Surface Morphology and Drug Release Studies on Repaglinide Controlled Release Pellets Prepared by Solution Layering Method with Blend of PEG - HPMC - EC Polymers

About Author: Patil Amol Vilas,* Madgulkar Ashwini R., Bhingare Chandrashekhar L., Bhalekar Mangesh R., Jamadar Shahaji Ambadas
Department of Pharmaceutics,
AISSMS College of Pharmacy,
Kennedy Road, Near R.T.O.,
Pune - 411 001, INDIA

Abstract
Pellets are of great interest to the pharmaceutical industry for a variety of reasons. Pelletized products not only offer flexibility in dosage form design and development, but are also utilized to improve safety and efficacy of bioactive agents. However, the single most important factor responsible for the proliferation of pelletized products is the popularity of controlled release technology in the delivery of drugs. This research article mainly focused on the Surface Morphology and Drug Release Studies on Repaglinide Controlled Release Pellets Prepared by Solution layering method with Blend of PEG - HPMC - EC Polymersof controlled release pellets of Repaglinide. Surface morphology study to revel morphological changes when pellets were exposed to dissolution study and correlate their relation from P1 to P9 batches. Amongst all batches, P1 batch selected for morphological evaluation by using Scanning Electron Microscopy (SEM). XRD study done for powder characterization of drug before and after manufacturing process. Cellulose derivative blend of Hydroxypropyl methylcellulose (HPMC-K100), Ethyl cellulose (EC) and PEG4000, due to their hydrophilic and hydrophobic properties and ease of application provide desired drug release profile Upto 12 Hrs, when used in optimum concentration P1 batch (1:1%).

Reference ID: PHARMATUTOR-ART-1121

Introduction
Repaglinide, (S) - (+) - 2 ethoxy – 4 (2 [ [3-methyl-1 [2 - (1-piperidinyl) phenyl]-butyl] amino]-2 oxoethyl] benzoic acid, is a novel blood glucose lowering agent from the class of carbamoyl-methylbenzoic acid ^[1].It lowers blood glucose by stimulating the release of insulin from the pancreas. It achieves this by closing ATP-dependent potassium channels in the membrane of the beta cells. This depolarizes the beta cells, opening the cells' calcium channels, and the resulting calcium influx induces insulin secretion ^[2].

Repaglinide is developed in attempts to overcome the adverse effects associated with existing antidiabetic compounds. These include hypoglycaemia, secondary failure and cardiovascular side effects ^[3]. Repaglinide, a fast and short acting Meglitinide analog, is chosen as the drug candidate for controlled release pellets formulation. As far as the specific properties of the repaglinide are concerned, though it possesses phenomenal anti-diabetic properties, it has only short half-life 1 h and has low bioavailability (50%) and poor absorption characteristics in the upper intestinal tract. Furthermore it produces hypoglycemia after oral administration ^[4]. Since these drugs are intended to be taken for a long period, patient compliance is also very important and is to be looked at ^[5]. Headache, gastrointestinal effects, and musculoskeletal pain are also been reported by repaglinide users ^[6]. Controlled release coated pellets enclosed anti-diabetic drug, may improve the therapeutic efficacy of the drug and also the coated pellets which releases the drug in a predetermined controlled manner for a prolonged duration. Thus the adverse effects, as mentioned earlier, due to conventional dose can be surmounted.

Material
Repaglinide received as gift sample from DR. Reddys Laboratories Ltd, Bollaram, AndhraPradesh,non Pareils obtained from Murli Krishna Pharmaceuticals, HPMC-K100 purchased from Colorcon Asia Ltd, Goa, Ethyl cellulose from Degussa Pharmaceutical Coating Pvt Ltd, and potassium dihydrogen orthophosphate  from S.D. Fine Chem. Ltd; India were received as gift samples. All other chemicals and reagents were of analytical grade.

Experimental methods
Drug solution layering
Repaglinide loaded pellets (5% weight gain) were prepared by layering a drug-binder solution onto non-pareil seeds using a pan coater. A solution of Repaglinide in Isopropyl alcohol (IPA) was prepared by dispersing 1.119 gm of Repaglinide in sufficient quantity of IPA. PVP K 30(1g) was separately dissolved in sufficient quantity of IPA and then mixed with the drug solution. Volume of this solution was made up to 100ml with IPA. The composition for solution layering of drug shown in table 1 and process parameters are listed in table 2.

Table 01: Composition for solution layering of drug

Table 02: Drug solution layering parameters for Nonpareils

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3.2 Coating of drug loaded pellets
Coating suspension was prepared from polymers mixture of HPMC and ethyl cellulose, plasticizer PEG 4000, Talc used for its Antiadherent property, PVPK30 used as binder to increase binding strength of coating films and IPA used as solvent. The weighed quantity HPMC soak in IPA and dissolve in hot water 20 ml (60-70º C) then add slowly in 30 ml IPA solution with stirring. Simultaneously weighed quantity of Ethyl cellulose dissolved in IPA. Both solution of HPMC-K100: EC (50:50) mixed with stirring at 100 RPM. Separately dispersed PEG 4000 and PVP K 30 were prepared in IPA and mix in previous solution of polymer. The polymer content of the mixture was then adjusted by Isopropyl Alcohol to 100 ml. To remove small undissolved residue of polymer blend removed by solution passing through mesh number 80 to get uniform polymer solution for coating process.

Different concentrations of HPMCK100 and ethyl cellulose from 1% w/v to 5 %w/v were used for preparing polymer solution for coating as indicated by P1 to P9 to get the desired drug release. The composition of coating solution is as shown in table 3. For coating 20gm drug loaded pellets were used. All the coating processes were performed using pan coater under the coating conditions shown in table 4

Table 03: Composition of coating solution for drug loaded pellets

Table 04: Coating parameters for pellets

3.3 Drug content
500mg of pellets was crushed. Powder equivalent to drug 9.325 mg present in the pellets was transferred to 20 ml methanol in volumetric flask. Further, from this prepared 1 ml solution diluted with 10 ml methanol volume. The solution was filtered and the drug content was determined spectrophotometrically at 244nm.

3.4 X-ray powder diffraction:
To understand XRD pattern of pure drug and optimized formulation, a Philips 1710 X-ray Diffractometer (XRD) with a copper target and nickel filter was used to obtain XRD result for the samples. Powder were mounted on aluminum stages with glass bottoms and smoothed to a level surface. The XRD pattern of each sample was measured from 10 to 50 degrees 2-theta using a step increment of 0.1 2 theta degree and a dwell time of 1 second at each step.^[7]

3.5 In vitroDrug release studies
Drug release was evaluated by conventional in vitro dissolution testing.^[8] The dissolution test for pellets was performed in triplicate using USP type I (Basket type) dissolution test apparatus (Veego DA-6D USP Standard). Phosphate buffer (pH 7.4) 900 ml was taken as the release medium at 100rpm and maintained at 37±0.5⁰C. Aliquots of samples were periodically withdrawn and the sample volume replaced with an equal volume of fresh dissolution medium. The samples were analyzed at 244 nm by UV Spectrophotometer (JASCO V-530, Japan). Percentage drug release was calculated using PCP Disso v2.08 Software (Poona College of Pharmacy, Pune).

3.6 Drug release models:
To describe the kinetics of the drug release from the controlled release pellets, mathematical models such as zero-order, first-order, Higuchi, Hixon-crowell, Koresmeyer-peppas models.^{[9, 10]}. The release data were evaluated by model-dependent (curve fitting) method using PCP Disso v2.08 software.

3.7 Scanning electron microscopy (SEM)
To understand changes in the surface morphology, the topography of pellets was analyzed with help of scanning electron microscopy (SEM; JEOL), (Pune University, Physics Department). Pellet surfaces were evaluated before and after coating.

The shape and surface morphology of the Repaglinide Pellets were examined using scanning electron microscopy (SEM) (JSM-T20, Tokyo, Japan). An appropriate sample of coated and uncoated was mounted on metal stubs, using double-sided adhesive tapes. Samples were gold coated and observed for morphology, at acceleration voltage of 15 kV. ^{[11, 12]}

4.0 Result and Discussion:
4.1 Drug Content:
Drug content of pellet formulations (P1 to P9) was found out to be 96.18±0.21 to 93.13±0.36 which reveals drug content was within the limits prescribed by I.P. Drug contents from all formulations are shown in Table No 07.

Table 07:Drug content and drug release of coated pellets

Mean ± SD; n = 3, no. of experiments conducted

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4.2 X-ray Diffraction (XRD) study:
X-ray diffraction of the bulk powder of Repaglinide yield characteristic crystalline intense peak at 25.25, 27.25, and 52.5, 157.21, at 2-theta because of its crystallinity Shown in Figure No 01.

Formulation P1 of Repaglinide with polymer combination shows an amorphous characteristic. The crystalline character of Repaglinide changed in amorphous form, due to combination with the polymer and PEG 4000 which may help to enhance the solubility of Repaglinide. However these peaks were not found in Repaglinide coated pellets. There were ill-defined peaks observed in the polymer matrix. Generally, XRD peaks depend upon the crystal size. But in the present study, the drug loaded pellets the characteristic peak of repaglinide overlapped with the noise of coated polymer. From this, it is evident that XRD signals of encapsulated Shown in figure No.02).

4.3 In vitro drug release study:
In vitro dissolution studies of Repaglinide from different batches of pellets was performed in phosphate buffer (7.4) for 12 hours using USP Type I dissolution test apparatus (Basket type) at 100 rpm in interval time 1 hour. The correct in vitro conditions required to study the release behaviour of a hydrophobic drug were maintained because Repaglinide showed 2 Pka values at 4.16 and 6.01. Solubility of Repaglinide increases 3-6 times using phosphate buffer (PH-7.4) as solvent. In water solubility of Repaglinide is 39.82 ug /ml. In Phosphate buffer (PH 7.4) the solubility of Repaglinide is 140.86 ug/ml.

It was found that formulations P4 showed 70.33 %, P7 and P4 showed 75.34 to 70.33 % releases in 12 hr. While, Formulations P1, P2, showed 90.58% to 80.11% ofreleasein 12 hr (Figure 03). Batch P3, P6, P5, P8 and P9 showed 50.86, 51.1, 64.82, 59.74, and 49.93. Dissolution study shows that as the concentration of ethyl cellulose increased the % drug release of Repaglinide decreased. This was due to hydrophobic nature of ethyl cellulose which results in increase diffusion path length and consequent retardation of drug release. On the other hand,increase in concentration of hydrophilic polymer HPMC showed diffusion controlled mechanism for drug release.

In case of controlled or sustained release formulations, diffusion, swelling and erosion are the three most important rate controlling mechanisms. Formulations containing swelling polymers showed swelling as well as diffusion mechanism,because in addition to diffusion, processes includes relaxation of polymer chains, imbibitions of water causing polymer to swell and changing them from initial glassy to rubbery state. Peppas (1985) described the concept of drug release mechanism, values of n i.e. release exponent for formulations are given in Table No 08,value of n as 0.5 indicates diffusion-controlled drug release and for the value 1.0 it indicates swelling-controlled drug release. Value of n between 0.5 and 1.0 can be regarded as an indicator for both the phenomenon (anomalous transport). As in Table No. 34 it was observed that value of n from 0.5to1 which indicates the release mechanism following anomalous transport.The value of n wasfound to be 0.9565, 0.6823, and 0.6324 for P2, P6, and P7 which followed Korsemeyer-Peppas for drug release. The value of n for P1, P3, P4, P5, P8 and P9 was found to be 0.6327, 0.6168, 0.4945, 0.6928, 0.6607 and 0.6897 which followed zero order models for drug release.

The release kinetics from various coating solution formulations for pellets may be explained on the hydrophilicity of the coating polymers. It is observed that as the concentration of hydrophilic polymer HPMC increases the diffusion controlled mechanism for drug release in low concentration batches like P1, P2, P4, P7 and result in Zero-order kinetics. As concentration of hydrophobic polymer ethyl cellulose increases the water penetration is slow and results in surface erosion thereby contributing controlled drug release resulting in Peppas model.        

r- Regression coefficient

n – Release exponent

k- Proportionality constant

4.4 Morphology of pellets:
4.4.1 Morphology of uncoated pellets:
Morphology of non pareils was examined by scanning electron microscopy. The view of the pellets showed a spherical structure with a rough surface morphology (Figure 4a) and exhibited a range of sizes within each batch (Figure 4b, 4c). 

The outer surface of the pellets was rough, while the inner structure was porous. The shell of the pellets also showed some porous structure (Figure 04 b, c). It may be caused by the evaporation of solvent entrapped within the shell of pellets. This spherical surface area of uncoated pellets enhances uniformity coating layer of coating material.   

4.4.2 Morphology of coated pellets:
Morphology of coated pellets was examined by scanning electron microscopy which showed smooth surface of coated film (Figure 05a).This uniformity may be due film forming agent PEG 4000, also uniform, fast evaporation of organic solvent IPA which is not seen in water evaporation drying method and exhibited a uniform range of sizes within each batch (Figure 05b, 05c).                                   

After coating of non pareils, smooth surface of pellets was observed. Even the surface morphological studies carried out by SEM supported the results of evaluation of flow properties of pellets. It was observed that if the surface of coated pellets become smooth then flow should be improved.

Morphology of the surfaces of coated pellets were examined prior to and in between dissolution study by scanning electron microscopy (shown in Figure No.06). SEMs of pellets revealed a rigid and tight network of both polymers before dissolution study, and exposed pellets to dissolution study shows decrease in diameter and rigidity with respect to time. This decrease in diameter with time due to different complex mechanisms like water diffusion, polymer swelling, polymer and drug dissolution, drug diffusion through the polymeric coating, creation of considerable hydrostatic pressure within the dosage form, formation of water-filled cracks in the film coating and subsequent drug diffusion through these cracks, changes in system size and coating thickness. This all considerable effect due to combination of HPMC, PEG 4000 and EC blend. PEG 4000 act as pore former agent with respect to drug release mechanism and film former with respect to coating mechanism which lead to uniform coating material. SEM study also reveals change in pore size from 1 Hr, 3Hr, 6Hr, 9Hr and 12 Hr. (shown in Figure No.07).

4.5 Release mechanism
The mass transport mechanisms controlling drug release from coated  pellets  are complex because of the considerable number of processes that are/might be involved (e.g. water diffusion, polymer swelling, polymer and drug dissolution, drug diffusion through the polymeric coating, creation of considerable  hydrostatic  pressure  within  the  dosage  form, formation of water-filled cracks in the film coating and subsequent drug diffusion through these cracks, changes in system size and coating thickness)^{[13, 14, 15]}. These processes become even more complex if not only one single polymer, but a blend of two different macromolecules is used as coating material. Yet,  only  little  is  known  on  the  underlying  mass transport  mechanisms  in this  type of drug  delivery systems.

In this study two polymers were coated on the pellets as a mixture rather a single coating solution. If pellets are coated separately as separate layers, desired controlled release may not be achieved. Instead of that either maximum retardation or no retardation may be observed in single coating of polymeric solution. HPMC, being hydrophilic is more permeable to water so it promotes release of drug. Ethyl cellulose is hydrophobic and retards drug release being less permeable to water. Hence the combination of a release promoting and retarding polymers was used to obtain controlled drug release. Hence combination of these release enhancing and release retarding polymers was used for controlled drug release.

4.6 Stability Study:
The stability studies were carried out on P1formulation .The samples were stored at 40⁰C ± 2­ºC/75% ± 5 % RH for three months to access their stability. The protocol of stability studies were in compliance with WHO guidelines for stability testing intended for the global market. After 30 days samples were withdrawn and retested for drug contentanddrug release studies.No significant difference in values of % drug release after 12 h and t50% observed during the stability studies.It indicates that irrespective of concentration of polymer, this formulation was able to retain its stability.(Result shown in Table 08)

Table 08: Results of stability studies of selectedformulation: Drugcontent and in vitro release during stability studies.

Conclusion:
Although Repaglinide is one of the emerging molecules in case of diabetes mellitus type 2. It is usually given orally with dosage regimen depending on patient, site of action and need. To maintain steady state concentration and patient compliance, controlled release of Repaglinide is desirable. Hence, in the present work an attempt has been made to prepare controlled release formulation of Repaglinide using Solution layering as a technique of Pelletization. Step by step studies were carried out to develop formulation of pellets using solution layering method. Non pareils (inert materials) were loaded with drug solution. Concentration of hydrophilic and hydrophobic polymers in coating solution was optimized by preliminary studies. Optimization search of the F1 formulation provided suitable concentration (1:1%) of the polymers for coating solution. Cellulose derivative blend of Hydroxypropyl methylcellulose (HPMC) and Ethyl cellulose (EC) due to their hydrophilic and hydrophobic properties and ease of application provide desired drug release profile Upto 12 Hrs, when used in optimum concentration(1:1%). This result are evaluated and confirmed by using drug content, Scanning Electron Microscopy (SEM) for morphological changes with respect to time in dissolution medium which revels the release mechanism of repaglinide from pellets, and XRD study confirms that drug convert from crystalline form to amorphous form which help to increase in solubility of drug in dissolution medium. Stability study concludes that normal room temperature is best for store the formulation.

Acknowledgements
The authors are grateful to Principal of AISSMS College of pharmacy, Pune, India, for providing all the Lab facilities during this study. The authors also would like to thank DR Reddys Laboratories Ltd, Bollaram, AndhraPradeshand Murli Krishna Pharmaceuticals for free supplying of Drug sample and non-pareil seeds respectively.

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