JP2003171315A - Injection agent containing medicine-polyethylene glycol combination for ocular tissue injection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare an injection which enables the effective stay of a medicine in an ocular tissue over a long period. <P>SOLUTION: When the injection containing a medicine-PEG combination is injected into the ocular tissue, the medicine stays in the ocular tissue such as margo ciliaris iridis, vitreous body, retina, or optic nerve, whereby the efficacy of the medicine can be sustained over a long period by one administration. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an ocular tissue injecting agent containing a drug-polyethylene glycol conjugate, which retains in ocular tissue for a long period of time.

[0002]

2. Description of the Related Art Many diseases in the eye tissues such as the iris ciliary body, retina, optic nerve, and vitreous body are intractable diseases, and development of effective treatment methods therefor is desired. For eye diseases, it is most common to administer the drug by instillation, but transfer of the drug is difficult depending on the eye tissue, and most of the drug is applied to the inner eye part such as the vitreous body and retina. Do not move. This makes the treatment of diseases in the inner eye more difficult. Further, it is difficult to obtain the drug sustainability by eye drop administration, and frequent administration is required.

Therefore, a method of directly administering a drug into an ocular tissue has been tried, and for example, a technique of administering a drug-containing liposome or microsphere to the inner ocular part of the vitreous body has been reported (patented). References 1 and 2, etc.).

However, it is not easy to control the drug release using liposomes, and because liposomes and microspheres have a large particle size, for example, when they are administered to the inner ocular part of the vitreous body, etc. Can lose transparency.

On the other hand, drugs and polyethylene glycol (P
It is known that the use of a conjugate in which (EG) is covalently bonded improves the retention of the drug in the body, and the drug-PEG conjugate is useful as a drug delivery system. Various specific drug-PEG conjugates have been synthesized, and insulin-PEG conjugates (see Patent Literature 3), taxol-PEG conjugates (see Patent Literature 4), interferon-PEG conjugates (see Patent Literature 5). , Asparaginase-PEG conjugate (see Patent Document 6), urate oxidase-PEG conjugate (see Patent Document 7) and the like are known. However, none of these are intended for use in the ophthalmic area.

Regarding the application of the drug-PEG conjugate in the ophthalmological field, it has been reported that the scleral permeability of the hydrocortisone-PEG conjugate is improved as compared with hydrocortisone alone (see Non-Patent Document 1). In addition, it has been reported that the superoxide dismutase (SOD) -PEG conjugate is intravenously administered to rats to improve the retention of SOD and suppress edema of the retina due to ischemia (see Non-Patent Document 2). ).

However, there has been no report on a technique for injecting a drug-PEG conjugate into an ocular tissue, and of course, the effect of the intraocular infusion has not been known at all.

[0008]

[Patent Document 1] Japanese Patent Publication No. 6-508369.

[0009]

[Patent Document 2] Japanese Patent Laid-Open No. 4-221322.

[0010]

[Patent Document 3] US Pat. No. 4,179,337.

[0011]

[Patent Document 4] International Publication WO93 / 24476 pamphlet.

[0012]

[Patent Document 5] International Publication WO99 / 48535 pamphlet.

[0013]

[Patent Document 6] Pamphlet of International Publication WO99 / 39732.

[0014]

[Patent Document 7] International publication WO00 / 7629 pamphlet.

[0015]

[Non-Patent Document 1] Int. J. Pharm., 182 (1), 79-92, 1999).

[0016]

[Non-Patent Document 2] Invest.Ophthalmol.Vis.Sci., 32, 1471
-1478, 1991).

[0017]

[Problems to be Solved by the Invention]
Although PEG conjugates are known to be useful as drug delivery systems, the conventional methods of intravenous administration, oral administration, and instillation of PEG conjugates treat various ocular tissues, especially internal ocular diseases. Has many problems. For example, an intravenously administered drug-PEG conjugate spreads throughout the body via the bloodstream, so the amount of drug that reaches the eye tissue is extremely small compared to the dose. Therefore, a large amount of drug must be administered to reach an effective amount of the drug to the eye tissue, but in that case, side effects on the whole body become a serious problem. Further, since the drug-PEG conjugate administered intravenously has a problem of being metabolized in blood, frequent administration is required during treatment. Oral administration further reduces the amount that reaches the eye tissues due to the addition of metabolic processes in the liver. Even in the case of topical administration by eye drop, the amount of the drug that penetrates the cornea and reaches the inner eye part such as the retina is about 1 / 10,000 of the dose due to the barrier mechanism of the corneal epithelium.

[0018]

Means for Solving the Problems The present inventors have proposed Drug-P.
If the EG conjugate is directly administered into the eye tissue, the iris ciliary body,
It has been found that the drug can directly reach the ocular tissues such as the vitreous body, retina, optic nerve, etc., and also allows the drug to stay in the ocular tissues for a long period of time, which is useful for treating diseases in various ocular tissues. It was When the drug-PEG conjugate is directly administered into the ocular tissue, the conjugate does not enter the systemic circulation, so almost all of the dose is used for the treatment of diseases in the ocular tissue, and there are also systemic side effects. It will be reduced. The drug is administered in a state of being bound to PEG, and the bond is gradually broken after injection into the ocular tissue to control the release of the drug and exert a therapeutic effect on the disease for a long period of time. Also,
The drug-PEG conjugate has excellent retention in the ocular tissue, and even if the drug and PEG bond is not broken in the ocular tissue and the bond is maintained, the drug-PEG conjugate may exert a therapeutic effect on diseases in various ocular tissues. it can. Therefore, the intraocular tissue injection of the present invention is
In particular, it is possible to treat diseases in various ocular tissues, which have been difficult to treat up to now, with a single administration over a long period of time.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is an intraocular tissue injection containing a substance in which a drug and PEG are covalently bound. Drug-P
If EG conjugate is injected into the eye tissue, it will stay in the eye tissue such as iris ciliary body, retina, optic nerve, vitreous body for a long period of time, so it is possible to maintain the drug effect for a long time with a single administration. Becomes

In the present invention, PEG is chain type, star type,
It may be a branched type. Since a drug is generally bound to chain type PEG by utilizing hydroxyl groups at both ends, the binding ratio between the drug and PEG is 1: 1 or 2: 1. Star,
Since a branched PEG has a plurality of hydroxyl groups, it is possible to covalently bond a plurality of drugs. The molecular weight of PEG is not particularly limited and can be appropriately selected in consideration of the type and properties of the drug to be covalently bonded, the period for which the drug is retained, etc., but is usually 300 to 200,000, and more preferably 1000 to 50,000.

By utilizing the hydroxyl group located at the terminal of PEG, PEG can be directly covalently bound to a drug having a functional group such as a carboxyl group. PEG may be previously derivatized into a form having a functional group such as an amino group, a thiol group and a carboxyl group so that a covalent bond with a drug can be easily formed depending on the kind of the drug. That is, depending on the type of drug, the hydroxyl group on the PEG side is used,
After deriving PEG in a form having a functional group such as an amino group, a thiol group and a carboxyl group, a drug can be covalently bonded to the obtained derivative.

Further, both of the hydroxyl groups located at the terminal of PEG may be used for covalent bond formation, but when only one is used, the hydroxyl group not involved in the bond is an alkyl group,
It may be protected by a protecting group such as an acyl group. When PEG is derivatized to have a functional group, both functional groups may be used for covalent bond formation, or only one may be used. .

These bonds are schematically shown below (wherein X represents a drug, R represents a hydrogen atom or an alkyl group, a protecting group such as an acyl group, or a carboxyalkyl group, an aminoalkyl group or the like). Represent each).

-Drug not bound (PEG only):

[Chemical 1]

One drug is bound to the hydroxyl group of PEG:

[Chemical 2]

Two drugs bound to the hydroxyl group of PEG:

[Chemical 3]

・ One drug is bound to the amino derivative of PEG

[Chemical 4]

1 drug in the carboxyl derivative of PEG
United

[Chemical 5]

One drug is bound to the thiol derivative of PEG

[Chemical 6]

A commonly used method may be used to form the above-mentioned covalent bond, for example, a method of esterifying the carboxylic acid derivative of PEG with the hydroxyl group of the drug.

The chemical structure of the drug covalently bonded to PEG is not particularly limited as long as it has a functional group capable of bonding to PEG. Specific examples are drugs having a hydroxy group, a carboxyl group, a carbonyl group, an amino group, an alkenyl group and the like. In order to easily form a covalent bond with PEG, the form having a functional group such as an amino group, a thiol group, a carboxyl group, and an isothiocyanate group can be derived in advance. The type of drug is not particularly limited as long as it has a therapeutic effect or a preventive effect on eye diseases, and examples thereof include an anti-inflammatory drug, an immunosuppressant drug, an antiviral drug, an antibacterial drug, an antifungal drug, and an antitumor drug. , Neuroprotective agents, blood flow improving agents, anti-glaucoma agents, analgesics, anesthetics, angiogenesis inhibitors, test agents and the like. Especially the iris ciliary body, retina,
Diseases of the inner eye such as the optic nerve and the vitreous body include inflammation of the inner eye due to various causes, infections of viruses and bacteria, proliferative vitreoretinopathy accompanied by neovascular and retinal cell proliferation changes, and various Drugs effective for retinal hemorrhage due to the cause, retinal detachment, retinoblastoma, etc. are mentioned. For example, in the case of inflammation associated with internal eye surgery, anti-inflammatory drugs such as betamethasone phosphate, in the case of autoimmune uveitis, immunosuppressive drugs such as cyclosporine, and in the case of viral infection ganciclovir. , Antibacterial agents such as ofloxacin in case of postoperative infection, antitumor agents such as doxorubicin hydrochloride, carmustine in case of proliferative vitreoretinopathy, ophthalmic examination for various tests Drugs are used.

Examples of the method for injecting the drug-PEG conjugate into the ocular tissue include subretinal injection, intravitreal injection, intrascleral injection, intracameral injection, Tenon's capsule injection and the like.

The effects of the present invention will be described in detail in the intraocular kinetic test and pharmacological test described below. Briefly described here, in the intraocular kinetic test, with respect to the fluorescein-PEG conjugate, the retention of the drug-PEG conjugate in the inner ocular region (vitreous and retina) after intravitreal injection was examined.
As a result, it was revealed that the drug for intraocular injection of the present invention allows the drug to stay in the vitreous and retina for a long period of time. Furthermore, in the pharmacological test, the betamethasone-PEG conjugate was injected once intravitreally or subconjunctivally,
We investigated the effect on krypton laser-induced choroidal neovascularization. As a result, it was clarified that choroidal neovascularization was suppressed by the intraocular injectable agent of the present invention, and that the drug-PEG conjugate was useful for the treatment of eye diseases.

From these test results, it is understood that various diseases in the eye can be effectively treated with a small number of administrations by appropriately selecting the drug to be bound with PEG. Further, by using the intraocular tissue injection of the present invention,
Since the drug can be efficiently retained in the intraocular tissues such as the iris ciliary body, retina, optic nerve, and vitreous body, the amount of the drug can be reduced, and the side effect reducing effect can be expected.

Drug-P in the ocular tissue injection of the present invention
The formulation form of the EG conjugate is preferably a liquid formulation. For example, the drug-PEG conjugate can be prepared by dissolving it in a BSS (Balanced Salt Solution) solution, a glycerin solution, a hyaluronic acid solution, or the like, and if necessary, a stabilizer, an isotonicity agent, a buffer, a pH adjustment. Agents, soothing agents, preservatives and the like can be added in appropriate amounts.

Examples of the stabilizer include sodium edetate and the like. As a tonicity agent, glycerin, propylene glycol, polyethylene glycol,
Examples thereof include sodium chloride, potassium chloride, sorbitol, mannitol and the like. Examples of the buffer include citric acid, sodium hydrogen phosphate, glacial acetic acid, trometamol and the like. As a pH adjusting agent, hydrochloric acid,
Examples thereof include citric acid, phosphoric acid, acetic acid, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate and the like.
Examples of soothing agents include benzyl alcohol and the like. Examples of antiseptics include benzalkonium chloride, benzethonium chloride, paraoxybenzoic acid ester, sodium benzoate, chlorobutanol and the like.

[0037]

The present invention will be described below with reference to examples.
These examples are intended to aid the understanding of the present invention and are not intended to limit the scope of the invention.

(Synthesis Example) Synthesis of betamethasone-PEG conjugate In a nitrogen atmosphere, methoxy-PEG-propionic acid [Shea
manufactured by rwater Polymers, average molecular weight of about 5,000] (1.
Methylene chloride (7 mL) was added to 00 g; about 0.20 mmol) and dicyclohexylcarbodiimide (49.2 mg; 0.23 mmol), and the mixture was stirred at 0 ° C. for 20 minutes. Then betamethasone (59.3 mg; 0.15 m
mol) and 4-dimethylaminopyridine (12.6
mg; 0.10 mmol) was added, and the whole was stirred overnight at room temperature. The reaction solution was poured into 30 times the amount of diethyl ether, the precipitate was collected by filtration, and washed with a small amount of cold acetone and diethyl ether.
2 mg was obtained as white crystals. The yield was about 70%.

[0039]

[Chemical 7]

(Intraocular Kinetics Test) Intraocular Kinetics Test by Rabbit Photometry in Rabbit It is difficult in terms of measurement technology to trace a trace amount of tissue migration using the drug-PEG conjugate prepared above. Therefore, a fluorescein-PEG conjugate (hereinafter referred to as FL-PEG) was synthesized using fluorescein, which has fluorescence and can be measured with high sensitivity, as a model drug, and an intraocular dynamic test was conducted. Fluorescein sodium (hereinafter F
L) was used.

Synthesis of FL-PEG: NH under nitrogen atmosphere
2-PEG-propionic acid [Shearwater Polymers] (1.00 g; about 0.20 mmol) and triethylamine (55.6 μL; 0.40 mmol) were dissolved in methanol (100 mL). PEG having an average molecular weight of about 5,000 was used. Then fluorescein isothiocyanate (233 mg; 0.60 mmol)
Was added and the whole was stirred overnight at room temperature. After the reaction solution was evaporated to dryness under reduced pressure, the solid substance was converted into chloroform / methanol (1 /
1, v / v), and unreacted fluorescein isothiocyanate was removed by column chromatography. The fractionated fraction was concentrated, the concentrate was thrown into 30 times the amount of diethyl ether, and the resulting precipitate was collected by filtration and washed with a small amount of diethyl ether.
86 g were obtained as pale yellow crystals.

[0042]

[Chemical 8]

Preparation of drug solution: 18 mg of FL-PEG was added with 10 mL of sterilized 2.6% glycerin solution, and FL-PEG was dissolved while stirring this solution to prepare an injection solution. The same operation was performed to prepare an injection solution of FL at 10 μg / mL.

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 to anesthetize the rabbit.

2) Tropicamide (0.5%) / phenylephrine hydrochloride (0.5%) ophthalmic solution was applied to the eyes to cause dilation.

3) Oxybuprocaine hydrochloride (0.5%)
The eye surface was anesthetized with eye drops.

4) Using a syringe with a 30 G needle from the pars plana of the pars plana, 100 μL of the above FL-PEG or FL drug solution was injected into the center of the vitreous in one eye.

5) Immediately after intravitreal administration, 1, 2 after administration,
After 4, 7, 15, 18, 23, 28, 35, 42, 49 and 56 days using a fluorophotometry device,
Intraocular fluorescence intensity was measured over time, and a calibration curve was created to determine the concentration transitions in the vitreous and retina. Next, each half-life was calculated from the change in concentration by the method of moments. The operation 2) was performed before measuring the intraocular fluorescence intensity.

Results: FIG. 1 shows the transition of the concentration in the vitreous body.
FL was not detected after 2 days after administration,
FL-PEG was detected even on the 56th day, indicating that it was present in the vitreous. FIG. 2 shows changes in the concentration in the retina. FL
Was detected only on the first day after administration, whereas FL-
It can be seen that PEG is detected even on the 56th day, and it is present in the retina.

Table 1 shows the half-lives of FL and FL-PEG in the vitreous and in the retina. The half-life of FL-PEG in the vitreous body was 3.4 days, whereas that of FL was less than 4 hours. Therefore, it can be said that FL-PEG significantly prolongs the retention period in the vitreous body. The half-life of FL-PEG in the retina was 11.0 days.
Since FL was only detected at one time point immediately after administration to the retina, the half-life could not be calculated, but it is clear that it is quite short. Therefore, it can be seen that FL-PEG migrates from the vitreous to the retina and stays in the retina for a long period of time.

[0051]

[Table 1]

(The numerical values in the table indicate the average value of each of the three examples.
The half-life of FL-PEG in the vitreous was measured on days 4 to 28, the half-life of FL-PEG in the retina was measured on days 1 to 56, and the half-life of FL-PEG in the vitreous was measured immediately after administration. And the measurement values on the first day after administration were used for calculation. )

(Pharmacological test) 1. Effect of intravitreal betamethasone-PEG conjugates on krypton laser-induced choroidal neovascularization in rats betamethasone-PEG prepared according to the examples.
The conjugate (hereinafter referred to as BM-PEG) was intravitreally administered to rats to examine the effect on krypton laser-induced choroidal neovascularization.

Preparation of drug solution: To 100 mg of BM-PEG, 1 mL of sterilized physiological saline was added, and BM-PEG was dissolved while stirring this solution to prepare an injection solution for intravitreal administration.

Administration and measurement methods: 1) 1 mL of a 7: 1 mixture of an aqueous solution of ketamine hydrochloride (50 mg / mL) and an aqueous solution of xylazine hydrochloride (50 mg / mL).
General anesthesia was performed by intramuscularly administering to the rat / kg.

2) Photocoagulation was carried out using 0.5% tropicamide-
After dilating with a 0.5% phenylephrine hydrochloride solution, using a krypton laser photocoagulator (red),
The spot size was 100 μm, the output was 100 mW, and the solidification time was 0.1 sec.

3) Using a tissue observation cover glass as a contact lens, eight photocoagulations were performed per eye in a scattered manner in the posterior region of the fundus avoiding thick retinal blood vessels. At this time, photocoagulation was performed by focusing on the deep layer of the retina for the purpose of rupturing Bruch's membrane. After photocoagulation, fundus photography was performed.

4) From the flat portion of the ciliary body of the eye, using a syringe with a 33 G needle, 5 μL of the above BM-PEG or the base material (physiological saline) was injected into the vitreous center of each eye.

5) On the 14th day after photocoagulation, 0.1 mL of 10% fluorescein was injected through the tail vein, and fluorescent fundus imaging was performed.

6) In the fluorescence fundus imaging, the photocoagulation site where no fluorescence leakage was observed was judged to be negative, and the one where obvious fluorescence leakage was recognized was judged to be positive. In addition, the photocoagulation site where some fluorescence leakage was observed was defined as positive when two photocoagulation sites were present. The neovascularization rate was calculated according to the following formula.

[0061]

[Formula 1] New blood vessel expression rate (%) = (number of positive photocoagulation sites /
Total photocoagulation sites) x 100

Results: Table 2 shows the effect of intravitreally administered BM-PEG on choroidal neovascularization. While the incidence of new blood vessels in the base-administered eye was 75.0%,
35.4% of eyes were treated with BM-PEG, and BM-PEG was used.
Intravitreal administration suppressed the expression of new blood vessels.

[0063]

[Table 2]

(Numerical values in the table are base = 7 eyes, BM-PE
G = The average value of 6 eyes is shown. )

2. Subconjunctivally administered betamethasone for krypton laser-induced choroidal neovascularization in rats-
Effect of PEG Conjugate The betamethasone-PEG conjugate (hereinafter referred to as BM-PEG) prepared according to the example was subconjunctivally administered to rats to examine the effect on krypton laser-induced choroidal neovascularization. Betamethasone phosphate (hereinafter referred to as BP) was used as a comparative substance.

Preparation of drug solution: 40 mg of BM-PEG was added with 1 mL of sterilized physiological saline, and B was added to the solution with stirring.
An injection solution for subconjunctival administration was prepared by dissolving M-PEG. By the same operation, an injection solution of BP at 2.8 mg / mL was prepared.

Administration method and measurement method: The administration method and measurement method were carried out by changing the item 4) of the above-mentioned intravitreal administration test as follows.

4) Using a 30 G needle injector, 50 μL of the above BM-PEG or BP was injected subconjunctivally into both eyes.

Results: Table 3 shows the effect of subconjunctivally administered BM-PEG on choroidal neovascularization. The incidence of new blood vessels in the base-administered eye was 71.9%, while B
The number of eyes administered with M-PEG was 35.4%, and the expression of new blood vessels was suppressed by the administration of subconjunctival administration of BM-PEG.
In addition, the expression rate of new blood vessels of BP not bound to PEG was 50.0%, and although BM-P had an inhibitory effect,
The inhibitory effect was weaker than that of EG. From this result, it was shown that the use of the PEG conjugate extended the retention period in the eye tissue and increased the inhibitory effect.

[0070]

[Table 3]

(Numerical values in the table indicate the average value of 8 eyes.)

[0072]

EFFECT OF THE INVENTION By using the injectable agent for intraocular tissue containing the drug-PEG conjugate of the present invention, the drug is retained in the ocular tissue such as iris ciliary body, vitreous body, retina and optic nerve for a long period of time. You can Therefore, the intraocular tissue injection of the present invention makes it possible to treat or prevent diseases in various ocular tissues with a single administration over a long period of time.

[Brief description of drawings]

FIG. 1 is a graph showing changes in concentration in the vitreous body (56 days).

FIG. 2 is a graph showing changes in concentration in the retina (56 days).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yoshihito Horibe             8916-16 Takayama-cho, Ikoma-shi, Nara Santen Pharmaceutical Co., Ltd.             Shikisha Institute (72) Inventor Mitsuaki Kuwano             8916-16 Takayama-cho, Ikoma-shi, Nara Santen Pharmaceutical Co., Ltd.             Shikisha Institute F-term (reference) 4C076 AA12 AA95 BB11 BB24 CC01                       CC04 CC07 CC10 CC11 CC14                       CC27 CC31 CC32 CC35 DD38                       EE59 FF11 FF31 FF68                 4C084 AA17 MA58 MA66 NA10 NA11                       NA12 NA13 ZA331

Claims (4)

[Claims] 1. An intraocular tissue injection containing a drug-polyethylene glycol conjugate. 2. The method for injecting into the eye tissue is subconjunctival injection,
The intraocular injectable composition according to claim 1, which is intravitreal injection, subretinal injection, intrascleral injection, intracameral injection or Tenon's capsule injection. 3. The drug is an anti-inflammatory drug, immunosuppressant drug, antiviral drug, antibacterial drug, antifungal drug, antitumor drug, neuroprotective drug, blood flow improving drug, antiglaucoma drug, analgesic drug, anesthetic drug, The intraocular tissue injection according to claim 1, which is an angiogenesis inhibitor or a test agent. 4. The intraocular tissue injection according to claim 1, wherein the drug is a drug for treating or preventing eye diseases.