Novel polymers and their use

Abstract

Disclosed herein are novel copolymers of poly(ε-caprolactone) (PCL) and polydopamine (PDA), optionally further comprising PEG chains, methods for their production, and their use in pharmaceutical preparations, particularly implants or in situ gelling depots, for the treatment of ocular disorders or diseases.

Description

[Technical Field]

[0001] This invention generally relates to the field of polymers for use as pharmaceutically acceptable carriers, particularly for application to the eyes. [Background technology]

[0002] It is estimated that approximately 39 million people worldwide are blind, and 246 million have visual impairments. The vast majority of these are people over 50 years of age. The main causes of partial and complete vision loss are uncorrected refractive errors and cataracts, respectively. 1 The main posterior segment vision disorders are age-related macular degeneration, diabetic retinopathy, and uveitis. Drugs such as corticosteroids treat these disorders. Some drugs are loaded into implantable polymer devices for long-term delivery. 2 In biodegradable polymer systems, drug release is controlled by diffusion and polymer degradation. For example, Ozurdex® is the first biodegradable intravitreal implant approved by the FDA for the treatment of diabetic macular edema and non-infectious uveitis. Dexamethasone is loaded into a poly(lactic-co-glycolic acid) (PLGA) matrix based on Novadur® technology. The drug is released rapidly over the first two months, and then slowly over the following four months. However, the degradation of PLGA leads to two possible causes of inflammation of ocular tissue: implant fragmentation and release of the acid portion. Several non-biodegradable polymers are known in the art, such as the Iluvien® intravitreal microimplant for 36-month drug release of fluocinolone acetonide in DME. Therefore, there is still a need to develop novel polymers with improved stability, tolerability, and release profiles. [Overview of the project]

[0003] In one embodiment, the present invention provides a novel copolymer comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA).

[0004] In one embodiment, the novel copolymer consisting of poly(ε-caprolactone) (PCL) and polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

[0005] In one embodiment, the present invention provides a method for producing the graft copolymers disclosed herein.

[0006] In another embodiment, the present invention provides a novel PCL-g-PDA copolymer for use as a carrier in pharmaceutical preparations, particularly for the sustained release of active ingredients.

[0007] In one embodiment, the active ingredient is a small molecule.

[0008] In another embodiment, the present invention provides a novel PCL-g-PDA copolymer for use in the treatment of eye diseases or disorders.

[0009] In another embodiment, the present invention provides a novel PCL-g-PDA copolymer for use as an intravitreous implant.

[0010] In another embodiment, the present invention provides a novel PCL-g-PDA in which two PCL-g-PDA chains are bonded to a PEG chain to form a polymer of the type (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA).

[0011] In another embodiment, the present invention provides a polymer of the type (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA), in which the PEG chain has a molecular weight as defined herein and both PCL-g-PDA chains have the same molecular weight.

[0012] In another embodiment, the present invention provides a (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) type polymer for use in a pharmaceutical preparation for use in a pharmaceutical preparation for use in situ to form a depot that preferably sustains release of a pharmaceutically active ingredient upon intravitreal injection, wherein the pharmaceutically active ingredient is an antibody. [Invention 1001] A copolymer consisting of poly(ε-caprolactone) (PCL) and polydopamine (PDA). [Invention 1002] The copolymer of the present invention 1001, wherein the copolymer comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA) is a graft copolymer (PCL-g-PDA). [Invention 1003] The PCL-g-PDA copolymer according to the present invention 1001 or 1002, wherein the PCL-g-PDA copolymer contains PCL having a molecular weight in the range of 1,000 g / mol to 200,000 g / mol. [Invention 1004] The PCL-g-PDA copolymer according to any one of the present invention 1001 to 1003, wherein the PCL-g-PDA copolymer comprises a PCL backbone having a molecular weight of 1,000 g / mol to 200,000 g / mol and branched PDA having a mass content of 0.1 to 50% by weight. [Invention 1005] A method for producing any of the PCL-g-PDA copolymers according to invention 1001 to 1004, characterized in that PCL having a molar percentage of halogenated PCL units in the range of 0.1 to 50 mol% reacts with a PDA precursor. [Invention 1006] A PCL-g-PDA copolymer according to any of the present invention 1001 to 1004, particularly for use in pharmaceutical preparations as a carrier for sustained release of active ingredients. [Invention 1007] PCL-g-PDA copolymer for use in the invention 1006, wherein the pharmaceutical preparation is an intravitreal implant. [Invention 1008] A PCL-g-PDA copolymer for use in the present invention 1006 or 1007, wherein the pharmaceutically active ingredient is a small molecule and is present in a pharmaceutically active product or intravitreal implant in an amount of 10% by weight or more. [Invention 1009] A PCL-g-PDA copolymer according to any of the present invention 1001 to 1004, for use in the treatment of eye diseases or eye disorders. [Invention 1010] A PCL-g-PDA copolymer according to any of invention 1001 to 1004, wherein two PCL-g-PDA chains are bonded to a PEG chain to form a polymer of the type (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA). [Invention 1011] The polymer of the present invention 1010, wherein the PEG chain has a molecular weight of up to 20,000 g / mol, and both PCL-g-PDA chains have the same molecular weight. [Invention 1012] Polymer of formula (II): TIFF0007872272000001.tif28147 [In the formula, p is 3 to 397, r is between 1 and 170. m is between 1 and 170. [Invention 1013] A polymer of any of the invention 1010 to 1012 for use in pharmaceutical preparations. [Invention 1014] A polymer for use in the present invention 1013, wherein the pharmaceutical preparation forms an in situ gelled depot for sustained release of the pharmaceutical active ingredient upon intraocular injection. [Invention 1015] A polymer for use in the present invention 1014, wherein the pharmaceutically active ingredient is an antibody. [Invention 1016] Novel polymers, methods, and uses substantially described herein. [Brief explanation of the drawing]

[0013] [Figure 1] 1H NMR spectrum of iodized PCL (PCL-I) in CDCl3. [Figure 2] Size exclusion chromatography of iodized PCL in THF using RI detection and UV detection at λ290 nm. [Figure 3] 1H NMR of PCL-g-PDA in DMSO-d6. [Figure 4] DOSY NMR of purified PCL-g-PDA in DMSO-d6. [Figure 5] Size exclusion chromatography in DMSO using UV detection at λ=350nm for early commercially available PCL, PCL-g-PDA copolymer, and oligo-PDA. [Figure 6] Thermogravimetric analysis (TGA) of PCL-g-PDA from 30°C to 700°C under a nitrogen atmosphere at 20°C / min. [Figure 7] Differential scanning calorimetry (DSC) thermogram of PCL-g-PDA: First heating lamp from -80°C to 150°C at 10°C / min, cooling lamp from 150°C to -80°C at 10°C / min. [Figure 8] PCL-g-PDA containing 30 wt% DEX was prepared by membrane casting and pressurized at 130°C and 4 tons for 15 minutes (one staple was equal to 1.2 cm, and the membrane thickness was approximately 500 μm). [Figure 9A] Cumulative release of dexamethasone (DEX30) from PCL and PCL-g-PDA implants as a function of implant composition. Data are expressed as the mean of results obtained from measurements by HPCL (mean ± SD; n=3). PDA content is estimated to be 5 wt% by TGA. [Figure 9B] Cumulative release of ciprofloxacin hydrochloride (CIP30) from PCL and PCL-g-PDA implants as a function of implant composition. Data are expressed as the mean of results obtained from measurements by HPCL (mean ± SD; n=3). PDA content is estimated to be 5 wt% by TGA. [Figure 10] Survival rate of L929 cells after 24 hours incubation with a PCL-g-PDA membrane. Percentages were obtained from fluorescence intensity after PrestoBlue testing. [Figure 11] Survival rate of human retinal epithelial cell line ARPE-19 (ATCC, CRL-2302) after incubation with PCL or PCL-g-PDA membrane for 48 hours. [Figure 12] Residual mass of PCL-g-PDA during degradation studies under standard conditions (PBS, 37°C, pH=7.4). [Figure 13] Residual molecular weight of PCL-g-PDA during degradation studies under standard conditions (PBS, 37°C, pH=7.4). [Figure 14] Photograph of a PCL-g-PDA implant after 110 days of immersion under standard conditions (PBS, 37°C, pH=7.4). [Figure 15] Swelling degree of PCL-g-PDA during degradation studies under standard conditions (PBS, 37°C, pH=7.4). [Figure 16] pH of the degradation medium during the study of PCL-g-PDA implant degradation under standard conditions (PBS, 37°C, pH=7.4). [Figure 17] Residual mass of PCL-g-PDA during decomposition studies under accelerated conditions (HCl(2M), 37°C, pH=1). [Figure 18] Residual molecular weight of PCL-g-PDA during decomposition studies under accelerated conditions (HCl (2M), 37°C, pH=1). [Figure 19] Photograph of a PCL-g-PDA implant after immersion in accelerated conditions (HCl (2M), 37°C, pH=1) for 60 days. [Figure 20] 1H NMR of T-PDA in DMSO-d6. [Figure 21]DOSY NMR of T-PDA in DMSO-d6. [Figure 22A] Size exclusion chromatography at 280 nm during a stability study of mAbs in a formulation composed of HBS:PEG400=1:1 T-HD. [Figure 22B] Size exclusion chromatography at 280 nm during a stability study of mAbs in a formulation composed of HBS:PEG400=1 and T / T-PDA-HD(2:1). [Figure 22C] Size exclusion chromatography at 280 nm during a stability study of mAbs in a formulation composed of HBS:PEG400=1:1 T-PDA-HD. [Figure 23A] Formation and development of appearance of the T-HD formulation during a 30-day in situ depot administration. [Figure 23B] Formation and development of appearance of the T / T-PDA-HD (2:1) formulation during a 30-day in-situ depot. [Figure 23C] Formation and development of appearance of the T-PDA-HD formulation during a 30-day in-situ depot. [Modes for carrying out the invention]

[0014] Detailed description of the invention Most eye diseases are conditions or impairments that impair the eye's ability to function properly and / or negatively affect visual clarity, posing a significant public health problem. Intravitreal (IVT) administration (including implantation of medical devices or injection of suspensions, solutions, or implants) is a routine method and the most efficient way to deliver APIs to the retina. The main challenges of IVT administration are reducing injection frequency to improve patient compliance and adherence to treatment, ensuring the formulation is tolerable in the eye, and ensuring the stability of biologics. Currently, meeting all specifications remains challenging, and polymer-based formulations, though recent and limited, have the potential to overcome these challenges. Scientists are focused on developing biocompatible, (biodegradable) or (biodegradable) formulations that result in long-term, sustained delivery of APIs with minimal surgical intervention, in order to improve patient health and reach therapeutic levels for the efficient treatment of eye diseases.

[0015] The object of this invention is to evaluate the advantages of incorporating PDA units in the design of novel ophthalmic copolymers that exhibit improved tolerability due to preferential drug-PDA interactions and superior sustained-release properties (for small or large molecules). Among polymers used in healthcare and / or drug delivery applications, biodegradable synthetic formulations exhibit interesting and promising properties. In particular, PCL is biodegradable, can be slowly degraded and functionalized, and is FDA approved for medical purposes (but not yet for ophthalmic use). PEG is also biodegradable (depending on its molecular weight), FDA approved for ophthalmic use, and provides tunable gelling properties when combined with PCL.

[0016] The inventors have developed two strategies: one is a solid formulation for the delivery of small molecules (Chapter I), and the other is an in situ gelation system for the delivery of biologics (Chapter II).

[0017] Chapter I: PCL-g-PDA Solid Implants The solid implant method provides PCL-g-PDA, a hydrophobic grafted copolymer. The copolymer was synthesized in a two-step process. First, PCL (Mn = 190,000 g / mol) was post-functionalized with iodine via electrophilic substitution to obtain iodized PCL (PCL-I). Next, PCL-I was functionalized with PDA under ATRP-like oxidizing and basic conditions to obtain a PCL-g-PDA copolymer containing approximately 3-5% by weight of grafted PDA. In vitro cytotoxicity assays showed that the implanted PCL-g-PDA was non-cytotoxic to mouse fibroblasts and retinal cells. Furthermore, the implants did not degrade for more than 110 days under physiological conditions, but they did degrade under accelerated conditions, demonstrating the ability of PCL-g-PDA implants to degrade slowly. In vitro, the PCL-g-PDA implant exhibited a sustained, constant, and complete release (zero-order kinetics) of non-water-soluble dexamethasone (DL=30% w / w) for 155 days. In contrast, the PCL-g-PDA implant showed a burst effect, followed by a sustained release of water-soluble ciprofloxacin hydrochloride (DL=30% w / w) for 125 days, with estimated complete release after 500 days. In all cases, the release kinetics of the PCL-g-PDA implant were slower compared to the PCL implant, thus demonstrating that PDA has the ability to retain the drug within the implant even with a small amount of grafted PDA. Furthermore, both PCL and PCL-g-PDA implants were shown to have longer dexamethasone release times compared to commercially available PLGA-based implants (Ozurdex®).

[0018] Therefore, according to the present invention, a novel intravitreous implant is provided that can provide sustained drug delivery over several months, avoids changes in the microenvironment medium, and degrades slowly to avoid fragmentation. In one embodiment, the sustained drug delivery is provided for at least two months, or at least three months, or at least six months, or at least twelve months, or at least eighteen months, or at least twenty-four months, or at least thirty-six months. The implant contains poly(ε-caprolactone) (PCL) and polydopamine. 3 It is made of (PDA). PCL is a biocompatible, hydrophobic, FDA-approved polymer. It breaks down slowly by hydrolysis and has a long release period. Melanin is located in retinal cells and is involved in biological functions. 4 PDA is biocompatible and likely exhibits similar properties to melanin, particularly drug-binding properties. 5 The present inventors discovered that combining both polymers improves biocompatibility or tolerability and results in sustained release with limited changes in the microenvironment.

[0019] Therefore, in one embodiment, the present invention provides a novel copolymer comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA). In another embodiment, the novel copolymer comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

[0020] As used herein, the term PCL means poly(ε-caprolactone). In one embodiment, the PCL has a molecular weight in the range of 1,000 g / mol to 200,000 g / mol. In another embodiment, the PCL has a molecular weight in the range of 10,000 g / mol to 100,000 g / mol.

[0021] As used herein, the term PDA refers to polydopamine.

[0022] The term graft copolymer (or graft polymer) refers to a segmented copolymer comprising a linear or branched backbone of one composite material (e.g., PCL) and randomly distributed branches of another composite material (e.g., PDA). In one embodiment, the PCL backbone is linear.

[0023] In one embodiment, the PCL-g-PDA copolymer according to the present invention comprises PCL with a molecular weight in the range of 1,000 g / mol to 200,000 g / mol; or 10,000 g / mol to 100,000 g / mol.

[0024] Prior to polymerization with PDA, PCL is chemically modified, for example by electrophilic substitution, to obtain halogenated PCL, such as iodized PCL. 6 Therefore, in one embodiment, the copolymer according to the present invention is obtained using halogenated PCL having a molecular weight in the range of 1,000 g / mol to 1,000,000 g / mol; or 2,500 g / mol to 50,000 g / mol, and a molar percentage of iodized ε-caprolactone units in the range of 0.1 to 50 mol%; or 1 to 20 mol%. The term "halogenated" has the usual meaning known to those skilled in the art. In one embodiment, the PCL is brominated, chlorinated, or iodized.

[0025] In another embodiment, the PDA-g-PCL of the present invention has a PDA mass content in the range of 0.1 to 50% by weight, or 1 to 20% by weight, or 1 to 15% by weight, or 1 to 10% by weight, or about 5% by weight of PDA.

[0026] The term wt% (or weight %) has the usual meaning for those skilled in polymer chemistry. Preferably, weight % means mass relative to the total mass of the graft copolymer. Unless otherwise specified, the weight % of the PDA content of the graft copolymer of the present invention is calculated after purification and measured by TG analysis, as described herein, for example.

[0027] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 1,000 g / mol to 200,000 g / mol and random branching of PDA having a mass content of 0.1 to 50% by weight.

[0028] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 1,000 g / mol to 200,000 g / mol and random branching of PDA having a mass content of 1 to 20% by weight.

[0029] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 1,000 g / mol to 200,000 g / mol and random branching of PDA having a mass content of 1 to 10% by weight.

[0030] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 1,000 g / mol to 200,000 g / mol and random branching of PDA having a mass content of about 5% by weight.

[0031] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 10,000 g / mol to 100,000 g / mol and random branching of PDA having a mass content of 0.1 to 50% by weight.

[0032] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 10,000 g / mol to 100,000 g / mol and random branching of PDA having a mass content of 1 to 20% by weight.

[0033] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 10,000 g / mol to 100,000 g / mol and random branching of PDA having a mass content of 1 to 10% by weight.

[0034] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 10,000 g / mol to 100,000 g / mol and random branches of PDA having a mass content of about 5% by weight.

[0035] In another embodiment, the graft copolymer according to the present invention consists of a PCL backbone having a molecular weight of 15,000 g / mol to 150,000 g / mol and random branches of PDA having a mass content of about 3% or 5% by weight.

[0036] In yet another embodiment, the present invention provides a polymer of formula (I): HO-[-(CH2)4-CH(PDA)-C(O)-O-] r -[-(CH2)5-C(O)-O-] p -H (I) Wherein p = 23 to 1580 and r = 1 to 395; and PDA is present at up to 5% by weight; or about 3 to 5% by weight, or about 3% or 5% by weight.

[0037] In another embodiment, the present invention provides a method for producing the present graft copolymer. In one embodiment, the PCL backbone is first chemically modified, for example iodinated, and subsequently reacted with a suitable PDA precursor to give the copolymer according to the present invention. Any suitable PDA precursor known to those skilled in polymer chemistry can be used. In one embodiment, the PDA precursor is dopamine hydrochloride. The polymerization is known to those skilled in the art and is carried out according to the conditions further disclosed in the attached examples. In one embodiment, the polymerization is carried out under oxidative and basic conditions to obtain PCL grafted with PDA (PCL-g-PDA) 7 and then further purified.

[0038] Accordingly, the present invention provides a method for producing the PCL-g-PDA copolymer of the present invention, characterized by reacting PCL having a molar percentage of iodized PCL units in the range of 0.1 to 50 mol%; or 1 to 20 mol% with a PDA precursor, such as dopamine hydrochloride. In one embodiment, this method is carried out under oxidizing and basic conditions, in the presence of copper(I) bromide, under an inert atmosphere, at about 70°C. The graft copolymer thus obtained is purified. Purification can be carried out according to methods known to those skilled in the art and / or according to the methods described in the appended examples. In one embodiment, purification is carried out by precipitation, preferably by precipitation from methanol. The precipitation step can be repeated several times, preferably up to three times. In yet another embodiment, purification is carried out by precipitation from methanol, followed by pulverizing the PCL-g-PDA twice from cold methanol.

[0039] In one embodiment, a method for producing the PCL-g-PDA copolymer of the present invention is as disclosed in the appendix examples or in schemes 1 and 2, using the specific starting materials, intermediates, and reaction conditions disclosed herein.

[0040] In another embodiment, the present invention discloses a PCL-g-PDA copolymer obtained by using the methods disclosed herein, particularly those disclosed in schemes 1 and 2.

[0041] Iodized PCL can be prepared by methods known to those skilled in the art, for example, 6 It can be obtained using the methods described herein. In one embodiment, iodized PCL is obtained according to the methods disclosed in Scheme 1 and the attached examples. In yet another embodiment, the present invention provides functionalization of the PCL by post-modification. In a preferred embodiment of the present invention, PCL having a high molecular weight, i.e., a molecular weight greater than 45,000 g / mol, is iodized. The use of such high molecular weight PCL is advantageous for the development of biodegradable solid preparations that combine the feasibility of synthesis with the good mechanical properties of the resulting implants, in the sense that the implants are more flexible and less brittle.

[0042] The PCL-g-PDA copolymer according to the present invention possesses valuable pharmaceutical properties. In particular, these properties are stable, highly tolerable, and suitable for the sustained release of pharmaceutical active ingredients.

[0043] Accordingly, in another embodiment, the present invention provides the copolymer for use in pharmaceutical preparations, for example, as a carrier for a pharmaceutically active ingredient. In one embodiment, according to the present invention, the pharmaceutical preparation is an implant. In another embodiment, the implant is suitable for intraocular use, for example, as an intravitreal implant. In yet another embodiment, the implant may further contain, together with the active ingredient, another polymer, such as PCL, PLA, or PLGA. In yet another embodiment, the intravitreal implant consists only of the copolymer and a suitable active ingredient.

[0044] As used herein, the term "pharmaceutical active ingredient" means any molecule having clinically meaningful pharmacological activity. In one embodiment, the pharmaceutical active ingredient is a small molecule as defined by Lipinski's Rule of Five. In another embodiment, the pharmaceutical active ingredient is a drug approved for the treatment of eye diseases such as glaucoma, cataracts, age-related macular degeneration, diabetic retinopathy, and uveitis. In yet another embodiment, the pharmaceutical active ingredient is selected from the group consisting of ganciclovir, dexamethasone, fluocinolone acetonide, and cyclosporine A.

[0045] According to the present invention, the pharmaceutically active ingredient is present in the implant in an amount of 10% by weight or more, or 10-60% by weight, or 10-30% by weight.

[0046] In another embodiment, the present invention provides the use of the copolymer for, for example, the preparation of pharmaceuticals such as implants for the treatment of eye diseases; or for intravitreal administration of drugs. In one embodiment, such administration is a sustained release over a period of time as defined herein.

[0047] In another embodiment, the present invention provides a method for treating an eye disease by placing an implant containing the copolymer of the present invention, loaded with a suitable pharmaceutically active ingredient or an approved drug, into the eye of a patient requiring such treatment.

[0048] General synthesis of PCL-g-PDA The PCL-g-PDA copolymer of the present invention can be obtained using the following general reaction scheme. In the first step, iodized PCL is obtained by post-modification of PCL with iodine. The post-modification method for functionalization of PCL was studied by Nottelet et al. 6 The method is based on the two-step one-pot reaction described in Scheme 1. The first step is the anionic activation of PCL in the presence of LDA, and the second step is the electrophilic substitution of iodine. TIFF0007872272000002.tif16141 Scheme 1: Synthesis scheme for iodized PCL (PCL-I). Here, n=43~1755; p=23~1580, and q=2~395.

[0049] Starting from commercially available PCL, a series of iodized PCLs were prepared targeting various molecular weights and copolymer masses, and SEC and 1 The samples were characterized by 1H NMR. The results are summarized in Table 1.

[0050] [Table 1]

[0051] In the second step, Cho et al. 7 Based on the conditions applied, PCL-g-PDA is obtained according to the following reaction scheme 2. TIFF0007872272000004.tif62141 Scheme 2: Synthesis of PCL-g-PDA. Here, p=23~1580; q=2~395, and r=1~395.

[0052] Briefly, dopamine was introduced at room temperature into a Schlenk flask containing DMSO, sodium carbonate, BPO, and PMDETA. Sodium carbonate is used to obtain basic conditions. BPO is an organic peroxide commonly used as a radical initiator to induce chain polymerization, and PDMETA (also called PMDTA) is a tridentate ligand that can bind to metal cations to form complexes. Immediately after introducing all these components (in less than 1 minute), the solution turned from white to black, suggesting oxidative polymerization of dopamine. Meanwhile, PCL-I was solubilized in DMSO at room temperature. The solution was stirred for 4 hours. The PCL-I macroinitiator solution was transferred to the first solution, and copper(I) bromide was added. Copper(I) bromide is a metallic agent that binds to ligands and, like ATRP, activates the dormant PCL-I macroinitiator, enabling the generation of dopamine monomers or free radical PCL bound to already grown PDA. The solution was heated at 70°C for 48 hours and then cooled by immersion in liquid nitrogen. The majority of the solvent was removed by evaporation, and the solution was then precipitated in methanol to collect the final copolymer. The change in methanol to black during precipitation suggests the presence of non-grafted PDA in the DMSO solution and that the non-grafted PDA compound was further solubilized in methanol.

[0053] Accordingly, in another embodiment, the present invention provides a method for producing PCL-g-PDA, the method comprising a reaction sequence including a starting material, an intermediate, a reaction partner, and conditions described in schemes 1 and 2 herein.

[0054] Materials and methods Chemicals and materials Poly(ethylene glycol) (PEG), toluene, diethyl ether, methanol, dichloromethane (DCM), tetrahydrofuran (THF), poly(ε-caprolactone) (PCL), iodine, hydrochloric acid (HCl, 37%), lithium diisopropylamide (LDA), sodium thiosulfate, benzenedimethanol, ε-caprolactone (εCL), stannous octanoate (Sn(Oct)2), dimethyl sulfoxide (DMSO), dopamine hydrochloride, benzoyl peroxide (BPO), copper(I) bromide, N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and ciprofloxacin hydrochloride (CIP.HCl) were purchased from Sigma-Aldrich. Ammonium chloride and polysorbate 20 (Tween 20) were purchased from Acros Organics. Sodium carbonate was purchased from Fisher Scientific. Dexamethasone (DEX) was purchased from either Sigma Aldrich or TCI.

[0055] Characterization nuclear magnetic resonance (NMR) Proton nuclear magnetic resonance spectroscopy ( 1 The functionalization rate of the polymer for iodized PCL was determined using a Bruker AMX-400MHz spectrometer in CDCl3 or DMSO-d6 by performing 1H-NMR. Diffusion order NMR (DOSY NMR) was performed to highlight the individual diffusion coefficients of species contained in the sample and to determine the presence of residual free species. Sample concentrations ranged from 5 to 15 mg / mL.

[0056] Size exclusion chromatography (SEC) SEC THF: The sample (5 mg / ml) was filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Shimadzu (Japan) instrument equipped with a RID-20A refractive index signal detector, an SPD-20A UV / VIS detector, a PLgel MIXED-C guard column (Agilent, 5 μm, 50 × 7.5 mm), and two PLgel MIXED-C columns (Agilent, 5 μm, 300 × 7.5 mm). The mobile phase flow rate was 1.0 mL / min.-1 The THF was 100 μL. The injection volume was 100 μL. The average molecular weight and degree of dispersion (D) were calculated using polystyrene (PS) as a standard.

[0057] SEC DMSO:PCL-g-PDA copolymer (1 mg / mL) was filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Waters 515 HPLC instrument equipped with a Waters 410 differential refractometer, Waters 2996 photodiode array detector, Polargel-M guard column (Agilent, 50 × 7.5 mm), and two Polargel-M columns (Agilent, 300 × 7.5 mm). The mobile phase was DMSO at a flow rate of 1.0 mL / min. The injection volume was 50 μL. PDA content was quantified by the area ratio between the PDA-containing copolymer and the oligo-PDA itself at a specific wavelength selected in the range of 254–400 nm.

[0058] In both of the above SEC methods, when analyzing PCL-g-PDA copolymers, DMF can also be used as the mobile phase under the conditions described.

[0059] Thermogravimetric analysis (TGA) The thermal decomposition of copolymers (0.1-10 mg) was studied using a thermogravimetric analyzer (TGA Q500 v20.13 build 39). Samples were heated under a nitrogen atmosphere at 20°C / min from 30°C to 700°C.

[0060] Differential Scanning Calorimetry (DSC) Samples (1–10 mg) of each copolymer were placed in an aluminum pot. Using a Mettler Toledo DSC 3, the samples were heated from -80°C to 300°C at 10°C / min. The glass transition temperature and melting temperature of each sample were determined during the first heating cycle. For crystalline PCL, the degree of crystallinity (χ) was calculated using the enthalpy of fusion, with a baseline value of ΔH = 139.5 J / g. 8 .

[0061] High-performance liquid chromatography (HPLC) The samples were filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Shimadzu (Japan) instrument equipped with an SPD-M20A diode array detector and an HPLC C18 column (Kinetex, 2.6 μm, 100 A, 100 × 4.6 mm). For drug detection, isocratic mode was applied with 40% ACN (0.1% TFA) + 60% H2O (0.1% TFA) for DEX detection and 13% ACN (0.1% TFA) + 87% H2O (0.1% TFA) for CIP detection. After detection, the column was washed using a linear gradient until 100% ACN was reached. The flow rate was 1.0 mL / min.

[0062] Chapter II: (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) The in-situ gelation system method according to the present invention provides an amphiphilic grafted copolymer of the type (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) based on the knowledge obtained in Chapter I. First, amphiphilic triblock PCL-b-PEG-b-PCL was synthesized with various PEG and PCL chain lengths, and an in-situ gel was produced in water at physiological temperature. In one embodiment, the PEG has a molecular weight of up to 20,000 g / mol. In another embodiment, the PEG has a molecular weight of 1,000 to 4,600 g / mol. In yet another embodiment, a triblock copolymer is provided in which the PEG and PCL chain lengths differ, but each PCL has the same chain length, in order to obtain an EG / CL ratio in the range of 0.30 to 2.13 and a molecular weight of 4,300 to 9,400 g / mol. In yet another embodiment, the two PCL chains have a molecular weight between 846 and 2,100 g / mol, and the PEG chain has a molecular weight between 1,000 and 4,600 g / mol. In yet another embodiment, the two PCL chains had a molecular weight of 855 or 890 g / mol, and the PEG chain had a molecular weight of 2000 g / mol. These two specific PCL-b-PEG-b-PCL exhibited good gelling ability at room temperature.

[0063] PCL-b-PEG-b-PCL having a distribution of 855-2000-855 (g / mol) was functionalized via iodine by electrophilic substitution to obtain (PCL-I)-b-PEG-b-(PCL-I), which was further functionalized with PDA under ATRP-like oxidizing and basic conditions. The unprocessed (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) contained approximately 40% by weight of PDA, including some free (ungrafted) PDA.

[0064] General synthesis of (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) The (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) type copolymer (also referred to herein as "T-PDA") according to the present invention can be synthesized based on the following general reaction schemes 3 to 5. TIFF0007872272000005.tif75139 Scheme 3: Synthesis of PCL-b-PEG-b-PCL (m=22~455, n=3~568)

[0065] The triblock copolymer PCL-b-PEG-b-PCL (also referred to herein as "T") is synthesized by ring-opening polymerization (ROP) of ε-Cl in anhydrous toluene using a commercially available PEG-diol (Scheme 3) as an initiator and Sn(Oct)2 as a catalyst. The solution is stirred at 100°C for 24 hours, precipitated in cold diethyl ether, filtered, and dried under vacuum.

[0066] Post-polymerization modifications leading to the functionalization of PCL by iodine were described by Nottelet et al. 6 This method is based on research described by [author's name] and is similar to the method presented in Chapter I. The method consists of a two-step one-pot reaction as described in Scheme 4. TIFF0007872272000006.tif44141 Scheme 4: Synthesis of (PCL-I)-b-PEG-b-(PCL-I) (m=22~455; n=3~568; p=3~397 and q=1~170)

[0067] The first step is anionic activation of the most electrophilic proton of the PCL skeleton in the presence of LDA, and the second step is electrophilic substitution with iodine. The resulting (PCL-I)-b-PEG-b-(PCL-I) type polymer is also referred to herein as "TI".

[0068] Finally, functionalization of TI with PDA was carried out under the same conditions as described in Chapter I. The reaction scheme is shown in Scheme 5, and the detailed conditions are described in Example 9. TIFF0007872272000007.tif40141 Scheme 5: Synthesis of (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) ("T-PDA"; p=3~397; m=1~170 and r=1~170)

[0069] For purification, a solution of DMSO containing T-PDA was precipitated in cold diethyl ether, but T-PDA adhered to the bottom, making product recovery complicated. In the second step, the solution of DMSO containing T-PDA was introduced into a dialysis bag, and the DMSO was replaced with water to recover the T-PDA. Dialysis was maintained at this stage as the preferred purification method to recover T-PDA for further analysis. T-polymer containing PDA (T-PDA) is a novel type of copolymer and is one embodiment of the present invention.

[0070] Accordingly, in one embodiment, the present invention provides a PCL-g-PDA copolymer as defined herein (Chapter I), in which two PCL-g-PDA chains are bonded to a PEG chain to give a grafted copolymer of the type (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA). In one embodiment, the PEG chain has a molecular weight as defined herein, and both PCL-g-PDA chains have the same molecular weight.

[0071] In another embodiment, the present invention provides a polymer of formula (II) of the T-PDA type as defined herein: TIFF0007872272000008.tif28147[In the formula, p is 3 to 397, r is between 1 and 170. m is between 1 and 170.

[0072] The present invention also provides a method for producing novel T-PDA polymers. In one embodiment, the present invention provides a method for producing a T-PDA type polymer, the method comprising a reaction sequence including starting materials, intermediates, reaction partners, and conditions described in schemes 3 to 5 herein.

[0073] In another embodiment, the present invention also provides T-PDA polymers obtained by using the reaction sequence (starting materials, intermediates, and conditions) according to schemes 3-5 herein.

[0074] The T-PDA copolymer according to the present invention, for example, of formula (II), possesses valuable pharmaceutical properties. In particular, they are known to be stable, highly tolerable, and suitable for the sustained release of pharmaceutical active ingredients.

[0075] Accordingly, in one embodiment, the present invention provides a T-PDA copolymer of formula (II), as defined herein, for use as a carrier for a pharmaceutically active ingredient in a pharmaceutical preparation. In one embodiment, according to the present invention, the pharmaceutical preparation is a depot formed by in situ gelation. In another embodiment, the in situ-formed depot is suitable for intraocular use, for example, for intravitreal injection. In yet another embodiment, the pharmaceutical preparation forms an in situ gelled depot for sustained release of the pharmaceutically active ingredient upon intraocular injection. In yet another embodiment, according to the present invention, the pharmaceutical preparation is an aqueous solution or a suitable buffer, such as a histidine buffer solution, comprising the T-PDA copolymer together with the pharmaceutically active ingredient, and further comprising PEG, preferably PEG400 as defined herein, as a cosolvent.

[0076] The term "pharmaceutical active ingredient" as used in relation to T-PDA copolymers means any molecule, preferably an antibody, that has clinically meaningful pharmacological activity. In this specification, the term "antibody" is used in its broadest sense and is not limited to a wide range of antibody classes or structures, including monoclonal antibodies (mAbs), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. In one embodiment, the antibody is a monoclonal, monospecific, or bispecific antibody, or an antigen-binding fragment thereof. In one embodiment, the antibody is a human antibody or a humanized antibody. In another embodiment, the antibody is one of the aforementioned antibodies that can be used to treat an eye disease. In yet another embodiment, the antibody is a molecule containing INN-falisimab.

[0077] According to the present invention, the pharmaceutically active ingredient is present in the depot in an amount of up to 45% by weight; or 5-45% by weight; or 15-45% by weight; or 20-45% by weight, where the weight percentage is relative to T-PDA.

[0078] In another embodiment, the present invention provides the use of the T-PDA copolymer of the present invention, for example, formula (II), for the preparation of pharmaceuticals. In one aspect, such pharmaceuticals are for the treatment of eye diseases; or for intravitreal administration of drugs.

[0079] In another embodiment, the present invention provides a method for treating an eye disease by injecting a pharmaceutical preparation comprising the T-PDA copolymer of the present invention, loaded with a suitable pharmaceutically active ingredient or an approved drug, into the eye of a patient requiring such treatment. In one embodiment, the injection is an intravitreal injection.

[0080] In another embodiment, an aqueous formulation is provided comprising PCL-b-PEG-b-PCL and / or (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) and a monoclonal antibody (mAb). In one embodiment, the formulation further comprises a soluble cosolvent, preferably PEG400. In yet another embodiment, the present invention provides a formulation comprising histidine buffer (HBS) and PEG400 (ratio HBS:PEG400 = 1:1 or 1:2) as solvents, comprising 5 to 15, preferably 5 or 10 wt% PCL-b-PEG-b-PCL or (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA), and loaded with 40 mg / mL of mAb. In one embodiment, these formulations are injectable through a 30G needle. Stability studies of the mAb demonstrated that (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) was able to interact with mAbs for 30 days without causing denaturation. In vitro, these formulations formed in-situ gelled depots via a solvent exchange process.

[0081] As used herein, particularly in the embodiments of Chapter II, the term "PEG" means poly(ethylene glycol). According to the present invention, PEG of different molecular weights can be used. In one embodiment, PEG has a molecular weight of up to 20,000 g / mol. In another embodiment, PEG has a molecular weight of 400; or 1,000; or 1,450; or 2,000; or 4,600; or 10,000; or 20,000 g / mol.

[0082] In particular, in Chapter II, the molecular weight of each polymer is described by an index number. For example, PEG with 1000 g / mol is defined as PEG1000, and PCL with 2000 g / mol is defined as PCL2000. Throughout this specification, a PCL-b-PEG-b-PCL type copolymer having, for example, 1000 g / mol of PEG and 2000 g / mol of PCL may also be represented as 1000-2000-1000.

[0083] Unless otherwise explicitly stated, the terms defined in Chapter I have the same meaning when used in relation to the embodiments defined in Chapter II.

[0084] Materials and methods Many of the materials referred herein for the preparation of PCL-g-PDA copolymers and implants based thereon (Chapter I) can also be used for the preparation of the triblock copolymers in this Chapter II by adding poly(ethylene glycol) (PEG, Mn 400 or 1000 or 1450 or 2000 or 4600 or 10000 or 20000 g / mol), toluene, stannous octanoate (Sn(Oct)2), ε-caprolactone (ε-Cl), and diethyl ether, purchased from Sigma-Aldrich. PEG was dried by azeotropic distillation of a toluene solution of PEG, and ε-Cl was dried with calcium hydride (CaH2) at room temperature for 48 hours and distilled under reduced pressure before use. PEG, ε-Cl, and Sn(Oct)2 were stored under an argon atmosphere.

[0085] Characterization Nuclear magnetic resonance spectroscopy (NMR spectroscopy), size exclusion chromatography (SEC) using THF as the mobile phase, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and the instruments used are the same as those described in Chapter I.

[0086] Size exclusion chromatography (SEC) using DMF as the mobile phase The number-average and weight-average molar masses (Mn and Mw, respectively) and dispersion (D, Mw / Mn) of the polymers were determined by SEC. The sample (5 mg / ml) was filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Shimadzu (Japan) instrument equipped with an SPD-20A UV / VIS detector, a RID-20A refractive index signal detector coupled to a PLgel MIXED-C guard column (Agilent, 5 μm, 50 × 7.5 mm), and two PLgel MIXED-C columns (Agilent, 5 μm, 300 × 7.5 mm). The mobile phase was DMF + 0.1% LiBr. The flow rate was 1.0 mL / min⁻¹, and the injection volume was 100 μL. Average molecular weight and dispersion (D) were expressed according to calibration using poly(ethylene glycol) (PEG) standards.

[0087] Aqueous size exclusion chromatography (aqueous SEC) The sample (1 mg / ml) was filtered through a 0.20 μm RC Millipore filter and analyzed using a Shimadzu (Japan) instrument equipped with an SPD-40 UV / VIS detector, a RID-20A refractive index signal detector coupled to a Biobasic SEC300 guard column (Thermo Scientific, 5 μm, 20 × 8 mm), and one Biobasic SEC300 column (Thermo Scientific, 5 μm, 150 × 7.8 mm). The mobile phase was an aqueous buffer solution consisting of HK2PO4 / KH2PO4 (0.1 M, pH=7). The flow rate was 0.80 mL / min⁻¹, and the injection volume was 100 μL.

[0088] Injectability test by compression measurement Injectability testing was performed using an Instron 3344 with a 500N captor in compression mode. This study used subcutaneous needles of size 27G-30G (Sterican®, for specific indications, B. Braun, Germany) and 1 mL disposable syringes (Omnifix®-F Luer, B. Braun, Germany). The syringes were filled with 1 mL of solution, and then the subcutaneous needles were attached. The injection rate was set to 0.5 or 1 mm / second, and the injection volume was 100 μL, corresponding to a plunger displacement of 6 mm. The ambient medium was air.

[0089] References (1) Pascolini, D.; Mariotti, SP Global Estimates of Visual Impairment: 2010. Br J Ophthalmol 2012, 96 (5), 614-618. https: / / doi.org / 10.1136 / bjophthalmol-2011-300539. (2) Yasin, MN; Svirskis, D.; Seyfoddin, A.; Rupenthal, ID. Implants for Drug Delivery to the Posterior Segment of the Eye: A Focus on Stimuli-Responsive and Tunable Release Systems. Journal of controlled release: official journal of the Controlled Release Society 2014, 196, 208-221. https: This may be considered. However, this issue was addressed on 14.09.030. (3) Liebscher, J.; Mrowczynski, R.; Scheidt, H. A.; Filip, C.; Hadade, N. D.; Turcu, R.; Bende, A.; Beck, S. Structure of Polydopamine: A Never-Ending Story? Langmuir: the ACS journal of surfaces and colloids 2013, 29 (33), 10539-10548. https: / / doi.org / 10.1021 / la4020288. (4) Rimpelae, A.-K.; Reinisalo, M.; Hellinen, L.; Grazhdankin, E.; Kidron, H.; Urtti, A.; Del Amo, E. M. Implications of Melanin Binding in Ocular Drug Delivery. Advanced drug delivery reviews 2018, 126, 23-43. https: / / doi.org / 10.1016 / j.addr.2017.12.008. (5) Liu, X.; Cao, J.; Li, H.; Li, J.; Jin, Q.; Ren, K.; Ji, J. Mussel-Inspired Polydopamine: A Biocompatible and Ultrastable Coating for Nanoparticles in Vivo. ACS nano 2013, 7 (10), 9384-9395. https: / / doi.org / 10.1021 / nn404117j. (6) Nottelet, B.; Coudane, J.; Vert, M. Synthesis of an X-Ray Opaque Biodegradable Copolyester by Chemical Modification of Poly (ε-Caprolactone). Biomaterials 2006, 27 (28), 4948-4954. https: / / doi.org / 10.1016 / j.biomaterials.2006.05.032. (7) Cho, JH; Shanmuganathan, K.; Ellison, CJ Bioinspired Catecholic Copolymers for Antifouling Surface Coatings. ACS applied materials & interfaces 2013, 5 (9), 3794-3802. https: / / doi.org / 10.1021 / am400455p. (8) Pitt, CG; Chasalow, FI; Hibionada, YM; Klimas, DM; Schindler, A. Aliphatic Polyesters. I. The Degradation of Poly(ε-Caprolactone) in Vivo. J. Appl. Polym. Sci. 1981, 26 (11), 3779-3787. https: / / doi.org / 10.1002 / app.1981.070261124.

[0090] Overall, the present invention provides novel PDA-based biomaterials using biocompatible, degradable synthetic copolymers to address the challenges of minimally invasive, long-acting intraocular delivery. This provides PDA-based implants suitable for long-term delivery of small molecules and PDA-based injectable in situ gelling systems promising for the formulation of biologics. The present invention will now be further illustrated by the following examples, but these are not intended to limit the scope of the invention. [Examples]

[0091] Example 1. Synthesis of initiator and precursor Synthesis of diethylene glycol bis(2-bromoisobutyrate) In a typical experiment, diethylene glycol (1 g, 9.42 mmol), triethylamine (3.94 mL, 28.3 mmol), and dry THF (40 mL) were added to a dry three-necked round-bottom flask and placed in an ice bath. Next, isobutyryl bromide (3.49 mL, 28.3 mmol) was slowly added to the flask through a dropping funnel. A guard tube filled with calcium chloride was placed in place to maintain anhydrous conditions. The solution was left standing overnight with stirring. The solution was filtered through diatomaceous earth and concentrated by evaporating the THF. The crude product was dissolved in a mixture of water and dichloromethane. The product was extracted from the solution by washing three times with dichloromethane using a separatory funnel. The organic phase was dried using MgSO4 powder, filtered, and dried under reduced pressure. The product was purified by using a mixture of ethyl acetate:heptane (30:70) as the solvent and filtering through silica. The fraction was collected and evaporated under reduced pressure. The pure fractions were collected and stored at 4°C for further use. Yield: 100 mol% 1 H NMR (300 MHz, CDCl3): δ = 4.28 (t, R-CH2-O-CO), 3.73 (t, O-CH2-R), 1.89 (s, R-CH3)

[0092] Synthesis of PDA oligomers In a typical experiment, dopamine hydrochloride (1.5 g, 7.91 mmol), PMDETA (130 μL, 0.63 mmol), Na2CO3 (402.0 mg), BPO (1.92 g, 7.91 mmol), and DMSO (76 mL) were added. The solution was left to stand for 4 hours with stirring and under an argon stream. Next, oxygen was removed by three freeze-pump thaw cycles. Diethylene glycol bis(2-bromoisobutyrate) (0.13 g, 0.32 mmol) and copper(I) bromide (0.09 mg, 0.63 mmol) were added. The flask was then immersed in an oil bath at 70°C and the reaction was carried out for 48 hours with vigorous stirring. The reaction was stopped by cooling in a liquid nitrogen bath. The solution was then concentrated by evaporating the DMSO under vacuum at 70°C. Finally, the polymer was precipitated, filtered, and dried under vacuum. Yield: 17% by weight 1 H NMR (600 MHz, DMSOd6): δ = 6.30-7.00 ppm (m, PDA)

[0093] Synthesis of iodized poly(ε-caprolactone) The PCL skeleton was anionically activated using LDA and modified with iodine after electrophilic substitution. In this synthesis, PCL(3g, M) n,SEC,THF, PCL (65000 g / mol, 26.3 mmol per εCL unit) and anhydrous THF (300 mL) were introduced into a dry conical reactor and left under an argon atmosphere until PCL was completely dissolved. Then, under argon, the solution was cooled to -50°C by immersion in a liquid nitrogen / ethanol mixture before adding LDA (13.16 mL, 1 equivalent per εCL unit, 26.3 mmol). After 30 minutes of reaction, the minimum amount of iodine solution in anhydrous THF (6.68 g, 1 equivalent per εCL unit, 26.3 mmol) was injected through the septum using a syringe, and the mixture was stirred and maintained at -50°C under an argon atmosphere. After 30 minutes, NH4Cl (aq) The reaction was stopped by adding an aqueous solution (2M, 200mL) of HCl, and after raising the temperature to 0°C, HCl (aq)(37%) was added to reach a neutral pH. The polymer was extracted from the solution by washing three times with dichloromethane (3 × 200 mL) in a separatory funnel. The organic phase was collected, washed three times with Na2S2O3 solution (3 × 100 mL, excess), dried using MgSO4 powder, filtered, and concentrated under reduced pressure using a rotary evaporator. The polymer was precipitated in cold methanol, filtered, and dried under reduced pressure.

[0094] Characterization: - In CDCL3 1 1H NMR: Determination of functionalization rate (Figure 1): - 1 H NMR (CDCl3, 300 MHz, ppm): 4.30 (m, R-CHI-CO), 4.05 (t, R-CH2-O-CO), 2.30 (t, R-CH2-CO-O), 2.00 (t, R-CH2-CHI), 1.64 (m, R-CH2-C-CO), 1.38 (m, R-CH2-R) - Determination of the number-average molecular weight of SEC in THF (Figure 2).

[0095] result: The degree of substitution was calculated by comparing the integral values ​​of the 4.30 ppm resonance peak corresponding to the iodine adjacent proton and the 4.05 ppm resonance peak corresponding to the unsubstituted methylene group (Figure 1, Table 2).

[0096] [Table 2]

[0097] Example 2: Synthesis of PCL-graft-PDA Typically, iodized PCL (1.5 g, 1.18 mmol iodized Cl units) and DMSO (20 mL) were added to Schlenk flask A. Dopamine-HCl (5.62 g, 25 equivalents per iodized Cl units, 29.6 mmol), PMDETA (370 μL, 1.5 equivalents per iodized Cl units, 1.78 mmol), Na2CO3 (300.0 mg), BPO (7.18 g, 25 equivalents per iodized Cl units, 29.6 mmol), and DMSO (37 mL) were added to Schlenk flask B. The solutions were stirred and left under an argon stream for 4 hours. Next, oxygen was removed by three freeze-pump-thaw cycles. The iodized PCL solution was transferred to flask B, and copper(I) bromide (255 mg, 1.5 equivalents per iodized Cl units, 1.78 mmol) was added. Next, the flask was immersed in an oil bath at 70°C under an inert atmosphere and vigorously stirred for 48 hours. The reaction was stopped by cooling in a liquid nitrogen bath. The solution was concentrated by evaporating DMSO under vacuum at 110°C. The copolymer was precipitated in methanol, filtered, and dried under vacuum.

[0098] Characterization: - During DMSO-d6 1 1H NMR: Highlights of chemical modification and purity of graft copolymer (Figure 3). - Grafting confirmed by a 4.58 ppm peak in DOSY NMR in DMSO-d6 (Figure 4). - SEC in DMSO (UV at λ=350nm): Confirmation of the presence of PDA in the copolymer (Figure 5). - Quantification of TGA:PDA content (Figure 6). - DSC: Determination of the melting temperature of the copolymer (Figure 7).

[0099] result: 1In 1H NMR, peaks at 4.58, 1.90, and 1.80 ppm are characteristic of iodized PCL modification under basic conditions, resulting from grafting onto PCL (Figure 4) (Figure 3). UV-SEC analysis showed that the PCL-g-PDA copolymer and PDA absorbed from 254 nm to 450 nm (Figure 5). To avoid residual noise, a wavelength of 350 nm was selected. Peak intensity is proportional to PDA content. PDA content was also measured by TGA analysis (Figure 6). The melting temperature of PCL-g-PDA was 49°C, measured with the first heating lamp by DSC analysis (Figure 7). A summary of the results is also shown in Table 3 below.

[0100] [Table 3]

[0101] The quantification of PDA content by TGA is based on the residual mass at 600°C of PCL, PCL-g-PDA, and oligo-PDA using the apparatus described herein. The PDA content is then calculated using the following formula: TIFF0007872272000011.tif14155

[0102] The proportion of PDA in the PCL-g-PDA copolymer after three purifications was approximately 3% by weight.

[0103] Another purification method involves grinding from cold methanol: after the initial purification step of precipitation from methanol, the copolymer can be further purified by grinding from cold methanol. More specifically, 300 mg of copolymer is placed in a Falcon tube. 45 mL of cold methanol is added, and the polymer powder is ground for several minutes, then recovered by centrifugation (0°C, 5000 rpm, 15 minutes). This step is repeated once more before drying. (Grounding step yield = 50%).

[0104] The data disclosed herein, particularly in Table 3, included only precipitation from methanol and did not involve grinding.

[0105] Example 3: Preparation of a drug-loaded implant The PCL(M) obtained in Example 2 n Approximately 60,000 g / mol of PCL-g-PDA (100-500 mg) and appropriate amounts of DEX or CIP.HCl (corresponding to 10% and 30% of the final weight) were dispersed in DMSO (5-30 mL) and thoroughly mixed. The DMSO was removed under reduced pressure at 110°C to obtain a thin film of copolymer / drug. The film was pulverized, and the resulting powder was deposited on a Teflon sheet. The powder was pressurized at 130°C and 4 tons for 15 minutes (see Figure 8).

[0106] Example 4: In vitro study of release Drug release from PCL and the PCL-g-PDA membranes obtained in Example 3 was evaluated in phosphate-buffered saline (PBS, pH 7.4) containing 0.05% v / v Tween 20 under constant orbital shaking (100 rpm) at 37°C. Typically, a 10 mg drug / polymer membrane was immersed in 20 mL of phosphate buffer containing 0.05% Tween 20 at 37°C. At specific time points, the entire release medium was removed and replaced with 20 mL of fresh buffer. The collected samples were analyzed by HPLC using UV detection at the maximum absorbance wavelength of the drug (in the range of 200–400 nm) with acetonitrile / TFA(1000 / 1) and water / TFA(1000 / 1) (10:90 to 40:60) as mobile phases.

[0107] result: Drug release kinetics vary as a function of the presence of PDA in the copolymer, drug selection, and the percentage of the drug. The PDA content in the copolymer is estimated to be between 1% and 20% by weight by TG analysis. The release kinetics of both dexamethasone and ciprofloxacin hydrochloride are slower in the PCL-g-PDA membrane compared to the PCL-loaded membrane and are regulated according to the proportion of PDA in the implant (Figures 9A and 9B).

[0108] Example 5: Biological (cytotoxic) research Fibroblasts The cytotoxicity of the PCL-g-PDA membrane was analyzed using the mouse fibroblast cell line L929 (NCTC clone 929, ECACC85011425). L929 cells were cultured at 37°C under humidified 5% CO2 in DMEM containing 1 mM L-glutamine, 5% v / v fetal bovine serum, and 100 U / mL penicillin and 100 μg / mL streptomycin in 4.5 g / L D-glucose. A polyurethane membrane containing 0.25% zinc dibutyldithiocarbamate (ZDBC) (Hadano Research Institute, FDSC, batch B-173K) was used as the positive standard (RM), and a high-density polyethylene membrane (Hadano Research Institute, FDSC, batch C-161) was used as the negative RM. Cells were seeded in 24-well plates at a density of 60,000 cells / well and incubated overnight at 37°C. To decontaminate PCL and PCL-g-PDA membranes (6 mm in diameter, less than 0.5 mm thick), each surface was irradiated with 254 nm light for 2 minutes. The membranes were added to the wells and incubated with cells for 24 hours. The membranes were removed, and the culture medium was replaced with 500 μL of PrestoBlue® (PB) solution (10% in the medium) and incubated for 30 minutes. The PB assay was performed using fluorescence (λex=558 nm, λem=593 nm). Each experiment was repeated three times. The PCL-g-PDA copolymer purified in methanol enabled cell survival after 24 hours of incubation (Figure 10).

[0109] Human retinal cells The cytotoxicity of PCL and PCL-g-PDA membranes was analyzed using the human retinal epithelial cell line ARPE-19 (ATCC, CRL-2302) (Figure 11). ARPE-19 cells were cultured at 37°C under humidified 5% CO2 in Dulbecco's modified Eagle medium / nutrient mixture F-12 (DMEM:F-12, ATCC30-2006) supplemented with 10% v / v fetal bovine serum. A polyurethane membrane containing 0.1% zinc diethyldithiocarbamate (ZDEC) (Hadano Research Institute, Food and Drug Safety Center, Japan, batch A-202K) was used as the positive standard (RM), and a high-density polyethylene membrane (Hadano Research Institute, Food and Drug Safety Center, Japan, batch C-141) was used as the negative RM. Cells were seeded at a density of 20,000 cells / well in 24-well plates and incubated overnight at 37°C under humidified 5% CO2. PCL and PCL-g-PDA membranes, along with RM control (7 mm in diameter, less than 0.5 mm thick), were irradiated twice on each side at λ=254 nm for 2 minutes to remove contamination. The membranes were added to the wells and incubated with cells for 48 hours. The membranes were removed, the culture medium was replaced with PrestoBlue® (PB) solution (10% in cell medium), and incubated for 30 minutes. The PB assay was performed using fluorescence (λex=558 nm, λem=593 nm). Each experiment was repeated four times.

[0110] The percentage of cell viability was calculated using the following formula (1): TIFF0007872272000012.tif13128

[0111] Example 6: In vitro degradation The degradation dynamics were studied in vitro on a polymer membrane under standard conditions (PBS at pH=7.4) and accelerated conditions (HCl aqueous solution (2M) at pH=1) at 37°C for 75 days. Following ISO-10993-13, the membrane was cut into implants (dimensions = 10 × 4 mm, thickness = 0.3~0.5 mm), and weighed (15 mg, w). dry,t0 The implant was immersed in 0.75 mL of culture medium while being stirred. At predetermined intervals, the implant was removed from the medium, washed with water, wiped dry, and its weight was measured to determine the wet mass (w we,t) is determined, and then dried under reduced pressure until a certain mass is reached to obtain the dry mass (w dry,tx The following was determined. These experiments were repeated three times. Water uptake was calculated from equation (2), the remaining weight from equation (3), and the remaining molecular weight from equation (4). pH was evaluated using a pH meter at 20°C. TIFF0007872272000013.tif46128

[0112] result: At pH=7.4, the PCL-g-PDA implant maintained its initial mass (Figure 12), molecular weight (Figure 13), and shape (Figure 14) without incorporating water (Figure 15), and the pH remained stable for 110 days (Figure 16). At pH=1, the PCL-g-PDA implant immediately lost its initial mass (Figure 17) and molecular weight (Figure 18), and became brittle and broken (Figure 19). The PCL-g-PDA implant is biodegradable and is expected to degrade very slowly in vitro under standard conditions without changing the local pH value.

[0113] Example 7: Synthesis of PCL-b-PEG-b-PCL("T") In a dry Schlenk flask, PEG (5.0 g, 2.5 mmol, Mn = 2000 g / mol) was solubilized in 55 mL of dry toluene under an argon atmosphere. Then, Sn(Oct)2 (0.20 g, 0.5 mmol) and ε-Cl monomer (4.99 g, 43.8 mmol, 17.5 equivalents) were added under an inert atmosphere. Water and oxygen were removed by three freeze-pump-thaw cycles. The reaction was carried out at 100°C for 24 hours under a flow of argon and strong stirring. The reaction was stopped by adding a few drops of HCl solution (0.1 M in methanol). The product was precipitated in cold diethyl ether, filtered, and dried under reduced pressure.

[0114] The molecular weight of the triblock copolymer was calculated according to the following equations (5) to (8): TIFF0007872272000014.tif34128

[0115] The molecular weight of the ethylene glycol unit is 44 g / mol, and the molecular weight of the ε-caprolactone unit is 114 g / mol.

[0116] The conversion rate is calculated by comparing the DPCL obtained by NMR after purification with the theoretical value. The yield (η) is calculated by comparing the mass of the obtained polymer with the theoretical mass value of the polymer obtained considering the conversion rate calculated by NMR. TIFF0007872272000015.tif27128 conversion: 86% Yield: 93% 1 H NMR (600 MHz, DMSOd6): δ = 3.99 (t, R-CH2-O-CO), 3.5 (m, R-CH2-O), 2.27 (t, R-CH2-CO-O), 1.54 (m, R-CH2-CH2-CO), 1.30 (m, R-CH2-R) SEC(THF, RI, PS): Mn=6143g / mol, D=1.09 SEC (DMF, RI, PEG): Mn = 3529 g / mol, D = 1.07

[0117] Similar to the method described above, the following PCL-b-PEG-b-PCL polymers were synthesized (see Table 4). In Table 4, a PCL-b-PEG-b-PCL type polymer consisting of 1000 g / mol of PEG and 2000 g / mol of PCL is defined as "1000-2000-1000". Furthermore, PCL-b-PEG-b-PCL copolymers are designated as "T" in this specification.

[0118] [Table 4]

[0119] Example 8: Synthesis of iodized (PCL-I)-b-PEG-b-(PCL-I) ("TI") In a typical experiment, the polymer obtained from Example 7, for example, PCL-b-PEG-b-PCL (4g, M), registration number 5 in Table 4, is used. n,NMR3710 g / mol (1.08 mmol, 16.2 mmol in CL units) and anhydrous THF (200 mL) were introduced into a dry conical reactor and placed under an argon flow until completely dissolved. Then, under argon, the solution was cooled to -50°C by immersion in a liquid nitrogen / ethanol mixture before adding LDA (8.09 mL, 16.2 mmol). After 30 minutes of reaction, the minimum amount of iodine solution in anhydrous THF (4.10 g, 1.62 mmol) was injected through the septum using a syringe, and the mixture was stirred and maintained at -50°C under an argon atmosphere. After 30 minutes, the reaction was stopped by adding aqueous solution of NH4Cl (2 M, 200 mL), the temperature was raised to 0°C, and HCl was added. (aq) (37%) was added to reach a neutral pH. The polymer was then extracted from the solution by washing three times with dichloromethane (3 × 200 mL) in a separatory funnel. The organic phase was collected, washed three times with Na2S2O3 solution (0.3 M, 3 × 100 mL), dried using MgSO4 powder, filtered, and concentrated under reduced pressure. The polymer was precipitated in cold diethyl ether, filtered, and dried under reduced pressure. Substitution: 23 mol% Yield: 50% by weight 1 H NMR (600 MHz, DMSOd6): δ = 4.44 (m, R-CHI-CO-O), 3.99 (t, R-CH2-O-CO), 3.50 (m, R-CH2-O), 2.27 (t, R-CH2-CO-O), 1.87 (m, R-CH2-CHI), 1.54 (m, R-CH2-CH2-CO), 1.30 (m, R-CH2-R) SEC(THF, RI, PS): Mn=5760g / mol, D=1.28 SEC (DMF, RI, PEG): Mn = 3500 g / mol, D = 1.24

[0120] Example 9: Synthesis of (PCL-g-PDA)-b-PEG-b-(PCL-g-PDA) ("T-PDA") In a typical experiment, the iodized polymer obtained from Example 8, for example, (PCL-I)-b-PEG-b-(PCL-I) (1.5 g, 1.07 mmol of iodized Cl units), was mixed with DMSO (20 mL) in the first Schlenk flask (Schlenk flask A). In the second Schlenk flask (Schlenk flask B), dopamine hydrochloride (5.09 g, 25 equivalents relative to iodized Cl units, 26.8 mmol), PMDETA (340 μL, 1.5 equivalents relative to iodized Cl units, 26.8 mmol), Na2CO3 (300.0 mg), BPO (6.49 g, 25 equivalents relative to iodized Cl units, 26.8 mmol), and DMSO (37 mL) were added. The solution was stirred and left under an argon stream for 4 hours. Then, oxygen was removed by three freeze-pump-thaw cycles. The iodized PCL solution was transferred to flask B, and copper(I) bromide (0.23 g, 1.5 equivalents relative to the iodized Cl, 1.61 mmol) was added. The flask was then immersed in an oil bath at 70°C and vigorously stirred for 48 hours. The reaction was stopped by cooling in a liquid nitrogen bath. The solution was concentrated by evaporating DMSO under vacuum at 70°C. The polymer was dialyzed in water and freeze-dried. PDA: 38~49% by weight 1 H NMR (600 MHz, DMSOd6): δ= 4.57 (m, R-CH(PDA)-CO), 3.99 (t, R-CH2-O-CO), 3.5 (m, R-CH2-O), 2.27 (t, R-CH2-CO-O), 1.96 (m, R-CH2-CH(PDA)), 1.84 (m, R-CH2-CH(PDA)), 1.54 (m, R-CH2-CH2-CO), 1.30 (m, R-CH2-R) SEC (DMF, RI, PEG): Mn = 3000 g / mol, D = 1.24

[0121] In the NMR spectrum, the disappearance of the 4.44 ppm and 1.87 ppm peaks, characteristic signals from iodine-assisted PCL functionalization (see Example 8), indicates a change in the polymer's chemical environment after dopamine introduction and polymerization. Furthermore, the appearance of peaks at 4.57 ppm, 1.96 ppm, and 1.84 ppm also supports this change in chemical environment. 1 The 1H-NMR spectra of TI and T-PDA based on polymer T, registration number 5 in Table 4, are shown in Figure 20. To confirm that the changes in chemical shift may be due to the effective grafting of PDA side chains onto the PCL backbone, diffusion-ordered NMR spectroscopy (DOSY NMR) analysis was performed. The peaks at 4.57 ppm, 1.96 ppm, and 1.84 ppm showed the same diffusion coefficient (D = -6.02 * 10⁻¹¹ m² s⁻¹) as the peaks attributed to T, demonstrating PDA grafting onto the PCL chain (Figure 21).

[0122] The molecular weight of this T-PDA was further analyzed by SEC in DMF using UV detection at 450 nm. It is important to note that while previous copolymers were analyzed by SEC in THF, the PDA-containing copolymer is insoluble in THF.

[0123] The PDA content is quantified using the following modified formula, according to the TGA method described in Example 2: TIFF0007872272000017.tif16133

[0124] The proportion of PDA is 38% by weight. This high value includes the mass of free and grafted PDA in the copolymer, which is consistent with the intensity of the peaks detected by NMR (Figures 20 and 21). The respective proportions of grafted PDA and free oligo-PDA are unknown.

[0125] Example 10: Protein Stability The stability of the mAb was evaluated using formulations consisting of 5% (w / v) copolymer (T or T-PDA) in HBS:PEG400 1:1 (v / v). The formulations contain either 40 mg / ml (high dose (HD)) or 13 mg / ml (low dose (LD)) of mAb. In this example, the formulations are defined as formulation XY, where X is a letter referring to the copolymer (T, T-PDA, T / T-PDA) and Y is a number referring to the mAb dose (LD, HD). The "T" polymer used herein refers to registration number 6 in Table 4 of Example 7, and the resulting T-PDA can be obtained by the method described in Example 9. The transition of the SEC characteristic parameters for each formulation is shown in Figure 22. Details of the formulations are shown in Table 5.

[0126] [Table 5]

[0127] For formulation T-HD, the sample solution was white due to the presence of the copolymer's white powder, but became transparent after the addition of the mobile phase. On days 0 and 3, the absorbance at 280 nm, the relative wavelength ratio, and the relative AUC were nearly constant. Subsequently, from day 3 onwards, the mAb intensity and relative AUC gradually decreased, but the relative wavelength ratio remained constant. These results suggest an interaction between the mAb and the copolymer that reduces the amount of mAb detected, which is consistent with the results of pre-formulation studies.

[0128] For formulation T / T-PDA-HD, the sample solution was black and became blurred after the addition of the mobile phase. From day 0 to day 30, the absorbance and relative AUC of the detected mAbs gradually decreased, but the relative wavelength ratio remained constant, suggesting interaction between the mAbs and the copolymer. Note that the absorbance of T / T-PDA-HD on day 0 was divided by 2 compared to the T-HD formulation, indicating a stronger initial interaction of mAbs with the T and T-PDA mixture. Furthermore, the decrease in the amount of mAbs detected from day 0 to day 30 was greater for formulation T / T-PDA-HD (93% mAb detection loss) than for formulation T-HD (63% mAb detection loss).

[0129] For the formulation T-PDA-HD, the absorbance was approximately 5% at 280 nm, but the wavelength ratio at 254 nm was insufficient to calculate the wavelength ratio on day 0. Subsequently, no mAbs were detected from day 3 to day 30. This suggests a strong interaction between the mAb and T-PDA. Regarding pre-formulation stability studies, a gradual decrease in mAbs was observed in the presence of the copolymer. These results support the strong affinity of the PDA-based copolymer for the mAb.

[0130] As a result, a comparison of SEC-UV spectra demonstrated that the three formulations tested, namely HBS:PEG400 (1:1) and 10% (w / v) copolymers (T, T / T-PDA, and T-PDA), interacted with the mAb without denaturing it. The greatest decrease in the amount of mAb was observed in the presence of T-PDA, demonstrating the high affinity of the mAb for PDA.

[0131] Example 11: In-situ formation of depot The in-situ depot formation and behavior of formulations T-HD, T / T-PDA-HD, and T-PDA-HD are shown in Figures 23-A, 23-B, and 23-C, respectively. Each LD formulation appeared similar. The names used in the examples (e.g., T-HD) are as established in Example 10.

[0132] Immediately after injection (day 0), in-situ depot formation at the bottom of the vial could be observed. Formulation T formed a gel-like precipitated white mass on day 3, due to a solvent exchange mechanism in which PEG400 diffused into PBS. The aggregate appeared to become smaller from day 5 to day 30. Formulation T / T-PDA formed a smaller (due to a lower amount of T) and black (due to T-PDA) mass on day 3, and its appearance remained similar until day 30. Slight discoloration of the releasing medium was observed, likely due to the release of PDA-based impurities (e.g., those detected by SEC-UV corresponding to additional peaks). Formulation T-PDA formed a thin film, some of which adhered to the bottom, on day 3, but its appearance remained similar until day 30. Discoloration of the medium induced by Formulation T / T-PDA and T-PDA may be a consideration for ocular administration. However, this problem appears to be solvable by introducing a further purification step for the T-PDA polymer used, and does not affect the overall proof of the principle demonstrated in this example.

[0133] Preliminary in vitro release of mAbs under physiological conditions demonstrated the ability of T-PDA to strongly interact with mAbs. mAbs not bound to the copolymer were released in a burst effect 3 days prior, while bound mAbs remained unreleased for up to 30 days. T formulations favored the stability of released mAbs, while T-PDA-based formulations tended to destabilize some mAbs at 37°C in the release medium.

[0134] In conclusion, this embodiment demonstrates that T-PDA offers an interesting perspective in terms of the injectability of monoclonal antibodies or their fragments (e.g., a 30G needle) for IVT administration, and in particular the stability of such antibodies during storage at 4°C.

Claims

1. A pharmaceutical preparation for use in the treatment of an eye disease or eye disorder, comprising a pharmaceutically active ingredient and a copolymer consisting of poly(ε-caprolactone) (PCL) and polydopamine (PDA).

2. The pharmaceutical preparation according to claim 1, wherein the copolymer comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

3. The pharmaceutical preparation according to claim 1 or 2, wherein the copolymer is a PCL-g-PDA copolymer, and the PCL-g-PDA copolymer contains PCL having a molecular weight in the range of 1,000 g / mol to 200,000 g / mol.

4. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the copolymer is a PCL-g-PDA copolymer, and the PCL-g-PDA copolymer comprises a PCL skeleton having a molecular weight of 1,000 g / mol to 200,000 g / mol and branches of PDA having a mass content of 0.1 to 50% by weight.

5. A method for producing a graft copolymer (PCL-g-PDA copolymer) comprising poly(ε-caprolactone) (PCL) and polydopamine (PDA), A method characterized in that PCL having a molar percentage of halogenated PCL units in the range of 0.1 to 50 mol% reacts with a PDA precursor.

6. The pharmaceutical preparation according to any one of claims 1 to 4, wherein the copolymer is a PCL-g-PDA copolymer, and the PCL-g-PDA copolymer is a carrier for the sustained release of the pharmaceutical active ingredient.

7. The pharmaceutical preparation according to claim 6, wherein the pharmaceutical preparation is an intravitreal implant.

8. The pharmaceutical preparation according to claim 6 or 7, wherein the pharmaceutically active ingredient is a small molecule and is present in the pharmaceutical preparation or intravitreal implant in an amount of 10% by weight or more.

9. A polymer of the (PCL-g-PDA) type, having a structure in which two PCL-g-PDA chains are bonded to a PEG chain.

10. The polymer according to claim 9, wherein the PEG chain has a molecular weight of up to 20,000 g / mol, and both PCL-g-PDA chains have the same molecular weight.

11. Polymer of formula (II): [In the formula, p is 3 to 397, r is between 1 and 170. m is between 1 and 170.

12. A pharmaceutical preparation comprising a pharmaceutically active ingredient and a polymer according to any one of claims 9 to 11.

13. The pharmaceutical preparation according to claim 12, wherein the pharmaceutical preparation forms an in situ gel depot for sustained release of the pharmaceutical active ingredient upon intraocular injection.

14. The pharmaceutical preparation according to claim 13, wherein the pharmaceutically active ingredient is an antibody.

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