Ocular drug delivery

Abstract

This invention relates to a Nanostructured Lipid Carrier (NLC) particle comprising a therapeutic agent encapsulated therein for ocular delivery of the therapeutic agent, wherein the Nanostructured Lipid Carrier comprises: (i) a solid outer shell comprising a solid lipid, and (ii) a liquid core comprising a liquid lipid; and wherein the core comprises the therapeutic agent. Compositions of the NLC particles, use of the NLC particles, methods of treatment or prevention of an eye disorder and an eye drop dispenser are also provided.

Claims

1. A Nanostructured Lipid Carrier (NLC) particle comprising a therapeutic agent encapsulated therein for ocular delivery of the therapeutic agent, wherein the NLC particle comprises: (i) a solid outer shell comprising a solid lipid, wherein the solid lipid comprises cholesterol, and (ii) a liquid core comprising a liquid lipid wherein the liquid lipid comprises a medium chain triglyceride (MCT), and wherein the core comprises the therapeutic agent; the NLC particle providing enhanced mucoadhesion and enhanced residence time on a cornea as compared to the therapeutic agent alone.

2. The NLC particle according to claim 1, wherein the medium chain triglyceride comprises triglycerides of fatty acids selected from the group consisting of caproic acid, enanthic acid, caprylic acid, nonanoic acid, capric acid, lauric acid, and combinations thereof.

3. The NLC particle according to claim 1, wherein the medium chain triglyceride comprises triglycerides of capric acid (C10) and/or caprylic acid (C8).

4. The NLC particle according to claim 1, wherein the liquid lipid of the liquid core comprises a lipid selected from the group consisting of paraffin oil, 2-octyl dodecanol, oleic acid, squalene, isopropyl myristate, vitamin E, triglycerides of the fractionated plant fatty acids C8 and C10, diethylene glycol monoethyl ether NF, medium-chain triglycerides of caprylic (C8) and capric (C10) acids, propylene glycol dicaprylocaprate, propylene glycol monolaurate, propylene glycol monocaprylate, and combinations thereof; or wherein the liquid lipid of the liquid core comprises coconut oil, or an equivalent, fraction or component thereof, and/or castor oil, or an equivalent, fraction or component thereof.

5. The NLC particle according to claim 1, wherein the liquid lipid of the liquid core comprises medium-chain triglycerides of caprylic (C8) and capric (C10) acids.

6. The NLC particle according to claim 1, wherein the therapeutic agent is suitable for treatment or prevention of an eye disorder.

7. The NLC particle according to claim 6, wherein the eye disorder is selected from any one of the group of disorders consisting of dry eye syndrome (keratoconjunctivitis sicca); conjunctivitis; keratitis; uveitis; scleritis; episcleritis; blepharitis; acanthamoeba keratitis; and iritis; or combinations thereof.

8. The NLC particle according to claim 1, wherein the therapeutic agent is hydrophobic.

9. The NLC particle according to claim 1, wherein the therapeutic agent is an anti-inflammatory agent.

10. The NLC particle according to claim 9, wherein the anti-inflammatory agent comprises of a corticosteroid.

11. The NLC particle according to claim 10, wherein the corticosteroid is selected from the group consisting of fluocinolone, difluprednate, loteprednol, fluorometholone, medrysone, dexamethasone, prednisolone, triamcinolone, rimexolone, and combinations thereof.

12. The NLC particle according to claim 1, wherein the therapeutic agent comprises an antihistamine and/or decongestant.

13. The NLC particle according to claim 1, wherein the therapeutic agent is provided at a concentration of between about 0.01 mg/ml and about 1 mg/ml.

14. The NLC particle according to claim 1, wherein the therapeutic agent is provided in combination with one or more other therapeutically active agents.

15. The NLC particle according to claim 1, wherein the NLC particle comprises between about 1:4 w:w and about 1:30 w:w of solid outer shell relative to liquid core.

16. A composition comprising a plurality of NLC particles comprising a therapeutic agent encapsulated therein in accordance with claim 1.

17. The composition according to claim 16, wherein the composition is an ophthalmically acceptable composition.

18. A method of treatment or prevention of an eye disorder in a subject comprising the administration of the NLC particle according to claim 1 to an eye of the subject.

19. The method according to claim 18, wherein the administration is topical to the surface of the eye or to the eyelid.

20. An eye drop dispenser or eye wash device comprising the NLC particle according to claim 1.

21. A method of treating or preventing an eye disorder in a subject comprising administering a Nanostructured Lipid Carrier (NLC) particle to an eye of the subject and allowing enhanced mucoadhesion and enhanced residence time of the NLC particle on the cornea, wherein the NLC particle comprises: (i) a solid outer shell comprising a solid lipid, wherein the solid lipid comprises cholesterol, and (ii) a liquid core comprising a liquid lipid wherein the liquid lipid comprises a medium chain triglyceride (MCT), and wherein the core comprises the therapeutic agent; and wherein the NLC particle provides enhanced mucoadhesion and enhanced residence time on a cornea as compared to the therapeutic agent alone.

Description

(1) Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

(2) FIG. 1. Schematic illustrating the structure of the DX encapsulated CHLF-NLCs. NLCs mainly consist of lipids e.g. Solid lipid (outer layer) and liquid lipid (core). The drug is encapsulated in Liquid lipid. Cholesterol is solid lipid and Labrafac Lipophile® WL 1349 is liquid lipid with drug is encapsulated in the liquid lipid.

(3) FIG. 2. SEM images of CHLF-NLCs with (CHLF-D) and without (CHLF) drug loading.

(4) FIG. 3. Calibration curve for the dexamethasone and calculated drug entrapment efficiency of CHLF-D. Percentage entrapment efficiency for CHLF—D (1:30) (1 mg/ml): 99.9%.

(5) FIG. 4. Dexamethasone release study from the CHLF-NLCs.

(6) FIG. 5. Cytotoxicity study of CHLF NLCs ratio (1:30) with MTT assay.

(7) FIG. 6. Cellular uptake study of CHLF-NLC with fluorescence microscopy.

(8) FIG. 7. Ex vivo mucoadhesion study with fluorescence Microscopy.

(9) FIG. 8. Permeation study with drug loaded CHLF NLCs and marketed formulation MAXIDEX.

(10) FIG. 9. Size and zeta potential of CHLF (without drug loaded) NLC and CHLF-D (with drug loaded) NLC of 1:4 ratio.

(11) FIG. 10. Calibration curve for the dexamethasone and calculated drug entrapment efficiency of CHLF-D. Percentage entrapment efficiency for CHLF—D (1:4) (1 mg/ml): 99.67%.

(12) FIG. 11. Dexamethasone release study from the CHLF-D (1:4) NLCs.

(13) FIG. 12. Cytotoxicity study of CHLF (1:4) NLCs with MTT assay.

(14) FIG. 13. Permeation study with drug loaded CHLF (1:4) NLCs and marketed formulation MAXIDEX.

(15) FIG. 14. Ex vivo mucoadhesion study by fluorescence Microscopy of without CHLF-NLC treated cornea.

(16) FIG. 15. Ex vivo mucoadhesion study by fluorescence Microscopy of Coumarin-6 labelled CHLF-NLC treated cornea.

(17) FIG. 16. Cellular uptake study by fluorescence microscopy of human corneal epithelium cells without CHLF-NLC treatment.

(18) FIG. 17. Cellular uptake study by fluorescence microscopy of human corneal epithelium cells with Cumarin 6 encapsulated CHLF-NLC treatment.

(19) FIG. 18. Cell viability study of pilot scale CHLF-D-NLCs.

(20) FIG. 19. (a) Untreated control HCEC cells (DIC mode), (b) control (FITC mode), (c) cumarine-6 labelled CHLF-NLCs treated cells (DIC mode), (d) cumarine-6 labelled CHLF-NLCs treated cells (FITC mode).

(21) FIG. 20. (a) Untreated control cornea (DIC mode), (b) control (FITC mode), (c) cumarine-6 labelled CHLF-NLCs treated cornea (DIC mode), (d) cumarine-6 labelled CHLF-NLCs treated cornea (FITC mode).

(22) FIG. 21. Drug release study of pilot scale CHLF-D-NLCs.

(23) FIG. 22. Permeation study of marketed formulation and pilot scale CHLF-D-NLCs.

(24) FIG. 23. Size stability study of lab scale samples.

(25) FIG. 24. Size stability study of Pilot scale samples.

(26) FIG. 25. Dexamethasone encapsulation efficiency stability study of Lab scale samples.

(27) FIG. 26. Dexamethasone encapsulation efficiency stability study of Pilot scale samples.

(28) FIG. 27. Drug release study is performed for sample PLSC-3 after 6 months.

(29) FIG. 28. Drug release study is performed for sample PLSC-4 after 6 months.

(30) FIG. 29. Permeation study is performed for sample PLSC-3 after 6 months.

(31) FIG. 30. Permeation study is performed for sample PLSC-4 after 6 months.

(32) FIG. 31. Cytotoxicity study of CHCACO-NLCs with MTT assay.

(33) FIG. 32. Cytotoxicity study of CHCACO-D-NLCs (Drug loaded NLCs) with MTT assay.

(34) FIG. 33. Cellular uptake study of CHCACO-NLCs.

(35) FIG. 34. Mucoadhesion study of CHCACO-NLCs.

(36) FIG. 35. Drug release study of CHCACO-D-NLCs, and

(37) FIG. 36. Permeation study of CHCACO-D-NLCs.

EXAMPLE 1

(38) Nanostructured Lipid Carriers (NLCs) of Cholesterol-Labrafac Lipophile® WL 1349 (CHLF)

(39) Nanostructured lipid carriers (NLCs) of cholesterol-Labrafac Lipophile® WL 1349 (CHLF) lypophile have been designed, optimised, synthesised and characterized. The prepared cholesterol-Labrafac Lipophile® WL 1349 NLCs encapsulated anti-inflammatory corticosteroid drug dexamethasone (DX). The CHLF-NLCs are transported transcellularly to cornea. The Labrafac Lipophile® WL 1349 acts as a penetration enhancer and facilitates the release of the drug in the cytoplasm of corneal cells. The CHLF-NLCs were further studied for cytotoxicity, cellular internalisation, ex vivo mucoadhesion with cornea, controlled extended drug release and permeation. The present invention discloses technology for the synthesis of NLCs of CHLF, which can be used for the patients suffering from ophthalmic inflammatory conditions such as acute and chronic dry eye.

(40) Cytotoxicity Assessment of the Prepared NLC.

(41) The cytotoxicity of the NLC was carried out on Human Corneal Epithelium Cell (HCEC). The cells with the initial density of 10,000 cells/well were seeded in a 96-well plate and were then cultured for 24 h in DMEM medium containing 10% FBS. The cells were then treated with varying concentrations of NLC, and NLC treated cells were incubated in a humidified environment with 5% CO.sub.2 at 37° C. for 4 h. After 4 h culture medium is replaced with the fresh medium, the cells were further maintained for another 20 h. After the specified time, MTT reagent (5 mg/mL) was added to each well, and the cells were incubated for another 4 h. The medium in each well was replaced with 200 μL of DMSO to dissolve the formazan crystals. The absorbance (O.D.) was recorded with a microplate reader. The cell viability was calculated by using the following equation:
Cell viability(%)=[O.D.(test)/O.D.(control)]×100

(42) Where O.D. (test) and O.D. (control) are the absorbance values of the cells cultured with and without NLC, respectively.

(43) The cytotoxicity study showed that prepared CHLF-NLC is non-toxic. In particular, the experiment using a 1:30 ratio was performed to a concentration of 200 microgram/ml where it showed cell viability. In the 1:4 ratio the experiment was performed to a concentration of 100 microgram/ml and showed cell viability.

(44) In Vitro Cellular Internalization of the NLCs were Examined with Fluorescence Microscopy.

(45) The cellular uptake of FITC labelled NLC was further examined using fluorescent microscopy. The cells were seeded in confocal imaging dishes at a density of 5×10.sup.4 cells per dish. The FITC labelled NLC were prepared and were exposed to the HCEC cells in serum free DMEM medium. After 4 h of incubation at 37° C., NLC treated serum free medium was removed and the cells were washed with without phenol red DMEM. Further 1 ml of without phenol red DMEM was added to the cells and the cellular uptake study was performed under fluorescent microscope. The data showed that the FITC labelled CHLF-NLC are internalised in the HCEC cells within 4 hours.

(46) Mucoadhesion Study was Performed Ex Vivo with Pig Cornea and Examined Under Fluorescence Microscope.

(47) The Mucoadhesion study of FITC labelled NLC on pig cornea was further examined using fluorescent microscopy. The pig cornea was excised and placed in the confocal imaging dishes containing without phenol red DMEM. The FITC labelled NLC were prepared and were exposed to the cornea in serum free DMEM medium. After 4 h of incubation at 37° C., NLC treated serum free medium was removed and the cornea was washed with without phenol red DMEM. Further, 1 ml of without phenol red DMEM was added to the cornea and the Mucoadhesion study on cornea was performed under fluorescent microscope.

(48) The study showed that the CHLF-NLC are mucoadhesive, as the data showed that the NLC particles stick to the cornea.

(49) Drug Release Study is Performed with Definite Concentration of Drug in CHLF-NLCs Over a Period of 18 Days.

(50) The drug release study was performed over 18 days for CHLF-NLC (1:30), and was performed by dialysis membrane. 1 ml of the drug loaded CHLF-NLC was taken in the dialysis membrane with concentration of drug 1 mg/ml. The membrane was clipped and dipped into the phosphate buffered saline (PBS) of pH=7.4 and temperature was maintained at 37° C. The PBS was stirred with a magnetic bead. 1 ml of the PBS was collected after different time intervals and 1 ml of fresh PBS was added to maintain the volume of the PBS. The collected sample was analysed with HPLC.

(51) The drug release data showed that the drug was released over time. In particular, the study was performed for the 1:30 ratio of CHLF-NLC for 18 days/381.2 h. In the case of the 1:4 ratio of CHLF-NLC, the study was performed for 81.5 hours.

(52) Permeation Study is Explored with Cornea Over the Definite Interval of Time

(53) The permeation study was performed ex-vivo with pig cornea. The excised cornea fit in the Franz diffusion cell. The donor chamber was filled with drug loaded CHLF-NLC (concentration of drug 1 mg/ml). In the acceptor chamber PBS of pH=7.4 was taken and stirred with a magnetic bead and the chamber was maintained at the temperature of 37° C. 1 ml of the PBS was collected at different time intervals and 1 ml of fresh PBS was added to maintain the volume of the PBS in the acceptor chamber. The collected sample was analysed with HPLC.

(54) The permeation study showed that the drug is efficiently released through the cornea.

(55) Size Measurement and Stability Study with Dynamic Light Scattering (DLS) (Table 1).

(56) The synthesised NLCs have a size range from 100-500 nm.

(57) TABLE-US-00001 TABLE 1 Size and zeta potential of CHLF NLCs with and without drug loading Dexamethasone Particle size Zeta potential Sample (mg/ml) (nm) (mV) CHLF 0 285 −25.6 CHLF-D 1 483 −23.6

(58) Zeta potential can be determined with instruments such as the MICROTRAC NANOWAVE II. Measurement of zeta-potential is also described in Salopek et al. (1992. Rudarsko-geolosko-naftni zbornik, Vo. 4, pp. 147-151, Zagreb), which provides the following guidelines of suspension stability depending on zeta-potential size:

(59) TABLE-US-00002 Zeta-Potential Assessment of stability (mV) Maximal agglomeration and precipitation  0 . . . +3   Region of strong agglomeration precipitation  +5 . . . −5  Beginning of agglomeration −10 . . . −15 Beginning of peptization (disperging) −16 . . . −30 Medium stability −31 . . . −40 Good stability −41 . . . −60 Very good stability −61 . . . −80 Extremely good stability  −81 . . . −100
Cytotoxicity Assessment of the Prepared CHLF-NLC (1:4).

(60) The cytotoxicity of the NLC was carried out on Human Corneal Epithelium Cell s (HCEC). The cells with an initial density of 10,000 cells/well were seeded in a 96-well plate and were then cultured for 24 h in DMEM medium containing 10% FBS. The cells were then treated with varying concentrations of NLC, and NLC treated cells were incubated in a humidified environment with 5% CO.sub.2 at 37° C. for 4 h. After 4 h the culture medium was replaced with fresh medium, the cells were further maintained for another 20 h. After the specified time, MTT reagent (5 mg/mL) was added to each well, and the cells were incubated for another 4 h. The medium in each well was replaced with 200 μL of DMSO to dissolve the formazan crystals. The absorbance (O.D.) was recorded with a microplate reader. The cell viability was calculated by using the following equation:
Cell viability (%)=[O.D.(test)/O.D.(control)]×100

(61) Where O.D. (test) and O.D. (control) are the absorbance values of the cells cultured with and without NLC, respectively

(62) The cytotoxicity study showed that prepared 1:4 ratio CHLF-NLC is non-toxic up to the concentration of 100 μg/ml.

(63) In Vitro Cellular Internalization of the NLCs was Examined with Fluorescence Microscopy.

(64) The cellular uptake of coumarin-6 labelled NLC was further examined using fluorescent microscopy. The cells were seeded in confocal imaging dishes at a density of 5×10.sup.4 cells per dish. The coumarin-6 labelled NLC were prepared and were exposed to the HCEC cells in serum free DMEM medium. After 4 h of incubation at 37° C., NLC treated serum free medium was removed and the cells were washed with without phenol red DMEM. Further 1 ml of without phenol red DMEM was added to the cells and the cellular uptake study was performed under fluorescent microscope. The data showed that the coumarin-6 labelled NLC are internalised in the HCEC cells within 4 h.

(65) Mucoadhesion study was performed ex vivo with pig cornea and examined under fluorescence microscope.

(66) The Mucoadhesion study of coumarin-6 labelled NLC on pig cornea was further examined using fluorescent microscopy. The pig cornea was excised and placed in the confocal imaging dishes containing without phenol red DMEM. The coumarin-6 labelled NLC were prepared and were exposed to the cornea in serum free DMEM medium. After 4 h of incubation at 37° C., NLC treated serum free medium was removed and the cornea was washed with without phenol red DMEM. Further 1 ml of without phenol red DMEM was added to the cornea and cellular mucoadhesion study was performed under fluorescent microscope.

(67) The study showed that the CHLF-NLC (1:4 ratio) are mucoadhesive, as the data showed that the NLC particles stick to the cornea.

(68) Drug Release Study is Performed with Drug Encapsulated CHLF-NLCs with the Drug Concentration 1 mg/ml Over the Time of 81.5 Hr.

(69) The drug release study was performed by dialysis membrane. 1 ml of the drug loaded CHLF-NLC was taken in the dialysis membrane with a drug concentration of 1 mg/ml. The membrane was clipped and dipped into the phosphate buffered saline (PBS) of pH=7.4 and temperature was maintained at 37° C. The PBS was stirred with a magnetic bead. 1 ml of the PBS was collected at different time intervals and 1 ml of fresh PBS was added to maintain the volume of the PBS. The collected sample was analysed with the HPLC.

(70) The drug release data showed that the drug is released over the time of 81.5 Hr.

(71) Permeation Study is Explored with Cornea Over the Definite Interval of Time.

(72) The permeation study was performed ex-vivo with pig cornea. The excised cornea fit in the Franz diffusion cell. The donor chamber was filled with drug loaded CHLF-NLC (concentration of drug 1 mg/ml). In the acceptor chamber PBS of pH=7.4 was taken and stirred with a magnetic bead and the chamber was maintained at the temperature of 37° C. 1 ml of the PBS was collected after different time intervals and 1 ml of fresh PBS was added to maintain the volume of the PBS in the acceptor chamber. The collected sample was analysed with HPLC.

(73) The permeation study showed that the drug is efficiently released through the cornea.

(74) Size Measurement and Stability Study with Dynamic Light Scattering (DLS) (Table 1).

(75) The synthesised NLCs have size range from 100-500 nm.

(76) TABLE-US-00003 TABLE 2 Size and zeta potential of CHLF NLCs with and without drug loading DRUG PARTICLE ZETA SAMPLE CONTENT SIZE POTENTIAL NAME (mg/ml) (nm) (mV) CHLF 1:4 0 336 −10.2 CHLF-D 1:4 1 153.4 −12.2

(77) CHLF-NLC and CHLF-D-NLC are characterised by DLS. It has been observed that CHLF-NLC have a particle size of 336 nm and zeta potential of −10.2 mV and CHLF-D-NLC have a particle size of 153.4 nm and zeta potential of −12.2 nm.

(78) Results & Discussion

(79) All studies showed a positive result for the synthesised NLCs indicating that these NLCs are a viable option for conventional eye drops for dry eye treatment. In the present invention of drug encapsulated NLCs, the drug is released from the carrier system and controlled by a diffusion and hydration. The drug release from the carrier can also be controlled by ionic interaction between the lipid and drug. In this invention, the drug molecule is present in the Labrafac Lipophile® WL 1349, which is a medium chain triglyceride. The Labrafac Lipophile® WL 1349 is entrapped in cholesterol. The NLCs are transported transcellularly to cornea. The Labrafac Lipophile® WL 1349 acts as penetration enhancer and facilitates the release of drug in the cytoplasm of corneal cells.

(80) The CHLF-NLC is mucoadhesive, as shown in the ex vivo experimental result. The NLC will show better bioavailability, because the ex vivo study showed that CHLF-NLC is mucoadhesive and the in vitro cellular uptake study showed that CHLF-NLC are internalised in the human corneal epithelium cell (HCEC).

(81) As cell membrane consists of lipid bilayer including cholesterol, better diffusion of NLC through the cell membrane is expected. The liquid lipid, e.g. Labrafac Lipophile® WL 1349, acts as a penetration enhancer and facilitates the release of the drug in the cytoplasm of corneal cells. In particular, the eye surface is covered with a mucus layer. The mucus glycoproteins on the surface of eye have various subdomains rich in specific amino acids. These amino acids are positively charged due to the amino group. Mucus glycoprotein is a protein that contains oligosaccharide chains covalently attached to polypeptide side chain. Peptides are short chain amino acids (e.g. cysteine, lysine), which are linked by amide bond. These amino acids are positively charged due to the amino group.

(82) The developed drug encapsulated NLCs are negatively charged and can interact with the mucus glycoproteins. This interaction leads to the enhanced residence time of NLCs and expected to enhance the drug bioavailability.

(83) NLCs have higher tolerability, permeability, sustained delivery, and storage stability.

(84) a) Tolerability: NLCs are expected to show good tolerability as the cholesterol component of NLCs is already there in the cell membrane, so it is expected to show good tolerability. The Labrafac Lipophile® WL 1349 is already used in skin cosmetics, so it is expected to show good tolerability.

(85) b) Permeability: Regarding permeability, the permeation study data shows that NLCs have good permeability.

(86) c) Sustained delivery: Drug release data shows that initially an increase in drug release is obtained which becomes nearly sustainable with time, with only mild variation. The NLCs have shown efficient controlled drug release over the period of 18 days for CHLF-NLC ratio 1:30 ratio and 81.5 hr for CHLF-NLC ratio 1:4.

(87) d) Storage stability: From the zeta potential study it showed that the prepared NLCs are stable in suspension.

(88) The present technology shields the encapsulated drug from the physiological barrier of the eye and facilitates the drug release at the targeted site. This technology will provide the therapeutic enhancement with the properties of enhanced biocompatibility, mucoadhesion, bioavailability, less drainage, increased residence time, reduced dosage frequency, and provides an efficient carrier for drug delivery.

(89) Due to these properties, the present invention is highly efficient for use as an eye drop based formulation. Due to the mucoadhesion property of the NLCs, the invention will have enhanced retention on the corneal surface with controlled released of drug over a prolonged duration. These NLCs can reduce the dosage frequency. The ex vivo permeation study also shows efficient release of drug. The studies have shown that present technology efficiently overcomes the problems which have been observed in already marketed formulations.

EXAMPLE 2

(90) Development of Dexamethasone Loaded Cholesterol-Labrafac Nanostructed Lipid Carrier (CHLF-D-NLCs)

(91) Pilot Scale Sample Preparation by High Pressure Homogeniser

(92) Cholesterol-labrafac of ratios 1:4 is selected, which was observed to be stable for the lab scale. The Cholesterol-labrafac ratio 1:4 is taken in a beaker 1 to this added acetone and ethanol in equal ratio. In a beaker 2, 1% tween 80 prepared in aqueous medium is taken. Both the beakers stirred and heated in a water bath at equal temperature (60-65° C.) for mixing and dissolving the component. After few min, when beaker 1 solvent is almost evaporated, hot 1% tween 80 is added to it, which results in white emulsion formation. The emulsion is stirred at the same temperature for another 5 mins. After 5 mins emulsion sample is cycled through High Pressure Homogeniser for 5 min, which result in the formation of the CHLF-NLCs.

(93) Preparation and Characterisation of Dexamethasone Loaded Cholesterol-Labrafac Nanostructed Lipid Carrier (CHLF-D-NLCs)

(94) Cholesterol-labrafac of ratios 1:4, dexamethasone 1 mg/ml (0.1% w/v) is taken in a beaker 1 to this added acetone and ethanol in equal ratio. In a beaker 2, 1% tween 80 prepared in aqueous medium is taken. Both the beakers stirred and heated in a water bath at equal temperature (60-65° C.) for mixing and dissolving the component. After few min, when beaker 1 solvent is almost evaporated, hot 1% tween 80 is added to it, which results in white emulsion formation. The emulsion is stirred at the same temperature for another 5 mins. After 5 mins emulsion sample is cycled through High Pressure Homogeniser for 5 min, which result in the formation of the CHLF-D-D-NLCs. The synthesised formulation is centrifuged and 0.01% of benzalkonium chloride is added to it. Further, formulation is sterilised by filtration through 0.2μ filter. The CHLF-D-NLCs is characterised by DLS.

(95) Particle Size Measurement and Zeta Potential Measurement:

(96) The particle size and zeta potential of the synthesised pilot scale sample is determined by DLS. The size and zeta potential of CHLF-NLCs is given in Table 3.

(97) TABLE-US-00004 SAMPLE DRUG PARTICLE ZETA NAME CONTENT SIZE POTENTIAL (Ratio) (mg/ml) (nm) (mV) CHLF-D-NLCs 1 20.64 −7.1 (1:4) CHLF-D-PILOT 1 19.51 9.8 SCALE STERLISED
Encapsulation Efficiency of Dexamethasone Loaded CHLF-D-NLCs

(98) Entrapment efficiency of dexamethasone entrapped in CHLF-NLC is determined by HPLC. For this 1 μl of CHLF-D-NLCs is added to the 999 μl of mobile phase—Phosphate Buffer (pH=3) and acetonitrile (ACN) in ratio 50:50. From the obtained area, the unknown concentration of the drug is determined by calibration curve and entrapment efficiency is calculated by the given formula

(99) $\mathrm{Encapsulation}\mathrm{Efficiency}\left(\%\right)=\frac{\begin{array}{c}(\mathrm{Drug}\mathrm{Loaded}-\\ \mathrm{free}\mathrm{untrapped}\mathrm{drug})\end{array}}{\mathrm{Drug}\mathrm{Loaded}}*100$

(100) It has been observed that the encapsulation efficiency (%) of CHLF-D-PILOT SCALE STERLISED formulation is 99.9%.

(101) Experimental Study Performed with Pilot Scale Sample CHLF-NLCs Cytotoxicity Assessment of the Prepared CHLF-NLC

(102) The cytotoxicity of the pilot scale CHLF-D-NLC was carried out on Human Corneal Epithelium Cell (HCEC). The cells with the initial density of 10,000 cells/well were seeded in a 96-well plate and were then cultured for 24 h in DMEM medium containing 10% FBS. The cells were then treated with varying concentrations of CHLF-D-NLC, and CHLF-D-NLC treated cells were incubated in a humidified environment with 5% CO2 at 37° C. for 4 h. After 4 h culture medium is replaced with the fresh medium, the cells were further maintained for another 20 h. After the specified time, MTT reagent was added to each well, and the cells were incubated for another 4 h. The medium in each well was replaced with 200 μl of DMSO to dissolve the formazan crystals. The absorbance (O.D.) was recorded with a microplate reader. The cell viability was calculated by using the following equation:

(103) $\mathrm{Cell}\mathrm{viability}\left(\%\right)=\left[\frac{O.D.\left(\mathrm{test}\right)}{O.D.\left(\mathrm{control}\right)}\right]*100$

(104) Where O.D. (test) and O.D. (control) are the absorbance values of the cells cultured with and without NLC, respectively. The cytotoxicity study showed that prepared 1:4 ratio CHLF-D-NLC is non-toxic up to the given concentration FIG. 18.

(105) Cellular Uptake Study

(106) In vitro cellular uptake of coumarin-6 labelled CHLF-D-NLC was further examined using fluorescent microscopy. The cells were seeded in confocal imaging dishes at a density of 5×10.sup.4 cells per dish. The coumarin-6 labelled CHLF-D-NLC were prepared and were exposed to the HCEC cells in medium. After 4 h of incubation at 37° C., CHLF-D-NLC treated medium is removed and the cells are washed. Further cellular uptake study is performed under fluorescent microscope. The data showed that the NLCs are internalised into the cells and coumarin-6, which is encapsulated in CHLF-D-NLCs is successfully released in the cytoplasm of the HCEC cells. Indicating that dexamethasone encapsulated in CHLF-D-NLCs will be released within the HCEC cells (FIG. 19).

(107) Mucoadhesion Study

(108) Mucoadhesion study was performed ex vivo with pig cornea. The mucoadhesion study of coumarin-6 labelled CHLF-NLC on pig cornea was further examined using fluorescent microscopy. The pig cornea is excised and placed in the confocal imaging dishes containing medium. The coumarin-6 labelled CHLF-NLC were prepared and were exposed to the cornea in medium. After 4 h of incubation at 37° C., CHLF-NLC treated medium is removed and the cornea is washed. Further mucoadhesion study is performed under fluorescent microscope. The mucoadhesion study showed that the CHLF-NLCs are mucoadhesive. As the CHLF-NLCs particles stick to the cornea (FIG. 20).

(109) Drug Release Study

(110) Drug release study is performed with drug encapsulated CHLF-NLCs with the drug concentration 1 mg/ml over the time of 65.25 h. The drug release study is performed by dialysis membrane. 1 ml of the drug loaded CHLF-NLC is taken in the dialysis membrane with concentration of drug 1 mg/ml. The membrane is clipped and dipped into the phosphate buffer saline (PBS) of pH=7.4 and temperature is maintained at 37° C. The PBS is stirred with magnetic bead. The 1 ml of the PBS is collected after different time interval and 1 ml of fresh PBS is added to maintain the volume of the PBS. The collected sample is analysed with the HPLC. The drug release data showed that drug is released over the time of 65.25 h (FIG. 21).

(111) Permeation Study

(112) The permeation study is performed ex-vivo with pig cornea. The excised cornea fit in the Franz diffusion cell. The donor chamber is filled with drug loaded CHLF-NLC (concentration of drug 1 mg/ml). In the acceptor chamber PBS of pH=7.4 is taken and stirred with magnetic bead and the chamber is maintained at the temperature of 37° C. The 1 ml of the PBS is collected after different time interval and 1 ml of fresh PBS is added to maintain the volume of the PBS in acceptor chamber. The collected sample is analysed with the HPLC. For the reference marketed formulation Maxidex is used for permeation. It has been observed that drug release percentage of the CHLF-D-NLCs is nearer to the value of marketed formulation. The study showed that dexamethasone is efficiently permeated through the cornea (FIG. 22).

(113) Sterilisation of CHLF-D-NLCs Lab Scale and Pilot Scale Samples:

(114) Lab scale and pilot scale samples are treated with benzalkonium chloride (BAK) (0.01% w/v). The BAK treated samples are filtered through 0.2 micron filter. The particle size and zeta potential of sterilised lab scale and pilot scale sample is determined with DLS. The study was carried out with lab scale and pilot scale samples.

(115) Stability Study of CHLF-D-NLCs Lab Scale and Pilot Scale Samples

(116) Stability study of sterilised lab scale and pilot scale sample is performed by Q1 Scientific Company for the period of 6 months. The analysis performed for the stability study sample is mainly size analysis by DLS to check the stability of size of CHLF-D-NLCs and drug encapsulation efficiency (%) analysis by HPLC to analyse the stability of encapsulation efficiency of CHLF-D-NLCs. For stability study four batches of samples (2 Batches for Lab Scale Sample (LBSC) and 2 Batches of pilot Scale Sample (PLSC)) are prepared, which are studied under two different temperature conditions. The stability study for all the four batches is performed for six months. After completion of 6 months, drug release and permeation study are performed for 6 months sample. Till the period of one-month pilot scale batch of samples are analysed weekly and after one month it has been analysed monthly. Lab scale samples are analysed monthly. The detail of the batches and their respective storage condition is given in Table 4.

(117) TABLE-US-00005 BATCH NAME STORAGE CONDITION LBSC-1 2-8° C. LBSC-2 25° C./Relative Humidity (RH) 60 PLSC-3 2-8° C. PLSC-4 25° C./Relative Humidity (RH) 60
Stability Study Data Analysis Results

(118) Data demonstrating the stability of the CHLF-NLC particles is presented in FIGS. 23-26.

(119) Drug Release Study of 6-Month Sample

(120) Data demonstrating the drug release from samples PLSC-3 and PLSC-4 are presented in FIGS. 27 and 28.

(121) Permeation Study of 6-Month Sample

(122) Data demonstrating the permeation of samples PLSC-3 and PLSC-4 are presented in FIGS. 29 and 30.

EXAMPLE 3

(123) Synthesis of Dexamethasone Encapsulated Cholesterol-Coconut Oil-Castor Oil Based Nanostructured Lipid Career (CHCACO-NLCs) for Dry Eye Treatment

(124) This project is based on dexamethasone encapsulated in cholesterol-castor oil-coconut oil based nanostructured lipid carriers (CHCACO-NLCs). This technology showed the advantageous release of hydrophilic drug over the period of time.

(125) This project was developed to treat acute and chronic dry eye with synthesised CHCACO-NLCs based eye drop. These NLCs is a viable option to replace conventional ointment, gels, eye drop. This material can lead to reduced dosage frequency and enhanced therapeutic output. The dexamethasone encapsulated CHCACO-NLCs were synthesised and characterised.

(126) The NLCs particle size is determined with DLS instrument, where the particle size is in the range of 5-500 nm.

(127) The cytotoxicity of the CHCACO-NLCs is determined with MTT assay.

(128) In vitro cellular internalization of the CHCACO-NLCs was examined with fluorescence Microscopy.

(129) The mucoadhesion study of CHCACO-NLCs is performed ex vivo with pig cornea and examined under fluorescence microscope.

(130) The drug release study is performed with definite concentration of dexamethasone in CHCACO-NLCs over the period of time.

(131) The permeation study is explored with cornea over the definite interval of time.

(132) All studies showed positive results for the synthesised CHCACO-NLCs and indicate that these CHCACO-NLCs are viable option for conventional eye drop for dry eye treatment.

(133) Optimisation of Cholesterol-Coconut Oil-Castor Oil based Nanostructured Lipid Carrier

(134) In the optimization steps samples of different ratio have been synthesised and characterised with DLS. Out of the different ratio of samples, a stable ratio has been selected and used for the study. The detail of the optimization ratio is given in the below table:

(135) TABLE-US-00006 Cholesterol- Total Coconut Oil- Coconut Castor volume Serial Castor Oil Cholesterol oil Oil Tween of No. ratio (mg) (μl) (μl) 80 (%) water 1 1:1 10 5.5 5.2 1 10 2 1:2 10 11 10.4 1 10 3 1:3 10 16.5 15.6 1 10 4 1:4 10 22 20.8 1 10 5 1:5 10 27.5 26 1 10
Cholesterol-Coconut Oil-Castor Oil Nanostructured Lipid Carrier (CHCACO-NLCs) Preparation Method:

(136) Cholesterol-Coconut Oil-Castor Oil of different ratios are taken in a beaker 1 to this added acetone and ethanol in equal ratio. In a beaker 2, 1% tween 80 prepared in aqueous medium is taken. Both the beakers were stirred and heated in a water bath at equal temperature (60-65° C.) for mixing and dissolving the component. After a few minutes, when beaker 1 solvent is almost evaporated, hot 1% tween 80 is added to it, which results in white emulsion formation. The emulsion is stirred at the same temperature for another 5 mins. After 5 mins the emulsion is ultrasonicated for 4 mins at the amplitude of 40%. Further the ultrasonicaticated sample is stirred at RT and high RPM for 1 hr, which results in the formation of the Cholesterol-Coconut Oil-Castor Oil nanostructured lipid carrier (CHCACO-NLCs).

(137) Cholesterol-Coconut Oil-Castor Oil Nanostructured Lipid Carrier (CHCAO-NLCs) Preparation Method (PILOT-SCALE):

(138) Cholesterol-Coconut Oil-Castor Oil of 1:1 ratio is taken in a beaker 1 to this was added acetone and ethanol in equal ratio. In a beaker 2, 1% tween 80 was prepared in aqueous medium. Both the beakers were stirred and heated in a water bath at equal temperature (60-65° C.) for mixing and dissolving the component. After a few minutes, when beaker 1 solvent is almost evaporated, hot 1% tween 80 is added to it, which results in white emulsion formation. The emulsion is stirred at the same temperature for another 5 mins. After 5 mins, emulsion is passed through High Pressure Homogenizer for 5 mins, which results in the formation of the Cholesterol-Castor Oil nanostructured lipid carrier (CHCAO-NLCs).

(139) Characterisation of Lab Scale Sample

(140) Particle size measurement and zeta potential measurement: The particle size and zeta potential of the synthesised sample is characterised by DLS. The characterisation showed that sample with Cholesterol-Coconut Oil-Castor Oil nanostructured lipid carrier (CHCACO-NLCs) ratio 1:1 is the most stable compared to other ratios. The size and zeta potential of CHCACO-NLCs is given in Table 6.

(141) TABLE-US-00007 Dexamethasone Particle size Zeta potential Sample (mg/ml) (nm) (mV) CHCACO 0 319 −13.8

(142) CHCACO-NLCs is characterised by DLS. It has been observed that CHCACO-NLCs have particle size of 319 nm and zeta potential of −13.8 mV.

(143) Preparation and Characterisation of Dexamethasone Loaded Cholesterol-Coconut Oil-Castor Oil Nanostructured Lipid Carrier (CHCACO-NLCs).

(144) Cholesterol-Coconut Oil-Castor Oil nanostructured lipid carrier of ratios 1:1, dexamethasone 1 mg/ml (0.1% w/v) is taken in a beaker 1 to this added acetone and ethanol in equal ratio. In a beaker 2, 1% tween 80 prepared in aqueous medium is taken. Both the beakers are stirred and heated in a water bath at equal temperature (60-65° C.) for mixing and dissolving the component. After a few minutes, when beaker 1 solvent is almost evaporated, hot 1% tween 80 is added to it, which results in white emulsion formation. The emulsion is stirred at the same temperature for another 5 mins. After 5 mins the emulsion is ultrasonicated for 4 mins at the amplitude of 40%. Further the ultrasonicaticated sample is stirred at room temperature and high RPM for 1 h, which results in the formation of the CHCACO-D-NLCs and characterised by DLS. The size and zeta potential of CHCACO-D-NLCs is given in Table 7.

(145) TABLE-US-00008 Dexamethasone Particle size Zeta potential Sample (mg/ml) (nm) (mV) CHCACO-D 1 294.4 −10.2

(146) CHCACO-D-NLCs is characterised by DLS. It has been observed that CHCACO-D-NLCs has particle size of 294.4 nm and zeta potential of −10.2 nm.

(147) Encapsulation Efficiency of Dexamethasone Loaded CHLF-D-NLCs

(148) Entrapment efficiency of dexamethasone entrapped in CHCACO-NLCs is determined by HPLC. For this 1 μl of CHCACO-D-NLCs is added to the 999 μl of mobile phase—Phosphate Buffer (pH=3) and acetonitrile (ACN) in ratio 50:50. From the obtained area, the unknown concentration of the drug is determined by calibration curve and entrapment efficiency is calculated by the given formula

(149) $\mathrm{Encapsulation}\mathrm{Efficiency}\left(\%\right)=\frac{\begin{array}{c}(\mathrm{Drug}\mathrm{Loaded}-\\ \begin{array}{c}\mathrm{free}\mathrm{unencapsualted}\\ \mathrm{drug})\end{array}\end{array}}{\mathrm{Drug}\mathrm{Loaded}}*100$

(150) It has been observed that the encapsulation efficiency (%) of CHCACO-D-NLCs is 97.3%

(151) Experimental Study Performed with CHCACO-NLCs

(152) a) Cytotoxicity Assessment of the Prepared CHCACO-NLC

(153) The cytotoxicity of the CHCACO-NLCs was carried out on Human Corneal Epithelium Cell (HCEC). The cells with the initial density of 10,000 cells/well were seeded in a 96-well plate and were then cultured for 24 h in DMEM medium containing 10% FBS. The cells were then treated with varying concentrations of CHCACO-NLCs, and CHCACO-D-NLCs treated cells were incubated in a humidified environment with 5% CO2 at 37° C. for 4 h. After 4 h culture medium is replaced with the fresh medium, the cells were further maintained for another 20 hours. After the specified time, MTT reagent was added to each well, and the cells were incubated for another 4 hours. The medium in each well was replaced with 200 μl of DMSO to dissolve the formazan crystals. The absorbance (O.D.) was recorded with a microplate reader. The cell viability was calculated by using the following equation:

(154) $\mathrm{Cell}\mathrm{viability}\left(\%\right)=\left[\frac{O.D.\left(\mathrm{test}\right)}{O.D.\left(\mathrm{control}\right)}\right]*100$

(155) Where O.D. (test) and O.D. (control) are the absorbance values of the cells cultured with and without NLC, respectively. The cytotoxicity study showed that prepared 1:1 ratio CHCACO-NLCs is non-toxic up to the given concentration FIG. 31,32.

(156) b) Cellular Uptake Study

(157) In vitro cellular uptake of coumarin-6 labelled CHCACO-NLCs was further examined using fluorescent microscopy. The cells were seeded in confocal imaging dishes at a density of 5×10.sup.4 cells per dish. The coumarin-6 labelled CHCACO-NLCs were prepared and were exposed to the HCEC cells medium. After 4 hours of incubation at 37° C., CHCACO-NLCs treated medium is removed and cells are washed. A further cellular uptake study was performed under fluorescent microscope. The data showed that NLCs are internalised in the cells and the coumarin-6, which is encapsulated in CHCACO-NLCs is successfully released in the cytoplasm of the HCEC cells. Indicating that dexamethasone encapsulated in CHCACO-NLCs will be released within the HCEC cells (FIG. 33).

(158) c) Mucoadhesion Study

(159) A mucoadhesion study was performed ex vivo with pig cornea. The mucoadhesion study of coumarin-6 labelled CHCACO-NLCs on pig cornea was further examined using fluorescent microscopy. The pig cornea is excised and placed in the confocal imaging dishes containing medium. The coumarin-6 labelled CHCACO-NLCs were prepared and were exposed to the cornea in medium. After 4 hours of incubation at 37° C., CHCACO-NLCs treated medium is removed and the cornea is washed. A further mucoadhesion study was performed under fluorescent microscope. The mucoadhesion study showed that the CHCACO-NLCs are mucoadhesive. As the CHLF-NLCs particles stick to the cornea (FIG. 34).

(160) d) Drug Release Study

(161) A drug release study was performed with drug encapsulated CHCACO-NLCs with the drug concentration 1 mg/ml over the time of 93.5 hours. The drug release study is performed by dialysis membrane. 1 ml of the drug loaded CHCACO-NLCs is taken in the dialysis membrane with concentration of drug 1 mg/ml. The membrane is clipped and dipped into the phosphate buffer saline (PBS) of pH=7.4 and temperature is maintained 37° C. The PBS is stirred with magnetic bead. The 1 ml of the PBS is collected after different time interval and 1 ml of fresh PBS is added to maintain the volume of the PBS. The collected sample is analysed with the HPLC. The drug release data showed that drug is released over the time of 93.5 hours (FIG. 35).

(162) e) Permeation Study

(163) The permeation study was performed ex vivo with pig cornea. The excised cornea fit in the Franz diffusion cell. The donor chamber is filled with drug loaded CHCACO—NLCs (concentration of drug 1 mg/ml). In the acceptor chamber PBS of pH=7.4 is taken and stirred with magnetic bead and the chamber is maintained at the temperature of 37° C. The 1 ml of the PBS is collected after different time interval and 1 ml of fresh PBS is added to maintain the volume of the PBS in acceptor chamber. The collected sample is analysed with the HPLC. For the reference marketed formulation Maxidex is used for permeation. The study showed that dexamethasone is efficiently permeated through the cornea (FIG. 36).