Claims:1. A niosomal in-situ gel forming system for ocular drug delivery comprises a tetrahydrocurcumin combination of natural polymer and a vehicle alongwith one or more optional components selected from active pharmaceutical ingredient(s), buffer, preservative with or without stabilizer, pH modifier (s) and tonicity modifiers.

2. The niosomal in-situ gel forming system as claimed in claim 1, wherein the natural polymer is selected from the group consisting of xanthan gum, locust bean gum, guar gum and tamarind seed polysaccharide, present in the range of 0.1 - 3% w/v.

3. The niosomal in-situ gel forming system as claimed in claim 1, wherein the thermo reversible polymers are selected from the group consisting of gelatin, poly(vinyl chloride), poly(acrylonitrile), polystyrene (atactic), poly(vinyl alcohol), agarose, carrageenans, benzohydroxamic acid, polysaccharides, methylcellulose, hydroxy propyl methylcellulose, hydroxy ethyl cellulose, carboxy methyl cellulose and its salts, and polaxomer.

4. The in-situ gel forming system as claimed in claim 3, wherein said thermo reversible polymer is methyl cellulose present in the range of 0.1-2.5%.

5. The in-situ gel forming system as claimed in claim 3, wherein said thermo reversible polymer is polaxomer present in the range of 0.1-20%.

6. The niosomal in-situ gel forming system as claimed in claim 1, wherein said composition comprises active pharmaceutical ingredient(s), natural polysaccharide with thixotropic behavior, thermo reversible polymer, vehicle, buffer, preservative with or without stabilizer, pH modifier (s) and tonicity modifiers, useful for diagnosis or treatment of various eye disorders.

8. The in-situ gel forming system as claimed in claim 7, wherein the pharmaceutical agents includes local anesthetics, antimicrobial agents, antifungals, anti-inflammatory, anti-cataract, antiglucoma, steroids, ß-blocker, antihistamics, immunosuppressant, cycloplegics, mydriatics, vasodilators, vasoconstrictors and lubricants, wherein said active ingredient is present in the range of 0.00001% to 10% w/v.

9. The niosomal in-situ gel forming system as claimed in claims 1 and 6, wherein the buffer used in the composition includes phosphate, citrate, citrophosphate and TRIS, preferably phosphate and TRIS buffer, in the range of 0.01% to 20% w/v.

10. The niosomal in-situ gel forming system as claimed in claims 1 and 6, wherein the preservative used in the composition includes benzalkonium chloride, sodium perborate, thiomersol, chlorbutanol, methyl paraben, propyl paraben, phenyl ethyl alcohol, disodium edetate and polyquard, preferably sodium perborate and benzalkonium chloride, in the range of 0.0001% to 1% w/v.

, Description:FIELD OF THE INVENTION
The present disclosure relates to a novel type of niosomal in-situ gel and their preparations. In particular, the present disclosure relates to methods and process of preparing niosomal in-situ gel for ocular drug delivery. One aspect of the present disclosure is method of preparation of niosomal in-situ gel comprising of tetrahydrocurcumin and other pharmaceutical excipients. In second part, evaluation of biodegradable neosomes is done by pH, Viscosity, Drug content and In-vitro drug release studies.

BACKGROUND OF THE INVENTION
Curcumin is one of the most valuable natural products due to its pharmacological activities. However, the low bioavailability of curcumin has long been a problem for its medicinal use. Large studies have been conducted to improve the use of curcumin; among these studies, curcumin metabolites have become a relatively new research focus over the past few years. Curcumin has been shown to have considerable potential health benefits in recent studies. A search yielded almost twenty thousand manuscripts on this topic from 2014 to 2019, most of which are related to the use of curcumin derivatives against cancer and cardiovascular diseases. Additionally, accumulating evidence suggests that curcumin or curcuminoid metabolites have similar or better biological activity than the precursor of curcumin. Curcumin is mainly metabolized into dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), and octahydrocurcumin (OHC), the final form of hydrogenated curcumin. Recent studies focus on the protective role of plasma tetrahydrocurcumin, a main metabolite of curcumin, against tumors and chronic inflammatory diseases. Nevertheless, studies of tetrahydrocurcumin in eye diseases have not yet been conducted. Since ophthalmic conditions play a crucial role in worldwide public health, the prevention and treatment of ophthalmic diseases are of great concern.
Topical drug delivery is a preferred method for the therapeutic treatment of most of the ocular problems and well-accepted route of administration for the treatment of various eye diseases. Ocular drug delivery has been a foremost challenge for researchers because of the distinctive anatomy and physiology of eye which contains different barriers such as different layers of cornea, sclera and retina in addition to blood retinal barriers, lachrymal fluid-eye barrier and also drug loss from the ocular surface. These obstacles cause a considerable challenge for delivery of a drug alone or in a dosage form, especially to the posterior segment of the eye. A major problem in conventional ophthalmic drug delivery systems is poor ocular bioavailability due to ocular anatomical and physiological constraints, which include the poor permeability of cornea, nasolacrimal drainage and short retention time in the precorneal area. The bioavailability of topically administered drug in the anterior chamber of eye is extremely low due to the protective barrier function of the cornea, its rapid clearance by the tear-fluid drainage, its absorption into the conjunctiva and its out by aqueous humor from the anterior chamber. Topically applied drugs can reach the intraocular tissues by either the corneal and/or the non-corneal pathways. Under normal conditions, the eye can accommodate only a very small volume of administered drugs without overflowing.
Ocular disorders are mainly treated by using drug formulations in the form of eye drops. Eye drops are inefficient means of delivering ophthalmic drugs because of limited bioavailability and these can cause significant side effects due to systemic uptake of the drug, when the drug gets into the gastrointestinal tract through nasolacrimal drainage. The drug contained in the drops is lost due to absorption through the conjunctiva or through the tear drainage. Attempts to improve ocular bioavailability have been focused on overcoming precorneal constraints through improving corneal penetration and prolonging the precorneal retention and reducing the nasolacrimal drainage.
U.S. Pat. No. 4,029,817 to Blanco et al. discloses a contact lens preserving solution including propylene glycol in combination with polysorbate 80 and/or polyvinyl pyrrolidone. Similarly, U.S. Pat. No. 5,141,665 to Sherman discloses a contact lens cleaning, wetting and storing solution which includes propylene glycol as a wetting agent. Also, U.S. Pat. No. 4,525,346 to Stark discloses a borate buffered, preserved contact lens solution including propylene glycol.
EP Patent No. 424043 describes liquid ophthalmic composition comprising at least one active principle and a sulphated polysaccharide or sulphated polysaccharide derivative in aqueous solution which undergoes a liquid-gel phase transition on interaction of the said sulphated polysaccharide or one of its derivatives with the proteins of the lachrymal fluid.
US Patent No. 5422116 describes formulation of pH sensitive in-situ gel for ophthalmic application using chitosan as a polymer. The formulated ophthalmic solution consists of chitosan having the pH range in between 3 to 6.2. The acidic pH of these formulations may not be suitable for the drugs which are unstable at acidic aqueous environment.
US Patent No. 5,591,426 to Dabrowski et al. discloses an ophthalmic solution useful as an artificial tear. The reference includes a specific example of a borate buffered, preserved (e.g. benzalkonium chloride), aqueous solution including the following three demulcents: 1) glycerin, 2) polyvinyl pyrrolidone, and 3) a cellulose derivative, e.g. hydroxypropyl methyl cellulose.
Sci. Pharm. 2008; 76:515-532 describes in-situ gum based ophthalmic drug delivery system of linezolid, wherein hydroxypropyl guar and xanthan are used as gum with combination of hydroxyethyl cellulose, carbopol and sodium alginate as viscosity enhancing agents. The pH of the formulation in the said study is maintained at 7.4, to retain the clarity of the formulation, however most of the drugs are stable in either weakly acidic and weakly basic drugs. The said article utilized more than 2 polymers in gel forming solutions, leading to complication during manufacturing process.
WO 93/00887 demonstrates use of carbomer as an aqueous gel forming agent in ophthalmic preparations. Carbomer are acid insoluble polymers and swell above their pKa value preferably at neutral pH. Carbomer based liquid formulations should be formulated with pH range of 6-7. These formulations are not suitable to load the drugs having physicochemical properties and chemical properties favorable to acidic environment.
The goal of designing an ophthalmic gel is to make the gel sufficiently flowable that the gel can be conveniently applied to the eye, while at the same time providing a gel that is viscous enough to prolong residence time (contact time) in the eye. But the viscosity at body temperature of known in situ gelling systems can be difficult to predict with certainty. Gels having viscosities above 55 centipoise (cps) can be uncomfortable and aesthetically unattractive in the eye. For this reason, it is critical to provide an ophthalmic gel that provides the desired residence time while avoiding the discomfort and unattractive cosmetic appearance of a substantially solidified gel.
Thus it would be desirable to provide an ophthalmic gel that improves the contact time between the target ocular tissue and an active pharmaceutical agent, while also overcoming the problems associated with high viscosity gels. It would also be desirable to improve the durability and the useful life of the gel composition once formed to further prolong contact time with the target ocular tissue
OBJECT OF THE INVENTION:
The primary objective of the present invention is to provide niosomal in-situ gel for ocular drug delivery comprising of tetrahydrocurcumin with other pharmaceutical acceptable excipients and process for preparation thereof.
Another objective of the invention is to provide in-situ gel formation by one or more combination of mechanisms such as thermal gelation, corneal mucoadhesion, lysosomal interaction and ionic gelation which provides prolonged corneal residential time, improved corneal absorption, patient compliance and clinical success.
Yet another objective of the invention is to provide niosomal in-situ gel having longer duration of action, and hence improved patient compliance and therapeutic response.

SUMMARY OF THE INVENTION:
Accordingly, to meet the above stated objectives, the present invention provides niosomal in-situ gel forming solution for ocular drug delivery, consisting of natural polymer and thermo reversible polymers using one or more combination of mechanisms such as thermal gelation, corneal mucoadhesion, lysosomal interaction and ionic gelation. The combination of natural polymer with thixo tropic behavior and thermo reversible polymers utilizing the said mechanisms provides in-situ gel formation in eye and leads to prolonged corneal residence time and improved corneal absorption to achieve patient compliance and clinical success.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
The phrase 'thixo-tropic behavior' as discussed herein refers to a property exhibited by certain compounds of becoming fluid when stirred or shaken, and returning to the semisolid state upon standing.
The phrase 'thermo reversible polymer' as discussed herein refers to a polymer whose properties or action can be reversed by heating.
In-situ system is an alternative and novel approach to overcome the problems of conventional dosage form
1. They overcome poor bioavailability problem of conventional ophthalmic solution by instilled gel into eyes as drops.
2. To increase the ocular bioavailability of drug by increasing the corneal contact time.
3. Drug effect is prolonged hence frequent instillation of drug is not required.
4. Reduced systemic absorption of drug drained through the nasolacrimal duct.
5. To provide sustained and controlled drug delivery
6. For patient compliance and enhance therapeutic performance of drug.
The present invention relates to niosomal in-situ gel forming solution for ocular drug delivery, comprising of natural polymer with thixotropic behavior and thermo reversible polymers, utilizing one or more combinations of mechanisms such as thermal gelation, corneal mucoadhesion, lysosomal interaction and ionic gelation. The combination of natural polysaccharide with thixotropic behavior and thermo reversible polymers, using the said mechanisms, provides in-situ gel formation and leads to prolonged corneal residence time, improved corneal absorption, patient compliance and clinic success.
Accordingly, the invention provides niosomal in-situ gel forming system for ocular drug delivery comprises a combination of natural polysaccharide having thixotropic behavior, thermo reversible polymer and a vehicle along with one or more optional components selected from active pharmaceutical ingredient(s), buffer, preservative with or without stabilizer, pH modifier(s) and tonicity modifiers
The active ingredient i.e. tetrahydrocurcumin is useful for diagnosis or treatment of various eye disorders, whereas the formulation without active ingredient is used to treat the dry eyes. The active ingredient is more effective in suppressing nuclear factor-?B (NF-?B) and inhibiting the expression of cyclooxygenase 2 (COX-2). The ophthalmically acceptable agents include without limitation thereto, from the categories of local anesthetics, antimicrobial agents, antifungals, anti-inflammatory, antiglucoma, steroids, ß-blocker, anti-cataract, antihistamics, immunosuppressant, cycloplegics, mydriatics, vasodilators, vasoconstrictors and lubricants. The active ingredient used in the formulation is ranging from 0.00001% to 10% w/v.
According to the present invention, the ophthalmic gel forming solution prepared by using any one of the systems consisting of natural polysaccharide with thixotropic behavior and thermo reversible polymers that provide in-situ gel formation, by one or more combinations of mechanism such as thermal gelation, corneal mucoadhesion, lysosomal interaction and ionic gelation, provides prolonged corneal residence time, improved corneal absorption, patient compliance and clinical success. Accordingly in a preferred embodiment the invention provides in-situ gel compositions comprising of:
• Polysaccharide with thixotropic behavior
• Thermo reversible polymer
• Vehicle
• Other one or more optional components selected from active pharmaceutical ingredient(s), buffer, preservative with or without stabilizer, pH modifier(s) and tonicity modifiers
In another preferred embodiment the invention provides in-situ gel composition prepared by process as detailed below:
The present invention further describes the use of thermo reversible polymers as rheological modifiers. The role of thermo reversible polymers/ Theological modifiers is to retain the high viscosity of the polymers in presence of shear stress at physiological conditions. The commonly used rheological modifiers are natural and semi-synthetic cellulose derivatives such as methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose and its salts and polaxomer; preferably rheological modifiers are methylcellulose and polaxomer. These polymers also act as a lubricant surviving the purpose of lubrication. The role of thermo reversible polymers is to maintain the rigidity of gel or to retain the gel strength against shear stress and dilution, which is achieved through thermo-gelation properties of thermo reversible polymers at physiological pH. The optimal concentration of methylcellulose present in the formulation is 0.1 - 2.5%.
A variety of viscosity enhancing agents are known in the art and include, but are not limited to: polyols such as, glycerol, glycerin, polyethylene glycol 300, polyethylene glycol 400, polysorbate 80, propylene glycol, and ethylene glycol, polyvinyl alcohol, povidone, and polyvinyl pyrrolidone; cellulose derivatives such hydroxy propyl methylcellulose (also known as hypromellose and HPMC), carboxymethyl cellulose sodium, hydroxypropyl cellulose, hydroxyethyl cellulose, and methyl cellulose; dextrans such as dextran 70; water soluble proteins such as gelatin; carbomers such as carbomer 934P, carbomer 941 ,carbomer 940 and carbomer 974P; and gums such as HP-guar, or combinations thereof. Other compounds may also be added to the formulations of the present invention to increase the viscosity of the carrier. Examples of viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers. Combinations and mixtures of the above agents are also suitable. According to some embodiments, the concentration of viscosity enhancing agent or combination of agents ranges from about 0.2% to about 10% w/v, or any specific value within said range.
In one a preferred embodiment of the invention, the viscosity enhancing component comprises hydroxy propyl methylcellulose (Hypromellose or HPMC). According to some embodiments, the concentration of HPMC ranges from about 0.5% to about 2% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.5% to about 1.5% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.5% to about 1% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.6% to about 1% w/v, or any specific value within said range. In a preferred embodiment, the concentration of HPMC ranges from about 0.7% to about 0.9% w/v, or any specific value within said range.
The composition optionally further includes at least one ophthalmically acceptable salt in an amount required to bring osmolality of the composition into an ophthalmically acceptable range. Such salt include those having sodium, potassium or ammonium cation and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anion; preferred salt include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate, with sodium chloride being especially preferred. Other solutes suitable for adjustment of osmolality include sugar, for example dextrose, lactose, xylitol, mannitol and glycerine.
Tonicity agents include, for example, mannitol, sodium chloride, xylitol, etc. These tonicity agents may be used to adjust the osmolality of the compositions. In one aspect, the osmolality of the formulation is adjusted to be in the range of about 250 to about 350 mOsmol/kg. In a preferred aspect, the osmolality of the formulation is adjusted to between about 280 to about 300 mOsmol/kg.
Accordingly, one aspect of this invention relates to a drug delivery system containing a therapeutic agent (e.g., protein, nucleic acid, or small molecule) and a delivery vehicle that includes phospholipid and cholesterol. The mole percent of cholesterol in the delivery vehicle (e.g., in lyophilized form) can be 5-40% (e.g., 10-33% or 20-25%). The delivery vehicle and the therapeutic agent can be either admixed or separate.
In the drug delivery system of this invention, 50-90% of the therapeutic agent is in non-associated form and the weight ratio of the phospholipid and cholesterol in combination to the therapeutic agent is 5-80. In one example, the therapeutic agent is an antibody against vascular endothelial growth factor (VEGF) and 60-90% of the antibody is in non-associated form and the weight ratio of the phospholipid and cholesterol in combination to the antibody is 5-40. In another example, the therapeutic agent is an anti-inflammatory molecule (e.g., a corticorsteroid).
Another aspect of this invention relates to a method of delivering a therapeutic agent to an eye of a subject. This method includes (i) providing the drug delivery system described above, which can be in aqueous suspension form, and (ii) administering it to an eye of a subject in need by, e.g., intravitreal injection. The delivery system can be prepared by mixing phospholipid, cholesterol, and one or more therapeutic agent to form a mixture; lyophilizing the mixture; and, before administration, suspending the mixture in an aqueous solution to form the aqueous suspension. Alternatively, it is prepared by mixing phospholipid and cholesterol to form a mixture; lyophilizing the mixture; and, before administration, suspending the mixture together with one or more therapeutic agent in an aqueous solution to form the aqueous suspension.
One preferred method of carrying out the dilutions involves overnight refrigeration, solubilizing the active drug and the other excipients. This is a well known technique for solubilizing drugs for use with poloxamers. However, other methods can also be used. The compositions of the invention may include various inactive ingredients commonly used in formulating topical compositions and that may improve stability of the formulation. For example, the compositions of the invention may include alcohols and/or surface active agents, including but not limited to polyglycol ether, polyethylene glycol-nonphenol ether, polyethylene glycol sorbitan monolaurate, polyethylene glycol sorbitan monooleate, polyethylene glycol sorbitan monooleate, polyethylene glycol sterarate, polyethylene glycol polypropylene glycol ether, polyvinyl alcohol, polyvinyl pyrrolidine, PEG and its derivatives, including but not limited to PEG 4000 or PEG 6000, in a total amount of 0.05% to 5% by mass of the composition.
Preferably the formulations of the present invention are preservative free. Such formulations would be useful for patients with dry eye, patients who wear contact lenses, or those who use several topical ophthalmic drops and/or those with an already compromised ocular surface (e.g. dry eye) wherein limiting exposure to a preservative may be more desirable.
In certain embodiments, however, the formulations of the invention may additionally comprise a preservative. A preservative may typically be selected from a quaternary ammonium compound such as benzalkonium chloride, benzoxonium chloride etc. Benzalkonium chloride is better described as: N-benzyl-N-(C8 -C 18 alkyl)-N,N dimethylammonium chloride. Examples of preservatives different from quaternary ammonium salts are alkyl-mercury salts of thiosalicylic acid, such as thiomersal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, sodium perborate, sodium chlorite, parabens, such as methylparaben or propylparaben, alcohols, such as chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine derivatives, such as chlorohexidine or polyhexamethylene biguanide, sodium perborate, Germal®II, sorbic acid, and stabilized oxychloro complexes (e.g., Purite®). Preferred preservatives are quaternary ammonium compounds, in particular benzalkonium chloride or its derivative such as Polyquad (see U.S. Pat. No. 4,407,791), alkyl-mercury salts, parabens and stabilized oxychloro complexes (e.g., Purite®). Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against secondary contaminations during use caused by bacteria and fungi.
METHOD OF PREPARATION OF NIOSOMES
Ether injection method: Niosomes prepared by using ether injection method in which Non-ionic surfactant (NIS) Sorbitan (e.g. Span20, span40, span60, span80.) and cholesterol is taken in the different ratios (1:1, 1:2, 1:3 etc.) and dissolved in organic solvent (diethyl ether). The organic phase is slowly injected using 24-gauge needle into preheated 10 ml phosphate buffer containing drug which is magnetically stirred and maintained at 650C for 45 min. Stirring is continued until all ether evaporating to get drug loaded niosome. Vaporization of ether leads to formation of vesicles.
Phase I: Formulation of Niosomes containing Tetrahydrocurcumin
Among 14 formulations of niosomes prepared by ether injection technique i.e.(N1-N14) based on the entrapment efficiency and in-vitro drug release, the optimized formulation (N8) containing 1:2 C/S ratio of span 60 and cholesterol is found to be effective and that formulation is selected as best formulation for the conversion of niosomal in-situ gel formulation. Formulations are carried out for developing niosomal in-situ gel using pH sensitive polymers like Carbopol 940 and HPMC K 15M.

Table No :01 formulations of niosomes
Sr.
No. Formul ation code Choles terol( mg) Surfactant used (mg) Drug (mg) C/S ratio Stear yl amin e(mg) Diethyl ether(ml) PBs(pH 7.4) ml
1 N1 100 Span 20 100 10 1:1 0.01 10 10
2 N2 100 200 10 1:2 0.01 10 10
3 N3 100 300 10 1:3 0.01 10 10
4 N4 100 Span40 100 10 1:1 0.01 10 10
5 N5 100 200 10 1:2 0.01 10 10
6 N6 300 10 1;3 0.01 10 10
7 N7 100 Span 60 100 10 1:1 0.01 10 10
8 N8 100 200 10 1:2 0.01 10 10
9 N9 100 300 10 1:3 0.01 10 10
10 N10 100 Span60+Span
40 100 10 1:1 0.01 10 10
11 N11 100 200 10 1:2 0.01 10 10
12 N12 100 Span 80
100 10 1:1 0.01 10 10
11 N13 100 200 10 1:2 0.01 10 10
14 N14 100 200 10 1:3 0.01 10 10

Phase II: Formulation of Niosomal pH induced in-situ gel containing Tetrahydrocurcumin
The in-situ gel of niosomes Tetrahydrocurcumin are prepared by using hydroxy propyl methyl cellulose K15M and Carbopol 940. Four formulations of the Niosomal in-situ gels are prepared by adding viscosifier (HPMC K15M) to the suspension and then gelling agent (Carbopol 940) is added and allowed to hydrate overnight. The solution is made isotonic with sodium chloride (0.9%). Benzalkonium chloride is added as a preservative. The prepared gels are filled in glass vials and stored in refrigerator at a temperature of 4 to 6°C.

Table No 2 – Gel formulations
Sr. No. Ingredients G1 G2 G3 G4
1 Niosomal dispersion 10ml 10ml 10ml 10ml
2 Carbopol 940 0.05 0.1 0.2 0.25
3 HPMC K15M 0.05 0.1 0.2 0.25
4 Sodium chloride (0.9%) 0.9 0.9 0.9 0.9
5 Triethanolamine q.s q.s q.s q.s
6 Benzalkonium Chloride 0.001 0.001 0.001 0.001

Evaluation of niosomal in-situ gel:
1. Appearance:
The formulations are observed for the presence of any particular matter. Clarity is one of the most important features of ophthalmic preparations. The appearance and clarity is determined by visually.
The appearance of niosomal in-situ gel is translucent and off- White in colour.
2. pH:
Ophthalmic formulations should have a pH ranging between 6 and 7.4. The developed formulations are evaluated for pH by using a digital pH meter.
pH is found to be 6-6.8
3. In vitro Gelation Studies:
The gelling capacity of the prepared system containing different concentrations of Carbopol 940 and HPMCK 15M is evaluated. It is performed by placing a drop of system in vials containing 1 ml of simulated tear fluid freshly prepared and equilibrated at 370C, the gel formation and time taken for gelation is assessed visually.
In-vitro gelation studies of the formulations G1, G2, G3, and G4 formed gels immediately which remained for extended period of time while G3 exhibited immediate gelation which remains for 2-3 hours.
4. Drug content:
Drug content of niosomal in-situ gel is determined by adding n-propanol to the formulation for lysis of the vesicles. 0.1 ml of niosomal in situ gel is then diluted to 100 ml with STF of pH 7.4. Drug content is estimated spectrophotometrically at 281nm.
The in-situ gel is analyzed for drug content spectrophotometrically at 281 nm. All the formulations exhibited fairly uniform drug content. This ensures intended delivery of drug to the site after administration of the gel formulation. The drug content is found to be in acceptable range for all the formulations. The results revealed that drug content of all developed formulations are in the range of 93 to 98.80%
5. Viscosity Studies:
Viscosity of the formulations is determined using Brookfield viscometer (Ametek DV2T ) fitted with S-6 spindle at 10, 20, 50 and 100 rpm.
The viscosity of the all gel formulations before and after gelation ranged from 190-1400 cps and 300-2025 cps and viscosity of optimized formulation is found to be 200-800 cps and 400-1415 cps
6. In-vitro of drug release:
The diffusion cell consisted of a hollow glass cylinder. One end of the cylinder is covered with dialysis membrane which is previously soaked in warm water for 24 hrs. The diffusion cell is placed in a 500ml beaker that served as the receptor cell and the temperature is maintained at 370C. Simulated tear fluid (100 ml) pH 7.4 is placed in the receptor cell. Sample withdrawn at specified time intervals and the medium volume is made up with fresh simulated tear fluid (pH7.4).
The prepared gel formulations released 65.40% (G1), 63.15 % (G2), and 71.55 % (G3), and 66.29 % (G4) of drug after 12 hours. In G3 formulation concentration of Carbopol 940 (0.2%w/v) and HPMC K15M (0.2%w/v) have shown good gelation characteristics and in-vitro drug release. The in-vitro release of G3 is then compared with marketed drops. In marketed drops the maximum drug (100.1%) is released at 4 hours when compared to niosomal in-situ gel of G3 formulation. The release studies of niosomal in-situ gel G3 (71.50 % at 12 hour) showed sustained release when compared to marketed drops
7. Isotonicity study:
Isotonicity is important characteristic of the ophthalmic preparations. Isotonicity should be maintained to prevent tissue damage or irritation of eye. All ophthalmic preparations undergo isotonicity testing, formulations mixed with few drops of blood and observed under microscope at 45X magnification and compared with standard marketed ophthalmic formulation.
Formulation is treated with hypotonic (0.5% NaCl), hypertonic (1.5% NaCl), or normal saline (0.9% NaCl) solutions. Increase in vesicle size is observed in formulation significantly in formulation incubated with hypotonic solution. In hypertonic solution, the formulation shrank uniformly, formulations incubated with saline no change in the shape of blood cells (bulging or shrinkage and formulation G3 exhibited which reveals the isotonic nature).