In-situ Gel Containing Cyclosporine Micelles as Sustained Ophthalmic Drug Delivery System

Abstract

The present invention provides aqueous ophthalmic formulations containing 0.01%-5% by weight of cyclosporine which exists in the form of micelles having a particle size not greater than 20 nm, and methods of making and using such formulations.

Claims

1. An aqueous ophthalmic formulation comprising cyclosporine A, a solubilizer, an osmotic pressure regulator, a pH regulator, a viscosity adjuster, and water, wherein micelles with particle size no greater than 20 nm are formed with cyclosporine and the solubilizer and contained in the formulation.

2. The aqueous ophthalmic formulation of claim 1, further comprising a gel-forming polysaccharide polymer, wherein a gel is formed in situ at the physiological temperature with instant viscosity increase upon instillation of the formulation into the eye.

3. The aqueous ophthalmic formulation of claim 1, wherein cyclosporine has a concentration of 0.01% to 5% by weight in the formulation.

4. The aqueous ophthalmic formulation of claim 1, wherein the solubilizer comprises Polyoxyl 20 Cetostearyl Ether, Polyoxyl 15 Hydroxystearate, Soluplus, Polyoxyethylene hydrogenated castor oil, Polyoxyethylene castor oil, Vitamin E Polyethylene Glycol Succinate, or any combination thereof.

5. The aqueous ophthalmic formulation of claim 1, wherein the solubilizer has a concentration of 0.01% to 10% by weight in the formulation.

6. The aqueous formulation of claim Zany of claim 2, wherein the polysaccharide is contained in the formation at a concentration of 0.1% to 0.6% by weight.

7. The aqueous ophthalmic formulation of claim 2, wherein the polysaccharide comprises deacetylated gellan gum (DGG), xanthan, sodium alginate, carrageenan, or any mixture thereof.

8. The aqueous ophthalmic formulation of claim 2, wherein the polysaccharide comprises deacetylated gellan gum (DGG).

9. The aqueous ophthalmic formulation of claim 1, wherein said osmotic pressure regulator comprises sodium chloride, mannitol, glucose, sorbitol, glycerin, polyethylene glycol, propylene glycol, or any combination thereof.

10. The aqueous ophthalmic formulation of claim 1, wherein the osmotic pressure regulator is in the formulation at a concentration of 0.01% to 10% by weight.

11. The aqueous ophthalmic formulation of claim 1, further comprising a preservative which comprises butylparaben, benzalkonium chloride, benzalkonium bromide, chlorhexidine, sorbate, chlorobutanol, or any combination thereof.

12. The aqueous ophthalmic formulation of claim 10, wherein the preservative in the formulation is at a concentration of 0.01% to 5% by weight.

13. The aqueous ophthalmic formulation of claim 1, wherein the pH adjuster comprises boric acid, sodium borate, phosphate buffer, tromethamine, tromethamine hydrochloric acid buffer, sodium hydroxide, hydrochloric acid, citric acid, sodium citrate, or any combination thereof.

14. The aqueous ophthalmic formulation of claim 1, wherein the pH adjuster in the formulation is at a concentration of 0.01% to 5% by weight.

15. The aqueous ophthalmic formulation of claim 1, wherein the viscosity adjuster in the formulation has a concentration of 0.01% to 5% by weight.

16. The aqueous ophthalmic formulation of claim 1, wherein the viscosity adjuster comprises carboxyl methyl cellulose, sodium cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, or any combination thereof.

17. The aqueous ophthalmic formulation of claim 1, wherein the average particle size of the micelles ranges from 10 nm to 20 nm.

18. A micelle comprising water, cyclosporine A, and a solubilizer, wherein the micelle has a particle size no greater than 20 nm.

19. The micelle of claim 18, wherein the solubilizer comprises Polyoxyl 20 Cetostearyl Ether, Polyoxyl 15 Hydroxystearate, Soluplus, Polyoxyethylene hydrogenated castor oil, Polyoxyethylene castor oil, Vitamin E Polyethylene Glycol Succinate, or any combination thereof; and the cyclosporine is cyclosporin A.

20. A method of treating or alleviating symptoms of dry eye disease or condition in a subject in need thereof, comprising topically administering to the eye of the subject a therapeutically effective amount of an aqueous ophthalmic formulation of claim 1.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0030] FIG. 1 shows the particle size and distribution of Sample 1 prepared in Example 1.

[0031] FIG. 2 shows the particle size and distribution of Sample 2 prepared in Example 1.

[0032] FIG. 3 shows the particle size and distribution of Sample 3 prepared in Example 1.

[0033] FIG. 4 shows the particle size and distribution of Sample 4 prepared in Example 1.

[0034] FIG. 5 shows the particle size and distribution of Sample 5 prepared in Example 1.

[0035] FIG. 6 shows the particle size and distribution of Sample 6 prepared in Example 1.

[0036] FIG. 7 shows the particle size and distribution of Sample 7 prepared in Example 1.

[0037] FIG. 8 shows the particle size and distribution of Sample 8 prepared in Example 1.

[0038] FIG. 9 shows the bar chart of viscosity changes of formulation Sample 1 to Sample 6 with gelling matrix DGG prepared in Example 2.

[0039] FIG. 10 shows the bar chart of viscosity changes of formulation Sample 7 to Sample 10 with gelling matrix xanthan gum prepared in Example 2.

[0040] FIG. 11 shows the bar chart of viscosity changes of formulation Sample 11 to Sample 14 with gelling matrix carrageenan prepared in Example 2.

[0041] FIG. 12 shows the bar chart of viscosity changes of formulation Sample 15 to Sample 18 with gelling matrix sodium alginate prepared in Example 2.

[0042] FIG. 13 shows the particle size and distribution of the sample prepared in Example 3.

[0043] FIG. 14 shows the particle size and distribution of RESTASIS.

[0044] FIG. 15 shows the particle size and distribution of CEQUA.

[0045] FIG. 16 shows in vitro release curve of the sample prepared in Example 3, RESTASIS®, CEQUA®.

[0046] FIG. 17 shows the particle size and distribution of the sample prepared in Example 4.

[0047] FIG. 18 shows the in vitro release curve of the sample prepared in Example 4, RESTASIS®, CEQUA®.

[0048] FIG. 19 shows the particle size and distribution of the sample prepared in Example 5.

[0049] FIG. 20 shows the in vitro release curve of the sample prepared in Example 5, RESTASIS®, CEQUA®.

[0050] FIG. 21 shows the particle size and distribution of the sample prepared in Example 6.

[0051] FIG. 22 shows the in vitro release curve of the sample prepared in Example 6, RESTASIS®, CEQUA®.

[0052] FIG. 23 shows the particle size and distribution of the sample prepared in Example 7.

[0053] FIG. 24 shows the in vitro release curve of the sample prepared in Example 7, RESTASIS®, CEQUA®.

[0054] FIG. 25 shows the in vitro dialysis release test of the sample prepared in Example 8 (Samples 1-3), RESTASIS®, CEQUA®.

[0055] FIG. 26 shows the in vitro dialysis release test of the sample prepared in Example 8 (Samples 4-6), RESTASIS®, CEQUA®.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The solubilizers that were used to prepare cyclosporine into micellar solutions as described in literature have been investigated, but were found that the particle sizes formed in those formulations were all above 20 nm. U.S. Pat. No. 2019/0060397A1 describes the use of HCO (i.e., polyoxyethylene hydrogenated castor oil) combined with octoxynol 40 to form a micellar solution, we have confirmed that the particle size of CEQUA® is 22 nm. U.S. Pat. No. 2009/0092665 describes micellar solutions prepared using vitamin E TPGS as a solubilizer and its particle size was larger than 20 nm. CN 103735495B describes the use of polyoxyethylene castor oil as a solubilizer to prepare a micellar solution. Similarly, the micellar solution forms particle size larger than 20 nm. In all the examples mentioned above as cyclosporine solubilizers, the particle sizes formed were all above 20 nm (See Table 1).

TABLE-US-00001 TABLE 1 The particle size of micelles prepared by solubilizers reported in prior arts Percentage Particle size Solubilizer (w/w %) (nm) 0.05% CsA Polyoxyethylene 1.0%/0.05% 22 nm hydrogenated 40 castor oil/Octoxynol 40 Polyoxyethylene 10% 60 nm castor oil 60 Vitamin E TPGS  3% 30 nm

[0057] In order to further increase the bioavailability of cyclosporine in the eye, we have conducted a large number of experiments. We have surprisingly found several solubilizers or combinations of some solubilizers unexpectedly resulted in formation of cyclosporine-containing micelles with particle size less than 20 nm.

[0058] In one aspect, one type of suitable solubilizers is Cetomacrogol 1000 series which has the formula of CH.sub.3[CH.sub.2].sub.m[OCH.sub.2CH.sub.3].sub.nOH, with n being 20.sup.˜24 and m being 15.sup.˜17. Based on the quantity of ethylene oxide (n), it has 2 CAS numbers: CAS 9004-95-9 (macrogol cetyl ethers); CAS 68439-49-6 (macrogol cetostearyl ethers). One representative ingredient of Cetomacrogol 1000 series, Polyoxyl 20 Cetostearyl Ether, belongs to polyoxyethylene (20) cetyl octadecyl ether (n=20) in the polycetol 1000 series. Polyoxyl 20 cetostearyl ether is used as an emulsifier in creams (Synalar®). It had never been reported as a solubilizer for ophthalmic preparations, and there is no research on it as a solubilizer for cyclosporine to form a micellar solution. We have surprisingly discovered that polyoxyl 20 cetostearyl ether (solubilizer A) can form a micellar solution with cyclosporine above its critical micelle concentration for ophthalmic application. Additionally, we have surprisingly found out that the sample's particle average size was extremely small at around 10 nm and maintains uniformity and stability. The particle sizes of these samples were much smaller than those of RESTASIS® and CEQUA®. We expect to have a higher corneal permeability compared to RESTASIS® and CEQUA®, therefore increasing the bioavailability.

[0059] In another aspect, Polyoxyl 15 Hydroxysterate is used as an emulsifier in microemulsion ophthalmic preparations. For example, the commercial product Xelpros® contains 0.25% of Polyoxyl 15 hydroxystearate. CN 201510785005.4 discloses use of Polyoxyl 15 hydroxystearate as an emulsifier at the concentration of 1.2%.sup.˜3.5%. In another prior art example, the particle size of microemulsions prepared with the emulsifier polyoxyl 15 hydroxysterate is 50±30 nm (See L. Gan et al., Int J Pharm., 2009; 365 (1-2): 143-149.). The cyclosporine microemulsion solution prepared by using polyoxyl 15 hydroxystearate as an emulsifier had a particle size greater than 20 nm. Polyoxyl 15 hydroxystearate was never reported to be used as a solubilizer for ophthalmic preparations to prepare micellar solution. The maximum safe dosage of polyoxyl 15 hydroxystearate as an emulsifier for ophthalmology is 0.25%. We have confirmed in our own experiments that 0.25% polyoxyl 15 hydroxystearate could only serve as an emulsifier and could not result in formation of a micellar solution with 0.05% CsA. But we were surprised to discover that polyoxyl 15 hydroxystearate at 1.0% resulted in formation of a micellar solution with cyclosporine above its critical micelle concentration. It was discovered that the sample's particle size was very small, ranging from 10 nm to 15 nm, therefore maintaining good uniformity and stability.

[0060] In another aspect, Soluplus (polyethylene caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer) is a new type of solubilizer, which is mostly used in oral solid preparations. Soluplus has not been used in any commercial eye drops. We surprisingly found out that Soluplus with a concentration of 0.9% and above resulted in forming a micellar solution with 0.05% CsA, and the micelles formed at different concentrations of Soluplus had a particle size of about 65 nm. On the basis of this micellar solution, we also surprisingly discovered that this micellar solution could be combined with the in-situ gel to form micellar in-situ gel eye drops which increased the retention time of micellar particles on the ocular surface and improved bioavailability, and the solution was stable.

[0061] Based on our experimental results, a suitable solubilizing system was found to be any combinations of polyoxyl 20 cetostearyl ether, polyoxyl 15 hydroxystearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, and vitamin E polyethylene glycol succinate. It was found that these combinations also had a good solubilizing capacity for cyclosporine which could form micelles with particle sizes smaller than 20 nm.

[0062] The above solubilizers or mixtures thereof were used with 0.09% cyclosporine to investigate their solubilizing ability. These solubilizers or their mixtures were also found to have a good solubilizing effect for cyclosporine. The particle size of the resultant micelles was much smaller than the particle size of micelles prepared with RESTASIS® or CEQUA®.

[0063] The in-situ gel forming cyclosporine nanoparticle carrier are formulated with one or more ion-sensitive in-situ gel forming materials such as polysaccharides to increase the residence time of the dosage form in the eyes. An in-situ gel topical drug delivery platform was developed by employing an ion-sensitive polysaccharide (e.g., gellan gum) as the gel-forming matrix. Different concentrations of gellan gum were used to determine the viscosity changes at 25° C. (without artificial tears) and 34° C. (with artificial tears), to produce in vitro release profile. Only such optimized gel matrix can potentially form an in-situ gel.

[0064] Deacetylated gellan gum (“DGG”, an exocellular polysaccharide of microbial origin, commercially available as Gelrite®) is an interesting in-situ gelling polymer that seems to perform very well in humans. DGG is an anionic linear polysaccharide comprised of a plurality of four-sugar units. Upon instillation of DGG solutions containing drugs into eyes, gel is formed in-situ after interaction of DGG with the electrolytes (Na.sup.+, K.sup.+, Ca.sup.2+, etc.) in the eye fluid. Since human eye fluid contains large amounts of ions (e.g., sodium, potassium, and calcium ions), ion-sensitive gel preparations are expected to achieve a solution-gel phase transition.

[0065] The current invention involves the incorporation of cyclosporine nano-micelles in the in-situ gel matrix and the formulations are further optimized with the following iterative approaches.

[0066] The current invention is further elucidated with specific examples. It is understood that these examples are included herein to illustrate, and not intended to limit the scope of, the invention. The experimental methods with no specific conditions in the following examples are usually prepared under conventional conditions as reported in the literature or according to the conditions suggested by the excipient's manufacturer. Unless specifically stated, all percentages, ratios, proportions or fractions in this invention are calculated on the weight-by-weight basis. Unless specifically defined in this invention, all professional and scientific terms used herein have the same meaning as well-trained personnel may be familiar with. In addition, any methods and materials similar or equivalent to those recorded in this invention can be applied to this invention. The preferred embodiments and materials described herein are used only for exemplary purposes.

Example 1: Determination of Concentration of Solubilizer

[0067] Samples of the micelle solution s containing 0.05% cyclosporin A are listed in Table 2 below:

TABLE-US-00002 TABLE 2 Sample Formulations of Cyclosporine A Nanomicelle Solutions Sample Active Concentration of Concentration of No. ingredients active ingredients Solubilizer solubilizer 1 Cyclosporine 0.05% Polyoxyl 20 Cetostearyl Ether 0.6% 2 A (Solubilizer A) 5.0% 3 Cyclosporine 0.05% Polyoxyl 15 hydroxystearate 0.6% 4 A (Solubilizer B) 5.0% 5 Cyclosporine 0.05% Soluplus 0.9% 6 A (Solubilizer C) 5.0% 7 Cyclosporine 0.05% Polyoxyl 15 hydroxystearate/ Polyoxyl 15 A Hydrogenated 40 castor oil hydroxystearate: (Solubilizer D) 5.0% Hydrogenated 40 castor oil: 0.1% 8 Polyoxyl 15 hydroxystearate: 0.1% hydrogenated 40 castor oil: 5.0%

Particle Size and Distribution Detection

[0068] Samples 1 to 8 prepared with the above formulations were tested with a particle size analyzer for their micelle particle size and distribution or polydispersity index (PDI) (Table 3). The results are shown in FIGS. 1-8 and confirm the particle sizes of micelles in Samples 1-8 prepared and tested as described are smaller than those in RESTASIS® or CEQUA®.

TABLE-US-00003 TABLE 3 Comparison of particle size of nanomicelles in Samples and RESTASIS ® and CEQUA ® Samples Particle size(nm) PDI Samples 1 10.54 0.013 Samples 2 10.19 0.023 Samples 3 12.43 0.014 Samples 4 12.45 0.015 Samples 5 64.29 0.012 Samples 6 60.90 0.008 Samples 7 12.23 0.010 Samples 8 13.83 0.018 RESTASIS ® 159.4 0.433 CEQUA ® 22.04 0.367

Example 2: Determination of Concentrations of Gelling Agent

[0069] Different in-situ gelling solution samples containing 0.05% cyclosporin A are listed below in Tables 4-7:

TABLE-US-00004 TABLE 4 Concentrations of Gelling Agent DGG Sample deacetylated gellan gum NaCl Cyclosporin A 1 0.2% 0.2% 0.05% 2 0.3% 0.05% 3 0.3% 0.2% 0.05% 4 0.3% 0.05% 5 0.4% 0.2% 0.05% 6 0.3% 0.05%

TABLE-US-00005 TABLE 5 Concentrations of gelling agent Xanthan gum Sample Xanthan gum NaCl Cyclosporine A 7 0.1% 0.2% 0.05% 8 0.3% 0.05% 9 0.3% 0.2% 0.05% 10 0.3% 0.05%

TABLE-US-00006 TABLE 6 Concentration of gelling agent Carrageenan Sample Carrageenan NaCl Cyclosporine A 11 0.1% 0.2% 0.05% 12 0.3% 0.05% 13 0.3% 0.2% 0.05% 14 0.3% 0.05%

TABLE-US-00007 TABLE 7 Concentration of Gelling Agent Sodium Alginate Sample Sodium alginate NaCl Cyclosporine A 15 0.1% 0.2% 0.05% 16 0.3% 0.05% 17 0.3% 0.2% 0.05% 18 0.3% 0.05%

Method for Preparation of Gel Solutions

[0070] Accurately weigh a certain amount of sodium chloride, slowly and evenly add the 85 g of ultrapure water. Stir the solution until sodium chloride was completely dissolved, then slowly and evenly add the gelling agent described above under continuous stirring. Put this solution in a 90° C. water bath and stir for 1 hour. Then cool the mixture to room temperature. Weigh 0.05 g of cyclosporin A and slowly add it to the cooled solution that is being stirred. Add water to the final quantity of 100 g.

Artificial Tear Preparation Method

[0071] Measure NaHCO.sub.3: 2.18 g; NaCl: 6.78 g; CaCl.sub.2.Math.2H.sub.2O: 0.084 g; KCl:1.38 g. respectively and dissolve in 1,000 mL deionized water.

Viscosity Testing Method

[0072] 20 mL of sample solution was loaded to the sample cylinder and was allowed to rest for 5 minutes. Then rotate the rotor to measure the initial viscosity value at 25° C. Under 34° C. (add artificial tears-40:7): 20 mL of sample solution was loaded to the sample cylinder and held it for 5 minutes. Then rotate the rotor to measure the initial viscosity value.

[0073] Viscosities of Samples 1 to 18 were measured for values before and after adding artificial tears using a viscometer respectively. Results are shown in Tables 8-11.

TABLE-US-00008 TABLE 8 Viscosity of Samples 1-6 25° C. 34° C. Viscosity Viscosity (artificial tears) Sample (mpa .Math. s) (mpa .Math. s) Sample 1 40.57 58.10 Sample 2 99.70 369.46 Sample 3 71.71 295.47 Sample 4 238.12 442.28 Sample 5 150.58 553.55 Sample 6 130.91 583.73

TABLE-US-00009 TABLE 9 Viscosity of Samples 7-10 25° C. 34° C. Viscosity Viscosity (artificial tears) Sample (mpa .Math. s) (mpa .Math. s) Sample 7 19.24 20.76 Sample 8 19.45 23.21 Sample 9 222.51 256.80 Sample 10 221.68 255.64

TABLE-US-00010 TABLE 10 Viscosity of Samples 11-14 25° C. 34° C. Viscosity Viscosity (Artificial tears) Sample (mpa .Math. s) (mpa .Math. s) Sample 11 0.00 16.16 Sample 12 2.89 16.58 Sample 13 3.20 19.41 Sample 14 3.17 23.73

TABLE-US-00011 TABLE 11 Viscosity of Samples 15-18 25° C. 34° C. Viscosity Viscosity (artificial tears) Sample (mpa .Math. s) (mpa .Math. s) Sample 15 4.18 17.84 Sample 16 4.94 16.91 Sample 17 6.87 26.98 Sample 18 9.81 18.33

[0074] Based on the data shown in Tables 8-11, we have generated histogram charts (see: FIG. 9 to FIG. 12) about the comparative analysis of viscosity changes before and after mixing with artificial tears for samples using different gelling matrix polymers. Comparing the viscosity value at 25° C. and the viscosity value at 34° C. after adding artificial tears indicated that DGG has shown optimal in-situ gel characteristics with the greatest viscosity changes. After adding artificial tears, the viscosity of the formulation greatly increased, and a larger viscosity value was achieved with a small amount of DGG; Xanthan gum, and Carrageenan, and sodium alginate also exhibited certain in-situ gel properties. After adding artificial tears, the viscosity value has also increased to a certain extent, however the viscosity change is not optimal comparing to gellan gum. Therefore, gellen gum is preferred choice as in-situ gelling matrix polymer.

Example 3: The In-situ Gel of Cyclosporine Micelles in the Present Invention

[0075] The formulation of the micellar ophthalmic gel containing 0.05% cyclosporin A is shown as follows:

[0076] Cyclosporine A 0.05 wt %, deacetylated gellan gum 0.25 wt %, Polyoxyl 20 Cetostearyl Ether 1.0 wt %, sodium chloride 0.15 wt %, mannitol 3.3 wt %, hydroxyparaben 0.02 wt %, appropriate amount of tromethamine-hydrochloric acid buffer, and injection water were added to make a 100 g ophthalmic gel containing 0.05% cyclosporine micelles(Table 12).

TABLE-US-00012 TABLE 12 The composition of example 3 nanomicelle in-situ gel Composition Percentage (wt %) Cyclosporine A 0.05 wt % Deacetylated gellan gum 0.25 wt % Polyoxyl 20 cetostearyl ether 1.0 wt % Sodium chloride 0.15 wt % Mannitol 3.3 wt % Hydroxyparaben 0.02 wt % Tromethamine hydrochloric acid buffer As needed Injection water 100%

Sample Preparation

[0077] Take a prescribed amount of water for injection into a beaker and stir at a uniform speed with a rotary stirrer. Spread the prescribed amount of deacetylated gellan gum in the above-mentioned water under stirring, and then put it into a 90° C. water bath under stirring for 1 h. The solution was taken out and filtered through 0.45 μm microporous filter membrane while it's hot to get sterilized. Solution 1: precisely weigh the prescribed amount of cyclosporin A, add the prescribed amount of Polyoxyl 20 Cetostearyl Ether to dissolve the cyclosporin A, then add the appropriate amount of sodium chloride, mannitol, hydroxybutyrate, and tromethamine hydrochloric acid buffer respectively. Then pass the solution through a 0.45 μm microporous membrane to obtain Solution 2. Mix Solution 1 and Solution 2 with agitation, and pack into eye drops bottles to obtain cyclosporine nanomicelle in-situ gel.

Particle Size and Distribution Detection

[0078] Measure the particle size and distribution of the 0.05% cyclosporine micelle in-situ gel prepared above using a particle size analyzer. Results are shown in FIG. 9 and Table 13.

[0079] Measure the particle size and distribution of RESTASIS® using a particle size analyzer. Results were shown in FIG. 10 and Table 13.

[0080] Measure the particle size and distribution of CEQUA® using a particle size analyzer. Results were shown in FIG. 11 and Table 13.

TABLE-US-00013 TABLE 13 Comparison of particle sizes of nanomicelles of Example 3 and RESTASIS ® and CEQUA ® Sample Particle size(nm) PDI Example 3 12.62 0.328 RESTASIS ® 159.4 0.433 CEQUE ® 22.04 0.367

[0081] From the results in Table 13, it can be seen that the particle size of the nano micelles prepared as sample 3 were smaller than those prepared with Restasis® and Cequa®.

In vitro Release Curve of 0.05% Cyclosporine Micelle Ophthalmic Gel

[0082] The in vitro release test was carried out by the dissolution method, using 100 mL artificial tears as the medium. The temperature was set at 34±0.5° C. The shaking frequency was 100 r/min. 1 mL of sample was added to the ampoule, then 4 mL of artificial tears was added, and the ampoule was placed into the constant temperature and humidity oscillator. At 0.5, 1, 2, 4, 8, 12, 24, 48 hours, 2 mL of each solution was taken, and 2 mL of fresh medium was added. The sample was filtered through a 0.45 μm microporous membrane filter, and 20 μm of the filtrate was injected into a liquid chromatography system to determine the content (amount) of cyclosporin A. The same method was used to measure the in vitro release profiles of nanomicelles prepared with RESTASIS® and CEQUA®. The release curve was plotted as a percentage of cumulative drug release versus time. We compared the cumulative release data of RESTASIS®, CEQUA® and the sample in Example 3. The release curve was shown in FIG. 12 and Table 14.

TABLE-US-00014 TABLE 14 Drug Release Profiles of Example 3 and RESTASIS ® and CEQUA ® Example 3 RESTASIS ® CEQUA ® Time (cumulative (cumulative (cumulative (h) release percent) release percent) release percent) 0.5 0.4% 80.4% 46.1% 1 10.2% 90.7% 87.5% 2 25.0% 91.2% 91.5% 4 36.7% 90.1% 91.2% 8 55.8% 90.1% 91.2% 12 66.7% 90.1% 91.2% 24 75.2% 90.1% 91.2% 30 88.0% 90.1% 91.2% 48 95.6% 90.1% 91.2%

[0083] The data listed in in FIG. 12 show that the 0.05% cyclosporine micelle ophthalmic gel forming formulation of Example 3 had a significantly sustained release profile than the formulations prepared with RESTASIS® and CEQUA®, as it slowly released 90% of cyclosporine after 30 hours, while formulations of both RESTASIS® and CEQUA® proved to be fast release formulations and released around 90% of cyclosporine within 2 hours. The release rate of the formulation of Example 3 was much slower than the release rates of RESTASIS® and CEQUA®, indicating that the in-situ gel matrix did provide a slow-release profile.

[0084] Stability study: 0.05% cyclosporin A micellar ophthalmic gel was prepared and divided into multi-dose eye drop bottles. Samples were stored in a 25° C. stability chamber. Samples were taken on 0, 10, 20 days, 30 days.

[0085] Characterization: property, pH, osmotic pressure, viscosity, content, particle size.

TABLE-US-00015 TABLE 15 The characterization and stability of the prepared nanomicelle in-situ gel 34° C. Viscosity Osmotic 25° C. with Artificial pressure Viscosity Tears (40:7) Content Particle Time Property pH (mOsmol/kg) (mPa .Math. s) (mPa .Math. s) (%) size (nm)  0 Day Clear and 6.86 299 95.60 141.27 101.19 12.62 transparent 10 Day Clear and 6.61 303 93.30 160.98 100.61 12.59 transparent 20 Day Clear and 6.58 303 87.18 159.33 100.23 12.64 transparent 30 Day Clear and 6.56 300 90.26 155.29 100.45 12.55 transparent

Example 4: The In-situ Gel of Cyclosporine Micelles in the Current Invention

[0086] The formulation of the micellar ophthalmic gel containing 0.05% cyclosporin A was shown as followed:

[0087] Cyclosporine A 0.05 wt %, DGG 0.3 wt %, HS-15 1.0 wt %, potassium chloride 0.2 wt %, glycerin 0.8 wt %, paraben 0.05%, propyl paraben 0.01%, appropriate amount of phosphate buffer solution, and injection water were added to make a 100 g ophthalmic gel containing 0.05% cyclosporine micelle (Table 16).

TABLE-US-00016 TABLE 16 Composition of Example 4 nanomicelle in-situ gel Composition Percentage (wt %) Cyclosporine A 0.05%  Deacetylated gellan gum 0.3% HS-15 1.0% Potassium chloride 0.2% Glycerin 0.8% Paraben/propyl paraben 0.05%/0.01% Phosphate buffer As needed Injection water 100% 

Sample Preparation

[0088] Take a prescribed amount of water for injection into a beaker and stir at a uniform speed with a rotary stirrer. Spread the prescribed amount of DGG in the above-mentioned water under stirring, and then put it into a 90° C. water bath under stirring for 1h. The solution was taken out and filtered through 0.45 μm microporous filter membrane while hot to get sterilized Solution 1. Precisely weigh the prescribed amount of cyclosporin A, add the prescribed amount of HS-15 to dissolve the cyclosporin A, add the prescribed amount of potassium chloride, glycerin, paraben, propyl paraben, and phosphate buffer. Then the solution was passed through a 0.45 μm microporous filter to obtain Solution 2. Mix Solution 1 and Solution 2 with agitation, and pack into eye drops bottles to obtain cyclosporine micelle ophthalmic gel.

Particle Size and Distribution Detection

[0089] Measure the particle size and distribution of the 0.05% cyclosporine micelle in-situ gel prepared above using a particle size analyzer. Results are shown in FIG. 13 and Table 17.

TABLE-US-00017 TABLE 17 Comparison of particle size of Example 4 nanomicelle with RESTASIS ® and CEQUA ® Sample Particle size (nm) PDI Example 4 13.25 0.111 RESTASIS ® 159.4 0.433 CEQUE ® 22.04 0.367

[0090] From the results in Table 19, it can be seen that the particle size is much smaller than that of RESTASIS® and CEQUA®.

[0091] In vitro release evaluation: The in vitro release of 0.05% cyclosporine micelle ophthalmic gel was tested.

[0092] The in vitro release test was carried out by the dissolution method, using 100 ml artificial tears as the medium. The temperature was set at 34±0.5° C. The shaking frequency was 100 r/min. 1 mL of sample was added to the ampoule, then 4 mL of artificial tears was added, and the ampoule was placed into the constant temperature and humidity oscillator; at 0.5, 1, 2, 4, 8, 12, 24, 48 hours 2 ml of each solution was taken, and 2 mL of fresh medium was added. The sample was filtered through a 0.45 μm membrane filter, and 20 μL was injected into the liquid chromatography system to determine the content of cyclosporin A. The release curve was plotted as a percentage of cumulative drug release versus time. We compared the cumulative release data of RESTASIS®, CEQUA® and the sample in Example 4. The release curve was shown in FIG. 14 and Table 18.

TABLE-US-00018 TABLE 18 Drug release of example 4 and RESTASIS ® and CEQUA ® Example 4 RESTASIS ® CEQUE ® Time (cumulative (cumulative (cumulative (h) release percent) release percent) release percent) 0.5 5.9% 80.4% 46.1% 1 40.9% 90.7% 87.5% 2 60.2% 91.2% 91.5% 4 70.0% 91.2% 91.5% 8 74.9% 91.2% 91.5% 12 78.6% 91.2% 91.5% 24 80.8% 91.2% 91.5% 30 82.6% 91.2% 91.5% 48 92.4% 91.2% 91.5%

[0093] From the results shown in FIG. 14, it can be seen that the 0.05% cyclosporine micelle ophthalmic gel forming formulation Example 4 comparing to RESTASIS® and CEQUA® has shown a significant sustained release profile and slowly release 90% of cyclosporine after 30 hours, while both RESTASIS® and CEQUA® proved to be fast release formulations and release around 90% of cyclosporine within 2 hours. The release rate is much slower than the release rate of RESTASIS® and CEQUA®, indicating that the in-situ gel matrix provided a slow-release profile.

[0094] Stability study: 0.05% cyclosporin A micellar ophthalmic gel was prepared and divided into multi-dose eye drop bottles. The bottles were stored in a 25° C. stability Chamber. Samples were taken on 0, 10, 20 days, 30 days.

[0095] Characterization: Appearance, pH, osmotic pressure, viscosity, content, particle size. The experimental results are shown in Table 19 below.

TABLE-US-00019 TABLE 19 Characterization and Stability of Prepared Nanomicelle In-Situ Gel 34° C. Viscosity Osmotic 25° C. with Artificial Time pressure Viscosity Tears (40:7) Content Particle (Day) Appearance pH (mOsmol/kg) (mpa .Math. s) (mpa .Math. s) (%) size (nm) 0 Clear and 6.84 297 82.37 151.88 99.68 13.04 transparent 10 Clear and 6.73 300 77.94 156.86 99.56 13.22 transparent 20 Clear and 6.71 302 73.80 163.84 99.48 13.24 transparent 30 Clear and 6.69 298 76.55 159.72 99.15 13.28 transparent

Example 5: In-situ Gel with Cyclosporine Micelles

[0096] The specific prescription of the micellar ophthalmic gel containing 0.05% cyclosporin A was shown as follows:

[0097] Cyclosporine A 0.05 wt %, deacetylated gellan gum 0.4 wt %, Soluplus 0.9 wt %, calcium chloride 0.2 wt %, propylene glycol 0.8 wt %, potassium sorbate 0.01 wt %, appropriate amount of borate buffer, and water for injection were added to make a 100 g of ophthalmic gel containing 0.05% cyclosporine micelles (see Table 20).

TABLE-US-00020 TABLE 20 The composition of nanomicelle-containing in-situ gel in Example 5 Composition Percentage(wt %) Cyclosporine A 0.05%  Deacetylated gellan gum 0.4% Soluplus 0.9% Calcium chloride 0.2% Propylene glycol 0.8% Potassium sorbate 0.01%  Borate buffer As needed Injection water 100% 

Sample Preparation

[0098] Soluplus in a prescribed amount was weighted into a 250 mL beaker. 10 mL of absolute ethanol was added to dissolve prescribed amount of cyclosporin A. The solution was heated at 80° C. to evaporate ethanol, and colorless and transparent film was obtained. 20 ml of deionized water was added to hydrate the film for 15 hours to make Solution 1. Propylene glycol, calcium chloride, potassium sorbate, deacetylated gellan gum were weighted according to the prescribed amounts, and added into 70 ml of deionized water, heated at 90° C. for 1 hour under stirring until gellan gum was completely dissolved. Solution 2 was obtained after cooling. Solution 2 was slowly added into Solution 1 under stirring, and finally the pH was adjusted with borate buffer. Deionized water was added to make the final weight of 100 g. Samples were filtered through 0.22 μm microporous membrane filter for sterilization.

Particle Size and Distribution Detection

[0099] Measure the particle size and distribution of the 0.05% cyclosporine micelle in-situ gel prepared above using a particle sizer. Results are shown in FIG. 15 and Table 21.

TABLE-US-00021 TABLE 21 Comparison of particle size of Example 5 nanomicelle with RESTASIS ® and CEQUA ® Sample Particle size(nm) PDI Example 5 71.93 0.125 RESTASIS ® 159.4 0.433 CEQUA ® 22.04 0.367

[0100] The results in Table 21 and FIG. 15 show that the micellar particle size of Example 5 was much smaller than that of RESTASIS® but bigger than CEQUA®.

[0101] In vitro release evaluation: The in vitro release curve of 0.05% cyclosporine micelle ophthalmic gel was generated.

[0102] The in vitro release test was carried out by the dissolution method, using 100 ml artificial tears as the medium. The temperature was set at 34±0.5° C. The shaking frequency was 100 r/min. 1ml of sample was added to the ampoule, then 4m1 of artificial tears was added, and the ampoule was placed into the constant temperature and humidity oscillator; at 0.5, 1, 2, 4, 8, 12, 24, 48 hours, 2 mL of each solution was taken and 2 ml of fresh medium was added. The sample was filtered through a 0.45 μm microporous membrane filter, and 20 μL was injected into the liquid chromatography system to determine the content of cyclosporin A. The release curve was plotted as a percentage of cumulative drug release versus time. We compared the cumulative release data of RESTASIS®, CEQUA® and the sample in Example 5. The release curve was shown in FIG. 16 and Table 22.

TABLE-US-00022 TABLE 22 Drug release of example 5 and RESTASIS ® and CEQUA ® Example 5 RESTASIS ® CEQUA ® Time (cumulative (cumulative (cumulative (h) release percent) release percent) release percent) 0.5 10.3% 80.4% 46.1% 1 20.2% 90.7% 87.5% 2 29.3% 91.2% 91.5% 4 36.8% 91.2% 91.5% 8 44.2% 91.2% 91.5% 12 60.3% 91.2% 91.5% 24 81.5% 91.2% 91.5% 30 86.7% 91.2% 91.5% 48 93.1% 91.2% 91.5%

[0103] It can be seen from the results in FIG. 16 that the 0.05% cyclosporine micelle ophthalmic gel forming formulation Example 5 comparing to RESTASIS® and CEQUA® has shown a significant sustained release profile and slowly release 90% of cyclosporine after 30 hours, while both RESTASIS® and CEQUA® proved to be fast release formulations and release around 90% of cyclosprine within 2 hours. The release rate is much slower than the release rate of RESTASIS® and CEQUA®, indicating that the in-situ gel matrix provided a slow-release profile.

[0104] Stability study: 0.05% cyclosporin A micellar ophthalmic gel was prepared and divided into multi-dose eye drop bottles. Samples were stored in a 25° C. stability chamber. Samples were taken on 0, 10, 20 days, 30 days.

[0105] Characterization: appearance, pH, osmotic pressure, viscosity, content, particle size.

Experimental results (Table 23):

TABLE-US-00023 TABLE 23 The characterization and stability of the prepared nanomicelle in-situ gel 34° C. Viscosity Osmotic 25° C. With Artificial Time pressure Viscosity Tears(40:7) Content Particle (Day) Appearance pH (mOsmol/kg) (mpa .Math. s) (mpa .Math. s) (%) size (nm) 0 Milky white 7.59 299 70.76 184.23 99.78 71.93 10 Milky white 7.44 300 67.99 183.59 98.83 71.36 20 Milky white 7.35 298 61.83 206.33 98.59 72.89 30 Milky white 7.28 301 68.29 198.55 98.66 71.43

Example 6: The In-situ Gel of Cyclosporine Micelles in the Current Invention

[0106] The formulation of the micellar ophthalmic gel containing 0.05% cyclosporin A is shown as follows:

[0107] Cyclosporine A 0.05 wt %, DGG 0.3 wt %, HS-15 0.25 wt %, RH-40 1.0 wt %, sodium chloride 0.25 wt %, mannitol 3.3 wt %, paraben fat 0.05%, Propylparaben 0.01 wt %, appropriate amount of tromethamine hydrochloric acid buffer solution, and water for injection were added to make a 100 g of ophthalmic gel containing 0.05% cyclosporine micelles (Table 24).

TABLE-US-00024 TABLE 24 The composition of example 6 nanomicelle in-situ gel Composition Percentage(wt %) Cyclosporine A 0.05 w % Deacetylated gellan gum  0.3 w % HS-15/RH-40 0.25 w %/1.0 t % Sodium chloride 0.25% Mannitol  3.3% Paraben fat/Propylparaben  0.05%/0.01% Tromethamine hydrochloric acid buffer As needed Injection water  100%

Sample Preparation

[0108] Take a prescribed amount of water for injection into a beaker and stir at a uniform speed with a rotary stirrer. Spread the prescribed amount of deacetylated gellan gum in the above-mentioned water under stirring, and then put it into a 90° C. water bath under stirring for 1 hour. The solution was taken out and filtered through 0.45 μm microporous filter membrane while hot to get sterilized Solution 1. Precisely weigh the prescribed amount of cyclosporin A, add the prescribed amounts of HS-15 and RH-40 to dissolve the cyclosporin A, Add the appropriate amount of sodium chloride, mannitol, paraben, propyl paraben, and tromethamine hydrochloride buffer. Then the solution was passed through a 0.45 μm microporous membrane filter to obtain Solution 2. Mix Solution 1 and Solution 2 with agitation to obtain cyclosporine micelle ophthalmic gel and pack into eye drops bottles.

Particle Size and Distribution Measurement

[0109] The particle size and distribution index of the 0.05% cyclosporine micelles-containing in-situ gel prepared above was measure using a particle size analyzer, and the results are listed below in FIG. 17 and Table 25.

TABLE-US-00025 TABLE 25 Comparison of particle size of nanomicelles in Example 6 and RESTASIS ® and CEQUA ® Sample Particle size(nm) PDI Example 6 14.57 0.168 RESTASIS ® 159.4 0.433 CEQUE ® 22.04 0.367

[0110] From the results in Table 25, it can be seen that the particle size is much smaller than that of RESTASIS® and CEQUA®.

[0111] In vitro release evaluation: The in vitro release curve of 0.05% cyclosporine micelle ophthalmic gel was generated.

[0112] The in vitro release test was carried out by the dissolution method, using 100 ml artificial tears as the medium. The temperature was set at 34±0.5° C. The shaking frequency was 100 r/min. 1 ml of sample was added to the ampoule, then 4 ml of artificial tears was added, and the ampoule was placed into the constant temperature and humidity oscillator; at 0.5, 1, 2, 4, 8, 12, 24, 48 hours, 2 ml of each solution was taken, and 2 ml of fresh medium was added. The sample was filtered through a 0.45 μm microporous membrane filter, and 20 μL was injected into the liquid chromatography system to determine the content of cyclosporine A. The release curve was plotted as a percentage of cumulative drug release versus time. We compared the cumulative release data of RESTASIS®, CEQUA® and the sample in Example 5.The release curve was shown in FIG. 18 and Table 26.

TABLE-US-00026 TABLE 26 Drug release of example 6 and RESTASIS ® and CEQUA ® Example 6 RESTASIS ® CEQUA ® Time (cumulative (cumulative (cumulative (h) release percent) release percent) release percent) 0.5 6.1% 80.4% 46.1% 1 29.8% 90.7% 87.5% 2 43.0% 91.2% 91.5% 4 55.2% 91.2% 91.5% 8 67.6% 91.2% 91.5% 12 72.8% 91.2% 91.5% 24 81.5% 91.2% 91.5% 30 84.5% 91.2% 91.5% 48 91.6% 91.2% 91.5%

[0113] It can be seen from the results in FIG. 18 that the 0.05% cyclosporine micelle ophthalmic gel forming formulation Example 6 comparing to RESTASIS® and CEQUA® has shown a significant sustained release profile and slowly release 90% of cyclosporine after 30 hours, while both RESTASIS® and CEQUA® proved to be fast release formulations and release around 90% of cyclosprine within 2 hours. The release rate is much slower than the release rate of RESTASIS® and CEQUA®, indicating that the in-situ gel matrix provided a slow-release profile.

[0114] Stability study: 0.05% cyclosporin A micellar ophthalmic gel was prepared and divide it into multi-dose eye drop bottles. Samples were stored in a 25° C. stability chamber. Samples were taken on 0, 10, 20 days, 30 days.

[0115] Characterization: Appearance, pH, osmotic pressure, viscosity, content, particle size. Experimental results are listed in Table 27 below.

TABLE-US-00027 TABLE 27 The characterization and stability of the prepared nanomicelle in-situ gel 34° C. Viscosity Osmotic 25° C. with Artificial Time pressure Viscosity Tears (40:7) Content Particle (Day) Appearance pH (mOsmol/kg) (mpa .Math. s) (mpa .Math. s) (%) size (nm) 0 Clear and 6.91 308 76.25 247.82 99.01% 14.57 transparent 10 Clear and 6.85 304 61.91 257.50 97.13 15.25 transparent 20 Clear and 6.74 309 60.77 241.18 98.11 15.85 transparent 30 Clear and 6.69 305 66.97 239.25 98.65 14.99 transparent

Example 7: The In-situ Gel of Cyclosporine Micelles in the Current Invention

[0116] The formulation of the micellar ophthalmic gel containing 0.09% cyclosporin A is shown as follows:

[0117] Cyclosporine A 0.09 wt %, DGG 0.3 wt %, HS-15 0.25 wt %, RH-40 1.0 wt %, sodium chloride 0.25 wt %, mannitol 3.3 wt %, paraben fat 0.05% ,propylparaben 0.01 wt %, appropriate amount of tromethamine hydrochloric acid buffer solution, and injection water were added to make a 100 g of ophthalmic gel containing 0.05% cyclosporine micelles (Table 28).

TABLE-US-00028 TABLE 28 Composition of Example 7 nanomicelle in-situ gel Composition Percentage(wt %) Cyclosporine A 0.09% Deacetylated gellan gum  0.3% HS-15/RH-40 0.25%/1.0%  Sodium chloride 0.25% Mannitol  3.3% Paraben fat/Propylparabe 0.05%/0.01% Tromethamine hydrochloric acid buffer As needed Injection water  100%

Sample Preparation

[0118] Take a prescribed amount of water for injection into a beaker and stir at a uniform speed with a rotary stirrer. Spread the prescribed amount of deacetylated gellan gum in the above-mentioned water under stirring, and then put it into a 90° C. water bath under stirring for 1 hour. The solution was taken out and filtered through 0.45 μm microporous membrane filter while hot to get sterilized Solution 1. Precisely weigh the prescribed amount of cyclosporin A, add the prescribed amounts of HS-15 and RH-40 to dissolve the cyclosporin A, Add the appropriate amount of sodium chloride, mannitol, paraben, propyl paraben, and tromethamine hydrochloride buffer. Then the solution was passed through a 0.45 μm microporous membrane filter to obtain Solution 2. Mix Solution 1 and Solution 2 with agitation to obtain cyclosporine micelle ophthalmic gel, and pack into eye drops bottles.

Particle Size and Distribution Measurement

[0119] Measure the particle size and distribution of the 0.09% cyclosporine micelle in-situ gel prepared above using a particle size analyzer. Results were shown in FIG. 19 and Table 29.

TABLE-US-00029 TABLE 29 Comparison of particle size of nanomicelles in Example 7 and RESTASIS ® and CEQUA ® Sample Particle size(nm) PDI Example 7 14.10 0.097 RESTASIS ® 159.4 0.433 CEQUE ® 22.04 0.367

[0120] The results in Table 29 show that the particle size of nanomicelles in Example 7 was smaller than that of RESTASIS® and CEQUA®.

[0121] In vitro release evaluation: The in vitro release curve of 0.09% cyclosporine micelle ophthalmic gel was tested.

[0122] The in vitro release test was carried out by the dissolution method, using 100 ml artificial tears as the medium. The temperature was set at 34±0.5° C. The shaking frequency was 100 r/min. 1 ml of sample was added to the ampoule, then 4 ml of artificial tears was added, and the ampoule was placed into the constant temperature and humidity oscillator; at 0.5, 1, 2, 4, 8, 12, 24, 48 hours, 2 ml of each solution was taken, and 2ml of fresh medium was added. The sample was filtered through a 0.45 μm microporous membrane filter, and 20 μL was injected into the liquid chromatography system to determine the content of cyclosporin A. The release curve was plotted as a percentage of cumulative drug release versus time. We compared the cumulative release data of RESTASIS®, CEQUA® and the sample in Example 5. The release curve was shown in FIG. 20 and Table 30.

TABLE-US-00030 TABLE 30 Drug release of Example 7 and RESTASIS ® and CEQUA ® Example 7 RESTASIS ® CEQUA ® Time (cumulative (cumulative (cumulative (h) release percent) release percent) release percent) 0.5 6.57% 80.4% 46.1% 1 26.6% 90.7% 87.5% 2 37.9% 91.2% 91.5% 4 60.5% 91.2% 91.5% 8 69.3% 91.2% 91.5% 12 75.8% 91.2% 91.5% 24 86.9% 91.2% 91.5% 30 89.6% 91.2% 91.5% 48 92.6% 91.2% 91.5%

[0123] From the results in FIG. 20, it can be seen that the 0.05% cyclosporine micelle ophthalmic gel forming formulation Example 7 comparing to RESTASIS® and CEQUA® has shown a significant sustained release profile and slowly release 90% of cyclosporine after 30 hours, while both RESTASIS® and CEQUA® proved to be fast release formulations and release around 90% of cyclosprine within 2 hours. The release rate is much slower than the release rate of RESTASIS® and CEQUA®, indicating that the in-situ gel matrix provided a slow-release profile.

[0124] Stability study: 0.09% cyclosporin A micellar ophthalmic gel was prepared and divide it into multi-dose eye drop bottles. Samples were stored in a 25° C. stability chamber. Samples were taken on 0, 10, 20 days, 30 days.

[0125] Characterization: appearance, pH, osmotic pressure, viscosity, content, particle size. The results are listed in Table 31 below.

TABLE-US-00031 TABLE 31 Characterization and stability of nanomicelle-containing in-situ gel 34° C. Viscosity Osmotic 25° C. with artificial Time pressure Viscosity Tears (40:7) Content Particle (Day) Property pH (mOsmol/kg) (mpa .Math. s) (mpa .Math. s) (%) size (nm) Clear and 6.86 291 83.79 166.56 98.37 14.10 transparent 10 Clear and 6.79 295 79.60 167.03 98.33 14.31 transparent 20 Clear and 6.76 293 80.55 172.66 98.26 14.26 transparent 30 Clear and 6.75 293 82.41 169.37 98.05 14.08 transparent

Example 8: In Vitro Dialysis Release Test

[0126] In vitro dialysis release test was conducted on Samples 1-6, RESTASIS®, and CEQUA®. The formulations/compositions of tested Samples 1-6 are listed below in Table 32.

TABLE-US-00032 TABLE 32 Compositions of the nanomicelles samples tested for dialysis release Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Cyclosporine  0.03%  0.05%  0.09% 0.03% 0.05% 0.09% Polyoxyl 20 0.6% 0.6% 1.0% — — — Cetostearyl Ether Polyoxyl 15 — — — 0.25% 0.25% 0.25% Hydroxystearate Polyoxyethylene — — — 1.0%  1.0% 1.0% 40 castor oil Mannitol 3.3% 3.3% 3.3% 3.3% 3.3% 3.3% Water for Up to 100 g Up to 100 g Up to 100 g Up to 100 g Up to 100 g Up to 100 g Injection

[0127] 2 mL of each of Samples 1-6, RESTASIS® and CEQUA® was taken and added to a 14 KDa dialysis bag, which was then put into 200 mL artificial tear (containing 30% ethanol) pre-warmed to 34.5° C. The sample was shaken in water bath shaker at 100 rpm, and , take out 5 ml release medium at certain time point (0.5, 1, 2, 4, 6, 8, 12, 18 h), and add same volume of release medium (pre-warm to 34.5° C.) quickly. The available cyclosporine concentration was determined using HPLC. The release curve is obtained by plotting the cumulative release percentage of the drug against time. We compared the cumulative release data of RESTASIS®, CEQUA® and Sample 1-3. The release curve is shown in Table 33 and FIG. 21. Additionally, we compared the cumulative release data of RESTASIS®, CEQUA® and the Sample 4-6. The release curve is shown in Table 33 and FIG. 22.

TABLE-US-00033 TABLE 33 Comparison of drug release from samples 1-6 and RESTASIS ® and CEQUA ® Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 RESTASIS ® CEQUA ® Time(h) Cumulative release (μg/mL) 0.5 0.515 0.669 0.433 0.209 0.291 0.294 0.304 0.286 1 0.656 0.847 0.601 0.348 0.513 0.842 0.403 0.877 2 0.809 0.952 1.389 0.622 0.846 1.741 0.570 1.504 4 1.125 1.542 3.612 1.035 1.384 3.208 0.911 2.896 8 1.756 2.028 5.694 1.514 1.976 5.247 1.566 4.211 12 2.138 2.869 7.553 1.966 2.625 7.189 1.825 6.189 18 2.612 4.125 8.972 2.374 3.975 8.687 2.183 7.569

[0128] Polyoxyl 20 cetostearyl ether was used as a solubilizer to prepare cyclosporine Sample 1 (0.03 % CsA), Sample 2 (0.05 % CsA) and Sample 3 (0.09 % CsA). The drug permeation from those samples was compared with that of RESTASIS® (0.05 % CsA) and CEQUA® (0.09 % CsA) using the semipermeable membrane as shown in FIG. 22. The cumulative release of Sample 2 (0.05% CsA) was significantly higher than that of RESTASIS® (0.05 % CsA) and the cumulative release of Sample 3 (0.09 % CsA) was significantly higher than that of CEQUA® (0.09 % CsA). The cumulative release of Sample 1 (0.03 % CsA) was similar to that of RESTASIS® (0.05 % CsA). The results demonstrated that in the simulated corneal penetration test using semi-permeable membrane, a smaller micelle particle size significantly increased the penetration of cyclosporine in the cornea and thus reduced the concentration of the drug in the ophthalmic preparation to achieve the same or even better therapeutic effect. This is a surprising discovery that potentially we can use less concentration of cyclosporine to achieve similar therapeutic effect with the smaller particle size nanomicelle formulation, and we can expect our formulation with same concentrations as RESTASIS® (0.05 % CsA) or CEQUA® (0.09 % CsA) can achieve much better therapeutic effect. In addition, reducing the concentration of the drug will also reduce the irritation of the drug to the eyes.

[0129] Polyoxyl 15 Hydroxystearate and Polyoxyethylene 40 Castor oil were used as solubilizers to prepare Sample 4 (0.03 % CsA), Sample 5 (0.05 % CsA) and Sample 6 (0.09 % CsA). The drug permeation from those Samples was compared with that of RESTASIS® (0.05% CsA) and CEQUA® (0.09 % CsA) using the semipermeable membrane as shown in FIG. 22. While the cumulative release of Sample 4 (0.03 % CsA) was similar to that of RESTASIS® (0.05 % CsA), the cumulative release of Sample 5 (0.05 % CsA) was significantly higher than that of RESTASIS® (0.05 % CsA) and the cumulative release of Sample 6 (0.09 % CsA) was significantly higher than that of CEQUA® (0.09 % CsA). This further confirmed that smaller micelle particle size greatly increased the penetration of cyclosporine in the cornea and further reduced the need for higher concentration of the drug in the ophthalmic preparation to achieve the same or even better therapeutic effect. These advantages may also help reduce the frequency of drug administration as well.