FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION Sec 10-Rule 13
A STABLE OPHTHALMIC NANOSUSPENSION OF BRINZOLAMIDE.
INDOCO REMEDIES LIMITED
Indian
R-92-93, T.T.C Industrial Area, Thane Belapur Road, Rabale MIDC
Navi Mumbai-400701
The following complete specification describes the invention and the manner
in which it is to be performed.

Technical Field:
The present invention relates to a stable ophthalmic nanosuspension containing a drug of low water solubility. Specifically, the present invention relates to a carbonic anhydrase inhibitor ophthalmic nanosuspension and a method of preparing same. More specifically, the present invention relates to a stable brinzolamide ophthalmic nanosuspension and a method of preparing same, which is chemically stable at room temperature and free of quaternary ammonium salt. Moreover, the composition of present invention is sterile aqueous ophthalmic nanosuspension.
Background of the Invention
Brinzolamide is a white powder insoluble in water, commercially formulated as a 1% ophthalmic suspension to reduce intraocular pressure (IOP). Pharmacologically, brinzolamide is a highly specific, non-competitive, reversible, and effective inhibitor of carbonic anhydrase II. The commercially available preparation of brinzolamide is 1% w/v suspension (Azopt), with a pH of approximately 7.5 and osmolarity of 300 mOsm/kg. Azopt is indicated to treat ocular hypertension and open-angle glaucoma.
Brinzolamide is chemically designated as (R)-(+)-4-Ethylamino-2-(3-methoxypropyl)-3, 4-dihydro-2H-thieno[3,2-e] -1,2-thiazine-6-sulfonamide-1,1-dioxide, first disclosed in US5378703. It has the following structural formula:


Brinzolamide is indicated in the treatment of elevated intraocular pressure in patients with ocular hypertension or open-angle glaucoma. The particle size of Azopt® formulation is between 1-5 Μ, as disclosed in US6071904.
Process to manufacture Brinzolamide ophthalmic suspension has also been disclosed in prior art, US6071904. The aforementioned patent describes a process to manufacture Brinzolamide suspensions by autoclaving brinzolamide and surfactant together followed by ball milling. This milled mixture is then added to the rest of the excipients to form a final suspension
WO2012053011 discloses process for preparing sterile ophthalmic suspension. The process involves solubilizing Brinzolamide to get a solution which is aseptically filtered to get a filtrate which is further precipitated to get slurry of brinzolamide. The sterile slurry of Brinzolamide is further ball milled or jet milled along with surfactants and further processed with suitable excipients.
Another approach disclosed in '011 discloses preparing aqueous solution of Brinzolamide, addition of surfactant to said aqueous solution, filtering, precipitating Brinzolamide, followed by ball milling or jet milling; further suspension vehicle is prepared and autoclaved, eventually added to slurry of Brinzolamide and surfactant.
WO2011067791 also describes another process to manufacture a Brinzolamide suspension. The process involves preparation of the slurry of Brinzolamide, followed by preparation of polymer slurry and preparation of a solution of the preservative along with the tonicity agent. The aforementioned preparations are homogenized and autoclaved followed by a sizing process. The sizing process employs a ball mill, colloidal mill or a microfluidiser. An alternately cited process involves autoclaving of Brinzolamide and the surfactant together followed by a micro fluidisation process followed by addition of the rest of the excipients in a sterile manner.
IJS20160279139 discloses improved process for manufacturing sterile ophthalmic pharmaceutical suspension of carbonic anhydrase inhibitors wherein the process

does not involve the use of any special equipment's such as ball mill, milling bottle and/or jet mill.
Therapeutic agents which exhibit relatively high degrees of hydrophobicity are formulated as suspensions, which can cause those agents to undesirably aggregate within an aqueous solution. In turn, the overall suspension can lack homogeneity and can, consequently, deliver inconsistent amounts of therapeutic agent to a target.
In efforts to accommodate these undesirable properties, many materials such as surfactants have been added to pharmaceutical vehicles with the objective of developing new stabilization systems. However, many of these new systems can lack biocompatibility and can cause irritation or other undesirable effects to human tissue.
It is known that poorly water-soluble drugs may be formulated as nanoparticles. Nanoparticles are of interest for a variety of reasons, such as to improve the bioavailability of poorly water-soluble drugs, to provide targeted drug delivery to specific areas of the body, to reduce side effects, or to reduce variability in vivo.
In view of the above, it would be desirable to provide a pharmaceutical nanosuspension, method of preparing the same, and/or materials suitable for preparing nanosuspension, which overcome one or more of the aforementioned difficulties, problems and drawbacks.
The inventors of the present invention have developed nanosuspension of brinzolamide with an aim to enhance bioavailability and thereby reduce the dosage of the active pharmaceutical ingredient (API) in the formulation as compared to the marketed product Azopt®
Object of the Invention:
The object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable
excipients.

Another object of the present invention is to provide a process of preparation of a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients.
Further object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients with an aim to enhance bioavailability.
Yet another object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients wherein the dose of brinzolamide is reduced as compared to the marketed formulation Azopt.
Yet another object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients for the treatment of elevated intraocular pressure in patients with ocular hypertension or open angle glaucoma.
Yet another object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients by reducing the particle size of brinzolamide in the composition to nano size to increase the corneal bioavailability. With this modification, the dose of brinzolamide can be reduced from 1% w/v to 0.5% w/v or 0.75% w/v.
Yet another object of the present invention is to provide a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients and thereby reduce the risk of undue toxicity due to increased bioavailability of the drug at the required site.
Further object of the present is to provide a stable ophthalmic nanosuspension comprising brinzolamide and a method of preparing same, which is chemically stable at room temperature, and free of quaternary ammonium preservative.

Summary of the Invention:
The present invention relates to a stable brinzolamide ophthalmic nanosuspension and a method of preparing same, which is chemically stable at room temperature and free of quaternary ammonium salt. Moreover, the composition of present invention is sterile aqueous ophthalmic nanosuspension.
Detailed Description:
The present invention relates to a stable brinzolamide ophthalmic nanosuspension and a method of preparing same, which is chemically stable at room temperature and free of quaternary ammonium salt.
Thus, in one aspect, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable
excipients.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, preservative, at least one polymer, surfactant and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, and pharmaceutically acceptable excipients, wherein the composition is free of quaternary ammonium preservative.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, sodium CMC and pharmaceutically acceptable excipients.

According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, combination of sodium CMC and HPMC, and pharmaceutical^ acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, tyloxapol, sodium CMC and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, Sodium CMC and pharmaceuticallyacceptable excipients wherein the composition is sterile and free of quaternary ammonium salt.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, sodium CMC, tyloxapol and pharmaceuticallyacceptable excipients wherein the composition is sterile and free of quaternary ammonium salt.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, sodium CMC, sodium chloride, hydrochloric acid and purified water.
According to one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate, sodium CMC, sodium chloride, hydrochloric acid and purified water, wherein the composition is sterile and free of quaternary ammonium salt.
According to one embodiment, the present invention provides a stable ophthalmic
nanosuspension comprising:
0.25% to 1 % (w/v) brinzolamide,
0.1 % to 0.4 % (w/v) surfactant,
1.8 % to 5.7 % (w/v) stabiliser,
3.76 % to 5.17 % preservative,
3.0 % to 6.0 % (w/v) viscosity enhancing agent, and,

6.0 % to 6.3 % (w/v) tonicity agent,
wherein the composition is sterile and free of quaternary ammonium preservative.
According to one specific embodiment, the present invention provides a stable
ophthalmic nanosuspension comprising:
0.25% to 1 % (w/v) brinzolamide,
0.1 % to 0.4 % (w/v) tyloxapol,
1.8 % to 5.7 % (w/v) sodium CMC,
3.76 % to 5.17 % potassium sorbate,
3.0 % to 6.0 % (w/v) xanthan gum, and,
6.0 % to 6.3 % (w/v) sodium chloride,
wherein the composition is sterile and free of quaternary ammonium preservative.
The pharmaceutically acceptable excipients contained in the aqueous ophthalmic composition of the present invention include surfactants, stabilizing agents, preservatives, viscosity imparting agents, tonicity agents, vehicles and other conventional agents that may be typically used in formulating an ophthalmic nanosuspension.
The ophthalmic nanosuspension of the present invention may optionally contain a preservative that is conventionally used in ophthalmic compositions such as eye drops. The preservatives that can be used in the aqueous ophthalmic composition include, without limitation, hydrogen peroxide, biquanides, sorbic acid, potassium sorbate, boric acid, borate, chlorohexidine, zinc chloride, propylene glycol, purite, polyquad, or the like.
In one aspect, the present invention provides an aqueous ophthalmic composition without using a preservative.
Accordingly, the present invention relates to a preservative free ophthalmic nanosuspension comprising brinzolamide and pharmaceutically acceptable excipients.

In one embodiment, the present invention relates to a preservative free ophthalmic nanosuspension comprising brinzolamide, tyloxapol, sodium CMC and pharmaceuticallyacceptable excipients.
Tonicity agents that can be used in the aqueous ophthalmic nanosuspension of the present invention include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, sodium bromide, mannitol, glycerol, sorbitol, propylene glycol, dextrose sucrose, and combinations thereof Tonicity agent is used to make the composition isotonic with respect to the ophthalmic fluids present in the human eye.
Surfactants that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to, propylene glycol, macrogol 15 hydroxystearate, polyethylene glycol, polypropylene glycol, polysorbate, tyloxapol, povidone, polyvinyl alcohol, hypromellose, polaxamer, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl-15- hydroxystearate, glycerine, ethanol, dimethyl acetamide, dimethyl sulfoxide, PEG 300 caprylic/capric glycerides, PEG 300 oleic glycerides, miglyol, soluplus, sorbitan monooleate, sodium lauryl sulfate, docusate sodium, polyoxyalkyl ethers, polyoxyalkyl phenyl ethers, caprylocaproyl polyoxyl 8- glycerides, Kolliphor, caprylyl capryl glucoside, heptyl glucosides, and lauroyl macrogol- 32 glycerides, macrogol glycerol hydroxystearate and glyceryl laurate and combinations thereof.
Viscosity enhancing agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to hydroxy ethyl cellulose, carrageenan, polycarbophil, polyvinyl alcohol, povidone, sodium alginate, locust bean gum, pullulan, sodium carboxy methyl cellulose, gellan gum, methyl cellulose, guar gum, xanthan gum and combinations thereof.
Stabilising agents that can be used in the ophthalmic nanosuspension of present invention include, but are not limited to sodium carboxy methyl cellulose, (sodium CMC), hydoxy propyl methyl cellulose (HPMC), polycarbophil, polaxamer, hydroxyl propyl cellulose and combinations thereof

Buffering agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, acetic acid, hydrochloric acid, sodium carbonate, sodium hydroxide and combinations thereof.
In the context of the present invention, the concentration of brinzolamide is between 0.25 % to 1 % (w/v), or any specific value within the said range.
In the context of the present invention the active ingredient brinzolamide also
includes its pharmaceuticallyacceptable salts, solvates, hydrates, polymorphs,
stereoisomers, esters, prodrugs, enantiomers, complexes and their metabolites
thereof.
According to another aspect, the present invention provides a method for the
preparation of a stable ophthalmic nanosuspension comprising brinzolamide and
pharmaceuticallyacceptable excipients,
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic nanosuspension, comprising:
a) Sterilization of brinzolamide by ethylene oxide sterilization.
b) Preparing a solution comprising at least one srfatant and at least one stabilizing agent in water.
c) Mixing of step (a) brinzolamide and step (b) solution.
d) Addition of step (c) mixture into suitable homogenizer or mill to reduce average particle size in the range of 100-400 nm.
c) Preparing a solution comprising of at least one viscosity enhancing agent suitable for ophthalmic use.
f) Preparing a solution comprising of at least one preservative and at least one tonicity agent.
g) Mixing a solution of step (e) and step (f) to form a slurry.
h) Aseptically mixing mixture of step (d) and slurry of step (g) until
homogeneity is obtained. i) Measuring and adjusting the volume using water for injection.

In one embodiment, the average particle size of wet milling phase or homogenizer phase is reduced 100 to 400 nm (nanometres), preferably 250 to 300 nm, more preferably around 285 nm.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic nanosuspension, comprising:
a) Sterilization of brinzolamide by ethylene oxide sterilization.
b) Preparing a solution comprising of tyloxapol and sodium carboxymethyl cellulose in water.
c) Mixing of step (a) brinzolamide and step (b) solution.
d) Addition of step (c) mixture into suitable homogenizer or mill to reduce average particle size in the range of 100-400 nm.
e) Preparing a solution comprising of xanthan gum suitable for ophthalmic use.
f) Preparing a solution comprising of potassium sorbate and sodium chloride
g) Mixing a solution of step (e) and step (f) to form a slurry.
h) Aseptically mixing mixture of step (d) and slurry of step (g) until
homogeneity is obtained. i) Measuring and adjusting the volume using water for injection.
The particle size of wet milling phase by laser diffraction is given in Table 1 Table 1: Particle size distribution of Brinzolamide

Milling time
(mins) Particle size in microns
d(10) d(50) d(90)
Initial 1.041 4.102 51.049
20 0.187 0.385 1.669
40 0.168 0.301 0.958
60 0.164 0.284 0.869
According to another aspect, the present invention provides a method of prevention or treatment of an ocular disease by administering to an affected eye a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaccutically acceptable excipients.

In one embodiment, the present invention provides a method of treating elevated intraocular pressure by administering to an affected eye a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients
In one embodiment, the present invention provides a method of treating elevated intraocular pressure in patients with open angle glaucoma by administering to an affected eye a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients.
In one embodiment, the present invention provides a method of treating elevated intraocular pressure in patients with ocular hypertension by administering to an affected eye a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients.
According to another aspect, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers, or polypropylene containers, or polyethylene containers.
In one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers, or polypropylene containers, or polyethylene containers with or without oxygen scavenger.
In one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers.
In one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients packaged in polypropylene containers.

In one embodiment, the present invention provides a stable ophthalmic nanosuspension comprising brinzolamide, potassium sorbate and pharmaceutically acceptable excipients packaged in polyethylene containers.
Sterilization of Different Components of the Nanosuspension:
Since the drug product is a suspension, intended to be filled in LDPE bottles, terminal sterilization of finished product by filtration is not possible and autoclaving the final filled bottles is also ruled out as LDPE bottles are heat labile. Thus, to make a sterile suspension product, different components / phases of the drug product need to be sterilized separately and then processed in the aseptic area as discussed below. a) Sterilization of Brinzolamide API
Various method were explored for sterilization of brinzolamide API, namely i) Dry Heat Sterilization
ii) Moist Heat Sterilization (Autoclaving)
iii) Gamma Irradiation
iv) Ethylene Oxide (ETO) Gaseous Sterilization i) Dry heat sterilization - Brinzolamide has a melting point of 131°C. Thus, the conventional method of dry heat sterilization at 160°C for 2 hours was not feasible.
ii) Moist heat sterilization - The temperature, time and pressure for conventional cycles of autoclave are 121 °C for at least 30 minutes at 15 psi pressure. In these conditions, part of brinzolamide gets partially solubilized and upon cooling, large crystals are generated. Since the objective of this work was to reduce the particle size to nano size, this crystallization phenomena would have caused variability in the starting material before milling. Thus, this approach was also ruled out.
iii) Gamma irradiation - Brinzolamide was irradiated using gamma irradiation (using the radioisotope Cobalt-60) at 3 different doses, namely, 8 kGy, 16 kGy and 24 kGy. To check the impact of gamma irradiation on the content of related substances in the API, the irradiated API was tested by the HPLC method for

related substances, listed in the USP monograph of brinzolamide. The results are tabulated in Table 2
Table 2: Effect of different doses of gamma irradiation on related compounds of brinzolamide

Dose of Gamma Sterilization
Sr.
No. Related compounds of brinzolamide Specifications (as per USP) 8 kGy 16 kGy 24 kGy
1. Brinzolamide related compd. A NMT 0.5% ND ND ND
2. Single maximum unknown impurity NMT 0.3% 0.29 % 0.42 % 0.46 %
3. Total impurities NMT 1.0% 1.25% 1.63% 1.71 %
ND - Not detected
At all 3 doses of gamma irradiation, the total impurities are higher than the specified limits of USP. At 16 and 24 kGy doses of gamma irradiation, the single maximum unknown impurity also failed as per the USP specifications. Thus gamma irradiation was not found suitable for sterilization of the API.
iv) Ethylene oxide (ETO) sterilization - Brinzolamide was packed in nylon bags which are permeable to gases and subjected to ETO sterilization cycles.
Table 3: Effect of ETO sterilization on related compounds of brinzolamide.

Sr.
No. Related compounds of brinzolamide Specifications (as per USP) Before
ETO
sterilization After
ETO
sterilization
1. Brinzolamide related compound A NMT 0.5% ND ND
2. Single maximum unknown impurity NMT 0.3% 0.06 0.06
3. Total impurities NMT 1.0% 0.24 0.35

Conclusion:
The impurity levels of brinzolamide after ETC) sterilization were well within the ICH limits. Hence the method of choice for sterilization of brinzolamide was ethylene oxide sterilization (ETC)).
b) Sterilization of sodium CMC and Xanthan gum
Both xanthan gum and sodium CMC turn brownish in colour when exposed to gamma irradiation and tend to lose their viscosities when subjected to heat. Hence, both the materials were subjected to ETO sterilization, using similar process as that for brinzolamide.
c) Sterilization of aqueous solution containing potassium sorbate and sodium
chloride
Since the solution containing potassium sorbate and sodium chloride is a clear, non-viscous solution, it is easily filterable through a sterile 0.2μ polyether sulfone membrane. Both the ingredients are inorganic compounds and hence not likely to adsorb onto the filter surfaces. Sterile xanthan gum is dispersed in this phase aseptically.
d) Sterilization of LDPE container closure system (CCS)
ETO sterilization was selected for the LDPE CCS, as gamma sterilization has the tendency to increase the degradation products of the drug product filled in the LDPE bottles, due to free radicals formed during gamma irradiation.
e) Selection of concentration of brinzolamide in the nanosuspension
Literature reference, Ying Zhang, Ke Ren, et.al, Development of inclusion complex of beinzolamide with hydroxylpropyl beta cyclodextrin, Carbohydrate Polymers, 2013, Volume 1, page 638 to 643, revealed that lower concentration of 0.2% w/v did not give encouraging results when brinzolamide was solubilized in an inclusion complex using hydroxypropyl beta cyclodextrin. However, 0.5% w/v of brinzolamide gave similar IOP-reducing efficacy when compared to that of Azopt® in the hydroxypropyl beta cyclodextrin inclusion complex.

Since no work was done on dose reduction of brinzolamide nano particles, it was decided to try two different strengths for formulating brinzolamide nano suspension, namely, 0.5% w/v and 0.75% w/v. The final formulae for both strengths of brinzolamide nano suspension is tabulated below as Example 1 (1A & IB).
Accordingly, the following examples arc intended to illustrate the present invention, but are not intended to limit the scope of the invention. Example 1 (1A & 1B)

1A 1B
Sr. No Ingredients % w/v % w/v
1 Brinzolamide 0.5 0.75
2 Tyloxapol 0.025 0.025
3 Sodium CMC 0.375 0.5625
4 Potassium sorbate 0.47 0.47
5 Xanthan gum 0.5 0.5
6 Sodium chloride 0.63 0.60
7 Hydrochloric acid q.s q.s
8 Water for injection q.s q.s
Manufacturing Process:
a) Brinzolamide was sterilized by ethylene oxide sterilization
b) A solution was prepared by adding tyloxapol and sodium carboxymethyl cellulose in water for injection.
c) The step (a) sterile brinzolamide was added to step (b) solution and transferred into the hopper of the bead mill fitted with accelerator type impellers and a fixed quantity of 0.5 mm ceramic beads inside the milling chamber.
d) A solution of xanthan gum was prepared by mixing it in water for injection.
e) A separate solution was prepared by mixing potassium sorbate and sodium chloride in water for injection
f) A slurry was prepared by mixing solution of step (d) and step (e)

g) The wet milled brinzolamide of step (c) and slurry of step (f) was mixed until
homogeneity is obtained. h) Volume make up was done and pH was adjusted 5 to 8 using hydrochloric
acid.
Eye Irritation Test (Draizc test)
A Draize test to assess the irritation potential was performed in rabbit eyes. The grades of ocular reaction (conjunctivae, cornea and iris) were recorded as per the Draize method.
Results: - No signs of eye irritation was observed up to 72 hours post-treatment with the nanosuspension.
Efficacy studies in rabbits for brinzolamide ophthalmic suspension in comparison with Azopt®
An efficacy study on both the formulation strengths, namely, 0.5% and 0.75% of brinzolamide ophthalmic suspension, was conducted on rabbits, and compared with Azopt®. The IOP (intra ocular pressure) was measured till 300 min of treatment at specific time intervals.
Results: - Single instillation of both the formulations of brinzolamide suspension resulted in a decrease in IOP at all time points of observation. Effect of test formulations, i.e. 0.5% and 0.75% , were significantly more at 90 and 120 min post-instillation as compared to the marketed brand product, Azopt®. The onset of action for IOP reduction in both the test formulations is faster than Azopt®. Upto 180 minutes, the 0.5%) nanosuspesnion showed superior efficacy than Azopt®. However, after 180 minutes the IOP reduction of the 0.5%o formulation is lower than Azopt®.
The 0.75%) nanosuspension of brinzolamide showed superior efiifcacy than Azopt® at all time points.
In comparison to Azopt, the 0.75%o nanosuspension showed faster onset of action and better efficacy than the 0.5%) nanosuspension.

Stability Data:
Two strengths of brinzolamide ophthalmic nanosuspension viz:, 0.5% w/v and 0.75% w/v were subjected to stability conditions as per the ICH guidelines at 40 °C/25% RH, 25 °C/40% RH and 2-8 °C. The stability data is tabulated below in Table 4, 5, 6 & 7.
Table 4: Stability data of brinzolamide ophthalmic nanosuspension 0.5% w/v at 40°C/25%RH.

Tests Specifications Brinzolamide nanosuspension 0.5%) w/v

Initial 40°C/25%RH

1M 2M 3M 6M
Appearance A white to off- Compli Does not comply. The colour has
white es changed from off-white to slightly
suspension reddish brown suspension.
% Assay of 90.0-110.0 101.79 100.89 95.53 95.18 96.96
brinzolamide
% Assay of 50.0-110.0 100.38 90.9 83.4 76.8 55.6
potassium sorbate
pH 5.3-6.3 5.81 5.85 5.9 6.1 6.4
Viscosity in cps 280-360 cps 320 322 318 319 316
(60 rpm; S00
spindle)
Osmolality 260-300 282 285 285 287 290
(mOsmols/Kg)
Related compounds of brinzolamide
Brinzolamide MMT 1.5% ND ND ND ND ND
related compound
A (%)
Single maximum NMT 0.5% 0.074 0.15 0.24 0.48 1.3
unknown impurity
(%)
Total Impurities NMT 2.0% 0.13 0.40 0.68 1.3 2.6

Table 5: Stability data of brinzolamide ophthalmic nanosuspension 0.5% w/v at 25°C/40% RH and 2 to 8 °C.

Tests Specifications Brinzolamide nanosuspension 0.5% w/v

Initial 25°C/45%RII 2-8 °C

6M 12M 6M 12M
Appearance A white to off- Compli Does not comply. Compli Compli
white suspension es The colour has changed to beige. es es
% Assay of 90.0-110.0 101.79 98.92 99.10 100.18 101.07
brinzolamide
% Assay of 50.0-110.0 100.38 72.6 66.1 99.6 91.1
potassium sorbate
pH 5.3-6.3 5.81 6.0 6.28 5.83 5.89
Viscosity in cps 280-360 cps 320 320 322 320 321
(60 rpm; S00
spindle)
Osmolality 260-300 282 283 285 282 285
(mOsmols/Kg)
Related compounds of brinzolamid e
Brinzolamide MMT1.5% ND ND ND ND ND
related compound
A (%)
Single maximum NMT 0.5% 0.074 0.16 0.16 0.07 0.13
unknown
impurity (%)
Total Impurities NMT 2.0% 0.13 0.25 0.67 0.13 0.25

Table 6: Stability data of brinzolamide ophthalmic nanosuspcnsion 0.75% w/v at 40°C/25%RH.

Tests Specifications Brinzolamide nanosuspension 0.75% w/v

Initial 40°C725%RH

1M 2M 3M 6M
Appearance A white to off- Complies Docs not comply. The colour has
white changed from off-white to slightly
suspension reddish brown suspension.
% Assay of 90.0-110.0 100.66 100.66 96.24 95.18 96.96
brinzolamide
% Assay of 50.0-110.0 100.56 91.9 84.4 76.8 55.6
potassium sorbate
pH 5.3-6.3 5.80 5.86 5.9 6.1 6.4
Viscosity in cps 280-360 cps 320 325 325 320 318
(60 rpm; S00
spindle)
Osmolality 260-300 280 280 281 282 285
(mOsmols/Kg)
Related compounds of brinzolamide
Brinzolamide MMT1.5% ND ND ND ND ND
related compound
A (%)
Single maximum NMT 0.5% 0.074 0.15 0.24 0.48 0.97
unknown
impurity (%)
Total Impurities NMT 2.0% 0.13 0.40 0.68 1.3 1.9

Table 7: Stability data of brinzolamide ophthalmic nanosuspension 0.5% w/v at 25°C/40% RH and 2 to 8 °C.

Tests Specifications Brinzolamide nanosuspension 0.5% w/v

Initial 25°C/40%RH 2-8 °C

6M 12M 6M 12M
Appearance A white to off- Comp Does not comply. Compli Compli
white suspension lies The colour has changed to beige. es es
% Assay of 90.0-110.0 100.6 98.08 97.71 99.9 99.65
brinzolamide 6
% Assay of 50.0-110.0 100.5 64.8 58.8 92.6 87.4
potassium sorbate 6
pH 5.3-6.3 5.80 6.1 6.35 5.84 5.93
Viscosity in cps 280-360 cps 320 330 337 320 321
(60 rpm; S00
spindle)
Osmolality 260-300 280 280 280 282 283
(mOsmols/Kg)
Related compounds of brinzolamid e
Brinzolamide MMT1.5% ND ND ND ND ND
related compound
A (%)
Single maximum NMT 0.5% 0.074 0.14 0.15 0.07 0.14
unknown
impurity (%)
Total Impurities NMT 2.0% 0.13 0.22 0.58 0.18 0.25

Conclusion:
1. Both the strengths of brinzolamide nanosuspension, namely, 0.5% w/v and
0.75% w/v exhibited similar stability behaviour at accelerated (40°C/25 % RH),
long-term (25°C/40 % RH), and at 2 - 8 °C. At accelerated conditions, the colour
of the suspension changed significantly from the initial appearance of 'white to
off-white suspension' to 'slightly reddish-brown suspension'. At long-term
conditions, the colour of the suspension turned 'beige' and is lighter than that at
accelerated conditions. At 2 - 8 °C, there is no change in appearance from the
initial samples.
2. At 6 months accelerated conditions of 40°C/25 % RH, both the batches showed a decrease in the content of brinzolamide. The drop in the assay of brinzolamide was around 4.8% for the 0.5% brinzolamide batch and around 7% for the 0.75% brinzolamide batch. At 6 months and 12 months long-term conditions of 25°C/40 % RH, the decrease in the assay of brinzolamide was not very significant ( around 2-3 %). At 6 and 12 months at 2 - 8 °C, there was no significant decrease in the assay of brinzolamide in both the batches.
3. At 6 months accelerated conditions of 40°C/25 % RH, both the batches showed a significant increase in the content of unknown impurities. However, the content of unknown impurities was well within the specifications at 25°C/40 % RH, and 2 - 8 °C, for 6 and 12 months stability time points. The content of total impurities at 2 - 8 °C was much lower at 12 months as compared to the 25°C/40 % RH samples.
4. At 6 months accelerated conditions of 40°C/25 % RH, both the batches showed a decrease in the content of potassium sorbate. The drop in the assay of potassium sorbate was around 45% for the 0.5% brinzolamide batch and around 48% for the 0.75% brinzolamide batch. At 6 months and 12 months long-term conditions of 25°C/40 % RH also, the decrease in the assay of potassium sorbate was very significant ( around 34 - 42 %). At 6 and 12 months at 2 - 8 °C, the decrease in the assay of potassium sorbate was around 9 -13% in both the batches. Since the content of potassium sorbate decreased across all stability conditions, it was

decided to perform a preservative effectiveness test for the product at a level of 50% content of potassium sorbate, to assess its functionality.
5. The pH values in both the batches increased significantly at accelerated
conditions and long-term conditions, but not so significantly at 2 - 8 °C.
6. There was no significant change in the viscosity and osmolality values across
all the stability conditions for both the batches.
Scanning Electron Microscopy (SEM) Studies:
The samples of brinzolamide nanosuspension 0.5% w/v (Batch No. BZO-14-2018) from 6 months stability samples at all three stability conditions, namely, 40°C/25 % RH, 25°C/40 % RH, and 2 - 8 °C were observed under an SEM at a magnification of 10000X.
The testing methodology was as follows:
The samples were tested by placing a small quantity of the sample on electrically conductive carbon tapes and air-dried for 30 mins. The sample holder with the carbon-taped sample was placed in a gold sputtering chamber, which coats the surface of the samples with a thin gold film that imparts conductivity to the surface of the sample. A maximum of 10 measurements per image as permitted by the instrument software was done for some images to assess the particle size. Results:
1. At all conditions of stability at 6 months, i.e., 2 - 8 °C, 40°C/25 % RH and 25°C/40 % RH, the nano particles are seen embedded in the xanthan gum matrix. No evidence of crystal growth was seen in any of the samples. At all conditions, the nano particles were in the stabilized state.
2. The Azopt® samples show a fibrous background, which is typical of the carbomer polymer with the brinzolamide particles which are quite separated from the polymer fibres and significantly larger in size than the developed nano formulation of brinzolamide.
Rheological Characterization of Brinzolamide Ophthalmic Nanosuspension: Rheological characterization of brinzolamide nanosuspension was carried out to determine following properties:

i) Viscosity of the Product ii) Flow Curve iii) Yield Stress
Viscosity of Brinzolamide Nanosuspension
Viscosity of brinzolamide suspension was determined for both the strengths of the formulation, namely, 0.5% w/v and 0.75% w/v, using a rotational viscometer (Make: Brookfield Viscometer LVT), using an S 00 spindle suitable to measure the viscosity of viscous solutions. The viscosity was found to be between 300-400 cps. Flow Curve
Rheology plays a very important role in determining the stability of the nano suspension and also in determining ocular residence time. At very low shear rates, the viscosity of a shear-thinning polymer-thickened eye-drop formulation plateaus at a maximum value. High zero-shear viscosity has been proposed as conducive to longer ocular surface residence times.
Flow curve was determined for brinzolamide nanosuspension 0.5% w/v, and compared with that of Azopt® using a rheometer (Make: Anton Paar; Model: MCR 302). The summary of data of rheological shear measurements is given in Table 8
Table 8: Comparative data for shear rate Vs viscosity for brinzolamide nanosuspension 0.5% w/v and Azopt®

Shear Rate
[l/s] Viscosity (mPa.s)

Azopt® (Lot 25269F) Brinzolamide nano suspension 0.5% w/v (Batch no. BZO-14-2018)
0.01 26295.3 50706.7
0.1 4981.6 14499.2
1 1042 2632.2
10 234 415
100 72.7 73.1
1000 28.2 20.2

Yield Stress
Yield stress quantifies the amount of stress that the fluid may experience before it yields and begins to flow. The yield stress was obtained from the shear stress Vs shear rate plot. Shear stress Vs shear rate curve was fitted using Herschel-Bulkley model21 to obtain yield stress. The summary of data for shear stress and shear rate for brinzolamide nanosuspension 0.5% w/v, and Azopt® is given in Table 9, Table 9: Comparative data of shear rate Vs shear stress for brinzolamide nanosuspension 0.5% w/v and Azopt®

Shear Rate
[1/s] Shear stress (Pa)

Azopt® (Lot 25269F) Brinzolamide nanosuspension
0.5% w/v
(Batch no. BZO-14-2018)
0.01 0.26 0.51
0.1 0.49825 1.4501
1 1.0419 2.6322
10 2.3402 4.1501
100 7.9547 7.3058
1000 28.156 20.192
Results of Rhcological Evaluation:
i) The viscosity of both strengths of brinzolamide ophthalmic nano
suspension, viz., 0.5% w/v and 0.75% w/v was around 320 cps.
ii) Comparative results of viscosity Vs shear rate for brinzolamide ophthalmic nano suspension 0.5%) w/v and brand product, Azopt® showed that both the products exhibited shear thinning behaviour.
iii) The initial viscosity of the test product was higher than that of Azopt® at a lower shear rate, whereas at the higher shear rate it was reverse. This result is desirable as initial higher viscosity would result in longer ocular residence time.
iv) The yield stress of Azopt was 0.95, and that of the test sample was 2.4 Pa, which is significantly higher than that of Azopt®, indicating that the test product is expected to exhibit greater mucoadhesion on the ocular surface

and hence, it is expected to give faster onset of action as compared to Azopt couplet with nano size.
Results of AET for brinzolamide ophthalmic nanosuspension at 100% and 50% of the labelled amount of potassium sorbate
Table 10: Results of AET at 100% and 50% label claim of potassium sorbate for brinzolamide nanosuspension 0.75% w/v

Name of the microorganism culture Log10 viable microorganism counts per raL at each incubation period Log I reduction

0 hour 7d 14 d 28 d 7d 14 d 28 d
For 100% of potassium sorbate (0.47% w/v)
Bacteria LIMIT s NLT 1.0 NLT
3.0 NI

l k5

Escherichia coli ATCC 8739 5.79 51 2.0413 0.0000 0.0000 3.8 5.8 NI
Pseudomonas aeruginosa ATCC 9027 5.82
73 2.0000 0.0000 0.0000 3.8 5.8 NI
Staphylococcus aureus ATCC 6538 5.79 51 2.2490 0.0000 0.0000 3.5 5.8 NI
Yeasts and Molds LIMITS — -> NI NI NI
Candida albicans ATCC 10231 5.81 15 2.2174 1.4771 0.0000 NI NI NI
Aspergillus brasiliensis ATCC 16404 5.79 93 2.2552 1.3010 0.0000 NI NI NI
For 50% of potassium so rbate (0.235% w/v)
Bacteria
Escherichia coli ATCC 8739 5.79
51 2.1000 0.0000 0.0000 3.7 5.7 NI
Pseudomonas aeruginosa ATCC 9027 5.82
73 2.2211 0.0000 0.0000 3.6 5.8 NI
Staphylococcus aureus ATCC 6538 5.79 51 2.2981 0.0000 0.0000 3.5 5.7 NI
Yeasts and Molds
Candida albicans ATCC 10231 5.81
15 2.0000 1.0100 0.0000 NI NI NI
Aspergillus brasiliensis ATCC 16404 5.79 93 2.2174 1.6020 0.0000 NI NI NI
NI - No increase from initial count ('0' hour); NLT - Not less than

Conclusion:
At 50% and 100% of the labelled amount of the preservative, potassium sorbate, the formulation of brinzolamide nanosuspension 0.75% w/v, passed the acceptance criteria as per the USP specifications. This proved that potassium sorbate would be effective in withstanding microbial contamination during use of this nanosuspension, even if its content reduces to 50% (0.235% w/v of potassium sorbate) of the label claim during its shelf life.

We Claim:
1. A stable ophthalmic nanosuspension comprising brinzolamide and
pharmaceuticallyacceptable excipients.
2. The stable ophthalmic nanosuspension as claimed in claim 1, wherein brinzolamide is used in the range of 0.25% to 1%.
3. The stable ophthalmic nanosuspension as claimed in claim 1, wherein the pharmaceuticallyacceptable excipients are selected from the group comprising of
surfactants, stabilizers, preservatives, viscosity enhancing agents, tonicity agents and combinations thereof.
4. The stable ophthalmic nanosuspension as claimed in claim 3, wherein the preservative is selected from the group comprising of hydrogen peroxide, biguanides, sorbic acid, potassium sorbate, boric acid, borate, chlorohexidine, zinc chloride, propylene glycol, purite, polyquad and combinations thereof.
5. The stable ophthalmic nanosuspension as claimed in claim 3, wherein the surfactant is selected from the group comprising of propylene glycol, macrogol 15 hydroxystearate, polyethylene glycol, polypropylene glycol, polysorbate, tyloxapol, povidone, polyvinyl alcohol, hypromellose, polaxamer, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl-15- hydroxystearate, glycerine and combinations thereof.
6. The stable ophthalmic nanosuspension as claimed in claim 3, wherein the stabiliser is selected from the group comprising of sodium carboxy methyl cellulose, (sodium CMC), hydoxy propyl methyl cellulose (HPMC), polycarbophil, polaxamer, hydroxyl propyl cellulose and combinations thereof.
7. The stable ophthalmic nanosuspension as claimed in claim 3, wherein the viscosity enhancing agent is selected from the group comprising of hydroxy ethyl cellulose, carrageenan, polycarbophil, polyvinyl alcohol, povidone, sodium alginate, locust bean gum, pullulan, sodium carboxy methyl cellulose, gellan gum, methyl cellulose, guar gum, xanthan gum and combinations thereof.

8. The stable ophthalmic nanosuspension as claimed in claim 3, wherein the tonicity agent is selected from the group comprising of sodium chloride, potassium chloride, calcium chloride, sodium bromide, mannitol, glycerol, sorbitol, propylene glycol, dextrose sucrose, and combinations thereof.
9. The stable ophthalmic nanosuspension as claimed in claim 1, wherein the said nanosuspension is free of quaternary ammonium salt.
10. The method for the preparation of a stable ophthalmic nanosuspension as
claimed in claim 1, comprising:
a) Sterilization of brinzolamide by ethylene oxide sterilization,
b) Preparing a solution comprising of tyloxapol and sodium carboxymethyl cellulose in water.
c) Mixing of step (a) brinzolamide and step (b) solution.
d) Addition of step (c) mixture into suitable homogenizer or mill to reduce average particle size in the range of 100-400 nm.
e) Preparing a solution comprising of xanthan gum suitable for ophthalmic use.
f) Preparing a solution comprising of potassium sorbate and sodium chloride
g) Mixing a solution of step (e) and step (f) to form a slurry.
h) Aseptically mixing mixture of step (d) and slurry of step (g) until homogeneity
is obtained. i) Measuring and adjusting the volume using water for injection.