Topical ocular delivery of cyclosporin

Abstract

The present disclosure relates to formulations for topical ocular delivery of high concentrations of cyclosporin. The formulation comprises at least 0.1 wt% cyclosporin supported in a plurality of nanostructures in the form of nanodroplets dispersed in a continuous aqueous phase.

Description

Technical Field

[0001] The present disclosure relates to ophthalmic formulations for the topical delivery of cyclosporine to the front of the eye. Background Art

[0002] Cyclosporine is well known for treating various ocular conditions. Cyclosporine is a cyclic oligopeptide from the anti-calcineurin family and has immunosuppressive and anti-inflammatory activities. Eye drop formulations of cyclosporine A are widely used to treat various ocular conditions such as vernal keratoconjunctivitis, corneal transplant rejection, dry eye syndrome (which is resistant to first-line treatment) and many other indications.

[0003] The high hydrophobicity of cyclosporin makes it difficult to stably formulate into ophthalmic preparations that require high concentrations of cyclosporin. Such preparations are reported to be based on high concentrations of oily components, which are themselves irritating and difficult for patients to tolerate. In addition, due to problems with physical stability and entrapment of high concentrations of cyclosporin in the formulations to date, high-loaded cyclosporin in various formulations has been reported to be highly irritating. Therefore, to date, most commercial preparations contain relatively low concentrations of cyclosporin, typically up to 0.05 wt%. Summary of the invention

[0004] The present disclosure provides ophthalmic topical formulations, particularly in the form of eye drops, containing high concentrations of cyclosporin with minimal irritation and high active delivery capabilities. Such formulations can be used to treat, for example, anterior eye disorders, such as dry eye.

[0005] The formulations disclosed herein contain at least 0.1 wt% cyclosporin and are formulated into stable nanostructures, uniformly dispersed in an aqueous phase, wherein the cyclosporin is captured and stabilized within the nanostructures. Since the nanostructures contain very small amounts of oily components, yet cyclosporin is still captured therein due to its unique combination of components, minimal irritation to the eye is observed while allowing for the delivery of a high effective dose of cyclosporin to the eye. The inventors have surprisingly discovered that the use of a combination of at least two nonionic hydrophilic surfactants enables the physical stabilization of high loadings of highly lipophilic cyclosporin within the nanostructures while maintaining very low amounts of oil.

[0006] Thus, in one aspect thereof, the present disclosure provides an ophthalmic formulation comprising a plurality of nanostructures dispersed in a continuous aqueous phase, the nanostructures being in the form of droplets having an average diameter of at most 50 nm, the nanostructures comprising:

[0007] a) cyclosporine at a concentration of at least 0.1 wt % of the formulation,

[0008] b) at least two nonionic hydrophilic surfactants,

[0009] c) at least one oil in a concentration of up to 2 wt % of the formulation, and

[0010] d) at least one co-surfactant.

[0011] The formulations of the present disclosure are designed for ophthalmic delivery of cyclosporine, i.e., delivery of cyclosporine to one or more parts of the eye, such as to the cornea, conjunctiva, aqueous humor, iris, vitreous humor, ciliary body, anterior chamber, posterior chamber, etc. The formulation is preferably a topical formulation in the form of a solution or suspension of the nanostructures in the continuous aqueous phase.

[0012] The nanostructures are droplets, which consist of at least the at least one oil, the nonionic hydrophilic surfactant and at least one co-surfactant, for capturing and stabilizing cyclosporin. The nanostructures are typically in the form of vesicles, with an average diameter of up to 50 nm (nanometers), wherein the nonionic hydrophilic surfactant and the co-surfactant form an interface between the continuous aqueous phase and the oil core. Without being bound by theory, cyclosporin is primarily located at the interface, where it is physically captured between the heads of the surfactant and the co-surfactant, interacting via hydrogen bonds and dipole-dipole interactions, thereby stabilizing it within the nanostructure.

[0013] The term mean size refers to the arithmetic mean of the measured diameters of the droplets. When the droplets are not spherical, the calculation of the mean size is based on an equivalent sphere of the largest dimension of the particle.

[0014] By some embodiments, the droplets are substantially monodisperse. Due to the monodisperse submicron nanostructure size of the formulation, the formulation is generally transparent (or substantially transparent), maintaining its transparency for a long period of time. This allows for easy detection of changes in formulation stability (since phase separation, bioactive precipitation, and / or oil droplet coalescence will result in detectable turbidity).

[0015] Cyclosporine is a cyclic oligopeptide from the anti-calcineurin family. Cyclosporine A is a cyclic hydrophobic 11-amino acid peptide containing 7N-methyl amino acid residues and the amino acid (4R)-4-([E]-2-butenyl)-4-N-methyl-(L)-threonine (MeBmt), as shown in formula (I):

[0016]

[0017] In the context of the present disclosure, the term cyclosporin refers to cyclosporin A, its salts, derivatives and analogs.

[0018] In some embodiments, the cyclosporin is cyclosporin A.

[0019] As previously described, the formulations of the present disclosure are highly loaded with cyclosporin. The formulations of the present disclosure, due to their unique compositional balance, are capable of stably loading cyclosporin into the formulation at a concentration far exceeding its solubility limit in water (which is 27.67 μg / ml at 25° C., or ˜0.027 wt %). In some embodiments, the concentration of cyclosporin is at least about 0.1 wt %, at least about 0.15 wt %, at least about 0.2 wt %, at least about 0.25 wt %, or even at least about 0.3 wt % (e.g., about 0.5 wt %) of the formulation.

[0020] The inventors have discovered that a combination of two or more nonionic hydrophilic surfactants is able to load cyclosporin to a high level into a formulation and stabilize it for a longer period of time. In the formulations of the present disclosure, the balance of ingredients not only allows for high loading and entrapment of cyclosporin in the formulation, but also achieves both kinetic and thermodynamic stability of the formulation, thereby allowing for a long shelf life while minimizing phase separation, sedimentation, and / or undesirable expulsion of cyclosporin from the nanostructures.

[0021] The term one or more nonionic hydrophilic surfactants refers to uncharged surfactants, and have a hydrophilic head group and one or more lipophilic tails, which can be arranged into nanostructures in aqueous media. The inventors have found that the combination of two or more such nonionic hydrophilic surfactants can form a stable nanostructure and dissolve cyclosporin into the nanostructure at a relatively high concentration. By adjusting the composition of the nanostructure, cyclosporin can be encapsulated between the surfactant tails, thereby mainly dissolving it in the interface, and also possibly dissolving it in the oil core. This specific combination is based on two nonionic hydrophilic surfactants, which are different in the geometric structure of their head groups and can form a head group complex. One of the surfactants has a linear hydrophilic head and the other surfactant has a bulky head. This combination separates the nanostructure interface, allowing cyclosporin to be encapsulated (due to the bulky head) while maintaining the curvature and integrity of the interface (due to the linear head).

[0022] According to some embodiments, the at least two nonionic hydrophilic surfactants include at least one first nonionic hydrophilic surfactant selected from ethoxylated fatty acids, and at least one second nonionic hydrophilic surfactant selected from ethoxylated castor oil and hydrogenated derivatives thereof.

[0023] According to some embodiments, the first nonionic hydrophilic surfactant can be selected from ethoxylated fatty acids (polyoxyethylene stearate, polyoxyethylene oleate, polyoxyethylene caprylate / caprate, polyoxyethylene laurate, etc.), ethoxylated alkyl ethers (polyoxyethylene cetyl ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl-octadecyl ether, polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, etc.), ethoxylated monoglycerides and combinations thereof. According to some embodiments, the second nonionic hydrophilic surfactant can be selected from polyoxyethylene castor oil (polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene 60 hydrogenated castor oil, polyoxyethylene 60 castor oil, polyoxyethylene 100 castor oil, polyoxyethylene 100 hydrogenated castor oil, polyoxyethylene 200 castor oil, polyoxyethylene 200 hydrogenated castor oil, etc.), polyoxyethylene dehydrated sorbitan fatty acid esters (polysorbate 20, polysorbate 60, polysorbate 80, etc.) and combinations thereof.

[0024] According to some embodiments, the weight ratio (w / w) of the first nonionic hydrophilic surfactant to the second nonionic hydrophilic surfactant in the formulation ranges from about 1:1 to 1:30. According to some other embodiments, the weight ratio (w / w) of the first nonionic hydrophilic surfactant to the second nonionic hydrophilic surfactant ranges from about 1:1 to 1:28.

[0025] According to some embodiments, the total concentration of the nonionic hydrophilic surfactant in the formulation ranges from about 1 wt % to about 7 wt %.

[0026] The formulation includes up to 2 wt % of oil. By some embodiments, the at least one oil is present in the formulation at a concentration of no more than 0.7 wt %. The relatively low oil content allows for a high loading capacity of cyclosporin on the one hand, and on the other hand acts as a stabilizer for the nanostructure at high temperatures in the presence of cyclosporin. At high temperatures, the polar portion of cyclosporin is oriented toward the core of the molecule, thereby reducing interaction with the hydrophilic head of the surfactant.

[0027] The term oil refers to an agent that is insoluble in water and is capable of forming different domains when introduced into an aqueous liquid. In some embodiments, the at least one oil is selected from the following: acylglycerols of fatty acids including triacetin, tributyrin, tricaprylin, triolein, medium chain triglycerides and mixed fatty acid triglycerides, olive oil, sesame oil, soybean oil, canola oil, castor oil, partially or fully hydrogenated castor oil, paraffin oil, mineral oil, non-saponifiable fatty derivatives, alkyl alcohols, including oleyl alcohol, lauryl alcohol, terpenoids and combinations thereof.

[0028] According to some embodiments, the weight ratio between the total nonionic hydrophilic surfactant and the oil in the formulation ranges between about 5:1 and about 50:1.

[0029] As mentioned above, the formulation also includes at least one co-surfactant. A co-surfactant is understood to encompass any lipophilic, hydrophilic or amphiphilic agent, different from the non-ionic hydrophilic surfactant, which helps (together with the surfactant) to reduce the interfacial tension between the oil phase and the aqueous phase to almost zero (or to zero), allowing the formation of thermodynamically stable nanostructures. Thus, the combination of surfactant and co-surfactant allows the nanostructure to be stabilized both kinetically and thermodynamically.

[0030] According to some embodiments, the co-surfactant is a hydrophilic co-surfactant or an amphiphilic co-surfactant.

[0031] By some embodiments, the at least one co-surfactant is at least one polyol.A polyol is an alcohol containing at least 2 hydroxyl groups.

[0032] By some embodiments, the at least one co-surfactant is selected from polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, propylene glycol, polypropylene glycol, diethylene glycol monoethyl ether (Transcutol), and combinations thereof.

[0033] According to some embodiments, the at least one co-surfactant is present in the formulation at a concentration ranging between about 0.5 wt % to about 5 wt %.

[0034] According to other embodiments, the weight ratio between the nonionic hydrophilic surfactant and the co-surfactant ranges from about 1:1 to 5:1.

[0035] The inventors unexpectedly found that the combination of low amounts of oil, the at least two nonionic hydrophilic surfactants and the at least one co-surfactant allows stabilization of cyclosporin at high loadings within the formulation at ambient and refrigerated temperatures (e.g., 4° C.), while when the formulation is warmed to about 35-40° C., the cyclosporin becomes more hydrophobic and is stabilized primarily by the surfactant tail and the oil. Upon contact with the ocular epithelium, the cyclosporin contained in the nanostructures can be released due to the fusion of the physiological membrane and the nanostructures. Thus, the formulations of the present disclosure were found to be highly stable at storage temperatures and have increased availability of cyclosporin after administration.

[0036] By some embodiments, the nanostructure further comprises at least one solvent. The solvent is an organic solvent, typically polar, miscible with water and suitable for helping cyclosporin dissolve into the nanostructure, and for regulating the osmotic pressure of the system. Introducing at least one such solvent into the formulation can promote the hydrophilic surfactant to completely cover the interface at a high water dilution of the formulation. In other words, the use of at least one solvent changes the effective critical packing parameter (ECPP) of the interface, which is conducive to controlling the hydrophilicity / hydrophobicity of the surfactant according to the amount of water in the formulation, thereby improving the stability of the formulation.

[0037] According to some embodiments, the at least one solvent is selected from glycerol, ethanol, methanol, propanol, isopropanol, diethanolamine, triethanolamine, and combinations thereof.

[0038] By some embodiments, the formulation includes the at least one solvent in a concentration ranging from about 0.05 wt % to about 1.5 wt %.

[0039] According to some other embodiments, the weight ratio between the nonionic hydrophilic surfactant and the solvent ranges from about 5:1 to 35:1.

[0040] In order to increase the residence time of the preparation in the eye, by some embodiments, the preparation further includes at least one film former in the aqueous phase. The term film former (or film former) refers to a material that can increase the viscosity of the preparation and temporarily form a film on the external mucous membrane of the eye to delay tear fluid from emptying the nanostructure from the eye. According to some embodiments, the at least one film former is selected from polyvinyl pyrrolidone, a block copolymer of polyoxypropylene and polyoxyethylene (poloxamer), carboxymethyl cellulose and its salt, hydroxypropyl methylcellulose (HPMC), poly (vinyl alcohol), poly (acrylic acid), hydrocolloids such as xanthan gum and combinations thereof.

[0041] By some embodiments, the at least one film former is present in the formulation at a concentration of up to about 0.75 wt%.

[0042] In some embodiments, the formulation may further include various additives approved for ophthalmic use, such as pH adjusters and buffers, neutralizers, emollients, humectants, preservatives, antioxidants, and the like.

[0043] The ophthalmic formulations of the present disclosure can be prepared from a concentrated form, typically a substantially water-free concentrate, which can be diluted by an aqueous medium. This allows the formation of a concentrate that is stable for a longer period of time, lacks a life-supporting environment for microorganisms, and is easily diluted to obtain nanostructures.

[0044] Thus, by another aspect thereof, the present disclosure provides a cyclosporine concentrate formulation suitable for use in the preparation of an ophthalmic formulation as described herein, the concentrate comprising:

[0045] a) cyclosporine, in a concentration of at least 0.5 wt % of the concentrate,

[0046] b) at least two nonionic hydrophilic surfactants,

[0047] c) at least one oil in a concentration of up to 12% by weight of the concentrate, and

[0048] d) at least one co-surfactant.

[0049] By some embodiments, the at least two nonionic hydrophilic surfactants include at least one first nonionic hydrophilic surfactant selected from ethoxylated fatty acids, and at least one second nonionic hydrophilic surfactant selected from ethoxylated castor oil and hydrogenated derivatives thereof.

[0050] In some embodiments, the weight ratio of the first nonionic hydrophilic surfactant to the second nonionic hydrophilic surfactant ranges from about 1:1 to 1:30.

[0051] By some embodiments, the at least one oil is present in the concentrate at a concentration of no more than 7 weight percent.

[0052] By some other embodiments, the total concentration of the nonionic hydrophilic surfactant in the concentrate ranges from about 20 wt % to about 75 wt %.

[0053] According to some embodiments, the at least one co-surfactant is present in the concentrate at a concentration ranging between about 20 wt % to about 45 wt %.

[0054] By some embodiments, the concentrate further comprises at least one solvent. In such embodiments, the concentrate comprises the at least one solvent in a concentration ranging from about 1 wt % to about 10 wt %.

[0055] The concentrate is substantially free of water, ie, comprises up to about 5 wt% water. By some preferred embodiments, the concentrate is anhydrous.

[0056] Another aspect of the present disclosure provides a method for preparing an ophthalmic formulation as described herein, comprising mixing a concentrate as described herein with an aqueous dispersion medium, thereby obtaining a plurality of nanostructures formed from the concentrate and dispersed in a continuous aqueous phase formed from the aqueous dispersion medium.

[0057] According to some embodiments, the aqueous dispersion medium includes at least one film former.

[0058] By other embodiments, the aqueous dispersion medium includes at least one buffer.

[0059] According to some embodiments, the concentrate is mixed with the aqueous dispersion medium in a weight ratio ranging between about 1:5 and 1:25.

[0060] By some embodiments, the mixing is performed under conditions where only mild to moderate shear is applied using a mechanical rotor or magnetic stirring. The system does not need to be subjected to high shear applied by homogenization, intense ultrasonic treatment, fluidization techniques, etc. Thus, by some embodiments, the mixing is performed under conditions that prevent high shear forces from being generated in the mixture.

[0061] By way of another aspect, the present disclosure provides a kit for preparing the ophthalmic formulations described herein, the kit comprising: at least one first container, the at least one first container comprising the concentrate described herein; at least one second container, the at least one second container comprising an aqueous dispersion medium; and instructions for use.

[0062] The first container and the second container may independently be rigid, semi-rigid or flexible and may have a suitable form.The first container and the second container may respectively comprise a concentrate and an aqueous dispensing medium in amounts suitable for preparing a single dose ophthalmic formulation or a multi-dose ophthalmic formulation.

[0063] By some embodiments, the first container and the second container are integrally formed and configured to mix the concentrate and aqueous dispersion medium as desired by the user (e.g., by introducing the contents of one of the containers into the other or by having a mixing zone that can conveniently mix the contents of the containers).

[0064] By way of another aspect, there is provided an ophthalmic formulation as disclosed herein for use in treating anterior ocular diseases or conditions.

[0065]

[0013] Another aspect provides a method of treating anterior ocular diseases or conditions comprising administering to a subject in need thereof an effective amount of an ophthalmic formulation described herein.

[0066] By some embodiments, the anterior ocular disease or condition is selected from dry eye disease, wet and dry age-related macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia, and strabismus.

[0067] As is well known, the effective amount for the purposes herein can be determined by considerations known in the art. The amount must effectively achieve the desired therapeutic effect, which depends in particular on the type and severity of the disease to be treated and the treatment regimen. The effective amount is usually determined in appropriately designed clinical trials (dose range studies), and those skilled in the art will know how to properly conduct such tests to determine the effective amount. As is well known, the effective amount depends on a variety of factors, including a variety of pharmacological parameters such as half-life in vivo, undesirable side effects (if any), factors such as age and gender, etc.

[0068] As used herein, the term treatment or any linguistic variation thereof refers to the administration of a therapeutic amount of a formulation of the present disclosure that is effective to ameliorate adverse symptoms associated with a disease, prevent the manifestation of such symptoms before they occur, slow the progression of a disease, slow the worsening of symptoms, enhance the onset of remission, slow the irreversible damage caused by the progressive chronic phase of a disease, delay the onset of the progressive phase, reduce the severity of a disease or cure a disease, increase survival or faster recovery, or prevent the occurrence of a disease, or a combination of two or more of the foregoing.

[0069] As used herein, the term about is meant to encompass deviations of ±10% from the specifically mentioned parameter value (such as temperature, concentration, etc.).

[0070] Unless expressly stated otherwise, all concentrations disclosed herein are provided as weight percent wt % based on the weight of the ophthalmic formulation or concentrate formulation, as the case may be.

[0071] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integer) within the indicated range. The phrases ranging (ranging) and ranges between a first indicated numeral and a second indicated numeral and a "range" from a first indicated numeral "to" a second indicated numeral are used interchangeably herein and are meant to include the first and second indicated numerals and all fractions and integers therebetween.

[0072] Unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to mean the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any integer or step or group of integers and steps.

[0073] The term ...at least one of ... when applied to any component of a formulation is to be understood to encompass one, two, three, four or more different occurrences of that component in the formulation.

[0074] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention in an appropriate manner. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment cannot function without those elements. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to better understand the subject matter disclosed herein and to illustrate how it can be implemented in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0076] Figures 1A-1K is a picture of exemplary formulations according to some examples of the present disclosure: OPH1c ( Figure 1A )、OPH4b( Figure 1B )、OPH5a( Figure 1C )、OPH1_D( Figure 1D )、OPH1_0.75D( Figure 1E-1F )、OPH1-D / E( Figure 1G )、OPH1-D / F( Figure 1H )、OPH1-D / G( Fig. 1I )、OPH1-D / H( Figure 1J )、OPH1-D / I( Figure 1K ).

[0077] Figure 2A-2E The droplet size distribution (by volume) is shown as: OPH1_0.5D ( Figure 2A )、OPH1_0.75D( Figure 2B )、OPH1_D( Figure 2C ), OPH1-D / G-placebo ( Figure 2D ) and OPH1-D / G 0.3% CsA( Figure 2E ).

[0078] Figures 3A-3H is the LUMiFuge test result of an example formulation loaded with cyclosporin A: OPH1_0.5D ( Figure 3A )、OPH1_0.5D( Figure 3B )、OPH1_D( Figure 3C )、OPH1-D / E( Figure 3D )、OPH1-D / F( Figure 3E )、OPH1-D / G( Figure 3F )、OPH1-D / H( Figure 3G) and OPH1-D / I( Figure 3H ).

[0079] Figure 4 The Draize scoring scale for preclinical studies conducted on rabbits is provided.

[0080] Figures 5A-5E The penetration of CsA into ocular structures is shown for OPH1c (◆), OPH5a (■), OPH5a' (▲), and Restasis (●), with error bars indicating SD: conjunctiva ( Figure 5A ),cornea( Figure 5B )、Aqueous humor( Figure 5C ), retina( Figure 5D ) and whole blood ( Figure 5E ). DETAILED DESCRIPTION

[0081] Exemplary Formulations

[0082] The empty concentrate was prepared by weighing all the concentrate components and mixing them at 40-60°C. Cyclosporin A was then dissolved into the concentrate at 50-60°C to obtain the loaded concentrate. The aqueous phase was prepared separately, first preparing the buffer component, adjusting the pH to 7.6±0.2, and then adding one or more polymers. The loaded concentrate and aqueous phase were then combined and mixed under mild mixing conditions to obtain the final formulation, as shown in Table 1.

[0083] Table 1: Exemplary Formulations

[0084]

[0085] *Polyoxyethylene 40 stearate

[0086] **Polyoxyl 35 Castor Oil

[0087] ***Polyoxyl 40 Hydrogenated Castor Oil

[0088] Table 1 (continued): Exemplary Formulations

[0089]

[0090]

[0091] *Polyoxyl 40 Stearate**Polyoxyl 35 Castor Oil***Polyoxyl 40 Hydrogenated Castor Oil

[0092] Table 1 (continued): Exemplary Formulations

[0093]

[0094] *Polyoxyethylene 40 stearate

[0095] **Polyoxyl 35 Castor Oil

[0096] ***Polyoxyl 40 Hydrogenated Castor Oil

[0097] Physical properties

[0098] The physical properties of selected formulations are shown in Table 2. All formulations were in the form of dispersions of nano-domains in a continuous aqueous phase, showing high clarity, homogeneity and thermodynamic stability. OPH5a1 is a non-tackified OPH5a formulation. Figures 1A-1K An exemplary visualization of the appearance of an exemplary formulation can be seen in .

[0099] The hydrodynamic radius of the droplets was measured by dynamic light scattering (DLS) using a Nano-ZS Zetasizer (Malvern, UK) at room temperature with water as the dispersant. Figure 2A-2E Exemplary size distribution curves are presented in .

[0100] Table 2: Characteristics of the selected preparations

[0101]

[0102] a. pH measurement: SevenEasy Meter Toledo

[0103] b. Micro-osmometer (Model 210)

[0104] c. Turbidity evaluation: HANNA Instruments HI 83414 turbidity and free / total chlorine meter

[0105] (Using calibration curve samples and 0.13 NTU of WFI as reference)

[0106] d. Droplet size inspection: ζ particle size analyzer, nano-particle size analyzer (nano-s), MALVERN instrument

[0107] Table 2 (continued): Characteristics of the selected preparations

[0108]

[0109] As clearly shown in Table 2, the formulations exhibited complete transparency, with nearly monodisperse nanodroplet size and uniform refractive index. In addition, from the comparison of OPH1-D / G with and without cyclosporin A (CsA), it can be seen that the incorporation of CsA does not affect the pH, osmotic pressure and refractive index of the formulations. In the presence of CsA, the droplet size increased by about 4 nm, and it can also be seen from the size distribution curve that the droplet size distribution of the CsA-loaded system is broader ( Figure 3D-3E ).

[0110] Long-term physical stability

[0111] To determine the long-term stability of the formulation, use The results were quickly measured by analytical centrifugation. Figures 3A-3H shown. The analysis is able to predict the shelf life of a formulation at its original concentration, even in the presence of slow destabilization processes such as sedimentation, flocculation, coalescence, and fractionation. During the measurement, parallel light illuminates the entire sample cell in the centrifugal field; the transmitted light is detected by sensors arranged linearly along the total length of the sample cell. Due to the change in light transmission over time, local changes in particles or droplets are detected. The results are presented in a diagram that plots the percentage of transmitted light (transmittance %) as a function of local position (mm), revealing the corresponding spectrum of transmittance over time.

[0112] Changes in transmittance indicate the stability of the formulation - when the transmittance profile remains constant, the sample is considered physically stable and its shelf life can be inferred based on the measurement conditions.

[0113] like Figures 3A-3H As shown, in all transmittance plots, the lines overlap, indicating that no transmittance changes were observed and all systems were physically stable and based on this analysis are expected to be stable for at least 2 years under storage conditions.

[0114] Chemical stability

[0115] The chemical stability of OPH1-0.5D was tested during storage at different temperatures (4°C and 25°C) in terms of CsA level, visual appearance, pH, osmotic pressure, droplet size, and refractive index. The results are provided in Tables 3-1 to 3-2.

[0116] It can be seen that the formulation remained stable at the storage temperature tested without any signs of changes in physical and chemical properties.

[0117] Table 3-1: Stability of OPH1-0.5D, 4 ℃

[0118]

[0119] Table 3-1 (continued): Stability of OPH1-0.5D, 4 ℃

[0120]

[0121] Table 3-2: Stability of OPH1-0.5D at room temperature

[0122]

[0123]

[0124] Table 3-2 (continued): Stability of OPH1-0.5D at room temperature

[0125]

[0126] Pharmacological tests

[0127] All preclinical studies detailed below were conducted on the exemplary formulations detailed in Table 4:

[0128] Table 4: Selected formulations for preclinical studies (wt %)

[0129] Components OPH1c OPH5a OPH5a' Myrj S40 2.369 1.185 1.185 Cremophor EL 2.211 3.158 3.158 HECO 40 0.396 PEG 400 0.552 0.947 0.947 Propylene glycol 1.184 1.423 1.423 glycerin 0.552 0.394 0.394 castor oil 0.236 0.394 0.394 C 0.500 0.500 0.500 PVP K30 0.184 --- --- CMC Sodium --- 0.46 --- Poloxamer 407 --- 0.184 --- Water for Injection (WFI) 91.816 91.356 92.000 total 100.0 100.0 100.0

[0130] Ocular tolerance evaluation

[0131] Study Design

[0132] Multiple ocular topical administrations (twice a day) were performed on male albino rabbits for five consecutive days to evaluate the tolerability of the formulations of Table 4. The therapeutic agent was instilled into the conjunctival sac of the right eye (RE) of the rabbit. The test design is summarized in Tables 5-1 and 5-2. At the end of the measurement period, animals treated with the test article were euthanized by intracardiac injection of an overdose of pentobarbital after anesthesia. Animals treated with the vehicle were reused for other studies.

[0133] Table 5-1: Study Groups and Dosing Regimens

[0134]

[0135] Table 5-2: Research Plan

[0136]

[0137] 1 General clinical examination included recording of general appearance and clinical signs as well as weight changes.

[0138] 2Perform an eye examination using a light source (ophthalmoscope) followed by a Draize examination of the conjunctiva, cornea, and iris. See the Draize scoring scale in Appendix A.

[0139] result

[0140] Tolerance was assessed from the following aspects (see Design section for details):

[0141] 1. General behavior and weight and

[0142] 2. Eye examination

[0143] 1. General behavior and weight

[0144] During the study period, no clinical signs were observed in any of the animals.

[0145] Body weight assessments of test article and vehicle treated animals (Table 6) were normal over the five day period. No differences were observed in mean body weights between test article and vehicle treated animals.

[0146] Table 6: Average weight of rabbits (±SD)

[0147]

[0148] 2. Eye examination

[0149] Individual data of ocular examinations of both eyes are summarized in Table 7. The effects in Table 7 are provided according to the Draize scoring scale ( Figure 4 ).

[0150] Table 7: Ocular Findings in All Treatment Groups

[0151]

[0152] 1 The indications are listed in Figure 5

[0153] 2 Scores are given according to the indications detailed in the Draize scale and expressed as n / n 最大 For example, an indication of iritis may receive a score of 1 / 2 or 2 / 2.

[0154] As seen in Table 7, the observed ocular findings occurred only in the treated eye (right eye). The vehicle-treated group did not develop ocular findings, while only mild and transient ocular findings were recorded in the other treatment groups, indicating that the three tested formulations were macroscopically as well tolerated as the vehicle.

[0155] Overall, all three tested formulations were found to be tolerated under the experimental conditions of multiple topical administration over 5 consecutive days.

[0156] Evaluation of ocular penetration of CsA

[0157] Study objectives and design

[0158] The purpose of this study was to evaluate the penetration of CsA into the conjunctiva (CJ), cornea (C), aqueous humor (AH), retina (R), and whole blood (WB) after a single intraconjunctival instillation of 50 μL of the test formulation in both eyes of pigmented rabbits (HY79b strain) compared with a commercially available Liyanda product (containing 0.05% CsA).

[0159] For the purpose of the study, 72 pigmented rabbits were divided into four groups of 18 subjects each, which were further subdivided into 6 time points (Table 8).

[0160] Table 8: Study Groups and Dosing Regimens

[0161]

[0162] Both were measured at baseline and end point using a light source (Dreize Scale, Figure 4 ) The treated eye of each rabbit was examined. At all time points, the rabbit was injected i.m. (xylazine) and The animals were anesthetized with a mixed solution of 1000 (ketamine). Whole blood was sampled into K3-EDTA anticoagulant tubes by intracardiac puncture and stored at -80±15°C before euthanasia. The animals were euthanized by intracardiac injection of an overdose of pentobarbital. Immediately after euthanasia, the cornea, conjunctiva, aqueous humor, and retina were removed from both eyes, weighed, and stored at -80±15°C. CsA was extracted from different structures and whole blood of both eyes, and its content was determined by RRLC-MS / MS. The analyzed CsA levels were further used to calculate PK parameters, including apparent Cmax, apparent Tmax, and AUC 0.25-8 hr. PK parameters were calculated using the mean values ​​of the groups.

[0163] result

[0164] The patterns of CsA penetration into CJ, C, AH, R, and WB are presented in Tables 9-1 to 9-5 (and Figures 5A-5E ), and the PK results are shown in Table 10.

[0165] Table 9-1: CsA penetrated into the conjunctiva in all treatment groups

[0166]

[0167] Table 9-2: CsA penetration into the cornea was observed in all treatment groups

[0168]

[0169] Table 9-3: CsA permeated into the aqueous humor in all treatment groups

[0170]

[0171] Table 9-4: CsA penetrated into the retina in all treatment groups

[0172]

[0173] Table 9-5: CsA penetration into whole blood in all treatment groups

[0174]

[0175] Table 10: PK parameters obtained for all test groups

[0176]

[0177] All tested preparations as well as Liyanda were tolerated microscopically based on the Draize scale.

[0178] Based on the permeation profiles and calculated PK parameters, mainly AUC values, which provide information on the cumulative permeation of CsA during the exposure period, we can conclude that the penetration of CsA into the conjunctiva was found to be in the order of OPH5a>OPH1c>OPH5a'>Liyanda in terms of absolute values.

[0179] However, in terms of % of applied dose, Liyanda showed the highest penetration, followed by OPH5a, OPH1c and finally OPH5a'.

[0180] It was found that the penetration of CsA into the cornea was in the order of OPH5a>OPH5a'≈OPH1c>Liyanda in absolute terms (or in terms of % of applied dose: OPH5a>Liyanda>OPH5a'≈OPH1c).

[0181] It was found that the order of CsA penetration into the aqueous humor in terms of absolute values ​​was: OPH5a>OPH5a'>OPH1c>Liyanda.

[0182] It was found that the order of CsA penetration into the retina and whole blood in terms of absolute values ​​was: OPH5a>OPH1c>OPH5a'>Liyanda.

[0183] Overall, all three tested formulations had higher bioavailability to the retina compared with Liyanda.

Claims

1. An ophthalmic preparation comprising a plurality of nanostructures dispersed in a continuous aqueous phase, the nanostructures being in the form of droplets having an average diameter of at most 50 nm, the nanostructures comprising: a) cyclosporine at a concentration of at least 0.1 wt % of the formulation, b) at least two nonionic hydrophilic surfactants, c) at least one oil in a concentration of up to 2 wt % of the formulation, and d) at least one co-surfactant.

2. The ophthalmic preparation according to claim 1, wherein The at least two nonionic hydrophilic surfactants include at least one first nonionic hydrophilic surfactant selected from ethoxylated fatty acids, and at least one second nonionic hydrophilic surfactant selected from ethoxylated castor oil and hydrogenated derivatives thereof.

3. The ophthalmic preparation according to claim 2, wherein The weight ratio of the first nonionic hydrophilic surfactant to the second nonionic hydrophilic surfactant ranges from about 1:1 to 1:

30.

4. The ophthalmic preparation according to any one of claims 1 to 3, wherein The total concentration of the nonionic hydrophilic surfactant in the formulation ranges from about 1 wt % to about 7 wt %.

5. The ophthalmic preparation according to any one of claims 1 to 4, wherein The at least one oil is selected from acylglycerols of fatty acids, triacetin, tributyrin, tricaprylin, triolein, medium chain triglycerides and mixed fatty acid triglycerides, olive oil, sesame oil, soybean oil, canola oil, castor oil, partially or fully hydrogenated castor oil, paraffin oil, mineral oil, non-saponifiable fat derivatives, alkyl alcohols including oleyl alcohol, lauryl alcohol, terpenoids and combinations thereof.

6. The ophthalmic preparation according to any one of claims 1 to 5, wherein The at least one oil is present in the formulation at a concentration of no more than 0.7 wt%.

7. The ophthalmic preparation according to any one of claims 1 to 6, wherein The at least one co-surfactant is present in the formulation at a concentration ranging between about 0.5 wt % and 5 wt %.

8. The ophthalmic preparation according to any one of claims 1 to 7, wherein The weight ratio between the nonionic hydrophilic surfactant and the co-surfactant ranges from about 1:1 to about 5:

1.

9. The ophthalmic preparation according to any one of claims 1 to 8, wherein The nanostructures further include at least one solvent.

10. The ophthalmic preparation according to claim 9, wherein The at least one solvent is selected from the group consisting of glycerol, ethanol, methanol, propanol, isopropanol, diethanolamine, triethanolamine, and combinations thereof.

11. The ophthalmic preparation according to claim 9 or 10, wherein The formulation includes the at least one solvent in a concentration range of between about 0.05 wt % and about 1.5 wt %.

12. The ophthalmic preparation according to any one of claims 9 to 11, wherein The weight ratio between the nonionic hydrophilic surfactant and the solvent ranges from about 5:1 to about 35:

1.

13. The ophthalmic preparation according to any one of claims 1 to 12, wherein The aqueous phase includes at least one film former.

14. The ophthalmic preparation according to claim 13, wherein The at least one film former is selected from polyvinyl pyrrolidone, block copolymers of polyoxypropylene and polyoxyethylene (poloxamers), carboxymethyl cellulose and its salts, hydroxypropyl methyl cellulose (HPMC), poly(vinyl alcohol), poly(acrylic acid), hydrocolloids such as xanthan gum, and combinations thereof.

15. The ophthalmic preparation according to claim 13 or 14, wherein The at least one film former may be present in the formulation in a concentration of up to about 0.75 wt%.

16. The ophthalmic preparation according to any one of claims 1 to 15, wherein The droplets are substantially monodisperse.

17. A cyclosporin concentrate formulation for preparing an ophthalmic preparation according to any one of claims 1 to 16, the concentrate comprising: a) cyclosporine, in a concentration of at least 0.5 wt % of the concentrate, b) at least two nonionic hydrophilic surfactants, c) at least one oil in a concentration of up to 12% by weight of the concentrate, and d) at least one co-surfactant.

18. The concentrate formulation according to claim 17, wherein The at least two nonionic hydrophilic surfactants include at least one first nonionic hydrophilic surfactant selected from ethoxylated fatty acids, and at least one second nonionic hydrophilic surfactant selected from ethoxylated castor oil and hydrogenated derivatives thereof.

19. The concentrate formulation according to claim 18, wherein The weight ratio of the first nonionic hydrophilic surfactant to the second nonionic hydrophilic surfactant ranges from about 1:1 to 1:

30.

20. The concentrate formulation according to any one of claims 17 to 19, wherein The total concentration of the nonionic hydrophilic surfactant in the concentrate ranges from about 20 wt % to about 75 wt %.

21. A concentrate formulation according to any one of claims 17 to 20, wherein The at least one oil is selected from acylglycerols of fatty acids, triacetin, tributyrin, tricaprylin, triolein, medium chain triglycerides and mixed fatty acid triglycerides, olive oil, sesame oil, soybean oil, canola oil, castor oil, partially or fully hydrogenated castor oil, paraffin oil, mineral oil, non-saponifiable fat derivatives, alkyl alcohols including oleyl alcohol, lauryl alcohol, terpenoids and combinations thereof.

22. A concentrate formulation according to any one of claims 17 to 21, wherein The at least one oil is present in the concentrate in a concentration of no more than 7 wt%.

23. A concentrate formulation according to any one of claims 17 to 22, wherein The at least one co-surfactant is present in the concentrate at a concentration ranging between about 20 wt % to about 45 wt %.

24. A concentrate formulation according to any one of claims 17 to 23, wherein The weight ratio between the nonionic hydrophilic surfactant and the co-surfactant ranges from about 1:1 to about 5:

1.

25. A concentrate formulation according to any one of claims 17 to 24, wherein The nanostructures further include at least one solvent.

26. The concentrate formulation of claim 25, wherein: The at least one solvent is selected from the group consisting of glycerol, ethanol, methanol, propanol, isopropanol, diethanolamine, triethanolamine, and combinations thereof.

27. The concentrate formulation according to claim 25 or 26, wherein The concentrate includes the at least one solvent in a concentration ranging from about 1 wt % to about 10 wt %.

28. A concentrate formulation according to any one of claims 25 to 27, wherein The weight ratio between the nonionic hydrophilic surfactant and the solvent ranges from about 5:1 to about 35:

1.

29. The concentrate formulation of any one of claims 17 to 28, being substantially free of water.

30. A method for preparing an ophthalmic preparation according to any one of claims 1 to 16, It comprises mixing the concentrate according to any one of claims 17 to 29 with an aqueous dispersion medium, thereby obtaining a plurality of nanostructures formed from the concentrate and dispersed in a continuous aqueous phase formed from the aqueous dispersion medium.

31. The method according to claim 30, wherein: The aqueous dispersion medium includes at least one film-forming agent.

32. The method according to claim 30 or 31, wherein: The aqueous dispersion medium includes at least one buffer.

33. The method according to any one of claims 30 to 32, wherein: The concentrate is mixed with the aqueous dispersion medium in a weight ratio ranging between about 1:5 and 1:

25.

34. The method according to any one of claims 30 to 33, wherein: The mixing is performed under conditions that prevent high shear forces from being generated in the mixture.

35. A kit for preparing an ophthalmic preparation according to any one of claims 1 to 16, comprising: at least one first container comprising a concentrate according to any one of claims 17 to 29; at least one second container comprising an aqueous dispersion medium; and Instructions for use.

36. The kit according to claim 35, wherein The aqueous dispersion medium includes at least one film-forming agent.

37. The kit according to claim 35 or 36, wherein The aqueous dispersion medium includes at least one buffer.

38. A kit according to any one of claims 35 to 37, wherein The first container and the second container are integrally formed and configured to mix the concentrate and the aqueous dispersion medium according to user requirements.

39. An ophthalmic preparation according to any one of claims 1 to 16 for use in treating anterior ocular diseases or conditions.

40. The ophthalmic preparation for use according to claim 39, wherein The anterior eye disease or condition is selected from dry eye disease, wet and dry age-related macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia and strabismus.

41. A method of treating anterior ocular diseases or conditions comprising administering to a subject in need thereof an effective amount of an ophthalmic formulation according to any one of claims 1 to 15.

42. The method according to claim 41, wherein: The anterior eye disease or condition is selected from dry eye disease, wet and dry age-related macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia and strabismus.

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