Compositions and methods for modifying macrophage phenotype

Abstract

Methods and compositions for repolarizing tumor-associated macrophages (TAMs) from M2 to M1 are disclosed, comprising administering to a subject in need thereof an effective dose of a compound comprising a dextran backbone and one or more CD206 targeting moieties attached thereto. In certain embodiments, the compound further comprises a therapeutic agent selected from paclitaxel, gemcitabine, lapatinib, and doxorubicin. In further embodiments, the therapeutic agent comprises a chelator and at least one metal ion. In certain implementations, the at least one metal ion comprises at least one Cu(II) ion.

Description

[Technical field]

[0001] The present disclosure relates to the field of compositions and methods for modifying macrophage phenotype. [Background technology]

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 62 / 824,853, filed March 27, 2019, and entitled "Altering the Phenotype of Macrophages with CD206 Targeted Drug Delivery Vehicles Carrying Appropriate Pharmacologically Active Payloads," which is incorporated herein by reference in its entirety.

[0003] Cancer is the second leading cause of death in the United States, with nearly one in four deaths due to cancer. Cancer is characterized by uncontrolled proliferation and cell division of cancer cells. However, cancer benefits greatly from a chronic maladaptive immune response against the tumor, and macrophages are key mediators of this maladaptive response. Macrophages generally respond to various stimuli in their local microenvironment by altering the expression pattern of many genes, potentially hundreds of genes. Macrophages that have changed their phenotype in this way are called activated macrophages. Depending on which stimuli the macrophages respond to, numerous phenotypic states of activation can be acquired. Genes that are differentially expressed upon macrophage activation include cell surface markers (e.g., macrophage mannose receptor, CD206) and various cytokines, enzymatic pathways leading to the generation of reactive oxygen species (ROS), as well as other signaling molecules that can control the behavior of other components of the immune system, such as T lymphocytes (T cells). When first reported, activated macrophages were divided into two phenotypes. There are two types of activated macrophages: the classical activated type, termed M1, which is a highly pro-inflammatory phenotype, and the alternative activated type, termed M2, which is immunosuppressive and promotes wound healing. Although it is now understood that the strict dichotomy of activated macrophages phenotypes is an oversimplification and does not represent the true flexibility of macrophage responses to stimuli from their microenvironment, the concept that activated macrophages can influence local immune responses by being either pro-inflammatory (M1-like) or immunosuppressive (M2-like) remains useful in explaining the role of macrophages in various pathological conditions.

[0004] Tumor associated macrophages (TAMs) are abundant in tumors and contribute significantly to the maladaptive immune responses associated with cancer. Although both M1-like and M2-like TAMs are known, the majority of TAMs residing in or near established tumors are immunosuppressive, i.e., M2-like activated macrophages. Importantly, these M2-like TAMs are frequently identified in immunohistochemical assessment of tumors by their high expression of CD206 (i.e., they are CD206+). M2-like TAMs suppress T cells by expressing IL-10, TGF-β, and PD-L1, and promote tumor angiogenesis and metastasis.

[0005] There is a need in the art for compositions and methods that induce a phenotypic change from M2-like TAMs to M1-like TAMs and treat cancer with high efficacy and low toxicity. Summary of the Invention

[0006] Disclosed herein are compositions and methods for repolarizing tumor-associated macrophages (TAMs) from M2 to M1. In an embodiment, the method comprises administering to a subject in need thereof an effective dosage of a compound comprising a dextran scaffold and one or more CD206 targeting moieties attached to the scaffold. In an exemplary embodiment, The compounds include those of formula (I):

[0007] [ka]

[0008] During the ceremony, each X is independently H, L1-A, or L2-R; each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent.

[0009] According to certain further embodiments, the therapeutic agent is selected from paclitaxel, gemcitabine, lapatinib, and doxorubicin. In further embodiments, the therapeutic agent is doxorubicin. In certain aspects, administration of the composition to a subject results in reduced toxicity compared to an equivalent dose of the therapeutic agent not bound to the composition.

[0010] According to certain embodiments, the therapeutic agent is a bisphosphonate. In further embodiments, the bisphosphonate is zoledronic acid. In certain embodiments, the therapeutic agent comprises a chelator and at least one metal ion. In certain implementations, the at least one metal ion comprises at least one Cu(II) ion. In further embodiments, the at least one Cu(II) ion comprises about 4 Cu(II) ions. In further embodiments, the at least one Cu(II) ion is between about 1 Cu(II) ion and a number of Cu(II) ions equal to the number of chelator moieties.

[0011] In certain aspects, the compound is administered in combination with at least one other treatment or therapy.In certain embodiments, the at least one other treatment or therapy is chemotherapy, radiation therapy, or immunotherapy.According to certain implementations, the at least one other treatment or therapy is anti-CTLA4 immunotherapy.In exemplary implementations, the combination administration of the compound and at least one treatment or therapy has a synergistic effect compared to administration of either alone.

[0012] According to certain alternative embodiments, the compound does not comprise a therapeutic agent. Further disclosed herein are compounds for repolarizing TAM from M2 to M1, including compounds of formula (I):

[0013] [ka]

[0014] During the ceremony, each X is independently H, L1-A, or L2-R; each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent, the therapeutic agent comprising a chelator and at least one Cu(II) ion.

[0015] In certain implementations, at least one L1 is -(CH2)pS(CH2) q It contains —NH—, where p and q are integers from 0 to 5. In a further embodiment, at least one L2 is a C2-12 hydrocarbon chain optionally interrupted by up to three heteroatoms selected from the group consisting of O, S and N.

[0016] In yet further embodiments, at least one L2 is -(CH2)pS(CH2) q Contains -NH-, wherein p and q are independently an integer of 0 to 5. Further disclosed herein are compounds for repolarizing TAM from M2 to M1, including compounds of formula (I):

[0017] [ka]

[0018] wherein each X is independently H, L1-A, or L2-R; each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent, wherein the therapeutic agent comprises doxorubicin.

[0019] In certain implementations, each L1 is a hydrazone linker. In further implementations, the ratio of the mannose-binding C-type lectin receptor targeting moiety to the doxorubicin moiety is about 18.5 to about 1.5, and in yet further implementations, the dextran backbone is about 10 kD.

[0020] Further disclosed herein is a method of treating an infectious disease comprising administering to a subject in need thereof an effective amount of any of the compounds disclosed herein, wherein the compound is administered in a dosage sufficient to induce M2 macrophages to repolarize to M1 macrophages. In certain implementations, the infectious disease is selected from the following: dengue fever, tuberculosis, leishmaniasis, and human immunodeficiency virus. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows a graph of the decrease in geometric mean CD206 fluorescence of M2 macrophages from three volunteers after 23 hours (volunteer A) or 48 hours (volunteers B and C) of exposure to increasing concentrations of Cu(II)-Tilmanocept. [Diagram 2] Figure 2 shows a graph of the geometric mean CD206 ratios comparing Cu(II)-tilmanocept to +0 control from three volunteers after 23 or 48 hours of exposure to increasing concentrations of Cu(II)-tilmanocept. [Diagram 3] FIG. 3 shows the changes in CD80 and CD86 expression by M2 macrophages upon exposure to increasing concentrations of Cu(II)-tilmanocept for either 23 or 48 hours. [Figure 4] FIG. 4 shows the changes in CD80 and CD86 expression normalized to the change in CD206 expression in CD206+ M2 macrophages treated with increasing concentrations of Cu(II)-tilmanocept for either 23 or 48 hours. [Diagram 5] FIG. 5 shows normalized MO CD86 expression data from six experiments derived from four donors. [Figure 6] FIG. 6 shows normalized MO CD163 expression data from seven experiments derived from four donors. [Figure 7] FIG. 7 shows normalized MO CD64 expression data from five experiments derived from four donors. [Figure 8] FIG. 8 shows normalized MO CD206 control data from eight experiments derived from five donors. [Figure 9] FIG. 9 shows the growth of 4TI tumors in response to the six treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, a further embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by use of the antecedent "about," the particular value forms a further embodiment. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. Additionally, there are a number of values ​​disclosed herein, and each value is also understood to be disclosed herein as "about" that particular value in addition to the value itself. For example, when a value of "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0023] As used herein and in the concluding claims, a residue of a chemical species refers to a moiety that is a product resulting from that chemical species, regardless of whether that moiety is actually obtained from that chemical species in a particular reaction scheme or subsequent formulation of a chemical product. Thus, a residue of ethylene glycol in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a residue of sebacic acid in a polyester refers to one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0024] As used herein, the term "substituted" is expected to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, can have hydrogen substituents that satisfy the valence of the heteroatom, and / or any of the permissible substituents of organic compounds described herein. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Furthermore, the terms "substituted" or "substituted with" include the implicit proviso that such substitution is in accordance with the permissible valences of the atom and substituent being substituted, and that such substitution results in a stable compound, e.g., a compound that does not undergo spontaneous transformation by rearrangement, cyclization, elimination, etc. It is further contemplated that in certain embodiments, unless expressly stated to the contrary, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted).

[0025] In the definition of various terms, the present specification uses "A1", "A2", "A3" and "A4" as general symbols to represent various specific substituents. These symbols may be any substituent and are not limited to those disclosed herein. Even when they are defined as specific substituents in one example, in another example, they may be defined as some other substituent.

[0026] "R1", "R2", "R3", "Rn", where n is an integer, as used herein, may independently carry one or more of the groups listed above. For example, if R1 is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may be optionally replaced with a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the group selected, the first group may be incorporated within the second group, or alternatively, the first group may be pendant (i.e., appended) to the second group. For example, if there is a phrase "an alkyl group containing an amino group", the amino group may be incorporated within the backbone of the alkyl group. Alternatively, the amino group may be appended to the backbone of the alkyl group. The nature of the group selected will determine whether the first group is incorporated or appended to the second group.

[0027] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at every substitutable position of the group, and multiple positions in any given structure may be substituted with multiple substituents selected from the specified group, and the substituents may be the same or different at each position. Combinations of substituents envisioned by the present invention are preferably those combinations that result in the formation of stable or chemically feasible compounds. Furthermore, in certain embodiments, it is also anticipated that individual substituents may be further optionally substituted (i.e., further substituted or not substituted), unless expressly indicated to the contrary.

[0028] The specific materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques known to those of skill in the art. For example, starting materials and reagents used in preparing the disclosed compounds and compositions are available from commercial suppliers such as, for example, Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics, Inc. (Morris Plains, N.J.), Fisher Scientific, Inc. (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.), or may be readily obtained from, for example, 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, 1999). The compounds are prepared according to procedures described in references such as (E. G., Inc., 1989) and by methods known to those skilled in the art.

[0029] Disclosed are the components used to prepare the compositions of the present invention, as well as the compositions themselves used in the methods disclosed herein. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific references to the various individual and collective combinations and arrangements of each of these compounds cannot be explicitly disclosed, but each is specifically anticipated and described herein. For example, when a particular compound is disclosed and contemplated, and a number of modifications that can be made to a number of molecules that include the compound are contemplated, all possible combinations and arrangements of compounds and modifications are specifically anticipated unless specifically indicated to the contrary. Thus, when a type of molecule A, molecule B, and molecule C, and a type of molecule D, molecule E, and molecule F, and an example of a combination molecule AD are disclosed, each individually and collectively, the anticipated meaningful combinations of AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed with any particular embodiment, or combination of embodiments, of the methods of the invention.

[0030] As used herein, the term "pharmaceutically acceptable carrier" or "carrier" refers to sterile aqueous or non-aqueous solutions, colloids, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution immediately prior to use into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose, and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may further contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the effects of microorganisms may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceuticals may be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin. Injectable depot forms are prepared by forming microencapsule matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). The rate of drug release may be controlled depending on the ratio of the drug to the polymer and the nature of the particular polymer employed. Depot injectable formulations are prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations may 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 medium immediately before use. Suitable inert carriers include sugars, such as lactose. It is desirable that 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.

[0031] The term "polarization" is used herein to indicate the phenotypic and functional properties of macrophages. Phenotype may be defined through surface markers expressed by macrophages. Functionality may be defined, for example, based on the nature and amount of chemokines and / or cytokines expressed and especially secreted by macrophages. Indeed, macrophages exhibit different phenotypic and functional properties depending on their status, either pro-inflammatory M1 type macrophages or anti-inflammatory M2 type macrophages. M2 type macrophages may be characterized, for example, by the expression of surface markers such as CD206, CD11b, PD-L1, and CD200R, followed by the secretion of cytokines such as CCL17. M1 type macrophages may be defined, for example, by the expression of surface markers such as CD86 and CCR7, and the secretion of cytokines such as IL-6, TNF-a, and IL12p40. In the context of the present disclosure, "repolarization" refers to inducing a phenotypic change of M1 macrophage population towards M1 type macrophages.

[0032] As used herein, the term "cancer" refers to cells capable of autonomous growth. Examples of such cells include cells with abnormal processes or conditions characterized by rapidly proliferating cell proliferation. The term is intended to include cancerous growths, such as, for example, tumors; neoplastic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. It also includes malignant tumors of various organ systems, such as, for example, the respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, hepatic system, digestive system, and endocrine system, as well as adenocarcinomas, including malignant tumors, such as, for example, most colon cancers, renal cell carcinomas, prostate cancer, and / or testicular tumors, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer. "Naturally occurring" cancer includes any cancer that is not experimentally induced by the implantation of cancer cells into a subject, including, for example, naturally occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from the insertion of a transgenic cancer gene or from the knockout of a tumor suppressor gene, and cancers caused by infection, such as, for example, viral infection. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. In some embodiments, the method may be used to treat a subject with an epithelial cancer, such as a solid tumor of epithelial origin, such as, for example, lung, breast, ovarian, prostate, kidney, pancreatic, or colon cancer.

[0033] As used herein, the term "subject" refers to a target of administration, such as an animal. Thus, the subject of the methods disclosed herein may be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein may 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, it is intended to cover adult and neonatal subjects, as well as fetuses, regardless of gender. In one embodiment, 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 embodiments of the methods of the present disclosure, the subject has been diagnosed as in need of treatment for one or more cancer disorders prior to the administration step.

[0034] As used herein, the term "synergistic" means that the effect achieved with the methods and combinations of the invention is greater than the sum of the effects resulting from the use of the compounds, compositions, treatments and / or methods, or pharma- ceutically acceptable salts thereof, separately. Such synergistic effects are beneficial in that they provide greater efficacy at the same dose and / or prevent or delay the development of multidrug resistance.

[0035] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorate a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed to remove the cause of the associated disease, pathological condition or disorder. The term further includes palliative treatment, i.e., treatment designed to relieve symptoms but not cure a disease, pathological condition or disorder, preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition or disorder, and supportive treatment, i.e., treatment employed to supplement another specific treatment aimed at ameliorating the associated disease, pathological condition or disorder. In various embodiments, the term encompasses any treatment of a subject, including a mammal (e.g., a human), and includes (i) preventing the disease from occurring in a subject susceptible to the disease but not yet diagnosed as having the disease, (ii) inhibiting the disease, i.e., arresting its progression, or (iii) alleviating the disease, i.e., causing regression of the disease. In one embodiment, the subject is a mammal, such as a primate, and in a further embodiment, the subject is a human. The term "subject" also includes pets (e.g., cats, dogs, etc.), farm animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.).

[0036] As used herein, the term "prevent" or "preventing" refers to eliminating, avoiding, forestalling, preventing, halting, or impeding an occurrence, especially by proactive measures. Unless specifically indicated otherwise, when reduce, inhibit, or prevent are used herein, it is understood that the use of the other two terms is also expressly disclosed.

[0037] As used herein, the term "diagnosed" means that one has undergone a physical examination by a skilled artisan, such as a physician, and has been found to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with cancer" means that one has undergone a physical examination by a skilled artisan, such as a physician, and has been found to have a condition that can be diagnosed or treated by a compound or composition that can reduce the size of a tumor or slow the rate of tumor growth. A subject having cancer, a tumor, or at least one cancer or tumor cell may be identified using methods known in the art. For example, the anatomical location, gross size, and / or cellular composition of a cancer cell or tumor may be determined using contrast-enhanced MRI or CT. Additional methods for identifying cancer cells 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-sensitive composition of the present invention may be used in combination with, for example, MRI or CT to identify cancer cells.

[0038] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are known to those skilled in the art and include, but are not limited to, oral, transdermal, inhalation, nasal, topical, vaginal, ocular, auricular, intracerebral, rectal, sublingual, buccal, and parenteral administration, including injection, such as intravenous, intraarterial, invasive to a particular organ, intramuscular, intratumoral, and subcutaneous. Administration may be continuous or intermittent. In various embodiments, the preparation may be administered therapeutically, i.e., to treat an existing disease or condition. In further various embodiments, the preparation may be administered prophylactically, i.e., for the prevention of a disease or condition.

[0039] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired outcome or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic outcome or to have an effect on an undesired condition, 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 particular composition employed, the patient's age, weight, general health, sex and diet, time of administration, route of administration, rate of excretion of the particular compound employed, duration of treatment, drugs used in combination with or concurrently with the particular compound employed, and similar factors known in the medical art. For example, it is within the skill of the art to begin administering the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dosage may be divided into multiple dosages for administration purposes. As a result, a single administration composition may contain such amounts or submultiples thereof to constitute a daily dosage. The dosage may be adjusted by the individual physician in the event of any contraindications. The dosage may vary and one or more doses may be administered daily over a single or multiple day period. Guidance on appropriate dosages for a given type of pharmaceutical product can be found in the literature. In further various embodiments, the preparation may be administered in a "prophylactically effective amount", i.e., in an amount effective for the prevention of a disease or condition.

[0040] Effective doses can be initially estimated from in vitro assays. For example, initial doses for use in animals can be formulated to achieve circulating or serum concentrations of active compounds equal to or greater than the IC50 of a particular compound as measured in in vitro assays. It is within the skill of the art to calculate doses that achieve such circulating or serum concentrations, taking into account the bioavailability of a particular active agent. For guidance, see Fingl & Woodbury, "General Principles," In: Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, pp.1-46, latest edition, Pergamagon Press, which and the references cited therein are incorporated herein by reference in their entirety.

[0041] The phrase "anti-cancer composition" may include compositions that exert antineoplastic, chemotherapeutic, antiviral, antimitotic, antitumorigenic, antiangiogenic, antimetastatic, and / or immunotherapeutic effect, e.g., directly on tumor cells by cytostatic or cytocidal action, and indirectly, e.g., through mechanisms such as modification of biological responses, inhibiting the development, maturation, or spread of neoplastic cells. There are numerous anti-proliferative agents available in commercial use, clinical evaluation, and preclinical development that may be included in the present application with combination drug chemotherapy. For ease of review, antiproliferative agents are divided into the following classes, subtypes, and species: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatins, anthracyclines / DNA intercalators, anticancer antibiotics or antibiotic-type agents, antimetabolites, antimetabolite compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase, endostatin, epipodophyllotoxins, genistein. , hormonal anticancer agents, hydrophilic bile acids (URSOs), immunomodulatory or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, mixed antitumor agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radiation / chemotherapeutic agent sensitizers / protectors, retinoids, selective inhibitors of endothelial cell proliferation and migration, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids.

[0042] The major categories into which some antiproliferative agents fall include the antimetabolite, alkylating, antibiotic-type, hormonal anticancer, immunological, interferon-type, and mixed antitumor categories. Some antiproliferative agents act via multiple or unknown mechanisms and may therefore be classified into multiple categories.

[0043] "Tilmanocept" refers to the non-radiolabeled active pharmaceutical ingredient (API) of the LYMPHOSEEK® diagnostic agent. Tilmanocept is a mannosylaminodextran. Tilmanocept has a dextran backbone with multiple amino-terminated leashes (-O(CH2)3S(CH2)2NH2) attached to a core glucose element. Additionally, mannose moieties may be attached to the amino groups of many of the leashes, and the chelator diethylenetriamine pentaacetic acid (DTPA) may be attached to the amino groups of other leashes that do not contain mannose. Tilmanocept generally has a dextran backbone in which multiple glucose residues include the following amino-terminated leashes:

[0044] [ka]

[0045] The mannose moiety is attached to the amino group of the chain via an amidine linker:

[0046] [ka]

[0047] The chelating agent diethylenetriaminepentaacetic acid (DTPA) is attached to the amino group of the chain via an amide linker:

[0048] [ka]

[0049] Tilmanocept has the chemical name dextran 3-[(2-aminoethyl)thio]propyl 17-carboxy-10,13,16-tris(carboxymethyl)-8-oxo-4-thia-7,10,13,16-tetraazaheptadec-1-yl 3-[[2-[[1-imino-2-(D-mannopyranosylthio)ethyl]amino]ethyl]thio]propyl ether complex and Tilmanocept Tc99m has the molecular formula: [CH 10 O5] n .(C 19 H 28 N4O9S 99m Tc) b .(C 13 H 24 N2O5S2) c .(C5H 11 N.S. a and contains 3-8 bound DTPA molecules (b); 12-20 bound mannose molecules (c); and 0-17 amine side chains (a) which remain free. Tilmanocept has the following general structure:

[0050] [ka]

[0051] Some of the glucose moieties may not have an amino terminal chain attached. The present disclosure describes compositions and methods that have utility for changing or transitioning the phenotype of activated macrophages from an immunosuppressive phenotype to a pro-inflammatory phenotype (referred to herein as "repolarization"). The ability to change or transition 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 allow targeted delivery of small molecules and / or metal ions to TAMs with the goal of repolarizing the TAMs. Targeted delivery of TAMs provides a higher dose of small molecules and ions to the TAMs, enhancing the effectiveness of the phenotypic change while limiting potential toxic exposure to non-targeted cells and tissues. Through the use of the compositions and methods of the present disclosure, M2-like (immunosuppressive) activated macrophages can be induced to switch their phenotype to an M1-like (pro-inflammatory) activated phenotype by exposure to various small molecules or metal ions.

[0052] compound In certain embodiments, the compounds disclosed herein employ carrier constructs that include a polymer (e.g., carbohydrate) backbone conjugated with a mannose-binding C-lectin type receptor targeting moiety (e.g., mannose) to deliver one or more active therapeutic agents. Examples of such constructs include mannosylaminodextran (MAD). MAD includes a dextran backbone with mannose molecules attached to glucose residues of the backbone and active pharmaceutical ingredients attached to glucose residues of the backbone. Tilmanocept is a specific example of MAD. Tilmanocept derivatives, which are tilmanocept without DTPA attached, are a further example of MAD.

[0053] In certain implementations, the present disclosure provides compounds that include a dextran-based moiety or scaffold having one or more mannose-binding C-type lectin receptor targeting moieties and one or more therapeutic agents attached thereto. The dextran-based moiety generally includes a dextran backbone similar to that described in U.S. Patent No. 6,409,990 (the '990 patent), which is incorporated herein by reference. The backbone thus includes a plurality of glucose moieties (i.e., residues) that are linked primarily by α-1,6 glycosidic linkages. Other linkages, such as, for example, α-1,4 and / or α-1,3 linkages, may also be present. In some embodiments, not all backbone moieties are substituted. In some embodiments, the mannose-binding C-type lectin receptor targeting moieties are attached to about 10% to about 50% of the glucose residues of the dextran backbone, or about 20% to about 45% of the glucose residues, or about 25% to about 40% of the glucose residues. In some embodiments, the dextran-based moiety is about 50-100 kD. The dextran-based moiety may be at least about 50 kD, at least about 60 kD, at least about 70 kD, at least about 80 kD, or at least about 90 kD. The dextran-based moiety may be less than about 100 kD, less than about 90 kD, less than about 80 kD, less than about 70 kD, or less than about 60 kD. Alternatively, in some embodiments, the dextran backbone has a MW of about 1 to about 50 kDa, while in other embodiments, the dextran backbone has a MW of about 5 to about 25 kDa. In yet other embodiments, the dextran backbone has a MW of about 8 to about 15 kDa, e.g., about 10 kDa. In other embodiments, the dextran backbone has a MW of about 1 kDa to about 5 kDa, e.g., about 2 kDa.

[0054] According to further aspects, the mannose-binding C-type lectin receptor targeting moiety is selected from, but not limited to, mannose, fucose, and n-acetylglucosamine. In some embodiments, the targeting moiety is attached to about 10% to about 50% of the glucose residues of the dextran backbone, or about 20% to about 45% of the glucose residues, or about 25% to about 40% of the glucose residues. The MW referred to herein, as well as the number and degree of attachment of receptor substrates, chains and diagnostic / therapeutic moieties attached to the dextran backbone, refer to the average amount of a given number of carrier molecules, as some variation occurs due to synthesis techniques.

[0055] According to certain embodiments, the one or more mannose-binding C-type lectin receptor targeting moieties and the one or more therapeutic agents are attached to the dextran-based moiety by a linker. The linker may be attached at about 50% to about 100%, 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 amino-terminated linker. In some embodiments, the linker may comprise -O(CH2)3S(CH2)2NH-. In some embodiments, the linker may be a chain of 1-20 atoms selected from carbon, oxygen, sulfur, nitrogen, and phosphorus. The linker may be a straight or branched chain. Linkers may also include, but are not limited to, halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, e.g., C1-4 alkyl, alkenyl groups, e.g., C1-4 alkenyl, alkynyl groups, e.g., C1-4 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 or heteroaralkoxy groups, HO-(C=O)- groups, , heterocyclic groups, cycloalkyl groups, amino groups, alkylamino groups 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, =NH, =N-alkyl, -SH, -S-alkyl, -NH-C(O)-, -NH-C(=N)-, etc. Other suitable linkers are possible, as will be apparent to one of skill in the art.

[0056] In some embodiments, one or more therapeutic agents are attached via a biodegradable linker. In some embodiments, the biodegradable linker comprises a pH-sensitive moiety, such as a hydrazone. At low (more acidic) pH, the hydrazone linker spontaneously hydrolyzes at a faster rate as the pH decreases. Once mannosylated dextran binds to CD206, it is internalized into endosomes and gradually becomes acidic. The therapeutic payload is then released into the cell.

[0057] In certain embodiments, the therapeutic agent, when added to a MAD carrier disclosed herein, is capable of repolarizing M2-macrophages to M1-macrophages, hi further embodiments, the therapeutic agent is capable of inducing T cell activation.

[0058] In a further embodiment, the therapeutic agent is a cytotoxic agent. In yet a further embodiment, the therapeutic agent is an anti-cancer agent. In certain alternative embodiments, the compound is any of the carrier constructs comprising a polymeric (e.g., carbohydrate) backbone to which is attached a mannose-binding C-lectin type receptor targeting moiety as disclosed herein, but no therapeutic moiety / agent. In exemplary implementations of these embodiments, such compounds are administered in conjunction with one or more therapeutic modalities or therapies, as detailed below.

[0059] According to certain further embodiments, the therapeutic agent is selected from paclitaxel, gemcitabine, lapatinib, and doxorubicin. In further embodiments, the therapeutic agent is doxorubicin. In certain aspects, administration of the composition to a subject results in reduced toxicity compared to an equivalent dose of the therapeutic agent not bound to the composition.

[0060] According to certain embodiments, the therapeutic agent is a bisphosphonate. In an exemplary implementation of these embodiments, the bisphosphonate is zoledronic acid. In certain aspects, the therapeutic agent is a metal ion, hi certain exemplary embodiments, a metal such as, for example, arsenic, silver antimony, cadmium, gallium, or gadolinium is the therapeutic agent.

[0061] In exemplary embodiments, the metal ion is Cu(II). In exemplary aspects of these embodiments, the Cu(II) ion is attached to a chelator (described further below) on one or more chains. In certain aspects, the therapeutic agent is comprised of one or more Cu(II) ions per molecule of compound. In further embodiments, the therapeutic agent is comprised of from one Cu(II) ion to a number of Cu(II) ions equal to the number of chelator moieties. In still further embodiments, the number of Cu(II) ions is between 1 and 12 Cu(II) ions. In further embodiments, the number of Cu(II) ions is between 3 and 8 Cu(II) ions. In further embodiments, the therapeutic agent is comprised of about 4 Cu(II) ions.

[0062] In certain embodiments, a chelator may be added to or incorporated into the disclosed compounds and used to chelate a therapeutic agent, such as, for example, Cu(II). Exemplary chelators include, but are not limited to, DTPA (e.g., Mx-DTPA), DOTA, TETA, NETA, or NOTA. According to certain exemplary implementations, the chelator is DOTA.

[0063] Further disclosed herein are compounds for repolarizing TAM from M2 to M1, including compounds of formula (I):

[0064] [ka]

[0065] During the ceremony, each X is independently H, L1-A, or L2-R; each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent, the therapeutic agent comprising a chelator and at least one Cu(II) ion.

[0066] In certain implementations, at least one L1 is -(CH2)pS(CH2) q It contains —NH—, where p and q are integers from 0 to 5. In a further embodiment, at least one L2 is a C2-12 hydrocarbon chain optionally interrupted by up to three heteroatoms selected from the group consisting of O, S and N.

[0067] In yet further embodiments, at least one L2 is -(CH2)pS(CH2) q Contains -NH-, wherein p and q are independently an integer of 0 to 5. Further disclosed herein are compounds for repolarizing TAM from M2 to M1, including compounds of formula (I):

[0068] [ka]

[0069] wherein each X is independently H, L1-A, or L2-R; each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent, wherein the therapeutic agent comprises doxorubicin.

[0070] In certain implementations, each L1 is a hydrazone linker. In further implementations, the ratio of the mannose-binding C-type lectin receptor targeting moiety to the doxorubicin moiety is about 18.5 to about 1.5, and in yet further implementations, the dextran backbone is about 10 kD.

[0071] In a further embodiment, the disclosed composition is of formula (II):

[0072] [ka]

[0073] where * indicates the point at which a therapeutic agent is attached. In certain embodiments, the therapeutic agent is attached via a linker. According to certain embodiments, the disclosed compounds may include a pharma- ceutically acceptable carrier and a compound disclosed herein or a pharma- ceutically acceptable salt of a compound disclosed herein. The disclosed compounds, or a pharma- ceutically acceptable salt thereof, may be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds.

[0074] The pharmaceutical carrier employed may be, for example, solid, liquid or gas.Examples of solid carrier include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.Examples of liquid carrier include sugar syrup, peanut oil, olive oil and water.Examples of gas carrier include carbon dioxide and nitrogen.

[0075] In preparing compositions for oral dosage form, any convenient pharmaceutical medium may be adopted.For example, water, glycol, oil, alcohol, flavoring agent, preservative, coloring agent, etc. may be used to form oral liquid preparations such as suspension, elixir and solution, while carriers such as starch, sugar, microcrystalline cellulose, diluent, granulating agent, lubricant, binder, disintegrant, etc. may be used to form oral solid preparations such as powder, capsule and tablet.Because of ease of administration, tablets and capsules are the preferred oral dosage units, and therefore solid pharmaceutical carriers are adopted.Optionally, tablets may be coated by standard aqueous or non-aqueous techniques.

[0076] Further disclosed herein are methods of using the disclosed compounds. In certain embodiments, disclosed herein are methods of repolarizing tumor-associated macrophages (TAMs) from M2 to M1, comprising administering to a subject in need thereof an effective amount of a compound disclosed herein.

[0077] 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 injecting the compound intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intraocularly, intratumorally, or administering it transdermally, or delivering it directly to the tumor organ by invasive techniques.

[0078] In yet further embodiments, the method further comprises administering the composition in combination with at least one other treatment or therapy. In further embodiments, the other treatment or therapy comprises the co-administration of an anti-cancer agent. In further embodiments, the other treatment or therapy is chemotherapy. In certain embodiments, the compound is administered alone or in combination with other chemical therapeutic agents, or in combination with radiation therapy or hyperthermia or physical therapy or dietary therapy.

[0079] According to further embodiments, the at least one other treatment or therapy is an immunotherapy, such as, for example, administration of an immunomodulatory agent. According to certain implementations, the at least one other treatment or therapy is an anti-CTLA4 immunotherapy. In certain implementations, 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, non-glucocorticoid steroids, cytostatic agents, alkylating agents, nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, or the like. substances, antimetabolites, purine analogs, azathioprine, mercaptopurine, mycophenolic acid, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogs, methotrexate, cytotoxic antibiotics, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies or fusions thereof, antithymocyte globulin, antilymphocyte globulin, anti-IL-2 receptor antibodies, anti-CD3 antibodies, OKT3 (muromonab), otelixizumab, teplizumab, bilirubin Silizumab, anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, anti-CD11a antibody, efalizumab, anti-CD18 antibody, elizumab, rovelizumab, anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, lumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, acelizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gabi Rimomab, B-lymphocyte stimulator (BLyS) inhibitor antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belatacept, ipilimumab, tremelimumab, anti-eotaxin 1 antibody, bertilimumab, anti-α4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, odulimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab,Nelerimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomilikimab, levrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox), bapaliximab, beparimomab, aflibercept, alefacept, rilonacept, immunophilin modulators, rapamycin, calcinulin inhibitors, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, TNF inhibitors, infliximab, adalimumab, certolizumab Pegol, golimumab, etanercept, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, IL-1 receptor antagonist, anakinra, anti-IL-5 antibody, mepolizumab, IgE inhibitor, omalizumab, talizumab, IL12 inhibitor, IL23 inhibitor, ustekinumab, opioid, IMPDH inhibitor, mycophenolic acid, myriocin, fingolimod, NF-κB inhibitor, raloxifene, drotrecogin alfa, denosumab, NF-κB signaling Cascade inhibitors, disulfiram, olmesartan, dithiocarbamates, proteasome inhibitors, bortezomib, MG132, Pro1, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), 13C (indole-3-carbinol) / DIM (di-indolemethane) (I3C / DIM), Bay 11-7082, luteolin, cell penetrating peptide SN-50, IκBα super repressor overexpression, NFκB decoy oligodeoxynucleotide (ODN), or any derivative or analog thereof.

[0080] In an exemplary implementation, the combined administration of the compound and at least one treatment or therapy has a synergistic effect compared to administration of either alone. According to certain embodiments, administration of a compound disclosed herein in combination with another therapy or treatment is associated with reduced toxicity compared to administration of the other therapy or treatment alone. In further embodiments, co-administration of the disclosed compound with the other therapy or treatment produces a synergistic effect. In yet further embodiments, co-administration of the disclosed compound provides a lower effective dosage than the other therapy or treatment.

[0081] The methods provided herein may be performed in an adjuvant setting. In some embodiments, the methods are performed in a neoadjuvant setting. That is, the methods may be performed prior to a primary / definitive therapy. In some embodiments, the methods are used to treat individuals who have been previously treated. Individuals who have not been previously treated may be treated using any of the treatment methods provided herein. In some embodiments, the methods are used as a first-line therapy. In some embodiments, the methods are used as a second-line therapy.

[0082] According to certain embodiments, the subject has been diagnosed with melanoma, breast cancer, lung cancer, pancreatic cancer, renal cancer, ovarian cancer, prostate cancer or cervical cancer, glioblastoma or colorectal cancer, brain and spinal cord cancer, head and neck cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, bladder cancer, testicular cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, or any combination thereof.

[0083] In certain embodiments, the method further comprises administering the composition as a bolus and / or at regular intervals. In certain embodiments, the method of the present disclosure further comprises administering the composition intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally.

[0084] 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. According to yet a further aspect, the method further comprises evaluating the effectiveness of the composition. In a still further aspect, evaluating the effectiveness of the composition comprises measuring a tumor size prior to administration of the composition and measuring a tumor size after administration of the compound. In a further aspect, evaluating the effectiveness of the composition is performed at regular intervals. According to certain aspects, the method of the present disclosure further comprises optionally adjusting at least one aspect of the method. In a still further aspect, 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.

[0085] According to certain alternative embodiments, the subject is diagnosed with a disease associated with increased levels of CD206+ macrophages and / or MDSCs.Such diseases or conditions include, but are not limited to, acquired immune deficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic disease, alopecia areata, 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 cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Baro's disease / Baroconcentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal bone disease Myelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, caput arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type I, diabetes mellitus type II, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus erythematosus, 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, gastrointestinal pemphigoid, Gaucher disease, glomerulonephritis, Goodpasture syndrome, Graves disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henoch-Schönlein purpura, herpes gestationis (also known as pemphigoid of gestationis), hidradenitis suppurativa, HIV infection, Hughes-Stovin syndrome,Hypogammaglobulinemia, infections (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), lupus erythematosus, lymphomatoid granulomatosis, Majeed syndrome, malignancies 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, localized scleroderma, Mucca-Habermann disease (aka pityriasis lichenoidis acute), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (aka Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disorder, seizure disorder Nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schneider-Henry syndrome, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia ... Mitt's syndrome, Schnitzler's syndrome, Scleritis, Scleroderma, Sepsis, Serum sickness, Sjogren's syndrome, Spondyloarthropathy, Still's disease (adult onset), Stiff body syndrome, Stroke, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome, Sydenham's chorea, Sympathetic ophthalmia, Systemic lupus erythematosus, Takayasu's arteritis, Temporal arteritis (aka Giant cell arteritis), Thrombocytopenia, Tolosa-Hunt syndrome, Transplant (e.g. heart / lung transplant) rejection, Transverse myelitis, Tuberculosis, Ulcerative colitis, Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Urticarial vasculitis, Vasculitis, Vitiligo,and Wegener's granulomatosis.

[0086] Working Example The following examples are set forth to provide those of skill in the art with a complete disclosure and description of specific examples of how to make and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, one of ordinary skill in the art will recognize in light of this disclosure that many changes can be made in the specific embodiments that are disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0087] Example 1: Copper(II) [Cu(II)] tilmanocept repolarizes M2-like macrophages towards an M1-like phenotype. CD206 is expressed at higher levels on most M2-like macrophages, including M2-like TAMs, than on M1-like macrophages. Thus, tilmanocept is localized to M2-like TAMs. In initial experiments, tilmanocept was loaded with Cu[II] ions via chelation to the DTPA moiety of tilmanocept at a ratio of approximately 3.9 copper ions per tilmanocept molecule. Human peripheral blood mononuclear cells from three human volunteers were induced to adopt an M2-like phenotype by seeding in RPMI-1640 medium supplemented with fetal bovine serum at a final concentration of 10%, 2.0 g / L glucose, 0.3 g / L L-glutamine, 2.0 g / L NaHCO3, and 1 mL sodium pyruvate (11 g / L). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was also added to the culture medium at a concentration of 50 ng / ml. Flasks containing monocytes in this culture medium were incubated for 3 days to induce differentiation into CD206-expressing macrophages with an M2 phenotype. These cells also express the myeloid cell surface marker CD14.

[0088] CD14+ CD206+ M2 macrophages were then incubated in the same culture medium containing various concentrations of Cu(II)-tilmanocept: 0, 1, 2, 4, 8, 16 (ug / ml). These concentrations of Cu(II)-tilmanocept are equivalent to approximately 0, 50, 100, 200, 400, 800 nM. Cultures were incubated for 23 or 48 hours and then evaluated by flow cytology for expression of CD206 and for cell surface markers of macrophages with an M1-like phenotype, CD80 and CD86.

[0089] Treatment with Cu(II)-tilmanocept reduced CD206 expression on macrophages. Figure 1 shows that although there were differences between macrophages from three volunteer donors, the amount of CD206 immunofluorescence decreased from approximately 40-60+% upon Cu(II)-tilmanocept exposure compared to the geometric mean fluorescence of CD206+ macrophages in untreated control macrophages. This indicates that these macrophages transitioned from an M2-like phenotype to a more M1-like phenotype (Figure 1). Note that the majority of the observed changes in CD206 expression occurred at Cu(II)-tilmanocept concentrations of 1.0 μg / ml or 2.0 μg / ml (50 nM or 100 nM), as shown in Figure 2, and that the majority of this change occurred within 23 hours. Figure 2. Percentage of CD206 expression observed in macrophages treated with increasing concentrations of Cu(II)-tilmanocept for either 23 or 48 hours, compared to untreated controls. Macrophages from three donors were evaluated: red; volunteer donor A (23 h), blue and green; volunteer donors B and C (48 h). At these low concentrations of Cu(II)-tilmanocept (50 and 100 nM), Cu(II)-tilmanocept did not significantly increase the rate of cell death over that observed in untreated control macrophages incubated in the same medium for the same time (data not shown).

[0090] CD80 and CD86 are cell surface markers that can be expressed by a variety of immune cells. M1-like activated macrophages express higher levels of CD80 and CD86 than M2-like macrophages. CD80 and CD86 form a receptor complex to bind to CD28 expressed on T cells, resulting in T cell activation. Expression of both CD80 and CD86 increases with exposure to Cu(II)-tilmanocept, especially after 48 hours of exposure (Figure 3).

[0091] Although the trends toward increased expression of CD80 and CD86 were not as strong as those observed for decreased expression of CD206, a significant portion of the increased expression was observed at the relatively low concentration of 2.0 μg / ml (100 n m ) of Cu(II)-tilmanocept.

[0092] Repolarization of M2-like macrophages to M1-like macrophages is manifested as a decrease in the expression of M2 markers and an increase in the expression of M1 markers. Thus, a measure of the efficiency of repolarization by Cu(II)-tilmanocept can be represented by the change in CD80 and CD86 expression on Cu(II)-tilmanocept-treated macrophages compared to untreated controls (Figure 3), normalized to the change in CD206 expression on similarly treated macrophages (Figure 2). The results of this analysis are shown in Figure 4. The results of these CD206 expression normalization analyses show that Cu(II)-tilmanocept repolarizes M2-like macrophages to a more M1-like macrophage phenotype in a concentration- and time-dependent manner.

[0093] Example 2: Mannosylated dextran constructs carrying a doxorubicin payload repolarize M2-like macrophages towards an M1-like phenotype. The promising therapeutic strategy described in Example 1 is not limited to delivering metal ions to CD206-expressing macrophages, but can be employed to deliver a wide variety of small molecule payloads to CD206+ macrophages to repolarize them or change their phenotype. In a second experimental setting, mannosylated dextran carrying the cytotoxic drug payload doxorubicin was synthesized and evaluated. The construct, such as tilmanocept in Example 1, was synthesized on a 10 kD dextran backbone, with mannose moieties attached to the backbone via amine-terminated chains. The empty amine-terminated chains were attached to hydrazone linkers to which doxorubicin was added. Hydrazone linkers are known to release their payload more rapidly at lower than neutral pH. When CD206 binds to a ligand, such as mannosylated dextran, it is internalized into endosomes by receptor-mediated endocytosis. These endosomes undergo acidification to accelerate the release of the drug payload from the hydrazone linker. The construct evaluated in the experiments described in this Example carried an average of 18.5 mannose moieties and 1.5 doxorubicin moieties per 10 kD dextran backbone and is conveniently referred to in this study as MT1001.8.

[0094] Similar to Cu(II)-tilmanocept, MT1001.8 was evaluated using macrophages differentiated from human peripheral blood mononuclear cells cultured in supplemented RPMI-1640 medium supplemented with GM-CSF. The resulting macrophages were then exposed to MT1001.8 for 23 hours at various concentrations ranging from 0 (control) to 6.68 μM. Free doxorubicin and a mannosylated dextran construct without doxorubicin payload were also evaluated as controls. After 23 hours of exposure to the drug construct, the drug-containing medium was removed and replaced with fresh supplemented RPMI-1640 with GM-CSF and incubated for an additional 72 hours. This final 72 hours of incubation in fresh medium allowed the macrophages to recover from the drug treatment and express any phenotypic changes that may have been induced by exposure to the drug construct. All experiments were performed in 5-8 duplicates using peripheral blood mononuclear cells obtained from 5 separate donors. After a final 72-hour incubation, the medium was collected and analyzed for the concentration of various cytokines.Cells were harvested and assessed by flow cytometry for the expression of various cell surface markers of differentiation (CD206, CD64, CD163, CD80, and CD86), dose-dependent uptake of doxorubicin, and cell death.

[0095] Similar to the results observed in Example 1, different human donors provided differentiated macrophages that varied in their extent of response to MT1001.8. However, macrophages from all donors showed the same directional dose-dependent response. For example, CD86 expression was elevated in all macrophage cultures exposed to MT1001.8, but the maximal response varied between a 2.5-fold and 3.2-fold increase in expression at the highest concentration of MT1001.8. For simplicity and clarity, the data is presented as the change in expression relative to the maximum and minimum levels observed in each experiment and is displayed as a percentage of the maximum change. Free doxorubicin control results are not shown, as free doxorubicin produced high levels of cell death at all concentrations tested. MT1001.8 was significantly less toxic to macrophages, and cell viability was high at the end of the 72-hour drug exposure incubation, even at the highest concentration. Except for two cases discussed in the following paragraphs, results from mannosylated dextran without doxorubicin are not shown as this construct did not alter the expression of the markers examined in this study, nor are results shown demonstrating that MT1001.8 is taken up by CD206+ macrophages in a dose-dependent manner and at a rate approximately 25-fold higher than that observed for nonspecific binding to lymphocytes that do not express CD206.

[0096] Two cell surface differentiation markers, CD86 and CD163, showed significant and reproducible increases in expression upon exposure to MT1001.8 in a dose-dependent manner. At the highest dose tested (6.68 μM), CD86 and CD163 were elevated in expression levels by approximately 2.9-fold and 2.5-fold, respectively, in all experiments and in macrophage cultures from all human donors. These results are summarized in Figures 5 and 6, where the percentage of maximum change observed in macrophages from each donor at each dose of MT1001.8 is shown. The dose-response curves shown in these figures were very similar in each experiment, showing a nearly linear response to the log10 of dose exposure over the range of MT1001.8 concentrations evaluated. As shown in Figures 5 and 6, macrophages from some donors were evaluated in duplicate. The differences in the individual evaluations of macrophages derived from the same donor suggest that the majority of the differences observed between donors may be explained by inter-examination variability, and further suggest that the macrophage dose response is stable, reproducible, and highly similar between macrophages obtained from different donors.In contrast to CD86 and CD163, CD64 showed a dose-dependent (~40%) decrease in expression upon exposure to MT1001.8 (Figure 7).

[0097] An unexpected result was observed in that drug-free mannosylated dextran without drug payload altered the expression of two cell surface differentiation markers, CD206 and CD80. The expression level of CD80 increased by approximately 60% at the highest concentration of either drug-free mannosylated dextran or MT1001.8, suggesting that it was the mannosylated dextran, and not the doxorubicin payload of MT1001.8, that was responsible for the change in CD80 expression. It is important to note that the degree of increase in CD80 expression was much lower than that observed for CD86 upon exposure to MT1001.8. However, it may be important to note that both CD80 and CD86 bind to CD28 and CTLA4. When bound to CD28, it activates the proinflammatory response of T cells. When their competing ligand, CTLA4, is bound, T cell activation is attenuated. CD206 expression was also altered by exposure to either drug-free mannosylated dextran or MT1001.8. However, unlike CD80, CD206 expression was reduced by 40-50% at the highest concentration of either construct. This effect is unlikely to be a simple consequence of ligand binding. Ligand binding may reduce the amount of CD206 receptor presented on the cell surface by internalization of the receptor-ligand complex. However, the 72-hour incubation after removal of drug-containing medium would have allowed time for replacement or recycling of CD206 receptor to the cell surface. This suggests that the reduction in CD206 expression is a phenotypic change and not a transient consequence of receptor binding and internalization. It is also noteworthy that, as in Example 1, 50% of the maximum response to the five surface markers was achieved at concentrations of 1-2 μM MT1001.8 or, where appropriate, drug-free mannosylated dextran.

[0098] At the end of the final 72-hour incubation, media was collected and assessed for concentrations of various cytokines to identify cytokines whose expression was increased as a result of exposure to MT1001.8. There was considerable variation between baseline (no drug-saline control) levels of cytokine expression in macrophage cultures derived from different donors. Therefore, data was analyzed as the ratio of cytokine concentrations in drug-treated cultures compared to concentrations observed in donor-specific drug-free controls (saline). Increased concentrations were observed at higher concentrations of MT1001.8 treatment for some of the cytokines evaluated. This was shown by the mean ratios observed in MT1001.8 macrophage cultures, which were greater than 1.0, with a standard deviation greater than 1.0. 1.0 is the ratio expected if no changes in cytokine expression occurred. Interferon gamma (IFN-g), interleukin 2b (IL2b), IL12p70, tumor necrosis factor alpha (TNFa), IL8, and IL10 were all observed to have increased expression levels by this criterion, whereas IL6, although elevated in expression, did not exceed the one standard deviation threshold due to high variance in the data. No increases were observed in medium concentrations of IL4, IL5, and IL7.

[0099] With the exception of IL10, all cytokines observed to have increased expression levels are pro-inflammatory and would be predicted to promote pro-inflammatory activation of various immune cell types, including lymphocytes. The observed changes in cell surface differentiation markers, particularly increased expression of CD86 and CD80 and decreased expression of CD206, also indicate a shift in macrophage gene expression toward a more pro-inflammatory pattern. The increased expression of CD163 is intriguing in the context of a recent publication showing that tumor-associated macrophages (TAMs) with increased expression of CD163 and decreased expression of CD206 were associated with improved outcomes in patients with gastric cancer (62). Taken together, the changes in expression of both cell surface markers and cytokines suggest that MT1001.8-treated macrophages are altering their overall phenotype to become more M1-like and pro-inflammatory. Although the observed decrease in CD64 and increase in IL10 expression is not entirely consistent with a pro-inflammatory phenotype, the large number of other genes whose expression was altered indicates that MT1001.8-treated macrophages are repolarized toward a much more pro-inflammatory phenotype.

[0100] The increased expression of CD80 and CD86, together with the increased expression of proinflammatory cytokines, suggests that MT1001.8 or other similar mannosylated dextran constructs carrying doxorubicin payloads may be effective as anti-cancer immunotherapies, and perhaps especially in combination with other cancer therapies. Such combinations include radiation therapy, other cytotoxic agents besides doxorubicin, and perhaps other cancer immunotherapies specifically designed to activate lymphocytes. Anti-CTLA4 antibodies are a regulatory approved anti-cancer immunotherapy designed to activate T-cell lymphocytes. Because of the interactions between CD80 and CD86, and between CTLA4 and CD28, it was hypothesized that MT1001.8 may improve the efficacy of anti-CTLA4 immunotherapy. To test this hypothesis, experiments were performed using a 4T1 syngeneic breast cancer model in Balb / c mice. The 4T1 tumor model is largely resistant to treatment with anti-CTLA4, and it was hypothesized that MT1001.8 might rescue the therapeutic efficacy of this immunotherapy. 4T1 cells were implanted into mice and grew to approximately 50 mm 3 Tumors were allowed to form in 1000 mice. Six treatment groups were selected: saline control, drug-free mannosylated dextran control (vehicle), MT1001.8, free doxorubicin, anti-CTLA4 antibody, or a combination of anti-CTLA4 and MT1001.8. Animals received tail vein injections of 500 μg of MT1001.8, vehicle control, saline control, or free doxorubicin (equimolar to MT1001.8) every 2 days. Anti-CTLA4 antibody was administered twice weekly at a dose of 500 μg as an intraperitoneal (IP) injection. Experimental treatments were performed for 13 days. Growth of 4T1 tumors in response to each treatment is shown in Figure 9.

[0101] As shown in Figure 9, except for the combination therapy of anti-CTLA4 + MT1001.8, none of the treatment groups had a mean tumor volume that was significantly different from either the saline control group or the vehicle control group. Compared to the vehicle control, the group treated with anti-CTLA4 + MT1001.8 had a mean tumor volume of 69% of the vehicle control at the end of the experiment. In a two-tailed T-test, this result was significant at p = 0.02. This demonstrated that MT1001.8 could partially rescue the efficacy of anti-CTLA therapy in this typically refractile tumor model. This cooperative effect between anti-CTLA4 and MT1001.8 is expected to be widely applicable to other cancer treatments other than anti-CTLA4, and is expected to be widely applicable to other mannosylated dextran drug delivery vehicles that deliver other small molecule payloads, such as copper-loaded DTPA (Example 1).

[0102] Infectious disease: In addition to cancer, CD206+ M2-like macrophages are important contributors to the pathobiology of numerous infectious diseases. Repolarization of macrophages from an M2-like to an M1-like activated phenotype may have therapeutic benefits in people with these diseases. Examples include dengue fever, caused by a vector-borne flavivirus, the bacterial infection tuberculosis, and the protozoan infection Leishmania. All of these pathogens replicate in CD206+ macrophages and / or invade these cells via interactions with CD206. Human immunodeficiency virus (HIV) causes acquired immune deficiency syndrome (AIDS). Currently, HIV viremia and many of the symptoms of AIDS can be controlled with combined antiretroviral therapy (cART). However, a persistent reservoir of cART-resistant cells exists in patients treated with cART, preventing curative treatment with cART. A critical reservoir of cART resistance consists of CD206+ macrophages. Furthermore, CD206+ macrophages are contributors to the pathobiology of several parasites.

Claims

1. A kit for repolarizing tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype, comprising: The compounds include compounds of formula (I) comprising a dextran backbone and one or more CD206 targeting moieties attached thereto: 【Chemistry 1】 During the ceremony, each X is independently H, L1-A, or L2-R; Each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and at least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine; at least one A comprises a therapeutic agent, said therapeutic agent being selected from paclitaxel, gemcitabine, lapatinib, doxorubicin, and a bisphosphonate; each R is mannose, fucose, n-acetylglucosamine, or H; and n is an integer greater than zero; at least one A is said therapeutic agent and at least one R is mannose; The compound is to be administered in an effective dosage to a subject in need thereof.

2. The kit of claim 1 , wherein administration of the compound to the subject has reduced toxicity compared to an equivalent dose of the therapeutic agent not conjugated to the compound.

3. 10. The kit of claim 1, wherein the therapeutic agent comprises a bisphosphonate or comprises a chelating agent and at least one metal ion, and the therapeutic agent is attached to the dextran backbone via a biodegradable linker.

4. The kit of claim 3, wherein the bisphosphonate is zoledronic acid.

5. 4. The kit of claim 3, wherein the at least one metal ion is between one Cu(II) ion and a number of Cu(II) ions equal to the number of chelator moieties.

6. 2. The kit of claim 1, wherein the compound is administered in combination with at least one other therapy or treatment, the at least one other therapy or treatment being chemotherapy, radiation therapy, or immunotherapy, and the combined administration of the compound with the at least one therapy or treatment has a synergistic effect compared to administration of either alone.

7. The kit of claim 6, wherein the at least one other treatment or therapy is an anti-CTLA4 immunotherapy.

8. The kit of claim 1 , wherein the compound does not include a therapeutic agent.

9. A composition for repolarizing tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype, comprising a compound of formula (I): 【Chemistry 2】 During the ceremony, each X is independently H, L1-A, or L2-R; Each of L1 and L2 is independently a linker; each A independently comprises a therapeutic agent or H; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, and At least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine, and at least one A comprises a therapeutic agent, said therapeutic agent comprising paclitaxel, gemcitabine, lapatinib, doxorubicin, a bisphosphonate, or a chelator and at least one metal ion; composition.

10. At least one L1 comprises -(CH2)pS(CH2)q-NH-, where p and q are integers from 0 to 5; and / or At least one L2 contains -(CH2)pS(CH2)q-NH-; The composition of claim 9, wherein p and q are independently integers from 0 to 5.

11. 10. The composition of claim 9, wherein at least one L2 is a C2-12 hydrocarbon chain optionally interrupted by up to three heteroatoms selected from the group consisting of O, S and N.

12. The therapeutic agent comprises doxorubicin, 10. The composition of claim 9, wherein each L1 is a biodegradable linker and the ratio of the mannose-binding C-type lectin receptor targeting moiety to the doxorubicin moiety is from 18.5 to 1.

5.

13. The composition of claim 9, wherein the dextran backbone is between 1 kDa and 50 kDa.

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