Ophthalmic preparations

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] 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. [Background technology]

[0002] Corneal transparency is maintained by the highly organized composition of collagen fibrils within the stroma (1). Common corneal surgeries, such as crosslinking (CXL) or excimer laser photoablation, can alter the structure of the corneal layers, resulting in a cascade of corneal haze formation. The remodeling process of corneal tissue during wound healing includes keratocyte apoptosis, swelling of the stromal matrix, and production of less organized collagen fibrils (2). Corneal haze induces increased light scattering and the resulting loss of transparency, which can lead to complaints such as glare, halos, or reduced contrast sensitivity and visual quality (3). The amount of corneal haze is usually highest early after surgery, but a significant increase can be seen even one year after treatment (4). Remodeling processes during the wound healing response can also occur in other corneal pathologies (e.g., after thermal or chemical injury, or infectious keratitis), leading to the development of permanent corneal haze. Corneal lesions with opacities can have a significant burden on the vision-related quality of life of the affected patients. (5) Generally, corticosteroid eye drops and application of mitomycin-C are used as topical medications in the treatment of corneal opacities, which can 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, has a central role in several physiological and metabolic functions in the human body. AA is necessary for the biosynthesis of collagen fibrils, since it has major antioxidant properties and is a necessary cofactor in hydroxylation. Lack of AA can result in the production of structurally unstable collagen molecules. Thus, AA deficiency can affect the tissue healing process (6). Intravenously administered AA has been shown to safely and effectively reduce the size of epithelial defects and corneal opacification in the treatment of infectious keratitis (13). The antioxidant effect of AA can reduce corneal neovascularization and postoperative stromal opacification after excimer photoablation (14, 15). Stojanovic and Ringvold found that the severity of corneal opacification after PRK treatment was significantly reduced in the AA-treated group (16). Thus, according to recently available data, AA may have a beneficial effect on corneal opacification. However, the permeability of AA through corneal tissue may be insufficient, since the cornea represents a primarily lipophilic diffusion barrier for hydrophilic substances such as AA. Evidence suggests that increasing the concentration of an aqueous solution of free AA increases the amount of AA that penetrates, up to a certain point (17).

[0004] There remains a need for compositions that effectively aid in the corneal wound healing process without side effects. Summary of the Invention

[0005] A pharmaceutical composition is provided that comprises or consists of an ester of ascorbic acid and a fatty acid, a C1-3 alkyl and / or C1-3 hydroxyalkyl substituted β-cyclodextrin, isotonic 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. Highly preferably, the ester of ascorbic acid with a fatty acid is L-ascorbyl 6-palmitate (ASP).

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

[0008] Preferably, the β-cyclodextrin substituted C1-3 hydroxyalkyl 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 comprises: - an ASP of about 800 to 1200 μM, preferably about 850 to 1150 μM, preferably about 900 to 1100 μM, preferably about 950 to 1050 μM, very preferably about 1000 μM, and about 5 to 30 mM RAMEB, preferably about 8 to 25 mM, more preferably about 9 to 22 mM RAMEB, or about 8 to 12 mM RAMEB, preferably about 8.5 to 11.5 mM, preferably about 9 to 11 mM, preferably about 9.5 to 10.5 mM, very preferably about 10 mM RAMEB, or about 15-25 mM RAMEB, preferably about 16-24 mM, preferably about 17-23 mM, preferably about 18-22 mM, preferably about 19-21 mM, very preferably about 20 mM RAMEB, or about 5 to 30 mM (2-hydroxypropyl)-β-cyclodextrin (HPBCD), preferably about 8 to 25 mM, more preferably about 9 to 22 mM HPBCD, or about 8 to 12 mM HPBCD, preferably about 8.5 to 11.5 mM, preferably about 9 to 11 mM, preferably about 9.5 to 10.5 mM, very preferably about 10 mM HPBCD, or about 15-25 mM HPBCD, preferably about 16-24 mM, preferably about 17-23 mM, preferably about 18-22 mM, preferably about 19-21 mM, very preferably about 20 mM HPBCD, or a mixture of about 5 to 30 mM RAMEB and HPBCD, preferably about 8 to 25 mM, more preferably about 9 to 22 mM RAMEB and HPBCD, or a mixture of RAMEB and HPBCD at about 8 to 12 mM, preferably about 8.5 to 11.5 mM, preferably about 9 to 11 mM, preferably about 9.5 to 10.5 mM, very preferably about 10 mM, or a mixture of RAMEB and HPBCD at about 15-25 mM, preferably about 16-24 mM, preferably about 17-23 mM, preferably about 18-22 mM, preferably about 19-21 mM, very preferably about 20 mM, and -isotonic saline, and optionally about 0.001-1 m / V% (mass / volume) BC, preferably about 0.002-0.009 m / V%, preferably about 0.003-0.009 m / V% BC, or optionally a BC of about 0.002 to 0.006 m / V%, preferably about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, It comprises or consists of:

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

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

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

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

[0017] Preferably, the pharmaceutical composition comprises: - about 950 to 1050 μM, very preferably about 1000 μM, of ASP; - about 9.5 to 10.5 mM, very preferably about 10 mM, HPBCD; isotonic saline; optionally about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, of BC; It comprises or consists of:

[0018] Preferably, the pharmaceutical composition comprises: - about 950 to 1050 μM, very preferably about 1000 μM, of ASP; - about 18 to 22 mM, very preferably about 20 mM, HPBCD; isotonic saline; optionally about 0.003 to 0.005 m / V%, very preferably about 0.004 m / V%, of BC; It comprises or consists of:

[0019] Highly preferably, the pharmaceutical composition comprises: -1000 μM ASP, -10 mM RAMEB; BC of 0.004m / V% and Isotonic saline; It consists of:

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

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

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

[0023] Preferably the isotonic saline solution is for pharmaceutical use, e.g. suitable for use in eye drops.

[0024] Preferably, the pharmaceutical composition is lyophilized. 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 haze, preferably the formation of corneal haze following eye surgery. Preferably, the corneal haze is acute corneal haze.

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

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

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

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

[0029] [Figure 1] Solubility profiles of ASP-CD (L-ascorbyl 6-palmitate-cyclodextrin) complexes with (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] [Diagram 2] Effect of benzalkonium chloride (BC) on the solubility profiles of ASP-HPBCD (A) and ASP-RAMEB (B) complexes in saline solution at 35°C

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

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

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

[0034] [Figure 6] Storage stability profile 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 15 min of treatment compared to ASP solutions

[0036] [Figure 8] Relative intensity values ​​of ASP obtained in Raman maps of concentrated formulations during 60 min of treatment compared to ASP solutions

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

[0038] [Figure 10] Corneal permeability (A) and aqueous humor permeability (B) flux of ASP DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] A composition containing ascorbic acid was developed that effectively aids in the corneal wound healing process without side effects. In order to create a useful formulation, the permeability of AA had to be increased.

[0040] With higher corneal permeability, AA levels can be achieved in deeper intraocular structures. This may have an important role in several ocular pathologies where oxidative stress influences the pathophysiology, such as age-related macular degeneration (AMD), diabetic retinopathy or maculopathy, or glaucoma. Depletion of AA in the vitreous may be associated with macular ischemia in patients with proliferative diabetic retinopathy (18). AA may prevent apoptotic loss of capillary pericytes and endothelial dysfunction, and thus has a potential role in preventing diabetic macular edema (19). It has been reported that oxidative stress may result in damage to the trabecular meshwork, leading to increased levels of intraocular pressure and loss of retinal ganglion cells, and thus vitamin C may also affect the pathogenesis of glaucoma (23, 24). Increasing AA levels in the aqueous humor or vitreous may have a potential therapeutic modality for the aforementioned diseases.

[0041] Free form AA has a hydrophilic profile and acidic properties. Therefore, 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 structural analogue of AA with lipophilic properties, may be more effective in treating corneal scars due to its higher corneal permeability, but the use of this drug alone is limited by its poor water solubility resulting in low local concentrations. Cyclodextrin (CD) complexation allows for the formulation of a predominantly lipophilic composition as an aqueous eye drop solution (25, 26).

[0042] Among the CDs tested, BCD, HPBCD and RAMEB showed promising water solubility. However, BCD was reported to extract cholesterol and other lipid components from cell membranes, resulting in cell destruction and enhanced drug permeation through the corneal epithelial membrane. On the other hand, HPBCD was better tolerated in ocular tissues and less likely to cause destruction of the corneal epithelial barrier. We 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 had a proportional effect on the solubility of ASP, therefore, to avoid any toxicological issues, the amount of CD applied had to be minimized to the range of 1–20 mM (less than 2.8% (m / V%)).

[0044] Results from cornea-specific parallel artificial membrane permeability assays showed that in both cases of HPBCD and RAMEB, 10 and 20 mM concentrations of CD, in addition to 0.004% BC, led to a significant increase in the donor-side concentration of ASP.

[0045] With increasing CD concentration, an inverse change in in vitro corneal permeability could be observed, which could be partially explained by different donor ASP concentrations and increased amounts of CD applied. Cyclodextrins can enhance permeation by increasing the solubility of the drug, but it has also been reported that excessive amounts of cyclodextrin can result in decreased absorption through the cornea. The corneal permeability of ASP increases until the maximum solubility of ASP is reached at the donor side.

[0046] Ex vivo porcine corneal permeation study Surprisingly, it has been found that formulations containing RAMEB provide superior permeation values ​​for ASP.

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

[0048] Preservatives Although BC is a preservative frequently used in eye drops, 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 high enough to ensure the required microbiological stability of ophthalmic products.

[0049] Storage conditions Stability studies have shown that storage in the cold has a detrimental effect on the ASP-CD complex, and the concentration-time curves show that the ASP content in the solute phase decreases with a higher tendency, while the decomposition rate of ASP is accelerated by increasing the temperature. Based on this result, it is preferable to store the final formulation at room temperature.

[0050] The term "about" as used herein when referring to a concentration has its usual meaning in the art, i.e., as accurate as the measurement method will allow. The term "about" can also refer to a concentration that is within ±25%, preferably within ±20%, or preferably within ±20% of a given particular value. EXAMPLES

[0051] Materials and Methods material L-Ascorbic acid 6-palmitate (ASP) (CAS number: 137-66-6), benzalkonium chloride (BC) (CAS number: 63449-41-2), L-α-phosphatidylcholine (CAS number: 97281-47-5), and modified Dulbecco's phosphate buffered saline (PBS, pH 7.4), which is free of calcium and magnesium chloride, liquid, sterile filtered, and suitable for cell culture, were 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 number: 7585-39-9), (2-hydroxypropyl)-β-cyclodextrin (HPBCD) (CAS number: 128446-35-5), γ-cyclodextrin (GCD) (CAS number: 17465-86-0), (2-hydroxypropyl)-γ-cyclodextrin (HPGCD) (CAS number: 128446-34-4), sulfobutylated β-cyclodextrin sodium salt (SBECD) (CAS number: 182410-00-0) and randomly substituted methyl-β-cyclodextrin (RAMEB) (CAS number: 128446-36-6) were kindly provided by Cyclolab Ltd. (Budapest, Hungary).

[0052] Phase Solubility Test Excess amounts of ASP were added to 5 mL of phosphate buffer (PBS, pH = 7.4) containing increasing concentrations of CD ranging from 1 to 50 mM in sealed glass vials. The resulting suspensions were electromagnetically stirred (500 rpm) at constant temperature (35 ± 0.5 °C) until equilibration (24 h). An aliquot was then removed and centrifuged at 16000 rpm for 30 min using a Hermle Z323K high-performance refrigerated centrifuge (Hermle AG, Goss-heim, Germany) to separate the solid ASP crystals from the ASP-CD solution, and the ASP concentration was assayed by HPLC-DAD.

[0053] Development of ophthalmic formulations Excess amounts of ASP were added to 5 mL of saline solution containing increasing concentrations of CD ranging from 1 to 20 mM in sealed glass vials. Different concentrations of benzalkonium chloride (BC) (0.004% and 0.008%) as a preservative were added to the formulation. The resulting suspension was magnetically stirred (500 rpm) at constant temperature (35 ± 0.5 °C) until equilibrium (24 h). An aliquot was then removed and centrifuged at 16000 rpm for 30 min using a Hermle Z323K high performance refrigerated centrifuge (Hermle AG, Goss-heim, Germany) to separate the solid ASP crystals from the ASP-CD solution, and the ASP concentration was assayed by HPLC-DAD.

[0054] High-Performance Liquid Chromatography (HPLC) The determination of ASP concentration was performed 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 applied as stationary phase. Isocratic elution with purified water and methanol 10:90 (v / v) was applied for 6 min at 25° C. with a flow rate of 1.0 mL / min. 10 μL of 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, with limit of detection (LOD) and limit of quantification (LOQ) of 1.11 μg / mL and 3.33 μg / mL, respectively.

[0055] In vitro corneal permeability measurement The cornea-specific parallel artificial membrane permeability assay (cornea-PAMPA) was used to determine the transcorneal permeability of ASP-CD formulations. 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 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. 150-150 μL of formulation and reference solutions were applied onto the membrane of the donor plate. It was then covered with a plate lid to reduce the possibility of solvent evaporation. The sandwich system was incubated at 35°C for 4 h (Heidolph Titramax 1000, Heidolph Instruments, Schwabach, Germany). The concentration of ASP that penetrated the acceptor plate was determined by HPLC. The effective permeability of ASP was calculated using the following equation (29):

number

number

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[0056] Stability testing The chemical stability of ASP and ASP-CD complexes in saline was investigated at room temperature (25±0.5°C) and cold conditions (4±0.5°C) for 5 days. The actual drug content was quantified using HPLC at predefined time intervals to predict the shelf life of the formulations.

[0057] Ex vivo permeation studies on porcine cornea The ex vivo permeation of ASP was investigated by HPLC and Raman microscopy. Freshly donated porcine eyes obtained from a slaughterhouse were placed on a sterile cotton pad moistened with saline solution and kept in a refrigerator during transport. The porcine eyes were placed in a Teflon cell, where the cornea was uncovered and surrounded by a Teflon ring to prevent the flow of eye drops. 1000 μL of saline solution was instilled on the cornea, and 50 μL of ASP-CD formulation was added to the covered saline, which allows to mimic the physiological dilution caused by tears, and this was incubated at 35 °C. After individual treatments of 15, 30 and 60 min, residual solution from the topical surface and aqueous humor were aspirated via corneal puncture 2 to 5 h after death. The corneas were excised and ASP was extracted with 2 mL of methanol:water 50:50 (v / v) using an orbital shaker (PSU-10i Orbital Shaker, Grant Instruments Ltd, Cambs, England) at 450 rpm for 60 min. The ASP content in the residual solution, aqueous humor and cornea was measured by HPLC. The corneal retention of AS was calculated according to Equation 2, while the apparent permeability was calculated by using the following equation:

number

[0058] Investigating corneal drug permeation by Raman spectroscopy In parallel with the HPLC permeability determination, the corneas were investigated by Raman mapping after 15 and 30 min of treatment. Treated corneas were frozen and sectioned into cross sections (15 μm thick) using a Leica CM1950 Cryostat (Leica Biosystems GmbH, Wetzlar, Germany). Aluminum-coated slides were 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 microscope lens with a magnification of 50x was used. Measurements were performed with a laser power of 24 mW and a slit aperture of 50 μm. Corneal mapping of an area of ​​150 × 1000 μm 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. The individual spectra of unformulated ASP were used as references when profiling the chemical maps.

[0059] result Screening of CD derivatives in PBS Solubility studies of ASP in the presence of different concentrations of CD derivatives showed enhanced solubility of ASP in PBS (pH = 7.4) with increasing concentration of CD, thus indicating successful drug complexation (Figure 1).

[0060] The solubility study clearly shows that the aqueous solubility of ASP was highest when complexed with BCD, HPBCD and RAMEB compared to GCD, HPGCD or SBECD. However, the selection of BCD was avoided because BCD has been proven to have the ability to extract cholesterol and other lipid components from cell membranes (31), resulting in cell destruction and enhanced drug permeation through the corneal epithelial membrane (32). Conversely, HPBCD is better tolerated in ocular tissues and is less likely to cause destruction of the corneal epithelial barrier (33). Therefore, HPBCD and RAMEB were selected as suitable candidates for further investigation. However, increasing the amount of CDs had a proportional effect on the solubility of ASP, and to avoid any toxicological issues, the amount of CDs applied was minimized to the 1-20 mM range (less than 2.8%) for further investigation. Ocular administration of RAMEB at concentrations of 5% and 12.5% ​​was irritating to the conjunctival and corneal surfaces of rabbit eyes, whereas HPBCD was well tolerated, even at a concentration of 12.5%.

[0061] Formulation development regarding the effect of electrolytes and preservatives on API solubility To meet the requirements of ophthalmic formulations, PBS was changed to saline solution and preservatives were added. For that purpose, BC seemed to be the most suitable, but BC may also have an undesirable effect on ASP solubility, i.e., competition with regard to complex formation. BC is a preservative frequently used in eye drops. Typical concentrations range from 0.004 to 0.01% (35). Therefore, to elucidate this, solubility studies were also performed in 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, to minimize the possibility of BC-related adverse reactions (e.g., irritation), its concentration was minimized to 0.004% in the final formulation, a concentration high enough 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 formulations to investigate the effect of CD and BC concentrations on permeability (Figure 3).

[0064] Corneal-PAMPA measurements confirm the results of the preformulation studies, by showing that 10 and 20 mM CD concentrations, in addition to 0.004% BC, showed a significant increase in the donor-side concentration of ASP in both the HPBCD and RAMEB cases. To clarify which CD concentration was the most promising, the effective permeability (Figure 4) and corneal permeation flux (Figure 5) of ASP as concentration-dependent factors were also calculated.

[0065] With increasing CD concentrations, an inverse change in in vitro corneal permeability could be observed, which could be partially explained by different donor ASP concentrations and increased amounts of CD applied. Cyclodextrins can enhance permeation by increasing the solubility of the drug, but it has also been reported that excessive amounts of cyclodextrin can result in decreased absorption through the cornea. The corneal permeability of ASP increases until the maximum solubility of ASP is reached at the donor side (36).

[0066] The flux values ​​of the corneal-PAMPA measurements of APS showed the highest increase in the case of 0.004% BC at 10 mM CD concentration as opposed to 20 mM, which could be explained by the different donor side concentration of APS (5-fold). In summary, based on the corneal-PAMPA results, the 20 mM CD formulations seemed promising and therefore were used for further characterization.

[0067] Stability testing Stability studies were performed to determine the optimal storage conditions and shelf life of the ASP-CD formulations. The time-dependent changes in ASP content of different ASP-CD complexes stored at 4° C. and 25° C. are shown in FIG. 6.

[0068] Stability tests show that storage in the cold has a negative effect on the ASP-CD complex, and the concentration-time curves show that the ASP content in the solute phase tends to decrease with a higher tendency, while the decomposition rate of ASP is accelerated by increasing the 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 of the liquid formulation. Based on this result, 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] The shelf-life data show that the CD complex is able to stabilize the ASP and the degradation process is sustained 3-5 times longer compared to the initial ASP, which improves the therapeutic applicability of the formulation. The BC does not show any significant effect on the shelf-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 the porcine cornea. Both concentrated formulations (Figure 7) and 10-fold diluted formulations (Figure 8) were tested to mimic the physiological dilution conditions of the living eye. For localization of the transmitted formulations, the Raman spectra of ASP were set as profiles and their occurrence frequency 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] The Raman maps after 5 min treatment clearly showed that both HPBCD and RAMEB complexes improved the ocular penetration of ASP compared to the reference ASP solution. RAMEB 0.004% BC showed a higher relative intensity compared to the other two studied samples, indicating a higher concentration of the penetrated drug.

[0072] After 15 min of treatment, the relative intensity of ASP also increased in the case of ASP reference and HPBCD, indicating higher permeation compared to 5 min of treatment. Physiologically, the first 15 min is associated with drug permeation due to elimination by ocular clearance.

[0073] After 30 min of treatment, both HPBCD and RAMEB showed higher relative intensities compared to the ASP solution, but in the case of RAMEB a significantly higher ASP concentration can be observed.

[0074] To make the experiments more biologically relevant, taking into account physiological clearance by tears, previous measurements were also performed using 10-fold diluted formulations.

[0075] The diluted formulation showed a similar trend in formulation permeation as the concentrated formulation, but after 15 minutes, the permeated drug still did not reach the interstitium. This is not important from a therapeutic point of view, since the expected site of action is the cornea, whose saturation corresponds to therapeutic purposes.

[0076] After 60 minutes, ASP permeation is similar to the concentrated formulation, with the RAMEB complex showing significantly higher relative intensity and ASP saturation in all cross sections 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] Donor site concentrations showed a slight decrease in ASP concentration, which could be explained by an increased permeation of the drug to the cornea. This finding is supported by the corneal concentration, which increases simultaneously by the time of treatment. Essentially, corneal concentrations are therapeutically important, and these results predict improved therapeutic efficacy of both CD complexes. Interestingly, an increase in ASP concentration could also be detected in the aqueous humor, which provides a large amount of ocular permeation of ASP. In all cases, RAMEB showed significantly increased ASP concentration, which could be explained by a 2.5-fold higher donor concentration relative to HPBCD. Corneal and aqueous humor permeation fluxes were also calculated (Figure 10).

[0079] RAMEB showed a significantly increased corneal flux value compared to HPBCD, and both CDs showed significantly higher flux values ​​for the first drug, proving their beneficial effect on improving the ocular delivery of ASP. The aqueous humor flux values ​​showed less clear results, with only slight differences being observable.

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Claims

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 saline, and optionally benzalkonium chloride (BC).

2. 2. The pharmaceutical composition of claim 1, comprising ASP, a β-cyclodextrin selected from the group consisting of RAMEB and HPBCD, isotonic saline, and BC.

3. about 800-1200 μM ASP, preferably about 850-1150 μM, preferably about 900-1100 μM, preferably about 950-1050 μM, very preferably about 1000 μM ASP, and about 5-30 mM RAMEB, preferably about 8-25 mM, more preferably about 9-22 mM RAMEB, or about 8-12 mM RAMEB, preferably about 8.5-11.5 mM, preferably about 9-11 mM, preferably about 9.5-10.5 mM, very preferably about 10 mM RAMEB, or about 15-25 mM RAMEB, preferably about 16-24 mM, preferably about 17-23 mM, preferably about 18-22 mM, preferably about 19-21 mM, very preferably about 20 mM RAMEB, or about 5-30 mM (2-hydroxypropyl)-β-cyclodextrin (HPBCD), preferably about 8-25 mM, more preferably about 9-22 mM HPBCD, or about 8-12 mM HPBCD, preferably about 8.5-11.5 mM, preferably about 9-11 mM, preferably about 9.5-10.5 mM, very preferably about 10 mM HPBCD, or about 15-25 mM HPBCD, preferably about 16-24 mM, preferably about 17-23 mM, preferably about 18-22 mM, preferably about 19-21 mM, very preferably about 20 mM HPBCD, and - isotonic saline, and optionally about 0.001-1 m / V% (mass / volume) BC, preferably about 0.002-0.009 m / V%, preferably about 0.003-0.009 m / V%, or optionally a BC of about 0.002-0.006 m / V%, preferably about 0.003-0.005 m / V%, very preferably about 0.004 m / V%, The pharmaceutical composition according to claim 1 or 2, comprising:

4. - about 800-1200 μM 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 ASP, and about 8-12 mM RAMEB, preferably about 8.5-11.5 mM, preferably about 9-11 mM, preferably about 9.5-10.5 mM, very preferably about 10 mM RAMEB, or about 8-12 mM HPBCD, preferably about 8.5-11.5 mM, preferably about 9-11 mM, preferably about 9.5-10.5 mM, very preferably about 10 mM HPBCD, and - isotonic saline, and optionally a BC of about 0.002-0.006 m / V%, preferably about 0.003-0.005 m / V%, very preferably about 0.004 m / V%, The pharmaceutical composition of claim 3 , comprising:

5. about 950 to 1050 μM, very preferably about 1000 μM, of ASP, - a β-cyclodextrin selected from about 9.5-10.5 mM RAMEB, preferably about 10 mM RAMEB, about 9.5-10.5 mM HPBCD, preferably about 10 mM HPBCD, - isotonic saline, optionally a BC of about 0.003-0.005 m / V%, very preferably about 0.004 m / V%, The pharmaceutical composition of claim 4, comprising:

6. about 950 to 1050 μM, very preferably about 1000 μM, of ASP, - a β-cyclodextrin selected from about 18-22 mM RAMEB, preferably about 20 mM RAMEB, about 18-22 mM HPBCD, preferably about 20 mM HPBCD, - isotonic saline, optionally a BC of about 0.003-0.005 m / V%, very preferably about 0.004 m / V%, The pharmaceutical composition of claim 3 , comprising:

7. about 1000 μM ASP, - a β-cyclodextrin selected from about 10 mM RAMEB and about 10 mM HPBCD; - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to any one of claims 1 to 5, comprising:

8. about 1000 μM ASP, about 10 mM RAMEB, - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to claim 7, comprising:

9. about 1000 μM ASP, about 10 mM HPBCD, - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to claim 7, comprising:

10. about 1000 μM ASP, - a β-cyclodextrin selected from about 20 mM RAMEB and about 20 mM HPBCD; - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to any one of claims 1 to 3 and 6, comprising:

11. about 1000 μM ASP, RAMEB at about 20 mM, - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to claim 10, comprising:

12. about 1000 μM ASP, about 20 mM HPBCD, - BC of about 0.004 m / V%; - isotonic saline, The pharmaceutical composition according to claim 10, comprising:

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition is lyophilized.

14. A pharmaceutical composition according to any one of claims 1 to 13 for use in the prevention or treatment of eye disorders.

15. 15. The pharmaceutical composition for use according to claim 14, wherein said eye disorder is selected from fibrosis, preferably corneal fibrosis, corneal haze, preferably corneal haze formation associated with ophthalmic surgery and / or acute corneal haze, wounds, preferably corneal wounds, and preferably said pharmaceutical composition is for use in promoting wound healing.

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