP-2-LPs are more effective in prolonging the lifespan than unconjugated CP-2. Notably, neither CP-2-LP nor CP-2 had a significant effect on the longevity of WT CL802 worms.

[0275] Fig. 3A is a Kaplan-Maier plot of % survival as a function of time in days. Transgenic CL2006 worms (expressing human A 42 in body-wall muscles) and wild-type CL802 worms were treated with heat-inactivated OP50 solution containing either the vehicle (5% DMSO), CP-2 (50 pM), or an equivalent dose of CP-2-LPs. Worms were transferred to fresh plates every second day, and their survival was monitored daily. Worms with internal hatching were removed and excluded from the survival analysis. The experiment was concluded when all worms were either deceased or censored (n = 3 experiments, each involving 100 animals each group). The same data is summarized as a bar graph in Fig. 3B

[0276] Fig. 3B is a bar graph of mean lifespan (days) for CL2006 (transgenic) and CL802 (WT) worms treated with CP-2-LPs, CP-2, or nothing. Values are mean ± SD from three experiments analyzed by one-way ANOVA followed by Tukey's multiple comparison test (ns=non-significant, *p<0.05, ***p<0.001).

[0277] A second line of transgenic C. elegans (CL2355) expresses pan-neuronal human Api-42 and exhibits difficulties in learning, chemotaxis and thrashing when temperature is up-shifted to 23- 25°C. At elevated temperature (25 °C), CL2355 worms exhibit a reduced bending rate of about 75 bends per minute compared to 82 bends per minute in WT CL2122 animals due to AP-induced toxicity (Fig. 3C). Feeding the CL2355 transgenic worms with increasing concentrations of CP-2 and CP-2-LPs increased the motility of CL2355 worms to the level of untreated WT worms (Fig 3C) in a dose dependent manner. Notably, while CP-2 was not effective at a low concentration (10 pM), CP-2-LPs induced a significant improvement at this dose. This confirms that CP-2 LPs have a greater physiologic effect than free CP-2 as suggested by the results presented above.

[0278] Chemotaxis behavior in C. elegans is essential for seeking food sources, avoiding toxic substances, finding mates, and laying eggs. The chemotaxis index (Cl) is a measure of the fraction of animals that reach the location of attractant using their chemosensory detection system.

[0279] In transgenic CL2355 worms expressing pan-neuronal AP42, chemotaxis is impaired due to AP-induced neurotoxicity. The Cl of the WT CL2122 and CL2355 worms is 0.31 ± 0.01 and 0.22 ± 0.01 (p< 0.001), respectively, indicating chemotactic dysfunction in the transgenic worms.

[0280] Feeding the CL2355 mutants with CP-2-LPs and CP-2 dose dependently increased the Cl. At 50 pM, CP-2-LPs increased the Cl to the WT level and neutralized the damaging effect of aggregated AP (Fig. 3D). Notably, CP-2-LPs demonstrated significant protective effects on the chemotaxis behavior of CL2355 even at low micromolar concentrations whereas CP-2 failed to induce a similar protective activity. The Cl levels of WT CL2122 worms were not affected by treatment with CP-2 or CP-2-LPs. Fig. 3C is a bar graph of number of body thrashes / min as a function of concentration (pm) of CP-2 or CP-2-LPs for transgenic CL2355 (neuronal AP-expressing) and WT CL2122 worms. Data are mean ± SD from three experiments (n=20) and statistical analysis was performed as described above (**p<0.01, ***p<0.001).

[0281] Fig. 3D is a bar graph of chemotaxis index (Cl) towards 0.1% benzaldehyde (in absolute Ethanol; V / V) at RT as a function of concentration (pM) of CP-2 or CP-2-LPs for transgenic CL2355 and WT CL2122 worms. Synchronized worms were fed with increasing amount of CP-2 (2-50 pM) or equivalent amount of CP-2-LPs at 16°C for 36 h and at 23°C for another 36 h. Results are mean ± SD from three independent experiments analyzed as described above (n=30; *p<0.05, ***p<0.001).

[0282] Collectively, these C. elegans studies demonstrate that CP-2-LPs effectively restore the impaired behavioral profiles of the transgenic worms. The observed effect was significantly greater than for CP-2 which was not bound to liposomes.

[0283] In order to explore the underlying physiologic mechanisms of these results, immunochemical dot-blot and western blot (WB) experiments were conducted to investigate the effect of CP-2-LPs and CP-2 on A species extracted from treated worms.

[0284] Fig. 3E is an immunoblot analysis of equal amounts of extracted proteins and shows that CP-2-LPs more effectively reduced overall A levels, with more pronounced effects on All- reactive oligomers than CP-2. This result suggests that CP-2-LPs are effective against the toxic oligomers. Fig. 3F is a western blot analysis of extracted proteins illustrating elevated levels of trimers (~14 kDa), hexamers (~ 27 kDa) and heptamers (~32 kDa) in the transgenic CL2006 animals. It is believed that rimers and hexamers of AP are building blocks for toxic aggregates, including AP-derived diffusible ligands, AP dodecamers and globulomers, all associated with AD pathology.

[0285] Fig. 3F and Fig. 3G show that both CP-2-LPs and CP-2 reduced the amount of trimers, tetramers, and hexamers in transgenic CL2006 worms while concomitantly increasing the levels of soluble AP dimers ("'8.5 kDa).

[0286] Fig. 3E is an immune-dot blot experiment developed by sequence and oligomer specific antibodies (6E10 and All, respectively) after spotting equal amounts of proteins extracted from transgenic CL2006 and WT CL802 worms treated with CP-2, CP-2-LPs or vehicle. Fig. 3F is a western immunoblot of AP species in transgenic CL2006 and WT CL802 worms fed with CP-2, CP-2-LPs or vehicle. Equal amounts of extracted proteins were loaded onto each lane and immunoblotted with an anti-Ap antibody (6E10). Anti-a-tubulin antibody served as a loading control.

[0287] Fig. 3G is a quantification of AP species in transgenic CL2006 worms treated with CP-2, CP-2-LPs, or the vehicle (control). The relative band intensity of AP species is presented as fold change compared to the corresponding a-tubulin bands, used as a loading control.

[0288] Fig. 3H presents in vivo fluorescence imaging of WT female C57BL / 6J mice intravenously injected with Cy5-CP-2-LPs demonstrates high fluorescence signals in the brain, indicating successful penetration of CP-2-LPs through the BBB.

[0289] Subsequently, the animals were perfused with saline prior to dissection of tissues to examine the uptake of fluorescent CP-2-LPs in various organs.

[0290] Fig. 31 shows Intense fluorescence in the liver, heart, spleen, kidney, and lungs 4 h post injection.

[0291] Fig. 3J presents the average fluorescence intensities of different organs at 4 and 24 h post Cy5-CP-2-LPs injection. The fluorescence intensity signals gradually decreased in most tissues after 24 h of injection, indicating efficient clearance of the liposomes upon injection. Liposome fusion and their internalization through endocytosis / phagocytosis followed by degradation within cells, exocytosis, or lysosomal pathways, along with potential interaction with interstitial fluid and cerebrospinal fluid, are likely responsible for the clearance and drainage of the liposomes. Notably, detectable fluorescence was also observed in the brains of WT mice 4 h after injection but the signal decreased after 24 h, suggesting that Cy5-CP-2-LPs penetrate the BBB in WT mice.

[0292] While Fig. 31 presents representative ex vivo fluorescence images showing the biodistribution of Cy5-CP-2-LPs in mice, Fig. 3J is a bar graph displaying the average fluorescence intensity from three independent experiments, including the data shown in Fig. 31, providing a quantitative assessment of the distribution across different tissues.

[0293] Fig. 3H is representative in vivo fluorescence images of ~6-month-old C57BL / 6J mice

[0294] 4 h post intravenous (LV.) injection with 0.1 mL of saline (Left image[animal]) or Cy5-labelled liposomes conjugated with CP-2 (Cy5-CP-2-LPs, 3.8 pM) (Right image[animal]). Images were captured using a MAESTRO II (CRi) in vivo fluorescence imaging system; Fig. 31 is a series of ex vivo fluorescence imagers showing biodistribution of Cy5-CP-2- LPs in mice. C57BL / 6J mice were perfused 4 or 24 h post injection of vehicle or Cy5-CP-2-LPs (3.8 pM), the mice were euthanized and tissues were removed and imaged.

[0295] Fig. 3J is a bar graph of average fluorescence intensity of Cy5-CP-2-LPs in different organs from three independent experiments, including the data shown in Fig. 31, providing a quantitative assessment of the distribution across different tissues.

[0296] Results from the experiments in mice confirm the ability of CP-2 according to an exemplary embodiment of the invention to transport liposome and / or CY5 across the blood brain barrier. Taken together with the experiments in transgenic C. elegans this example suggests that CP-2-LPs should reduce aggregation of Ap in mammalian brains.

[0297] EXAMPLE 4:

[0298] Evaluation of CP-2-LPs in a Transgenic Mouse Model

[0299] In order to further evaluate the potential therapeutic effect of CP-2-LPs in mammalian brains additional experiments were conducted in a transgenic mouse model.

[0300] 5xFAD transgenic mice were employed. 5xFAD transgenic mice rapidly develop severe amyloid pathology due to the presence of the Swedish (K670N / M671L), Florida (1716V) and London (V717I) mutations in Amyloid Precursor Protein (APP) and the M146L and L286V mutations in Presenuilin-1 genes. At an age of about 1.5 months, these mice begin to accumulate intraneuronal A 42 aggregates in the cortex, hippocampus and thalamus followed by extracellular amyloid deposition at an age of about 2 months. The extracellular amyloid deposition progressively increases with age. Initial memory impairment is observed at an age of 4 to 5 months.

[0301] Fig.4A and Fig. 4B present results from In vivo fluorescence imaging of ~5-month-old female 5xFAD mice brains at 4 h post-injection with Cy5-CP-2-LPs. The results show significantly higher (p<0.05) fluorescence in the transgenic mice relative to WT mice. Remarkably, while the fluorescence decreased over time in WT brains, it increased over 24 h and remained stable for up to 72 h post-injection in 5xFAD brains, enabling clear visualization of AD brains with minimal background signal.

[0302] Collectively, the imaging studies demonstrate the effective penetration of Cy5-CP-2-LPs through the BBB in both WT and 5xFAD mice. However, the clearance of Cy5-CP-2-LPs from the brains of 5xFAD mice was significantly slower relative to WT mice (p<0.05), leading to their accumulation in the brain. Although the precise mechanism behind the prolonged retention of Cy5-CP-2-LPs in the brain of 5xFAD mice is still under investigation, two possibilities are currently being considered. First, the prolonged retention of Cy5-CP-2-LPs in the brain may be attributable to a compromised BBB in the transgenic mice. Second, the prolonged retention of Cy5-CP-2-LPs in the brain may be due to binding of Cy5-CP-2-LPs to AP species in the brains of 5xFAD mice, enabling the effective visualization of amyloid species in the young transgenic 5xFAD mice brains.

[0303] Fig. 4A is a series of fluorescence images obtained at different time intervals (4, 24 and 72 h) from WT and 5xFAD mice brains (aged 5-6 months) after single LV. injection of 0.1 mL of Cy5- CP-2-LPs.

[0304] Fig. 4B is a plot of average fluorescence signal (X106photons / CM2 / S) as a function of time (h) for fluorescence signals obtained from brain samples from 3 independent experiments (n=3) including data from Fig 4A. Results are mean ± SD analyzed using student's unpaired t-test (n = 3 each; *p < 0.05, ***p <0.001).

[0305] Fig. 4C and Fig. 4D present results from fluorescence imaging of brains from perfused WT and 5xFAD mice. The fluorescence images of Fig. 4C reveal the accumulation of Cy5-CP-2- LPs in the brains of 5xFAD and confirm that the liposomal formulations cross the BBB. Since the limited ability of naked liposomes to cross the BBB has previously been reported it was hypothesized that CP-2 plays a specific role in facilitating BBB penetration. In order to test that hypothesis liposomes were conjugated to p-alanine ethyl ester to generate p-Ala-LPs and further conjugated to Cy5 probe. As illustrated in Fig. 4C and Fig. 4D, P-Ala-LPs do not penetrate the BBB nearly as well as Cy5-CP-2-LPs. This result indicates that the penetration of the BBB by Cy5-CP- 2-LPs in AD mice is not solely due to a compromised BBB in AD and suggests an ability of CP-2 to enhance the BBB permeability of the liposomes.

[0306] Fig. 4C is a series of representative fluorescence images of 5xFAD mice brains after intravenous administration of 0.1 mL of Cy5-CP-2-LPs or control Cy5-LPs conjugated to p-alanine ethyl ester (Cy5- -Ala-LPs).

[0307] Fig. 4D is a bar graph of average fluorescence signal (X106photons / CM2 / S) based on 3 independent experiments (n=3) including data from Fig 4C; Results are mean ± SD analyzed as described above (n = 3 each; ***p <0.0001).

[0308] After fluorescence imaging, perfused brains from Cy5-CP-2-LPs treated mice were sectioned and stained with sequence-specific anti-A 6E10 to assess the localization of AP species and Cy5-CP-2-LPs. Confocal fluorescence microscopy from the hippocampus, cortex and thalamus regions demonstrated that the 6E10 antibody colocalized with Cy5-CP-2-LPs, indicating that the latter bind specifically to AP species (See Fig. 4E and Fig. 4F). These in vivo experiments demonstrate that CP-2-LPs can efficiently penetrate the BBB and bind early A species present in the brains of young AD mice.

[0309] Fig. 4E is a series of representative immunofluorescence images of cortex, thalamus, and hippocampus of a 5xFAD mouse brain stained with Cy5-CP-2-LPs (Left column) and sequence-specific monoclonal 6E10 antibody (Second column from left); mice were injected intravenously with 0.1 mL of Cy5-CP-2-LPs, perfused after 24 h with saline and sacrificed; sections were blocked with 5% BSA, incubated with 6E10 antibody followed by incubation with Alexa Fluor 488 goat anti-mouse IgG secondary antibody, and stained with DAPI (Second column from right). The right column presents Cy5-CP-2-LPs, AP (6E10) and DAPI data fused together in composite images.

[0310] Fig. 4F is a plot of pixel intensity (a.u). as a function of pixel distance (a.u.) using ImageJ software for analysis of co-localization intensity profiles; Line scan graphs show the intensity obtained from the black (Cy5-CP-2-LPs) and grey (6E10) fluorescence channels using the ImageJ Plot Profile tool.

[0311] These results demonstrate that Cy5-CP-2-LPs cross the BBB and bind to molecules in the brain that are associated with clinical progression of AD. These findings suggest a potential clinical role for Cy5-CP-2-LPs in both diagnosis and treatment of AD and related disorders. As well as a role for CP-2-LPs in treatment of AD and related disorders.

[0312] EXAMPLE 5:

[0313] Synthesis and Characterization of Gold Nanoparticles Conjugated to CP-2

[0314] Gold nanoparticles (GNPs) were synthesized as described hereinabove and conjugated to CP-2. Unmodified (naked) gold nanoparticles (GNPs), PEG-conjugated GNPs, and CP-2- conjugated GNPs were characterized using UV-Vis spectroscopy.

[0315] Fig. 5 is a histogram of normalized absorption (a.u.) as a function of wavelength (nm) for unmodified (naked) gold nanoparticles (GNPs), PEG-conjugated GNPs, and CP-2-conjugated GNPs as indicated.

[0316] A broad absorption peak at 520 nm confirms the successful generation of GNPs. Dynamic light scattering (DLS) and zeta potential measurements revealed that the citrate-coated GNPs had an average size of 23 nm and a zeta potential of -22.3 mV, consistent with literature values (data not shown). Subsequently, the GNPs were modified with bifunctional thiolated polyethylene glycol (HS-PEG-COOH) and methoxy PEG (HS-mPEG) in a 70:30 ratio. This modification caused a red shift in the UV-Vis spectrum to 522 nm, an increase in particle size to 55.5 nm, and a reduction in the zeta potential to -2.13 mV.

[0317] The PEG-functionalized GNPs were then conjugated with CP-2 by activating the carboxyl groups of PEG-COOH with EDC / NHS chemistry. The conjugation of CP-2 to the GNPs was confirmed by UV-Vis and DLS, which showed a further red shift in the absorbance to 524 nm, an increase in size to 61.0 nm, and a zeta potential of -3.14 mV. The concentration of CP-2 conjugated to the GNPs was quantified by high-performance liquid chromatography (HPLC) analysis of the supernatant after centrifugation of the reaction mixture.

[0318] EXAMPLE 6:

[0319] Effect of CP-2-GNPs on Af} Aggregation

[0320] In order to assess the effect of CP-2-GNPs on AP aggregation the thioflavin T (ThT) assay was employed. Incubation of increasing concentrations of CP-2-GNPs (4-22 pM) with A 40 (20 pM) revealed a dose-dependent inhibition of A aggregation. At a 1:1 molar ratio of AP to CP-2-GNPs, significant inhibition (~50%) of amyloid formation was observed. In contrast, unconjugated CP- 2 did not exhibit anti-aggregation activity under the same conditions.

[0321] Fig. 6 is a histogram of ThT fluorescence as a function of incubation time for A 40 alone and A 40 plus increasing amounts of CP-2-conjugated GNPs (4; 22 and 44 pM as indicated).

[0322] These results are consistent with the results from liposomes conjugated to CP-2 presented hereinabove.

[0323] EXAMPLE 7:

[0324] In-Vivo Studies of CP-2-GNPs in C. elegans

[0325] In order to ascertain whether the observed in-vitro effects with CP-2 GNPs would translate to an in-vitro system, CP-2-GNPs were evaluated using two C. elegans strains: GMC101, a transgenic strain expressing human AP42 in muscle cells under temperature control (25°C), and CL2122, a GFP-expressing control strain.

[0326] Worms were synchronized by bleaching and cultured on agar plates with dead E. coli OP50 as a food source. At the L4 larval stage, GMC101 worms were exposed to 25 and 50 pM CP-2-GNPs or vehicle (DDW) for 24 hours, followed by induction of AP expression at 25°C. Motility was assessed using the thrashing assay, measuring the number of body bends (thrashes) per 30 seconds. GMC101 worms exhibited reduced motility (44 thrashes) compared to control CL2122 worms (60 thrashes) due to AP-induced toxicity (P < 0.0001). Feeding GMC101 worms with CP- 2-GNPs resulted in dose-dependent improvements in motility, restoring it to levels comparable to untreated CL2122 worms. No effect on motility was observed in CL2122 worms, confirming the lack of toxicity of CP-2-GNPs.

[0327] Fig. 7 is a bar graph illustrating the Effect of CP-2-GNPs on the motility of C. elegans strains GMC101 and CL2122. Data are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey's multiple comparison test (n=20 worms per group; ***P <0.001, ns = not significant). Experiments were repeated 3 times.

[0328] Fig. 8A is a western blot analysis of total proteins from treated and untreated transgenic GMC101 C. elegans treated with CP-2-GNPs (25 or 50 pM) or vehicle (0 pM); total proteins were analyzed using anti-A 6E10 antibody and anti-a-tubulin antibody was used for normalization; left lane is a MW marker.

[0329] Fig. 8B is a bar graph showing relative intensity (folds) of AP bands at ~15 kDa for the western blot of Fig. 8A as a function of CP-2-GNP concentration in pM as quantified using IMAGEJ software.

[0330] Fig. 8C is a dot blot analysis of total proteins from treated GMC101 C. elegans worms using the All antibody, which specifically detects AP oligomers.

[0331] In order to determine the effect of CP-2-GNPs on AP oligomer formation in vivo, a dot blot assay was performed using the All antibody that specifically detects AP oligomers. Protein extracts from treated C. elegans were lysed and analyzed.

[0332] Results presented in Fig 8C show the intensity of All antibodies decreased dose- dependently upon incubation of the worms with increasing concentrations of CP-2-GNPs. These findings corroborate the results obtained from the Western blot analysis and further confirm that CP-2-GNPs inhibit AP aggregation and oligomer formation.

[0333] Results of Example 7 are consistent with results from examples 2 through 4 and suggest that CP-2 conjugated particles (whether metal or liposomal) are potentially useful as a therapeutic agent in mammals suffering from AP mediated disorders (e.g. AD) .

[0334] Results of Example 7 also suggest that GNPs are a suitable delivery system for CP-2.

[0335] EXAMPLE 8:

[0336] In-Vivo comparison ofCP-2-GNPs to CP-2-LPs in additional

[0337] Transgenic Worm Models, WT Mice and Transgenic Mice In order to ascertain the relative in-vivo effects of CP-2-GNPs and CP-2-LPs, it is proposed to assay CP-2-GNPs in the experimental systems described hereinabove in Example 3 and Example 4. These results will provide information about whether GNPs or LPs are a better potential delivery system for CP-2 in humans. It may be necessary to compare CP-2-GNPs and CP-2-LPs head-to-head in the experimental systems described hereinabove in Example 3 and Example 4 in order to ascertain differences in potential efficacy. Based on the currently available data, it is expected that both CP-2-GNPs and CP-2-LPs will be promising candidates for development as therapeutic agents in humans.

Claims

CLAIMS:

1. A composition comprising cyclic D,L-a-peptide according to Formula IA or Formula IBFormula IA Formula IB coupled to liposomes for use as a medicament; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

2. A composition for use according to claim 1, wherein said medicament is formulated for treatment of Alzheimer's disease (AD).

3. A composition for use according to claim 1 or claim 2, wherein said medicament is formulated for intravenous injection.

4. A composition for use according to any one of claims 1 to 3, wherein said liposomes comprise1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);Cholesterol; and1.2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH).

5. A composition for use according to claim 4, wherein a molar ratio of DSPCxholesterol:DSPE-PEG2k-COOH is 55:40:5.

6. A composition for use according to claim 4, wherein a molar ratio of DSPCxholesterol:DSPE-PEG2k-COOH is 45:50:5.

7. A composition for use according to claim 4, wherein a molar ratio of DSPC:cholesterol:DSPE-PEG2k-COOH is 65:35:5.

8. A composition for use according to any one of claims 1 to 7, formulated as a medicament for Alzheimer's Disease (AD).

9. A composition for use according to any one of claims 1 to 8, comprising dye molecules according to Formula II coupled to said liposomes.Formula II10. A composition for use according to claim 9, wherein said dye molecules are polyvalently coupled to said liposomes. ll. A method of treatment comprising: administering a physiologically effective amount of a cyclic D,L-a-peptide according toFormula IA or Formula IBcoupled to liposomes to a subject in need thereof; wherein D and L indicate an enantiomeric state of the amino acids in the cyclic peptide.-Si12. A method of treatment according to claim 11, wherein said physiologically effective amount is at least 2.0 nanomoles.

13. A method of treatment according to claim 11 or claim 12, wherein said administering is via intravenous injection.

14. A method of treatment according to any one of claims 11 to 13, wherein said liposomes comprise1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);Cholesterol; and1.2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH).

15. A method of treatment according to claim 14, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 55:40:5.

16. A method of treatment according to claim 14, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 45:50:5.

17. A method of treatment according to claim 14, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 65:35:5.

18. A method of treatment according to any one of claims 11 to 17, directed towards Alzheimer's Disease (AD).

19. A method of treatment according to any one of claims 11 to 18, comprising administering a label comprising dye molecules according to Formula II coupled to said liposomes.Formula II20. A method according to claim 19, wherein said dye molecules are polyvalently coupled to said liposomes.

21. A method of diagnosis or treatment comprising administering a cyclic D,L-a-peptide according to Formula IA or Formula IBcoupled to metal nanoparticles to a subject in need thereof; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

22. A method of treatment according to claim 21, wherein the metal nanoparticles comprise gold nanoparticles.

23. A method of treatment according to claim 22, wherein said gold nanoparticles have an average diameter of not more than 65 nm.

24. A method of treatment according to claim 22, wherein said gold nanoparticles have an average diameter of at least 45 nm.

25. A method according to any one of claims 21-24, directed towards treatment of Alzheimer's Disease (AD).

26. A composition comprising a cyclic D,L-a-peptide according to Formula IA or Formula IBFormula IA Formula IB coupled to metal nanoparticles for use as a medicament; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

27. A composition for use according to claim 26, wherein the metal nanoparticles comprise gold nanoparticles.

28. A composition for use according to claim 27, wherein said gold nanoparticles have an average diameter of not more than 65 nm.

29. A composition for use according to any one of claim 27 or claim 28, wherein said gold nanoparticles have an average diameter of at least 45 nm.

30. A composition for use according to any one of claims, 26-29 formulated as a medicament for Alzheimer's Disease (AD).

31. A pharmaceutical composition comprising:(a) a cyclic D,L-a-peptide according to Formula IA or Formula IB;Formula IA Formula IB(b) a liposomal delivery vehicle; and(c) diluents, excipients and carriers; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

32. A pharmaceutical composition according to claim 31, formulated for intravenous injection.

33. A pharmaceutical composition according to claim 31 or claim 32, wherein said liposomal delivery vehicle comprises:1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);Cholesterol; and1.2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH).

34. A pharmaceutical composition according to any one of claims 31 to 33, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 55:40:5.

35. A pharmaceutical composition according to claim 33, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 45:50:5.

36. A pharmaceutical composition according to claim 33, wherein a molar ratio of DSPCxholesterol: DSPE-PEG2k-COOH is 65:35:5.

37. A pharmaceutical composition according to any one of claims 31 to 36, comprising a label.

38. A pharmaceutical composition according to claim 37, wherein said label comprises dye molecules according to Formula II.Formula II39. A pharmaceutical composition according to claim 37, wherein said label is polyvalently coupled to liposomes of said liposomal delivery vehicle.

40. A pharmaceutical composition according to any one of claims 31 to 39, wherein said cyclic D,L-a-peptide according to Formula IA or Formula IB is polyvalently coupled to liposomes of said liposomal delivery vehicle.

41. A method of synthesis comprising:(a) dissolving l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH) in chloroform-methanol (4:1, v / v);(b) evaporating the chloroform-methanol and drying to form a film;(c) hydrating the film to form a suspension; and(d) co-extruding l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG-carboxylic acid (DSPE-PEG2k-COOH) through 100 nm pores to produce liposomes;(e) activation of carboxylic acid groups on a surface of said liposomes with EDC / NHS; and(f) conjugation of a molecule according to Formula IA or Formula IB to the activated carboxylic acid groups.Formula 1A Formula IB42. A method according to claim 41, wherein a molar ratio of DSPCxholesterokDSPE- PEG2k-COOH is 55:40:5.

43. A method according to claim 41 or claim 42, wherein a molar ratio of DSPCxholesterok DSPE-PEG2k-COOH is 45:50:5.

44. A method according to any one of claims 41 to 43, wherein a molar ratio of DSPCxholesterok DSPE-PEG2k-COOH is 65:35:5.

45. A method according to claim 41, comprising: including a DSPE-PEG2k-NH2 in said dissolving to introduce free amino groups on a surface of said liposomes.

46. A method according to claim 41, comprising: reacting said liposomes with Cy5-NHS.

47. A method any one of claims 41 to 46, wherein a molar ratio of DSPCxholesterokDSPE- PEG2k-COOH: DSPE-PEG2k-NH2 is 55:40:4.5:0.5.

48. A method comprising:(a) coating gold nanoparticles (GNPs) with citrate to produce citrate coated GNPs;(b) applying HS-PEG-COOH to a surface of said citrate coated GNPs to produce PEGylated GNPs;(c) activating carboxyl groups on the PEGylated GNPs with N-hydroxysuccinimide and 1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide to produce activated GNPs;(d) contacting a solution of CP-2 in dimethyl sulfoxide (DMSO) with the activated GNPs to produce CP-2 conjugated GNPs.

49. A method according to claim 48, comprising: purifying the CP-2-conjugated GNPs by centrifugation.

50. A method according to claim 48 or claim 49, wherein said coating includes adding sodium citrate solution to a boiling solution of auric chloride (HAuCI4) in deionized water and cooling.

51. A method according to any one of claims 48 to 50, wherein said applying includes mixing the citrate coated GNPs with HS-PEG-COOH and HS-mPEG to a final PEG ratio of 2.33:1.

52. A method according to any one of claims 48 to 50, wherein said activating includes prereacting N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in DDW for at least 2 hours and adjusting the pH to 9.

53. A pharmaceutical composition comprising:(a) a cyclic D,L-a-peptide according to Formula IA or Formula IB;Formula IA Formula IB(b) metal nanoparticles conjugated thereto; and(c) diluents, excipients and carriers; wherein D and L indicate enantiomeric state of the amino acids in the cyclic peptide.

54. A pharmaceutical composition according to claim 53, wherein the metal nanoparticles comprise gold nanoparticles.

55. A pharmaceutical composition according to claim 53 or claim 54, wherein said gold nanoparticles have an average diameter of not more than 65 nm.

56. A pharmaceutical composition according to any one of claims 53 to 55, wherein said gold nanoparticles have an average diameter of at least 45 nm.

57. A pharmaceutical composition according to any one of claims 53 to 56, formulated as a medicament for Alzheimer's Disease (AD).

58. A pharmaceutical composition according to any one of claims 53 to 57, formulated for intravenous injection.

59. A pharmaceutical composition according to any one of claims 53 to 58, wherein said cyclic D,L-a-peptide according to Formula IA or Formula IB is polyvalently coupled to said metal nanoparticles.

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