Claims:Title: Novel ophthalmic compositions of Cyclosporin A for the
treatment of dry eye syndrome

Statement of claims

We claim:

1. The lyophilized nanoparticles formulation and implant nano fibres formulation compositions of cyclosporin A, containing cyclosporin A as the active constituent, polycaprolactone and hyaluronic acid as biodegradable polymers, polyvinyl alcohol as emulsifier, mannitol as cryoprotectant and the process for their preparation involving mixing of suitable quantities of cyclosporine A and polycaprolactone in dichloromethane, and emulsifying the mixture with a solution of suitable quantities of hyaluronic acid and polyvinyl alcohol in water using diffusion method or by sonication or some suitable technique to obtain an emulsion, which is lyophilized at a suitable temperature to obtain a lyophilized powder, which could be directly dispersed in tear fluid, and converting the lyophilized powder into implant nano fibres using an electro-charge mixture (13%) of Polyvinyl alcohol through electro-spinning technique which are suitable for dispersion in tear fluid for instillation into eyes for the management of dry eye syndrome.
2. The lyophilized nanoparticles formulation and implant nano fibres formulation compositions of cyclosporin A as claimed in claim 1, wherein the biodegradable polymer used is polycaprolactone and the mucuadhesive agent also as ocular comforting agent used is hyaluronic acid and polyvinyl alcohol is used as the emulsifier for the formulation.
3. The lyophilized nanoparticles formulation and implant nano fibres formulation compositions of cyclosporin A as claimed in claim 1, wherein mannitol is used as cryoprotectant and Polyvinyl alcohol, as electrocharge mixture (8-13%) for obtaining the nano fibres.
4. The process for the formulation of the lyophilized nanoparticles formulation and implant nano fibres formulation compositions of cyclosporin A as claimed in claim 1 which consisted of first making a nano emulsion either by diffusion method or by emulsification method, lyophilizing the nano emulsion and then converting the lyophilized powder into nano fibres by electorspinning method.
5. The product and process for formulations of above said composition claimed in claim 1 provides extended release of Cyclosporin A for chronic treatment of dry eye to reduce dosing frequency and improve patient compliance.
, Description:Field of Invention

Dry eye syndrome (DES) affects 5-34 % of people around the world and has a much higher prevalence in females. Current lack of understanding makes the diagnosis of dry eye syndrome difficult. The treatment approach for dry eye syndrome includes oral omega-3 fatty acid, artificial tears, autologous serum, and various tear retention devices such as Lipiflow® or Punctal plugs, anti-inflammatory drugs, corticosteroids and immunosuppressants. These are associated with one or the other serious side effects. Cyclosporine A (CsA) is an anti-inflammatory agent of choice for the treatment of DES acting as an immunosuppressant, because it can be used for a long period of duration without eliciting adverse effects commonly associated with other anti-inflammatory agents, such as corticosteroids (increased intraocular pressure, risk of infection, cataract formation etc). Furthermore, unlike corticosteroids, the activity of CsA resulting from specific and reversible action on T-cells, makes it safe for prolonged use. Unless the active constituents of the eye drops penetrate through ocular surface tissues into the intraocular tissues to offer appropriate concentrations of the drug, the patient would not get relief from the problem. The present invention discloses biodegradable nanoparticles and implant nanofibres for ocular delivery of poorly aqueous soluble/insoluble anti-inflammatory therapeutic agents in the management of dry eye syndrome.

Background of the Invention
Although ophthalmic formulations are available in the market for the treatment of DES, but they have the disadvantage of instilling the eye drops frequently time and again into the eyes. A sustained-release drug delivery system with minimal side effects and invasion could be an ideal method for addressing this problem. Delivery of a drug using intraocular implants can overcome most of the disadvantages, as they can keep the drug in the vicinity of the target tissues, in concentrations within the therapeutic window for extended periods of time, at least theoretically. Implants manufactured from biodegradable polymers are degraded into non–toxic products that are metabolized or excreted by the body. In this regard, the surgical removal of the implant after completion of therapy is not needed, thereby enhancing the acceptance and patient compliance. Polycaprolactone (PCL) is a biodegradable and biocompatible polymer that has a slow degradation rate, making it suitable for the development of long-term drug delivery systems. One study showed that PCL implants offered a controlled and prolonged delivery of drugs, releasing 25% of the drug in 21 weeks. This result showed that PCL implants could be of interest when long-term sustained intraocular delivery of a drug is required. Topical administration of a drug is the most convenient method of drug delivery for the treatment of various ocular surface diseases. Eye drops account for 90 % of all conventional ophthalmic formulations, even though they suffer from several disadvantages, like very poor bioavailability (due to mechanisms such as nasolacrimal drainage, loss in conjunctival blood circulation, tear dilution, normal tear drainage and reflux blinking etc). This justifies the development and application of novel and more efficient drug delivery systems for ophthalmic problems. Drug loaded biodegradable nanoparticles and implant nanofibres are such novel drug delivery solutions to address the above discussed problems. .

US 2516804 (1950) Artificial Eye Implant: Implant was made up of Tantalum and muscle ring is molded from a suitable plastic material in desired shape (David E., Rolf University Heights, 1950, Ohio; US patent number: 2516804)

US 4,839,342 (1989) Method of increasing tear production by topical administration of cyclosporine: Cyclosporine was formulated in olive oil as a solution for ophthalmic administration. Concentration range is of 0.01 to 50 weight percent, preferably 0.1 to 20 weight percent, of cyclosporine. High amount of cyclosporine was observed in the lacrimal glands by liquid scintillation analysis (Renee Kaswan, Anthens, Ga. University of Georgia Research Foundation, Athens; 1989, US patent number: 4839342).

US 4997652 (1991) Biodegradable ocular implants: The microcapsule shaped implant is formed using biodegradable polymers (polyesters/ethers) or liposomes, wherein the drug is surrounded by a barrier to impede immediate release of the drug upon introduction into the chamber of the eye (Vernon G. Wong, Rockville, Md; Visionex, California,1991, US patent number: 4997652)

WO2006050837A2 (2006) Ophthalmic emulsions containing an immunosuppressive agent: Ophthalmic o/w emulsion comprising at least one immunosuppressive agent, preferably selected from the group consisting of cyclosporine, sirolimus, tacrolimus have been reported. (Nova Gali Pharma SA; 2006, PCT/EP2005/011649, WO2006/050837A2)

US 8,034,366 B2 (2011) Ocular implant made by a double extrusion process: Micronized PLGA (50:50) ester and acid were blended with DEX to form extrudes. These extrudes were pelletized followed by extrudization of the pellets and cut them out into desired size in rod shape. (Jane-Guo Shiah, Rahul Bhagat; Allergan Inc. 2011; US 8034366B2)

Objectives of the Invention
One of the objectives of this invention was to assess the solubility of polymers and drugs in different types of solvents to obtain higher drug solubility so that the maximum therapeutic concentration of the drug could be availed for better therapeutic effect.
It was also aimed to invent a process for the development of lyophilized PCL-HA laden biodegradable nanoparticles containing CsA using the design of experiments approach.
Another objective of the invention was to load the drug into previously prepared biodegradable nanoparticles, in aqueous-polyvinyl alcohol solution (5-20 %).
The main objective of the invention was to formulate biodegradable nano fibres containing CsA by Electrospinning technique with the help of an instrument like E-SPIN NANOTECH SUPER ES-0.
It was also planned to load the biodegradable nanoparticles directly into PVA solution and lyophilize the system so that the lyophilized composition could be directly immersed into simulated tear fluid for instillation into the eyes for DES treatment.
The developed novel biodegradable implant nanofibres were characterized and evaluated by performing various studies including in-vitro drug release, In-vivo pharmacokinetic study on Wister albino rat, Osmolarity, Sterility, Ocular irritancy and Short-term stability study.

Summary of the Invention
The present invention relates to in depth description on the enhancement in solubility of active therapeutics (BCS class II) with thorough screening of polymers, stabilizers, solvents and co-solvents. Formulations of biodegradable nanoparticles of poorly water soluble or practically insoluble drugs using suitable concentrations of polymers have been invented by formation of o/w emulsion with the help of solvents in suitable ratio in which they got solubilized. Along with this, the biodegradable implant nanofibres have been developed using previously prepared PCL-HA laden biodegradable nanoparticles and evaluate and assessed them for various parameters.

The present invention provides o/w emulsion composition formulation of PCL-HA laden biodegradable nanoparticles containing CsA. The resulting nanoparticles were evaluated for the Particle size distribution, Zeta potential, Entrapment of poorly soluble drugs and in-vitro characteristics. The dissolution study was used for the comparison of nanoparticles with the nanofibres developed by this novel technique. Further comparison was done with a marketed formulation to assess the superiority of our finished dosage forms over the marketed formulation.
In an embodiment of the invention, solubility study and selection of one or more excipients for the formulation of emulsion, such as different polymers like PLGA, PCL, Pluronic F 127as well as short to medium chain triglycerides were used to determine the highest solubility of the lipophilic drugs in the selected solvents.
Concentration of various polymers like Hyaluronic acid in o/w emulsion varying in the ranges of 0.15-5.0% w/v of the formulation were used in order to prepare & maintain stable o/w emulsion.
The solvents and co-solvents were added to obtain emulsion systems to get stable emulsion preparations. Short- to medium-chain-length alcohols (C3–C8) are commonly added as co-solvents, which further reduce the interfacial tension and increase the fluidity at the interface. Alcohols may also increase the miscibility of the aqueous and oily phases due to its partitioning between both these phases. Polyvinyl alcohol may reduce the interfacial tension and help in drug solubilization.
Drug polymeric nanodispersions were sonicated using ultrasonicator followed by lyophillization process at initial drying and freezing temp of -10 °C upto 24 hour and secondary drying at -70 °C for another 36 hours, to get lyophilized nanoparticles
Experimental designs are frequently used to establish an empirical relationship in terms of a mathematical model between dependent variables, and a number of factors or independent variables. Physical properties of biodegradable nanoparticles were mainly affected by two independent factors (i.e. concentration of PCL and concentration of HA). Thus, first attempt was made to evaluate the effect of these parameters on formulation of biodegradable nanoparticles. It is desirable to develop acceptable pharmaceutical formulation in shortest possible time using minimum number of hours and raw material. Central composite design (CCD), an experimental design method of Response Surface Methodology, is composed of two-level factorial design with axial point and central point. CCD enables simultaneous investigation of the effect of each factor and their interaction over the experimental responses, and reduces the number of experimental runs that are necessary to establish a mathematical functional relationship in the experimental design region. Nine experimental points are required in a two-factor CCD. In order to estimate pure experimental uncertainty of CCD, it is important to measure repeatedly the response function to the conditions determined by the central points. Minitab 17 software was used for the generation of the mathematical models. ANOVA test was performed to evaluate the level of signi?cance of the tested factors on the release of the drug in the specified formulation as well as the interactions between these factors.

The PCL-HA laden nanoparticle compositions were subjected to different characterization tests as particle size determination using dynamic light scattering technique with particle size of lyophilized PCL-HA laden biodegradable nanoparticles in nano range (206nm - 460nm), zeta potential measurements (-4.10mV - 0.688mV), Entrapment efficiencies (22 - 78%) and for ophthalmic irritancy behavior.
Further, the developed novel biodegradable implant fibers were prepared by addition of lyophilized PCL-HA laden biodegradable nanoparticles and subjected to different evaluation tests as particle size determination using Scanning electron microscopic technique with size of 264.2nm of nanofibres.
The in vitro drug release of PCL HA laden biodegradable nanoparticles was found to be 86.11 ± 1.95 % after 24 hours, which shows sustained release of CsA due to biodegradation of PCL at pH 7.4 (Artificial tear fluid) through hydrolysis and diffusion of the drug. This would lead to decrease in the frequency of administration of the drug resulting in better patient compliance for relief from dry eye syndrome.
The pharmacokinetic parameters were evaluated by determining the drug concentration; AUC (area under the curve) and MRT (mean residence time).
In-vivo studies were performed to compare the release profiles of poorly soluble plain drug and the newly developed implant fiber formulation. The drug release from plain drug solution and from the implant fibers at specific time interval in plasma was quantified using HPLC.
In the in vivo study the maximum concentration (Cmax) of Nanofibre formulation was found to be 19.45 mg, with Mean Residence time at 8 hrs, and Area under curve (AUC) 117.0. Autoclave sterilization method was used for sterilizing the product, and direct inoculation technique was used for the test of sterility which showed no growth of microorganisms in the medium. The non-irritant behavior of the formulation was assessed in rat eye without any significant symptoms of redness or swelling. The resulting implant fibers increase the retention time of the drug in the eye which leads to its enhanced absorption and increased bioavailability.

Detailed Description of the Invention
Experiments were performed for optimization of different criteria as given below:
Criterion-1: Solubility studies and selection of excipients for the formulation of PCL-HA laden biodegradable nanoparticles and NPs loaded implant fibres: In this study, different solvents were used to identify the solvents with the highest solubility of the lipophilic drug (CsA) and the polymers in the selected solvents.

Criterion-2: Determination of ideal concentrations of the polymers for the nanoparticles: The concentration of polymers in biodegradable nanoparticles prepared by o/w emulsification technique varies for each polymer. Polycaprolactone selected as a biodegradable polymer was varied in a concentration range from 0.5-1.5% w/v, whereas Hyaluronic acid was varied in a concentration range from 0.15-0.25 % w/v of the final formulation in order to prepare PCL-HA laden biodegradable nanoparticles by o/w emulsification technique.

Criterion-3: Selection of co-solvent: A co-solvent is added to obtain stable emulsion systems. Short to medium chain-length alcohols (C3–C8) are commonly added as co solvents, which further reduce the interfacial tension and increase the fluidity at the interface. Alcohols may increase the miscibility of the aqueous and oily phases due to its partitioning between these phases.

The present invention will now be described with reference to the following examples, which along with the foregoing preferred embodiments are illustrative and should not be construed as limiting the scope of the invention. Various alterations and modifications will fall within the scope of the invention herein. Typical most preferred isotropic mixtures in accordance with the present invention were prepared as follows with the amount of each ingredient being as indicated below.

Example-1: Preparation of drug laden PCL-HA biodegradable nanoparticle composition by solvent diffusion method: Briefly, polycaprolactone (PCL) at different concentration (0.5 - 0.15%) was dissolved in dichloromethane (DCM) (5.0 ml). After complete dissolution of PCL, Cyclosporin-A (0.5 %) was added into it. An appropriate concentration of Hyaluronic acid (0.15-0.25 %) was also added into (5.0 ml) cold water. The organic and aqueous phases were mixed with the help of ultrasonication probe using ultrasonication time (5 min and 10 min) and ultrasonication amplitude (20% and 30%) that led to diffusion of the organic solvent into aqueous phase. The resulting nanosuspension was then cooled down to -20 °C immediately and lyophilized using 5-10 % mannitol as cryoprotectant. The obtained drug loaded lyophilized nanoparticles were stored at 4 °C till further characterization.

Example-2: Preparation of drug laden PCL-HA biodegradable nanoparticle composition by emulsification method: PCL-HA laden biodegradable nanoparticles containing Cyclosporine-A was prepared by o/w solvent emulsification followed by lyophilization. Briefly, Polycaprolactone (PCL) at different concentration (0.15 - 0.25%) was dissolved in dichloromethane (DCM) (5.0 ml). After complete dissolution of PCL, Cyclosporine-A (0.5 %) was added into it. Simultaneously, an appropriate amount of hyaluronic acid (0.15-0.25 %) was dissolved in 1% aqueous solution of polyvinyl alcohol. The organic phase of dichloromethane which contained Drug and PCL was added to aqueous phase of PVA (5.0 ml) and HA (0.20 %) with the help of ultrasonic probe (60 W, 60 sec) to form an emulsion. The resulting nanoemulsion was then cooled down to -20 °C immediately and lyophilized using 5-10% mannitol as a cryoprotectant. The obtained drug loaded lyophilized nanoparticles were stored at 4 °C for carrying out further studies.

Example-3: Preparation of nano fibers of the drug loaded biodegradable polymers:
Preparation of biodegradable implant fibers using electrospinning technique:
Preparation of PVA aqueous solution:
PVA (13 %) was dissolved in de-ionized water at 80°C with vigorous magnetic stirring. Stirrer hot plate was used and mixing duration was 3 hours. The well dissolved solutions became transparent and colorless. Electrospinning set up was used for production of nanofibers. The parameters of electrospinning were optimized as follows:
o Flow rate- 0.5 ml/hr
o Applied voltage- 22.3 kV
o The distance between spinneret and collector- 20 cm
o Drum speed- 400-500 rpm
Preparation of Polymeric nanoparticles laden implant fibres:
PCL-HA laden biodegradable nanofiber implants were prepared by the electrospinning technique. The E-Spin was performed using the electrospinning of aqueous polyvinyl alcohol solution containing the lyophilized powder from the needle tips. The polyvinyl alcohol aqueous solution (13%) was prepared to make fiber structures. In the above PVA polymer solution, PCL-HA laden biodegradable nanoparticles i.e. appropriate quantity calculated based as per entrapment of drug in prepared biodegradable nanoparticles (0.5 gm) were added and mixed thoroughly to get a clear transparent solution. The PVA solution containing biodegradable nanoparticles were delivered at a constant feed rate of 0.5 ml/hr. The solutions were electrospun on the collecting plate at a distance of 20 cm, generating an electric field of 22.3 kV. The switch of UV light which is provided on the E-spin instrument, was ON at the time of delivery of solution to sterilize the nano fibers (Fig 3). On the collecting plate implant fibers were formed. These implant fibers were collected and used for further analysis and study.

The PCL-HA laden biodegradable nanoparticle compositions were subjected to different characterization test as particle size determination using dynamic light scattering technique, zeta potential measurements as shown in figure 1 and 2, and Entrapment efficiencies by using validated HPLC method as shown in figure 3.

The developed novel biodegradable implant fibres prepared by addition of lyophilized PCL-HA laden biodegradable nanoparticles were subjected to different evaluation tests as determination of particle size using Scanning electron microscopic technique with average diameter of size 264.2 nm of nano fibers shown in figure 4.

The developed novel biodegradable implant fibres were subjected to in-vitro drug dissolution study. The results obtained for in-vitro study indicated 72.30 % drug release after 72 hours, which showed sustained release of this poorly soluble molecule, from the developed implant fibre formulation. This proved that the invented formulation would be able to release the drug continuously upto 72 hrs avoiding frequent instillation of the formulation into eye.

The novel biodegradable implant fibers were subjected to in-vivo pharmacokinetic study. The study was performed to compare the release profiles of this poorly soluble drug (CsA) and the invented implant nano-fiber formulation. The drug release from the plain drug solution and from the invented implant fibers at specific time interval in plasma was quantified using HPLC. The measured drug concentration was plotted against time, and AUC (area under the curve) and MRT (mean residence time) were calculated. The GraphPad Prism software was used to determine the area under curve as, shown in figure 5.
List of illustrations
Figure 1. Particle size distribution analysis of PCL-HA laden biodegradable nanoparticles using dynamic light scattering.
Figure 2. Zeta potential analysis of PCL-HA laden biodegradable nanoparticles to predict kinetic stability Malvern Inc.
Figure 3. Entrapment efficiency analysis of PCL-HA laden biodegradable nanoparticles using validated HPLC method.
Figure 4. Surface analysis and Particle size distribution analysis of biodegradable implant fibres using scanning electron microscopy.
Figure 5. Comparison of In-vivo pharmacokinetic profile of Implant fibres and pure drug using GraphPad Prism software.

SPECIFIC EMBODIMENT
Concentration of Hyaluronic acid had a prominent effect (P<0.05) on particle size than Polycaprolactone. As the concentration of Hyaluronic acid is increasing it resulted in signi?cant increase in the particle size, whereas concentration of Polycaprolactone has a negative influence on the particle size of the nanoparticles formulation. Both the polymers - Polycaprolactone and Hyaluronic acid showed least effect (P<0.05) effect on zeta potential of the formulation. The effect of entrapment of CsA is dependent on the concentration of Polycaprolactone while hyaluronic acid is negatively influencing the entrapment efficiency. Release of the drug from the PCL-HA laden biodegradable Nanoparticles, and the implant fibre formulation, is being shown in the drug dissolution profile as shown in table 1. It becomes clear from this study that the implant fiber formulation enhances the drug dissolution behavior of a poorly soluble drug like cyclosporine A and offers a sustained release of the active constituent.

Table 1.Comparison of dissolution profile of PCL-HA laden biodegradable nanoparticles and implant fibres:
Time (hr) % drug release of PCL-HA laden biodegradable nanoparticles % drug release of implant fibres
0.5 11.90 ± 1.207 1.644±0.012
1 24.003 ± 2.694 7.88 ±0.08
2 26.99 ± 1.003 16.06±0.055
4 32.75 ± 0.961 19.98 ±0.456
6 54.84 ± 0.0859 22.092±1.237
8 64.854 ± 0.103 25.44 ±2.8963
12 71.92 ± 0.0625 26.44 ±1.5684
24 86.11 ± 1.95 30.92±0.639
48 - 42.1 ±3.4893
72 - 72.30 ±1.725
(mean±SD, n=3)