Formulation and Development of Ocusert for Gemifloxacin Mesylate with Corticosteroid

 

Mrs. Swati Mayur Keny1,2, Dr. Ketan Shah3

1PhD Scholar, RK University, Rajkot

2PES’s Rajaram and Tarabai Bandekar College of Pharmacy, Farmagudi-Ponda, Goa.

3 Professor, Parul Institute of Pharmacy & Research, Parul University, Vadodara, Gujarat, India

*Corresponding Author E-mail: swati_pharmaco@yahoo.co.in

ABSTRACT:

Ocular drug delivery is one of the most challenging and fascinating task faced by the Pharmaceutical Researchers. The challenges to the formulators are to circumvent the protective barriers of the eye without causing permanent tissue damage, maintain a stable therapeutic level at the site of action for a prolonged period of time. Frequent instillation to maintain a continuous sustained therapeutic level is required which often results in local or systemic side effects. The objective of the present investigation is to formulate a novel drug delivery system for ophthalmic use for the treatment of ocular conjunctivitis. Ocusrets of Gemifloxacin Mesylate and Dexamethasone were formulated and evaluated. Materials and Methods: Initially preformulation studies of Gemifloxacin Mesylate, Dexamethasone, polymer and excipients were performed to check their compatibility. Different combinations of Gemifloxacin Mesylate, Dexamethasone, Carbopol 974, 980 981, PEG 400 and glycerine were formulated by solvent cast method and evaluated. Results and Discussion: The formulated ocusert was evaluated for clarity, smoothness, surface pH, drug content, and in-vitro drug release study. Conclusion: Formula GD 74 fulfilled the needs of all organoleptic parameters and also the in vitro release study. These Ocusert will be surely an alternative for conventional eye drops and boon to patients in future.

KEY WORDS: Gemifloxacin Mesylate, Dexamethasone, Carbopol 974, 980, 981, Ocular inserts and Betacyclodextrin complex.

 

INTRODUCTION:

Delivering a drug to eye is a complex process and has emphasized scientists to work in multi-disciplinary areas related to the eye including chemical, biochemical, pharmaceutical, medical, clinical, and toxicological sciences. In the recent years, attention has been increased on two main objectives:

(A)            To find effective and safe drug molecules for various ocular conditions and diseases that are poorly controlled.

(B)             To improve existing ocular dosage forms and to develop newer delivery systems for improving the ocular bioavailability of existing molecules.[1]

Ophthalmic preparations are specialized dosage forms designed to be instilled onto the external surface of the eye (topical), administered inside (intraocular) or adjacent (periocular) to the eye or used in conjunction with an ophthalmic device.

Ophthalmic preparations are at par with the parental dosage forms in terms of sterility as well as considerations for osmotic pressure (tonicity), preservation, tissue compatibility, pyrogen free intraocular dosage forms, particulate matter and suitable packaging.[2]


 

As eye imposes various physiological and anatomical constraints, only a small fraction of the administered drug, effectively 1% or even less of the instilled dose is ocularly absorbed. This becomes the driving force for the clinician to recommend a frequent dosing at an extremely high concentration which may results in several side effects of ophthalmic products. Newer ophthalmic delivery systems are being explored in order to overcome the problems of conventional ocular therapy and to improve the ocular bioavailability of the drug.

 

Topical Therapeutic dosage forms have customarily been restricted to solutions, suspensions and ointments. But with recent advancements in material science, the range of ophthalmic dosage forms has expanded significantly which includes gels, either preformed or spontaneous gels responsive to the ocular environment and ocular inserts, both forms reducing dosage frequency[3].

 

Thus the aim of the present work was to formulate ocusert with a definite concentration of Gemifloxacin and Dexamethasone for the treatment of ocular conjunctivitis and compared for the sustained release of active. The formulation was formulated with the objective of increasing the residence time of the drug, reducing the dosing frequency by combining with Carbopol 974, 980, 981, PEG 400, Polyvinyl alcohol and glycerine.

 

MATERIALS AND METHODS:

Materials:

Gemifloxacin Mesylate was obtained as a gift sample from Glenmark Pharmaceuticals, Solan, Himachal Pradesh and Dexamethasone was from Symbiotec Pharma Private Limited. Carbopol 974, 980 and 981 were gifted by Lubrizol Pvt, Ltd, Mumbai. PVA, PEG 400 and Beta cyclodextrins used were procured from Hi-Media. All chemicals used were of analytical grade.

 

Methods:

Preformulation Studies:

Preformuation studies were performed on the procured drug samples and excipients with respect to description, melting point, solubility, IR spectras, ultra -violet (UV) spectroscopic studies and Differential Scanning Calorimetry (DSC) [4].

 

UV Spectroscopy Study:

Determination of wavelength of maximum absorption:

Pure Gemifloxacin Mesylate and Dexamethasone were weighed and diluted with distilled water. The prepared solutions were scanned in the wavelength region of 200 – 400 nm. This procedure was conducted using UV-visible spectrophotometer (UV- Shimadzu make).

 

Determination of Linearity and Range:

25 mg of Gemifloxacin Mesylate was weighed and transferred to 25-ml volumetric flask, dissolved and diluted up to mark with Methanol to give a stock solution having strength of 1 mg/ml. Similarly accurately weighed 50mg of Dexamethasone was transferred into 50 ml volumetric flask, dissolved and diluted up to mark with Methanol to give a stock solution having strength of 1 mg/ml. It is further diluted to get a stock solution of strength 100 µg/ml.

 

Aliquots of 0.3 ml, 0.6 ml, 0.9 ml, 1.2 ml, 1.5 ml of working standard solution of individual drugs were transferred to a series of 10 ml standard volumetric flask and diluted with Phosphate buffer pH 6.8 to get 3, 6, 9, 12 and 15 μg/ml of Gemifloxacin Mesylate and Dexamethasone respectively.

 

Absorbance was recorded at their respective working λ max of 263.8 nm and 242 nm against the blank solution prepared using methanol and phosphate buffer pH 6.8 without drug. The Beer’s Lambert law was verified from the calibration curve by plotting the graph of concentration against absorbance [5].

 

DSC:

DSC (DSC-60 Shimadzu, TA-60 WS collection software) was employed to study the thermal property of drug and excipient alone and in the combination. Endothermic and exothermic parameters of the drug and polymer were subsequently obtained.


 

IR

The FT-IR spectrum of the obtained sample was compared with the reference standard FT-IR spectrum of Gemifloxacin Mesylate and Dexamethasone by potassium bromide method.

 

Preparation of Ocusert:

Preparation of Beta cyclodextrin and dexamethasone complex.

Dexamethasone is a poorly soluble drug in water and hence to enhance its solubility complexes of drug with β- cyclodextrin were prepared. Six different molar ratios were prepared and evaluated. The solubility profile of the drug was checked and the ratio to be used (Drug: β cyclodextrins) was finalized based on % cumulative drug release

[6].

 

Preparation of Ocusert of Gemifloxacin Mesylate and Dexamethsaone.

Petri Dish of size 9 cm was chosen and area of the same was calculated. Depending on the area of the petri dish, drug to be incorporated was calculated. Proportion of carbopol: PVA i.e 1:9 ratio was dissolved in 20ml of distilled water the previous night, followed by incorporation of drug and PEG 400 and Glycerin with stirring on Magnetic Stirrer for 6 hours. On completion of 6 hours the preparation was poured in the mentioned perti dishes and was dried at 500c for 4 hours.1cm x 1cm areas of the prepared films were used for the evaluation purpose[7].

 

Table 1: Composition of Ocusert

Ingredients

Quantity

GD 74

GD 80

GD 81

Gemifloxacin Mesylate

19 mg equivalent to 0.3 mg Gemifloxacin

Dexamethasone: βeta CycloDextrin

398 mg equivalent to 0.7 mg Dexamethasone

Carbopol 974

60 mg

--

--

Carbopol 980

--

60 mg

--

Carbopol 981

--

--

60 mg

Poly Vinyl Alcohol

540 mg

540 mg

540 mg

Poly Ethylene Glycol 400

0.5 ml

0.5 ml

0.5 ml

Glycerin

25 mg

25 mg

25 mg

Distilled Water

20 ml

20 ml

20 ml

 

Surface pH:

Ocular insert should be non-irritating to eye and should be compatible with lacrymal fluid. The prepared films were dissolved in 0.1 ml of double distilled water at room temperature. The swollen films were placed in a digital pH meter and the surface pH was recorded[8,9].

 

Drug Content:

1cm x 1 cm film were cut and dissolved in 10 ml phosphate buffer pH 6.8, further 1ml is diluted to 10 ml and analyzed using UV-visible spectrophotometer at the absorbance value of 263.8 nm and 242 nm respectively[10,11].

 

In Vitro drug release study:

The bi-chambered donor-receiver compartment model (Franz Diffusion Cell) was employed to determine the in  vitro drug release. The set up was placed on the magnetic stirrer with room temperature value maintained. Semi- permeable membrane (dialysis membrane 50, HIMEDIA) was used at the receptor site. Stirring rpm was minimum just closely relating to eye blinking movement. At periodic intervals, 1ml sample was withdrawn and replaced by 1 ml Phosphate buffer. Withdrawn sample were analyzed and drug release was calculated[12].

 

Antimicrobial activity:

The cup-plate technique with agar diffusion medium was used. The cup was bored at the centre of the plate. The developed film and standard solution of pure drug were taken separately into soyabean casein digest medium earlier seeded with Staphylococcus Aureus organism. On placing the film and standard solution in the plate, they were incubated for a day at 370c. Compared with the standard, the zone of inhibition (ZOI) was calculated[6,13].

 

Sterility Testing:

This is performed as per Indian Pharmacopoeia 1996, wherein fluid thioglycolate and soyabean casein digest media are used. Experiment is to be performed under laminar air flow, wherein the formulated films were cut into two


 

equal halves and dropped in the two test tubes simultaneously. Both the media were incubated at 370c for seven days and checked for microbial growth. The results were compared with positive and negative control samples[14].

 

Isotonicity evaluation:

Tissue damages if the tonicity of the film is not maintained, hence isotonicity of the film is a mandatory parameter. Sodium chloride solutions of three different concentrations namely hypertonic (HT - 3% w/v), hypotonic (HP - 0.2% w/v) and isotonic (IS - 0.9% w/v) concentrations were prepared. Four clean slides were taken and labeled as HT,  HP, IS and Test. A drop of blood with heparin (1% w/v) was taken to prevent coagulation, further placed on all slides. Optimized film drop was placed on test slide and all four slides were covered with cover slip and checked under 45x magnification microscope. Morphology of RBC’s was studied[15].

 

Antibacterial Activity:

Microbiological assay was carried out on serial dilution method. Test organism employed was S.aureus. Two samples tested for minimum inhibitory concentration (MIC) were coded as A (film) and B (pure sample). For MIC the compounds are tested against S.aureus. The concentration of pure drug taken is 5mg/ml. 51μl of this drug solution contains 256μg of the drug. Series of 14 test tubes were taken and numbered as 1 – 14. To 1st test tube 2000 μl of broth is added. And from 2nd test tube till 14th test tube 1000μl broth was added. 51μl of broth from test tube 1st was withdrawn and discarded and replaced with drug solution. Concentration corresponds to 128μg of drug. 1000μl of the content from 1st test tube is transferred to 2nd and so on. This procedure is repeated till second last test tube corresponding to 128μg/ml, 64μg/ml, 32μg/ml, 16μg/ml, 8μg/ml, 4μg/ml, 2μg/ml, 1μg/ml, 0.5μg/ml and 0.25μg/ml. The last test tube serves as negative control. 10μl of S.aureus broth is added in each tube except negative and kept for incubation at 370C for 24hours. Further MIC was calculated and results were tabulated [16].

 

Short term Stability Studies:

The purpose of the stability testing is to ensure efficacy, safety and quality of active used in the product. Optimized film was subjected to stability studies at room temperature 250c for a period of three months. The samples were withdrawn at 30 days, 60 days and 90 days’ time period and evaluated for parameters like surface pH, Drug content and in vitro drug release[17].

 

RESULTS AND DISCUSSION:

Results of preformulation studies performed on drug and excipients are represented in table 2.

 

Table 2: Preformulation study on Drug and Excipients

Observed parameters

Gemifloxacin Mesylate

Dexamethasone

Description

Gemifloxacin is off-white, amorphous solid form. The mesylate salt is a white to light brown solid

Dexamethasone is off-white, powder form

Melting Point

234.5 °C

267 °C

Solubility

Freely soluble 0.21 mg/ml distilled water, freely soluble in DMSO, DMF, Ethanol and Methanol

Insoluble in water, freely soluble in DMSO, DMF, Methanol and sparingly soluble in Ethanol

 

IR Spectra:

IR spectras of pure drugs and excipients were plotted and compared with standard samples. Drugs and excipients were found to be compatible with each other as represented in Table 3.

 

Table 3: IR interpretation

Functional groups

Gemifloxacin mesylate (a)

Dexamethasone (b)

A + b + carbopol 974

A+b+ carbopol 980

A+b+

Carbopol 981

O-H

3248.13

3419.79

3421.72

3421.72

3421.72

N-H

3431.8

--

3246.20

3246.20

3246.20

AROMATIC C-H

2929.87

2941.44

3066.82

3066.82

3064.89

ALIPHATIC C-H

2821.86

2875.86

2931.80

2931.80

2927.94

C=O (KETONE)

1724.36

1714.72

1724.36

1718.58

1724.36

C-F

1376.53

1107.14

1367.53

1367.53

1367.53


 

DSC:

DSC was employed to understand thermal properties of Dexamethasone and Beta cyclodextrins. Due to glass transition endothermic peaks were observed for Dexamethasone at 267.20C, Betacyclodextrins at 121.10C and Complex of Dexamethasone with Betacyclodextrins at 105.70C, 291.50C respectively as shown in Figure 1, 2, 3.

 

Figure 1: Thermal Analysis of Dexamethasone.                                       

 

Figure 2: Thermal Analysis of Beta Cyclodextrin.

 

Figure 3: Thermal Analysis of Dexamethasone + Beta Cyclodextrin

 

Determination of Linearity and range:

Linear calibration curve was obtained for Gemifloxacin Mesylate and Dexamethasone in the concentration range of 3-15 μg/ml at λ max 263.8 nm and 242 nm respectively. It followed Beer’s Lamberts Law with regression coefficient (R2) value of 0.999 for both Gemifloxacin and Dexamethasone.

 

Preparation of Beta cyclodextrin and dexamethasone complex.

 

Dexamethasone was complexed with β-cyclodextrinsin six different molar ratios before incorporating in the occusert development. The solubility profile of the drug was checked and the ratio 1:1.5 (Drug: β cyclodextrins) was finalized based on % cumulative drug release as shown in figure 4


 

Figure 4 graph of absorbance v/s concentration

The Drug content was determined based on the UV-simultaneous estimation method developed for the combined dosage form. Other evaluated parameters of the prepared occuserts with respect to surface pH, Tensile strength, thickness is recorded in following tabular column, Table 4

Table 4: Evaluated parameters

Formulation code

Surface texture

Thickness (mm)*

Weight (mg)*

Tensile strength (g/cm2)*

% Drug Content (± SD*)

DRUG A

DRUG B

GD 74

Smooth

0.128 ± 0.05

210 ± 0.02

420 ± 0.05

98.57

83.33

GD 80

Smooth

0.112 ± 0.07

183 ± 0.02

430 ± 0.09

85.71

66.67

GD 81

Smooth

0.136 ± 0.03

185± 0.06

450 ± 0.05

71.43

67

In vitro Release Study:

It was performed using Franz Diffusion Cell and it was found that Formulation GD 74 gave best results compared to other two formulations. The values are shown in Table 5 and graphical representation in figure 5, 6

Table 5: % Cumulative drug diffusion profile

Time (hrs)

GD74

GD80

GD81

% cumulative drug release

263.8 nm

242 nm

263.8 nm

242 nm

263.8 nm

242 nm

01

17.95

12.83

11.91

6.14

14.69

8.42

02

31.95

26.66

25.19

18.22

22.97

16.27

03

38.54

37.14

33.67

23.42

31.09

26.31

04

45.96

46.96

39.58

26.89

32.82

30.69

05

45.25

50.15

46.49

34.02

36.23

37.88

06

42.27

58.95

46.75

31.05

30.77

33.37

07

47.49

61.12

47.30

30.94

32.25

37.05

08

45.59

61.49

48.25

34.13

33.16

41.83

09

50.28

73.72

56.21

44.45

34.60

49.18

10

47.16

70.63

36.57

43.44

50.68

40.31

11

61.96

87.27

40.13

49.20

63.44

72.06

12

97.15

100.33

63.81

89.63

90.54

100.90

 

Figure 5: % cumulative release of Gemifloxacin.

 

Figure 6: % cumulative release of Dexamethasone.


 

Antimicrobial activity:

Measurement of ZOI by cup plate method:

 

Zone of inhibitions of the formulated films were compared with that of pure drug against a positive and negative control. Readings of this study is tabulated and images on ZOI’s are depicted in table 6 and figure 7.

a                                                                                                               b                                        c

 

d                                                                                                             e                                         f

 

Formula

Zone of inhibition

Negative Control

--

--

Positive Control

--

--

Gemifloxacin Mesylate

Present

4 cm

Dexamethasone

Absent

0 cm

Gemifloxacin Mesylate + Dexamethasone

Present

4.8 cm

GD 74

Present

4 cm

GD 80

Present

3.5 cm

GD 81

Present

3.3 cm

 

 

Figure 7: a) Gemifloxacin, b) Dexamethasone,c) Gemifloxacin + dexamethasone , d) GD 74, e) GD 80 and GD 81 Table 6: Zone of Inhibition value

 

 

 

 

 

 

 

 

Sterility Testing:

When the formulation was incubated for prescribed time and temperature, no turbidity was observed. This indicates it passes the test for sterility as shown in figure 8.

 

a                                 b

Figure 8 a) fluid thioglycolate media, b soyabean casein media.


 

Isotonicity evaluation:

 

Isotonicity test proved that the optimized film produces no change in the blood cells, neither bulging nor shrinking as shown in figure 9. This proves the formulated film is isotonic in nature.

a                                          b                                       c                                      d

Figure 9: a) Isotonic standard, b) Isotonic film, c) Hypertonic standard and d) Hypotonic standard

Antibacterial Activity:

The MIC concentration was found to be 1µg/ml for the film and 8µg/ml for the pure drug as shown in table 7. Turbidity below the mentioned concentration indicates growth of organism.

Table 7: MIC determination of Film GD 74 and pure drug

Concentration μg/ml

128

64

32

16

8

4

2

1

0.5

0.25

0.125

NC

MC

PC

Turbidity in GD 74

-

-

-

-

-

-

-

-

+

+

+

-

-

+

Turbidity in pure drug

-

-

-

-

-

+

+

+

+

+

+

-

-

+

NC- Negative Control, MC – Media Control, PC – Positive Control

 

Short term Stability Studies:

Stability studies proved that the formulations GD 74, GD 80 and GD 81 showed no significant variations. There was a slight decrease in drug content but were not significant. The variation in drug content can be attributed to moisture content. Thus from the results we can interpret that films can be stored at room temperature for a short term.

 

CONCLUSION:

The formulated ocular films proves to be a novel drug delivery system with a promising approach in achieving greater drug absorption in comparison to the conventional ocular drops. The results concluded that film GD 74 was the best amongst the three formulation in terms of drug content, in vitro drug release and anti-microbial activity. The optimized film showed no interactions between drugs, excipients and also beta cyclodextrin complex when characterized with IR and DSC studies. Hence combination of Gemifloxacin and Dexamethasone as an ocular film serves as a boost for the researchers and boon to the patients in the future over the conventional ocular dosage forms.

 

ACKNOWLEDGEMENT:

We are thankful to Glenmark Pharmaceuticals Solan, Himachal Pradesh, Symbiotec Pharma Private Limited and Lubrizol Pvt, Ltd, Mumbai for providing with Gemifloxacin, Dexamethasone and Carbopol as gift samples for my work. I am also grateful to KLE’s College of Pharmacy for providing Laboratory services to carry out DSC studies.

 

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Received on 10.09.2019       Modified on 28.09.2019

Accepted on 14.10.2019      ©A&V Publications All right reserved

Research J. Science and Tech. 2019; 11(4):227-235.

DOI: 10.5958/2349-2988.2019.00034.2