Pharmaceutical compositions containing polysaccharides

Abstract

A pharmaceutical composition comprising a conjugate, the conjugate comprising a polysaccharide shell and a hydrophobic core, the polysaccharide shell having amphiphilic properties and stabilizing the oil-water interface to form emulsions and nanoprecipitation-based nanoparticles.

Description

[Technical field]

[0001] The present invention relates to the field of polysaccharide applications, in particular to pharmaceutical compositions comprising polysaccharides. [Background technology]

[0002] Fucoidan is a sulfated polysaccharide with multiple biological activities, including antibacterial activity, antiviral activity, antitumor activity, anticoagulant activity, and antioxidant activity.Fucoidan also shows high affinity to p-selectin, which allows for targeted delivery of therapeutic compounds to sites that overexpress p-selectin, such as tumors or unstable atherosclerotic plaques.However, the retention time of fucoidan after administration is very short, which hinders the accumulation of pure compounds at the target site.Thus, even if fucoidan has biological activity, its therapeutic potential is limited due to the above-mentioned obstacles to accumulation.

[0003] Engineered drug delivery systems (DDS) are technologies for targeted delivery and / or controlled release of therapeutic agents in desired tissues / organs. Polysaccharides, such as fucoidan, allow for the formation of complexes with other oppositely charged molecules. Polyelectrolyte complexation is the most commonly used technique to obtain fucoidan-based particles. One of the most commonly used materials to form complexes with fucoidan is chitosan. Positively charged chitosan can interact with fucoidan to form self-assembled or multi-layered DDS. Other methods are coacervation, ionic cross-linking, self-assembly, and spray drying. Summary of the Invention [Problem to be solved by the invention]

[0004] However, even though fucoidan-based particles may be synthesized, fucoidan is not a preferred material for stabilizing the water-oil interface of emulsions or nanoprecipitated nanostructures. Most of the side chains of fucoidan are substituted with sulfate groups, so the polysaccharide structure is extremely hydrophilic and lacks amphiphilicity to stabilize the water-oil interface. Considering that emulsification and nanoprecipitation are the most environmentally efficient and mature technologies in the pharmaceutical industry, it is important to develop technologies that overcome these problems. [Means for solving the problem]

[0005] In one aspect, the invention provides a complex comprising a polysaccharide shell and a hydrophobic core, wherein the polysaccharide shell comprises a sulfated polysaccharide and a complement having an affinity for sulfated polysaccharides, and the hydrophobic core comprises hydrophobic molecules, and the complex has amphiphilic properties to reduce surface tension and stabilize the water-oil interface, particularly between the polysaccharide shell and the hydrophobic core.

[0006] In one aspect, the invention provides a pharmaceutical composition comprising a conjugate described herein.

[0007] In one embodiment, the pharmaceutical composition is an emulsion-based nanoparticle. In a further embodiment, the nanoparticle is a nanoprecipitated nanostructure.

[0008] In some embodiments of the invention, the sulfated polysaccharide is fucoidan.

[0009] In some embodiments of the invention, the complex has affinity for p-selectin or a modified version thereof.

[0010] In one embodiment, the peak molecular weight of the fucoidan used in the present invention is in the range of 10 to 200 kDa. Specific embodiments of fucoidan include, but are not limited to, fucoidan derived from Fucus vesiculosus, Cladosiphon okamuranus Tokida, Ascophyllum nodosum, Fucus evanescens, Fucus ceranoides, Fucus distichus, Fucus serratus, Fucus spiralis, Ascophyllum mackaii, Pelvetia canaliculata, Silvetia babingtonii, and Undaria pinnatifida.

[0011] In one embodiment, the purity of the fucoidan used in the present invention is in the range of about 60% to about 99%, about 65% to about 95%, about 70% to about 90%, about 75% to about 85%, or about 70% to about 80%.

[0012] In one embodiment, the sulfate content of the fucoidan used in the present invention is in the range of about 15% to about 40%, about 18% to about 38%, about 20% to about 35%, about 22% to about 32%, or about 25% to about 30%.

[0013] In some embodiments of the invention, the complements described herein have a positively charged functional group. In some embodiments of the invention, the complements are positively charged amino acids. Examples of positively charged amino acids include, but are not limited to, lysine or a polymer / copolymer thereof, arginine or a polymer / copolymer thereof, histidine or a polymer / copolymer thereof, and glutamine or a polymer / copolymer thereof. In some embodiments of the invention, the complements having a positively charged functional group further comprise a hydrophobic domain. Examples of complements having a positively charged functional group and a hydrophobic domain include, but are not limited to, zein, chitosan, protamine, or polyethyleneimine.

[0014] In some embodiments of the invention, the molar ratio of sulfated polysaccharide to its complement having a positively charged functional group is about 1:0.005 to about 1:200, about 1:0.01 to about 1:180, about 1:0.02 to about 1:160, about 1:0.03 to about 1:140, about 1:0.04 to about 1:120, about 1:0.05 to about 1:100, about 1:0.06 to about 1:80, about 1:0.07 to about 1:60, about 1:0.08 to about 1:50, about 1:0.09 to about 1:20, about 1:0.1 to about 1:10, about 1:0.2 to about 1:8, about 1:0.3 to about 1:7, about 1:0.4 to about 1:6, about 1:0.5 to about 1:5, about 1:0.6 to about 1:6, about 1:0.7 to about 1:5, about 1:0.8 to about 1:4, about 1:0.9 to about 1:3, or about 1:1 to about 1:2.

[0015] In some embodiments of the invention, the ratio of negative charges in the sulfated polysaccharide to positive charges in the complement is in the range of about 1:0.05 to about 1:3, about 1:0.06 to about 1:2.5, about 1:0.07 to about 1:2, about 1:0.08 to about 1:15, about 1:0.09 to about 1:1, about 1:0.1 to about 1:0.95, about 1:0.2 to about 1:0.9, about 1:0.3 to about 1:85, about 1:0.4 to about 1:0.8, about 1:0.5 to about 1:0.7, or about 1:0.6 to about 1:0.65 to form an electrically stable complex.

[0016] In some embodiments of the invention, the complement binds to the sulfated polysaccharide by hydrogen bonding. In some embodiments of the invention, the complement comprises an amine-containing ligand, a carboxylic acid group, or an oxygen acceptor. Examples of complements that bind to sulfated polysaccharides by hydrogen bonding include, but are not limited to, oxidized dextran, polyethylene glycol (PEG), chemically modified PEG, polydextrose, polysorbate 20, polysorbate 80, polyvinyl acetate, polyvinyl alcohol (PVA), PLURONIC® F68, PLURONIC® F123, PLURONIC® F127, polyvinyl alcohol, and propylene glycol alginate. Examples of chemically modified PEG include, but are not limited to, NH2-PEG or COOH-PEG.

[0017] In some embodiments of the invention, the molar ratio of sulfated polysaccharide to complement that is bound to the sulfated polysaccharide by hydrogen bonds is about 1:0.01 to about 1:100, about 1:0.01 to about 1:90, about 1:0.02 to about 1:80, about 1:0.03 to about 1:70, about 1:0.04 to about 1:60, about 1:0.05 to about 1:50, about 1:0.06 to about 1:40, about 1:0.07 to about 1:100, about 1:0.08 to about 1:100, about 1:0.09 to about 1:20, about 1:0.10 to about 1:20, about 1:0.11 to about 1:30, about 1:0.12 to about 1:30, about 1:0.13 to about 1:40, about 1:0.14 to about 1:50, about 1:0.15 to about 1:30, about 1:0.16 to about 1:40, about 1:0.17 to about 1:50, about 1:0.18 to about 1:50, about 1:0.19 to about 1:60, about 1:0.19 to about 1:70, about 1:0.19 to about 1:80, about 1:0.19 to about 1:90, about 1:0.19 to about 1:10 ... 1:0.5 to about 1:5, about 1:0.6 to about 1:6, about 1:0.7 to about 1:5, about 1:0.8 to about 1:4, about 1:0.9 to about 1:3, or about 1:1 to about 1:2.

[0018] In some embodiments of the invention, the complement described herein is p-selectin or a modified version thereof.

[0019] In some embodiments of the invention, the molar ratio of sulfated polysaccharide to complement as p-selectin is in the range of about 1:0.1 to about 1:100, about 1:0.5 to about 1:95, about 1:1 to about 1:90, about 1:5 to about 1:85, about 1:10 to about 1:80, about 1:15 to about 1:75, about 1:20 to about 1:70, about 1:25 to about 1:65, about 1:30 to about 1:60, about 1:35 to about 1:55, or about 1:40 to about 1:50.

[0020] The hydrophobic core comprises as described herein. In some embodiments of the present invention, the hydrophobic core is a lipid, an oil, a hydrophobic polymer, or a polypeptide. In one embodiment of the present invention, the hydrophobic core is co-encapsulated with a therapeutic agent in a pharmaceutical composition. Examples of oils include, but are not limited to, vegetable oil, labrafac, soybean oil, castor oil, olive oil, Nigella sativa oil, garlic oil, echium oil, cottonseed oil, peanut oil, sesame oil, aniseed oil, cinnamon oil, coconut oil, corn oil, PEG-60 hydrogenated castor oil, and polyoxyl 35 castor oil.

[0021] In some embodiments, the hydrophobic core comprises one or more types of lipids. Examples of lipids include, but are not limited to, non-ionic / ionic lipids such as tristearin, phosphate lipids, egg phospholipids, stearic acid, lecithin, cholesterol, hydrogenated soy phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), DSPE-PEG, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dimethyldioctadecylammonium (DDA), and 1,2-dimyristoyl rac-glycero-3 (DMG)-PEG.

[0022] In some embodiments, the hydrophobic core comprises one or more types of hydrophobic polymers. Examples of hydrophobic polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA).

[0023] In some embodiments, the hydrophobic core described herein comprises more than one material. In some embodiments of the invention, the hydrophobic core comprises a lipid and a hydrophobic polymer.

[0024] In some embodiments, the polysaccharide shells described herein comprise a multi-complementary polysaccharide shell. Examples of polysaccharide shells include, but are not limited to, a complex comprising fucoidan, PVA, and lysine. For example, based on 10 mg of fucoidan, the weight of PVA may be about 0.01 mg to about 2 mg, 0.05 mg to about 1.95 mg, 0.10 mg to about 1.90 mg, 0.15 mg to about 1.85 mg, 0.2 mg to about 1.8 mg, 0.25 mg to about 1.75 mg, 0.30 mg to about 1.70 mg, 0.35 mg to about 1.75 mg, 0.40 mg to about 1.70 mg, 0.45 mg to about 1.65 mg, 0.45 mg to about 1.65 mg, 0.5 mg to about 1.5 mg, 0.6 mg to about 1.5 mg, 0.7 mg to about 1.5 mg, 0.8 mg to about 1.5 mg, 0.9 mg to about 1.6 mg, 10 mg to about 1.6 mg, 1.8 mg to about 1.7 mg, 1.8 mg to about 1.6 ... g, 0.50 mg to about 1.60 mg, 0.55 mg to about 1.55 mg, 0.60 mg to about 1.50 mg, 0.65 mg to about 1.55 mg, 0.70 mg to about 1.50 mg, 0.75 mg to about 1.45 mg, 0.80 mg to about 1.4 mg, 0.85 mg to about 1.35 mg, 0.9 mg to about 1.3 mg, 0.95 mg to about 1.25 mg, 1.0 mg to about 1.20 mg, or 1.05 mg to about 1.15 mg. For example, based on 10 mg of fucoidan, the weight of lysine is about 0.3 mg to about 5 mg, about 0.35 mg to about 4.95 mg, about 0.40 mg to about 4.90 mg, about 0.45 mg to about 4.85 mg, about 0.5 mg to about 4.8 mg, about 0.55 mg to about 4.75 mg, about 0.6 mg to about 4.7 mg, about 0.65 mg to about 4.65 mg, about 0.70 mg to about 4.60 mg, about 0.7 The range is from about 0 mg to about 4.55 mg, from about 0.75 mg to about 4.5 mg, from about 0.80 mg to about 4.45 mg, from about 0.85 mg to about 4.40 mg, from about 0.90 mg to about 4.45 mg, from about 0.95 mg to about 4.50 mg, from about 1.0 mg to about 4.45 mg, from about 1.5 mg to about 4.4 mg, from about 2.0 mg to about 4.3 mg, from about 2.5 mg to about 4 mg, or from about 3.0 mg to about 4 mg.

[0025] In one embodiment of the invention, the complex comprises fucoidan, PLGA, and lysine. In some embodiments of the invention, the molar ratio between fucoidan and PLGA is about 1:3 to about 1:25, about 1:4 to about 1:24, about 1:5 to about 1:23, about 1:6 to about 1:22, about 1:7 to about 1:21, about 1:8 to about 1:20, about 1:9 to about 1:19, about 1:10 to about 1:18, about 1:11 to about 1:17, about 1:12 to about 1:16, about 1:13 to about 1:17, about 1:14 to about 1:16, or about 1:15. In some embodiments of the invention, the molar ratio between fucoidan and lysine is about 1:40 to about 1:160, about 1:50 to about 1:150, about 1:60 to about 1:140, about 1:70 to about 1:130, about 1:80 to about 1:120, about 1:90 to about 1:110, or about 1:100 to about 1:105. In some embodiments of the invention, the complex comprising fucoidan, PLGA, and lysine further comprises soybean oil. In some embodiments of the present invention, the molar ratio between fucoidan and soybean oil is about 1:6 to about 1:26, about 1:7 to about 1:25, about 1:8 to about 1:24, about 1:9 to about 1:23, about 1:10 to about 1:22, about 1:11 to about 1:21, about 1:12 to about 1:20, about 1:13 to about 1:19, about 1:14 to about 1:18, about 1:15 to about 1:17, or about 1:16.

[0026] In some embodiments, the pharmaceutical composition further comprises a therapeutic agent. In one embodiment of the invention, the therapeutic agent is encapsulated in the complex. Examples of therapeutic agents include, but are not limited to, anti-cancer agents, anti-inflammatory agents, agents for stroke medication, immunomodulatory agents, nucleic acid molecules, antibacterial agents, antiviral agents, anticoagulants, or antioxidant agents.

[0027] Examples of anti-cancer drugs include, but are not limited to, bleomycin, cisplatin, carboplatin, cytarabine, docetaxel, doxorubicin, daunorubicin, epirubicin, fluorouracil, gemcitabine, irinotecan, leuprorelin, oxaliplatin, paclitaxel, pemetrexed, topotecan, vinorelbine, or vinblastine.

[0028] Examples of anti-inflammatory drugs include, but are not limited to, ibuprofen, naproxen sodium, diclofenac potassium, celecoxib, sulindac, oxaprozin, piroxicam, or indomethacin.

[0029] Examples of drugs for stroke medication include, but are not limited to, tissue plasminogen activator (tPA), warfarin, clopidogrel, aspirin, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin.

[0030] Examples of immunomodulatory agents include, but are not limited to, cytokines, thalidomide, lenalidomide, pomalidomide, or imiquimod.

[0031] Examples of nucleic acids include, but are not limited to, plasmid DNA, messenger RNA (mRNA), RNA inhibitor (RNAi), small interfering RNA (siRNA), aptamer, or microRNA. In some embodiments of the invention, the nucleic acid is plasmid DNA, siRNA, or aptamer.

[0032] In some embodiments of the invention, the loading capacity of the conjugate for a therapeutic agent ranges from about 1% to about 30%, from about 2% to about 28%, from about 3% to about 26%, from about 4% to about 24%, from about 6% to about 22%, from about 8% to about 20%, from about 10% to about 18%, from about 12% to about 16%, from about 12% to about 15%, or from about 12% to about 14%.

[0033] In some embodiments of the invention, the pharmaceutical compositions exhibit the ability to target CD62P (p-selectin) within the tumor microenvironment and improve the therapeutic efficacy of the encapsulated therapeutic agent in CD62P-positive cancer types, such as breast cancer, lymphoma, lung cancer, bladder cancer, ovarian cancer, and pancreatic cancer.

[0034] The present invention also provides a method of treating a disease in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described herein.

[0035] The invention also provides the use of a pharmaceutical composition described herein in the manufacture of a medicament for treating a disease in a subject in need of such treatment.

[0036] In some embodiments of the invention, the therapeutic agent is an anti-cancer agent and the disease is selected from the group consisting of breast cancer, lymphoma, lung cancer, bladder cancer, ovarian cancer, and pancreatic cancer. [Brief description of the drawings]

[0037] [Figure 1] FIG. 2 shows the particle size of the pharmaceutical composition comprising fucoidan, lysine, and PLGA and docetaxel of Example 1. [Diagram 2] FIG. 1 shows the colloidal stability of the pharmaceutical composition of Example 3 containing fucoidan, lysine, PLGA, and docetaxel. [Figure 3A] FIG. 1 shows that the fucoidan-based DDS described in Example 1 extends the median survival rate of 4T1-3-bearing mice. [Figure 3B] FIG. 1 shows that the fucoidan-based DDS described in Example 1 extends the median survival rate of SKOV-3-bearing mice. [Figure 4] FIG. 1 shows the particle size of the pharmaceutical composition of Example 4 containing fucoidan, lysine, soybean oil, PLGA, and docetaxel. [Diagram 5] FIG. 1 shows the stability of the pharmaceutical composition of Example 4 containing fucoidan, lysine, soybean oil, PLGA, and docetaxel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Unless otherwise defined, all scientific or technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be understood and used by one of ordinary skill in the art to practice the present invention.

[0039] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and claims are approximations and may vary depending upon the desired properties sought to be obtained by the present invention.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms, including "at least one," unless the content clearly indicates otherwise. It is further understood that the terms "comprises" and / or "comprising," or "includes" and / or "including," as used herein, specify the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0041] As used herein, "about" is inclusive of the stated value and means within an acceptable range of deviation of the particular value as determined by one of ordinary skill in the art, taking into account the measurement in question and the error associated with the measurement of the particular quantity (i.e., measurement system limitations). For example, "about" can mean within one or more standard deviations, or within ±30%, 20%, 10%, or 5% of the stated value.

[0042] As used herein, the term "pharmaceutical composition" means a mixture containing therapeutic agents that is administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or condition from which the mammal suffers.

[0043] As used herein, the term "composite" refers to a material comprising two or more materials having different physical or chemical properties, where the composite has properties that differ from the individual materials that make up the composite, and where the individual materials are macroscopically or microscopically separate and distinguishable from one another in the final structure of the composite.

[0044] As used herein, the term "amphiphilic" refers to the property of a substance having both hydrophobic and hydrophilic sites. For example, when the medium is water, a substance having amphiphilic properties will form micellar particles, which can be observed. In some embodiments of the present invention, molecules having amphiphilic properties can reduce surface tension and stabilize the water-oil interface.

[0045] As used herein, the term "affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner.

[0046] As used herein, the term "loading capacity" refers to the ratio of a therapeutic agent loaded into a pharmaceutical composition to the total pharmaceutical composition.

[0047] As used herein, the terms "treat" or "treatment" mean to reverse, alleviate, inhibit the progression of, or ameliorate the disorder, disease, or condition to which such term applies, or one or more symptoms of such disorder, disease, or condition.

[0048] As used herein, the term "therapeutic agent" means any compound, substance, drug, drug moiety or active ingredient that has a therapeutic or pharmacological effect suitable for administration to a mammal, e.g., a human.

[0049] As used herein, the term "subject" refers to any mammal that may be treated with the disclosed compositions. The subject may be a vertebrate, such as a mammal. In some embodiments, the subject may be, for example, a human, a primate, a dog, a cat, a horse, a cow, a pig, a rodent, such as a rat or a mouse, etc. Typically, the subject may be a human. The subject may or may not exhibit symptoms. The term does not denote a particular age or sex. Thus, it is intended to cover adult and newborn subjects, whether male or female. The subject may include a control subject or a test subject.

[0050] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, nurse practitioner, or individual in the case of a human, a veterinarian in the case of an animal, including a non-human mammal) that a subject is in need of treatment or would benefit from treatment from the caregiver. This judgment is made based on a variety of factors that are within the expertise of the caregiver, but includes the recognition that the subject is ill or will become ill as a result of a condition treatable by a compound of the invention.

[0051] As used herein, a "polysaccharide" can refer to a naturally occurring full-length polysaccharide molecule, a mixture of any combination of hydrolysis products of a full-length polysaccharide molecule (including monosaccharide, oligosaccharide and polysaccharide species), any chemically modified or functionalized derivative of a full-length polysaccharide molecule or its hydrolysis products, or any combination thereof. A polysaccharide can be linear or branched, and can be a single species or a mixture of related species (e.g., molecules having the same basic monosaccharide unit but with different repeating numbers). As used herein, a "sulfated polysaccharide" refers to a polysaccharide in which at least one monosaccharide is replaced with a sulfate group. In one embodiment, a sulfated polysaccharide is a polysaccharide in which at least one sugar ring is replaced with a sulfate group.

[0052] Fucoidan, a sulfated polysaccharide, has multiple biological effects. Fucoidan is also highly biocompatible. Therefore, fucoidan has been used as a component of DDS to improve drug delivery. However, fucoidan lacks amphiphilicity, making it a poor surfactant for stabilizing oil-in-water (O / W) or water-in-oil (W / O) interfaces. The structure of fucoidan mainly contains α-fucose residues, while the negative charge of this biopolymer comes from the presence of sulfate groups substituted mainly at C-2 and C-4 positions, and occasionally at C-3 position. Chemical modification of the molecular structure of fucoidan has demonstrated improved amphiphilicity. However, once the molecular structure is altered, biological functions, such as p-selectin targeting and immunomodulatory effects, are impaired. Therefore, new strategies are urgently needed to stabilize oil-water interfaces using fucoidan without impairing its biological functions. In some embodiments of the present invention, fucoidan is not chemically modified.

[0053] In some embodiments of the invention, the fucoidan may be produced by Bladderwrack, Cladophora okamuranus, Cladophora okamuranus, Ascophyllum nodosum, Fucus fucus, Fucus serranoides, Fucus distichus, Fucus serratus, Fucus spiralis, Ascophyllum mackaii, Pelvetia canaliculata, Sclerotinia niger, and Undaria pinnatifida, and may be purified or partially purified from cultures of the organisms. In some embodiments of the invention, the peak molecular weight of the fucoidan ranges from 10 kDa to 200 kDa, 20 kDa to 180 kDa, 30 kDa to 160 kDa, 40 kDa to 140 kDa, 50 kDa to 120 kDa, 60 kDa to 100 kDa, 80 kDa to 90 kDa.

[0054] The purity of the fucoidan can vary, in one embodiment, the purity of the fucoidan ranges from about 60% to about 99%, from about 65% to about 95%, from about 70% to about 90%, from about 75% to about 85%, or from about 70% to about 80%.

[0055] Without wishing to be bound by theory, it is believed that the sulfate content of the sulfated polysaccharide may play a role in the complexes of the present invention. In one embodiment, the sulfate content of the fucoidan ranges from about 15% to about 40%, from about 18% to about 38%, from about 20% to about 35%, from about 22% to about 32%, or from about 25% to about 30%.

[0056] Thus, the present invention provides a pharmaceutical composition comprising a conjugate comprising a polysaccharide shell and a hydrophobic core, wherein the polysaccharide shell comprises a sulfated polysaccharide and a complement having an affinity for sulfated polysaccharides, and the hydrophobic core comprises one or more hydrophobic molecules.

[0057] The present invention also provides a method of treating a disease in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described herein.

[0058] Complement as used herein refers to a substance that has a high affinity for sulfated polysaccharides, such as fucoidan, and can form the complexes described herein. The formation of complexes between sulfated polysaccharides and complements can modulate the physiological properties of sulfated polysaccharides, thereby further endowing the complexes with the ability to stabilize the oil-water interface. Furthermore, the formation of complexes between sulfated polysaccharides and complements does not change the molecular structure of sulfated polysaccharides, thereby theoretically not impairing the biological functions of sulfated polysaccharides. Thus, this strategy shows the possibility of forming a stable drug delivery system with uniform size distribution, while retaining or even enhancing the biological functions of fucoidan, including antibacterial activity, antiviral activity, antitumor activity, anticoagulant activity, antioxidant activity, and p-selectin targeting ability. Thus, by promoting the inherent therapeutic properties, the sulfated polysaccharide-based drug delivery system has the ability to deliver drugs to the affected area and further enhance the therapeutic effect.

[0059] By using complement, the complex can stabilize the oil-water interface. Thus, the pharmaceutical composition can be emulsion-based or nanoprecipitated nanoparticles.

[0060] The complement disclosed herein is physically, chemically, or biologically complementary to sulfated polysaccharides, and the complement can form a complex with the sulfated polysaccharide to stabilize the interface without compromising the biological effect. By forming a complex with the complement, sulfated polysaccharide-based DDS can be formed by a simple emulsification process, and can obtain improved stability and broader application.

[0061] The complement can be in the form of a small molecule, protein, polymer, or combination thereof that exhibits high affinity to sulfated polysaccharides due to physical, chemical, or biological interaction forces. In one embodiment, the complement also has a hydrophobic domain. Thus, when the complement is mixed with sulfated polysaccharides in a certain range of ratios and at a certain defined pH value, the formation of the complex confers amphiphilic ability and stabilizes the interface. Thus, the complement can compensate for the weakness of using hydrophilic sulfated polysaccharides alone. It is noted that in the process of emulsion or nanoprecipitation, shear stress is usually present to mix solutions of different phases into a homogeneous solution. The complement and its interaction force with the sulfated polysaccharide must be higher than the shear stress, so that the formation of sulfated polysaccharide-complement complex can promote the stabilization of the interface without tearing them apart from each other during emulsification.

[0062] Examples of complements include, but are not limited to, a physical complement, a chemical complement, or a biological complement.

[0063] Examples of physical forces applied in physical complementation include, but are not limited to, electrostatic interactions or hydrophobic interactions.

[0064] Sulfated polysaccharides contain sulfate salts, which makes them molecules with strong negative charges. Basic complements containing positive charges and optionally hydrophobic domains are the preferred choice to interact with sulfated polysaccharides to form complexes. Positively charged amino acids including lysine, arginine, histidine, glutamine, and their polymeric / copolymeric molecules can form complexes with sulfated polysaccharides by electrostatic forces to stabilize the O / W and W / O interfaces and provide smaller particle sizes after emulsification. Zein, chitosan, protamine, polyethyleneimine (PEI), amine polyethylene glycol (PEG), amine-terminated poly(ethylene oxide) (PEO), and poly(epsilon-caprolactone) (PCL) and other materials / molecules containing positively charged functional groups with hydrophobic domains can also act as complements for sulfated polysaccharides to form complexes to stabilize the particle interface.

[0065] In some embodiments of the present invention, the molar ratio range between fucoidan and lysine that can stabilize the O / W interface and the W / O interface to provide a stable formulation is about 1:10 to about 1:160, the molar ratio range between fucoidan and arginine is about 1:0.005 to about 1:5, the molar ratio range between fucoidan and histidine is about 1:0.005 to about 1:5, the molar ratio range between fucoidan and glutamine is about 1:0.005 to about 1:5, the molar ratio range between fucoidan and zein is about 1:0.002 to about 1:10, the molar ratio range between fucoidan and chitosan is about 1:0.05 to about 1:50, the molar ratio range between fucoidan and protamine is about 1:0.02 to about 1:100, and the molar ratio range between fucoidan and polyethyleneimine is about 1:0.01 to about 1:100.

[0066] In some embodiments of the invention, the ratio of negative charges in the sulfated polysaccharide to positive charges in the complement is from about 1:0.05 to about 1:3.

[0067] In one embodiment of the present invention, hydrogen bonds are applied to chemical complements. Sulfated polysaccharides are rich in hydroxyl groups and have both hydrogen bond donor and acceptor sites that simultaneously form two types of hydrogen bonds. Thus, hydrogen bonds can be easily formed between sulfated polysaccharides and a wide variety of materials / molecules that contain hydrogen bond donor and acceptor sites. For example, sulfated polysaccharides can form OH···:N with amine-containing ligands / molecules (i.e., hydrogen donors). Sulfated polysaccharides can also form OH···:O with other molecules that contain acceptor atoms, such as carboxylic acids and oxygen acceptors. It is noted that hydrogen bonds are relatively weak, and therefore chemical complements can only attach to sulfated polysaccharides and contribute to the stabilization of the water-oil interface if sulfated polysaccharides form complexes with hydrogen bond donors or acceptors or combinations thereof in a certain ratio to generate sufficient forces between the molecules. Molecules with hydrogen donors or hydrogen acceptors, such as oxidized dextran, polyethylene glycol (PEG), chemically modified PEG, polydextrose, polysorbate 20, polysorbate 80, polyvinyl acetate, polyvinyl alcohol, Pluronic® F68, Pluronic® F123, Pluronic® F127, polyvinyl alcohol, and propylene glycol alginate, may form complexes with fucoidan to stabilize the O / W and W / O interfaces.

[0068] Regarding biological complements, fucoidan is known to be a ligand of P-selectin, a type 1 transmembrane protein encoded by the human SELP gene, or its modified forms. P-selectin as a protein has a hydrophobic domain in its structure. There is a high affinity between fucoidan and p-selectin, and therefore the formation of their complexes can be an amphiphilic material that stabilizes the oil-water interface. Similarly, since fucoidan has some affinity for selectins, it is predicted that the biological interaction between them can form complexes and stabilize the oil-water interface. P-selectin can be further chemically modified to increase the portion of hydrophobic side chains or provide functional groups. The modification of P-selectin leads to a stronger interaction with fucoidan, providing a cross-linking point for the fixation of target ligands or target molecules. The formation of a complex between fucoidan and P-selectin, or between fucoidan and chemically modified P-selectin, may stabilize the O / W and W / O interfaces.

[0069] The above mentioned materials / molecules can be used to form complexes with sulfated polysaccharides, providing additional or even synergistic effects in stabilizing the interface and forming nano / microparticles with tunable size and structure.

[0070] For example, PVA and lysine were used as different kinds of complements and simultaneously formed a complex with fucoidan. After emulsification, the formed nanoparticles showed smaller size (i.e., more compact), more uniform size distribution, and higher aqueous colloidal stability. Therefore, different kinds of complements can be combined with sulfated polysaccharides in a certain ratio to form a composition to optimize the ability to stabilize the O / W and W / O interfaces to form the required particle size. The weight ratio of sulfated polysaccharides and different kinds of complements that can obtain stabilization of the O / W and W / O interfaces varies depending on the type and number of complements and their relative composition. For example, the formation of a complex of sulfated polysaccharides with PVA and lysine can stabilize the O / W and W / O interfaces. For example, based on 10 mg of fucoidan, the weight of PVA ranges from about 0.01 mg to about 2 mg. For example, based on 10 mg of fucoidan, the weight of lysine ranges from about 0.3 mg to about 5 mg.

[0071] In one embodiment of the present invention, the complex comprises fucoidan, PLGA, and lysine. In some embodiments of the present invention, the molar ratio between fucoidan and PLGA is about 1:3 to about 1:25, particularly about 1:3.04 to about 1:22.8. In some embodiments of the present invention, the molar ratio between fucoidan and lysine is about 1:40 to about 1:160, particularly about 1:38.94 to about 1:160. In some embodiments of the present invention, the complex comprising fucoidan, PLGA, and lysine further comprises soybean oil. In some embodiments of the present invention, the molar ratio between fucoidan and soybean oil is about 1:6 to about 1:26, particularly about 1:6.49 to about 1:26.

[0072] As described herein, the hydrophobic core comprises a therapeutic agent. In some embodiments of the present invention, the hydrophobic core is a lipid or a hydrophobic polymer. Without wishing to be bound by theory, it is believed that the addition of lipids to the composition causes the emulsion to exhibit improved surface properties, improved drug delivery, and improved cellular uptake into desired cells / tissues.

[0073] In some embodiments of the present invention, the hydrophobic core comprises oil. In one embodiment of the present invention, the hydrophobic core is co-encapsulated with a therapeutic agent in a pharmaceutical composition. Without wishing to be bound by theory, it is believed that the addition of oil to the composition can allow the therapeutic agent to be loaded more efficiently. Examples of oil include, but are not limited to, vegetable oil, labrafac, soybean oil, castor oil, olive oil, nigella sativa oil, garlic oil, echium oil, cottonseed oil, peanut oil, sesame oil, aniseed oil, cinnamon oil, coconut oil, corn oil, PEG-60 hydrogenated castor oil, and polyoxyl 35 castor oil.

[0074] In some embodiments, the hydrophobic core comprises one or more types of lipids. Examples of lipids include, but are not limited to, non-ionic / ionic lipids such as tristearin, phosphate lipids, egg phospholipids, stearic acid, lecithin, cholesterol, hydrogenated soy phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), DSPE-PEG, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dimethyldioctadecylammonium (DDA), and 1,2-dimyristoyl rac-glycero-3 (DMG)-PEG.

[0075] In some embodiments, the hydrophobic core comprises one or more types of hydrophobic polymers or polypeptides. Examples of hydrophobic polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and poly-L-leucine.

[0076] In some embodiments, the hydrophobic core described herein comprises more than one material. In some embodiments of the invention, the hydrophobic core comprises a lipid and a hydrophobic polymer.

[0077] DDS based on sulfated polysaccharides can deliver drugs, change their pharmacokinetic behavior, improve the biodistribution of drugs, and further improve the therapeutic effect. Thus, the pharmaceutical compositions described herein further comprise one or more therapeutic agents. In one embodiment of the present invention, the therapeutic agent is encapsulated in the complex. Examples of therapeutic agents that can be incorporated into the fucoidan-complement formulation include anticancer drugs (e.g., bleomycin, cisplatin, carboplatin, cytarabine, docetaxel, doxorubicin, daunorubicin, epirubicin, fluorouracil, gemcitabine, irinotecan, leuprorelin, oxaliplatin, paclitaxel, pemetrexed, topotecan, vinorelbine, vinblastine), anti-inflammatory drugs (e.g., ibuprofen, naproxen sodium, diclofenac potassium, celecoxib, sulindac, oncolytic drugs, steroids ... xaprozin, piroxicam, indomethacin), drugs for stroke medication (e.g., tissue plasminogen activator (tPA), warfarin, clopidogrel, aspirin, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), immunomodulatory drugs (e.g., cytokines, thalidomide, lenalidomide, pomalidomide, and imiquimod), messenger RNA (mRNA), RNA inhibitor (RNAi), or microRNA.

[0078] The following examples are provided to aid those of ordinary skill in the art in practicing the present invention. EXAMPLES

[0079] [Example 1] Complexes containing polysaccharide shells with electrostatic complements

[0080] The particle size of the emulsions having a polysaccharide shell with electrostatic complement and a hydrophobic PLGA core was analyzed using dynamic light scattering (DLS) and is shown in Table 1.

[0081] [Table 1]

[0082] Positively charged amino acids, including lysine, arginine, histidine, glutamine, and their polymerized molecules, were shown to form complexes with sulfated polysaccharides through electrostatic forces to stabilize the O / W and W / O interfaces, resulting in smaller particle sizes after emulsification.

[0083] Additionally, suitable ratios of fucoidan to lysine molecules for forming emulsions or nanoprecipitates are shown in Table 2.

[0084] [Table 2]

[0085] [Example 2] Complexes containing polysaccharide shells containing different types of complements

[0086] The particle sizes of emulsions with polysaccharide shells and hydrophobic PLGA cores containing different types of complements were analyzed using dynamic light scattering (DLS) and are shown in Table 3.

[0087] [Table 3]

[0088] [Example 3] Pharmaceutical Compositions Comprising the Conjugate and a Therapeutic Agent

[0089] The complex containing fucoidan and a polysaccharide shell containing lysine that stabilizes the O / W interface to the hydrophobic core containing PLGA can encapsulate docetaxel in the fucoidan-based DDS. The loading capacity of docetaxel can reach about 15% to about 50%. The particle size of the emulsion was analyzed using DLS and is shown in Figure 1.

[0090] The fucoidan-based DDS described in Example 1 could exist in colloidal form at room temperature for at least 2 weeks without precipitation. The stability was observed using DLS and the results are shown in Figure 2.

[0091] The fucoidan-based DDS described in Example 1 showed stronger cytotoxicity in triple-negative breast cancer cell lines (MDA-MB-231 and 4T1) and pancreatic cancer cell line (CFPAC-1) compared to unformulated DTX. The IC50 results are shown in Table 4.

[0092] [Table 4]

[0093] The fucoidan-based DDS described in Example 1 showed improved therapeutic efficacy in the syngeneic 4T1 triple-negative breast cancer animal model and the SKOV3 ovarian cancer animal model, prolonging the median survival rate when compared with docetaxel, as shown in Figures 3A and 3B.

[0094] [Example 4] Pharmaceutical Compositions Comprising the Conjugate and a Therapeutic Agent

[0095] The complex containing fucoidan and a polysaccharide shell containing lysine that stabilizes the O / W interface to the hydrophobic core containing PLGA and soybean oil can encapsulate docetaxel in the fucoidan-based DDS. The loading capacity of docetaxel can reach about 15% to about 45%. The particle size of the emulsion was analyzed using DLS and is shown in Figure 4.

[0096] The fucoidan-based DDS could remain in colloidal form at room temperature for at least two weeks without precipitation. The stability was observed using DLS, and the results are shown in Figure 5.

[0097] The fucoidan-based DDS showed more potent cytotoxicity compared to unformulated DTX in triple-negative breast cancer cell lines (MDA-MB-231 and 4T1), pancreatic cancer cell lines (CFPAC-1 and BxPC3), and ovarian cancer cell line (SKOV3). The IC50 results are shown in Table 5.

[0098] [Table 5]

[0099] The above description of example embodiments of the invention has been presented for purposes of illustration and description only, and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

1. A complex comprising a polysaccharide shell and a hydrophobic core, wherein the polysaccharide shell comprises fucoidan and a complement having an affinity for fucoidan, and the hydrophobic core comprises a hydrophobic molecule; A complex in which the peak molecular weight of fucoidan ranges from 30 to 120 kDa.

2. 2. The complex of claim 1, wherein the purity of the fucoidan ranges from about 60% to about 99% and / or the sulfate content of the fucoidan ranges from about 15% to about 40%.

3. 2. The complex of claim 1, wherein the complement is selected from the group consisting of a molecule having a positively charged functional group, a molecule having a hydrophobic domain, a molecule having an amine-containing ligand, a molecule having a carboxylic acid group, and a molecule having a hydrogen bond acceptor.

4. The complex described in claim 1, wherein the ratio of negative charges in the fucoidan to positive charges in the complement is in the range of about 1:0.05 to about 1:

3.

5. The complex of claim 1, wherein the complement binds to the fucoidan by hydrogen bonds.

6. 2. The complex of claim 1, wherein the complement is selected from the group consisting of lysine or a polymer / copolymer thereof, arginine or a polymer / copolymer thereof, histidine or a polymer / copolymer thereof, glutamine or a polymer / copolymer thereof, zein, chitosan, protamine, polyethyleneimine, amine polyethylene glycol (PEG), amine-terminated poly(ethylene oxide) (PEO), poly(epsilon-caprolactone) (PCL), oxidized dextran, polyethylene glycol (PEG), chemically modified PEG, polydextrose, polysorbate 20, polysorbate 80, polyvinyl acetate, Pluronic® F68, Pluronic® F123, Pluronic® F127, polyvinyl alcohol, propylene glycol alginate, and p-selectin or modified p-selectin.

7. The complex described in claim 1, wherein the molar ratio of fucoidan to complement is in the range of about 1:0.005 to about 1:

100.

8. The complex of claim 1 , wherein the hydrophobic core is a lipid, oil, hydrophobic polymer, or polypeptide.

9. The hydrophobic core is made up of vegetable oil, Lavrafac, soybean oil, castor oil, olive oil, nigella sativa oil, garlic oil, echium oil, cottonseed oil, peanut oil, sesame oil, aniseed oil, cinnamon oil, coconut oil, corn oil, PEG-60 hydrogenated castor oil, polyoxyl 35 castor oil, tristearin, phosphate lipids, egg phospholipids, stearic acid, lecithin, cholesterol, hydrogenated soy phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-s 9. The conjugate of claim 8, wherein the conjugate is selected from the group consisting of n-glycero-3-phosphorylethanolamine (DSPE), DSPE-PEG, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl rac-glycero-3 (DMG)-PEG, polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and poly-L-leucine.

10. 2. The complex of claim 1, comprising fucoidan, PLGA, and lysine.

11. 11. The complex of claim 10, wherein the molar ratio between fucoidan and PLGA is about 1:3 to about 1:25, and / or the molar ratio between fucoidan and lysine is about 1:40 to about 1:

160.

12. 10. The complex of claim 1, comprising fucoidan, PLGA, lysine, and soybean oil.

13. 13. The complex of claim 12, wherein the molar ratio between fucoidan and PLGA is about 1:3 to about 1:25, the molar ratio between fucoidan and lysine is about 1:40 to about 1:160, and / or the molar ratio between fucoidan and soybean oil is about 1:6 to about 1:

26.

14. A pharmaceutical composition comprising the conjugate of claim 1 and a therapeutic agent.

15. 15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is in the form of an emulsion or nanoprecipitated nanoparticles.

16. 15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is encapsulated in a complex.

17. 15. The pharmaceutical composition of claim 14, wherein the loading capacity of the conjugate for the therapeutic agent ranges from about 1% to about 30%.

18. 15. Use of the pharmaceutical composition of claim 14 in the manufacture of a medicament for treating a disease in a subject in need of such treatment.

19. 19. The use of claim 18, wherein the therapeutic agent is an anticancer agent and the disease is selected from the group consisting of breast cancer, lymphoma, lung cancer, bladder cancer, ovarian cancer, and pancreatic cancer.

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