Eye drop formulations

Abstract

Ophthalmic formulations comprising L-ascorbic acid 6-palmitate (ASP) are provided. The formulations are useful in situations where the maintenance of corneal transparency is at risk, such as during or after corneal surgery.

Description

[Technical Field]

[0001] An ophthalmic formulation containing L-ascorbic acid 6-palmitate (ASP) is provided. The formulation is useful in situations where the maintenance of corneal transparency is at risk, such as during or after corneal surgery. [Background technology]

[0002] Corneal transparency is maintained by the highly organized composition of collagen fibrils in the interstitium (1). Common corneal surgeries, such as cross-linking (CXL) or excimer laser photoexcision, can alter the structure of the corneal layers and lead to a cascade of corneal opacity formation. The remodeling process of corneal tissue during wound healing includes apoptosis of corneal stromal cells, swelling of the interstitium matrix, and production of less organized collagen fibrils (2). Corneal opacity induces increased light scattering and consequently loss of transparency, which can lead to complaints such as glare, halos, or decreased contrast sensitivity and visual quality (3). The amount of corneal opacity is usually highest in the early postoperative period, but a significant increase may also be seen one year after treatment (4). The remodeling process during the wound healing response also occurs in other corneal pathologies (e.g., after thermal or chemical injury or infectious keratitis) and can lead to the development of permanent corneal opacity. Corneal lesions with opacity can impose a significant burden on the visual quality of life of the affected patients (5). Generally, the use of corticosteroid eye drops and mitomycin-C is used as topical medication in the treatment of corneal opacity, which may be associated with the development of serious long-term complications, such as cataracts, secondary glaucoma, scleroderma, or perforation (5).

[0003] L-ascorbic acid (AA, ascorbic acid, or vitamin C), an essential water-soluble vitamin, plays a central role in several physiological and metabolic functions in the human body. AA is essential for collagen fibril biosynthesis because it possesses major antioxidant properties and is a necessary cofactor in hydroxylation. AA deficiency can lead to the production of structurally unstable collagen molecules. Therefore, AA deficiency can affect the tissue healing process (6). Intravenous administration of AA has been shown to safely and effectively reduce the size of epithelial defects and corneal opacities in the treatment of infectious keratitis (13). The antioxidant effect of AA can reduce corneal neovascularization and postoperative interstitial opacity after excimer photoablation (14, 15). Stojanovic and Ringvold found that the severity of corneal dysopagnosia after PRK treatment was significantly reduced in the AA treatment group (16). Therefore, according to recently available data, AA may have a beneficial effect on corneal dysopagnosia formation. However, because the cornea acts primarily as a lipophilic diffusion barrier to hydrophilic substances like AA, the permeability of AA through corneal tissue may be insufficient. There is evidence that increasing the concentration of an aqueous solution of free AA increases the amount of AA that permeates up to a certain point (17).

[0004] There is still a need for compositions that effectively support the corneal wound healing process without side effects. [Overview of the project]

[0005] A pharmaceutical composition is provided comprising or consisting of an ester of ascorbic acid and a fatty acid, a β-cyclodextrin substituted with C1-3 alkyl and / or C1-3 hydroxyalkyl, isotonic physiological saline, and optionally a preservative.

[0006] Preferably, the ester of ascorbic acid with a fatty acid is selected from ascorbyl laurate, ascorbyl myristate, ascorbyl stearate, ascorbyl palmitate, ascorbyl oleate, ascorbyl linoleate, and any mixture thereof. Very preferably, the ester of ascorbic acid with a fatty acid is L-ascorbic acid 6-palmitate (ASP).

[0007] Preferably, the β-cyclodextrin substituted with C1-3 alkyl is methyl-β-cyclodextrin (randomly methylated β-cyclodextrin; RAMEB).

[0008] Preferably, the β-cyclodextrin-substituted C1-3 hydroxyalkyl group is (2-hydroxypropyl)-β-cyclodextrin (HPBCD).

[0009] Preferably, the preservative is benzalkonium chloride (BC).

[0010] A pharmaceutical composition is provided comprising or consisting of ASP, a β-cyclodextrin selected from RAMEB, HPBCD and mixtures thereof, isotonic saline, and optionally BC.

[0011] Preferably, the pharmaceutical composition consists of ASP, a β-cyclodextrin selected from RAMEB, HPBCD and mixtures thereof, isotonic saline, and BC.

[0012] Preferably, the pharmaceutical composition is -ASP of about 800-1200 μM, preferably about 850-1150 μM, preferably about 900-1100 μM, preferably about 950-1050 μM, very preferably about 1000 μM, and - RAMEB of approximately 5-30 mM, preferably approximately 8-25 mM, more preferably approximately 9-22 mM, or - RAMEB of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, or - RAMEB of approximately 15-25 mM, preferably approximately 16-24 mM, preferably approximately 17-23 mM, preferably approximately 18-22 mM, preferably approximately 19-21 mM, very preferably approximately 20 mM, or - Approximately 5 to 30 mM (2-hydroxypropyl)-β-cyclodextrin (HPBCD), preferably about 8 to 25 mM, more preferably about 9 to 22 mM HPBCD, or -HPBCD of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, or -HPBCD of approximately 15-25 mM, preferably approximately 16-24 mM, preferably approximately 17-23 mM, preferably approximately 18-22 mM, preferably approximately 19-21 mM, very preferably approximately 20 mM, or - A mixture of RAMEB and HPBCD containing approximately 5-30 mM, preferably about 8-25 mM, more preferably about 9-22 mM, or - A mixture of RAMEB and HPBCD of about 8-12 mM, preferably about 8.5-11.5 mM, preferably about 9-11 mM, preferably about 9.5-10.5 mM, very preferably about 10 mM, or - A mixture of RAMEB and HPBCD containing approximately 15-25 mM, preferably approximately 16-24 mM, preferably approximately 17-23 mM, preferably approximately 18-22 mM, preferably approximately 19-21 mM, very preferably approximately 20 mM, and -Isotonic saline solution, - Depending on the case, BC of about 0.001 to 1 m / V% (mass / volume), preferably about 0.002 to 0.009 m / V%, preferably about 0.003 to 0.009 m / V%, or -optionally, about 0.002~0.006 m / V% BC, preferably about 0.003~0.005 m / V% BC, very preferably about 0.004 m / V% BC, comprises or consists of the foregoing.

[0013] Preferably, the pharmaceutical composition: -about 800~1200 μM L-ascorbic acid 6-palmitate (ASP), preferably about 850~1150 μM ASP, more preferably about 900~1100 μM ASP, more preferably about 950~1050 μM ASP, most preferably about 1000 μM ASP, and -about 8~12 mM RAMEB, preferably about 8.5~11.5 mM RAMEB, more preferably about 9~11 mM RAMEB, more preferably about 9.5~10.5 mM RAMEB, most preferably about 10 mM RAMEB, or -about 8~12 mM HPBCD, preferably about 8.5~11.5 mM HPBCD, more preferably about 9~11 mM HPBCD, more preferably about 9.5~10.5 mM HPBCD, most preferably about 10 mM HPBCD, or -a mixture of RAMEB and HPBCD at a total concentration of about 8~12 mM, preferably about 8.5~11.5 mM, more preferably about 9~11 mM, more preferably about 9.5~10.5 mM, most preferably about 10 mM, and -isotonic physiological saline, and -optionally, about 0.002~0.006 m / V% BC, preferably about 0.003~0.005 m / V% BC, very preferably about 0.004 m / V% BC, comprises or consists of the foregoing.

[0014] Preferably, the pharmaceutical composition: -about 800~1200 μM L-ascorbic acid 6-palmitate (ASP), preferably about 850~1150 μM ASP, more preferably about 900~1100 μM ASP, more preferably about 950~1050 μM ASP, most preferably about 1000 μM ASP, and - about 15 to 25 mM RAMEB, preferably about 16 to 24 mM, preferably about 17 to 23 mM, preferably about 18 to 22 mM, preferably about 19 to 21 mM, very preferably about 20 mM RAMEB, or - about 15 to 25 mM HPBCD, preferably about 16 to 24 mM, preferably about 17 to 23 mM, preferably about 18 to 22 mM, preferably about 19 to 21 mM, very preferably about 20 mM HPBCD, or - a mixture of RAMEB and HPBCD at about 15 to 25 mM, preferably about 16 to 24 mM, preferably about 17 to 23 mM, preferably about 18 to 22 mM, preferably about 19 to 21 mM, very preferably about 20 mM of the mixture of RAMEB and HPBCD, and - isotonic saline, and - optionally, BC at about 0.002 to 0.006 m / V%, preferably about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, comprises or consists of the above components.

[0015] Preferably, the pharmaceutical composition is - ASP at about 950 to 1050 μM, very preferably about 1000 μM, and - RAMEB at about 9.5 to 10.5 mM, very preferably about 10 mM, and - isotonic saline, and - optionally, BC at about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, and comprises or consists of the above components.

[0016] Preferably, the pharmaceutical composition is - ASP at about 950 to 1050 μM, very preferably about 1000 μM, and - RAMEB at about 18 to 22 mM, very preferably about 20 mM, and - isotonic saline, and - optionally, BC at about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, and comprises or consists of the above components.

[0017] Preferably, the pharmaceutical composition is - Approximately 950-1050 μM, very preferably about 1000 μM of ASP, -Approximately 9.5~10.5 mM, very preferably about 10 mM HPBCD, -Isotonic saline solution, - Depending on the case, approximately 0.003 to 0.005 m / V%, very preferably approximately 0.004 m / V%, BC and It includes or consists of.

[0018] Preferably, the pharmaceutical composition is - Approximately 950-1050 μM, very preferably about 1000 μM of ASP, -Approximately 18-22 mM, very preferably about 20 mM HPBCD, -Isotonic saline solution, - Depending on the case, approximately 0.003 to 0.005 m / V%, very preferably approximately 0.004 m / V%, BC and It includes or consists of.

[0019] Very preferably, the pharmaceutical composition is -1000μM ASP and, -10mM RAMEB and BC of 0.004 m / V%, Isotonic saline solution and It consists of.

[0020] Preferably, the pharmaceutical composition is -1000μM ASP and, -10mM HPBCD and BC of 0.004 m / V%, Isotonic saline solution and It consists of.

[0021] Preferably, the pharmaceutical composition is -1000μM ASP and, -20mM RAMEB and BC of 0.004 m / V%, Isotonic saline solution and It consists of.

[0022] Preferably, the pharmaceutical composition is -1000μM ASP and, -20mM HPBCD and BC of 0.004 m / V%, Isotonic saline solution and It consists of.

[0023] Preferably, isotonic saline solution is for medicinal use and is suitable for use in eye drops, for example.

[0024] Preferably, the pharmaceutical composition is freeze-dried. Preferably, the pharmaceutical composition is for ophthalmic (e.g., intraocular) use. Preferably, the pharmaceutical composition is for use in the prevention or treatment of corneal opacity, preferably in the prevention or treatment of corneal opacity formation associated with eye surgery. Preferably, the corneal opacity is acute corneal opacity.

[0025] Preferably, the pharmaceutical composition is intended for use in the prevention or treatment of fibrosis in the eye, preferably the cornea.

[0026] Preferably, the pharmaceutical composition is intended for use in promoting wound healing in the eye, preferably the cornea.

[0027] Preferably, the lyophilized pharmaceutical composition is dissolved in isotonic saline before administration. Preferably, the lyophilized pharmaceutical composition is dissolved in, for example, 10 mL of isotonic saline. Preferably, the pharmaceutical composition should be administered immediately after eye surgery and 1 to 20 times a day, preferably 1 to 10 times a day, preferably 3 to 8 times, and very preferably 5 times. The pharmaceutical composition can be used for 30 days, or 25 days, 20 days, 15 days, 10 days, or 5 days after surgery. Preferably, one drop is administered to one eye at a time (for example, one drop is administered to one eye 1 to 10 times a day).

[0028] Preferably, the average degree of substitution (methyl group) is 1.6 to 2.0 per glucose unit in RAMEB. Preferably, the average degree of substitution (hydroxypropyl group) is 2.5 to 7.0 per glucose unit in HPBCD. [Brief explanation of the drawing]

[0029] [Figure 1] Solubility profiles of ASP-CD (L-ascorbic acid 6-palmitate-cyclodextrin) complexes for (2-hydroxypropyl)-γ-cyclodextrin (HPGCD), γ-cyclodextrin (GCD), sulfobutylated β-cyclodextrin sodium salt (SBECD) (A); (2-hydroxypropyl)-β-cyclodextrin (HPBCD), β-cyclodextrin (BCD) (B), and randomly substituted methyl-β-cyclodextrin (RAMEB) (C).

[0030] [Figure 2] Effect of benzalkonium chloride (BC) on the solubility profiles of ASP-HPBCD(A) and ASP-RAMEB(B) complexes in physiological saline solution at 35°C.

[0031] [Figure 3] Solubility of ASP-CD complex in physiological saline solution at 35°C (on the donor side) and corneal permeability profile

[0032] [Figure 4] In vitro corneal permeability of ASP-CD complex at 35°C

[0033] [Figure 5] Corneal flux profile of ASP-CD complex at 35°C

[0034] [Figure 6] Storage stability profiles of ASP-CD complexes stored at 4°C (A) and 25°C (B).

[0035] [Figure 7] Relative intensity values ​​of ASP obtained in Raman maps of concentrated formulations during a 15-minute treatment period compared with ASP solution.

[0036] [Figure 8] Relative intensity values ​​of ASP obtained in Raman maps of concentrated formulations during a 60-minute treatment period compared with ASP solution.

[0037] [Figure 9] ASP concentrations in the donor site (A), cornea (B), and aqueous humor (C) after treatment with a 10-fold diluted preparation.

[0038] [Figure 10] ASP corneal permeability (A) and aqueous humor permeability (B) flux [Modes for carrying out the invention]

[0039] A composition containing ascorbic acid that effectively assists the corneal wound healing process without side effects has been developed. To create a useful formulation, it was necessary to increase the permeability of AA (agar-agar).

[0040] Higher corneal permeability may lead to AA levels being achieved in deeper intraocular structures. This may play an important role in several ocular conditions where oxidative stress affects the pathophysiology, such as age-related macular degeneration (AMD), diabetic retinopathy or maculopathy, or glaucoma. Depletion of AA in the vitreous humor may be associated with macular ischemia in patients with proliferative diabetic retinopathy (18). AA may play a potential role in the prevention of diabetic macular edema because it can prevent apoptotic loss of capillary pericytes and endothelial dysfunction (19). Oxidative stress can lead to damage to the trabecular network, resulting in elevated intraocular pressure levels and loss of retinal ganglion cells; therefore, vitamin C has been reported to also influence the pathogenesis of glaucoma (23, 24). Increased levels of AA in aqueous humor or vitreous humor may have a potential therapeutic role in the aforementioned diseases.

[0041] Free AA has a hydrophilic profile and acidic properties. Therefore, its permeability through the corneal epithelium, which provides a relatively negatively charged lipophilic lipid barrier, may be insufficient. L-ascorbic acid 6-palmitate (ASP or 6-O-palmitoyl-L-ascorbic acid), used as a lipophilic structural analog of AA, may be more effective in treating corneal scars due to its higher corneal permeability; however, the use of this drug alone is limited due to its poor water solubility and resulting low local concentrations. Cyclodextrin (CD) complexation primarily enables the formulation of lipophilic compositions as aqueous eye drop solutions (25, 26).

[0042] Among the tested chloroforms (CDs), BCD, HPBCD, and RAMEB showed promising water solubility. However, BCD was reported to extract cholesterol and other lipid components from cell membranes, leading to cell disruption and enhanced drug permeability across the corneal epithelial membrane. HPBCD, on the other hand, was better tolerated in ocular tissue and less likely to cause disruption of the corneal epithelial barrier. The inventors also found that ocular administration of RAMEB at concentrations of 5% and 12.5% ​​was irritating to the conjunctiva and corneal surface of rabbit eyes, whereas HPBCD was well tolerated even at a concentration of 12.5%.

[0043] Increasing the amount of CD has a proportional effect on the solubility of ASP; therefore, to avoid any toxicological problems, the amount of CD applied had to be kept to a minimum, in the range of 1-20 mM (less than 2.8% (m / V%)).

[0044] Results from corneal-specific parallel artificial membrane permeability assays showed that in both HPBCD and RAMEB cases, 0.004% BC, along with 10 and 20 mM concentrations of CD, resulted in a significant increase in donor-side ASP concentrations.

[0045] Increasing the CD concentration may lead to the opposite change in in vitro corneal permeability, which can be partially explained by different donor ASP concentrations and increased amounts of applied CD. Cyclodextrins can enhance permeability by increasing drug solubility, but it has also been reported that excessive amounts of cyclodextrin can lead to decreased absorption through the cornea. Corneal permeability of ASP increases until the maximum solubility of ASP is achieved on the donor side.

[0046] Ex vivo porcine corneal permeation study Surprisingly, formulations containing RAMEB were found to provide excellent permeability of ASP.

[0047] The degree of substitution does not appear to affect the ability of a particular CD to increase permeability.

[0048] Preservatives BC is a preservative frequently used in eye drops; however, our solubility results showed that BC has a negative effect on solubility. Therefore, its concentration was minimized to 0.004% in the final formulation. This concentration is sufficiently high to ensure the required microbiological stability of the ophthalmic product.

[0049] Storage conditions Stability tests indicate that storage in cold temperatures adversely affects the ASP-CD complex, and the concentration-time curve shows that the ASP content in the solute phase decreases at a higher rate, although the degradation rate of ASP is accelerated by increasing the temperature. Based on these results, it is preferable to store the final formulation at room temperature.

[0050] As used herein, the term "approximately" has the common sense in this art, i.e., the precise sense permitted by the measurement method, when referring to concentration. The term "approximately" may also refer to a concentration within ±25%, preferably ±20%, of a given particular value, or preferably within ±20%. [Examples]

[0051] Materials and methods material Modified Dulbecco's phosphate-buffered saline (PBS, pH 7.4), a liquid, sterile-filtered, cell culture-suitable solution that does not contain L-ascorbic acid 6-palmitate (ASP) (CAS No.: 137-66-6), benzalkonium chloride (BC) (CAS No.: 63449-41-2), L-α-phosphatidylcholine (CAS No.: 97281-47-5), calcium chloride, or magnesium chloride, was purchased from Sigma Aldrich Co. Ltd. (Budapest, Hungary). Analytical-grade solvents methanol, hexane, dodecane, and chloroform were purchased from Merck KGaA (Darmstadt, Germany). β-cyclodextrin (BCD) (CAS No.: 7585-39-9), 2-hydroxypropyl)-β-cyclodextrin (HPBCD) (CAS No.: 128446-35-5), γ-cyclodextrin (GCD) (CAS No.: 17465-86-0), (2-hydroxypropyl)-γ-cyclodextrin (HPGCD) (CAS No.: 128446-34-4), sulfobutylated β-cyclodextrin sodium salt (SBECD) (CAS No.: 182410-00-0), and randomly substituted methyl-β-cyclodextrin (RAMEB) (CAS No.: 128446-36-6) were kindly provided by Cyclolab Ltd. (Budapest, Hungary).

[0052] Phase solubility test An excess amount of ASP was added to 5 mL of phosphate buffer (PBS, pH=7.4) containing progressively increasing concentrations of CD in the range of 1–50 mM in a sealed glass vial. The resulting suspension was electromagnetically stirred at a constant temperature (35±0.5℃) (500 rpm) until equilibrium was reached (24 hours). Aliquots were then taken and centrifuged at 16000 rpm for 30 minutes using a Hermle Z323K high-performance refrigerated centrifuge (Hermle AG, Goss-heim, Germany) to separate solid ASP crystals from the ASP-CD solution, and the ASP concentration was assayed by HPLC-DAD.

[0053] Development of ophthalmic formulations An excess amount of ASP was added to 5 mL of physiological saline solution containing gradually increasing concentrations of CD in the range of 1–20 mM in a sealed glass vial. Different concentrations of benzalkonium chloride (BC) (0.004% and 0.008%) were added to the formulation as a preservative. The resulting suspension was electromagnetically stirred at a constant temperature (35±0.5℃) (500 rpm) until equilibrium was reached (24 hours). Aliquots were then taken and centrifuged at 16000 rpm for 30 minutes using a Hermle Z323K high-performance refrigerated centrifuge (Hermle AG, Goss-heim, Germany) to separate solid ASP crystals from the ASP-CD solution, and the ASP concentration was assayed by HPLC-DAD.

[0054] High-performance liquid chromatography (HPLC) ASP concentration was determined by HPLC using an Agilent 1260 (Agilent Technologies, Santa Clara, USA). A Zorbax Eclipse® C18 column, 100 × 4.6 mm, 5 μm (Phenomenex, Torrance, CA, USA) was used as the stationary phase. Consistent elution with purified water and methanol in a 10:90 (v / v) ratio was performed at 25°C at a flow rate of 1.0 mL / min for 6 minutes. A 10 μL sample was injected to determine the ASP concentration. Chromatograms were detected at 255 nm using a UV-VIS diode array detector. Data were evaluated using ChemStation B.04.03 software (Agilent Technologies, Santa Clara, USA). The linear regression of the calibration curve was 0.9998, and the limit of detection (LOD) and limit of quantification (LOQ) were 1.11 μg / mL and 3.33 μg / mL, respectively.

[0055] In vitro corneal permeability measurement The transcorneal permeability of ASP-CD preparations was determined using a corneal-specific parallel artificial membrane permeability assay (cornea-PAMPA). A filter donor plate (Multiscreen™-IP, MAIPN4510, pore size 0.45 μm; Millipore, Merck Ltd., Budapest, Hungary) was coated with 5 μL of phosphatidylcholine (16 mg) dissolved in 600 μL of a solvent mixture consisting of 70% (v / v) hexane, 25% (v / v) dodecane and 5% (v / v) chloroform. An acceptor plate (MSSACCEPTOR; Millipore, Merck Ltd., Budapest, Hungary) was filled with 300 μL of PBS solution at pH 7.4. 150 μL of the test preparation and 150 μL of the reference solution were applied onto the membrane of the donor plate. The plate was then covered with a plate lid to reduce the possibility of solvent evaporation. This sandwich system was incubated at 35° C. for 4 hours (Heidolph Titramax 1000, Heidolph Instruments, Schwabach, Germany). The concentration of ASP that permeated to the acceptor plate was determined using HPLC. The effective permeability of ASP was calculated using the following formula (29).

Formula

number

number

[0056] Stability testing The chemical stability of ASP and ASP-CD complex in physiological saline was investigated for 5 days under room temperature conditions (25±0.5℃) and cold storage conditions (4±0.5℃). To predict the shelf life of the formulations, the actual drug content was quantified using HPLC at predetermined time intervals.

[0057] Ex vivo transmission studies of porcine corneas Ex vivo permeability of ASP was investigated by HPLC and Raman microscopy. Freshly provided pig eyes obtained from the slaughterhouse were placed on sterile cotton beds moistened with physiological saline solution and stored in a refrigerator during transport. The pig eyes were placed in Teflon cells, where the corneal covering was removed and surrounded with a Teflon ring to prevent the flow of eye drops. 1000 μL of physiological saline solution was dropped onto the cornea, and 50 μL of ASP-CD formulation was added to the covering saline to mimic the physiological dilution caused by tears, and this was incubated at 35°C. After individual treatments at 15, 30, and 60 minutes, residual solution and aqueous humor from the local surface were aspirated via corneal puncture 2–5 hours postmortem. The cornea was excised, and ASP was extracted with 2 mL of methanol:water 50:50 (v / v) at 450 rpm for 60 minutes using an orbital shaker (PSU-10i Orbital Shaker, Grant Instruments Ltd, Cambs, England). The ASP content in the residual solution, aqueous humor, and cornea was measured by HPLC. Corneal retention of AS was calculated according to Equation 2, while apparent permeability was calculated using the following formula.

number

[0058] Investigation of corneal drug penetration using Raman spectroscopy In parallel with HPLC permeability determination, the cornea was investigated by Raman mapping after 15 and 30 minutes of treatment. The treated cornea was frozen and divided into 15 μm thick cross sections using a Leica CM1950 Cryostat (Leica Biosystems GmbH, Wetzlar, Germany). An aluminum-coated slide was used under the 15 μm thick cross sections. Raman spectroscopy was performed using a Thermo Fisher DXR Dispersive Raman Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a CCD camera and a diode laser operating at 780 nm. A 50x magnification microscope lens was used. Measurements were performed with a laser output of 24 mW and a 50 μm slit aperture. Corneal mapping of a 150 × 1000 μm area was captured vertically and horizontally with a step size of 50 μm. OMNIC for Dispersive Raman 8.2 software (Thermo Fisher Scientific) was used for chemical evaluation. When profiling the chemical map, the individual spectra of the unblended ASP were used as references.

[0059] result Selection (screening) of CD derivatives in PBS Solubility tests of ASP in the presence of different concentrations of CD derivatives showed that increasing the concentration of CD enhanced the solubility of ASP in PBS (pH=7.4), thus demonstrating successful drug conjugation (Figure 1).

[0060] Solubility tests clearly showed that the water solubility of ASP was highest when complexed with BCD, HPBCD, and RAMEB compared to GCD, HPGCD, or SBECD. However, BCD was not selected because it has been shown to have the ability to extract cholesterol and other lipid components from cell membranes (31), leading to cell disruption and enhanced drug permeability through the corneal epithelial membrane (32). Conversely, HPBCD is better tolerated in ocular tissue and is less likely to cause disruption of the corneal epithelial barrier (33). Therefore, HPBCD and RAMEB were selected as suitable candidates for further investigation. However, increasing the amount of CD has a proportional effect on the solubility of ASP, and to avoid any toxicological problems, the amount of CD applied was minimized to the range of 1–20 mM (less than 2.8%) for further investigation. Administration of RAMEB at concentrations of 5% and 12.5% ​​to the eyes was irritating to the conjunctival and corneal surfaces of rabbit eyes, while HPBCD was well tolerated even at a concentration of 12.5%.

[0061] Development of formulations related to the effects of electrolytes and preservatives on API solubility To meet the requirements for ophthalmic formulations, PBS was replaced with physiological saline solution and a preservative was added. For this purpose, BC seemed the most suitable, but BC may also have an undesirable effect on ASP solubility, namely competition with respect to complex formation. BC is a preservative frequently used in eye drops. Typical concentrations range from 0.004 to 0.01% (35). Therefore, to clarify this, solubility tests were also performed in physiological saline solution and in the presence of BC (0.004 and 0.008%) (Figure 2).

[0062] The results confirmed that BC has a negative effect on solubility, and furthermore, its concentration was minimized to 0.004% in the final formulation to minimize the possibility of adverse reactions (e.g., irritation) associated with BC. This concentration is sufficiently high to ensure the required microbiological stability of the ophthalmic product.

[0063] In vitro corneal permeability results Corneal-PAMPA measurements were performed in saline solutions containing both HPBCD and RAMEB preparations to investigate the effects of CD and BC concentrations on permeability (Figure 3).

[0064] Corneal-PAMPA measurements, in both HPBCD and RAMEB cases, showed a significant increase in donor concentrations of ASP with 10 and 20 mM CD in addition to 0.004% BC, thus supporting the results of the preliminary formulation study. To determine which CD concentration was most promising, the effective permeability (Figure 4) and corneal transmission flux (Figure 5) of ASP were also calculated as concentration-dependent factors.

[0065] Increasing the CD concentration may lead to the opposite change in in vitro corneal permeability, which can be partially explained by different donor ASP concentrations and increased amounts of applied CD. Cyclodextrins can enhance permeability by increasing drug solubility, but it has also been reported that excessive amounts of cyclodextrins can lead to decreased absorption through the cornea. Corneal permeability of ASP increases until the maximum solubility of ASP is achieved on the donor side (36).

[0066] The flux values ​​of corneal-PAMPA measurements in APS showed the highest increase at 0.004% BC with a 10 mM CD concentration, in contrast to 20 mM, which can be explained by the different donor concentrations (5x) of ASP. In summary, based on the corneal-PAMPA results, 20 mM CD formulations appeared promising and were used for further characterization.

[0067] Stability testing Stability tests were conducted to determine the optimal storage conditions and shelf life of the ASP-CD formulation. Figure 6 shows the time-dependent change in ASP content of different ASP-CD complexes stored at 4°C and 25°C.

[0068] Stability tests indicate that storage at low temperatures adversely affects the ASP-CD complex, and the concentration-time curve shows a higher tendency for ASP content in the solute phase to decrease, although the rate of ASP degradation is accelerated by increasing temperature. This phenomenon can be explained by the presence of a saturated ASP solution due to the applied CD. By lowering the temperature to 4°C, ASP tends to be released from the CD complex and precipitates in the aqueous medium, reducing the ASP concentration in the liquid formulation. Based on these results, it is preferable to store the final formulation at room temperature. The shelf life of the ASP-CD complex was determined based on the storage stability profile shown in Table 1. [Table 1] m / v%

[0069] Storage life data shows that the CD complex can stabilize ASP, and the degradation process lasts 3 to 5 times longer compared to the initial ASP, which improves the therapeutic applicability of the formulation. BC does not show a significant impact on storage life.

[0070] Ex vivo porcine corneal permeation study Raman mapping was performed to investigate the distribution of selected ASP-CD formulations and initial ASP on porcine corneas. Both concentrated formulations (Figure 7) and 10-fold diluted formulations (Figure 8) were tested to mimic the physiological dilution conditions of living eyes. To determine the location of the transmitted formulations, the Raman spectrum of ASP was set as a profile, and its incidence was determined by measuring the relative intensity of ASP in the Raman map using ImageJ 1.4 software (National Institutes of Health, Bethesda, MD, USA).

[0071] Raman maps taken after 5 minutes of treatment clearly showed that both HPBCD and RAMEB complexes improved ocular penetration of ASP compared to the reference ASP solution. RAMEB 0.004%BC showed a higher relative intensity compared to the other two investigated samples, indicating a higher concentration of the drug that was permeated.

[0072] After 15 minutes of treatment, the relative intensity of ASP increased in both the ASP reference and HPBCD cases, showing higher permeability compared to 5 minutes of treatment. Physiologically, the first 15 minutes are related to drug permeability due to ocular clearance and rejection.

[0073] After 30 minutes of treatment, both HPBCD and RAMEB showed higher relative intensity compared to the ASP solution, but in the case of RAMEB, significantly higher ASP concentrations could be observed.

[0074] To make the experiment more biologically relevant, taking into account physiological clearance by tears, the same measurements were performed using a 10-fold diluted formulation.

[0075] The diluted formulation showed a similar trend to the concentrated formulation in terms of drug permeation, but after 15 minutes, the permeated drug still did not reach the interstitial space. Since the cornea is the expected site of action, this is not significant from a therapeutic standpoint, and its saturation is consistent with the therapeutic objective.

[0076] After 60 minutes, ASP permeation was similar to that of the concentrated formulation, and the RAMEB complex showed significantly higher relative intensity and ASP saturation across the entire cross-section of the porcine cornea.

[0077] Ex vivo permeation was also quantitatively investigated by determining ASP concentrations in the cornea and aqueous humor using HPLC (Figure 9).

[0078] The donor site concentration showed a slight decrease in ASP concentration, which can be explained by increased drug permeability to the cornea. This finding is supported by the simultaneous increase in corneal concentration by the time of treatment. Essentially, corneal concentration is therapeutically important, and these results predict improved therapeutic efficacy of both CD complexes. Interestingly, the increase in ASP concentration can also be detected in the aqueous humor, which provides a large amount of ocular permeability of ASP. In all cases, RAMEB showed a significantly increased ASP concentration, which can be explained by a 2.5-fold higher donor concentration compared to HPBCD. The fluxes of corneal and aqueous humor permeability were also calculated (Figure 10).

[0079] RAMEB showed significantly increased corneal flux values ​​compared to HPBCD, and both CDs showed significantly higher flux values ​​than the first drug, demonstrating their favorable effect on improving ocular delivery of ASP. Aqueous humor flux values ​​did not show a clearer result, with only slight differences observed. Examples of embodiments of the present invention are listed in the following sections [Aspect 1] to [Aspect 15]. [Aspect 1] A pharmaceutical composition comprising L-ascorbic acid 6-palmitate (ASP), a β-cyclodextrin selected from the group consisting of randomly methylated β-cyclodextrin (RAMEB), (2-hydroxypropyl)-β-cyclodextrin (HPBCD), and mixtures thereof, isotonic physiological saline, and optionally benzalkonium chloride (BC). [Aspect 2] The pharmaceutical composition according to embodiment 1, wherein the pharmaceutical composition comprises ASP, a β-cyclodextrin selected from the group consisting of RAMEB and HPBCD, isotonic saline, and BC. [Aspect 3] -ASP of about 800-1200 μM, preferably about 850-1150 μM, preferably about 900-1100 μM, preferably about 950-1050 μM, very preferably about 1000 μM, and - RAMEB of approximately 5-30 mM, preferably approximately 8-25 mM, more preferably approximately 9-22 mM, or - RAMEB of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, or - RAMEB of approximately 15-25 mM, preferably approximately 16-24 mM, preferably approximately 17-23 mM, preferably approximately 18-22 mM, preferably approximately 19-21 mM, very preferably approximately 20 mM, or - Approximately 5 to 30 mM (2-hydroxypropyl)-β-cyclodextrin (HPBCD), preferably about 8 to 25 mM, more preferably about 9 to 22 mM HPBCD, or -HPBCD of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, or -HPBCD of approximately 15-25 mM, preferably approximately 16-24 mM, preferably approximately 17-23 mM, preferably approximately 18-22 mM, preferably approximately 19-21 mM, very preferably approximately 20 mM, and -Isotonic saline solution, - Depending on the case, BC of about 0.001 to 1 m / V% (mass / volume), preferably about 0.002 to 0.009 m / V%, preferably about 0.003 to 0.009 m / V%, or - Depending on the case, approximately 0.002 to 0.006 m / V% BC, preferably approximately 0.003 to 0.005 m / V%, and very preferably approximately 0.004 m / V% BC. A pharmaceutical composition according to embodiment 1 or 2, comprising: [Aspect 4] - Approximately 800-1200 μM of L-ascorbic acid 6-palmitate (ASP), preferably about 850-1150 μM, preferably about 900-1100 μM, preferably about 950-1050 μM, very preferably about 1000 μM of ASP, and - RAMEB of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, or -HPBCD of approximately 8-12 mM, preferably approximately 8.5-11.5 mM, preferably approximately 9-11 mM, preferably approximately 9.5-10.5 mM, very preferably approximately 10 mM, and -Isotonic saline solution, - Depending on the case, approximately 0.002 to 0.006 m / V% BC, preferably approximately 0.003 to 0.005 m / V%, and very preferably approximately 0.004 m / V% BC. A pharmaceutical composition according to embodiment 3, comprising: [Aspect 5] - Approximately 950-1050 μM, very preferably about 1000 μM of ASP, - A β-cyclodextrin selected from approximately 9.5-10.5 mM RAMEB, preferably approximately 10 mM RAMEB, and approximately 9.5-10.5 mM HPBCD, preferably approximately 10 mM HPBCD, -Isotonic saline solution, - Depending on the case, approximately 0.003 to 0.005 m / V%, very preferably approximately 0.004 m / V%, of BC, A pharmaceutical composition according to embodiment 4, comprising: [Aspect 6] - Approximately 950-1050 μM, very preferably about 1000 μM of ASP, - A β-cyclodextrin selected from approximately 18-22 mM RAMEB, preferably approximately 20 mM RAMEB, and approximately 18-22 mM HPBCD, preferably approximately 20 mM HPBCD, -Isotonic saline solution, - Depending on the case, approximately 0.003 to 0.005 m / V%, very preferably approximately 0.004 m / V%, of BC, A pharmaceutical composition according to embodiment 3, comprising: [Aspect 7] -Approximately 1000 μM of ASP, - A β-cyclodextrin selected from approximately 10 mM RAMEB and approximately 10 mM HPBCD, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to any one of embodiments 1 to 5. [Aspect 8] -Approximately 1000 μM of ASP, - Approximately 10mM RAMEB and, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to embodiment 7, comprising the above. [Aspect 9] -Approximately 1000 μM of ASP, -Approximately 10 mM HPBCD, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to embodiment 7, comprising the above. [Aspect 10] -Approximately 1000 μM of ASP, - A β-cyclodextrin selected from approximately 20 mM RAMEB and approximately 20 mM HPBCD, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to any one embodiment of embodiments 1 to 3 and 6. [Aspect 11] -Approximately 1000 μM of ASP, - Approximately 20mM RAMEB, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to embodiment 10, comprising the above. [Aspect 12] -Approximately 1000 μM of ASP, -Approximately 20mM HPBCD, - Approximately 0.004 m / V% BC, -Isotonic saline solution, A pharmaceutical composition according to embodiment 10, comprising the above. [Aspect 13] The pharmaceutical composition according to any one of embodiments 1 to 12, wherein the pharmaceutical composition is freeze-dried. [Aspect 14] A pharmaceutical composition according to any one of embodiments 1 to 13, for use in the prevention or treatment of eye disorders. [Aspect 15] The pharmaceutical composition for use according to embodiment 14, wherein the eye disorder is selected from fibrosis, preferably corneal fibrosis, corneal opacity, preferably corneal opacity formation associated with ophthalmic surgery and / or acute corneal opacity, wound, preferably corneal wound, and preferably the pharmaceutical composition is for use in promoting wound healing.

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Claims

1. A pharmaceutical composition for use in the prevention or treatment of corneal opacity, wherein the pharmaceutical composition comprises L-ascorbic acid 6-palmitate (ASP), a cyclodextrin selected from randomly methylated β-cyclodextrin (RAMEB) and (2-hydroxypropyl)-β-cyclodextrin (HPBCD), and isotonic saline, or the pharmaceutical composition comprises ASP, a cyclodextrin selected from RAMEB and HPBCD, isotonic saline, and benzalkonium chloride (BC).

2. The pharmaceutical composition according to claim 1, wherein the corneal opacity is the formation of corneal opacity associated with ophthalmic surgery.

3. -800 to 1200 μM ASP, and RAMEB of -5 to 30 mM, or HPBCD at -5 to 30 mM, and - Isotonic physiological saline, A pharmaceutical composition according to claim 1 or 2, comprising:

4. -800 to 1200 μM ASP, and RAMEB of -8 to 12 mM, or HPBCD at -8 to 12 mM, and - Isotonic physiological saline, A pharmaceutical composition according to claim 1 or 2, comprising:

5. -ASP in the range of -950 to 1050 μM, - A cyclodextrin selected from RAMEB at -9.5 to 10.5 mM and HPBCD at -9.5 to 10.5 mM, - Isotonic saline solution, A pharmaceutical composition according to claim 1 or 2, comprising:

6. -ASP in the range of -950 to 1050 μM, A cyclodextrin selected from -18 to 22 mM RAMEB and 18 to 22 mM HPBCD, - Isotonic saline solution, A pharmaceutical composition according to claim 1 or 2, comprising:

7. -1000 μM ASP and, A cyclodextrin selected from -10 mM RAMEB and 10 mM HPBCD, BC of -0.004 m / V%, - Isotonic saline solution, A pharmaceutical composition according to claim 1 or 2, comprising the above.

8. -1000 μM ASP and, A cyclodextrin selected from -20 mM RAMEB and 20 mM HPBCD, BC of -0.004 m / V%, - Isotonic saline solution, A pharmaceutical composition according to claim 1 or 2, comprising the above.

9. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is freeze-dried.

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