Amide linkage of the sugar moiety to an amine-terminated leash attached to a carbohydrate polymer

Abstract

Methods are provided for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold using an amide bond, which can be found between a leash, such as an amine-terminated leash, and the mannose-binding C-type lectin receptor targeting moiety. The disclosed compounds and compositions utilizing this amide bond provide highly stable compounds that significantly reduce loss of the mannose-binding C-type lectin receptor targeting moiety from the polymeric carbohydrate scaffold.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. Section 119(e) to and the benefit of U.S. Provisional Application No. 63 / 412,804, filed October 3, 2022, entitled "Amide Linkages of Sugar Moieties to Amine Terminated Linkers Attached to Carbohydrate Polymers," which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates to therapeutic and diagnostic constructs, methods for making and using the same, particularly to the attachment of sugar moieties to polymeric carbohydrate backbones via amide bonds. [Background technology]

[0003] Mannosylated amine dextrans (MADs) have been developed as targeted imaging agents and drug delivery vehicles that bind to C-type lectins, particularly the mannose receptor (MR or CD206). One example of an MAD, tilmanocept, has been approved by the FDA and EMA as the active pharmaceutical ingredient in the imaging agent Lymphoseek®. Lymphoseek® is approved for use in the identification of sentinel lymph nodes (SLNs) during surgical procedures to remove cancerous tumors. Tilmanocept is a synthetic molecular construct built on a 10 kDa (MW) backbone of the carbohydrate dextran, a polymer of glucose. This dextran backbone contains a variable number of amine-terminated leashes, typically 30–40, attached to the hydroxyl groups of the glucose moieties of the 10 kDa dextran. These amine-terminated linkers are conjugated with typically 3–8 moieties of the chelator DTPA. The DTPA moiety allows localization of Tc99m-tilmanocept, detected by handheld gamma probe or SPECT. 99mTilmanocept can be radiolabeled with various radioactive metal ions, such as technetium (Tc99m). After DTPA addition, the remaining amine-terminated linker is unoccupied and serves as a substrate for the addition of the sugar mannose. Tilmanocept has 12 to 20 mannose moieties attached to some of these unoccupied linkers.

[0004] In current clinical practice, 99m Technetium-labeled tilmanocept (i.e., Lymphoseek®) is injected into the tumor bed or tissues near the tumor. From there, it enters the tumor's lymphatic drainage and is transported via lymphatic vessels to the first lymph node encountered, the SLN. Lymph nodes contain large numbers of CD206-expressing macrophages and dendritic cells. Tc 99m -Tilmanocept binds to CD206 on these cells and is internalized by receptor-mediated endocytosis. Retained radioactivity is detected by the oncological surgeon, who removes the SLN, which is then examined by a pathologist who determines whether the SLN contains tumor nodal metastasis.

[0005] The synthesis of the prototype tilmanocept construct is described in U.S. Patent No. 5,629,499 (hereinafter the "Vera Patent"), which is incorporated herein by reference in its entirety. The synthesis protocol described in the Vera Patent is still used to make the commercial tilmanocept used in Lymphoseek®. For simplicity, Tc 99mA stylized structure of tilmanocept is shown in Figure 1. However, tilmanocept can differ from the simplified structure shown in Figure 1 in the following ways: (1) an amine-terminated leash may be attached to any hydroxyl group on the glucose moiety of the dextran backbone, (2) some of the glucose moieties in the dextran backbone may not have an amine-terminated leash attached, and (3) some glucose moieties may have two or more amine-terminated leashes attached to different hydroxyl groups. While tilmanocept may incorporate the above modifications, current synthetic protocols do not provide for a modification that attaches a mannose moiety to an amine-terminated leash.

[0006] Under the current synthetic protocol, the mannose moiety of tilmanocept is attached to an amine-terminated leash via the formation of an amidine bond. However, the amidine bond of the mannose moiety presents several challenges. The amidine bond is unstable in aqueous solution, especially at alkaline pH. The products of hydrolysis of the amidine bond include regenerated amine-terminated leashes and undesirable mannose-related impurities. Furthermore, purifying the mannosylation reaction product by ultrafiltration with water leads to the continuous generation of undesirable impurities and a continuous decrease in the number of mannose moieties retained on the MAD construct. In many production batches, it has been observed that the number of mannose moieties retained on MAD is reduced sufficiently by hydrolysis of the amidine bond to impair the binding of MAD to CD206. This result necessitates repeated mannosylation reactions with MAD. This repeated mannosylation reaction is not only expensive but also time-consuming.

[0007] Therefore, current technology faces challenges in attaching the same or similar number of mannose moieties to the MAD backbone. This lack of control in the chemical synthesis process results in undesirable product variability in the final MAD product (i.e., tilmanocept). The lack of reliability and reproducibility of the amidine attachment is highly problematic for the scalability of the production of MAD-based therapeutic and diagnostic drugs, and would require orders of magnitude more product than is currently required for the use of tilmanocept as an imaging agent. Furthermore, the instability of the amidine mannose bond reduces the shelf life of tilmanocept and Lymphoseek® drug products, potentially resulting in waste. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 6,409,990 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there remains a need for compositions and methods that improve the attachment of mannose to carbohydrate backbones to address the current problems identified above. [Means for solving the problem]

[0010] Provided herein are compounds and compositions comprising a carbohydrate polymer backbone and a mannose moiety attached thereto via an amine terminated leash and an amide linker, as well as methods of making and using such compounds.

[0011] In Example 1, the compound comprises a polymeric carbohydrate backbone comprising at least one amine-terminated leash attached thereto, and one or more mannose-binding C-type lectin receptor targeting moieties, wherein the one or more mannose-binding C-type lectin receptor targeting moieties are attached to the amine-terminated leash via an amide linker.

[0012] Example 2 relates to compounds according to Example 1, wherein the carbohydrate backbone comprises a monosaccharide, a disaccharide, or a polysaccharide. Example 3 relates to compounds according to Examples 1 or 2, wherein the carbohydrate backbone comprises dextran, cellulose, mannan, chitin, or hyaluronic acid.

[0013] Example 3 illustrates a compound having the formula (I):

[0014] [ka]

[0015] and During the ceremony, each X is independently H, L1-A, L1-YA, L2-R, or L3-H, and each X is bonded to any OH group; L1, L2, and L3 are each independently a leash; each A independently comprises a therapeutic agent, a diagnostic agent, or a theranostic agent; each Y independently comprises a chelating agent; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety; n is an integer greater than zero, and each unit of n may be the same or different; At least one R is present, The compound according to any one of Examples 1 to 3, wherein L2 and R are linked via an amide linker.

[0016] Example 5 relates to a compound according to any one of Examples 1-4, wherein the amine-terminated leash comprises a linear or branched chain having from about 1 to about 20 member atoms selected from the group consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus.

[0017] Example 6 is where each L2 has the following formula:

[0018] [ka]

[0019] wherein bond 1A is attached to a hydroxide group of the polymeric carbohydrate backbone and y is an integer from 1 to 6. Example 7 is a group in which L1, L2, or L3 are each independently -(CH2) p S(CH2) q The compound according to any one of Examples 1 to 5 contains -NH-, wherein p and q are integers from 0 to 5.

[0020] Example 8 relates to a compound according to any one of Examples 1 to 7, wherein L2 does not contain an amidine bond. Example 9 relates to a compound according to any one of Examples 1 to 7, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, a high mannose glycan or mannose oligosaccharide, fucose, n-acetylglucosamine, a peptide, galactose, or a combination thereof.

[0021] Example 10 is a compound in which at least one subunit of formula (I) is

[0022] [ka]

[0023] The present invention relates to a compound according to any one of Examples 1 to 9, including Example 11 illustrates a compound having the formula (Ia):

[0024] [ka]

[0025] wherein * indicates the point at which a therapeutic, diagnostic, or theranostic agent is attached, and y is an integer from 1 to 6.

[0026] Example 12 relates to a compound according to any one of Examples 1 to 11, wherein the polymeric carbohydrate backbone has a molecular weight of from about 1 kD to about 150 kD. In Example 13, the pharmaceutical composition comprises a compound according to any one of Examples 1-10 and a pharmaceutically acceptable carrier.

[0027] Example 14 illustrates a compound having the formula (I):

[0028] [ka]

[0029] and During the ceremony, each X is independently H, L1-A, L1-YA, L2-R, or L3-H, and each X is bonded to any OH group; L1, L2, and L3 are each independently a leash; each A independently comprises a therapeutic agent, a diagnostic agent, or a theranostic agent; each Y independently comprises a chelating agent; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety; n is an integer greater than zero, and each unit of n may be the same or different; At least one R is present, Relating to the composition according to Example 13, wherein L2 and R are linked via an amide linker.

[0030] Example 15 relates to a composition according to Examples 13 or 14, wherein the amine-terminated leash comprises a linear or branched chain having from about 1 to about 20 member atoms selected from the group consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus.

[0031] Example 16 is where each L2 has the following formula:

[0032] [ka]

[0033] wherein bond 1A is attached to a hydroxide group on the polymeric carbohydrate backbone and y is an integer from 1 to 6. In Example 17, a method for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold includes: (a) synthesizing a mannose-binding C-type lectin receptor targeting moiety comprising an anomeric thiocarboxylic acid moiety; (b) converting the mannose-binding C-type lectin receptor targeting moiety comprising the anomeric thiocarboxylic acid moiety into an activated N-hydroxysuccinimide carboxylic acid ester; and (c) reacting the activated N-hydroxysuccinimide carboxylic acid ester with a polymeric carbohydrate scaffold having one or more amine-terminated leashes attached thereto, wherein reaction of the activated N-hydroxysuccinimide carboxylic acid ester with the polymeric carbohydrate scaffold having one or more amine-terminated leashes attached thereto forms an amide bond between the one or more amine-terminated leashes and the carbonyl carbon of the activated N-hydroxysuccinimide carboxylic acid ester.

[0034] Example 18 relates to a method according to Example 17, wherein the method results in retention of about 80% of the mannose-binding C-type lectin receptor targeting moiety on the polymeric carbohydrate scaffold after about 20 hours. Example 19 relates to a method according to Example 17 or 18, wherein the dextran backbone has a molecular weight of about 1 kD to about 150 kD.

[0035] Example 20 relates to a method according to any one of Examples 17-19, wherein the polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto is formed before step (a) or at a point between step (a) and step (c).

[0036] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 shows a diagram of the current prior art structure of tilmanocept, which includes the amidine linkage of mannose to amine-terminated leashes on a dextran backbone. [Figure 2] 1 shows the chemical structures of MAD constructs, illustrating a comparison of amidine and amide bonds for attaching the mannose moiety to the amine-terminated leash of MAD. [Figure 3] The synthesis of reaction compounds 1 and 2 is shown. [Figure 4] The synthesis of reaction compounds 3, 4, 5, and 6 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0038] Various embodiments of the present disclosure will now be described in detail with reference to the drawings. Reference to various embodiments does 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 purposes of the present disclosure.

[0039] The embodiments of the present disclosure are not limited to specific polymeric carbohydrate compositions, which may vary and are understood by those skilled in the art. Furthermore, it should be understood that all terms used herein are intended to describe particular embodiments only and are not intended to be limiting in any manner or scope. In order that the present disclosure may be more readily understood, certain terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the embodiments of the present disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, and preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terms will be used in accordance with the definitions set forth below.

[0040] Numerical ranges recited herein are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of the disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges, fractions, and individual numbers within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6, as well as decimals and fractions, e.g., 1.2, 3.8, 1 1 / 2, and 4 3 / 4. This applies regardless of the breadth of the range.

[0041] As used herein, the term "about" refers to variations in numerical quantities that may occur with typical measurement techniques and equipment for any quantifiable variable, including, but not limited to, mass, volume, temperature, and time. Furthermore, given real-world solid and liquid handling procedures, certain unintentional errors and variations likely exist due to differences in manufacture, source, or purity of ingredients used to make a composition or perform a method, etc. Whether modified by the term "about," the claims include equivalents to the quantities.

[0042] The terms "active" or "percent active" or "weight percent active" or "active concentration" are used interchangeably herein and refer to the concentration of ingredients participating in cleaning expressed as a percent minus inactive ingredients such as water or salt. This is sometimes indicated as a percentage in parentheses, e.g., "chemical (10%)."

[0043] Disclosed are the components used to prepare the disclosed compositions, and the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, when a particular compound is disclosed and discussed, and numerous modifications that can be made to some molecules, including the compound, are discussed, any and all combinations and permutations of the compound and possible modifications are specifically contemplated unless specifically indicated to the contrary.

[0044] Thus, if a class of molecules A, B, and C, and a class of molecules D, E, and F are disclosed, and an example of a combination molecule AD is disclosed, it is intended to mean that, even if each is not individually listed, each individually and collectively, the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the subgroups A-E, B-F, 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 in any specific embodiment or combination of embodiments of the methods of the invention.

[0045] 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 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, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, 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 also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by the inclusion of 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 and sodium chloride. Prolonged absorption of injectable pharmaceutical forms can be achieved by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The rate of drug release can be controlled depending on the drug-to-polymer ratio and the nature of the particular polymer used. Depot injectable formulations are also prepared by entrapping 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 sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before 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.

[0046] The term "polarization" is used herein to refer to the phenotypic and functional characteristics of macrophages. Phenotype can be defined through the surface markers expressed by macrophages. Functionality can be defined, for example, based on the nature and quantity of chemokines and / or cytokines expressed, particularly secreted, by macrophages. Indeed, depending on their state, macrophages display distinct phenotypic and functional characteristics as either pro-inflammatory (M1 type) or anti-inflammatory (M2 type) macrophages. M2 type 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 type macrophages can be defined by the expression of surface markers such as CD86 and CCR7, and the secretion of cytokines such as IL-6, TNF-α, and IL12p40. In the context of the present disclosure, the term "repolarize" is used herein to refer to the induction of a phenotypic change of an M1 macrophage population towards M1-type macrophages.

[0047] As used herein, the term "cancer" refers to cells capable of autonomous growth. Examples of such cells include cells with an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, including most colon, renal cell, prostate, and / or testicular cancers, non-small cell lung, small intestine, and esophageal cancers. "Spontaneously occurring" cancers include any cancer not experimentally induced by the implantation of cancer cells into a subject, including, for example, spontaneously induced cancers, cancers caused by patient exposure to carcinogen(s), cancers resulting from the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancers caused by infection, e.g., viral infection. The term "cancer" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. In some embodiments, the methods can be used to treat a subject with an epithelial cancer, e.g., a solid tumor of epithelial origin, e.g., lung cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer, or colon cancer.

[0048] 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, it is intended to encompass adult and neonatal (newborn) subjects, as well as fetuses (embryos), whether male or female. 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 with a need for treatment of one or more cancer disorders prior to the administering step.

[0049] As used herein, the term "synergistic" or "synergistically" means that the effect achieved with the methods and combinations of the invention is greater than the sum of the effects resulting from using the compounds, compositions, treatments, and / or pharmaceutically acceptable salts thereof separately. Advantageously, such a synergistic effect provides greater efficacy at the same dose and / or prevents or slows the development of multidrug resistance.

[0050] 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, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment directed at minimizing or partially or completely suppressing the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed at ameliorating the associated disease, pathological condition, or disorder. In various aspects, 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 who may be predisposed to the disease but has not yet been diagnosed as having it, (ii) inhibiting the disease, i.e., arresting its onset, or (iii) relieving the disease, i.e., causing regression of the disease.

[0051] As used herein, the term "diagnosed" means having undergone a physical examination by a skilled artisan, e.g., a physician, and 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 having undergone a physical examination by a skilled artisan, e.g., a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can reduce tumor size or slow the rate of tumor growth. A subject having cancer, a tumor, or at least one cancer or tumor cell can be identified using methods known in the art. For example, the anatomical location, total size, and / or cellular composition of cancer cells or tumors can be determined using contrast-enhanced MRI or CT. Additional methods for identifying cancer cells may 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 in combination with MRI or CT, for example, to identify cancer cells.

[0052] 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 of skill in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration (including injection, such as intravenous administration, intraarterial administration, administration to a specific organ by infiltration, intramuscular administration, intratumoral administration, and subcutaneous administration). Administration can be continuous or intermittent. In various aspects, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various aspects, the preparation can be administered prophylactically, i.e., administered for the prevention of a disease or condition.

[0053] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired result or to affect an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to have an effect on undesired symptoms, but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on various factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex, and diet; the timing of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concomitantly with the specific compound used; and similar factors well known in the medical field. For example, it is well within the skill of the art to start a dose of a 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, an effective daily dose can be divided into multiple doses for administration. Consequently, a single-dose composition can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical product. In further various embodiments, the preparations can be administered in a "prophylactically effective amount," i.e., an amount effective for the prevention of a disease or condition.

[0054] An effective dose may be estimated initially from in vitro assays. For example, an initial dose for use in animals may be determined based on the IC 50The active compound may be formulated to achieve a circulating blood or serum concentration of the active compound that is equal to or greater than 100 mg / kg. Calculating the dosage to achieve such a circulating blood or serum (scrum) concentration, taking into account the bioavailability of a particular active agent, is well within the ability of one skilled 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 (incorporated herein by reference in its entirety), and the references cited therein.

[0055] The phrase "anti-cancer composition" can include compositions that exert antineoplastic, chemotherapeutic, antiviral, antimitotic, antitumor, antiangiogenic, anti-metastatic, and / or immunotherapeutic effects, e.g., compositions that can prevent the development, maturation, or spread of tumor cells directly, e.g., by cytostatic or cytocidal effects on tumor cells, and indirectly through mechanisms such as biological response modification. There are numerous anti-proliferative agents available in commercial use, clinical evaluation, and preclinical development, which may be included in this application through combination drug chemotherapy. For convenience of discussion, anti-proliferative agents are divided into the following classes, subtypes, and species: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracycline / DNA intercalators, anti-cancer antibiotics or antibiotic-type agents, antimetabolites, anti-metastatic compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerases, endostatin, epipodophyllotoxins, Genistein, hormonal anticancer agents, hydrophilic bile acids (URSOs), immunomodulatory or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, various antineoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radiosensitizers / chemosensitizers / protectants, retinoids, selective inhibitors of endothelial cell proliferation and migration, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids. 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 agents. Some antiproliferative agents function via multiple or unknown mechanisms and therefore can be classified into more than one category.

[0056] The terms "weight percent," "wt. %," "wt-%," "percent by weight," "% by weight," and variations thereof, as used herein, refer to the concentration of a substance as calculated by dividing the weight of that substance by the total weight of the composition and multiplying by 100.

[0057] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein 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.

[0058] The present disclosure provides novel compositions for mannosylated polymeric carbohydrate scaffolds containing MADs, such as tilmanocept, and methods for synthesizing these novel compositions. The present disclosure differs from current state-of-the-art MAD constructs, such as those described in the Vera patent, by replacing the amidine bond used to attach the mannose moiety to the amine-terminated leash of the MAD with an amide bond. Illustrative examples of currently used amidine and amide bonds in the present disclosure are shown in Figure 2. As described herein, the currently used amidine bond between the mannose and the amine-terminated leash presents numerous stability and production challenges. Therefore, in some embodiments, it would be preferable to load mannose or some other mannose-binding C-type lectin receptor targeting moiety onto a polymeric carbohydrate scaffold with specific and reproducible targeting range. In beneficial embodiments, amide-linked mannose-binding C-type lectin receptor targeting moieties are more stable than amidine-linked mannose-binding C-type lectin receptor targeting moieties, thereby correcting the deficiencies of the current technology regarding the instability of amidine bonds. In embodiments, the use of an amide linker allows for increased control over the number of mannose-binding C-type lectin receptor targeting moieties that can be attached to the polymeric carbohydrate backbone.

[0059] Compounds and Compositions In certain embodiments, the compounds disclosed herein deliver one or more active therapeutic or diagnostic agents using a carrier construct comprising a polymeric (i.e., carbohydrate) backbone with a conjugated mannose-binding C-lectin-type receptor targeting moiety (i.e., mannose). Examples of such polymeric carbohydrate constructs include mannosylated amine dextran (MAD), which comprises a dextran backbone with mannose molecules conjugated to glucose residues of the backbone and an active pharmaceutical ingredient conjugated to glucose residues of the backbone. Tilmanocept is a specific example of a MAD. A tilmanocept derivative, i.e., tilmanocept without DTPA conjugation, is a further example of a MAD.

[0060] MADs are synthetic molecules purposefully designed to be high-affinity ligands for mannose-binding C-lectin-type receptors, such as CD206. MADs are described in U.S. Patent No. 5,629,499, the entire contents of which are incorporated herein by reference. Thus, the backbone comprises multiple glucose moieties (i.e., residues or subunits) that are originally linked by α-1,6 glycosidic bonds. Other linkages, such as α-1,4 and / or α-1,3 linkages, may also be present.

[0061] Some embodiments may include a backbone that is not 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 multiple monosaccharide residues (i.e., sugar residues or modified sugar residues). In certain embodiments, the glycan backbone has sufficient monosaccharide residues to provide a MW of 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 substituted in the compounds described herein. Additional description of carbohydrate-based carrier molecules used to target CD206 is provided in PCT Application No. US / 2017 / 055211, which is incorporated herein by reference in its entirety.

[0062] In some embodiments, not all backbone moieties are substituted. In some embodiments, one or more leashes are attached to the backbone. In embodiments, a mannose-binding C-type lectin receptor targeting moiety is attached to one or more leashes. In certain embodiments, the mannose-binding C-type lectin targeting moiety is attached to about 15% to about 70%, about 17% to about 65%, or about 20% to about 60% of the glucose residues via one or more leashes. In some embodiments, and as further described herein, one or more leashes may be amine-terminated leashes. In further embodiments, the mannose-binding C-type lectin targeting moiety is attached 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 via one or more leashes. In certain aspects, the percentage may vary depending on the size of the dextran backbone. In further embodiments, one or more therapeutic, diagnostic, or theranostic agents may optionally be attached to the glucose residues via one or more leashes. In certain embodiments, the therapeutic agent is attached to about 1% to about 30%, about 2% to about 25%, or about 5% to about 20% of the glucose residues via one or more leashes and optional degradable linkers, as described herein.

[0063] 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 an example of a C-type lectin receptor expressed on macrophages, dendritic cells, and mesangial cells. CD206 binds to molecules displaying multiple terminal mannose moieties. Without being limited to any particular mechanism or theory, it is contemplated that upon CD206 binding to its ligand, the receptor / ligand complex is internalized into endosomes by receptor-mediated endocytosis, and endosomes naturally acidify 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 a polymeric carbohydrate construct provides numerous advantages in delivering therapeutic, diagnostic, or theranostic agents to targets such as macrophages.

[0064] According to some embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose, a high-mannose glycan or mannose oligosaccharide, fucose, n-acetylglucosamine, a peptide, or galactose. In further embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose. In other embodiments, the at least one targeting ligand can be sialic acid. In embodiments, the mannose-binding C-type lectin receptor targeting moiety is attached to the amine-terminated leash via an amide linker. As previously described herein, illustrative examples of amide linkers can be found in FIG. 2. In embodiments, a carboxylic acid-bearing mannose moiety can be converted to an activated N-hydroxysuccinimide (NHS) carboxylic acid ester and then reacted with the amine group of the amine-terminated leash to form an amide-linked mannose moiety. In embodiments, when the mannose moiety is linked to the amine-terminated leash, the amide linker and the amine-terminated leash share the same nitrogen, as shown in FIG. 2.

[0065] As will be understood by one of skill in the art, the number of carbon atoms between the amide portion of the amide linker and the sulfur atom attached to the C-type lectin receptor targeting moiety (e.g., mannose) can vary, as shown in Figure 2. In some embodiments, y ranges from about 1 to about 6. In further embodiments, y is at least 2. In even further embodiments, y is 1 to 5. Examples of mannosylated polymeric carbohydrate constructs having an amide bond connecting the mannose moiety to an amine-terminated leash have been further synthesized as described in the Examples provided within this disclosure.

[0066] As will be understood by those skilled in the art, any sugar, disaccharide, or carbohydrate may be attached to an amine-terminated leash on a polymeric carbohydrate backbone using the same chemical methods. By modifying the sugars, disaccharides, or carbohydrates attached to the polymeric carbohydrate backbone, high-affinity ligands can be generated for a wide variety of receptors other than CD206. As will be understood by those skilled in the art, amine-terminated leashes can be attached to any carbohydrate using the chemistry described in the Vera patent for attaching amine-terminated leashes (or "linkers") to dextran. For example, amine-terminated leashes can be attached to cellulose, mannan, chitin, or hyaluronic acid by the methods of the Vera patent, which are incorporated herein by reference in their entirety.

[0067] In further embodiments, one or more optional therapeutic, diagnostic, or theranostic agents are attached to the glucose residue via a degradable linker. In some embodiments, the degradable linker may include an acid-sensitive moiety such as a hydrazone. The use of an acid-sensitive linker allows the therapeutic, diagnostic, or theranostic agent to be transported into the cell and allows for release of the agent substantially inside the cell. In further embodiments, one or more therapeutic agents are attached to the glucose residue via an amine-terminated leash and a hydrazone linker that is linked to the backbone using thiol-maleimide conjugation. In still further embodiments, the therapeutic, diagnostic, or theranostic agent is attached to the glucose residue via a degradable linker comprising one or more carbonate and / or disulfide moieties. In still further embodiments, the therapeutic, diagnostic, or theranostic agent may be attached to the glucose residue via click chemistry or an azide-alkyne cycloaddition reaction. As provided herein, selected degradable linkers are further conjugated to amine-terminated leashes that are attached to the polymeric carbohydrate backbone described herein.

[0068] According to certain embodiments, and as further described throughout this disclosure, one or more mannose-binding C-type lectin receptor targeting moieties and one or more optional therapeutic, diagnostic, or theranostic agents are each independently attached to a polymeric carbohydrate-based backbone by a leash. As described in more detail below, one or more additional moieties may be present between the leash and the mannose-binding C-type lectin receptor targeting moiety or therapeutic, diagnostic, or theranostic agent. In some aspects, the one or more additional moieties present between the leash and the mannose-binding C-type lectin receptor targeting moiety or therapeutic, diagnostic, or theranostic agent comprise a linker, i.e., a moiety that connects the mannose-binding C-type lectin receptor targeting moiety or therapeutic, diagnostic, or theranostic agent to the leash.

[0069] In further embodiments, the leash is not conjugated to a mannose-binding C-type lectin receptor targeting moiety or a therapeutic, diagnostic, or theranostic agent, but instead is provided as a free-standing leash attached to the polymeric carbohydrate-based backbone via connection to one or more hydroxyl groups on the polymeric carbohydrate-based backbone. The leash can be attached to about 50% to about 100% of the backbone portion, or about 70% to about 90% of the backbone portion. The leashes can be the same or different. In some embodiments, the leash is an amine-terminated leash. In some embodiments, the leash has the formula -(CH2) p S(CH2) q In further embodiments, the leash comprises the formula -(CH2)3S(CH2)2NH-. In embodiments where the leash is not attached to a mannose-binding C-type lectin receptor targeting moiety or a therapeutic agent, the leash may comprise the formula -(CH2) p S(CH2) q -NH2, where p and q are integers from 0 to 5.

[0070] In further embodiments, the leash can be a chain of about 1 to about 20 member atoms selected from carbon, oxygen, sulfur, nitrogen, and phosphorus. The leash can be straight or branched. The leash can be a halo group, a perfluoroalkyl group, a perfluoroalkoxy group, a C 1-4 Alkyl groups such as alkyl, C 1-4 Alkenyl groups such as alkenyl, C 1-4The linker 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 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—NH, ═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. In some embodiments, the leash used contains an amine group at one end of the leash for the purpose of forming an amide bond with the mannose-binding C-type lectin receptor targeting moiety.

[0071] The size of the MAD construct can be varied by varying the size of the initial carbohydrate backbone as the construct is assembled. In some embodiments, the backbone of the polymeric carbohydrate-based moiety is between about 1 and 150 kDa. In other embodiments, the backbone of the polymeric carbohydrate-based moiety is between about 1 and 100 kDa. In some aspects, the backbone of the polymeric carbohydrate-based 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. In other aspects, the polymeric carbohydrate-based 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 a MW of between about 1 kDa and about 50 kDa, and in other embodiments, the polymeric carbohydrate backbone has a MW of between about 5 kDa and about 25 kDa. In still other embodiments, the polymeric carbohydrate scaffold has a MW of about 8 kDa to about 15 kDa, such as about 10 kDa. In other embodiments, the polymeric carbohydrate scaffold has a MW of about 1 kDa to about 5 kDa, such as about 3 kDa. Advantageously, the smaller size of the disclosed constructs allows for greater tumor penetration and greater localization to tumor-associated macrophages (TAMs) than is possible with other, larger constructs.

[0072] As provided throughout the present disclosure, the discovery of using an amide linker to attach a mannose-binding C-type lectin receptor targeting moiety to a leash is significant. In embodiments, the use of an amide linker allows for greater control over the number of mannose-binding C-type lectin receptor targeting moieties that can be added to the polymeric carbohydrate backbone, increasing the stability of the polymeric carbohydrate-based construct and thereby improving scalability. Thus, the compounds and methods of the present disclosure can be incorporated into any polymeric carbohydrate construct that requires attachment of a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate backbone. While any polymeric carbohydrate-based construct is contemplated, the following optional components may be further attached to the polymeric carbohydrate-based compounds of the present disclosure:

[0073] In some embodiments, the therapeutic, diagnostic, or theranostic agent can be any compound known to be useful in the treatment or diagnosis of macrophage-mediated diseases. In some aspects, a chelating agent can be attached to or incorporated into the disclosed mannosylated carbohydrate polymer-based compounds. In embodiments, the chelating agent allows for the labeling of the mannosylated carbohydrate polymer diagnostic (or imaging) compound with a radioactive metal ion. In further embodiments, the chelating agent can be used to chelate the therapeutic or theranostic agent. Exemplary chelating agents include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), triethylenetetramine (TETA), {4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]-triazonan-1-yl}-acetic acid (NETA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), mercaptoacetylglycylglycyl-glycine (MAG3), dimercaptosuccinic acid, diphenylethylenediamine, porphyrin, iminodiacetic acid, and ethylenediaminetetraacetic acid (EDTA). In embodiments, the chelating agent may be attached to the polymeric carbohydrate backbone via an amine-terminated leash. The chelating agent may be further attached to the amine-terminated leash via an amide linker. In some embodiments, the chelating agent attached to the carbohydrate polymeric backbone is not further attached to any therapeutic, diagnostic, or theranostic agent. In a further embodiment, the mannosylated polymeric carbohydrate compound may be free of a chelating agent.

[0074] According to embodiments, the therapeutic agent is a cytotoxic agent. In further embodiments, the therapeutic agent is a chemotherapeutic or anti-cancer agent. In further embodiments, the therapeutic agent may include, but is not limited to, anti-inflammatory agents such as doxorubicin, paclitaxel, bisphosphonates, metal ions, and dexamethasone. For example, the therapeutic agent may further include anti-infective agents, such as antibiotics (e.g., tetracycline, streptomycin, and isoniazid), antiviral agents, antifungal agents, and antiparasitic agents; immunological adjuvants; steroids; nucleotides, such as DNA, RNA, RNAi, siRNA, CpG, or poly(I:C); peptides; or proteins. In some aspects, the metal ion may include copper, iron, arsenic, antimony silver, cadmium, gallium, or gadolinium. In further aspects, the therapeutic agent is 99m Tc, 210 / 212 / 213 / 214 Bi, 131 Ba, 140 Ba, 11 / 14 C. 51 Cr, 67 / 68 Ga, 153 Gd, 88 / 90 / 91 Y, 123 / 124 / 125 / 131 I, 111 / 115m In, 18 F, 13 N, 105 Rh, 153 Sm, 67 Cu, 166 Ho, 177 Lu, 223 Ra, 62 Rb, 186 / 188 Re, 32 / 33 P, 46 / 47 Sc, 72 / 75 Se, 35 S, 89 Sr, 182 Ta, 123m Te, 127 Te, 129 Te, 132 Te, 65 Zn, 89 / 95 It may further include, but is not limited to, Zr, or other chelatable isotope(s). In embodiments, the concentration of the radioisotope may be present in an amount of at least about 100 mCi (millicuries).

[0075] In certain embodiments, the therapeutic agent is an antibacterial agent selected from the group including or consisting of antibiotics; antituberculosis antibiotics (such as isoniazid, streptomycin, or ethambutol); antiviral or antiretroviral agents, such as reverse transcription inhibitors (such as zidovudine) or protease inhibitors (such as indinavir); and drugs effective against leishmaniasis (such as meglumine antimoniate). In certain embodiments, the therapeutic agent is an antimicrobial active agent, such as amoxicillin, ampicillin, tetracycline, aminoglycosides (e.g., streptomycin), macrolides (e.g., erythromycin and its analogs), chloramphenicol, ivermectin, rifamycins and polypeptide antibiotics (e.g., polymyxins, bacitracin), and zwittermicins. In certain embodiments, the therapeutic agent is selected from isoniazid, doxorubicin, streptomycin, and tetracycline.

[0076] In other embodiments, the therapeutic agent comprises a non-radioactive species selected from the group consisting of, but not limited to, Bi, Ba, Mg, Ni, Au, Ag, V, Co, Pt, W, Ti, Al, Si, Os, Sn, Br, Mn, Mo, Li, Sb, F, Cr, Ga, Gd, I, Rh, Cu, Fe, P, Se, S, Zn, and Zr.

[0077] In still further embodiments, the therapeutic agent is selected from the group consisting of cytostatic agents, alkylating agents, antimetabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, anthracycline drugs, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine drugs, diynenes, podophyllotoxins, toxic enzymes, and radiosensitizing agents. More specific examples of therapeutic agents include mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, triazicon, nitrosourea compounds, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, isoniazid, indomethacin, gallium (III), 68 gallium (III), aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, etoposide ... and / or benzocaine, ...

[0078] In embodiments where the therapeutic agent is a hormone or hormone antagonist, the therapeutic agent may be selected from the group consisting of prednisone, hydroxyprogesterone, medroprogesterone, diethylstilbestrol, tamoxifen, testosterone, and aminoglutethimide.

[0079] In embodiments where the therapeutic agent is a prodrug, the therapeutic agent may be selected from the group consisting of phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, (-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosineme, and 5-fluorouridine prodrugs that can be converted to more active, non-cytotoxic drugs.

[0080] According to further embodiments, any of a variety of detectable moieties can be directly or indirectly bound to a carrier molecule for a variety of purposes. As used herein, the terms "detectable moiety," "diagnostic moiety," or "diagnostic agent" (these terms may be used interchangeably) refer to an atom, isotope, or chemical structure that: (1) is capable of binding to a carrier molecule; (2) is non-toxic to humans; and (3) provides a directly or indirectly detectable signal, particularly a signal that is not only measurable but whose intensity is related (e.g., proportional) to the amount of detectable moiety. The signal can be detected by any suitable means, including spectroscopic, electrical, optical, magnetic, acoustic, radio signal, or tactile detection means, and the measurement processes described herein.

[0081] Suitable detectable moieties include, but are not limited to, radioisotopes (radionuclides), fluorophores, chemiluminescent agents, bioluminescent agents, magnetic moieties (including paramagnetic moieties), metals (e.g., for use as imaging agents), RFID moieties, enzymatic reactants, colorimetric emitting agents, dyes, and particle forming agents. In some embodiments, the radioisotope may be present in an amount of from about 0.01 mCi to about 40 mCi, from about 0.05 mCi to about 35 mCi, or from about 0.1 mCi to about 30 mCi.

[0082] By way of specific example, suitable diagnostic moieties include, but are not limited to: - contrast agents suitable for magnetic resonance imaging (MRI), such as gadolinium (Gd3+), paramagnetic materials, and superparamagnetic materials (such as superparamagnetic iron oxide); - contrast agents suitable for computed tomography (CT) imaging, such as iodinated molecules, ytterbium, and dysprosium; - 99m Tc, 210 / 212 / 213 / 214 Bi, 131 Ba, 140 Ba, 11 / 14 C. 51 Cr, 67 / 68 Ga, 153 Gd, 88 / 90 / 91 Y, 123 / 124 / 125 / 131 I, 111 / 115m In, 18 F, 13 N, 105 Rh, 153 Sm, 67 Cu, 166 Ho, 177 Lu, 223 Ra, 62 Rb, 186 / 188 Re, 32 / 33 P, 46 / 47 Sc, 72 / 75 Se, 35 S, 89 Sr, 182 Ta, 123m Te, 127 Te, 129 Te, 132 Te, 65 Zn, 89 / 95 a radioisotope suitable for scintigraphic imaging (or scintigraphy), such as Zr, or other chelatable isotope(s); - 99m Tc, 111 In, and 123 gamma-ray emitting agents suitable for single photon emission computed tomography (SPECT), such as I; dyes and fluorescent agents suitable for optical imaging; and - 18 Agents suitable for positron emission tomography (PET), such as F.

[0083] Diagnostic moieties can be attached to carrier molecules in a variety of ways, including direct attachment or using a chelator attached to the carrier molecule. In some embodiments, diagnostic moieties can be attached using leashes attached to the carrier backbone. In some embodiments, chelators can be conjugated to the amino groups of one or more leashes and used to attach diagnostic moieties thereto. Note that in some cases, no leashes may be attached to the glucose moieties of the carbohydrate polymer backbone. Certain embodiments may include a single type of diagnostic moiety or a mixture of different diagnostic moieties. For example, embodiments of the compounds disclosed herein may include a contrast agent suitable for MRI and a radioisotope suitable for scintigraphic imaging, as well as further combinations of diagnostic moieties described herein. In embodiments, after administration of the diagnostic or imaging compound to an animal or human subject, the radioactive metal ion enables single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of the anatomical location or site where the labeled imaging agent is present.

[0084] According to certain embodiments, the disclosed polymeric carbohydrate-based compounds have the formula (I):

[0085] [ka]

[0086] may include the formula provided in During the ceremony, each X is independently H, L1-A, L1-YA, L2-R, or L3-H, and each X is bonded to any OH group; L1, L2, and L3 are each independently a leash; each A independently comprises a therapeutic agent, a diagnostic agent, or a theranostic agent; each Y independently comprises a chelating agent; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety; n is an integer greater than zero, and each unit of n may be the same or different; At least one R is present, L2 and R are linked via an amide linker.

[0087] In certain embodiments, L1, L2, and L3 each comprise a degradable leash. In other embodiments, L1, L2, and L3 each comprise a degradable leash comprising a straight or branched chain of about 1 to about 20 member atoms selected from carbon, oxygen, sulfur, nitrogen, and phosphorus. In further embodiments, L1, L2, and L3 each comprise a group of the formula -(CH2) p S(CH2) q It may contain -NH-, where p and q are independently integers from 0 to 5.

[0088] According to further embodiments, at least one of L, L, or L is a C hydrocarbon chain optionally interrupted by up to three heteroatoms selected from the group consisting of O, S, and N.

[0089] According to some embodiments, each L2 has the following formula:

[0090] [ka]

[0091] wherein linkage 1A is attached to a hydroxide group of the polymeric carbohydrate backbone and y is an integer from 1 to 6. In some embodiments, n is an integer greater than zero. In other embodiments, n is an integer greater than 1. In further embodiments, n can be an integer from 1 to about 50, from about 5 to about 40, or from about 5 to about 30. As will be understood by one of skill in the art, each unit of n can be the same or different. Each X can independently be H, L1-A, L1-YA, L2-R, or L3-H, such that each unit of n can consist of any combination of X selected from H, L1-A, L1-YA, L2-R, or L3-H.

[0092] By way of example only, mannosylated carbohydrate polymer therapeutic and diagnostic compounds may be prepared according to formula (Ia):

[0093] [ka]

[0094] where * indicates the point at which a therapeutic or diagnostic agent is attached. In certain embodiments, a therapeutic or diagnostic agent is attached to the end of the amino-terminated leash, with or without a linker. In further embodiments, a chelator may be attached to the end of the amino-terminated leash, with or without a linker. The chelator may optionally be further attached to a diagnostic, therapeutic, or theranostic agent. In embodiments, y is an integer from 1 to 6.

[0095] As a further example, one or more of the subunits of formula (I) may be

[0096] [ka]

[0097] Although the structure shown contains two carbon atoms between the sulfur atom and the amide bond, as described herein, the carbon chain can range from 1 to 6 or more. In certain aspects, the disclosed compounds are present in the form of pharmaceutical compositions in the presence of pharmaceutically acceptable carriers.In embodiments, the pharmaceutically acceptable carriers used can be, for example, solid, liquid, or gaseous.Examples of solid carriers include lactose, mannitol, microcrystalline cellulose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.Examples of liquid carriers include liquid sugar, peanut oil, olive oil, ethanol, propylene glycol, and water.Examples of gaseous carriers include carbon dioxide, nitrogen, and compressed air.

[0098] Any convenient pharmaceutical medium can be used when preparing the composition into a dosage form. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations such as suspensions, elixirs, and solutions, while carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used to form oral solid preparations such as powders, capsules, and tablets. Due to their ease of administration, tablets and capsules are preferred oral dosage units, and therefore solid pharmaceutical carriers are used. Optionally, tablets can be coated by standard aqueous or non-aqueous techniques. In further aspects, aerosol carriers, such as sugars including glucose, fructose, mannitol, sucrose, lactose, and cellulose, propellants, liquid carriers, and gaseous carriers can be used to form preparations suitable for inhalation. In certain aspects, the composition is administered intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally. In a preferred embodiment, the composition is administered intravenously.

[0099] In embodiments, the composition may include any therapeutically effective amount of the mannosylated polymeric carbohydrate compound as would be understood by one of skill in the art. In further embodiments, the composition may include any prophylactically effective amount of the mannosylated polymeric carbohydrate compound as would be understood by one of skill in the art.

[0100] method In some embodiments, methods are provided for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold disclosed herein. Advantageously, the mannose-binding C-type lectin receptor targeting moiety can be attached to the polymeric carbohydrate scaffold via an amine-terminated leash, and an amide linker is used to link the mannose-binding C-type lectin receptor targeting moiety to the amine-terminated leash. In certain embodiments, a method for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold may include the following steps: (a) synthesizing a polymeric carbohydrate scaffold having one or more amine-terminated leashes attached thereto; (b) obtaining a mannose-binding C-type lectin receptor targeting moiety and reacting it with mercaptopropionic acid to derivatize the mannose-binding C-type lectin receptor targeting moiety to include an anomeric thiocarboxylic acid moiety; (c) optionally converting the anomeric thiocarboxylic acid moiety to an activated N-hydroxysuccinimide (NHS) carboxylic acid ester; and (d) coupling the activated N-hydroxysuccinimide (NHS) carboxylic acid ester to the polymeric carbohydrate scaffold having one or more amine-terminated leashes to form an amide bond (or "linker") between the carbonyl carbon of the activated N-hydroxysuccinimide (NHS) carboxylic acid ester and the amine group of the amine-terminated leash.

[0101] In certain aspects, steps (a)-(d) need not be performed in the exact same order. In further aspects, additional steps may be performed between each of steps (a)-(d). In some embodiments, step (a) may be performed before step (b), (c), or (d), or after step (b) or (c).

[0102] In a further embodiment, a method for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold may comprise the following steps: (a) synthesizing a mannose-binding C-type lectin receptor targeting moiety comprising an anomeric thiocarboxylic acid moiety; (b) converting the mannose-binding C-type lectin receptor targeting moiety comprising the anomeric thiocarboxylic acid moiety into an activated N-hydroxysuccinimide carboxylic acid ester; and (c) reacting the activated N-hydroxysuccinimide carboxylic acid ester with a polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto, wherein the reaction of the activated N-hydroxysuccinimide carboxylic acid ester with the polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto forms an amide bond between the one or more amine-terminated leashes and the carbonyl carbon of the activated N-hydroxysuccinimide carboxylic acid ester.

[0103] In some embodiments, the polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto is formed before step (a) or at a point between step (a) and step (c).

[0104] In further embodiments, a therapeutic, diagnostic, or theranostic agent can be conjugated to the polymeric carbohydrate scaffold before, after, or simultaneously with attachment of the mannose-binding C-type lectin receptor targeting moiety to the polymeric carbohydrate scaffold. Additional discussion regarding methods for attaching mannose-binding C-type lectin receptor targeting moieties to the polymeric carbohydrate scaffold can be found within the non-limiting examples provided herein.

[0105] In certain embodiments, the methods of the present disclosure provide improved stability of mannosylated polymeric carbohydrate compounds. In some embodiments, the methods provide mannosylated polymeric carbohydrate compounds that retain at least 80%, at least 90%, at least 95%, or at least 98% of the original mannose-binding C-type lectin receptor targeting moiety attached to the polymeric carbohydrate backbone. In some aspects, the methods achieve such retention of the mannose-binding C-type lectin receptor targeting moiety after about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours, or about 40 hours. In embodiments, stability is measured at a temperature of about 20°C to about 50°C, about 30°C to about 45°C, or about 37°C to about 42°C.

[0106] In certain embodiments, the disclosed methods further comprise administering to a subject in need thereof an effective dose of a compound or composition disclosed herein. In further embodiments, the methods comprise administering a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties attached to the polymeric carbohydrate backbone via an amide linker, and a therapeutic, diagnostic, or theranostic agent coupled to the polymeric carbohydrate backbone. In yet further embodiments, the compound comprises a subunit provided by Formula (I) disclosed herein.

[0107] In certain embodiments, the compound is administered in a therapeutically effective amount. In further embodiments, the compound is administered in a prophylactically effective amount. In still further aspects, the method further comprises administering the compound or composition intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intraocularly, by intratumoral injection, or transdermally, or delivering directly to the tumor organ by an invasive technique.

[0108] In still further embodiments, the method further comprises administering the composition in combination with at least one other treatment or therapy. In some embodiments, the other treatment or therapy comprises an anti-inflammatory agent. In further embodiments, the other treatment or therapy comprises co-administering 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-based therapies, or in combination with radiation therapy, heat therapy, physical therapy, or dietary therapy.

[0109] According to further embodiments, the at least one other treatment or therapy is an immunotherapy, such as administration of an immunomodulatory agent. According to certain embodiments, the 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 selected from the group consisting of glucocorticoids, hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone (doca) acetate, aldosterone, non-glucocorticoid steroids, cytostatics, alkylating agents, nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and antimetabolites. Antibodies, purine analogues, azathioprine, mercaptopurine, mycophenolic acid, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogues, methotrexate, cytotoxic antibiotics, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies or fusions thereof, antithymocyte globulin, antilymphocyte globulin, anti-IL-2 receptor antibody, anti-IL6 antibody, anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, biciclear Lisumab, anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, anti-CD11a antibody, efalizumab, anti-CD18 antibody, erlizumab, 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-CD 80 antibodies, galiximab, anti-CD147 antibodies, gavilimomab, B-lymphocyte stimulator (BLyS) inhibitor antibodies, belimumab, CTLA4-Ig fusion proteins, abatacept, belatacept, ipilimumab, tremelimumab, anti-eotaxin 1 antibodies, bertilimumab, anti-α4-integrin antibodies, natalizumab, anti-IL-6R antibodies, tocilizumab, anti-LFA-1 antibodies, odulimomab, anti-CD25 antibodies, basiliximab, daclizumab,Inolimomab, anti-CD5 antibodies, zolimomab, anti-CD2 antibodies, siplizumab, nerelimomab, faralimomab, atlizumab, atolilimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, immunophilin modulators, rapamycin, calcincurin inhibitors, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, rilonacept Daforolimus, everolimus, temsirolimus, zotarolimus, TNF inhibitors, infliximab, adalimumab, certolizumab pegol, golimumab, etanercept, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, talizumab , IL12 inhibitors, IL23 inhibitors, ustekinumab, opioids (opioids), IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-κB inhibitors, 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), Bay11-7082,Luteolin, cell-penetrating peptide SN-50, IκBα-super-repressor overexpression, NFκB decoy oligodeoxynucleotide (ODN), or any derivative or analog thereof.

[0110] In an exemplary embodiment, the combined administration of the compound and at least one treatment or therapy is synergistically effective compared to administration of either alone. According to certain embodiments, administration of a compound disclosed herein in conjunction 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 a compound disclosed herein with the other therapy or treatment produces a synergistic effect. In yet further embodiments, co-administration of a compound disclosed herein provides a lower effective dose of the other therapy or treatment.

[0111] The methods provided herein can be performed in an adjuvant setting. In some embodiments, the methods are performed in a neoadjuvant setting, i.e., the methods can be performed before primary / definitive treatment. In some embodiments, the methods are used to treat individuals who have been previously treated. Any of the treatment methods provided herein can be used to treat individuals who have not been previously treated. In some embodiments, the methods are used as first-line treatment. In some embodiments, the methods are used as second-line treatment.

[0112] In further embodiments, methods of treating a disease are 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 disease, inflammatory disorder, nonalcoholic steatohepatitis (NASH), acute respiratory distress syndrome (ARDS), sepsis, coronavirus infection, influenza infection, cytokine storm, and other macrophage-associated diseases.

[0113] According to other aspects, the subject has been diagnosed with melanoma, breast cancer, lung cancer, pancreatic carcinoma, kidney cancer, ovarian cancer, prostate cancer or cervical carcinoma, glioblastoma, or colorectal cancer, brain and 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 tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, or any combination thereof.

[0114] In certain embodiments, the methods further comprise administering the composition as a bolus and / or at regular intervals. In certain embodiments, the disclosed methods further comprise administering the composition intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally.

[0115] 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 assessing the effectiveness of the composition. In a still further aspect, assessing the effectiveness of the composition comprises measuring tumor size prior to administering the composition and measuring tumor size after administering the compound. In a still further aspect, assessing the effectiveness of the composition is performed at regular intervals. According to certain aspects, the disclosed methods further comprise, 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 dose of the composition, the frequency of administration of the composition, or the route of administration of the compound.

[0116] According to certain alternative embodiments, the subject has been diagnosed with a disease associated with elevated levels of CD206+ macrophages and / or MDSCs, including acquired immune deficiency 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 disorders, 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 / Baro's concentric sclerosis, Behçet's disease, Buerger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multiple osteomyelitis, 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, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diabetes mellitus type 2, diffuse cutaneous systemic sclerosis, Dressler's 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, peptic 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 gestationis), hidradenitis suppurativa, HIV infection, Hughes-Stobin 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), lupus erythematosus, lymphomatoid granulomatosis, Majeed syndrome, malignancies including cancer (e.g., sarcomas) , 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), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, Ord's thyroiditis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria Pneumocystis candida (PNH), Parry-Romberg syndrome, Parsonage-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 neuropathies, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome syndrome, Schnitzler syndrome, scleritis, scleroderma, sepsis, serum sickness, Sjögren's syndrome, spondyloarthropathy, 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's 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 spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo,and Wegener's granulomatosis, but are not limited to.

[0117] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein 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. [Example]

[0118] Embodiments of the present disclosure are further defined by the following non-limiting examples. It should be understood that these examples, while illustrating certain embodiments of the present disclosure, are provided by way of example only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present disclosure and can make various changes and modifications to the embodiments of the present disclosure to adapt to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the embodiments of the present disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0119] Example 1 An exemplary method for attaching mannose moieties to a dextran backbone was performed. Without being limited to any particular polymeric carbohydrate compound, such a method was performed to demonstrate how mannose moieties can be attached to a dextran backbone.

[0120] 3.0 g (7.7 mmol) of pentaacetyl-α-D-mannose and 2.7 ml (31 mmol) of 3-mercaptopropionic acid were combined in 40 ml of anhydrous dichloromethane under nitrogen and cooled on an ice bath. 3.0 ml (29 mmol) of boron trifluoride dietherate was added, and the solution was stirred for 15 hours. The reaction was diluted with dichloromethane and washed carefully with water, followed by a wash with saturated sodium bicarbonate. The organics were dried over sodium sulfate, filtered, and concentrated. As shown in Figure 3, 2.85 g of compound 1 was isolated after silica chromatography using 40% ethyl acetate and 1% acetic acid in hexane. 1.0 g of the tetraacetate salt 1 was dissolved in 16 ml of anhydrous methanol at room temperature, and 0.5 ml of 25 wt% sodium methoxide was added. The reaction was confirmed to be complete by TLC, and the pH was adjusted to 3 by adding acidic resin. The resin was removed by filtration and the filtrate was concentrated to collect crude 2 (512 mg). As will be appreciated by those skilled in the art, the number of carbons in the anomeric thiocarboxylic acid moiety in compounds 1 and 2 can vary.

[0121] Compounds 1 or 2 in Figure 3 can be converted to activated N-hydroxysuccinimide (NHS) carboxylic acid esters 3 and 4, as shown in Figure 4, and coupled to amine leashes on dextran to form MADs 5 and 6. Coupling of compound 3 required the removal of the acetate protecting group on the mannose while attaching it to the dextran polymer in a subsequent step.

[0122] NHS ester 4 was prepared by dissolving 512 mg (1.9 mmol) of carboxylic acid 2 in 6 ml of anhydrous methanol and adding 14 ml of anhydrous tetrahydrofuran and 14 ml of anhydrous DCM. To this cloudy solution, 264 ml (2.29 mmol) of NHS and 473 mg (2.29 mmol) of N,N'-dicyclohexylcarbodiimide were charged. The mixture was stirred at room temperature under a nitrogen overlay for 15 hours. The reaction was filtered through a glass frit, concentrated, and purified on a silica column using 20% ​​methanol in DCM. 300 mg was isolated as a mixture of activated ester 4 and starting 2.

[0123] NHS ester 3 was prepared by dissolving 1.0 g (2.29 mmol) of carboxylic acid 1 and 316 mg (2.75 mmol) of NHS in 15 ml of anhydrous DCM. 527 mg (2.75 mol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was added to the solution and stirred for 15 hours. The reaction was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated. This process afforded 1.25 g of sufficiently pure NHS ester 3.

[0124] Conjugation of activated ester 4 to amine dextran to form MAD5: 150 mg of amine dextran (MW 5492) prepared from 3.5 kD dextran was dissolved at 25 mg / ml in 0.1 M pH 8.3 sodium carbonate buffer. 300 mg of crude NHS ester 4 in 2 ml of methanol was added and stirred overnight. The crude MAD was concentrated and washed with purified water by ultrafiltration using a 3 kDa MWCO membrane, and the residue was frozen and lyophilized. 137 mg of mannose aminodextran was isolated as a white foam, with an average of 12.9 mannoses attached per MAD based on H NMR integration.

[0125] Conjugation of activated ester 3 to amine dextran to form 6 and removal of mannose acetate to obtain 5: 100 mg of amine dextran (MW 5492) prepared from 3.5 kD dextran was dissolved at 25 mg / ml in 1:1 0.1 M pH 8.3 sodium carbonate buffer and dimethyl sulfoxide (DMSO) using brief sonication. 194 mg (0.36 mmol) of crude NHS ester 3 in 0.7 ml of DMSO was added in two portions to the amino dextran solution over 4 h and then stirred overnight. The opaque crude MAD solution was washed with 90% methanol, followed by pure methanol via ultrafiltration using a 3 kDa MWCO membrane to obtain a clear MAD residue solution. 6.25 wt% sodium methoxide in methanol was added dropwise until a basic solution was obtained. The solution became cloudy after approximately 10 min, and the mixture was stirred for an additional 1 h. The mixture was clarified by dilution with purified water and concentrated by ultrafiltration using four separate volumes of water. After lyophilization, 79 mg of mannose aminodextran 5 was isolated as a white foam, with an average of 17 mannoses per MAD based on H NMR integration.

[0126] Example 2 The stability of mannose attached to the polymeric carbohydrate backbone was further evaluated by comparing amide and amidine bonds between mannose and the amine-terminated leash attached to the dextran backbone of MAD. The stability of three mannosylated dextrans (MADs) with different mannose linkages to 10 kDa dextran, as shown in Table 1, was compared in distilled water and 0.1 M aqueous sodium carbonate solution at pH 9.74 at 40 °C. MAD was dissolved at 20 mg / ml, sealed in a borosilicate glass tube, and placed in a heating block with shaking. At each time point, the tube was removed from the heating block, and the contents were concentrated in a centrifuge using a 3 kDa MWCO spin filter. The retentate containing the mannosylated dextran was washed three times with distilled water by spin filtration to remove hydrolyzed mannose no longer attached to the dextran polymer. The washed retentate was removed from the top of the membrane, frozen, and lyophilized. The number of mannose molecules in each sample was determined by setting the integral of the anomeric hydrogen of the dextran polymer between 4.92 and 5.24 ppm to 1 (an average of 61.7 hydrogens in a 10 kDa dextran) and integrating the anomeric hydrogen of the bound mannose at 5.36 ppm. 1 Determined in D2O by 1 H NMR.

[0127] [Table 1]

[0128] As shown in Table 1, MAD compounds using amide bonds to attach mannose resulted in remarkable stability of the mannose moiety remaining attached to the dextran backbone. The method used to develop tilmanocept, which utilizes amidine bonds, reduced the mannose from 25.3 to 14.2 after 20 hours. Therefore, multiple steps are required to reattach additional mannose to the polymeric carbohydrate backbone to maintain the appropriate number of mannose moieties on the backbone.

[0129] On the other hand, both MAD compounds utilizing amide bonds to attach mannose to dextran resulted in little loss of mannose from the dextran backbone. Similar results were observed between thiomethylamide and thioethylamide, indicating that the number of carbon atoms between the sulfur atom and the amide bond does not affect the stability of mannose. The ability of the mannose moiety to remain attached to the dextran backbone provides excellent predictability for the resulting compounds, increased efficacy for therapeutic and diagnostic uses of compounds for binding to C-type lectin receptors, and increased scalability of production.

[0130] The above specification provides a description of how to make and use the disclosed compositions and methods. Since many embodiments can be made without departing from the spirit and scope of the disclosure, the disclosure resides in the claims.

Claims

1. A compound comprising: a polymeric carbohydrate backbone comprising at least one amine-terminated leash attached thereto; one or more mannose-binding C-type lectin receptor targeting moieties; The compound, wherein the one or more mannose-binding C-type lectin receptor targeting moieties are attached to the amine-terminated leash via an amide linker.

2. The compound of claim 1 , wherein the carbohydrate backbone comprises a monosaccharide, a disaccharide, or a polysaccharide.

3. The compound of claim 1 or 2, wherein the carbohydrate backbone comprises dextran, cellulose, mannan, chitin, or hyaluronic acid.

4. The compound has the formula (I): 【Chemical 1】 and During the ceremony, each X is independently H, L1-A, L1-YA, L2-R, or L3-H, and each X is bonded to any OH group; L1, L2, and L3 are each independently a leash; each A independently comprises a therapeutic agent, a diagnostic agent, or a theranostic agent; each Y independently comprises a chelating agent; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety; n is an integer greater than zero, and each unit of n may be the same or different; at least one R is present, 2. The compound of claim 1, wherein L2 and R are linked via the amide linker.

5. 5. The compound of any one of claims 1 to 4, wherein the amine-terminated leash comprises a straight or branched chain having from about 1 to about 20 member atoms selected from the group consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus.

6. Each L2 has the following formula: 【Chemistry 2】 6. The compound of claim 5, comprising: wherein bond 1A is attached to a hydroxide group of the polymeric carbohydrate backbone; and y is an integer from 1 to 6.

7. L1, L2, or L3 each independently represent -(CH ２ ) ｐ S (CH ２ ) ｑ The compound of any one of claims 4 to 6, comprising -NH-, wherein p and q are integers from 0 to 5.

8. The compound according to any one of claims 4 to 7, wherein L2 does not contain an amidine bond.

9. 9. The compound of any one of claims 1 to 8, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling reagent, mannose, a high mannose glycan or mannose oligosaccharide, fucose, n-acetylglucosamine, a peptide, galactose, or a combination thereof.

10. At least one subunit of formula (I) is 【Chemistry 3】 The compound according to any one of claims 4 to 9, comprising:

11. The compound has the formula (Ia): 【Chemistry 4】 wherein * indicates the point at which the therapeutic, diagnostic, or theranostic agent is attached, and y is an integer from 1 to 6.

12. The compound of any one of claims 1 to 11, wherein the polymeric carbohydrate backbone has a molecular weight of about 1 kD to about 150 kD.

13. 1. A pharmaceutical composition comprising: A compound according to any one of claims 1 to 12; and a pharmaceutically acceptable carrier.

14. The compound has the formula (I): 【Chemistry 5】 and During the ceremony, each X is independently H, L1-A, L1-YA, L2-R, or L3-H, and each X is bonded to any OH group; L1, L2, and L3 are each independently a leash; each A independently comprises a therapeutic agent, a diagnostic agent, or a theranostic agent; each Y independently comprises a chelating agent; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety; n is an integer greater than zero, and each unit of n may be the same or different; at least one R is present, 14. The composition of claim 13, wherein L2 and R are linked via the amide linker.

15. 15. The composition of claim 13 or 14, wherein the amine-terminated leash comprises a straight or branched chain having from about 1 to about 20 member atoms selected from the group consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus.

16. Each L2 has the following formula: 【Chemistry 6】 16. The composition of claim 14 or 15, comprising: wherein linkage 1A is attached to a hydroxide group of the polymeric carbohydrate backbone; and y is an integer from 1 to 6.

17. 1. A method for attaching a mannose-binding C-type lectin receptor targeting moiety to a polymeric carbohydrate scaffold, comprising: (a) synthesizing a mannose-binding C-type lectin receptor targeting moiety comprising an anomeric thiocarboxylic acid moiety; (b) converting the mannose-binding C-type lectin receptor targeting moiety containing the anomeric thiocarboxylic acid moiety into an activated N-hydroxysuccinimide carboxylic acid ester; (c) reacting the activated N-hydroxysuccinimide carboxylic acid ester with a polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto; wherein the reaction of the activated N-hydroxysuccinimide carboxylic acid ester with the polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto forms an amide bond between the one or more amine-terminated leashes and the carbonyl carbon of the activated N-hydroxysuccinimide carboxylic acid ester.

18. 18. The method of claim 17, wherein the method results in retention of about 80% of the mannose-binding C-type lectin receptor targeting moiety on the polymeric carbohydrate scaffold after about 20 hours.

19. 19. The method of claim 17 or 18, wherein the dextran backbone has a molecular weight of about 1 kD to about 150 kD.

20. 20. The method of any one of claims 17 to 19, wherein the polymeric carbohydrate backbone having one or more amine-terminated leashes attached thereto is formed before step (a) or at a point between step (a) and step (c).

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