Cyclodextrin dimers, compositions thereof, and uses thereof

Abstract

A new class of synthetic cyclodextrin dimers is described. Exemplary cyclodextrin dimers are capable of treating atherosclerotic plaques by targeting various forms of cholesterol both intracellularly and extracellularly. Methods of depleting atherosclerotic plaques of cholesterol, cholesterol esters, 7-ketocholesterol, and 7-ketocholesterol esters by treatment with such cyclodextrins are also provided. A sub-class of dimers with high specificity for 7-ketocholesterol is also described.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of U.S. Provisional Application Serial No. 62 / 787,869 (Agent No. 48731.1600), filed January 3, 2019, and U.S. Provisional Application Serial No. 62 / 850,334 (Agent No. 48731.1601), filed May 20, 2019, each of which is incorporated herein by reference in its entirety. Background Technology

[0003] 7-Ketocholesterol (7KC) is an oxosterol produced by the non-enzymatic reaction of cholesterol with oxygen free radicals. 7KC can be formed in organisms or consumed in food, but it is potentially toxic and is considered useless to humans and other eukaryotes. Like cholesterol, 7KC is present in atherosclerotic plaques. 7KC is the most abundant non-enzymatically produced oxosterol in atherosclerotic plaques and can be a contributing factor to the pathogenesis of atherosclerosis and other age-related diseases. 7KC is also believed to be a contributing factor to lysosomal storage diseases such as Niemann-Pick Type C (NPC).

[0004] Cyclodextrin (CD) is a cyclic oligosaccharide polymer composed of 6 (αCD), 7 (βCD), or 8 (γCD) sugar rings. Figure 1A α, β, and γ cyclodextrins are the most common forms and have many medical, industrial, consumer, and food-related applications. Cyclodextrins have been used in a variety of applications, including as a food additive form of dietary fiber. Cyclodextrins are also commonly combined with active pharmaceutical ingredients, used in pharmaceutical compositions as atomizing agents, and as excipients for slightly hydrophobic drugs.

[0005] Hydroxypropyl-β-cyclodextrin (HPβCD) is a β-cyclodextrin in which a certain number of hydroxypropyl (HP) groups have been added to some or all of the seven glucose monomers constituting βCD on the O2, O3, or O6 oxygen atoms (or added to atoms that replace said oxygen). Hydroxypropylation of cyclodextrin improves its water solubility and safety, making it suitable for a variety of uses in humans, particularly as an excipient for active pharmaceutical ingredients; this has earned it a place on the FDA's GRAS (Generally Recognized As Safe) list for HPβCD. Most commercially available HPβCDs have an average degree of HP substitution between 4 and 9, and all existing products contain a combination of substitution number and position, which is typically reflected in the advertised average degree of substitution (DS).

[0006] Other CD substitutions include methyl, succinyl, sulfobutyl, maltose, carboxymethyl, and quaternary ammonium, which can produce CDs that are highly soluble in water and have low cytotoxicity, regardless of whether they are charged or neutral groups. Commercially available βCDs can have different degrees of substitution, ranging from only about 1 to up to fully substituted (degree of substitution 21), depending on the specific substituent and supplier. Summary of the Invention

[0007] This disclosure describes the design and testing of various dimers of cyclodextrin (CD), including HPβCD dimers, methyl-βCD dimers, succinoyl-βCD dimers, sulfobutyl-βCD dimers, and quaternary ammonium dimers. It shows that some dimers exhibit significantly increased affinity for 7KC and cholesterol compared to the monomeric CD. Exemplary dimers represent a new class of linked and substituted cyclodextrin dimers with improved properties, including the ability to selectively interact with and solubilize sterols. Molecular modeling experiments described below demonstrate the predicted interaction mechanisms. Furthermore, working examples confirm the predictive ability of novel substituted cyclodextrin dimers to solubilize sterols, including selective solubilization of 7KC compared to cholesterol.

[0008] In one aspect, this disclosure provides a CD dimer having the structure CD-L-CD, wherein each CD is a β-cyclodextrin, L is attached to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers are substituted with at least one functional group, such as methyl, hydroxypropyl (HP), sulfobutyl (SB), succinyl (SUCC), quaternary ammonium (QA) (such as -CH2CH(OH)CH2N(CH3)3). + (or combinations thereof). Typically, each CD monomer consists of a glucose monomer in the D configuration. The CD dimer is functionalized, typically having a degree of substitution (DS) between 1 and 28, where the degree of substitution refers to the total number of functional group substitutions present on both CD subunits. The substitutions can be present on any one or both of the CD subunits. The linker length can be between 2 and 8 atoms long, such as between 4 and 8 atoms long, along the shortest path through which the two CD subunits of the cyclodextrin dimer are linked. The linker can include an alkyl (e.g., butyl) linker and / or a triazole linker, which may optionally be substituted. Exemplary CD dimers have formulas I-IX (respectively as shown below). Figure 3B-3J (As shown). Optionally, the CD dimer is further substituted.

[0009] In another aspect, this disclosure provides a βCD dimer having the structure CD-L-CD, wherein each CD is a β-cyclodextrin, L is attached to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers are substituted with at least one hydroxypropyl group. Typically, each CD monomer consists of a glucose monomer with a D configuration. The βCD dimer is substituted with hydroxypropyl (HP) groups and typically has a degree of substitution (DS) between 1 and 40, wherein the degree of substitution refers to the total number of substitutions present on both CD subunits. The substitutions can be present on any one or both of the CD subunits. The linker length can be between 4 and 8 atoms long along the shortest path of the linker connecting the two CD subunits of the cyclodextrin dimer. The linker can include an alkyl (e.g., butyl) linker and / or a triazole linker, which may optionally be substituted. Exemplary βCD dimers have formula I, II, or III (respectively as shown below). Figure 3B-3D (As shown). Optionally, the βCD dimer is further substituted.

[0010] 7KC is believed to be associated with heart disease, cystic fibrosis, liver damage and failure, and complications of hypercholesterolemia. When someone is affected by hypercholesterolemia, 7KC can diffuse across the cell membrane, where it affects receptor and enzyme function; the increased incidence of hypercholesterolemic dementia is associated with 7KC accumulation. In the liver, 7KC affects tissue perforation and porosity, which increases with age. 7KC also promotes the transport of cytoplasmic NADPH oxidase components from neutrophils (leukocytes) to the membrane and enhances the rapid production of reactive oxygen species. Increased 7KC levels are also associated with the pathogenesis of other age-related diseases such as age-related macular degeneration (AMD-dry), Alzheimer's disease, and lysosomal storage diseases such as Niemann-Pick disease type C (NPC). Oxysterols (including 7KC) are also associated with increased free radical levels, which in turn affects lipid cycling in cystic fibrosis. It is believed that the increase in free radicals induced by oxosterols (such as 7KC) is associated with apoptosis, cytotoxicity, endothelial dysfunction, and regulation of enzymes involved in inflammation and fatty acid metabolism.

[0011] 7KC is formed from the non-enzymatic reaction of oxygen free radicals with cholesterol, suggesting that its formation may not be beneficial. In fact, 7KC is believed to enhance free radical production throughout the body, but is of particular concern in the heart and vascular tissues. Free radicals affect cellular and enzymatic reactions, which are important for cholesterol-mediated tissue damage, especially in these tissues; this is believed to enhance inflammation in the vascular system. 7KC is believed to cause dysfunction of mitochondria and lysosomes by disrupting the function of cell and organelle membranes, and is thought to be associated with an increased frequency of foam cell formation from macrophages in atherosclerotic plaques. The scavenging function of these macrophages is expected to help improve plaques, but when they are filled with cholesterol and oxosterols, they can become part of the plaque.

[0012] Exemplary implementations provide treatment for diseases associated with and / or exacerbated by 7KC accumulation, such as atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis due to calcified coronary artery lesions, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lemli-Opitz syndrome, infantile neuronal ceroid lipofuscin deposition, and lysosomal acid lipase. Deficiency syndromes, tendon xanthomas, X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher disease, Stargardt disease, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and / or hypercholesterolemia or hypercholesterolemia-related dementia. Preferred cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers are selective for 7KC (compared to cholesterol). Preferably, the CD dimer preferentially solubilizes 7KC while minimizing or avoiding potentially harmful or toxic effects that may result from excessive removal of cholesterol.

[0013] Exemplary embodiments of the present invention provide the use of cyclodextrin (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) dimers for solubilization and / or removal at 7KC, said use being applicable to in vitro or in vivo conduct.

[0014] In exemplary embodiments, the cyclodextrin dimers (e.g., HPβCD, MeβCD, SUCCβCD, QAβCD, or SBβCD) exhibit a greater binding affinity for 7KC and / or a greater solubilizing effect than that for cholesterol. At subsaturated concentrations, the specificity for 7KC relative to cholesterol is most pronounced, while at higher concentrations, the solubilization of both sterols can approach 100%. This specificity allows the use of such cyclodextrin dimers to preferentially solubilize and remove 7KC.

[0015] In an exemplary embodiment, this disclosure provides a cyclodextrin dimer having the following structure:

[0016] CD—L—CD

[0017] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0018] Where CD has the structure of formula X:

[0019] (Formula X)

[0020] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the 8 atoms are preferably each of C, N, O or S;

[0021] Furthermore, the CD is composed of between 1 and 40 groups, such as between 1 and 28 groups, optionally between 2 and 15 groups, or between 4 and 20 groups. The substitution number refers to the non-H R... 1 R 2 and / or R 3 The total number of groups. The CD may have one or more additional substitutions.

[0022] The R 1 R 2 and R 3 Each group can be independently selected from H, methyl, hydroxypropyl, sulfonyl, succinyl, quaternary ammonium such as -CH2CH(OH)CH2N(CH3)3 +Alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, azide, bromine, chlorine, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl The group includes cycloalkyl, alkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclic alkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic group, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodine, urea, carbamate, carboxyl, sulfate, sulfonyl, sulfonamide, nitro, nitrite, cyano, phosphate, phosphoryl, phenoxy, acetyl group, fatty acid such as palmitoyl group, monosaccharide or disaccharide. In an exemplary embodiment, the substitution is preferably a maltose group or a carboxymethyl group.

[0023] In an exemplary implementation, the R 1 R 2 and / or R 3 The functional groups can be independently selected from H, methyl, hydroxypropyl, sulfonyl, succinoyl, maltose, carboxymethyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3). + ), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azide, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromine, chlorine, wherein one or more of the following are present: between 1 and 40, such as between 1 and 28, or optionally between 2 and 15, or between 4 and 20. 1 R 2 and R 3 The group is not H.

[0024] In an exemplary implementation, the R 1 R 2 and R 3 The functional groups can be independently selected from H, methyl, hydroxypropyl, sulfonyl, succinoyl, maltose, carboxymethyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3). + ), where the number of R is between 1 and 40, such as between 1 and 28. 1R 2 and R 3 The R group is not H, and optionally consists of between 2 and 15 or between 4 and 20 groups. 1 R 2 and R 3 The group is not H. The R group... 1 R 2 and R 3 The group may include one or more maltodextrin or carboxymethyl groups.

[0025] In another exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0026] CD—L—CD

[0027] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0028] Where CD has the structure of formula X:

[0029] (Formula X)

[0030] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the not more than 8 atoms are preferably each of C, N, O or S;

[0031] The CD is substituted with hydroxypropyl (HP) groups between 1 and 28, optionally between 2 and 15, or between 4 and 20, preferably between 2 and 5, and optionally the CD has one or more additional substitutions. The CD may contain between 2 and 4 HP groups, or may contain 2, 3, 4, or 5 HP groups.

[0032] In another exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0033] CD—L—CD

[0034] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0035] Where CD has the structure of formula X:

[0036] (Formula X)

[0037] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the 8 atoms are preferably each of C, N, O or S;

[0038] The CD is methylated by between 1 and 40 Me groups, optionally between 1 and 28 Me groups, optionally between 2 and 15 Me groups, or preferably between 4 and 20 Me groups, and optionally between 2 and 10 Me groups, and optionally the CD has one or more additional substitutions. Not wishing to be theoretically limited, it is believed that the methyl groups are particularly suitable for being substituted with a large number of substituents on such CD dimers because the methyl groups are particularly small and therefore do not interfere with the entry of guests (such as 7KC or cholesterol) into the CD dimer binding cavity. Additionally, it is contemplated that one or more methyl substitutions may be added to any cyclodextrin dimer of this disclosure, including in amounts greater than those specified in the general formula herein, for example, up to a total of 40 non-hydrogen substituents when both non-methyl substituents and added methyl substituents are included.

[0039] In another exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0040] CD—L—CD

[0041] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0042] CD has the structure of formula X:

[0043] (Formula X)

[0044] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the 8 atoms are preferably each of C, N, O or S;

[0045] The CD is substituted with 1 to 28 sulfobutyl groups, such as 1 to 14 sulfobutyl groups, optionally 2 to 10 sulfobutyl groups, preferably 2 to 5 sulfobutyl groups, and optionally the CD has one or more additional substitutions. The CD may have 2 to 4 sulfobutyl groups, or may have 2, 3, 4, or 5 sulfobutyl groups.

[0046] In another exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0047] CD—L—CD

[0048] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0049] CD has the structure of formula X:

[0050] (Formula X)

[0051] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the 8 atoms are preferably each of C, N, O or S;

[0052] The CD is succinyl-substituted with between 1 and 28 succinyl groups, optionally between 2 and 15 succinyl groups, or between 4 and 20 succinyl groups, preferably between 2 and 5 succinyl groups, and optionally the CD has one or more additional substitutions. The CD may contain between 2 and 4 succinyl groups, or may contain 2, 3, 4, or 5 succinyl groups.

[0053] In another exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0054] CD—L—CD

[0055] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0056] CD has the structure of formula X:

[0057] (Formula X)

[0058] Wherein, in the shortest path through the connector connecting the two CD subunits of the dimer, the length of L does not exceed 8 atoms, wherein the 8 atoms are preferably each of C, N, O or S;

[0059] The CD is substituted with between 1 and 28 quaternary ammonium groups, optionally between 2 and 15 quaternary ammonium groups, or between 4 and 20 quaternary ammonium groups, preferably between 2 and 5 quaternary ammonium groups, wherein the quaternary ammonium groups include -CH2CH(OH)CH2N(CH3)3 + Such as -CH2CH(OH)CH2N(CH3)3Cl, and optionally the CD has one or more additional substitutions. The CD may contain between two and four quaternary ammonium groups, or may contain two, three, four, or five quaternary ammonium groups. It should be understood that any pharmaceutically acceptable salt of the quaternary ammonium is included within the scope of this disclosure.

[0060] L can have the following structure:

[0061]

[0062] Each R is independently selected from H, X, SH, NH, NH2, or OH, or may not exist;

[0063] The connection between each CD and the connector is achieved independently through the O, S, or N of the C2 or C3 carbons connected to them, or through the acetal connection of two adjacent oxygens of the CD.

[0064] Each X is a substituted or unsubstituted alkane, alkene, or alkyne;

[0065] Each A is independently selected from single, double, or triple covalent bonds, S, N, NH, O, or substituted or unsubstituted alkanes, alkenes, or alkynes; and

[0066] B is a substituted or unsubstituted 5- or 6-membered ring, S, N, NH, NR, O, or it is not present.

[0067] The length of the connector can be between 2 and 7, between 3 and 6, between 4 and 7, between 4 and 6, between 4 and 5, or 4, or between 2 and 3.

[0068] The connector can be an unsubstituted alkyl group, such as an unsubstituted butyl group.

[0069] The connector may be a substituted or unsubstituted butyl connector.

[0070] The connector may include triazole.

[0071] The connector may include the following structure: -(CH2) n1 (CH2) n2- (Formula XI), where n1 and n2 are each between 1 and 8 or between 1 and 4, preferably where n1 is 1 and n2 is 3.

[0072] In an exemplary embodiment, when the connector L comprises a triazole, for example having the structure of formula XI, the connector L can be connected to the O2 position of each CD monomer, wherein n1 and n2 can each be between 0 and 8, such as between 1 and 4; preferably, the total length of the connector can be 8 or less, such as 8, 7, 6, 5, 4, 3 or any numerical range thereof; and in a preferred embodiment, n1 is 1 and n2 is 3.

[0073] In an exemplary embodiment, when the connector L comprises a substituted or unsubstituted alkyl group, preferably having a length not exceeding 8 atoms, such as between 2 and 7, between 2 and 6, or between 4 and 7, or between 4 and 6, or between 4 and 5, or with a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range thereof, the connector L may be connected to the O2 position of each CD monomer, the O2 position of one CD monomer and the O3 position of another CD monomer, or the O3 positions of two CD monomers; wherein preferably the connector is a substituted or unsubstituted butyl group, more preferably an unsubstituted butyl group.

[0074] The connector may include a single connection point with each CD player. The connector may include a single connection point with one CD player and multiple (two or more) connection points with another CD player. The connector may include multiple connection points with each CD player (two or more connection points per CD player). The connector may include... Figure 8D Any of the connectors shown. It should be understood that the connectors shown include oxygen atoms at each end, which form part of the cyclodextrin to which they are attached; these oxygen atoms are not considered part of the connector for determining the length of the connector. Furthermore, regarding connectors that connect to one or two cyclodextrin monomers at multiple locations, the connection shown on the left connects to one monomer, while the connection shown on the right connects to another monomer.

[0075] In an exemplary embodiment, this disclosure provides a CD dimer having the following structure:

[0076] CD—L—CD

[0077] Where L is through the C2 carbon of each CD subunit (in place of R) 1 ) and / or C3 carbon (in place of R) 2 ) are attached to the large (sub)surface of each CD molecule;

[0078] CD has the structure of formula X:

[0079]

[0080] (Formula X)

[0081] Wherein L is a triazole and has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each of C, N, O or S;

[0082] The CD is substituted with between 0 and 28 groups, optionally with 0 groups, or optionally the CD has one or more substitutions.

[0083] The connector may include the following structure: -(CH2) n1 (CH2) n2 - (Formula XI), where n1 and n2 are each between 1 and 8 or between 1 and 4, preferably where n1 is 1 and n2 is 3.

[0084] The length of the connector can be between 3 and 7, between 3 and 6, between 4 and 7, between 4 and 6, or between 5 and 6.

[0085] The length of the connector can be between 4 and 5.

[0086] The cyclodextrin may be further substituted with the following groups: (a) at least one methyl, hydroxypropyl, sulfobutyl, or succinyl group, and / or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl, heteroalkyl, heteroaryl, heteroaryl Arylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclic alkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic group, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, urea, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, acetyl group, ammonium, ammonia, azide, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, sulfonyl, nitrite, phosphate, phosphoryl, fatty acid such as palmitoyl group, monosaccharide or disaccharide, and / or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltose, carboxymethyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3) + ), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azide, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromine or chlorine group.

[0087] Cyclodextrin dimers can have properties according to formulas I-IX (as shown below) Figure 3B-3J The structure of any of them (as shown).

[0088] Each R 1 Each R 2 and each R 3It may be independently selected from (a) methyl, H, hydroxypropyl, sulfobutyl ether, succinoyl, succinoyl-hydroxypropyl, quaternary ammonium, carboxymethyl, carboxymethyl-hydroxypropyl, hydroxyethyl, maltose, acetyl, carboxyethyl, sulfate, sulfopropyl, sodium phosphate, or glucosyl; and / or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylamino alkylsulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl, cycloalkylenealkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocycloalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic, heterocyclic alkyl, heterocyclic alkyl, heterocyclic alkyloxy, heterocyclic alkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, urea, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy or acetyl group.

[0089] L can be attached to the C2 carbon of each CD monomer, to the C3 carbon of each CD monomer, or to the C2 carbon of one CD monomer and the C3 carbon of another CD monomer. For adapters having multiple attachment points to a single CD monomer, these attachment points can be attached to the C2, C3, or a combination of C2 and C3 carbons of that monomer; specific arrangements may be advantageous depending on the reaction, purification steps employed in their formation, and / or on the structure of the adapter.

[0090] The cyclodextrin dimer can exhibit a greater affinity for 7KC than for cholesterol. This greater affinity can be determined using the turbidity test disclosed herein.

[0091] The cyclodextrin dimer may exhibit an affinity for 7KC that is at least 1.1, 1.5, 2, 3, 4, 5, or 10 times greater than that for cholesterol. In turbidity tests, the cyclodextrin dimer may exhibit a relative turbidity of 7KC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more lower than that of cholesterol.

[0092] In an exemplary embodiment, this disclosure provides a composition comprising a mixture of cyclodextrin dimers as disclosed herein, wherein optionally the average degree of substitution may be between 2 and 10, such as between 2 and 8, such as between 3 and 7, or between 2 and 5. The composition may comprise a mixture of CD dimers having a degree of substitution of hydroxypropyl, sulfonyl, succinic, or quaternary ammonium groups between 2 and 5, such as about 2, about 3, about 4, or about 5 of the said substituents. The composition may comprise a mixture of CD dimers having a degree of substitution of methyl groups between 2 and 10. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry (such as MALDI).

[0093] In exemplary embodiments, this disclosure provides a cyclodextrin dimer comprising, for example, according to formulas I-III (each as follows) Figure 3B-3D A composition of a mixture of cyclodextrin dimers (as shown).

[0094] In exemplary embodiments, this disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer or a combination thereof as disclosed herein, and a pharmaceutically acceptable carrier. The cyclodextrin dimer may be the sole active ingredient in the composition. The pharmaceutical composition may consist of, or substantially consist of, the cyclodextrin dimer and the pharmaceutically acceptable carrier.

[0095] In an exemplary embodiment, this disclosure provides a treatment method comprising administering to a subject in need an effective amount of a cyclodextrin dimer, as disclosed herein, or a combination thereof. The subject in need may suffer from the harmful or toxic effects of 7KC.

[0096] In an exemplary embodiment, this disclosure provides a method for reducing the amount of 7KC in a subject in need, the method comprising administering an effective amount of a cyclodextrin dimer as disclosed herein to the subject in need.

[0097] The cyclodextrin dimer can be administered to the patient via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenously.

[0098] The method may include administering the cyclodextrin dimer to the patient in amounts between about 1 mg and 10 g, such as between 10 mg and 1 g, between 50 mg and 200 mg, or 100 mg. In an exemplary embodiment, the cyclodextrin dimer may be administered in amounts between 1 g and 10 g, such as about 2 g, about 3 g, about 4 g, or about 5 g. In an exemplary embodiment, the cyclodextrin dimer may be administered in amounts between 50 mg and 5 g, such as between 100 mg and 2.5 g, between 100 mg and 2 g, between 250 mg and 2.5 g, for example, about 1 g.

[0099] The method described can prevent, treat, and / or improve symptoms of one or more of the following conditions: atherosclerosis, arteriosclerosis, coronary atherosclerosis due to calcified coronary artery disease, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Limeley-Optz syndrome, infantile neuronal ceroid lipofuscin deposition, lysosomal acid lipase deficiency, and cerebral tendon xanthomas. X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher disease, Staggart disease, age-related macular degeneration (dry), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and / or hypercholesterolemia, preferably atherosclerosis.

[0100] The method may further include administering a second therapy to the patient, wherein the second therapy may be administered simultaneously or sequentially in any order.

[0101] The second therapy may include one or more of the following: anticholesterol drugs (such as fibrates or statins), antiplatelet drugs, antihypertensive drugs, or dietary supplements. The statin drugs mentioned may include ADVICOR(R) (nicotinic acid extended-release / lovastatin), ALTOPREV(R) (lovastatin extended-release), CADUET(R) (amlodipine and atorvastatin), CRESTOR(R) (rosuvastatin), JUVISYNC(R) (sitagliptin / simvastatin), LESCOL(R) (fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR(R) (atorvastatin), LIVALO(R) (pitavastatin), MEVACOR(R) (lovastatin), PRAVACHOL(R) (pravastatin), SIMCOR(R) (nicotinic acid extended-release / simvastatin), and VYTORIN(R). (ezetimibe / simvastatin) or ZOCOR(R) (simvastatin).

[0102] The second therapy may include anticholesterol drugs and antihypertensive drugs.

[0103] In an exemplary embodiment, this disclosure provides a method for purifying an oxosterol, the method comprising: contacting a composition containing an oxosterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxosterol in the cyclodextrin dimer; and recovering the cyclodextrin dimer and the solubilized oxosterol. The oxosterol comprises or is composed of 7KC. The method may further include measuring the concentration of 7KC in the solubilized oxosterol to determine the relative concentration of 7KC in the composition. The composition may include a patient sample. The method may be used to determine the concentration of 7KC in a patient sample, the 7KC concentration being used for diagnostic and / or treatment planning.

[0104] In an exemplary embodiment, this disclosure provides a method for removing oxosterols from a sample. in vitro A method comprising: contacting a sample containing an oxosterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxosterol in the cyclodextrin dimer; and separating the sample from the cyclodextrin dimer and the solubilized sterol.

[0105] In an exemplary embodiment, this disclosure provides a method for producing a cholesterol-reduced product, the method comprising: contacting a cholesterol-containing product with a cyclodextrin dimer as disclosed herein, thereby solubilizing the cholesterol in the cyclodextrin dimer; and removing the cyclodextrin dimer and the solubilized cholesterol from the product. The product may be a food product, such as meat and / or milk.

[0106] In another aspect, this disclosure provides a method for preparing cyclodextrin dimers as described herein (such as cyclodextrin dimers comprising unsubstituted or substituted alkyl links), the method comprising: (a) reacting a β-cyclodextrin protected on a major side with a dialkylating agent to generate a major side-protected βCD dimer linked by a subsurface, and optionally purifying the major side-protected βCD dimer; (b) deprotecting the major side-protected βCD dimer to generate a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD to generate a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. The β-cyclodextrin protected on the major side may include hepta(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may include a dibromoalkane, optionally 1,4-dibromobutane. Step (a) can be carried out under anhydrous conditions and / or with sodium hydride as a base. The purification in step (a) may include direct phase chromatography and isocratic elution. Step (b) may be carried out in tetrahydrofuran (THF) with tetrabutylammonium fluoride. The purification in step (b) may include direct phase chromatography and isocratic elution. Step (c) may include reacting the deprotected βCD dimer with a hydroxypropylating agent (such as propylene oxide), a methylating agent (such as iodomethane), a succinylation agent (such as succinic anhydride), a sulfobutylating agent (such as 1,4-butanesulfonate lactone), and / or a quaternary ammonium linker (such as glycidyltrimethylammonium chloride).

[0107] Step (c) can be carried out under aqueous conditions, optionally including sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, charcoal clarification, and dialysis.

[0108] In another aspect, this disclosure provides a method for preparing cyclodextrin dimers as described herein (such as cyclodextrin dimers containing a triazole linker), the method comprising: (a) reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkynyl)-βCD to form a βCD-triazole-βCD dimer having the structure βCD-alkyl1-triazole-alkyl2-βCD, and optionally (b) purifying the βCD-triazole-βCD dimer. Step (a) may be carried out using a copper (I) catalyst, optionally about 15 mM copper (I). Step (a) may be carried out in an aqueous solution. The aqueous solution may contain dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-azidoalkyl)-βCD by: (1) reacting an n-azido-1-bromo-alkane with a β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to generate the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-ynyne)-βCD by: (i) reacting an n-bromo-1-ynyne with a β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to generate the 2-O-(n-ynyne)-βCD; and (ii) optionally purifying the 2-O-(n-ynyne)-βCD. Step (2) may include silica gel chromatography. Step (1) may be carried out in dry DMSO. The reaction in step (1) may involve lithium hydride. The βCD-triazole-βCD dimer may contain the following structure: CD-(CH2)n1 (CH2) n2 -CD (Formula XII), wherein n1 can be between 1 and 8, and / or n2 can be between 1 and 8, optionally n1 can be 1, 2, 3 or 4, and / or n2 can be 1, 2, 3 or 4, preferably wherein n1 is 1 and n2 is 3. The length of the triazole linker can be between 5 and 8. The method may further include hydroxypropylating the βCD-triazole-βCD dimer to generate a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. Step (c) may include reacting the βCD-triazole-βCD dimer with a hydroxypropylating agent (such as propylene oxide), a methylating agent (such as iodomethane), a succinylation agent (such as succinic anhydride), a sulfobutylating agent (such as 1,4-butanesulfonate lactone), and / or a quaternary ammonium linker (such as glycidyltrimethylammonium chloride).

[0109] Step (c) can be carried out under aqueous conditions, optionally including sodium hydroxide as a base. The purification in step (c) may include one or more of ion exchange resin treatment, charcoal clarification, membrane filtration, and dialysis.

[0110] Embodiments of the present invention provide compositions and methods for treating or preventing atherosclerosis. 7KC is the most abundant non-enzymatically generated oxosterol in atherosclerotic plaques and is believed to be a contributing factor to the pathogenesis of atherosclerosis. Treatment with the CD dimer of the present invention (such as HPβCD or another CD disclosed herein) is expected to be advantageous for the prevention and / or reversal of atherosclerotic plaque formation.

[0111] Embodiments of the present invention provide compositions and methods for treating or preventing diseases and conditions related to 7KC. These diseases and conditions include, but are not limited to, age-related diseases such as atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis caused by calcified coronary artery lesions, heart failure (all stages), Alzheimer's disease, Parkinson's disease, vascular dementia, chronic obstructive pulmonary disease, non-alcoholic fatty liver disease, and / or hypercholesterolemia or hypercholesterolemia-related dementia. Other sporadic and / or congenital diseases associated with 7KC accumulation include Huntington's disease, multiple sclerosis, Smith-Limeley-Opz syndrome, infantile neuronal ceroid lipofuscin deposition, lysosomal acid lipase deficiency, amyotrophic lateral sclerosis, tendon xanthomas, X-linked adrenoleukodystrophy, sickle cell anemia, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher disease, Staggart disease, idiopathic pulmonary fibrosis, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, ulcerative colitis, Crohn's disease, and other irritable bowel syndromes.

[0112] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a degree of substitution of substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituents are selected from methyl, hydroxypropyl, sulfonyl, succinyl, quaternary ammonium such as -CH2CH(OH)CH2N(CH3)3 +Alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, azide, bromine, chlorine, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclic alkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic, heterocyclic The composition comprises a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or C3 carbon (replacing R2); wherein each CD has the structure X, wherein the substituent is located at one or more of R1, R2 and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably C, N, O or S. The substituent may be carboxymethyl or maltodextrin. The substituent is preferably methyl, hydroxypropyl, sulfonyl, succinyl, or quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3). + The degree of substitution can be determined by NMR. The degree of substitution can also be determined by mass spectrometry (such as MALDI).

[0113] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a degree of substitution of substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituents are selected from methyl, hydroxypropyl, sulfonyl, succinoyl, maltose, carboxymethyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3) +The composition comprises a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has the structure X, wherein the substituent is located at one or more of R1, R2 and / or R3, and wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each of C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0114] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a degree of substitution of substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituents are selected from methyl, hydroxypropyl, sulfonyl, succinoyl, maltodextrin, carboxymethyl, or quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3). + The composition comprises a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each of C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0115] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a degree of substitution of hydroxypropyl substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4. The composition comprises a cyclodextrin dimer having the structure CD—L—CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has a structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0116] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a methyl substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 10, the composition comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each of C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0117] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a sulfobutyl substituent with a degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4. The composition comprises a cyclodextrin dimer having the structure CD—L—CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has a structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0118] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a succinyl substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4. The composition comprises a cyclodextrin dimer having the structure CD—L—CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has a structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0119] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a quaternary ammonium substituent (preferably -CH2CH(OH)CH2N(CH3)3). +The degree of substitution is between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4. The composition comprises a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituent is located at one or more of R1, R2, and / or R3, and wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O, or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0120] In another exemplary embodiment, this disclosure provides a cyclodextrin dimer composition having a degree of substitution between 0 and 40, the composition comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub)surface of each CD molecule via a C2 carbon (replacing R1) and / or a C3 carbon (replacing R2) of each CD subunit; wherein each CD has a structure of formula X, which is optionally substituted by one or more substituents, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O, or S. The cyclodextrin dimer composition can be used to synthesize cyclodextrin dimer compositions substituted by one or more substituents. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry (such as MALDI).

[0121] The connector L may have the following structure:

[0122]

[0123] Each R is independently selected from H, X, SH, NH, NH2, or OH, or may not exist;

[0124] The connection between each CD and the connector is achieved independently through the O, S, or N of the C2 or C3 carbons connected to them, or through the acetal connection of two adjacent oxygens of the CD.

[0125] Each X is a substituted or unsubstituted alkane, alkene, or alkyne;

[0126] Each A is independently selected from single, double, or triple covalent bonds, S, N, NH, O, or substituted or unsubstituted alkanes, alkenes, or alkynes; and

[0127] B is a substituted or unsubstituted 5- or 6-membered ring, S, N, NH, NR, O, or it is not present.

[0128] The length of the connector can be between 2 and 7. The length of the connector can be between 3 and 6. The length of the connector can be 2 or 3. The length of the connector can be between 4 and 7. The length of the connector can be between 4 and 6. The length of the connector can be between 4 and 5. The length of the connector can be 4.

[0129] The connector can be a substituted or unsubstituted alkyl group, such as an unsubstituted alkyl group, for example, an unsubstituted butyl group. The connector may include triazoles.

[0130] The connector may include the following structure: -(CH2) n1 (CH2) n2 - (Formula XI). n1 and n2 can each be between 0 and 8, such as between 1 and 4 respectively. Preferably, the total length of the connector can be 8 or less, such as 8, 7, 6, 5, 4 or any numerical range thereof. In a preferred embodiment, n1 is 1 and n2 is 3.

[0131] In an exemplary embodiment, when the connector L comprises a triazole, for example having the structure of formula XI, the connector can be connected to the O2 position of each CD monomer, wherein n1 and n2 can each be between 0 and 8, such as between 1 and 4; preferably, the total length of the connector can be 8 or less, such as 8, 7, 6, 5, 4 or any numerical range thereof; and in a preferred embodiment, n1 is 1 and n2 is 3.

[0132] In an exemplary embodiment, when the connector L comprises a substituted or unsubstituted alkyl group, preferably having a length not exceeding 8 atoms, such as between 2 and 7, between 2 and 6, or between 4 and 7, or between 4 and 6, or between 4 and 5, or with a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range thereof, the connector L may be connected to the O2 position of each CD monomer, the O2 position of one CD monomer and the O3 position of another CD monomer, or the O3 positions of two CD monomers; wherein preferably the connector is a substituted or unsubstituted butyl group, more preferably an unsubstituted butyl group.

[0133] The connector may include Figure 8D Any of the connectors shown, wherein the oxygen atom shown at each end of each connector forms part of the cyclodextrin monomer connected by the connector.

[0134] The cyclodextrin dimer composition may further comprise the cyclodextrin dimer being substituted with: (a) at least one methyl, hydroxypropyl, sulfonyl, succinoyl, or quaternary ammonium group (such as -CH2CH(OH)CH2N(CH3)3). + (a) and / or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaryloxy Alkyloxy, heterocyclic alkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic group, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, urea, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, acetyl, ammonium, ammonia, azide, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, sulfonyl, nitrite, phosphate, phosphoryl, fatty acids such as palmitoyl group, monosaccharide or disaccharide, and / or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinoyl, maltose, carboxymethyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3) + ), glucosyl, palmitoyl, phosphate, phosphoryl, amino, azide, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromine, chlorine.

[0135] The cyclodextrin dimer composition may contain components having formulas I-IX (as shown in the figures below) Figure 3B-3J Cyclodextrin dimers of any of the structures shown.

[0136] Unless otherwise specified, each R1, each R2, and each R3 may be independently selected from (a) methyl, H, hydroxypropyl, sulfonyl ether, succinoyl, succinoyl-hydroxypropyl, quaternary ammonium (such as -CH2CH(OH)CH2N(CH3)3). +), carboxymethyl, carboxymethyl-hydroxypropyl, hydroxyethyl, maltose, acetyl, carboxyethyl, sulfate, sulfopropyl, sodium phosphate or glucosyl; and / or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylthioalkyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, Aryl, arylalkyl, arylsulfonyl, aryloxy, arylalkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylenealkyl, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocycloalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclic amino, heterocyclic group, heterocyclic alkyl, heterocyclic oxy, heterocyclic alkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, urea, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy or acetyl group.

[0137] The connector L can be connected to the C2 carbon of each CD monomer. The connector L can be connected to the C3 carbon of each CD monomer. The connector L can be connected to the C2 carbon of one CD monomer and the C3 carbon of another CD monomer.

[0138] The cyclodextrin dimer composition can exhibit a greater affinity for 7KC than for cholesterol, wherein optionally the greater affinity is determined by a turbidity test.

[0139] The cyclodextrin dimer composition can exhibit an affinity for 7KC that is at least 1.1, 1.5, 2, 3, 4, 5, or 10 times greater than that for cholesterol. In turbidity tests, the cyclodextrin dimer can exhibit a relative turbidity of 7KC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more lower than that of cholesterol.

[0140] The degree of substitution can be 2. The degree of substitution can be 3. The degree of substitution can be 4. The degree of substitution can be 5. The degree of substitution can be 6. The degree of substitution can be 7. The degree of substitution can be 8. The degree of substitution can be 9. The degree of substitution can be 10.

[0141] The cyclodextrin dimer composition may comprise a mixture of cyclodextrin dimer molecules having different numbers of substituents and / or different linker sites, wherein the average degree of substitution of the composition is specified.

[0142] In another aspect, this disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for administration to a subject, for example, parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenous or subcutaneous administration, more preferably intravenous administration. The cyclodextrin dimer composition may be the sole active ingredient in the composition. The pharmaceutical composition may consist of, or substantially consist of, the cyclodextrin dimer and the pharmaceutically acceptable carrier.

[0143] In another aspect, this disclosure provides a treatment method comprising administering to a subject in need an effective amount of a cyclodextrin dimer composition as disclosed herein. The subject may be suffering from adverse or toxic effects of 7KC or conditions related to such adverse or toxic effects.

[0144] In another aspect, this disclosure provides a method for reducing the amount of 7KC in a subject in need, the method comprising administering to the subject an effective amount of a cyclodextrin dimer composition as disclosed herein or a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein.

[0145] The cyclodextrin dimer composition can be administered to the subject via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenous administration.

[0146] The method may include administering to the subject (a) a cyclodextrin dimer composition between about 1 mg and 20 g, such as between 10 mg and 1 g, between 50 mg and 200 mg, or 100 mg; or (b) a cyclodextrin dimer composition between 1 g and 10 g, such as about 2 g, about 3 g, about 4 g, or about 5 g; or (c) a cyclodextrin dimer composition between 50 mg and 5 g, such as between 100 mg and 2.5 g, between 100 mg and 2 g, or between 250 mg and 2.5 g.

[0147] The method described can be used to prevent, treat, or improve symptoms of one or more of the following diseases: atherosclerosis / coronary artery disease, arteriosclerosis, coronary atherosclerosis due to calcified coronary artery lesions, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Limeley-Opz syndrome, infantile neuronal ceroid lipofuscin deposition disease, lysosomal acid lipase deficiency, and cerebral tendon xanthomas. X-linked adrenoleukodystrophy, sickle cell disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Gaucher disease, Staggart disease, age-related macular degeneration (dry), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and / or hypercholesterolemia; wherein optionally, the treatments described herein are administered in combination with another therapy. The method may include administering a second therapy to the subject, wherein the second therapy is administered simultaneously or sequentially in any order.

[0148] The method can be used to prevent, treat, or improve the symptoms of atherosclerosis. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of atherosclerosis, such as anticholesterol drugs, antihypertensive drugs, antiplatelet drugs, dietary supplements, or surgical or behavioral interventions, including but not limited to those described herein. The anticholesterol drugs may include fibrates or statins, antiplatelet drugs, antihypertensive drugs, or dietary supplements. The statins may include ADVICOR(R) (nicotinic acid extended-release / lovastatin), ALTOPREV(R) (lovastatin extended-release), CADUET(R) (amlodipine and atorvastatin), CRESTOR(R) (rosuvastatin), JUVISYNC(R) (sitagliptin / simvastatin), LESCOL(R) (fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR(R) (atorvastatin), LIVALO(R) (pitavastatin), MEVACOR(R) (lovastatin), PRAVACHOL(R) (pravastatin), SIMCOR(R) (nicotinic acid extended-release / simvastatin), VYTORIN(R) (ezetimibe / simvastatin), or ZOCOR(R) (simvastatin).

[0149] The method can be used to prevent, treat, or improve symptoms of dry age-related macular degeneration. The method can also be used to prevent, treat, or improve symptoms of Staggart's disease. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of dry AMD or Staggart's disease, such as LBS-008 (Belite Bio) (a non-retinoic acid antagonist of retinol-binding protein 4), AREDS supplement formulations (containing vitamins C and E, beta-carotene, zinc, and copper), or AREDS2 supplement formulations (including supplement formulations containing vitamins C and E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof).

[0150] The method can be used to prevent, treat, or improve the symptoms of Niemann-Pick disease. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of Niemann-Pick disease, such as miglustat (ZAVESCA(R)), HPβCD (TRAPPSOL CYCLO, VTS-270), and one or more physical therapies.

[0151] The method can be used to prevent, treat, or improve the symptoms of Alzheimer's disease. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of Alzheimer's disease, such as cholinesterase inhibitors (ARICEPT(R), EXELON(R), RAZADYNE(R)) and NAMENDA(R)) or combinations thereof.

[0152] The method can be used to prevent, treat, or improve symptoms of heart failure. The cyclodextrin dimer composition can be administered in combination with another therapy for the treatment or prevention of heart failure, such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNIs (angiotensin receptor-neprilysin inhibitors), β-blockers, vasodilators, calcium channel blockers, digoxin, diuretics, heart pump drugs, potassium, magnesium, selective sinoatrial node inhibitors, or combinations thereof.

[0153] In another aspect, this disclosure provides a method for preparing cyclodextrin dimer compositions as described herein (such as cyclodextrin dimer compositions comprising unsubstituted or substituted alkyl links), the method comprising: (a) reacting a β-cyclodextrin protected on a major side with a dialkylating agent to generate a major side-protected βCD dimer linked by a subsurface, and optionally purifying the major side-protected βCD dimer; (b) deprotecting the major side-protected βCD dimer to generate a deprotected βCD dimer, and optionally purifying the deprotected βCD dimer; and (c) hydroxypropylating the deprotected βCD to generate a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. The β-cyclodextrin protected on the major side may include hepta(6-O-tert-butyldimethylsilyl)-β-cyclodextrin. The dialkylating agent may include a dibromoalkane, optionally 1,4-dibromobutane. Step (a) can be carried out under anhydrous conditions and / or with sodium hydride as a base. The purification in step (a) may include direct phase chromatography and isocratic elution. Step (b) may be carried out in tetrahydrofuran (THF) with tetrabutylammonium fluoride. The purification in step (b) may include direct phase chromatography and isocratic elution. Step (c) may include reacting the deprotected βCD dimer with a hydroxypropylating agent (such as propylene oxide), a methylating agent (such as iodomethane), a succinylation agent (such as succinic anhydride), a sulfobutylating agent (such as 1,4-butanesulfonate lactone), and / or a quaternary ammonium linker (such as glycidyltrimethylammonium chloride). The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have substituents with a degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5 or between 2 and 10.

[0154] Step (c) can be carried out under aqueous conditions, optionally including sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, charcoal clarification, and dialysis.

[0155] In another aspect, this disclosure provides a method for preparing cyclodextrin dimer compositions as described herein (such as cyclodextrin dimer compositions comprising a triazole linker), the method comprising: (a) reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkynyl)-βCD to form a βCD-triazole-βCD dimer having the structure βCD-alkyl1-triazole-alkyl2-βCD; and optionally (b) purifying the βCD-triazole-βCD dimer. Step (a) may be carried out using a copper (I) catalyst, optionally about 15 mM copper (I). Step (a) may be carried out in an aqueous solution. The aqueous solution may contain dimethylformamide (DMF), optionally about 50% DMF (v / v). Step (b) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-azidoalkyl)-βCD by: (1) reacting an n-azido-1-bromo-alkane with a β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to generate the 2-O-(n-azidoalkyl)-βCD; and (2) optionally purifying the 2-O-(n-azidoalkyl)-βCD. Step (2) may include chromatography. The method may further include, prior to step (a), generating the 2-O-(n-ynyne)-βCD by: (i) reacting an n-bromo-1-ynyne with a β-cyclodextrin, optionally using a catalytic amount of lithium iodide, to generate the 2-O-(n-ynyne)-βCD; and (ii) optionally purifying the 2-O-(n-ynyne)-βCD. Step (2) may include silica gel chromatography. Step (1) may be carried out in dry DMSO. The reaction in step (1) may involve lithium hydride. The βCD-triazole-βCD dimer composition may contain the following structure: CD-(CH2) n1 (CH2) n2-CD (Formula XII), wherein n1 can be between 1 and 8, and / or n2 can be between 1 and 8, optionally n1 can be 1, 2, 3 or 4, and / or n2 can be 1, 2, 3 or 4, preferably wherein n1 is 1 and n2 is 3. The length of the triazole linker can be between 5 and 8. The method may further include hydroxypropylating the βCD-triazole-βCD dimer composition to produce a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. Step (c) may include reacting the βCD-triazole-βCD dimer with a hydroxypropylating agent (such as propylene oxide), a methylating agent (such as iodomethane), a succinylation agent (such as succinic anhydride), a sulfobutylating agent (such as 1,4-butanesulfonate lactone), and / or a quaternary ammonium linker (such as glycidyltrimethylammonium chloride). The cyclodextrin dimer composition may be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have substituents with a degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5 or between 2 and 10.

[0156] Step (c) can be carried out under aqueous conditions, optionally including sodium hydroxide as a base. The purification in step (c) may include one or more of ion exchange resin treatment, charcoal clarification, membrane filtration, and dialysis.

[0157] In another aspect, this disclosure provides a pharmaceutical composition comprising a dimer of the CD (such as HPβCD or another CD of this disclosure).

[0158] In another aspect, this disclosure provides pharmaceutical compositions comprising a cyclodextrin dimer as disclosed herein and a hydrophobic drug. The hydrophobic drug may include hormones or sterols, such as estrogens, estrogen analogs, etc. The cyclodextrin dimer may be present in an amount that effectively solubilizes the hydrophobic drug.

[0159] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for entry into a living organism or living tissue, preferably without significant toxicity, irritation, or allergic reactions, to a reasonable extent of medical judgment. The present invention includes methods comprising administering a cyclodextrin dimer to a patient, wherein the cyclodextrin dimer is contained within a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with pharmaceutically acceptable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerability, etc. Many suitable formulations may be present in formulations known to pharmaceutical chemists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbon wax (polyethylene glycol of various molecular weights) emulsions, semi-solid gels, and semi-solid mixtures containing carbon waxes. See also (Powell […]). et al. ], J. Pharm. Sci. Technol. ,52:238-311, (1998)).

[0160] As used herein, the phrase "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or stearic acid), or a solvent encapsulation material for introducing an active agent into the body. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating material (such as lecithin), in terms of the dispersion, by maintaining the desired particle size, and by using a surfactant.

[0161] Other examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, (10) Safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (11) Diols, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Atherless water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) pH buffer solutions; (22) Polyesters, polycarbonates, and / or polyanhydrides; and (23) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0162] Various excipients (such as wetting agents, emulsifiers, lubricants (e.g., sodium dodecyl sulfate and magnesium stearate), colorants, separating agents, coating agents, sweeteners, flavoring agents, preservatives, and antioxidants) may also be included in the pharmaceutical composition. Some examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. In some embodiments, the pharmaceutical formulation comprises excipients selected from, for example, cellulose, liposomes, micelle forming agents (e.g., bile acids) and polymer carriers (e.g., polyesters and polyanhydrides). In addition to the active compound, the suspension may also contain suspending agents, such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and astragalus gum, and mixtures thereof. Prevention of microbial action on the active compound can be ensured by incorporating various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). Including isotonic agents (such as sugars, sodium chloride, etc.) in the composition is also desirable. Furthermore, prolonged absorption of injectable drug forms can be achieved by incorporating agents that delay absorption (such as aluminum monostearate and gelatin).

[0163] The pharmaceutical formulations of the present invention can be prepared by any of the methods known in the pharmaceutical field. The amount of the active ingredient (i.e., a CD dimer, such as HPβCD dimer or another CD dimer of this disclosure) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. The amount of the active compound can range from about 0.1% to 99.9%, more generally from about 80% to 99.9%, and more generally from about 99%. The amount of the active compound can range from about 0.1% to 99%, more generally from about 5% to 70%, and more generally from about 10% to 30%. In one exemplary embodiment, a dosage form for intravenous administration in an aqueous solution having a concentration between 0.5% and 0.001%, such as between 0.12% and 0.0105%, for example, about 0.01% (w / v). In one exemplary embodiment, a dosage form for intravenous administration in an aqueous solution having a concentration between 2.5% and 0.25%, such as between 2% and 0.5%, for example, about 1% (w / v). In one exemplary embodiment, the dosage form provides intravenous administration of up to 500 mL of a 1% solution (w / v), thereby producing a dose of up to 5 grams.

[0164] In an exemplary embodiment, the cyclodextrin dimer may be administered to a patient in an amount between 1 mg and 10 g, such as between 10 mg and 1 g, or between 100 mg and 500 mg. In an exemplary embodiment, about 400 mg of the cyclodextrin dimer may be administered. In an exemplary embodiment, the cyclodextrin dimer may be administered in an amount between 1 g and 10 g, such as about 2 g, about 3 g, about 4 g, or about 5 g. In an exemplary embodiment, the cyclodextrin dimer may be administered in an amount between 50 mg and 5 g, such as between 100 mg and 2.5 g, between 100 mg and 2 g, between 250 mg and 2.5 g, for example, about 1 g.

[0165] Exemplary embodiments provide a single dosage form that may contain the aforementioned amount of cyclodextrin dimer, which may be packaged for single administration and optionally additionally contain a pharmaceutically acceptable carrier or excipient. The total amount of the cyclodextrin dimer in the single dosage form may be provided as described above, for example, between 1 mg and 10 g, such as between 10 mg and 1 g, between 100 mg and 500 mg, between 1 g and 10 g of cyclodextrin dimer, between about 50 mg and 5 g, between 100 mg and 2.5 g, between 100 mg and 2 g, between 250 mg and 2.5 g, such as about 1 g, 2 g, about 3 g, about 4 g, or about 5 g.

[0166] Formulations of the present invention suitable for oral administration may be in the form of capsules, flat capsules, pills, tablets, lozenges (using flavoring agents, typically sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions, or as elixirs or syrups, or as soft lozenges (using an inert matrix, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes, etc., each form containing a predetermined amount of the compound of the present invention as the active ingredient. The active compound may also be administered in the form of large pills, granules, or pastes.

[0167] Methods for preparing these formulations or compositions generally include the step of blending the compounds of the present invention with a carrier and optionally one or more excipients. With regard to solid dosage forms (e.g., capsules, tablets, pills, powders, granules, tablets containing tablets, etc.), the active compound may be blended with a subdivided solid carrier and is typically shaped such as by pelleting, tableting, granulation, pulverization, or coating. Typically, solid carriers may include, for example, sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicate; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; (5) slowing agents, such as paraffin; (6) (7) Absorption enhancers, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (8) wetting agents, such as, for example, cetyl alcohol, glyceryl monostearate and nonionic surfactants; (9) absorbents, such as kaolin and bentonite; (10) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (11) colorants; and (22) controlled-release agents, such as crospovidone or ethyl cellulose. For capsules, tablets and pills, pharmaceutical compositions may also contain buffers. Similar types of solid compositions may also be used as fillers in soft-shell and hard-shell gelatin capsules using excipients such as lactose or toffee and high molecular weight polyethylene glycol.

[0168] Tablets can be prepared by compression or molding, optionally using one or more excipients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants, or dispersants.

[0169] Other solid dosage forms of tablets and active agents, such as capsules, pills, and granules, may optionally be prepared using coatings and shells (such as enteric coatings and other coatings well known in the field of pharmaceutical formulation). Dosage forms may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, varying proportions of hydroxypropyl methylcellulose (to provide a desired release profile), other polymer matrices, liposomes, and / or microspheres. Alternatively, dosage forms may be formulated for rapid release, such as lyophilization.

[0170] Typically, the dosage form must be sterile. For this purpose, the dosage form can be sterilized, for example, by filtration with a bacterial trapping filter, or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved in sterile water or some other sterile injection medium before use. The pharmaceutical composition may also contain a light-blocking agent, and may be a light-blocking agent of a composition that optionally releases one or more active ingredients in a delayed manner in certain portions of the gastrointestinal tract, either alone or preferably. Examples of embedding compositions that can be used include polymeric substances and waxes. The active compound may also be (if suitable) in a microencapsulated form having one or more of the excipients described above.

[0171] Liquid dosage forms are typically pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, or elixirs of the active ingredient. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizers, and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, as well as mixtures thereof.

[0172] Dosage forms specifically intended for topical or transdermal application may be, for example, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, or patches. Ophthalmic formulations, such as ophthalmic ointments, powders, solutions, etc., are also contemplated herein. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that may be necessary. In addition to the active compound of the present invention, topical or transdermal dosage forms may also contain one or more excipients, such as those selected from animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth gum, cellulose derivatives, polyethylene glycols, organosilicones, bentonite, silicic acid, talc, and zinc oxide, and mixtures thereof. Sprays may also contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane or propane).

[0173] For the purposes of this invention, transdermal patches can provide the advantage of allowing controlled delivery of the compounds of this invention into the body. Such dosage forms can be prepared by dissolving or dispersing the compounds in a suitable medium. Absorption enhancers may also be included to increase the flux of the compounds across the skin. The rate of this flux can be controlled by providing a rate-controlled membrane or by dispersing the compounds in a polymer matrix or gel.

[0174] Pharmaceutical compositions of the present invention suitable for parenteral administration typically comprise one or more of the compounds of the present invention in combination with one or more of the following: pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions before use, which may contain sugars, alcohols, antioxidants, buffers, antibacterial agents or solutes that make the formulation isotonic with the blood of the intended recipient.

[0175] In some cases, to prolong the action of a drug, it may be desirable to slow its absorption after subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous substance. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, delayed absorption of parenteral administration can be achieved by dissolving or suspending the drug in an oil-based medium.

[0176] Injectable reservoir formulations can be prepared by forming microcapsule matrices of active compounds within biodegradable polymers, such as polylactide-polyglycolic acid. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir-type injectable formulations can also be prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions.

[0177] Pharmaceutical compositions can also be in the form of microemulsions. In microemulsion form, the bioavailability of the active agent can be improved. See (Dorunoo [ et al. ], Drug Development and Industrial Pharmacy ,17(12):1685-1713 (1991)) and (Sheen [ et al. ], J. Pharm. Sci. ,80(7):712-714, (1991)), the content of that document is incorporated into this paper in its entirety by way of citation.

[0178] The pharmaceutical composition may also contain micelles formed from the compounds of the present invention and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. In some embodiments, the micelles have an average diameter of less than about 50 nm, or an average diameter of less than about 30 nm, or an average diameter of less than about 20 nm.

[0179] While any suitable amphiphilic carriers are considered herein, amphiphilic carriers are generally those that have been granted Generally Recognized As Safe (GRAS) status and that both solubilize the compounds of the invention and microemulsify them later when the solution comes into contact with a complex aqueous phase, such as a complex aqueous phase present in living biological tissue. Typically, amphiphilic components that meet these requirements have an HLB (hydrophilic-lipophilic balance) value of 2-20 and their structure contains straight-chain aliphatic groups in the C-6 to C-20 range. Some examples of amphiphilic agents include polyethylene glycol-modified fatty glycerides and polyethylene glycol.

[0180] Particularly preferred amphiphilic carriers are saturated and monounsaturated polyethylene glycol-modified fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. These oils can advantageously consist of triglycerides, diglycerides, and monoglycerides of the corresponding fatty acids, with particularly preferred fatty acid compositions including 4%-10% decanoic acid, 3%-9% decanoic acid, 40%-50% lauric acid, 14%-24% myristic acid, 4%-14% palmitic acid, and 5%-15% stearic acid. Another class of useful amphiphilic carriers includes partially esterified dehydrated sorbitol and / or sorbitol, as well as saturated or monounsaturated fatty acids (SPAN series) or their corresponding ethoxylated analogs (TWEEN series). Specifically, commercially available amphiphilic carriers are envisioned, including the Gelucire® series, Labrafil®, Labrasol® or Lauroglycol®, PEG-monoleate, PEG-dioleate, PEG-monolauroate and diolauroate, lecithin, and polysorbate 80.

[0181] CD (such as HPβCD or another CD disclosed herein) dimer can be administered by any suitable means. Preferred routes of administration include parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration. Administration may be ocular (e.g., in the form of eye drops), intravitreal, retro-orbital, subretinal, or subscleral, and is preferred in relation to ocular disorders such as AMD.

[0182] CD (such as HPβCD or another CD of this disclosure) dimer can be administered to a subject, or can be in vitro Use, for example, by applying to cells or tissues that have been removed from an animal. The cells or tissues can then be introduced into a subject, whether the subject from whom the cells or tissues were removed or another individual (preferably of the same species).

[0183] The subjects receiving treatment (i.e., patients) are typically animals, usually mammals, and preferably humans. Subjects can be non-human animals, including all vertebrates such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. In some embodiments, subjects are livestock such as cattle, pigs, sheep, poultry, and horses, or companion animals such as dogs and cats. Subjects can be genetically male or female. Subjects can be of any age, such as older adults (typically at least or older than 60, 70, or 80 years), subjects in transition from old age to adulthood, adults, subjects in transition from adulthood to pre-adulthood, and pre-adult subjects, including adolescents (e.g., 13 to a maximum of 16, 17, 18, or 19 years), children (typically under 13 years of age or before the onset of puberty), and infants. Subjects can also be humans of any race or genotype.

[0184] This disclosure includes further substitution of the dimer CDs described herein (such as HPβCD or another CD of this disclosure). Chemical modification can be performed before or after dimerization. Chemical modification of cyclodextrins can be performed directly on the natural β-cyclodextrin ring by reacting a chemical reagent (nucleophile or electrophile) with a suitably functionalized cyclodextrin (Adair-Kirk[ et al. ], Nat. Med. ,14(10):1024-5, (2008)); (Khan, [ et al. ], Chem. Rev. ,98(5):1977-1996, (1998)). To date, more than 1,500 cyclodextrin derivatives have been prepared by chemical modification of natural cyclodextrins. Cyclodextrins can also be prepared from pyranose-linked oligopyranosides. From the beginning Synthetic preparation. This synthesis can be accomplished using various chemical reagents or biological enzymes (such as cyclodextrin transglycosylase). An overview of chemically modified cyclodextrins as drug carriers in drug delivery systems is provided in, for example, Stella, [ et al. ], Toxicol. Pathol.,As described in 36(1):30-42, (2008), the disclosure of which is incorporated herein by reference in its entirety. Electroneutral cyclodextrins are described in U.S. Patent Nos. 3,453,259 and 3,459,731, the disclosure of which is incorporated herein by reference in its entirety. Other derivatives include cyclodextrins with cationic properties, as disclosed in U.S. Patent No. 3,453,257; insoluble cross-linked cyclodextrins, as disclosed in U.S. Patent No. 3,420,788; and cyclodextrins with anionic properties, as disclosed in U.S. Patent No. 3,426,011, the disclosure of which is incorporated herein by reference in its entirety. In cyclodextrin derivatives with anionic properties, carboxylic acids, phosphorous acid, hypophosphonic acid, phosphonic acid, phosphoric acid, thiophosphonic acid, thiosulfinic acid, and sulfonic acid have been attached to the parent cyclodextrin, as disclosed, for example, in U.S. Patent No. 3,426,011. Sulfonyl ether cyclodextrin derivatives are also described, for example, in U.S. Patent No. 5,134,127, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the cyclic oligosaccharide may have two or more monosaccharide units replaced by a triazole ring, which can be synthesized via an azide-alkynyl Huisgen cycloaddition reaction (Bodine, [ et al. ], J. Am. Chem. Soc .,126(6):1638-9, (2004)).

[0185] The dimeric cyclodextrins disclosed herein are linked via a linker. Methods for linking CD subunits to the linker are described in the working examples. Other methods for linking CD subunits to the linker are known in the art. (Georgeta [ wait people ], J. Bioact. Compat. Pol., 16:39-48. (2001)), (Liu [ et al. ], Acc. Chem. Res., 39:681-691. (2006)), (Ozmen [ et al. ], J. Mol. Catal. B-Enzym., 57:109-114.(2009)), (Trotta [ et al. ], Compos. Interface ,16:39-48. (2009)), each of these references is incorporated herein by reference in its entirety. For example, a linker group containing a portion that reacts with a hydroxyl group (e.g., a carboxyl group, which can be activated by a carbodiimide) can react with a cyclodextrin to form a covalent bond thereon. In another instance, one or more hydroxyl groups of a cyclodextrin can be activated by known methods (e.g., toluenesulfonation) to react with a reactive group (e.g., an amino group) on the linker.

[0186] Typically, the linker initially contains two reactive moieties that react and bond with each CD monomer. In one embodiment, the linker is first attached to a cyclodextrin to produce a separate linker-cyclodextrin compound, and then the remaining reactive moieties of the linker in the linker-cyclodextrin compound subsequently react with a second cyclodextrin. The second reactive moieties of the linker can be protected during the reaction of the first reactive group, although protection may not be required if the first and second reactive moieties of the linker react differently with the two molecules. The linker can react with two molecules simultaneously to link them together. In other embodiments, the linker may have additional reactive groups for attachment to other molecules.

[0187] Many connectors are known in the art. Such connectors can be used to link any of a number of groups together when the group has or has been functionalized to have a group that can react and connect with a reactive connector. Some groups capable of reacting with a bireactive connector include amino, thiol, hydroxyl, carboxyl, ester, and haloalkyl groups. For example, when each group to be linked has at least one amino group, an amino-amino coupling agent can be used to link a cyclic oligosaccharide to a polysaccharide (or, for example, any of these groups to be linked to a fluorophore or to each other). Some examples of amino-amino coupling agents include diisocyanates, alkyl dihalides, dialdehydes, disuccinimide octanoate (DSS), disuccinimide tartrate (DST), and disulfosuccinimide tartrate (sulfon-DST), all of which are commercially available. In other embodiments, an amino-thiol coupling agent can be used to link a molecule of thiol group to an amino group of another molecule. Some examples of amino-thiol coupling agents include 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid succinimide ester (SMCC) and 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfon-SMCC). In other embodiments, thiol-thiol coupling agents can be used to connect groups having at least one thiol group.

[0188] In some embodiments, the length of the connector is as small as a single atom (e.g., --O--, --CH2--, or --NH--) or two or three atoms (e.g., amino, urea, carbamate, ester, carbonate, sulfone, ethylene, or trimethylene). In other embodiments, the connector provides greater degrees of freedom of movement through lengths of at least four, five, six, seven, or eight atoms, and up to, for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 atoms. Preferred connector lengths are between 2 and 12 atoms, or between 4 and 8 atoms. In an exemplary embodiment, the connector is a C4 alkyl group, which may be unsubstituted. In an exemplary embodiment, the connector comprises a triazole.

[0189] Atherosclerosis

[0190] The exemplary cyclodextrin dimers described herein can be used to prevent or treat diseases such as atherosclerosis. Combinations of cyclodextrin dimers and one or more active agents (such as those described herein, e.g., lipid-lowering agents, such as statins) can be used to treat any atherosclerosis and its signs, symptoms, or complications. Atherosclerosis (also known as atherosclerotic vascular disease or ASVD, or coronary artery disease or CAD) is a condition that thickens the walls of arteries due to the accumulation of fatty substances such as cholesterol. Atherosclerosis is a chronic disease that can remain asymptomatic for decades. It is a syndrome affecting the arteries, a chronic inflammatory response of the arterial walls, thought to be primarily caused by the accumulation of macrophages (white blood cells) and promoted by low-density lipoproteins (a plasma protein carrying cholesterol and triglycerides) when functional high-density lipoprotein (HDL) is unable to properly remove fat and cholesterol from macrophages. It is commonly referred to as arteriosclerosis or occlusion. It is caused by the formation of multiple plaques within the arteries.

[0191] The pathobiology of atherosclerotic lesions is complex, but generally, stable atherosclerotic plaques are asymptomatic, rich in extracellular matrix and smooth muscle cells, while unstable plaques are rich in macrophages and foam cells, and the extracellular matrix separating the lesion from the arterial lumen (also known as the fibrous cap) is usually weak and prone to rupture. Rupture of the fibrous cap exposes thrombus-forming substances (such as collagen) to the circulation, eventually leading to thrombus formation within the lumen. After formation, intraluminal thrombi can completely block the artery (e.g., coronary artery blockage), but more often they detach, enter the circulation, and can eventually block smaller downstream branches, leading to thromboembolism (e.g., stroke is often caused by thrombus formation in the carotid artery). In addition to thromboembolism, chronic dilated atherosclerotic lesions can lead to complete luminal closure. Chronic dilated lesions are usually asymptomatic until the luminal narrowing becomes severe enough to cause insufficient blood supply to one or more downstream tissues, resulting in local ischemia.

[0192] These complications of advanced atherosclerosis are chronic, slowly progressive, and cumulative. In some cases, soft plaques rupture suddenly, leading to the formation of a thrombus that rapidly slows or blocks blood flow, causing the death of tissue supplied by the artery (infarction). Coronary thrombosis of the coronary arteries is also a common complication, which can lead to myocardial infarction. Blockage of an artery in the brain can cause a stroke. In advanced atherosclerotic disease, claudication due to insufficient blood supply to the legs may occur, often caused by a combination of narrowing and clotted aneurysm segments.

[0193] Atherosclerosis can affect the entire arterial tree, but the risk is generally higher in larger, high-pressure vessels such as the coronary arteries, renal arteries, femoral arteries, cerebral arteries, and carotid arteries.

[0194] Signs, symptoms, and complications of atherosclerosis include, but are not limited to, increases in plasma total cholesterol, VLDL-C, LDL-C, free cholesterol, cholesterol esters, triglycerides, phospholipids, and the presence of lesions in the arteries (e.g., plaques), as discussed above. In some cases, increases in cholesterol (e.g., total cholesterol, free cholesterol, and cholesterol esters) can be seen in one or more of the plasma, aortic tissue, and aortic plaques.

[0195] Some individuals may be predisposed to atherosclerosis. Therefore, this disclosure relates to methods of administering cyclodextrin dimers alone or in combination with one or more other therapeutic agents (e.g., lipid-lowering agents, such as statins) to subjects to prevent atherosclerosis or its signs, symptoms, or complications. In some embodiments, subjects predisposed to atherosclerosis may exhibit one or more of the following characteristics: advanced age, family history of heart disease, biological conditions, and high blood cholesterol. In some embodiments, biological conditions include high levels of low-density lipoprotein cholesterol (LDL-C), low levels of high-density lipoprotein cholesterol (HDL-C), hypertension, insulin resistance, diabetes, overweight, obesity, sleep apnea, one or more contributing lifestyle choices and / or one or more contributing habitual behaviors. In some embodiments, behavioral habits include smoking and / or alcohol consumption. In some embodiments, lifestyle choices include an inactive lifestyle and / or high stress levels.

[0196] Exemplary embodiments provide the administration of the cyclodextrin dimer of this disclosure to a patient suffering from atherosclerosis, optionally in combination with one or more other reagents. The patient may exhibit one or more signs or symptoms of atherosclerosis. Atherosclerosis can be diagnosed based on one or more of the following methods: Doppler ultrasound, ankle-brachial index, electrocardiogram, pressure test, angiography (optionally with cardiac catheterization), computed tomography (CT), magnetic resonance angiography (MRA), or other methods that image the arteries or measure blood flow.

[0197] Exemplary embodiments provide combinations of therapies comprising the cyclodextrin dimer of this disclosure with one or more other therapies. These combination therapies for treating atherosclerosis may include the cyclodextrin dimer of this disclosure and another therapy for treating or preventing atherosclerosis, such as an anticholesterol drug, an antihypertensive drug, an antiplatelet drug, a dietary supplement, or a combination of surgery or behavioral intervention, said other therapy including, but not limited to, those described below. Other combination therapies include the CD dimer of this disclosure and another therapy for treating heart failure, said other therapy such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNIs (angiotensin receptor-neprilysin inhibitors), beta-blockers, vasodilators, calcium channel blockers, digoxin, diuretics, heart pump drugs, potassium, magnesium, selective sinoatrial node inhibitors, or combinations thereof. Combination therapies for treating dry age-related macular degeneration (AMD) or Staggart's disease include the CD dimer of this disclosure and another therapy for treating AMD, such as LBS-008 (Belite Bio) (a non-retinoic acid antagonist of retinol-binding protein 4), AREDS supplement formulations (containing vitamins C and E, beta-carotene, zinc, and copper), and AREDS2 supplement formulations (including supplement formulations containing vitamins C and E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof). Combination therapies for treating Alzheimer's disease include the CD dimer of this disclosure and one or more cholinesterase inhibitors (ARICEPT®, EXELON®, RAZADYNE®) and NAMENDA®, or combinations thereof. Combination therapies for Niemann-Pick disease include one or more of the CD dimer and megroot (ZAVESCA®), HPβCD (TRAPPSOLCYCLO, VTS-270), and physical therapy disclosed herein. Combination therapies can be administered simultaneously, substantially simultaneously, or sequentially in any order. Combination therapies can be administered as a single formulation, or individually, optionally in combination, in a dosing kit or package containing each drug, for example, in a convenient pre-measured form (where one or more single doses of each drug are provided in the combination). Combination therapies can exhibit synergistic effects, where the effect of the combination therapy exceeds that of the individual treatments alone. While combination therapies typically involve the administration of an effective amount of CD dimer and the combination therapy, combination therapies can allow for effective treatment with lower doses of CD and / or the combination therapy, which can advantageously reduce conventional (non-combination) dose-related side effects.

[0198] Combination therapy may include treatments for the treatment or prevention of diseases or conditions associated with atherosclerosis, such as coronary artery disease, angina pectoris, heart attack, cerebrovascular disease, transient ischemic attack, and / or peripheral artery disease. Combination therapy may also include treatments for the treatment or prevention of conditions that may lead to the formation of atherosclerosis and / or poor prognosis, such as hypertension, hypercholesterolemia, hyperglycemia, and diabetes.

[0199] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with an anticholesterol drug, such as fibrates or statins, for example ADVICOR(R) (nicotinic acid extended-release / lovastatin), ALTOPREV(R) (lovastatin extended-release), CADUET(R) (amlodipine and atorvastatin), CRESTOR(R) (rosuvastatin), JUVISYNC(R) (sitagliptin / simvastatin), LESCOL(R) (fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR(R) (atorvastatin), LIVALO(R) (pitavastatin), MEVACOR(R) (lovastatin), PRAVACHOL(R) (pravastatin), SIMCOR(R) (nicotinic acid extended-release / simvastatin), VYTORIN(R) (ezetimibe / simvastatin) and / or ZOCOR(R). (Simvastatin). Anticholesterol drugs can be administered in doses that are effective in preventing or treating hypercholesterolemia.

[0200] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with an antiplatelet drug (e.g., aspirin).

[0201] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with an antihypertensive drug. Exemplary antihypertensive drugs include beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, and / or diuretics.

[0202] In an exemplary embodiment, the cyclodextrin dimer of the present invention is co-administered with a dietary supplement such as one or more of the following: alpha-linolenic acid (ALA), barley, β-sitosterol, black tea, psyllium husk, calcium, cocoa, cod liver oil, coenzyme Q10, fish oil, folic acid, garlic, green tea, niacin, oat bran, omega-3 fatty acids (such as eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA)), sitosterol, and / or vitamin C.

[0203] Exemplary combination therapies also include interventions in patient behavior and / or lifestyle, including advice and / or support for smoking cessation, exercise, and a healthy diet (such as a diet low in low-density lipoprotein (LDL) and optionally high in high-density lipoprotein (HDL)).

[0204] Exemplary combination therapies also include surgical interventions such as angioplasty, stent placement, or both.

[0205] The methods of the present invention can be used to treat or prevent atherosclerosis in human subjects. In some cases, the patient is healthy except for exhibiting atherosclerosis. For example, the patient may not exhibit any other risk factors for cardiovascular, thrombotic, or other diseases or disorders at the time of treatment. However, in other cases, patients are selected based on a disease or disorder diagnosed as being caused by or related to atherosclerosis, or having a risk of developing such disease or disorder. For example, at or before the administration of the pharmaceutical composition of the present invention, the patient may be diagnosed or identified as having a risk of developing a cardiovascular disease or disorder, such as coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, peripheral artery occlusive disease, etc. In some cases, the cardiovascular disease or disorder is hypercholesterolemia.

[0206] In other cases, at the time of or prior to the administration of the pharmaceutical composition of the present invention, a patient may be diagnosed or identified as having a risk of developing atherosclerosis.

[0207] In other cases, patients to be treated using the method of the present invention are selected based on one or more factors, which are selected from age (e.g., age greater than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years), race, sex (male or female), exercise habits (e.g., regular exerciser, non-exerciser), other pre-existing medical conditions (e.g., type II diabetes, hypertension, etc.) and current medication status (e.g., currently taking statins such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc., beta blockers, niacin, etc.). Attached Figure Description

[0208] In the following figures, the following abbreviations are used: Me or ME or me or met: methyl; SB: sulfonyl; QA = quaternary ammonium, for example, -CH2CH(OH)CH2N(CH3)3 + Examples include -CH2CH(OH)CH2N(CH3)3Cl; SUCC: succinyl; DMSO: dimethyl sulfoxide.

[0209] Figure 1A Cyclodextrins (CDs) are cyclic oligosaccharide polymers composed of 6 (αCD), 7 (βCD), or 8 (γCD) sugar rings (from left to right). All sugar rings in all CDs are D-glucose molecules.

[0210] Figure 1B-1J The structure of the replaced CD.

[0211] Figure 1B Among them, R 1 R 2 and R 3 It is a substituent.

[0212] Figure 1C βCD (DS0), i.e., each R 1 R 2 and R 3 It's hydrogen.

[0213] Figure 1D Hydroxypropyl CD (DS4),

[0214] Figure 1E .Methyl βCD (DS6),

[0215] Figure 1F .Sulfobutyl BCD (DS4),

[0216] Figure 1G Quaternary ammonium (DS 3),

[0217] Figure 1H .succinyl (DS 1),

[0218] Figure 1I Carboxymethyl (DS4), and

[0219] Figure IJ. Maltose syl (DS 1) groups are substituted at the C2, C3, or C6 positions of the BCD.

[0220] Figure 2A The solubilization of various cholesterol derivatives by HPβCD (DS 4.5) monomers was assessed by relative turbidity, where 100 was defined as the absorbance of an aqueous suspension containing 300 μM sterols tested in PBS. Figure 2A The results for cholesterol (diamond), 7KC (square), vitamin D2 (triangle), vitamin D3 (X), and steroids (+) are shown. In this and subsequent plots, the data points are connected by smooth curves to aid in the visualization of the results.

[0221] Figure 2B The solubilization of various sterols by hydroxypropyl-β-cyclodextrin (DS 4.5) monomers was assessed by relative turbidity, where 100 was defined as the absorbance of an aqueous suspension containing 300 μM sterols tested in PBS. Figure 2BResults were described for 7-ketocholesterol (7KC (X with a straight line)), 4-β-hydroxycholesterol (4-BOH (square)), 25-hydroxycholesterol (25OH (triangle)), cholesterol epoxide (rhombus) and 27-hydroxycholesterol (27OH (circle)).

[0222] Figure 2C Solubilization of 7KC by various forms of hydroxypropyl-β-cyclodextrin monomers as assessed by relative turbidity. DS = average degree of hydroxypropyl substitution per molecule.

[0223] Figure 2D The solubilization of cholesterol by various forms of hydroxypropyl-β-cyclodextrin monomers was assessed by relative turbidity. DS = mean degree of hydroxypropyl substitution per molecule.

[0224] Figure 2E The predicted relative affinity of the HPβCD molecule, calculated via molecular docking. DS represents the degree of hydroxypropyl substitution per molecule.

[0225] Figure 2F MeβCD of various degrees of substitution assessed by relative turbidity in vitro Solubilizes cholesterol.

[0226] Figure 2G MeβCD of various degrees of substitution assessed by relative turbidity in vitro Solubilization of 7KC.

[0227] Figure 2H Various monomers of βCD were assessed by relative turbidity. in vitro Solubilizes cholesterol.

[0228] Figure 2I Various monomers of βCD were assessed by relative turbidity. in vitro Solubilization of 7KC.

[0229] Figure 3A The structure of the HPβCD dimer disclosed herein. The β-cyclodextrin monomers are linked via large (secondary) surface areas. Right now The connector is attached to the C2 or C3 carbon of each CD subunit. The HP is replaced by a connection to the C2, C3 and / or C6 carbons (usually connected in combination).

[0230] Figure 3B Formula I. C2-C2 cyclodextrin dimer with a triazole linker.

[0231] Figure 3C Formula II. C2-C3 cyclodextrin dimers with triazole linkers.

[0232] Figure 3DFormula III. C3-C3 cyclodextrin dimers with triazole linkers.

[0233] Figure 3E Formula IV. Methyl-substituted BCD with a subsurface-linked connector L.

[0234] Figure 3F Formula V. A sulfonyl-substituted BCD with a subsurface connection of a connector L. The figure depicts the sodium salt, but other salts are also included in the compounds disclosed herein.

[0235] Figure 3G Formula VI. Succinyl-substituted BCD with a subsurface connection of connector L.

[0236] Figure 3H Formula VII. Maltose-substituted BCD with a subsurface connection of connector L.

[0237] Figure 3I Formula VIII. Quaternary ammonium-substituted BCD with a subsurface connection of a connector L.

[0238] Figure 3J Formula IX. Carboxymethyl-substituted BCD with a subsurface-linked connector L. The figure depicts the sodium salt, but other salts are also included in the compounds disclosed herein.

[0239] Figure 4A Structural models of HPβCD monomers associated with sterols (top) or HPβCD butyl-linked dimers associated with sterols (bottom). This is illustrated using diagrams of monomer-sterol and dimer-sterol host-guest interactions.

[0240] Figure 4B Predicted relative affinities of butyl- and triazole-linked dimers for cholesterol and 7KC. Docking calculations were performed on HPβCD dimers linked at various degrees of hydroxypropylation.

[0241] Figure 4C Description of measurements used in molecular dynamics simulations. The figure includes the nomenclature of cyclodextrins and sterols to define the O4 atom of the CD (marked with arrows), the subsurface and main surface of the CD, and the head and tail groups of the sterol. The angle between the O4 plane and the ligand indicates the degree of nesting of the ligand within the CD cavity. 30 degrees corresponds to the solubilized "upward" configuration (the head of the sterol associates with the subsurface of the CD, and the tail with the main surface), while 150 degrees corresponds to the solubilized "downward" configuration (the tail of the sterol associates with the subsurface of the CD, and the head with the main surface).

[0242] Figure 4DMD simulations of DS0 βCD: In the GROMOS force field, distances between the centroids of all O4 oxygen atoms and the centroids of the ligands for native (i.e., unsubstituted) monomer βCD under up and down ligand orientations (top); the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligand (middle); and the Lennard-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligand (bottom). In the figures included between 4D and 4LL, the light-colored lines represent the results for cholesterol, while the dark-colored lines represent the results for 7KC.

[0243] Figure 4E In the GROMOS force field, the natural DS0 monomer βCD solubilizes the ligand.

[0244] Figure 4F Visual trajectories of 7KC and cholesterol in two orientations that are complexed with the natural DS0 βCD (GROMOS force field).

[0245] Figure 4G In the AMBER force field, for the natural monomer DS0 β-cyclodextrin, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0246] Figure 4H In the AMBER force field, the natural DS0 monomer βCD solubilizes the ligand.

[0247] Figure 4I Visual trajectories of 7KC and cholesterol in two orientations, compounded with the natural DS0 βCD (AMBER force field). Abbreviation used: "ms": microsecond.

[0248] Figure 4J In the GROMOS force field, for the translated natural monomer β-cyclodextrin (DS0), the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0249] Figure 4K In the GROMOS force field, the translational monomer βCD solubilizes the ligand.

[0250] Figure 4L Visual trajectories of 7KC and cholesterol in the GROMOS force field, with translation of the natural (DS0) βCD complex.

[0251] Figure 4M In the AMBER force field, for the translated natural monomer β-cyclodextrin (DS0), the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0252] Figure 4N In the AMBER force field, the translational monomer βCD solubilizes the ligand.

[0253] Figure 4O Visual trajectories of 7KC and cholesterol in two orientations that are complexed with the natural DS0 βCD (AMBER force field).

[0254] Figure 4P In the GROMOS force field, for the natural DS0 monomer β-cyclodextrin, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0255] Figure 4Q In the GROMOS force field, the natural monomer βCD solubilizes the ligand.

[0256] Figure 4R Visual trajectories of 7KC and cholesterol complexed with the natural monomer βCD (GROMOS force field) in two orientations.

[0257] Figure 4S In the AMBER force field, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by O4 atoms perpendicular to CD and the principal axis of the ligands; the Lanner-Jones and Coulomb energies of the interaction between HPβCD DS5 and cholesterol or 7KC.

[0258] Figure 4T In the AMBER force field, HPβCD DS5 solubilizes the ligand.

[0259] Figure 4U Visual trajectories of 7KC and cholesterol in two orientations associated with HPβCD DS5 (AMBER force field).

[0260] Figure 4VIn the GROMOS force field, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under translational and upward and downward ligand orientations; the angle between the vector of the plane formed by O4 atoms perpendicular to CD and the principal axis of the ligands; the Lanner-Jones and Coulomb energies of the interaction between HPβCD DS5 and cholesterol or 7KC.

[0261] Figure 4W In the GROMOS force field, the translational monomer HPβCD solubilizes the ligand.

[0262] Figure 4X Visual trajectories of 7KC and cholesterol in two orientations, combined with translational monomers HPβCD DS5 (GROMOS force field).

[0263] Figure 4Y In the AMBER force field, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under translational and upward and downward ligand orientations; the angle between the vector of the plane formed by O4 atoms perpendicular to CD and the principal axis of the ligands; the Lanner-Jones and Coulomb energies of the interaction between HPβCD DS5 and cholesterol or 7KC.

[0264] Figure 4Z In the AMBER force field, the translational monomer HPβCD DS5 solubilizes the ligand.

[0265] Figure 4AA Visual trajectories of 7KC and cholesterol in two orientations, combined with the translational monomer DS5 HPβCD (AMBER force field).

[0266] Figure 4BB In the GROMOS force field, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by O4 atoms perpendicular to CD and the principal axis of the ligands; the Lanner-Jones and Coulomb energies of the interaction between butyl dimerized HPβCD DS5 and cholesterol or 7KC.

[0267] Figure 4CC In the GROMOS force field, butyl dimerized HPβCD DS5 solubilizes 7KC and cholesterol.

[0268] Figure 4DD Visual trajectories of 7KC and cholesterol complexed with butyl dimerized DS5 HPβCD (GROMOS force field) in two orientations.

[0269] Figure 4EEIn the AMBER force field, for butyl dimerized HPβCD DS5, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0270] Figure 4FF In the AMBER force field, butyl dimerized HPβCD DS5 solubilizes the ligand.

[0271] Figure 4GG Visual trajectories of 7KC and cholesterol complexed with butyl dimerized HPβCD DS5 (AMBER force field) in two orientations.

[0272] Figure 4HH In the GROMOS force field, under translational conditions and with upward and downward ligand orientations, for dimerized DS5 hydroxypropyl β-cyclodextrin, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0273] Figure 4II In the GROMOS force field, the translational butyl dimerization DS5 HPβCD solubilizes 7KC and cholesterol.

[0274] Figure 4JJ Visual trajectories of 7KC and cholesterol in two orientations, combined with translational butyl dimerized DS5 HPβCD (GROMOS force field).

[0275] Figure 4KK In the AMBER force field, under translational conditions and with upward and downward ligand orientations, for butyl dimerized DS5 hydroxypropyl β-cyclodextrin, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0276] Figure 4LL In the AMBER force field, the translational butyl dimerization DS5 HPβCD solubilizes 7KC and cholesterol.

[0277] Figure 4MM Visual trajectories of 7KC and cholesterol in two orientations, combined with translational butyl dimerized DS5 HPβCD (AMBER force field).

[0278] Figure 4NNIn the GROMOS force field, for unsubstituted (DS0) butyl dimerized β-cyclodextrin, the distance between the centroids of all O4 oxygen atoms and the centroids of the ligands under upward and downward ligand orientations; the angle between the vector of the plane formed by the O4 atoms perpendicular to CD and the principal axis of the ligands; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligands.

[0279] Figure 400 Visual trajectories of 7KC and cholesterol complexed with unsubstituted (DS0) butyl dimerized βCD (AMBER force field) in two orientations.

[0280] Figure 4PP MD analysis of triazole-linked DS0 cyclodextrins. The angle between the vector of the plane formed by O4 atoms perpendicular to CD and the principal axis of the ligand in the GROMOS force field, with upward and downward ligand orientations; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligand.

[0281] Figure 4 QQ Visual trajectories of 7KC and cholesterol in two orientations associated with triazole dimerized DS0 βCD complexes.

[0282] Figure 4RR MD analysis of triazole-linked DS4 HPβCD. The angle between the vector perpendicular to the plane formed by the O4 atoms of CD and the principal axis of the ligand in the GROMOS force field for the translated dimerized DS4 hydroxypropyl β-cyclodextrin under upward and downward ligand orientations; and the Lanner-Jones and Coulomb energies of the interaction between the cyclodextrin and the ligand.

[0283] Figure 4SS Visual trajectory of the 100ns interaction between the triazole-linked DS4 hydroxypropyl βCD dimer and 7KC / cholesterol in two orientations.

[0284] Figure 5A Predicted relative affinities of many possible dimerized MeβCD molecules calculated via molecular docking. Affinity of sterols to dimers linked by butyl (left) and triazole (right). Docking calculations for MeβCD dimers linked at various degrees of methylation. Cholesterol (dotted line) versus 7KC (solid line).

[0285] Figure 5B MD simulations depict the 100 ns interaction between the butyl-linked DS4 methyl βCD dimer and 7KC / cholesterol in both upward and downward orientations. Legend: 7KC (dark line) and cholesterol (light gray line), dashed lines indicate downward orientation, solid lines indicate upward orientation.

[0286] Figure 5CVisual trajectories of DS4 methyl βCD dimer and 7KC / cholesterol linked by butyl groups in both upward and downward orientations.

[0287] Figure 5D MD simulations depict the 100 ns interaction between the triazole-linked DS4 methyl βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0288] Figure 5E Visual trajectories of DS4 methyl βCD dimer and 7KC / cholesterol linked by triazole in both upward and downward orientations.

[0289] Figure 6A Predicted relative affinities of many possible dimerized sulfonated βCD molecules calculated via molecular docking. Affinities of butyl and triazole-linked dimers to sterols. Docking calculations for linked SBβCD dimers at various degrees of sulfonation. Cholesterol (dotted line) versus 7KC (solid line).

[0290] Figure 6B MD simulations depict a 100 ns interaction between the butyl-linked DS4 sulfobutyl βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0291] Figure 6C Visual trajectories of DS4 sulfobutyl βCD dimer and 7KC / cholesterol linked by butyl in both upward and downward orientations.

[0292] Figure 6D MD simulations depict a 100 ns interaction between the triazole-linked DS4 sulfobutyl βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0293] Figure 6E Visual trajectories of DS4 sulfobutyl βCD dimer and 7KC / cholesterol linked by triazole in both upward and downward orientations.

[0294] Figure 7A MD simulations depict a 100 ns interaction between the butyl-linked DS4 quaternary ammonium βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0295] Figure 7B Visual trajectories of DS4 quaternary ammonium βCD dimer and 7KC / cholesterol linked by butyl groups in both upward and downward orientations.

[0296] Figure 7C MD simulations depict a 100 ns interaction between the triazole-linked DS4 quaternary ammonium βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0297] Figure 7D Visual trajectories of the triazole-linked DS4 quaternary ammonium βCD dimer and 7KC / cholesterol in both upward and downward orientations. (See figure for example.) Figure 5B As shown.

[0298] Figure 8A Different hydroxypropylation sites in the triazole and butyl-linked dimers of DS8 and DS4, including hydroxypropylation on only the small surface or the large surface. Docking calculations were performed on various hydroxypropylation sites in the HPβCD dimer to determine the effect of changing the position of the hydroxypropyl group on sterol binding. The hydroxypropylation sites are variable in practice due to the randomness of substitution onto a generally symmetrical molecule. The labels “C,” “D,” and “E” refer to different (distinct from each other) variant structures with an equal distribution of HP groups on both the small and large surfaces of the CD monomer. Legend: The upper (light gray) bar represents cholesterol values, and the lower (dark) bar represents 7KC values.

[0299] Figure 8B Different lengths of alkyl-linked HPβCD DS5 dimers. Butt joint calculations were performed for carbon-only joints of various degrees of hydroxypropylation and lengths. The bars in each group, from top to bottom, are DS20, DS16, DS12, DS8, DS4, and DS0.

[0300] Figure 8C Different lengths of triazole-linked HPβCD DS5 dimers. Butt joint calculations were performed for triazole joints of varying lengths by changing the number of carbon atoms on either side of the triazole ring. The length of each side of the joint is distinguished by n1 or n2. Cholesterol is represented by striped bars, while 7KC is a solid bar. Within each group, the bars, from top to bottom, are: N1=2 and 7KC; N1=2 and cholesterol; N1=3 and 7KC; N1=3 and cholesterol; N1=4 and 7KC; N1=4 and cholesterol.

[0301] Figure 8D Testing the joint through mating calculations ( Figure 8E To determine the linker-dependent changes in sterol binding, the HPβCD dimer combination linked by hydroxypropyl DS4 and DS8 dimers was determined based on the addition of various side chains, rings, double bonds, and / or substitution of sulfur, nitrogen, and / or oxygen atoms in the linker combination, compared to the four-carbon linker (linker W, where n = 3 carbons) and triazole-linked dimers (linker U, where n = 1 carbon, and linker V, where n = 1 carbon).

[0302] Figure 8E Docking results for various HPβCD dimers with different joints. Compared with the tetracarbon joint (joint W, where n=3 carbons) and the triazole-linked dimers (joint U, where n=1 carbon, and joint V, where n=1 carbon), the docking results based on joint AW ( Figure 8D The figure shows the preference of the HPβCD dimer 7KC for the hydroxypropyl DS4 and DS8 dimers. Legend: The upper (light gray) bar represents cholesterol values, and the lower (dark) bar represents 7KC values.

[0303] Figure 8F The effect of CD attachment sites on molecular docking projections of triazole-linked and butyl-linked dimers on the projection affinities of cholesterol and 7KC. Docking calculations were performed on dimers linked by symmetrical butyl and triazole linkers, resulting in three possible linkages: C2-C2, C3-C3, and C2-C3. Due to the symmetry of the linkers, the C2-C3 dimer is identical to the C3-C2 linked dimer. Legend: The upper (light gray) bar represents cholesterol values, and the lower (dark) bar represents 7KC values.

[0304] Figure 8G Asymmetric joint variations at connection points. Docking calculations are performed on dimers connected by asymmetric tetraatomic joints C, D, K, N, and R. See Figure 8D For these asymmetric joints, there are four possible connections: C2-C2, C3-C3, C2-C3, and C3-C2. In these cases, C3-C2 differs from C2-C3 due to the asymmetry of the joint. Legend: Each group of bars, from top to bottom, represents cholesterol with a C3 / C2 connection; cholesterol with a C2 / C3 connection; 7KC with a C3 / C2 connection; and 7KC with a C2 / C3 connection.

[0305] Figure 8H MD simulations depict the 100 ns interaction (connector O) between nitrogen-linked DS4 hydroxypropyl βCD dimers and 7KC / cholesterol in two orientations. (See figure for example.) Figure 5B As shown.

[0306] Figure 8I Visual trajectories (connector O) of nitrogen-linked DS4 hydroxypropyl BCD dimers and 7KC / cholesterol in two orientations.

[0307] Figure 9APredicted 7KC specificity for many linked dimers determined by molecular docking. 7KC specificity is maintained across a variety of linkers and substitution types in the βCD dimer. The order of the bars within each group from left to right is: sulfobutyl (DS4); hydroxypropyl (DS4); methyl (DS4); quaternary ammonium (DS4); succinyl (DS4); carboxymethyl (DS4); maltose (DS4).

[0308] Figure 9B Sterol affinities (DS4) for alkyl links of various lengths with hydroxypropyl, methyl, and sulfobutyl substitutions; as modeled by molecular docking. The order of the bars within each group from top to bottom is: methyl, sulfobutyl, and hydroxypropyl.

[0309] Figure 9C Sterol affinities (DS4) for triazole linkers of various lengths with hydroxypropyl, methyl, and sulfonbutyl substitutions; as modeled by molecular docking. The order of the bars is as follows: Figure 9B As shown.

[0310] Figure 9D Predicted 7KC specificity of βCD dimers linked to butyl and triazole for multiple substitution sites; as modeled by molecular docking. The X-axis represents the fold affinity of 7KC relative to cholesterol. In each group, the upper column represents triazole and the lower column represents butyl.

[0311] Figure 9E Docking screening of other βCD variants. 7KC specificity was observed for butyl and triazole-linked βCD dimers, and even for combinations of substitutions; as modeled by molecular docking. The X-axis represents the fold affinity of 7KC relative to cholesterol. The order of the bars is as follows: Figure 9D As shown.

[0312] Figure 10A A synthetic strategy involving the hydroxypropylated dimer linked to a 1,4-dibromobutane-based linker unit (resulting in a butyl-linked HPβCD dimer).

[0313] Figure 10B A synthetic strategy involving the hydroxypropylated dimer linked to a 3-azido-1-bromo-propane-based linker unit (resulting in a triazole-linked HPβCD dimer).

[0314] Figure 10C TLC analysis is used to assess reaction progress and conversion rate.

[0315] Figure 10D MALDI spectrum of TBDMS-βCD-BUT-βCD-TBDMS.

[0316] Figure 10E TLC analysis is used to assess reaction progress and conversion rate.

[0317] Figure 10F MALDI spectra of synthesized large-surface butyl-linked β-cyclodextrin (βCD-BUT-βCD) with DS=0.

[0318] Figure 10G MALDI spectra of the synthesized large-surface butyl-linked hydroxypropyl β-cyclodextrin HP(βCD-BUT-βCD) DS~3 were obtained. Some peaks were unlabeled due to crowding, but they indicate the expected molecular weight.

[0319] Figure 10H MALDI spectra of the synthesized large-surface butyl-linked hydroxy-propyl β-cyclodextrin HP(βCD-BUT-βCD) DS~6 were obtained. Some peaks were unlabeled due to crowding, but they indicate the expected molecular weight.

[0320] Figure 10I MALDI spectra of synthesized butyl-linked hydroxy-propyl β-cyclodextrin HP(βCD-BUT-βCD) DS~8.

[0321] Figure 10J HP(βCD-BUT-βCD) 1 H-NMR spectrum (D2O, 298K), and the signal was labeled.

[0322] Figure 10K The structure of a prospective isomer of HP(βCD-BUT-βCD) DS8, and the nomenclature of the connector.

[0323] Figure 10L HSQC spectra of HP(βCD-BUT-βCD) edited by DEPT (D2O, 298K).

[0324] Figure 10M HSQC spectrum of HP(βCD-BUT-βCD) edited by DEPT, and the allocation of junction frequencies determined by thermogram (D2O, 298K).

[0325] Figure 10N HSQC spectra of HP(βCD-BUT-βCD) edited by DEPT, and complete partition (D2O, 298K).

[0326] Figure 10O MALDI spectra of synthesized large-surface triazole-linked β-cyclodextrin (βCD-(triazole)1-BCD, DS=0).

[0327] Figure 10PMALDI spectra of the synthesized large-surface triazole-linked β-cyclodextrin HP (βCD-triazole-βCD) DS~3 were obtained. Some peaks were unlabeled due to crowding, but they indicate the expected molecular weight.

[0328] Figure 10Q MALDI spectra of the synthesized large-surface triazole-linked β-cyclodextrin HP (βCD-triazole-βCD) DS~7 were obtained. Some peaks were unlabeled due to crowding, but they indicate the expected molecular weight.

[0329] Figure 10R DEPT-edited HSQC spectra of HP(βCD-triazole-βCD), and linker fractionation (D2O, 298K). DS~7 (left) and TLC, and linker fractionation (right).

[0330] Figure 10S TLC plates show reaction monitoring and spot allocation.

[0331] Figure 10T MALDI spectrum of 2-O-propynyl-β-CD.

[0332] Figure 10U 2-O-propynyl-β-CD 1 H-NMR spectrum, and some peak picking (DMSO-d6, 298K).

[0333] Figure 10V BCD-(triazole)1-BCD dimer 1 H-NMR spectrum (D2O, 298K).

[0334] Figure 10W HP (βCD-triazole-βCD) 1 H-NMR spectrum (D2O, 298K), and the signal is labeled. Corresponding to Figure 16B And the molecule elsewhere labeled as CD-triazole-CD DS3.

[0335] Figure 10X HP (βCD-triazole-βCD) 1 H-NMR spectrum (D2O, 298K), and the signal is labeled. Corresponding to Figure 16B The molecule is labeled as CD-triazole-CD DS6.

[0336] Figure 10Y HP (βCD-triazole-βCD) 1 H-NMR spectrum (D2O, 298K), and the signal is labeled. Corresponding to Figure 16B The molecule is labeled as CD-triazole-CD DS7.

[0337] Figure 11A Synthetic scheme for methylated βCD dimer.

[0338] Figure 11B TLC analysis is used to evaluate reaction processes and conversion rates.

[0339] Figure 11C MALDI spectra of the final compound obtained by the reaction in (A).

[0340] Figure 11D MALDI spectra of the final compound obtained by the reaction in (B).

[0341] Figure 11E MALDI spectra of the final compound obtained by the reaction in (C).

[0342] Figure 11F MALDI spectra of the final compound obtained by the reaction in (D).

[0343] Figure 11G Superimposed MALDI spectra of reaction traces. Reaction A (DS0), reaction B (DS1), reaction C (DS2), and reaction D (DS4, 5, 6).

[0344] Figure 11H MALDI spectrum of Me-(βCD-triazole-βCD) dimer.

[0345] Figure 11I Magnification of the MALDI spectrum of Me-(βCD-triazole-βCD) dimer.

[0346] Figure 11J The structure and atom numbering of a possible isomer of Me-(βCD-triazole-βCD) dimer.

[0347] Figure 11K The HNMR spectrum of Me-(βCD-triazole-βCD) dimer, and the complete frequency allocation.

[0348] Figure 11L HNMR spectrum and integral of Me-(βCD-triazole-βCD) dimer.

[0349] Figure 11M DEPT-edited HSQC spectra of Me-(βCD-triazole-βCD) dimer, and complete partitioning.

[0350] Figure 11N COSY-NMR spectrum and partition of Me-(βCD-triazole-βCD) dimer.

[0351] Figure 12A Synthetic scheme for sulfonated βCD dimer.

[0352] Figure 12B TLC analysis was used to evaluate the reaction progress and conversion rate in the SB-βCD assay.

[0353] Figure 12C Overlap fingerprint chromatographic analysis was used to evaluate the DS of reaction A in the SB-βCD test.

[0354] Figure 12D Overlap fingerprint chromatographic analysis was used to evaluate the DS of reaction B in the SB-βCD test.

[0355] Figure 12E MALDI of SB-βCD dimer (low DS).

[0356] Figure 12F A possible isomer of the SB-βCD dimer, and its atom number.

[0357] Figure 12G HNMR spectra of the sulfonated dimer (low DS) and complete partition (D20; 298K).

[0358] Figure 12H HNMR spectra and integrals (D20; 298K) of the sulfonated dimer (low DS). The figure shows the DS values ​​calculated based on NMR.

[0359] Figure 12I DEPT-edited HSQC spectra of SB dimer (low DS), and complete partition (D2O, 298K).

[0360] Figure 12J COSY spectrum of SB dimer (low DS), and complete partition (D2O, 298K).

[0361] Figure 12K MALDI spectrum of SB dimer (high DS).

[0362] Figure 12L The structure and atom numbering of a possible isomer of SB dimer (DS3).

[0363] Figure 12M HNMR spectrum of SB dimer (high DS) and complete partition (D20, 298K).

[0364] Figure 12N . HNMR spectrum and integral (D20, 298K) of SB dimer (high DS). The figure shows the DS value calculated based on NMR.

[0365] Figure 12O HSQC spectra of SB dimer (high DS) edited by Dept, and complete partition (D20, 298K).

[0366] Figure 12P COSY spectrum of SB dimer (high DS) and complete partition (D20, 298K).

[0367] Figure 13A Synthetic scheme for quaternary ammonium β-cyclodextrin dimers.

[0368] Figure 13B MALDI spectra of quaternary ammonium β-cyclodextrin dimer reaction A.

[0369] Figure 13C MALDI spectra of quaternary ammonium β-cyclodextrin dimer reaction B.

[0370] Figure 13D MALDI spectra of quaternary ammonium β-cyclodextrin dimer reaction C.

[0371] Figure 13E MALDI spectra of quaternary ammonium β-cyclodextrin dimer reaction D.

[0372] Figure 13F MALDI spectra of quaternary ammonium β-cyclodextrin dimers.

[0373] Figure 13G The structure and atom numbering of a possible QA dimer isomer (DS3).

[0374] Figure 13H HNMR spectrum of QA dimer and complete partition (D20, 298K).

[0375] Figure 13I The HNMR spectrum and integral (D20, 298K) of the QA dimer are shown in the figure. The DS value is calculated based on NMR.

[0376] Figure 13J DEPT-edited HSQC spectrum of QA dimer and complete partition (D20, 298K).

[0377] Figure 13K COSY spectrum of QA dimer and partial distribution (D20, 298K).

[0378] Figure 14A Synthetic scheme for succinylated dimers.

[0379] Figure 14B MALDI of succinylated dimer A.

[0380] Figure 14C MALDI of succinylated dimer B.

[0381] Figure 14D MALDI of succinylated dimer C.

[0382] Figure 14E MALDI of succinylated dimer reaction D.

[0383] Figure 14F .Succinylated dimer MALDI.

[0384] Figure 14G The structure and atom numbering of a possible SUCC dimer isomer (DS3).

[0385] Figure 14H HNMR spectra of the succinylated dimer and complete partition (D20, 298K).

[0386] Figure 14I The ¹H NMR spectrum and integral (D20, 298K) of the succinylated dimer are shown in the figure. The DS values ​​are calculated based on NMR.

[0387] Figure 14J DEPT-edited HSQC spectra of the succinylated dimer and complete partition (D20, 298K).

[0388] Figure 14K COSY spectrum of succinylated dimer and partial partition (D20, 298K).

[0389] Figure 15A Blood cell efflux concentration after incubation with DS8 HPβCD dimer for 7 kC.

[0390] Figure 15B Blood cell efflux concentration after incubation with HPβCD monomer for 7 kC.

[0391] Figure 15C Plasma cholesterol is not affected by incubation with HPβCD dimers. Plasma cholesterol is measured by mass spectrometry to determine the efflux of cholesterol from blood cells caused by incubation with HPβCD dimers.

[0392] Figure 15D Hemolysis assay, as a measure of the potential cytotoxicity of various butyl and triazole-linked HPβCD and methyl dimers.

[0393] Figure 15EHemolysis assay as a measure of the potential cytotoxicity of various triazole-linked βCD dimers: unsubstituted βCD, SBβCD (low and high DS), QAβCD, and succinylated βCD dimers.

[0394] Figure 16A Butyl-linked HPβCD dimers are significantly superior to monomeric HPβCD in solubilizing 7KC and cholesterol. Dimers with approximately 3, 6, and 8 degrees of substitution were tested.

[0395] Figure 16B Triazole-linked HPβCD dimers are significantly superior to monomeric HPβCD in solubilizing 7KC and cholesterol. Dimers with substitution degrees of 0, approximately 3, approximately 5, and approximately 6 were tested. HPBCD represents the monomeric HPβCD, while CD-triazole-CD represents a triazole-linked dimer with a specified degree of substitution.

[0396] Figure 16C Butyl-linked HPβCD dimer (DS~8) solubilizes various cholesterol derivatives and oxosterols. The figure depicts the results for cholesterol, 7-ketocholesterol (7KC), vitamin D2, vitamin D3, sterols, 27-hydroxycholesterol (27OH), 4-β-hydroxycholesterol (4BOH), 25-hydroxycholesterol (25OH), and cholesterol epoxides.

[0397] Figure 16D The butyl-linked HPBCD dimer (DS-8) solubilized the compound. The sterol hormones tested were estradiol, estriol, estrone, pregnenolone, and progesterone.

[0398] Figure 16E The butyl-linked HPβCD dimer (DS~3) (“DS3 butyl dimer”) has affinity and specificity for 7KC. HPBCD represents the monomer HPβCD.

[0399] Figure 16F The triazole-linked HPβCD dimer (DS~3) has both affinity and specificity for 7KC.

[0400] Figure 16G The triazole-linked MeβC dimer (DS~3) (“methyl dimer DS3”) has a similar effect to HPβCD dimer (DS~3) (“HPBCD dimer DS3”) in solubilizing 7KC and cholesterol.

[0401] Figure 16HTriazole-linked unsubstituted βCD (“CD-triazole-CD DS 0”), triazole-linked SBβCD dimer (DS~3.4”) (“SB CD-triazole-CD DS 3.4”), triazole-linked QaβCD dimer (DS~2”) (QA CD-triazole-CDDS 2”), and triazole-linked succinylated βCD dimer (DS~2) (“SUCC CD-triazole-CD DS 2”) all exhibit specificity for 7KC relative to cholesterol in vitro. Triazole-linked SBβCD dimer (DS~14.6) (“SB CD-triazole-CD DS14.6”) shows lower affinity for both cholesterol and 7KC.

[0402] definition

[0403] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given herein.

[0404] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used in this specification and the appended claims include plural references.

[0405] Linkage length. As used herein, the term "linkage length," or the interchangeable term "linkage length," refers to the number of atoms in the linker along the shortest path connecting the two CD subunits of the cyclodextrin dimer. For clarity, the linkage length does not include the oxygen atom (or other atom that may replace said oxygen) of each CD subunit to which the linker connects. For example, in Figure 3B In this context, the joint length is 3+n1+n2, which reflects the shortest path through the triazole ring. For a joint that connects to one or both cyclodextrin monomers at multiple points, the joint length is the shortest path connecting the two cyclodextrins across all possible paths, which can begin and terminate at different locations within each cyclodextrin.

[0406] Head-to-head cyclodextrin dimers. As used herein, the term “head-to-head cyclodextrin dimer” refers to a CD dimer in which two CD monomers are linked by the large (or small) surface of the cyclodextrin, typically via the C2 and / or C3 carbons of each CD monomer.

[0407] Tail-to-tailed cyclodextrin dimers. As used herein, the term “tail-to-tailed cyclodextrin dimer” refers to a CD dimer in which two CD monomers are linked on the small (major) surface of the cyclodextrin molecule, typically via the C6 carbon of each CD monomer.

[0408] Head-to-tail cyclodextrin dimers. As used herein, the term “head-to-tail cyclodextrin dimer” refers to a CD dimer in which two CD monomers are linked at opposite ends, i.e., one monomer is linked from a small (major) surface, typically via C6 carbon, and the other monomer is linked from a large (minor) surface, typically via C2 and / or C3 carbon.

[0409] Degree of Substitution (DS). As used herein, “degree of substitution” or “DS” refers to the number of given daughter groups bonded to a monomer or dimer. For example, MeβCD DS3 refers to a βCD with an average of 3 methyl R groups bonded to CD at O2, O3, or O6, while HPβCD DS3 represents a monomer or dimer with an average of 3 hydroxypropyl groups bonded to CD at O2, O3, or O6. When referring to a CD dimer, unless otherwise specified, DS is used to refer to the total average substitution of the two constituent monomers including all substituents (e.g., in the case of mixed substituents such as mixed hydroxypropyl and methyl substituents, all are included). Terms such as “degree of substitution of substituent X” refer to the average number of substituents X in each CD dimer, i.e., excluding any other substituents that may be present. DS can be measured by mass spectrometry (e.g., matrix-assisted laser desorption / ionization, “MALDI”) or by NMR. MALDI is preferred for cyclodextrin derivatives containing substituents that give a more typical Gaussian distribution of ions in the mass spectrum, for example, Figure 10G-10IThe methyl, hydroxypropyl, and sulfobutyl substituents in 10P-10Q, 11C-11G, 11I, 12E, and 12K are shown. The average DS determined by MALDI is calculated by averaging the peak heights corresponding to each DS class of the CDs under discussion. In other cases, such as due to the formation of various addition, cleavage, elimination products, etc., less regular ion peak patterns may occur. These problems can be potentially circumvented by using other mass spectrometry techniques. Alternatively, NMR can be used to determine the DS values, which is preferred for succinyl and quaternary ammonium groups given the greater complexity of the MS spectra observed by MALDI. The average degree of substitution (DS) is then calculated by identifying the peaks corresponding to the protons from the core dimer and scaling the measurements initially so that the peak areas correspond to the known number of such protons in the structure. The signals corresponding to the protons in the substituent groups are then examined and appropriately scaled to obtain the average degree of substitution. In a simpler case, the clearly resolved peaks corresponding to the substituent protons are identified and scaled as described above, then divided by the number of protons represented by that peak to obtain the average number of substituents. For example, with hydroxypropyl substituents, the peak corresponding to 14 protons in the core structure (the anomeric region of pyranose) is identified and its signal is normalized to 14. Then, the peak corresponding to 3 protons for the methyl substituent is identified, and finally, the peak area is divided by 3 to obtain the average number of hydroxypropyl groups present in each molecule. In other cases, the substituent peaks and the cyclodextrin core peaks may be very close or overlap. In this case, the number of contributing protons in the cyclodextrin core structure is determined, then subtracted from the peak area (the peak area has been scaled to the integral area of ​​1 per proton), and the remaining area is divided by the number of contributing protons to obtain the average degree of substitution. For example, with methyl substituents (such as...), the peaks corresponding to the substituent protons are identified and scaled as the integral area of ​​1 per proton, then the remaining area is divided by the number of contributing protons to obtain the average degree of substitution. Figure 11K-11L As shown), peak clusters corresponding to the three methyl hydrogens of the substituent and a set of 86 protons in the core cyclodextrin dimer structure were identified. For example, in the case of the hydroxypropyl substituent, peaks corresponding to 14 protons in the core structure (the anomeric region of pyranose) were identified and their signals were normalized to 14; the peak area containing the methyl hydrogen and the core cyclodextrin hydrogen was determined to be 92.77, which, after subtracting the signal from the 86 protons in the core cyclodextrin structure, yielded 6.77; and after dividing by the 3 protons of each methyl group, the estimated average degree of substitution was 2.26. For HP and ME-substituted CDs, the integral was divided by 3; for QA, by 9; for SB, by 2; and for SUCC, by 4. Based on the identification of peaks corresponding to protons in the substituent structure, the above calculations are directly applicable to other substituent types. DS calculations using NMR are shown below. Figure 10X-Y, 11L, 12H, 12N, 13I, and 14I are shown. CD compositions (such as CD dimer compositions (as defined below)) may comprise a mixture of individual molecules substituted with different numbers of substituents, in which case the DS value is expressed as the average (median) number of substitutions. Decimal DS values ​​reflect cases where the median value can be between integer substitutions. Unless otherwise specified, integer DS values ​​represent CD compositions having that DS number when rounded to the nearest integer. For example, DS4 refers to a DS value of at least 3.5 and less than 4.5.

[0410] Average degree of substitution of the hydroxypropyl group. As used herein, the term "average degree of substitution of the hydroxypropyl group" refers to the degree of substitution excluding any substituents other than the hydroxypropyl group, as defined above. Similarly, references to the average degree of substitution of a specified substituent refer to the average degree of substitution excluding other types of substituents, as defined above.

[0411] Hydroxypropyl (HP or Hp)-substituted cyclodextrins (CDs). As used herein, the terms "hydroxypropyl-substituted cyclodextrin" or "HP-substituted CD" refer to cyclodextrins attached to a hydroxypropyl group (i.e., -CH2-CH(OH)-CH3). Typically, the HP group is attached to an oxygen atom bonded to the C2, C3, and / or C6 carbon atom of the CD (most commonly a combination of these bonding sites).

[0412] Hydroxypropyl-β-cyclodextrin, abbreviated as HPβCD, HPBCD, HPβCD, HPBCD, HP-BCD, HP-BCD, HP-βCD, HP-βCD, 2-HPβCD and similar terms, refers to a β-cyclodextrin substituted with one or more hydroxypropyl groups (i.e., -CH2-CH(OH)-CH3), typically linked to an oxygen atom attached to the C2, C3 and / or C6 carbon of CD (most commonly a combination of these linkage sites).

[0413] Hydroxypropyl-β-cyclodextrin dimers, abbreviated as HP(CD-L-CD), HP(CD-L-CD), HP(βCD-L-βCD), or HP(βCD-L-βCD), HP and similar terms, refer to hydroxypropyl-β-cyclodextrin dimers covalently linked by a linker L. Specific average substitution numbers may be present; for example, DS4 indicates an average of 4 HP groups. Further substitutions may be present as further described herein.

[0414] Similar conventions are used for other substituted cyclodextrins and cyclodextrin dimers, such as methyl (Me), quaternary ammonium (QA), succinyl (SUCC), sulfobutyl (SB), etc. Thus, for example, MeβCD refers to methyl-β-cyclodextrin. Similarly, methyl-β-cyclodextrin dimers are sometimes abbreviated as Me(CD-L-CD) or Me(CD-L-CD) or Me(βCD-L-βCD) or Me(βCD-L-βCD)Me and similar terms, which refer to methyl-β-cyclodextrin dimers covalently linked by the linker L. Specific average substitution numbers can be present; for example, DS4 indicates an average of 4 Me groups. Further substitutions can be present, as further described herein.

[0415] Cyclodextrin dimer compositions. As used herein, the term "cyclodextrin dimer composition" or "CD dimer composition" refers to a mixture of cyclodextrin dimers, such as CD dimers substituted with different numbers of the same substituents. Typically, CD dimer compositions are characterized by having a specified degree of substitution for the specified substituents. CD dimer compositions can be produced by a synthetic process in which substituents are added to the CD dimer in a random manner due to the generally symmetrical nature of the CD molecules, such that the number and position of substituents in individual CD molecules will vary. Additionally, CD dimer compositions may comprise a mixture of individual molecules with different linker sites (e.g., O2 to O2, O2 to O3, O3 to O2, or O3 to O3), or the linker sites may be homogeneous (e.g., only O2 to O2, only O2 to O3, only O3 to O2, or only O3 to O3). The degree of substitution of a CD dimer composition can be determined by NMR and / or mass spectrometry, for example, as described above.

[0416] The term "specific binding" refers to the formation of a relatively stable complex under physiological conditions by a molecule (e.g., the cyclodextrin dimer of this disclosure) and a binding partner (e.g., cholesterol (such as oxosterols, such as 7KC)). Methods for determining whether a molecule specifically binds to a binding partner are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. In exemplary embodiments, the cyclodextrin dimer of this disclosure is present in amounts between about 5 μM and about 100 μM, between about 10 μM and about 90 μM, between about 20 μM and about 80 μM, between about 30 μM and about 70 μM, between about 40 μM and about 60 μM, between about 0.5 μM and about 50 μM, between about 1 μM and about 40 μM, between about 2 μM and about 30 μM, between about 3 μM and about 20 μM, between about 4 μM and... Km is between approximately 10 μM, less than approximately 1000 μM, less than approximately 500 μM, less than approximately 300 μM, less than approximately 200 μM, less than approximately 100 μM, less than approximately 90 μM, less than approximately 80 μM, less than approximately 70 μM, less than approximately 60 μM, less than approximately 50 μM, less than approximately 40 μM, less than approximately 30 μM, less than approximately 20 μM, less than approximately 10 μM, less than approximately 5 μM, less than approximately 4 μM, less than approximately 3 μM, less than approximately 2 μM, less than approximately 1 μM, or less than approximately 0.5 μM. D It combines with cholesterol, oxosterol, or 7KC.

[0417] The affinity for 7KC is greater than that for cholesterol. As used herein, the term "greater affinity for 7KC than for cholesterol" means that a compound (e.g., cyclodextrin) has a greater ability to solubilize 7KC than it has to solubilize cholesterol. This greater affinity can also be predicted by molecular docking, by molecular dynamics simulations, or by calorimetry. In an exemplary embodiment, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, or at least 50 times greater than its binding affinity for cholesterol. This can optionally be determined by comparing concentrations that solubilize 50% of the 7KC in a suspension, for example, using the procedures described in the working examples herein. In an exemplary embodiment, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.1, 1.5, 2, 3, 4, 5, or 10 times greater than its binding affinity for cholesterol, which may optionally be determined by calculating or measuring the binding affinity for cholesterol (KC). D The binding affinity is determined by dividing by the calculated affinity for 7KC.

[0418] A greater affinity for one compound than for another—for example, a greater affinity for 7KC than for cholesterol—can be determined using a turbidity test. This test involves an aqueous suspension in PBS containing 3% ethanol, 300 μM sterol, and 1 mM of the cyclodextrin to be tested. This single concentration of cyclodextrin is used to standardize the test results. For the test, the sample is incubated at 37°C for 30 minutes, and then the absorbance is measured at 350 nm, for example, using a spectrophotometer plate reader. Relative turbidity is determined by dividing the turbidity measured in the presence of cyclodextrin by the baseline turbidity without cyclodextrin. If the relative turbidity of the 7KC suspension is less than that of the cholesterol solution, then the given cyclodextrin has a greater affinity for 7KC than for cholesterol.

[0419] Hydrophobic drugs. As used herein, the term "hydrophobic drug" refers to a drug that is insoluble in water in the absence of a detergent or other solvent. Hydrophobic drugs include, but are not limited to, hormones such as estrogen, progesterone, and testosterone. The cyclodextrin dimer of this disclosure can be used as an excipient for hydrophobic drugs. Other exemplary hydrophobic drugs include dextromethorphan hydrobromide (DXM), diphenhydramine hydrochloride (DPH), lidocaine hydrochloride (LDC), heparin, benzfluthiazide, acyclovir, revivalazine, curcumin, and testosterone propionate (TP). The cyclodextrin dimer may be present in an amount sufficient to increase the solubility of the molecule and / or contribute to better drug delivery. The molecular ratio of the drug to the cyclodextrin may be 1:1 or greater than 1:1.

[0420] The amount that effectively solubilizes the hydrophobic drug. As used herein, the phrase "the amount that effectively solubilizes the hydrophobic drug" refers to the concentration of a substance (e.g., cyclodextrin dimer) that typically solubilizes the hydrophobic drug in an aqueous composition (such as phosphate-buffered saline (PBS)) or in water. Solubilization can be determined by spectrophotometry or other means known in the art. Solubilization can be determined at room temperature, physiological temperature (37°C), or another suitable temperature (e.g., between 0 and 4°C).

[0421] "Alkyl" refers to a monovalent straight-chain or branched saturated hydrocarbon moiety consisting only of carbon and hydrogen atoms, with one to twelve carbon atoms.

[0422] "Lower alkyl" refers to an alkyl group having one to six carbon atoms, i.e., C3 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, etc.

[0423] "alkylene" refers to a straight-chain or branched saturated divalent hydrocarbon group having one to twelve carbon atoms or a branched saturated divalent hydrocarbon group having three to six carbon atoms, such as methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, etc.

[0424] "Alkenyl" refers to a straight-chain monovalent hydrocarbon group containing at least one double bond and having two to twelve carbon atoms, or a branched monovalent hydrocarbon group having three to twelve carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), and 2-propenyl (allyl, -CH-CH=CH2) moieties, including but not limited to methoxy, ethoxy, and isopropoxy groups.

[0425] "Alkoxyalkyl" refers to the part of the formula Ra-O-Rb-, where Ra is an alkyl group and Rb is an alkylene group as defined herein. Exemplary alkoxyalkyl groups include, for example, 2-methoxyethyl, 3-methoxypropyl, 1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and 1-(2-methoxyethyl)-3-methoxypropyl.

[0426] "Alkoxyalkoxyalkyl" means a group of the formula -RO-R'-OR", where R and R' are each alkylene groups and R" is an alkyl group as defined herein.

[0427] "alkylcarbonyloxyalkyl" means a group of the formula -ROC(O)-R', where R is an alkylene group and R' is an alkyl group as defined herein.

[0428] "alkyl carbonyl" refers to the part of the formula -R'-R", where R' is -C(=O)- and R" is an alkyl group as defined herein.

[0429] "alkylsulfonyl" refers to the part of the formula -R'-R", where R' is -SO2- and R" is an alkyl group as defined herein.

[0430] "alkylsulfonylalkyl" refers to the part of the formula -R'-R"-R'", where R' is an alkyl group, R" is -SO2-, and R'" is an alkyl group as defined herein.

[0431] "alkylamino" refers to the part of the formula -NR-R', where R is hydrogen or alkyl, and R' is an alkyl group as defined herein.

[0432] "Alkoxyamino" refers to the part of the formula -NR-OR', where R is hydrogen or alkyl and R' is an alkyl as defined herein.

[0433] "alkylthioalkyl" refers to the part of the formula -SR, where R is an alkyl group as defined herein.

[0434] "Alkali metal ions" refers to monovalent ions of Group I metals, such as lithium, sodium, potassium, rubidium, or cesium, preferably sodium or potassium.

[0435] "Alkaline earth metal ions" refers to divalent ions of Group II metals, such as beryllium, magnesium, calcium, strontium, or barium, preferably magnesium or calcium.

[0436] “Amino” means the group -NR'R", where R' and R” are each independently hydrogen or alkyl. Therefore, as used herein, “amino” encompasses both “alkylamino” and “dialkylamino”.

[0437] "alkylaminoalkyl" refers to the group -R-NHR', where R is alkylene and R' is alkyl. Alkylaminoalkyl includes methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminoethyl, etc.

[0438] "Dialkylaminoalkyl" means the group -R-NR'R", where R is an alkylene group and R' and R" are alkyl groups as defined herein. Dialkylaminoalkyl groups include dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, N-methyl-N-ethylaminoethyl, etc.

[0439] "Aminoalkyl" means the group -R-R', where R' is an amino group and R is an alkylene group as defined herein. "Aminoalkyl" includes aminomethyl, aminoethyl, 1-aminopropyl, 2-aminopropyl, etc.

[0440] "Aminoalkoxy" means the group -OR-R1, where R' is an amino group and R is an alkylene group as defined herein.

[0441] "alkylsulfonylamino" refers to the part of the formula -NR'SO2-R, where R is an alkyl group and R' is hydrogen or an alkyl group.

[0442] "Aminocarbonyloxyalkyl" or "carbamoylalkyl" means the group –ROC(=O)-R', where R' is an amino group and R is an alkylene group as defined herein.

[0443] "Aminosulfonyl" means the group -SO2-NR'R", where R' and R" are each independently hydrogen or alkyl. Therefore, as used herein, "aminosulfonyl" encompasses both "alkylaminosulfonyl" and "dialkylaminosulfonyl".

[0444] "Alkynylalkoxy" refers to a group of the formula -OR-R', where R is an alkylene group and R' is an alkynyl group as defined herein.

[0445] "Aryl" refers to the monovalent cyclic aromatic hydrocarbon moiety consisting of a monocyclic, bicyclic, or tricyclic aromatic ring. The aryl group may be optionally substituted, as defined herein. Examples of aryl moieties include, but are not limited to, optionally substituted phenyl, naphthyl, phenanthryl, fluorenyl, indene, pentenyl, azulel, oxadiphenyl, biphenyl, methylenediphenyl, aminodiphenyl, diphenylthio, diphenylsulfonyl, diphenylisopropylidene, benzodioxane, benzofuranyl, benzodioxy, benzopyranyl, benzooxazinyl, benzooxazinoneyl, benzopiperidinyl, benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl, methylenedioxyphenyl, ethylenedioxyphenyl, etc., including their partially hydrogenated derivatives.

[0446] "Arylalkyl" and "arylalkyl" are used interchangeably, meaning the group -RaRb, where Ra is an alkylene group and Rb is an aryl group as defined herein; examples of arylalkyl are, for example, phenylalkyl, such as benzyl, phenethyl, 3-(3-chlorophenyl)-2-methylpentyl, etc.

[0447] "Arylsulfonyl" refers to a group of the formula -SO2-R, where R is an aryl group as defined herein.

[0448] "Aryloxy group" refers to a group of the formula -OR, where R is an aryl group as defined herein.

[0449] "Arylalkyloxy" or "arylalkyloxy" refers to a group of the formula -ORR", where R is an alkylene group and R' is an aryl group as defined herein.

[0450] “Cyanoalkyl” refers to the part of the formula -R'-R”, where R' is an alkylene group as defined herein, and R” is a cyano or nitrile group.

[0451] "Cycloalkyl" refers to a monovalent saturated carbocyclic moiety consisting of a monocyclic or bicyclic ring. A cycloalkyl moiety may optionally be substituted with one or more substituents, wherein each substituent is independently a hydroxyl, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino group unless otherwise specified. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and their partially unsaturated derivatives.

[0452] "Cycloalkenyl" refers to a monovalent unsaturated carbocyclic moiety consisting of a monocyclic or bicyclic ring containing at least one double bond. The cycloalkenyl moiety may optionally be substituted with one or more substituents, wherein each substituent is independently a hydroxyl, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino group unless otherwise specified. Examples of cycloalkenyl moieties include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0453] “Cycloalkylalkyl” refers to the part of the formula -R'-R”, where R' is alkylene and R” is cycloalkyl as defined herein.

[0454] "Cycloalkylene" refers to a divalent saturated carbocyclic group consisting of a monocyclic or bicyclic ring. Cycloalkylene groups may optionally be substituted with one or more substituents, wherein each substituent is independently a hydroxyl, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino group unless otherwise specified.

[0455] "Cycloalkylalkylene" refers to the part of the formula -R'-R"-, where R' is alkylene and R" is cycloalkylene as defined herein.

[0456] "Heteroalkyl" means an alkyl group as defined herein, in which one, two, or three hydrogen atoms have been replaced by a substituent independently selected from -ORa, -NRbRc, and -S(O)nRd (where n is an integer from 0 to 2), wherein the heteroalkyl group is connected by a carbon atom, wherein Ra is hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl; Rb and Rc are independently hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl; and when n is 0, Rd is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl; and when n is 1 or 2, Rd is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino, or dialkylamino. Representative examples include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxypropyl, 1-hydroxymethylethyl, 3-hydroxybutyl, 2,3-dihydroxybutyl, 2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl, 2-methylsulfonylethyl, aminosulfonylmethyl, aminosulfonylethyl, aminosulfonylpropyl, methylaminosulfonylmethyl, methylaminosulfonylethyl, methylaminosulfonylpropyl, etc.

[0457] "Heteroaryl" refers to a monocyclic or bicyclic group having 5 to 12 ring atoms, wherein at least one aromatic ring contains one, two, or three cyclic heteroatoms selected from N, O, or S, and the remaining ring atoms are C, wherein the heteroaryl group is attached to the aromatic ring. The heteroaryl ring may be optionally substituted, as defined herein. Examples of heteroaryl moieties include, but are not limited to, optionally substituted imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thiophenyl, benzothiophenyl, phenylthio, furanyl, pyranyl, pyridyl, pyrroleyl, pyrazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, benzothiaranyl, benzoimidazolyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isindolyl, triazolyl, triazinyl, quinoxolinyl, purinyl, quinazolinyl, quinazinyl, naphridinyl, pteridinyl, carbazoleyl, azazolyl, diazazolyl, acridineyl, etc., including their partially hydrogenated derivatives.

[0458] "Heteroarylalkyl" or "heteroarylalkyl" means a group of the formula -R-R', where R is an alkylene group and R' is a heteroaryl group as defined herein.

[0459] "Heteroarylsulfonyl" refers to a group of the formula -SO2-R, where R is a heteroaryl group as defined herein.

[0460] "Heteroaryloxy" refers to a group of the formula -OR, where R is a heteroaryl group as defined herein.

[0461] "Heteroarylalkyloxy" refers to a group of the formula -ORR", where R is an alkylene group and R' is a heteroaryl group as defined herein.

[0462] "Heterocyclic alkoxy" refers to a group of the formula –OR-R', where R is an alkylene group and R' is a heterocyclic group as defined herein.

[0463] The terms “halogenated,” “halogen,” and “halide” are used interchangeably and refer to substituents such as fluorine, chlorine, bromine, or iodine. In some embodiments, halogenation refers to a fluorine substituent.

[0464] "Halogenated alkyl" means alkyl as defined herein, wherein one or more hydrogen atoms have been substituted with the same or different halogens. In some embodiments, the halogenated alkyl is a fluoroalkyl; in some embodiments, the halogenated alkyl is a perfluoroalkyl. Exemplary halogenated alkyls include -CH2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl (e.g., -CF3), etc.

[0465] "Haloalkoxy" refers to the portion of the formula -OR, where R is a haloalkyl portion as defined herein. In some embodiments, the haloalkoxy is a fluoroalkoxy; in some embodiments, the haloalkoxy is a perfluoroalkoxy. An exemplary haloalkoxy is a difluoromethoxy.

[0466] "Heterocyclic amino" refers to a saturated ring in which at least one ring atom is N, NH or N-alkyl, and the remaining ring atoms form an alkylene group.

[0467] "Heterocyclic group" refers to a monovalent saturated moiety consisting of one to three rings doped with one, two, three, or four heteroatoms (selected from nitrogen, oxygen, or sulfur). The heterocyclic rings may be optionally substituted, as defined herein. Examples of heterocyclic moieties include, but are not limited to, optionally substituted piperidinyl, piperazinyl, homopiperazinyl, azathioyl, pyrrolyl, pyrazolyl, imidazolinyl, imidazolinyl, pyridinyl, pyridinyl, pyrimidinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolinyl, isothiazolyl, quininecycloyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazolyl, benzothiazolinyl, benzopyrroleyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0468] "Heterocyclic alkyl" refers to the part of the formula -R-R', where R is an alkylene group and R' is a heterocyclic group as defined herein.

[0469] "Heterocyclic oxygen group" refers to the part of the formula -OR, where R is a heterocyclic group as defined herein.

[0470] "Heterocyclic alkoxy" refers to the part of the formula -OR-R', where R is an alkylene group and R' is a heterocyclic group as defined herein.

[0471] "Hydroxyalkoxy" refers to the part of the formula -OR, where R is a hydroxyalkyl group as defined herein.

[0472] "Hydroxyalkylamino" refers to the part of the formula -NR-R', where R is hydrogen or alkyl, and R' is a hydroxyalkyl as defined herein.

[0473] "Hydroxyalkylaminoalkyl" refers to the part of the formula -R-NR'-R", where R is an alkylene group, R' is hydrogen or an alkyl group, and R" is a hydroxyalkyl group as defined herein.

[0474] "Hydroxyalkyl" means an alkyl moiety as defined herein that is substituted with one or more, preferably one, two or three hydroxyl groups, provided that the same carbon atom carries no more than one hydroxyl group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl.

[0475] "Hydroxycarbonylalkyl" or "carboxyalkyl" refers to a group of the formula -R-(CO)-OH, where R is an alkylene group as defined herein.

[0476] "Hydroxyalkyloxycarbonylalkyl" or "hydroxyalkoxycarbonylalkyl" refers to a group of the formula -RC(O)-OR-OH, where each R is an alkylene group and can be the same or different.

[0477] "Hydroxyalkyl" means an alkyl moiety as defined herein that is substituted with one or more, preferably one, two or three hydroxyl groups, provided that the same carbon atom carries no more than one hydroxyl group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxy-5-methyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl.

[0478] "Hydroxycycloalkyl" means, as defined herein, a cycloalkyl moiety in which one, two, or three hydrogen atoms of the cycloalkyl group have been replaced by a hydroxyl substituent. Representative examples include, but are not limited to, 2-hydroxy-cyclohexyl, 3-hydroxy-cyclohexyl, or 4-hydroxy-cyclohexyl.

[0479] “Urea” or “urea group” refers to a group of the formula -NR'-C(O)-NR''R''', where R, R" and R''' are each independently hydrogen or alkyl.

[0480] "Carbamate" refers to a group of the formula -OC(O)-NR'R", where R' and R" are each independently hydrogen or alkyl.

[0481] "Carboxyl group" refers to a group with the formula -C(O)OH.

[0482] "Sulfanamide group" refers to a group of the formula -SO2-NR'R", where R', R" and R" are each independently hydrogen or alkyl.

[0483] "Nitro" refers to -NO2.

[0484] “Cyanogen” refers to -CN.

[0485] "Phenoxy" refers to a benzene ring that is substituted with at least one -OH group.

[0486] "Acetyl" refers to -C(=O)-CH3.

[0487] "Cn-m-" is used as a prefix before a functional group, where "n" and "m" represent integer values ​​(i.e., 0, 1, 2, 12), such as C1-12-alkyl or C5-12-heteroaryl. The prefix indicates the number or range of carbon atoms present in the functional group. In the case of ring systems, regardless of whether the ring atom is a carbon atom or a heteroatom, the prefix indicates the number or range of ring atoms. In the case where the functional group constitutes both a cyclic and acyclic portion (i.e., "arylalkyl" consists of an aryl portion and an alkyl portion), the prefix is ​​used to indicate the total number of carbon atoms and ring atoms present. For example, for arylalkyl, "C7-arylalkyl" can be used to represent "phenyl-CH2-". For some functional groups, zero carbon atoms can be present, such as C0-aminosulfonyl (i.e., -SO2-NH2, where both potential R groups are hydrogen), where "0" indicates the absence of carbon atoms.

[0488] The term "peptide" refers to an amide derived from two or more amino acids through the combination of an amino group and a carboxyl group of an acid. A "monopeptide" refers to a single amino acid, a "dipeptide" refers to an amide compound containing two amino acids, a "tripeptide" refers to an amide compound containing three amino acids, and so on. The C-terminus of a peptide can be linked to another part via an ester functional group.

[0489] When used in conjunction with "aryl", "phenyl", "heteroaryl", "cyclohexyl" or "heterocyclic", "optionally substituted" means an aryl, phenyl, heteroaryl, cyclohexyl or heterocyclic group optionally and independently substituted by one to four substituents, preferably one or two substituents, wherein the substituents are selected from alkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, hydroxyalkyl, halogen, nitro, cyano, hydroxy, alkoxy, amino, acylamino, monoalkylamino, dialkylamino, haloalkyl, haloalkoxy, heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl), -(CR'R")n-COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R")n-CONRaRb (where n is an integer from 0 to 5, R' and R” are independently hydrogen or alkyl, and Ra and Rb are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl).

[0490] "Leaving group" refers to a group that has the meaning commonly associated with it in synthetic organic chemistry, namely, an atom or group that can be replaced under substitution reaction conditions. Examples of leaving groups include, but are not limited to, halogens, alkane- or arylsulfonyloxy groups, such as methanesulfonyloxy, ethanesulfonyloxy, thiomethyl, benzenesulfonyloxy, toluenesulfonyloxy, and thiophenoxy, dihalophosphonooxy, optionally substituted benzyloxy, isopropoxy, acyloxy, etc.

[0491] "Regulator" refers to a molecule that interacts with a target. Interactions include, but are not limited to, agonists and antagonists as defined in this article.

[0492] "Optional" or "optionally" means that the event or situation described below may, but not necessarily, occur, and the description includes both cases where the event or situation occurs and cases where it does not.

[0493] "Disease" and "disease state" refer to any disease, symptom, condition, illness, or indication.

[0494] "Inert organic solvent" or "inert solvent" means a solvent that is inert under the reaction conditions described herein, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, chloroform, dichloromethane (methylene chloride or dichloromethane), dichloroethane, diethyl ether, ethyl acetate, acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, etc. Unless otherwise stated, the solvents used in the reactions of this disclosure are inert solvents.

[0495] "Pharmaceutical acceptable" means that the pharmaceutical composition used to prepare the pharmaceutical composition is generally safe, non-toxic, and not biologically or otherwise desirable, and includes veterinary and human pharmaceuticals that are acceptable.

[0496] The term "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable as defined herein and has the desired pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, etc.; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth metal ion, or aluminum ion; or complexes formed with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucosamine, triethanolamine, trimethylamine, tris(hydroxymethyl)aminomethane, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Preferred pharmaceutically acceptable salts are salts formed from acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium, potassium, calcium, zinc, and magnesium. All references to pharmaceutically acceptable salts include the same acid addition salts in their solvation form (solvent) or crystalline form (polymorph) as defined herein. Generally, when a particular salt is included in the structure or formula herein, it should be understood that other pharmaceutically acceptable salts may be substituted within the scope of this disclosure; for example, in the case of the quaternary ammonium salt of formula VIII, a chloride or another anion or combination of ions may be included, and similarly, in the sodium carboxymethyl salt of formula IX, the sodium shown may be substituted with another cation.

[0497] The term "protective group" or "protecting group" refers to a group that selectively blocks one reaction site in a multifunctional compound, allowing the chemical reaction in synthetic chemistry to proceed selectively at another unprotected reaction site in a generally associated sense. Certain processes of this disclosure rely on protecting groups to block reactive nitrogen and / or oxygen atoms present in the reactants. For example, the terms "amino protecting group" and "nitrogen protecting group" are used interchangeably herein and refer to those organic groups intended to protect nitrogen atoms from undesirable reactions during synthesis. Exemplary nitrogen protecting groups include, but are not limited to, trifluoroacetyl, acetamipridyl, benzyl (Bn), benzyloxycarbonyl (benzyloxycarbonyl, CBZ), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl (BOC), etc. Those skilled in the art will know how to select groups that are easily removable and capable of withstanding subsequent reactions.

[0498] "Subject" refers to both mammals and non-mammals. Mammals mean any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cows, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; etc. Examples of non-mammals include, but are not limited to, birds. The term "subject" does not indicate a specific age or sex.

[0499] "Therapeutic effective amount" refers to the amount of a compound that, when administered to a subject to treat a disease state, is sufficient to affect that disease state. "Therapeutic effective amount" will vary depending on the compound, the disease state being treated, the severity of the disease, the subject's age and relative health condition, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0500] When referring to variables, the terms "those defined above" and "those defined herein" are incorporated by reference into the generalized definition of the variable as well as the preferred, more preferred, and most preferred definitions (if any).

[0501] “Treating” a disease state includes: (i) preventing the disease state, that is, preventing the development of clinical symptoms of the disease state in subjects who may be exposed to or are susceptible to the disease state, but who have not yet experienced or shown symptoms of the disease state; (ii) suppressing the disease state, that is, preventing the development of the disease state or its clinical symptoms; or (iii) alleviating the disease state, that is, temporarily or permanently resolving the disease state or its clinical symptoms.

[0502] In the structure of this paper, any open valence appearing on a carbon, oxygen, sulfur, or nitrogen atom indicates the presence of a hydrogen atom.

[0503] Example

[0504] Example 1. Solubilization of compounds by HPβCD

[0505] Example 1 The ability of HPβCD (DS4.5) monomers to solubilize various sterols, vitamins, oxosterols, and steroid hormones has been demonstrated. Figure 2A -B). Lower turbidity indicates a greater ability to solubilize a given sterol. Figure 2A -B shows the solubilization of various sterols and sterol derivatives by the HPβCD (DS4.5) monomer as assessed by relative turbidity.

[0506] We also tested HPβCD variants by measuring the number of hydroxypropyl groups on the HPβCD. We tested a range from 3.7 to 21 (the maximum possible number of substitutions). Although the data were noisy, with greater substitution, the ability to solubilize 7KC and cholesterol decreased. Figure 2C-2D This is supported by molecular docking with extensive substitutions on the monomer HPβCD. Figure 2E Based on known chemical characteristics, monomers and sterols were designed in PyMOL. The most probable position of each hydroxypropyl group was used, and the top 20 conformations were considered when determining the affinity score for each pair. If any atom of the sterol passed through a plane formed by the O4 oxygen of the cyclodextrin, the conformation was included in the calculation. The HPβCD of lower DS showed a greater preference for solubility at 7KC relative to cholesterol, indicating their specificity for 7KC. Not limited by theory, a possible explanation is the availability of the maximum number of hydroxyl groups that can hydrogen bond with the ketone group at position 7 on 7KC; this theory is not required to implement the present invention.

[0507] Example 2. Computational modeling of the interactions between cyclodextrin monomers and dimers and cholesterol and 7KC.

[0508] Overview

[0509] This embodiment describes molecular modeling and computational simulations aimed at investigating the mechanism of CD binding to sterols, predicting the relative binding affinity of cyclodextrin dimers to cholesterol and 7KC, and identifying cyclodextrin dimers predicted to have a higher affinity for 7KC than for cholesterol. It is hypothesized that a configuration in which the sterol is completely surrounded by CD or CD dimers isolates the hydrophobic sterol from the hydrophilic solvent, thereby allowing the sterol to enter the solution.

[0510] For initial docking analysis ( Figure 2E [Monomer], 4B [Dimer]), HPβCD monomers and dimers of various substitution levels were constructed using the computer modeling program PyMOL (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC.), and then the extended package AutoDock Vina (Trott [ et al. ], J. Comput.Chem.,31(2):455-61. (2010))(Developed by Scripps Research Institute (La Jolla, CA, USA)) to simulate the interactions between hypothetical CD molecules and 7KC or cholesterol. Autodock Vina is a molecular docking software with significant improvements in accuracy and speed compared to the previous Autodock 4. The software predicts non-covalent binding between molecules, thus using a scoring function to approximate the standard chemical potential of the system to predict energy-favorable conformations and binding affinity. Typically, hydroxypropyl dimers and DS2-6 monomers are found to show the best specificity for 7KC.

[0511] Using GROMACS 2018 (University of Groningen, Groningen, Netherlands; Bekker [ et al. ], World Scientific (1993); and Berendsen [ et al. ], Comp. Phs. Comm. Molecular dynamics simulations were performed using AutoDock Vina, 91:43-56. (1995) et al. Docking simulations were also conducted using AutoDock Vina to simulate the binding of three β-cyclodextrin derivatives to 7KC or cholesterol: natural monomer (DS0) β-cyclodextrin (βCD), monomeric hydroxypropyl-β-cyclodextrin (DS5, HPβCD), and dimerized DS5 hydroxypropyl-β-cyclodextrin, where two HPβCD monomers are linked via a butyl chain through the O2 oxygen of DS2 monomer to the O3 oxygen of DS3 monomer, resulting in a total DS of 5. Both ligands are asymmetric, therefore simulations were performed for both upward and downward orientations of the ligands. These simulations were then repeated in the AMBER force field and at translational positions to determine which position / force field produces the most informative data for these novel molecules (initial MD analysis). Figure 4D -MM). The GROMOS force field with the initial location determined most effectively captures the interaction between the CD dimer and the sterol, therefore this force field and location are used for subsequent abbreviation MD simulations of other CD dimers (subsequent MD analyses, Figure 4NN -SS; 5B-C; 6B-7B).

[0512] Typically, the addition of hydroxypropyl groups has been found to lead to instability in the complex; however, it exhibits higher specificity for 7KC than for cholesterol, compared to observations of native, unsubstituted βCD. This is because 7KC can form and reform a stable complex in both up and down orientations, while cholesterol is less likely to form a stable complex. This may be because βCD does not appear to be as fully encapsulated by 7KC, especially in the down orientation. βCD dimerization significantly enhances the affinity for sterol targets such as 7KC and cholesterol. This is clearly evident in the formation of stable dimer complexes with strong interaction energies across all ligands and orientations, where the ligands are nested within the hydrophobic core of the CD dimer, thus allowing the ligands to be solubilized in aqueous solutions.

[0513] To further analyze the effect of minor modifications on βCD dimers, additional docking and molecular dynamics simulations were performed for various linkers and degrees of substitution of HPβCD (Figure 8). We extended this analysis to include other alternative substitution types and other alternative linkers (Figure 9) and found that, in general, DS showed the best specificity for 7KC at ~2–6, applicable to a wide range of substitution and linker types.

[0514] Based on this extended computational analysis, we propose that the dimerization of βCD is crucial for forming robust, soluble complexes with sterols, regardless of the type or position of the substituents or linkers used. A wide range of dimerized βCD molecules have been tested, and it has been shown that even with significant chemical differences between various forms of substitution and linker, and compared to the monomeric βCD form, a higher affinity for sterols is maintained.

[0515] Calculation method

[0516] Initial docking simulation

[0517] We have developed a method using AutoDock Vina that allows for faster and easier analysis of the entire trajectory. On the computer Predicting the binding of cyclodextrins to various sterols is extremely time-consuming and computationally intensive. Applying this technique to cyclodextrin systems allowed us to perform hundreds of docking simulations with many different cyclodextrins we designed. This type of computational modeling shows us the possible interactions between different cyclodextrins and different sterols, while generating spatial information and binding affinity data.

[0518] These conformational predictions can be modeled for various sterols and / or derivatives of CD to reveal potential mechanical characteristics. We have several preliminary theories of binding, which we hope to test using computational techniques. We have developed different models for HPβCD to test our binding theories:

[0519] Monomer-sterol association: We tested the affinity of monomers for sterols to compare with dimer association to help determine whether sterols are more likely to bind to monomers or dimers of HPβCD, and whether these monomers exhibit specificity for 7KC or cholesterol. Figure 4A ).

[0520] Linked dimers-sterols: To eliminate the need for multiple steps and to test novel potential molecules, two monomers are covalently linked to various types of linkers and associated with sterols to investigate the affinity and specificity for these pre-linked dimers. Figure 4A ).

[0521] To make the outputs of these documents comparable, a scoring system for sterol complexes was developed, where the most favorable affinity is adjusted based on whether the dimer is head-to-head (where applicable) and whether the sterol is actually in the barrel of the HPβCD cavity. The number of “complex conformations” (up to twenty configurations) is then added to the absolute value of the most favorable affinity; that is, an association producing a 15 / 20 configuration of complex with sterol (head-to-head and / or sterol within the CD cavity) and an optimal affinity of -10 kJ / mol will receive a score of 25 (|-10|+15=25). For this calculation, if any atom on the ligand crosses the plane formed by the O4 atoms of the CD, regardless of the angle or extent of insertion into the cavity, the ligand is considered to be in the complex. The resulting value is called the “affinity score.”

[0522] We then expanded the docking analysis to include various types of substitutions (including those with charged groups) and linkers to determine whether 7KC specificity was affected by these factors. Sulfobutyl and methyl substitutions with triazole and butyl linkers were tested across the entire DS range of 0–20 and showed a similar pattern to hydroxypropyl, with the highest 7KC specificity observed at a DS of approximately 4 (…). Figure 5A and 6A Therefore, other cyclodextrins (such as quaternary ammonium and carboxymethylated cyclodextrins) were only tested at low DS (~4).

[0523] Initial molecular dynamics simulations ( Figure 4D -MM)

[0524] An initial set of simulations was performed using GROMACS 2018 (University of Groningen, Groningen, Netherlands) in the force fields of GROMOS 54a7 and AMBER 99SB, with two repetitions of these simulations to help determine the consistency of the observed interactions. Then, two repetitions of each of the three CD molecules and each orientation of the ligands were repeated with different initial structures in which the ligands were displaced to determine the dependence of these calculations on the initial structure and the force field. The resulting 48 hydroxypropyl dimer trajectories were then analyzed using GROMACS tools.

[0525] Unlike docking, molecular dynamics allows simulated molecules to interact in a time-dependent manner, rather than taking a simple snapshot of an energy-favorable conformation as docking provides. For the initial three CD-sterol complexes, the simulation time was extended to one microsecond (a very long time for MD simulations), thus allowing sufficient time for the complexes to stabilize. The analysis output was then used to determine the distance between the centroids of all O4 atoms (for dimers and monomers, the center of the CD cavity) and the centroids of the ligands, the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the principal axis of the ligands (see [link to analysis]). Figure 4C ), as well as the Lanner-Jones and Coulomb energy of the interaction between cyclodextrins and ligands.

[0526] Thus, distance represents the proximity of the ligand to the cyclodextrin, angle represents the degree of nesting of the ligand within the CD cavity, and the energy of the interaction represents the strength of the interaction between the two molecules (the stronger the negative interaction energy, the stronger the interaction). Figure 4C The "angle" measurement indicates how the ligand is shielded from surrounding water molecules: zero or 180 degrees indicates the ligand is completely perpendicular to the cyclodextrin plane, while 90 degrees indicates the ligand is parallel to the CD plane and therefore does not recombine within the cavity. For these simulations, we chose 30 degrees for the initial, recombinating "upward" configuration (the sterol head associated with the CD subsurface, the tail associated with the main surface, and the entire ligand inserted into the CD cavity) and 150 degrees for the initial, recombinating "downward" configuration (the sterol tail associated with the CD subsurface, the head associated with the main surface, and the entire ligand inserted into the CD cavity). Note that for dimers, only the plane of a single CD monomer is considered as the angle between the CD and the ligand, but if the dimer is fully formed, this plane will reflect the plane of the sister monomer.

[0527] The number of water molecules within 3 Å of the ligand was determined over time to ascertain the degree to which CD shields the ligand from the surrounding solvent. It was hypothesized that more water molecules around the ligand would indicate that it was not adequately shielded by the surrounding water and therefore not in solution. All these simulations were extended to 1 microsecond (1000 ns), a time long enough to accurately characterize the interaction between CD and the sterol.

[0528] Preliminary long-term analysis provides evidence that the simulation correctly captures the interactions between CD monomers and dimers and sterol ligands, and therefore can be extended to other CD monomers and dimers without this laborious approach.

[0529] Other molecular dynamics simulations ( Figure 4NN -SS, 5B-C, 6B-C, 7A-B, 8H-I)

[0530] Based on the initial HPβCD simulations, it was concluded that the GROMOS force field at the non-translational position yielded the best and most dynamic results for these complexes. This lengthy initial analysis is crucial for establishing precedents for modeling these novel molecules, thus enabling shorter and more targeted simulations for other types of dimers. Therefore, the expansion of molecular dynamics analysis with various types of linkers and substitutions showed promise. Firstly, for methyl ( Figure 5A ) and sulfobutyl ( Figure 6A Docking calculations were performed on the DS range of the βCD dimer. This indicates that low DS (~4) shows the most promising results for the dimer with the best 7KC specificity. Therefore, additional MD simulations were performed for the DS4 βCD dimer with triazole and butyl linkers. Figure 4RR -SS, 5B-C, 6B-C, 7A-B). We also simulated the DS0 βCD dimer ( Figure 4NN These simulations were performed for 100 ns, and only the angles and energies of the interactions were analyzed to assess the key differences or similarities between these molecular interactions and the interactions of the hydroxypropyl dimer linked to the butyl group.

[0531] Other docking simulations

[0532] After initial simulations demonstrated similar promise for the feasible range of substitutions and linkers, further screening of linkers, substitutions, and even substitution sites was conducted using the same docking technique described above. This analysis showed that, regardless of linker or substitution type, the actual dimerization of βCD largely (if not completely) transmits the effectiveness of these molecules.

[0533] Calculation results and conclusions

[0534] Connection:

[0535] We first examined whether HPβCD could act as a monomer to bind cholesterol and 7KC ( Figure 2E Then we examined whether HPβCD could act as a dimer to bind cholesterol and 7KC ( Figure 4B ).

[0536] We discovered HPβCD monomer ( Figure 2E At low degrees of substitution (DS), it exhibits high affinity for both cholesterol and 7KC, but appears to show decreasing affinity for both sterols with increasing DS. This is likely due to the crowding of the hydroxypropyl group, which prevents the sterol from entering the core of the monomer. Additionally, fewer hydroxyl groups on the inner surface of the CD can be used to bond to the carbonyl group on 7KC via hydrogen bonding. Optimal specificity (rather than optimal affinity) is considered to be spiking at DS4, with a preference for 7KC extending from DS2 to DS6, and shifting to cholesterol for DS7 and higher. After DS10, almost no affinity is observed in these models.

[0537] Compared to the monomeric CD, the butyl-linked dimer exhibits a higher affinity for sterols, with the dimerized DS10 and DS4 showing the best affinity / specificity for 7KC. Figure 4B However, for these calculations, this specificity appears to exist only in dimers of specific DSs, and the variation between different DSs shown in these calculations is significant. Except for DS6, triazole-linked dimers generally exhibit better specificity, with similar affinity to butyl-linked dimers. This specificity is presumably due to additional hydrogen bonding between the contributing nitrogen and the hydrogen-accepting ketone at 7KC.

[0538] Initial molecular dynamics analysis:

[0539] Figure 4D -O supports the hypothesis that the natural (unsubstituted, DS0) monomer βCD is capable of complexing with 7KC and cholesterol in both up and down orientations, although 7KC maintains a more stable complexation in the down orientation than cholesterol, and vice versa in the up orientation. Cholesterol exhibits less variation throughout the up trajectory, showing how cholesterol can leave and reassociate with CD multiple times in the up orientation (note the large angular change around approximately 150 ns, around which cholesterol rotates to associate with the relative orientations). Figure 4D This angular change indicates that the downward orientation is significantly more stable, to the point that cholesterol leaves the cavity and rotates 180 degrees before reassociating, and the overall affinity for cholesterol is very high because it is able to accomplish this large movement in the simulation.

[0540] On the other hand, once the complex breaks in either orientation, 7KC does not reassociate, but the downward orientation is significantly more stable at more than half the trajectory, supporting the hypothesis that 7KC favors downward orientation. This suggests that both 7KC and cholesterol favor downward orientation, where the head group associates with the main surface and the tail group with the subsurface, but in this favorable conformation, only cholesterol can actually leave CD and reassociate with CD. This could explain why native CD solubilizes cholesterol and its derivatives well, but not specifically for 7KC. This slight preference of the native monomer βCD for cholesterol is expected and consistent with published experimental results (Zidovetzki [ et al. ], Biochim. Biophys. Acta., 1768(6):1311-1324. (2007)), and further enhanced by the number of water molecules around the ligand ( Figure 4E ); cholesterol has much less water than 7KC, especially in the "upward" orientation.

[0541] AMBER force field ( Figure 4G-4I The AMBER force field shows a significantly enhanced interaction between native βCD and the sterol. Throughout the trajectory, both ligands at both orientations remain inside the cyclodextrin ring, with little observed preference for 7KC or cholesterol. Compared to the GROMOS force field, the AMBER force field shows a stronger and longer interaction between the two molecules, and the solubilization of native βCD for the sterol in the AMBER force field appears to be the same for both upward and downward orientations between the two ligands. Despite this strong and stable interaction, the AMBER force field may not fully capture the interaction between βCD and the sterol, as the complex does not break down at all. Some shifts are necessary to fully elucidate the interaction that occurs, but this is good evidence that a strong complex does indeed form between the two molecules.

[0542] Even when the ligand translates deeper within the CD cavity ( Figure 4J The native βCD complex still forms efficiently in both force fields, although the consistency is less pronounced for GROMOS than for AMBER. The GROMOS force field shows a significant preference for the "up" orientation of 7KC and the "down" orientation of cholesterol; however, only AMBER shows strong interactions between both ligands and CD. This suggests that 7KC and cholesterol have similar and strong interactions with native βCD, consistent with experimental data, but the orientation of the ligands does appear to differ in the observed complexation. These subtle differences in trajectory are detailed below.

[0543] Monomer DS5 HPβCD ( Figure 4P-AA) showed an interaction between CD and sterols in the GROMOS force field that was inconsistent with that of natural CD, but as Figure 4P As shown, this also appears to favor the downward orientation of 7KC. AMBER force field ( Figure 4S Y) again showed stronger and more consistent interactions, but the stable complexes formed in both force fields remained the same. In general, we can see that the addition of a hydroxypropyl group to the cyclodextrin monomer reduces the likelihood of complex formation between the two ligands in both force fields, but 7KC is able to form and reform stable complexes more stably than cholesterol. Generally, cholesterol appears to be less likely to form complexes with HPβCD than 7KC in both force fields, and more water molecules are available for contact with cholesterol, indicating a preference of HPβCD for 7KC. This is in Figure 4R The pattern is clear because the 7KC complex forms and reforms in a "downward" orientation, while cholesterol does not. The visual trajectory also shows how strongly 7KC prefers an "upward" orientation, but still forms complexes in a "downward" orientation at approximately 500 ns.

[0544] When the ligand translates, HPβCD can more effectively complex with both sterols because the initial position of the ligand is more deeply embedded in the cavity of the CD. This translational trajectory in GROMOS ( Figure 4V Compared to previous simulations, the preference for 7KC over cholesterol is more pronounced because 7KC can form stable complexes in both orientations, while cholesterol can only form stable complexes in the "downward" orientation. Furthermore, the upward-oriented 7KC... external of the cavity Initially, it is able to associate with the cavity and form a very stable complex within 300 ns. The AMBER force field again shows a significantly stronger interaction between HPβCD and the sterol, but still forms the same stable complex and favors the upward orientation of both ligands, with slightly less water around 7 KC overall throughout the trajectory (see [link to relevant documentation]). Figure 4T This is presumably because the "downward" orientation shows a greater protrusion of the sterol head group in the cavity compared to the "upward" orientation. This is consistent with our experimental data (Figure 2), as the HPβCD monomer has been shown to have some specificity for 7KC while still forming stable, clearly soluble complexes with 7KC and cholesterol. All of these simulations will be detailed below.

[0545] Then, our novel butyl-linked DS5 hydroxypropyl-β-cyclodextrin dimer was modeled in the GROMOS and AMBER force fields using 7KC and cholesterol, as shown below. Figure 4BB-MM. The comparison of these trajectory plots with those of monomeric HPβCD and natural βCD provides clear evidence that the dimer form binds sterols significantly more reliably than its corresponding hydroxypropylated or non-hydroxypropylated monomers. This is consistent with our experimental data (Fig. 16). Compared to monomeric simulations, the angles, distances, and energies around the ligand, as well as the water molecules, are more stable, and it is in a configuration with significantly stronger solubility. When the complex is fully formed in a downward orientation, the GROMOS force field shows less than five Å between the ligand centroid and CD ( Figure 4BB When forming complexes, monomers in the GROMOS force field consistently exhibit an upward orientation of 5–10 Å between molecules. The AMBER force field also shows a very strong interaction between the sterol and the dimerized CD, with the interaction energy approaching -300 kJ / mol in the downward orientation compared to approximately -150 kJ / mol for the monomers. Figure 4BB This indicates that the dimer forms a very strong, stable complex with the two ligands, especially in the downward orientation, and particularly compared to the monomer βCD.

[0546] AMBER force field results ( Figure 4EE The findings from GROMOS force field simulations (KK) support the conclusion that the dimerization of HPβCD produces a stronger and more stable interaction between CD and sterol, with a small distance between the two molecules and a large interaction energy. Dimeric CD also consistently shows fewer than five water molecules surrounding the ligand, particularly in a downward orientation, while monomeric CD shows ten water molecules surrounding the ligand in an upward orientation. Figure 4CC While this can sometimes be achieved with monomers containing 7KC and cholesterol, dimerization has significantly reduced the overall presence of water surrounding the sterol. Dimerization of HPβCD also transmits some specificity for 7KC, which is evident because 7KC... Always At least one of the two connected CDs along the entire trajectory Maintaining Agreement Regardless of the force field or translation, cholesterol in at least a portion of the trajectory typically dissociates from both monomers and may even form a distorted head-to-tail dimer configuration, in which cholesterol cannot be completely encapsulated by the dimer. These trajectories will be discussed in detail in the next section.

[0547] These simulations provide strong evidence that the dimerization of HPβCD promotes complexation with sterols by forming an encapsulated complex that isolates the hydrophobic sterol from the surrounding water molecules. The data indicate that dimerized HPβCD generally has a higher sterol affinity than the monomer, and it exhibits a preference for 7KC, as the association time of 7KC with at least one CD is significantly longer than that with cholesterol. From this approach, we can conclude that while the strong complex formation in the AMBER force field provides good evidence for the plausibility of our complex formation and stability, more valuable information can be gleaned from the GROMOS force field. This is because, unlike AMBER, the GROMOS force field reveals dynamic interactions between molecules, rather than simply an incredibly (and potentially impractical) stable complex.

[0548] The details of the 48 trajectories of the hydroxypropyl-β-cyclodextrin dimer are described below, with each trajectory being one microsecond in length.

[0549] Detailed description of the initial molecular dynamics trajectory (Figure 4):

[0550] Natural monomers βCD and 7KC, upward orientation, GROMOS force field:

[0551] exist Figure 4F In the process, 7KC begins at the head base inserted into the CD cavity and the tail of the extending subsurface. At 134 ns, the complex breaks off, and 7KC moves toward the subsurface, rotating out of the cavity. It then remains associated with the subsurface, moving the head base in and out of the cavity until the complex completely dissociates at 150 ns, and 7KC moves around the frame, reassociating with the primary surface. 7KC continues to associate and dissociate with the primary surface, but does not re-enter the cavity for the remainder of the trajectory.

[0552] Natural monomers βCD and cholesterol, upward orientation, GROMOS force field:

[0553] 4F shows cholesterol (upward) initially inserting into the CD cavity at the tail end, with the head end extending outwards from the subsurface. The complex breaks down at approximately 150 ns, and the "angle" of the cholesterol changes significantly as it leaves the cavity and rotates outwards, parallel to the cyclodextrin, before recassociating in the opposite direction, with the tail end extending outwards from the subsurface. The cholesterol then reinserts into the head end, cycling for approximately 200 ns between insertion and becoming parallel to the CD. Figure 4D The changes in the angle, energy, and distance (upward) of cholesterol are visible. At approximately 300 ns, the complex completely breaks down (corresponding to...). Figure 4DThe cholesterol is spiked and moves randomly around the CD molecule. The two molecules briefly re-associate for about one nanosecond at 310 ns, with the cholesterol parallel to the main surface of the CD. The cholesterol then resumes random movement until it re-associates with the subsurface for about two nanoseconds at 330 ns, while the cholesterol tail loosely inserts into the CD cavity. The cholesterol then flips at about 400 ns to associate the head group with the CD cavity, and this configuration remains relatively stable with the periodic association and dissociation of the head group until the complex breaks again at about 560 ns. At this point, the cholesterol briefly moves randomly around the CD and then associates the tail with the subsurface of the CD. At 580 ns, the tail of the cholesterol is tightly inserted into the CD molecule, with its head group extending from the CD subsurface. The complex then breaks again at 582 ns until 610 ns, at which point the complex reforms again with the head group inserted from the subsurface. The complex breaks again at approximately 680 ns, reforms at 750 ns, then breaks again at 880 ns, reforms at 920 ns, and continues to break and reform approximately every 10 ns (always associating as shown at 920 ns) until the trajectory ends. The fact that cholesterol completely leaves the CD cavity and then reassociates within the simulation time indicates that the procedure is capable of spontaneously associating two molecules, unaffected by any external environment. This provides strong evidence that this interaction is plausible, repeatable, and can be effectively captured through simulation.

[0554] Natural monomers βCD and 7KC, downward orientation, GROMOS force field:

[0555] 7KC begins by inserting into the CD chamber at the tail and extending from the head base. Figure 4F The primary surface of the CD chamber. As 7KC moves back and forth and tilts within the cavity, the complex remains in this conformation. The complex breaks down only after 600 ns, at which point 7KC rapidly leaves the cavity and rotates to the secondary surface. 7KC continues to float near the simulation frame, periodically and briefly binding with the CD within the conformation, similar to 720 ns. Overall, the complex remains in a dissociated state until the simulation ends. Despite this dissociation, the complex remains stable for 600 ns, indicating that once 7KC enters the CD chamber, it is held within by interaction forces. This trajectory can be observed... Figure 4D The quantification is based on the fact that the 7KC (upward) curve remains relatively flat until about 600 ns, which is where the complex breaks down and is assumed to move randomly.

[0556] Natural monomers βCD and cholesterol, downward orientation, GROMOS force field:

[0557] exist Figure 4FIn the process, cholesterol begins at a head base inside the CD cavity in a downward position and extends outwards to the subsurface. This remains stable until approximately 125 ns when cholesterol rotates out of the cavity, but cholesterol continues to periodically insert its head base from the subsurface into the CD cavity over the next 200 ns. At approximately 340 ns, the complex completely breaks down, and cholesterol flies around the simulation frame until it re-associates with the subsurface in the same manner as before at approximately 560 ns. Cholesterol then dissociates after approximately 30 ns and re-associates parallel to the primary surface. Cholesterol then oscillates between associating with the primary surface in this manner and randomly floating for the remainder of the trajectory.

[0558] Natural monomers βCD and 7KC, upward orientation, AMBER force field:

[0559] exist Figure 4I The interactions observed in the AMBER force field support the strong solubilization of sterols by the natural monomer βCD. Throughout the trajectory, both ligands in both orientations remain within the cyclodextrin ring, with almost no preference for 7KC or cholesterol. 7KC (upward) originates from the molecular center within the CD cavity, with the head group extending slightly beyond the subsurface and the tail group extending slightly beyond the main surface. Throughout the trajectory, 7KC remains tightly bound within the CD cavity. Figure 4G Slight back-and-forth swaying is visible, and the overall flattened lines show slight changes, indicating a stable conformation that will not break. This is consistent with experimental data, although the AMBER force field shows a stronger and longer interaction between the two molecules than the GROMOS force field.

[0560] Natural monomers βCD and cholesterol, upward orientation, AMBER force field:

[0561] Cholesterol (upwards) originates from the molecular center within the CD cavity, with the head base extending slightly from the subsurface, and the tail base from... Figure 4I The main surface extends slightly outwards. The complex remains stable throughout its trajectory; cholesterol never leaves the lumen or changes orientation; it merely oscillates back and forth within the lumen. These small positional changes correspond to... Figure 4G Small protrusions, especially at the corners.

[0562] Natural monomers βCD and 7KC, downward orientation, AMBER force field:

[0563] Figure 4I In this complex, 7KC (downward) originates from the molecular center within the CD cavity, with the head group extending slightly beyond the main surface and the tail group extending slightly beyond the subsurface. The complex remains stable throughout its trajectory; 7KC never leaves the cavity or changes orientation; it merely oscillates within it. These small positional changes correspond to… Figure 4G Small protrusions, especially at the corners.

[0564] Natural monomers βCD and cholesterol, downward orientation, AMBER force field:

[0565] Figure 4I The cholesterol (downward) structure begins at the molecular center within the CD cavity, with the head group extending slightly beyond the primary surface and the tail group extending slightly beyond the secondary surface. The complex remains stable throughout its trajectory; cholesterol never leaves the cavity or changes orientation; it merely oscillates back and forth within it. These small positional changes correspond to… Figure 4G Small protrusions, especially at the corners.

[0566] The translated natural monomers βCD and 7KC are oriented upwards, with a GROMOS force field.

[0567] exist Figure 4L In the process, 7KC begins at the molecular center within the CD cavity, with the head extending slightly beyond the subsurface and the tail extending slightly beyond the main surface. As 7KC is removed from the subsurface and rotated to associate parallel to it, the complex remains stable for approximately 710 ns. Then, 7KC rotates fully to insert the head, causing it to extend toward the main surface at 715 ns, while the tail extends from the subsurface. 7KC then associates and dissociates the head with the CD cavity several times until the complex completely breaks apart at approximately 850 ns. For the remainder of the trajectory, the complex remains in a dissociated state.

[0568] The translated natural monomers βCD and cholesterol are oriented upwards, within a GROMOS force field.

[0569] Figure 4L The diagram shows cholesterol initially associating with CD, with the head extending from the subsurface and the tail extending from the main surface. As cholesterol migrates to the main surface of CD, the complex dissociates at approximately 120 ns, inserts into the head, and rotates in and out of the cavity on the main side until it completely dissociates again at approximately 160 ns. At approximately 163 ns, cholesterol reassociates with the subsurface portion, rotating back to the main surface after 5 ns. Cholesterol then alternates between association with the subsurface or main surface and random movement until the complex reforms at the very end of the trajectory in the final three nanoseconds. This continuous formation and deformation of the complex on the computer suggests a strong tendency for formation in reality.

[0570] The translated natural monomers βCD and 7KC are oriented downwards, with a GROMOS force field.

[0571] Figure 4LThe diagram shows a 7KC starting from a downward position, with the head base extending from the main surface. At 40 ns, the 7KC withdraws from the cavity and associates parallel to the subsurface, reinserting the head base after 2 ns, then withdrawing again. The complex completely breaks at 45 ns, at which point the 7KC floats around the analog frame and again associates with the main surface at 47 ns, briefly inserting the head base, then rotating back to be parallel to that surface until the complex breaks again at 51 ns. The complex reforms at 210 ns, with the head base inserting from the subsurface and the tail extending outward, just like the initial conformation, and remains stable until 268 ns when the 7KC withdraws from the CD again and associates parallel to the subsurface. The complex breaks completely again but briefly reforms at 360 ns. Thereafter, the 7KC occasionally associates parallel to one of the two surfaces in the conformation, as at 710 ns, but does not re-enter the CD cavity. The trajectory is somewhat obscure because 7KC associates with the cavity in just 100 ns, but the complex still forms freely in the simulation, suggesting that it is likely to form in reality, even if the interaction forces seem inconsistent.

[0572] The translated natural monomers βCD and cholesterol are oriented downwards, within a GROMOS force field.

[0573] exist Figure 4L In the process, cholesterol begins association with the head base on the primary side, while the tail extends out onto the subsurface. Cholesterol oscillates back and forth within the cavity until the complex breaks apart at approximately 15 ns. Cholesterol reinsertes into the head base at 17 ns and continues to rotate between parallel to the subsurface of the CD and from the subsurface (always the head base) into the cavity until the complex truly dissociates at approximately 675 ns. This indicates a strong interaction and tendency for cholesterol to form a surface-stable complex with native β-CD, but once cholesterol has completely dissociated from the subsurface of the CD at 675 ns, the complex does not reassociate.

[0574] The translated natural monomers βCD and 7KC are oriented upwards, with an AMBER force field.

[0575] exist Figure 4O In the middle, 7KC begins with a head base extending from the subsurface and a tail extending from the main surface. The complex remains stable throughout the trajectory, but 7KC does exhibit greater flexurality than the down-oriented type—for most of the trajectory, 7KC remains flexed around the CD ring.

[0576] The translated natural monomers βCD and cholesterol are oriented upwards, within the AMBER force field.

[0577] Figure 4OThis shows how cholesterol begins at the head base extending from the subsurface and the tail extending from the main surface. The complex remains stable throughout its trajectory. Cholesterol moves essentially back and forth within the lumen, but the angle within the lumen remains relatively constant.

[0578] The translated natural monomers βCD and 7KC are oriented downwards, with an AMBER force field.

[0579] 7KC begins with a head base extending from the primary surface and a tail extending from the secondary surface, as... Figure 4O As shown. The complex remains stable throughout the trajectory, and as... Figure 4M As shown in the horizontal and stable curves, 7KC does not bend significantly within the CD chamber.

[0580] The translated natural monomers βCD and cholesterol are oriented downwards, within the AMBER force field.

[0581] Figure 4O This shows how cholesterol begins from the head base extending from the main surface and the tail extending from the subsurface. The complex remains stable throughout its trajectory, and as... Figure 4M As shown in the level and stability curves, cholesterol does not bend significantly within the CD cavity.

[0582] Monomers hydroxypropyl βCD and 7KC, upward orientation, GROMOS force field:

[0583] 7KC is in the upward position ( Figure 4R The process begins at the tail end inside the HPβCD cavity and the head extending from the subsurface. At approximately 13 ns, 7KC rotates out of the subsurface and associates parallel to it. At 28 ns, the head base of 7KC reassociates with the cavity, but after several rotations back, 7KC remains associated parallel to the subsurface until the complex completely breaks down at approximately 47 ns. Then, 7KC rotates between associating parallel to one surface and randomly moving around the frame until the remainder of the trajectory is reached. No stable complex forms.

[0584] Monomer hydroxypropyl βCD and cholesterol, upward orientation, GROMOS force field:

[0585] exist Figure 4R In the process, cholesterol begins at the tail end inserted into the CD cavity and extends from the subsurface. As the cholesterol rotates out from the subsurface, parallel to the CD, and then around the primary surface for 7 ns, the complex remains stable until about 3 ns. Then, the cholesterol moves randomly around the simulated frame, occasionally associating parallel to the primary or subsurface, but except for a brief period of about 300 ns, it remains unstable within the cavity. Figure 4P The strong changes in the data clearly demonstrate the lack of strong association, and this is supported by experimental evidence.

[0586] Monomers hydroxypropyl βCD and 7KC, downward orientation, GROMOS force field:

[0587] Figure 4R The 7KC pattern begins slightly outside the monomeric cavity and initially flies randomly around the simulation frame. At 29 ns, the 7KC has associated the head group within the HPβCD cavity, with the tail extending from the subsurface. This state remains stable until 35 ns as the complex completely dissociates. The complex remains dissociated until 320 ns, when it reforms with the head group within the cavity and the tail extending from the subsurface. The complex remains associated until approximately 470 ns, when it dissociates again until the trajectory ends.

[0588] Monomer hydroxypropyl βCD and cholesterol, downward orientation, GROMOS force field:

[0589] Figure 4R The trajectories show cholesterol entering the cavity from the tail and extending outwards from the main surface in a downward orientation. This complex remains stable until approximately 300 ns, at which point cholesterol rotates out of the subsurface and associates parallel to the CD, at which point the complex completely breaks apart and dissociates from the CD. Cholesterol then moves around the CD, sometimes associating parallel to the subsurface, and finally associates with the main surface at approximately 100 ns. Cholesterol then continues to move randomly around the CD, sometimes associating with or rotating with the surface, similar to its conformation at 275 ns, just as it entered the cavity, but cholesterol does not completely re-enter the cavity within any considerable timeframe. These trajectories indicate a preference for an upward orientation, with the only stable complexes formed being 7KC-upward (forming independently in the simulation after complete dissociation) and cholesterol-downward (remaining stable from the initial conformation). This suggests a strong preference for 7KC in the upward orientation and interaction with cholesterol in the downward orientation.

[0590] Monomers hydroxypropyl βCD and 7KC, upward orientation, AMBER force field:

[0591] 7KC (upward) begins at the molecular center within the CD cavity and extends slightly beyond the subsurface at the head base, while the tail base extends slightly beyond the main surface. Figure 4U This shows how 7KC remains within the cavity of HPβCD throughout the trajectory, swinging slightly upwards and downwards, but never extending either end away from the cavity. The complex does not break down.

[0592] Monomer hydroxypropyl βCD and cholesterol, upward orientation, AMBER force field:

[0593] Compared to the GROMOS force fields of natural and HPβCD, the AMBER force field exhibits more consistent interactions and a more stable complex. Figure 4UIn the complex, cholesterol (upward) begins at the molecular center within the CD cavity and extends slightly beyond the subsurface as a head group, while the tail group extends slightly beyond the main surface. This complex remains stable throughout its trajectory; cholesterol never leaves the cavity or changes orientation; it merely oscillates back and forth within it. Figure 4S As shown, the most favorable conformation occurs within 500-700 ns, but cholesterol and CD still recombine along the entire trajectory. These small positional changes correspond to... Figure 4S Small protrusions, especially in angular sections.

[0594] Monomers hydroxypropyl βCD and 7KC, downward orientation, AMBER force field:

[0595] 7KC (downward) begins at the molecular center within the CD cavity and extends slightly outward from the head base of the main surface, while the tail base extends slightly outward from the subsurface. Figure 4U This shows how the head of 7KC protrudes from the cavity rather than being oriented upwards, yet the complex remains intact throughout the trajectory. Figure 4S This preference for upward orientation is evident, as the "upward" graph shows less variation compared to the "downward" graph. However, in GROMOS, the variation between the two is still significantly smaller than that of HPβCD.

[0596] Monomer hydroxypropyl βCD and cholesterol, downward orientation, AMBER force field:

[0597] Figure 4U The cholesterol (downward) complex begins inside the CD cavity with a head base extending beyond the primary surface, while the tail base extends slightly beyond the subsurface. Notably, the head base of cholesterol sometimes extends significantly further out of the cavity in an upward orientation, but the complex remains stable throughout its trajectory. Cholesterol never completely leaves the cavity or changes orientation. These small positional changes correspond to... Figure 4S Small protrusions in the CD chamber, especially at the corners. Compared to other compounds, it exhibits significantly more lateral movement and less radial rocking through the CD cavity.

[0598] The translated monomers hydroxypropyl βCD and 7KC are oriented upwards, under the influence of the GROMOS force field.

[0599] Figure 4XThe diagram shows a 7KC beginning with an upwardly oriented translation at the tail of the CD cavity and a head base extending from the subsurface. The 7KC rotates out of the cavity at approximately 105 ns, and then oscillates approximately every 5–10 ns between the head base inserting into the CD and being parallel to it, seemingly spending more time in the conformation where the head base is in the cavity. At approximately 415 ns, the structure rests the inserted head base until it breaks off again and completely dissociates at 700 ns. Then, as the head base of the 7KC itself inserts into the large surface of the CD, the complex remains dissociated for the remainder of the trajectory, except for a brief reassociation at 726 ns. The interaction energy here is briefly comparable to the interaction energy at 400 ns when the complex forms. Because the complex appears to form and break off readily, this interaction is likely real, strong, and could be captured by simulation.

[0600] The translated monomers hydroxypropyl βCD and cholesterol are oriented upwards in a GROMOS force field.

[0601] Cholesterol begins with a head base inserted into the cavity and a tail extending from the primary surface. This complex remains stable for 60 ns until cholesterol rotates out of the subsurface and associates parallel to the CD. Cholesterol then completely leaves the CD and moves randomly around the simulation frame until, at approximately 215 ns, it re-associates its tail with the CD cavity, and the head base again extends from the subsurface. This remains stable for approximately 30 ns until cholesterol leaves the CD again and then rapidly re-associates its head base within the CD cavity at 280 ns, this time with both the head base in the cavity and the tail extending from the subsurface. The complex remains stable for the remainder of the trajectory. This suggests that the complex formed at the end of the trajectory is very stable and likely as... Figure 4X As shown in the diagram.

[0602] The translated monomers hydroxypropyl βCD and 7KC are oriented downwards, under the influence of the GROMOS force field.

[0603] The 7KC, oriented downwards and translated, begins with a head base inserted into the CD cavity and a tail extending from the subsurface. The 7KC rotates out of the cavity at approximately 105 ns, then oscillates approximately every 5–10 ns between the head base being inserted into the CD and parallel to it, seemingly spending more time in the conformation where the head base is in the cavity. At approximately 415 ns, the structure rests with the inserted head base until it breaks again and completely dissociates at 700 ns. Then, for the remainder of the trajectory, the complex remains dissociated, as... Figure 4X As shown.

[0604] The translated monomers hydroxypropyl βCD and cholesterol are oriented downwards in a GROMOS force field.

[0605] In the downward orientation of cholesterol in HPβCD translation, cholesterol begins with a tail inserted into the CD cavity and a head base extending from the main surface. The complex breaks at 50 ns, but cholesterol remains associated with the main surface, periodically entering and exiting the cavity before the tail completely dissociates at 88 ns. The cholesterol molecule then associates with the subsidence side of the CD and resumes random movement around the simulated frame. The trajectory cycles between association with one of the two surfaces and random movement until 215 ns, at which point the cholesterol tail re-enters the cavity from the main side, continuing for the next 25 ns. Cholesterol then resumes random movement around the CD. At 275 nanoseconds, the cholesterol head base enters the cavity from the main surface and remains there until the complex completely dissociates at approximately 410 ns. At this point, cholesterol moves randomly around the simulated frame until approximately 490 ns, when it rotates to the subsidence side and inserts its head base into the cavity. As cholesterol removes from the cavity, rotates, and reinserts its tail base from the subsidence side into the cavity, the complex remains in this conformation until approximately 530 ns. At 540 ns, cholesterol resumes its random movement. Cholesterol never reinsertes into the lumen but typically associates tightly with either surface of the CD. Because cholesterol does not form a stable complex with HPβCD at any significant time, the interaction between HPβCD and cholesterol appears to be transient, unlike the strong interaction between HPβCD and 7KC, even in cases such as... Figure 4X The same applies to the translation positions shown.

[0606] The translated monomers hydroxypropyl βCD and 7KC are oriented upwards, with an AMBER force field.

[0607] Figure 4AA The 7KC structure begins with a head base extending from the primary surface and a tail portion where the surface diverges from the secondary surface, while the center of the 7KC is located within the cavity of the CD. The complex remains stable throughout its trajectory, and as... Figure 4Y As shown in the horizontal and stable curves, 7KC does not bend significantly within the CD chamber.

[0608] The translated monomers hydroxypropyl βCD and cholesterol are oriented upwards, using the AMBER force field.

[0609] Figure 4AA The cholesterol complex begins at a head base extending from the main surface and a tail portion facing away from the subsurface, with its center located within the cavity of the CD. The complex remains stable throughout its trajectory and, as... Figure 4Y As shown in the level and stability curves, cholesterol does not bend or move around significantly within the CD cavity.

[0610] The translated monomers hydroxypropyl βCD and 7KC are oriented downwards, with an AMBER force field.

[0611] Figure 4AAThe 7KC structure begins with a head base that significantly extends beyond the main surface and a tail that deviates from the subsurface, but the tail remains entirely within the cavity. The complex remains stable throughout its trajectory and, as... Figure 4Y As shown in the horizontal and stability curves, 7KC does not bend significantly within the CD cavity. 7KC does not exhibit greater lateral movement in this orientation compared to an upward orientation.

[0612] The translated monomers hydroxypropyl βCD and cholesterol are oriented downwards, using the AMBER force field.

[0613] Figure 4AA The cholesterol complex begins with a head base extending from the primary surface and a tail portion extending away from the subsurface, with its center located within the lumen of the CD. The complex remains stable throughout its trajectory, but cholesterol does move significantly within the lumen, typically only the tail portion associates with the head base extending out of the CD. This can be observed in… Figure 4Y It was observed that the downward orientation changed more than the upward orientation, especially in terms of distance.

[0614] Dimerized hydroxypropyl βCD and 7KC, upward orientation, GROMOS force field:

[0615] exist Figure 4DD In this configuration, 7KC begins within the dimer and remains well-cage-like. The dimer begins to stretch at approximately 100 ns, and despite this stretching, 7KC remains trapped within the barrels of the two CDs. At 111 ns, the head group dissociates from its monomer (in this discussion, the term "monomer" refers to the CD subunit, although it is part of the covalently linked dimer), while the tail remains associated with the cavity of the other monomer. After 5 ns, the head group of 7KC continues to interact with the large surface area (not the cavity) of one monomer, while the tail remains anchored within the other monomer. At 120 ns, the tail releases its monomer, and the head group itself inserts into the cavity of the other monomer. This configuration remains stable, with the sterol-associated monomer oscillating around the empty monomer until the trajectory ends.

[0616] Dimerized hydroxypropyl βCD and cholesterol, upward orientation, GROMOS force field:

[0617] The cholesterol (upward) trajectory begins with cholesterol encased in a dimer. The dimer begins to bend at approximately 22 ns, but the cholesterol moves and remains within the dimer cavity. At approximately 200 ns, the monomer associated with the cholesterol head breaks off and dissociates from the dimer, but the cholesterol remains associated with one of the monomers (head aligned with the subsurface, tail aligned with the primary surface). This configuration remains unchanged until the cholesterol is completely dissociated from the cavity and rotates toward the subsurface at 355 ns. The cholesterol then remains between the two monomers, occasionally with the head loosely associated with one monomer until it completely leaves and floats around the simulation frame. The cholesterol continues to interact intermittently with one of the CD monomers, but as... Figure 4DD As shown, the dimer-cholesterol complex never fully reformed.

[0618] Dimerized hydroxypropyl βCD and 7KC, downward orientation, GROMOS force field:

[0619] 7KC is in a downward position, such as Figure 4DD As shown, the dimer initially remains cage-like within itself. As one monomer stretches away from the other, the dimer begins to deform until approximately 600 ns, while 7KC remains between them. At approximately 820 ns, 7KC dissociates the tail from one monomer, while the head remains within the cavity of the other. This configuration remains stable, with the sterol-associated monomer oscillating around the empty monomer until the trajectory ends.

[0620] Dimerized hydroxypropyl βCD and cholesterol, downward orientation, GROMOS force field:

[0621] Cholesterol in the downward position ( Figure 4DD The process begins inside the dimer cage. At approximately 50 ns, the complex begins to stretch and twist, but cholesterol remains anchored inside the dimer throughout its trajectory. This... Figure 4BB The curve is clear because the cholesterol angle curve is very horizontal and stable throughout the entire trajectory. This is the only complex in GROMOS force field analysis that remains intact throughout the entire trajectory. Figure 4BBMolecular dynamics analyses show the formation of highly stable complexes between our novel butyl-linked hydroxypropyl DS5 β-cyclodextrin dimer and 7KC and cholesterol. The comparison between these plots and those of the monomeric HPβCD provides clear evidence that the dimer form reliably and consistently binds to sterols more firmly than its monomeric counterpart. Very consistent and minimal variation is observed for the down orientation, energy, angle, and distance, indicating the existence of stable, clearly soluble complexes between 7KC and cholesterol that do not change significantly over time. Similar variation is seen in the up orientation, but with slightly greater variation, especially for cholesterol. This suggests that 7KC binds most efficiently in the down orientation, strongly favoring it, as indicated by the angle reversal at approximately 350 ns in the up orientation. This is where 7KC leaves the dimer and reassociates in the down orientation. Cholesterol also does this, but its stability is lower than that formed by 7KC, suggesting that cholesterol does not appear to have the ability to form more stable down complexes from less stable up complexes, while 7KC appears to do so.

[0622] Dimerized hydroxypropyl βCD and 7KC, upward orientation, AMBER force field:

[0623] Figure 4GG The method for maintaining 7KC within a cavity formed by the two monomers throughout the trajectory is explained in detail. The complex is somewhat curved, and 7KC moves slightly within the cavity, but it remains complexed with the CD dimer throughout the trajectory.

[0624] Dimerized hydroxypropyl βCD and cholesterol, upward orientation, AMBER force field:

[0625] exist Figure 4GG In this process, cholesterol remains nested between two monomers throughout its trajectory. The monomers remain associated with each other, and the cholesterol-complex bends but never breaks.

[0626] Dimerized hydroxypropyl βCD and 7KC, downward orientation, AMBER force field:

[0627] Figure 4GG The study shows that 7KC remains nested within a cavity formed by the two monomers throughout the trajectory. The complex is somewhat curved, and 7KC moves slightly within the cavity, but it remains complexed with the CD dimer throughout the trajectory.

[0628] Dimerized hydroxypropyl βCD and cholesterol, downward orientation, AMBER force field:

[0629] Figure 4GG The study shows that cholesterol remains nested within a cavity formed by two monomers throughout the trajectory. The complex is somewhat curved, and cholesterol moves slightly within the cavity, but it remains complexed with the CD dimer throughout the trajectory.

[0630] Translated dimerized hydroxypropyl βCD and 7KC, upward orientation, GROMOS force field:

[0631] Figure 4JJ It shows that the dimerized complex starts with translated 7KC in upward orientation, which is tightly nested in the cavities of two CD monomers. The complex stretches at about 140 ns, thus Figure 4GG produces the first change at this time in , but it quickly re-forms. As shown by the changes in Figure 4GG , the complex continues to periodically stretch and deform, but 7KC remains in the two cavities until the tail is released from its monomer at about 700 ns, and 7KC does not re-enter both cavities simultaneously for the rest of the trajectory.

[0632] Translated dimerized hydroxypropyl βCD and cholesterol, upward orientation, GROMOS force field:

[0633] Figure 4JJ It shows that cholesterol translated in upward orientation starts to complex with the dimer, which begins to distort at about 100 ns (much larger than the 7KC complex). When one monomer associated with the head cholesterol via the secondary surface completely flips to the other side of the second monomer, so that the slightly deformed secondary surface of the second monomer (associated with cholesterol) associates with the slightly deformed primary surface, producing a slightly deformed head-to-tail dimer, a large angle change for cholesterol occurs at about 180 ns in Figure 4GG . However, this head-to-tail dimer never completely forms a complex with cholesterol, and the head group of cholesterol remains associated with the monomer with which it was initially associated. This is the only trajectory that produces a head-to-tail dimer, and this configuration does not appear to efficiently form a complex with cholesterol.

[0634] Translated dimerized hydroxypropyl βCD and 7KC, downward orientation, GROMOS force field:

[0635] Translated 7KC in the downward position starts to associate with two monomers at the center of the CD dimer. Figure 4JJ It shows that one monomer stretches away from the tail of 7KC at 230 ns, and then 7KC completely leaves the dimer at 355 ns (note that this is also Figure 4GG where the consistent fracture occurs in ). At 400 ns, 7KC re-associates the head group with one monomer. For the rest of the trajectory, the head of 7KC remains associated with this monomer, but the tail never re-inserts into the second monomer again.

[0636] Translated dimerized hydroxypropyl βCD and cholesterol, downward orientation, GROMOS force field:

[0637] Cholesterol migrates downwards, forming a complex with the CD dimer for approximately 162 ns. At this point, the dimerized complex begins to stretch and deform, and then the head group of cholesterol releases its monomers at 190 ns. At 210 ns, cholesterol does not associate with the cavity of either monomer, but remains between the two individual monomers. Cholesterol remains tightly associated with the dimer until complete dissociation at 320 ns. Figure 4JJ As shown, cholesterol does not re-enter both cavities, nor does the dimerized complex completely reform on the remainder of the trajectory. However, cholesterol can sometimes associate the head group with the subsurface of a monomer, as in the configuration at 640 ns.

[0638] The translated dimerized hydroxypropyl βCD and 7KC are oriented upwards, with an AMBER force field.

[0639] Figure 4MM The method of nesting 7KC within a cavity formed by two monomers is described in detail throughout the trajectory. The complex curves slightly around 7KC, but 7KC remains in almost exactly the same position throughout the trajectory.

[0640] The translated dimerized hydroxypropyl βCD and cholesterol are oriented upwards, using the AMBER force field.

[0641] Figure 4MM The process is detailed in how cholesterol is nested within a cavity formed by two monomers throughout the trajectory. The complex and cholesterol move slightly within the trajectory, particularly the monomer associated with the cholesterol head, but the cholesterol never completely dissociates from either monomer. Throughout the trajectory, cholesterol complexes with the CD dimer.

[0642] Translational dimerized hydroxypropyl βCD and 7KC, downward orientation, AMBER force field:

[0643] Figure 4MM The process is detailed in explaining how 7KC is nested within a cavity formed by the two monomers throughout the trajectory. The complex bends slightly around 7KC, but 7KC remains in almost exactly the same position throughout the trajectory. 7KC complexes with the CD dimer throughout the trajectory.

[0644] Translational dimerized hydroxypropyl βCD and cholesterol, downward orientation, AMBER force field:

[0645] Figure 4MM The diagram details how cholesterol is nested within a cavity formed by two monomers throughout the trajectory. The complex curves slightly around the cholesterol, but the cholesterol remains in almost exactly the same position throughout the trajectory. Cholesterol complexes with the CD dimer throughout the trajectory.

[0646] Additionally, for DS0 βCD dimers with butyl and triazole linkers ( Figure 4NN -QQ) and hydroxypropyl dimer with a triazole linker ( Figure 4RR Short-term analysis was performed using the DS0 model. DS0 simulations showed that the triazole linker somewhat disrupted the stability of the complex; however, this allowed for the transfer of certain additional specificities of 7KC. Slightly different but still strong and favorable interactions were observed for both linker types, indicating promising results.

[0647] Triazole-linked HPβCD dimer ( Figure 4RR The interaction was slightly weaker than that of the butyl-linked hydroxypropylated dimer, and showed a strong preference for 7KC in the down-orientation. The cholesterol interaction was weaker than with 7KC, indicating a certain specificity for 7KC, while 7KC was the most stable complex formed to date in the down-orientation. The addition of the triazole group stabilized 7KC in the down-orientation, while all other complexes broke at some point.

[0648] Other MD analysis

[0649] In addition, simplified MD analysis was performed on triazole and butyl-linked methyl βCD, sulfobutyl βCD, and quaternary ammonium βCD using DS4. Figure 5B -C, 6B-C, 7A-B). Methyl dimers showed the most stable complexes with the butyl linker, and both linkers appeared to favor upward orientation; however, the interactions were very similar for the two methyl dimers tested. It was difficult to distinguish which was more effective in practice, but both types of linkers readily formed complexes with the two ligands used for methyl substitution. The trajectory indicated that the 7KC head group was not entirely within the dimer cavity but remained stable between the two sister monomers. The complex with 7KC in the downward orientation remained associated for approximately 50 ns before 7KC moved out of the cavity, and in the remaining trajectories, only the head group associated with one monomer.

[0650] The negatively charged sulfonyl dimer exhibits a similar pattern to that of the methyl and hydroxypropyl dimers, where the triazole linker produces a slightly unstable complex that then allows for 7KC specificity. The bulky, charged sulfonyl group appears to interact very favorably with both 7KC and cholesterol, but in the case of both linkers, the only complex that is disrupted is the cholesterol complex. This suggests that the sulfonyl dimer may have very good specificity for 7KC compared to methyl and hydroxypropyl dimers.

[0651] To further evaluate the use of charged substituents, MD analysis was performed on the positively charged quaternary ammonium βCD of DS4. These trajectories elucidated the strong binding between QA βCD and sterols, as no sterol was released at any point along either linker. The strong energies of interaction and association with at least one sister monomer throughout the ligand and linker trajectories suggest that DS4 QA βCD is well-suited for binding sterols and solubilizing them, much like other types of substitution.

[0652] In the final MD analysis, HPβCD with a single O-connector was tested. Figure 8H O-linked dimers ( Figure 8H The O-linked group exhibits good 7KC specificity because only the upward-oriented 7KC remains complexed throughout the entire 100 ns. The O-linked group shows slightly lower interaction energies compared to the butyl-linked group, but overall specificity for the O-linker appears to be better since the cleavage of both cholesterol complexes is 100 ns. The interactions are similar to those of the butyl-linked dimers, but they appear to have better 7KC specificity, which is clearly due to the interaction between the nitrogen in the linker and the carbonyl group of the 7KC.

[0653] Other connection screening

[0654] Docking simulations allow us to quickly model many different possible molecules without synthesizing them. For this reason, we use these docking techniques to “screen” many different substitution types, linker types, substitution numbers, and substitution positions (Figures 8-9). This screening allows us to determine whether certain modifications produce better or worse specificity for 7KC.

[0655] Figure 8E This paper describes the assessment of the dependence of our HPβCD dimer on the composition and linkage sites of the linker, variations in hydroxypropylation sites, changes in linker length, and different chemical compositions of the linker. Linker linkage sites have been... On the computer Tests were conducted because linker sites are not easily controlled during the chemical synthesis of cyclodextrins. Various hydroxypropylation sites ( Figure 8A Carbon-only connectors of various lengths (chain lengths of two to eight carbons), Figure 8B ) and triazole linkers (different n1 and n2 values ​​around the triazole ring, Figure 8C ), and the different binding sites of O2 and / or O3 oxygen to the dimerized HPβCD ( Figure 8F -G) and different connector types ( Figure 9A Docking calculations were performed. The results showed that changing the position of the hydroxypropyl group had little effect on 7KC preference and minimal effect on total sterol binding. Linkage lengths between 3 and 5 carbons exhibited the highest affinity and specificity for 7KC. Figure 8B ).

[0656] Various triazole connectors modeled in AutoDock are displayed. Figure 8C In the context of these linked dimers, n1 refers to the number of carbon atoms on the right side of the ring, while n2 refers to the number of carbon atoms on the left side of the azide ring. Based on these results, the predicted length of the triazole linker varies less than 4 on each side of the ring, with the pair exhibiting the greatest affinity for 7KC.

[0657] exist Figure 8E In this section, we address 23 possible alternative connectors ( Figure 8D Docking calculations were performed on the HPβCD dimer with 7KC (as shown in the figure). Based on these results, it is predicted that most of the tested linker dimers will maintain a good affinity for 7KC.

[0658] We also considered the fact that the linker can attach to the subsurface of the cyclodextrin at either the C2 or C3 carbon atom. We tested whether this would affect the predicted affinity by molecular docking. Figure 8F We also investigated whether there could be more significant differences in the affinity of sterols linked via asymmetric linkers and variable linker sites. These calculations showed that for all three possible linker sites, there was a tendency to bind 7KC and cholesterol, which are present in roughly equal amounts in typical synthesis. These calculations showed the tendency to bind 7KC and cholesterol for all four possible links present in the synthesis of dimers linked by five different asymmetric linkers. Overall, we observed no significant differences between the C2 and C3 linker sites.

[0659] Our molecular modeling revealed varying levels of specificity for 7KC across different substitution numbers. Of particular interest were HPβCDs linked with 3, 4, or 5 hydroxypropyl groups, which exhibited the highest specificity for 7KC in any of the butyl dimers we modeled. Figure 4B We synthesized several butyl- and triazole-linked HPβCD dimers, including DS3. Consistent with our predictions, HPβCD-butyl-DS3 and HPβCD-triazole-DS3 exhibited greater specificity for 7KC than cholesterol. Figure 16A -C).

[0660] After completing the hydroxypropyl CD dimer docking analysis, various CD dimers with different degrees of substitution and various linkers targeting 7KC and cholesterol were docked to observe how these factors affect the binding of 7KC and cholesterol. Figure 5A , 6A And 9). Methyl and sulfobutyl substitutions from DS1 to DS20 were tested using butyl and triazole linkers. Figure 5A , 6AThe results were encouraging enough to stimulate further molecular dynamics analyses and ultimately lead to synthesis.

[0661] We are Figure 5A and 6A It was observed that for sulfobutyl and methyl substitutions, 7KC specificity was optimal at low DS (2–6). DS4 MeβCD and SBβCD behaved most similarly to HPβCD DS5, with good 7KC solubilization, but not cholesterol. It appears that 7KC specificity becomes less pronounced with increasing DS for both linkers and all substitutions. When ~DS4 appears to achieve the highest 7KC specificity for all tested substitution types, DS4 is tested only with other linker types.

[0662] Substitutions other than hydroxypropyl, methyl, or sulfobutyl were tested only at low DS using butyl connectors, triazole connectors, connector O, and connector R. Figure 9A While some linker or substitution types do show more or less specificity than others, the vast majority still show at least some specificity for 7KC. This suggests that, in the compounds tested, 7KC specificity depends not on the type of linker or substitution, but on the number of substitutions on the βCD ring. Although some substitution types do show negative specificity for a few linker types, the average 7KC specificity remains well greater than 0 for all 23 linkers and seven low-DS(4) substitution types.

[0663] Using molecular docking, we were able to test how the length of the triazole or alkyl linker affects the 7KC specificity of cyclodextrin dimers containing hydroxypropyl, methyl, and sulfobutyl substituents. Figure 9B -C). We show that specificity decreases with increasing linker length. Without intending to be bound by theory, it is believed that for longer linkers, the CD subunits can be separated by a greater distance, thus spending less time in conformations capable of efficiently encapsulating molecules the size of 7KC or cholesterol. Based on these results, we conclude that dimers with linker lengths that allow the guest (7KC or cholesterol) to fit between the two CD subunits will exhibit greater solubility for such molecules, e.g., linker lengths of 7 atoms or less.

[0664] We also tested whether the specificity of the CD dimer for 7KC depended on the substitution position by using sulfobutyl, hydroxypropyl, and methyl substitutions, as well as combinations of these three, to produce many different substitution patterns. Figure 9D-E). We found that when a CD dimer has a single or even multiple substitution types, 7KC specificity is largely maintained when DS is approximately 4. The type and position of these substitutions have little effect on the specificity of 7KC. Docking simulations indicate that while both the linker and the composition of the substitutions affect the ability of a given CD to solubilize a guest, the degree of specificity for 7KC depends primarily on the number of substitutions on the CD ring. For example, in Figure 4B and 5A As shown in -B, the butyl-linked dimer exhibits the highest specificity for 7KC at approximately DS 2–5 for methyl, sulfobutyl, and hydroxypropyl substitution. This also applies to the triazole linker, supporting the idea that multiple linkers and substitution types exhibit similar specificity for 7KC at substitution degrees between 2 and 5. Furthermore, docking was performed on 23 different linkers and 14 different substitution patterns / combinations to determine whether the linker or substitution pattern affects 7KC specificity. Figure 9A Both analyses showed differences in the specificity of 7KC, but the average specificity was still much greater than zero.

[0665] Docking and molecular dynamics screening were performed to determine whether certain linker types or substitution numbers, types, and positions affected 7KC specificity. The only modification that significantly affected binding (affinity) was the actual dimerization of the cyclodextrin (compared to the docked monomer). Figure 2E (The dimer exhibits much better sterol binding). In contrast, the number of substitutions present on the dimer has the greatest impact on 7KC binding specificity. Docking simulations show that once βCD is dimerized and substituted with approximately four compatible functional groups, specificity for 7KC is maintained in most cases for many different substitution types, patterns, and linkers.

[0666] Since the methyl, sulfobutyl, and hydroxypropyl groups are completely distinct from each other, and the range of linkers tested contains significant variability, we believe it is reasonable that other substitution types with linker lengths similar to sterol guests behave similarly to butyl-linked CD dimers with hydroxypropyl groups. While substitution and linker types can have some influence on other properties such as solubility and toxicity, specificity for 7KC is expected for other molecules of this type as well.

[0667] Example 3. Synthesis of HPβCD-substituted cyclodextrin dimers

[0668] Figure 3A -D shows the molecule to be synthesized in Figure 10 below.

[0669] This embodiment describes the synthesis of substituted cyclodextrin dimers, which are first linked via a butyl linker and then via a triazole-containing linker.

[0670] For DS measurements, the residual solvent signal was used as an internal reference, and 1H and 2D NMR spectra were recorded on a Varian VXR-600 at 600 MHz. The sample was dissolved in DMSO-d6 / D2O to elucidate the structure. The FID signal was recorded with at least 16 scans to obtain a spectral window at least between 0 ppm and +10 ppm. The average degree of substitution (DS) was calculated by setting the integral of the anomeric region to fourteen (fourteen is the number of anomeric protons in the β-cyclodextrin dimer) and dividing the integral of the alkyl region by three (see [reference]). Figure 10J ).

[0671] Overview of Synthesis and Characterization

[0672] HP(βCD-BUT-βCD)

[0673] The preparation of hydroxypropylated β-cyclodextrin dimers was accomplished through a three-step synthesis (see [link]). Figure 10A ). Starting materials It is a monomeric β-cyclodextrin (TBDMS-βCD, CycloLab, Budapest, Hungary) whose main side is protected by tert-butyldimethylsilyl groups.

[0674] Subsurface dimerization was achieved using TBDMS-βCD, anhydrous conditions, and sodium hydride as a base. The dialkylating agent was added dropwise to the heterogeneous reaction mixture, and the reaction was allowed to proceed completely at room temperature.

[0675] The host-side protected βCD dimer (TBDMS-βCD-BUT-βCD-TBDMS) was purified by chromatography using isocratic elution (chloroform:methanol:water = 50:8:0.8 (v / v / v) as eluent). MALDI analysis of the compound confirmed the product's identity. Figure 10D ).

[0676] Desilylation was performed in THF with tetrabutylammonium fluoride at room temperature. The βCD dimer (βCD-BUT-βCD) was purified by chromatography using isocratic elution (1,4-dioxane:NH3 = 10:7 (v / v) as eluent). MALDI and TLC analyses confirmed the identity of the product. Figure 10E-10F ).

[0677] Hydroxypropylation of the βCD dimer was achieved under aqueous conditions using sodium hydroxide as a base at room temperature. Purification of the hydroxypropylated βCD dimer (HP(βCD-BUT-βCD)) was based on ion-exchange resin treatment, charcoal clarification, and extensive dialysis. MALDI and NMR analyses of the compound confirmed the identity and structure of the product. Figure 10G-10N ).

[0678] HP(βCD-triazole-βCD)

[0679] The preparation of hydroxypropylated β-cyclodextrin dimers linked to a triazole moiety via a subsurface can be performed using a four-part procedure. Figure 10B The first part concerns the preparation of the azide linker (3-azido-1-bromopropane), as this reagent is not commercially available. The second part concerns the preparation of two βCD monomers, namely 2- O -propynyl-β-CD and 2- O -(3-Azidepropyl)-βCD. The third part of the synthesis involves the stacking of the dimer core formed by a copper-assisted azido-alkyne cycloaddition reaction, and the final part involves the preparation of a series of 2-hydroxypropylated triazole-linked dimers according to the classical alkylation method.

[0680] Specifically, the azide linker can be prepared by strictly limiting the amount of sodium azide and extending the addition time of the limiting reagent. The azide linker is then characterized by NMR spectroscopy and TLC. Figure 10R ).

[0681] Two monomers were synthesized using lithium hydride as a selective base for sub-side deprotonation. Specifically, according to this method, only the hydroxyl group at C2 was activated. As a result, the monomers prepared by this method were substituted only at O2 (they are monoisomers). Both monomers were characterized by MALDI NMR spectroscopy and TLC. Figure 10S -U).

[0682] The dimer-core was then prepared by reacting two monomers. The NMR spectroscopy was then used to analyze the results. Figure 10V ) and MALDI ( Figure 10O The obtained compound was characterized as a monoisomer (BCD-(triazole)1-BCD DS=0).

[0683] The hydroxypropylation of BCD-triazole-BCD was accomplished using propylene oxide and an alkaline aqueous solution. The series of hydroxypropylated compounds was characterized by MALDI. Figure 10P -Q).

[0684] Detailed description of the synthesis (HP(βCD-BUT-βCD))

[0685] Step 1: Subsurface dimerization of TBDMS-βCD

[0686] Dry TBDMS-βCD (10 g, 5.17 mmol) was solubilized in THF (400 mL) under an inert atmosphere, and sodium hydride (2.5 g, 50 mmol) was carefully added in portions (over 30 minutes). The addition of sodium hydride resulted in hydrogen formation and strong foaming of the suspension. After stirring for 15 minutes, the reaction mixture gelled and became difficult to stir. To break the gel, the reaction mixture was heated until mild reflux occurred and maintained under reflux for 30 minutes. The pale yellow heterogeneous suspension became easier to stir, and the gel structure disappeared. The reaction mixture was cooled to room temperature in a water bath. The alkylating agent 1,4-dibromobutane (1.25 mL, 2.25 g, 10.5 mmol) was added dropwise (over 15 minutes), and the color of the reaction mixture turned deep orange.

[0687] The brown suspension was stirred overnight under an inert atmosphere. The conversion was estimated by TLC to be between 10% and 15% (eluent: chloroform:methanol:water = 50:10:1, v / v / v, see [reference]). Figure 10C (and is considered acceptable for post-processing).

[0688] The reaction mixture was quenched with methanol (30 mL), concentrated under reduced pressure (about 20 mL), and precipitated with water (200 mL). The crude product was filtered through a sintered glass filter and washed thoroughly with water (3 × 300 mL). The crude product was dried to constant weight in an oven in the presence of KOH and P₂O₅ (12.1 g).

[0689] The crude product was purified by chromatography, and fractions containing the product were collected based on TLC analysis and evaporated under reduced pressure until dry. Figure 10C The white substance was obtained and dried in a drying oven containing KOH and P2O5 to constant weight (TBDMS-βCD-BUT-βCD-TBDMS, 3.5g).

[0690] Step 2: Deprotection of the TBDMS-βCD butyl-linked dimer

[0691] Dry TBDMS-βCD-BUT-βCD-TBDMS (3.5 g, 0.89 mmol) was solubilized in THF (250 mL) under an inert atmosphere, and tetrabutylammonium fluoride (8.75 g, 33.47 mmol) was added in a single addition to the pale yellow solution. After stirring at room temperature for 30 minutes, the reaction mixture turned dark green. The reaction mixture was stirred overnight at room temperature. TLC analysis (1,4-dioxane:NH3 = 10:7 (v / v)) indicated that the reaction was incomplete, and a second portion of tetrabutylammonium fluoride (4 g, 13.3 mmol) was added to the vessel. The reaction mixture was heated to mild reflux and refluxed for two hours. The conversion at this stage was complete, as no starting material was detected by TLC. The reaction mixture was cooled to room temperature, concentrated under reduced pressure (to about 10 mL), and methanol (200 mL) was added, yielding a white precipitate. The solid was filtered off, analyzed by TLC, and dried to constant weight in an oven in the presence of KOH and P₂O₅ (1.2 g). TLC analysis showed that the substance contained negligible (≤3%) amounts of tetrabutylammonium fluoride. The mother liquor was concentrated under reduced pressure (to approximately 10 mL) and purified by chromatography (eluent: 1,4-dioxane:NH₃ = 10:7 v / v). The fraction containing the product was collected and evaporated under reduced pressure until dry to obtain a white substance. The white substance was dried to constant weight (βCD-BUT-βCD, 0.55 g) in an oven in the presence of KOH and P₂O₅.

[0692] Step 3: Hydroxypropylation of βCD-BUT-βCD

[0693] βCD-BUT-βCD (0.5 g, 0.21 mmol) was suspended in water (10 mL). Sodium hydroxide (0.1 g, 2.5 mmol) was added to the reaction vessel, and the mixture turned into a pale yellow solution. The reaction mixture was cooled in a water bath (10 °C), and propylene oxide (0.5 mL, 0.415 g, 7.14 mmol) was added in one go. The reaction vessel was flushed with argon, sealed at room temperature, and stirred for two days. The reaction mixture was concentrated under reduced pressure until a viscous slurry was obtained, which was precipitated with acetone (50 mL). The white solid was filtered through a sintered glass filter and washed thoroughly with acetone (3 × 15 mL). The substance was solubilized with water (50 mL), treated with an ion exchange resin (to remove salts), clarified with charcoal, filtered through a membrane, and dialyzed against pure water for one day. The retentate was evaporated under reduced pressure until dry to give a white solid (0.8 g).

[0694] Detailed synthesis instructions (HP(βCD-triazole-βCD))

[0695] Step 1: Preparation of azide-connector

[0696] 1,3-Dibromopropane (10 mL, 20.18 g, 0.1 mol) was dissolved in 40 mL of DMSO under vigorous stirring. A solution of sodium azide (6.7 g, 0.1 mol) was prepared in DMSO (240 mL) and added dropwise (over 2 hours) to the dihalopropane solution. The solution was stirred overnight at room temperature. Then... n-Hexane Extract the crude product from the reaction mixture using 3 × 100 mL, and back-extract the collected product using water using 3 × 50 mL. n-Hexane The organic phase was carefully evaporated under reduced pressure (strictly at 40°C and 400 mbar, otherwise the target compound may distill off). The residual oil was purified by chromatography. n-Hexane -EtAc=98:2 as eluent, isocratic elution. Collect appropriate fractions and concentrate under reduced pressure to obtain the target compound as a viscous oil (which can be stored in a dark, refrigerated container under an inert atmosphere). The compound was observed using the following steps: immersing a TLC plate in a 10% triphenylphosphine solution in dichloromethane for approximately 15 s, drying the TLC plate below 60°C, immersing the TLC plate in a 2% ninhydrin ethanol solution for approximately 15 s, and finally drying the TLC plate below 60°C. The target compound appeared as purple spots on the TLC plate.

[0697] Step 2.1: Preparation of 2-O-propyne-βCD

[0698] Lithium hydride (212 mg, 26.432 mmol) was added to a solution of β-cyclodextrin (20 g, 17.62 mmol) in anhydrous DMSO (300 mL). The resulting suspension was stirred at room temperature under N2 until clear (12–24 h). Propylene bromide (1.964 mL, 17.62 mmol) and a catalytic amount of lithium iodide (approximately 20 mg) were then added, and the mixture was stirred at 55 °C under darkness for 5 h. The product was characterized by TLC (10:5:2 CH3CN-H2O-25% v / v NH3 aqueous solution), showing spots corresponding to monopropynized and non-propynized β-cyclodextrin, respectively. The solution was poured into acetone (3.2 L), the precipitate was filtered, and thoroughly washed with acetone. The resulting solid was transferred to a round-bottom flask and dissolved in a minimum volume of water. Silica gel (40 g) was added, and the solvent was removed under vacuum until a powdery residue was obtained. The crude mixture was applied to the top of a silica gel column (25 × 6 cm) and subjected to chromatographic separation (10:5:2 CH3CN-H2O-25% v / v NH3 aqueous solution). After freeze-drying, a solid 2- O -O-propynyl-β-CD. Analysis of 2-O-propynyl-β-CD by MALDI and NMR (… Figure 10T and Figure 10U ).

[0699] Step 2.2: Synthesis of 2-O-(3-azidopropyl)-βCD

[0700] Lithium hydride (212 mg, 26.432 mmol) was added to a solution of β-cyclodextrin (20 g, 17.62 mmol) in anhydrous DMSO (300 mL). The resulting suspension was stirred at room temperature under N2 until clear (12–24 h). Then, 3-azido-1-bromopropane (3 mL) and a catalytic amount of lithium iodide (approximately 20 mg) were added, and the mixture was stirred at 55 °C under darkness for 5 h. TLC (10:5:2 CH3CN-H2O-25% v / v NH3 aqueous solution) was used to characterize the product, showing a result corresponding to 2- O -(3-azidopropyl)-βCD and βCD spots. The solution was poured into acetone (3.2 L), the precipitate was filtered and washed thoroughly with acetone. The resulting solid was transferred to a round-bottom flask and dissolved in a minimum volume of water. Silica gel (40 g) was added, and the solvent was removed under vacuum until a powdery residue was obtained. This crude mixture was applied to the top of a silica gel column and subjected to chromatographic separation (10:5:2 CH3CN-H2O-25% v / v NH3 aqueous solution), yielding a solid 2- O -(3-azidopropyl)-β-CD.

[0701] Step 3: Synthesis of βCD-triazole-βCD dimer

[0702] 2- O -propynyl-β-CD and 2- O -(3-azidopropyl)-β-CD was suspended in water (300 mL) (each concentration between approximately 8-12 mM). Dimethylformamide (DMF) (approximately 300 mL) was added to the suspension to completely dissolve the heterogeneous mixture (the addition of DMF is a slightly exothermic process). Copper bromide (2 g, 13.49 mmol) was added to the solution. The suspension was stirred at room temperature for 1 hour. The reaction was monitored by TLC and was expected to be completed in approximately 1 hour (eluent: CH3CN:H2O:NH3 = 10:5:2). The crude product was filtered, and the mother liquor was concentrated under reduced pressure (60 °C). The gel-like substance was diluted with water, and silica (15 g) was added. The heterogeneous mixture was concentrated to dryness under reduced pressure. The crude mixture was applied to the top of a silica gel column and separated chromatographically (10:5:2 CH3CN-H2O-25% v / v NH3 aqueous solution), yielding BCD-(triazole)1-BCD dimer upon drying. The preparation of BCD-(triazole)1-BCD dimer was characterized by NMR. Figure 10V ).

[0703] Step 4: HP (βCD-triazole-βCD)

[0704] βCD-(triazole)1-βCD dimer (1 g, 0.418 mmol), obtainable according to steps 1-3 above or by other methods, was suspended in water (50 mL). Sodium hydroxide (DS3 = 0.32 g, 8 mmol; DS6 = 0.74 g, 18.5 mmol; DS7 = 0.87 g, 21.75 mmol) was added to the reaction vessel, and the mixture turned into a pale yellow solution. The reaction mixture was cooled by a water bath (10 °C), and propylene oxide (DS3 = 0.49 mL, 0.42 g, 7.25 mmol; DS6 = 1.21 mL, 1.04 g, 17.9 mmol; DS7 = 1.46 mL, 1.7 g, 29.3 mmol) was added all at once. The reaction vessel was flushed with argon, sealed at room temperature, and stirred for two days. The solution was concentrated under reduced pressure until a viscous slurry was obtained, which was precipitated with acetone (50 mL). The white solid was filtered through a sintered glass filter and washed thoroughly with acetone (3 × 15 mL). The substance was dissolved in water (50 mL), treated with an ion exchange resin (to remove salts), clarified with charcoal, filtered through a membrane, and dialyzed against pure water for one day. The residue was evaporated under reduced pressure until dry to give a white solid (0.8 g). The HP(βCD-triazole-βCD) product was analyzed by NMR (…). Figure 10W , Figure 10X and Figure 10Y ), and calculate their respective degrees of substitution as shown in the figure.

[0705] Example 4. Synthesis of methyl-substituted cyclodextrin dimers

[0706] Figure 3E The molecule to be synthesized is shown.

[0707] This example describes the synthesis of a methyl-substituted cyclodextrin dimer with a triazole linker.

[0708] Methyl (βCD-(triazole)1-βCD) dimer (exemplary synthesis)

[0709] The preparation of methylated β-cyclodextrin dimers was carried out in a one-step reaction (see [link]). Figure 11A The βCD-(triazole)1-βCD dimer core was prepared according to the synthesis strategy described in Example 3 above.

[0710] synthesis

[0711] 1.1 g (0.46 mmol) of βCD-(triazole)1-βCD dimer core was suspended in 100 mL of deionized H₂O under vigorous stirring, and sodium hydroxide (0.35 g, 8.8 mmol) was added. The resulting pale yellow suspension was stirred for 30 minutes until completely solubilized. When the temperature of the pale yellow transparent solution stabilized at approximately 20 °C, iodomethane (0.5 mL, 1.14 g, 8.03 mmol) was added in a single addition under vigorous stirring (Note: Iodomethane is immiscible with the reaction mixture; therefore, vigorous stirring improves efficiency). The reaction mixture was stirred at room temperature for 24 hours, and then treated with ion exchange resin: H⁺ resin (6 g) and OH⁻ resin (6 g) were added to the solution, stirred for 15 minutes, and filtered off (the resin was washed with 3 × 15 mL of deionized water). The resulting filtrate (final pH=7) was clarified with activated carbon: 0.2 g of activated carbon was added to the solution with vigorous stirring, stirred for 30 minutes, and then filtered off (the activated carbon pad was washed with 3 × 15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40 °C) to give the title compound (approximately 1 g) as a white powder.

[0712] Characterization

[0713] The reaction process was monitored by TLC. Figure 11B The obtained substances were characterized by MALDI-TOF and NMR analyses, such as... Figure 11C -N is shown.

[0714] Example 5. Synthesis of sulfonyl-substituted cyclodextrin dimers

[0715] Figure 12F The molecule to be synthesized is shown.

[0716] This example describes the synthesis of a sulfobutyl-substituted cyclodextrin dimer with a triazole linker.

[0717] The preparation of SB-dimer was completed in one step. Figure 12A ).

[0718] Synthetic (SB low DS)

[0719] βCD-(triazole)1-βCD dimer core (1.2 g, 0.5 mmol) was suspended in deionized H₂O (60 mL) under vigorous stirring. Sodium hydroxide (0.39 g, 9.75 mmol) was added to the mixture, and the resulting solution was heated at 60 °C. Butanesulfonate lactone (0.88 mL, 1.17 g, 8.6 mmol) was added dropwise at 60 °C, and the solution was heated at the same temperature for 3 hours. The reaction was then heated to 90 °C and continued for 1 hour to destroy any remaining butanesulfonate lactone. The reaction mixture was cooled and treated with ion exchange resins. Cation exchange resin (H⁺ resin, 2 g) and anion exchange resin (OH⁻ resin, 2 g) were added to the solution, stirred for 15 minutes, and filtered off (the resin was washed with 3 × 15 mL of deionized water). Clarify the resulting filtrate (final pH=7) with activated carbon: Add 0.3 g of activated carbon to the solution with vigorous stirring, stir for 30 minutes and filter (wash the activated carbon pad with 3 × 15 mL of deionized water).

[0720] The colorless solution was evaporated under reduced pressure (40°C) to obtain a white powder (1.47 g).

[0721] Characterization

[0722] The reaction was monitored by TLC analysis. Figure 12B The obtained substances were characterized by MALDI-TOF and NMR analyses, such as... Figure 12C -K is shown.

[0723] Synthesis (high DS)

[0724] The (βCD-(triazole)1-βCD) dimer core (1.2 g, 0.5 mmol) was suspended in deionized H₂O (60 mL) under vigorous stirring. Sodium hydroxide (1.22 g, 30.5 mmol) was added to the mixture, and the resulting solution was heated at 60 °C. Butanesulfonate lactone (2.8 mL, 3.72 g, 27.35 mmol) was added dropwise at 60 °C, and the solution was heated at the same temperature for 3 hours. The reaction was then heated at 90 °C for another 1 hour to destroy any remaining butanesulfonate lactone. The reaction mixture was cooled and treated with ion exchange resins. Cation exchange resin (H⁺ resin, 4 g) and anion exchange resin (OH⁻ resin, 4 g) were added to the solution, stirred for 15 minutes, and filtered off (the resin was washed with 3 × 15 mL of deionized water). The resulting filtrate (final pH=7) was clarified with activated carbon: 0.5 g of activated carbon was added to the solution with vigorous stirring, stirred for 30 minutes, and then filtered out (the activated carbon pad was washed with 3 × 15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40 °C) to give a white powder (1.51 g).

[0725] Characterization

[0726] The obtained substances were characterized by MALDI-TOF and NMR analysis, such as Figure 12M -P is shown.

[0727] Example 6. Synthesis of Quaternary Ammonium-Substituted Cyclodextrin Dimers

[0728] Figure 3I and 13G The molecule to be synthesized is shown.

[0729] This example describes the synthesis of a quaternary ammonium-substituted cyclodextrin dimer with a triazole linker.

[0730] Quaternary ammonium (βCD-(triazole)1-βCD) dimer (exemplary synthesis)

[0731] The preparation of QA-dimer was carried out in a one-step reaction (see [link]). Figure 13A The βCD-(triazole)1-βCD dimer core was prepared according to the synthesis strategy described in Example 2 above.

[0732] synthesis

[0733] The (BCD-(triazole)1-BCD) dimer core (1.2 g, 0.5 mmol) was suspended in 100 mL of deionized H₂O under vigorous stirring, and sodium hydroxide (0.39 g, 9.8 mmol) was added. The resulting pale yellow suspension was stirred for 30 minutes until completely solubilized. The temperature of the pale yellow transparent solution was stabilized at 5–10 °C, and glycidyltrimethylammonium chloride (1.17 mL, 1.32 g, 8.7 mmol) was added in a single batch under vigorous stirring. The reaction mixture was stirred at room temperature for 24 hours, and then the temperature of the solution was stabilized at 5–10 °C, and a second batch of glycidyltrimethylammonium chloride (0.4 mL, 0.45 g, 3 mmol) was added. The reaction mixture was heated at 50 °C for 3 hours, then cooled and treated with ion exchange resin: H⁺ resin (6 g) and OH⁻ resin (6 g) were added to the solution, stirred for 15 minutes, and filtered (the resin was washed with 3 × 15 mL of deionized water). The resulting filtrate (final pH=7) was clarified with activated carbon: 0.2 g of activated carbon was added to the solution with vigorous stirring, stirred for 30 minutes, and then filtered off (the activated carbon pad was washed with 3 × 15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40 °C) to give the title compound (approximately 800 mg) as a white powder.

[0734] Characterization

[0735] The obtained substances were characterized by MALDI-TOF and NMR analysis, such as Figure 13B -K is shown.

[0736] For QA-BCD derivatives, the typical Gaussian distribution with the regular patterns observed in randomly substituted derivatives is lacking, while irregular fragmentation patterns are detected. The identification / assignment of these irregular peaks is highly complex because simple fragmentation patterns cannot be predicted. The irregular patterns observed in MALDI spectra are likely due to the instability of the trimethylammonium moiety under the experimental conditions. Specifically, the elimination product (see Figure 2) is the result of trimethylammonium moiety cleavage, while the demethylation product (see Figure 2) is the result of stepwise cleavage of the methyl group from the cationic side chain. It is reasonable to conclude that MALDI conditions are unsuitable for determining the DS of QA-BCD derivatives because non-informative peaks are generated during laser desorption. However, the DS of QA-BCD derivatives can be determined by NMR. Figure 13I ), and is estimated to be about 2.1.

[0737] Example 7. Synthesis of succinyl-substituted cyclodextrin dimers

[0738] Figure 3G and 14G The molecule to be synthesized is shown. The preparation of the succinyl-substituted dimer (Succ-dimer) is completed in a one-step reaction. Figure 14A ).

[0739] synthesis

[0740] The (βCD-(triazole)1-βCD) dimer core (1.2 g, 0.5 mmol) was suspended in pyridine (23 mL) under vigorous stirring and an inert atmosphere. However, to increase the solubility of the (βCD-(triazole)1-βCD) dimer, the suspension was heated at 40 °C for 1 hour, but complete solubilization was not achieved. A second portion of pyridine (23 mL) was added to the suspension, but dilution did not further improve the solubility of the (βCD-(triazole)1-βCD) dimer. Succinic anhydride (0.1 g, 1 mmol) was added at room temperature, and the reaction mixture was stirred for 24 hours. The crude product was concentrated under reduced pressure, solubilized in water (not a clear solution) (50 mL), and treated with ion exchange resin: H+ resin (2 g) and OH- resin (2 g) were added to the solution, stirred for 15 minutes, and filtered (the resin was washed with 3 × 15 mL of deionized water). The resulting filtrate (final pH=7) was clarified with activated carbon: 0.5 g of activated carbon was added to the solution with vigorous stirring, stirred for 30 minutes, and then filtered off (the activated carbon pad was washed with 3 × 15 mL of deionized water). The colorless solution was evaporated under reduced pressure (40 °C) to give the title compound (approximately 900 mg) as a white powder.

[0741] Characterization

[0742] The obtained substances were characterized by MALDI-TOF and NMR analysis, such as Figure 14B -K is shown.

[0743] As with QA-dimer, MALDI analysis has proven unfavorable for determining DS, and DS is determined by NMR. Figure 14I ), and is estimated to be about 2.1.

[0744] Example 8. Extraction of 7KC and cholesterol from blood cells using βCD dimer and monomers

[0745] method

[0746] Blood was collected from healthy volunteers by a licensed phlebotomist. The test substance or PBS (negative control) was added to the whole blood at various concentrations and incubated at 37°C for 3 hours. The blood was then centrifuged and the serum was collected. The serum was frozen and then processed for mass spectrometry analysis.

[0747] Plasma free 7-ketocholesterol was determined by LC-MS / MS after protein precipitation, extraction with acetonitrile, and derivatization with a novel quaternary aminooxygenated (QAO) mass marker (Amplifex Keto Reagent (AB Sciex, Framingham, MA, USA)). This mass marker has been used in the analysis of testosterone (Star-Weinstock). et al. ], Analytical Chemistry ,84(21):9310-9317. (2012)).

[0748] A 50 µL plasma sample was spiked with 0.5 ng of internal standard d7-7-ketocholesterol (Toronto Research Chemicals, North York, Ontario, CA), prepared in ethanol at 0.1 ng / µL. The sample was treated with 250 µL of acetonitrile, vortexed, and centrifuged at 12,000 × g for 10 min to remove proteins. The supernatant was vacuum dried and then treated with 75 µL of QAO reagent. The working reagent was prepared by mixing 0.7 mL of Amplifex ketone reagent with 0.7 mL of Amplifex ketone diluent to prepare a 10 mg / mL stock solution. The stock solution was then diluted 1:4 with 5% acetic acid in methanol to a final working concentration of 2.5 mg / mL. The mixture was allowed to react at room temperature for two days before LC-MS / MS analysis.

[0749] 7-Ketocholesterol standards ranging from 1 to 100 ng / ml were prepared in charcoal-leached plasma SP1070 (Golden West Biological, Temecula, CA, USA) and phosphate-buffered saline (Toronto Research Chemicals, North York, Ontario, CA). Residual 7-ketocholesterol was detected in the leached plasma; therefore, standards from PBS were used.

[0750] QAO-7-ketocholesterol derivatives were analyzed using a 4000 Q-TRAP tandem / triple quadrupole linear ion trap mass spectrometer (SCIEX, Framingham, MA, USA) in positive mode electrospray ionization (ESI). The mass spectrometer was connected to a Shimadzu (Columbia, MD) SIL-20AC XR autosampler, and then to two LC-20AD XR LC pumps.

[0751] The instrument was operated with the following settings: power supply voltage 4500 kV, GS1 50, GS2 50, CUR 20, TEM 550, and CAD gas medium. Compounds were quantified using multiple reaction monitoring (MRM), and conversions were optimized by infusing purified derivatized compounds, as shown in Table 1 below. Conversions in bold were used for quantification.

[0752]

[0753] Separation was performed using a Gemini 3µ C6-phenyl 110Å, 100×2mm column (Phenomenex, Torrance, CA, USA), maintained at 35°C in a Shimadzu (Columbia, MD) CTO-20AC column oven. The gradient mobile phase flow rate was 0.5 mL / min and consisted of two solvents: A: 0.1% formic acid in water, and B: 0.1% formic acid in acetonitrile. The initial concentration of solvent B was 20%, then linearly increased to 60% B over 10 min, then linearly increased to 95% B over 0.1 min, held for 3 min, then decreased back to the initial 20% B over 0.1 min, and held for 4 min. The retention time for 7-ketocholesterol was 8.46 min.

[0754] Data were acquired using Analyst 1.6.2 (SCIEX, Framingham, MA, USA) and analyzed using Multiquant 3.0.1 (SCIEX, Framingham, MA, USA). Sample values ​​were calculated based on a standard curve derived from the ratio of the peak area ratio of the analyte to the internal standard to the analyte concentration, fitted to a 1 / x weighted linear equation. The limit of quantitation was 1 ng / mL, with an accuracy of 102% and a precision (relative standard deviation) of 8.5%. The signal-to-noise ratio (S / N) was 19:1. At a concentration of 100 ng / mL, the accuracy was 98%, the precision was 0.5%, and the S / N was 24:1.

[0755] result

[0756] Figure 15A and 15B HPβCD dimer (DS~8) was determined by MALDI and NMR, see [link to MALDI and NMR data]. Figure 10I and 10J It can remove 7KC from blood cells (whole blood) more effectively than HPβCD monomer. This is Ex vivo of human subjects This measurement allows us to obtain results that can more accurately predict the effects on human patients than experiments on non-human animals. Figure 15C This indicates that it does not significantly affect plasma cholesterol levels. This means that HPβCD dimer cannot remove large amounts of cholesterol from blood cells. Removing too much cholesterol from cells can lead to cell and organelle membrane rupture and cell death. We wanted to investigate this directly, so we performed a hemolysis assay.

[0757] Example 9. Hemolysis caused solely by high concentrations of cyclodextrin dimers

[0758] method

[0759] For the test solution, the amount of PBS varied depending on the concentration of the cyclodextrin being tested. Samples were tested in triplicate. 50 µL of blood was added to each sample along with PBS and a stock solution (also made from PBS) to achieve a final volume of 200 µL for the appropriate concentration. 5% Triton X-100 was used as a positive control, and PBS was used as a negative control. Once all samples were mixed, they were incubated at 37°C for three hours with stirring. The positive control was 100% hemolyzed using Triton X-100 detergent. Once the samples were removed from the incubation, they were diluted by the same factor in a 96-level plate and normalized to the positive control absorbance (approximately 1.1). The absorbance was read at 540 nm. The mean of the samples was then corrected by subtracting the negative control. The experiment was performed three times, and the error bars represent the standard error of the mean (Melanga […]). et al. ], Journal of Pharmaceutical Sciences ,105(9):2921-31. (2016)), (Kiss [ et al. ], European Journal of Pharmaceutical Sciences, 40(4):376-80. (2010)).

[0760] Figure 15D-15E This indicates that the butyl- and triazole-linked dimer remains low in toxicity to blood cells and has no significant toxicity in the pharmacological range of less than 1 mM. Figure 15D Hemolysis of butyl-linked HP-dimers of three different DS was shown. Figure 10G-10I The DS in the middle is determined by MALDI, and Figure 10J DS (confirmed by NMR), DS~3 triazole-linked HP dimer (in Figure 10P and 10W Characterization; based on MALDI labeling) and DS~3 triazole-linked Me dimers (in Figure 11I and 11L (Characteristics). At higher concentrations, the dimer with only three butyl groups showed measurable hemolysis. Figure 15E In our study, we tested hemolysis with various other substitutions of the triazole-linked βCD dimer. We also tested hemolysis with the unsubstituted quaternary ammonium (DS2) in… Figure 13I Characterization was performed in [the following], succinyl (DS~2, in [the following]). Figure 14I Characterization in (in the process) and sulfobutyl (DSes in Figure 12E , 12H Characterized by NMR and MALDI in 12K and 12N; MALDI DSes were used in the labeling. Only unsubstituted dimers up to 7.5 mM were tested, at which concentrations approximately 5% hemolysis was detected. Other dimers were tested at a maximum of 5 mM, and no significant hemolysis was detected at any of the tested concentrations.

[0761] It appears that at high concentrations, the hemolytic activity of the triazole dimer form of βCD is lower than that of the tested HPβCD butyl dimer, but the linker and all substitution types showed extremely low cleavage, indicating low toxicity.

[0762] Example 10. Solubilization of sterols and sterol-like compounds by cyclodextrin dimers

[0763] The solubilization of lipophilic compounds was tested using the dimers described in Examples 2-6. The tested compounds included cholesterol precursors (chain sterols), other oxidized sterols, steroid hormones, and sterol vitamins.

[0764] Methods for in vitro solubility determination (turbidity determination)

[0765] Sterol stock solutions (including oxosterols, hormones, and vitamins) were suspended in 100% ethanol. The final suspension concentration was: 3% ethanol, 300 μM sterols, dissolved in PBS, and various concentrations of cyclodextrin. Samples were incubated at 37°C for 30 minutes, and absorbance was measured at 350 nm using a spectrophotometer plate reader. Samples were prepared in quadruplicate using a Beckman Biomek 2000 liquid processor, and plates with a hydrophilic coating were used to minimize sterol binding to the pore surface. All experiments were performed three or more times, and the error bars represent the standard error of the mean.

[0766] Turbidity values ​​are normalized to percentages of turbidity measured in the absence of cyclodextrin.

[0767] result

[0768] We are outside the body Our novel dimer was tested for 7-ketocholesterol in spectroscopic measurements. Figure 16A In this context, DS3 is a dimer with an average of about 3 hydroxypropyl groups linked by butyl groups (in...). Figure 10G DS6 is a dimer with an average of about 6 substituted butyl links (quantified by MALDI), and is a butyl-linked dimer with an average of about 6 substitutions. Figure 10H ), and DS8 is a dimer with an average of about 8 hydroxypropyl-substituted butyl links (MALDI). Figure 10I HPβCD dimers were tested at various concentr...

Claims

1. A cyclodextrin (CD) dimer having the following structure: CD—L—CD The connector L is connected to the subsurface of each CD molecule, wherein the connection of each CD to the connector is achieved independently by O, S or N connected to the C2 or C3 carbon of each CD subunit, or by acetal connection of two adjacent oxygens of the CD. Each CD has the structure of formula X: (Formula X) Where L includes a butyl connector; Furthermore, R 1 R 2 and R 3 Each of the R groups is independently selected from H and hydroxypropyl, wherein there are between 1 and 3 of them. 1 R 2 and R 3 The group is hydroxypropyl.

2. A cyclodextrin (CD) dimer having the following structure: CD—L—CD The connector L is connected to the subsurface of each CD molecule, wherein the connection of each CD to the connector is achieved independently by O, S or N connected to the C2 or C3 carbon of each CD subunit, or by acetal connection of two adjacent oxygens of the CD. Each CD has the structure of formula X: (Formula X) Where L includes the following structure: -(CH2) n1 (CH2) n2 - (Equation XI), where n1 and n2 are each between 1 and 8; Furthermore, in which: (a) R 1 R 2 and R 3 Each of the R molecules is independently selected from H and methyl groups, wherein one to three of them are present. 1 R 2 and R 3 The group is methyl; (b) R 1 R 2 and R 3 Each of the R groups is independently selected from H and hydroxypropyl, wherein there are between 1 and 7 of them. 1 R 2 and R 3 The group is hydroxypropyl; (c) R 1 R 2 and R 3 Each of the R groups is independently selected from H and sulfonyl, wherein one to three of the R groups are present. 1 R 2 and R 3 The group is sulfobutyl; (d) R 1 R 2 and R 3 Each of the R groups is independently selected from H and succinyl groups, wherein one or two of the R groups are selected from H and succinyl groups. 1 R 2 and R 3 The group is succinyl; (e) R 1 R 2 and R 3 Each is independently selected from H and contains -CH2CH(OH)CH2N(CH3)3Cl or -CH2CH(OH)CH2N(CH3)3 + The quaternary ammonium group, wherein there are one to two of the R groups. 1 R 2 and R 3 The group is a quaternary ammonium group; or (f) R 1 R 2 and R 3 It is H.

3. The cyclodextrin dimer of claim 1, wherein the connector is a substituted or unsubstituted butyl connector.

4. The cyclodextrin dimer of claim 2, wherein the length of the linker is between 5 and 7 atoms.

5. The cyclodextrin dimer of claim 2, wherein the length of the linker is between 5 and 6 atoms.

6. The cyclodextrin dimer of claim 2, wherein the length of the linker is 7 atoms.

7. The cyclodextrin dimer of any one of claims 1-6, wherein L is attached to the C2 carbon of each CD monomer.

8. The cyclodextrin dimer of any one of claims 1-6, wherein L is attached to the C3 carbon of each CD monomer.

9. The cyclodextrin dimer according to any one of claims 1-6, wherein L is linked to the C2 carbon of one CD monomer and the C3 carbon of another CD monomer.

10. The cyclodextrin dimer according to any one of claims 1-6, wherein the cyclodextrin dimer exhibits a greater affinity for 7-ketocholesterol (7KC) than for cholesterol.

11. The cyclodextrin dimer of claim 10, wherein the cyclodextrin dimer exhibits an affinity for 7-ketocholesterol (7KC) of at least 1.1, 1.5, 2, 3, 4, 5, or 10 times that of cholesterol.

12. A composition comprising a mixture of cyclodextrin dimers according to any one of claims 1-11.

13. A pharmaceutical composition comprising a cyclodextrin dimer according to any one of claims 1-11 and a pharmaceutically acceptable carrier, or substantially consisting of a cyclodextrin dimer according to any one of claims 1-11 and said pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein the cyclodextrin dimer is the sole active ingredient in the pharmaceutical composition.

15. Use of the cyclodextrin dimer according to any one of claims 1-11 in the preparation of a medicament for treating a subject in need.

16. The use as claimed in claim 15, wherein the subject in need suffers from the harmful or toxic effects of 7-ketocholesterol (7KC).

17. Use of the cyclodextrin dimer according to any one of claims 1-11 in the preparation of a medicament for reducing the amount of 7-ketocholesterol (7KC) in a subject in need.

18. The use as described in claim 17, wherein the cyclodextrin dimer is administered to the subject via parenteral, subcutaneous, intramuscular, intravenous, topical, transdermal, oral, sublingual, or sublingual administration.

19. The use as claimed in claim 17, wherein the drug comprises between 1 mg and 20 g of the cyclodextrin dimer.

20. The use as claimed in claim 19, wherein the drug comprises between 10 mg and 1 g of the cyclodextrin dimer.

21. The use as claimed in claim 19, wherein the drug comprises between 50 mg and 200 mg of the cyclodextrin dimer.

22. The use as claimed in claim 19, wherein the drug comprises 100 mg of the cyclodextrin dimer.

23. The use as claimed in claim 17, wherein the drug comprises between 1 g and 10 g of the cyclodextrin dimer.

24. The use as claimed in claim 23, wherein the drug comprises 2g, 3g, 4g or 5g of the cyclodextrin dimer.

25. The use as claimed in claim 17, wherein the drug comprises between 50 mg and 5 g of the cyclodextrin dimer.

26. The use as claimed in claim 25, wherein the medicament comprises between 100 mg and 2.5 g of the cyclodextrin dimer.

27. The use as claimed in claim 25, wherein the drug comprises between 100 mg and 2 g of the cyclodextrin dimer.

28. The use as claimed in claim 25, wherein the medicament comprises between 250 mg and 2.5 g of the cyclodextrin dimer.

29. The use as claimed in claim 17, wherein the medicament is used to prevent, treat, or improve symptoms of one or more age-related diseases and conditions directly related to 7-ketocholesterol (7KC), said diseases and conditions including: Atherosclerosis, coronary artery disease, heart failure at all stages, Alzheimer's disease, vascular dementia, Smith-Limeley-Opz syndrome, Niemann-Pick disease type C, dry form of age-related macular degeneration, chronic obstructive pulmonary disease, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and / or hypercholesterolemia.

30. The use as described in claim 17, wherein the drug is used to prevent, treat or improve symptoms of arteriosclerosis in which 7-ketocholesterol (7KC) is directly related.

31. The use as described in claim 17, wherein the drug is used to prevent, treat or improve symptoms of atherosclerosis.

32. The use as claimed in claim 31, wherein the drug is used in combination with a second therapy, wherein the second therapy is used simultaneously or sequentially in any order.

33. The use as described in claim 32, wherein the second therapy comprises one or more of an anticholesterol drug, a fibrate or statin, an antiplatelet drug, an antihypertensive drug, or a dietary supplement.

34. The use as described in claim 33, wherein the statin includes ADVICOR(R) (nicotinic acid extended-release / lovastatin), ALTOPREV(R) (lovastatin extended-release), CADUET(R) (amlodipine and atorvastatin), CRESTOR(R) (rosuvastatin), JUVISYNC(R) (sitagliptin / simvastatin), LESCOL(R) (fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR(R) (atorvastatin), LIVALO(R) (pitavastatin), MEVACOR(R) (lovastatin), PRAVACHOL(R) (pravastatin), SIMCOR(R) (nicotinic acid extended-release / simvastatin), VYTORIN(R) (ezetimibe / simvastatin), or ZOCOR(R) (simvastatin).

35. A method for purifying oxosterols, the method comprising: The composition containing oxosterol is contacted with the cyclodextrin dimer according to any one of claims 1-11, thereby solubilizing the oxosterol in the cyclodextrin dimer; and the cyclodextrin dimer and the solubilized oxosterol are recovered.

36. The method of claim 35, wherein the oxosterol comprises or is composed of 7-ketocholesterol (7KC).

37. The method of claim 36, further comprising measuring the amount or concentration of 7-ketocholesterol (7KC) in the solubilized oxosterol to determine the relative concentration of 7-ketocholesterol (7KC) in the composition.

38. An in vitro method for removing oxysterols from a sample, the method comprising: The sample containing oxosterol is contacted with a cyclodextrin dimer according to any one of claims 1-11, thereby solubilizing the oxosterol in the cyclodextrin dimer; and the sample is separated from the cyclodextrin dimer and the solubilized sterol.

39. A method for producing a cholesterol-reducing product, the method comprising: Contact a product containing cholesterol with a cyclodextrin dimer according to any one of claims 1-11, thereby solubilizing the cholesterol in the cyclodextrin dimer; And to remove the cyclodextrin dimer and solubilized cholesterol from the product.

40. The method of claim 39, wherein the product is a food product.

41. The method of claim 40, wherein the food product comprises meat and / or dairy products.

42. A method for preparing the cyclodextrin dimer according to claim 1, comprising: (a) Reacting β-cyclodextrin protected on the main side with a dialkylating agent to generate a βCD dimer protected on the main side by subsurface linkage; (b) Deprotecting the βCD dimer protected by the main side to generate a deprotected βCD dimer; as well as (c) Linking the deprotected βCD to one or more hydroxypropyl groups to generate the cyclodextrin dimer.

43. The method of claim 42, wherein the β-cyclodextrin protected on the main side comprises hepta(6-O-tert-butyldimethylsilyl)-β-cyclodextrin.

44. The method of claim 42, wherein the dialkylating agent comprises dibromoalkane.

45. The method of claim 42, wherein (i) Step (a) is carried out under anhydrous conditions and / or with sodium hydride as the base; (ii) Step (b) is carried out in tetrahydrofuran (THF) containing tetrabutylammonium fluoride; (iii) Step (c) includes reacting the deprotected βCD dimer with a hydroxypropylating agent or propylene oxide; or (iv) Step (c) is carried out under aqueous conditions, which include using sodium hydroxide as a base.

46. ​​The method of claim 42, wherein: (i) Step (a) includes purifying the main-side protected βCD dimer by direct phase chromatography and isocratic elution; (ii) Step (b) includes purifying the deprotected βCD dimer by direct phase chromatography and isocratic elution; or (iii) Step (c) includes purifying the cyclodextrin dimer by one or more of ion exchange resin treatment, charcoal clarification, and dialysis.

47. A method for preparing the cyclodextrin dimer according to claim 2, comprising: (a) Reacting 2-O-(n-azidoalkyl)-βCD with 2-O-(n-alkynyl)-βCD to form a βCD-triazole-βCD dimer; and (b) The βCD-triazole-βCD dimer is prepared by reacting the βCD-triazole-βCD dimer with a hydroxypropylating agent, a methylating agent, a succinylation agent, a sulfobutylating agent, a quaternary ammonium linker, or glycidyltrimethylammonium chloride.

48. The method of claim 47, wherein in step (b), the βCD-triazole-βCD dimer is prepared by reacting the βCD-triazole-βCD dimer with propylene oxide, iodomethane, succinic anhydride, 1,4-butanesulfonate lactone, a quaternary ammonium linker, or glycidyltrimethylammonium chloride.

49. The method of claim 47, wherein: (i) Step (a) is carried out using a copper (I) catalyst; (ii) Step (a) is carried out in an aqueous solution; and / or (iii) Step (a) is carried out in an aqueous solution containing dimethylformamide (DMF).

50. The method of claim 47, wherein step (a) further comprises purifying the βCD-triazole-βCD dimer by silica gel chromatography.

51. The method of claim 47, further comprising generating the 2-O-(n-azidoalkyl)-βCD prior to step (a) by a method comprising: (1) Reaction of n-azido-1-bromo-alkane with β-cyclodextrin using a catalytic amount of lithium iodide to generate the 2-O-(n-azidoalkyl)-βCD; and (2) Purification of the 2-O-(n-azidoalkyl)-βCD.

52. The method of claim 51, wherein step (2) comprises silica gel chromatography.

53. The method of claim 47, further comprising generating 2-O-(n-yyn)-βCD prior to step (a) by a method comprising: (1) Using a catalytic amount of lithium iodide, n-bromo-1-yne is reacted with β-cyclodextrin to generate the 2-O-(n-yne)-βCD, and (2) The 2-O-(n-yne)-βCD is purified.

54. The method of claim 53, wherein step (2) comprises silica gel chromatography.

55. The method of claim 53, wherein step (1) is carried out in dry DMSO.

56. The method of claim 53, wherein the reaction in step (1) comprises lithium hydride.

57. The method of claim 47, wherein the βCD-triazole-βCD dimer comprises the following structure: CD-(CH2)n1 (CH2) n2 -CD (Formula XII), where n1 and n2 are each between 1 and 8.

58. The method of claim 57, wherein n1 is 1, 2, 3 or 4, and / or n2 is 1, 2, 3 or 4, or wherein the length of the triazole connector is between 5 and 8.

59. The method of claim 47, wherein step (b) comprises hydroxypropylating the βCD-triazole-βCD dimer to generate the cyclodextrin dimer.

60. The method of claim 59, wherein: (i) Step (b) is carried out under aqueous conditions, which include using sodium hydroxide as a base; and / or (ii) Step (b) further includes purifying the cyclodextrin dimer by one or more of ion exchange resin treatment, charcoal clarification, membrane filtration and dialysis.

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