CN116601211A - Novel polymers and their use - Google Patents

Abstract

Disclosed herein are novel copolymers with poly (epsilon-caprolactone) (PCL) and Polydopamine (PDA), optionally further comprising PEG chains, methods for preparing the same, and their use in pharmaceutical formulations, especially implants or in situ gelling reservoirs, for treating ocular disorders or ocular diseases.

Description

Novel polymers and their use

The present invention relates generally to the field of polymers for use as pharmaceutical carriers, in particular for application in the eye.

Worldwide, it is estimated that about 3900 tens of thousands of people are blind, and 2.46 hundred million people are visually impaired. Most of them are people over 50 years old. The main causes of partial and total vision loss are from uncorrected refractive errors and cataracts, respectively ¹ . The major vision posterior segment disorders are age-related macular degeneration, diabetic retinopathy and uveitis. Drugs such as corticosteroids treat these conditions. Some drugs are loaded into implantable polymeric devices for delivery over an extended period of time ² . In biodegradable polymer systems, drug release is controlled by diffusion and polymer degradation. For example, the number of the cells to be processed,is the first biodegradable intravitreal implant approved by the FDA for the treatment of diabetic macular edema, non-infectious uveitis. Based on- >Technology, dexamethasone was loaded into poly (lactic-co-glycolic acid) (PLGA) matrix. The drug is released rapidly in the first two months and then released slowly in the next four months. However, PLGA degradation results in implant fragmentation and acid partial release, which are two possible factors for ocular tissue inflammation. Some non-biodegradable polymers are known in the art, e.g. for 36 months drug release of fluocinolone acetonide in DME +.>Intravitreal micro-implants. There is thus still a need to develop novel polymers with improved stability, tolerance and release profile.

Disclosure of Invention

In one embodiment, the present invention provides novel copolymers composed of poly (epsilon-caprolactone) (PCL) and Polydopamine (PDA).

In one embodiment, the novel copolymer consisting of poly (epsilon-caprolactone) (PCL) and Polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

In one embodiment, the present invention provides a process for preparing a graft copolymer as disclosed herein.

In another embodiment, the present invention provides novel PCL-g-PDA copolymers for use in pharmaceutical formulations, particularly as carriers for sustained release of active ingredients.

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

In another embodiment, the present invention provides novel PCL-g-PDA copolymers for use in treating ocular diseases or disorders.

In another embodiment, the present invention provides novel PCL-g-PDA copolymers for use as intravitreal implants.

In another embodiment, the present invention provides a novel PCL-g-PDA, wherein two PCL-g-PDA chains are linked to a PEG chain to form a (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) type polymer.

In another embodiment, the invention provides a polymer of the type (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA), wherein the PEG chains have a molecular weight as defined herein, and the PCL-g-PDA chains all have the same molecular weight.

In another embodiment, the present invention provides a polymer of the type (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) for use in a pharmaceutical formulation, preferably for use in forming an in situ gelling reservoir for use in a pharmaceutical formulation that continuously releases an active pharmaceutical ingredient upon injection into the eye, and wherein the active pharmaceutical ingredient is an antibody.

Drawings

Fig. 1: iodinated PCL (PCL-I) in CDCL ₃ In (a) and (b) ¹ H NMR spectrum.

Fig. 2: size exclusion chromatography in THF on iodinated PCL using RI detection and uv detection at λ290 nm.

Fig. 3: PCL-g-PDA in DMSO-d ₆ In (a) and (b) ¹ H NMR。

Fig. 4: PCL-g-PDA after purification in DMSO-d ₆ DOSY NMR in (a).

Fig. 5: size exclusion chromatography in DMSO using UV detection at λ=350 nm for the initial commercial PCL, PCL-g-PDA copolymer and oligomeric PDA.

Fig. 6: thermogravimetric analysis (TGA) of PCL-g-PDA was performed at 20 ℃/min from 30 ℃ to 700 ℃ under nitrogen atmosphere.

Fig. 7: differential Scanning Calorimetry (DSC) thermogram of PCL-g-PDA: firstly heating and ramping up from-80 ℃ to 150 ℃ at 10 ℃/min, and then cooling and ramping down from 150 ℃ to-80 ℃ at 10 ℃/min.

Fig. 8: PCL-g-PDA (one pin equals 1.2cm, film thickness about 500 μm) containing 30wt.% of DEX prepared by film casting and pressed at 130 ℃ during 15mn at 4 tons.

Fig. 9: cumulative release of (A) dexamethasone (DEX 30) and (B) CIP 30) CIP from PCL and PCL-g-PDA implants as a function of implant composition. Data are expressed as the mean of the results obtained from HPCL measurements (mean ± SD; n=3). PDA content was estimated to be 5wt.% by TGA.

Fig. 10: survival of L929 cells after 24 hours incubation with PCL-g-PDA membrane. Percentage obtained from fluorescence intensity after PrestoBlue test.

Fig. 11: survival of human retinal epithelial cell line ARPE-19 (ATCC, CRL-2302) after 48 hours incubation with PCL or PCL-g-PDA membranes.

Fig. 12: residual mass of PCL-g-PDA during standard condition (PBS, 37 ℃, ph=7.4) degradation study

Fig. 13: residual molecular weight of PCL-g-PDA during standard condition (PBS, 37 ℃, ph=7.4) degradation study

Fig. 14: photographs of PCL-g-PDA implants after 110 days of immersion in standard conditions (PBS, 37 ℃, ph=7.4)

Fig. 15: swelling degree of PCL-g-PDA during degradation study under standard conditions (PBS, 37 ℃, ph=7.4)

Fig. 16: degradation study of the pH of the degradation medium during PCL-g-PDA implants under standard conditions (PBS, 37 ℃, ph=7.4)

Fig. 17: residual mass of PCL-g-PDA during accelerated condition (HCl (2M), 37 ℃, ph=1) degradation study

Fig. 18: residual molecular weight of PCL-g-PDA during accelerated condition (HCl (2M), 37 ℃, ph=1) degradation study

Fig. 19: photographs of PCL-g-PDA implants after 60 days of immersion under accelerated conditions (HCl (2M), 37 ℃, ph=1)

Fig. 20: T-PDA in DMSO-d ₆ In (a) and (b) ¹ H NMR。

Fig. 21: T-PDA in DMSO-d ₆ DOSY NMR in (C)

Fig. 22: size exclusion chromatography at 280nm during stability studies of mabs in formulations consisting of HBS: pe400=1:1 containing (a) T-HD, (B) T/T-PDA-HD (2:1) and (C) T-PDA-HD.

Fig. 23: the in situ depot of formulations (A) T-HD or (B) T/T-PDA-HD (2:1) and (C) T-PDA-HD developed and evolved in appearance over a 30 day period.

Detailed Description

Most ocular diseases are conditions or disorders that interfere with the proper functioning of the eye and/or negatively affect visual clarity and are a major public health problem. Intravitreal (IVT) administration (including implantation of medical devices or injection of suspensions, solutions or implants) is the conventional method and the most effective method of delivering APIs to the retina. The main challenges of IVT administration are reducing the frequency of injection to improve patient compliance and compliance with the treatment, ocular tolerance of the formulation, and stability of the biologic. Currently, meeting all specifications remains challenging, and formulations based on polymer technology are non-mainstream, emerging recently, but may solve these problems. Scientists are working to develop biocompatible (bio) degradable or non-degradable formulations to provide long and sustained API delivery with minimal surgical procedures to increase patient well-being and reach therapeutic levels effective in treating ocular diseases.

The object of the present invention is to evaluate the benefits of incorporating PDA units into the design of novel copolymers for ophthalmic use under improved tolerability (of small or large molecules) and excellent sustained release characteristics that benefit from preferential drug-PDA interactions. Among polymers used in medical and/or drug delivery applications, formulations based on degradable compositions exhibit interesting and promising properties. In particular, PCL is biodegradable, slowly degrading, can be functionalized and is FDA approved for medical purposes (but not yet used in ophthalmology). Furthermore, PEG is bioremovable (in terms of its molecular weight), FDA approved for ophthalmic applications, and provides tunable gelation properties in combination with PCL.

The inventors developed two strategies: one is a solid formulation for delivery of small molecules (chapter I) and the other is an in situ gelling system for delivery of biological agents (chapter II).

Chapter I: PCL-g-PDA solid implant

The solid implant approach provides a hydrophobic graft copolymer PCL-g-PDA. The copolymer is synthesized in a two-step process. First, PCL (mn=190 000 g/mol) was post-functionalized by iodine via electrophilic substitution to give iodinated PCL (PCL-I). Next, PCL-I was functionalized by PDA under ATRP-like oxidative and basic conditions to give PCL-g-PDA copolymers containing about 3-5wt% of grafted PDA. In vitro cytotoxicity assays showed that the implant PCL-g-PDA was not cytotoxic to mouse fibroblasts and retinal cells. Furthermore, the implant did not degrade under physiological conditions within 110 days, but degraded under accelerated conditions, demonstrating the ability of PCL-g-PDA implants to degrade slowly. In vitro, PCL-g-PDA implants showed sustained, constant and complete release (zero order kinetics) of water insoluble dexamethasone (dl=30% w/w) over a period of 155 days. In contrast, PCL-g-PDA implants showed an abrupt effect over a period of 125 days, followed by sustained release of the water-soluble salt ciprofloxacin (DL =30%w/w), it is inferred that the release is complete after about 500 days. In all cases, the release kinetics of the PCL-g-PDA implant were slower compared to the PCL implant, thus demonstrating the ability of the PDA to retain the drug inside the implant (even with a small amount of grafted PDA). In addition, with the commercial PLGA-based implants (Ozurdex ^TM ) In comparison, PCL implants and PCL-g-PDA implants exhibit longer release times of dexamethasone.

Thus, according to the present invention, a new intravitreal implant is provided which can provide sustained drug delivery over several months and has slow degradation to avoid changing the microenvironment medium and to avoid fragmentation. In one embodiment, the sustained drug delivery is provided for at least 2 months or at least 3 months or at least 6 months or at least 12 months or at least 18 months or at least 24 months or at least 36 months. The implant is made of poly (epsilon-caprolactone) (PCL) and polydopamine ³ (PDA) preparation. PCL is a biocompatible, hydrophobic and FDA approved polymer. It slowly degrades by hydrolysis, covering extended and longer release periods. Melanin is located in retinal cells and is involved in biological functions ⁴ . PDA is a biocompatible that may exhibit the same properties as melanin, particularly drug binding properties ⁵ Synthesis of melanin-like polymers. The inventors have found that combining these two polymers results in improved biocompatibility or tolerability and sustained release with limited microenvironment changes.

Thus, in one embodiment, the present invention provides novel copolymers composed of poly (ε -caprolactone) (PCL) and Polydopamine (PDA). In another embodiment, the novel copolymer consisting of poly (. Epsilon. -caprolactone) (PCL) and Polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

As used herein, the term PCL means poly (epsilon-caprolactone). In one embodiment, the PCL has a molecular weight in the range of 1000g/mol to 200 g/mol. In another embodiment, the PCL has a molecular weight in the range of 10 to 100 000 g/mol.

As used herein, the term PDA means polydopamine.

The term graft copolymer (or graft polymer) means a multiblock copolymer having a linear or branched backbone of one complex (e.g., PCL) and randomly distributed branches of another complex (e.g., PDA). In one embodiment, the PCL backbone is linear.

In one embodiment, the PCL-g-PDA copolymer according to the present invention comprises PCL having a molecular weight in the range of 1000g/mol to 200 g/mol or 10 g/mol to 100 g/mol.

Prior to polymerization with PDA, PCL is chemically modified (e.g., by electrophilic substitution) to produce a halide, e.g., iodinated PCL ⁶ . Thus, in one embodiment, the copolymer according to the invention is obtained using a halogenated PCL having a molecular weight in the range of 1000g/mol to 100 g/mol or 2500g/mol to 50 g/mol and a molar percentage of iodinated epsilon-caprolactone units in the range of 0.1 to 50mol.% or 1 to 20 mol.%. The term "halogenation" has the ordinary meaning known to those skilled in the art. In one aspect, the PCL is brominated, chlorinated, or iodinated.

In another embodiment, the PDA mass content of the PDA-g-PCL of the present invention ranges from 0.1 to 50wt.% or from 1 to 20wt.% or from 1 to 15wt.% or from 1 to 10wt.% or about 5wt.% PDA.

The term wt.% (or wt%) has its ordinary meaning to those skilled in the art of polymer chemistry. Preferably, wt.% means mass relative to the total mass of the graft copolymer. Unless explicitly stated otherwise, the wt.% of PDA content in the graft copolymers of the present invention is calculated after purification and measured by TG analysis, as described herein, for example.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of 1000g/mol to 200 g/mol and random branches of PDA in a mass content of 0.1 to 50 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of 1000g/mol to 200 g/mol and random branches of PDA in a mass content of 1 to 20 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of 1000g/mol to 200 g/mol and random branches of PDA in a mass content of 1 to 10 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of 1000g/mol to 200 g/mol and random branches of PDA in a mass content of about 5 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of from 10 to 100 g/mol and random branches of PDA in a mass content of from 0.1 to 50 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of from 10 to 100 g/mol and random branches of PDA in a mass content of from 1 to 20 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of from 10 to 100 g/mol and random branches of PDA in a mass content of from 1 to 10 wt.%.

In another embodiment, the graft copolymer according to the invention consists of a PCL backbone having a molecular weight of from 10 to 100 g/mol and random branches of PDA in a mass content of about 5 wt.%.

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

In yet another embodiment, the present invention provides a polymer of formula (I)

HO-[-(CH ₂ ) ₄ -CH(PDA)-C(O)-O-] _r -[-(CH ₂ ) ₅ -C(O)-O-] _p -H (I)

Wherein p=23-1580 and r=1-395; and wherein the PDA is present at up to 5wt.% or at about 3 or 5 wt.%.

In another embodiment, the present invention provides a process for preparing the graft copolymers of the present invention. In one embodiment, the PCL backbone is first chemically modified (e.g., iodinated) and then reacted with a suitable PDA precursorTo obtain the copolymer according to the invention. Any suitable PDA precursor known to those skilled in the art of polymer chemistry may be used. In one embodiment, the PDA precursor is dopamine hydrochloride. The polymerization is carried out according to conditions known to the person skilled in the art and further disclosed in the accompanying working examples. In one embodiment, the polymerization is conducted under oxidizing and alkaline conditions to produce PDA grafted PCL (PCL-g-PDA) ⁷ The PDA-grafted PCL was then further purified.

Accordingly, the present invention provides a process for preparing the PCL-g-PDA copolymers of the present invention characterized in that PCL having a mole percent of iodinated PCL units in the range of 0.1 to 50 mole percent or 1 to 20 mole percent is reacted with a PDA precursor (e.g., dopamine hydrochloride). In one aspect, the process is carried out under an inert atmosphere at about 70 ℃ under oxidizing and alkaline conditions and in the presence of copper (I) bromide. The graft copolymer thus obtained was purified. Purification may be carried out according to methods known to the person skilled in the art and/or as described in the accompanying working examples. In one embodiment, the purification is performed by precipitation, preferably by precipitation from methanol. The precipitation step may be repeated several times, preferably up to three times. In yet another embodiment, purification is performed by precipitation from methanol, followed by 2 trituration of PCL-g-PDA from cold methanol.

In one embodiment, the process for preparing the PCL-g-PDA copolymers of the present invention uses the specific starting materials, intermediates and reaction conditions disclosed therein as disclosed in the accompanying working examples or as disclosed in schemes 1 and 2.

In another embodiment, the present invention discloses PCL-g-PDA copolymers obtained by using the methods disclosed herein, particularly the methods disclosed in schemes 1 and 2.

Iodinated PCL may be known to those skilled in the art and as such, for example ⁶ Obtained by the method described in (a). In one embodiment, the iodinated PCL is obtained according to the method disclosed in scheme 1 and in the accompanying working examples. In yet another embodiment, the present invention provides functionalization of the PCL by post-modification. In the best mode according to the inventionIn alternative embodiments, PCL with a high molecular weight (i.e., greater than 45 g/mol) is iodinated. The use of such high molecular weight PCL is advantageous for the development of degradable solid formulations, combining the synthetic feasibility and good mechanical properties of the resulting implant in the sense that the implant is more flexible and less fragile.

The PCL-g-PDA copolymers according to the invention have valuable pharmaceutical properties. In particular, the copolymers have been found to be stable, well tolerated and suitable for sustained release of pharmaceutically active ingredients.

Thus, in another embodiment, the present invention provides copolymers of the present invention for use in pharmaceutical formulations, for example as carriers for active pharmaceutical ingredients. In one aspect, according to the invention, the pharmaceutical formulation is an implant. In another aspect, the implant is suitable for use in an eye, for example as an intravitreal implant. In another aspect, the implant may further contain another polymer, such as PCL or PLA or PLGA, and an active ingredient. In yet another aspect, the intravitreal implant consists only of the copolymers of the invention together with a suitable active ingredient.

As used herein, the term active pharmaceutical ingredient means any molecule having a clinically significant pharmacological activity. In one embodiment, the active pharmaceutical ingredient is a small molecule, as defined by the rule of five (rule of five) of librisky (Lipinski). In another embodiment, the active pharmaceutical ingredient is a drug approved for the treatment of ocular diseases (e.g., glaucoma, cataract, age-related macular degeneration, diabetic retinopathy, and uveitis). In another embodiment, the active pharmaceutical ingredient is selected from the group consisting of ganciclovir, dexamethasone, fluocinolone acetonide, and cyclosporine a.

According to the invention, the active pharmaceutical ingredient is present in the implant in an amount of not less than 10% by weight or 10 to 60% by weight or 10 to 30% by weight.

In another embodiment, the invention provides the use of the copolymers of the invention for the preparation of a medicament (e.g., an implant for the treatment of an ocular disease) or for intravitreal administration of a medicament. In one aspect, such administration is sustained release over time as defined herein.

In another embodiment, the present invention provides a method for treating an ocular disease by placing an implant comprising the copolymer of the present invention or an approved drug loaded with a suitable active pharmaceutical ingredient into the eye of a patient in need of such treatment.

General Synthesis of PCL-g-PDA

The PCL-g-PDA copolymers of the present invention can be obtained using the following general reaction scheme. In a first step, iodinated PCL is obtained by post-modification of PCL with iodine. Post-modification methods for functionalizing PCL are based on Nottelet et al ⁶ Is provided. The process comprises a two-step one-pot reaction as 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.

Scheme 1: synthetic protocol for iodinated PCL (PCL-I), where n=43-1755; p=23-1580 and q=2-395.

Starting from commercial PCL, a series of iodinated PCLs were prepared by targeting different molecular weights and copolymer masses, and by SEC and ¹ h NMR was used to characterize the series of PCLs. The results are summarized in table 1.

Table 1: characterization of iodinated PCL (PCL-I) prepared in THF by anionic activation with LDA and electrophilic substitution with iodine.

^a Determined by SEC in THF using PS standards for calibration; ^b by CDCl ₃ In (a) and (b) ¹ Determination by H NMR

In a second step, based on the method of Cho et al ⁷ The adapted conditions PCL-g-PDA was obtained according to the following reaction scheme 2:

scheme 2: synthesis of PCL-g-PDA, wherein p=23-1580; q=2-395 and r=1-395

Briefly, dopamine was introduced into Schlenk flasks containing DMSO, sodium carbonate, BPO, and PMDETA at room temperature. Sodium carbonate is used to obtain alkaline conditions. BPO is an organic peroxide that is often used as a free radical initiator to induce chain growth polymerization, and PDMETA (also written as PMDTA) is a tridentate ligand that can bind to metal cations to form complexes. After all of these components were introduced (less than one minute), the solution quickly changed from white to black, indicating oxidative polymerization of dopamine. At the same time, PCL-I was dissolved 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 reagent that binds to a ligand and allows activation of PCL-I dormant macroinitiator as in ATRP to generate free radical PCL coupled to dopamine monomer or grown PDA. The solution was heated at 70 ℃ for 48 hours and then cooled by plunging into liquid nitrogen. Most of the solvent was removed by evaporation, and the solution was then precipitated in methanol to collect the final copolymer. During precipitation, methanol blackens, indicating the presence of non-grafted PDA in DMSO solution and further dissolution of the non-grafted PDA compound in methanol.

Thus, in another embodiment, the present invention provides a process for the preparation of PCL-g-PDA, wherein the process comprises a reaction sequence comprising starting materials, intermediates, reaction partners and conditions as described in schemes 1 and 2 herein.

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 were purchased from Sigma Aldrich Co., ltd(HCl, 37%), lithium Diisopropylamide (LDA), sodium thiosulfate, benzenedimethanol, epsilon-caprolactone (epsilon CL), stannous octoate (Sn (Oct) ₂ ) Dimethyl sulfoxide (DMSO), dopamine hydrochloride, benzoyl Peroxide (BPO), copper (I) bromide, N, N, N ', N ', N ' -pentamethyl diethylenetriamine (PMDETA) and ciprofloxacin hydrochloride (CIP.HCl). Ammonium chloride, polysorbate 20 (Tween) 20 were purchased from aclos Organics. Sodium carbonate was purchased from feishier technologies (Fisher Scientific). Dexamethasone (DEX) was purchased from sigma aldrich corporation or TCI.

Characterization of

Nuclear Magnetic Resonance (NMR)

For iodinated PCL in CDCl ₃ Or DMSO-d ₆ Proton nuclear magnetic resonance spectroscopy using Bruker AMX-400MHz spectrometer ¹ H-NMR) to determine the functionalization ratio of the polymer. Diffusion ordered spectroscopy NMR (DOSY NMR) was performed to highlight individual diffusion coefficients of the substances contained in the sample and to determine the presence of residual free substances. The sample concentration was 5 to 15mg/mL.

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) apparatus equipped with a RID-20A refractive index signal detector, an SPD-20A UV/VIS detector, a PLgel MIXED-C guard column (Agilent, 5 μm, 50X 7.5 mm) and two PLgel MIXED-C columns (Agilent, 5 μm, 300X 7.5 mm). The mobile phase was THF, with a flow rate of 1.0mL. Min ^-1 . The sample loading was 100. Mu.L. Calculation of average molecular weight and dispersity Using Polystyrene (PS) as Standard

SEC DMSO: PCL-g-PDA copolymer (1 mg/mL) was filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Waters 515HPLC apparatus equipped with a Waters 410 differential refractometer, a Waters 2996 photodiode array detector, a polar-M guard column (Agilent Corp., 50X7.5 mm) and two polar-M columns (Agilent Corp., 300X7.5 mm). The mobile phase was DMSO at a flow rate of 1.0mL/min. The sample loading was 50. Mu.L. Quantification of PDA content was determined from the area ratio of the copolymer containing PDA to the oligomeric PDA itself at a specific wavelength selected in the range 254 to 400 nm.

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

Thermogravimetric analysis (TGA)

Thermal decomposition of the copolymer (0.1 to 10 mg) was studied using a thermogravimetric analyzer (TGA Q500 v20.13 build 39). The sample was heated from 30 ℃ to 700 ℃ at 20 ℃/min under a nitrogen atmosphere.

Differential Scanning Calorimetry (DSC)

Samples (1 to 10 mg) of each copolymer were placed in an aluminum pan. The sample was heated from-80 ℃ to 300 ℃ using Mettler Toledo DSC at 10 ℃/min. The glass transition temperature and melting temperature of each sample were determined during the first heating cycle. Use of crystalline PCL ⁸ The melting enthalpy was used to calculate the crystallinity (χ) with reference to Δh= 139,5J/g.

High Performance Liquid Chromatography (HPLC)

The samples were filtered through a 0.45 μm PTFE Millipore filter and analyzed using Shimadzu (japan) equipment equipped with an SPD-M20A diode array detector and an HPLC C18 column (Kinetex, 2.6 μm,100A,100 x 4.6 mm). Isocratic mode was used to detect drug, 40% ACN (0.1% tfa) +60% H ₂ O (0.1% TFA) was used to detect DEX and 13% ACN (0.1% TFA) +87% H ₂ O (0.1% TFA) was used to detect CIP. After detection, the column was washed using a linear gradient until 100% ACN. The flow rate was 1.0. Mu.l/min.

Chapter II: (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA)

Based on the knowledge obtained in chapter I, the in situ gelling system method according to the present invention provides amphiphilic graft copolymers of the type (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA). First, amphiphilic triblock PCL-b-PEG-b-PCL was synthesized at various PEG and PCL chain lengths to generate in situ formed gels in water at physiological temperatures. In one embodiment, the PEG has a molecular weight of up to 20000 g/mol. In another embodiment, the PEG has a molecular weight of 1000 to 4600 g/mol. In yet another embodiment, triblock copolymers having different PEG and PCL chain lengths but the same chain length for each PCL are provided to achieve EG/CL ratios ranging from 0.30 to 2.13 and molecular weights of 4300 to 9400 g/mol. In yet another embodiment, the two PCL chains have a molecular weight between 846 and 2100g/mol, and the PEG chain has a molecular weight between 1000 and 4600 g/mol. In yet another embodiment, the two PCL chains have a molecular weight of 855 or 890g/mol, and the PEG chain has a molecular weight of 2000 g/mol. These two specific PCL-b-PEG-b-PCL showed good gelling ability at room temperature.

PCL-b-PEG-b-PCL having a distribution of 855-2000-855 (g/mol) was functionalized via iodine by electrophilic substitution to give (PCL-I) -b-PEG-b- (PCL-I), (PCL-I) -b-PEG-b- (PCL-I) which was further functionalized by PDA under ATRP-like oxidizing and basic conditions. Original (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) contained about 40wt.% PDA, including some free (ungrafted) PDA.

General Synthesis of (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA)

Copolymers of the type (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) according to the invention (chapter II) (also denoted herein as "T-PDA") can be synthesized based on the following general schemes 3 to 5.

Scheme 3: synthesis of PCL-b-PEG-b-PCL (m=22-455, n=3-568)

Commercial PEG-diol was used as initiator (scheme 3) and Sn (Oct) ₂ As a catalyst, a triblock copolymer PCL-b-PEG-b-PCL (also denoted herein as "T") was synthesized by Ring Opening Polymerization (ROP) in anhydrous toluene of epsilon-CL. The solution was stirred at 100 ℃ for 24 hours and precipitated in cold diethyl ether, filtered and dried under vacuum.

Post-polymerization modifications leading to functionalization of PCL with iodine are based on Nottelet et al ⁶ Described work and classSimilar to the method presented in chapter I. The process consisted of a two-step one-pot reaction as described in scheme 4.

Scheme 4: ( Synthesis of PCL-I) -b-PEG-b- (PCL-I) (m=22-455; n=3-568; p=3-397 and q=1-170 )

The first step is anion activation in the presence of LDA of the most electrophilic proton of the PCL backbone, and the second step is electrophilic substitution with iodine. The resulting (PCL-I) -b-PEG-b- (PCL-I) type polymer is also denoted herein as "T-I".

Finally, functionalization of T-I with PDA is performed under conditions similar to those described in chapter I. The reaction scheme is shown in scheme 5 and the detailed conditions have also been described in example 9.

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 )

For purification, a DMSO solution containing T-PDA was precipitated in cold diethyl ether, but T-PDA was stuck to the bottom, complicating the collection of the product. In the second step, a DMSO solution containing the T-PDA is introduced into the dialysis bag and DMSO is exchanged with water to collect the T-PDA. Dialysis was maintained at this stage as the preferred purification method to collect T-PDA for further analysis. The PDA-containing T polymer (T-PDA) is a novel type of copolymer and is one embodiment of the present invention.

Thus, in one embodiment, the present invention provides a PCL-g-PDA copolymer as defined herein (chapter I), wherein two PCL-g-PDA chains are linked to a PEG chain to obtain a graft copolymer of the type (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA). In one aspect, the PEG chain has a molecular weight as defined herein, and both PCL-g-PDA chains have the same molecular weight.

In another embodiment, the present invention provides a polymer of the T-PDA type as defined herein having formula (II)

Wherein the method comprises the steps of

p is 3 to 397

r is 1 to 170

m is 1 to 170.

The invention also provides a process for preparing the novel T-PDA polymer. In one embodiment, the present invention provides a process for preparing a polymer of the T-PDA type, wherein the process comprises a reaction sequence comprising a starting material, an intermediate, a reaction partner and the conditions as described in schemes 3 to 5 herein.

In another embodiment, the present invention also provides a T-PDA polymer obtained when using the reaction sequence (starting materials, intermediates and conditions) according to schemes 3 to 5 herein.

The T-PDA copolymers according to the invention, e.g. having formula (II), have valuable pharmaceutical properties. In particular, the copolymers have been found to be stable, well tolerated and suitable for sustained release of pharmaceutically active ingredients.

Thus, in one embodiment, the invention provides a T-PDA copolymer as defined herein (e.g. having formula (II)) for use in a pharmaceutical formulation, e.g. as a carrier for an active pharmaceutical ingredient. In one aspect, according to the invention, the pharmaceutical formulation is a depot formed by in situ gelation. In another aspect, the in situ formed reservoir is suitable for use in an eye (e.g., for intravitreal injection). In yet another aspect, this pharmaceutical formulation forms an in situ gelling reservoir for sustained release of the active pharmaceutical ingredient upon injection into the eye. In still another aspect, according to the invention, the pharmaceutical formulation is an aqueous solution or a suitable buffer (e.g. histidine buffer) comprising the T-PDA copolymer and the active pharmaceutical ingredient and further comprising PEG as co-solvent, preferably PEG400 as defined herein.

The term active pharmaceutical ingredient used in combination with a T-PDA copolymer means any molecule, preferably an antibody, having clinically significant pharmacological activity. The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody classes or structures, including, but not limited to, monoclonal antibodies (mabs), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. In one embodiment, the antibody is a monoclonal monospecific or bispecific antibody or antigen binding fragment thereof. In one embodiment, the antibody is a human antibody or a humanized antibody. In another embodiment, the antibody is any of the foregoing antibodies that may be used to treat an ocular disorder. In yet another embodiment, the antibody is a molecule with INN fariximab (faricimab).

According to the invention, the active pharmaceutical ingredient is present in the reservoir in an amount of up to 45wt.% or 5 to 45wt.% or 15 to 45wt.% or 20 to 45wt.%, wherein wt.% is relative to the T-PDA.

In another embodiment, the invention provides the use of the T-PDA copolymer of the invention (e.g., having formula (II)) for the preparation of a medicament. In one aspect, such agents are used for the treatment of ocular diseases or for intravitreal administration of drugs.

In another embodiment, the present invention provides a method for treating an ocular disease by injecting a pharmaceutical formulation comprising the T-PDA copolymer or approved drug of the present invention loaded with a suitable active pharmaceutical ingredient into the eye of a patient in need of such treatment. In one aspect, the injection is intravitreal injection.

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 aspect, this formulation further comprises a soluble co-solvent, preferably PEG400. In yet another aspect, the invention provides a formulation consisting of Histidine Buffer (HBS) and PEG400 (ratio HBS: PEG400 = 1:1 or 1:2) as solvents, the formulation containing 5 to 15, preferably 5 or 10wt.% PCL-b-PEG-b-PCL or (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) and loaded with 40mg/mL mAb. In one aspect, these formulations may be injected through a 30G needle. Stability studies of mabs showed the ability of (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) to interact with mabs during 30 days without causing denaturation thereof. In vitro, these formulations form an in situ gelling reservoir through a solvent exchange process.

As used herein, especially for the examples in chapter II, the term "PEG" means poly (ethylene glycol). According to the present invention, PEG of different molecular weights may be used. In one embodiment, the PEG has a molecular weight of up to 20000 g/mol. In another embodiment, the PEG has a molecular weight of 400 or 1000 or 1450 or 2000 or 4600 or 10000 or 20000 g/mol.

In particular for chapter II, the molecular weight of each polymer will be written in the index number. For example, 1000g/mol of PEG will be defined as PEG1000, and 2000g/mol of PCL will be defined as PCL2000. Throughout the specification, a copolymer of the PCL-b-PEG-b-PCL type (e.g., having 1000g/mol PEG and 2000g/mol each PCL) may also be denoted as 1000-2000-1000.

Unless explicitly mentioned, the terms defined in chapter I have the same meaning when used in connection with the embodiments as defined in chapter II.

Materials and methods

Many of the materials mentioned herein for the preparation of PCL-g-PDA copolymers and implants based thereon (chapter I) can also be used for the preparation of this triblock copolymer in chapter II, to which is added poly (ethylene glycol) (PEG, mn 400 or 1000 or 1 or 450 or 2 or 4600 or 10000 or 20 g/mol), toluene, stannous octoate (Sn (Oct) purchased from Sigma Aldrich Co ₂ ) Epsilon-caprolactone (epsilon-CL) and diethyl ether. PEG was dried by azeotropic distillation of toluene solution of PEG and epsilon-CL was dried with calcium hydride (CaH) at room temperature prior to use ₂ ) Dried for 48 hours and distilled under reduced pressure. PEG, ε -CL and Sn (Oct) were maintained under an argon atmosphere ₂ 。

Characterization of

Nuclear magnetic resonance spectroscopy (NMR spectroscopy), size Exclusion Chromatography (SEC) using THF as the mobile phase, differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA) methods and apparatus are similar to those described in chapter I.

Size Exclusion Chromatography (SEC) using DMF as mobile phase

Determination of the number and weight average molar masses (Mn and Mw respectively) and the dispersity of the polymers by SECMw/Mn). The sample (5 mg/ml) was filtered through a 0.45 μm PTFE Millipore filter and analyzed using a Shimadzu (Japan) device equipped with a RID-20A refractive index signal detector coupled to an SPD-20A UV/VIS detector and PLgel MIXED-C guard columns (Agilent, 5 μm, 50X 7.5 mm) and two PLgel MIXED-C columns (Agilent, 5 μm, 300X 7.5 mm). The mobile phase was DMF+0.1% LiBr. The flow rate was 1.0mL.min-1, and the sample loading was 100. Mu.L. Average molecular weight and dispersity expressed according to calibration using poly (ethylene glycol) (PEG) standards

Size exclusion chromatography of aqueous phase (aqueous phase SEC)

Samples (1 mg/ml) were filtered through a 0.20 μm RC Millipore filter and analyzed using a Shimadzu (Japan) device equipped with a RID-20A refractive index signal detector coupled to an SPD-40UV/VIS detector and a Biobasic SEC 300 guard column (Sesamer technologies (Thermo Scientific), 5 μm, 20X 8 mm) and a Biobasic SEC 300 column (Sesamer technologies, 5 μm, 150X 7.8 mm). The mobile phase is composed of HK ₂ PO ₄ /KH ₂ PO ₄ (0.1 m, ph=7). The flow rate was 0.80mL.min-1, and the sample loading was 100. Mu.L.

Injectability testing by compression measurement

Injectability tests were performed in compressed mode using an Instron 3344 with 500N trap. In the study, use was made ofHypodermic needle with size of 27G-30GFor special indications, below company (B. Braun, gremany) and 1mL disposable syringe ()>F Luer, belgo, germany). The syringe was filled with 1mL of solution and then the hypodermic needle was attached. The injection speed was set to 0.5 or 1mm/s and the injection volume was 100 μl, corresponding to a plunger displacement of 6 mm. The surrounding medium is air.

Reference to the literature

(1)Pascolini,D.；Mariotti,S.P.Global Estimates of Visual Impairment:2010.Br J Ophthalmol 2012,96(5),614–618.https://doi.org/10.1136/bjophthalmol-2011-300539.

(2)Yasin,M.N.；Svirskis,D.；Seyfoddin,A.；Rupenthal,I.D.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://doi.org/10.1016/j.jconrel.2014.09.030.

(3)Liebscher,J.；Mrówczyński,R.；Scheidt,H.A.；Filip,C.；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)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,J.H.；Shanmuganathan,K.；Ellison,C.J.Bioinspired Catecholic Copolymers for Antifouling Surface Coatings.ACS applied materials&interfaces2013,5(9),3794–3802.https://doi.org/10.1021/am400455p.

(8)Pitt,C.G.；Chasalow,F.I.；Hibionada,Y.M.；Klimas,D.M.；Schindler,A.Aliphatic Polyesters.I.The Degradation of Poly(∈-Caprolactone)in Vivo.J.Appl.Polym.Sci.1981,26(11),3779–3787.https://doi.org/10.1002/app.1981.070261124.

In general, the present invention provides novel PDA-based biomaterials to address the challenges of minimally invasive long-lasting ocular delivery using biocompatible degradable synthetic copolymers. It provides PDA-based implants and PDA-based injectable in situ gelling systems suitable for long term delivery of small molecules, thereby being promising for formulating biological agents. The invention will now be further elucidated by means of the attached working examples, which are in no way intended to limit the scope of the invention.

Examples

Example 1 Synthesis of initiators and precursors

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 anhydrous THF (40 mL) were added to a dry three-necked round bottom flask and placed in an ice bath. Isobutyryl bromide (3.49 mL,28.3 mmol) was then slowly added to the flask via the dropping funnel. A protective tube filled with calcium chloride was placed to maintain anhydrous conditions. Stirring in the presence ofThe solution was left overnight with stirring. The solution was filtered through celite and concentrated by evaporation of 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 separating funnel. The organic phase was treated with MgSO ₄ The powder was dried, filtered and dried under reduced pressure. The product was purified by filtration through silica using a mixture of ethyl acetate: heptane (30:70) as solvent. Fractions were collected and evaporated under reduced pressure. The pure fractions were collected and stored at 4 ℃ for further use.

Yield: 100 mol%

¹ H NMR(300MHz,CDCl ₃ ):δ＝4.28(t,R-CH2-O-CO),3.73(t,O-CH2-R),1.89(s,R-CH3)

Synthesis of PDA oligomers

In a typical experiment, dopamine hydrochloride (1.5 g,7.91 mmol), PMDETA (130. Mu.L, 0.63 mmol), na were added ₂ CO ₃ (402.0 mg), BPO (1.92 g,7.91 mmol) and DMSO (76 mL). The solution was left under stirring and argon flux for 4 hours. Oxygen is then removed by three freeze pump defrost 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 put in an oil bath at 70℃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 DMSO under vacuum at 70 ℃. Finally, the polymer was precipitated, filtered and dried under vacuum.

Yield: 17wt.%

¹ H NMR(600MHz,DMSOd6):δ＝6.30-7.00ppm(m,PDA)

Synthesis of iodinated Poly (epsilon-caprolactone)

The PCL backbone was anionically activated using LDA and then modified with iodine after electrophilic substitution. For this synthesis, PCL (3 g, M _n,SEC,THF, =65 g/mol,26.3mmol CL units) and anhydrous THF (300 mL) were introduced into a dry conical reactor and placed under an argon atmosphere until the PCL was completely dissolved. Then, by adding the solution to a liquid nitrogen/ethanol mixtureThe solution was cooled at-50 ℃ before LDA (13.16 mL, 1 equivalent relative to epsilon CL units, 26.3 mmol) was added under argon. After 30 minutes of reaction, a minimum amount of anhydrous THF solution of iodine (6.68 g, 1 equivalent to epsilon CL unit, 26.3 mmol) was injected through the septum with a syringe and the mixture was maintained at-50 ℃ under stirring and argon atmosphere. After 30 minutes, by addition of NH ₄ Cl _(aq) Aqueous solution (2M, 200 mL) was used to stop the reaction and the temperature was increased to 0℃after which HCl was added _(aq) (37%) to reach 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 using Na ₂ S ₂ O ₃ Solution (3X 100mL, excess) was washed three times using MgSO ₄ The powder was dried, filtered and concentrated under reduced pressure using a rotary evaporator. The polymer was precipitated in cold methanol, filtered and dried under vacuum.

Characterization:

-CDCL ₃ in (a) and (b) ¹ H NMR: determination of the functionalization ratio (fig. 1):

¹ H NMR(CDCl ₃ ,300MHz,ppm):4.30(m,R-CHI-CO),4.05(t,R-CH ₂ -O-CO),2.30(t,R-CH ₂ -CO-O),2.00(t,R-CH ₂ -CHI),1.64(m,R-CH ₂ -C-CO),1.38(m,R-CH ₂ -R)

SEC in THF: determination of number average molecular weight (FIG. 2).

Results:

the degree of substitution was calculated by comparison between formant integrals at 4.30ppm (corresponding to the ortho-proton of iodine) and at 4.05ppm (corresponding to the unsubstituted methylene group) (FIG. 1, table 2).

Table 2. Results of commercial PCL modified with iodine after precipitation in methanol. [ εCL]/[LDA]/[I ₂ ]＝1/1/1.m _PCL ＝5g。

^a The integration of the peak at 4.30ppm and the peak at 4.05ppm was used as reference for the passage through CDCl ₃ In (a) and (b) ¹ Determination by H NMR ^b Determined by SEC in THF using PS standards for calibration (fig. 2).

Example 2: synthesis of PCL-graft-PDA

Typically, in Schlenk flask A, PCL iodide (1.5 g,1.18mmol of CL unit iodide) and DMSO (20 mL) are added. In a Schlenk flask B, dopamine-HCl (5.62 g, 25 equivalents relative to the iodinated CL units, 29.6 mmol), PMDETA (370. Mu.L, 1.5 equivalents relative to the iodinated CL units, 1.78 mmol), na were added ₂ CO ₃ (300.0 mg), BPO (7.18 g, 25 equivalents relative to the iodinated CL units, 29.6 mmol) and DMSO (37 mL). The solution was left under stirring and argon flux for 4 hours. Oxygen is then removed by three freeze pump defrost cycles. The PCL iodide solution was transferred to flask B, and copper (I) bromide (255 mg, 1.5 equivalents, 1.78mmol relative to CL iodide) was added. The flask was then placed in an oil bath at 70 ℃ 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 ℃. The copolymer was precipitated in methanol, filtered and dried under vacuum.

Characterization:

-DMSO-d ₆ in (a) and (b) ¹ H NMR: chemical modification and purity highlighting of the graft copolymer (fig. 3).

-DMSO-d ₆ DOSY NMR in (a): confirmation of the interface at peak 4.58ppm (FIG. 4).

SEC in DMSO (UV at λ=350 nm): confirmation of the presence of PDA in the copolymer (fig. 5).

-TGA: quantification of PDA content (fig. 6).

-DSC: determination of the melting temperature of the copolymer (FIG. 7).

Results:

at the position of ¹ In H NMR, peaks at 4.58, 1.90 and 1.80ppm are characteristic of iodinated PCL modifications under alkaline conditions (FIG. 3) resulting from grafting onto PCL (FIG. 4). In UV-SEC analysis, PCL-g-PDA copolymer and PDA absorbed from 254 to 450nm (FIG. 5). 350nm was chosen as the wavelength to avoid residual noise. The peak intensity is proportional to the PDA content. Also measured by TGA analysisPDA content (fig. 6). The melting temperature of PCL-g-PDA was 49 ℃, as determined by DSC analysis on the first heating ramp (fig. 7). A summary of the results can be found in table 3 below.

Table 3: characteristics of PCL-g-PDA copolymer after purification in methanol as a function of initial molecular weight and degree of substitution of iodinated PCL.

^a By DMSO-d ₆ In (a) and (b) ¹ H NMR; ^b determined by SEC in THF using PS standards for calibration; ^c determined by TGA after 3 purification steps; ^d Determination by SEC in DMF using PS standards for calibration

Quantification of PDA content by TGA was based on the residual mass of PCL, PCL-g-PDA and oligomeric PDA at 600 ℃ using the apparatus as described herein. The PDA content was then calculated using the following equation:

the proportion of PDA in the three-time purified PCL-g-PDA copolymer was about 3wt.%.

Another purification method involves trituration from cold methanol: after the first purification step by precipitation from methanol, the copolymer may be further purified by trituration from cold methanol. More specifically, 300mg of copolymer was placed in a falcon tube. 45mL of cold methanol was added and the polymer powder was milled for several minutes, after which it was recovered by centrifugation (0 ℃,5000rpm,15 minutes). This step was repeated once before drying.

(trituration step yield = 50%)

The data disclosed herein (especially in table 3) do not relate to milling, but only to precipitation from methanol.

Example 3: preparation of drug-loaded implants

PCL (M) obtained in example 2 _n About 60 g/mol) or PCL-g-PDA (100 to 500 mg) and appropriate amounts of DEX or CIP.HCl (corresponding to 10% and 30% of the final weight) were dispersed in DMSO (5 to 30 mL) for intimate mixing. DMSO was removed under vacuum at 110 ℃ to produce copolymer/drug films. The film was ground and the resulting powder was deposited on a teflon sheet. The powder was pressed at 130 ℃ for 15 minutes at 4 tons (see fig. 8).

Example 4: in vitro Release study

Drug release from PCL and PCL-g-PDA membranes obtained in example 3 was assessed at 37℃in phosphate buffered saline (PBS, pH 7.4) containing 0.05% v/v Tween 20 with constant orbital shaking (100 rpm). Typically, 10mg of drug/copolymer membrane is put into 20mL of phosphate buffer solution containing 0.05% tween 20 at 37 ℃. At a particular time point, the entire release medium was removed and replaced with 20mL of fresh buffer solution. The collected samples were analyzed by HPLC using a ratio of acetonitrile/TFA (1000/1) and water/TFA (1000/1) (10:90 to 40:60) as mobile phases using UV detection at the maximum absorption wavelength of the drug (in the range of 200 to 400 nm).

Results:

the kinetics of drug release was modified based on the presence of PDA, drug selection and drug percentage in the copolymer. The PDA content in the copolymer was estimated to be between 1wt.% and 20wt.% by TG analysis. Compared to PCL-loaded membranes, the release kinetics of dexamethasone and ciprofloxacin hydrochloride were slowed in the PCL-g-PDA membrane and adjusted according to the proportion of PDA moieties in the implant. (FIGS. 9A and 9B).

Example 5: biological (cytotoxicity) study

Fibroblast cells

Cytotoxicity of PCL-g-PDA membrane was analyzed on the mouse fibroblast cell line L929 (NCTC-clone 929,ECACC 85011425). At 37℃in wet 5% CO ₂ L929 cells were cultured in 4.5g/L D-glucose DMEM supplemented with 1mM L-glutamine, 5% v/v fetal bovine serum, 100U/mL penicillin and streptomycin 100. Mu.g/mL. Polyurethane films (Bodoku institute (Hatano Research Institute), FDSC, lot B-173K) containing 0.25% Zinc Dibutyldithiocarbamate (ZDBC) were used as the filmPositive Reference Material (RM), and high density polyethylene film (bomulti field institute, FDSC, lot C-161) was used as negative RM. Cells were seeded into 24-well plates at a density of 60 000 cells/well and incubated overnight at 37 ℃. PCL and PCL-g-PDA films (6 mm diameter, less than 0.5mm thickness) were irradiated at 254nm for 2 minutes, twice on each side, for decontamination. Membranes were added to the wells and incubated with cells for 24 hours. The membrane was removed and the medium was replaced with 500. Mu.L(PB) solution (10% of the medium) and incubated for 30 min. PB assay was performed using fluorescence (λex=558 nm, λem=593 nm). Each experiment was performed in a triplicate. PCL-g-PDA copolymer purified in methanol gave cells with viability after 24 hours incubation (FIG. 10).

Human retina cells

Cytotoxicity of PCL and PCL-g-PDA membranes was analyzed on the human retinal epithelial cell line ARPE-19 (ATCC, CRL-2302) (FIG. 11). At 37℃in wet 5% CO ₂ ARPE-19 cells were cultured in Dulbecco's modified eagle's medium (Dulbecco's Modified Eagle Medium)/nutrient mixture F-12 (DMEM: F-12, ATCC 30-2006) supplemented with 10% v/v fetal bovine serum. A polyurethane film (Bodof institute, food and drug safety center (Food and Drug Safety Center), japan, lot A-202K) containing 0.1% Zinc Diethyldithiocarbamate (ZDEC) was used as a positive Reference Material (RM), and a high-density polyethylene film (Bodof institute, food and drug safety center, japan, lot C-141) was used as a negative RM. Cells were seeded into 24-well plates at a density of 20,000 cells per well and at 37 ℃ in wet 5% CO ₂ Incubate overnight. PCL and PCL-g-PDA membranes and RM controls (diameter 7mm, thickness less than 0.5 mm) were irradiated at λ=254 nm for 2 minutes, twice on each face for decontamination. Membranes were added to the wells and incubated with cells for 48 hours. Removing the membrane and replacing the culture medium with(PB) solution (10% of cell culture medium)) And incubated for 30 minutes. PB assay was performed using fluorescence (λex=558 nm, λem=593 nm). Each experiment was performed in a quadruplicate.

The percentage of cell viability was calculated using the following formula (1):

Example 6: in vitro degradation

The polymer films were subjected to in vitro studies of degradation kinetics for 75 days at 37 ℃ under standard conditions (PBS at ph=7.4) and accelerated conditions (aqueous HCl at ph=1 (2M)). The films were cut into implants (size=10×4mm, thickness=0.3-0.5 mm), weighed (15 mg, w) according to ISO-10993-13 _{Dry, t0} ) And immersed in 0.75mL of the medium with stirring. At predetermined time points, the implants were removed from the culture medium, rinsed with water, wiped and weighed to determine the wet mass (w _we,t ) And then dried under vacuum to a constant mass to determine the dry mass (w _{Dry, tx} ). These experiments were performed in a triplicate. The water absorption is calculated from equation (2), the residual weight is calculated from equation (3), and the residual molecular weight is calculated from equation (4). The pH was evaluated at 20℃using a pH meter.

Results:

at ph=7.4, the PCL-g-PDA implant maintained its initial mass (fig. 12), molecular weight (fig. 13) and shape (fig. 14) without any water absorption (fig. 15) and pH remained stable (fig. 16) during 110 days. At ph=1, PCL-g-PDA implants immediately lost their initial mass (fig. 17) and molecular weight (fig. 18), and were broken and fragile (fig. 19). PCL-g-PDA implants are degradable and are expected to degrade very slowly in vitro under standard conditions without changing the local pH.

Example 7: synthesis of PCL-b-PEG-b-PCL ("T")

In a dry Schlenk flask, PEG (5.0 g,2.5mmol, mn=2 g/mol) was dissolved in 55mL of dry toluene under an argon atmosphere. Then, sn (Oct) is added still under an inert atmosphere ₂ (0.20 g,0.5 mmol) and ε -CL monomer (4.99 g,43.8mmol,17.5 eq.). Water and oxygen were removed by three freeze pump defrost cycles. The reaction was carried out at 100℃for 24 hours under vigorous stirring with a flux of argon. The reaction was stopped by adding a few drops of HCl solution (0.1M in methanol). The product was precipitated in cold diethyl ether, filtered and dried under vacuum.

The molecular weight of the triblock copolymer was calculated according to the following equations (5) - (8):

M _{n,NMR PCL} ＝DP _CL ×114 (7)

M _{n，NMR，PCL-PEG-PCL} ＝M _n，PEG +M _n，PCL (8)

the molecular weight of the ethylene glycol unit was 44g/mol, and the molecular weight of the epsilon-caprolactone unit was 114g/mol.

The conversion was calculated by comparing DPCL obtained by NMR after purification with the theoretical value. The yield (. Eta.) is calculated by comparing the obtained polymer mass with a value of the polymeric physical theoretical mass obtained in consideration of the conversion calculated by NMR.

m _{th，PCL-PEG-PCL} ＝m _PEG +m _PCL ×T _conv (10)

Conversion rate: 86%

Yield: 93%

¹ H NMR(600MHz，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，

SEC(DMF、RI、PEG)：Mn＝3529g/mol，

Similar to the above procedure, 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 1000g/mol of PEG and 2000g/mol of PCL will be defined as "1000-2000-1000". Furthermore, the PCL-b-PEG-b-PCL copolymer will be denoted herein as "T".

Table 4: characterization of T

^a By CDCl using equation (8) ₃ In (a) and (b) ¹ H NMR; ^b determined by SEC in THF using PS standards for calibration; ^c using equation (11).

Example 8: synthesis of iodinated (PCL-I) -b-PEG-b- (PCL-I) ("T-I")

In a typical experiment, the polymer obtained from example 7,for example PCL-b-PEG-b-PCL (4 g, M) of entry No. 5 in Table 4 _n,NMR =3710 g/mol,1.08mmol,16.2mmol CL units) and anhydrous THF (200 mL) were introduced into a dry conical reactor and placed under a flux of argon until completely dissolved. The solution was then cooled at-50 ℃ by pouring the solution into a liquid nitrogen/ethanol mixture, after which LDA (8.09 ml,16.2 mmol) was added under argon. After 30 minutes of reaction, a solution of iodine in a minimum amount of anhydrous THF (4.10 g,1.62 mmol) was injected through a septum with a syringe and the mixture was maintained at-50 ℃ under stirring and argon atmosphere. After 30 minutes, by addition of NH ₄ Aqueous Cl (2M, 200 mL) was used to stop the reaction and the temperature was increased to 0deg.C before HCl was added _(aq) (37%) to reach neutral pH. Then, the polymer was extracted from the solution by washing three times with dichloromethane (3×200 mL) in a separating funnel. The organic phase was collected using Na ₂ S ₂ O ₃ The solution (0.3M, 3X 100 mL) was washed three times with MgSO ₄ The powder was dried, filtered and concentrated under reduced pressure. The polymer was precipitated in cold diethyl ether, filtered and dried under vacuum.

Substitution: 23 mol%

Yield: 50wt.%

¹ H NMR(600MHz,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＝5 760g/mol，

SEC(DMF、RI、PEG)：Mn＝3 500g/mol，

Example 9: synthesis of (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) ("T-PDA")

In a typical experiment, the iodinated polymer obtained from example 8, e.g. (PCL-I) -b-PEG-b- (PCL-I) (1.5 g,1.07mmol of iodinated CL units), was mixed with DMSO (20 mL) in a first Schlenk flask (Schlenk flask A). In a second Schlenk flask (Schlenk flask B), dopamine hydrochloride (5.09 g, 25 equivalents relative to the CL unit of iodination, 26.8 mmol), PMDETA (340. Mu.L, 1.5 equivalents relative to the CL unit of iodination, 26.8 mmol), na were added ₂ CO ₃ (300.0 mg), BPO (6.49 g, 25 equivalents relative to the CL-iodide unit, 26.8 mmol) and DMSO (37 mL). The solution was left under stirring and argon flux for 4 hours. Oxygen is then removed by three freeze pump defrost cycles. The PCL iodide solution was transferred to flask B, and copper (I) bromide (0.23 g, 1.5 equivalents, 1.61mmol relative to CL iodide) was added. The flask was then placed in an oil bath at 70 ℃ 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 ℃. The polymer was dialyzed in water and freeze dried.

PDA：38-49wt.％

¹ H NMR(600MHz,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＝3 000g/mol，

In the NMR spectra, the disappearance of the peaks at 4.44ppm and 1.87ppm, which are characteristic signals of the iodine-functionalized PCL (see example 8), indicate that the chemical environment of the polymer was modified after the incorporation and polymerization of dopamine. In addition, the appearance of peaks at 4.57ppm, 1.96ppm and 1.84ppm also confirms this modification of the chemical environment. Corresponding to ¹ The H-NMR spectra (i.e., T-I and T-PDA for polymer T based on entry number 5 in Table 4) are shown in FIG. 20. To confirm that the change in chemical shift can be attributed to efficient grafting of PDA side chains onto PCL backbone, diffusion ordered NMR spectroscopy (DOSY NMR) analysis was performed. Peaks at 4.57ppm, 1.96ppm and 1.84ppm showed the same diffusion coefficient as the peaks attributed to T (d= -6.02 x 10-11m2. S-1), demonstrating the grafting of PDA onto PCL chain (fig. 21).

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 the previous copolymers were analyzed by SEC in THF, but copolymers containing PDA were insoluble in THF.

PDA content was quantified according to the TGA method as explained in example 2 using the modified formula:

the proportion of PDA was 38wt.%. This high value includes the mass of free and grafted PDA in the copolymer, which is consistent with the intensity of the peaks detected in NMR (fig. 20 and 21). The respective proportions of grafted PDA and free oligomeric PDA are unknown.

Example 10: protein stability

The stability of the mAb was evaluated in a formulation consisting of HBS: PEG 400:1 (v/v) with 5% (w/v) copolymer (T or T-PDA). The formulation will contain 40mg/ml (high dose (HD)) or 13mg/ml (low dose (LD)) mAb. In this example, the formulation will be defined as formulation X-Y, where X is the letter referring to the copolymer (T, T-PDA, T/T-PDA) and Y is the number referring to the mAb dose (LD, HD). "T" polymer as used herein refers to entry number 6 of Table 4 in example 7 and the resulting T-PDA as obtainable by the process described in example 9. The evolution of the characteristic parameters of SEC is shown in figure 22 for each formulation. The formulations are detailed in table 5.

Table 5: formulation composition for stability and in vitro release of mAbs

For formulation T-HD, the sample solution is white due to the presence of the white copolymer powder, but becomes clear after the mobile phase is added. The absorbance at 280nm, relative wavelength ratio and relative AUC were almost constant at day 0 and day 3. Then, from day 3, the intensity and relative AUC of the mAb gradually decreased, but the relative wavelength ratio remained constant. These results indicate that the interaction of mAb with the copolymer reduced the amount of mAb detected, consistent with the results of the pre-formulation study.

For formulation T/T-PDA-HD, the sample solution was black and then obscured after the mobile phase was added. From day 0 to day 30, the absorbance and relative AUC of the detected mAb gradually decreased, but the relative wavelength ratio remained constant, indicating mAb interaction with the copolymer. It should be noted that the absorbance of T/T-PDA-HD at day 0 divided by 2 compared to the formulation T-HD, thus indicating that mAb has a stronger initial interaction with the mixture of T and T-PDA. Furthermore, the decrease in the amount of mAb detected from day 0 to day 30 was higher in formulation T/T-PDA-HD (93% mAb detection loss) than in formulation T-HD (63% mAb detection loss).

For formulation T-PDA-HD, the absorbance at 280nm was about 5%, but the absorbance at 254nm was insufficient to calculate the wavelength ratio on day 0. No mAb was then detected from day 3 to day 30. This suggests a strong interaction between mAb and T-PDA. A study of the stability of the pre-formulation showed that a gradual decrease in mAb was observed in the presence of the copolymer. These results demonstrate that PDA-based copolymers exhibit strong affinity for mabs.

Thus, comparison of SEC-UV spectra indicated that the formulations tested (i.e., 3 formulations T, T/T-PDA and T-PDA consisting of HBS: PEG400 (1:1) and 10% (w/v) copolymer) interacted with mAb without denaturation of the mAb. The greatest decrease in mAb amount was observed in the presence of T-PDA, demonstrating the high affinity of mAb for PDA.

Example 11: in situ formation of reservoirs

The formation and behavior of in situ reservoirs based on formulations T-HD, T/T-PDA-HD and T-PDA-HD are shown in FIGS. 23-A, 23-B and 23-C, respectively. The corresponding LD formulations look similar. The names used in the examples (e.g., T-HD) are established in example 10.

In situ reservoir formation at the bottom of the vial was observed immediately after injection (day 0). Formulation T formed a gelatinous precipitated white mass on day 3 due to the solvent exchange mechanism of PEG400 diffusing into PBS. From day 5 to day 30, the aggregates appear to be smaller. The formulation T/T-PDA formed smaller (due to the lower amount of T) and black (due to the T-PDA) blocks on day 3, and the appearance remained similar until day 30. The slight coloration of the release medium was observed probably due to the release of PDA-based impurities (e.g. impurities corresponding to additional peaks detected in SEC-UV). Formulation T-PDA formed a film on day 3 with a portion adhered to the bottom and the appearance remained similar until day 30. For administration into the eye, the medium staining induced by the formulations T/T-PDA and T-PDA can be taken into account. However, this problem appears to be solved by introducing a further purification step of the T-PDA polymer used, and does not affect the overall demonstration of the principles presented in this example.

Preliminary in vitro release of mAbs under physiological conditions showed the ability of T-PDA to generate strong interactions with mAbs. It shows that mAb not bound to the copolymer was released with a burst effect before 3 days, while bound mAb remained unreleased for 30 days. T-formulations favor stability of released mAb, while T-PDA based formulations tend to destabilize a small fraction of mAb in the release medium at 37 ℃.

As a conclusion, the examples of the present invention demonstrate that T-PDA provides an interesting view on injectability of IVT administration of monoclonal antibodies or fragments thereof (e.g. through a 30G needle) and stability of such antibodies during storage (in particular at 4 ℃).

Claims (16)

1. A copolymer consisting of poly (epsilon-caprolactone) (PCL) and Polydopamine (PDA).

2. The copolymer of claim 1, wherein the copolymer consisting of poly (epsilon-caprolactone) (PCL) and Polydopamine (PDA) is a graft copolymer (PCL-g-PDA).

3. PCL-g-PDA copolymer according to any of claims 1 or 2, wherein the PCL-g-PDA copolymer comprises PCL with a molecular weight in the range of 1000g/mol to 200000 g/mol.

4. A PCL-g-PDA copolymer according to any one of claims 1 to 3, wherein the PCL-g-PDA copolymer comprises a PCL backbone having a molecular weight of 1000g/mol to 200 g/mol and a branched chain of PDA having a mass content of 0.1 to 50 wt.%.

5. A process for preparing a PCL-g-PDA polymer according to any of claims 1 to 4, characterized in that PCL with a mole percentage of halogenated PCL units in the range of 0.1 to 50mol.% is reacted with PDA precursors.

6. PCL-g-PDA copolymer according to any one of claims 1 to 4 for use in pharmaceutical formulations, in particular as carrier for sustained release of active ingredient.

7. PCL-g-PDA copolymer for use according to claim 6, wherein the pharmaceutical formulation is an intravitreal implant.

8. PCL-g-PDA copolymer for use according to claim 6 or 7, wherein the active pharmaceutical ingredient is a small molecule and is present in the pharmaceutical formulation or intravitreal implant in an amount of not less than 10% by weight.

9. PCL-g-PDA copolymer according to any one of claims 1 to 4, for use in the treatment of ocular diseases or disorders.

10. PCL-g-PDA copolymer according to any of claims 1 to 4, wherein two PCL-g-PDA chains are connected to PEG chains to form a (PCL-g-PDA) -b-PEG-b- (PCL-g-PDA) -type polymer.

11. The polymer of claim 10, wherein the PEG chain has a molecular weight of up to 20000g/mol and the two PCL-g-PDA chains each have the same molecular weight.

12. A polymer of the formula (II),

wherein the method comprises the steps of

p is 3 to 397

r is 1 to 170

m is 1 to 170.

13. The polymer according to any one of claims 10 to 12 for use in a pharmaceutical formulation.

14. The polymer for use according to claim 13, wherein the pharmaceutical formulation forms an in situ gelling reservoir for sustained release of the active pharmaceutical ingredient upon injection into the eye.

15. The polymer for use according to claim 14, wherein the active pharmaceutical ingredient is an antibody.

16. The novel polymers, methods and uses substantially as described herein.