A mannosylated amine dextran drug delivery vehicle having a cleavable disulfide / carbonate linker that targets the payload to CD206-expressing cells

Abstract

Provided are compounds, compositions, and methods for re-polarizing tumor-associated macrophages (TAMs), reducing macrophage-mediated inflammation, and treating diseases. The compound or pharmaceutical composition can be administered to a subject in need thereof, the compound comprising a polymeric carbohydrate backbone and one or more mannose-binding C-type lectin receptor targeting moieties, and the therapeutic agent comprising one or more reactive hydroxyl groups and being attached to the polymeric carbohydrate backbone via a cleavable linker. The cleavable linker can comprise one or more carbonate and / or disulfide moieties. Diseases to be treated can include cancer, autoimmune diseases, inflammatory disorders, non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, and other macrophage-related diseases.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 388,777, filed on July 13, 2022, entitled "Mannosylated Amine Dextran Drug Delivery Vehicles with Degradable Disulfide / Carbonate Linkers Targeting Payloads to CD206 Expressing Cells", the entire content of which is incorporated herein by reference. The present disclosure relates to the targeted delivery of therapeutic payloads, more specifically, therapeutic payloads having reactive hydroxyl groups, and to therapeutic constructs, methods of making the same, and methods of use thereof for releasing the therapeutic payload when internalized in cells expressing mannose - binding C - type lectin receptors and / or shifting the phenotype towards a pro - inflammatory state.

Background Art

[0002] Various C - type lectin receptors, such as the mannose receptor (CD206), an example of a C - type lectin receptor, can be expressed on macrophages, dendritic cells, and mesangial cells. The CD206 receptor typically binds to molecules presenting multiple terminal mannose moieties, and when CD206 binds to a ligand, the receptor / ligand complex is internalized into endosomes via receptor - mediated endocytosis, and the endosomes are naturally acidified to a pH of about 4 - 5. At this low pH level, CD206 releases its ligand and recycles to the cell surface. In addition to pH, various enzymes are released into the endosomes and can act on the contents of the endosomes.

[0003] Macrophages are a common cell type in all tissues of the body and are an important component of innate immunity. Macrophages also contribute significantly to the maintenance of tissue homeostasis and wound repair. An important feature of macrophages is their ability to adopt many phenotypic states in response to various stimuli from their local environment. Furthermore, macrophages can change their phenotypic states when the stimuli from their local environment change. When macrophages change their phenotype in response to environmental stimuli, they are referred to as activated macrophages. Activated macrophages alter the expression of hundreds of genes. Furthermore, genes whose expression is changed in response to one set of environmental stimuli may differ significantly from genes whose expression is altered in response to a different set of stimuli. One of the genes that often undergoes increased expression during macrophage activation is CD206. Research efforts to understand the gene expression changes of macrophages in response to different stimuli in various in vivo and in vitro situations have been the subject of thousands of scientific publications over the past few decades.

[0004] A large number of activated phenotypes exist, and it has been found in the extensive literature on macrophage activation that the expression of only one or a few genes cannot accurately identify any one specific phenotype. However, these activated phenotypes can be characterized in terms of their overall immune state and can be placed on a continuum with a highly pro-inflammatory phenotype at one end and an immunosuppressive and wound-healing phenotype at the other end. Conventionally, as referred to in the historical macrophage phenotype literature, activated macrophages are divided into two phenotypes: (1) a classical activated type called M1 that is highly pro-inflammatory, and (2) an alternative activated type called M2 that is immunosuppressive and promotes wound healing. Although it is now understood that the strictly dichotomous classification of activated macrophage phenotypes is overly simplistic and does not represent the true flexibility of macrophage responses to stimuli from their microenvironment, the concept that activated macrophages can influence the local immune response by being either pro-inflammatory (M1-like) or immunosuppressive (M2-like) continues to be useful in explaining the role of macrophages in various pathological conditions.

[0005] In living humans and animals, activated macrophages with a mixed activated phenotype having characteristics of both M1-like and M2-like phenotypic states have been observed. Examples of stimuli that can induce an M1-like phenotype in macrophages include tumor necrosis factor (TNF), interferon gamma (INFγ), and toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS). Examples of stimuli that can induce an M2-like phenotype in macrophages include interleukin 4 (IL4), interleukin 13 (IL13), tumor growth factor beta (TGFβ), and glucocorticoids such as dexamethasone. There are also many other hormones, cytokines, chemokines, and environmental factors that can affect the macrophage phenotype.

[0006] Tumor-associated macrophages (TAMs) are abundant in tumors and are highly important contributing factors to maladaptive immune responses associated with cancer and other conditions. TAMs are the most numerous immune cells infiltrating tumors and can constitute over about 5% to 30% of all cells in a tumor. Both M1-like and M2-like TAMs are known, but the majority of TAMs that reside within or near established tumors are immunosuppressive, i.e., M2-like activated macrophages.

[0007] Therefore, there remains a need for compositions and methods that induce a phenotypic change from M2-like TAMs to M1-like TAMs in order to treat cancer with higher efficacy and lower toxicity.

[0008] Other objects, advantages, and features of the present disclosure will become apparent from the following specification when interpreted in conjunction with the accompanying drawings. SUMMARY OF THE INVENTION

[0009] In Example 1, the compound includes a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent bound to the polymeric carbohydrate backbone via a cleavable linker, the therapeutic agent including one or more reactive hydroxyl groups.

[0010] Example 2 is where the compound includes subunits represented by formula (I),

[0011]

Chemical formula

[0012] wherein each X is, independently, H, L1-A-Z, or L2-R, each X is bonded to an OH group, each of L1 and L2 is, independently, an amine-terminated linker, each A includes a cleavable linker containing one or more carbonate and / or disulfide moieties, each Z includes a therapeutic agent containing one or more reactive hydroxyl groups, Each R independently contains a mannose-binding C-type lectin receptor targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different, regarding the compound according to Example 1.

[0013] Example 3 relates to a compound according to Example 1 or 2, wherein at least one X is L1-A-Z, at least one X is L2-R, and R contains a mannose-binding C-type lectin receptor targeting moiety.

[0014] Example 4 relates to a compound according to any one of Examples 1 to 3, wherein the polymeric carbohydrate backbone has a molecular weight of about 1 kDa to about 50 kDa. Example 5 relates to a compound according to any one of Examples 1 to 4, wherein the mannose-binding C-type lectin receptor targeting moiety contains a mannosyl coupling reagent, mannose, high-mannose glycan or mannooligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof.

[0015] Example 6 relates to a compound according to any one of Examples 2 to 5, wherein at least one L1 and / or at least one L2 contains -(CH2) p S(CH2) q- NH-, wherein p and q are integers from 0 to 5.

[0016] Example 7 relates to a compound according to any one of Examples 2 to 6, wherein the therapeutic agent is conjugated to a cleavable linker before being bound to the polymeric carbohydrate backbone. Example 8 relates to a compound according to any one of Examples 1 to 7, wherein the cleavable linker contains one or more carbonate and / or disulfide moieties.

[0017] Example 9 relates to a compound according to any one of Examples 1 to 7, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone,

[0018]

Chemical Formula

[0019] Relates to a compound according to any one of Examples 2 to 8, wherein x is an integer from 1 to 5 and y is an integer from 1 to 5. Example 10 relates to a compound according to any one of Examples 2 to 8, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone:

[0020]

Chemical formula

[0021] Relates to a compound according to any one of Examples 2 to 8. Example 11 relates to a compound according to any one of Examples 2 to 10, wherein A has the following formula:

[0022]

Chemical formula

[0023] Relates to a compound according to any one of Examples 2 to 10, wherein x is an integer from 1 to 5 and y is an integer from 1 to 5. Example 12 relates to a compound according to any one of Examples 2 to 11, wherein A has the following formula:

[0024]

Chemical formula

[0025] Relates to a compound according to any one of Examples 2 to 11. Example 13 relates to a compound according to any one of Examples 1 to 12, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

[0026] Example 14 relates to a compound according to any one of Examples 1 to 13, wherein the therapeutic agent comprises dexamethasone or paclitaxel. In Example 15, the pharmaceutical composition comprises a compound according to any one of Examples 1 to 14 and a pharmaceutically effective carrier.

[0027] Example 16 is that the compound contains the subunit represented by formula (I),

[0028]

Chemical formula

[0029] wherein, each X is independently H, L1-A-Z, or L2-R, and each X is bonded to an OH group, each of L1 and L2 is independently an amine-terminated linker, each A independently contains a cleavable linker containing one or more carbonate and / or disulfide moieties, each Z independently contains a therapeutic agent containing one or more reactive hydroxyl groups, each R independently contains a mannose-binding C-type lectin targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different, and relates to the composition according to Example 15.

[0030] In Example 17, the compound contains a therapeutic agent containing reactive hydroxyl groups, a cleavable linker containing one or more carbonate and / or disulfide moieties, and a second compound containing a primary amine, and the cleavable linker is bonded to the reactive hydroxyl groups of the therapeutic agent and to the primary amine of the second compound.

[0031] Example 18 is that the compound has the following formula (II),

[0032]

Chemical formula

[0033] wherein, Z is a therapeutic agent containing reactive hydroxyl groups, Y is a second compound containing a primary amine, x is an integer from 1 to 5, Regarding the compound according to Example 17, wherein y is an integer from 1 to 5.

[0034] Example 19 relates to the compound according to Example 17 or 18, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

[0035] Example 20 relates to the compound according to any one of Examples 17 to 19, wherein the therapeutic agent comprises dexamethasone or paclitaxel. In Example 21, a method for preparing the compound according to any one of Examples 1 to 14 comprises: (a) synthesizing a polymeric carbohydrate backbone to which one or more amine-terminated lysines are attached; (b) synthesizing a cleavable linker comprising one or more carbonate and / or disulfide moieties; (c) reacting the cleavable linker with a reactive hydroxyl group of the therapeutic agent to form a therapeutic agent-linker compound; and (d) reacting the therapeutic agent-linker compound with one of the one or more amine-terminated lysines on the polymeric carbohydrate backbone.

[0036] Example 22 relates to the method according to Example 21, wherein step (a) can be performed before step (b), (c), or (d), or after step (b) or (c).

[0037] Example 23 relates to the method according to Example 21 or 22, wherein the therapeutic agent is conjugated to the cleavable linker before being attached to the polymeric carbohydrate backbone. Example 24 relates to the method according to any one of Examples 21 to 23, wherein at least one X is L1-A-Z, at least one X is L2-R, and R comprises a mannose-binding C-type lectin receptor targeting moiety.

[0038] Example 25 relates to the method according to any one of Examples 21 to 24, wherein the polymeric carbohydrate backbone has a molecular weight of about 1 kDa to about 50 kDa. Example 26 relates to a method according to any one of Examples 21 to 25, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, high mannose glycan or mannooligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof.

[0039] Example 27 relates to a method according to any one of Examples 21 to 26, wherein at least one L1 and / or at least one L2 comprises -(CH2) p S(CH2) q- NH-, where p and q are integers from 0 to 5.

[0040] Example 28 relates to a method according to any one of Examples 21 to 27, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone,

[0041]

Chemical formula

[0042] where x is an integer from 1 to 5 and y is an integer from 1 to 5. Example 29 relates to a method according to any one of Examples 21 to 28, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone,

[0043]

Chemical formula

[0044] relates to a method according to any one of Examples 21 to 28. Example 30 relates to a method according to any one of Examples 21 to 29, wherein A has the following formula,

[0045]

Chemical formula

[0046] Regarding a method according to any one of Examples 21 to 29, where x is an integer from 1 to 5 and y is an integer from 1 to 5. Example 31 relates to a method according to any one of Examples 21 to 30, where A has the following formula:

[0047] [Chemical formula]

[0048] Regarding a method according to any one of Examples 21 to 30. Example 32 relates to a method according to any one of Examples 21 to 31, where the therapeutic agent includes a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

[0049] Example 33 relates to a method according to any one of Examples 21 to 32, where the therapeutic agent includes dexamethasone, paclitaxel, or a combination thereof. In Example 34, a method of re-polarizing tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype includes administering to a subject in need thereof an effective dose of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent bound to the polymeric carbohydrate backbone via a cleavable linker and comprising one or more reactive hydroxyl groups.

[0050] Example 35 relates to a compound comprising a subunit represented by formula (I),

[0051] [Chemical formula]

[0052] wherein, each X is independently H, L1-A-Z, or L2-R, and each X is bonded to an OH group, each of L1 and L2 is independently an amine-terminated lysine, Each A independently comprises a cleavable linker comprising one or more carbonate and / or disulfide moieties, Each Z independently comprises a therapeutic agent comprising one or more reactive hydroxyl groups, Each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different, and relates to the method according to Example 24.

[0053] Example 36 relates to the method according to Example 34 or 35, wherein the compound is administered in combination with at least one other therapy or treatment, and the at least one other therapy or treatment is chemotherapy, radiotherapy, or immunotherapy.

[0054] Example 37 relates to the method according to any one of Examples 34 to 36, wherein the therapeutic agent comprises paclitaxel. Example 38 relates to the method according to any one of Examples 34 to 37, wherein the subject in need thereof suffers from cancer.

[0055] Example 39 relates to the method according to any one of Examples 34 to 38, wherein the therapeutic agent is released from the polymeric carbohydrate backbone in the presence of a reducing agent. Example 40 relates to the method according to any one of Examples 34 to 39, wherein the method does not suppress the antitumor activity of lymphocytes.

[0056] In Example 41, a method of reducing macrophage-mediated inflammation comprises administering to a subject in need thereof an effective dose of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent bound to the polymeric carbohydrate backbone via a cleavable linker and comprising one or more reactive hydroxyl groups.

[0057] Example 42, the compound comprises the subunit represented by formula (I),

[0058]

Chemical formula

[0059] In the formula, each X is independently H, L1-A-Z, or L2-R, and each X is attached to an OH group, each of L1 and L2 is independently an amine-terminated linker, each A independently contains a cleavable linker containing one or more carbonate and / or disulfide moieties, each Z independently contains a therapeutic agent containing one or more reactive hydroxyl groups, each R independently contains a mannose-binding C-type lectin receptor targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different, and relates to the method according to Example 41.

[0060] Example 43 relates to the method according to Example 41 or 42, wherein the compound is administered in combination with at least one other therapy or treatment. Example 44 relates to the method according to any one of Examples 41 to 43, wherein the therapeutic agent contains dexamethasone.

[0061] Example 45 relates to the method according to any one of Examples 41 to 44, wherein the subject in need thereof suffers from non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, other macrophage-related diseases, or a combination thereof.

[0062] Example 46 relates to the method according to any one of Examples 41 to 45, wherein the therapeutic agent is released from a polymeric carbohydrate backbone in the presence of a reducing agent. Example 47 relates to the method according to any one of Examples 41 to 46, wherein the method does not suppress the anti-tumor activity of lymphocytes.

[0063] In Example 48, a method of treating a disease comprises administering to a subject in need thereof an effective amount of a compound according to any one of Examples 1 to 14, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory disorders, non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, and other macrophage-related diseases.

[0064] Example 49 relates to the method according to Example 48, wherein the compound is administered in combination with at least one other therapy or treatment. Example 50 relates to the method according to Example 49, wherein at least one other treatment or therapy is chemotherapy, radiotherapy, or immunotherapy.

[0065] Example 51 relates to the method according to any one of Examples 48 to 50, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

[0066] Although multiple embodiments are disclosed, still other embodiments of the present disclosure will be apparent to those skilled in the art from the following detailed description, which illustrates and describes exemplary embodiments of the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0067]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

[0068] Various embodiments of the present disclosure are described in detail with reference to the drawings. References to various embodiments do not limit the scope of the present disclosure. The drawings presented herein are not limited to the various embodiments according to the present disclosure, but are presented for illustrative examples of the present disclosure.

Embodiments for Carrying Out the Invention

[0069] Embodiments of the present disclosure are not limited to specific compounds, compositions, and methods, which can vary and are understood by those skilled in the art. Furthermore, it is understood that all terms used herein are for the purpose of describing only specific embodiments and are not intended to limit in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a", "an", and "the" can include plural referents unless the context clearly dictates otherwise. Additionally, all units, prefixes, and symbols can be expressed in their SI acceptable forms.

[0070] The numerical ranges recited within this specification include the numbers defining the range and include each integer within the defined range. Throughout the present disclosure, various aspects of the present disclosure are presented in range format. The description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to specifically disclose all possible sub-ranges, fractions, and individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and decimals and fractions, such as 1.2, 3.8, 1 1 / 2, and 4 3 / 4. This applies regardless of the breadth of the range.

[0071] To make the present disclosure more readily understandable, certain terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present disclosure pertain. Many methods and materials similar to or equivalent to those described herein can be used in the practice of embodiments of the present disclosure without undue experimentation, and preferred materials and methods are described herein. In the description and claims of embodiments of the present disclosure, the following terms will be used according to the definitions set forth below.

[0072] As used herein, the term "about" refers to variations in numerical quantities that can occur through typical measuring techniques and equipment for any quantifiable variable, including but not limited to, for example, mass, volume, and time. Further, considering the handling procedures of solids and liquids used in the real world, there are likely to be certain unintentional errors and variations through differences in the manufacture, source, or purity of the components used to make a composition or carry out a method, etc. The term "about" also encompasses these variations. Whether or not modified by the term "about", the claims include equivalents to the quantity.

[0073] The methods and compositions of the present disclosure can include, consist essentially of, or consist of the components and ingredients of the present disclosure and other ingredients described herein. As used herein, "consist essentially of" means that a method, system, apparatus, and composition can include additional steps, components, or ingredients, but only if the additional steps, components, or ingredients do not substantially alter the basic and novel features of the claimed method, system, apparatus, and composition.

[0074] The terms "active", or "percent active", or "weight percent active", or "active concentration" are used interchangeably herein and refer to the concentration expressed as a percentage of these components, minus any inert components such as water or salts involved in the wash. Also, sometimes the percentage is indicated in parentheses, for example, "chemistry (10%)".

[0075] As used herein, an alkyl or alkyl group refers to a saturated hydrocarbon having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or cycloalkyl or alicyclic or carbocyclic groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups).

[0076] Unless otherwise specified, the term "alkyl" includes both "unsubstituted alkyl" and "substituted alkyl". As used herein, the term "substituted alkyl" refers to an alkyl group having a substituent that replaces one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.

[0077] In some embodiments, substituted alkyl can include a heterocyclic group. As used herein, the term "heterocyclic group" includes a closed-ring structure similar to a carbocyclic group in which one or more of the carbon atoms in the ring are elements other than carbon, such as nitrogen, sulfur, or oxygen. The heterocyclic group can be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxide, oxirane), thiirane (episulfide), dioxolane, azetidine, oxetane, thietane, dioxetane, dithietane, dithieto, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan.

[0078] As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of the organic compound. Exemplary substituents include, for example, those described below. Permissible substituents for a suitable organic compound can be one or more and can be the same or different. For the purposes of the present disclosure, a heteroatom such as nitrogen can have a hydrogen substituent of the organic compound described herein and / or any permissible substituent that satisfies the valence of the heteroatom. The present disclosure is not intended to be limited in any way by the permissible substituents of the organic compound. Also, the terms "substituted" or "substituted with" include the implicit condition that such substitution results in a stable compound, e.g., a compound that does not undergo spontaneous transformation such as rearrangement, cyclization, elimination, etc., in accordance with the permissible valences of the substituted atom and the substituent. Also, in certain embodiments, unless expressly indicated to the contrary, each individual substituent is further optionally substitutable (i.e., can be further substituted or unsubstituted).

[0079] As described herein, the compounds of the present invention can contain "optionally substituted" moieties. In general, the term "substituted" means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, whether or not the term "optionally" is prefixed. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when two or more positions in any given structure are substituted with two or more substituents selected from a particular group, the substituents can be the same or different at all positions. Combinations of substituents contemplated by the present invention preferably result in the formation of stable or chemically feasible compounds. In certain embodiments, unless expressly indicated otherwise, each individual substituent is also further optionally substitutable (i.e., can be further substituted or unsubstituted).

[0080] In the definitions of various terms, "A1", "A2", "A3", "A4", "X1", "X2", "Y1", "Y2", etc. are used in this specification as general symbols to represent various specific substituents. These symbols are not limited to those disclosed in this specification and can be any substituent, and if they are defined as a certain specific substituent in one example, they can be defined as some other substituents in another example.

[0081] "R1", "R2", "R3", "R n ", where n is an integer and when used in this specification, can independently have one or more of the groups listed above. For example, when R1 is a linear alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the group selected, the first group can be incorporated within the second group or, alternatively, the first group can be pendant (i.e., attachable) to the second group. For example, in the phrase "an alkyl group containing an amino group", the amino group can be incorporated within the alkyl group skeleton. Alternatively, the amino group can be attached to the alkyl group skeleton. The nature of the group(s) selected will determine whether the first group is embedded within or attached to the second group.

[0082] Certain materials, compounds, compositions, and components disclosed herein are commercially available or can be readily synthesized using techniques generally known to those of skill in the art. For example, starting materials and reagents used in the preparation of the disclosed compounds and compositions are available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.), or can be prepared by methods known to those of skill in the art according to procedures described in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0083] Disclosed are the components used to prepare the compositions of the present disclosure, as well as the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific mention of each of the various individual and collective combinations and permutations of these compounds cannot be explicitly disclosed, but each is understood to be specifically contemplated and described herein. For example, if a particular compound is disclosed and considered, and numerous modifications that can be made to form several molecules containing the compound are considered, then every combination and permutation of the compound and possible modifications is specifically contemplated, unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C, and classes of molecules D, E, and F are disclosed, and A-D, an example of a combination molecule, is disclosed, then each of A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is considered to be disclosed, even if each is not individually described. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are considered to be disclosed. This concept applies to all aspects of this application, including without limitation the steps in methods of making and using the compositions of the present invention. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the method of the present invention.

[0084] As used herein, the terms "pharmaceutically acceptable carrier" or "carrier" refer to sterile aqueous or non-aqueous solutions, colloids, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin that delay absorption. Injectable depot forms are prepared by forming a microcapsule matrix of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The drug release rate can be controlled depending on the drug-to-polymer ratio and the nature of the specific polymer used. Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media immediately prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0085] The term "polarization" is used herein to indicate phenotypic and functional characteristics of macrophages. The phenotype can be defined through surface markers expressed by macrophages. Functionality can be defined, for example, based on the nature and amount of chemokines and / or cytokines expressed, particularly secreted, by macrophages. In fact, macrophages present different phenotypic and functional characteristics of either pro-inflammatory (M1 type) macrophages or anti-inflammatory (M2 type) macrophages depending on their state. M2 macrophages can be characterized by the expression of surface markers such as CD206, CD11b, PD-L1, and CD200R, followed by the secretion of cytokines such as CCL17. M1 macrophages can be defined by the expression of surface markers such as CD86 and CCR7, as well as the secretion of cytokines such as IL-6, TNF-α, and IL12p40. In the context of the present disclosure, the term "re-polarize" is used herein to refer to the induction of a change in the phenotype of an M1 macrophage population to M1 macrophages.

[0086] As used herein, the term "cancer" refers to cells having the ability of autonomous growth. Examples of such cells include cells having an abnormal condition or condition characterized by the growth of rapidly proliferating cells. This term means to include cancerous growth, such as tumors, carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasion. It also includes malignant tumors of various organ systems such as the respiratory system, cardiovascular system, renal system, genital system, blood system, nervous system, hepatic system, gastrointestinal system, and endocrine system, as well as adenocarcinomas including most colorectal cancers, renal cell cancers, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers. "Spontaneously occurring" cancer includes any cancer not experimentally induced by transplantation of cancer cells into a subject, for example, cancers spontaneously induced, cancers caused by exposure of a patient to carcinogen(s), cancers resulting from insertion of a transgenic cancer gene or knockout of a tumor suppressor gene, and cancers caused by infection, such as viral infection. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues. In some embodiments, the method can be used to treat a subject having an epithelial cancer, such as a solid tumor of epithelial origin, such as lung cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer, or colorectal cancer.

[0087] As used herein, the term "subject" refers to the target of administration, e.g., an animal. Thus, the subject of the methods disclosed herein can be a vertebrate such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses regardless of sex, are intended to be included. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed as having a need for treatment of one or more cancer disorders prior to the administration step.

[0088] As used herein, the term "synergistic" means that the effects achieved by the methods and combinations of the present invention are greater than the sum of the effects resulting from using the compound, composition, treatment, and / or pharmaceutically acceptable salts thereof separately. Advantageously, such synergistic effects provide greater efficacy at the same dose and / or prevent or delay the accumulation of multidrug resistance.

[0089] As used herein, the term "treatment" refers to the medical management of a patient intended to cure, ameliorate, stabilize, or prevent a disease, pathologic condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward the improvement of a disease, pathologic condition, or disorder, and causal treatment, i.e., treatment directed toward the elimination of the cause of the related disease, pathologic condition, or disorder. Additionally, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, pathologic condition, or disorder; prophylactic treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the development of a related disease, pathologic condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment modality directed toward the improvement of a related disease, pathologic condition, or disorder. In various aspects, this term encompasses any treatment of a subject, including a mammal (e.g., a human), to (i) prevent the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (ii) inhibit a disease, i.e., prevent its onset, or (iii) alleviate a disease, i.e., cause regression of the disease.

[0090] As used herein, the term "prevent" or "preventing" refers to impeding, avoiding, excluding, precluding, stopping, or hindering something from happening, particularly by prior action. It is understood that the use of the other two terms, "reduce" and "inhibit", is also expressly disclosed when "reduce", "inhibit", or "prevent" is used herein, unless specifically indicated otherwise.

[0091] As used herein, the term "diagnosed" means having been found, by a person skilled in the art, such as a physician, to have undergone a physical examination and to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with cancer" means having been found, by a person skilled in the art, such as a physician, to have undergone a physical examination and to have a condition that can be diagnosed or treated by a compound or composition capable of reducing the size of a tumor or slowing the growth rate of a tumor. A subject having cancer, a tumor, or at least one cancerous or tumor cell can be identified using methods known in the art. For example, the anatomical location, total size, and / or cell composition of cancer cells or tumors can be determined using contrast-enhanced MRI or CT. Additional methods for identifying cancer cells can include, but are not limited to, ultrasound, bone scans, surgical biopsies, and biological markers (e.g., serum protein levels and gene expression profiles). An imaging solution containing the cell-sensitizing composition of the present invention can be used, for example, in combination with MRI or CT to identify cancer cells.

[0092] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including intravenous administration, intraarterial administration, administration to a particular organ by infiltration, intramuscular administration, intratumoral administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be therapeutically administered, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation can be prophylactically administered, i.e., administered to prevent a disease or condition.

[0093] As used herein, the terms "effective amount" and "effective quantity" refer to an amount sufficient to achieve a desired result or to have an effect on an undesirable condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to have an effect on an undesirable symptom, but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start the dosage of a compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dosage can be divided into multiple dosages for administration. Consequently, a single-dose composition can contain such an amount or an approximation thereof to constitute a daily dosage. The dosage can be adjusted by the individual physician in any case of contraindication. The dosage can vary and can be administered in one or more doses per day for one or more days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical products. In further various aspects, the preparation can be administered in a "preventively effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0094] An effective dosage can initially be estimated from in vitro assays. For example, an initial dosage for use in animals can be the IC of a specific compound measured in an in vitro assay 50It can be formulated to achieve the circulating blood or serum concentration of the active compound as described above. Taking into account the bioavailability of a particular active agent, calculating the dosage to achieve such a circulating blood or serum concentration is well within the ability of one of ordinary skill in the art. For guidance, the reader is referred to Fingl & Woodbury, “General Principles,” In: Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46 (latest edition), Pergamagon Press (which is hereby incorporated by reference in its entirety), and the references cited therein.

[0095] The term "anticancer composition" can include compositions that exhibit an anti - malignant tumor effect, a chemotherapy effect, an antiviral effect, an anti - mitotic effect, an antitumor action, an anti - angiogenesis effect, an anti - metastasis effect, and / or an immunotherapy effect. For example, compositions that directly on tumor cells, e.g., by a cytostatic or cytocidal effect, and indirectly via mechanisms such as biological response modification, to prevent the generation, maturation, or spread of tumor cells. There are numerous antiproliferative agents available for commercial use, clinical evaluation, and pre - clinical development, and these can be included in the present application by combination drug chemotherapy. For the sake of convenience of discussion, antiproliferative agents are classified into the following classes, subtypes, and species: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracycline / DNA intercalators, anticancer antibiotics or antibiotic - type agents, antimetabolites, antimetastatic compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase - 2 inhibitors, DHA derivatives, DNA topoisomerase, endostatin, epipodophyllotoxin, genistein, hormonal anticancer agents, hydrophilic bile acids (URSO), immunomodulators or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, various antineoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT radiosensitizers / chemosensitizers / protectors, retinoids, selective inhibitors of endothelial cell proliferation and migration, endothelin growth inhibitors, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids. The major categories to which some antiproliferative agents belong include antimetabolites, alkylating agents, antibiotic - type agents, hormonal anticancer agents, immunological agents, interferon - type agents, and various antineoplastic drugs. Some antiproliferative agents function via multiple or unknown mechanisms and can thus be classified into more than one category.

[0096] The terms "weight percent", "wt.%", "wt-%", "percent by weight", "% by weight", and variations thereof, as used herein, refer to the concentration of that substance, obtained by dividing the weight of that substance by the total weight of the composition and multiplying by 100.

[0097] The present disclosure describes compounds, compositions, methods having utility for altering or shifting the phenotype of activated macrophages from an immunosuppressive phenotype to a pro-inflammatory phenotype (referred to herein as "re-polarization"), and methods having utility for increasing the target specificity of a compound or composition in a subject. The ability to alter or shift the phenotype of activated macrophages from immunosuppressive to pro-inflammatory constitutes a therapeutic modality for cancer, various infectious diseases, and other medical conditions. The present disclosure further describes drug delivery vehicles and methods of use that enable targeted delivery of a therapeutic agent to TAMs, including the intention to re-polarize TAMs. TAM-targeted delivery provides a higher mass dose of a therapeutic agent to TAMs, increasing the phenotypic alteration effect while limiting potential toxic exposure to off-target cells and tissues. Through the use of the disclosed compounds, compositions, and methods, M2-like (immunosuppressive) activated macrophages can be induced to switch their phenotype to an M1-like (pro-inflammatory) activated phenotype. In a further embodiment, the present disclosure provides a method for reducing macrophage-mediated inflammation in a subject.

[0098] Compounds and Compositions In certain embodiments, the compounds disclosed herein deliver one or more active therapeutic agents using a carrier construct that includes a polymeric (e.g., carbohydrate) backbone conjugated to a mannose-binding C-lectin type receptor targeting moiety (e.g., mannose). Examples of such polymeric carbohydrate constructs include mannosylated aminodextran (MAD), which includes a dextran backbone with mannose molecules conjugated to the glucose residues of the backbone and active pharmaceutical ingredients conjugated to the glucose residues of the backbone. Tylomannoside is a specific example of MAD. Tylomannoside derivatives that are tylomannoside without DTPA conjugated thereto are further examples of MAD.

[0099] MAD is a synthetic molecule that is intentionally designed to be a high-affinity ligand for mannose-binding C-lectin type receptors such as, for example, CD206. MAD is described in U.S. Patent No. 6,409,990, which is hereby incorporated by reference in its entirety. Thus, the backbone includes a plurality of glucose moieties (i.e., residues or subunits) that are originally linked by α-1,6 glycosidic bonds. Other bonds, such as α-1,4 and / or α-1,3 bonds, may also be present.

[0100] Some embodiments may include a backbone other than a dextran backbone. Some embodiments may have a monosaccharide-based backbone that does not include dextran. The backbone of the carbohydrate-based carrier molecules described herein may include a glycan other than dextran, where the glycan includes a plurality of monosaccharide residues (i.e., sugar residues or modified sugar residues). In certain embodiments, the glycan backbone has a sufficient number of monosaccharide residues to provide an MW of from about 1 to about 50 kDa, as well as optional groups such as one or more amino acids, polypeptides, and / or lipids. Given the disclosure contained herein, as would be understood by one of ordinary skill in the art, when referring to a "dextran" backbone, other monosaccharide residues may be considered to be substituted in the compounds described herein. Additional description of the carbohydrate-based carrier molecules used to target CD206 is described in PCT Application No. US / 2017 / 055211, which is hereby incorporated by reference in its entirety.

[0101] In some embodiments, not all of the backbone moieties are replaced. In some embodiments, one or more amine-terminated lysines are attached to the backbone. In further embodiments, a mannose-binding C-type lectin targeting moiety is attached to one or more amine-terminated lysines. In certain embodiments, the mannose-binding C-type lectin targeting moiety is attached via the amine-terminated lysine to about 15% to about 70%, about 17% to about 65%, or about 20% to about 60% of the glucose residues. In further embodiments, the mannose-binding C-type lectin targeting moiety is attached via the amine-terminated lysine to up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to about 100% of the glucose residues. In certain aspects, the percentage may vary depending on the size of the dextran backbone. In still further embodiments, one or more therapeutic agents are attached to the glucose residues via a cleavable linker that includes one or more carbonate and / or disulfide moieties. The cleavable linker is further conjugated to the amine-terminated lysine, which is linked to the backbone described in more detail herein. In certain embodiments, the therapeutic agent is attached via the amine-terminated lysine and the cleavable linker to about 1% to about 30%, about 2% to about 25%, or about 5% to about 20% of the glucose residues as described herein.

[0102] The size of the polymeric carbohydrate construct can be varied by changing the size of the initial polymeric carbohydrate backbone when the construct is assembled. In some embodiments, the polymeric carbohydrate moiety is from about 50 to 100 kDa. The polymeric carbohydrate moiety can be at least about 50 kDa, at least about 60 kDa, at least about 70 kDa, at least about 80 kDa, or at least about 90 kDa. The polymeric carbohydrate moiety can be less than about 100 kDa, less than about 90 kDa, less than about 80 kDa, less than about 70 kDa, or less than about 60 kDa. Alternatively, in some embodiments, the polymeric carbohydrate backbone has an MW of from about 1 kDa to about 50 kDa, and in other embodiments, the polymeric carbohydrate backbone has an MW of from about 5 kDa to about 25 kDa. In still other embodiments, the polymeric carbohydrate backbone has an MW of from about 8 kDa to about 15 kDa, such as about 10 kDa. In other embodiments, the polymeric carbohydrate backbone has an MW of from about 1 kDa to about 5 kDa, such as about 3 kDa. Advantageously, the disclosed constructs of smaller size enable higher tumor penetration and higher localization to tumor-associated macrophages (TAMs) than is possible with other larger constructs.

[0103] Since all tumors contain TAMs, one clinically important class of activated macrophages consists of TAMs. TAMs are the most numerous immune cells infiltrating tumors and can constitute over 5% to 30% of all cells in a tumor. Tumors have both M1-like and M2-like TAMs, however, in established tumors, M2-like TAMs have a dominant influence on the immune state of the tumor microenvironment. CD206 is generally highly expressed in the majority of TAMs. Under the influence of M2-like TAMs, the tumor immune microenvironment becomes tumor-promoting and immunosuppressive, suppressing the antitumor activity of other immune cells such as lymphocytes. The tumor-promoting and immunosuppressive activities of M2-like TAMs reduce the effectiveness of anticancer therapies and, perhaps most notably, anticancer immunotherapies. For these reasons, TAMs are well recognized as therapeutic targets for cancer. TAM-targeted cancer treatment strategies include (1) killing or removing TAMs, (2) blocking the recruitment of TAMs to tumors, or (3) causing TAMs to switch their phenotype from M2-like to M1-like. This third strategy is sometimes referred to as TAM repolarization or TAM re-education. M1-like TAMs attack tumor cells and stimulate other types of immune cells such as lymphocytes to attack tumor cells.

[0104] Mannosylated amine dextran (MAD, see U.S. Patent Nos. 6,409,990 and 10,806,803) is a synthetic molecule intentionally designed to have a high affinity ligand for CD206. The size of MAD can be varied by changing the size of the initial dextran when the MAD construct is assembled. In some embodiments, described within this disclosure are polymeric carbohydrate constructs or MAD constructs synthesized on dextran backbones of various molecular weights that carry any therapeutic agent on a degradable linker containing carbonate and / or disulfide bonds. In some examples, dexamethasone is used as a potent anti-inflammatory drug through its activity as a corticosteroid hormone receptor agonist. In a further example, paclitaxel changes the inflammatory phenotype of macrophages from an immunosuppressive M2-like phenotype towards a more pro-inflammatory M1-like phenotype, improving the effectiveness of other anti-cancer therapies. Under neutral pH and oxidative conditions, the construct retains a payload long enough to be carried within endosomes. Upon entering an acidified endosome with potentially reducing conditions and / or appropriate enzymatic activity, the drug payload is released. These constructs have low toxicity towards human macrophages but have a remarkable ability to alter their phenotype. In the case of the dexamethasone-bearing MAD construct (MAD-DEX), MAD-DEX induced macrophages to adopt a more immunosuppressive M2-like phenotype. In some embodiments, a therapeutic agent containing paclitaxel, MAD-PAC, induced macrophages to adopt a highly pro-inflammatory M1-like phenotype. In some embodiments, the adoption of a highly pro-inflammatory M1-like phenotype is not fully replicated by unbound (free) paclitaxel.

[0105] According to certain embodiments and as further described throughout the present disclosure, one or more mannose-binding C-type lectin receptor targeting moieties and one or more therapeutic agents are each independently conjugated to a polymeric carbohydrate-based backbone by a linker. As described in more detail below, one or more additional moieties may be present between the linker and the mannose-binding C-type lectin receptor targeting moiety or therapeutic agent. In further embodiments, the linker is provided as an independent linker conjugated to the polymeric carbohydrate-based backbone rather than conjugated to the mannose-binding C-type lectin receptor targeting moiety or therapeutic agent. The linker may be conjugated to about 50% to about 100% of the backbone moiety, or about 70% to about 90% of the backbone moiety. The linkers may be the same or different. In some embodiments, the linker is an amine-terminated linker. In some embodiments, the linker may comprise the formula -(CH2) p S(CH2) q- NH-, where p and q are integers from 0 to 5. In further embodiments, the linker comprises the formula -(CH2)3S(CH2)2NH-. In embodiments where the linker is not conjugated to the mannose-binding C-type lectin receptor targeting moiety or therapeutic agent, the linker may comprise the formula -(CH2) p S(CH2) q- NH2, where p and q are integers from 0 to 5.

[0106] In some embodiments, the linker may be a chain of about 1 to about 20 constituent atoms selected from carbon, oxygen, sulfur, nitrogen, and phosphorus. The linker may be linear or branched. The linker may also be substituted with halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, C 1-4 alkyl groups such as C 1-4 alkenyl groups such as C 1-4It may be substituted with one or more substituents including, but not limited to, alkynyl groups such as alkynyl, hydroxy groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups, azidoalkyl groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy groups or heteroaralkoxy groups, HO-(C=O)- groups, heterocyclic groups, cycloalkyl groups, amino groups, alkyl- and dialkylamino groups, carbamoyl groups, alkylcarbonyl groups, alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylaminocarbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups, -NH-NH2; =N-H; =N-alkyl; -SH; -S-alkyl; -NH-C(O)-; -NH-C(=N)-, etc. As will be apparent to those skilled in the art, other suitable linkers are possible.

[0107] According to certain embodiments, the polymeric carbohydrate constructs disclosed herein contain at least one targeting moiety. In some embodiments, the targeting moiety can be a mannose-binding C-type lectin receptor targeting moiety. In further embodiments, the polymeric carbohydrate constructs disclosed herein contain at least one targeting ligand. CD206 is a C-type lectin receptor expressed on macrophages, dendritic cells, and mesangial cells. CD206 binds to molecules presenting multiple terminal mannose moieties. Without being limited to any particular mechanism or theory, it is contemplated that when CD206 binds to a ligand, the receptor / ligand complex is internalized into endosomes by receptor-mediated endocytosis, and the endosomes are naturally acidified to a pH of about 4-5. At this low pH, CD206 releases its ligand and recycles to the cell surface. In some aspects, including a mannose-binding C-type lectin receptor targeting moiety within the polymeric carbohydrate construct provides many advantages for delivering therapeutic agents to targets such as macrophages.

[0108] According to some embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose, high mannose glycans or mannooligosaccharides, fucose, or N-acetylglucosamine, peptides, or galactose. In further embodiments, the mannose-binding C-type lectin receptor targeting moiety is attached to the amine-terminated leish via a mannosyl coupling reagent described in U.S. Patent No. 6,409,990, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, high mannose glycans or mannooligosaccharides, fucose, N-acetylglucosamine, peptides, galactose, or combinations thereof. In further embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose. In other embodiments, at least one targeting ligand can be sialic acid.

[0109] In some embodiments, one or more therapeutic agents are attached to a polymeric carbohydrate backbone via a cleavable linker. In some embodiments, the cleavable linker comprises one or more carbonate and / or disulfide moieties. The cleavable linker releases the therapeutic agent payload when a free thiol group reductively cleaves the disulfide moiety of the linker. In some embodiments, when the mannosylated polymeric carbohydrate construct binds to CD206, it is internalized into an endosome, and the endosome becomes increasingly acidic over time, thereby releasing the therapeutic agent payload intracellularly. In one aspect, the cleavable linker has the following formula below before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone.

[0110]

Chemical formula

[0111] wherein x is an integer from 1 to 5 and y is an integer from 1 to 5. In a further aspect, the cleavable linker has the following formula below before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone.

[0112] [Chem.]

[0113] In some embodiments, the cleavable linker is not a hydrazone linker. In some embodiments, one or more therapeutic agents are attached to the polymeric carbohydrate construct. In some aspects, one or more therapeutic agents are attached to the polymeric carbohydrate construct via a cleavable linker disclosed herein. In some aspects, the cleavable linker is further conjugated to an amine-terminated lysine on the polymeric carbohydrate backbone. In certain embodiments, the therapeutic agent, when attached to the MAD backbone disclosed herein, is capable of reprogramming M2-like macrophages to M1-like macrophages. In a further embodiment, the therapeutic agent is capable of inducing T cell activation. In some embodiments, the therapeutic agent includes a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof. According to a further embodiment, the therapeutic agent is a cytotoxic agent. In yet a further embodiment, the therapeutic agent is an anti-cancer agent.

[0114] In certain embodiments, the therapeutic agent comprises one or more reactive hydroxyl groups. In further embodiments, the therapeutic agent can be dexamethasone or paclitaxel. Dexamethasone is a potent anti-inflammatory drug through its activity as a corticosteroid hormone receptor agonist. Paclitaxel can alter the inflammatory phenotype of macrophages from an immunosuppressive M2-like phenotype towards a more pro-inflammatory M1-like phenotype and improve the efficacy of other anti-cancer therapies. Under neutral pH and oxidative conditions, the construct retains a payload long enough to be carried within endosomes. Upon entering acidic endosomes with potentially reducing conditions and / or appropriate enzymatic activity, the drug payload is released. These dexamethasone- and paclitaxel-loaded MADs have low toxicity towards human macrophages but have a remarkable ability to alter their phenotypes. In the case of the dexamethasone-loaded MAD construct (MAD-DEX), MAD-DEX induced macrophages to adopt a more immunosuppressive M2-like phenotype. The paclitaxel-loaded MAD construct (MAD-PAC) induced macrophages to adopt a highly pro-inflammatory M1-like phenotype, which could not be fully replicated by unbound (free) paclitaxel.

[0115] According to some embodiments, there is provided a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent comprising one or more reactive hydroxyl groups and attached to the polymeric carbohydrate backbone via a degradable linker as discussed herein. In further embodiments, the compound comprises a subunit represented by the following formula (I),

[0116] [Chemical formula]

[0117] wherein, each X is independently H, L1-A-Z, or L2-R, and each X is attached to an OH group, each of L1 and L2 is independently an amine-terminated leash, Each A independently comprises a cleavable linker comprising one or more carbonate and / or disulfide moieties, each Z independently comprises a therapeutic agent comprising one or more reactive hydroxyl groups, each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different.

[0118] According to some embodiments, at least one X is L1-A-Z, at least one X is L2-R, and R comprises a mannose-binding C-type lectin receptor targeting moiety as contemplated herein. In some aspects, at least one L1 comprises -(CH2) p S(CH2) q- NH-, wherein p and q are integers from 0 to 5. In some further aspects, at least one L2 comprises -(CH2) p S(CH2) q- NH-, wherein p and q are integers from 0 to 5. In some embodiments, the therapeutic agent is conjugated to the cleavable linker before being conjugated to the polymeric carbohydrate backbone.

[0119] In some aspects, n is an integer greater than zero. In other aspects, n is an integer greater than 1. In further aspects, n can be an integer from 1 to about 50, about 5 to about 40, or about 5 to about 30. As will be appreciated by those skilled in the art, the order of each X attached to the polymeric carbohydrate backbone can be the same or different for each subunit of n, so each subunit of n can be the same or different.

[0120] In some embodiments, the cleavable linker has the following formula below before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone,

[0121]

Chemical formula

[0122] In the formula, x is an integer from 1 to 5, and y is an integer from 1 to 5. In a further embodiment, the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone.

[0123]

Chemical formula

[0124] In an embodiment, A has the following formula,

[0125]

Chemical formula

[0126] In the formula, x is an integer from 1 to 5, and y is an integer from 1 to 5. In a further embodiment, A has the following formula.

[0127]

Chemical formula

[0128] In some embodiments, a compound is provided that includes a therapeutic agent containing a reactive hydroxyl group, a cleavable linker containing one or more carbonate and / or disulfide moieties, and a second compound containing a primary amine, wherein the cleavable linker is bonded to the reactive hydroxyl group of the therapeutic agent and to the primary amine of the second compound. In one aspect, the compound has the formula (II):

[0129]

Chemical formula

[0130] and includes a compound according to, wherein Z is a therapeutic agent containing a reactive hydroxyl group, Y is a second compound containing a primary amine, x is an integer from 1 to 5, y is an integer from 1 to 5.

[0131] In certain embodiments, Z is a therapeutic agent contemplated herein. In further embodiments, Y is a second compound containing a primary amine group. The compound according to formula (II) further comprises a carbonate moiety and a disulfide moiety. In some embodiments, the hydrogen groups within formula (II) are linear or branched alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.

[0132] In a further embodiment, the therapeutic agents disclosed herein can be conjugated to the polymeric carbohydrate constructs disclosed herein. In some embodiments, the therapeutic agent is modified with a cleavable linker that includes a carbonate and / or disulfide linker prior to being conjugated to the polymeric carbohydrate backbone via one of the amine-terminated lysines. In certain embodiments where the therapeutic agent modified with the carbonate / disulfide linker is conjugated to the MAD construct, the polymeric carbohydrate backbone already includes one or more lysines attached to the backbone. Without being limited to any particular theory or mechanism, the drug payload is released when cleaved by a reducing agent. Examples of reducing agents can include L-glutathione (GSH), or other free thiol groups.

[0133] According to certain embodiments, the disclosed compounds can comprise a pharmaceutically acceptable carrier and a compound disclosed herein, or a pharmaceutically acceptable salt of the compound. The disclosed compounds, or pharmaceutically acceptable salts thereof, can be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds. In certain embodiments, the compound is administered in a therapeutically effective amount. In a further embodiment, the compound is administered in a prophylactically effective amount. In some embodiments, the pharmaceutical composition is prepared in a form suitable for intravenous, intraperitoneal, or intramuscular injection.

[0134] The molecular weights referred to herein, as well as the number and degree of conjugation of receptor substrates, lysines, and therapeutic moieties attached to the polymeric carbohydrate backbone, refer to the average amount of a given amount of carrier molecules since the synthetic techniques give rise to some variability.

[0135] Method In living humans and animals, activated macrophages with a mixed activation phenotype characterized by both M1-like and M2-like phenotypic states have been observed. Examples of stimuli that can induce an M1-like phenotype in macrophages include tumor necrosis factor (TNF), interferon gamma (INFγ), and toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS). Examples of stimuli that can induce an M2-like phenotype in macrophages include interleukin 4 (IL4), interleukin 13 (IL13), and tumor growth factor beta (TGFβ). There are also many other hormones, cytokines, chemokines, and environmental factors that can affect the macrophage phenotype.

[0136] Table 1 shows examples of cell surface markers and secreted proteins whose expression levels are often altered in M1-like and M2-like activated macrophages. In addition, two immune checkpoint receptors, PD-1 and SIRPα, can be expressed on the surface of macrophages. When these immune checkpoint receptors bind to their respective ligands, PD-L1 and CD47, signals are generated that suppress the phagocytic activity of macrophages. M1-like macrophages are expected to attack and phagocytose perceived pathogens or tumor cells. However, if M1-like macrophages express PD-1 and / or SIRPα bound to their ligands, phagocytosis is suppressed.

[0137] Table 1: Examples of proteins that increase expression in activated macrophages with M1-like and M2-like phenotypes

[0138]

Table 1

[0139] Under the influence of M2-like TAMs, the tumor immune microenvironment becomes tumor-promoting and immunosuppressive, suppressing the antitumor activity of other immune cells such as lymphocytes. In some aspects, the tumor-promoting and immunosuppressive activities of M2-like TAMs reduce the effectiveness of anticancer therapies and, perhaps most notably, anticancer immunotherapies. For these reasons, TAMs are an important therapeutic target for cancer. TAM-targeted cancer treatment strategies include (1) killing or removing TAMs, (2) blocking the recruitment of TAMs to tumors, or (3) causing TAMs to switch their phenotype from M2-like to M1-like. This third strategy is sometimes referred to as TAM repolarization or TAM re-education. M1-like TAMs attack tumor cells and stimulate other types of immune cells such as lymphocytes to attack tumor cells.

[0140] In certain embodiments, methods are disclosed for repolarizing TAMs from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype. Further embodiments provide methods for reducing macrophage-mediated inflammation. In certain aspects, a polymeric carbohydrate construct carrying a therapeutic agent contains carbonate and / or disulfide bonds and is attached via a cleavable linker linked to the polymeric carbohydrate backbone using amine-terminated lysine. In certain aspects, at neutral pH, the construct retains a payload (i.e., a therapeutic agent) of sufficient length to be delivered to mannose-binding C-type lectin receptors (e.g., CD206, etc.). When the mannose-binding C-type lectin receptor binds to the construct, the receptor / ligand complex is internalized into endosomes by receptor-mediated endocytosis, and the endosomes are naturally acidified to a pH of about 4 - 5. Upon entering the acidified endosome, the drug payload is released. In some aspects, the polymeric carbohydrate constructs have low toxicity to human macrophages but have a remarkable ability to change their phenotype to be more pro-inflammatory and antitumor.

[0141] In certain embodiments, the method comprises administering to a subject in need thereof an effective dose of a compound disclosed herein. In further embodiments, the method comprises administering a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent attached to the polymeric carbohydrate backbone via a carbonate / disulfide bond linker. In still further embodiments, the compound comprises a subunit provided by formula (I) disclosed herein.

[0142] In certain aspects, the compound is administered in a therapeutically effective amount. In further aspects, the compound is administered in a prophylactically effective amount. In still further aspects, the method further comprises administering the compound intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intravitreally, by intratumoral injection, or transdermally, or delivering directly to a tumor organ by invasive techniques.

[0143] In still further aspects, the method further comprises administering the composition in combination with at least one other treatment or therapy. In some aspects, the other treatment or therapy comprises an anti-inflammatory agent. In further aspects, the other treatment or therapy comprises co-administering an anti-cancer agent. In further aspects, the other treatment or therapy is chemotherapy. In certain aspects, the compound is administered alone, or in combination with other chemotherapeutic agents, or in combination with radiotherapy or hyperthermia or physical therapy or dietary therapy.

[0144] According to a further embodiment, at least one other treatment or therapy is an immunotherapy such as the administration of an immunomodulatory agent. According to certain embodiments, at least one other treatment or therapy is an anti-CTLA4 immunotherapy. In certain embodiments, the immunomodulatory agent is an immunostimulant. In some embodiments, the immunomodulatory agent is a glucocorticoid, hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a cytostatic agent, an alkylating agent, nitrogen mustard (cyclophosphamide), a nitrosourea, a platinum compound, an antimetabolite, a purine analogue, azathioprine, mercaptopurine, mycophenolic acid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a folic acid analogue, methotrexate, a cytotoxic antibiotic, dactinomycin, an anthracycline, mitomycin C, bleomycin, mitramycin, an antibody or a fusion thereof, antithymocyte globulin, antilymphocyte globulin, an anti-IL-2 receptor antibody, an anti-IL6 antibody, an anti-CD3 antibody, OKT3 (muromonab), otrexizumab, teprotumumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanilimumab, an anti-CD11a antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, ofatumumab, ocrelizumab, obinutuzumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD40L antibody, rupatuzumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, galiximab, an anti-CD147 antibody, gavilimomab, a B lymphocyte stimulator (BLyS) inhibitory antibody, belimumab, a CTLA4-Ig fusion protein, abatacept, belatacept, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-α4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab,Inolimomab, an anti-CD5 antibody, Zolimomab, an anti-CD2 antibody, Cipilizumab, Neralimomab, Pharalimomab, Atrizumab, Atorolimumab, Cedelizumab, Dorlimomab Aritox, Dorlixizumab, Fontolizumab, Gantenerumab, Gomiliximab, Lebrilizumab, Maslimomab, Morolimumab, Pexelizumab, Reslizumab, Robelizumab, Talizumab, Telimomab Aritox, Vapaliximab, Vepalimomab, Aflibercept, Alefacept, Rilonacept, Immunophilin modulators, Rapamycin, a calcineurin inhibitor, Tacrolimus, Cyclosporine, Pimecrolimus, Abetimus, Gusperimus, Ridafolimus, Everolimus, Temsirolimus, Zotarolimus, TNF inhibitors, Infliximab, Adalimumab, Certolizumab Pegol, Golimumab, Etanercept, Thalidomide, Lenalidomide, Pentoxifylline, Bupropion, Curcumin, Catechins, IL-1 receptor antagonists, Anakinra, anti-IL-5 antibodies, Mepolizumab, IgE inhibitors, Omalizumab, Talizumab, IL12 inhibitors, IL23 inhibitors, Ustekinumab, opioids, IMPDH inhibitors, Mycophenolic acid, Milosins, Fingolimod, NF-κB inhibitors, Raloxifene, Drotrecogin Alpha, Denosumab, NF-κB signaling cascade inhibitors, Disulfiram, Olmesartan, Dithiocarbamate, Proteasome inhibitors, Bortezomib, MG132, Pro1, NPI-0052, Curcumin, Genistein, Resveratrol, Parthenolide, Thalidomide, Lenalidomide, Flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), Arsenic trioxide, Dehydroxymethyl epoxyquinomycin (DHMEQ), 13C (Indole-3-carbinol) / DIM (Di-indolylmethane) (I3C / DIM), Bay11-7082, Luteolin,It is a cell-permeable peptide SN-50, overexpression of IκBα-super repressor, NFκB decoy oligodeoxynucleotide (ODN), or any derivative or analog thereof.

[0145] In an exemplary embodiment, the combined administration of the compound with at least one treatment or therapy is synergistically effective compared to either administration alone. According to certain embodiments, the administration of the compounds disclosed herein in combination with another therapy or treatment is associated with reduced toxicity compared to the administration of the other therapy or treatment alone. In further embodiments, the co-administration of the compounds of the present disclosure with other treatments or therapies results in a synergistic effect. In still further embodiments, the co-administration of the compounds of the present disclosure provides a lower effective dose of the other therapy or treatment.

[0146] The methods provided herein can be carried out in an adjuvant setting. In some embodiments, the method is carried out in a neoadjuvant setting, i.e., the method can be performed prior to primary / definitive treatment. In some embodiments, the method is used to treat an individual who has been previously treated. Any of the treatment methods provided herein can be used to treat an individual who has not been previously treated. In some embodiments, the method is used as a first-line treatment. In some embodiments, the method is used as a second-line treatment.

[0147] In further embodiments, a method of treating a disease is provided. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a compound disclosed herein. In some aspects, the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory disorders, non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, and other macrophage-related diseases.

[0148] According to another aspect, the subject is diagnosed with melanoma, breast cancer, lung cancer, pancreatic cancer, kidney cancer, ovarian cancer, prostate cancer or cervical cancer, glioblastoma, or colorectal cancer, spinal cord tumor, head and neck cancer, thymic cancer, mesothelioma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, bladder cancer, testicular cancer, germ cell cancer, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, or any combination thereof.

[0149] In certain embodiments, the method further comprises administering the composition as a bolus and / or at regular intervals. In certain embodiments, the disclosed method further comprises administering the composition intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally.

[0150] According to certain further embodiments, the method further comprises diagnosing the subject with cancer. In a further aspect, the subject is diagnosed with cancer prior to administration of the composition. In still further aspects, the method further comprises evaluating the effectiveness of the composition. In even further aspects, evaluating the effectiveness of the composition comprises measuring the tumor size before administering the composition and measuring the tumor size after administering the compound. In even further aspects, evaluating the effectiveness of the composition is performed at regular intervals. According to certain embodiments, the disclosed method further comprises optionally adjusting at least one aspect of the method. In even further aspects, adjusting at least one aspect of the method comprises changing the dosage of the composition, the frequency of administration of the composition, or the route of administration of the compound.

[0151] According to certain alternative embodiments, the subject is diagnosed with a disease associated with an elevated level of CD206+ macrophages and / or MDSCs. Such diseases or conditions include acquired immunodeficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic diseases, alopecia, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disorder, asthma, atherosclerosis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune myocarditis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Baló disease / Baló concentric sclerosis, Behçet's disease, Burger's disease, Bickerstaff encephalitis, Braun syndrome, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type I diabetes mellitus, type II diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, pemphigoid gestationis (also known as gestational pemphigoid), hidradenitis suppurativa, HIV infection, Hughes-Stovin syndrome, hypogammaglobulinemia, infectious diseases (including bacterial infections),Idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonia, juvenile idiopathic arthritis (also known as juvenile rheumatoid arthritis), Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis (also known as autoimmune hepatitis), systemic lupus erythematosus, lymphomatoid granulomatosis, Majid syndrome, malignant tumors including cancer (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma, and melanoma), Meniere's disease, microscopic polyangiitis, Miller Fisher syndrome, mixed connective tissue disease, morphea, Mucha Habermann disease (also known as acute pityriasis lichenoides et varioliformis acuta), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, Hashimoto's thyroiditis, recurrent rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Persistent Turner syndrome, pars planitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, erythroleukemia, Rasmussen encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, sepsis, serum sickness, Sjogren's syndrome, spondyloarthritis, Still's disease (adult-onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa Hunt syndrome, transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthritis, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis, among others, but not limited to these.

[0152] Also provided is a method of making a compound disclosed herein. In certain embodiments, a method of making a compound of formula (I) may comprise the following steps: (a) synthesizing a polymeric carbohydrate backbone having one or more amine-terminated lysines attached thereto; (b) synthesizing a cleavable linker comprising one or more carbonate and / or disulfide moieties; (c) reacting the cleavable linker with a reactive hydroxyl group of a therapeutic agent to form a therapeutic agent-linker compound; and (d) reacting the therapeutic agent-linker compound with one of the one or more amine-terminated lysines on the polymeric carbohydrate backbone.

[0153] In one aspect, steps (a)-(d) need not be performed in exactly the same order. In a further aspect, additional steps may be performed between each of steps (a)-(e). In some embodiments, step (a) may be performed before step (b), (c), or (d), or after step (b) or (c).

[0154] In further embodiments, the therapeutic agent is conjugated to the cleavable linker before being attached to the polymeric carbohydrate backbone. Additional considerations regarding methods of making the compounds can be found in the non-limiting illustrative examples provided herein.

[0155] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference and to the extent that such disclosure is relevant to the ordinary skill level in the relevant art. EXAMPLES

[0156] Embodiments of the present disclosure are further defined by the following non-limiting examples. It should be understood that these examples illustrate certain embodiments of the present disclosure but are provided for illustrative purposes only. From the above discussion and these examples, one of ordinary skill in the art can identify the essential features of the present disclosure and make various changes and modifications to the embodiments of the present disclosure to adapt to various applications and conditions without departing from its spirit and scope. Accordingly, in addition to what is illustrated and described herein, various modifications of the embodiments of the present disclosure will be apparent to those of ordinary skill in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0157] As drug delivery constructs targeting CD206-expressing cells, mannosylated amine dextran (MAD) carrying a dexamethasone or paclitaxel payload (MAD-DEX or MAD-PAC, respectively) was synthesized in three parts that need not be in a consecutive order: (1) synthesis of the MAD backbone, (2) a degradable linker and a part that forms a therapeutic agent-linker compound by reacting the reactive hydroxyl group of dexamethasone or paclitaxel, and (3) a part that reacts the therapeutic agent-linker compound with one or more amine-terminated lysines on the MAD backbone. The constructs were evaluated to determine their ability to release the therapeutic payload when cleaved by a reducing agent and their effect on the expression of inflammatory markers in human macrophages.

[0158] Example 1: Synthesis of MAD Delivery Constructs Synthesis of the MAD backbone: Starting from dextran of 10 kDa or 3.5 kDa (Mw), the MAD backbone was synthesized as described in U.S. Patent No. 6,409,990 (which is hereby incorporated by reference in its entirety), except that the conjugation of the chelating agent (i.e., DTPA) was omitted. The resulting construct had a combination of (1) a glucose moiety modified by the attachment of an amine-terminated lysine conjugated with a mannose moiety, (2) a glucose moiety having an amine-terminated lysine with no conjugated mannose moiety (i.e., a free amine-terminated lysine), and (3) an unmodified glucose moiety having neither an amine-terminated lysine nor a conjugated mannose. The resulting construct had the following structure:

[0159] [Chemical Formula]

[0160] The free amine-terminated lysine can be later utilized as a binding site for a drug payload via a cleavable linker. For the sake of simplicity of the above structure, the amine-terminated lysine is shown attached to the C2 hydroxyl group of the glucose moiety in the dextran polymer; however, these lysines can be distributed to any of the hydroxyl groups of the MAD backbone.

[0161] A MAD scaffold suitable for drug delivery of a therapeutic payload to CD206-expressing cells can be constructed using an initial dextran polymer in the range of 1 kDa to greater than 150 kDa in average molecular weight (Mw). By varying the initial Mw of the starting dextran, the final size (Mw) of the final drug delivery construct is determined. Drug delivery vehicles of different Mws have variable biodistributions when administered to either animal or human subjects. These differences in biodistribution can enable optimization of the drug delivery vehicle for varying pharmacological applications. Additionally, the number of amine-terminated lysines and / or the number of conjugated mannose moieties can theoretically be intentionally varied from zero to the number of hydroxyl groups on the glucose moieties of the initial dextran. By varying the number of free amines and mannose moieties, the biodistribution and CD206 receptor affinity also vary, thereby enabling optimization of the drug delivery vehicle for varying pharmacological applications.

[0162] Example 2: Conjugation of Dexamethasone to the MAD Scaffold To conjugate dexamethasone to the MAD scaffold, various linkers were evaluated. Those that enabled release of the therapeutic agent / payload upon endosomal internalization were determined to be successful conjugations.

[0163] Modification of the MAD scaffold with dexamethasone via a hydrazone linker: Using hydrazone formation, dexamethasone was conjugated to the MAD scaffold. Hydrazone formation was accomplished via condensation of the carbonyl group on dexamethasone with a hydrazide. The dexamethasone-hydrazone linker compound was then conjugated to the MAD scaffold to prepare a MAD-dexamethasone construct (also described herein as the MAD-dexamethasone conjugate). The reaction method using the hydrazone linker was previously disclosed in U.S. Patent No. 10,806,803, the entirety of which is hereby incorporated by reference. The reaction between dexamethasone and the hydrazide to form various dexamethasone-hydrazone linker compounds is shown in FIG. 1.

[0164] The hydrazide was successfully conjugated to dexamethasone, but when the dexamethasone-hydrazone linker compound was conjugated to the MAD backbone, the release profile of dexamethasone from the MAD backbone under the aqueous low pH conditions found within endosomal and lysosomal compartments was found to be unacceptable. The reaction of the hydrazide with dexamethasone produced a mixture of hydrazones as shown in Figure 1. The formation at the C3 ketone resulted in a stable thermodynamic product. Thus, without being limited to any particular mechanism or theory, in some embodiments, the conjugation of the hydrazide at the C3 ketone position of dexamethasone results in a high percentage of hydrazone formation that cannot be released from the construct, even under highly acidic conditions, due to the stable thermodynamic product formed. Thus, hydrazone linker chemistry can be used to attach dexamethasone to the MAD backbone, but the resulting construct, for example, CD206 + Upon internalization into endosomes by cells, does not release a significant portion of the dexamethasone payload and renders the compound pharmacologically inert.

[0165] Modification of the MAD backbone with a reductant-sensitive disulfide linker conjugated to dexamethasone via an ester bond: The synthesis and drug release profile of the MAD-dexamethasone construct were further evaluated with a symmetric disulfide linked to the C21 hydroxyl group of dexamethasone and the amine-terminated lysine of the MAD backbone (Figure 2). The first application of this method resulted in the successful synthesis of the MAD-drug conjugate via an ester bond to dexamethasone (not shown in Figure 2). Furthermore, dexamethasone in the ester bond conjugate was separated from the MAD backbone by disulfide cleavage at a pH of 4.65 in a 10 mM aqueous solution of glutathione. Without being limited to any particular theory or mechanism, it is contemplated that the dexamethasone payload linked in this manner was released upon intracellular uptake of the MAD construct when an abundance of free thiols reductively cleaved the disulfide linker. However, the ester bond remained intact with dexamethasone included, and free dexamethasone could not be detected after more than 24 hours.

[0166] Modification of the MAD backbone with a reducing agent-sensitive disulfide linker conjugated to dexamethasone via a carbonate linkage: As shown in Figure 2, after conjugating the symmetric disulfide / carbonate linker 3 to dexamethasone as a carbonate, the MAD backbone was further evaluated. As shown in Figure 2, the dexamethasone and disulfide / carbonate linker 3 compound (which may be further referred to herein as the "dexamethasone-carbonate compound") was synthesized as follows: 2,2'-Dithiobisethanol (1.0 g, 6.5 mmol) was dissolved in 26 ml of anhydrous dichloromethane (DCM). Triethylamine (3.6 ml, 25.9 mmol), and a solution of p-nitrophenyl chloroformate (2.87 g, 14.3 mmol) in 8.5 ml of DCM were added, and the reaction was stirred overnight under nitrogen at room temperature. The solution was concentrated and purified by silica column chromatography using a gradient of 0 - 30% ethyl acetate in hexane to obtain 1.86 g (59%) of dinitrophenyl carbonate 2 as shown in Figure 2. To 500 mg (1.03 mmol) of dinitrophenyl carbonate 2 in 30 ml of anhydrous dichloromethane, under nitrogen, 10 ml of a 1:1 anhydrous N,N-dimethylformamide (DMF) and DCM solution of diisopropylamine (0.27 ml, 1.55 mmol), and 203 mg (0.516 mmol) of dexamethasone were slowly added. Then, 13 mg (20 mol%, 0.103 mmol) of dimethylaminopyridine in 5 ml of DCM was further added, and the solution was stirred under nitrogen at room temperature and protected from light. The reaction was monitored by TLC for the disappearance of the starting dexamethasone, diluted with ethyl acetate, extracted with saturated ammonium chloride and brine, dried over sodium sulfate, and concentrated. Silica column chromatography using a gradient of 10 - 60% ethyl acetate in hexane yielded 203 mg (53%) of the dexamethasone-carbonate compound as an off-white crystalline solid. Surprisingly, these dexamethasone-carbonate compounds efficiently conjugated to the MAD backbone, yielding MAD-dexamethasone constructs with an appropriate release profile under reducing conditions (Figure 2). These beneficial effects are further described within the examples of this specification.

[0167] To prepare the MAD-dexamethasone construct on a 10 kDa MAD backbone (25 mannoses, 12 available amines), 100 mg of MAD (5.0 μmol) was dissolved in 4 ml of anhydrous dimethyl sulfoxide (DMSO) using short sonication and mild heating. The solution was cooled to ambient temperature and 6 equivalents (4.2 μl, 30 μmol) of triethylamine (TEA) were added, followed by 6 equivalents (22.3 mg, 30 μmol) of the solid dexamethasone-carbonate compound 3. After 12 h, samples of the reaction solution were tested and compared to the time zero sample for loss of amine content, which was equivalent to the addition of 3.8 dexamethasone-carbonate compounds bound as carbamates to the MAD backbone. The reaction solution was cooled on an ice bath and slowly diluted with 18 ml of purified water. The solution was concentrated with a centrifugal spin filter containing a 10 kDa MWCO (molecular weight cut-off) membrane and subsequently diluted and concentrated with 2 volumes of purified water, 2 volumes of 50% ethanol, and 2 volumes of purified water. The residue containing the product was removed from the membrane with purified water and then frozen and lyophilized, yielding 60 mg of the MAD-dexamethasone conjugate as a light yellow foam. Total dexamethasone in the conjugate was determined by UV at 239 nm and free unbound dexamethasone (0.36 wt%) was determined by standard curve and HPLC analysis. Bound dexamethasone was determined to be 6.91 wt%, which corresponds to an average of 3.92 dexamethasone moieties per MAD on a 10 kDa backbone.

[0168] To prepare the MAD-dexamethasone construct on a 3.5 kDa MAD backbone (11 mannoses, 6 available amines), 100 mg of MAD (12.4 μmol) was dissolved in 4 ml of anhydrous DMSO and 3 equivalents (5.2 μl, 37 μmol) of TEA, followed by 3 equivalents (27.4 mg, 37 μmol) of solid dexamethasone-carbonate 3. Over 12 h, the reaction solution was cooled on an ice bath and slowly diluted with 18 ml of purified water. The solution was concentrated with a centrifugal spin filter containing a 3 kDa MWCO membrane, followed by dilution and concentration with 5 volumes of purified water. The residue containing the product was removed from the membrane with purified water and then frozen and lyophilized, yielding 98 mg of the MAD-dexamethasone conjugate as a bright yellow foam. Total dexamethasone in the conjugate was determined by UV at 239 nm, and free unbound dexamethasone (0.74 wt%) was determined by standard curve and HPLC analysis. Bound dexamethasone was determined to be 7.77 wt%, corresponding to an average of 1.88 dexamethasone moieties per MAD with respect to the 3.5 kDa backbone.

[0169] Example 3: Release of Dexamethasone from the MAD-Dexamethasone Conjugate (10 mM GSH, 37 °C) After the synthesis of MAD-dexamethasone on the 10 kDa dextran backbone was completed, the release of dexamethasone from the samples was evaluated over time. Two 1 mg / ml solutions of the construct were prepared: one in 0.1 M PBS buffer at pH 7.1 and the other in 0.1 M sodium acetate buffer at pH 4.65 and 10 mM glutathione. Each sample was sealed and shaken at 37 °C and monitored by HPLC from time zero to 48 hours. The amount of dexamethasone released from the construct was determined from a dexamethasone standard curve. Dexamethasone released under pH 4.65 and reducing conditions increased from 3 to 20 hours (Figure 3), and at 21 hours, 4.5 wt% free dexamethasone (2.5 drug moieties per dextran chain or 71% of the conjugated drug) was detected by HPLC. In comparison, approximately one dexamethasone was released at pH 7.1 under non-reducing conditions at 21 hours, which is far beyond the time frame in which the MAD construct would circulate in the blood. This is an important feature of the MAD constructs disclosed herein as it allows for a gradual and continuous release of the drug payload to CD206-expressing cells, which would not have been possible if the drug was not delivered on the MAD backbone.

[0170] Example 4: Evaluation in a MAD-dexamethasone conjugate-human macrophage cell culture assay The MAD-dexamethasone conjugate (MAD-DEX) was evaluated in a human macrophage cell culture assay. In this assay, human peripheral blood monocytes (hPBMC) were incubated for 5 days in RPMI + 10% FBS + 1× penicillin / streptomycin / L-glutamine + 50 ng / ml of GM-CSF (complete medium) at a concentration of 500,000 monocytes per well in 48-well tissue culture plates. During this 5-day incubation, the monocytes differentiated into macrophages. GM-CSF induces macrophages to adopt an activated phenotype that is intermediate between the two extremes of M1 or M2. After 5 days of incubation, the medium was removed and replaced with complete medium supplemented with various concentrations of MAD-DEX or unbound dexamethasone (free dexamethasone). As negative controls, saline and vehicle (MAD without drug payload, 23.3 μg / ml) were added as alternative supplements to other cultures. The macrophage cell cultures were incubated for 24 hours with the supplemented complete medium, after which the medium was removed and replaced with fresh complete medium. The macrophage cultures were then incubated for an additional 3 days. To enable assessment of persistent changes in the macrophage phenotype, an incubation of 3 days after treatment was performed.

[0171] After an additional 3-day incubation in fresh complete medium, cells were harvested and evaluated by flow cytometry for viability (DAPI) or expression of macrophage surface markers. These markers are often considered indicators of either an M1-like or M2-like phenotype, or are representative of markers that are known immune checkpoint receptors. Evaluated cell surface markers included CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD-1, using antibodies specific for each marker. The observed amounts of each surface marker varied considerably between markers and between treatment groups, but almost all live cells expressed detectable amounts of all markers. The marker with the lowest expression in the saline and vehicle treatment controls was PD-1. The output of the flow cytometry assay was the mean fluorescence intensity (MFI). Macrophages differentiated from monocytes collected from three separate donors were evaluated for all treatments. For macrophages from each donor, all experiments were performed three times.

[0172] Results: In all replicates of this example, most macrophages treated with saline control survived until the end of the experiment (9 days). For cell viability and all surface markers evaluated, the results of the drug-free vehicle treatment control were not statistically different from those observed in the saline (drug-free) control, indicating that the drug-free vehicle had no pharmacologic activity observed in this study. Furthermore, neither MAD-DEX nor free dexamethasone decreased cell viability at any of the test concentrations.

[0173] Figure 4 shows a summary representation of how various treatments with either MAD-DEX3.5 (MAD-DEX constructed on a 3.5 kDa dextran backbone) or free dexamethasone affected the expression levels of eight evaluated surface markers. The values shown are the mean fold change in expression (MFI) compared to the MFI values observed in the saline control. The statistical significance of these changes in expression was measured by the Z-test.

[0174] The data shown in Figure 4 shows the fold change in the expression of the indicated surface markers in cells treated with either an equimolar amount of free dexamethasone not bound to 11.7 μg / ml of MAD-DEX3.5 or MAD vehicle. A fold change of 1.0 indicates no change in expression, i.e., the mean expression level (MFI) observed in the treated cells was the same as the MFI observed in the saline control. A value of 2.0 means that the mean MFI of the treated cells was twice that observed in the saline-treated control. Conversely, a value of 0.5 means that the MFI observed in the treated cells was half that observed in the saline control.

[0175] Several important observations can be made from the data presented in Figure 4. First, MAD-DEX3.5 and free dexamethasone (at the same concentration as the amount of dexamethasone loaded onto the MAD-DEX3.5 backbone) induced similar changes in the macrophage phenotype. Both MAD-DEX3.5 and free dexamethasone significantly increased the expression of CD163, which is typically considered a marker of the M2-like phenotype. CD163 expression can be increased by treatment with corticosteroids such as dexamethasone. Although not statistically significant, both also reduced the expression of CD86, which is typically considered a marker of the M1-like phenotype. Also interestingly, both significantly decreased the expression of SIRPα and induced a non-significant increase in the expression of MHC2. Furthermore, the expression of PD-1 was increased by MAD-DEX3.5, but the very low level of PD-1 expression in macrophages treated with the saline control suggests that the modest but significant increase in PD-1 expression induced by MAD-DEX3.5 may not be phenotypically significant.

[0176] The results shown in Figure 4 are remarkable for several reasons. Free dexamethasone is known to be highly anti-inflammatory, and the observation that MAD-DEX3.5 induces the same phenotypic changes as free dexamethasone suggests that MAD-DEX3.5 is also highly anti-inflammatory. Furthermore, MAD-DEX3.5 was found to be equally potent as an equimolar dose of free dexamethasone. Dexamethasone is lipophilic and freely crosses cell membranes. The MAD construct is highly hydrophilic and can enter cells when transported across the cell membrane by CD206. Thus, the observation that MAD-DEX3.5 efficiently modifies the macrophage phenotype suggests that it is being transported into macrophages by CD206. As a result, cells that do not express CD206 would not be expected to receive dexamethasone delivered by MAD-DEX3.5.

[0177] To test the hypothesis that MAD-DEX constructs, such as MAD-DEX3.5, selectively deliver dexamethasone to CD206-expressing cells, such as CD206-positive macrophages, MAD-DEX3.5 was labeled with the fluorescent marker Alexa Fluor™ 488 (AF488), which enabled localization of MAD-DEX3.5 to various cell populations and was evaluated by flow cytometry. As described previously, differentiated CD206-positive macrophages of peripheral blood monocytes and fresh peripheral blood mononuclear cells (mainly lymphocytes) that do not express CD206 in complete medium were exposed to increasing concentrations of AF488-MAD-DEX3.5. The cells were then washed and evaluated by flow cytometry for AF488-mediated fluorescence. The control groups consisted of cells exposed to an AF488-labeled anti-CD206 monoclonal antibody or an antibody isotype control, and untreated cells. The results are shown in Figure 5. Figure 5 demonstrates that AF488-MAD-DEX3.5 selectively binds to macrophages and that only macrophages bind to the anti-CD206 antibody, indicating that MAD-DEX3.5, and by inference all other MAD-DEX constructs, preferentially deliver dexamethasone to CD206-expressing cells. This finding is important for the purpose of utilizing MAD-DEX to reduce macrophage-mediated inflammation while reducing off-target side effects. Targeted delivery of dexamethasone to CD206-expressing cells on the MAD construct provides clinical utility for treating diseases such as macrophage-related autoimmune diseases that are not limited to NASH, ARDS, coronavirus infection, sepsis, cytokine storm, and rheumatoid arthritis.

[0178] Example 5: Conjugation of Paclitaxel to the MAD Skeleton Modification of the MAD backbone by a reducing agent-sensitive disulfide linker conjugated to paclitaxel via a carbonate linkage: The chemical pathway described in Example 2 for the dexamethasone payload on the MAD backbone was further evaluated with different drug payloads. As would be contemplated by one of ordinary skill in the art, the methods discussed herein can then be adapted to additional drug products bearing freely accessible and reactive hydroxyl groups and having a wide variety of polymer backbones. To demonstrate this chemical utility, paclitaxel was modified with dinitrophenyl carbonate 2, as shown in Figure 6, in the same manner as used for dexamethasone and disulfide / carbonate linker compound 3 (Figure 2) to prepare paclitaxel and disulfide / carbonate linker compound 4 (which may be further referred to herein as the "paclitaxel-carbonate compound"). Then, in a similar process as described in Example 2, paclitaxel was conjugated to 3.5 kDa and 10 kDa MAD backbones to obtain paclitaxel-MAD constructs having the desired release of paclitaxel under reducing conditions.

[0179] The paclitaxel-carbonate compound 4 was prepared as follows: 352 mg (0.727 mmol) of dinitrophenyl carbonate 2 was weighed into a vial equipped with a stir bar and dissolved in 18.6 mL of anhydrous DCM under nitrogen. To this clear solution was added 620 mg (0.727 mmol) of paclitaxel (Accela SY016928), followed by 381 μL (2.18 mmol) of diisopropylethylamine (DIPEA), and 20 mol% (17.8 mg, 0.145 mmol) of DMAP. The reaction solution immediately turned yellow upon addition of the base. The vial was protected from light and stirred at ambient temperature for 12 hours. Completion of the reaction was confirmed by TLC by consumption of the starting paclitaxel. The reaction solution was diluted with DCM, extracted with saturated ammonium chloride and brine, dried over anhydrous sodium carbonate, and concentrated. Purification by silica gel chromatography using a gradient of 20 - 80% ethyl acetate in hexanes afforded 391 mg of pure paclitaxel-carbonate compound 4.

[0180] To prepare a MAD-paclitaxel conjugate (MAD-PAC) on a 10 kDa MAD backbone (18 mannoses, 20 available amines), 100 mg of MAD (5.38 μmol) was dissolved in 4 ml of anhydrous DMSO using short sonication and mild heating. The solution was cooled to ambient temperature, 6 equivalents (4.5 μl, 32 μmol) of TEA were added, followed by 6 equivalents (39 mg, 32 μmol) of solid paclitaxel-carbonate compound 4, whereupon the solution turned bright yellow. After stirring overnight, the reaction was cooled on an ice bath and slowly diluted with purified water. The solution was concentrated with a centrifugal spin filter and subsequently diluted and concentrated with 3 volumes of purified water. The residue containing the product was removed from the membrane with purified water and then frozen and lyophilized, yielding 90 mg of the MAD-paclitaxel conjugate as a yellow foam. Free unbound paclitaxel (0.94 wt%) in the conjugate was determined by standard curve and HPLC analysis. Bound paclitaxel was determined using the same HPLC method under release conditions (50 mM TCEP at 37 °C for 4 h) and found to be 12.6 wt%, corresponding to an average of 3.53 paclitaxel moieties per MAD with respect to the 10 kDa backbone.

[0181] To prepare the MAD-paclitaxel conjugate on a 3.5 kDa MAD backbone (11 mannoses, 6 available amines), 600 mg of MAD (74.2 μmol) was dissolved in 24 ml of anhydrous DMSO using short sonication and mild heating. The solution was cooled to ambient temperature and 4 equivalents (41 μl, 0.30 mmol) of TEA were added, followed by 4 equivalents (356 mg, 0.30 mmol) of solid paclitaxel-carbonate compound 4, at which point the solution turned bright yellow. After stirring overnight, the reaction was cooled on an ice bath and slowly diluted with 125 ml of purified water. With the aid of additional purified water, the solution was transferred to stirred cells attached to a 3 kDa MWCO membrane and concentrated from 250 ml to approximately 10 ml, then diluted and concentrated with an additional 3 volumes of purified water. The residue containing the product was removed from the membrane with purified water, filtered through a 0.2 μm vacuum filter, and then frozen and lyophilized to yield 525 mg of the MAD-paclitaxel conjugate as a yellow foam. Free unbound paclitaxel (3.16 wt%) in the conjugate was determined by standard curve and HPLC analysis. Bound paclitaxel was determined using the same HPLC method under release conditions (50 mM TCEP at 37 °C for 22 h) and found to be 16 wt%, corresponding to an average of 2.0 paclitaxel moieties per MAD on the 3.5 kDa backbone.

[0182] Similar to the described experiments evaluating AF488-MAD-DEX3.5, the MAD-PAC3.5 conjugate (constructed on a 3.5 kDa dextran backbone) was labeled with AF488 to generate AF488-MAD-PAC3.5, and its ability to selectively bind to macrophages expressing CD206 but not to peripheral blood lymphocytes that do not express CD206 was evaluated (see Figure 7). Similar to the results observed for AF488-MAD-DEX3.5, AF488-MAD-PAC3.5 preferentially bound only to macrophages and macrophages expressing CD206 in this assay. Thus, MAD-PAC3.5 and similar MAD-PAC constructs built on dextran backbones of different molecular weights preferentially deliver paclitaxel to CD206-expressing cells such as tumor-promoting TAMs while avoiding or limiting target exposure and toxicity to cells that do not express CD206.

[0183] Example 6: Evaluation in a Human Macrophage Cell Culture Assay of MAD-Paclitaxel Conjugates The MAD-PAC3.5 conjugate was further evaluated in a human macrophage culture assay similar to the analysis completed in Example 4 for the MAD-DEX conjugate. Unlike previous examples of MAD-DEX3.5 and free dexamethasone, MAD-PAC3.5 altered the surface marker expression pattern such that, as shown in Figure 8, it was different from that observed for an equal molar equivalent concentration of free paclitaxel. MAD-PAC3.5 decreased the expression of CD206 and CD163, whereas free paclitaxel gently increased the expression of these markers. MAD-PAC3.5 also increased the expression of CD80 and CD86. In conjunction with the increase in the expression of CD80 and CD86, the decrease in the expression of CD206 and CD163 indicated that MAD-PAC3.5 strongly shifted the immune state of macrophages towards a more M1-like pro-inflammatory phenotype. Free paclitaxel also increased the expression of CD80 and CD86; however, these changes were more gentle than those observed for MAD-PACX3.5. Importantly, the changes in surface marker expression induced by MAD-PAC3.5 and free paclitaxel were significantly different from each other. Surprisingly, the changes in surface marker expression observed for MAD-PAC3.5 could not be replicated by free paclitaxel at any drug concentration. The expression levels of MHC1, MHC2, and SIRPα were not significantly altered by either agent. PD1 was significantly increased by MAD-PAC3.5 but not by free paclitaxel.

[0184] These results demonstrate that targeted delivery of paclitaxel on the MAD construct to CD206-expressing cells, by altering the M2-like, CD206-expressing TAM phenotype towards a more M1-like phenotype, has a high likelihood of providing clinical utility for treating cancer.

[0185] Example 7: MAD-PAC3.5 reduces tumor growth when combined with anti-CTLA4 immunotherapy Using a CT26 / Balb-c syngeneic mouse tumor model in an experiment where tumor-bearing mice were treated with MAD-PAC3.5 alone or in combination with anti-CTLA4 immunotherapy, the hypothesis that MAD-PAC3.5 can provide clinical utility for treating cancer was evaluated. CT26 cells grown and established until they reached a tumor volume of 80 - 100 mm 3 were transplanted into mice. Test articles were administered to tumor-bearing mice (n = 10 / treatment group) by intravenous injection on 4 days (treatment days 1, 4, 7, and 11). Treatments were saline, MAD without payload (127 μg / injection), MAD-PAC3.5 alone (127 μg / injection), a molar equivalent dose of free paclitaxel, anti-CTLA4 antibody treatment alone, combination therapy of free paclitaxel (equimolar dose) + anti-CTLA4 therapy, and combination therapy of MAD-PAC3.5 (127 μg / injection) + anti-CTLA4 therapy. Tumor volumes were measured and recorded at regular intervals. The results of the mean tumor volume per treatment group observed on day 14 of treatment are shown in Figure 9. The MAD treatment group without payload showed results similar to the saline treatment (not shown). Treatment with both free paclitaxel and MAD-PAC3.5 caused a gradual decrease in the mean tumor volume, but these results did not reach statistical significance. In this experiment, treatment with the anti-CTLA4 antibody alone almost halved the tumor volume on day 14 on average and reached statistical significance (p = 0.01). Combination therapy of the anti-CTLA4 antibody with either free paclitaxel or MAD-PAC3.5 further reduced the tumor volume on day 14 on average (p = 0.005 and p = 0.0008, respectively, compared to the saline control group). The treatment group that received the combination of anti-CTLA4 and MAD-PAC3.5 had the smallest mean tumor volume on day 14, which was 76% lower than the saline control.

[0186] Enhanced tumor control observed in mice treated with the combination of anti-CTLA4 and MAD-PAC3.5 can be achieved without exposing cells and tissues that do not express CD206 to the potential off-target cytotoxic effects of paclitaxel. Thus, this experiment shows, in combination with the results of the selective binding assay, that the safety of combination therapies consisting of immunotherapies such as MAD-PAC3.5 (or other MAD-PAC constructs) + anti-CTLA4 is increased. Similar results are expected to be observed when MAD-PAC3.5 (or a similar MAD-PAC construct) is combined with anti-PD1 or anti-PD-L1 immunotherapy. Similarly, since MAD-PAC3.5 was observed to shift the macrophage phenotype towards a more M1-like immune state, it is expected that MAD-PAC constructs can enhance the efficacy of other anti-cancer therapies such as radiation-based therapies and conventional chemotherapy.

[0187] Although the present disclosure has been described in this manner, it will be apparent that these can be modified in many ways. Such modifications are not to be regarded as a departure from the spirit and scope of the present disclosure, and it is intended that all such modifications be included within the scope of the following claims.

Claims

1. A compound comprising: a polymeric carbohydrate backbone; one or more mannose-binding C-type lectin receptor targeting moieties; a therapeutic agent comprising one or more reactive hydroxyl groups and attached to the polymeric carbohydrate backbone via a cleavable linker.

2. The compound according to claim 1, wherein the compound comprises a subunit represented by formula (I), 【Chemical Formula 1】 wherein: Each X is independently H, L １ -A-Z, or L ２ -R, and each X is bonded to an OH group L １ and L ２ each of which is, independently, an amine-terminated leash each A independently comprises a cleavable linker comprising one or more carbonate and / or disulfide moieties; each Z independently comprises a therapeutic agent comprising one or more reactive hydroxyl groups; each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H; n is an integer greater than zero, and each unit of n can be the same or different.

3. At least one X is L １ -A-Z, and at least one X is L ２ -R, wherein R comprises the mannose-binding C-type lectin receptor targeting moiety, the compound according to claim 2

4. The compound according to any one of claims 1 to 3, wherein the polymeric carbohydrate backbone has a molecular weight of about 1 kDa to about 50 kDa.

5. The compound according to any one of claims 1 to 4, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, high-mannose glycan or mannooligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof.

6. At least one L １ and / or at least one L ２ is / are —(CH ２ ) ｐ S(CH ２ ) ｑ－ NH—, wherein p and q are integers from 0 to 5, the compound according to any one of claims 2 to 5.

7. The compound according to any one of claims 2 to 5, wherein the therapeutic agent is conjugated to the cleavable linker before being attached to the polymeric carbohydrate backbone.

8. The compound according to any one of claims 1 to 7, wherein the cleavable linker comprises one or more carbonate and / or disulfide moieties.

9. The compound according to any one of claims 2 to 8, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone: [Chemical Formula 2] wherein x is an integer from 1 to 5 and y is an integer from 1 to 5.

10. The compound according to any one of claims 2 to 8, wherein the cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone: [Chemical Formula 3] The compound according to any one of claims 2 to 8.

11. A has the following formula: 【Chemical Formula 4】 wherein x is an integer from 1 to 5 and y is an integer from 1 to 5. The compound according to any one of claims 2 to 8.

12. A has the following formula: [Chemical Formula 5] The compound according to any one of claims 2 to 8.

13. The compound according to any one of claims 1 to 12, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

14. The compound according to any one of claims 1 to 13, wherein the therapeutic agent comprises dexamethasone or paclitaxel.

15. A pharmaceutical composition comprising: a compound according to any one of claims 1 to 14; and a pharmaceutically effective carrier.

16. The composition according to claim 15, wherein the compound comprises a subunit represented by formula (I), 【Chemical Formula 6】 wherein: Each X is independently H, L １ -A-Z, or L ２ -R, and each X is bonded to an OH group L １ and L ２ each is independently an amine-terminated leash, each A independently comprises the cleavable linker comprising one or more carbonate and / or disulfide moieties; each Z independently comprises the therapeutic agent comprising one or more reactive hydroxyl groups; each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and each unit of n can be the same or different.

17. A compound comprising: a therapeutic agent comprising a reactive hydroxyl group; a cleavable linker comprising one or more carbonate and / or disulfide moieties; and a second compound comprising a primary amine, wherein the cleavable linker is bonded to the reactive hydroxyl group of the therapeutic agent and to the primary amine of the second compound.

18. The compound according to claim 17, wherein the compound has the following formula (II): 【Chemical Formula 7】 wherein: Z is the therapeutic agent comprising a reactive hydroxyl group; Y is the second compound comprising a primary amine; x is an integer from 1 to 5; and y is an integer from 1 to 5.

19. The compound according to claim 17 or 18, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

20. The compound according to any one of claims 17 to 19, wherein the therapeutic agent comprises dexamethasone or paclitaxel.

21. A method for preparing a compound according to any one of claims 1 to 14, the method comprising: a) synthesizing a polymer carbohydrate backbone to which one or more amine-terminated leeches are attached; and b) synthesizing a cleavable linker comprising one or more carbonate and / or disulfide moieties. (c) reacting the cleavable linker with a reactive hydroxyl group of the therapeutic agent to form a therapeutic agent-linker compound; (d) reacting the therapeutic agent-linker compound with one of the one or more amine-terminated lysines on the polymeric carbohydrate backbone, a method comprising. **Claim 22** The method according to claim 21, wherein step (a) can be performed before step (b), (c), or (d), or after step (b) or (c). **Claim 23** The method according to claim 21 or 22, wherein the therapeutic agent is conjugated to the cleavable linker before being bound to the polymeric carbohydrate backbone. **Claim 24** At least one X is L １ -A-Z, and at least one X is L ２ -R, and R contains the mannose-binding C-type lectin receptor targeting moiety, the method according to any one of claims 21 to 23. **Claim 25** The method according to any one of claims 21 to 24, wherein the polymeric carbohydrate backbone has a molecular weight of about 1 kDa to about 50 kDa. **Claim 26** The method according to any one of claims 21 to 25, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, high-mannose glycan or mannooligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof. **Claim 27** at least one L １ and / or at least one L ２ is / are —(CH ２ ) ｐ S(CH ２ ) ｑ－ NH—, wherein p and q are integers from 0 to 5, the method according to any one of claims 21 to 26 **Claim 28** The cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone: 【Chemical 8】 wherein x is an integer from 1 to 5 and y is an integer from 1 to 5, the method according to any one of claims 21 to 27. **Claim 29** The cleavable linker has the following formula before being conjugated to the therapeutic agent and the polymeric carbohydrate backbone: 【Chemical Formula 9】 The method according to any one of claims 21 to 28. **Claim 30** A has the following formula: 【Chemical 10】 wherein x is an integer from 1 to 5 and y is an integer from 1 to 5, the method according to any one of claims 21 to 29. **Claim 31** A has the following formula: 【Chemical 11】 The method according to any one of claims 21 to 30. **Claim 32** The method according to any one of claims 21 to 31, wherein the therapeutic agent comprises a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof. **Claim 33** The method according to any one of claims 21 to 32, wherein the therapeutic agent comprises dexamethasone, paclitaxel, or a combination thereof. **Claim 34** A method for reprogramming tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype, comprising: administering to a subject in need thereof an effective dose of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent comprising one or more reactive hydroxyl groups and attached to the polymeric carbohydrate backbone via a cleavable linker.

35. The compound comprises a subunit represented by formula (I), 【Chemical 12】 wherein Each X is independently H, L １ -A-Z, or L ２ -R, and each X is bonded to an OH group L １ and L ２ each of which is, independently, an amine-terminated leash each A independently comprises a cleavable linker comprising one or more carbonate and / or disulfide moieties, each Z independently comprises a therapeutic agent comprising one or more reactive hydroxyl groups, each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H, n is an integer greater than zero, and each unit of n can be the same or different, the method according to claim 34.

36. The compound is administered in combination with at least one other therapy or treatment, and the at least one other therapy or treatment is chemotherapy, radiotherapy, or immunotherapy, the method according to claim 34 or 35.

37. The therapeutic agent comprises paclitaxel, the method according to any one of claims 34 to 36.

38. The subject in need thereof has cancer, the method according to any one of claims 34 to 37.

39. The therapeutic agent is released from the polymeric carbohydrate backbone in the presence of a reducing agent, the method according to any one of claims 34 to 38.

40. The method does not suppress the anti-tumor activity of lymphocytes, the method according to any one of claims 34 to 39.

41. A method for reducing macrophage-mediated inflammation, comprising: administering to a subject in need thereof an effective dose of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a therapeutic agent comprising one or more reactive hydroxyl groups and attached to the polymeric carbohydrate backbone via a cleavable linker.

42. The compound comprises a subunit represented by formula (I), 【Chemical Formula 13】 wherein Each X is independently H, L １ -A-Z, or L ２ -R, and each X is bonded to an OH group L １ and L ２ each is independently an amine-terminated leash each A independently comprises a cleavable linker comprising one or more carbonate and / or disulfide moieties, each Z independently comprises a therapeutic agent comprising one or more reactive hydroxyl groups, Each R independently contains the mannose-binding C-type lectin receptor targeting moiety or H, The method according to claim 41, wherein n is an integer greater than zero, and each unit of n can be the same or different. **Claim 43** The method according to claim 41 or 42, wherein the compound is administered in combination with at least one other therapy or treatment. **Claim 44** The method according to any one of claims 41 to 43, wherein the therapeutic agent contains dexamethasone. **Claim 45** The method according to any one of claims 41 to 44, wherein the subject in need thereof suffers from non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, other macrophage-related diseases, or a combination thereof. **Claim 46** The method according to any one of claims 41 to 45, wherein the therapeutic agent is released from the polymeric carbohydrate backbone in the presence of a reducing agent. **Claim 47** The method according to any one of claims 41 to 46, wherein the method does not suppress the anti-tumor activity of lymphocytes. **Claim 48** A method of treating a disease, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 14, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory disorders, non-alcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, and other macrophage-related diseases. **Claim 49** The method according to claim 48, wherein the compound is administered in combination with at least one other therapy or treatment. **Claim 50** The method according to claim 49, wherein the at least one other treatment or therapy is chemotherapy, radiotherapy, or immunotherapy. **Claim 51** The method according to any one of claims 48 to 50, wherein the therapeutic agent contains a corticosteroid, cortisol, glucocorticoid receptor ligand, chemotherapeutic agent, toll-like receptor agonist or antagonist, or a combination thereof.

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