Targeted contrast agents for MRI of amyloid deposits

Abstract

To provide a liposome composition used in a method of imaging amyloid deposit.SOLUTION: A method of imaging an amyloid deposit includes: (A) introducing a detectable amount of a liposome composition to a patient, where the liposome composition includes (1) macrocyclic gadolinium-based imaging agent conjugated with specific phospholipid or salt thereof, and (2) a targeted ligand represented by the following formula; (B) giving enough time, and associating a liposome composition with one or more amyloid deposit; and (C) detecting a liposome composition associated with one or more amyloid deposit.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 967,295, filed January 29, 2020, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under Contract Nos. R44AG051292, U01DE028233 and R01HD094347 awarded by the National Institutes of Health. The United States Government has certain rights in the invention. [Background technology]

[0003] Definitive diagnosis of Alzheimer's disease ("AD") requires postmortem neuropathological demonstration of β-amyloid plaques and neurofibrillary tau tangles. However, advances in the development of positron emission tomography ("PET") imaging probes for these biomarkers have prompted a new research framework to study and characterize the disease in vivo. Although not approved for clinical diagnosis, this framework, advanced by the National Institute of Aging and Alzheimer's Associated, defines biological AD by either in vivo PET imaging or other biomarker evidence of β-amyloid plaques and neurofibrillary tau tangles. The use of targeted PET tracers in clinical studies has dramatically improved understanding of the evolution of AD biomarkers in the context of dementia. Clinical deployment of such noninvasive imaging AD biomarkers may enable earlier diagnosis of AD-related dementia and facilitate early intervention.

[0004] Accumulation of beta-amyloid plaques in the brain is one of the earliest pathogenic events in AD. Preclinical and clinical studies using PET probes have demonstrated that parenchymal deposition of amyloid plaques begins decades before the clinical presentation of cognitive impairment in AD-related dementia. Furthermore, amyloid plaque formation is causally linked to the pathogenesis of neurofibrillary tau tangles. Amyloid imaging PET probes such as 18F-florbetaben, 18F-florbetapir, and 18F-flutemetamol have substantially advanced the understanding of AD pathophysiology leading to cognitive impairment and play a key role in clinical trials for the evaluation of disease-modifying investigational therapies, but accessibility of PET modalities to the general population remains a global issue. Amyloid imaging agents for use with magnetic resonance imaging ("MRI") could be transformative due to their ease of accessibility and relatively low cost.

[0005] A high T1 relaxivity amyloid-targeted liposomal-gadolinium (Gd) nanoparticle contrast agent (containing a first linear Gd chelate, Gd-DTPA bis(stearylamide) ("Gd-DTPA-BSA"), conjugated to the inner and outer surfaces of the liposomal bilayer, and a second linear Gd chelate, gadobenate dimeglumine ("Gd-BOPTA"), within the liposomal core) enabled in vivo MRI of amyloid plaques in a transgenic mouse model of AD. See WO2016057812A1 and Ghaghada KB, Ravoori M, Sabapathy D, Bankson J, Kundra V, et al. (2009) New Dual Mode Gadolinium Nanoparticle Contrast Agent for Magnetic Resonance Imaging, PLoS ONE 4(10);e7628 Doi:10.1371 / journal.pone.0007628, each of which is incorporated herein by reference in its entirety. However, evidence of brain deposition of Gd dissociated from such linear chelates has emerged. Thus, there is a need for a more stable targeted liposomal Gd contrast agent for MRI of amyloid plaques. Summary of the Invention

[0006] In one aspect, a liposomal composition ("ADx-001") is provided. ADx-001 comprises a first phospholipid, a sterically bulky excipient capable of stabilizing the liposomal composition, a second phospholipid derivatized with a first polymer, a macrocyclic gadolinium-based imaging agent, and a third phospholipid derivatized with a second polymer, the second polymer being conjugated to a targeting ligand, the targeting ligand being: [ka] It is expressed as During the ceremony, Pyrimidine "P" is optionally substituted with zero, one or more -OH, O-alkyl, and NH; R 2 is a linking group containing C1-C6 alkyl or C1-C6 alkoxyalkyl, R 3 is hydrogen, C1-C6 alkyl, or C1-C6 alkoxyalkyl, and R other than hydrogen 3 comprises a third phospholipid that is substituted with zero, one or more -OH.

[0007] In a further aspect, the first phospholipid comprises hydrogenated soy L-α-phosphatidylcholine ("HSPC"), the sterically bulky excipient capable of stabilizing the liposomal composition comprises cholesterol ("Chol"), the second phospholipid derivatized with the first polymer comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) ("DSPE-mPEG2000"), the macrocyclic gadolinium-based imaging agent comprises gadolinium(3+) 2-[4,7,10-tris(carboxylatomethyl)-1,4,7,10 tetrazacyclododec-1-yl]acetate ("gadoterate" or "Gd(III)-DOTA"), and the fourth phospholipid, e.g., [ka] or a salt thereof (e.g., sodium salt). In some embodiments, the variable x can be one of 12, 13, 14, 15, 16, 17, or 18. In one embodiment, the variable x is 16 (conjugate: "Gd(III)-DOTA-DSPE"). In some embodiments, the third phospholipid derivatized with a second polymer, the second polymer being conjugated to a targeting ligand, is [ka] or a salt thereof (e.g., an ammonium phosphate salt). In some embodiments, the variable n can be any integer from about 10 to about 100, e.g., about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m can be one of 12, 13, 14, 15, 16, 17, or 18. For example, n can be 77 and m can be 14. n can be 79 and m can be 14. n can be 77 and m can be 16. n can be 79 and m can be 16.

[0008] In one aspect, the targeting ligand is [ka] Includes.

[0009] In one embodiment, n is 79 and m is 16 ("DSPE-PEG3500") and the targeting ligand is compound iii: [ka] (conjugate: "ET3-73"), and compound iii is included as a salt thereof (e.g., an ammonium phosphate salt).

[0010] In one embodiment, a method for imaging amyloid deposits in a subject is provided.The method can include introducing a detectable amount of liposome composition into the subject.The method can include allowing the liposome composition to be associated with one or more amyloid deposits for a sufficient period of time.The method can include detecting the liposome composition associated with one or more amyloid deposits.

[0011] In one embodiment, the liposomal composition of the method of imaging amyloid deposits in a subject may include ADx-001. In one embodiment, the liposomal composition of the method of imaging amyloid deposits in a subject may include Gd(III)-DOTA-DSPE and ET3-73. In one embodiment, the liposomal composition of the method of imaging amyloid deposits in a subject may include HSPC, Chol, DSPE-mPEG2000, Gd(III)-DOTA-DSPE, and ET3-73.

[0012] In one embodiment, the liposome composition is suitable for use in imaging amyloid deposits in a patient, the use comprising introducing a detectable amount of the liposome composition into the patient, allowing a sufficient time for the liposome composition to associate with one or more amyloid deposits, and detecting the liposome composition associated with the one or more amyloid deposits, hi one embodiment, the use comprises detecting using MRI.

[0013] In one embodiment, the use includes identifying a patient as likely to have AD by detecting a liposomal composition associated with one or more amyloid deposits, subjecting the patient to analysis for tau neurofibrillary tangles, and diagnosing the patient as having AD upon determining that tau neurofibrillary tangles are present in conjunction with detecting the liposomal composition associated with one or more amyloid deposits. [Brief description of the drawings]

[0014] [Figure 1] 1 provides an exemplary cross-sectional view of a liposome containing a targeted contrast agent for MRI of amyloid deposits.

[0015] [Diagram 2] FIG. 1 shows a representative schematic diagram of a surface-conjugated macrocyclic Gd-based imaging agent ("SC-Gd") described herein compared to prior art dual-Gd liposomes ("Dual-Gd").

[0016] [Diagram 3] FIG. 1 shows a comparison of the T1 relaxivity of the Gd(III) forms described herein at 1 T field strength between "free" Gd(III)-DOTA (i.e., not conjugated to liposomes), prior art Gd(III)-DTPA-BSA liposomes, and Gd(III)-DOTA-DSPE liposomes (i.e., ADx-001 liposomes).

[0017] [Figure 4] 1 provides an exemplary synthetic scheme for synthesizing ET3-73 ammonium salt.

[0018] [Diagram 5] 1 provides an exemplary synthetic scheme for synthesizing Gd(III)-DOTA-DSPE sodium salt.

[0019] [Figure 6] FIG. 1 shows an exemplary process flow diagram for the preparation of ADx-001.

[0020] [Figure 7] Demonstration of the identification of cortical regions of interest (ROIs) in axial MR images of the brain. Cortical ROIs are outlined on pre-contrast (top) and post-contrast (bottom) FSE-IR brain images.

[0021] [Figure 8]Figure 1 shows pre- and post-ADx-001 T1-weighted spin-echo ("T1w-SE") axial images of the brains of wild-type ("WT") mice (amyloid negative) at a dose of 0.20 mmol Gd / kg, and Tg APPswe / PSEN1dE9 ("Tg") mice (amyloid positive) at doses of b) 0.20 mmol Gd / kg, c) 0.15 mmol Gd / kg, d) 0.10 mmol Gd / kg. Arrows indicate areas of signal enhancement in the cortex and hippocampus.

[0022] [Figure 9] Fast spin-echo inversion recovery ("FSE-IR") axial images of the brains of a) WT mice at a dose of 0.20 mmol Gd / kg, and b) Tg mice at doses of 0.20 mmol Gd / kg, c) 0.15 mmol Gd / kg, and d) 0.10 mmol Gd / kg, pre- and post-ADx-001 administration. Arrows indicate areas of signal enhancement in the cortex and hippocampus.

[0023] [Figure 10] Figure 1 shows that Tg mice exhibit MR signal enhancement in cortical brain regions compared to corresponding WT at all dose levels of ADx-001. Box plots show statistically significant differences in signal change (expressed as percentages) between pre-contrast and delayed contrast T1w-SE images for ADx-001 dose levels a) 0.20 mmol Gd / kg, b) 0.15 mmol Gd / kg, and c) 0.10 mmol Gd / kg, and between pre-contrast and delayed contrast FSE-IR images for dose levels d) 0.20 mmol Gd / kg, e) 0.15 mmol Gd / kg, f) 0.10 mmol Gd / kg.

[0024] [Figure 11]Signal intensity means and ranges for (a) T1w-SE and (b) FSE-IR sequences for pre-contrast scans of both WT (n=18) and Tg (n=18) mice. ROIs were drawn in the cortex of each animal. No significant differences were found between signal intensities of WT and Tg mice for either T1w-SE or FSE-IR sequences (NS).

[0025] [Figure 12] Cortical brain signal changes as a function of time in WT (n=3) and Tg (n=3) mice in a) T1w-SE and b) FSE-IR sequences. Maximum signal enhancement was seen 4 days after imaging (arrows). Tg animals showed signal enhancement compared to WT animals in both sequences. Signal returned to near baseline levels by day 21.

[0026] [Figure 13] Representative fluorescence microscopy images of ADx-001 binding to amyloid plaques in a) mouse cortex and b) hippocampal regions of Tg animals compared to representative images of c) WT cortex and d) hippocampal regions of WT animals provide post-mortem confirmation of ADx-001 binding to β-amyloid plaques.

[0027] [Figure 14] Plasma Gd concentrations determined using inductively coupled plasma mass spectrometry ("ICP-MS") at various time points following administration of ADx-001 in dogs (▲) and monkeys (·) are shown.

[0028] [Figure 15] Biodistribution of ADx-001 in a) spleen, b) liver, c) kidney, d) bone, e) skin, and f) brain of rats 4 and 28 days after intravenous administration of ADx-001 at the 0.15 mmol Gd / kg dose level (▲) and the 0.3 mmol Gd / kg dose level (·) as determined by ICP-MS analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] A novel amyloid-targeting liposomal-Gd contrast agent, ADx-001, was developed based on the highly stable macrocyclic Gd-DOTA imaging moiety. ADx-001 can generally be seen as shown in cross-sectional form in FIG. FIG. 2 shows a representative schematic of the conjugation of a macrocyclic gadolinium-based imaging agent described herein ("SC-Gd") with Gd-chelates conjugated to the inner and outer surfaces of the liposome bilayer compared to the double Gd-liposomes of WO2016057812A1, which contain both a core-encapsulated Gd-chelate and a surface-conjugated Gd-chelate, as described, for example, in WO2016057812A1 and / or Tanifum EA, Ghaghada K, Vollert C, Head E, Eriksen JL, Annapragada AA Novel Liposomal Nanoparticle for the Imaging of Amyloid Plaque by Magnetic Resonance Imaging. J Alzheimer's Dis. 2016. doi:10.3233 / JAD-151124, each of which is incorporated herein by reference in its entirety.

[0030] Contrast agents with higher T1 relaxivity provide stronger enhancement. Figure 3 shows a comparison of the T1 relaxivity of the Gd(III) forms described herein between "free" Gd(III)-DOTA (i.e., not conjugated to liposomes), prior art Gd(III)-DTPA-BSA liposomes, and Gd(III)-DOTA-DSPE liposomes (i.e., ADx-001 liposomes) at 1 T field strength. ADx-001 shows approximately 3-fold higher T1 relaxivity compared to prior art Gd(III)-DTPA-BSA liposomes.

[0031] More specifically, liposomal Gd-DOTA with Gd DOTA conjugated to phospholipids exhibits about three times higher T1 relaxivity (about 31 mM-1 S-1 on a Gd basis and about 2,295,000 mM-1 S-1 on a nanoparticle basis at 1 T field strength) than liposomal Gd-DTPA with Gd-DTPA conjugated to bis(stearylamide) (about 9.0 mM-1 S-1 on a Gd basis and about 668,000 mM-1 S-1 on a nanoparticle basis at 1 T field strength). Gd conjugation is important for at least three reasons. First, conjugation of Gd chelates to macromolecules slows down the rotational correlation of the Gd atom, thus increasing the rotational correlation time. Longer rotational correlation times result in higher T1 relaxivity. Secondly, conjugation of the Gd chelate to a phospholipid (here, DSPE) (Gd-DOTA-DSPE) further increases the rotational correlation time compared to the Gd chelate conjugated to bis(stearylamide) (Gd-DTPA-BSA), and thus Gd-DOTA-DSPE liposomes perform better (higher T1 relaxivity) compared to Gd-DTPA-BSA liposomes. Finally, by being conjugated to a true phospholipid, the insertion into the bilayer becomes more stable. In contrast, the absence of a phosphatidyl group in Gd-DTPA-BSA reduces the amphiphilicity of the molecule and therefore the stability of insertion.

[0032] Thus, in one aspect, ADx-001 comprises a first phospholipid, a sterically bulky excipient capable of stabilizing the liposomal composition, a second phospholipid derivatized with a first polymer, a macrocyclic gadolinium-based imaging agent, and a third phospholipid derivatized with a second polymer, where the second polymer is conjugated to a targeting ligand. The macrocyclic gadolinium-based imaging agent may be conjugated to a fourth phospholipid.

[0033] Phospholipids In some embodiments, suitable phospholipids include those in which the two hydrocarbon chains are about 14 to about 24 carbon atoms in length and have various degrees of unsaturation. In some embodiments, suitable phospholipids include HSPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine ("DPPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DSPC"), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine ("DSPE"), and mixtures of two or more thereof. Suitable phospholipids may be naturally occurring or synthetic.

[0034] In some embodiments, suitable phospholipids may include any of those listed in International Publication No. WO2005107820A1, the contents of paragraphs

[0031] to

[0033] of which are incorporated herein by reference in their entirety.

[0035] Polymer-derivatized phospholipids In some embodiments, the liposomes of the liposome composition may contain flexible water-soluble (hydrophilic) polymer chains or may have a surface coated with flexible water-soluble (hydrophilic) polymer chains. These polymer chains may prevent the interaction between the liposomes and plasma components, which play a role in the uptake of liposomes by blood cells and their removal from blood. Liposomes may avoid uptake by organs of the mononuclear phagocyte system, mainly the liver and spleen (reticuloendothelial system).

[0036] In one embodiment, the polymer in the derivatized phospholipid can be polyethylene glycol ("PEG"). The PEG can have any of a variety of molecular weights. In one example, the PEG chains can have a molecular weight of about 1,000 to 10,000 daltons. When liposomes are formed, the PEG chains can provide a surface coating of hydrophilic chains sufficient to extend the blood circulation time of the liposomes in the absence of such a coating.

[0037] In some embodiments, the second phospholipid derivatized with a first polymer comprises DSPE-mPEG 2000. In some embodiments, the third phospholipid derivatized with a second polymer, where the second polymer is conjugated to a targeting ligand, comprises: [ka] or a salt thereof (e.g., an ammonium phosphate salt), where the variable n can be any integer from about 10 to about 100, e.g., about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m can be one of 12, 13, 14, 15, 16, 17, or 18. For example, n can be 77 and m can be 14. n can be 79 and m can be 14. n can be 77 and m can be 16. n can be 79 and m can be 16. In some embodiments, the third phospholipid derivatized with the second polymer comprises DSPE-PEG3500.

[0038] In some aspects, suitable polymers can include any of those listed in WO2005107820A1, the contents of paragraphs

[0034] to

[0038] of which are incorporated herein by reference in their entirety. In some embodiments, the phospholipids derivatized with the polymer can be any of the combinations disclosed in WO2016057812A1.

[0039] 3D bulky excipients In some embodiments, the liposomes may include stabilizing excipients. For example, the liposome composition may be formulated to include Chol. In other embodiments, the liposome composition may include fatty alcohols, fatty acids, cholesterol esters, other pharma- ceutically acceptable excipients, and mixtures thereof.

[0040] Macrocyclic gadolinium-based imaging agents The liposome composition comprises a macrocyclic Gd-based imaging agent. In some embodiments, the macrocyclic gadolinium-based imaging agent is Gd(III)-DOTA conjugated to a phospholipid, e.g., [ka] or a salt thereof (e.g., a sodium salt). In some embodiments, the variable x can be one of 12, 13, 14, 15, 16, 17, or 18. In one embodiment, the variable x is 16 and the conjugate is Gd(III)-DOTA-DSPE.

[0041] In another aspect, the macrocyclic gadolinium-based imaging agent is [Table 1] Includes.

[0042] Targeting Ligands The liposome composition comprises at least one phospholipid derivatized with a polymer, the polymer being conjugated to a targeting ligand. Thus, in some embodiments, the phospholipid is modified to include a spacer chain. The spacer chain may be a hydrophilic polymer. The hydrophilic polymer may typically be end-functionalized for coupling to a targeting ligand. The functionalized end group may be, for example, a maleimide group, a bromoacetamide group, a disulfide group, an activated ester or an aldehyde group. The hydrazide group is reactive to aldehydes that can be generated in many biologically relevant compounds. The hydrazide may also be acylated by an active ester or a carbodiimide-activated carboxyl group. The acyl azide group reactive as an acylation species can be easily obtained from the hydrazide, allowing the attachment of amino-containing ligands.

[0043] In some embodiments, the targeting ligands may be accessible from the surface of the liposome and may, for example, specifically bind or attach to one or more molecules or antigens. These targeting ligands may direct or target the liposome to a particular cell or tissue, such as amyloid-β plaques, and may bind to molecules or antigens on or associated with the cells or tissue.

[0044] The targeting ligand is [ka] It is expressed as During the ceremony, Pyrimidine "P" is optionally substituted with zero, one or more -OH, O-alkyl, and NH; R 2 is a linking group containing C1-C6 alkyl or C1-C6 alkoxyalkyl, R 3 is hydrogen, C1-C6 alkyl, or C1-C6 alkoxyalkyl, and R other than hydrogen 3 is substituted with zero, one or more -OH.

[0045] In one embodiment, the targeting ligand is compound iii. In one embodiment, the phospholipid polymer targeting ligand conjugate is ET3-73.

[0046] In a further aspect, the targeting ligand is any of compounds i-xiii disclosed in WO2016057812A1. In yet a further aspect, the targeting ligand is compounds ii, iii, xi, and xiii disclosed in WO2016057812A1.

[0047] Liposomes "Liposome" generally refers to a spherical or nearly spherical particle that contains an internal cavity. The wall of the liposome may comprise a bilayer of lipids. These lipids may be phospholipids. A number of lipids and / or phospholipids may be used to make liposomes. One example is an amphipathic lipid with hydrophobic and polar head group moieties, which may spontaneously form into bilayer vesicles in water, as exemplified by phospholipids, or may stably incorporate into a lipid bilayer with its hydrophobic moiety in contact with the hydrophobic region at the interior of the bilayer membrane and its polar head group moiety facing the exterior polar surface of the membrane. Liposomes may be prepared by any known method, including those described in the examples herein, WO2016057812A1 and WO2012139080A1, which are incorporated herein by reference in their entireties. Figure 1 provides an exemplary cross-sectional view of a liposome containing a targeted contrast agent for MRI of amyloid deposits.

[0048] In one embodiment, ADx-001 comprises HSPC, Chol, DSPE-mPEG2000, ET3-73, and Gd(III)-DOTA-DSPE. In some embodiments, the first phospholipid may comprise DPPC, DSPC, or a mixture of DPPC and DSPC. In one embodiment, the lipid composition and molar ratio (%) of the components in ADx-001 is HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73=about 31.5:about 40:about 2.5:about 25:about 1. In some embodiments, the molar ratio of any one of HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73 may be adjusted by up to 10%, thus 31.5±10%:40±10%:2.5±10%:25±10%:1±10%. In one embodiment, the lipid composition and molar ratios (%) of the components in ADx-001 are HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73=about 32.5:about 40:about 2:about 25:about 0.5.

[0049] In one embodiment, the HSPC content in ADx-001 is about 24 mg / mL to about 32 mg / mL (total lipids). In one embodiment, the Chol content in ADx-001 is about 14 mg / mL to about 19 mg / mL. In one embodiment, the DSPE-mPEG2000 content in ADx-001 is about 5 mg / mL to about 7 mg / mL. In one embodiment, the Gd(III)-DOTA-DSPE content in ADx-001 is 30 mg / mL to 45 mg / mL. In one embodiment, the ET3-73 content in ADx-001 is about 2 mg / mL to about 3 mg / mL. In one embodiment, the free gadolinium content in ADx-001 is 100 μg / mL or less, including less than 2.5 μg / mL.

[0050] In one embodiment, the liposome composition has a pH of 6.4 to 8.4. In a further embodiment, the liposome has an osmolality of 200 to 400 mOsmol / kg. In a further embodiment, the liposome has a viscoelasticity of 150 nm (D) as measured by dynamic light scattering. 50 ) or less than about 140 nm (D 50 ) and about 120 nm (D 50 ) including 200 nm (D 50 ) vesicle size (Z average).

[0051] The term "about" in connection with a number is intended to include ±10% of that number. This is true whether "about" is modifying an individual number or a number at either or both ends of a numerical range. In other words, "about 10" means 9 to 11. Similarly, "about 10 to about 20" contemplates 9 to 22 and 11 to 18. In the absence of the term "about," the exact number is intended. In other words, "10" means 10.

[0052] example The dose response, pharmacokinetics, and biodistribution of ADx-001 in animal models were studied. A dose-ranging efficacy study was performed in a Tg mouse model of early-onset AD. Imaging was performed on a 1 Tesla permanent magnet MR scanner using T1w-SE and FSE-IR sequences. ADx-001 was tested at three dose levels: 0.10, 0.15, and 0.20 mmol Gd / kg. Tg and age-matched WT control animals (n=6 / dose level / genotype) were imaged pre-contrast and 4 days after administration of ADx-001 (delayed post-contrast). Pre-contrast and delayed post-contrast images were analyzed qualitatively and quantitatively to determine the sensitivity, specificity, and accuracy for post-mortem histological identification of amyloid-β plaques. The pharmacokinetics of ADx-001 was studied in monkeys and dogs. Blood samples were taken at multiple time points after administration of ADx-001, and Gd levels were analyzed by ICP-MS. The biodistribution of ADx-001 was studied in rats. Gd levels in target organs (liver, spleen, kidney, skin, bone and brain) were measured by ICP-MS on days 4 and 28 after administration of ADx-001.

[0053] Example 1: Preparation of ET3-73 Referring to FIG. 4, the starting material (E)-2-((2-aminoethyl)(4-(2-(pyrimidin-4-yl)vinyl)phenyl)amino)ethan-1-ol (SM1) was reacted with DIPEA to form ET3-73 intermediate 1. ET3-73 intermediate 1 was reacted with TBDMS-OTf (TBDMS-triflate) to give ET3-73 intermediate 2. ET3-73 intermediate 2 was coupled with HO-PEG3500-NH2 (SM2) to form ET3-73 intermediate 3. ET3-73 intermediate 3 was reacted with bis-pentafluorophenyl-carbonate, and the activated ET3-73 intermediate 3 was combined with DSPE that was silylated using bis-trimethylsilyl-acetamide to give ET3-73 intermediate 4. Finally, the ET3-73 ammonium salt was formed by deprotection using TBAF followed by reaction with sodium ammonium acetate.

[0054] Example 2: Preparation of Gd(III)-DOTA-DSPE Referring to FIG. 5, the synthetic scheme begins with further esterification of DOTA-tris(tert-butyl ester) (SM1) with pentafluorophenol (HOpFP). The tert-butyl ester protecting group was removed using TFA and trimethylchlorosilane (TMSC1). The pentafluorophenyl ester was coupled to DSPE in the presence of BSA and NMM to form DSPE-DOTA. Gadolinium chelation and salt formation occurred by addition of gadolinium [Gd(OAc)3] as acetate salt, followed by addition of scavenger SiliaMetS TAAcONa (triaminetetraacetate, sodium salt functionalized silica gel) to form the final DSPE-DOTA-Gd sodium salt.

[0055] Example 3: Preparation of ADx-001 With reference to FIG. 6, ADx-001 was prepared as follows.

[0056] Step 1 (Buffer solution): Sodium chloride and histidine were dissolved in water with mixing and filtered through a 0.2 μm filter. The solution was nominally pH 7.5.

[0057] Step 2 (lipid solution): DSPE-DOTA-Gd, HSPC (Lipoid Inc., Newark, NJ, USA), DSPE-mPEG 2000 (Corden Pharma, Liestal, Switzerland), Chol (Lipoid Inc., Newark, NJ, USA) and ET3-73 (31.5:40:2.5:25:1 molar ratio) were dissolved in tert-butyl alcohol with mixing.

[0058] Step 3 (liposome formation): The lipid solution (step 2) was added to a portion of the buffer solution (step 1). If necessary, the pH was adjusted to a nominal pH of 6.5-7.0 using sodium hydroxide solution. This step produced liposomes of indeterminate size and lamellar nature. The drug substance (DSPE-DOTA-Gd) and other lipid components (HSPC, DSPE-mPEG 2000, cholesterol and ET3-73) are present within the lipid bilayer of the liposome.

[0059] Step 4 (Extrusion): To reduce the liposome vesicle size, the process material was extruded at high pressure through a track-etched polycarbonate filter. The process was continued until the desired vesicle size (approximately 140 nm nominal size) was achieved, as measured by dynamic light scattering studies during the process.

[0060] Step 5 (Ultrafiltration): Ultrafiltration was performed on the process material using a tangential flow filtration assembly with a molecular weight cutoff (MWCO) rating of 500,000. During ultrafiltration, the liposomal nanoparticles were recirculated and retained by the ultrafilter, while a portion of the carrier solution (buffer solution + solvent tert-butyl alcohol) passed through the ultrafilter into the permeate waste stream. The ultrafiltration step consisted of three parts. First, the process material was concentrated by discarding the permeate from the ultrafilter. Second, the ultrafiltration assembly was operated in diafiltration mode. In diafiltration mode, a constant concentration was maintained by replenishing the permeate waste stream with buffer solution (step 1). Third, the process material was concentrated to reach a concentration level slightly higher than the nominal 98 mg / mL total lipid composition of the final formulation.

[0061] Step 6 (Prefiltration): The process material was passed through a track-etch polycarbonate filter until the desired filterability was achieved.

[0062] Step 7 (Clarification Filtration): The process material was passed through a 0.2 μm filter (Sartorius Sartopore® 2 XLI containing a polyethersulfone membrane).

[0063] Step 8 (Dilution): The process material was diluted with buffer solution (Step 1) to a target labeling strength of 38.7 mg / mL of DSPE-DOTA-Gd. The nominal concentration was 98 mg / L total lipid.

[0064] Step 9 (Sterile Filtration): Under aseptic conditions, the process material was passed through a 0.2 μm sterilizing grade filter (Sartorius Sartopore® 2 XLI containing a polyethersulfone membrane).

[0065] Step 10 (Aseptic Filling): Process materials were filled into vials, stoppered, and sealed under aseptic conditions. Fill weight checks were performed during the filling operation, and filled vials were 100% visually inspected for particulates and container closure defects. Batch data for several exemplary batches is shown in Table 1. [Table 2]

[0066] Example 4: MRI study The study was performed in the APPswe / PSEN1dE9 (C57BL / 6J background, 11-18 months old) double Tg mouse model (JAX MMRRC Stock#005864) of early onset AD. Tg mice develop amyloid plaques in the brain around 6-7 months of age. ADx-001, as prepared in Example 3, was tested at three dose levels, 0.10, 0.15 and 0.20 (mmol Gd / kg). At each dose level, ADx-001 was tested in Tg mice (n=6) and WT mice (mice lacking both mutations) (n=6). ADx-001 was administered intravenously as a slow bolus injection via the tail vein.

[0067] Imaging was performed in a 1T permanent MRI scanner (M7 system, Aspect Imaging, Shoham, Israel). Animals were sedated using 2.5 or 3% isoflurane and placed on a custom-made sled with an integrated face-cone for continuous anesthesia delivery by inhalation (1–2% isoflurane). Respiratory rate was monitored by a pneumatically controlled pressure pad placed under the abdominal region. Two MRI sequences were tested: a T1w-SE sequence and a 2D FSE-IR approximating fluid-attenuated inversion recovery sequence. SE parameters were TR=600ms, TE=11.5ms, slice thickness=1.2mm, matrix=192×192, FOV=30mm, slices=16, NEX=4. FSE-IR parameters were TR=6500ms, TE=80ms, TI=2000ms, slice thickness=2.4mm, matrix=192x192, FOV=30mm, slices=6, NEX=6. Coil calibration, RF calibration, and shimming were performed for each subject at the beginning of the study. All animals underwent a pre-contrast scan followed by intravenous administration of ADx-001. A delayed post-contrast scan was acquired 4 days after contrast administration. Pre- and post-contrast scans were acquired using both T1w-SE and FSE-IR sequences.

[0068] Animals were euthanized after the post-contrast scan and perfused with 0.9% saline followed by 4% formalin solution. Brains were excised, fixed in 4% formalin solution for 24 hours, and transferred to 30% sucrose for cryoprotection. Brains were embedded in OCT and stored at -80°C until ready for sectioning. Brain sections 15 μm thick were cut and used for postmortem phenotypic confirmation of amyloid deposits. Sections were incubated in 5% bovine serum albumin ("BSA") for 1 hour, followed by incubation with fluorescently tagged anti-amyloid-β antibody (AF647-4G8, BioLegend, San Diego, CA) in 3% BSA overnight at 4°C. Sections were further stained with a nuclear marker (DAPI), washed, mounted, coverslipped using Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and imaged on a confocal microscope with appropriate filter sets. The presence of amyloid-bound ADx-001 nanoparticles was analyzed by imaging in the FITC channel.

[0069] Qualitative and quantitative analysis of MRI images was performed in OsiriX (version 5.8.5, 64-bit) and MATLAB® (version 2015a). Brain extraction was performed in OsiriX by a combination of thresholding and manual segmentation. Signal change between pre-contrast and delayed post-contrast images was assessed by quantification of signal intensity in cortical regions near the center of the image stack (see FIG. 7). Amyloid-positive animals were identified by qualitative assessment of signal enhancement between pre-contrast and delayed post-contrast assessments of the cortex and hippocampus. Signal change between pre-contrast and post-contrast images was quantified by integration of signal in a ROI encompassing the cortical tissue in the central slice of the MRI volume. Signal change (%) was calculated using the formula (1):

number

[0070] Observations of signal enhancement in MRI of amyloid-positive animals (as determined by immunofluorescence) were counted as true-positive results. Conversely, lack of signal enhancement between pre-contrast and delayed post-contrast images for amyloid-negative animals was considered a true-negative. Sensitivity was determined by the ratio of true positives identified by MRI to the total number of true positives identified by the gold standard of postmortem amyloid plaque staining by immunofluorescence analysis. Specificity was determined as the ratio of true negatives identified by MRI to the total number of true negatives. Overall accuracy was calculated as the total number of animals correctly identified by MRI compared to amyloid true positives determined by postmortem immunofluorescence.

[0071] WT mice (amyloid negative) did not show brain signal enhancement in delayed post-contrast images acquired using T1w-SE or FSE-IR at any dose level of ADx-001, but Tg mice (amyloid positive) showed moderate to high MR signal enhancement in cortical and hippocampal regions in T1w-SE delayed post-contrast images at ADx-001 doses of 0.20 and 0.15 mmol Gd / kg. Thus, as shown in FIG. 8, a) WT animals administered 0.2 mmol Gd / kg ADx-001 show no signal enhancement 4 days after injection, b) Tg animals show high enhancement in the cortical (upper arrow) and hippocampal (lower arrow) regions 4 days after administration of 0.2 mmol Gd / kg ADx-001, c) Tg animals show moderate enhancement in the cortical (upper arrow) and hippocampal (lower arrow) regions 4 days after administration of 0.15 mmol Gd / kg ADx-001, and d) Tg animals show low enhancement in the cortical region (arrow) 4 days after administration of 0.10 mmol Gd / kg ADx-001.

[0072] Similarly, Tg mice showed moderate to high signal enhancement in delayed post-contrast FSE-IR images at ADx-001 doses of 0.20 and 0.15 mmol Gd / kg, and relatively mild signal enhancement at 0.10 mmol Gd / kg. As shown in Figure 9, FSE-IR axial images show MR signal enhancement in ADx-001 delayed post-contrast scans of Tg APPswe / PSEN1dE9 mice, but not age-matched WT control mice. Specifically, (a) WT animals administered 0.20 mmol Gd / kg ADx-001 show no signal enhancement in delayed post-contrast images, (b) Tg animals administered 0.20 mmol Gd / kg ADx-001 show high signal enhancement in the cortical (upper arrow) and hippocampal (lower arrow) regions in delayed post-contrast images, (c) Tg animals administered 0.15 mmol Gd / kg ADx-001 show moderate signal enhancement in the cortical region (upper arrow) and low enhancement in the hippocampal region (lower arrow) in delayed post-contrast images, and (d) Tg animals administered 0.10 mmol Gd / kg ADx-001 show low signal enhancement in the cortical region (arrow) in delayed post-contrast images. All delayed post-contrast images were acquired 4 days after ADx-001 administration.

[0073] Quantitative analysis of cortical ROIs confirmed the qualitative observation of MR signal enhancement in delayed post-contrast images and found statistically significant differences in post-contrast signal changes between Tg and WT animals at all dose levels. As shown in FIG. 10, Tg mice show MR signal enhancement in cortical brain regions compared to corresponding WT at all dose levels of ADx-001. Specifically, box plots show the signal change (expressed as percentage) between pre-contrast and delayed post-contrast T1w-SE images for dose levels of ADx-001 of a) 0.20 mmol Gd / kg, b) 0.15 mmol Gd / kg, and c) 0.10 mmol Gd / kg. Similar signal changes are shown between pre-contrast and delayed post-contrast FSE-IR images for dose levels of d) 0.20 mmol Gd / kg, e) 0.15 mmol Gd / kg, and f) 0.10 mmol Gd / kg. Wilcoxon rank sum statistical test was applied to compare group differences. Values ​​of p≦0.05 were considered statistically significant. In Figure 10, p<0.05 (*) and p<0.005 (**).

[0074] After establishing that the pre-contrast signals of WT and Tg mice were indistinguishable, signal difference thresholds were estimated from the pre-contrast (baseline) scans of all test mice (see FIG. 11). Estimated baseline signal thresholds were 5.1% (FSE-IR) and 5.6% (T1w-SE). Amyloid-positive mice were identified if they showed signal enhancement above these cutoffs.

[0075] Using these thresholds, ADx-001 demonstrated excellent specificity (100%) at all dose levels using both T1w-SE and FSE-IR sequences. As shown in Table 2 below, for T1w-SE imaging, ADx-001 demonstrated high sensitivity (>80%) at the 0.20 and 0.10 mmol Gd / kg dose levels, while for FSE-IR imaging, ADx-001 demonstrated high sensitivity (>80%) at the 0.20 and 0.15 mmol Gd / kg dose levels. For both T1w-SE and FSE-IR, ADx-001 demonstrated the highest accuracy (>90%) at the highest dose level (0.20 mmol Gd / kg). [Table 3]

[0076] Longitudinal imaging studies in WT and Tg mice demonstrated that signal enhancement was optimal 4 days after post-contrast administration, with signal returning to near baseline levels by 21 days after post-contrast administration (see FIG. 12).

[0077] Immunofluorescence microscopy confirmed the preferential concentration of ADx-001 in the cortical and hippocampal regions of Tg mice and its colocalization with amyloid plaque deposits. In comparison, WT animals did not show amyloid plaque deposits or the presence of bound ADx-001 nanoparticles. Figure 13 shows representative fluorescence microscopy images of ADx-001 binding to amyloid plaques in mouse a) cortical and b) hippocampal regions in Tg animals. Representative images for WT c) cortical and d) hippocampal regions are also shown. WT mice showed no evidence of amyloid plaque deposits (4G8 antibody staining) or the presence of bound ADx-001 (observed for amyloid ligand fluorescent signal). Images were taken at 60x magnification.

[0078] Example 5: Pharmacokinetic studies The pharmacokinetics ("PK") of ADx-001 was evaluated in cynomolgus monkeys and beagle dogs. Non-naive male cynomolgus monkeys (n=3, 2-5 years old, 2.3-3.1 kg body weight) were administered ADx-001 intravenously at 0.30 mmol Gd / kg over approximately 60 minutes using a calibrated infusion pump. Blood samples were collected from all animals pre-dose, immediately after the end of the infusion, and at 4, 8, 24, 48, 96, 168, 336, and 672 hours after start of injection ("SOI"). For PK analysis in beagle dogs, animals (n=5, 5-7 months old, 6.2-7.9 kg body weight) were infused intravenously with ADx-001 at 0.30 mmol Gd / kg over approximately 60 minutes. Blood samples were processed to plasma and stored frozen until ready for analysis.

[0079] Gd concentrations in plasma samples were determined using ICP-MS. Plasma samples (100 μL) were digested in 90% concentrated HNO3 (750 μL) at 90 °C for 15 min. The digested samples were diluted with deionized ("DI") water, centrifuged at 3000 rpm for 15 min, and the supernatant was further diluted for ICP-MS analysis such that the Gd concentration was within the range of the ICP-MS calibration standards (1-500 ppb).

[0080] ADx-001 was well tolerated in dogs and monkeys without adverse effects. Figure 14 shows the long blood half-life of ADx-001. Plasma Gd concentrations were determined using ICP-MS at various time points after administration of ADx-001 to dogs (▲) and monkeys (·). Assuming first-order kinetics, the elimination rate was 0.017 h -1 with a blood half-life of approximately 41 hours in monkeys. Plasma levels of Gd were reduced by approximately 80% at 96 hours and by more than 99% at 336 hours after SOI in monkeys. There is no literature on blood half-lives of comparable liposomal MRI contrast agents in monkeys, but studies in mice have shown blood half-lives in the range of 14-24 hours. In dogs, the elimination rate was 0.0297 hours. -1 , resulting in a blood half-life of approximately 23 hours. Plasma levels of Gd were reduced by approximately 85% at 96 hours and by approximately 99% at 168 hours after SOI in dogs.

[0081] Example 6: Tissue distribution The biodistribution of ADx-001 was studied in a rat model. Wistar Han rats (10 weeks old, 257-296 g body weight, n=13 / treatment group) were administered ADx-001 as a single intravenous bolus injection at 0.15 mmol Gd / kg (n=13) or 0.30 mmol Gd / kg. Animals were euthanized on days 4 (n=7 / dose level) and 28 (n=6 / dose level) after administration of ADx-001. Tissues were harvested to determine Gd levels in target organs (liver, spleen, kidney, skin, bone and brain). Tissue samples were immediately frozen in liquid nitrogen and stored at -20°C until ready for analysis.

[0082] Gd concentrations in tissue samples were quantified using ICP-MS. Wet tissue (approximately 100 mg) was digested in 90% concentrated HNO3 (approximately 750 μL) at 90 °C for 10-15 min. Digested samples were diluted with DI water, vortexed vigorously, and centrifuged at 3500 rpm for 15 min. The supernatant was separated and further diluted as necessary to ensure that the Gd concentration was within the range of the calibration standards (1-500 ppb). Quality control samples (50 and 100 ppb) were included at the beginning, middle, and end of the analytical run.

[0083] ADx-001 was well tolerated in rats at doses up to 0.30 mmol Gd / kg with no observable adverse effects on clinical toxicity, clinical pathology, or histopathology endpoints (data not shown). Tissue Gd levels showed a dose-related increase in all organs. Thus, FIG. 15 shows ICP-MS analyses illustrating Gd levels in a) spleen, b) liver, c) kidney, d) bone, e) skin, and f) brain at 4 and 28 days after intravenous administration of ADx-001 at the 0.15 mmol Gd / kg dose level (▲) and the 0.3 mmol Gd / kg dose level (·). Tissue Gd content is expressed as mg Gd per gram wet tissue. The highest Gd tissue levels were observed in the liver and spleen, consistent with known organs for clearance of PEGylated liposomal agents. The lowest Gd levels were observed in the brain. Tissue levels of Gd on day 28 were reduced by more than 90% compared to day 4 Gd tissue levels in all organs.

[0084] Animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee. Studies followed the NC3RS-ARRIVE guidelines.

[0085] ADx-001-enhanced MRI showed significantly (p<0.05) higher brain signal enhancement in Tg mice (amyloid positive) compared to WT (amyloid negative) mice at all dose levels. ADx-001-enhanced T1w-SE imaging showed high sensitivity (>80%) at 0.10 and 0.20 mmol Gd / kg, whereas ADx-001-enhanced FSE-IR imaging showed high sensitivity (>80%) at 0.15 and 0.20 mmol Gd / kg. Excellent specificity (100%) was observed at all dose levels of ADx-001. Pharmacokinetic studies showed a long blood half-life (23 hours in dogs and 41 hours in monkeys). Biodistribution studies demonstrated systemic clearance of ADx-001 primarily via the mononuclear phagocyte system (also known as the reticuloendothelial system). Tissue Gd levels in all organs at day 28 were reduced by >90% compared to day 4, suggesting ongoing clearance.

[0086] In brief, the amyloid-targeted liposomal macrocyclic gadolinium contrast agent, ADx-001, demonstrated high sensitivity and excellent specificity for in vivo imaging of β-amyloid plaques in mouse brain. No signs of toxicity were detected, and the pharmacokinetics followed the pattern expected for PEGylated nanoparticles.

[0087] Unless otherwise noted, "a," "an," "the," "one or more of," and "at least one" are used interchangeably. The singular forms "a," "an," and "the" include the plurals thereof. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). The terms "comprising" and "including" are intended to be equivalent and open ended. The phrase "consisting essentially of" means that the composition or method may include additional components and / or steps, but only if the additional components and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. The phrase "selected from the group consisting of" is intended to include mixtures of the recited group.

Claims

1. A liposome composition for use in a method of imaging amyloid deposits in a patient, the method comprising: (A) introducing into a patient a detectable amount of a liposomal composition, wherein the liposomal composition comprises: (1) A macrocyclic gadolinium-based imaging agent, conjugated to a phospholipid or a salt thereof, including: 【Chemistry 1】 The variable x is one of 12, 13, 14, 15, 16, 17, or 18; and (2) a targeting ligand, wherein the targeting ligand is 【Chemistry 2】 is; (B) allowing a sufficient period of time for the liposome composition to associate with one or more amyloid deposits; and (C) detecting the liposomal composition associated with one or more amyloid deposits.

2. The liposome composition described in claim 1 for use in a method for imaging amyloid deposits in a patient, wherein the detection step includes detecting using magnetic resonance imaging.

3. The liposome composition of claim 1 for use in a method for imaging amyloid deposits in a patient, further comprising diagnosing the patient for Alzheimer's disease by detecting the liposome composition associated with one or more amyloid deposits.

4. The liposome composition of claim 1 for use in a method for imaging amyloid deposits in a patient, further comprising: identifying a patient with possible Alzheimer's disease by detecting the liposomal composition associated with one or more amyloid deposits; subjecting the patient to analysis of neurofibrillary tau tangles; and Diagnosing the patient as having Alzheimer's disease if the presence of neurofibrillary tau tangles is determined in conjunction with detection of the liposomal composition associated with one or more amyloid deposits.

5. A liposome composition for use in a method of imaging amyloid deposits in a patient, the method comprising: (A) introducing into a patient a detectable amount of a liposomal composition, wherein the liposomal composition comprises: (1) A macrocyclic gadolinium-based imaging agent conjugated to a phospholipid, wherein said macrocyclic gadolinium-based imaging agent is: 【Chemistry 3】 or a salt thereof, wherein the variable x is one of 12, 13, 14, 15, 16, 17, or 18; and (2) a second phospholipid derivatized with a polymer, wherein the polymer is conjugated to a targeting ligand, the conjugate of the second phospholipid, the polymer, and the targeting ligand comprises: 【Chemistry 4】 or a salt thereof, where the variable n is any integer between 70 and 90, The variable m is one of 12, 13, 14, 15, 16, 17, or 18; (B) allowing a sufficient period of time for the liposome composition to associate with one or more amyloid deposits; and (C) detecting the liposomal composition associated with one or more amyloid deposits.

6. The liposome composition of claim 5 for use in a method for imaging amyloid deposits in a patient, wherein the detecting step comprises detecting using magnetic resonance imaging.

7. The liposome composition of claim 5 for use in a method for imaging amyloid deposits in a patient, further comprising diagnosing the patient for Alzheimer's disease by detecting the liposome composition associated with one or more amyloid deposits.

8. The liposome composition of claim 5 for use in a method for imaging amyloid deposits in a patient, further comprising: identifying a patient with possible Alzheimer's disease by detecting the liposomal composition associated with one or more amyloid deposits; subjecting the patient to analysis of neurofibrillary tau tangles; and Diagnosing the patient as having Alzheimer's disease when the presence of neurofibrillary tau tangles is determined in conjunction with detection of the liposomal composition associated with one or more amyloid deposits.

9. A liposome composition for use in a method of imaging amyloid deposits in a patient, the method comprising: (A) introducing into a patient a detectable amount of a liposomal composition, wherein the liposomal composition comprises: (1) A liposome comprising: (a) hydrogenated soy L-α-phosphatidylcholine; (b) cholesterol; (c) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000); (d) 【Chemistry 5】 and (e) 【Chemistry 6】 (B) allowing a sufficient period of time for the liposome composition to associate with one or more amyloid deposits; and (C) detecting the liposomal composition associated with one or more amyloid deposits.

10. The liposome composition of claim 9 for use in a method for imaging amyloid deposits in a patient, wherein the detecting step comprises detecting using magnetic resonance imaging.

11. The liposome composition of claim 9 for use in a method for imaging amyloid deposits in a patient, further comprising diagnosing the patient for Alzheimer's disease by detecting the liposome composition associated with one or more amyloid deposits.

12. The liposome composition of claim 9 for use in a method for imaging amyloid deposits in a patient, further comprising: identifying a patient with possible Alzheimer's disease by detecting the liposomal composition associated with one or more amyloid deposits; subjecting the patient to analysis of neurofibrillary tau tangles; and Diagnosing the patient as having Alzheimer's disease if the presence of neurofibrillary tau tangles is determined in conjunction with detection of the liposomal composition associated with one or more amyloid deposits.

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