Targeted contrast agents for MRI of amyloid deposition

Abstract

The present invention provides a liposome composition ("ADx-001") comprising: a first phospholipid; a sterically hindered bulky excipient capable of stabilizing the liposome composition; a second phospholipid derivatized with the first polymer; a macrocyclic gadolinium-based imaging agent; and a third phospholipid derivatized with the second polymer, the second polymer being coupled to a targeting ligand. The macrocyclic gadolinium-based imaging agent may be coupled to a fourth phospholipid.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 975,295, filed on January 29, 2020, which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with U.S. Government support under Contract Nos. R44AG051292, U01DE028233, and R01HD094347 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] A definitive diagnosis of Alzheimer's disease ("AD") requires autopsy neuropathological demonstration of beta-amyloid plaques and neurofibrillary tau tangles. However, advances in the development of positron emission tomography ("PET") imaging probes for these biomarkers have provided a new research framework for studying and characterizing the disease in vivo. Although not approved for clinical diagnosis, this framework, proposed by the National Institute of Aging and Alzheimer's Associated, defines the biology of AD through in vivo PET imaging or other biomarker evidence of beta-amyloid plaques and neurofibrillary tau tangles. The use of targeted PET tracers in clinical studies has greatly improved the understanding of the evolution of AD biomarkers in the context of dementia. Clinical deployment of such non-invasive imaging AD biomarkers may enable early diagnosis of AD-related dementia and facilitate early intervention.

[0006] The formation of β-amyloid plaques in the brain is one of the earliest pathogenic events in AD. Preclinical and clinical studies using PET probes have demonstrated that substantial deposition of amyloid plaques begins decades before the clinical manifestation of cognitive impairment in AD-related dementia. Furthermore, amyloid plaque formation is causally linked to the pathogenesis of neurofibrillary tau tangles. Although amyloid-imaging PET probes, such as 18F-florbetaben, 18F-florbetapir, and 18F-flutemetamol, have significantly advanced our understanding of the pathophysiology of AD leading to cognitive impairment and play a key role in clinical trials evaluating investigational disease-modifying therapies, access to PET modalities remains a worldwide issue for the general population. Amyloid-imaging agents for magnetic resonance imaging ("MRI") have been revolutionary due to their easy availability and relatively low cost.

[0007] A high T1 relaxivity, amyloid-targeted liposome-gadolinium (Gd) nanoparticle contrast agent (containing: a first linear Gd chelate, Gd-DTPA bis(stearamide) ("Gd-DTPA-BSA"), coupled to the inner and outer surfaces of the liposome bilayer, and a second linear Gd chelate, gadobenate dimeglumine ("Gd-BOPTA"), inside the liposome core) enables in vivo MRI of amyloid plaques in a transgenic AD mouse model. See WO2016057812A1 and Ghaghada KB, Ravoori M, Sabapathy D, Bankson J, Kundra V, et al. (2009) New DualMode 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, there is evidence that Gd dissociated from such linear chelates is deposited in the brain. Therefore, a more stable targeted liposomal Gd contrast agent is needed for MRI of amyloid plaques. Summary of the Invention

[0008] In one aspect, a liposome composition ("ADx-001") is provided, comprising: a first phospholipid; a sterically hindered bulky excipient capable of stabilizing the liposome composition; a second phospholipid derivatized with a first polymer; a macrocyclic gadolinium-based imaging agent; and a third phospholipid derivatized with a second polymer, wherein the second polymer is coupled to a targeting ligand, wherein the targeting ligand is represented by:

[0009]

[0010] in,

[0011] Pyrimidine "P" may be substituted by 0, 1 or more of -OH, O-alkyl and -NH2;

[0012] R 2 is a linking group comprising a C1-C6 alkyl group or a C1-C6 alkoxyalkyl group; and

[0013] R 3 is hydrogen, C1-C6 alkyl or C1-C6 alkoxyalkyl, and R other than hydrogen 3 Substituted with 0, 1 or more -OH groups.

[0014] In another aspect, the first phospholipid comprises hydrogenated soy L-α-phosphatidylcholine (“HSPC”); the sterically hindered bulky excipient capable of stabilizing the liposome 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”); and the macrocyclic gadolinium-based imaging agent comprises 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]gadolinium(3+) acetate (“gadoterate” or “Gd(III)-DOTA”) and is coupled to a fourth phospholipid, for example:

[0015]

[0016] or a salt thereof (e.g., a sodium salt). In some aspects, the variable x can be one of 12, 13, 14, 15, 16, 17, or 18. In one aspect, the variable x is 16 (conjugate: "Gd(III)-DOTA-DSPE"). In some aspects, the second polymer is conjugated to a targeting ligand, and the third phospholipid derivatized with the second polymer can comprise:

[0017]

[0018] or a salt thereof (e.g., ammonium phosphate). In some aspects, the variable n can be any integer from about 10 to about 100, for example, from about 60 to about 100, from about 70 to about 90, from 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.

[0019] In one aspect, the targeting ligand comprises:

[0020]

[0021] In one aspect, n is 79, m is 16 ("DSPE-PEG3500"), and the targeting ligand comprises:

[0022]

[0023] ("Conjugate: ET3-73"), including salts thereof (eg, ammonium phosphate salt).

[0024] In one aspect, a method for imaging amyloid deposits in a subject is provided. The method may comprise introducing a detectable amount of a liposome composition into the subject. The method may comprise allowing sufficient time for the liposome composition to bind to one or more amyloid deposits. The method may comprise detecting the liposome composition bound to the one or more amyloid deposits.

[0025] In one aspect, a liposomal composition for use in a method of imaging amyloid deposits in a subject can comprise ADx-001. In one aspect, a liposomal composition for use in a method of imaging amyloid deposits in a subject can comprise Gd(III)-DOTA-DSPE and ET3-73. In one aspect, a liposomal composition for use in a method of imaging amyloid deposits in a subject can comprise HSPC, Chol, DSPE-mPEG2000, Gd(III)-DOTA-DSPE, and ET3-73.

[0026] In one aspect, 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 sufficient time for the liposome composition to bind to one or more amyloid deposits; and detecting the liposome composition bound to the one or more amyloid deposits. In one aspect, the use comprises detecting using MRI.

[0027] In one aspect, the use further comprises: identifying whether the patient is likely to have AD based on the detection of a liposome composition associated with one or more amyloid deposits; performing a tau neurofibrillary tangle analysis on the patient; and diagnosing the AD patient after determining the presence of tau neurofibrillary tangles in combination with the detection of the liposome composition associated with one or more amyloid deposits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Exemplary cross-sectional views of liposomes containing targeted contrast agents for use in MRI of amyloid deposits are provided.

[0029] Figure 2 Shown are representative schematics of the surface-conjugated macrocyclic Gd-based imaging agents described herein ("SC-Gd") compared to prior art dual Gd liposomes ("Dual-Gd").

[0030] Figure 3 Shown is a comparison of the T1 relaxation rates of Gd(III) formed at a field strength of 1 T between "free" Gd(III)-DOTA (i.e., not coupled to liposomes), prior art Gd(III)-DTPA-BSA liposomes, and the Gd(III)-DOTA-DSPE liposomes described herein (i.e., ADx-001 liposomes).

[0031] Figure 4 An exemplary synthetic scheme for the synthesis of the ammonium salt of ET3-73 is provided.

[0032] Figure 5 An exemplary synthetic scheme for the synthesis of Gd(III)-DOTA-DSPE sodium salt is provided.

[0033] Figure 6 An exemplary process flow diagram for preparing ADx-001 is shown.

[0034] Figure 7 Figure 2. Demonstration of cortical region of interest (ROI) identification in axial MR images of the brain. The outline of the cortical ROI is shown on FSE-IR brain images before (top) and after (bottom) contrast agent administration.

[0035] Figure 8 Shown are T1-weighted spin echo ("T1w-SE") axial images of the brain before and after administration of ADx-001: a) wild-type ("WT") mice (amyloid negative) at a dose of 0.20 mmol Gd / kg; and TgAPPswe / PSEN1dE9 ("Tg") mice (amyloid positive) at the following doses: b) 0.20 mmol Gd / kg; c) 0.15 mmol Gd / kg; and d) 0.10 mmol Gd / kg. Arrows point to areas of increased signal in the cortex and hippocampus.

[0036] Figure 9Shown are fast spin echo inversion recovery ("FSE-IR") axial images of the brain before and after administration of ADx-001: a) WT mice at a dose of 0.20 mmol Gd / kg; and, Tg mice at the following doses: b) 0.20 mmol Gd / kg; c) 0.15 mmol Gd / kg; and d) 0.10 mmol Gd / kg. Arrows point to areas of increased signal in the cortex and hippocampus.

[0037] Figure 10 Tg mice demonstrated enhanced MR signal in the cerebral cortex relative to WT mice at all dose levels of ADx-001. Box plots show statistically significant differences in signal change (expressed as a percentage) between delayed T1w-SE images before and after contrast administration for the following ADx-001 dose levels: a) 0.20 mmol Gd / kg; b) 0.15 mmol Gd / kg; and c) 0.10 mmol Gd / kg, as well as statistically significant differences in signal change between delayed FSE-IR images before and after contrast administration for the following dose levels: d) 0.20 mmol Gd / kg; e) 0.15 mmol Gd / kg; and f) 0.10 mmol Gd / kg.

[0038] Figure 11 Figure 2 shows the mean and range of signal intensities for (a) T1w-SE and (b) FSE-IR sequences of WT (n=18) and Tg (n=18) mice scanned before contrast administration. ROIs were drawn in the cortex for each animal. No significant differences (NS) were found between the signal intensities of WT and Tg mice for either T1w-SE or FSE-IR sequences.

[0039] Figure 12 Figure 2 shows the signal changes in the cerebral cortex of WT (n=3) and Tg (n=3) mice as a function of time in a) T1w-SE and b) FSE-IR sequences. The maximum signal enhancement (arrow) was observed on day 4 after contrast agent administration. Tg animals showed signal enhancement relative to WT animals in both sequences. By day 21, the signal had returned to near baseline levels.

[0040] Figure 13 Postmortem confirmation of ADx-001 binding to β-amyloid plaques is provided by representative fluorescence microscopy images of ADx-001 binding to amyloid plaques in a) mouse cortex and b) hippocampus regions in Tg animals, compared with representative images of c) cortex and d) hippocampus regions in WT animals.

[0041] Figure 14Shown are plasma Gd concentrations measured at various time points following administration of ADx-001 in dogs (p) and monkeys (·) using inductively coupled plasma mass spectrometry ("ICP-MS").

[0042] Figure 15 Shown are the biodistribution of ADx-001 in rats at a) spleen, b) liver, c) kidney, d) bone, e) skin, and f) brain as determined by ICP-MS analysis on days 4 and 28 after intravenous administration of ADx-001 at dose levels of 0.15 mmol Gd / kg (p) and 0.3 mmol Gd / kg (·). DETAILED DESCRIPTION

[0043] Based on the highly stable macrocyclic Gd-DOTA imaging structure, a new amyloid-targeted liposome-Gd contrast agent ADx-001 was developed. Figure 1 The cross-sectional form of . Figure 2 Representative schematics are shown of a macrocyclic gadolinium-based imaging agent ("SC-Gd") as described herein coupled to Gd chelates coupled to both the inner and outer surfaces of the liposome bilayer, in contrast to the dual Gd liposomes of WO2016057812A1 containing both a core-encapsulated Gd chelate and a surface-coupled Gd chelate, e.g., as described 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.

[0044] Contrast agents with higher T1 relaxation rates produce greater enhancement. Figure 3 Shown is a comparison of the T1 relaxivity of the Gd(III) form at a field strength of 1 T between "free" Gd(III)-DOTA (i.e., not coupled to liposomes), prior art Gd(III)-DTPA-BSA liposomes, and the Gd(III)-DOTA-DSPE liposomes described herein (i.e., ADx-001 liposomes). Compared to prior art Gd(III)-DTPA-BSA liposomes, ADx-001 exhibited approximately 3-fold higher T1 relaxivity.

[0045] More specifically, liposomal Gd-DOTA, in which Gd-DOTA is conjugated to a phospholipid (~31 mM-1s-1 on a Gd-based basis and ~2,295,000 mM-1s-1 on a nanoparticle-based basis at a field strength of 1 T), exhibits a T1 relaxation rate approximately three times higher than liposomal Gd-DTPA, in which Gd-DTPA is conjugated to bis(stearamide) (~9 mM-1s-1 on a Gd-based basis and ~668,000 mM-1s-1 on a nanoparticle-based basis at a field strength of 1 T). The Gd conjugates are important in at least three ways. First, the conjugation of the Gd-chelate to the macromolecule slows down the rotational correlation of the Gd atoms, thereby increasing the rotational correlation time. Higher rotational correlation times result in higher T1 relaxation rates. Secondly, the coupling of the Gd-chelate to a phospholipid (here, DSPE) (Gd-DOTA-DSPE) further increases the rotational correlation time compared to the coupling of the Gd-chelate to bis(stearylamide) (Gd-DTPA-BSA). Consequently, Gd-DOTA-DSPE liposomes perform better (higher T1 relaxivity) than Gd-DTPA-BSA liposomes. Finally, by binding to a genuine phospholipid, the insertion into the bilayer is stabilized even more. In contrast, the absence of the phosphatidyl group in Gd-DTPA-BSA reduces the amphiphilicity of the molecule, thereby reducing the stability of the insertion.

[0046] Thus, in one aspect, ADx-001 comprises: a first phospholipid; a sterically hindered bulky excipient capable of stabilizing the liposome composition; a second phospholipid derivatized with the first polymer; a macrocyclic gadolinium-based imaging agent; and a third phospholipid derivatized with the second polymer, the second polymer being coupled to a targeting ligand. The macrocyclic gadolinium-based imaging agent may be coupled to a fourth phospholipid.

[0047] phospholipids

[0048] In some respects, suitable phospholipid includes the length of wherein two hydrocarbon chains between about 14 and about 24 carbon atoms and those phospholipids with varying degrees of unsaturation. In some respects, suitable phospholipid includes HSPC, 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (" DPPC "), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (" DSPC "), 1,2-distearoyl-sn-glyceryl-3 phosphoethanolamine (" DSPE ") and two or more mixtures thereof. Suitable phospholipid can be naturally occurring or synthetic.

[0049] In some aspects, suitable phospholipids may include any of the phospholipids listed in WO2005107820A1, the contents of paragraphs

[0031] to

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

[0050] Polymer-derivatized phospholipids

[0051] In some aspects, the liposomes of the liposome composition can include a surface containing or coated with flexible water-soluble (hydrophilic) polymer chains. These polymer chains can prevent interaction between the liposomes and plasma components, which play a role in the uptake of the liposomes by blood cells and their removal from the blood. The liposomes can avoid uptake by organs of the mononuclear phagocyte system, primarily the liver and spleen (reticuloendothelial system).

[0052] In one aspect, the polymer in the derivatized phospholipid can be polyethylene glycol ("PEG"). PEG can have any of a variety of molecular weights. In one example, the PEG chain can have a molecular weight of about 1,000 to 10,000 Daltons. Once the liposomes are formed, the PEG chains can provide a surface coating of hydrophilic chains that is sufficient to extend the blood circulation time of the liposomes in the absence of such a coating.

[0053] In some aspects, the second phospholipid derivatized with the first polymer comprises DSPE-mPEG2000. In some aspects, the second polymer is coupled to a targeting ligand, and the third phospholipid derivatized with the second polymer comprises:

[0054]

[0055] or a salt thereof (e.g., an ammonium phosphate salt), wherein the variable n can be any integer from about 10 to about 100, such as 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 aspects, the third phospholipid derivatized with the second polymer comprises DSPE-PEG3500.

[0056] In some aspects, suitable polymers may 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 aspects, the phospholipid derived from the polymer may be any of the combinations disclosed in WO2016057812A1.

[0057] Steric bulky excipients

[0058] In some aspects, the liposomes can include stabilizing excipients. For example, the liposome composition can be formulated to include cholesterol. In other aspects, the liposome composition can include fatty alcohols, fatty acids, cholesterol esters, other pharmaceutically acceptable excipients, and mixtures thereof.

[0059] Macrocyclic gadolinium-based imaging agents

[0060] The liposome composition comprises a macrocyclic gadolinium In some aspects, the macrocyclic gadolinium-based imaging agent comprises Gd(III)-DOTA coupled to a phospholipid, such as:

[0061]

[0062] or a salt thereof (e.g., a sodium salt). In some aspects, the variable x can be one of: 12, 13, 14, 15, 16, 17, or 18. In one aspect, the variable x is 16 and the conjugate is Gd(III)-DOTA-DSPE.

[0063] In other aspects, the macrocyclic gadolinium-based imaging agent comprises:

[0064]

[0065] Targeting ligands

[0066] The liposome composition comprises at least one phospholipid derivatized with a polymer to which a targeting ligand is coupled. The hydrophilic polymer is typically end-functionalized to facilitate coupling with the targeting ligand. The functionalized end group can be, for example, a maleimido group, a bromoacetamide group, a disulfide group, an active ester group, or an aldehyde group. Hydrazide groups are reactive toward aldehydes, which can be generated on many biologically relevant compounds. Hydrazides can also be acylated with active ester or carbodiimide-activated carboxyl groups. Acyl azide groups, which are reactive as acylation species, are readily obtained from hydrazides and allow for the attachment of amino-containing ligands.

[0067] In some aspects, the targeting ligands are accessible from the surface of the liposomes and can specifically bind or attach to, for example, one or more molecules or antigens. These targeting ligands can direct or target the liposomes to specific cells or tissues, such as beta-amyloid plaques, and can bind to molecules or antigens on or associated with cells or tissues.

[0068] The targeting ligand can be represented as:

[0069]

[0070] in,

[0071] Pyrimidine "P" may be substituted by 0, 1 or more of -OH, O-alkyl and -NH2;

[0072] R 2 is a linking group comprising a C1-C6 alkyl group or a C1-C6 alkoxyalkyl group; and

[0073] R 3 is hydrogen, C1-C6 alkyl or C1-C6 alkoxyalkyl, and R other than hydrogen 3 Substituted with 0, 1 or more -OH groups.

[0074] In one aspect, the targeting ligand is compound iii. In one aspect, the phospholipid-polymer-targeting ligand conjugate is ET3-73.

[0075] In another aspect, the targeting ligand is any one of compounds i to xiii disclosed in WO2016057812A1. In yet another aspect, the targeting ligand is compounds ii, iii, xi and xiii disclosed in WO2016057812A1.

[0076] liposomes

[0077] "Liposome" generally refers to a spherical or roughly spherical particle containing an internal cavity. The wall of the liposome can include a lipid bilayer. These lipids can be phospholipids. Many lipids and / or phospholipids can be used to prepare liposomes. One example is an amphiphilic lipid having a hydrophobic and polar head group portion, which can spontaneously form a bilayer vesicle in water, such as a phospholipid, or can be stably incorporated into a lipid bilayer, with its hydrophobic portion in contact with the inner hydrophobic region of the bilayer membrane and the polar head group portion facing the outer polar surface of the membrane. Liposomes can be prepared by any known method, including as described in the Examples herein and in WO2016057812A1 and WO2012139080A1, the entire contents of which are incorporated herein by reference. Figure 1 Exemplary cross-sectional views of liposomes containing targeted contrast agents for use in MRI of amyloid deposits are provided.

[0078] In one aspect, ADx-001 comprises: HSPC; Chol; DSPE-mPEG2000; ET3-73; and Gd(III)-DOTA-DSPE. In some aspects, the first phospholipid comprises: DPPC, DSPC, or a mixture of DPPC and DSPC. In one aspect, the lipid composition and molar ratio (%) of the components in ADx-001 is HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73 = approximately 31.5:approximately 40:approximately 2.5:approximately 25:approximately 1. In some aspects, the molar ratio of any one of HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73 can be adjusted by up to 10%, thus being 31.5±10%:40±10%:2.5±10%:25±10%:1±10%. In one aspect, the lipid composition and the molar ratio (%) 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.

[0079] On the one hand, the HSPC content in ADx-001 is between about 24mg / mL and about 32mg / mL (total lipids). On the one hand, the Chol content in ADx-001 is between about 14mg / mL and about 19mg / mL. On the one hand, the DSPE-mPEG2000 content in ADx-001 is between about 5mg / mL and about 7mg / mL. On the one hand, the Gd (III)-DOTA-DSPE content in ADx-001 is between 30mg / mL and 45mg / mL. On the one hand, the ET3-73 content in ADx-001 is between about 2mg / mL and about 3mg / mL. On the one hand, the free gadolinium content in ADx-001 is ≤100g / mL, including <2.5g / mL.

[0080] In one aspect, the pH of the liposome composition is between 6.4 and 8.4. In another aspect, the osmolarity of the liposomes is between 200 and 400 mOsmol / kg. In another aspect, the liposome vesicle size (Z-average) as measured by dynamic light scattering is less than about 200 nm (D 50 ), including less than 150nm (D 50 ), including about 140nm (D 50 ), and including about 120nm (D 50 ).

[0081] For clarity, the term "about" used with a number is intended to include ±10% of that number. This is true whether "about" modifies 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" means 9 to 22 and 11 to 18. If the term "about" is omitted, the exact number is intended. In other words, "10" means 10.

[0082] Example

[0083] The dose-response, pharmacokinetics, and biodistribution of ADx-001 were studied in animal models. A dose-ranging efficacy study was conducted in the 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 before contrast administration and 4 days after ADx-001 administration (delayed post-contrast administration). Qualitative and quantitative analysis of pre-contrast and delayed post-contrast images was performed to determine sensitivity, specificity, and accuracy for amyloid plaques identified by autopsy histology. The pharmacokinetics of ADx-001 were studied in monkeys and dogs. Blood samples were collected at multiple time points after ADx-001 administration and analyzed for Gd levels 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 determined by ICP-MS on days 4 and 28 after administration of ADx-001.

[0084] Example 1: Preparation of ET3-73

[0085] Reference Figure 4 The starting material (E)-2-((2-aminoethyl)(4-(2-(pyrimidin-4-yl)vinyl)phenyl)amino)ethan-1-ol (SM1) reacts with DIPEA to produce ET3-73 intermediate 1. ET3-73 intermediate 1 reacts with TBDMS-OTf (TBDMS-Triflate) to produce ET3-73 intermediate 2. ET3-73 intermediate 2 is coupled with HO-PEG3500-NH2 (SM2) to form ET3-73 intermediate 3. ET3-73 intermediate 3 reacts with bis-pentafluorophenyl-carbonate, and the activated ET3-73 intermediate 3 is combined with DSPE silylated with bis-trimethylsilyl-acetamide to produce ET3-73 intermediate 4. Finally, the ammonium salt of ET3-73 is formed by deprotection with TBAF and then reaction with sodium ammonium acetate.

[0086] Example 2: Preparation of Gd(III)-DOTA-DSPE

[0087] Reference Figure 5 The synthesis scheme begins with further esterification of DOTA-tri(tert-butyl ester) (SM1) with pentafluorophenol (HOpFP). The tert-butyl ester protecting group is removed using TFA and trimethylsilyl chloride (TMSCl). The pentafluorophenyl ester is coupled with DSPE in the presence of BSA and NMM to form DSPE-DOTA. The final DSPE-DOTA-Gd sodium salt is formed by the addition of gadolinium in its acetate form [Gd(OAC)3], followed by the scavenger SiliaMetS TAAcONa (Triaminetetraacetate, sodium salt functionalized silica gel), thereby forming the final DSPE-DOTA-Gd sodium salt.

[0088] Example 3: Preparation of ADx-001

[0089] Reference Figure 6 , ADx-001 was prepared as follows:

[0090] Step 1 (Buffer Solution): Dissolve sodium chloride and histidine in water and filter through a 0.2 μm filter. The nominal pH of the solution is 7.5.

[0091] Step 2 (lipid solution): DSPE-DOTA-Gd, HSPC (Lipoid Inc., Newark, NJ, USA), DSPE-mPEG 2000 A mixture of ELISA (Corden Pharma, Liestahl, Switzerland), Chol (Lipoid Inc., Newark, NJ, USA), and ET3-73 (31.5:40:2.5:25:1 molar ratio) was dissolved in tert-butyl alcohol.

[0092] Step 3 (Liposome Formation): Add the lipid solution (Step 2) to a portion of the buffer solution (Step 1). If necessary, adjust the pH to a nominal pH of 6.5-7.0 with sodium hydroxide solution. This step produces liposomes of indeterminate size and lamellarity. The drug (DSPE-DOTA-Gd) and other lipid components (HSPC, DSPE-mPEG 2000 , cholesterol and ET3-73) are present in the lipid bilayer of liposomes.

[0093] Step 4 (Extrusion): The material was extruded through a track-etched polycarbonate filter at elevated pressure to reduce the size of the liposome vesicles. The process was continued until the desired vesicle size (~140 nm nominal size) was reached, as measured by dynamic light scattering during the process.

[0094] Step 5 (Ultrafiltration): The treated material is ultrafiltered using a tangential flow filtration module with a molecular weight cutoff (MWCO) rating of 500,000. During the ultrafiltration process, the liposomal nanoparticles are recycled and retained by the ultrafiltration media, while a portion of the carrier solution (buffer plus solvent tert-butyl alcohol) passes through the ultrafiltration media into the permeate waste stream. The ultrafiltration step consists of three parts. First, the treated material is concentrated by discarding the permeate from the ultrafiltration media. Second, the ultrafiltration module is run in diafiltration mode, where a constant concentration is maintained by replenishing the permeate waste stream with buffer solution (Step 1). Third, the treated material is concentrated to achieve a concentration level slightly higher than the nominal 98 mg / mL total lipid composition of the final drug product.

[0095] Step 6 (Pre-filtration): Pass the treated material through track-etched polycarbonate filter media until the desired filterability is achieved.

[0096] Step 7 (clarification filtration): The treated material is passed through a 0.2 μm filter (Sartorius 2XLI, with polyethersulfone membrane).

[0097] Step 8 (Dilution): The treated material was diluted with the buffer solution (Step 1) to a target label strength of 38.7 mg / mL DSPE-DOTA-Gd. The nominal concentration was 98 mg / L total lipids.

[0098] Step 9 (Sterile Filtration): Under sterile conditions, the treated material was filtered through a 0.2 μm sterilizing grade filter (Sartorius 2XLI, with polyethersulfone membrane).

[0099] Step 10 (Aseptic Filling): Under aseptic conditions, the processed material is filled into vials, stoppered, and sealed. Fill weight checks are performed during the filling operation, and 100% of the filled vials are visually inspected for particulates and container closure defects. Batch data for several example batches are listed in Table 1:

[0100]

[0101]

[0102] Table 1

[0103] Example 4: MRI study

[0104] Studies were conducted in the APPswe / PSEN1dE9 (C57BL / 6J background, 11-18 months old) double Tg mouse model of early-onset AD (JAXMMRRC Stock #005864). Tg mice develop amyloid plaques in the brain at approximately 6-7 months of age. ADx-001, prepared in Example 3, was tested at three dose levels (mmol Gd / kg): 0.10, 0.15, and 0.20. At each dose level, ADx-001 was tested in Tg (n=6) and WT (mice lacking both mutations) mice (n=6). ADx-001 was administered intravenously via a slow bolus injection into the tail vein.

[0105] Imaging was performed on a 1T permanent MRI scanner (M7 system, Aspect Imaging, Shoham, Israel). Animals were sedated with 2.5% or 3% isoflurane and placed on a custom-made sled with an integrated tip cone for continuous anesthetic delivery by inhalation (1–2% isoflurane). Respiratory rate was monitored using a pneumatically controlled pressure pad placed beneath the abdominal area. Two MRI sequences were tested: a T1w-SE sequence and a 2D FSE-IR sequence, which approximates a fluid-attenuated inversion recovery sequence. SE parameters were: TR = 600 ms, TE = 11.5 ms, slice thickness = 1.2 mm, matrix = 192 × 192, field of view = 30 mm, number of slices = 16, and NEX = 4. FSE-IR parameters were: TR = 6500 ms, TE = 80 ms, TI = 2000 ms, slice thickness = 2.4 mm, matrix = 192 × 192, field of view = 30 mm, number of slices = 6, and NEX = 6. Coil calibration, RF calibration, and shimming were performed on each subject at the start of the study. All animals underwent a pre-contrast scan followed by intravenous administration of ADx-001. Post-contrast delayed scans were acquired 4 days after contrast administration. Pre-contrast and post-contrast scans were acquired using T1w-SE and FSE-IR sequences.

[0106] Following post-contrast scans, animals were euthanized and perfused with 0.9% saline followed by 4% formalin. Brain tissue was excised, fixed in 4% formalin for 24 hours, and transferred to 30% sucrose for cryoprotection. Brain tissue was embedded in OCT and stored at -80°C until ready for sectioning. 15 μm thick brain tissue sections were cut for postmortem phenotypic confirmation of amyloid deposition. Sections were incubated in 5% bovine serum albumin ("BSA") for 1 hour and then incubated with fluorescently labeled anti-β-amyloid antibody (AF647-4G8, Biolegend, San Diego, CA) in 3% BSA at 4°C overnight. Sections were further stained with a nuclear marker (DAPI), washed, mounted, mounted using Vectashield mounting medium (Vector Laboratories, Burlingame CA), and imaged on a confocal microscope with an appropriate filter set. The presence of amyloid-bound ADx-001 nanoparticles was analyzed by imaging in the FITC channel.

[0107] Qualitative and quantitative analysis of MRI images was performed in OsiriX (version 5.8.5, 64-bit) and MATLAB (version 2015a). Brain tissue extraction was performed in OsiriX by a combination of thresholding and manual segmentation. Signal changes between delayed images before and after contrast administration were assessed by quantifying the signal intensity of cortical regions near the center of the image stack (see Figure 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. The signal change between pre-contrast and post-contrast images was quantified by integrating the signal in the ROI containing the cortical tissue in the central slice of the MRI volume. Signal change (%) was calculated according to Equation 1:

[0108]

[0109] The observation of signal enhancement in amyloid positive animals (determined by immunofluorescence) MRI is considered a true positive result. Conversely, the absence of signal enhancement between delayed images before and after the administration of contrast agent in amyloid negative animals is considered a true negative. Sensitivity is determined by the ratio of the true positives identified by MRI to the total number of true positives identified by the gold standard of autopsy amyloid plaque staining performed by immunofluorescence analysis. The ratio of the true negatives identified by MRI to the total number of true negatives is determined as specificity. The total number of animals correctly identified by MRI compared to the true positives determined by autopsy immunofluorescence is used as overall accuracy.

[0110] At any dose level of ADx-001, WT mice (amyloid-negative) did not show brain signal enhancement in delayed images acquired after contrast administration using either T1w-SE or FSE-IR. However, at doses of 0.20 and 0.15 mmol Gd / kg of ADx-001, Tg mice (amyloid-positive) showed moderate to high MR signal enhancement in the cortex and hippocampus on delayed images acquired after contrast administration using T1w-SE. Figure 8 Shown are: a) WT animals administered 0.2 mmol Gd / kg of ADx-001 showed no signal enhancement four days after injection; b) Tg animals showed high enhancement in the cortex (upper arrow) and hippocampus (lower arrow) four days after administration of 0.2 mmol Gd / kg of ADx-001; c) Tg animals showed moderate enhancement in the cortex (upper arrow) and hippocampus (lower arrow) four days after administration of 0.15 mmol Gd / kg of ADx-001; and d) Tg animals showed low enhancement in the cortex (arrow) four days after administration of 0.10 mmol Gd / kg of ADx-001.

[0111] Similarly, Tg mice showed moderate to high signal enhancement in FSE-IR images delayed after contrast agent administration at ADx-001 doses of 0.20 and 0.15 mmol Gd / kg, and relatively mild signal enhancement at 0.10 mmol Gd / kg. Figure 9 As shown, FSE-IR axial images demonstrate MR signal enhancement in delayed scans after administration of ADx-001 contrast agent in Tg APPswe / PSEN1dE9 mice but not in age-matched WT control mice. Specifically: (a) WT animals administered 0.20 mmol Gd / kg ADx-001 showed no signal enhancement in delayed images after contrast administration; (b) Tg animals administered 0.20 mmol Gd / kg ADx-001 showed high signal enhancement in the cortex (upper arrow) and hippocampus (lower arrow) in delayed images after contrast administration; (c) Tg animals administered 0.15 mmol Gd / kg ADx-001 showed moderate signal enhancement in the cortex (upper arrow) and low enhancement in the hippocampus (lower arrow) in delayed images after contrast administration; and (d) Tg animals administered 0.10 mmol Gd / kg ADx-001 showed low signal enhancement in the cortex (arrow) in delayed images after contrast administration. All delayed images after contrast administration were acquired 4 days after ADx-001 administration.

[0112] Quantitative analysis of the cortical ROI confirmed the qualitative observations of MR signal enhancement in delayed images after contrast administration and revealed statistically significant differences in signal changes after contrast administration between Tg and WT animals at all dose levels. Figure 10 As shown, at all ADx-001 dose levels, Tg mice exhibited MR signal enhancement in the cerebral cortical region relative to WT mice. Specifically, the box plots show the signal change (expressed as a percentage) between delayed T1w-SE images before and after contrast administration for the following ADx-001 dose levels: a) 0.20 mmol Gd / kg; b) 0.15 mmol Gd / kg; and c) 0.10 mmol Gd / kg. Similar signal changes were shown between delayed FSE-IR images before and after contrast administration for the following dose levels: d) 0.20 mmol Gd / kg; e) 0.15 mmol Gd / kg; and f) 0.10 mmol Gd / kg dose levels. The Wilcoxon rank sum statistical test was used to compare the differences between the groups. Values ​​of p ≤ 0.05 were considered statistically significant. Figure 10 In the mean, p<0.05(*) and p<0.005(**).

[0113] After confirming that the pre-contrast signal was indistinguishable between WT and Tg mice, the signal variance threshold was estimated based on the pre-contrast (baseline) scans of all tested mice (see Figure 11 The 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.

[0114] 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, ADx-001 demonstrated high sensitivity (>80%) at dose levels of 0.20 and 0.10 mmol Gd / kg in T1w-SE imaging, and high sensitivity (>80%) at dose levels of 0.20 and 0.15 mmol Gd / kg in FSE-IR imaging. In both T1w-SE and FSE-IR, ADx-001 demonstrated the highest accuracy (>90%) at the highest dose level (0.20 mmol Gd / kg).

[0115]

[0116] Table 2

[0117] Longitudinal imaging studies in WT and Tg mice showed that signal enhancement was optimal 4 days after contrast administration and that the signal returned to near baseline levels 21 days after contrast administration (see Figure 12 ).

[0118] Immunofluorescence microscopy analysis confirmed the preferential concentration and colocalization of ADx-001 with amyloid plaque deposits in the cortex and hippocampus of Tg mice. In contrast, WT animals did not exhibit amyloid plaque deposits or the presence of bound ADx-001 nanoparticles. Figure 13 Representative fluorescence microscopy images of ADx-001 binding to amyloid plaques in the mouse cortex a) and hippocampus b) of Tg animals are shown. Representative images also show WT cortex c) and hippocampus d) areas. WT mice did not show evidence of amyloid plaque deposition (4G8 antibody staining) or the presence of bound ADx-001 (observation of amyloid ligand fluorescence signal). Images were collected at 60x magnification.

[0119] Example 5: Pharmacokinetic Study

[0120] The pharmacokinetics ("PK") of ADx-001 were evaluated in cynomolgus monkeys and beagle dogs. Non-naive male cynomolgus monkeys (n=3, age 2-5 years, body weight 2.3-3.1 kg) were administered ADx-001 intravenously over ~60 minutes using a calibrated infusion pump at a dose of 0.30 mmol Gd / kg. Blood samples were collected from all animals before administration, immediately after the end of the infusion, and at 4, 8, 24, 48, 96, 168, 336, and 672 hours after the start of injection ("SOI"). For PK analysis in beagle dogs, animals (n=5, age 5-7 months, body weight 6.2-7.9 kg) were infused intravenously with ADx-001 at 0.30 mmol Gd / kg over ~60 minutes. Blood samples were processed into plasma and stored frozen until ready for analysis.

[0121] Gd concentrations in plasma samples were determined using ICP-MS. Plasma samples (100 μL) were digested in 90% concentrated HNO (750 μL) at 90°C for 15 minutes. The digested samples were diluted in deionized ("DI") water and centrifuged at 3000 rpm for 15 minutes. The supernatant was further diluted for ICP-MS analysis such that the Gd concentration fell within the ICP-MS calibration standard range (1 to 500 ppb).

[0122] ADx-001 was well tolerated in dogs and monkeys without adverse reactions. Figure 14ADx-001 showed a long blood half-life. Plasma Gd concentrations were measured by ICP-MS at different time points after administration of ADx-001 to dogs (p) and monkeys (·). Assuming first-order kinetics, the elimination rate was 0.017 h -1 , resulting in a blood half-life of approximately 41 hours in monkeys. In monkeys, plasma Gd levels decreased by ∼80% by 96 hours after SOI and by more than 99% by 336 hours after SOI. Although literature on the blood half-life of comparable liposomal MRI contrast agents in monkeys is lacking, studies in mice have shown blood half-lives in the 14–24 hour range. In dogs, the elimination rate was 0.0297 h. -1 , resulting in a blood half-life of approximately 23 hours. In dogs, plasma Gd levels decreased by ∼85% by 96 hours after SOI and by ∼99% by 168 hours after SOI.

[0123] Example 6: Tissue Biodistribution

[0124] The biodistribution of ADx-001 was studied in a rat model. Wistar Han rats (10 weeks old, weighing 257-296 g; n=13 per treatment group) were administered ADx-001 as a single intravenous bolus at 0.15 mmol Gd / kg (n=13) or 0.30 mmol Gd / kg. Animals were euthanized on day 4 (n=7 / dose level) and day 28 (n=6 / dose level) after administration of ADx-001. Tissues were collected for determination of 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.

[0125] Gd concentrations in tissue samples were quantified using ICP-MS. Wet tissue (~100 mg) was digested in 90% concentrated HNO3 (~750 μL) at 90°C for 10-15 minutes. The digested samples were diluted in DI water, vortexed vigorously, and centrifuged at 3500 rpm for 15 minutes. The supernatant was separated and further diluted as needed to ensure that the Gd concentration was within the calibration standard range (1-500 ppb). Quality control samples (50 and 100 ppb) were included at the beginning, middle, and end of the analytical run.

[0126] 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 dose-related increases in all organs. Figure 15Shown are ICP-MS analyses illustrating Gd levels in the following organs: a) spleen; b) liver; c) kidney; d) bone; e) skin; and f) brain, on days 4 and 28 following intravenous administration of ADx-001 at dose levels of 0.15 mmol Gd / kg (p) and 0.3 mmol Gd / kg (·). Tissue Gd content is expressed as mg Gd per gram of wet tissue. The highest Gd tissue levels were observed in the liver and spleen, consistent with organs known to clear PEGylated liposome formulations. The lowest Gd levels were observed in the brain. In all organs, Gd tissue levels were reduced by >90% on day 28 compared to day 4 Gd tissue levels.

[0127] Animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee and were in compliance with NC3RS-ARRIVE guidelines.

[0128] ADx-001-enhanced MRI showed significantly greater brain signal enhancement in Tg mice (amyloid-positive) relative to WT (amyloid-negative) mice at all dose levels (p<0.05). ADx-001-enhanced T1w-SE imaging demonstrated high sensitivity (>80%) at 0.10 and 0.20 mmol Gd / kg, while ADx-001-enhanced FSE-IR imaging demonstrated 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 demonstrated a long blood half-life (23 hours in dogs and 41 hours in monkeys). Biodistribution studies demonstrated that ADx-001 was primarily cleared systemically through the mononuclear phagocyte system (also known as the reticuloendothelial system). Tissue Gd levels in all organs were reduced by more than 90% on day 28 compared to day 4, indicating ongoing clearance.

[0129] In summary, the amyloid-targeting liposomal macrocyclic gadolinium contrast agent ADx-001 demonstrated high sensitivity and excellent specificity for in vivo imaging of β-amyloid plaques in the mouse brain. No signs of toxicity were detected, and the pharmacokinetics followed the expected pattern for PEGylated nanoparticles.

[0130] Unless otherwise indicated, "a", "an", "the", "one or more" and "at least one" are used interchangeably. The singular forms "a", "an" and "the" include their plural forms. Numerical ranges recited by endpoints include 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 equivalent and open-ended. The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, but only when the additional ingredients and / or steps do not materially change the basic and novel characteristics of the claimed composition or method. The phrase "selected from" refers to a mixture of the listed groups.

Claims

1. A liposome composition comprising: where the variable x is 16; and 2. The liposome composition according to claim 1, further comprising: HSPC; Chol; and DSPE-mPEG2000.

3. The liposome composition according to claim 2, wherein the molar ratio of the components in the liposome composition is HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:ET3-73=31.5:40:2.5:25:1.

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