KR20150110974A - Dendrimer-based retinal drug delivery systems with facile retinal penetration properties by adjusting their surface functionalities - Google Patents

Abstract

The present invention relates to a dendrimer-based retinal disease treatment medicine carrier which can easily penetrate to the choroid of retinal layers by using an intravitreal injection method or an eye drop method. According to the present invention, the retinal disease treatment medicine carrier modifies a terminal functional group of a core to a surface functional group for enhancing the ocular penetration performance. Accordingly, the carrier can effectively pass through the outer layer of an eye such as the cornea or sclera by being dropped or injected into the eye in order to penetrate to choroid of the retinal layers in the vitreous cavity as well as the vitreous cavity itself, thereby being useful as a carrier for the retinal disease treatment medicine.

Description

[0002] Dendrimer-based retinal drug delivery systems for treating retinal diseases are well known in the art,

The present invention relates to a dendrimer-based drug delivery system for treating retinopathy which is easy to penetrate into the choroid of retinal layers by an intravitreal injection method or an eye drop method.

The nerve tissue located in the center of the inner retina of the eye is called the macula. Most of the photoreactive cells responding to the light stimuli are gathered here, and the phase of the object is also the center of the macula, and plays a very important role in sight. Age-related macular degeneration (AMD) is a chronic disease characterized by macular degeneration of retinal pigment epithelium, Bruch's membrane, and choroidal capillaries. Anatomically, the sensory retina is located in the anterior part of the retinal pigment epithelium, and the treatment of nutrition, support, recirculation and waste products depends on the retinal pigment epithelium. Bruch's membrane is a five-layered structure that is interposed between the choroid and the retinal pigment epithelium. The innermost layer is the basement membrane of the retinal pigment epithelium and the outermost layer is the basement membrane of the choroidal capillary endothelial cells. In other words, it is a metamorphosis of the retinal pigment epithelium, Bruch's membrane and choroidal capillary complex.

This disease occurs mainly in the age group of 50 years or older. In the West, it is the main cause of blindness in the population over 60 years of age and it is increasing in Korea. The cause of old age-related macular degeneration has not yet been elucidated. However, it is known that risk factors are aging (especially after 75 years of age), high-blood pressure, obesity, genetic predisposition, excessive UV exposure , Low serum antioxidant concentration, and the like.

There are two types of macular degeneration: dry (non-exudative) macular degeneration and habitual (exudative) macular degeneration. Dry AMD, nonexudative AMD, and nonneovascular AMD accumulate yellow deposits called drusen underneath the retina, which build up to accumulate and block blood flow to the retina, especially the macula, The failure starts to come. Dry AMD does not cause rapid visual loss, but it can progress to hatching macular degeneration. Under the retina, vascular aggregation (wet AMD, exudative AMD, neovascular AMD) in the fibrous tissue is caused by the neovascularization of the choroid below the retina. These weak neovasions are bleeding and bleeding and exudation, causing macular degeneration in the retina, resulting in visual disturbances. It is known that macular degeneration progresses very quickly, so the visual acuity rapidly deteriorates within weeks and blindness may occur between two months and three years.

In order to treat the above-mentioned diseases, drugs have to reach choroids in retinal layers, and accordingly, various drug delivery systems have been studied.

In Non-Patent Document 1, experiments were conducted to inject dispersion of polymeric nanoparticles having different surface properties into a glass body to measure the degree of dispersion according to the surface properties.

Specifically, positively charged polymer nanoparticles have strong interaction with negatively charged crystalline collagen fibers, and because of the anti-fouling effect, they easily pass through the lens wall and reach the inner-limiting layer But does not reach the retinal structure through the physical pores of the inner confinement layer.

In addition, in the case of the polymer nanoparticles having negative charge, it was confirmed that the polymer nanoparticles reach the RPE (retinal pigment epithelium) layer through all the retinal layers unlike the positively charged polymer nanoparticles. This is due to the interaction with Muller cells, which is an important mechanism for reaching internal retinal structures.

Non-Patent Document 2 discloses a drug delivery system for delivering an integrin-antagonist peptide to the inner retina surface, and encapsulates an aqueous soluble internal integrin-antagonist peptide Although nanoparticles have been used to examine the inhibitory effect of CNV, the cause of AMD, it has not been able to achieve a sustained therapeutic effect due to its short life span, thus describing the importance of a sustained release drug delivery system .

Photodynamic therapy (PDT) and anti-VEGF (anti-VEGF) injection have been used for the treatment of macular degeneration. Photodynamic therapy is a selective treatment by injecting a special laser that responds only to photosensitized substances when a photosensitizer reaches the neovascularization of the retina after a period of time following injection of the photosensitizer, Visudyne, through the blood vessels. It is a way to destroy new blood vessels. However, there are many cases of recurrence even after treatment, so it is often necessary to repeat treatment, and repeated treatment may cause damage to the retina itself.

Antibody injection therapy is an intravitreal injection of an antibody (anti-VEGF) that inhibits the production and proliferation of new blood vessels by selective binding to VEGF, which is important for the formation and progression of new blood vessels injection.

Lucentis and Avastin are used in antibody injections. Lucentis is a drug approved by the FDA for treatment of macular degeneration. Avastin is approved for use in the treatment of ocular diseases I am using it.

The protein antibody injection therapy is costly, and it is impossible to administer eye drops. Therefore, it is necessary to inject the protein antibody directly into the eye and periodically administer it once a month to prevent bleeding, pain, infection, retinal detachment There is a danger.

To address these drawbacks, one of the proposed methods is a small molecule drug based on a low molecular weight substance. Among the low-molecular-based macular degeneration drugs known to date, few drugs reach the choroid within the retina, There are no drugs approved by the KFDA as a treatment.

Therefore, there is a need to develop a drug delivery system that helps infiltrate small molecule drugs or antibody therapeutic agents into the choroid within the retina by instillation of a low molecular substance.

Accordingly, the present inventors have studied intravitreal injection methods or delivery vehicles for drugs for treatment of retinal diseases that can be administered by eye drops. As a result of modifying the terminal functional group of dendrimer-based central nucleus to a surface functional group for improving ocular penetration, It is not only possible to penetrate the outer layer of the eye such as cornea or sclera directly into the vitreous cavity by injection or directly into the vitreous cavity as well as to penetrate the choroid of the retinal layers choroid), and completed the present invention.

1. H. Koo et al. / Biomaterials 33 (2012) 3485-3493. 2. Journal of controlled release 142 (2010) 286-293.

It is an object of the present invention to provide a delivery vehicle for a drug for treating retinal diseases in which penetration into the choroid of the retinal layers is easy.

In order to achieve the above object,

The present invention relates to a dendrimer-based core; And

And a surface functional group which is introduced into the terminal functional group of the core to improve the ocular penetration ability.

The carrier of the medicament for treating retinopathy according to the present invention is modified into a surface functional group which enhances the ocular penetration ability of the core of the medicament. Therefore, the carrier of the present invention can be used as a cornea or a sclera ) Penetrate into the vitreous cavity of the eye as well as penetrate into the choroid of the retinal layers within the vitreous body. Thus, the drug for the treatment of retinal diseases May be useful as a delivery vehicle.

FIG. 1 shows a structure of dendrimer conjugates 1a - 1h , 12c , and 12f whose surface is modified with different functional groups and fluorescent dyes are labeled according to an embodiment of the present invention.
FIG. 2 is a graph showing cytotoxicity test results according to the concentrations of dendrimer conjugates 1a, 1b, 1c, 1d, 1e, 1f, and 1h according to an embodiment of the present invention (A: Time culture).
FIG. 3 is a graph showing the results of cryosectioned eyeballs extracted at 24 hours after administration of dendrimer conjugates 1c, 12c, and 12f according to an embodiment of the present invention to the eye of mice by intravitreal injection Eyes were observed with a confocal fluorescence laser scanning microscope.
FIG. 4 is a graph showing the results of cryosectioned eyes (eyes) taken at 24 hours after administration of dendrimer conjugates 1c and 1f according to an embodiment of the present invention to eyes of a mouse by intravitreal injection or eye dropping ) Was observed with a confocal fluorescence laser scanning microscope.
3 and 4, the IPL is an inner plexiform layer; INL is the inner nuclear layer; OPL is an outer plexiform layer; ONL is the outer nuclear layer; RPE is the retinal pigment epithelium.
5 is a view showing an anatomical structure of the eyeball.

Hereinafter, the present invention will be described in detail.

The present invention relates to a dendrimer-based core; And

And a surface functional group which is introduced into the terminal functional group of the core to improve the ocular penetration ability.

In the delivery of a drug for treating a retinal disease according to the present invention, the dendrimer-based core is preferably biodegradable, but not limited thereto.

Examples of the dendrimer-based core include polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer , Polyglycerol dendrimer, and polymelamine dendrimer. Of these, dendrimers comprising two or more copolymers may be used.

In the delivery vehicle for a drug for treating retinopathy according to the present invention, the surface functional group may be formed by effectively passing through the outer layer of the eye such as a cornea or a sclera by the delivery of the present invention, (see FIG. 5), as well as penetrating into the vitreous cavity, as well as penetrating the choroid of the retinal layers within the vitreous body.

The ends of the surface functional group is _{-OH, -OR, -NH 2, -NR} 2, -NHC (= O) CH 3, -NHC (= O) CR 3, -SH, -SR, -C (= O ) OH, -C (= O) OR, -C (= O) R, -C (= O) NR 2, -NHC (= O) NR 2, -C (= S) NR 2, -NHC (= _{S) NR 2, - (OCH} 2 CH 2) n OCH 3, - (OCH 2 CH 2) n oR, can be used, such as glucosamine, wherein wherein R is H, or C _{1 -6} straight or branched chain alkyl of , and n is an integer of 1-100.

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등을 사용할 수 있다. 여기서, 표면작용기 중 글루코사민기는 염증 치료에 효과가 있는 것으로 잘 알려져 있으므로, 본 발명의 전달체 자체만으로도 망막의 염증 치료제로 사용할 수 있다.
Specifically, the above-mentioned surface functional group introduced into the terminal functional group of the core

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Etc. may be used. Herein, since the glucosamine group among the surface functional groups is well known to be effective for the treatment of inflammation, the carrier of the present invention itself can be used as a therapeutic agent for inflammation of the retina.

As described above, the carrier of the drug for treating retinopathy according to the present invention can be prepared by introducing a surface functional group that improves the ocular penetration by modifying the terminal functional group of the dendrimer-based core, .

First administration method

The carrier of the drug for treating retinal diseases according to the present invention may be administered by intravitreal injection.

There are few drugs that can penetrate into the choroid of the retinal layers among known drugs for the treatment of the retinal diseases so far. It is known that only a few are used as drugs for the treatment of retinal diseases.

The carrier of the drug for treating a retinal disease according to the present invention easily penetrates from the vitreous cavity to the choroid of the retinal layers when administered by the intravitreal injection method, May be useful for treating retinal disease (see Experimental Example 1 and Fig. 3-4).

Second administration method

The carrier of the drug for treating retinal disease according to the present invention can be administered by an eye drop method.

It is known that there are few drugs that can effectively pass through the outer layer of the eye, such as cornea or sclera, among known drugs for treating retinal diseases.

The delivery vehicle for a drug for the treatment of retinal diseases according to the present invention is a drug delivery system that effectively penetrates the outer layer of the eye such as a cornea or a sclera to penetrate into the vitreous cavity, , It is easy to penetrate into the choroid of the retinal layers within the vitreous body. Therefore, when a drug for treating retinal diseases is introduced into the carrier of the present invention, it can be useful for treating retinal diseases (Experimental Examples 1 and 2) 3-4).

On the other hand, in the case of age-related macular degeneration (AMD), it is well known that drugs can penetrate into the choroid of the retinal layers, The drug delivery system may be particularly useful for age-related macular degeneration.

In the delivery agent for a drug for treating retinopathy according to the present invention, the drug may be introduced into any one or two or more parts of the dendrimer-based core, the terminal functional group and the surface functional group of the core.

Specifically, as a method for introducing a drug into the core, it is preferable that a drug is introduced into the core by a covalent bond through two or more functional groups in one drug unit during or after the synthesis of the dendrimer, The drug can be introduced by substitution.

Also, as a method of introducing a drug into the terminal functional group of the core, the drug can be introduced into the terminal functional group of the core before the modification of the terminal functional group of the core, as a generally known organic chemical reaction.

Furthermore, as a method for introducing a drug into the surface functional group of the core, the drug can be introduced into a generally known organic chemical reaction on the surface functional group obtained by modifying the terminal functional group of the core.

In the carrier of the drug for treating retinopathy according to the present invention, the drug is controlled to be released to the sustained release upon administration to the eye, or the drug introduced into the surface of the central nucleus without releasing the drug, May be combined in a multivalent manner to express the drug effect by a mechanism such as signal transduction.

Specifically, when the drug is controlled to release slowly in the carrier according to the present invention, the drug introduced into the terminal functional group or the surface functional group of the central nucleus is slowly released from the core by a chemical reaction that may occur in the living body environment such as hydrolysis And when the drug is introduced into the inner core of the core or introduced into the inner core of the core by covalent bonding through two or more functional groups in one drug unit, the core is synthesized as a biodegradable dendrimer So that the drug is released while the core is slowly degraded in the in vivo environment.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

≪ Example 1 > Preparation of dendrimer conjugate (1a)

G3 polyamidoamine (PAMAM) dendrimer whose core is ethylenediamine and whose terminal functional group is amine (purchased from Dendritech, 2 , About 92.3 [mu] L) was dried under vacuum for 11 hours to remove methanol. The compound 2 (15.7 mg, 2.28 μmol) of dimethyl sulfoxide (DMSO) (1.53 mL) 5 -carboxytetramethylrhodamine N -hydroxy succinimidyl (5-TAMRA NHS) was dissolved, was dissolved in DMSO- d ₆ in the stirred solution to the ester ( N , N -diisopropylethylamine (DIEA, 0.60 μL, 3.44 μmol) was added slowly after the addition of 3 , 99% purity, 122 μL, 1.15 μmol, and 9.44 mM. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, acetic anhydride (Ac ₂ O, 4 ) solution (130 μL, 41.3 μmol, 0.318 M) dissolved in DMSO- d ₆ was slowly added to the stirring solution. The reaction was stirred at room temperature for 68 hours while the light was blocked. Short size exclusion chromatography of the crude product in a light shielding state (size-exclusion chromatography, SEC) column ^{(Model: Bio-Beads ® S-X1} , H 4 cm × OD 0.7 cm, Manufacturer: Bio-Rad) As the first filtration. Next, the crude product SEC column (model ^{name: Bio-Beads ® S-X1} , H 38.5 cm × OD 4.5 cm, Manufacturer: Bio-Rad) with N, N - purified in dimethyl formamide (DMF). The purified products were combined and filtered through a short SEC column (Sephadex LH-20, H 5 cm x OD 0.7 cm) using methanol to remove residual DMF and further eluted with a short SEC column Sephadex G-25, H 4.3 cm x OD 0.7 cm). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufactured by Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 18.6 mg of the desired compound 1a .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 7.98-7.82 (m, 80.46H, NH G0, NH G1, NH G2, NH G3, and NH Ac), 6.52, 6.42 (m, 2.24H, H A _{, H C, and H d)} , 3.07 (s, 173.43H, H d, H f, H fAc, HgAc, H fTAMRA, and H gTAMRA), 2.94, 2.91 (m, 6.85H, H B), 2.64- _{2.54 (m, 132.62H, H b} and H g), 2.42 (s, 59.82H, H e and H a), 2.19-2.18 (m, J = 5.00 Hz, 120H, H c), 1.79 (s, 58.28 _H, H _h);

MS (MALDI-TOF, THAP matrix) M _n 7473.19, M _w 7631.77, PDI 1.02.

≪ Example 2 > Preparation of dendrimer conjugate (1b)

G3 PAMAM Dendrimer 2 (about 93.0 [mu] L) was dried under vacuum for 11 hours to remove methanol. 5-TAMRA NHS ester 3 (121 μL, 1.14 μmol, 9.44 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (15.7 mg, 2.27 μmol) in DMSO (1.52 mL) DIEA (0.60 [mu] L, 3.44 [mu] mol) was added. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, Ac ₂ O 4 solution (680 μL, 216 μmol, 0.318 M) dissolved in DMSO- d ₆ was slowly added to the stirred solution. The reaction was stirred at room temperature for 68 hours while the light was blocked. In the light shielding state to the crude product short SEC column was first filtered with (model: Bio-Rad: Bio-Beads ® S-X1, H 7.5 cm × OD 0.7 cm, Manufacturer). It was purified from the DMF Next, the crude product SEC column (Bio-Rad Model:: Bio-Beads S-X1 ^®, H 38 cm × 4.5 cm OD, manufacturer). The purified products were combined and filtered through a short SEC column (Sephadex LH-20, H 4 cm x OD 0.7 cm) using methanol to remove residual DMF and further eluted with a short SEC column Sephadex G-25, H 5 cm x OD 0.7 cm). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 23.3 mg of the desired compound 1b .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 7.96-7.81 (m, 100.22H, NH G0, NH G1, NH G2, NH G3, and NH Ac), 6.50, 6.42 (m, 3.34H, H A _{, H C, and H d)} , 3.07 (m, 184.06H, H d, H f, H fAc, H gAc, H fTAMRA, and H gTAMRA), 2.94, 2.91 (m, 7.53H, H B), 2.65 -2.64 (m, 123.63H, H _b ), 2.42 (m, 60.45H, H _e and H _a ), 2.19-2.17 (m, 120H, H _c ), 1.79 (s, 87.39H, H _h );

MS (MALDI-TOF, THAP matrix) M _n 7612.70, M _w 7757.08, PDI 1.02

< Example  3> Dendrimer Of the conjugate 1c  Produce

G3 PAMAM Dendrimer 2 (about 280 [mu] L) was dried under vacuum for 12 hours to remove methanol. 5-TAMRA NHS ester 3 (710 μL, 3.59 μmol, 5.05 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (49.3 mg, 7.13 μmol) in DMSO (4.80 mL) DIEA (2.48 [mu] L, 14.2 [mu] mol) was added. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Subsequently, succinic anhydride 5 (68.9 mg, 688 μmol) was slowly added to the stirring solution, and then DIEA (80.0 μL, 46.0 μmol) was added. The reaction mixture was stirred at room temperature for 29 hours while the light was blocked, and then dialyzed (Model: Spectra / Por Regenerated Cellulose (RC) membrane, MWCO 1000 , Manufacturer: Spectrum Laboratories). Next, the crude product obtained by removing the solvent under reduced pressure SEC column was purified in the distilled water to the (model name ^{Bio-Beads ® S-X1,} H 38.5 cm × OD 3 cm). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 87.6 mg of the desired compound 1c .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 8.02-7.91 (m, 94.9H, NH G0, NH G1, NH G2, NH G3, and NH SA), 6.50, 6.42 (m, 2.30H, H A _{, H C, and H D)} , 3.08 (m, 183.66H, H d, H f, H fSA, H gSA, H fTAMRA, and H gTAMRA), 2.94, 2.91 (m, 7.10H, H B), 2.65 _{(s, 120H, H b)} , 2.43-2.37 (m, 120.58H, H e, H a, and H i), 2.30-2.27 (m, 67.72H, H h), 2.20 (s, 116.26H, H _c );

MS (MALDI-TOF, THAP matrix) M _n 9560.13, M _w 9651.57, PDI 1.01.

< Example  4> Dendrimer In the conjugate 1d  Produce

G3 PAMAM Dendrimer 2 (about 93.0 [mu] L) was dried under vacuum for 11 hours to remove methanol. 5-TAMRA NHS ester 3 (120 μL, 1.13 μmol, 9.44 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (15.7 mg, 2.27 μmol) in DMSO , DIEA (0.60 [mu] L, 3.44 [mu] mol) were added. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Then, m-dPEG ₄ -NHS ester (purchased from Quanta BioDesign, MW 333.33; 6 , 460 μL, 214 μmol) dissolved in DMSO- d ₆ was slowly added to the stirring solution, and DIEA (25.0 μL, 143 μmol ) Was added. The reaction was stirred at room temperature for 68 hours while the light was blocked. In the light shielding state to the crude product short SEC column was first filtered with (model: Bio-Rad: Bio-Beads ® S-X1, H 4 cm × OD 0.7 cm, Manufacturer). It was purified from the DMF Next, the crude product SEC column (Bio-Rad Model:: Bio-Beads S-X1 ^®, H 38.5 cm × 4.5 cm OD, manufacturer). The purified products were combined and filtered through a SEC column (Sephadex LH-20, H 39 cm x OD 4.5 cm) using methanol to remove residual DMF. The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufactured by Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 18.1 mg of the title compound 1d .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 7.94-7.80 (m, 109H, NH G0, NH G1, NH G2, NH G3, and NH TEG), 6.50, 6.42 (m, 4.23H, H A, _{H C, and H D),} 3.59-3.41 (m, 418.03H, H i, H j, H k, H l, H m, H n, and H o), 3.23 (s, 93.59H, H p) , 3.08 (m, 184.84H, H d, H f, H fTEG, H gTEG, H fTAMRA, and H gTAMRA), 2.94, 2.91 (m, 7.34H, H b), 2.65 (m, 120.16H, H b ), 2.42 (m, 60.04H, H _e and H _a ), 2.30 (t, J = 6.60 Hz, 64.30H, H _h ), 2.19-2.18 (m, 120H, H _c );

MS (MALDI-TOF, THAP matrix) M _n 11985.38, M _w 12209.90, PDI 1.02.

< Example  5> Dendrimer The conjugate (1e)  Produce

G3 PAMAM Dendrimer 2 (about 110 [mu] L) was dried under vacuum for 23 hours to remove methanol. 5-TAMRA NHS ester 3 (300 μL, 1.44 μmol, 4.79 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (19.9 mg, 2.88 μmol) in DMSO , And DIEA (5.00 [mu] L, 28.7 [mu] mol) were added thereto. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, m-dPEG ₃₇ -NHS ester (purchased from Quanta BioDesign, MW 1787.07; 7 , 905 μL, 18.6 μmol) dissolved in DMSO- d ₆ was slowly added to the stirring solution. The reaction was stirred at room temperature for 66 hours while the light was blocked. In the light shielding state to the crude product short SEC column was first filtered with (model: Bio-Rad: Bio-Beads ® S-X1, H 4 cm × OD 0.7 cm, Manufacturer). It was purified from the DMF Next, the crude product SEC column (Bio-Rad Model:: Bio-Beads S-X1 ^®, H 38.5 cm × 4.5 cm OD, manufacturer). The purified products were combined and filtered through a short SEC column (Sephadex LH-20, H 4.2 cm x OD 0.7 cm) using methanol to remove residual DMF and further eluted with a short SEC column Sephadex G-25, H 4.3 cm x OD 0.7 cm). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 39.0 mg of the desired compound 1e .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 8.04-7.94 (m, 88.72H, NH G0, NH G1, NH G2, NH G3, and NH PEG), 6.50, 6.42 (m, 3.20H, H A _{, H C, and H d)} , 3.63-3.35 (m, 1237.04H, H i, H j, and H k), 3.24 (s, 27.26H, H l), 3.07 (m, 173.43H, H d, _{H f, H fPEG, H gPEG} , H fTAMRA, and H gTAMRA), 2.94, 2.91 (m, 8.70H, H B), 2.65-2.58 (m, 158.63H, H b, and H g), 2.43 (m _{, 60.27H, H e, and H} a), 2.30 (t, J = 6.30 Hz, H h), 2.20 (m, 120H, H c);

MS (MALDI-TOF, THAP matrix) M _n 17584.39, M _w 18307.65, PDI 1.04.

< Example  6> Dendrimer Of the conjugate 1f  Produce

G3 PAMAM Dendrimer 2 (about 93.0 [mu] L) was dried under vacuum for 12 hours to remove methanol. 5-TAMRA NHS ester 3 (121 μL, 1.14 μmol, 9.44 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (15.9 mg, 2.30 μmol) in DMSO , DIEA (0.60 [mu] L, 3.44 [mu] mol) were added. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, 3- (2-hydroxyethoxy) propanoic acid 8 (purchased from Broadpharm, 830 μL, 144 μmol) and DIEA (25.0 μL, 143 μmol) dissolved in DMSO- d ₆ were added to the stirring solution. Next, N -oxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP, 38.4 mg, 73.8 μmol) was slowly added to the stirring solution. The reaction was stirred at room temperature for 68 hours while the light was blocked. In the light shielding state to the crude product short SEC column was first filtered with (model: Bio-Rad: Bio-Beads ® S-X1, H 4.5 cm × OD 0.7 cm, Manufacturer). It was purified from the DMF Next, the crude product SEC column (Bio-Rad Model:: Bio-Beads S-X1 ^®, H 38.5 cm × 4.5 cm OD, manufacturer). The purified product was combined and filtered through a short SEC column (Sephadex LH-20, H 3.9 cm x OD 0.7 cm) using methanol to remove residual DMF and further purified by short SEC column Sephadex G-25, H 4.5 cm x OD 0.7 cm). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 16.5 mg of the desired compound 1f .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 8.00-7.78 (m, 90.75H, NH G0, NH G1, NH G2, NH G3, and NH PEA), 6.50, 6.42 (m, 3.15H, H A _{, H C, and H D)} , 4.63 (brs, 23.46H, H l), 3.59 (t, J = 6.60 Hz, 53.91H, H i), 3.47-3.46 (m, 55.10H, H j), 3.38 (t, J = 5.10 Hz, 63.56H, H _k ), 3.08 (m, 181.96H, H _d , H _f , H _{f PEA} , H _{g PEA} , H _{f TAMRA} and H _{g TAMRA} ), 2.94, _{, H b), 2.65 (m} , 120.12H, H b), 2.42 (m, 62.93H, H e and H a), 2.31 (t, J = 6.60 Hz, 58.99H, H h), 2.20-2.19 ( m, 120H, H &lt; _c &gt;);

MS (MALDI-TOF, THAP matrix) M _n 9129.60, M _w 9294.03, PDI 1.02.

< Manufacturing example  1> Preparation of glucosamine (10) with succinic acid introduced

D - (+) - glucosamine hydrochloride 9 (500 mg, 2.32 mmol) was dissolved in DMSO (2 mL), succinic anhydride 5 (466 mg, 4.66 mmol) was slowly added to the stirring solution, and anhydrous acetone ) Was added and sonicated to vigorously dissolve. Next, triethylamine (0.39 mL, 2.80 mmol) was slowly added to the stirred solution. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 16 hours. The crude product is concentrated under reduced pressure and recrystallized at low temperature (4 ° C) using ethanol (15 mL). The resulting white solid product was washed with cold diethyl ether and dried under vacuum to obtain 214 mg of the target compound 10 .

^{1 H NMR (600 MHz, D} 2 O) δ 5.21 (d, J = 3.6 Hz, 0.62H, H α 1), 4.74 (d, J = 8.4 Hz, 0.38H, H β 1), 3.94-3.86 ( m, 2.53H, H _{? 2} , H _{? 5} , and H _{? 5} ), 3.82-3.75 (m, 1.84H, H _{? 6} , H _{? 6} , and H _{? 3} ), 3.72-3.60 _{, H β 2), 3.59-3.56 (} m, 0.38H, H β 3), 3.53-3.46 (m, 1.45H, H α 4 and H β 4), 2.72-2.62 (m, 4.37H, H α 8 , H _{? 8} , H _{? 7} , and H _{? 7} );

_{HRMS (ESI) Calcd for C 10} H 18 NO 8 (M + H) +: 280.1032, Found: 280.1032.

< Example  7> Dendrimer Conjugate (1g)  Produce

G3 PAMAM Dendrimer 2 (about 280 [mu] L) was dried under vacuum for 6 hours to remove methanol. The compound 2 (48.7 mg, 7.05 μmol) was dissolved in DMSO (4.70 mL), and 5-TAMRA NHS ester 3 (600 μL, 3.54 μmol, 5.90 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution , And DIEA (3.00 [mu] L, 17.2 [mu] mol) were added thereto. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, the glucosamine derivative 10 (37.8 mg, 128 μmol) and DIEA (40.0 μL, 230 μmol) prepared in Preparation Example 1 were added to the stirring solution. Next, PyBOP (58.7 mg, 113 μmol) was slowly added to the stirring solution. The reaction was stirred at room temperature for 26 hours while the light was blocked. Next, succinic anhydride 5 (23.6 mg, 236 μmol) and DIEA (20.0 μL, 115 μmol) were added to the stirring solution, and the mixture was stirred at room temperature for 19 hours. The crude product was subjected to first filtration with a short SEC column (Sephadex G-25, H 3.7 cm x OD 0.7 cm) using tertiary distilled water. Next, the crude product was purified with a SEC column (Sephadex G-25, H 38.5 cm x OD 3 cm) in tertiary distilled water. The purified product obtained above was combined, concentrated under reduced pressure, and dried under vacuum to obtain 25.1 mg of the desired compound ( 1 g ).

^{1 H NMR (600 MHz, 4} : 1 DMSO d 6 / D 2 O) δ 8.28 (s, 0.66H, H G), 5.24-5.20 (m, 3.16H, H α j), 4.78-4.76 (m, 6.76H, _{β j} H), 3.91-3.66 (m, 31.36H, H _{α k, β k} H, H _{l α,} H _{β l, m α} H, _β H _m, H _{α n, β n} H, H _{α o, and H β o)} , 3.56-3.04 (m, 372.23H, H d, H f, H fGSA, H gGSA, H fSA, H gSA, H fTAMRA, and H gTAMRA), 2.76-2.48 (m, 245.36H, H _c , H _i , and H _h ).

< Example  8> Dendrimer Of the conjugate 1h  Produce

G3 PAMAM Dendrimer 2 (about 280 [mu] L) was dried under vacuum for 6 hours to remove methanol. 5-TAMRA NHS ester 3 (600 μL, 3.54 μmol, 5.90 mM) dissolved in DMSO- d ₆ was slowly added to the stirred solution of Compound 2 (49.2 mg, 7.12 μmol) in DMSO (4.75 mL) , And DIEA (3.00 [mu] L, 17.2 [mu] mol) were added thereto. After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. Next, the glucosamine derivative 10 (125 mg, 449 μmol) and DIEA (80.0 μL, 459 μmol) prepared in Preparation Example 1 were added to the stirring solution. Next, PyBOP (120 mg, 230 μmol) was slowly added to the stirring solution. The reaction was stirred at room temperature for 24 hours while the light was blocked. The crude product was subjected to first filtration with a short SEC column (Sephadex G-25, H 4.7 cm x OD 0.7 cm) using tertiary distilled water. Next, the crude product was purified with a SEC column (Sephadex G-25, H 38 cm x OD 3 cm) in tertiary distilled water. The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 99.1 mg of the desired compound 1h .

^{1 H NMR (600 MHz, D} 2 O) δ 8.48 (s, 0.65H, H G), 5.21 (d, J = 3.6 Hz, 14.12H, H α j), 3.92-3.87 (m, 50.57H, H _{α k, H α o, and} H β o), 3.81-3.74 (m, 48.82H, H α p, H β p, and H α l), 3.72-3.67 (m, 11.20H, H β k), _{3.59-3.56 (m, 16.20H, H β} l), 3.52-3.47 (m, 39.54H, H α m and H β m), 3.33 (m, 157.79H, H d, H f, H fGSA, H gGSA _{, H fTAMRA, and H gTAMRA)} , 2.94, 2.91 (m, 7.84H, H B), 2.90-2.87 (m, 120.59H, H b), 2.69-2.61 (m, 101.25H, H e, H a, and H _i ), 2.57-2.55 (m, 50.42H, H _h ), 2.50-2.43 (m, 120H, H _c );

MS (MALDI-TOF, THAP matrix) M _n 12827.15, M _w 13030.65, PDI 1.02.

< Example  9> Dendrimer The conjugate 12c  Produce

G3 PAMAM Dendrimer 2 (about 860 [mu] L) was dried under vacuum for 3 hours to remove methanol. The compound 2 (150 mg, 21.8 μmol) was dissolved in a DMSO (14.5 mL), obtained from Cy3 mono NHS ester (Life Technologies dissolved in DMSO- d ₆ in the stirred solution, 11, 82% purity, 100 μL, 10.7 μmol, 107 mM) was added slowly, followed by DIEA (10.0 μL, 57.4 μmol). After the reaction vessel was filled with argon gas, the reaction mixture was stirred at room temperature for 24 hours while the light was blocked. The reaction mixture was divided into nine portions, and succinic anhydride 5 (22.2 mg, 222 μmol) was slowly added to one of the portions 12 c ' (1.53 mL, 2.29 μmol) and DIEA (25.5 μL, 146 μmol) . The reaction was stirred at room temperature for 24 hours while the light was blocked. The crude product was subjected to first filtration with a short SEC column (Sephadex G-25, H 7 cm x OD 0.7 cm) using tertiary distilled water. Next, the crude product was purified by SEC column (Sephadex G-25, H 42 cm x OD 3.5 cm) in tertiary distilled water. The purified product was concentrated under reduced pressure and dried under vacuum to obtain 25.9 mg of the desired compound 12c .

^{1 H NMR (600 MHz, D} 2 O) 7.95-7.89 (m, 1.42H, H 5 and H 11), 6.49-6.40 (m, 0.93H, H 7, H 8, and H 9), 4.21 (brs , 1.33H, H ₂ and H ₁₄ ), 3.63-3.56 (m, 53.69H, H _d , H _{fCy 3} , and H _fSA ), 3.42-3.30 (m, 234.72H, H _b ), 3.16-3.10 36.18H, He), 2.74-2.65 (m , 145.04H, H b), 2.47 (m, 99.0H, H h), 1.80 (s, 3.19H, H 6 and H 10), 1.44-1.42 (m, 0.74H, H _1);

MS (MALDI-TOF, THAP matrix) M _n 10137.47, M _w 10282.43, PDI 1.01.

< Example  10> Dendrimer In the case of the conjugate 12f  Produce

PEA 8 (100 μL, 153 μmol) and DIEA (25.5 μL, 146 μmol) dissolved in DMSO- d ₆ were added to a potion of 12 c ' (1.53 mL, 2.29 μmol) in the potion prepared in Example 9. Next, PyBOP (38.9 mg, 74.7 μmol) was slowly added to the stirred solution. The reaction was stirred at room temperature for 24 hours while the light was blocked. In the light shielding state to the crude product short SEC column was first filtered with (model: Bio-Rad: Bio-Beads ® S-X1, H 7 cm × OD 0.7 cm, Manufacturer). It was purified from the DMF Next, the crude product SEC column (Bio-Rad Model:: Bio-Beads S-X1 ^®, H 38 cm × 4.5 cm OD, manufacturer). The purified product was dissolved in ethanol and filtered through a 0.45 μm syringe filter (Model: Puradisc 25 Syringe Filter, manufacturer: Whatman). The filtrate was concentrated under reduced pressure and dried under vacuum to obtain 31.0 mg of the title compound 12f .

^{1 H NMR (600 MHz, DMSO-} d 6) δ 7.93-7.81 (m, 93.54H, NH G0, NH G1, NH G2, NH G3, and NH PEA), 6.54-6.52 (m, 1.21H, H 7 _{, H 8, and H 9)} , 4.58 (brs, 27.84H, H l), 4.17-4.10 (m- 2.46H, H 2, and H 14), 3.59 (t, J = 6.30 Hz, 56.96H, H _{i), 3.48-3.46 (m, 57.51H} , H j), 3.38 (t, J = 5.10 Hz, 61.51H, H k), 3.08 (m, 180.39H, H d, H f, H fPEA, H gPEA _{, H fCy3, and H gCy3)} , 2.65 (m, 121.86H, H b), 3.42 (m, 60.37H, H e and H a), 2.31 (t, J = 6.30 Hz, 59.37H, H h), 2.20-2.19 (m, 120 H, H _c ), 1.66 (m, 5.56H, H ₆ and H ₁₀ ), 1.22-1.19 (m, 3.03H, H ₁ );

MS (MALDI-TOF, THAP matrix) M _n 10137.47, M _w 10282.43, PDI 1.01.

< Experimental Example  1> Cytotoxicity test ( Cytotoxicity Assays )

To evaluate the toxicity of the dendrimer conjugates 1a, 1b, 1c, 1d, 1e, 1f, and 1h prepared in Examples 1 to 6 and 7 according to the present invention to cells, the following experiment was conducted.

Specifically, human-derived retinal pigment epithelium ARPE-19 cell line was purchased from ATCC (American Type Culture Collection, Manassas, Va.) And incubated at 37 ° C in a 5% CO ₂ atmosphere with 10% fetal bovine serum Ml of DMEM / F12 (a mixture of Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient) supplemented with U / mL penicillin and 100 μg / mL streptomycin. The mixed medium DMEM / F12 was mixed with serum-free DMEM and Ham's F-12 nutrient at a volume ratio of 1: 1.

The dendrimer conjugates 1a-1f prepared in Examples 1 to 6 and 7 dissolved in distilled water and the mother liquor at a concentration of 600 μM at 1 h were diluted stepwise with DMEM / F12 to obtain 10 ^-8 , 10 ^-7.5 , 10 ^-7 , 10 ^-6.5 , 10 ^-6 , 10 ^-5.5 , And a sample solution having a concentration of 10 ^-5 M were prepared. ARPE-19 cells were plated at a density of 2 x 10 ³ cells per well in two 96-well microculture plates with flat bottom. Each well was filled with 100 [mu] L of culture medium and cultured at 37 [deg.] C for 24 hours to allow the cells to be adhered to the bottom of the plate. Next, the processing cells of each well in the diluted solution or DMEM / F12 (control) 100 μL, and 37 ℃, 5% CO ₂ Lt; / RTI &gt; for 24 or 48 hours, respectively. After each incubation period, each well was removed and rinsed once with DPBS (Dulbecco's Phosphate-Buffered Saline), and then 100 μL of fresh DMEM / F12 and 10 μL of Cell Counting Kit-8 (CCK- 8, purchased from Dojindo) (total 110 μL) and incubated for an additional 3 hours. Subsequently, the absorbance of each well, which is a value proportional to the number of living cells, was measured at 450 nm, and the cell viability was measured by calculating the relative value of the absorbance at 100% of the prepared control under the same conditions. All experiments were repeated 5 times under the same conditions, and the results are shown in FIG. 2 as the mean ± standard deviation (SD).

As shown in FIG. 2, the dendrimer conjugates according to the present invention exhibited cell survival rates as high as about 80% to 100% at 10 ^-8 and 10 ^-7.5 M, 10 ^-6 , 10 ^-6.5 and 10 ^-5 M , The cell survival rate is about 40% or more even at a very high concentration, and the toxicity to cells is relatively low. Furthermore, even when the dendrimer conjugate according to the present invention is incubated with the cells for 24 hours and 48 hours, the cell survival rate is maintained at a comparatively high level and the toxicity to cells is relatively low.

Therefore, the dendrimer conjugate according to the present invention has relatively low toxicity to cells and is thus excellent in safety when administered in vivo, so that it can be effectively used as a drug delivery vehicle for treating retinal diseases.

< Experimental Example  2> In vivo Retinal layer  Evaluation of penetration into

Effects of the surface functional groups of the dendrimer conjugates 1c, 1f, 12c, and 12f labeled with the fluorescent dyes according to the present invention on the penetration and distribution into the retina layer were examined using 8 week old male C57BL / 6 mice. The mother liquor at 5.00 mM concentration of the above-mentioned dendrimer conjugates 1c, 1f, 12c, and 12f dissolved in distilled water was diluted with PBS to prepare a 500 μM solution. Mice were anesthetized with tribromoethanol (purchased from Sigma-Aldrich, 1 mg per 1 kg body weight), 500 μM of each dendrimer conjugate solution (0.50 μL) was injected into Micro4 micro Was injected into the vitreous through the corneal rim using a NanoFil 34-gauge needle attached to a syringe pump (purchased from World Precision Instruments). For topical administration, 500 μM of each dendrimer conjugate solution (2.0 μL) was administered to the eye around the inferior fornix using a micropipette. At 24 hours after administration by each method, mice were euthanized and the extracted eyeballs were placed in a freezing mold and frozen with an optimum cutting temperature (OCT) compound (purchased from Sakura Finetek). Then, the frozen block was taken out of the mold, cut into 12 mu m-thick frozen flakes, and fixed on a microscope slide glass, respectively. After 2-3 drops of To-Pro3 iodide (purchased from Molecular Probes) were added to each flask for nuclear staining, the distribution of the retinal layer of the dendrimer conjugates labeled with the fluorescent dye of the above example was measured with FluoView FV1000 Spectral Were observed with a confocal fluorescence laser scanning microscope (purchased from Olympus).

3, the dendrimer conjugates 1c and 12c according to Example 3 and Example 9 of the present invention were labeled with different fluorescent dyes TAMRA and Cy3, respectively, but their surface was modified with the same anionic succinic acid group. In Example 10 12f according to the present invention is labeled with a fluorescent dye, TAMRA, and is a surface modified with a neutral hydroxy group. In the case of a retinal disease, a photoreceptor layer near the choroid layer, (Red fluorescence), and it was confirmed that the penetration into the retina of 12f having the neutral hydroxyl surface functional group was the most excellent among them.

FIG. 4 is a graph showing the penetration into the retina by two different dosing methods of the dendrimer conjugates 1c and 1f according to Example 3 and Example 6 of the present invention in which the surface functional group was labeled with a fluorescent dye TAMRA and the surface functional group was a succinic acid group or a hydroxy group , Both materials are similar or better in the degree of penetration into the retina when administered by intravitreal injections, which is the usual mode of administration for the treatment of AMD, I could confirm.

Accordingly, the dendrimer conjugate according to the present invention can be prepared by modifying a terminal functional group with a surface functional group that enhances the ocular penetration ability, so that the carrier of the present invention can be directly or indirectly injected into the eye, such as a cornea or a sclera It is effective to penetrate the outer layer through the vitreous cavity and penetrate into the choroid of the retinal layers within the vitreous body. Therefore, it is useful as a carrier of drugs for treating retinal diseases .

Claims (13)

A dendrimer-based core; And
And a surface functional group introduced into the terminal functional group of the core to enhance the ocular penetration ability.
The method according to claim 1,
The dendrimer-based core may be selected from the group consisting of polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer, A dendrimer selected from the group consisting of a polyglycerol dendrimer and a polymelamine dendrimer, or a dendrimer consisting of two or more copolymers.
The method according to claim 1,
Ends of the surface functional group is _{-OH, -OR, -NH 2, -NR} 2, -NHC (= O) CH 3, -NHC (= O) CR 3, -SH, -SR, -C (= O) OH, -C (= O) OR , -C (= O) R, -C (= O) NR 2, -NHC (= O) NR 2, -C (= S) NR 2, -NHC (= S ) NR _2, - the _{(OCH 2 CH 2) n oR} , and at least one element selected from the group consisting of glucosamine, wherein the R is H, or _{C 1 -6 - (OCH 2 CH} 2) n OCH 3, And n is an integer of 1-100.
The method of claim 3,
The surface functional group

,

,

,

,

, And

Wherein the drug is at least one selected from the group consisting of:
The method according to claim 1,
Wherein the dendrimer-based core is biodegradable.
The method according to claim 1,
Wherein the carrier is administered by an intravitreal injection method.
The method according to claim 6,
When the carrier is administered by intravitreal injection, the carrier penetrates from the vitreous cavity to the choroid of the retinal layers. Carrier.
The method according to claim 1,
Wherein the carrier is administered by an eye drop method.
9. The method of claim 8,
When the carrier is administered by an eye drop method, the transporter primarily penetrates into the vitreous cavity and secondly penetrates into the choroid of the retinal layers in the vitreous cavity &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.
The method according to claim 1,
Wherein the carrier is a drug for treating retinal disease, wherein the drug is introduced into at least one of the inner core, the terminal functional group, and the surface functional group of the core.
The method according to claim 1,
Wherein the drug for treating a retinal disease is a sustained release drug for the treatment of a retinal disease upon administration to the eye.
The method according to claim 1,
Wherein the delivery agent is multivalent to the drug when the drug is administered to the eye, and the drug introduced into the surface of the central core does not release the drug.
The method according to claim 1,
Wherein the retinal disease is macular degeneration.

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