Development and Characterization of Perindopril Erbumine Loaded Proniosomal Gel

 

Ashish Kute, Prakash Goudanavar*, Doddayya Hiremath, S.R. Reddy

Department of Pharmaceutics, NET Pharmacy College, Raichur-584 103, Karnataka, India

*Corresponding Author E-mail: pgoudanavar01@gmail.com

 

 

ABSTRACT:

The present work deals with the preparation of perindopril erbumine proniosomal gel by coaservation phase separation method by using different surfactants in different ratios. The prepared proniosomal formulation were evaluated for Vesicle size analysis, Surface morphological studies, Rate of spontaneity, encapsulation efficiency, In vitro release Stability studies  kinetic data analysis. The vesicle of proniosome varied between 15.13±4.65µm to24.05±2.10µm (without agitation) from 4.65±5.89µm to 9.7±0.15µm (with agitation), Rate of spontaneity between 9.10±0.65mm3×1000 to 15.26±5.35mm3×1000, % encapsulation efficiency between 70.72±0.39% to 76.43±5.56%. In vitro release profile indicated that, increases in liphophilicity of surfactants decreases release of perindopril erbumine from proniosomal formulations. Among the various formulation studied, proniosomal gel with T20:T60 in ratio of 900:100showed the highest % of drug release (80.03%) over period of 24 hrs. Cumulative amount of drug permeated through rat abdominal skin for optimized formulation was found to be 75.263%. The mechanism of drug release was Non-Fickin diffusion controlled zero order kinetics for optimized formulations PNG3, PNG6, PNG9,  PNG12. Stability studies indicated that, the prepared proniosomal gel remained more stable at room  refrigeration temperature than oven temperature.

 

KEY WORDS: Perindopril erbumine, Spans Tweens, Cholesterol  In vitro release.

 

 


INTRODUCTION:

Drug delivery systems using vesicular carriers such as liposomes and niosomes have distinct advantages over conventional dosage forms. They may serve as a solubilization matrix, as local depot, as permeation enhancer or as a rate limiting membrane barrier for the modulation of systemic absorption of drugs via the skin. [1] In recent years, non ionic surfactants vesicle also referd to as niosomes, have been studied as alternative to conventional liposomes in drug delivery.[2] Compared to liposomes (phospholipids vesicle), they offer higher chemical stability, lower cost and greater choice of surfactants. However, even though niosomes exhibit good chemical stability during storage, there may be problem of physical stability in niosomal dispersions. Aqueous suspensions of niosomes may exhibit aggregation, fusion, leaking of entrapped drug or hydrolysis of encapsulated drug, thus limiting the shelf life of the dispersion. [3]

        

The proniosomal approach minimizes the above mentioned problems, as it involves a dry product or a liquid crystalline gel that can be hydrated immediately before use. [4-5] Ease of transfer, distribution, measuring and storage makes proniosomes a versatile delivery system. Proniosomes are water soluble carrier particals that are coated with surfactants and can be hydrated to form niosomal dispersion immediately before use in hot aqueous media. [6] Proniosomes offer a versatile drug delivery concept with potential for delivery of drugs via transdermal route. This would be possible if proniosome form niosomes upon hydration with water from skin following topical application under occlusive conditions. [7]

 

Perindopril erbumine is an ACE inhibitor, used in treatment of hypertension and congestive heart failure, perindopril is converted in body into active metabolites perindoprilate, ACE inhibition is reported to occur within 1 hrs of dose, to be maintained for 24 hrs. Perindopril is given by mouth as erbumine salt and should be taken before food. In treatment of hypertension perindopril erbumine is given in an initial dose of 4 mg once daily, having biological half life 1-3 hrs, plasma protein binding of 60%, with peak plasma concentration occurring in 0.6-1.9 hrs. [8]

 

 


TABLE 1. Composition of perindopril erbumine proniosomal gel formulations.

Formulation code

Surfactant Types

Ratio

(mg)

Lecithin

(mg)

Cholesterol

(mg)

Alcohol

(ml)

Water

(ml)

Observations

PNG1

T20:T60

100:900

100

100

0.5

0.18

Yellowish gel

PNG2

T20:T60

500:500

100

100

0.5

0.18

Yellowish gel

PNG3

T20:T60

900:100

100

100

0.5

0.18

Creamish semisolid

PNG4

S20:S40

100:900

100

100

0.5

0.18

White semisolid

PNG5

S20:S40

500:500

100

100

0.5

0.18

Light brownishsemisolid

PNG6

S20:S40

900:100

100

100

0.5

0.18

Brown transparent liquid

PNG7

S20:S60

100:900

100

100

0.5

0.18

White semisolid

PNG8

S20:S60

500:500

100

100

0.5

0.18

White semisolid

PNG9

S20:S60

900:100

100

100

0.5

0.18

Brown liquid

PNG=Proniosomal gel, S=Span, T=Tween

Drug concentration used in each formulation is 4mg.

 


 

Perindopril erbumine shows 65-75% bioavilabity but presence of food reduces the conversion of perindopril to perindoprilate. According to previous research, the oxidation rate of perindopril in dermal homogenate is significantly lower than intestinal homogenate because the oxidative product perindopril erbumine a perindoprilate shows poor absorption from the intestine.[9] Perindopril erbumine when administered initially causes hypotension, which can prove to be harmful in diuretic treated and congestive heart failure patients. Persistent hypotension may cause some trouble in myocardial infarction patients.[10] Therefore, the use of transdermal drug delivery system can reduce the side effects associated with perindopril erbumine. Niosomes carrier, well known for their potential in topical drug delivery, have been used to transport perindopril erbumine molecule in the skin layer.

 

The aim of present study was to determine the factors influencing the encapsulation efficiency of perindopril erbumine proniosomal gel and to optimize encapsulation parameters in order to achieve a suitable drug delivery system.

 

MATERIALS AND METHODS:

Materials:

Peridopril erbumine was obtained as gift sample from Glenmark pharmaceuticals (Goa, India). Span20, 40, 60, 80, Tween 20, 60 and cholesterol were procured from S. D. Fine chemicals Pvt. Ltd, Mumbai. Soya lecithin was procured from High Media lab, Mumbai. All other reagents used were of analytical grades.

 

Preparation of Proniosomal Gel:

Proniosomal gel was prepared by a coacervation phase separation method Precisely weighed amounts of surfactant, lecithin, cholesterol and drug were taken in a clean and dry wide mouthed glass vial of 5.0 ml capacity and alcohol (0.5 ml) was added to it. After warming, all the ingredients were mixed well with a glass rod; the open end of the glass bottle was covered with a lid to prevent the loss of solvent from it and warmed over water bath at 60-70°C for about 5 min until the surfactant mixture was dissolved completely. Then the aqueous phase (0.1% glycerol solution) was added and warmed on a water bath till a clear solution was formed which was converted into proniosomal gel on cooling. The gel so obtained was preserved in the same glass bottle in dark conditions for characterization.[11] Compositions of proniosomal gel formulations are given in (Table 1).

 

Evaluation of proniosomal gel formulations [7]

Vesicle Size Analysis:

Hydration of proniosomal gel (100mg) was done by adding saline solution (0.9% solution) in a small glass vial with occasional shaking for 10 min. The dispersion was observed under optical microscope (Olympus, New Delhi) at 45 x magnification. The sizes of 200-300 vesicles were measured using a calibrated ocular and stage micrometer (Erma, Tokyo) fitted in the optical microscope.

 

Rate of Spontaneity:

Approximately 10 or 20 mg of proniosomal gel was transferred to the bottom of a clean stoppered glass bottle and spread uniformly around the wall of the glass bottle with the help of a glass rod. At room temperature, 2 ml of phosphate saline (0.154 M NaCl) was added carefully along the walls of the glass bottle and left in a test-tube stand After 20 minutes, a drop of this saline solution was withdrawn and placed on Neubauers Chamber (Marienfeld, Germany) to count the number of vesicles. The number of niosomes eluted from proniosomes was counted.

 

Surface morphological studies:

The surface morphology of noisome derived from proniosomal gel was studied using scanning electron microscopy. SEM revealed that the niosomes formed were spherical and homogeneous.

 

Encapsulation Efficiency:

To evaluate the loading capacity of proniosomal systems for Perindopril, proniosomal gel (100mg) was dispersed in distilled water and warmed a little for the formation of niosomes. Then the dispersion was centrifuged at 18000 rpm for 40 min at 5oC (Remi CPR-24 centrifuge) the clear fraction was used for the determination of free drug at 224 nm spectrophotometrically. The percentage encapsulation efficiency was calculated from following Equation. 

% Encapsulation Efficiency = (Total drug- Free drug / Total drug) × 100

 

In Vitro Release study:

In vitro release studies on proniosomal gel were performed using locally manufactured Franz-diffusion cell. The capacity of receptor compartment was 15 ml. The area of donor compartment exposed to receptor compartment was 1.41cm2. The dialysis cellophane membrane (MMCO 14KDC) was mounted between the donor and receptor compartment. A weighed amount of proniosomal gel was placed on one side of the dialysis membrane. The receptor medium was phosphate saline buffer pH 7.4. The receptor compartment was surrounded by a water jacket to maintain the temperature at 37±1oC. Heat was provided using a thermostatic hot plate with a magnetic stirrer. The receptor fluid was stirred by a Teflon-coated magnetic bead fitted to a magnetic stirrer at each sampling interval; samples were withdrawn and were replaced by equal volumes of fresh receptor fluid on each occasion. Samples withdrawn were analyzed spectrophotometrically (Shimadzu-1700) at 224 nm.

 

Stability Studies:

The ability of vesicles to retain the drug (Drug Retention Behavior) was assessed by keeping the proniosomal gel at three different temperature conditions, i.e., Refrigeration Temperature (4-80C), Room Temperature (25±20C) and oven (45±20C).Throughout the study, proniosomal formulations were stored in aluminium foil-sealed glass vials. The samples were withdrawn at different time intervals over a period of one month and drug leakage from the formulations was analyzed for drug content spectrophotometrically.

 

In vitro permeation study :

The permeation of Perindopril erbumine from proniosomal formulations was determined by using Franz diffusion cell. The shave abdominal skin of rat (0.8±0.1 mm thickness and 3.14 cm2exposed surface areas) was mounted on the receptor compartment with the stratum corneum side facing upwards towards the donor compartment. The receptor compartment was filled with 15.0 ml of pH 7.4 phosphate buffer maintained at 37. 8˚C and stirred by a magnetic bar at 600 rpm. One gram of proniosomal gel formulation was placed on the skin and the top of the diffusion cell was covered with paraffin paper. At appropriate time intervals (3, 6, 9, 12, 18, 21, 24, 27, and 30 h), 1 ml aliquots of the receptor medium were withdrawn and immediately replaced by an equal volume of fresh receptor solution to maintain sink conditions Samples withdrawn were analyzed spectrophotometrically at 224 nm. [12]

 

Drug Release Kinetic Data Analysis:

The release data obtained from various formulations were studied further for their fitness of data in different kinetic models like Zero order, Higuchi’s and Peppa’s. [13]

 

RESULTS AND DISCUSSION:

Results of Vesicle size of perindopril erbumine proniosome are presented in (Table2), which indicated that Vesicle formed with Span is smaller in size than vesicle formed with Tweens; this is due to grater hydrophobicity of Spans than Tweens. It is indicated that increasing in hydrophobicity decreases surface energy of surfactants resulting in smaller vesicle size. [12] Size of vesicle was reduced when dispersion was agitated. The reason for this is the energy applied in agitation which results in breakage of larger vesicles to smaller vesicles. The size range was found to be 15.13±4.65µm to24.05±2.10µm (without agitation) and from 4.65±5.89µm to 9.7±0.15µm (with agitation). Surface morphological studies revealed that proniosomes formed were spherical and homogeneous [7] as shown in (Fig. 1).

 

For rate of spontaneity studies proniosomal formulation were treated with ethanol, propanol, butanol and isopropanol. It was found that proniosome with isopropanol and butanol shows higher value than propanol and ethanol as result of faster phase separation which is due to their lower solubility in water [14] (Table 2 ).

 

Entrapment efficiency was found to be higher in case of proniosome prepared with Span40 and Span60 than proniosome prepared with Tween this is due to fact that Span 40 and Span 60 is more hydrophobic than Tween, which act as solid at room temperature and showed higher phase transition temperature (Tc), low HLB value and long alkyl chain length [15] and results are shown in (Table 2).

 

In vitro release studies are often performed to predict how a delivery system might work in an ideal situation as well as give some indications of its in vivo performance since drug release dictates the amount of drug available for absorption. The amount of drug released from different proniosomal gel formulation was found in the order of PNG3 > PNG6 > PNG9 > PNG2 > PNG5 > PNG8>PNG1>PNG4>PNG7 as shown in (Fig. 2).  It was found that PNG3 showed controlled release property from 10 to 24 hrs. The cumulative release found to 80.03% at the 24th hrs, respectively. The release rate was constant from 10thto 24th hrs. Thus the formulation exhibited zero order release over this period. [7]

 

Stability studies of all prepared niosomes were performed by storing 4°, 25° and 37° for a period of one month. The residual drug content was determined at the end of the month. It was observed that the drug leakage from the vesicles was least at 4° followed by 25° and 37°as shown in (Fig. 3). This may be attributed to phase transition of surfactant and lipid causing vesicles leakage at higher temperature during storage. Hence it is concluded from the obtained data that the optimum storage condition for niosomes was found to be 4°. [16]

 

In vitro permeation for optimized formulation through rat abdominal skin was found to be 75.263% after 24 hrs of release and data presented in (Fig. 4) respectively. It was found that permeation of perindopril from proniosomal gel formulation prepared with Tween is slower as compared to proniosomal gel formulation prepared with span. This was expected due to the larger size of the vesicles and the less lipophilic nature of the former, which makes it more difficult for these vesicles to penetrate or fuse with the   skin. [12]



TABLE 2. Results of vesicle size analysis, rate of spontaneity and encapsulation efficiency of proniosomal gel formulation loaded with perindopril erbumine.

Sr. No.

Formulation code

Vesicle size (µm)

Rate of spontaneity

(mm3×1000)

Encapsulation efficiency (%)

Without agitation  (µm)

With agitation (µm)

1

PNG1

22.50±1.58

7.76±0.56

9.10±0.65

73.93±0.24

2

PNG2

23.28±4.50

8.90±2.85

11.35±1.39

72.08±1.35

3

PNG3

24.05±2.10

9.75±0.15

12.89±0.75

70.72±0.39

4

PNG4

18.22±5.96

5.43±3.65

10.44±2.33

74.29±1.65

5

PNG5

19.40±2.56

6.59±1.75

13.54±0.39

73.00±0.89

6

PNG6

20.95±0.96

7.37±2.95

14.45±3.89

71.58±3.45

7

PNG7

15.13±4.65

4.65±5.89

12.15±4.81

76.43±5.56

8

PNG8

16.68±3.36

5.04±.12

13.89±1.79

75.15±7.68

9

PNG9

17.46±7.56

6.20±3.15

15.26±5.35

74.93±9.68

Average of three determination ± SD.

 


 

 

Figure 1. Scanning electron micrograph of optimized Perindopril erbumine proniosomal gel formulation (PNG3). (500X magnification).

Figure 2. Comparative in vitro release study of different perindopril erbumine proniosomal gel formulations in P.B. of PH 7.4.

Formulations PNG1 (--), PNG2 (--), PNG3 (-▲-), PNG4 (--), PNG5 (-×-), PNG6 (-●-), PNG7 (-+-), PNG8 (--), PNG9 (-).

Figure 3. Stability study of optimized perindopril proniosomal gel formulation (PNG3) at different temperatures conditions. 4-8°c (), 25±2°c(■) and 45±2°c ().

Figure 4. In vitro permeation profile of optimized perindopril erbumine proniosomal gel formulation (PNG3) (-♦-) through rat abdominal skin. 

 

CONCLUSION:

The results of investigation demonstrated that proniosomes offers an alternative colloidal carrier approach in transdermal drug delivery. The results obtained from the present study clearly revealed that proniosomal gel containing perindopril erbumine which is prepared by using coaservation phase separation method are capable of releasing drug for the extended period of time.

 

ACKNOWLEDGMENT:

The authors are thankful to VGST Government of Karnataka for providing financial support to carry out this project. We also thank Glenmark Ltd. Goa for providing gift Sample of perindopril erbumine.

 

 

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Received on 09.03.2012       Accepted on 21.04.2012     

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Asian J. Pharm. Tech. 2(2): April-June 2012; Page 54-58