Claims:1. A pharmaceutical in-situ gelling ophthalmic solution comprising;
brinzolamide;
dimethyl sulfoxide; and
a thickener.

2. The solution of claim 1, wherein the thickener is a polymer material having an ion-sensitive characteristics selected from the group consisting of, sodium alginate, gellan gum, guar gum, pectin, sodium hyaluronate, hydroxypropyl methylcellulose, methyl cellulose, polyvinyl pyrrolidone, polyvinylalcohol, poly(acrylic acid) polymers or combination thereof .

3. The solution of claim 2, wherein thickener is gellan gum.

4. The solution of claim 1, wherein the solution further comprises one or more component selected from the group consisting of isotonizing agents, viscosity modifying agents, stabilizers, buffers, preservatives, surfactants and antioxidants.

5. The solution of claim 1, wherein the viscosity of composition is about 10 cps to 100 cps.

6. The solution of claim 1, wherein has a pH between 6 and 9 and an osmolality between 200 and 600 mOsm/kg.

7. The solution of claim 4, wherein the surfactant is selected from the group consisting of sorbitan esters, glycerol esters, polyethylene glycol esters, block polymers, acrylic polymers, ethoxylated fatty esters, ethoxylated alcohols, ethoxylated fatty acids, monoglycerides, silicon based surfactants or combination thereof.

8. A pharmaceutical in-situ gelling ophthalmic solution comprising;
brinzolamide;
about 15%w/v to 60% w/v of dimethyl sulfoxide; and
about 0.10% w/v to 0.50%w/v of gellan gum.

9. The solution of claim 8, further comprises a surfactant is selected from the group consisting of polyoxyl 35 castor oil, polysorabte 80 or combination thereof.

10. The solution of claim 9, further comprises tromethamine and benzalkonium chloride.
, Description:FIELD OF THE INVENTION

This invention relates to a pharmaceutical in-situ gelling ophthalmic solution compositions comprising brinzolamide as active ingredient alone and/or in combination with other active ingredients. The invention also relates to the processes for making such compositions and use of these compositions in patient populations including pediatric populations.

BACKGROUND OF THE INVENTION

Conventional drops of prostaglandins, beta-blockers, carbonic anhydrase inhibitors, alpha-adrenergic agonist and their combinations are available for treatment of glaucoma. Amongst them, carbonic anhydrase inhibitor plays an important role in treatment of glaucoma for reduction of ocular hypertension.

One such carbonic anhydrase inhibitors (CAIs) is R 4-ethylamino-3,4-dihydro-2-(3-methoxy)propyl-2H-thieno [3,2-e]-1,2-thiazine-6-sulfonamide 1,1 dioxide, which is known as brinzolamide. This compound is disclosed in U.S. Pat. No. 5,378,703 (Dean, et al.). Brinzolamide is highly specific, no-competitive, reversible and effective inhibitor of carbonic anhydrase II, able to suppress formation of aqueous humor which reduces intraocular pressure

It is a drug of choice in case of glaucoma patients with a vascular dysregulation, it has double effect of reducing elevated intraocular pressure (IOP) with improving ocular blood flow. Brinzolamide can be used as first line medication due to fewer side effects and as monotherapy in patients unresponsive to beta-blockers or in patients in whom beta-blockers are contra-indicated, or as adjunctive therapy to beta blockers and prostaglandins. However, because of the poor aqueous solubility of brinzolamide, the clinical application is extremely limited.

US Pat. No. 6,071,904 discloses ophthalmic suspensions containing brinzolamide and processes for manufacturing the suspensions.

Azopt® (brinzolamide) commercial preparation of Brinzolamide available in the market is an aqueous multiple use suspension composed of 1% w/v Brinzolamide. Unfortunately, this formulation is associated with side effects, such as blurred vision, pain, discomfort (stinging and burning), eye discharge, blepharitis, dry eye, and taste perversion and the limitations of requirement of multiple dosing required (3 to 4 time per day) and dosing inaccuracy.

It is well known in the art that, the bioavailability of the suspension formulation is reduced because of the fact that the drug must be dissolved in order to be absorbed before being eliminated from the eye surface. The suspension formulation also suffer the drawback that the particulates have a potential to cause irritation, increasing the likelihood that they would be rubbed or washed from the eye. Additionally, manufacturing of sterile suspension formulation for ophthalmic use has its own challenges like particle size reduction, degree of homogenization required and ability of the formulation to remains in to the suspended state without particle aggregation throughout the shelf life.

Other types of vehicles have been developed for the purpose of increasing the residence time of the drug on the eye surface. Among these are those that increase the bioavailability by means of an increase of the viscosity such as hydrogels or ophthalmic ointments. In the case of hydrogels, an important increase of the bioavailability of the drug has not been achieved. On their part, ophthalmic ointments have the large inconvenience of the awkwardness of their application and blurred vision that is produced after their application, the use thereof being more appropriate at night.

Likewise, a large number of novel vehicles have been developed such as liposomes, nanoparticles, etc., though most of them have problems of stability, tolerance, difficulties for industrialization thereof and limited success as far as the increase of bioavailability is concerned.

The inventors of the present invention have developed the in-situ gelling ophthalmic solution compositions of brinzolamide which can deliver drug at the right dosage to the eye with the convenience of dosing just like any other ophthalmic solution. The in-situ gelling ophthalmic solution of the present invention provides the controlled release of brinzolamide over the longer period of time. Application method of in-situ gelling solution is simple like conventional drop and also avoids the discomfort associated with the other dosage forms.

SUMMARY OF THE INVENTION

This invention relates to a pharmaceutical in-situ gelling ophthalmic solution compositions comprising brinzolamide and process of preparing the same.

In an embodiment the present invention provides a pharmaceutical in-situ gelling ophthalmic solution compositions which provides an ease of administration, increases bioavailability in the eye, the said solution is stable during storage.

In another embodiment the present invention provides a pharmaceutical in-situ gelling ophthalmic solution compositions comprising; brinzolamide, dimethyl sulfoxide and a thickener.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Fig. 1: Graph of Comparative ?IOP (mm Hg) vs Time (h) profile for Example-A(0.5% w/v brinzolamide) and Example-C (1.0 % w/v brinzolamide) of brinzolamide in situ gelling solution composition in comparison with commercial eyedrops (Azopt® 1.0 % w/v brinzolamide) in glaucomatous rabbits.

Fig. 2 Graph of Comparative ?IOP (mm Hg) vs Time (h) profile for Example-D (0.5% w/v brinzolamide) and Example-F (1.0 % w/v brinzolamide) of brinzolamide in situ gelling solution composition in comparison with commercial eyedrops (Azopt® 1.0 % w/v brinzolamide) in glaucomatous rabbits.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a pharmaceutical in-situ gelling ophthalmic solution compositions comprising brinzolamide and process of preparing the same. Preferably the ophthalmic solution compositions is a sterile composition.

The invention further relates to a pharmaceutical in-situ gelling ophthalmic solution compositions comprising brinzolamide optionally in combination with other active ingredients and process of preparing the same.

In an embodiment the present invention provides a pharmaceutical in-situ gelling ophthalmic solution compositions comprising; brinzolamide, dimethyl sulfoxide and a thickener.

In another embodiment the present invention provides a pharmaceutical in-situ gelling ophthalmic solution compositions comprising; brinzolamide, dimethyl sulfoxide, surfactant and a thickener.

Although the present invention is described with respect to brinzolamide, the invention is not limited thereto. The present invention can also be utilized for manufacturing the pharmaceutical ophthalmic compositions of the active ingredients having ophthalmic applications such as corticosteroid, anti-inflammatory, beta-adrenergic receptor agonist, beta-blockers, prostaglandins, carbonic anhydrase inhibitors, alpha 2 adrenergic receptor agonist, antibiotics, antibacterial, antihistaminic/ mast cell stabilisers or combination thereof.

In some embodiments, the corticosteroid is selected from the group consisting of prednisolone, methylprednisolone, difluprednate, prednisone acetate, prednisolone sodium phosphate, triamcinolone, fluocinolone; fluorometholone, betamethasone, medrysone, rimexolone, dexamethasone, hydrocortisone, loteprednol and a combination thereof. In other embodiments, the anti-inflammatory is selected from a group consisting of a corticosteroid, a non-steroidal anti-inflammatory drug (“NSAID”), thymosin beta 4, and a combination thereof. In one particular instances, the NSAID is selected from the group consisting of diclofenac, flubiprofen, ketorolac, ketorolac thromethamine, bromfenac, nepafenac, flurbiprofen, and a combination thereof. In yet another embodiment, the beta-adrenergic receptor agonist is selected from the group consisting of dopexamine, epinephrine, isoprenaline, isoproterenol, levalbuterol, salbutamol, albuterol, and a combination thereof. Still in other embodiments, the beta-blocker is selected from the group consisting of timolol, propranolo, sotalol, nadolol, betaxolol, levobetaxolol and a combination thereof. Yet still in other embodiments, the prostaglandins analog is selected from the group consisting of latanoprost, bimatoprost, travoprost, tafluprost, and a combination thereof. Yet in other embodiments, the carbonic anhydrase inhibitor is selected from the group consisting of dorzolamide, methazolamide, dichlorphenamide, and a combination thereof. Yet in other embodiments, the alpha 2 adrenergic receptor agonists include, but are not limited to, brimonidine, 4-NEMD, 7-Me-marsanidine, agmatine, apraclonidine, cannabigerol, clonidine, detomidine, dexmedetomidine, fadolmidine, guanabenz, guanfacine, lofexidine, marsanidine, medetomidine, methamphetamine, mivazerol, rilmenidine, romifidine, talipexole, tizanidine, tolonidine, xylazine, xylometazoline, and the like including pharmaceutically acceptable salts thereof. Yet in other embodiments, antibiotics and/or antibacterial includes besifloxacin, neomycin; polymyxin b, tobramycin, sulfacetamide sodium, gentamicin, oxytetracycline, natamycin, chloramphenicol, tetracycline and a combination thereof. Yet in other embodiments, the antihistamine/mast cell stabiliser include, but are not limited to, levocabastine, alcaftadine, azelastine, bepotastine, emedastine, epinastine, ketotifen, olopatadine and a combination thereof.

The concentration of the brinzolamide present in the pharmaceutical in-situ gelling ophthalmic solution compositions according to the present invention is in the range of about 0.02% to 5% w/v of composition; preferably 0.01, 0.5, 0.75, 1, 2, 3, 4 or 5% w/v of composition or intermediate fraction thereof.

In accordance with the present invention “In-situ gel solution” are in solution that undergoes gelation in situ (at physiological site of application), to form a gel within 30 seconds of administration. Wherein the viscosity of gel formed in-situ is at least twice of the initial formulation. In a preferred embodiment viscosity of gel formed in-situ is increased at least 3 to 9 times of the initial formulation. In most preferred embodiment viscosity of gel formed in-situ is increased at least 5-7 times of initial solution.

In accordance with the present invention the in-situ gelling ophthalmic solution compositions according is cost effective, stable and provide a controlled release of brinzolamide.

In accordance with the present invention, the formulation has a pH between 6 and 9 and an osmolality between 200 and 600 mOsm/kg.

In accordance of the present invention the formulation comprises of dimethyl sulfoxide (DMSO). In a preferred embodiment the dimethyl sulfoxide is used for solubilizing the brinzolamide in the composition. In accordance with the present invention the dimethyl sulfoxide is present at least 15% w/v of the total composition. In a preferred embodiment dimethyl sulfoxide is present in about 15% w/v- about 60% w/v of the composition. In a more preferred embodiment dimethyl sulfoxide is about 25% w/v to 50 % w/v of the composition.

In accordance with the present invention, the thickener is a polymer material having an ion-sensitive characteristics. In accordance with the present invention Ionic strength dependent in-situ gel polymers are polymers that undergo a sol-gel transition upon contact with physiological ions (Na+, K+, Ca++, Mg++ etc.) in the site of application or site of action. Example of ionic strength in-situ gel polymer include various types of sodium alginate, gellan gum, guar gum, pectin, sodium hyaluronate, In a preferred embodiment the thickener is gellan gum. In an embodiment of present invention Ionic Strength in-situ gel polymer may be used alone or in combination. In further embodiment of the present invention in-situ gel polymer can be combined with other thickening polymers like One or more of hydroxypropyl methylcellulose (HPMC), methyl cellulose (MC), polyvinyl pyrrolidone (PVP), polyvinylalcohol (PVA), and Poly(acrylic acid) polymers such as carbomers and the like can also be added.

In accordance with the present invention, thickener is present in concentration preferably about 0.10% w/v to about 0.50% w/v of the composition. In a preferred embodiment thickener is present in about 0.15% w/v to about 0.30% w/v of the composition. In more preferred embodiment thickener is present in about 0.20 to about 0.25%w/v of the composition. In one of the most preferred embodiments the present invention comprises gellan gum as thickener in concentration about 0.20 to about 0.25%w/v of total composition.

In accordance with the present invention, the composition may optionally further comprise of one or more of the following components; isotonizing agents, stabilizers, buffers and/or pH modifying agent, preservatives, surfactants and/or antioxidants.

The composition of the present invention may contain an isotonizing agent such as mannitol, glycerin, glycerol, sorbitol, glucose or combination thereof. In preferred embodiment of the present invention the isotonizing agent is glycerin, glycerol or combination thereof. The composition of present invention may contain stabilizers such as sodium edatate, citric acid or combination thereof; buffers such as tris(hydroxymethyl)aminomethane, sodium phosphate and potassium phosphate, sodium citrate, sodium carbonate and sodium bicarbonate or combination thereof; preservatives such as quaternary ammonium compound like benzalkonium chloride, chlorobutanol, sodium perborate or combination thereof. In a preferred embodiment of present invention the preservative used is benzalkonium chloride.

In a preferred embodiment the surfactants is selected from the group consisting of, but not limited to, sorbitan esters (such as Span or Arlacel), glycerol esters (such as glycerin monostearate), polyethylene glycol esters (such as polyethylene glycol stearate), block polymers (such as poloxamers (Pluronics®)), acrylic polymers (such as Pemulen®), ethoxylated fatty esters (such as polyoxyl 35 castor oil ,Cremophor® RH-40), ethoxylated alcohols (such as Brij®), ethoxylated fatty acids (such as polysorbate 80, Tween or Tween 20), monoglycerides, silicon based surfactants alone or in combination. In a preferred embodiment, the surfactant is polyoxyl 35 castor oil and polysorbate 80 or combination thereof. In more preferred embodiment, the surfactant is polyoxyl 35 castor oil. In accordance with the present invention, surfactant is present in concentration preferably about 1% w/v to about 10 % w/v of the composition. In a preferred embodiment surfactant is present in about 2% w/v to about 7% w/v of the composition.

The compositions of the present invention may be sterilized by filtration, terminal sterilization or may be obtained by sterilization of the excipients and subsequently mixing in aspectic conditions.

In an embodiment disclose a pharmaceutical in-situ gelling ophthalmic solution compositions comprising;
brinzolamide;
dimethyl sulfoxide;
surfactant; and
a thickener.

In an embodiment disclose a pharmaceutical in-situ gelling ophthalmic solution compositions comprising;
brinzolamide;
dimethyl sulfoxide;
surfactant selected from the group comprising of polyoxyl 35 castor oil, polysorabte 80 or combination thereof; and
about 0.10% w/v to about 0.50%w/v of the thickener.

In an embodiment disclose a pharmaceutical in-situ gelling ophthalmic solution compositions comprising;
brinzolamide;
dimethyl sulfoxide;
surfactant selected from the group comprising of polyoxyl 35 castor oil, polysorabte 80 or combination thereof; and
about 0.10% w/v to about 0.50%w/v of the gellan gum.

In an another embodiment disclose a pharmaceutical in-situ gelling ophthalmic solution compositions comprising;
about 0.02% to 5% w/v of brinzolamide;
about 15% w/v to 50% w/v of dimethyl sulfoxide;
about 1% w/v to 10% w/v of polyoxyl 35 castor oil; and
about 0.10% w/v to about 0.50%w/v of the gellan gum.

Viscosity of the composition was determined by Rheocalc T 1.2.19 (Brookfield Engineering Labs Inc) using spindly SC4-18 at 25.0 ± 0.5°C. The rotation speed was 50 rpm and the viscosity was determined in centipoise from rheogram plotted using shear stress (dyn/cm2) and shear rate (1/s).
In accordance of the present invention, the viscosity of the present invention is 5cps to 200cps. In preferred embodiment of the present invention viscosity of the present invention is 10cps to 100 cps. In preferred embodiment of present invention, the viscosity of present invention is 25cps to 75cps.

In accordance with the present invention the viscosity of gel formed in-situ is at least twice of the initial formulation. In a preferred embodiment viscosity of gel formed in-situ is increased at least 3 to 9 times of the initial formulation. In most preferred embodiment viscosity of gel formed in-situ is increased at least 5-7 times of initial solution.

In-situ gelling of the present invention was determined by in-vitro study by placing 4mL of brinzloamide formulation of present invention in a vial containing about 2mLof pH 7.4 stimulated tear fluid (STF). Viscosity of in-situ gel was determined by Rheocalc T 1.2.19 (Brookfield Engineering Labs Inc) using spindly SC4-18 at 25.0 ± 0.5°C. The rotation speed was 50 rpm and the viscosity was determined in centipoise from rheogram plotted using shear stress (dyn/cm2) and shear rate (1/s).

In situ gelling time was determined by placing a drop of brinzolamide formulation of present invention in a vial containing about 2 mL of pH 7.4 stimulated tear fluid (STF) and time required for in-situ gel formation was noted down by visual observations.

In accordance with the present invention, in-situ gelling solutions were prepared by dissolving brinzolamide in dimethyl sulfoxide under stirring to obtain a drug solution phase. The aqueous phase was by dissolving gellan gum in water. The drug solution phase was then added drop wise to the aqueous phase under continues stirring process. The stirring was continued till clear solution was obtained. The in-situ gelling solution, thus obtained was filtered.

The in-vivo pharmacodynamic studies were carried out using glaucomatous rabbits. The commercially available ophthalmic drops (Azopt®) containing 1.0 % w/v (10 mg/mL) of brinzolamide were administered as a reference at the same dose as test formulation. IOP measurement carried out by using calibrated Schiotz tonometer at different time intervals. The change in IOP (?IOP) at each time point from the stabilised IOP (zero time) was determined by using following equation;
Equation 1 ?IOP = IOP _(zero time)-?IOP ?_(time t)
The AUC (?IOP vs.t) of ?IOP vs. time curve calculated using trapezoid rule and the AUCRel was calculated using the following equation;
Equation 2 ?AUC?_Rel=(?AUC?_(?IOP vs.t) Test (designed formulation))/(?AUC?_(?IOP vs.t) Reference (marketed eye drops))

In accordance with the present invention a pharmaceutical in-situ gelling ophthalmic solution composition shows a decrease in elevated IOP in glaucomatous rabbits for a longer period of time as compared to reference formulation. The AUC (?IOP vs. t) of the present solution was about 2-9 times higher than that of reference product

Example-1
Sr.No. Ingredients Example-A
Brinzolamide 0.5 % w/v Example-B
Brinzolamide 0.75 % w/v Example-C
Brinzolamide 1.0 % w/v
A) Drug Solution Phase
1 Brinzolamide 0.50 0.75 1.00
2 Dimethyl Sulfoxide 20.00 30.00 40.00
B) Aqueous Phase
3 Polyoxyl 35 Castor Oil * 3.00 3.00 3.00
4 Dimethyl Sulfoxide 10.00 10.00 10.00
5 Tromethamine 0.15 0.15 0.15
6 Gellan Gum 0.20 0.20 0.20
7 Benzalkonium Chloride 0.05 0.05 0.05
8 Orthophosphoric acid (1M) Q.S. Q.S. Q.S.
9 Purified Water Q.S. to make 100 % w/v
* Polyoxyl 35 Castor Oil was obtained from BASF SE Germany which is manufactured in Germany

Manufacturing Process:
1. The drug solution phase was prepared by dissolving brinzolamide in dimethyl sulfoxide under stirring using magnetic stirrer.
2. The aqueous phase was prepared by mixing, tromethamine (Tris buffer) in purified water and pH was adjusted in between 6.8 to 7.2 using 1M orthophosphoric acid.
3. The aqueous phase was heated at 70-80°C and dimethylsulfoxide, polyoxyl 35 castor oil, gellan gum and benzalkonium chloride were added.
4. Drug solution phase was added drop wise in aqueous phase under stirring and stirring was continued for 20 minutes and solution was cooled to room temperature.
5. Prepared solution then filtered through 1.0 micron filter to remove particulate matter present in solution.
6. Sterilization of above in-situ gelling solution was performed using both filtration techniques using 0.45 micron followed by 0.20 micron filter and using autoclave (121°C, 15 LB pressure for 15 minutes).

The in-vivo pharmacodynamic studies were carried out using glaucomatous rabbits. The commercially available ophthalmic drops (Azopt®) containing 1.0 % w/v (10 mg/mL) of brinzolamide were administered as a reference at the same dose as test formulation. The result of this study is shown in Table 1 and Fig 1.

TABLE 1: In-vivo Pharmacodynamic Study Results:

Formulation I max / Dose
(mm Hg) tmax (h) AUC (?IOP vs. t) /Dose
(h mm Hg) MRT (h) AUCRel
Azopt® 13.03 ± 1.72 1 35.97 ± 3.20 4.90 -
Example-A 28.06 ± 3.60 2 192.56 ± 5.92 11.2 5.4
Example-C 13.67 ± 1.75 4 137.58 ± 3.20 15.8 3.8

Imax/ Dose is ?IOP (mm Hg) normalized to dose, tmax time taken for ?IOP (h). AUC (?IOP vs. t)/Dose area under the ?IOP vs. time curve normalized to dose, MRT is mean residence time. AUCRel ratio of AUC (?IOP vs. t) test (designed formulations) to AUC (?IOP vs. t) reference (marketed eyedrops).

The pharmacodynamic studied conducted with optimized formulations of brinzolamide in-situ gelling solution showed decrease in elevated IOP in glaucomatous rabbits for a longer period of time as compared to reference formulation. The AUC (?IOP vs. t) for the solution were about 3-5 times higher than that of reference product.

Example-2
Sr.No Ingredients Example-D
Brinzolamide 0.5 % w/v Example-E
Brinzolamide 0.75 % w/v Example-F
Brinzolamide 1.0 % w/v
A) Drug Solution Phase
1 Brinzolamide 0.50 0.75 1.00
2 Dimethyl Sulfoxide 20.00 30.00 40.00
B) Aqueous Phase
3 Polyoxyl 35 Castor Oil* 3.00 3.00 5.00
4 Dimethyl Sulfoxide 10.00 10.00 10.00
5 Tromethamine 0.15 0.15 0.15
6 Gellan Gum 0.25 0.25 0.25
7 Benzalkonium Chloride 0.05 0.05 0.05
8 Orthophosphoric acid (1M) Q.S. Q.S. Q.S.
9 Purified Water Q.S. to make 100 % w/v
* Polyoxyl 35 Castor Oil was obtained from BASF SE Germany which is manufactured in Germany

In-situ gelling solution was prepared by using the similar manufacturing process as disclosed in example 1. The result of this study is shown in Table 2 and Fig 2.

TABLE 2: In-vivo Pharmacodynamic Study Results:
Formulation I max / Dose
(mm Hg) tmax (h) AUC (?IOP vs. t) /Dose
(h mm Hg) MRT (h) AUCRel
Azopt® 13.03 ± 1.72 1 35.97 ± 3.20 4.90 -
Example-D 28.14 ± 3.60 2 328.60 ± 8.15 17.5 9.1
Example-F 13.70 ± 1.75 6 164.39 ± 3.20 17.7 4.6
Imax/ Dose is ?IOP (mm Hg) normalized to dose, tmax time taken for ?IOP (h). AUC (?IOP vs. t)/Dose area under the ?IOP vs. time curve normalized to dose, MRT is mean residence time. AUCRel ratio of AUC (?IOP vs. t) test (designed formulations) to AUC (?IOP vs. t) reference (marketed eyedrops).

The pharmacodynamic studied conducted with optimized formulations of brinzolamide in-situ gelling solution showed decrease in elevated IOP in glaucomatous rabbits for a longer period of time as compared to reference formulation. The AUC (?IOP vs. t) for the selected solution were about 4-9 times higher than that of reference product.

Example-3
Sr.No Ingredients Example-G
Brinzolamide 0.5 % w/v Example-H
Brinzolamide 0.75 % w/v Example-I
Brinzolamide 1.0 % w/v
A) Drug Solution Phase
1 Brinzolamide 0.50 0.75 1.00
2 Dimethyl Sulfoxide 20.00 30.00 40.00
B) Aqueous Phase
3 Polysorbate 80 3.00 4.00 6.00
4 Dimethyl Sulfoxide 10.00 10.00 10.00
5 Tromethamine 0.15 0.15 0.15
6 Gellan Gum 0.25 0.25 0.25
7 Benzalkonium Chloride 0.05 0.05 0.05
8 Orthophosphoric acid (1M) Q.S. Q.S. Q.S.
9 Purified Water Q.S. to make 100 % w/v

In-situ gelling solution of example 3 was prepared by using the similar manufacturing process as disclosed in example 1.