In Vivo studies of Ophthalmic Ocular Insert Containing Aciclovir

 

S. Valarmathi1*, S. Shanmugam2 , S. Satheesh Kumar3, P. Shanmugasundaram3

1Research Scholar, School of Pharmacy, Vels University, (VISTAS), Pallavaram, Chennai, Tamil Nadu

2Adhiparasakthi College of Pharmacy, Melmaruvathur, Tamil Nadu

3School of Pharmacy, Vels University, (VISTAS), Pallavaram, Chennai, Tamil Nadu

*Corresponding Author E-mail: sahanashree2012@gmail.com

 

ABSTRACT:

Aciclovir is an antiviral drug used to treat viral infections like herpes simplex virus. The aim of the present research work was to develop ocular inserts of aciclovir and evaluate their drug release for both in vitro and in vivo. The draw backs of other ophthalmic formulations (solutions, suspensions, ointment) like poor bioavailability, patient non compliance, quick drug elimination was overcome by formulating the drug in ocusert formulation.1,2 The ocuserts was prepared by solvent casting technique using hydrophilic and hydrophobic polymers. Eight formulations was prepared with formulation code of F1, F2, F3, F4, F5, F6, F7, F8.The satisfied drug release was achieved in F2 formulation. The selected formulation F2 was subjected to sterilization before performing in vivo studies.3,46 healthy rabbits are used for in vivo study. Draize irritation studies are carried out to check the compatibility of ocusert in the eyes. After 21 days of irritation study the eyes are examined. Drug content of the ocuserts are calculated at predetermined time intervals and subtracted from the initial value.5Theinvivo drug release results are correlating with invitro drug release, which shows better therapeutic efficacy.

 

KEYWORDS:  Aciclovir, in vivo, Draize irritation, Rabbits, drug content.

 

 

 

 

INTRODUCTION:

Eye is an important organ in the human body. Aciclovir drug is having half life of 2-4 hrs which is necessary for controlled release formulation.1The factors to be considered while formulating controlled drug release was shown in table no 1

 

Table No 1 Properties of drug for controlled release formulation

S.No

Properties of drug

Limits allowed

1

Molecular weight

Less than 600 Daltons

2

Partition coefficient

1-2

3

Elimination half life

2-6 hrs

 

Mechanism of action aciclovir is shown in the Fig 1

 

Fig No 1 Mechanism of action of aciclovir

Criteria for controlled release ocular insert are comfort, convenient to use, sterility, stability, easy to formulate, excipients used non irritant to the eyes

 

MATERIALS AND METHODS:

Aciclovir was received as a gift sample from Kaushik Therapeutics Pvt. Ltd. Chennai. The polymers hydroxy propyl cellulose, Polyethylene glycol 400, Eudrajit RS 100, Ethyl cellulose was purchased from Nice Pharmaceuticals, Kerala. All other ingredients were of analytical grade.

 

Preparation of ocular inserts:

Preparation of drug reservoir7,8:

Polymeric solutions were prepared by dissolving hydrophilic polymer HPC/PVA along with aciclovir and polyethylene glycol 400 in doubly distilled water. The solutions were poured into a glass ring of 8.9 cm diameter placed in a Teflon coated petridish. The solvent was allowed to evaporate by placing it inside an oven maintained at 35 °C for 24 hrs. The formulation of aciclovir ocular insert was shown in table no 2

 

Preparation of rate controlling films:

To prepare the rate controlling films hydrophobic polymer (ethylcellulose, eduragit RL 100) along with plasticizer Dibutyl phthalate was dissolved in ethanol/acetone (80:20). The solutions were poured into a glass ring 8.9 cm diameter petriplate. The solvent was allowed to evaporate at 25°C for 24 hrs. Placing rate controlling film around, the drug reservoir and sealing them to obtain ocular inserts. The two rate controlling membranes containing the reservoir film between them were placed over a beaker saturated with ethanol/acetone vapors 60:40 for two minutes. The ophthalmic ocusert formulation is shown in Fig no 2

 

Fig No 2 Different layers of ocuserts

1. Transparent polymer membrane 2.Polymeric membrane ring 3. Drug reservoir 4.Transparent polymer membrane.

 

Table No:2 Formulation of aciclovir ocular inserts

In gredients

F1

F2

F3

F4

F5

F6

F7

F8

PREPARATION OF DRUG RESERVOIR

Aciclovir*

10

10

10

10

10

10

10

10

HPC*

2

 

-

-

2

4

-

-

PVA**

-

-

2

4

-

-

2

4

PEG 400**

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

Distilled water

10

10

10

10

10

10

10

10

Preparation Of Rate Controlling Membrane

Ethyl cellulose*

4

2

4

2

-

-

-

-

Eudragit RL 100*

-

-

-

-

4

2

4

2

PEG400**

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

Acetone

10

10

10

10

10

10

10

10

*Quantity in %**quantity in ml

 

In-vitro diffusion studies:

Studied using standard open ended cylindrical glass tube. The pre hydrated cellophane was tied to one end of cylindrical tube which act as a donor compartment. An ophthalmic insert was placed inside the donor compartment. The receptor compartment containing 25 ml of simulated tear fluid in 100 ml beaker. The content of the receptor compartment was stirred using magnetic stirrer at 370C.±0.5 0C .At specific time interval 1ml of simulated fluid from receptor compartment was withdrawn and replaced with fresh simulated tear fluid. The withdrawn samples are diluted and analyzed using UV at 254 nm.

 

Drug content uniformity:

Ocular inserts were dissolved individually in methanol/ethanol in a 100 ml volumetric flask. Then required volume of solution was taken out and further dilutions were made with STF pH 7.4. Similarly, a blank was carried out using a drug free insert. Then absorbance was taken at 254.0 nm by UV spectroscopy.

 

In vivo drug release studies:

Eye irritancy Test (Draize eye irritancy test) 8:

The selected formulations F2 were sterilized by UV radiation before performing animal studies. The institutional animal ethical committee (IAEC) approval was obtained before performing invivo drug release with the reference No: XIX/VELS/P.COL/12/2000/ CPCSEA/IAEC/O3.10.2016 .Eye irritation test was done to detect the damage of cornea, iris and conjuctivia caused by the formulation in the eye.

 

Animal:                              Rabbits

Species:                              Albino

Gender:                              Male

Number of animals:         Six

 

 

 

 

Housing:

The rabbits were housed in standard cages, in a light controlled room at 28 ± 2°C and 60 ± 15 % relative humidity, with no restriction of food or water.

 

During the experiments, the rabbits were placed in restraining boxes, where they could move their eyes and head freely. Selected sterilized ocusert was instilled in cul-de-sac of right eye and left eye kept as control. Both eyes were periodically observed by naked eye or by means of a pen torch for Redness, Swelling andwatering of the eye. Test eye (right eye) will be compared with control eye (left eye).

 

Table no: 3 Ocular irritation scoring system

S.

No

Eye part

Ocular reaction

Score

1

Cornea

No ulceration

0

2

Scattered or diffuse area

1

3

Easily discernible translucent area

2

4

Iris

Normal

0

5

Swelling

1

6

Hemorrhage

2

7

Conjuctivia

Blood vessels are normal

0

8

Blood vessels are hyperemic

1

9

Diffuse crimson color

2

 

This test was repeated for 21 days and finally reported as ocular safety of the selected formulation. At the time of examination period each rabbit was scored for ocular reaction given in Table 3. The test may be considered positive if three or more animal exhibit positive reactions at any observation period.

 

In vivo drug release studies 9 10:

Six healthy rabbits were used to study the in vivo drug release studies. The rabbits used for the in vivo study was kept in good condition in order to exclude any diseases. The selected ocuserts are placed in cul-de-sac of each rabbit and another eye served as a control. At predetermined time intervals (2, 4, 6, 12 and 24 hrs) the inserts were taken out carefully from the eyes of each rabbit and analyzed for the remaining drug content. The drug content remaining was subtracted from the initial drug content of the insert, which gives the amount of drug released at the specific time in the rabbit eye.

 

RESULTS AND DISCUSSION:

In vitro diffusion studies7,2,3

The F2 formulation containing Hydroxypropylcellulose (4mg) in the drug reservoir and Ethylcellulose (2mg) was showing satisfied controlled release when compared to remaining formulations. After 24 hrs the percentage drug release from F2 formulation was found to be 97.16%.The percentage drug release is shown in Table 4.

 

 

Table No 4 In vitro drug release studies of formulated ocular inserts

S. No

Time in hrs

F1

F2

F3

F4

F5

F6

F7

F8

1

0

0

0

0

0

0

0

0

0

2

2

18.1±0.9

10.0±0.9

8.3±0.1

22.8±0.4

20.0±0.8

33.3±0.9

33.3±0.8

37.5±0.7

3

4

30.6±0.2

20.8±0.8

30.6±0.2

33.3±0.2

39.4±0.8

66.7±0.5

55.6±0.8

56.7±0.3

4

6

50.0±03

43.3±1.2

51.7±0.5

45.6±0.2

58.3±0.3

79.2±0.3

77.8±0.5

77.2±0.8

5

8

55.0±0.1

61.1±0.6

54.4±0.4

56.1±0.1

77.8±0.2

82.7±0.7

82.0±0.4

90.5±0.4

6

12

69.4±02

77.8±0.3

72.2±0.3

71.4±0.2

80.5±0.4

83.8±0.4

96.1±0.2

93.4±0.2

7

24

80.6±0.5

97.16±0.1

82.8±0.2

83.9±0.1

82.0±0.2

93.3±0.6

96.13±0.2

94.7±0.2

All the values are expressed as mean ±SD, n=3

 

In vivo Study:

Eye Irritation Test5:

The selected formulation F2 which shows promising in vitro drug release was subjected to eye irritation test after sterilized by UV radiation. The results shows that there was no irritation, redness of eye, and swelling of eyes .so it was concluded that the prepared ocular inserts were compatible to the eyes. The ocular inserts was not expelled from the eye during eye irritation study. The ocusert insertion in to rabbit eye are shown in Figures 3,4,5,and 6.As the prepared inserts does not show any inflammation and abnormal discharge the excipients used in the formulation also found to be compatible with the eye. The eye irritation test results are shown in table no 5

 

Table No 5 Results of eye irritation study

S. No

PART OF EYE

SCORING VALUE

1

Cornea

0

2

Iris

0

3

Conjuctivia

0

 

 

 

 

 

 

Fig No 3 Normal rabbit eye 

 

Fig No 4 Inserting ocusert in to rabbit eye

 

Fig No 5 Ocusert inserted in rabbit eye

 

Fig No 6 Rabbit eye after irritation study

 

 In vivo drug release study:

After obtaining approval from institutional animal ethical committee (IAEC) under Ref No. XIX/VELS/P.COL/12/2000/CPCSEA/IAEC/O3.10.2016 the animal studies were carried out. The in vivo studies were performed using healthy rabbits. The drug content of the inserts was calculated at specific time intervals. The drug release was found to be as shown in table no 4.The in vitro drug release was correlating with in vivo drug results. The in vivo drug release results are shown in table no.6. Graphical representation of in vivo drug release was shown in Fig No 7

 

Table No 6 In vivo drug release for F2 formulation

S. No

Time intervals in hrs

In vivo % Drug release

1

0

0

2

2

9.6±0.9

3

4

19.5±0.7

4

6

42.5±0.9

5

8

60.5±0.5

6

12

76.2±0.6

7

24

98.5±0.2

 

Fig No 7 Graph showing in vivo drug release

CONCLUSION:

The ocular inserts were prepared by solvent casting technique by enclosing the drug reservoir membrane in between two rate controlling membranes. The drug reservoir was made up of hydrophilic polymer and the rate controlling membrane was made up of hydrophobic polymer. Eight formulations containing different ratios of polymers were prepared. The in vitro drug release results shows that F2 was the best formulation containing Hydroxypropylcellulose 4mg and Ethylcellulose 2mg, The F2 formulation meets all the controlled release formulation criteria’s .The selected formulation F2 was subjected to eye irritation study and in vivo study. The formulation passes Draize eye irritation test as there was no swelling, redness of the eye. The in vivo results are correlating with in vitro results, so the prepared formulation F2 shows better therapeutic efficacy.

 

ACKNOWLEDGEMENT:

The authors are grateful to Kaushik Pharmaceuticals Chennai for providing the gift sample of drug Aciclovir and Department of Pharmacology Dr. Santhosh Vels University (VISTAS) for providing assistance in animal studies.

 

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Received on 08.04.2017           Modified on 17.05.2017

Accepted on 23.05.2017          © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(7): 2139-2142.

DOI: 10.5958/0974-360X.2017.00376.6