DESC:

FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention A NOVEL IN VITRO DRUG RELEASE METHOD OF DEXAMETHASONE IN OPHTHALMIC SUSPENSION
2. Applicant(s)
NAME: ACULIFE HEALTHCARE PRIVATE LIMITED
NATIONALITY: INDIAN
ADDRESS: ACULIFE HEALTHCARE PRIVATE LIMITED, Village: Sachana, Taluka: Viramgam, District: Ahmedabad – 382150, Gujarat, India.
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed.

A NOVEL IN VITRO DRUG RELEASE METHOD OF DEXAMETHASONE IN OPHTHALMIC SUSPENSION

FIELD OF THE INVENTION
The present invention is related to a novel in vitro drug release method of dexamethasone in ophthalmic suspension. The present invention is related to a novel in vitro drug release method of dexamethasone in ophthalmic suspension which is comprising a dissolution medium consisting of an artificial tear solution of pH 7.4 and a surfactant using USP apparatus II paddle at 100 RPM, with 900 mL of medium volume (37 ± 0.5°C). The present invention is in particular related to a novel in vitro drug release method of dexamethasone in ophthalmic suspension for which uses micro-dialysis technique.

BACKGROUND OF THE INVENTION
Ophthalmic formulations (eye preparations) are sterile, liquid, semi-solid, or solid preparations that may contain one or more active pharmaceutical ingredient(s) intended for application to the conjunctiva, the conjunctival sac or the eyelids. One of the major challenges for topical ocular drug delivery is the limited precorneal retention time of active pharmaceutical ingredients (APIs), which results in low bioavailability. The impermeability of human corneas as well as the biological barriers of the other parts of the human eye limits drug absorption. Following eye drop instillation, up to 95% of the drug contained in the drops is lost as a result of tearing and nonproductive absorption. Tremendous effort has been made to improve ocular drug bioavailability using different mechanisms such as in situ gelling, contact lenses, viscosity enhancers, and mucoadhesive.

The in vitro release study is a critical test to assess the safety, efficacy, and quality of the formulation. In the pharmaceutical industry, dissolution testing is an important tool in both drug development and quality control. The release profile of the active ingredient from the formulation allows for optimization of the physical characteristics of the formulation during product development. A well-established IVRT method can also provide support during the stages of clinical development by establishing sameness as minor formulation changes are implemented during the course of clinical assessment. Besides aiding in product development, the IVRT can serve as a quality control tool to confirm batch-to-batch uniformity of the product. It helps in comparing the in vitro release profiles of test and reference products, though it is not expected to correlate or be predictive of in vivo bioavailability or bioequivalence. With the establishment of in vitro in vivo correlation for the developed method, the prediction of in vivo bioequivalence could also be possible.

Release testing of ophthalmic formulation is an effective approach to monitor post-approval changes, scale-up, lot-to-lot changes and stability studies in the pharmaceutical industry. Traditional methods and apparatus for the measurement of drug release profiles are designed to simulate the gastrointestinal tract and are inappropriate for ophthalmic dosage forms. It is always challenging to develop a reliable in vitro release testing method which determines whether difference in drug release rate exists between qualitatively and quantitatively equivalent generic ophthalmic formulations. There is a lack of regulatory guidance (US, Europe etc.) on in vitro release testing methods for ophthalmic formulations. Different in vitro release testing methods being developed and used which commonly uses USP apparatus IV with semisolid adapters, Franz diffusion cells etc.

Microdialysis is currently being explored as one of the alternative techniques for IVRT evaluation of dosage forms. Microdialysis has contributed with very important knowledge to the understanding of target- specific concentrations and their relationship to pharmacodynamic effects from a systems pharmacology perspective, aiding in the global understanding of drug effects. Therefore, an important role of microdialysis in systems pharmacology is to measure and identify target site concentrations that may differ from plasma concentrations due to distributional consequences of transporters and other processes and in relating these concentrations to pharmacodynamic measurements. The microdialysis probe is designed to mimic a blood capillary and consists of a shaft with a semipermeable hollow fiber membrane at its tip, which is connected to inlet and outlet tubing.

Microdialysis can offer significant advantages compared to other IVRT methods. For example, since microdialysis probes are very small, they can be placed directly into small “receivers” for in-vitro systems. Also, it enables quick and frequent sampling during a shorter duration of time.

Hence, there is a need to develop in vitro release testing method for ophthalmic suspensions which must have dissolution conditions capable of distinguishing even minor changes in the formulation or production process required for product development. It should measure the number of active substances released during the dissolution test in an accurate, robust, selective and repeatable manner.

In vitro drug release testing has been usually carried out with one or more of the following aims: (i) to predict the drug availability in preliminary stages of product development, (ii) to meet batch specifications, (iii) to assess formulation factors and manufacturing methods of dosage form, (iv) to support labeling claims of the product, and (v) to meet a compendial standards and regulatory requirement. In addition, in vitro drug release testing plays an important role in providing insight into the product’s in vivo performance.

Tobramycin-Dexamethasone Suspension Drops is a medication which is used to treat or prevent eye infections. This medication contains two drugs. Tobramycin belongs to a class of drugs called aminoglycoside antibiotics. It works by stopping the growth of bacteria. Dexamethasone belongs to a class of drugs called corticosteroids. It works by reducing swelling. Currently there is no any method available for in vitro release of dexamethasone in tobramycin and dexamethasone ophthalmic suspension using USP apparatus 2.

Hence, the inventors of the present invention developed a novel in vitro drug release method of dexamethasone in ophthalmic suspension which uses micro-dialysis technique in USP apparatus 2.

The objective of the present invention is as described herein.

OBJECTIVE OF THE INVENTION

The main object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension.

Another object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which uses microdialysis technique in USP apparatus II.

Another object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which is simple and easy to conduct.

Another object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which is precise and accurate.

Yet another object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which helps to fasten drug development process.

Yet another object of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension that can be used to reduce the development time and cost by preventing a lengthy and expensive clinical trial.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension with micro-dialysis technique.

The other aspect of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which is comprising a dissolution medium consisting of an artificial tear solution of pH 7.4 and a surfactant using USP apparatus II paddle at 100 RPM, with 900 mL of medium volume (37 ± 0.5°C).

The one other aspect of the present the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension and the process of preforming the same.

DESCRIPTION OF THE INVENTION
Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

As used herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

The term “about” is used herein to means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” or “approximately” is used herein to modify a numerical value above and below the stated value by a variance of 20%.

As used herein, whether in a transitional phase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components, but may also include additional features or components.

The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

The term “in-vitro” used herein refers to an artificial environment and to processes or reactions that occur within an artificial environment.

The term “dissolution apparatus” used herein refers to a device which may be used to determine dissolution characteristics of a drug product.

The term “dissolution” used herein refers to a test used to evaluate the rate of release of a drug substance from the dosage form. Dissolution is the process by which active ingredient enters into a solvent to yield a solution.

The term “Microdialysis” used herein refer to a technique that measures extracellular concentrations by sampling soluble molecules from the interstitial space of body tissue (so-called extra-cellular fluid, ECF) through a semipermeable membrane.

The term “microdialysis membrane” refers to a membrane which mimics a blood capillary which continuously perfused with a sample solution that closely resembles the (ionic) composition of the surrounding tissue.

The term “sinker” used herein refer to an element which is like glass beads and it is typically used to prevent floating of the dosage form in the media thereby facilitate sinking of dosage forms.

The term “High-performance liquid chromatography” (HPLC) used herein refer to a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.

The term “High-performance liquid chromatography” and HPLC can be interchangeably used herein.

The main embodiment of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension.

As per another main embodiment of the present invention, a novel in vitro drug release method of dexamethasone in ophthalmic suspension is USP apparatus type II paddle with Micro-dialysis technique.

Another main embodiment of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension comprising steps:
a) Activating dialysis tubing cellulose membrane by soaking in purified water for 30 minutes then rinsing thoroughly in purified water, and soaking in dissolution medium for 30 min before use;
b) Tying one side of activated membrane of step (a) with thread;
c) Perfusing sample solution in the membrane of step (b) and tying other side of membrane with thread;
d) Tying the membrane of step (c) from both side with sinker to prevent floating of membrane in dissolution vessel of apparatus;
e) Filing the dissolution vessel of step (e) with warm sonicated and degassed dissolution medium
f) Placing activated and perfused membrane of step (d) in dissolution vessel of apparatus;
g) Adjusting temperature of dissolution medium of step (f) at 37ºc and stirring of dissolution medium at 100 rpm;
h) Allowing diffusion of sample from the membrane to dissolution medium till the completion of dissolution testing;
i) Collecting samples from dissolution media of step (h) at intervals of 30 min, 1 hour, 3 hour, 6 hour, 12 hour;
j) Replenishing fresh dissolution media of equal quantity of sample withdrawn in step (i) in the vessel;
k) Analyzing collected samples by high performance liquid chromatography (HPLC) method to determine the % amount drug release;
Characterized by said in vitro release method is micro-dialysis technique in USP apparatus type 2 paddle.

The Dialysis tubing cellulose membrane used was procured from Sigma-Aldrich.

As per one more embodiment of the present invention, said dialysis tubing cellulose membrane of 10 cm length and 33 mm width.

Another main embodiment of the present invention is to provide a novel in vitro drug release method of dexamethasone in ophthalmic suspension which is comprising a dissolution medium consisting of an artificial tear solution of pH 7.4 and a surfactant using USP apparatus II paddle at 100 RPM, with 900 mL of medium volume (37 ± 0.5°C).

As per one embodiment of the present invention, said collected samples subjected to a HPLC column using a mobile phase to quantify dexamethasone at a flow rate of 1.5 mL/minute, wherein said mobile phase which comprises a mixture of acetonitrile and water in ratio of 35:65 %V/V, wherein, the quantification of the dexamethasone achieved within retention time of 3.8 Minute.

As per one more embodiment of the present invention, said HPLC column is a C18 column.

As per one more embodiment of the present invention, said chromatography carried out at temperature 25ºc.

As per one more embodiment of the present invention, said collected sample quantified at a wavelength of 254 nm.

As per one more embodiment of the present invention, said dissolution medium is artificial tear solution comprising sodium chloride, sodium bicarbonate and calcium chloride dihydrate, and surfactant.

As per one other embodiment the surfactant as mentioned in the present is defined as a substance which tends to reduce the surface tension of a liquid in which it is dissolved. They are mainly substance such as a detergent that, when added to a liquid, reduces its surface tension, thereby increasing its spreading and wetting properties. The surface-active molecule must be partly hydrophilic (water-soluble) and partly lipophilic (soluble in lipids, or oils). It concentrates at the interfaces between bodies or droplets of water and those of oil, or lipids, to act as an emulsifying agent, or foaming agent.

As per one preferred embodiment the non-limiting examples of the surfactant used in dissolution media can selected from sodium dodecyl sulfate, Sodium lauryl sulfate, N-dimethyldodecylamine oxide and sodium decyl-, sodium dodecyl-, sodium tetra-decylsulfate, Dioctyl sodium sulfosuccinate, Perfluorooctanesulfonate, Lignosulfonates, Sodium stearate. Most preferably the surfactant is sodium lauryl sulfate.

According to one embodiment the surfactant is present in range from 1-15 gm, preferably 1-10 gm and most preferably 1-7 gm.

As per one embodiment of the present invention, said dissolution medium is artificial tear solution comprising 1-10 gm of sodium chloride, 1-10 gm of sodium bicarbonate and 20-100 mg of calcium chloride dihydrate, and 1-15 gm of surfactant.

As per one embodiment of the present invention, an artificial tear solution of dissolution media comprises 5-8 gm of sodium chloride, 1-5 gm of sodium bicarbonate and 50-100 mg of calcium chloride dihydrate, and 1-10 gm of surfactant.

As per preferred embodiment of the present invention, said dissolution medium is artificial tear solution comprising 6.7 gm of sodium chloride, 2.0 gm of sodium bicarbonate and 80 mg of calcium chloride dihydrate, and 5.0 gm of Sodium lauryl sulphate (SLS).

According to one embodiment of the present invention, the pH of the dissolution media is 7.0 to 8.0, preferably the pH 7.0 to 7.8, more preferably the pH is 7.0 to 7.5.

As per preferred embodiment of the present invention, the pH of the dissolution media maintained 7.4 with dilute hydrochloric acid.

As per one embodiment of the present invention, an in-vitro release method of Dexamethasone uses tobramycin and dexamethasone ophthalmic suspension formulation in the release study.

As per one embodiment of the present invention, tobramycin and dexamethasone ophthalmic suspension formulation comprises tobramycin and dexamethasone along with excipients like Benzalkonium chloride, Tyloxapol, Disodium edetate, Sodium chloride, Hydroxyethylcellulose (Natrasol HX 250), Sodium sulfate anhydrous and water for injection.

As per one more embodiment of the present invention, a novel in vitro drug release method of dexamethasone in ophthalmic suspension validated by evaluating parameters like specificity, repeatability, intermediate precision, and accuracy.

Stability indicating evaluation: Stability of the final formulation was demonstrated by storing of formulation under different storage conditions, 40°C/25%RH and 25°C/40%RH for 3 month and 6 months.

The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLES

Example 1: Trial Batches of tobramycin and dexamethasone ophthalmic suspension
Ingredients Batch 1 Batch 2 Batch 3
Dexamethasone 1.00 1.00 1.00
Tobramycin 3.00 3.00 3.00
Benzalkonium Chloride 0.10 0.10 0.10
Tyloxapol 0.50 0.60 0.60
Edetate Disodium 0.10 0.10 0.10
Sodium Chloride 3.00 3.00 3.00
Hydroxyethyl Cellulose (Natrosol HX) 3.50 2.50 3.50
Sodium Sulfate Anhydrous 12.00 12.0 12.00
Water for Injection q.s to ml q.s to ml q.s to ml
Table 1: Trial Batches of tobramycin and dexamethasone ophthalmic suspension

Process of preparation of tobramycin and dexamethasone ophthalmic suspension:
A) Polymer Phase Preparation:
1) 40% Water for Injections (WFI) of the batch size was taken in the mixing vessel.
2) The WFI of step 1 was cooled up to temperature 60°C to 70°C by circulating cooling water in the jacket of the mixing vessel.
3) The required quantity of Hydroxyethyl Cellulose (Natrosol HX) was gradually added in mixture of step 2 in mixing vessel with constant stirring under vortex. The solution was stirred for minimum 30 minutes or till the complete dissolution of added quantity.
4) The mixture of step 4 was autoclaved at 121°C for 15 min.
5) The bulk solution step- 4 was cooled up to 25°C to 30°C by circulating cooling water in the jacket of mixing vessel.

B) API Slurry Preparation:
1) 4% Water for Injections (WFI) of the batch size was taken in the Slurry vessel.
2) The required quantity of Tyloxapol was added gradually in slurry vessel with constant stirring and Nitrogen flushing. The solution was stirred for minimum 10 minutes or till the complete dissolution of added quantity.
3) The required quantity of Dexamethasone was added gradually in the mixture of step 2 in the slurry vessel with constant stirring and Nitrogen flushing. The solution was stirred for minimum 15 minutes or till the complete dissolution of added quantity.
4) The mixture of Step -3 was autoclaved at 121°C for 15 min.
5) The bulk solution Step- 4 was cooled up to 25°C to 30°C by circulating cooling water in the jacket of mixing vessel.

C) Aqueous Phase Preparation:
1) 30% Water for Injections (WFI) of the batch size was taken in the mixing vessel.
2) The required quantity of Benzalkonium Chloride was added gradually in mixing vessel of step 1 with constant stirring and Nitrogen flushing. The solution was stirred for minimum 15 minutes or till the complete dissolution of added quantity.
3) The required quantity of Disodium Edetate was added gradually in mixing vessel of step 2 with constant stirring and Nitrogen flushing. The solution was stirred for minimum 5 minutes or till the complete dissolution of added quantity.
4) The required quantity of Sodium Chloride was added gradually in mixing vessel of step 3 with constant stirring and Nitrogen flushing. The solution was stirred for minimum 5 minutes or till the complete dissolution of added quantity.
5) Add gradually required quantity of Sodium Sulphate in mixing vessel of step 4 with constant stirring and Nitrogen flushing. The solution was stirred for minimum 5 minutes or till the complete dissolution of added quantity.
6) The required quantity of Tobramycin in mixing vessel of step 5 with constant stirring and Nitrogen flushing. Stir for minimum 20 minutes or till the added quantity dissolve completely.
7) The pH of bulk solution of step 6 was checked and adjusted with H2SO4 solution or NaOH solution.

D) Mixing Phase Preparation:
1) API slurry was added to Aqueous Phase with continuous stirring for 10 min.
2) The solution of step 1 was homogenized with IKA Ultra Turax disperser.
3) The solution of step 2 was passed through Panda Plus homogenizer.
4) Polymer phase added to solution of step 3 with continuous stirring.
5) The final volume of mixture of step 4 was adjusted with WFI.
6) The final formulation was filled in 5ml LDPE Opaque Bottle.

Example 2: Particulars for In-vitro release study
2.1: Dissolution media preparation (Artificial tear pH: 7.40 +0.5% W/V SLS):
About 6.7 gm of sodium chloride, 2.0 gm of sodium bicarbonate and 80 mg of calcium chloride dihydrate, and 5.0 gm of Sodium lauryl sulphate (SLS) was dissolved into 1000 mL water. The pH was adjusted to 7.40 with dilute hydrochloric acid and mixed well, sonicated to degassed.

2.2: Membrane Details
Name Dialysis tubing cellulose membrane
Make Sigma-Aldrich
Width 33 mm
Batch No. D9652-100FT
Lot 3110
Table 2: Membrane details
2.3: Membrane preparation:
1) The membrane was soaked in purified water for 30 minutes at room temperature to remove the preservative agent (sodium azide);
2) Then, it was rinsed thoroughly in purified water, and soaked in dissolution medium for 30 minutes before use.

2.4: Sample preparation:
1) About 1 mL sample was taken with 2 mL disposable syringe;
2) The syringe was weighed with sample. (Initial weight);
3) The sample suspension was added to a well-rinse section of dialysis membrane.
4) The tubing was sealed at both ends and tested for the leakage prior to immersion in dissolution vessel containing dissolution media and transferred carefully in the dissolution vessel and ensured that the membrane is not float on the dissolution media.
5) The syringe was weighed again. (Final weight);
6) The sample weight was calculated by subtracting the final weight to initial weight.

Example 3: HPLC analytical method for determination of Dexamethasone
3.1: Mobile phase preparation
A mixture of acetonitrile and water in ratio of 35:65 %V/V was prepared, mixed and degassed.

3.2: Standard solution set 01
About 50.0 mg of dexamethasone micronized standard was weighed and transferred into 50 mL volumetric flask. Dissolved and diluted to volume with methanol and mixed. Further 5.0 mL of this solution was transferred into 50 mL volumetric flask. Diluted to volume with dissolution media.

3.3: Standard solution set 02
About 52.0 mg of dexamethasone micronized standard was weighed and transferred into 50 mL volumetric flask. Dissolved and diluted to volume with methanol and mixed. Further 5.0 mL of this solution was transferred into 50 mL volumetric flask. Diluted to volume with dissolution media.

3.4: Sample solution
Sample solution was used from in-vitro release study.

3.5: Chromatographic parameter
Column: Zorbax Eclipse C18 (150 mm x 4.6 mm) 5µm
Make: Agilent
Column oven temperature: 25°C
Sample temperature: 25°C
Wave length: 254 nm
Flow rate: 1.5 mL/minute
Injection volume: 100 µL
Retention time: About 3.8 Minute
Run time: 6 minutes
Rinsing solution: Mobile phase

3.6: HPLC Procedure
Diluent, Standard solution and Sample Solution was injected separately at each time interval and the Chromatograms were recorded. The sequence was prepared as per below mention table.

Sr. No. Sample No. of Injection
1 Diluent 1
2 Standard solution 6
3 Sample solution 1
4 Standard solution (Bracketing) 2
Table 3: HPLC Sample sequence details
3.7: System suitability criteria
Relative standard deviation for Dexamethasone peak area obtained from six replicate injections of standard solution should not be more than 2.0%.
Tailing of Dexamethasone is not more than 2.0.

Example 4: Dissolution Study
4.1: Dissolution testing:
The dissolution/in-vitro release study was conducted using 900 mL of dissolution media maintained at 37°C, using the paddle apparatus and stirred at 100 rpm. At predetermine time point a 10 mL of sample was withdrawn and replenished with fresh media. The collected samples were analyzed on the HPLC instrument by a method mentioned under example 3.

4.2: Dissolution parameters
RPM: 100
Time point: 30 min, 1 hour, 3 hour, 6 hour, 12 hour
Media volume: 900 mL
Replenish volume: 10 mL
Sample volume: 10 mL
Temperature: 37°C

Result of In-vitro release study:

Time Points Batch 1 (%) Batch 2 (%) Batch 3 (%)
30 Minute 16.14 19.22 13.36
1 Hour 25.27 34.63 25.21
3 Hours 59.38 72.83 6.030
6 Hours 72.33 92.08 85.58
12 Hours 72.66 97.36 97.01
Table 4: Result of In-vitro release study
Based on the in-vitro release study Batch 2 is optimized batch which is reproduced and further used for method validation and stability study.
Example 5: Evaluation of optimized formulation batch
The optimized formulation was evaluated for particle size and viscosity of the final formulation.

Results:
Parameter Reading
Particle Size Distribution
D (0.1) 1.31 µm
D (0.5) 2.44 µm
D (0.9) 4.63 µm
Viscosity 50rpm-3.92 cps
Table 5: Results of evaluation study

Example 6: Results of method validation:
Parameters Observation Conclusion
Specificity No interference is observed at the retention time of Dexamethasone peak from diluent and Placebo. Method is specific for dexamethasone.
Precision Batch 2 Batch 2 Method is precise and accurate for the estimation of Dexamethasone release in Tobramycin and Dexamethasone eye drops, suspension.
Precision Intermediate
35.50
51.21
80.33
88.91
91.62 30.68
48.48
81.73
93.30
97.39
Difference Factor (F1): 6
Similarity Factor (F2): 70
Table 6: Results of method validation

The method validation parameters like specificity, repeatability, intermediate precision, and accuracy were found to be within acceptance criteria.

Example 7: Results of Stability
Condition Batch 2 3M_40°C/25%RH 3M_25°C/40%RH 6M_40°C/25%RH 6M_25°C/40%RH
Variable Initial Stability Stability Stability Stability
Time points In vitro drug release data
30 Minute 19.22 26.18 22.92 34.74 35.50
1 Hours 34.63 43.70 39.37 50.27 51.21
3 Hours 72.83 79.17 74.78 78.57 80.33
6 Hours 92.08 93.63 90.07 87.06 88.91
12 Hours 97.36 97.23 94.40 90.25 91.62
Table 7: Results of Stability
The formulation was found stable when stored at 40°C/25%RH and 25°C/40%RH for 3 month and 6 months.

Example 8: Comparison of formulation batches of the same composition prepared by the same process and marketed formulation
To assess the discriminatory power of the method, the sameness among batches of the same composition prepared by the same process was tested and compared those results with two different batches of marketed formulation, TOBRADEX® (Tobramycin and Dexamethasone ophthalmic suspension).

The 3 different batches were prepared using similar composition of optimized batch 2 and in vitro release study of these batches along with marketed formulation as reference was done as per procedure mentioned in above examples.

Results:
Time Points Batch 2.1 (%) Batch 2.2 (%) Batch 2.3 (%) Reference batch 1 (%) Reference batch 2 (%)
30 Minute 19.22 21.33 26.32 23.94 18.186
1 Hour 34.63 36.91 46.35 42.87 33.61
3 Hours 72.83 76.12 88.54 85.87 72.66
6 Hours 92.08 92.73 98.25 96.65 90.13
12 Hours 97.36 99.25 99.54 98.30 93.88
Table 8: Results of comparative study
The above results confirming sameness between batches of the same composition prepared by the same process. It also confirms similar release profile with the reference or marketed product.
,CLAIMS:CLAIMS
We claim,
1) A novel in vitro drug release method of dexamethasone in ophthalmic suspension comprising steps:
a) Activating dialysis tubing cellulose membrane by soaking in purified water for 30 minutes then rinsing thoroughly in purified water, and soaking in dissolution medium for 30 min before use;
b) Tying one side of activated membrane of step (a) with thread;
c) Perfusing sample solution in the membrane of step (b) and tying other side of membrane with thread;
d) Tying the membrane of step (c) from both side with sinker to prevent floating of membrane in dissolution vessel of apparatus;
e) Filing the dissolution vessel of step (e) with warm sonicated and degassed dissolution medium;
f) Placing activated and perfused membrane of step (d) in dissolution vessel of apparatus;
g) Adjusting temperature of dissolution medium of step (f) at 37ºc and stirring of dissolution medium at 100 rpm;
h) Allowing diffusion of sample from the membrane to dissolution medium till the completion of dissolution testing;
i) Collecting samples from dissolution media of step (h) at intervals of 30 min, 1 hour, 3 hour, 6 hour, 12 hour;
j) Replenishing fresh dissolution media of equal quantity of sample withdrawn in step (i) in the vessel;
k) Analyzing collected samples by high performance liquid chromatography (HPLC) method to determine the % amount drug release;
Characterized by said in vitro release method is micro-dialysis technique in USP apparatus type 2 paddle.

2) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said dialysis tubing cellulose membrane of 10 cm length and 33 mm width.

3) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said dissolution medium is artificial tear solution comprising 5.0 gm of Sodium lauryl sulphate (SLS), 6.7 gm of sodium chloride, 2.0 gm of sodium bicarbonate and 80 mg of calcium chloride dihydrate.

4) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein pH of said dissolution medium maintained 7.4 with dilute hydrochloric acid.

5) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said HPLC method comprises subjecting said collected samples to a HPLC column using a mobile phase to quantify dexamethasone at a flow rate of 1.5 mL/minute, wherein said mobile phase which comprises a mixture of acetonitrile and water in ratio of 35:65 %V/V,
Wherein, the quantification of the dexamethasone achieved within retention time of 3.8 Minute.

6) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said HPLC column is a C18 column.

7) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said chromatography carried out at temperature 25ºc.

8) The novel in vitro drug release method of dexamethasone in ophthalmic suspension as claimed in claim 1 wherein said collected sample quantified at a wavelength of 254 nm.