JP2007262078A - Deliverable substance and drug delivery system using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a substance which is excellent in deliverability and can effectively stay a drug in a living body over a long period, and to build a drug delivery system using the substance. <P>SOLUTION: When a deliverable substance produced by reacting a polyalkylene glycol or its reactive derivative, a phospholipid, and a drug to form covalent bonds is systemically or topically administered, pharmaceutical effects can be sustained at a dose over a long period, because the substance is stayed at a target site in the living body for the long period. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is a substance having an excellent delivery property in which a polyalkylene glycol or a reactive derivative thereof, a phospholipid, and a drug are reacted to form a covalent bond, and the delivery substance is administered systemically or locally. The present invention relates to a drug delivery system that enables a drug to stay in a specific part of the body for a long period of time.

  There are many intractable diseases in diseases of the inner eye such as the retina, optic nerve, and vitreous body, and the development of an effective treatment method is desired. For eye diseases, it is most common to treat the eye drop by administering a drug, but the drug hardly transfers to the inner eye such as the retina. This makes treatment of diseases in the inner eye more difficult.

  Therefore, a method of directly administering a drug to a specific part of the body has been attempted. For example, a technique for administering a liposome or microsphere containing a drug to an inner eye part such as a vitreous body has been reported (Patent Literature). 1, Patent Document 2, etc.).

  However, it is not easy to control the drug release using liposomes, and since liposomes and microspheres have a large particle size, they are transparent when administered to the inner eye such as vitreous. May not be maintained.

On the other hand, when a drug is orally administered, it is difficult to transfer the drug to a specific site until the drug is easily absorbed and metabolized in the stomach, small intestine, large intestine, liver and the like, and has a concentration that exerts a medicinal effect.
Japanese Patent Publication No. 6-508369 JP-A-4-221322

  For these reasons, it is an important issue to create a substance with excellent deliverability capable of effectively allowing a drug to exist in a living body for a long period of time and to construct a drug delivery system using the substance. .

  As a result of diligent research focusing on the deliverable substance and the drug delivery system using the deliverable substance, the present inventors have made deliverability in which polyethylene glycol or a reactive derivative thereof, phospholipid and a drug are reacted to form a covalent bond. Created an excellent material. Further, when the delivery substance is administered into the vitreous body, it stays in the retina and the vitreous body for a long period of time. Therefore, the delivery substance and the systemic or local drug delivery system using the delivery substance are various in the body. It was found that it can be used to treat diseases at various sites.

  The delivery substance of the present invention is a substance having excellent delivery ability by reacting polyalkylene glycol or a reactive derivative thereof, phospholipid and a drug to form a covalent bond. The drug delivery system using the delivery substance of the present invention can retain the delivery substance for a long period of time in the posterior eye portion such as the vitreous body, the retina, and the optic nerve. Therefore, the drug delivery system in which the deliverable substance of the present invention is administered systemically or locally makes it possible to treat or prevent various diseases in a specific part of the body over a long period of time by a single administration.

  The present invention is a delivery substance obtained by covalently bonding polyalkylene glycol or a reactive derivative thereof, phospholipid and a drug, and a drug delivery system using the substance. When the delivery substance of the present invention is administered systemically or locally, the substance stays at the target site in the living body for a long period of time, so that the drug effect can be maintained for a long time by one administration.

The present invention is a therapeutic agent, preventive agent, or test agent for ophthalmic diseases containing a delivery substance represented by the following general formula [1] in which a polyalkylene glycol or a reactive derivative thereof, a phospholipid, and a drug are covalently bonded. .

(In the formula, A and B are the same or different and are residues of the drug, X is an aminoalkyl group, carboxyalkyl group, mercaptoalkyl group, hydrazide alkyl group at one or both ends of polyalkylene glycol or polyalkylene glycol) , A maleimidoalkyl group, a sulfonylalkyl group, a vinylsulfonylalkyl group or a vinylcarbonyl group introduced, Y is a phospholipid residue, m is 0 or an integer of 1 or more, and n is 0 or 1. (At least one of m and n is not 0, and A, B, X and Y are all covalently bonded, that is,-represents a covalent bond.)
Here, the polyalkylene glycol is a polymer containing [—O-alkylene-] as a repeating unit, and the alkylene may be substituted with a lower alkyl group or a hydroxy group. Examples include those in which alkylene is formed of 2 to 3 carbon chains, and more preferable specific examples include polyethylene glycol or polypropylene glycol. Further, the reactive derivative of polyalkylene glycol refers to a product obtained by chemically modifying the terminal of polyalkylene glycol so that polyalkylene glycol can be covalently bonded to a drug or phospholipid. Preferable examples of the reactive derivative of polyalkylene glycol include aminoalkyl group, carboxyalkyl group, mercaptoalkyl group, hydrazide alkyl group, maleimide alkyl group, sulfonylalkyl group, vinylsulfonylalkyl at one or both ends of polyalkylene glycol. And a group introduced with a vinylcarbonyl group, and more preferred examples include those introduced with an aminoethyl group, aminopropyl group, carboxymethyl group, carboxyethyl group, mercaptoethyl group, hydrazidemethyl group.

  In the general formula [1], when m is 0, the OH group located at one end of the polyalkylene glycol may be protected with an alkyl group, an acyl group, or the like.

  In the present invention, the polyalkylene glycol or a reactive derivative thereof may be any of a chain type, a star type, and a branched type, and is appropriately determined in consideration of the concentration of the deliverable substance at the target site, the period during which the deliverable substance is present, etc. You can choose. By using a star-shaped or branched polyalkylene glycol or a reactive derivative thereof, a plurality of drugs can be covalently bonded to one delivery substance.

  The binding form is preferably bonded in the form of drug-polyalkylene glycol-phospholipid, but can also be bonded in the form of polyalkylene glycol-phospholipid-drug, and drug-polyalkylene glycol- It can also be bound in the form of a phospholipid-drug.

  In addition, a plurality of drugs can be bonded to the polyalkylene glycol by selecting the type of polyalkylene glycol. Furthermore, a plurality of drugs can be bound to the delivery substance by binding the drug to the phospholipid.

  The molecular weight of the polyalkylene glycol or the reactive derivative constituting the delivery substance of the present invention is not particularly limited, and is a drug delivery site in the living body, the kind and property of the covalently bonded drug, the required concentration of the delivery substance, Although it can select suitably considering the period etc. in which a delivery substance is made to stay, it is 500-200000 normally, More preferably, it is 1000-50000.

  In the present invention, the chemical structure of the drug to be covalently bonded to the polyalkylene glycol or its reactive derivative is not particularly limited as long as it has a functional group that can be bonded to the polyalkylene glycol or its reactive derivative. Specific examples are drugs having a hydroxy group, a carboxyl group, a carbonyl group, an amino group, an alkenyl group, and the like. The type of drug is not particularly limited as long as it is a systemic or local drug that has a therapeutic or preventive effect on various diseases. Examples include fungal drugs, antitumor drugs, neuroprotective drugs, blood flow improving drugs, antiglaucoma drugs, analgesics, anesthetics, angiogenesis inhibitors, and test drugs. In addition, drugs used for the treatment or prevention of diseases such as the retina, optic nerve, and vitreous body are accompanied by internal eye inflammation due to various causes, viral and bacterial infections, and changes in proliferation of new blood vessels and retinal cells. Drugs effective for proliferative vitreoretinopathy, retinal hemorrhage due to various causes, retinal detachment, retinoblastoma types, and the like. For example, in the case of inflammation associated with intraocular surgery, an anti-inflammatory drug such as betamethasone phosphate is used. In the case of autoimmune uveitis, an immunosuppressive drug such as cyclosporine is used in the case of a viral infection. Antiviral drugs such as ganciclovir, antibacterial drugs such as ofloxacin in the case of postoperative infection, antitumor drugs such as doxorubicin hydrochloride and carmustine in proliferative vitreoretinopathy, ophthalmic test drugs, etc. Is used.

  In order to covalently bond a polyalkylene glycol or a reactive derivative thereof to a drug, a chemical reaction may be performed in consideration of the functional group of the drug and the functional group of the polyalkylene glycol or the reactive derivative thereof. Can be combined. Covalent bonds can be formed with polyalkylene glycols as they are, but covalent bonds can be easily formed with various drugs by using reactive derivatives thereof. For example, reactive derivatives of polyalkylene glycols having various functional groups such as amino, thiol, carboxyl, succinimidyl carboxylate, epoxide, aldehyde, isocyanate, maleimide, acrylate, vinyl sulfone are commercially available. A covalent bond can be formed by chemically reacting a functional derivative and a drug having a functional group.

  Examples of the covalent bond formed in the delivery substance include an ester bond, an amide bond, an ether bond, a carbamate bond, a urea bond, a thiourea bond, a sulfide bond, a disulfide bond, a sulfone bond, a carbonate bond, and a carbon-carbon bond. . Synthesis of a delivery substance having a desired covalent bond in consideration of a functional group of a drug, a functional group of a polyalkylene glycol or a reactive derivative thereof, a functional group of a phospholipid, a residence time in a living body, a site of an affected part, etc. Can do.

In the present invention, the phospholipid covalently bonded to the polyalkylene glycol or its reactive derivative is not particularly limited, and examples thereof include a compound represented by the following general formula [2] or a salt thereof.

(Wherein R ¹ and R ² are the same or different and each represents a hydrogen atom, an alkyl group, an alkylcarbonyl group, an alkenyl group or an alkenylcarbonyl group, Z represents an aminoalkyl group, a diaminoalkyl group, a hydroxyalkyl group or a dihydroxy group) Each represents an alkyl group.)
The phospholipid is not particularly limited as long as it has low toxicity and is excellent in safety, and examples thereof include soybean lecithin, egg yolk lecithin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and synthetic lecithin. Examples of R ¹ and R ² in the compound represented by the general formula [2] include a lauroyl group, myristoyl group, palmitoyl group, stearoyl group, oleoyl group, linoleoyl group, a drug residue, and the like. Examples include aminoethyl group, hydroxyethyl group, 2,3-dihydroxypropyl group, and the like.

  In order for the polyalkylene glycol or its reactive derivative and the phospholipid to be covalently bonded, a functional group having reactive activity on the phospholipid is required, but the functional group is not particularly limited. For example, functional groups having reaction activity such as amino group in phosphatidylethanolamine, hydroxyl group in phosphatidylglycerol, carboxyl group in phosphatidylserine, and the like can be mentioned. Specific examples of particularly preferred phospholipids include phosphatidylethanolamine.

  The method of covalently bonding polyalkylene glycol or its reactive derivative and phospholipid includes a method using an acid anhydride, a method using cyanuric chloride, a method using carbodiimide, and a method using glutaraldehyde. A compound having a polyalkylene glycol or a reactive derivative thereof and a phospholipid can be covalently bonded by appropriately selecting a method.

  The chemical structure of the drug that can be covalently bonded to the phospholipid is not particularly limited as long as it has a functional group capable of binding to the phospholipid. Examples of the drug include the above-mentioned drugs that are covalently bonded to polyalkylene glycol or a reactive derivative thereof. The drug to be covalently bonded to the phospholipid may be the same drug as the drug to be covalently bonded to the polyalkylene glycol or its reactive derivative, or may be a different drug, taking into account the disease, symptoms, drug efficacy, etc. Can be combined as appropriate.

The delivery substance of the present invention can be produced by various methods. For example, as shown in the following formula, when the compound [A] is reacted with N-hydroxysuccinimide in the presence of a condensing agent (for example, N, N-dicyclohexylcarbodiimide), the active ester form of the compound [B] is obtained. can get. Subsequently, when the phospholipid which has an amino group is made to react with the active ester part of compound [B], the amide body of compound [C] will be obtained. T-Butoxycarbonyl introduced as a protecting group for the amide form of compound [C] is deprotected under acidic conditions to obtain an amine form of compound [D], and an active carbonyl form (for example, isothiocyanate) is added to this amine form. By reacting, the delivery substance [E] of the present invention can be obtained.

(In the formula, R ¹ and R ² are as defined above, and t represents an integer of 1 or more.)
When the delivery substance of the present invention is administered systemically or locally, the delivery substance stays in a specific part of the body and is less likely to be metabolized. Exhibits therapeutic and preventive effects for diseases. In addition, the deliverable substance of the present invention that stays in a specific part of the body may exert a therapeutic / preventive effect on the disease. Therefore, the drug delivery system of the present invention makes it possible to treat a specific part of the body, which has been difficult to treat, for a long time with a single administration.

  The drug delivery system of the present invention can be used for the treatment or prevention of various diseases in specific parts of the body by administering the delivery substance of the present invention systemically or locally. Specific diseases include inflammation due to various causes, viral and bacterial infections, immunodeficiency, tumors, proliferative vitreoretinopathy with neovascular and retinal cell proliferation changes, optic nerve diseases, retinal bleeding, retinal detachment And retinoblastoma. In addition, if a delivery substance covalently bonded to various test agents is administered systemically or locally, it can be used for various tests.

  It is desirable that the drug content in the delivery substance is an appropriate content to maintain the effective concentration of the drug over time.

  The effect of the present invention will be described in detail in the intraocular retention test described later. For a delivery substance obtained by covalently binding fluorescein as a model drug, the intraocular part (vitreous body and retina) after intravitreal injection is concerned. As a result of studying the retention of the delivery substance, it was found that the delivery substance of the present invention stays not only in the vitreous body but also in the retina for a long period (56 days or more). In addition, the intraocular kinetics after intravitreal injection of Dizocilpine (drug) and a delivery substance covalently bound to Dizocilpine, which have been reported to have an optic neuroprotective action, were compared. The concentrations in the body, retina choroid and optic nerve were found to be over 100 times higher than with dizocilpine and the elimination half-life was significantly prolonged.

  Based on the results of these tests, various diseases systemically or locally can be effectively treated with a small number of administrations by appropriately selecting the drug to be covalently bonded to the polyalkylene glycol of the present invention or its reactive derivative and / or phospholipid. It becomes possible. Further, by using the drug delivery system of the present invention, the delivery substance can be efficiently retained in a specific part of the body including the retina, optic nerve and vitreous body, so that polyalkylene glycol or its reactive derivative, phospholipid It is possible to reduce the amount of the drug to be covalently bonded to the drug, etc., and the effect of reducing the side effects can be expected.

  Since the deliverable substance of the present invention efficiently stays at a specific site in the living body, it is particularly effective for the treatment of local diseases, and the preparation form is not particularly limited, but is an injection, infusion solution, tablet, ointment. Agents, emulsions, suspensions and the like. For example, for eye diseases, various dosage forms such as eye drops, injections, perfusate, iontophoresis, needleless injection, and administration methods can be used. The delivery substance in the drug delivery system of the present invention can be prepared in a dosage form suitable for its administration method (intraocular administration, etc.) by a widely used formulation. For example, specific preparation examples of injections are described in the Examples section, but the delivery substance produced by the above production method is dissolved in a BSS (Balanced Salt Solution) solution, a glycerin solution, a hyaluronic acid solution, or the like. A stabilizer, an isotonic agent, a buffering agent, a pH adjuster, a preservative, etc. can be added as needed.

  Examples of the stabilizer include edetic acid and sodium edetate. Examples of the isotonic agent include glycerin, propylene glycol, polyethylene glycol, sodium chloride, potassium chloride, sorbitol, mannitol and the like. Examples of the buffer include citric acid, boric acid, sodium hydrogen phosphate, glacial acetic acid, trometamol, epsilon-aminocaproic acid, and the like. Examples of the pH regulator include hydrochloric acid, citric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate and the like. Examples of the preservative include sorbic acid, potassium sorbate, benzalkonium chloride, benzethonium chloride, paraoxybenzoic acid ester, sodium benzoate, dibutylhydroxytoluene, chlorobutanol, chlorhexidine gluconate and the like.

  The present invention will be described below with reference to examples, but these examples are intended to help understanding of the present invention and do not limit the scope of the invention.

a. Production of Deliverable Substance An example of producing a deliverable substance that can be used in the drug delivery system of the present invention is shown below.

Example 1
(1) Deliverable substance A:
i) one in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 5000) is substituted with thioureidoethyl and the other hydrogen atom is substituted with carbonylethyl;
ii) fluorescein and
iii) Covalently bonded L-α-distearoylphosphatidylethanolamine [Chemical Formula 6]
An active ester in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 5000) is substituted with fluoresceinylthioureidoethyl and the other hydrogen atom is substituted with succinimidyloxycarbonylethyl [Fluor-NHS- 5k: manufactured by NOF Corporation (0.20 g, about 40 μmol) and L-α-distearoylphosphatidylethanolamine (61 mg, 82 μmol), methylene chloride (10 ml), chloroform (5 ml), triethylamine (25 μl, 0.18 mmol) After stirring overnight at room temperature, tosylic acid (40 mg, 0.21 mmol) was added and concentrated under reduced pressure. 2-Propanol (5 ml) was added, and the mixture was stirred at room temperature for 30 minutes. The crystals were collected by filtration, methanol (10 ml) was added to the crystals, and insolubles were removed by filtration. The filtrate was concentrated under reduced pressure, and 2-propanol was added to the residue and collected by filtration to obtain 151 mg of the delivery substance A as orange crystals.

mp: 56.5-64.5 ° C
IR (KBr, cm ⁻¹ ): 2886, 1741, 1611, 1468, 1344

(2) Deliverable substance B:
i) one in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 5000) is substituted with thioureidoethyl and the other hydrogen atom is substituted with carbonylethyl;
ii) fluorescein and
iii) Covalently bonded L-α-dioleoylphosphatidylethanolamine mp: 49.0-51.0 ° C
IR (KBr, cm ⁻¹ ): 2889, 1741, 1613, 1468, 1344
(3) Deliverable substance C:
i) one in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 1000) is substituted with thioureidoethyl and the other hydrogen atom is substituted with carbonylethyl;
ii) fluorescein and
iii) Covalently bonded L-α-distearoylphosphatidylethanolamine mp: 55.0-65.0 ° C
IR (KBr, cm ⁻¹ ): 3313, 2917, 2850, 1748, 1617, 1540, 1468, 1349
(4) Deliverable substance D:
i) one in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 10,000) is substituted with thioureidoethyl and the other hydrogen atom is substituted with carbonylethyl;
ii) fluorescein and
iii) Covalently bonded L-α-distearoylphosphatidylethanolamine mp: 55.0-60.0 ° C
IR (KBr, cm ⁻¹ ): 2885, 1745, 1614, 1468, 1343
(5) Deliverable substance E:
i) one in which one hydrogen atom of a terminal alcohol of polyethylene glycol (molecular weight 5000) is substituted with thioureidoethyl and the other hydrogen atom is substituted with carbonylethyl;
ii) fluorescein and
iii) Covalently bonded L-α-dimyristoylphosphatidylethanolamine mp: 65.0-75.0 ° C
IR (KBr, cm ⁻¹ ): 2886, 1774, 1618, 1467, 1344
Example 2
Deliverable substance F:
i) one in which hydrogen atoms of both terminal alcohols of polyethylene glycol (molecular weight 5000) are substituted with carbonylethyl,
ii) (±) -3,4-dihydro-2- [5-methoxy-2- [3- [2- (3,4-methylenedioxy) phenoxyethylamino] propoxy] phenyl] -4-methyl-3 -Oxo-2H-1,4-benzothiazine and
iii) Covalently bonded L-α-distearoylphosphatidylethanolamine [Chemical Formula 7]
(±) -3,4-dihydro-2- [5-methoxy-2- [3- [2- (3,4-methylenedioxy) phenoxyethylamino] propoxy] phenyl] -4-methyl-3-oxo -2H-1,4-benzothiazine monooxalate [This compound is disclosed in Japanese Patent Application Laid-Open No. 62-123181. ] (54 mg, 88 μmol) was added chloroform (5 ml) and stirred at room temperature. Triethylamine (0.04 ml, 0.3 mmol), then one hydrogen atom of the terminal alcohol of polyethylene glycol (molecular weight 5000) is replaced with L-α-distearoylphosphatidyloxyethylaminocarbonylethyl and the other hydrogen atom is succin An active ester substituted with nimidyloxycarbonylethyl [DSPE-NHS-5000: manufactured by NOF Corporation] (0.30 g, about 50 μmol) was added. One hour later, paratoluenesulfonic acid monohydrate (0.20 g, 1.1 mmol) was added, and the mixture was concentrated under reduced pressure. 2-Propanol (20 ml) was added, and the mixture was stirred at room temperature for 15 minutes. The insoluble material was collected by filtration to obtain 0.28 g of the delivery substance F as colorless crystals.

mp: 51.7-56.1 ℃
IR (KBr, cm ⁻¹ ): 2887, 1742, 1467, 1113

Example 3
Deliverable substance G:
i) one in which hydrogen atoms of both terminal alcohols of polyethylene glycol (molecular weight 5000) are substituted with carbonylethyl,
ii) [5R, 10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cycloheptene-5,10-imine [Dizocilpine] and
iii) L-α-distearoylphosphatidylethanolamine covalently bonded [Chemical Formula 8]
Under a nitrogen atmosphere, [5R, 10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cycloheptene-5,10-imine [Dizocilpine] maleate (0.12 g, 0.36 mmol) was added methylene chloride (6.4 ml) and stirred at room temperature. Triethylamine (0.18 ml, 1.3 mmol), then one hydrogen atom of the terminal alcohol of polyethylene glycol (molecular weight 5000) is replaced with L-α-distearoylphosphatidyloxyethylaminocarbonylethyl and the other hydrogen atom is succin The active ester substituted with nimidyloxycarbonylethyl [DSPE-NHS-5000: manufactured by NOF Corporation] (1.9 g, about 0.32 mmol) was added and stirred overnight. The reaction solution was concentrated under reduced pressure, 0.1N hydrochloric acid (100 ml) was added, the mixture was extracted 3 times with chloroform (100 ml), and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was separated and purified by silica gel column chromatography, and the resulting crystals were collected by filtration with diethyl ether to give 0.27 g of delivery substance G as colorless crystals.

mp: 52.4-56.9 ° C
IR (KBr, cm ⁻¹ ): 3434, 2885, 1742, 1715, 1467, 1344, 1149, 1120

b. Formulation example (injection)
10 mL of a sterilized 2.6% glycerin solution was added to 30 mg of the delivery substance G, and this liquid was heated to 60 ° C. with stirring to prepare an injection solution in which the delivery substance G was dissolved. A desired injection solution can be obtained by appropriately changing the kind of the delivery substance of the present invention and the mixing ratio of additives.

c. Intraocular retention test by fluorophotometry The intraocular retention test was performed by the following method using the delivery substances A and B containing fluorescent dyes.

Delivery material preparation:
10 mL of a sterilized 2.6% glycerin solution was added to each 36 mg of deliverable substances A and B, and this liquid was heated to 60 ° C. with stirring to prepare an injection solution by dissolving the deliverable substances A and B. For comparison, fluorescein sodium was used in place of the delivery substances A and B, and the same operation as described above was performed to prepare a 10 μg / mL injection solution of sodium fluorescein.

Administration method and measurement method:
1) A 7: 3 mixed solution of a ketamine hydrochloride aqueous solution (50 mg / mL) and a xylazine hydrochloride aqueous solution (50 mg / mL) was intramuscularly administered to a white rabbit and anesthetized.

  2) Both eyes were instilled with tropicamide (0.5%) / phenylephrine hydrochloride (0.5%) ophthalmic solution to make mydriasis.

  3) The eyes were anesthetized with oxybuprocaine hydrochloride (0.5%) ophthalmic solution in both eyes.

  4) Each injection solution was administered to the central part of the vitreous body using a 30G needle syringe from the flat part of the eye ciliary body.

  5) Immediately after intravitreal administration, 1, 4, 7, 15, 35 and 56 days after administration, the intraocular fluorescence intensity was measured over time using a fluorophotometer, and a calibration curve was prepared. The concentration transition in the retina was obtained, and the half-life of each was calculated. Before measuring the intraocular fluorescence intensity, the above operations 1) and 2) were performed.

result:
The intravitreal half-lives of deliverable substances A and B and sodium fluorescein are shown in Table 1, and the half-lives in these retinas are shown in Table 2. In addition, the numerical value in Table 1 and Table 2 shows the average value of each 3 examples.

(The values in the table are values calculated using the method of moments from data for 1 to 35 days after vitreous injection.)

(The values in the table are the values calculated using the method of moments for 1 to 56 days after injection of the vitreous.)
Discussion:
As is apparent from Table 1, the intravitreal half-lives of deliverable substances A and B of the present invention are 5.0-7.0 days, whereas sodium fluorescein injection is less than 5 hours. Therefore, the delivery substance of the present invention significantly extends the residence time in the vitreous. Further, according to Table 2, since the half-life in the retina of the delivery substance of the present invention is 16.5 to 19.5 days, the injection solution of sodium fluorescein is only less than 2.5 hours. It can be seen that the delivery substance of the present invention administered into the body moves to the retina and stays in the retina for a long time.

d. Intraocular retention test using radioactive isotopes In order to investigate the retention effect of the delivery substance G in the internal eye (vitreous body, retina, optic nerve, etc.), the intraocular retention test using radioactive isotopes by the following method Went.

Chemical preparation:
9 mg of the delivery substance G was weighed and dissolved in a 2.6% glycerin solution in a 5 mL volumetric flask to make a total volume of 5 mL. In another test tube, a compound obtained by replacing the delivery substance G with tritium [ ³ H] (hereinafter referred to as “delivery substance G [ ³ H]”) 37 MBq / mL in toluene / ethanol solution (1: 1) 200 μL And toluene / ethanol was distilled off under a nitrogen stream. To this test tube, the previously prepared 5 mL solution of the delivery substance G was added and stirred to prepare an administration solution. On the other hand, [5R, 10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cycloheptene-5,10-imine [Dizocilpine] maleate (hereinafter referred to as “Comparative Substance X”) 0.96 mg was weighed and dissolved in a 2.6% glycerol solution in a 10 mL volumetric flask to make a total volume of 10 mL. In another test tube, add 400 μL of a 37 MBq / mL ethanol solution of the compound (hereinafter referred to as “Comparative Substance X [ ³ H]”) obtained by substituting Comparative Substance X with tritium [ ³ H], and add ethanol under a nitrogen stream. Distilled off. To this test tube, 10 mL of the previously prepared solution of Comparative Substance X was added and stirred to give a dosing solution. In the preparation, all sterilized instruments were used.

Intravitreal injection:
Japanese white rabbits were anesthetized by intramuscular injection of a 7: 3 mixed solution of ketamine hydrochloride and xylazine hydrochloride at a rate of 1 mL / kg. Next, the surface of the eye was anesthetized with oxybuprocaine hydrochloride (0.5%) ophthalmic solution, and then each test substance administration solution (100 μL / eye) was injected into the vitreous using a 30G needle. The injection was performed with a stopper attached to the needle so as not to pierce the retina. Table 3 shows the concentration and dosage of each administration solution.

Sample collection:
On the predetermined day after administration, 5 mL of a pentobarbital sodium aqueous solution (50 mg / mL) was administered to the ear vein of a Japanese white rabbit to kill anesthesia. After the eyeball was washed with about 10 mL of raw food, scissors were added to the eyes or the corners of the eyes to cut the periphery of the eyeball, and the eyeball was removed. The eyeball was washed twice with physiological saline, and excess water was wiped off with paper. After removing the eyeball conjunctiva, about 0.2 mL of aqueous humor was collected using a 1 mL syringe. Next, the eyeball is immersed in liquid nitrogen and frozen, and divided into two parts from the equator with a razor, and the vitreous, crystalline lens, iris / ciliary body, and cornea from the front, and the vitreous, choroid, and optic nerve from the rear. .

  Preparation of sample for measurement: Wet weight of the collected vitreous body, retina choroid and optic nerve was measured. After the measurement, the tissue was dissolved using a tissue solubilizer, and then a liquid scintillator was added.

Standard radioactivity sample preparation:
The delivery substance G [ ³ H] administration liquid and the comparative substance X [ ³ H] administration liquid were each diluted 1000 times and used as standard radioactivity samples.

薬物動態パラメーターの算出：
硝子体内に注入後１〜２１日間の眼内組織における各被験物質濃度推移から、モーメント法により消失半減期を算出した。 Quantitation method:
The radioactivity concentration of the prepared measurement sample and standard radioactivity sample was measured with a liquid scintillation counter. The radioactivity A (dpm / pmol) per ng of the test substance was determined from the radioactivity of each standard radioactivity sample, and the radioactivity concentration in each tissue was calculated from the following formula.

Calculation of pharmacokinetic parameters:
The elimination half-life was calculated by the moment method from the change in the concentration of each test substance in the intraocular tissue for 1 to 21 days after injection into the vitreous.

result:
The respective concentration transitions in the vitreous body, retina choroid, and optic nerve after intravitreal injection of the delivery substance G and the comparative substance X are shown in FIG. 1, FIG. 2, and FIG. 3, respectively. Tables 4 and 5 show the half-lives of the delivery substance G and the comparative substance X in the vitreous body and retina, respectively. In addition, the numerical value in Table 4 and Table 5 shows the average value of each 3 examples.

Discussion:
As is apparent from FIGS. 1 to 3, when the delivery substance G is administered to the vitreous body, the delivery substance migrates to the posterior eye part such as the vitreous body, the retina choroid, and the optic nerve and is high for a long period of time. Residual concentration. From Tables 4 and 5, the half-life of the delivery substance is extended by about 6 to 10 times that of the comparison substance X.

It is a figure which shows a time-dependent density | concentration change (21 days) in a vitreous tissue. It is a figure which shows a time-dependent density | concentration change (21 days) in a retina choroid tissue. It is a figure which shows a time-dependent density | concentration change (21 days) in an optic nerve tissue.

Claims (4)

A therapeutic, prophylactic or test agent for eye diseases comprising a delivery substance represented by the following general formula [1] wherein a polyalkylene glycol, a phospholipid and a drug are covalently bonded.

(In the formula, A and B are the same or different and are residues of the drug, X is an aminoalkyl group, a carboxyalkyl group, a mercaptoalkyl group, a hydrazide alkyl group at one or both ends of polyalkylene glycol or polyalkylene glycol) , A maleimidoalkyl group, a sulfonylalkyl group, a vinylsulfonylalkyl group or a vinylcarbonyl group introduced, Y is a phospholipid residue, m is 0 or an integer of 1 or more, and n is 0 or 1. (At least one of m and n is not 0, and A, B, X and Y are all covalently bonded.) The therapeutic agent, prophylactic agent or test agent according to claim 1, wherein the molecular weight of the polyalkylene glycol is 500 to 200,000. The therapeutic agent, preventive agent or test agent according to claim 1, wherein the eye disease is a retina, optic nerve or vitreous disease. Drug is anti-inflammatory, immunosuppressive, antiviral, antibacterial, antifungal, antitumor, neuroprotective, blood flow improving, antiglaucoma, analgesic, anesthetic, angiogenesis inhibitor or The therapeutic agent, prophylactic agent or test agent according to claim 1, which is a test agent.

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