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In-Vitro and In-Vivo evaluation of Transdermal prolonged release proniosomal gel formulations of Propranolol HCL

Jeevana Jyothi B* , Guru Lakshmi. Gz
Institute of Pharmaceutical Technology, Sri Padmavati. Mahila Visvavidyalayam, Tirupati – 517502, AP
Corresponding Author: Jeevana Jyothi B, Email: jeevanajyothib@yahoo.com
Date of Submission: 12-06-2014
Date of Acceptance: 29-06-2014
Conflict of Interest: NIL
Source of Support: NONE
 

Abstract

Propranolol HCl, an antihypertensive drug was formulated as transdermal prolonged release proniosomal gel due its drawback of short life when given through oral route. Nine formulations of proniosomes were prepared using different polymers viz. Maltodextrin, span 40, span 60 and cholesterol by slurry, slow stirring method. All were in turn prepared as proniosomal gels by using carbapol 940 by simple stirring. Gels were evaluated for entrapment efficiency, In-vitro skin permeation studies, scanning electron microscopy (SEM) analysis, vesicle size analysis, drug excipient interaction studies. Different release models like zero order, first order, Higuchi, Korsmeyer-peppas etc were applied to in-vitro drug release data in order to evaluate the drug release mechanisms and kinetics. Among all proniosomal transdermal gel formulation, F5 produced from pronisomal powder containing 10mg of Propranolol HCl , 100mg of cholesterol, 100mg of maltodextin, Span 40 and Span 60 each of 250mg exhibited ideal diffusion characteristics of 20.25% release at 2nd hr followed by controlled release for 12 hrs with 80.5% release at 12th hr. In-vivo evaluation of pharmacokinetic evaluation exhibited remarkable enhancement of elimination half life up to 13.08 hrs.

Keywords

Propranolol HCl, prolonged release, proniosomes, proniosomal gel, Maltodextrin, cholesterol, carbopol 940, Scanning electron microscopy (SEM) analysis, In-vitro skin permeation studies, Half- life.

INTRODUCTION

Propranolol HCL (PPLH) is an antihypertensive drug useful for treatment of angina pectoris. It is absorbed from GI tract completely but subjected to considerable hepatic tissue binding and first pass metabolism and possess short half life of 3-6 hrs. The daily dose of the drug is 40-80 mg twice daily and up to160-640mg and hence lead to more side effects[1]. Therefore the present research work is aimed to prepare transdermal controlled and prolonged release proniosomal gel containing 20 mg of PPLH for treatment of angina pectoris with enhanced half life, pharmacokinetic parameters[2] less side effects with one time comfortable application and. Proniosomes are novel drug delivery systems for oral short acting drugs. They are the dry formulations of niosomes, a nonionic surfactant vescicles and are coated with surfactant. They get converted to noisome dispersions upon hydration and are more stable drug delivery systems for development of both highly lipopholic drugs[3]. In turn proniosomal gels are semisolid liquid crystal gels produced by dissolving proniosomes in minimal amount of suitable solvent like ethanol and hydration with minimum amount of water to form gel. They are liquid crystalline compact hybrids converted into niosomes immediately upon hydration. Use in topical/dermal delivery does not require hydration prior to application, but they can be applied as such or loaded on a base material of emulsion, gel, ointment, etc. prior to application[4].

MATERIALS AND METHODS

Pure and certified sample of propranolol HCL was gifted by Aurobindo Pharma Ltd, Hyderabad. Span 60, span 40, cholesterol, maltodextrin were obtained from S.D. Fine Chemicals Pvt. Ltd Mumbai. All other chemicals and reagents were of analytical grade.

Preparation and evaluation of propranolol HCl transdermal proniosomal gel:

Proniosomes containing 20 mg of PPLH were prepared by slurry method. Nine formulations Viz., F1 to F9 as given in Table 1 were prepared. PPLH, Span 40, Span 60 and cholesterol were taken in 100 ml beaker and chloroform: ethanol (2:1) solution was added in small quantities with mixing at each addition till smooth slurry is produced. The slurry was transferred to 100 ml round bottom flask containing the maltodextrin carrier. Additional chloroform: ethanol solution was added to form slurry in case of lower surfactant loading. The flask was attached to rotary flash evaporator maintained at temperature of 45 ± 2ºC and reduced pressure of 600 mm Hg. It was rotated at 60 to 70 rpm to evaporate solvent and continued to obtain dry and free flowing product. These materials were further dried overnight in a dessicator under vacuum at room temperature. This dry preparation referred as ‘proniosomes’ is stored in air tight container for further use.
All formulations, F1 to F9 were further prepared as proniosomal gels by using carbapol 934. The proniosomal powder was dispersed in 10 ml of 2% Carbapol 934 solution with stirring at 100 rpm for 1 hour on magnetic stirrer. This was further neutralized with few drops of 0.5% triethanolamin amine and few drops of 10% glycerine slowly with constant stirring to obtain proniosomal gel.

EVALUATION[5] :

Trial formulations of proniosomes were observed for spherical morphology and nine spherical shaped proniosomes were observed under SEM analysis an d further evaluated conerted in to gels evaluated for entrapment efficiency, FTIR analysis, in vitro skin permeation studies, vesicle size analysis and pharmacokinetic evaluation of

SCANNING ELECTRON MICROSCOPY (SEM) ANALYSIS:

All formulations of proniosomes F1-F9 were coated uniformly with gold palladium by using Sputter coater under vacuum (0.1 mm Hg), after fixing the sample in individual stabs and was randomly examined for surface morphology (roundness, smoothness, and formation of aggregates).

ENTRAPMENT EFFICIENCY

500 mg of the proniosomal gel was weighed and dispersed in distilled water phosphate buffer saline pH 7.4 and warmed for formation of niosomes. The niosome dispersion so obtained was centrifuged at 18,000 rpm for 30 min. The clear fraction obtained was used for the determination of free drug at 288 nm UV spectrophotometrically. [%EE = [(Ct-Cf)/Ct] X 100 where, Ct is the concentration of total drug and Cf is the concentration of free drug.

In vitro SKIN PERMEATION STUDIES

In vitro SKIN PERMEATION STUDIES The full-thickness male albino Wistar rat skin removed with a clipper was rinsed with with physiological saline and clamped between the donor and the receptor chamber of vertical diffusion cell with the stratum corneum surface facing donor compartment of vertical diffusion cell. The effective diffusion area of the cell was 2.0 cm2 and had a receptor volume of 25 ml. The receptor chamber was filled with phosphate buffer saline pH 7.4. The diffusion cell was maintained at 37 ± 1°C and the solution in the receptor chambers was stirred continuously at 600 rpm with the help of magnetic bead. 1 gm of proniosomal gel of Propranolol HCL was gently placed in the donor chamber and spread evenly. 2 ml of the solution in the acceptor chamber was removed for drug content determination and replaced immediately with an equal volume of receptor media. Drug concentration was determined UV spectrophotometrically at 288 nm (y = 0.007x - 0.005, r = 0.997).

VESICLE SIZE ANALYSIS

Vesicle size of promising formulation F5 that exhibited highest drug release was determined. 100mg of proniosomal gel was hydrated 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 at 100X magnification. The size of 100 vescicles was measured using a calibrated ocular and stage micrometer fitted in optical microscope.

In-vitro DRUG RELEASE KINETICS

In-vitro release kinetics were assessed using zero order [C = kot] , first order [LogC = LogCo kt / 2.303], Fickian diffusion [Q = Kt1/ 2], Hixson-Crowell cube root law [Q0 1/3 – Qt 1/3 = KHC t]. Mechanism of drug release was assessed by Korsmeyer eq.that describes drug release from a polymeric system [Mt M = Ktn]. The plots were made by considering : cumulative % drug release vs. time (zero order kinetic model); log cumulative of % drug remaining vs. time (first order kinetic model); cumulative % drug release vs. square root of time (higuchi model) log cumulative % drug release vs. log time (korsmeyer model) and cube root of drug % remaining in matrix vs. time (hixsoncrowell cube root law).

DRUG EXCIPIENT INTERACTION STUDIES BY FTIR spectroscopy:

FTIR spectra of pure drug and the promising drug loaded proniosomal powder F5 were obtained on a Perkin-Elmer 841 model FTIR Spectrophotometer equipped with a DTSG detector. Samples were prepared by KBr pressed pellet technique. The scanning range was 4000- 400 cm-1 and the resolution was 1 cm-1

PHARMACOKINETIC EVALUATION

Pharmacokinetic studies were performed for promising proniosomal gel formulation, F5 exhibited good performance at In-vitro level. Male Wistar rats were stored under standard laboratory conditions (temperature25 ± 2°C and relative humidity of 55 ± 5%). The rats were kept in polypropylene cages (3/cage) with free access to standard laboratory diet. About 2 cm2 of skin was shaved on the abdominal side of rats. They were fasted for the period of 12 h for observations of any unwanted effects. Then they were applied with 1 g proniosomal gel in 2% carbopol.
Blood samples were withdrawn at time intervals of 1, 2, 4, 8, 12, 24hrs from retro-orbital venous plexus under ether anesthesia using glass capillaries into sodium citrate containing eppendorf micro-centrifuge tubes. Plasma was separated by centrifugation using Centrifuge and stored in vials at -70°C until further analysis. Propranolol HCL in plasma was determined by RP-HPLC method. Animal studies were approved by animals ethics committee, SPSP, Tirupati.

RESULTS AND DISCUSSION

Propranolol HCl proniosomal formulations many were prepared using different non ionic surfactants ratio and were observed for spherical shape by SEM analysis for surface morphology. Nine formulations possessing spherical shape were selected as given in Table 1. SEM image of all formulations proniosomes have evidenced smooth spherical surface and the SEM image of F5 is shown in Fig.1 and Fig.2. Hence all nine pronisomal powders were converted to proniosomal gels and further subjected to evaluation parameters such as visualization of vesicles by optical microscope, encapsulation efficiency, In-vitro skin permeation studies, drug release kinetics and FTIR specrtoscipy, pharmacokinetic studies.

ENTRAPMENT EFFICIENCY:

Entrapment efficiency values for of F1to F9 are given in Table 2. The drug entrapment characteristics are good for Span 40 and Span 60 and produced least leaky niosomes that may be due to their highest phase transition temperature. It is indicated that, formulations F5 &F9 possess high EE values of 85.47 and 88.71% respectively which may be due to optimum ratio of surfactant to maltodextrin to provide a high entrapment of drug.

In vitro Skin permeation studies:

Drug diffusion data indicating skin permeation is shown in Table 3, 4 and in Fig.2 and 3. Diffusion profiles for all formulations were found to be linear within a period of 12 hours. Data in terms of %drug release isd shown in Table5. All formulations exhibited similar diffusion characteristics, but F5 was considered as promising formulation because 20% amount of drug released in 2hrs which is deemed to be required for providing initial loading dose with in 2hrs for controlled drug delivery system and also . Formulation F5 evidenced 81.26% of drug release and it is considered as promising formulation with ideal prolonged release for 12 hours.

Vesicle size analysis:

Vesicle size of promising formulation F5 examined by optical microscopy was found to be 4.16 ± 2.1 μm.

IN-VITRO DRUG RELEASE KINETICS

The in-vitro release profiles for all formulations were assessed by zero order, first order, Higuchi and Korsmeyer equation and were shown in table5. Relevant plots are shown in fig 6 to 9. Fig. 6: ZERO ORDER PLOTS Jeevana Jyothi. B et al; In-Vitro and In-Vivo evaluation of Transdermal prolonged release proniosomal gel formulations of Propranolol HCL Linear plots in case of zero order with correlation coefficient values nearing one in Fig. 6 indicated that the drug release is zero order kinetics. Correlation values shown in Table 5 of Higuchi’s plot revealed that the mechanism of drug release was diffusion. The in vitro kinetic data subjected to log time vs log drug release transformation plot (peppa’s model),for the best formulation the value lies were found to be0.78 which lies between 0.45 and 0.89, this revealed that the drug release follows non fickian diffusion

Drug excipient interaction studies (FTIR-ATIR STUDIES):

FTIR spectra of pure PPHL (FIG.) main functional groups in drug as N-H(3323.99), AROMATIC C=C(2967.37), c-n(1323.78) and C-O(1267.51and f5 and in formulation N-H(3382.51), AROMATIC C=C(2921.34), c-n(1359.98) and C-O(1027.62) (Fig.) revealed that there is no appreciable change characteristic absorption peaks of puxtrin, span30 etc when produced as proniosomes.

PHARMACOKINETIC EVALUATION:

The pharmacokinetic parameters were calculated from the plasma concentrations of the drug and Peak plasma concentration, Cmax was found to be 526.24 ng/ml and tmax was 12 h. Area under the plasma concentration-time curve , AUC024 was found to be 9403.58 ng-h/ml andAUC0 was 14581.86 ng-h/ml. Elimination rate constant (Ke) calculated from semi logarithmic plot was found to be 0.053/h. Elimination half life (T1/2) was found to be 13.08 h

CONCLUSION:

Propranolol HCl was developed successfully as prolonged release transdermal gel using pronisomal powder containing 10mg of Propranolol HCl , 100mg of cholesterol, 100mg of maltodextin, Span 40 and Span 60 each of 250mg optimized after several trials. The prepared gel exhibited of ideal prolonged drug release characteristics of 20.25% release at 2nd hr continued up to 12 hrs. The prepared gel exhibited remarkable enhancement of elimination half life up to 13.08hrs.

Tables at a glance

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Table 1 Table 2 Table 3 Table 4 Table 5
 

Figures at a glance

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Figure 11 Figure 12 Figure 13
 

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