Transmucosal delivery system for active pharmaceutical ingredients to the submucosal tissue of the urinary bladder

Abstract

As a system enabling effective delivery of a medicinal active ingredient to the submucosa of bladder, provided is a transmucosal delivery system that comprises a complex of a hydrophobic compound containing the medicinal active ingredient and chondroitin sulfate.

Description

[Technical field]

[0001] The present invention relates to a transmucosal delivery system for an active pharmaceutical ingredient to the submucosal tissue of the bladder, a method for screening drug candidate compounds for treating bladder diseases, and a method for imparting a medicinal effect to the submucosal tissue of the bladder. [Background technology]

[0002] Efforts are being made to treat bladder diseases using glycosaminoglycans. For example, a chondroitin sulfate derivative in which a group derived from steroid is bonded to a group derived from chondroitin sulfate is known to be used as a bladder disease treatment agent (e.g., Patent Document 1). It has also been reported that chondroitin sulfate itself can be used as a treatment agent for interstitial cystitis (IC), a bladder disease (e.g., Non-Patent Document 1). Furthermore, Patent Document 2 describes an invention relating to a derivative of hyaluronic acid, a type of glycosaminoglycan, as a compound that exhibits high retention in the mucosal epithelial layer, and bladder mucosa is exemplified as one of the mucous membranes.

[0003] Some of the above prior arts state that bladder disease treatment agents injected into the bladder reach and adhere to the bladder wall. However, none of the above prior arts disclose or suggest that active pharmaceutical ingredients administered into the bladder cavity can be delivered to the submucosal tissue of the bladder. In other words, it cannot be said that a method for effectively delivering active pharmaceutical ingredients to treat inflammation, etc., in the submucosal tissue of the bladder has been established. The present invention has been made to solve the above-mentioned problems, and has an objective of providing a system that can achieve effective delivery of an active pharmaceutical ingredient administered to the bladder cavity to the submucosal tissue. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 111356 [Patent Document 2] International Publication No. 2007 / 043702 [Non-patent literature]

[0005] [Non-Patent Document 1] Paul J. Hauser et al., "Restoring Barrier Function to Acid-Damaged Bladder by Intravesical Chondroitin Sulfate," The Journal of Urology, Vol. 182, pp. 2477-2482, 2009 Summary of the Invention

[0006] As a result of intensive research aimed at solving the above problems, the inventors discovered that an active pharmaceutical ingredient derivatized with chondroitin sulfate is effective in achieving effective delivery of the active pharmaceutical ingredient to the submucosal tissue of the bladder, and thus completed the present invention.

[0007] That is, the present invention provides a transmucosal delivery system for a pharmaceutical active ingredient other than a steroid to the submucosal tissue of the bladder, comprising a conjugate of chondroitin sulfate and a hydrophobic compound (hereinafter, the conjugate is also referred to as a "chondroitin sulfate derivative"); The hydrophobic compound is a compound in which the active pharmaceutical ingredient and a spacer-forming molecule are bonded by an ester bond, and has a ClogP of 1 or more, and the spacer-forming molecule is bonded by an amide bond with the chondroitin sulfate. , a delivery system. [Brief description of the drawings]

[0008] [Figure 1] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 1, and FIG. 1B is a corresponding fluorescent image. [Diagram 2] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 2, and FIG. 1B is a corresponding fluorescent image. [Diagram 3]1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 3, and FIG. 1B is a corresponding fluorescent image. [Figure 4] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 4, and FIG. 1B is a corresponding fluorescent image. [Diagram 5] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 5, and FIG. 1B is a corresponding fluorescent image. [Figure 6] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Example 6, and FIG. 1B is a corresponding fluorescent image. [Figure 7] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Comparative Example 1, and FIG. 1B is a corresponding fluorescent image thereof. [Figure 8] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Comparative Example 2, and FIG. 1B is a corresponding fluorescent image thereof. [Figure 9] 1A is a bright field image of a frozen section taken one day after administration of a test substance using the fluorescently labeled compound of Comparative Example 3, and FIG. 1B is a corresponding fluorescent image thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] According to the present invention, a delivery system capable of achieving effective delivery of an active pharmaceutical ingredient administered into the bladder cavity to the submucosal tissue, and various methods related to this delivery system can be provided. It is known that the smaller the molecular weight of a compound, the higher the transmucosal penetration of the compound. The present invention is based at least in part on the surprising discovery that administration of a conjugate of a polymer, chondroitin sulfate, and an active pharmaceutical ingredient into the bladder cavity enables more effective delivery to the submucosal tissue than administration of the active pharmaceutical ingredient itself as a single agent. In this specification, the term "process" includes not only an independent process, but also a process that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, a numerical range indicated using "~" indicates a range that includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively. Furthermore, when a composition contains multiple substances corresponding to each component, the content of each component in the composition means the total amount of the multiple substances present in the composition, unless otherwise specified. Hereinafter, the embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and it should be understood that modifications and improvements to the following embodiments, which are made based on the ordinary knowledge of those skilled in the art, fall within the scope of the present invention, without departing from the spirit of the present invention.

[0010] (Embodiment 1) In one aspect, embodiment 1 of the present invention relates to a transmucosal delivery system for an active pharmaceutical ingredient to the submucosal tissue of the bladder. That is, embodiment 1 of the present invention provides a transmucosal delivery system for an active pharmaceutical ingredient to the submucosal tissue of the bladder, the delivery system comprising a conjugate of chondroitin sulfate and a hydrophobic compound, the hydrophobic compound comprising the active pharmaceutical ingredient. Another aspect of the present invention relates to a targeting system for a pharmaceutical active ingredient to a bladder submucosa. That is, the present invention provides a targeting system for a pharmaceutical active ingredient to a bladder submucosa, the targeting system comprising a conjugate of chondroitin sulfate and a hydrophobic compound, the hydrophobic compound comprising the pharmaceutical active ingredient. In yet another aspect, the first embodiment of the present invention relates to a method for transmucosally delivering an active pharmaceutical ingredient to the submucosal tissue of the bladder, the method comprising administering a conjugate of chondroitin sulfate and a hydrophobic compound to the bladder cavity of a subject. That is, the first embodiment of the present invention also provides a method for transmucosally delivering an active pharmaceutical ingredient to the submucosal tissue of the bladder, the method comprising administering a conjugate of chondroitin sulfate and a hydrophobic compound to the bladder cavity of a subject, the hydrophobic compound comprising the active pharmaceutical ingredient.

[0011] In yet another aspect, the first embodiment of the present invention relates to a conjugate for use in transmucosal delivery of an active pharmaceutical ingredient to the submucosal tissue of the bladder. That is, in the first embodiment of the present invention, there is also provided a conjugate for use in transmucosal delivery of an active pharmaceutical ingredient to the submucosal tissue of the bladder, the conjugate being a conjugate of chondroitin sulfate and a hydrophobic compound, the hydrophobic compound comprising the active pharmaceutical ingredient. In yet another aspect, the first embodiment of the present invention relates to a method for transmucosally delivering an active pharmaceutical ingredient to the submucosal tissue of the bladder by binding a hydrophobic compound containing an active pharmaceutical ingredient to chondroitin sulfate, and for sustained release of the active pharmaceutical ingredient in the submucosal tissue. That is, the first embodiment of the present invention also provides a method for transmucosally delivering (permeating) the active pharmaceutical ingredient to the submucosal tissue of the bladder by binding a hydrophobic compound containing an active pharmaceutical ingredient to chondroitin sulfate, and for sustained release of the active pharmaceutical ingredient in the submucosal tissue, comprising administering a conjugate of chondroitin sulfate and a hydrophobic compound to the bladder cavity of a subject. In yet another aspect, the first embodiment of the present invention relates to a prodrug of an active pharmaceutical ingredient. That is, the first embodiment of the present invention also provides a prodrug of an active pharmaceutical ingredient, the prodrug being a conjugate of chondroitin sulfate and a hydrophobic compound, the hydrophobic compound releasably containing the active pharmaceutical ingredient, and used for transmucosal delivery of the active pharmaceutical ingredient to the submucosal tissue of the bladder.

[0012] (1) Chondroitin sulfate derivatives (also called "conjugates") The chondroitin sulfate derivative has a structure in which chondroitin sulfate (hereinafter sometimes abbreviated as "CS") is bound to a hydrophobic compound. The hydrophobic compound also includes an active pharmaceutical ingredient (hereinafter sometimes abbreviated as "API"). Here, the active pharmaceutical ingredient refers to any type of pharmacologic active compound useful for the prevention, diagnosis, stabilization, treatment, etc. of a condition, disorder, or disease. Preferably, the active pharmaceutical ingredient is a compound used for the purpose of treating a disease. In one embodiment, the API may be a compound that has been approved for pharmaceutical purposes by the pharmaceutical authorities of any country or region, such as the U.S. Food and Drug Administration or the Ministry of Health, Labor, and Welfare of Japan. When administered to the bladder lumen of a subject, the chondroitin sulfate derivative facilitates effective delivery of the API to the submucosa of the bladder. In one embodiment, the chondroitin sulfate derivative can be a prodrug of the API. Typically, a prodrug is a compound that is metabolized or converted to an active or highly active form of the API after administration.

[0013] In chondroitin sulfate derivatives, CS exists as a group derived from CS.Therefore, chondroitin sulfate derivatives contain at least one group derived from CS.In one embodiment, the group derived from CS is a group formed by removing a hydroxyl group from the carboxyl group of CS.In chondroitin sulfate derivatives, the group derived from CS can correspond to a carrier.

[0014] CS is not particularly limited as long as it has a structure in which a sulfate group is held in a glycan skeleton formed by repeating disaccharide units of D-glucuronic acid (or L-iduronic acid) and N-acetyl-D-galactosamine. In addition, the acidic functional groups of CS, including a carboxyl group and a sulfate group, may be in a free state without forming a salt, or in a state of forming a pharma- ceutically acceptable salt.

[0015] Examples of pharma- ceutically acceptable salts include salts with alkali metal ions such as sodium salts and potassium salts, and salts with alkaline earth metal ions such as magnesium salts and calcium salts. From the viewpoint of applicability and affinity to the living body, CS is preferably a salt with a pharma- ceutically acceptable alkali metal ion, and more preferably a sodium salt.

[0016] CS can be produced by known methods depending on the type, including, for example, extraction and purification from animal-derived raw materials, culture and purification from chondroitin-producing bacteria, glycosylation, and glycosyl synthesis.

[0017] The weight average molecular weight of CS is not particularly limited and can be appropriately selected depending on the purpose, etc. The weight average molecular weight of CS is, for example, 4,000 or more, preferably 6,000 to 300,000, and more preferably 10,000 to 100,000. The weight average molecular weight of CS can be measured by a light scattering method. In one embodiment, the weight average molecular weight of the CS is 10 times or more than the molecular weight of the API.

[0018] In the chondroitin sulfate derivative, the API exists as a group derived from the API. Thus, the chondroitin sulfate derivative contains at least one type of group derived from the API. In one embodiment, the group derived from the API is a group formed by removing a hydrogen atom from a hydroxy group or a hydroxy group from a carboxy group of the API. The position of the hydroxy group from which the hydrogen atom is removed is not particularly limited. In the chondroitin sulfate derivative, the group derived from the API is preferably linked to a group derived from CS or a spacer described below via a covalent bond that can be decomposed in the living body (e.g., a covalent bond such as an ester bond, a thioester bond, or an amide bond that can be decomposed by hydrolysis or an enzyme in the living body such as carboxyesterase).

[0019] Examples of APIs are not particularly limited, but are preferably compounds used for the treatment of bladder diseases, such as steroids such as prednisolone, betamethasone, triamcinolone, triamcinolone acetonide, budesonide, fluticasone, etc. Examples of APIs other than steroids include non-steroidal anti-inflammatory drugs such as naproxen and diclofenac. The molecular weight of the API may be, for example, 600 or less, and preferably 500 or less. In one embodiment, an API with a molecular weight of 600 or less and a CS with a weight average molecular weight of 5,000 or more are used. In another embodiment, an API with a molecular weight of 500 or less and a CS with a weight average molecular weight of 5,000 or more are used.

[0020] In one embodiment, the hydrophobic compound is an API or a compound in which the API is covalently bound to a spacer-forming molecule. The spacer-forming molecule is preferably covalently bound to the API to form the hydrophobic compound. The spacer-forming molecule is preferably covalently bound to a CS. The hydrophobic compound preferably has a ClogP of 1 or more. That is, in a preferred embodiment, a compound having a ClogP of 1 or more is used as the hydrophobic compound. By making the ClogP of the hydrophobic compound 1 or more, when the chondroitin sulfate derivative is administered to the bladder cavity of a subject, it is possible to facilitate effective delivery of the API to the submucosa of the bladder. The upper limit of ClogP is not limited, but may be, for example, 10 or less, or 5 or less. Here, in this specification, "logP" is a partition coefficient based on the ratio of the concentrations of a compound distributed in a two-phase system of water and octanol. logP is an index indicating the hydrophobicity of a compound, and the greater the hydrophobicity, the greater the logP value. "ClogP" is a logP value calculated by calculation using the fragment method (A. Leo, Comprehensive Medicinal Chemistry; Hansch C., Ed., Pergamon Press, Oxford, 1990, Vol.4, p.315; Leo AJ, Chemical Reviews, 1993, Vol.93, p.1281-1306). ClogP can be calculated using Chem Draw Professional ver.18.1.2.18 (PerkinElmer).

[0021] As will be understood by those skilled in the art, the transitional epithelium (also called the mucosal epithelial layer) present at the innermost lumen of the bladder is a stratified layer consisting of three layers, from the lumen side: the superficial layer (including epithelial cells), the intermediate layer (intermediate cell layer), and the basal layer (basal cell layer). In this specification, the "submucosa" of the bladder refers to the part on the parenchymal side of the transitional epithelium (the parenchymal side refers to the direction farther from the lumen in a tissue cross-sectional image obtained by cutting the bladder perpendicularly to the axis connecting the bladder dome and the urethra), and includes the lamina propria, submucosa, muscularis, and serosa from the lumen side. A chondroitin sulfate derivative administered to the lumen of the bladder can deliver an API to the lamina propria or to the parenchymal side of the lamina propria. The API is preferably released from the chondroitin sulfate derivative after administration into the bladder cavity. By releasing the API from the chondroitin sulfate derivative after administration into the bladder cavity, the API can be gradually released after penetrating into the submucosa of the bladder. As a result, in this embodiment, a particularly excellent effect can be obtained as a bladder disease treatment agent. From the viewpoint of obtaining such a function, the bond between the API and the spacer-forming molecule is preferably a covalent bond that can be decomposed in the living body, and is preferably a covalent bond formed by a condensation reaction, and more preferably an ester bond. After the chondroitin sulfate derivative is administered into the bladder cavity, the covalent bond between the API and the CS or the spacer-forming molecule is decomposed (preferably hydrolysis), thereby releasing the API. Note that the covalent bond between the API and the spacer-forming molecule may be decomposed after the bond between the CS and the spacer-forming molecule is decomposed, thereby releasing the API. In one embodiment, the API is released from the chondroitin sulfate derivative over a period of 168 hours (7 days) or more after administration into the bladder cavity.

[0022] In one embodiment, the chondroitin sulfate derivative has an API covalently bonded to a spacer-forming molecule via an ester bond or an amide bond. In this case, the spacer-forming molecule is more preferably a compound having two or more functional groups selected from a carboxy group, a hydroxy group, and an amino group in its molecular structure. In the chondroitin sulfate derivative, the spacer-forming molecule is preferably covalently bonded to CS via an amide bond. In addition, from the viewpoint of controlling the release rate of API, the API is preferably covalently bonded to the spacer-forming molecule via an ester bond. Therefore, the spacer-forming molecule is preferably a compound containing a carboxyl group and an amino group, or a compound containing a hydroxyl group and an amino group in the molecular structure. When the spacer-forming molecule is a compound containing a carboxy group and an amino group in its molecular structure, the carboxy group of CS and the amino group of the spacer-forming molecule form an amide bond, and the hydroxy group of API and the carboxy group of the spacer-forming molecule form an ester bond. On the other hand, when the spacer-forming molecule is a compound containing a hydroxy group and an amino group in its molecular structure, the carboxy group of CS and the amino group of the spacer-forming molecule form an amide bond, and the carboxy group of API and the hydroxy group of the spacer-forming molecule form an ester bond.

[0023] An example of the spacer-forming molecule can be represented by the following general formula (I). H2N-(CH2) p -X (I) In the formula, p represents an integer of 1 to 12; each -CH2- may independently have a substituent; and X represents a hydroxyl group, a carboxyl group, or an amino group. p is preferably an integer of 1 to 6, more preferably 2 to 6. The substituent may be a group selected from the group consisting of an electron-donating group and a sterically hindering group. In one embodiment, -NH represented by general formula (I) and the carboxyl group of CS form an amide bond.

[0024] As used herein, the term "electron-donating group" refers to a group (e.g., an alkyl group, an aryl group, etc.) that is more likely to donate electrons than a hydrogen atom. As used herein, the term "sterically hindered group" refers to a bulky group that causes steric hindrance to chemical reactions such as solvolysis. When the methylene group in general formula (I) is substituted with an electron-donating group or a sterically hindered group, the covalent bond between the API and the spacer-forming molecule becomes difficult to decompose, and the release rate of the API decreases. The decrease in the release rate can be appropriately measured by the method described below.

[0025] The electron donating group or sterically hindered group is not particularly limited as long as it can be substituted for the methylene group of the general formula (I), and can be appropriately selected from commonly used electron donating groups or sterically hindered groups. Specifically, it can be exemplified by linear alkyl groups having 1 to 12 carbon atoms, such as methyl groups, ethyl groups, propyl groups, and butyl groups; branched alkyl groups having 3 to 12 carbon atoms, such as isopropyl groups, isobutyl groups, sec-butyl groups, and tert-butyl groups; cyclic alkyl groups having 3 to 6 carbon atoms, such as cyclohexyl groups; aryl groups having 6 to 10 carbon atoms, such as phenyl groups and tolyl groups; alkenyl groups having 2 to 12 carbon atoms, such as vinyl groups and allyl groups; alkynyl groups having 2 to 12 carbon atoms; and aralkyl groups having 7 to 12 carbon atoms, such as benzyl groups. Among these, it is preferable to use a group selected from the group consisting of methyl groups, branched alkyl groups having 3 to 4 carbon atoms, and benzyl groups. Specific examples of spacer-forming molecules that can be used include amino acids such as β-alanine, isoleucine, alanine, valine, leucine, and phenylalanine; and aminoalkyl alcohols having 2 to 12 carbon atoms such as 2-aminoethanol and 3-aminopropanol.

[0026] The spacer-forming molecule is present in the chondroitin sulfate derivative as a group derived from the spacer-forming molecule (i.e., a spacer). Thus, the chondroitin sulfate derivative contains at least one type of group derived from the spacer-forming molecule. In one embodiment, the group derived from the spacer-forming molecule is a linking group formed by removing a hydrogen atom from an amino group of the spacer-forming molecule and a hydroxyl group from a carboxyl group of the spacer-forming molecule or a hydrogen atom from a hydroxyl group of the spacer-forming molecule. As the group derived from the spacer-forming molecule, an amino acid residue represented by -HN-(CH2)2-CO-, -HN-CH(CH(CH3)CH2CH3)-CO-, -HN-CH(CH(CH3)2)-CO- or -HN-CHCH2(CH(CH3)2)-CO- is preferred; an amino alcohol represented by -HN-(CH2)2-O-, -HN-CH(CH(CH3)CH2CH3)-O-, -HN-CH(CH(CH3)2)-O- or -HN-CHCH2(CH(CH3)2)-O- is more preferred, and -HN-(CH2)2-CO-, -HN-(CH2)2-O-, -HN-CH(CH(CH3)CH2CH3)-CO- or -HN-CH(CH(CH3)CH2CH3)-O- is more preferred.

[0027] The release rate of the API from the chondroitin sulfate derivative can be appropriately selected depending on the purpose. For example, the release rate of the API is preferably 0.1 to 5% / day, more preferably 0.1 to 3% / day, in 10 mM phosphate buffer (pH 7.4) at 36°C. Here, the release rate of the API is the daily change in the release rate (%), and is defined as the slope of a graph when the horizontal axis is time and the vertical axis is the release rate (%), which is the ratio of the amount of API released (mol) to the total number of moles (100%) of groups derived from the API contained in the chondroitin sulfate derivative. Therefore, when the change in the release rate remains constant during the measurement period, the graph will be a monotonically increasing straight line. In this case, for example, when all the API contained in the chondroitin sulfate derivative is released in 10 days in 10 mM phosphate buffer (pH 7.4) at 36°C, the release rate is expressed as 10% / day. On the other hand, when the change in the release rate does not remain constant but decreases over time (the graph shows a gentle curve that monotonically increases upwards), the release rate is calculated by linear approximation of the change in the release rate during the initial rise period (e.g., 3 days). The amount of released API (amount of released API) is measured by a method appropriately selected depending on the type of API.

[0028] (2) Method for producing chondroitin sulfate derivatives The method for producing the chondroitin sulfate derivative is not particularly limited. For example, the chondroitin sulfate derivative can be obtained by covalently bonding a functional group of CS with a functional group of a hydrophobic compound (e.g., a spacer-forming molecule or an API). In one embodiment, the chondroitin sulfate derivative can be obtained by covalently bonding a carboxy group of CS with a hydroxy group or an amino group of a hydrophobic compound. An example of a method for producing a chondroitin sulfate derivative will be described below, but the method is not limited to the following method.

[0029] A chondroitin sulfate derivative in which CS and API are covalently bonded via a spacer-forming molecule can be produced, for example, by a production method including the following steps. (A) a step of condensing a hydroxy group of the API with a carboxy group of the spacer-forming molecule to form a covalent bond (ester bond), or condensing a carboxy group of the API with a hydroxy group of the spacer-forming molecule to form a covalent bond (ester bond), to obtain a hydrophobic compound; (B) a step of condensing a carboxy group of CS with an amino group of the spacer-forming molecule to form a covalent bond (amide bond).

[0030] In step (A), a hydrophobic compound is obtained by condensing a hydroxy group of the API with a carboxy group of the spacer-forming molecule to form a covalent bond, or by condensing a carboxy group of the API with a hydroxy group of the spacer-forming molecule to form a covalent bond (ester bond). In this case, the amino group of the spacer-forming molecule to react with CS may be protected by a commonly used method as necessary. In step (B), the carboxy group of CS and the amino group of the spacer-forming molecule are covalently bonded by condensation. At this time, the carboxy group or hydroxy group of the spacer-forming molecule that is to react with the API may be protected by a commonly used method as necessary. The method for producing a chondroitin sulfate derivative is only required to include steps (A) and (B), and the order in which each step is carried out is not limited.

[0031] The condensation (esterification, amidation) method may be appropriately selected from commonly used methods. Examples of the condensation method include a method using a condensing agent such as water-soluble carbodiimide (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or dicyclohexylcarbodiimide, a symmetric acid anhydride method, a mixed acid anhydride method, or an active ester method. The condensation reaction conditions are appropriately selected depending on the condensation reaction to be applied.

[0032] The spacer-forming molecule is preferably selected to obtain a desired release rate of the API. In one embodiment, the spacer-forming molecule has a carboxy or hydroxy group that forms an ester bond with the API, an amino group that forms an amide bond with CS, and a linking group that connects the two reactive functional groups. The linking group is preferably selected to control the decomposition rate of the ester bond with the API.

[0033] (3) Method of administration The chondroitin sulfate derivative is administered to the bladder cavity of the subject. By administering the chondroitin sulfate derivative in this manner, the API can be delivered transmucosally to the submucosal tissue of the bladder. A specific administration method includes, for example, inserting a urethral catheter into the bladder cavity under sterile conditions, draining residual urine, and then injecting the API through the catheter. The bladder may have a damaged mucosa. The subject may be a human or a non-human animal such as a mouse, rat, rabbit, dog, or pig. Chondroitin sulfate derivatives injected into the bladder lumen reach the bladder lumen and adhere to the mucosa while maintaining the covalent bond between CS and the hydrophobic compound. The chondroitin sulfate derivatives then penetrate into the bladder submucosa.

[0034] When the chondroitin sulfate derivative is administered in the form of a liquid pharmaceutical composition as described below, the concentration of the chondroitin sulfate derivative may be, for example, a concentration that allows the derivative to pass through a urethral catheter, and can be appropriately adjusted by a person skilled in the art.

[0035] (4) Pharmaceutical composition The chondroitin sulfate derivatives can be used as pharmaceutical compositions. The pharmaceutical compositions contain at least one chondroitin sulfate derivative, and may further contain other components such as pharma- ceutical acceptable excipients, as necessary.

[0036] (5) Bladder disease treatment The chondroitin sulfate derivatives can be used as a bladder disease treatment agent. The bladder disease treatment agent is preferably a pharmaceutical composition containing at least one chondroitin sulfate derivative, and this pharmaceutical composition is used for the treatment of bladder diseases. As used herein, "treatment" refers to any treatment given to a disease, such as cure, improvement, and inhibition of progression (prevention of deterioration) of a disease. In addition, as used herein, "bladder disease" refers to any disease or other abnormality occurring in the bladder, such as cystitis. The form of the agent for treating a bladder disease is not particularly limited as long as it is a formulation or pharmaceutical form that can be administered into the human bladder cavity, but the form when administered is preferably liquid, such as a solution, suspension, etc. The solution and suspension can also be administered by dissolving the powder of the chondroitin sulfate derivative when used.

[0037] (6) Methods for Treating Bladder Disorders The method for treating a bladder disease includes a step of administering the above-mentioned delivery system to the bladder cavity of a subject. Specifically, the method for treating a bladder disease includes a step of administering a chondroitin sulfate derivative used as a bladder disease treatment agent to the bladder cavity of a subject. The method for treating a bladder disease may further include other steps as necessary. The administration of the bladder disease treatment agent can be carried out in the same manner as described in "(3) Administration method".

[0038] According to embodiment 1 of the present invention, effective delivery of an active pharmaceutical ingredient administered into the bladder lumen to the submucosal tissue can be achieved.

[0039] (Embodiment 2) The second embodiment of the present invention relates to a method for screening a drug candidate compound for treating bladder disease, the method includes the steps of providing a compound, administering the compound into the bladder lumen, and analyzing the presence of the compound in the submucosa of the bladder, and the presence of the compound in the submucosa of the bladder is indicative of the drug candidate compound.

[0040] The compound (test substance) subjected to this method may be any compound, but is preferably a compound that is expected to have a medicinal effect on bladder diseases. The intravesical cavity to which the compound is administered may be the intravesical cavity of an animal (eg, rat, mouse, guinea pig, dog, monkey, etc.) in which a bladder disease has been induced. The method of administering the compound to the bladder cavity is not particularly limited, but may include, for example, inserting a urethral catheter into the bladder cavity under sterile conditions, draining residual urine, and then injecting the compound through the catheter. The amount of the compound administered may be an amount sufficient to cover the entire mucosa of the bladder cavity, and may be appropriately determined depending on the type of animal.

[0041] The method for analyzing the presence of a compound in the submucosa of the bladder is not particularly limited, and a known method can be used. The analysis method can be appropriately selected from imaging, mass spectrometry, immunoassay, radioactivity measurement, etc. according to the type of compound. These methods can be used alone or in combination of two or more.

[0042] Here, by way of example, we describe a method for analyzing the presence of compounds in the submucosa of the bladder using imaging, in particular fluorescence imaging. When using fluorescent imaging, the luminescence of the compound in the submucosal tissue is optically detected. In order to detect the luminescence of the compound, it is necessary to fluorescently label the compound of the test substance. The method of fluorescent labeling is not particularly limited, but a method of introducing a fluorescent dye compound into the compound of the test substance may be used. For example, when a chondroitin sulfate derivative having an API is used as the compound of the test substance, a fluorescent dye such as fluorescein may be introduced into the functional group of the API or CS. The test substance preferably has a polymer structure such as a polymer.

[0043] The presence of the compound in the submucosal tissue can be analyzed by taking the bladder of the animal, preparing a frozen section, and then observing the frozen section under a fluorescence microscope. The excitation wavelength and absorption wavelength of the fluorescence microscope can be appropriately adjusted depending on the type of the fluorescent dye introduced. The presence of the compound in the submucosa is preferably analyzed 6 hours or more after administration of the compound to the bladder lumen, which makes it easier to confirm the presence of the compound that has penetrated into the submucosa from the bladder mucosa.

[0044] The presence of a compound in the submucosal tissue may be evaluated by calculating an integrated value of brightness from a fluorescent image. For example, an integrated value of brightness from a fluorescent image of a compound effective for a bladder disease may be calculated in advance as an evaluation criterion, and a compound having an integrated value of brightness greater than this evaluation criterion may be determined to be a drug candidate compound.

[0045] The drug candidate compounds screened by the above method can be used as drugs for treating bladder diseases (agents for treating bladder diseases).

[0046] The above method can be used in a method for producing a drug for treating a bladder disease (a bladder disease treatment agent). That is, the method for producing a drug for treating a bladder disease (a bladder disease treatment agent) is included in a method for screening a drug candidate compound for treating a bladder disease (hereinafter referred to as a "screening step"). Furthermore, the method for producing a drug for treating a bladder disease (a bladder disease treating agent) may include a step of synthesizing or extracting a compound as a test substance prior to the screening step.

[0047] According to the second embodiment of the present invention, drug candidate compounds effective for treating bladder diseases can be appropriately screened.

[0048] (Embodiment 3) A third embodiment of the present invention relates to a method for imparting a medicinal effect to the submucosa of the bladder, comprising the step of administering to the bladder cavity of a subject a chondroitin sulfate derivative in which chondroitin sulfate is bound to a hydrophobic compound. The chondroitin sulfate derivative may be the same as the chondroitin sulfate derivative described in embodiment 1 of the present invention. The administration method of the chondroitin sulfate derivative may be the same as the administration method described in embodiment 1 of the present invention.

[0049] The third embodiment of the present invention can be used in a method for treating bladder disease. That is, the method for treating bladder disease includes a method for imparting a medicinal effect to the submucosa of the bladder. Specifically, the method for treating bladder disease includes a step of administering a chondroitin sulfate derivative used as a bladder disease treatment agent to the bladder cavity of a subject. The method for treating bladder disease may further include other steps as necessary.

[0050] According to the third embodiment of the present invention, the active pharmaceutical ingredient administered into the bladder cavity can effectively exert its efficacy in the submucosal tissue. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples and test examples, which, however, are not intended to limit the technical scope of the present invention. Among the examples shown below, Examples 1 and 4 are reference examples.

[0052] Example 1 (1-1) Preparation of 21-(Boc-β-alanyl)-triamcinolone acetonide 2.63 g of Boc-β-alanine (manufactured by Watanabe Chemical Industry Co., Ltd.) was dissolved in 40 mL of dichloromethane and 10 mL of dimethylformamide, and 5.01 g of triamcinolone acetonide (manufactured by Wako Pure Chemical Industries, Ltd.; hereinafter, also abbreviated as "TA") was added. Then, 426 mg of 4-dimethylaminopyridine and 3.32 g of water-soluble carbodiimide were added under ice cooling and stirred overnight at room temperature. After confirming the disappearance of the raw material by thin layer chromatography, the reaction was stopped by adding a saturated aqueous ammonium chloride solution. Separation and extraction were performed using toluene and water, and the collected organic layer was washed successively with a saturated aqueous ammonium chloride solution, a saturated aqueous sodium bicarbonate solution, and a saturated saline solution. Then, after drying with magnesium sulfate and filtration, the solvent was distilled off under reduced pressure to obtain 6.66 g of the desired 21-(Boc-β-alanyl)-triamcinolone acetonide (yield 96%).

[0053] (1-2) Preparation of 21-β-alanyl-triamcinolone acetonide hydrochloride 6.66g of 21-(Boc-β-alanyl)-triamcinolone acetonide was dissolved in 90mL of tetrahydrofuran, and 90mL of 4M hydrochloric acid / ethyl acetate was added under ice cooling, followed by stirring at room temperature for 3 hours. After confirming the disappearance of the raw material by thin layer chromatography, the solvent was distilled off under reduced pressure. The concentrate was washed with diethyl ether, and the solvent was distilled off under reduced pressure to obtain 5.78g of the desired 21-β-alanyl-triamcinolone acetonide hydrochloride (yield 97%).

[0054] 21-(β-alanyl)-triamcinolone acetonide has the following structure, and the ClogP was calculated to be 2.17 using Chem Draw Professional ver. 18.1.2.18 (PerkinElmer).

[0055] [ka]

[0056] (1-3) Preparation of 21-β-alanyl-triamcinolone acetonide-introduced CS (hereinafter abbreviated as "CS-TA-conjugate") 524 mg of CS (sodium salt, Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 458 mg of 21-β-alanyl-triamcinolone acetonide hydrochloride and 839 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hydrate (hereinafter, DMT-MM) (Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 3 mL), and stirred overnight. Then, 0.1 g of sodium chloride and 50% (v / v) acetone / distilled water for washing were added, and the reaction solution in which sodium chloride was dissolved was added to 90% (v / v) ethanol / distilled water 300 mL in sequence to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water, and dried overnight under reduced pressure to obtain 819 mg of the desired CS-TA-conjugate.

[0057] (1-4) Preparation of fluorescently labeled 21-β-alanyl-triamcinolone acetonide-introduced CS (hereinafter abbreviated as "fluorescently labeled CS-TA-conjugate") 500 mg of CS-TA-conjugate was dissolved in 5.5 mL of distilled water, and 5.5 mL of acetone was added and stirred. 5 mg of fluoresceinyl glycine amide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 10 mL), and stirred overnight. Then, 5.0 g of sodium chloride was dissolved in 37.5 mL of distilled water, and 125 mL of 2-propanol was added to the solution, and the reaction solution was added while washing with 50% (v / v) acetone / distilled water 9 mL, and then 25 mL of 2-propanol was added to form a precipitate, and the supernatant was discarded. Then, the precipitate was washed sequentially with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 471 mg of the desired fluorescently labeled CS-TA-conjugate.

[0058] Example 2 (2-1) Preparation of (Boc-β-alanyl)-hydroxyzine 2.00 g of hydroxyzine dihydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.; hereinafter also abbreviated as "HYD"), 1.09 g of 4-dimethylaminopyridine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.846 g of Boc-β-alanine (manufactured by Watanabe Chemical Industry Co., Ltd.) were added and dissolved in 20 mL of dichloromethane. Then, 1.71 g of water-soluble carbodiimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred overnight at room temperature. After confirming the disappearance of the raw material by thin layer chromatography, the stirring was stopped and the mixture was washed successively with a 5% (w / v) aqueous sodium hydrogen carbonate solution and a 20% (w / v) aqueous sodium chloride solution. Then, the mixture was dehydrated with sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The concentrate was purified with a silica gel column (chloroform:methanol) to obtain 1.99 g of the desired (Boc-β-alanyl)-hydroxyzine (yield 82%).

[0059] (2-2) Preparation of β-alanyl-hydroxyzine trihydrochloride 1.93 g of (Boc-β-alanyl)-hydroxyzine was dissolved in 71 mL of dichloromethane, and 19.4 mL of 4 mol / L-HCl / AcOEt (Watanabe Chemical Industry Co., Ltd.) was added under ice cooling, and the mixture was stirred for 110 minutes under ice cooling. After confirming the disappearance of the raw material by thin layer chromatography, the solvent was distilled off under reduced pressure. The obtained oily substance was dissolved in dichloromethane, and methyl tert-butyl ether was added to precipitate crystals. The crystals were dried under reduced pressure at 40°C overnight, and 1.45 g of the desired β-alanyl-hydroxyzine trihydrochloride was obtained (yield 74%).

[0060] β-alanyl-hydroxyzine has the following structure, and the ClogP calculated using ChemDraw Professional ver. 18.1.2.18 (PerkinElmer) was 4.31.

[0061] [ka]

[0062] (2-3) Preparation of β-alanyl-hydroxyzine-introduced CS (hereinafter abbreviated as "CS-HYD-conjugate") 1.04 g of CS (sodium salt, manufactured by Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 111 mg of β-alanyl-hydroxyzine trihydrochloride and 167 mg of DMT-MM (manufactured by Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 20 mL) and stirred overnight. After that, 2.00 g of sodium chloride was added and dissolved, and the reaction solution was added to 80 mL of 95% (v / v) ethanol / distilled water while washing with 15 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 903 mg of the desired CS-HYD-conjugate.

[0063] (2-4) Preparation of fluorescently labeled β-alanyl-hydroxyzine-introduced CS (hereinafter referred to as “fluorescently labeled CS-HYD-conjugate”) 500 mg of CS-HYD-conjugate was dissolved in 5 mL of distilled water, and 5 mL of acetone and 5 mL of 50% (v / v) acetone / distilled water were added and stirred. 5 mg of fluoresceinyl glycinamide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 5 mL) and stirred overnight. Then, 0.75 g of sodium chloride was added and dissolved. The reaction solution was added to 40 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 448 mg of the desired fluorescently labeled CS-HYD-conjugate.

[0064] Example 3 (3-1) Preparation of (Boc-2-aminoethyl)-naproxen 1.06 g of N-Boc-ethanolamine (Tokyo Chemical Industry Co., Ltd.), 1.50 g of (S)-naproxen (manufactured by Fujifilm Wako Pure Chemical Industries Co., Ltd.; hereinafter, also abbreviated as "NAP") and 1.59 g of 4-dimethylaminopyridine (manufactured by Fujifilm Wako Pure Chemical Industries Co., Ltd.) were added and dissolved in 28 mL of dichloromethane. Then, 2.50 g of water-soluble carbodiimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred overnight at room temperature. After confirming the disappearance of the raw material by thin layer chromatography, the stirring was stopped and the mixture was washed successively with a 10% (w / v) aqueous potassium hydrogen sulfate solution, a 5% (w / v) aqueous sodium hydrogen carbonate solution, and a 20% (w / v) aqueous sodium chloride solution. Then, the mixture was dehydrated with magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 2.32 g of the desired (Boc-2-aminoethyl)-naproxen (yield 95%).

[0065] (3-2) Preparation of 2-aminoethyl-naproxen hydrochloride 2.15g of (Boc-2-aminoethyl)-naproxen was dissolved in 115mL of dichloromethane, and 31.6mL of 4mol / L-HCl / AcOEt (Watanabe Chemical Industry Co., Ltd.) was added under ice cooling, and the mixture was stirred at room temperature for 1.5 hours. After confirming the disappearance of the raw material by thin layer chromatography, the crystals precipitated in the reaction solution were filtered. The crystals were dried under reduced pressure at 40°C overnight, and 1.42g of the desired 2-aminoethyl-naproxen hydrochloride was obtained (yield 80%).

[0066] 2-aminoethyl-naproxen has the following structure, and the ClogP was calculated to be 2.62 using Chem Draw Professional ver. 18.1.2.18 (PerkinElmer).

[0067] [ka]

[0068] (3-3) Preparation of 2-aminoethyl-naproxen-introduced CS (hereinafter abbreviated as "CS-NAP-conjugate") 1.04 g of CS (sodium salt, manufactured by Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 60 mg of 2-aminoethyl-naproxen hydrochloride and 167 mg of DMT-MM (manufactured by Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 20 mL) and stirred overnight. After that, 2.00 g of sodium chloride was added and dissolved, and the reaction solution was added to 80 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 1.03 g of the desired CS-NAP-conjugate.

[0069] (3-4) Preparation of fluorescently labeled 2-aminoethyl-naproxen-introduced CS (hereinafter referred to as "fluorescently labeled CS-NAP-conjugate") 500 mg of CS-NAP-conjugate was dissolved in 5 mL of distilled water, and 5 mL of acetone and 5 mL of 50% (v / v) acetone / distilled water were added and stirred. 5 mg of fluoresceinyl glycinamide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 5 mL), and stirred overnight. After that, 0.75 g of sodium chloride was added and dissolved, and the reaction solution was added to 40 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed sequentially with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 568 mg of the desired fluorescently labeled CS-NAP-conjugate.

[0070] Example 4 (4-1) Preparation of (Boc-2-aminoethyl)-dehydrocholic acid ester 2.00 g of dehydrocholic acid (manufactured by Tokyo Chemical Industry Co., Ltd.; hereinafter also abbreviated as "DEH"), 0.80 g of N-Boc-ethanolamine (manufactured by Tokyo Chemical Industry Co., Ltd.), and 1.22 g of 4-dimethylaminopyridine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added and dissolved in 21 mL of dichloromethane. Then, 1.90 g of water-soluble carbodiimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred overnight at room temperature. Stirring was stopped, and the mixture was washed successively with a 10% (w / v) aqueous potassium hydrogen sulfate solution, a 5% (w / v) aqueous sodium hydrogen carbonate solution, and a 20% (w / v) aqueous sodium chloride solution. Then, the mixture was dehydrated with magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 2.20 g of the desired (Boc-2-aminoethyl)-dehydrocholic acid ester (yield 81%).

[0071] (4-2) Preparation of 2-aminoethyl-dehydrocholic acid ester hydrochloride 2.16 g of (Boc-2-aminoethyl)-dehydrocholic acid ester was dissolved in 79 mL of dichloromethane, and 21.7 mL of 4 mol / L-HCl / AcOEt (Watanabe Chemical Industry Co., Ltd.) was added under ice cooling, and the mixture was stirred at room temperature for 90 minutes, and the crystals precipitated in the reaction solution were filtered out. The crystals were dried under reduced pressure at 40°C overnight, and 1.25 g of the desired 2-aminoethyl-dehydrocholic acid ester hydrochloride was obtained (yield 66%).

[0072] 2-aminoethyl-dehydrocholic acid has the following structure, and the ClogP was calculated to be 2.03 using Chem Draw Professional ver. 18.1.2.18 (PerkinElmer).

[0073] [ka]

[0074] (4-3) Preparation of 2-aminoethyl-dehydrocholic acid-introduced CS (hereinafter abbreviated as "CS-DEH-conjugate") 1.04 g of CS (sodium salt, manufactured by Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 96 mg of 2-aminoethyl-dehydrocholic acid ester hydrochloride and 167 mg of DMT-MM (manufactured by Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 20 mL) and stirred overnight. After that, 2.00 g of sodium chloride was added and dissolved, and the reaction solution was added to 80 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 ° C overnight to obtain 1.05 g of the desired CS-DEH-conjugate.

[0075] (4-4) Preparation of fluorescently labeled 2-aminoethyl-dehydrocholic acid-introduced CS (hereinafter referred to as "fluorescently labeled CS-DEH-conjugate") 500 mg of CS-DEH-conjugate was dissolved in 5 mL of distilled water, and 5 mL of acetone and 5 mL of 50% (v / v) acetone / distilled water were added and stirred. 5 mg of fluoresceinyl glycine amide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 5 mL), and stirred overnight. After that, 0.75 g of sodium chloride was added and dissolved, and the reaction solution was added to 40 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed sequentially with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The obtained precipitate was dried under reduced pressure at 40 ° C overnight to obtain 571 mg of the desired fluorescently labeled CS-DEH-conjugate.

[0076] Example 5 (5-1) Preparation of Boc-2-aminoethyl bromide 2.155g of 2-bromoethylamine hydrobromide was dissolved in 20mL of dichloromethane, and 1.463mL of triethylamine was added under ice cooling. 5mL of a dichloromethane solution of 2.299g of Boc2O was further added and stirred. After stirring at room temperature for 90 minutes, ethyl acetate was added and the mixture was washed with 5% (w / v) aqueous citric acid, water, and saturated saline in that order. After dehydration with sodium sulfate, the solvent was distilled off under reduced pressure to obtain 2.287g of the desired Boc-2-aminoethyl bromide (yield 97%).

[0077] (5-2) Preparation of 2-aminoethyl-diclofenac hydrochloride 5mL of a DMF solution of 2.287g of Boc-2-aminoethyl bromide was ice-cooled, and 6mL of a DMF solution of 3.255g of diclofenac sodium was added and stirred at room temperature overnight. The mixture was stirred at 60°C for 11 hours and then at room temperature overnight. Ethyl acetate was added, and the mixture was washed with 5% (w / v) aqueous sodium bicarbonate, water, and saturated saline in that order. After dehydration with sodium sulfate, the ethyl acetate was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (toluene:ethyl acetate=20:1 (v / v), 0.5% (v / v) triethylamine) to obtain 2.675g of Boc-2-aminoethyl-diclofenac. Next, 2.108 g of Boc-2-aminoethyl-diclofenac was dissolved in 5 mL of dichloromethane, and 20 mL of 4 M hydrochloric acid / ethyl acetate was added under ice cooling and stirred for 2.5 hours. Diethyl ether and hexane were added to cause precipitation, and the precipitate was dried under reduced pressure to obtain 1.775 g of the desired 2-aminoethyl-diclofenac hydrochloride (yield 98%).

[0078] 2-aminoethyl-diclofenac has the following structure, and the ClogP was calculated to be 4.38 using Chem Draw Professional ver. 18.1.2.18 (PerkinElmer).

[0079] [ka]

[0080] (5-3) Preparation of 2-aminoethyl-diclofenac-introduced CS (hereinafter abbreviated as "CS-DIC-conjugate") 1.04 g of CS (sodium salt, manufactured by Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 73 mg of 2-aminoethyl-diclofenac hydrochloride and 167 mg of DMT-MM (manufactured by Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 20 mL) and stirred overnight. After that, 2.00 g of sodium chloride was added and dissolved, and the reaction solution was added to 80 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 1.08 g of the desired CS-DIC-conjugate.

[0081] (5-4) Preparation of fluorescently labeled 2-aminoethyl-diclofenac-introduced CS (hereinafter referred to as "fluorescently labeled CS-DIC-conjugate") 500 mg of CS-DIC-conjugate was dissolved in 5 mL of distilled water, and 5 mL of acetone and 5 mL of 50% (v / v) acetone / distilled water were added and stirred. 5 mg of fluoresceinyl glycinamide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% acetone / distilled water (total 5 mL) and stirred overnight. After that, 0.75 g of sodium chloride was dissolved, and the reaction solution was added to 40 mL of 95% (v / v) ethanol / distilled water while washing with 5 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 572 mg of the desired fluorescently labeled CS-DIC-conjugate.

[0082] Example 6 (6-1) Preparation of (Boc-2-aminoethyl)-probenecid 1.51 g of probenecid (manufactured by Tokyo Chemical Industry Co., Ltd.; hereinafter also abbreviated as "PRO"), 0.85 g of N-Boc-ethanolamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and 1.29 g of 4-dimethylaminopyridine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added and dissolved in 23 mL of dichloromethane. Then, 2.02 g of water-soluble carbodiimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred overnight at room temperature. After confirming the disappearance of the raw material by thin layer chromatography, the stirring was stopped and the mixture was washed successively with a 1% (w / v) aqueous potassium hydrogen sulfate solution, a 5% (w / v) aqueous sodium hydrogen carbonate solution, and a 20% (w / v) aqueous sodium chloride solution. Then, the mixture was dehydrated with magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 1.96 g of the desired (Boc-2-aminoethyl)-probenecid (yield 86%).

[0083] (6-2) Preparation of 2-aminoethyl-probenecid hydrochloride 1.89 g of (Boc-2-aminoethyl)-probenecid was dissolved in 88 mL of dichloromethane, and 24.2 mL of 4 mol / L-HCl / AcOEt (Watanabe Chemical Industry Co., Ltd.) was added under ice cooling and stirred at room temperature for 80 minutes. After confirming the disappearance of the raw material by thin layer chromatography, the solvent was distilled off under reduced pressure. The concentrate was washed with methyl tert-butyl ether, and the solvent was distilled off under reduced pressure. The obtained crystals were dissolved in methanol, and then methyl tert-butyl ether was added to precipitate the crystals. The crystals were dried under reduced pressure at 40°C overnight to obtain 1.36 g of the desired 2-aminoethyl-probenecid hydrochloride (yield 85%).

[0084] 2-aminoethyl-probenecid has the following structure, and the ClogP was calculated to be 2.61 using ChemDraw Professional ver. 18.1.2.18 (PerkinElmer).

[0085] [ka]

[0086] (6-3) Preparation of 2-aminoethyl-probenecid-introduced CS (hereinafter abbreviated as "CS-PRO-conjugate") 1.04 g of CS (sodium salt, Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 10 mL of distilled water, and 10 mL of acetone was added and stirred. 71 mg of 2-aminoethyl-probenecid hydrochloride and 167 mg of DMT-MM (Tokuyama Corporation) were added while washing with 50% (v / v) acetone / distilled water (total 20 mL) and stirred overnight. After that, 2.00 g of sodium chloride was added and dissolved, and the reaction solution was added to 80 mL of 95% (v / v) ethanol / distilled water while washing with 10 mL of 50% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in that order. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 916 mg of the desired CS-PRO-conjugate.

[0087] (6-4) Preparation of fluorescently labeled 2-aminoethyl-probenecid-introduced CS (hereinafter referred to as "fluorescently labeled CS-PRO-conjugate") 500 mg of CS-PRO-conjugate was dissolved in 7.5 mL of distilled water, and 7.5 mL of acetone was added and stirred. 5 mg of fluoresceinyl glycine amide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 5 mL), and stirred overnight. After that, 0.75 g of sodium chloride was added and dissolved, and the reaction solution was added to 40 mL of 95% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. The precipitate was then washed sequentially with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 462 mg of the desired fluorescently labeled CS-PRO-conjugate.

[0088] Comparative Example 1 (1-1) Preparation of fluorescently labeled CS 500 mg of CS (sodium salt, manufactured by Seikagaku Corporation, weight average molecular weight measured by light scattering method: about 20,000) was dissolved in 7.5 mL of distilled water, and 7.5 mL of acetone was added and stirred. 5 mg of fluoresceinyl glycine amide (manufactured by Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) acetone / distilled water (total 1.5 mL) and stirred overnight. 175 mg of sodium chloride was added while washing with 2 mL of 50% (v / v) acetone / distilled water, and then 53 mL of ethanol was added to form a precipitate, and the supernatant was discarded. The precipitate was redissolved in 20 mL of distilled water, and 100 mg of sodium chloride and 100 mL of 95% (v / v) ethanol / distilled water were added to form a precipitate again, and the supernatant was discarded. The precipitate was then washed successively with 95% (v / v) ethanol / distilled water and ethanol. The resulting precipitate was dried under reduced pressure at 40°C overnight to obtain 380 mg of the desired fluorescently labeled CS.

[0089] Comparative Example 2 (2-1) Preparation of 21-β-alanyl-triamcinolone acetonide-introduced HA (hereinafter abbreviated as "HA-TA-conjugate") 2.17g of HA (sodium hyaluronate, Seikagaku Corporation) was dissolved in 200mL of distilled water, and 200mL of ethanol was added and stirred. 272mg of 21-β-alanyl-triamcinolone acetonide and 431mg of DMT-MM (Tokuyama Corporation) were added while washing with 50% (v / v) ethanol / distilled water (total 20mL) and stirred overnight. Then, 11mL of distilled water in which 0.75g of sodium bicarbonate was dissolved was added to half of the reaction solution. After leaving it for 4 hours, 0.2mL of acetic acid was added. 5.0g of sodium chloride was dissolved in the reaction solution, and then added to 483mL of 90% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. Then, the precipitate was washed successively with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The resulting precipitate was dried overnight under reduced pressure at 40° C. to obtain 929 mg of the desired HA-TA conjugate.

[0090] (2-2) Preparation of fluorescently labeled 21-β-alanyl-triamcinolone acetonide-introduced HA (hereafter referred to as "fluorescently labeled HA-TA conjugate"). 500 mg of HA-TA-conjugate was dissolved in 50 mL of distilled water, and 50 mL of ethanol was added and stirred. 5 mg of fluoresceinyl glycinamide (Setare Biotech) and 24 mg of DMT-MM were added while washing with 50% (v / v) ethanol / distilled water (total 3 mL), and stirred overnight. Then, 5 mL of distilled water in which 375 mg of sodium bicarbonate was dissolved was added to the reaction solution. After leaving it for 4 hours, 0.10 mL of acetic acid was added while washing with 1 mL of distilled water. After dissolving 2.5 g of sodium chloride in the reaction solution, it was added to 242 mL of 90% (v / v) ethanol / distilled water to form a precipitate, and the supernatant was discarded. Then, the precipitate was washed with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water in sequence. The resulting precipitate was dried overnight under reduced pressure at 40°C to obtain 567 mg of the desired fluorescently labeled HA-TA conjugate.

[0091] Comparative Example 3 (3-1) Preparation of 3-aminopropyl-cinnamic acid-introduced HA (hereinafter abbreviated as "HA-CIN-conjugate") A 172 mg / 5 mL aqueous solution of N-hydroxysuccinimide (HOSu: Watanabe Chemical Industry Co., Ltd.), a 143 mg / 5 mL aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (Watanabe Chemical Industry Co., Ltd.), and a 181 mg / 5 mL aqueous solution of 3-aminopropyl cinnamate hydrochloride (Tokyo Chemical Industry Co., Ltd.) were added to a solution of sodium hyaluronate (1.06 g, Seikagaku Corporation) in water (115 mL) / dioxane (144 mL). The mixture was stirred for 3 hours and a 750 mg / 10 mL aqueous solution of sodium bicarbonate (Japanese Pharmacopoeia) was added. After stirring for an additional 2 hours and 30 minutes, the reaction was quenched with acetic acid (214 mg) and sodium chloride (1.0 g). Ethanol (300 mL) was added, and the resulting precipitate was filtered off and washed twice with 80% (v / v) ethanol and 95% (v / v) ethanol, respectively. The solid was dried overnight at 40° C. in vacuum to give 1.06 g of HA-CIN-conjugate.

[0092] 3-Aminopropyl-cinnamic acid has the following structure, and the ClogP was calculated to be 2.14 using Chem Draw Professional ver. 18.1.2.18 (PerkinElmer).

[0093] [ka]

[0094] (3-2) Preparation of fluorescently labeled 3-aminopropyl-cinnamic acid-introduced HA (hereafter referred to as "fluorescently labeled HA-CIN-conjugate") 500 mg of HA-CIN-conjugate was dissolved in 50 mL of distilled water, and 50 mL of ethanol was added and stirred. 5 mg of fluoresceinyl glycinamide (Setare Biotech) and 24 mg of DMT-MM were dissolved in 50% (v / v) acetone / distilled water (total 14 mL) and added, and stirred overnight. Then, 375 mg of sodium bicarbonate was dissolved in 5 mL of distilled water and added. 0.10 mL of acetic acid was dissolved in 3 mL of distilled water and added. After adding and dissolving 2.5 g of sodium chloride, 242 mL of 90% (v / v) ethanol / distilled water was added to form a precipitate, and the supernatant was discarded. Then, the precipitate was washed sequentially with 90% (v / v) ethanol / distilled water and 95% (v / v) ethanol / distilled water. The obtained precipitate was dried under reduced pressure at 40 °C overnight to obtain 462 mg of the desired fluorescently labeled HA-CIN-conjugate.

[0095] <Release test> In the examples relating to the release test, the release rate of active pharmaceutical ingredients (API) from each of the non-fluorescently labeled chondroitin sulfate derivatives and hyaluronic acid derivatives was evaluated as follows. (1) Preparation of phosphate buffer 780 mg (5.0 mmol) of sodium dihydrogen phosphate dihydrate was dissolved in 500 mL of purified water to prepare solution A. Separately, 1.79 g (5.0 mmol) of disodium hydrogen phosphate dodecahydrate was dissolved in 500 mL of purified water to prepare solution B. Solutions A and B were mixed in a ratio of 3:7 to prepare a phosphate buffer solution with a pH of 7.4.

[0096] (2) Evaluation method The chondroitin sulfate derivatives of Examples 1 to 6 were dissolved in a phosphate buffer solution of pH 7.4 to a concentration of 5 mg / 10 mL, and then heated in a thermostatic bath at 36°C for one week. The release rate of API during that time was evaluated every 24 hours by high performance liquid chromatography (HPLC). A size exclusion chromatography column (TSKgel α, manufactured by Tosoh Corporation) was used, and the release rate was calculated from the ratio of the HPLC peak area corresponding to the amount of API in the derivative and the amount of released API. Release rate (%) = peak area of ​​released API / (peak area of ​​API in each derivative + peak area of ​​released API) x 100 The detailed conditions for liquid chromatography are as follows. Analysis time: 40 minutes Flow rate: 0.5mL / min Gradient: isocratic Eluent: acetonitrile (for HPLC): physiological saline = 1:2 (v:v) Detector: UV detector (210nm, 230nm, 240nm or 280nm) Temperature: 36℃. The hyaluronic acid derivatives of Comparative Examples 2 and 3 were also evaluated in accordance with the Examples.

[0097] (3) Evaluation results The evaluation results of the release test are shown in Table 1.

[0098] [Table 1]

[0099] As shown in Table 1, all of the chondroitin sulfate derivatives of Examples 1 to 6 showed a slow release rate. In contrast, the hyaluronic acid derivative of Comparative Example 2 was confirmed to have a large release of active ingredient at the start of the test. In addition, the hyaluronic acid derivative of Comparative Example 3 was not confirmed to have a release of cinnamic acid.

[0100] <Evaluation using rats 1> (1) Preparation of test substances Test substances were prepared by dissolving the fluorescently labeled compounds of Examples 1 to 6 and Comparative Examples 1 to 3 in phosphate buffered saline containing 2.3% (v / v) glycerin. The concentration of each fluorescently labeled compound in the test substances was 1% (w / v). (2) Pretreatment of test animals Under isoflurane inhalation anesthesia (concentration: 2.0-4.0%, flow rate: 2-3 L / min), the lower abdomen of rats (SD (SPF), female, 7-9 weeks old) was compressed to drain residual urine from the rat's bladder. A catheter (PE50) was inserted transurethrally into the bladder cavity, and the bladder cavity was irrigated with 0.2 mL of physiological saline. Then, 0.2 mL of 0.4 mol / L hydrochloric acid was injected into the bladder cavity and left in place for 3 minutes and 15 seconds. After the hydrochloric acid was left in place, the bladder cavity was irrigated four times with 0.4 mL of physiological saline.

[0101] (3) Evaluation method One day after pretreatment, 0.2 mL of each test substance was administered transurethrally into the bladder cavity using a catheter (PE50) under isoflurane inhalation anesthesia, and left in place for 30 minutes. One day after administration of the test substance, the rat bladder was collected and frozen sections were prepared perpendicular to the axis connecting the bladder top and the urethra. Bright field images (visible light) and fluorescent images (excitation wavelength 470 nm, absorption wavelength 525 nm) of the frozen sections were taken using a fluorescent microscope. The obtained fluorescent images were analyzed using the hybrid cell count of the software (BZ-X Analyzer) attached to the fluorescent microscope. The fluorescence extraction threshold was set to 40, and the integrated value of the brightness of each fluorescent image was calculated. For each test, CS-TA-conjugate (Example 1) was used as a positive control compound, and the relative value was calculated based on this.

[0102] (4) Evaluation results Bright field images (a) of the frozen sections taken 1 day after administration of the test substance and corresponding fluorescent images (b) are shown in Figures 1 to 6 (Examples) and Figures 7 to 9 (Comparative Examples). In the fluorescent images (b), the white areas indicate areas emitting fluorescence. As shown in Figures 1 to 6, in the chondroitin sulfate derivative administration group (Example), strong and widespread fluorescence was observed in the transitional epithelium and submucosa of the bladder, indicating high permeability to the submucosa of the bladder. In contrast, in the chondroitin sulfate administration group in Figure 7 and the hyaluronic acid derivative administration group in Figures 8 to 9 (Comparative Example), the fluorescence was weak in the transitional epithelium and submucosa of the bladder, indicating low permeability to the submucosa of the bladder. Table 2 also shows the integrated luminance values ​​(relative values ​​based on Example 1) on the first day after administration of each test substance.

[0103] [Table 2]

[0104] As shown in Table 2, the fluorescently labeled compounds (chondroitin sulfate derivatives) of Examples 1 to 6 had larger integrated brightness values ​​on the first day after administration than the fluorescently labeled compound (chondroitin sulfate) of Comparative Example 1 and the fluorescently labeled compounds (hyaluronic acid derivatives) of Comparative Examples 2 and 3. From this result, it can be said that the fluorescently labeled compounds (chondroitin sulfate derivatives) of Examples 1 to 6 can increase the permeability of active pharmaceutical ingredients (APIs) into the submucosal tissue of the bladder.

[0105] <Evaluation using rats 2> 1. Comparison of chondroitin sulfate derivatives and chondroitin sulfate According to the method described in the above Evaluation 1, the fluorescent labeled compound (chondroitin sulfate derivative) of Example 1 and the fluorescent labeled compound (chondroitin sulfate) of Comparative Example 1 were administered, and then the fluorescent images were measured on the 7th day, and the integrated brightness values ​​were calculated. The results are shown in Table 3.

[0106] [Table 3]

[0107] As shown in Table 3, the fluorescently labeled compound (chondroitin sulfate derivative) of Example 1 had a significantly larger integrated brightness value on the 7th day after administration than the fluorescently labeled compound (chondroitin sulfate) of Comparative Example 1. From this result, it can be said that the chondroitin sulfate derivative is present in greater amounts than chondroitin sulfate in the submucosal tissue of the bladder.

[0108] 2. Comparison of chondroitin sulfate derivatives with active pharmaceutical ingredients (single agents) (1) Preparation of test substances The chondroitin sulfate derivative (CS-TA-conjugate) of Example 1 was dissolved in 5 mM phosphate buffer containing 2.3% (v / v) glycerin to prepare a test substance. A commercially available triamcinolone acetonide preparation (Kenacort) was used as a comparative compound (single agent). In both the examples and comparative examples, the dose of TA was set to 0.8 mg / mouse. (2) Pretreatment of test animals Evaluation was carried out in the same manner as in 1.

[0109] (3) Evaluation method One day after pretreatment, 0.2 mL of each test substance was administered transurethrally into the bladder using a catheter (PE50) under isoflurane inhalation anesthesia, and left in place for 30 minutes. Seven days after administration of the test substance, the rats were anesthetized by isoflurane inhalation, and the abdominal aorta was cut and the rats were euthanized by exsanguination, after which the bladder was collected. The bladder was homogenized in a mixture of methanol and buffer (10 mmol / L ammonium formate buffer (pH 6.0):methanol = 3:2 (v / v)) to prepare a measurement sample with a tissue concentration of 0.05 g / mL. Next, the amount of TA in the measurement sample was quantified by LC / MS / MS analysis under the following conditions using Triamcinolone-6-d1Acetonide-d6 as an internal standard substance. ·Measuring equipment HPLC: Nexera X2 (Shimadzu Corporation) MS: QTRAP 6500 (AB SCIEX) ·HPLC conditions Analytical column: Reverse phase column (Inertsil ODS, 2.1 mm x 150 mm, GL Science) Analysis time: 7 minutes Flow rate: 0.4mL / min Mobile phase: Mobile phase A: 0.1% (v / v) formic acid aqueous solution, Mobile phase B: Methanol Gradient Temperature: 40℃ MS conditions Ionization mode: ESI (Turbo Spray Ion Drive) Measurement mode: MRM Detection mode: Positive (4) Evaluation results The results of the API (TA) concentration on day 7 after administration of the test substance are shown in Table 4.

[0110] [Table 4]

[0111] As shown in Table 4, the CS-TA conjugate had a higher TA concentration on the 7th day after administration than TA. In other words, the group administered the chondroitin sulfate derivative (CS-TA conjugate) contained a larger amount of the active pharmaceutical ingredient (TA) than the group administered the active pharmaceutical ingredient alone. These results suggest that chondroitin sulfate derivatives incorporating an API can deliver the API to the submucosal tissue of the bladder more effectively than the API alone.

[0112] As can be seen from the above results, the present invention can provide a system that can achieve effective delivery of an active pharmaceutical ingredient (API) administered into the bladder lumen to the submucosal tissue.

[0113] This application claims priority based on Japanese Patent Application No. 2019-183082, filed with the Japan Patent Office on October 3, 2019, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A transmucosal delivery system for an active pharmaceutical ingredient other than a steroid to the submucosal tissue of the bladder, comprising: A conjugate of chondroitin sulfate and a hydrophobic compound, the hydrophobic compound is a compound in which the active pharmaceutical ingredient and a spacer-forming molecule are ester-bonded, and has a ClogP of 1 or more; A delivery system wherein the spacer-forming molecule is amide bonded to the chondroitin sulfate.

2. The delivery system of claim 1 , wherein the active pharmaceutical ingredient is released from the conjugate after being administered to the bladder lumen.

3. 3. The delivery system of claim 1 or 2, wherein the release rate of the active pharmaceutical ingredient from the conjugate in 10 mM phosphate buffer (pH 7.4) at 36° C. is 0.1% / day to 5% / day.

4. The spacer-forming molecule has the following general formula (I): H 2 N-(CH 2 ) p -X (I) (In the formula, p represents an integer of 1 to 12, and each -CH 2 4. The delivery system according to claim 1, wherein each of - may independently have a substituent; and X represents a hydroxy group, a carboxy group, or an amino group.

5. The delivery system according to any one of claims 1 to 4, wherein the weight average molecular weight of the chondroitin sulfate is 10 times or more the molecular weight of the active pharmaceutical ingredient.

6. The delivery system according to any one of claims 1 to 5, wherein the weight average molecular weight of the chondroitin sulfate is 4,000 or more.

7. The delivery system of any one of claims 1 to 6, wherein the active pharmaceutical ingredient has a molecular weight of 600 or less.

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