Description

CONJUGATE COMPRISING PHARMACEUTICAL ACTIVE

COMPOUND COVALENTLY BOUND TO MUCO ADHESIVE

POLYMER AND TRANSMUCOSAL DELIVERY METHOD OF

PHARMACEUTICAL ACTIVE COMPOUND USING THE SAME

Technical Field

[1] The present invention relates to a conjugate comprising a pharmacologically active substance covalently bound to a mucoadhesive polymer and a method for transmucosal delivery of a pharmacologically active substance using the same. Background Art

[2] With great advances in genetic engineering and bioprocess technologies, it became possible to achieve industrial-scale production of various peptide and protein drugs, e.g. biopharmaceutical products (hereinafter, also referred to as "biodrugs"), which have suffered from difficulties in chemical synthesis. However, most proteins exhibit non-absorptive tendencies through the mucous membranes of organisms due to huge molecular weight and specific molecular structure, thereby suffering from difficulties in application thereof for oral preparations. Therefore, an administration route of proteins is confined to injection, which is accompanied by various problems, such as difficulty of medication upon chronic administration of drugs and fear and rejection of injection therapy to patients. Therefore, development of oral preparations having no burden of injection administration on patients by increasing an enteric absorption rate of biodrugs will obviate fear and rejection of injection and enables the patients to easily take a drug in compliance with medication instructions, thereby leading to improvements in the short- and long term- life quality of patients.

[3] For these reasons, various attempts have been actively made to enhance in vivo stability and absorption rate of therapeutic proteins. Among such trials, the most well- known approach is PEGylation, the process by which polyethylene glycol (PEG) chains are chemically attached to proteins or peptides. At the early stage of introduction, this technique was used to reduce antigenicity of target materials. Now, PEGylation is largely employed for improvement of in vivo stability and absorption rate of target proteins by increasing an in vivo residence time of the proteins.

[4] In addition to PEGylation, a great deal of research has been focused lately on a method of using the biodrug in conjunction with a substance that is capable of enhancing the permeability of an intestinal epithelial cell membrane, such as a fatty acid, a bile acid, or the like, a method of using a substance (for example, Vitamin B 12

and Fc receptor) that is capable of selectively binding to a receptor of the intestinal epithelial cell membrane, a method of increasing a drug absorption rate from the intestinal mucosa via a conjugate of insulin with a fat-soluble substance including lipid and bile acid through the direct chemical bonding therebetween, a method of drug delivery by inclusion of protein drugs into microparticles or nanoparticle of biodegradable polymers.

[5] However, these methods still suffer from disadvantages such as very low in vivo absorption and bioavailability upon oral administration and potential safety risk due to the use of additives in conventional formulations for oral applications that may exhibit toxicity upon chronic administration.

[6] In recent years, there is a great deal of interest in developing a method for delivery of an anti-cancer drug that is poorly water-soluble, particularly paclitaxel, an antineoplastic agent effective against a wide range of cancers including breast cancer and ovarian cancer. Meanwhile, paclitaxel has a very low solubility in conventional aqueous vehicles including water and therefore is formulated into a vehicle containing ethanol and Cremophor EL. For this reason, administration of the anti-caner drug paclitaxel via intravenous infusion causes severe side effects such as hypersensitivity reactions. In order to overcome such shortcomings of paclitaxel therapy, a variety of attempts has been made including micellular formulation, conjugation with a variety of water-soluble macromolecules and prodrug approaches. Meanwhile, with increases in such research and study, there has been a great deal of focus in recent years on the development of the oral delivery system of paclitaxel. This is because such an oral formulation of paclitaxel is preferable for treatment of chronic diseases including cancers and is greatly beneficial for patients by providing easy and convenient administration without a need to go to the hospital for an intravenous infusion. However, oral administration of paclitaxel poses a disadvantage of low bioavailability. According to recent research publication reports in the scientific articles and journals, the bioavailability of paclitaxel was increased to a clinically valuable level. One of the those research papers reported that the combined use of paclitaxel with a P- glycoprotein (P-gp) inhibitor such as cyclosporin A (CsA) or Valspodar resulted in a very high increase in the bioavailability of paclitaxel (ca. 50-60% vs. ca. 4-10% with PTX only). Despite such a favorable result, P-gp is known to protect gastrointestinal tract, cerebrum and excretory organs against xenotoxin and therefore use of the P-gp inhibitor may potentially cause adverse side effects. Furthermore, other studies were reported including methods of preparing emulsions of paclitaxel using surfactants and methods of encapsulating paclitaxel into biodegradable polymer nanoparticles. However, use of excessive amounts of surfactants may bring about the toxicity to the subjects and the above methods have a drawback of low bioavailability

[7] Therefore, there is a strong need for the development of a pharmaceutical formulation that can provide administration of an anti-cancer drug, such as paclitaxel, via an oral route capable of exerting high bioavailability of the drug.

[8] Transmucosal delivery is a method for administration of pharmacologically-active substance and provides great advantages. Owing to an ability of transmucosal delivery that can achieve systemic and local drug effects on target sites, the transmucosal delivery system has received a great deal of attention as an attractive drug delivery system which can cope with specific regimens of drugs. Transmucosal delivery not only rapidly exerts therapeutic effects but also exhibits rapid drug clearance, consequently increasing bioavailability of the drug. In addition, the transmucosal delivery system is superior in patient medication compliance, as compared to other administration methods.

[9] Due to the aforementioned advantages of the transmucosal delivery system, many efforts have been made to develop more advanced transmucosal delivery systems. International Publication Nos. WO 2005/032554 and WO 2005/016321, US Patent Nos. 6896519, 6564092 and 6506730 disclose transmucosal delivery systems. In particular, US application Ser. No 07/579,375 (US Patent No. 5194594) discloses antibodies which have been modified by chemical conjugation with succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and U.S. application Ser. No. 08/167,611 (US Patent No. 5554388) discloses a composition for administration to the mucosa which comprises a pharmacologically active compound and a polycationic substance. In addition, US Patent No. 6,913,746 describes complexes consisting of immunoglobulins and polysaccharides for oral and transmucosal use, and US Patent Application No. 2005/0175679 Al describes a composition for transmucosal administration, comprising morphine and a water-soluble polymer.

[10] However, most of attempts to develop methods capable of achieving oral administration of protein drugs or anti-cancer drugs via transmucosal delivery of drugs were found futile with little successful results, particularly resulting in unsatisfactory therapeutic efficacy of drugs. Disclosure of Invention Technical Problem

[11] The inventors of the present invention have made many efforts to develop a drug delivery system that can realize transmucosal delivery, particularly oral transmucosal delivery of drugs while overcoming side effects and disadvantages which were suffered by conventional drug delivery systems of pharmacologically active substances. As a result of a variety of extensive and intensive studies and experiments to solve the problems as described above, the inventors of the present invention have

surprisingly discovered that it is possible to elicit excellent pharmacological efficacy of desired drugs in vivo by selection of a mucoadhesive polymer, exhibiting an excellent in vivo mucosal absorption rate, safety and in vivo degradability, as a delivery system capable of achieving the above-mentioned purposes, and oral administration of a conjugate comprising a pharmacologically active substance covalently bound to the thus-selected mucoadhesive polymer. The present invention has been completed based on these findings.

[12] Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a conjugate comprising a pharmacologically active substance and a mucoadhesive polymer covalently bound to each other via a linker.

[13] It is another object of the present invention to provide a pharmaceutical composition for transmucosal administration of a drug, comprising the aforementioned conjugate and a pharmaceutically acceptable carrier.

[14] It is a further object of the present invention to provide a method for in vivo delivery of a pharmacologically active substance via a transmucosal route, by covalent binding of the active substance with a mucoadhesive polymer via a linker.

[15] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings and claims. Technical Solution

[16] In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a conjugate comprising a pharmacologically active substance and a mucoadhesive polymer covalently bound to each other via a linker.

[17] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for transmucosal administration of a drug, comprising the aforementioned conjugate and a pharmaceutically acceptable carrier.

[18] In accordance with yet another aspect of the present invention, there is provided a method for in vivo delivery of a pharmacologically active substance via a transmucosal route, by covalent binding of the active ingredient with a mucoadhesive polymer via a linker.

Advantageous Effects

[19] The conjugate of the present invention exhibits excellent absorption rate and bio- compatibility in biological mucous membranes, particularly mucous membranes of alimentary canal (especially the gastrointestinal tract), in vivo degradability, and superior bioavailability even with oral administration, thus enabling treatment of

diseases via oral administration of a drug. Brief Description of the Drawings

[20] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[21] FIG. 1 is a graph showing changes in the relative blood glucose levels of animals after intravenous injection of an insulin-chitosan conjugate of the present invention into the tail veins of diabetes -induced male rats;

[22] FIG. 2 is a graph showing changes in the relative blood glucose levels of animals after oral administration of an insulin-chitosan conjugate solution of the present invention to diabetes -induced male rats;

[23] FIG. 3 is a bar graph showing results of MTT assay for cytotoxic effects of a paclitaxel-chitosan conjugate of the present invention on tumor cells. Black: paclitaxel; Diagonal line: paclitaxel-chitosan (MW: 3000) conjugate of the present invention; White: paclitaxel-chitosan (MW: 6000) conjugate of the present invention;

[24] FIG. 4 is a graph showing analysis results of allograft experiments for in vivo anticancer effects of a paclitaxel-chitosan conjugate of the present invention; and

[25] FIG. 5 is a graph showing a survival rate of animals after oral administration of a paclitaxel-chitosan conjugate to mice. Best Mode for Carrying Out the Invention

[26] Herein after, the present invention will be described in more detail.

[27]

[28] Pharmacologically active substance

[29] As used herein, the term "pharmacologically active substance" refers to a protein or peptide having pharmacological activity or a functional equivalent compound thereof. The pharmacologically active substance includes recombinantly, or synthetically synthesized substances, and other substances isolated from nature.

[30] As used herein, the term "protein" refers to a polymer of amino acids in peptide linkages and the term peptide refers to an oligomer of amino acids in peptide linkages.

[31] Examples of the protein or peptide that is used as the pharmacologically active substance in the present invention may include, but are not limited to, hormones, hormone analogues, enzymes, enzyme inhibitors, signaling proteins or fragments thereof, antibodies or fragments, single-chain antibodies, binding proteins or binding domains thereof, antigens, attachment proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulatory factors, blood coagulation factors, and anti-cancer drugs. Preferably, the pharmacologically active substance of the present invention may include materials that can be used as a protein drug, for example

insulin, insulin-like growth factor 1 (IGF-I), growth hormones, interferons (IFNs), erythropoietins, granulocyte-colony stimulating factors (G-CSFs), granulocyte/ macrophage-colony stimulating factors (GM-CSFs), interleukin-2 (IL-2) or epidermal growth factors (EGFs). More preferred is insulin or IGF-I. Most preferred is insulin.

[32] Further, the pharmacologically active substance of the present invention may include any anti-cancer drug that is used as an anti-cancer chemotherapeutic agent, for example preferably cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, Dactinomycin (actinomycin-D), daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, adriamycin, vincristine, vinblastine and methotrexate. Most preferred is paclitaxel.

[33]

[34] Mucoadhesive polymers

[35] As used herein, the term mucoadhesive polymer refers to a polymer having a good in vivo mucosal absorption rate, safety and degradability. The mucoadhesive polymer used in the present invention may be synthesized or may be naturally-occurring materials.

[36] Examples of naturally-occurring mucoadhesive polymers may include, but are not limited to, chitosan, hyaluronate, alginate, gelatin, collagen, and derivatives thereof.

[37] Examples of synthetic mucoadhesive polymers may include, but are not limited to, poly(acrylic acid), poly(methacrylic acid), poly( -lysine), poly(ethylene imine), poly (2-hydroxy ethyl methacrylate), and derivatives or copolymers thereof.

[38] Preferably, the mucoadhesive polymer of the present invention may be chitosan.

Chitosan is made by deacetylation of chitin. Next to cellulose, chitin is one of the most abundant organic polymers in nature, with as much as ten billion tons of chitin and its derivatives estimated to be produced from living organisms each year. Chitin is quantitatively found in the epidermis or exoskeletons of crustaceans such as crabs and shrimps and insects such as grasshoppers and dragonflies, and in the cell walls of fungi, mushrooms such as Enoki Mushroom (Flammulina velutipes) and Shiitake mushrooms (Lentinus edodes) and bacteria. From a viewpoint of a chemical structure, chitin is a linear polymer of beta 1-4 linked N-acetyl-D-glucosamine units composed of mucopolysaccharides and amino sugars (amino derivatives of sugars). Chitosan is formed by removal of acetyl groups from some of the N-acetyl glucosamine residues (Errington N, et al., Hydrodynamic characterization of chitosan varying in molecular weight and degree of acetylation. Int J Biol Macromol. 15:1123-7 (1993)). Due to removal of acetyl groups that were present in the amine groups, chitosan is present as polycations in acidic solutions, unlike chitin. As a result, chitosan is readily soluble in an acidic aqueous solution and therefore exhibits excellent processability and relatively

high mechanical strength after drying thereof. Due to such physicochemical properties, chitosan is molded into various forms for desired applications, such as powders, fibers, thin films, gels, beads, or the like, depending desired applications (E. Guibal, et al., Ind. Eng. Chem. Res., 37:1454-1463 (1998)). Chitosan is divided into a chitosan oligomer form composed of about 12 monomer units and a chitosan polymer form composed of more than 12 monomer units, depending upon the number of constituent monomer units. In addition, the chitosan polymer is subdivided into three different types, low-molecular weight chitosan (LMWC, molecular weight of less than 150 kDa), high-molecular weight chitosan (HMWC, molecular weight of 700 to 1000 kDa), and medium-molecular weight chitosan (MMWC, molecular weight between LMWC and HMWC).

[39] Due to excellent stability, environmental friendliness, biodegradability and biocom- patibility, chitosan is widely used for a variety of industrial and medical applications. Further, it is also known that chitosan is safe and also exhibits no immunoenhancing side effects. The in vivo degradation of chitosan molecules by lysozyme produces N- acetyl-D-glucosamine which is used in the synthesis of glycoproteins and finally excreted in the form of carbon dioxide (CO ) (Chandy T, Sharma CP. Chitosan as a biomaterial. Biomat Art Cells Art Org. 18:1-24 (1990)).

[40] Chitosan that can be used in the present invention may include any type of chitosan conventionally used in the art. Chitosan of the present invention has a molecular weight of preferably 500 to 20000 Da, more preferably 500 to 15000 Da, particularly preferably 1000 to 10000 Da, and most preferably 3000 to 9000 Da. If the molecular weight of chitosan is lower than 500 Da, this may result in poor function of chitosan as a carrier. On the other hand, if the molecular weight of chitosan is higher than 20000 Da, this may lead to a problem associated with formation of self-aggregates in an aqueous solution. The preferred chitosan used in the present invention is oligomeric chitosan.

[41]

[42] Pharmacologically active substance-mucoadhesive polymer conjugate

[43] The conjugate of the present invention is characterized in that the pharmacologically active substance and the mucoadhesive polymer are covalently bound to each other via a linker.

[44] The covalent bonding between the pharmacologically active substance of the present invention and the mucoadhesive polymer may be formed depending upon various kinds of bonds. Examples of covalent bonds may include disulfide bonds, peptide bonds, imine bonds, ester bonds and amide bonds.

[45] Further, the covalent bonding is formed largely by two types: direct bonding and indirect bonding.

[46] According to the direct bonding method, a covalent bond may be formed by direct reaction of a functional group (for example, -SH, -OH, -COOH, and NH ) on the pharmacologically active substance with a functional group (for example, -OH and -NH ) on the mucoadhesive polymer. According to the indirect bonding method, the pharmacologically active substance-mucoadhesive polymer complex may be formed by the medium of a compound conventionally used as a linker in the art.

[47] In the preferred embodiment, the conjugate of the present invention is covalently bound via the linker.

[48] The linker used in the present invention may be any compound that is conventionally used as a linker in the art. The linker may be appropriately selected depending upon kinds of the functional groups present on the pharmacologically active substance.

[49] Specific examples of the linker may include, but are not limited to, N-succinimidyl iodoacetate, N-hydroxysuccinimidyl bromoacetate, m-maleimi- dobenzoyl-N-hydroxysuccinimide ester, m-maleimi- dobenzoyl-N-hydroxysulfosuccinimide ester, N-maleimidobutyryloxysuccinamide ester, N-maleimidobutyryloxy sulfosuccinamide ester, E-maleimidocaproic acid hydrazideDHCl, [N-(E- maleimidocaproyloxy)-succinamide] , [N-(E-maleimidocaproyloxy)-sulfosuccinamide], maleimidopropionic acid N- hydroxysuccinimide ester, maleimidopropionic acid N-hydroxysulfosuccinimide ester, maleimidopropionic acid hydrazideDHCl, N-suc- cinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-(4-iodoacetyl) aminobenzoate, succinimidyl-(N-maleimidomethyl)cyclohexane-l-carboxylate, suc- cinimidyl-4- (p-maleimidophenyl)butyrate, sulfosuccinimidyl- (4-iodoacetyl)aminobenzoate, sulfosuccinimidyl- 4- (N-maleimidomethyl)cyclohexane- 1 -carboxylate, sulfosuccinimidyl- 4-(p-maleimidophenyl)butyrate, m-maleimidobenzoic acid hydrazideDHCl, 4- (N-maleimidomethyl)cyclohexane-l-carboxylic acid hydrazideDHCl, 4-(4-N-maleimidophenyl)butyric acid hydrazideDHCl, N-succinimidyl 3-(2-pyridyldithio)propionate, bis(sulfosuccinimidyl)suberate, l,2-di[3'-(2'-pyridyldithio)propionamido]butane, disuccinimidyl suberate, disuc- cinimidyl tartrate, disulfosuccinimidyl tartrate, dithio-bis-(succinimidylpropionate), 3,3'-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(succinimidylsuccinate) and ethylene glycol bis(sulfosuccinimidylsuccinate).

[50] In the preferred embodiment of the present invention, the covalent bonding of the protein or peptide and chitosan involves interposition of the linker of -CO-(CH 2 ) n -

S-S-(CH 2 ) n -CO- therebetween. Here, -NH 2 of chitosan and -NH 2 of the ^r protein are re- spectively bound to the linker via the amide bond. In the Formula I, n is an integer of 1

to 5.

[51] In a specific embodiment of the present invention, the conjugate of the protein or peptide (e.g. insulin) and chitosan has a structure wherein -CO-(CH ) -S-S-(CH ) -CO- is interposed between two components and -NH of chitosan and -NH of the protein are respectively covalently bound to the linker via the amide bond.

[52] Further, in the preferred embodiment of the present invention, covalent bonding of an anti-cancer drug and chitosan involves interposition of a succinyl group therebetween. Here, the succinyl group and chitosan forms an amide bond, and the succinyl group and the anti-cancer drug forms an ester bond.

[53] In a specific embodiment of the present invention, the succinyl group (-C0-CH -

CH -CO-) is interposed between the anti-cancer drug (e.g. paclitaxel) and chitosan, and the succinyl group and chitosan are covalently bound to each other via the amide bond.

[54] The conjugate of the present invention is characterized by being capable of delivering the pharmacologically active substance via transmucosal routes. For example, administration routes for transmucosal delivery of the conjugate may include, but are not limited to, mucous membranes of buccal cavity, nasal cavity, rectum, vagina, urethra, throat, alimentary canal, peritoneum and eyes. The conjugate of the present invention enables oral administration of the drug by delivery of the pharmacologically active substance via a mucous membrane of the alimentary canal.

[55]

[56] Pharmaceutical compositions

[57] In another aspect, the present invention also provides a pharmaceutical composition for transmucosal administration of a drug, comprising a therapeutically effective amount of the conjugate of the present invention and a pharmaceutically acceptable carrier.

[58] As used herein, the term therapeutically effective amount refers to an amount enough to achieve inherent therapeutic effects of the pharmacologically active substance.

[59] As used herein, the term tharmaceutically acceptable refers to a formulation of a compound that is physiologically acceptable and does not cause allergic response or similar response such as gastric disorder, vertigo, and the like, when it is administered to a human.

[60] The pharmaceutically acceptable carrier may be a material that is conventionally used in preparation of a pharmaceutical formulation. Examples of the pharmaceutically acceptable carrier that can be used in the present invention may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydrox-

ybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Besides the aforesaid ingredients, the pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative or the like. Details for formulation and suitable pharmaceutically acceptable carriers may be found in Remington's Pharmaceutical Sciences(19th ed., 1995).

[61] Further, the pharmaceutical composition of the present invention is characterized in that it is administered via transmucosal routes. For example, administration routes for transmucosal delivery of the composition may include, but are not limited to, buccal, nasal, rectal, vaginal, urethral, throat, alimentary canal, peritoneal and ocular mucosae. Most preferably, the pharmaceutical composition of the present invention enables oral administration of the drug by delivery of the pharmacologically active substance via the alimentary canal mucosa.

[62] A suitable dose of the pharmaceutical composition of the present invention may vary depending upon various factors such as formulation method, administration mode, age, weight and sex of patients, pathological conditions, diet, administration time, administration route, excretion rate and sensitivity to response. For oral administration, the composition is administered at a dose of preferably 0.001 to 100 mg/kg BW/day.

[63] According to a method that can be easily practiced by a person having ordinary knowledge in the art to which the invention pertains, the pharmaceutical composition of the present invention may be formulated into a unit dosage form, or may be prepared in the form of a multi-dose form, using a pharmaceutically acceptable carrier and/or excipient. Here, the resulting formulation may be in the form of a solution, suspension or emulsion in oil or an aqueous medium, or otherwise may be in the form of an extract, a powder, a granule, a tablet or a capsule. The formulation may additionally comprise a dispersant or a stabilizer.

[64] In the most preferred embodiment, the present invention provides a pharmaceutical composition for oral administration of insulin, comprising (a) a conjugate comprising a therapeutically effective amount of insulin covalently bound to chitosan, and (b) a pharmaceutically acceptable carrier.

[65] The pharmaceutical composition for treatment of diabetes according to the present invention enables oral administration of insulin. Generally, diabetic patients are given an insulin injection. Such an administration method is very inconvenient to patients in several aspects. However, the pharmaceutical composition for treatment of diabetes according to the present invention may lead to remarkable improvement in diabetic treatment regimens due to the possibility of oral administration.

[66] Upon comparing an in vivo blood glucose-lowering effect of the insulin-chitosan conjugate prepared according to the present invention with that of free insulin not

bound to chitosan, it was confirmed through an experimental example of the present invention that the conjugate of the present invention exerts significantly higher blood glucose-lowering effects.

[67] Further, it was also confirmed that the insulin-chitosan conjugate of the present invention exhibits an excellent absorption rate through a mucous membrane (particularly, the gastrointestinal mucosa).

[68] In another most preferred embodiment, the pharmaceutical composition of the present invention provides a pharmaceutical composition for oral administration of paclitaxel, comprising (a) a conjugate comprising a therapeutically effective amount of paclitaxel covalently bound to chitosan, and (b) a pharmaceutically acceptable carrier.

[69] The pharmaceutical composition comprising the paclitaxel-chitosan conjugate of the present invention exerts an excellent anti-cancer effects even by transmucosal administration, particularly oral transmucosal administration..

[70] Upon comparing an in vivo anti-cancer effect of the paclitaxel-chitosan conjugate of the present invention with that of a free anti-cancer drug not bound to chitosan, it was confirmed through an experimental example of the present invention that the conjugate of the present invention exerts significantly higher anti-cancer effects.

[71] Further, it was also confirmed that the paclitaxel-chitosan conjugate of the present invention exhibits an excellent absorption rate from a mucous membrane (particularly, gastrointestinal mucous membrane).

[72]

[73] Transmucosal delivery of pharmacologically active substances

[74] In yet another aspect, the present invention provides a method for in vivo delivery of a pharmacologically active substance via a transmucosal route, by covalent binding of the active ingredient with a mucoadhesive polymer via a linker.

[75] Preferably, the method of the present invention comprises (a) binding the pharmacologically active substance to the linker, and (b) conjugating the pharmacologically active substance of Step (a) with the mucoadhesive polymer via the linker.

[76] Preferably, the method of the present invention comprises (a) binding the pharmacologically active substance to the linker, (b) binding the linker to the mucoadhesive polymer, and (c) conjugating the pharmacologically active substance of Step (a) with the mucoadhesive polymer of Step (b) via the linker.

[77] Hereinafter, technical accomplishment and advantages of the conjugate of the present invention and the transmucosal delivery of the pharmacologically active substance using the same will be summarized as follows:

[78] (i) The conjugate of the present invention exhibits an excellent absorption rate in biological mucous membranes, particularly mucous membranes of the alimentary canal (especially the gastrointestinal tract).

[79] (ii) Because the mucoadhesive polymer used as the carrier of a target drug is highly biocompatible and biodegradable in vivo, the conjugate of the present invention is safe and also exhibit excellent safety even with chronic administration.

[80] (iii) Consequently, the pharmaceutical composition of the present invention exhibits superior bioavailability even upon oral administration, thus making it possible to achieve treatment of diseases via oral administration.

[81] (iv) Oral administration of the pharmaceutical composition of the present invention leads to significant improvements in medication compliance of the patients, as compared to conventional injection medications. Mode for the Invention

[82] EXAMPLES

[83] Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

[84]

[85] I. Insulin-chitosan conjugate

[86]

[87] Example 1 : Preparation of insulin intermediate having insulin bound to linker

[88] 0.1 g (17.22x10 mol) of insulin (Serologicals Corp.) was dissolved in 10 mL of a hydrochloric acid solution, and 0.008 g (25.83x10 mol) of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pierce) was dissolved in 0.2x10 mL of DMF (Sigma) which was then added to the insulin solution. In order to achieve re- gioselective conjugation of SPDP with the 29th amino acid lysine on the B chain ( B29) of an insulin molecule, the aforementioned mixed solution was adjusted to a range of pH 9 to 10 using aqueous NaOH and stirred at room temperature for 30 min. The resulting stirred solution was subjected to reverse-phase HPLC (Shimadzu) separation and freeze-drying (lyophilization) to thereby prepare an insulin intermediate product (see Reaction Scheme 1).

[89]

[90] Example 2: Preparation of chitosan intermediate having chitosan bound to linker

[91] Each 0.1 g (16.67x10 mol, moles of monomer = 0.67x10 mol) of chitosan with a different molecular weight of 3000, 6000 and 9000 (KITTOLIFE, Co., Ltd., Seoul, Korea) was dissolved in 2 mL of a phosphate buffer solution (PBS), and 0.016 g (50.0IxIO ^'6 mol) of SPDP was dissolved in 0.2xl0 ^'3 mL of DMF which was then added to the aforementioned chitosan solution, followed by stirring at room temperature for 2 hours. Acetone was added to the resulting stirred solution to thereby precipitate pellets.

The resulting pellets were dissolved in distilled water and freeze-dried to thereby prepare a chitosan intermediate product (see Reaction Scheme 1).

[92] [Reaction Scheme 1] [93]

H ₂ N-GIy* ¹ - Asn ^A OH ^SPDP H ₂ N-Gly ^A1 -Asn ^A OH

H ₂ N Phe ^b Lys ^B Thr ^B3 ° — OH R.T. 1 h H ₂ N Phe ^B Lys ^B29 τhr ^B3 ° — OH

[94] Example 3: Construction of insulin-chitosan conjugate [95] In order to reduce the chitosan intermediate, 0.008 g (1.24x10 -v- ^" 6° mol) of the chitosan intermediate prepared in Example 2 and 0.3 mL of DTT (24.9 xlO mol) (Pierce) were dissolved in 0.3 mL of PBS and stirred at room temperature for 4 hours. 0.005 g (0.83x10 mol) of the insulin intermediate prepared in Example 1 was dissolved in a citrate buffer solution (500 D), the reduced chitosan intermediate solution (100 D) was added thereto, and the resulting mixture was stirred at room temperature for 12 to 24 hours. The stirred mixture was subjected to reverse-phase HPLC separation and freeze- drying to thereby prepare an insulin-chitosan conjugate (see Reaction Scheme 2).

[96] [97] [Reaction Scheme 2] [98]

[99] Experimental Example 1 : Determination of substitution degree with linker in chitosan intermediate [100] H NMR analysis was carried out to determine the SPDP-substituted degree in the chitosan intermediate of the present invention. The substitution degree of the linker was calculated in a D O solvent by integral calculus. The number of substituted molecules thus measured is given in Table 1 below.

[101] Table 1

[102] [103] As shown in Table 1, it can be seen that low-molecular weight chitosan is more easily substituted with the linker since the substitution degree of the linker decreases in proportion to an increase in the molecular weight of chitosan.

[104] [105] Experimental Example 2: Determination of insulin content in insulin-chitosan

conjugate

[106] In order to determine an amount of insulin contained in an insulin-chitosan conjugate of the present invention (a conjugate using chitosan of MW 6000), 1 mg of the insulin-chitosan conjugate was dissolved in 1 mL of a citrate buffer solution and an absorbance was measured at a wavelength of UV 275 nm. The standard curve was plotted by dissolving insulin (0.1, 0.5, 1 and 2 mg) in 1 mL of a citrate buffer solution and measuring the absorbance at the given wavelength. Using the thus-obtained standard curve, the amount of insulin contained in the insulin-chitosan conjugate was calculated. As a result, the content of insulin in the conjugate was 44%.

[107]

[108] Experimental Example 3: Determination of in vivo insulin activity using insulin- chitosan conjugate

[109] An insulin-chitosan conjugate of the present invention (a conjugate using chitosan of MW 6000) was dissolved in a citrate buffer solution and then diluted with physiological saline to prepare an insulin-chitosan conjugate solution at an insulin concentration of 1 U/mL. Diabetes-induced male Wistar rats (6 to 7-weeks old) were fasted for 6 hours prior to administration of insulin, and blood was collected from the tail veins of the animals and the blood glucose level was determined. The thus- obtained value was used as an initial value. Immediately after determination of the blood glucose level, a 0.5 IU/kg insulin- or 1 IU/kg insulin-chitosan conjugate (Insulin-6K LMWC) was intravenously injected to the tail veins of the animals. 0.5 IU is equivalent to 17.4 D of insulin. In addition, animals were given subcutaneous (s.c.) injection of 0.5 IU/kg insulin (control).

[110] As shown in FIG. 1, a physiological activity of insulin contained in the insulin- chitosan conjugate solution of the present invention (-

V

-) exhibited about 40% of the insulin solution control, thus confirming that the conjugate of the present invention has a normal physiological activity.

[I l l]

[112] Experimental Example 4: In vivo oral administration studies of insulin-chitosan conjugate

[113] An insulin-chitosan conjugate (a conjugate using chitosan of MW 3000, 6000 or

9000 Da) was dissolved in a citrate buffer solution and then diluted with physiological saline to prepare an insulin-chitosan conjugate solution at an insulin concentration of 100 U/mL. Diabetes -induced rats were fasted for 6 hours, and blood was collected from the tail veins of animals and the blood glucose level was determined. The thus- obtained value was used as an initial value prior to administration of the drug. The experimental animals were given oral administration of the above-prepared insulin-

chitosan conjugate solution at a dose of 50 IU/kg using a gastric sonde (50 IU is equivalent to 1.77 mg of insulin). As a control, animals were given oral administration of 50 IU/kg insulin and chitosan of MW 9000 Da in the same manner as above. On time points of 1, 2, 3, and 4 hours after administration of the drug, blood was collected from the tail veins of animals and the blood glucose level was determined. The blood glucose level at each time point was calculated by taking the initial value prior to administration of the drug to be 100%.

[114] As shown in FIG. 2, an experimental group of rat with administration of the insulin- chitosan conjugate solution of the present invention at a dose of 50 IU insulin/kg exhibited more than a 40% decrease in the blood glucose level 2 hours later, as compared to the initial blood glucose level. Whereas, animal groups with oral administration of insulin-free saline, insulin itself and chitosan itself exhibited no lowering of the blood glucose levels.

[115] [116] Then, the bioavailability of conjugates were calculated from the degree of blood glucose control (area under curve, AUC) obtained in FIG.2 after oral administration of each insulin-chitosan conjugate. The results thus obtained are summarized in Table 2 below. Analysis was conducted by administering insulin to homologous rats via IV and SC injection and taking the degree of blood glucose control thus obtained to be 100% bioavailability. In addition, as a control, known bioavailability of insulin, a protease- chitosan conjugate and a thiolated chitosan-insulin tablet preparation containing glutathione (a reducing agent) (Krauland AH, et al., J. Control Release, 24;95(3):547-555 (2004)) was compared.

[117] Table 2

[118] Group with administration of a thiolated chitosan-insulin tablet

[119] [120] As can be confirmed from the results of Table 2, the insulin-chitosan conjugate of the present invention also exhibited excellent bioavailability.

[121] [122] II. Paclitaxel-chitosan conjugates [123] Example 4: Preparation of paclitaxel intermediate having paclitaxel bound to linker [124] 0.1 g (0.117x10 mol) of paclitaxel (Samyang Genex Corp., Daejeon, Korea) was dissolved in 5 mL of a dichloromethane solution, and 0.015 g (0.152x10 mol) of a succinic anhydride (Sigma, St. Louis, MO) and 12.9x10 mL (0.160x10 mol) of pyridine (Sigma) were added to the paclitaxel solution. The resulting mixture was stirred at room temperature for 3 days. The resulting stirred solution was purified by silica column chromatography and dried to prepare a paclitaxel/succinic acid derivative.

[125] [126] Example 5: Construction of paclitaxel-chitosan conjugate [127] 0.1 g (0.105x10 mol) of a paclitaxel/succinic acid derivative, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma) and N- hydroxysuccinimide (NHS) (Sigma) were dissolved in 3 mL of DMF, and the resulting mixture was stirred at room temperature for 4 hours (see Reaction Scheme 3). 0.2 g (66.67xlO ^"6 mol) of chitosan of MW 3000 and 6000 (KITTOLIFE, Co., Ltd., Seoul, Korea) was dissolved in a borate buffer solution (3 mL) and DMF (9 mL), which was then added to the above stirred solution and stirred at room temperature for 4 hours (see Reaction Scheme 3). The reaction solution was dialyzed against distilled water and freeze-dried to thereby obtain a paclitaxel-chitosan conjugate.

[128] [129] [Reaction Scheme 3] [130]

[131] [Reaction Scheme 4]

[132]

[133] Experimental Example 6: Determination of paclitaxel content in paclitaxel-chitosan conjugate [134] In order to determine an amount of paclitaxel contained in a paclitaxel-chitosan conjugate of the present invention, 0.1 mg of the paclitaxel-chitosan conjugate obtained in Example 5 was dissolved in 1 mL of acetonitrile/water and an absorbance was measured at a wavelength of UV 227 nm. The standard curve was plotted by dissolving paclitaxel (5, 10, 12.5, 20 and 25 mg) in 1 mL of acetonitrile/water and measuring the absorbance at the given wavelength. Using the thus-obtained standard curve, the amount of paclitaxel contained in the paclitaxel-chitosan conjugate was calculated. As a result, the content of paclitaxel in the conjugate was 15-20% and 10-15% for chitosan of MW 3000 and 6000, respectively.

[135] [136] Experimental Example 7: In vitro cytotoxicity test using paclitaxel-chitos an conjugate

[137] A paclitaxel-chitosan conjugate of the present invention (3000 and 6000 Da) was

dissolved in dimethyl sulfoxide (DMSO) and diluted with a cell culture medium to prepare paclitaxel-chitosan conjugate solutions at a paclitaxel concentration of 0.01, 0.05, 0.1, 0.25, 0.5 and 1 D/mL. B16F10 melanoma cells (KTCC) were cultured in a 96- well plate at a cell density of 5x10 cells/well for 24 hours and were treated with the above-prepared paclitaxel solution for 48 hours. Thereafter, the cell viability was measured using an MTT cell viability kit (Molecular Probe, Netherlands). 50 D of MTT was added to cells which were then cultured at 37 ⁰ C for 4 hours. Then, the su- pernatants were completely eliminated and 100 D/well of DMSO was added to the 96-well plate. The absorbance was measured using a microplate reader. The cell viability was calculated according to the following Math Figure (1):

[138] [Math Figure 1]

[139] Cell viability (%) = (OD (Sample)/0D (Control)) x 100

[140] A non-conjugated paclitaxel solution was used as a control.

[141]

[142] As shown in FIG. 3, it was confirmed that the cytotoxicity of paclitaxel contained in the paclitaxel-chitosan conjugate solution of the present invention was similar to that of the non-conjugated paclitaxel.

[143]

[144] Experimental Example 8: Inhibitory effects of oral administration of paclitaxel- chitosan conjugate on tumor

[145] B16F10 melanoma cells were subcutaneously transplanted at a cell density of 5x10 cells/mice into a dorsal region of C57BL6 male mice (mean body weight: 25 g). When the tumor mass has reached a desired size of about 50 to 100 mm , animals were divided into a treatment group and a control group. Experiments were carried out for mouse groups, each consisting of 5 to 6 animals having the tumor, simultaneously with observation of changes. Animals were given oral administration of the drug or physiological saline for about 30 days, starting on day 10 after tumor transplantation. Paclitaxel and the paclitaxel-chitosan conjugate were administered to animals at a dose of 25 mg/kg for 5 days, with no administration for following two days. The control group was administered physiological saline, paclitaxel and chitosan. In order to confirm the degree of tumor growth, the size of tumor was daily measured using a calibrator. The tumor size was calculated according to the following Math Figure (2):

[146] [Math Figure 2]

[147] Tumor volume (mm ) = (Length x Width )/2

[148]

[149] FIG. 4 is a graph showing an anti-cancer activity in mice with administration of paclitaxel and the paclitaxel-chitosan conjugate, respectively. The paclitaxel-ad- ministered group exhibited no significant difference in the tumor size, as compared to

that of the control group. However, it can be seen that the group with the administration of the paclitaxel-chitosan conjugate of the present invention exhibited a significant decrease in the tumor size, as compared to the control group.

[150] The survival rate of mice was also monitored simultaneously with measurement of the tumor size. When the tumor mass reached a size of more than 8000 mm , the animals were euthanized.

[151] As shown in FIG. 5, the mice of the group with the administration of the paclitaxel- chitosan conjugate of the present invention exhibited a 100% survival rate for about 30 days, whereas the mice of the control group exhibited a 0% survival rate prior to 30 days. Industrial Applicability

[152] A conjugate of the present invention exhibits excellent absorption rate and biocom- patibility in biological mucous membranes, particularly mucous membranes of alimentary canal (especially the gastrointestinal tract), in vivo degradability, and superior bioavailability even with oral administration, thus enabling treatment of diseases via oral administration of a drug.

[153] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[154]