Targeted contrast agent for MRI of α-synnuclein deposits

Abstract

A liposomal composition ("ADx-003") is provided, comprising 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 contrast agent, and a third phospholipid derivatized with a second polymer, wherein the second polymer is conjugated to a targeting ligand, the targeting ligand having a structure represented by Formula I: [Formula 1] JPEG2023514991000053.jpg3889 wherein X is -CH-, -CH-CH-, -CHO- or -O-CO-, and Y is -CH-CH=CH- or [chemical 2] JPEG2023514991000054.jpg2131 and a third phospholipid represented by: wherein A and B are independently selected from C and N; R1, R2, R3, and R4 are independently selected from -H, halogen, -OH, and -CH3; and R5, R6, and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, a C1-C6 alkyl, or a substituted or unsubstituted G4-C6 aryl group, with the proviso that when A and / or B are N, then the adjacent R5 and / or R7 are -H) or a pharmaceutically acceptable salt thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 975,265, filed on 12 February 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Neurodegenerative disorders such as Parkinson's disease ("PD") and Alzheimer's disease ("AD") are characterized by pathological deposition of misfolded protein aggregates in different locations in the brain. These misfolded protein aggregates include α-synuclein ("α-syn") aggregates in the form of Lewy bodies ("LB") and Lewy neurites ("LN") in PD, and amyloid-beta ("Aβ") plaques and hyperphosphorylated τ-tangles in AD. PD is the second most common neurodegenerative disease after AD and is clinically characterized by motor symptoms including bradykinesia, rigidity, tremor, and postural instability. The motor symptoms are caused by degeneration of dopaminergic neurons in the substantia nigra, accompanied by cytoplasmic deposition of Lewy lesions. The regional distribution of α-syn in postmortem studies of PD suggests that Lewy lesions originate in the olfactory bulb and lower brainstem and gradually spread to other areas of the central nervous system. High levels of LB and LN are observed in the medulla oblongata / pontine tegmentum and preolfactory structures (Braak stages 1 and 2) prior to the onset of PD-related motor symptoms in patients. PD-related motor symptoms only begin to appear in the intermediate stages (Braak stages 3 and 4) when the disease has spread to the substantia nigra and other nuclei within the basal midbrain and forebrain. Apart from PD, the pathogenesis of several other neurodegenerative disorders (collectively referred to as "synucleinopathy"), including PD dementia ("PDD"), dementia with LB ("DLB"), and multiple system atrophy ("MSA"), is also characterized by misfolded α-syn aggregates.

[0003] The correlation between Lewy body lesions and autopsy studies involving nigrostriatal degeneration, cognitive impairment, and motor dysfunction suggests that techniques enabling non-invasive detection and quantification of α-syn aggregates are valuable tools for early diagnosis and clinical evaluation of LB disorders in surviving individuals. Early detection can provide better opportunities for recruiting a rich patient cohort for clinical trials, evaluation of disease reversal therapies, and validation of the therapeutic efficacy of new drug candidates. However, LB disorders often present with multiple protein disorders. For example, a study focusing on PD patients who developed dementia revealed that, apart from α-syn accumulation in the neocortex, approximately 60% of patients also had widespread Aβ accumulation. In addition, approximately 3% of cases showed τ accumulation along with α-syn and Aβ.

[0004] The recent approval of several Aβ positron emission tomography ("PET") contrast agents has significantly improved the cohort size for AD drug clinical trials. This has also stimulated the search for similar drugs for other protein disorders (τ and synucleinopathy). While various molecular scaffolds with moderate to high binding affinity to α-syn fibrils (Figure 1) have been reported over the past decade, none have achieved successful clinical interpretation, at least partially due to the low selectivity of α-syn to Aβ fibrils. Indeed, the development of contrast agents for in vivo detection of α-syn pathologies faces several challenges, one of which is the lack of diverse molecular scaffolds with high affinity and selectivity for α-syn fibrils for in vitro screening assays.

[0005] Furthermore, if such scaffolds are suitable for use in magnetic resonance imaging (MRI), the results may vary due to ease of access and lower cost (compared to PET). High T1 relaxation ability, an amyloid-targeted liposome-gadolinium (Gd) nanoparticle contrast agent (a very stable macrocyclic gadolinium-based contrast agent containing (3+)2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic acid gadolinium (referred to as "gadoterate" or "Gd(III)-DOTA") conjugated to phospholipids and the inner and outer surfaces of the liposome bilayer) enabled in vivo MRI of amyloid plaques in a transgenic mouse model of AD. See U.S. Patent Application No. 17 / 162,126, which is incorporated herein by reference in its entirety.

[0006] A stable targeted liposome Gd contrast agent for MRI of α-syn deposits is urgently needed.

Summary of the Invention

[0007] In one aspect, a liposome composition ("ADx-003") is provided, wherein ADx-003 comprises a first phospholipid, a sterically bulky excipient capable of stabilizing the liposome composition, a second phospholipid derivatized with a first polymer, a macrocyclic gadolinium-based contrast agent, and a third phospholipid derivatized with a second polymer, wherein the second polymer is conjugated to a targeting ligand, and the targeting ligand has the formula I,

Chemical Formula

Chemical Formula

[0008] In a further embodiment, the first phospholipid comprises hydrogenated soybean L-α-phosphatidylcholine ("HSPC"), the sterically 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"), the macrocyclic gadolinium-based contrast agent comprises Gd(III)-DOTA, and the fourth phospholipid, for example, [ka] or it is conjugated to a salt thereof (e.g., a sodium salt). In some embodiments, the variable x may be one of 12, 13, 14, 15, 16, 17, or 18. In one embodiment, the variable x is 16 (conjugated: "Gd(III)-DOTA-DSPE").

[0009] In some embodiments, a third phospholipid is derivatized with a second polymer, the second polymer being conjugated with a targeted ligand, [ka] or a salt thereof (e.g., ammonium phosphate salt) may be included. In some embodiments, the variable n may be any integer between about 10 and about 100, for example, about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m may be one of 12, 13, 14, 15, 16, 17, or 18. For example, n may be 77 and m may be 14, n may be 79, m may be 14, n may be 77 and m may be 16, n may be 79 and m may be 16.

[0010] In one embodiment, the targeted ligand of the phospholipid-polymer-targeted ligand conjugate is [ka] Includes.

[0011] In one embodiment, n is 77 and m is 16 ("DSPE-PEG3400"), and the phospholipid-polymer-targeted ligand conjugate is [ka] Includes ("DSPE-PEG3400-XW-01-11 conjugate").

[0012] Alternatively, n is 79 and m is 16 ("DSPE-PEG3500"), and the phospholipid-polymer-targeted ligand conjugate is [ka] Includes ("DSPE-PEG3500-XW-01-11 conjugate").

[0013] In one embodiment, a method is provided for imaging target α-syn deposits. This method may include introducing a detectable amount of liposome composition to the target. This method may include giving the liposome composition sufficient time to associate with one or more α-syn deposits. This method may include detecting the liposome composition associated with one or more α-syn deposits.

[0014] In one embodiment, the liposome composition for a method of imaging α-syn deposits in a target may include ADx-003. In one embodiment, the liposome composition for a method of imaging α-syn deposits in a target may include Gd(III)-DOTA-DSPE and DSPE-PEG3400-XW-01-11 conjugate or DSPE-PEG3500-XW-01-11 conjugate. In one embodiment, the liposome composition for a method of imaging α-syn deposits in a target may include HSPC, Chol, DSPE-mPEG2000, Gd(III)-DOTA-DSPE, and DSPE-PEG3400-XW-01-11 conjugate or DSPE-PEG3500-XW-01-11 conjugate.

[0015] In one embodiment, the liposome composition is suitable for use in imaging α-syn deposits in a patient, the use comprising introducing a detectable amount of the liposome composition into the patient, giving sufficient time for the liposome composition to associate with one or more α-syn deposits, and detecting the liposome composition associated with one or more α-syn deposits. In one embodiment, detection includes detection using MRI.

[0016] In one embodiment, this use further includes identifying a patient having PD by detecting a liposome composition associated with one or more α-syn deposits.

[0017] In one embodiment, a phospholipid-polymer-targeted ligand conjugate is provided, and the phospholipid-polymer aspect of the phospholipid-polymer-targeted ligand conjugate is [ka] or a salt thereof (e.g., ammonium phosphate salt). In some embodiments, the variable n may be any integer between about 10 and about 100, for example, about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m may be one of 12, 13, 14, 15, 16, 17, or 18. For example, n may be 77 and m may be 14, n may be 79, m may be 14, n may be 77 and m may be 16, n may be 79 and m may be 16.

[0018] In one embodiment, the targeted ligand of the phospholipid-polymer-targeted ligand conjugate is [ka] (In the formula, X is -CH2-, -CH2-CH2-, -CHO- or -O-CO-, and Y is -CH-CH=CH- or [ka] A and B are independently selected from C and N, R1, R2, R3 and R4 are independently selected from -H, halogen, -OH and -CH3, and R5, R6 and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, C1-C6 alkyl or substituted or unsubstituted C4-C6 aryl groups, provided that if A and / or B is N, the adjacent R5 and / or R7 is -H) or represented by a pharmaceutically acceptable salt thereof.

[0019] In one embodiment, the phospholipid-polymer-targeted ligand conjugate comprises a DSPE-PEG3400-XW-01-11 conjugate or a DSPE-PEG3500-XW-01-11 conjugate.

[0020] In one embodiment, [ka] (In the formula, X is -CH2-, -CH2-CH2-, -CHO- or -O-CO-, and Y is -CH-CH=CH- or [ka] A compound comprising the following elements, or a pharmaceutically acceptable salt thereof, is provided: A and B are independently selected from C and N; R1, R2, R3 and R4 are independently selected from -H, halogen, -OH and -CH3; and R5, R6 and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, C1-C6 alkyl or substituted or unsubstituted C4-C6 aryl groups, provided that if A and / or B is N, the adjacent R5 and / or R7 is -H.

[0021] In one embodiment, the compound has a structure [ka] It holds.

[0022] In another embodiment, a method for detecting α-syn aggregates is provided. This method is [ka] (In the formula, X is -CH2-, -CH2-CH2-, -CHO- or -O-CO-, and Y is -CH-CH=CH- or [ka] The method comprises introducing an effective amount of a compound containing (where A and B are independently selected from C and N, R1, R2, R3 and R4 are independently selected from -H, halogen, -OH and -CH3, and R5, R6 and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, C1-C6 alkyl or substituted or unsubstituted C4-C6 aryl groups, provided that if A and / or B is N, then adjacent R5 and / or R7 is -H) or a pharmaceutically acceptable salt thereof into a sample or subject, giving sufficient time for the compound to associate with α-syn aggregates in the sample or subject, and detecting the compound associated with α-syn aggregates in the sample or subject. Although this may overlap with other descriptions, the various aspects of the present invention are shown below. However, the present invention is not limited to the following. [1] Compounds according to formula I: [C1] JPEG0007846011000016.jpg3990 (In the formula, X is -CH 2 -, -CH 2 -CH 2 -, -CHO- or -O-CO-, Y is -CH-CH=CH- or [C2] JPEG0007846011000017.jpg2026 And, A and B are independently selected from C and N. R 1 、R 2 、R 3 and R 4 These are independently selected from -H, halogen, -OH, and -Me. R 5 、R 6 and R 7 These are independently -H, halogen, -OH, -OMe, and -NO 2 , -NMe 2 、C 1 ~C 6 Alkyl or substituted or unsubstituted C 4 ~C 6 Selected from aryl groups, provided that A and / or B are N, adjacent R 5 and / or R 7 (is -H), or a pharmaceutically acceptable salt thereof. [2] The above compound has the following structure [3] JPEG0007846011000018.jpg2974 The compound described in [1], having the following properties. [3] The above compound has the following structure [C4] JPEG0007846011000019.jpg46105 The compound according to claim 1, having the following characteristics. [4] The above compound has the following structure [5] JPEG0007846011000020.jpg2985 The compound described in [1], having the following properties. [5] A phospholipid-polymer-targeted ligand conjugate, The phospholipid polymer is [6] JPEG0007846011000021.jpg54113 (In the formula, the variable n is any integer between approximately 70 and approximately 90, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.) The aforementioned targeted ligand is a compound according to formula I: [7] JPEG0007846011000022.jpg3991 (In the formula, X is -CH 2 -, -CH 2 -CH 2 -, -CHO- or -O-CO-, Y is -CH-CH=CH- or [8] JPEG0007846011000023.jpg2026 And, A and B are independently selected from C and N. R 1 、R 2 、R 3 and R 4 These are independently selected from -H, halogen, -OH, and -Me. R 5 、R 6 and R 7 These are independently -H, halogen, -OH, -OMe, and -NO 2 , -NMe 2 、C 1 ~C 6 Alkyl or substituted or unsubstituted C 4 ~C 6 Selected from aryl groups, provided that A and / or B are N, adjacent R 5 and / or R 7 (is -H), or a pharmaceutically acceptable salt thereof, Phospholipid-polymer-targeted ligand conjugate. [6] n is 77 or 79. [9] JPEG0007846011000024.jpg54167 A phospholipid-polymer-targeted ligand conjugate according to claim 5, comprising: [7] n is 77 or 79. [C10] JPEG0007846011000025.jpg61167 A phospholipid-polymer-targeted ligand conjugate according to claim 5, comprising: [8] n is 77 or 79. [C11] JPEG0007846011000026.jpg76167 A phospholipid-polymer-targeted ligand conjugate as described in [5], comprising: [9] A liposome composition, The first phospholipid and, A sterically bulky excipient that can stabilize liposome compositions, A second phospholipid derivatized with the first polymer, Macrocyclic gadolinium-based contrast agents, A third phospholipid derivatized with a second polymer, wherein the second polymer is conjugated to a targeting ligand, and the targeting ligand is a compound according to formula I: [C12] JPEG0007846011000027.jpg3992 (In the formula, X is -CH 2 -, -CH 2 -CH 2 -, -CHO- or -O-CO-, Y is -CH-CH=CH- or [C13] JPEG0007846011000028.jpg2026 And, A and B are independently selected from C and N. R 1 、R 2 、R 3 and R 4 These are independently selected from -H, halogen, -OH, and -Me. R 5 、R 6 and R 7 These are independently -H, halogen, -OH, -OMe, and -NO 2 , -NMe 2 、C 1 ~C 6 Alkyl or substituted or unsubstituted C 4 ~C 6 Selected from aryl groups, provided that A and / or B are N, adjacent R 5 and / or R 7 A third phospholipid represented by -H, or its pharmaceutically acceptable salt, and A liposome composition containing the following:

[10] The liposome composition according to [9], wherein the first phospholipid comprises hydrogenated soybean L-α-phosphatidylcholine ("HSPC").

[11] The liposome composition according to [9], wherein the sterically bulky excipient capable of stabilizing the liposome composition comprises cholesterol ("Chol").

[12] The liposome composition according to [9], wherein the second phospholipid derivatized with the first polymer comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) ("DSPE-mPEG2000").

[13] The aforementioned macrocyclic gadolinium-based contrast agent, [C14] JPEG0007846011000029.jpg5060 The liposome composition according to [9], comprising:

[14] The aforementioned macrocyclic gadolinium-based contrast agent is conjugated to a fourth phospholipid, [C15] JPEG0007846011000030.jpg53137 The liposome composition according to [9], comprising or a salt thereof, wherein the variable x is one of 12, 13, 14, 15, 16, 17, or 18.

[15] The liposome composition according to

[14] , wherein the variable x is 16 (conjugate: "Gd(III)-DOTA-DSPE").

[16] The targeted ligand is [C16] JPEG0007846011000031.jpg2985 A liposome composition according to claim 9, comprising:

[17] The targeted ligand is [C17] JPEG0007846011000032.jpg46103 The liposome composition according to [9], comprising:

[18] The targeted ligand is [C18] JPEG0007846011000033.jpg3094 The liposome composition according to [9], comprising:

[19] The third phospholipid, derivatized with the second polymer, [C19] JPEG0007846011000034.jpg55114 The liposome composition according to [9], comprising or a salt thereof, wherein the variable n is any integer between about 70 and about 90, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.

[20] The conjugate of the third phospholipid, the second polymer, and the targeted ligand is

[20] JPEG0007846011000035.jpg55166 Includes, The liposome composition according to [9], wherein n is 77 or 79.

[0023] The present invention can be more easily understood by referring to the following figures. [Brief explanation of the drawing]

[0024] [Figure 1] This figure shows the chemical structure of a representative example of prior art for α-syn aggregate-binding ligands.

[0025] [Figure 2] This figure provides an exemplary cross-sectional view of a liposome containing a targeted contrast agent for MRI of α-syn deposits.

[0026] [Figure 3] This figure provides the chemical structures of novel α-syn ligands, including 1-indanonyl-, 1,3-indandionyl-, α-tetralonyl-, and 4-oxocmarinyl-diene derivatives.

[0027] [Figure 4] This figure provides a schematic diagram of the molecular design of a novel α-syn ligand.

[0028] [Figure 5] This figure provides synthetic formulas for novel α-syn ligands, including 1-indanonyl-, 1,3-indandionyl-, α-tetralonyl-, and 4-oxocmarinyl-diene derivatives.

[0029] [Figure 6] This figure provides a schematic diagram of the nuclear Overhauser effect ("NOE") in the E,E configuration of diene derivatives.

[0030] [Figure 7] This figure provides synthetic formulas for novel α-syn ligands, including 1-indanonyl- and 1,3-indandionyl-diene derivatives.

[0031] [Figure 8] This figure provides the chemical structures of novel α-syn ligands, including 1-indanonyl- and 1,3-indandionyl-diene derivatives in which thiophene is inserted into the diene crosslink.

[0032] [Figure 9] This figure provides a schematic diagram of the NOE interaction in compounds 36, 45, and 48.

[0033] [Figure 10] This figure provides synthetic formulas for various derivatives in which one of the double bonds of a cross-linked diene is masked within the ring system to increase the rigidity within the compound.

[0034] [Figure 11] This figure provides the chemical structures of novel α-syn ligands, including various derivatives in which the second double bond of the cross-linked diene is masked.

[0035] [Figure 12] Table 1 shows the emission spectra and binding affinity (Kd) data of novel α-syn ligands.

[0036] [Figure 13] This figure shows representative absorption / emission spectra of free ligands versus ligands bound to α-syn or Aβ fibrils, using ligands 8 (XW-01-11) and 32 (XW-01-64).

[0037] [Figure 14] Table 2 shows the observed depth shift of the maximum fluorescence and emission values, the increase in fluorescence, and the fluorescence quantum yield during fibril binding with a novel α-syn ligand.

[0038] [Figure 15] Table 3 shows a comparison of the dissociation constants of novel α-syn ligands relative to Aβ fibrils, compared to α-syn fibrils.

[0039] [Figure 16] This figure shows confocal microscopy images of PD brain tissue sections co-stained with anti-α-syn antibody and ligands 8 (XW-01-11) and 32 (XW-01-64).

[0040] [Figure 17] This figure shows confocal microscope images of PD brain tissue sections co-stained with anti-α-syn antibody and ligand 8 (XW-01-11).

[0041] [Figure 18]This figure shows comparative confocal microscopy images of PD and AD brain tissue sections co-stained with ligand 8 (XW-01-11) and their respective anti-α-syn counter Aβ antibodies.

[0042] [Figure 19] This figure shows an exemplary synthesis scheme for preparing the DSPE-PEG3400-XW-01-11 conjugate.

[0043] [Figure 20] The dynamic light scattering ("DLS") graphs of ligand 8 (XW-01-11)-labeled liposome nanoparticles versus control liposomes when incubated with α-syn fibrils are shown. [Modes for carrying out the invention]

[0044] A novel α-syn-targeted liposome-Gd contrast agent, ADx-003, was developed based on a highly stable macrocyclic Gd-DOTA imaging moiety. ADx-003 can be generally understood as shown in its cross-sectional morphology in Figure 2.

[0045] Accordingly, in one embodiment, ADx-003 comprises a first phospholipid, a sterically bulky excipient capable of stabilizing the liposome composition, a second phospholipid derivatized with the first polymer, a macrocyclic gadolinium contrast agent, and a third phospholipid derivatized with the second polymer, wherein the second polymer is conjugated to a targeting ligand. The macrocyclic gadolinium contrast agent may be conjugated to a fourth phospholipid.

[0046] Phospholipids In some embodiments, suitable phospholipids include those in which the two hydrocarbon chains have a length of about 14 to about 24 carbon atoms and exhibit a variety of 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.

[0047] In some embodiments, suitable phospholipids could be any of those listed in International Publication No. 2005107820, the contents of paragraphs

[0031] to

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

[0048] Polymer-derivativeized phospholipids In some embodiments, the liposomes of the liposome composition may contain flexible, water-soluble (hydrophilic) polymer chains or have a surface 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 liposomes by blood cells and the removal of liposomes from the blood. The liposomes can avoid uptake by mononuclear phagocytic organelles, mainly the liver and spleen (reticular endothelial system).

[0049] In one embodiment, the polymer in the derivatized phospholipid may be polyethylene glycol ("PEG"). PEG can have any of a range of molecular weights. For example, the PEG chain can have a molecular weight of about 1,000 to 10,000 daltons. Once liposomes are formed, the PEG chain can provide a hydrophilic surface coating sufficient to extend the blood circulation time of the liposomes compared to the absence of such a coating.

[0050] In some embodiments, the second phospholipid derivatized with the first polymer comprises DSPE-mPEG2000. In some embodiments, the third phospholipid derivatized with the second polymer, the second polymer conjugated with a targeted ligand, [ka] or a salt thereof (e.g., ammonium phosphate salt), where the variable n may be any integer between about 10 and about 100, for example, about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m may be one of 12, 13, 14, 15, 16, 17, or 18. For example, n may be 77 and m may be 14, n may be 79, m may be 14, n may be 77 and m may be 16, n may be 79 and m may be 16. In some embodiments, the third phospholipid derivatized with the second polymer comprises DSPE-PEG3400 or DSPE-PEG3500.

[0051] In some embodiments, suitable polymers could be any of those listed in International Publication No. 2005107820, the contents of paragraphs

[0034] to

[0038] thereof being incorporated herein by reference in their entirety. In some embodiments, the phospholipids derivatized by the polymers may be any of the combinations disclosed in International Publication No. 2016057812 and U.S. Patent Application No. 17 / 162,126, each of which is incorporated herein by reference in its entirety.

[0052] Three-dimensionally bulky excipients In some embodiments, liposomes may contain stabilizing excipients. For example, a liposome composition may be formulated to contain Chol. In other embodiments, a liposome composition may contain fatty alcohols, fatty acids, cholesterol esters, other pharmaceutically acceptable excipients, and mixtures thereof.

[0053] Macrocyclic gadolinium contrast agents The liposome composition comprises a macrocyclic Gd-based contrast agent. In some embodiments, the macrocyclic gadolinium-based contrast agent is Gd(III)-DOTA conjugated to a phospholipid, for example, [ka] or a salt thereof (e.g., a sodium salt). In some embodiments, the variable x may 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. The preparation of Gd(III)-DOTA-DSPE is described in U.S. Patent Application No. 17 / 162,126.

[0054] In other embodiments, macrocyclic gadolinium contrast agents are [ka] Includes.

[0055] Phospholipid-polymer-targeted ligand conjugate Another aspect of the present invention is Formula II: PL-AL-HP-X-TL II This provides a phospholipid-polymer-targeted ligand conjugate having the structure described above.

[0056] In the formula, PL is a phospholipid, AL is an aliphatic bond, HP is a hydrophilic polymer, and X is a bond, -O-, -R i O-, -R i O(C=O), R i -N(R ii )O(C=O), R i -N(R ii )(C=O)-, or R i -N(R ii ) and TL is a targeted ligand having the structure of formula I.

[0057] A phospholipid-polymer-targeted ligand conjugate contains a phospholipid-polymer region that facilitates the incorporation of the conjugate into a membrane, such as those present in liposomes. The phospholipid is an amphiphilic compound whose structure is known to those skilled in the art. In some embodiments, the phospholipid (PL) in the phospholipid-polymer-targeted ligand conjugate has the following structural formula [ka] It is represented by [this].

[0058] The formula represents a hydrophilic phosphate group and two hydrophobic fatty acid chains commonly found in phospholipids. The variable s may be one of 12, 13, 14, 15, 16, 17, or 18. For example, s may be 14 or 16. In various embodiments, the phospholipid group in the phospholipid-polymer-targeted ligand conjugate may be one of HSPC, DPPC, DSPE, DSPC, or DPPE. Suitable phospholipids and polymer-derivative phospholipids are also disclosed separately herein.

[0059] The conjugate also includes a hydrophilic polymer (HP). Hydrophilic polymers are polymers that contain polar or charged functional groups that make them soluble in water. Examples of hydrophilic polymers include polyacrylamide, polyethyleneimine, polyacrylic acid, polyvinyl alcohol, and polyalkylene oxide. In some embodiments, the hydrophilic polymer is a poly(alkylene oxide) polymer. A hydrophilic poly(alkylene oxide) may contain about 10 to about 100 repeating units and may have a molecular weight in the range of, for example, 500 to 10,000 Daltons. Examples of hydrophilic poly(alkylene oxides) include PEG, poly(ethylene oxide), and poly(propylene oxide). Hydrophilic polymers HP can be conjugated to the phospholipid moiety via amide or carbamate groups, as described herein. HP in phospholipid-polymer-targeted ligand conjugates can be conjugated to the aromatic moiety via amides, carbamates, poly(alkylene oxides), triazoles, or combinations thereof.

[0060] In some embodiments, hydrophilic polymers (HP) have the following structural formula [ka] It is represented by one of the following.

[0061] In some embodiments, the variable r may be any integer between approximately 10 and approximately 100, for example, approximately 60 to approximately 100, approximately 70 to approximately 90, approximately 75 to approximately 85, approximately 77, or approximately 79.

[0062] In some embodiments, the phospholipid-polymer moiety PL-HP- in the phospholipid-polymer-targeted ligand conjugate has the following structural formula [ka] It can be represented by one of the following.

[0063] In some embodiments, the variable r can be any integer from about 10 to about 100, such as, 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 s can be one of 12, 13, 14, 15, 16, 17, or 18. For example, r can be 77, s can be 14, r can be 79, s can be 14, r can be 77, s can be 16, r can be 79, and s can be 16.

[0064] As used herein, the "aliphatic linkage" represented by AL includes any aliphatic group useful in the linkage between the phospholipid PL and the hydrophilic polymer HP. Such aliphatic linkages can include, for example, heteroatoms via one or more moieties such as amide, carbamate, etc., and can be C2 - C 10 and can include an alkylene group. For example, in the following conjugate

Chemical formula

Chemical formula

[0065] ​Such aliphatic linkages are known in the art for linking phospholipids with hydrophilic polymers and can be found, for example, in commercial sources of phospholipid-PEG compounds and functionalized phospholipid-PEG conjugation precursors, which can be represented as PL-AL-PEG-NH2, PL-AL-PEG-CO2H, etc. When the presence of an aliphatic linkage is implied, it is common in the art and commercial sources to refer to such compounds in a simplified form without mentioning the aliphatic linkage. For example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] CAS number 147867-65-0, whose aliphatic linkage group is the amide-containing group -CH2CH2NH(C=O)CH2O-, is commonly known in the art and commercially as "DSPE-mPEG-2000". Commercial materials listed herein in conventional simplified forms such as "DSPE-mPEG-2000" should be understood to contain the corresponding aliphatic bond.

[0066] Therefore, in various embodiments, the aliphatic linker represented by AL may include a carbamate or an amide. The liposomes, methods, and conjugates described herein may include phospholipid-polymer-targeted ligand conjugates in which AL comprises a carbamate, an amide, or a mixture of such conjugates.

[0067] In a particular embodiment, a phospholipid-polymer-targeted ligand conjugate is provided, and the phospholipid-polymer aspect of the phospholipid-polymer-targeted ligand conjugate is [ka] or a salt thereof (e.g., ammonium phosphate salt). In some embodiments, the variable n may be any integer between about 10 and about 100, for example, about 60 to about 100, about 70 to about 90, about 75 to about 85, about 77, or about 79. The variable m may be one of 12, 13, 14, 15, 16, 17, or 18. For example, n may be 77 and m may be 14, n may be 79, m may be 14, n may be 77 and m may be 16, n may be 79 and m may be 16. In some embodiments, the phospholipid-polymer embodiment of the phospholipid-polymer-targeted ligand conjugate includes DSPE-PEG3400 or DSPE-PEG3500.

[0068] The phospholipid-polymer-targeted ligand conjugate of formula II also contains a targeted ligand (TL) as discussed below.

[0069] Targeted ligand Liposome compositions comprise at least one phospholipid derivatized with a polymer, the polymer being conjugated with a targeted ligand. Thus, in some embodiments, the phospholipid is modified to include a spacer chain. The spacer chain may be a hydrophilic polymer. Hydrophilic polymers may typically be terminally functionalized for coupling to a targeted ligand. The functionalized terminal groups may be, for example, maleimide groups, bromoacetamide groups, disulfide groups, activated esters, or aldehyde groups. Hydrazide groups are reactive to aldehydes, which can be produced in many biologically relevant compounds. Hydrazides can also be acylated with activated esters or carbodiimide activated carboxyl groups. Acylazide groups, which are reactive as acylated species, can be readily obtained from hydrazides and enable the binding of amino-containing ligands.

[0070] In some embodiments, the targeting ligand may be accessible from the surface of the liposome and, for example, can specifically bind to or attach to one or more molecules or antigens. These targeting ligands can direct or target the liposome to specific cells or tissues, such as α-syn plaques, and can bind to molecules or antigens on or associated with cells or tissues.

[0071] In one embodiment, the compound is of formula (I), [ka] In the formula, X is -CH2-, -CH2-CH2-, -CHO-, or -O-CO-, and Y is -CH-CH=CH- or [ka] Compounds of formula I are provided, wherein A and B are independently selected from C and N, R1, R2, R3 and R4 are independently selected from -H, halogen, -OH and -CH3, and R5, R6 and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, C1-C6 alkyl or substituted or unsubstituted C4-C6 aryl groups, provided that if A and / or B is N, the adjacent R5 and / or R7 is -H, or pharmaceutically acceptable salts thereof. Compounds of formula I may be interchangeably referred to herein as “targeting ligand” and “binding ligand”. Targeting ligands exhibit high affinity binding to α-syn, particularly misfolded α-syn, such as those found in deposits (also referred to herein as plaques) or fibrils.

[0072] The compounds in formula I can vary at position X to provide different heterocyclic compounds. In some embodiments, X is -CH2- providing a 1-indanone heterocyclic group. In some embodiments, X is -CH2-CH2- providing a tetralone heterocyclic group. In some embodiments, X is -CHO- providing a 1,3-indanedione heterocyclic group. In some embodiments, X is -O-CO- providing a 4-hydroxycoumarin heterocyclic group. This heterocyclic group may be referred to herein as the “first aromatic group”.

[0073] The compounds contained in formula I differ at position Y and can provide different dienes. Thus, in one embodiment, Y is -CH-CH=CH- and a diene bridge is provided. In one embodiment, the diene bridge has an E,E configuration. In another embodiment, Y is [ka] This allows for the realization of a diene in the form of an electron-rich thiophene group.

[0074] In some embodiments, the second (rightmost) aromatic group of the compound can be modified. Intraring modification may include substituting a nitrogen atom for methyldyne at position A or B to provide pyridine as the second aromatic group, or it may include substituting nitrogen atoms for methyldyne at positions A and B to provide pyrimidine as the second aromatic group. If positions A, B, or both are substituted with nitrogen, the substituted position does not have substituents outside the ring.

[0075] Compounds in formula I may also include compounds in which one or more substituents are added around the first and / or second aromatic ring. Suitable substituents include halogens, hydroxyls, methoxyls, nitrols, dimethylamines, and lower alkyl or aryl moieties. For example, in some embodiments, hydrogen atoms along the circumference of the second aromatic ring are substituted with para-substituted phenyl groups.

[0076] Suitable compounds included in Formula I include, for example, compound 8, ((E)-2-((E)-3-(4-hydroxy-3-methoxyphenyl)alilidene)-2,3-dihydro-1H-inden-1-one), 32, (4'-((E)-3-((E)-6-hydroxy-1-oxo-1,3-dihydro-2H-inden-2-ylidene)propa-1-en-1-yl)-[1,1'-biphenyl]-4-carboxylic acid), and 37, ((Z)-2-((5-(4-(hydroxymethyl)phenyl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one).

[0077] In some embodiments, the compound is compound 8: [ka] That is the case.

[0078] Examples of binding ligands for Formula I include compounds that have a high affinity for α-syn, such as α-syn present in the deposits and fibrils. In particular, since α-syn present in the fibrils and deposits is typically aggregated α-syn, the binding ligand has a high affinity for aggregated α-syn. In some embodiments, the compound is α-syn specific. In some embodiments, the compound has a higher affinity for α-syn than for Aβ. α-Syn specificity, as used herein, refers to the fact that the contrast agent binds exclusively or preferentially to α-syn compared to other proteins associated with the disease and impairment of misfolded proteins. As used herein, the term “specifically binds” refers to the interaction between the binding ligand and a second chemical species, and the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope). For example, a targeted ligand generally recognizes and binds to a specific protein structure of α-syn rather than the protein itself.

[0079] Compounds within the range of Formula I have various different binding affinities to α-syn (e.g., aggregated α-syn). In some embodiments, the compound has a K of about 500 nM or less. d It has binding affinity to α-syn aggregated with . In some embodiments, the compound has a K of about 200 nM or less. d It has binding affinity to α-syn aggregated with . In other embodiments, the compound has a K of about 100 nM or less. d It has binding affinity to α-syn aggregated with . In a further embodiment, the compound has a K of about 50 nM or less. d It has binding affinity to α-syn aggregated with [unclear].

[0080] In some embodiments, the compound further includes a radiolabel. The radiolabeled compound has one or more atoms substituted with a radionuclide. Examples of radiolabels include: 3 H, 14 C, 35 S, 125 I, 121 I, 112 In, 99 mTc is one example. The compound is 18 F, 11 C, and 15 It can also be modified to include atoms useful for positron emission tomography, such as oxygen (O).

[0081] Suitable compounds in formula I can exist as acid addition salts, including pharmaceutically acceptable salts, such as those formed with organic and inorganic acids. Such acid addition salts are generally pharmaceutically acceptable. However, salts of pharmaceutically unacceptable salts may be useful in the preparation and purification of the compound in question. Basic addition salts can also be formed and are pharmaceutically acceptable.

[0082] The term "pharmaceutically acceptable salt," as used herein, refers to a salt or zwitterionic form of a compound of Formula I that is soluble in water, oil, or dispersible, as defined herein, and therapeutically acceptable. Salts can be prepared during the final isolation and purification of the compound, or separately by reacting a suitable compound in the form of a free base with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisinate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, and malonic acid. Examples of basic compounds include salts, DL-mandelate, mesitylene sulfonate, methanesulfonate, naphthylene sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate (p-tosylate), and undecanoate. Basic groups in the compounds may be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dimethyl, diethyl, dibutyl, and diamyl sulfate, decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides, as well as benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Salts can also be formed by coordination of the compound with alkali metal or alkaline earth ions. Thus, sodium, potassium, magnesium, and calcium salts of the compound of formula I are conceived.

[0083] Liposomes "Liposomes" generally refer to spherical or nearly spherical particles containing an internal cavity. The wall of a liposome may include a lipid bilayer. These lipids may be phospholipids. Numerous lipids and / or phospholipids can be used to construct liposomes. One example is an amphiphilic lipid having a hydrophobic portion and a polar head portion, which can spontaneously form in bilayer vesicles in water, as exemplified by phospholipids, or can be stably incorporated into a lipid bilayer, with its hydrophobic portion in contact with the hydrophobic region inside the bilayer membrane and its polar head portion oriented towards the polar surface outside the membrane. Liposomes can be prepared by any known method, including the examples herein, and those described in U.S. Patent Application No. 17 / 162,126, International Publication No. 2016057812 and International Publication No. 2012139080, which are incorporated herein in whole by reference, respectively. Figure 2 provides an exemplary cross-sectional view of a liposome containing a targeted contrast agent for MRI of α-syn deposits.

[0084] In one embodiment, ADx-003 comprises HSPC, Chol, DSPE-mPEG2000, DSPE-PEG3400-XW-01-11 conjugate, and Gd(III)-DOTA-DSPE. In one embodiment, ADx-003 comprises HSPC, Chol, DSPE-mPEG2000, DSPE-PEG3500-XW-01-11 conjugate, and Gd(III)-DOTA-DSPE. In some embodiments, the first phospholipid may include DPPC, DSPC, or a mixture of DPPC and DSPC. In one embodiment, the lipid composition and molar ratio (%) of components in ADx-003 is approximately HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:DSPE-PEG3400 / 3500-Formula I conjugate = approximately 31.5:approximately 40:approximately 2.5:approximately 25:approximately 1. In some embodiments, the molar ratio of any one of the HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:DSPE-PEG3400 / 3500-Formula I conjugate can 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 ratio (%) of components in ADx-003 is HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:DSPE-PEG3400 / 3500-Formula I conjugate = approximately 32.5:approximately 40:approximately 2:approximately 25:approximately 0.5. In one embodiment, the lipid composition and molar ratio (%) of components in ADx-003 are approximately 32:40:2.5:25:0.5 for HSPC:Chol:DSPE-mPEG2000:Gd(III)-DOTA-DSPE:DSPE-PEG3400 / 3500-Formula I conjugate.

[0085] In one embodiment, the HSPC content in ADx-003 is approximately 24 mg / mL to approximately 32 mg / mL (total lipids). In one embodiment, the Chol content in ADx-003 is approximately 14 mg / mL to approximately 19 mg / mL. In one embodiment, the DSPE-mPEG2000 content in ADx-003 is approximately 5 mg / mL to approximately 7 mg / mL. In one embodiment, the Gd(III)-DOTA-DSPE content in ADx-003 is 30 mg / mL to 45 mg / mL. In one embodiment, the DSPE-PEG3400 / 3500-Formula I conjugate content in ADx-003 is approximately 2 mg / mL to approximately 3 mg / mL. In one embodiment, the free gadolinium content in ADx-003 is 100 μg / mL or less, including less than 2.5 μg / mL.

[0086] In one embodiment, the pH of the liposome composition is 6.4 to 8.4. In a further embodiment, the weight osmolality of the liposomes is 200 to 400 mOsmol / kg. In a further embodiment, the vesicle size (Z-mean) of the liposomes is 150 nm (D) when measured by dynamic light scattering. 50 ) less than approximately 140nm (D 50 ), and approximately 120 nm (D 50 ) including approximately 200nm (D 50 It is less than ).

[0087] To clarify, the term "approximately" when combined with a number is intended to include ±10% of that number. This is true whether "approximately" modifies an independent number or modifies a number at either or both ends of a range. In other words, "approximately 10" means 9 to 11. Similarly, "approximately 10 to approximately 20" means 9 to 22 and 11 to 18. When the term "approximately" is not present, the exact number is intended. In other words, "10" means 10.

[0088] α-Syn detection method A method is provided for detecting α-syn (e.g., aggregated α-syn). This method involves detecting an effective amount of a compound according to formula I: [ka] (In the formula, X is -CH2-, -CH2-CH2-, -CHO- or -O-CO-, and Y is -CH-CH=CH- or [ka] The method comprises introducing a compound (where A and B are independently selected from C and N, R1, R2, R3 and R4 are independently selected from -H, halogen, -OH and -CH3, and R5, R6 and R7 are independently selected from -H, halogen, -OH, -OCH3, -NO2, -N(CH3)2, C1-C6 alkyl or substituted or unsubstituted C4-C6 aryl groups, provided that if A and / or B is N, the adjacent R5 and / or R7 is -H), or a pharmaceutically acceptable salt thereof, into a sample or object. The method also comprises the steps of providing sufficient time for the compound to associate with α-syn in the sample or object, and detecting the compound associated with α-syn in the sample or object.

[0089] As used herein, α-syn refers to the full-length 140-amino acid α-synuclein protein, e.g., "α-syn-140". Other isoforms or fragments may include, for example, "α-syn-126", α-synuclein-126, which lacks residues 41-54 due to the deletion of exon 3, and "α-syn-112", α-synuclein-112, which lacks residues 103-130 due to the deletion of exon 5.

[0090] α-Syn aggregates form insoluble fibrils in pathological conditions characterized by LB, such as PD, DLB, and MSA. α-Syn is a major structural component of LB fibrils. α-Syn may be present in the brains of individuals with or suspected of having PD. Various α-syn peptides may be associated with neuronal damage related to PD. Examples of disease-related α-syn isoforms include, but are not limited to, α-syn-140, α-syn-126, and α-syn-112.

[0091] The compound used in the detection method may be any of the α-syn targeting ligands of formula I. For example, in some embodiments, the targeting ligand is a compound of formula I where Y is -CH-CH=CH-, and in further embodiments, the targeting ligand is a compound of formula I where both A and B are carbon atoms. In further embodiments, the targeting ligand is selected from compound 8, compound 32, and compound 37 in Figures 3 and 8, and in even further embodiments, the compound has the following structure, [ka] It holds.

[0092] In some embodiments, the compound used in the detection method is linked to a phospholipid polymer to form a phospholipid-polymer-targeted ligand conjugate of formula II. The phospholipid-polymer-targeted ligand conjugate may include either a phospholipid (PL) or a hydrophilic polymer (HP) as described herein. The phospholipid-polymer-targeted ligand conjugate can be incorporated into liposomes. The compound of formula I can be detected directly by fluorescence, modified to include a radiolabeled compound, or detected by other means, but incorporating the compound into liposomes can increase the detection options.

[0093] This method includes the step of introducing an effective amount of the compound according to formula I into a sample or subject. The sample may be a part of tissue that may contain α-syn, for example, a tissue sample obtained from a subject (e.g., a nerve tissue sample), in which case the method is used for ex vivo analysis. Introducing the compound into the sample simply means bringing the sample into contact with the compound. Alternatively, the compound may be introduced into a subject in order to perform in vivo analysis. As used herein, “subject” may be any animal, and may also be called a patient. The subject may be a vertebrate, a mammal, for example, a research animal (e.g., a mouse or rat), livestock (e.g., a cattle, horse, or pig), or a pet (e.g., a dog or cat). In some embodiments, the subject is a human.

[0094] This method may also include a step of providing sufficient time for the compound to associate with α-syn (e.g., aggregated α-syn) in the sample or object. The binding ligand of formula I has an affinity for α-syn, particularly aggregated α-syn, and therefore associates with α-syn present in the sample or object. The amount of time required for the compound to associate with α-syn may vary depending on several variables, such as the properties of the sample, the method of administration, and the affinity of the compound used. The amount of time sufficient for the compound to associate with α-syn can be easily determined by those skilled in the art. For example, sufficient time for the compound to associate with α-syn in the sample or object may be at least 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or at least 8 hours.

[0095] This method may include the step of detecting α-syn-related compounds (e.g., aggregated α-syn) in a sample or object. In some embodiments, detection may include detection using MRI. In another example, detection may include detection by fluorescence imaging ("FI"). Detection may include detection by SPECT imaging and / or PET imaging using a radiocontrast agent. Examples of radiocontrast agents include those considered suitable for use in SPECT imaging and / or PET imaging from the Molecular Imaging and Contrast Agent Database of the National Institutes of Health. Any other suitable type of imaging methodology known to those skilled in the art, including but not limited to PET imaging, may be considered.

[0096] In some embodiments, an image is generated showing the location of α-syn (e.g., aggregated α-syn) detected in the target. Therefore, in some embodiments, a method is provided for generating an image of a target tissue region by administering an effective amount of contrast agent (i.e., a targeted ligand or phospholipid-polymer-targeted ligand conjugate) to the target and generating an image of the tissue region of the target to which the contrast agent is distributed. To generate an image of the tissue region, a detectably effective amount of contrast agent must reach the target tissue region, but the contrast agent does not need to be localized in this region alone. However, in some embodiments, the contrast agent is targeted or locally administered so that it is primarily present in the target tissue region. Examples of images include two-dimensional cross-sectional images and three-dimensional images. In some embodiments, a computer is used to analyze the data generated by the contrast agent in order to generate visual images. An example of an image generation method is MRI. MRI scanners use strong magnetic fields, magnetic field gradients, and radio waves to generate images of organs within the body.

[0097] An imaging system typically includes three basic components: (1) a suitable source for inducing excitation of an imaging agent, (2) a system for separating or distinguishing luminescence from the contrast agent, and (3) a detection system. The detection system may be handheld or integrated into other useful imaging devices such as an intraoperative microscope. Exemplary detection systems include endoscopes, catheters, tomography systems, handheld imaging systems, or intraoperative microscopes.

[0098] Many targeted ligands exhibit higher affinity for α-syn (e.g., aggregated α-syn) than for other proteins involved in protein misfolding disorders, such as Aβ or τ proteins. Due to this higher affinity, the binding ligand, either alone or in a phospholipid-polymer-targeted ligand conjugate, can distinguish the level of α-syn from the level of other misfolding-prone proteins. We are particularly interested in distinguishing the level of aggregated α-syn from the level of aggregated Aβ. Thus, in some embodiments, α-syn is detected with higher specificity than Aβ. In other embodiments, α-syn is detected with more than 1.5 times, more than 2 times, more than 3 times, more than 5 times, or more than 10 times higher specificity than Aβ.

[0099] In some embodiments, a method for detecting and / or imaging α-syn (e.g., aggregated α-syn) in a sample or subject can be used to diagnose whether the subject has a disease related to misfolded α-syn, or to assess the progression of a disease in the subject. In some embodiments, the subject may be at risk of developing PD, having PD, or being treated for PD, or may be at risk of having a disease related to α-syn dysregulation, misfolding, aggregation, or disposal, such as MSA, or may be undergoing treatment for a disease related to α-syn dysregulation, misfolding, aggregation, or disposal.

[0100] This method may include diagnosing PD in a subject based on the detection of α-syn protein (e.g., aggregated α-syn). α-Syn misfolding and aggregation have been shown to be associated with the pathogenesis of PD. The diagnosis of PD may also include comparing images or the amount of detected α-syn protein with images taken from a control sample or a healthy subject. This method may also include determining or diagnosing the presence of a disease associated with α-syn aggregation in a subject based on the presence of soluble misfolded α-syn protein in the sample or subject. This method may also include determining or diagnosing the presence of MSA in a subject based on the presence of soluble misfolded α-syn protein in the sample or subject.

[0101] In some embodiments, the method includes treating a subject diagnosed with a disease associated with α-syn aggregation with an α-syn modulating therapy. Several novel therapies targeting α-syn homeostasis are currently under development via various mechanisms. α-syn modulating therapies can include, for example, inhibiting the production of α-syn, inhibiting the aggregation of α-syn, using appropriate inhibitors, active or passive immunotherapy approaches, etc. Therapeutic approaches targeting α-syn homeostasis can include active immunization such as PD01A+ or PD03A+, or passive immunization such as PRX002. Using the methods described herein for detecting the presence of soluble misfolded α-syn, it is possible to determine which patients can be treated with an α-syn modulating therapy. Currently, there is no cure for PD, but various drugs such as levodopa, dopamine agonists, and monoamine oxidase B inhibitors are useful for treating the motor symptoms of PD.

[0102] The lipid-polymer-targeting ligand compound containing a contrast agent can be administered with a pharmaceutically acceptable carrier. Pharmaceutically suitable forms for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. This must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0103] Kit for α-Syn detection Another aspect of the present invention provides a kit for detecting and / or imaging α-syn in a subject (e.g., aggregated α-syn). The kit generally comprises a package having one or more containers holding a targeting ligand and other components and reagents, as one or more separate compositions or optionally, as a mixture as far as reagent compatibility permits. The kit can include instructions and a liposomal composition. The instructions can direct the user to introduce a detectable amount of the liposomal composition into a sample or subject. The instructions can direct the user to allow sufficient time for the liposomal composition to associate with α-syn. The instructions can direct the user to detect the liposomal composition associated with α-syn. The kit can include a targeting ligand of Formula I and / or a lipid-polymer-targeting conjugate represented by Formula II.

[0104] The instructions included in the kit can be attached to the packaging material or included as an insert. The instructions are usually in writing or printed, but are not limited thereto. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROM), etc. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions.

[0105] The components of the kit may be in different physical states. For example, some components may be lyophilized, some in aqueous solution, and some frozen. Individual components may be packaged separately within the kit. The kit may also include other useful tools for performing the methods of the present invention or related tests, treatments, or calibrations, which may include buffers, enzymes, fluorescent reagents, MRI enhancers (e.g., paramagnetic ions), gels, plates, detectable labels, containers, and the like. The kit may also include sampling devices for obtaining biological samples from subjects, such as syringes or needles.

[0106] The term "effective dose" is intended to limit the number or amount of compound (e.g., α-syn targeted ligand) that is effective in carrying out the relevant method. For example, an effective dose of α-syn targeted ligand, or a conjugate containing α-syn targeted ligand, associates with α-syn present in the sample or subject at a detectable level. When used in a subject, the effective dose may be low enough to minimize undesirable side effects associated with the administration. The therapeutic effective dose may be administered in one or more doses.

[0107] The present invention includes compounds described herein in any pharmaceutically acceptable form, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, etc. In particular, where the compound is optically active, the present invention specifically includes each enantiomer of the compound and racemic mixtures of enantiomers. The term “compound” includes any or all of such forms, whether explicitly stated or not (sometimes “salt” is explicitly stated).

[0108] The term “diagnosis” can encompass determining the likelihood of a subject developing a disease or the presence or nature of a disease in a subject. The term “diagnosis” can also encompass determining the severity and possible outcome of a disease or episode of a disease, or the likelihood of recovery, commonly referred to as prognosis. “Diagnosis” can also encompass diagnosis in the context of rational treatment, which guides treatment, including initial treatment selection and treatment modifications (e.g., adjustment of dosage or medication plan).

[0109] Where a range of values ​​is provided, unless explicitly indicated in the context, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, and any other stated or intermediate values ​​within the stated range are included in the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in the invention.

[0110] All scientific and technical terms used in this application have their meanings as commonly used in the art unless otherwise specified. The definitions provided herein are for the purpose of facilitating the understanding of certain terms that are frequently used herein and are not intended to limit the scope of this application.

[0111] The present invention will be illustrated by the following examples. Specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0112] example All reagents were obtained from Sigma-Aldrich, TCI, Alfa Aesar, or Acros Organics and used without further purification. Proton nuclear magnetic resonance ( 1 ¹H NMR was recorded at 600 MHz or 500 MHz using a Bruker 600 or 500 NMR spectrometer. Carbon nuclear magnetic resonance ( 13The 3C NMR spectrum was recorded at 75 MHz or 125 MHz using a Bruker 300 or 500 NMR spectrometer, respectively. 1 For 1H NMR, report the chemical shift from the internal standard of acetone (2.05 ppm), chloroform (7.26 ppm), or dimethyl sulfoxide (2.50 ppm) in parts per million (ppm). 13 For 13C NMR, the chemical shift from one of the following internal standards—residual acetone (206.26 ppm), chloroform (77.00 ppm), or dimethyl sulfoxide (39.52 ppm)—is reported in parts per million (ppm). NMR peak multiplicity is expressed as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), dd (doublet of doublet), td (triplet of doublet), dt (doublet of triplet), and m (multilet). The coupling constant (J) is given in Hertz (Hz). High-resolution mass spectra were obtained from the Ohio State University Mass Spectrometry and Proteomics Facility. TLC was performed on silica gel 60 F254 plates from EMD Chemical Inc., and components were visualized using ultraviolet light (254 nm) and / or a 20 wt% phosphomolybdic acid solution in ethanol. SiliFlash silica gel (230-400 mesh) was used for all column chromatography.

[0113] Example 1: Molecular design of targeted ligands Compound 1 from the prior art was selected as a scaffold for structure-activity relationship ("SAR") studies aimed at developing novel structures with high affinity and selectivity for α-syn aggregates. Molecular design (Figure 4) focused on three parts of the molecule: the first aromatic group (A), the bridge (B), and the second aromatic group (C). For (A), 1-indanone and 1,3-indanedione were selected as starting points for novel derivatives. For (B), the diene was maintained in several derivatives. Derivatizations in which one of the double bonds was replaced with an electron-rich thiophene moiety were also introduced to increase the electron density within the molecule. Furthermore, derivatives with increased overall intramolecular rigidity were introduced by "locking" the second double bond in two different ring systems. Derivatization around the second aromatic group (C) included both electron-rich and electron-deficient aromatic rings, as well as heterocycles.

[0114] Example 2: Chemical synthesis of targeted ligands Referring to Figure 5, a first series of derivatives in which ring A is replaced with one of the following moieties: 1-indanonyl-(formula i for generating compounds 7-15, as shown in Figure 3), 1,3-indazionyl-(formula ii for generating compounds 16-22, as shown in Figure 3), α-teralonyl-(formula iii for generating compounds 23-24, as shown in Figure 3), or coumarinyl-(formula iv for generating compounds 25-28, as shown in Figure 3), were obtained by acid- or base-catalyzed aldol condensation reactions of the desired keto substrate with the corresponding cinnamaldehyde derivative, while maintaining the diene crosslink (B).

[0115] Therefore, 37% HCl (0.5 mL) was slowly added to a solution of aldehyde (1.0 equivalent) and indolinone (1.0 equivalent) in acetic acid (10 mL). The reaction mixture was stirred overnight at 110 °C and cooled to room temperature. The cooled solvent was poured into ice water and filtered off. The solid was recrystallized with methanol.

[0116] Alternatively, ethylenediamine dihydrochloride (0.25 mmol) was slowly added to a solution of aldehyde (1.0 equivalent) and indolinone (1.0 equivalent) in dichloromethane / methanol (1:2, 10 mL). The reaction mixture was stirred at room temperature for 5 hours. The solid was filtered off and recrystallized with methanol.

[0117] In particular, with respect to compound 8, (E)-2-((E)-3-(4-hydroxy-3-methoxyphenyl)alilidene)-2,3-dihydro-1H-inden-1-one, the compound was prepared by an acidic protocol using 1-indanone (250 mg, 1.89 mmol) and 4-hydroxy-3-methoxycinnamaldehyde (337 mg, 1.89 mmol) to obtain the desired product (8) as a red solid (436 mg, 79% yield). 1H NMR(600 MHz,DMSO-d6)δ 9.52(s,1H),7.74(d,J=7.8 Hz,1H),7.69(td,J1=1.2 Hz,J2=7.2 Hz,1H),7.64(d,J=7.8 Hz,1H),7.47(t,J=7.2 Hz,1H),7.29(dt,J1=1.8 Hz,J2=10.2 Hz,1H),7.28(s,1H),7.13(d,J=15.6 Hz,1H),7.09(dt,J1=10.2 Hz,J2=15.6 Hz,1H),7.06(d,J=8.4 Hz,1H),6.81(d,J=8.4 Hz,1H),3.93(s,2H),3.86(s,3H);13C NMR(150 MHz,DMSO-d6)δ 192.9,149.6,148.9,148.4,143.3,139.3,135.1,134.9,134.2,128.4,127 .9,127.1,123.7,122.6,122.5,116.1,111.0,56.2,30.7.HRMS(ESI)calcd for C19H17O3 [M+H]+293.1172,found,293.1171.

[0118] Early experiments suggested that mono-keto substrates yielded cleaner reaction products and better yields under acidic conditions, while di-keto substrates preferred basic conditions. Therefore, subsequent reactions involving these substrates were carried out under similar reaction conditions. The resulting diene 1 H and 13 Both 13C NMR spectra showed peaks consistent with a single product, suggesting that only one of the two possible isomers (E,E or Z,E) was formed. Further analysis of heteronuclear multibond correlation ("HMBC") and NOE spectra suggested that the isolated product had an E,E configuration due to NOE enhancement observed between the enhanced protons (Figure 6).

[0119] Derivatives of the cross-linked diene system, in which one of the double bonds is replaced with an electron-rich thiophene moiety to increase the intramolecular electron density, were synthesized in two steps as shown in formulas vii to ix (Figure 7). First, 5-bromo-2-thiophenecarboxyaldehyde was exposed to 1-indanone (or 6-hydroxyl-1-indanone) under aldol condensation conditions to obtain thiobromo intermediate 36, which was then exposed to various arylboronic acid esters under Suzuki coupling conditions (formula vii) to produce compounds 37 to 44. Similarly, other derivatives of this series were prepared by aldol condensation of 1-indanone (formulas vii and vii) and α-tetralone with 4-bromo-2-thiophenecarboxyaldehyde and 5-bromo-2-thiophenecarboxyaldehyde, respectively, to produce the corresponding thiobromide intermediates 45 and 48. These intermediates were then exposed to different arylboronic acid esters to obtain compounds 46 and 47 and compounds 49 to 51, respectively.

[0120] More specifically, referring to Figures 7 and 8, a second series of 1-indanonyl- and 1,3-indanedionyl-diene derivatives were generated by adding a second ring to 1-indanonyl-dien bromide (7 and 11) and 1,3-indanedionyl-dien bromide (17) via Suzuki coupling of their respective arylboronic acid esters, thereby producing compounds 29-35 as shown in formulas v and vi. Accordingly, a solution of indolinone derivative (1.0 equivalent), boronic acid derivative (2.0 equivalent), and K2CO3 (1.0 equivalent) in 1,4-dioxane / H2O (4:1, 10 mL) was degassed with argon for 20 minutes, and Pd(PPh3)4 (0.1 equivalent) was added. The reaction mixture was degassed again (5 minutes) and stirred overnight at 110°C. The reaction mixture was quenched with water (5 mL), the aqueous layer was extracted with ethyl acetate (30 mL), washed with saturated NaHCO3 (10 mL), and then washed with brine (10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography.

[0121] Analysis of the NOE (Figure 9) and HMBC spectra of compounds 36, 45, and 48 showed that all double bonds resulting from their respective aldol condensation reactions were in the Z conformation.

[0122] As shown in Figure 10, one of the double bonds of the cross-linked diene was masked within the ring system, allowing access to various derivatives to increase intramolecular rigidity. All members of this series were accessed through a single aldol condensation reaction between their respective keto derivatives and the corresponding aldehydes. Figure 11 shows the chemical structures of the various derivatives.

[0123] The structural elucidation of all compounds is 1 H and 13 This was achieved by 13C NMR and analysis of high-resolution mass spectra of each individual compound. UV / VIS absorption and emission spectra of all compounds were recorded in phosphate-buffered saline ("PBS"). All compounds with fluorescence properties suitable for fluorescence microscopy studies were selected for synthetic fibril binding studies.

[0124] Example 3: Binding affinity (K d ) for synthetic α-Syn fibrils All synthesized compounds (except 19 and 28) showed good emission spectra in PBS (Figure 12, Table 1). Binding affinity was determined. Binding affinity is the strength of the binding interaction between a single biomolecule and its ligand / binding partner. Binding affinity is measured and reported by the equilibrium dissociation constant (K d ). The smaller the K d value, the greater the binding affinity of the ligand for its target. The larger the K d value, the more weakly the target molecule and the ligand are attracted to each other and bind to each other. <00009​​​​​​​​​​Ligand solutions of various concentrations, from 0.1 nM to 10 μM, in PBS (pH=7.5, 197 μL) were added to microcentrifuge tubes containing α-syn fibrils (3 μL, final concentration 2.5 μM). The mixture was incubated at 37°C for 1 hour with shaking. The fibrils were separated by spinning down the mixture at 21,000 g / s for 15 minutes. The precipitate was washed twice with Tris-HCl and resuspended in 200 μL of buffer. Fluorescence was measured using a SpectraMax-384 plate reader with the molecular excitation and emission maximum values. All data points were obtained in triplicate. K at the binding site. d The value of the maximum number (Bmax) is obtained by nonlinear regression using MATLAB® software (R2019B) and the equation Y = Bmax × X / (X + K d This was determined by fitting it to the specified size.

[0127] K d All compounds showing a value of ≥2 μM (compounds 7, 11, 16-18, 28, 52-54, and 56) were considered to have insufficient binders and were reported as "no binding" ("NB").

[0128] In general, 1-indanone-diene derivatives were considered better binders than the corresponding 1,3-indanedione-dienes, as exemplified by 8:20 and 10:22. Any aromatic substitution on the 1-indanone-diene moiety (activated, 13 or inactivated, 14 and 15) reduced binding affinity compared to the unsubstituted derivatives 10 and 8, respectively. All α-tetralone-diene and coumarin-diene derivatives showed inferior binding compared to the corresponding 1,3-indanone-diene and 1,3-indanone-diene derivatives, as exemplified by 8, 20, 23 and 25. d Apart from compound 32, which has a molecular weight of 18.8 nM, adding a second ring to the second aromatic group (C) is not thought to improve the bond affinity of either the 1-indanone-diene or 1,3-indanedione-diene systems. Similarly, replacing one of the double bonds in the diene bridge with an electron-rich thiopenyl moiety (compounds 8 vs. 39) is thought to improve the bond affinity of several moderate K dExcept for the values ​​(compounds 37, 39, and 42), there is no positive effect on the ligand binding affinity to the α-syn fibril. Making the system more rigid by masking the second double bond of the cross-linked diene with a condensed ring with C (compounds 52-58) reduces the bond strength and eventually eliminates the bond.

[0129] Example 4: Fluorescence properties and ligand binding selectivity for α-Syn fibril vs. Aβ fibril α-syn aggregates represent the most prevalent misfolded protein aggregates encountered in PD and other synucleinopathy, although several studies suggest that Aβ and τ aggregates often overlap with α-syn. Potential α-syn agents for in vivo application must be both highly sensitive and highly selective (especially for Aβ) to minimize false positives in such cases. Preliminary α-syn fibril binding studies of 11 ligands have shown high to moderate affinity (K d The fluorescence properties and binding affinity of these ligands to α-syn were further evaluated in comparison to Aβ fibrils. The maximum absorption and emission values ​​and fluorescence quantum yield of the free ligands were determined in the presence of either α-syn or Aβ fibrils. As exemplified by the data for ligands 8 (XW-01-11) and 32 (XW-01-64) (Figure 13), all ligands showed minimal fluorescence at concentrations of ≤0.5 μM in aqueous media, which increased significantly upon the addition of either α-syn or Aβ fibrils.

[0130] The increase in fluorescence is accompanied by a depth shift in both absorbance and emission maximum from the free molecule to the ligand-fibril complex, with an 8- to 15-fold increase in fluorescence quantum yield upon ligand binding to α-syn fibrils and a further 2- to 3-fold increase upon binding to Aβ fibrils (Figure 14, Table 2).

[0131] The observed depth shift of fluorescence and emission maximums, the increase in fluorescence, and the fluorescence quantum yield upon fibril binding with these ligands are consistent with other observations of β-sheet binding ligands.

[0132] The binding affinity to Aβ fibril was evaluated using a saturation binding assay.

[0133] Aβ fibril formation β-amyloid(1-40) peptides were purchased from R-Peptide (Bogart, Georgia). Fibrils were prepared according to the protocol outlined by Eric et al. (PLoS One, 2012, 7(10), e48515). Aβ(1-40) was dissolved in PBS (pH 7.4) to a final concentration of 433 μg / ml (100 μM). The solution was stirred at 700 rpm for 4 days at room temperature using a magnetic stirrer to promote fibril formation. The stock solution was divided into equal parts and stored at -80°C for future use. After thoroughly stirring the stock solution to maintain a homogeneous suspension of fibrils, aliquots for the binding assay were removed.

[0134] Aβ fibril / ligand binding assay Ligand solutions of various concentrations, from 1 nM to 100 μM, in PBS (pH=7.5, 180 μL) were added to microcentrifuge tubes containing β-amyloid fibrils (20 μL, final concentration 10 μM). The mixture was incubated at 37°C for 1 hour with shaking. The mixture was spun down at 21,000 g / s for 12 minutes to separate the fibrils. The precipitate was washed twice with Tris-HCl and resuspended in 200 μL of buffer. Fluorescence was measured using a SpectraMax-384 plate reader with the molecular excitation and emission maximum values. All data points were obtained in triplicate. K at the binding site. d The value of the maximum number (Bmax) is obtained by nonlinear regression using MATLAB® software (R2019B) and the equation Y = Bmax × X / (X + K d This was determined by fitting it to the specified size.

[0135] The results (Figure 15, Table 3) show that, with the exception of compound 29, all other compounds have a three-digit dissociation constant to nanomolar-range Aβ fibrils compared to a two-digit α-syn fibril. The K between each compound of two protein aggregatesd The comparison of values ​​is based on the highest affinity (K d Compound 8, which has a concentration of 9.7 nM, is suggested to have 14.4 times the selectivity for Aβ. Compound 32 (K) is a slightly more moderate α-syn binder. d Compound 37 (K = 18.8 nM) has 26-fold selectivity for Aβ. Compound 37 (K = 18.8 nM) is also a moderate α-syn binder. d (=34.9nM) exhibits 11.2 times selectivity for Aβ.

[0136] Example 5: Fluorescent human PD and AD tissue staining Considering sufficient fluorescence properties, high affinity, and selectivity for α-syn aggregates, ligands 8, 32, and 37, exhibiting α-syn selectivity relative to Aβ selectivity of 14.4-fold, 26-fold, and 11.2-fold, respectively, were further evaluated by in vitro fluorescence staining of neuropathologically validated postmortem brain samples from human PD and AD patients. Sections from the pons and frontal cortex of PD brains were permeabilized and sequentially treated with antibodies [anti-α-synuclein (aa121-125) antibody, clone Syn211] and 1 μM solutions of each compound, and visualized by confocal microscopy. Column I of Figure 16 shows fluorescent Hoechst staining highlighting the cell nucleus, thereby providing a perspective view of the cell body within the tissue. Column II shows ligand fluorescence. Column III shows fluorescence from the antibody. Column IV is a composite image created by mixing the first three images. Row A shows an image obtained from a section of the frontal cortex of a PD brain treated with compound 8 (XW-01-11). As seen in the image, the ligand strongly labels Lewy lesions in the tissue. The pattern and labeling intensity appear similar in the antibody channel. The composite image shows colocalization of the ligand and antibody signals, confirming that they bind to the same pathology. Similarly, the section treated with ligand 32 (XW-01-64) in row B shows strong labeling of Lewy lesions by the ligand, which is supported by the staining pattern and intensity of the antibody. As observed with ligand 8, the complex imaged from a mixture of ligand 32 and the antibody image also shows colocalization of both signals, confirming the efficiency of these ligands in labeling Lewy lesions in postmortem human PD brain sections. In the adjacent Z-stacked image of the treated tissue (row C), the lesion appears to surround the ligand and antibody channel dark spots (arrows). The composite image created by mixing nuclear stain, ligand, and antibody signals shows that the ligand and antibody channel dark spots are actually spots occupied by more prominent nuclei at high magnification (row D). The proximity and location of the nuclei suggest, as expected, that the observed lesions are cytoplasmic inclusions and not extracellular aggregates.

[0137] To further characterize the sites labeled by ligand and antibody Syn211 tissue staining experiments, adjacent cortical sections were treated with compound 8 and then with either Syn211 or Syn303. As expected, sections treated with compound 8 and Syn211 (Figure 17, first row) show identical ligand and antibody labeling patterns co-localized in the composite image. Both ligand and antibody are thought to label all forms of pathology present in the tissue. On the other hand, in tissue sections treated with ligand and antibody Syn303 (Figure 17, second row), both small neurites (upper arrow) are effectively labeled by the ligand channel, but only mature Lewy bodies (lower arrow) are labeled by the antibody channel. These findings suggest that the labeled pathology is α-syn and that the ligand labels all conformations of the pathology.

[0138] To evaluate the selectivity observed for α-syn vs. Aβ fibril binding on aggregates in human tissue, equimolar concentrations of ligands 8, 32, and 37 were further evaluated on PD tissue as described above, along with cortical sections from neuropathologically validated postmortem brain samples from AD patients. Figure 18 shows data from the top binder (8), which exhibits a 14.4-fold selectivity for α-syn vs. Aβ. As can be observed in row A, the PD tissue shows strong labeling of both large and fine deposits of pathology (column II). Similar labeling patterns and efficiencies are observed in the antibody channel (column III). A composite image (column IV) generated by mixing both signals with the Hoechst signal shows co-localization of ligand and antibody signals. Unlike the PD tissue, the fluorescence image from AD tissue (row B) shows mostly dense core Aβ plaques in the ligand channel (column II), but not finer aggregates consisting of diffuse plaques. The antibody, anti-β-amyloid, 17-24 antibody (4G8), highlights both high-density core and diffuse Aβ pathology (column III). A synthetic image generated by mixing both signals with the Hoechst signal shows overlap of ligand and antibody signals from high-density core plaques (column IV). This data suggests that the ligand labels fibril α-syn with higher efficiency than fibril Aβ. High-magnification images from treated AD tissue (column C) show, as expected, that the observed Aβ lesions are extracellular, unlike the intracellular Lewy lesions observed in PD tissue.

[0139] Acquisition of human tissue Human PD brain tissue and AD brain tissue were obtained from the NIH Neurobiobank.

[0140] Labeling of α-syn pathology in human PD brain tissue Midbrain tissue (frontal cortex 5469) was embedded in Tissue-Tek OCT. The compound was held in liquid nitrogen for 30 minutes. The embedded tissue was sliced ​​into 30 μm thick sections using Lecia Biosystems Cryostat at -20°C and mounted on pre-washed microscope slides. The sections were washed twice with 1×PBST and loaded with 10% formalin solution for 20 minutes. The sections were washed three times with 1×PBS and permeabilized with 0.1% Triton-X100 for 10 minutes. The sections were washed twice with 1×PBS and incubated with 2% normal donkey serum at room temperature for 1 hour. The sections were incubated overnight at 4°C with anti-α-synuclein (aa121-125) antibody, clone Syn211 (Ascites free) (1:1000 in 1% donkey serum), and washed three times with 1×PBS. Sections were incubated with a fluorescent secondary antibody labeled with Alexa Fluor488 (1:200 in PBS) at room temperature for 2 hours. Sections were washed three times with 1×PBST and treated with the compound of test. Each tissue section was incubated with 5 μM of the test compound dissolved in PBS at room temperature for 30 minutes. Sections were washed three times with 1×PBST and loaded with TrueBlack Lipofuscin Autofluorescence Quencher (1:20 in ethanol) for 2 minutes. Sections were washed three times with 1×PBS and covered with coverslips. Tissues were imaged using a Lecia DMi8 motorized fluorescence microscope with standard excitation / emission filters for Alexa Fluor488 or Alexa Fluor647.

[0141] Staining of β-amyloid plaques in human AD brain tissue Midbrain tissue (frontal cortex 5590) was embedded in Tissue-Tek OCT. The compound was held in liquid nitrogen for 30 minutes. The embedded tissue was sliced ​​into 30 μm thick sections using Lecia Biosystems Cryostat at -20°C and mounted on pre-washed microscope slides. The sections were washed twice with 1×PBST and then loaded into 10% formalin solution for 20 minutes. The sections were washed three times with 1×PBS and permeabilized with 0.1% Triton-X100 for 10 minutes. The sections were washed twice with 1×PBS and incubated with 2% normal donkey serum at room temperature for 1 hour. The sections were incubated overnight at 4°C with purified anti-β-amyloid and 17-24 antibody (4G8) (1:500 in 1% donkey serum) and washed three times with 1×PBS. Sections were incubated with a fluorescent secondary antibody labeled with Alexa Fluor488 (1:200 in PBS) at room temperature for 2 hours. Sections were washed three times with 1×PBST and treated with the compound of test. Each tissue section was incubated with 5 μM of the test compound dissolved in PBS at room temperature for 30 minutes. Sections were washed three times with 1×PBST and loaded with TrueBlack Linpofuscin Autofluorescence Quencher (1:20 in ethanol) for 2 minutes. Sections were washed three times with 1×PBS and covered with coverslips. Tissues were imaged using a Lecia DMi8 motorized fluorescence microscope with standard excitation / emission filters for Alexa Fluor488 or Alexa Fluor647.

[0142] Human brain tissue staining with HRP-DAB Midbrain tissue (frontal cortex 5469 or frontal cortex 5590) was fixed with Tissue-Tek OCT. The compound was held in liquid nitrogen for 30 minutes. The frozen tissue was sliced ​​into 30 μm thick sections using Lecia Biosystems Cryostat at -20°C and mounted on pre-washed microscope slides. The sections were washed twice with 1×PBST and loaded with 10% formalin solution for 20 minutes. The sections were washed three times with 1×PBS, 50 μl of peroxide block was applied to each section, and incubated at room temperature for 10 minutes. The sections were washed twice with 1×PBS and incubated with 2% normal donkey serum at room temperature for 1 hour. Sections were incubated overnight at 4°C with anti-α-synuclein (aa121-125) antibody, clone Syn211 (Ascites free) (1:1000 in 1% donkey serum) or purified anti-β-amyloid, 17-24 antibody (4G8) (1:500 in 1% donkey serum), and washed three times with 1×PBS. Sections were incubated with biotinylated secondary antibody at room temperature for 2 hours and washed three times with 1×PBST. Sections were incubated with streptavidin / HRP labeling for 30 minutes and washed three times with 1×PBST. Sections were incubated with distilled water for 10 minutes and incubated with DAB Chromagen (50 μL of DAB Chromagen combined with 1 ml of DAB substrate). Sections were washed three times with distilled water and dehydrated through alcohol for 10 minutes. Finally, the sections were washed with xylene down to the coverslip, stored at room temperature for 2 days, and imaged using a bright-field microscope.

[0143] Example 6: Synthesis of the DSPE-PEG3400-XW-01-11 conjugate Referring to Figure 19, ethyl bromo (2.3 g, 13.5 mmol) was added in one step to a solution of 6-hydroxy-1-indanone (1.0 g, 6.8 mmol), K2CO3 (2.8 g, 20.2 mmol), and KI (112 mg, 0.7 mmol) in DMF (10 mL). The reaction mixture was stirred overnight at 90°C. At this point, TLC (silica, 1:3 siRNA) indicated that the reaction was complete. The reaction mixture was cooled to room temperature and filtered through a Celite pad using ethyl acetate (50 mL) as the eluent, and the filtrate was concentrated. The residue was purified by column chromatography to obtain the desired product (1.4 g, 90%) as a white solid. 1 H NMR(600 MHz,CDCl3)δ 7.38(d,J=8.4 Hz,1H),7.26(dd,J1=2.4 Hz,J2=8.4 Hz,1H),7.10(d,J=2.5 Hz,1H),4.64(s,2H),4.26(q,J=7.8 Hz,2H),3.06(t,J=6.0 Hz,2H),2.69(t,J=6.0 Hz,2H),1.29(t,J=7.8 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ 206.7,168.4,157.6,148.9,138.2,127.7,124.4,105.8,65.3,61.5,36.9,25.1,14.1.

[0144] 37% HCl (0.2 mL) was slowly added to the product (700 mg, 3.0 mmol) and a solution of 4-hydroxy-3-methoxycinnamaldehyde (588 mg, 3.3 mmol) in acetic acid (10 mL). The reaction mixture was stirred overnight at 120 °C and cooled to room temperature. The cooled solvent was poured into ice water and filtered off. The solid was recrystallized in methanol to obtain the desired product (768 mg, 70%) as a brown solid. 1H NMR(600 MHz,DMSO-d6)δ 13.09(s,1H),9.52(s,1H),7.55(d,J=8.4 Hz,1H),7.32-7.24(m,3H),7.13(dd,J1=6.0 Hz,J2=9.0 Hz,1H),7.08(d,J=5.4 Hz,1H),7.06(td,J1=3.0 Hz,J2=7.8 Hz,1H),6.81(d,J=8.4 Hz,1H),4.79(s,2H),3.85(s,3H),3.84(s,2H); 13 C NMR(150 MHz,DMSO-d6)δ 192.7,170.6,148.4,142.6,140.4,135.8,134.2,127.9,123.7,122.4,106.3,65.3,56.2,29.9.

[0145] HSTU (160 mg, 0.4 mmol) was added to a solution of the product (120 mg, 0.3 mmol) and DSPE-PEG3400-NH2 (500 mg, 0.1 mmol) in dried DMF (8 mL). The reaction mixture was stirred at room temperature for 2 days and concentrated under reduced pressure. The residue was diluted with a methanol / water mixture (1:1, 8 ml), loaded into a 2000 MWCO dialysis cassette, dialyzed with MES buffer (10 mM, 2 × 5 liters) for 8 hours, and dialyzed with water (3 × 5 liters) for 2 days. Water was removed by lyophilization to obtain DSPE-PEG3400-XW-01-11 (242 mg, 48%) as a yellow solid. 1H NMR(600 MHz, CDCl3)δ 7.45(d,J=8.4 Hz,1H),7.23(dd,J1=2.4 Hz,J2=5.6 Hz,1H),7.19-7.17(m,2H),7.15(d,J=2.4 Hz,1H),7.02-6.99(m,3H),6.78(dd,J1=8.4 Hz,J2=10.8 Hz,1H),5.09(brs,1H),4.48(s,2H),4.30(dd,J1=2.4 Hz,J2=12.0 Hz,1H),4.12-4.06(m,2H),4.01(t,J=4.2 Hz,2H),3.98(brs,2H),3.81-3.77(m,5H),3.73(s,2H),3.49(dd,J1=2.4 Hz,J2=7.8 Hz,2H),3.48-3.46(m,3H),3.35-3.32(m,4H),3.20-3.18(m,2H),2.21-2.18(m,4H),1.93-1.92(m,4H),1.50-1.47(m,4H),0.761(t,J=6.0 Hz,6H);HRMS(MALDI)calcd for C 219 H 410 N2O 92 P [M+H] + 4571.7203,found,4571.7069.

[0146] Example 7: Compound 8 (XW-01-11) identification manufactured by Rino A lipid mixture containing HSPC:DSPE-mPEG2000:Chol:Gd-DOTA-DSPE:DSPE-PEG3400-XW-01-11 in a molar ratio (%) of 32:2.5:40:25:0.5 was dissolved in ethanol (600 μL) at 60-65°C. DHPE-rhodamine (1 mg in 200 μL) dissolved in ethanol was added, and the resulting solution was hydrated with histidine-buffered saline (HBS) (10 mM histidine, 140 mM NaCl, approximately pH 7.6) at 60-65°C for 45 minutes. Hydrated lipid solutions were continuously extruded at 60–65°C using a high-pressure extruder (Northern Lipids, Vancouver, British Columbia, Canada) through nucleopore membranes of 400 nm (5 passes) followed by 200 nm (8 passes) to form liposomes of the desired size (dynamic light scattering (DLS) system, Brookhaven Instruments Corp., Holtzville, New York, USA). The liposome suspension was dialyzed against histidine-buffered saline (HBS) using a 300 kDa molecular weight cutoff membrane (Spectrum Laboratories Inc., California, USA) to remove unencapsulated material. Control liposomes (untargeted liposomes) were prepared using the same protocol, but the DSPE-PEG3400-XW-01-11 fraction was replaced with mPEG2000.

[0147] Example 8: In vitro evaluation of the binding of compound 8 (XW-01-11)-targeted liposomes to α-Syn fibrils. Figure 20 shows (a) a schematic diagram illustrating the reaction between the targeted liposomes of the present invention and α-syn fibrils, (b) a DLS graph showing that the solution of ligand 8 (XW-01-11) labeled liposomes prepared in Example 7 has an average hydrodynamic diameter of 194 nm, and (c) a DLS graph showing a large increase in hydrodynamic diameter (2346 nm) after incubation of the targeted liposomes of Example 7 with 1 nM α-syn fibrils for 1.5 hours, which is attributed to the formation of aggregates between the nanoparticles and fibrils and the high affinity of XW-01-11 for fibrils. (d) A DLS graph showing that the particles are retained together by affinity, an exemplary solution of untargeted nanoparticles (i.e., the DSPE-PEG3400-XW-01-11 fraction replaced with mPEG2000) has an average hydrodynamic diameter of 169 nm, and (e) a DLS graph showing that when an exemplary solution of untargeted nanoparticles is incubated with 1 nM α-syn fibril for 1.5 hours, no particles with a hydrodynamic diameter >500 nm are obtained, indicating no interaction between the untargeted nanoparticles and α-syn fibril.

[0148] The complete disclosure of all patents, patent applications, and publications, as well as electronically available materials, cited herein is incorporated by reference. The above detailed descriptions and examples are given solely for the purpose of clarifying understanding and are not to be understood as being unnecessarily limited therefrom. The present invention is not limited to the exact details illustrated and described, and modifications that are obvious to those skilled in the art are included within the present invention as defined by the claims.

Claims

1. Compounds according to the following formula: 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ and R ４ These are independently selected from -H, halogen, -OH, and -Me. R5 and R7 are independently -H, halogen, -OH, -OMe, -NO ２ , -NMe ２ , C １ ~C ６ alkyl or substituted or unsubstituted C ４ ~C ６ selected from aryl groups, R6 is selected from halogens, -OH, -NO2, C1-C6 alkyl groups, or substituted or unsubstituted C4-C6 aryl groups, or a pharmaceutically acceptable salt thereof.

2. The above compound has the following structure 【Chemistry 2】 The compound according to claim 1, having the following characteristics.

3. The above compound has the following structure 【Transformation 3】 The compound according to claim 1, having the following characteristics.

4. The above compound has the following structure 【Chemistry 4】 The compound according to claim 1, having the following characteristics. 【Request Item 5】 【Chemistry 5】 or 【Transformation 6】 (In the formula, R1, R3, and R4 are independently selected from -H, halogen, -OH, and -Me. R5 and R7 are independently selected from -H, halogen, -OH, -OMe, -NO2, -NMe2, C1-C6 alkyl, or substituted or unsubstituted C4-C6 aryl groups. R6 is selected from halogen, -OH, -NO2, C1-C6 alkyl, or substituted or unsubstituted C4-C6 aryl groups. The variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18), or a pharmaceutically acceptable salt thereof. Phospholipid-polymer-targeted ligand conjugate.

6. n is an integer that is 77 ± 10% or 79 ± 10%. 【Transformation 7】 The phospholipid-polymer-targeted ligand conjugate according to claim 5, comprising:

7. n is an integer that is 77 ± 10% or 79 ± 10%. 【Transformation 8】 The phospholipid-polymer-targeted ligand conjugate according to claim 5, comprising:

8. n is an integer that is 77 ± 10% or 79 ± 10%. 【Chemistry 9】 The phospholipid-polymer-targeted ligand conjugate according to claim 5, comprising:

9. A liposome composition, The first phospholipid and, An excipient selected from the group consisting of cholesterol, cholesterol esters, fatty alcohols, fatty acids, and mixtures thereof, A second phospholipid derivatized with the first polymer, Macrocyclic gadolinium-based contrast agents, A third phospholipid derivatized with a second polymer, wherein the second polymer is conjugated with a targeting ligand, and is represented by a compound of the following formula: 【Chemistry 10】 (In the formula, R1, R3 and R ４ These are independently selected from -H, halogen, -OH, and -Me. R5 and R7 are independently -H, halogen, -OH, -OMe, and -NO ２ , -NMe ２ , C １ ~C ６ Alkyl, substituted, or unsubstituted C ４ ~C ６ Selected from aryl groups, R6 is selected from halogen, -OH, -NO2, C1-C6 alkyl, or substituted or unsubstituted C4-C6 aryl groups. A third phospholipid represented by a pharmaceutically acceptable salt thereof (where the variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18) and A liposome composition containing the following:

10. The liposome composition according to claim 9, wherein the first phospholipid comprises hydrogenated soybean L-α-phosphatidylcholine.

11. The liposome composition according to claim 9, wherein the excipient comprises cholesterol.

12. The liposome composition according to claim 9, wherein the second phospholipid derivatized with the first polymer comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000).

13. The aforementioned macrocyclic gadolinium-based contrast agent, 【Chemistry 11】 A liposome composition according to claim 9, comprising:

14. The aforementioned macrocyclic gadolinium-based contrast agent is conjugated to a fourth phospholipid, 【Chemistry 12】 The liposome composition according to claim 9, comprising or a salt thereof, wherein the variable x is one of 12, 13, 14, 15, 16, 17, or 18.

15. The liposome composition according to claim 14, wherein the variable x is 16.

16. The third phospholipid described above is 【Chemistry 13】 (In the formula, the variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.) A liposome composition according to claim 9, comprising:

17. The third phospholipid described above is 【Chemistry 14】 (In the formula, the variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.) A liposome composition according to claim 9, comprising:

18. The third phospholipid described above is 【Chemistry 15】 (In the formula, the variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.) A liposome composition according to claim 9, comprising:

19. The third phospholipid, derivatized with the second polymer, 【Chemistry 16】 The liposome composition according to claim 9, comprising or a salt thereof, wherein the variable n is any integer between 70 ± 10% and 90 ± 10%, and the variable m is one of 12, 13, 14, 15, 16, 17, or 18.

20. The conjugate of the third phospholipid, the second polymer, and the targeted ligand is 【Chemistry 17】 Includes, The liposome composition according to claim 9, wherein n is an integer of 77 ± 10% or 79 ± 10%.

21. A liposome composition according to any one of claims 9 to 20 for use in a method for imaging target α-synchen deposits, wherein the method is (a) Introducing a detectable amount of the liposome composition into the patient, (b) Allow sufficient time for the liposome composition to associate with one or more α-syn deposits. (c) generating images of the target brain via magnetic resonance imaging, and (d) To detect a liposome composition associated with one or more α-syn deposits, A liposome composition containing the following:

22. The liposome composition according to claim 21, wherein the method comprises determining the magnetic resonance image contrast signal intensity generated by the association of the liposome composition with the target α-syn deposit, and comparing it with a baseline signal intensity detected in a control sample or an image taken from a healthy subject.

23. The liposome composition according to claim 22, wherein the step of determining a statistically significant difference in signal intensity between the subject and the baseline sample indicates the presence of α-syn deposition pathology in the subject.

24. The liposome composition according to claim 23, further comprising the method of identifying the subject as having Parkinson's disease based on the presence of α-syn deposit pathology in the subject.

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