INTRDUCTION:
The BCS is a scientific framework for classifying a drug substance based on its aqueous solubility and intestinal permeability. When combined with the in vitro dissolution characteristics of the drug product, the BCS takes into account three major factors: solubility, intestinal permeability, and dissolution rate, all of which govern the rate and extent of oral drug absorption from IR solid oral-dosage forms1. It classifies drugs into four classes (Fig. 1)
Solubility
Fig. 1: Biopharmaceutical classification system
Poorly water-soluble drug candidates often emerge from contemporary drug discovery programs, and present formulators with considerable technical challenges2. The poor solubility and low dissolution rate of poorly water soluble drugs in the aqueous gastro-intestinal fluids often cause insufficient bioavailability. Especially for class II substances according to the Biopharmaceutics Classification System (BCS), the bioavailability may be enhanced by increasing the solubility and dissolution rate of the drug in the gastro-intestinal fluids3. Consideration of the modified Noyes-Whitney equation4 provides some hints as to how the dissolution rate of Even very poorly soluble compounds might be improved to minimize the limitations to oral availability:
dC = AD. (Cs - C.)
dt
h
Where, dC/dt is the rate of dissolution, A is the surface area available for dissolution, D is the diffusion coefficient of the compound, Cs is the solubility of the compound in the dissolution medium, C is the concentration of drug in the medium at time t, h is the thickness of the diffusion boundary layer adjacent to the surface of the dissolving compound.
The main possibilities for improving dissolution according to this analysis are to increase the surface area available for dissolution by decreasing the particle size of the solid compound and/or by optimizing the wetting characteristics of the compound surface, to decrease the boundary layer thickness, to ensure sink conditions for dissolution and, last but definitely not least, to improve the apparent solubility of the drug under physiologically relevant conditions.
Larger the surface area, higher will be the dissolution rate. Since the surface area increases with decreasing particle size, which can be accomplished by conventional methods like trituration, grinding, ball milling, fluid energy micronization, salt formation and controlled precipitation. Although these conventional methods have been used commonly to increase dissolution rate of drug, there are practical limitation with these techniques as the desired bioavailability enhancement may not always be achieved. Therefore, formulation approaches are being explored to enhance bioavailability of poorly soluble drugs. One such formulation approach that has been shown to significantly enhance absorption of such dugs is to formulate solid dispersion5.
SOLID DISPERSIONS
Simple Eutectic Mixtures
No review of solid dispersions would be complete without a brief description of eutectic mixtures, which are the cornerstone of this approach to improving bioavailability of poorly soluble compounds. A simple eutectic mixture consists of two compounds which are completely miscible in the liquid state but only to a very limited extent in the solid state (Fig. 2). When, a mixture of A and B with composition E is cooled, A and B crystallize out simultaneously, whereas when other compositions are cooled, one of the components starts to crystallize out before the other. Solid eutectic mixtures are usually prepared by rapid cooling of a comelt of the two compounds in order to obtain a physical mixture of very fine crystals of the two components. When a mixture with composition E, consisting of a slightly soluble drug and an inert, highly water soluble carrier, is dissolved in an aqueous medium, the carrier will dissolve rapidly, releasing very fine crystals of the drug. The large surface area of the resulting suspension should result in an enhanced dissolution rate and thereby improved bioavailability.
Fig. 2: Phase diagram of a simple eutectic mixture with negligible solid solubility
The concept of solid dispersions was originally proposed by Sekiguchi and Obi, who investigated the generation and dissolution performance of eutectic melts of a sulfonamide drug and a water-soluble carrier in the early 1960s6. Solid dispersions represent a useful pharmaceutical technique for increasing the dissolution, absorption and therapeutic efficacy of drugs in dosage forms.
The term solid dispersion refers to the dispersion of one or more active ingredients in an inert carrier or matrix at solid state prepared by the melting (fusion), solvent, or melting-solvent method.
CLASSIFICATION8
The solid dispersions may also be called solid-state dispersions, as first used by Mayersohn and Gibaldi. The term “coprecipitates” has also been frequently used to refer to those preparations obtained by the solvent methods. Since the dissolution rate of a component from a surface is affected by the second component in a multiplecomponent mixture, the selection of the carrier has an ultimate influence on the dissolution characteristics of the dispersed drug. Therefore, a water-soluble carrier results in a fast release of the drug from the matrix, and a poorly soluble or insoluble carrier leads to a slower release of the drug from the matrix7.
STRATEGIES FOR SOLUBILITY ENHANCEMENT
Melting Method
The main advantages of this direct melting method is its simplicity and economy. The melting or fusion method was first proposed by Sekiguchi and Obi to prepare fast release solid dispersion dosage forms6. The physical mixture of a drug and a water-soluble carrier was heated directly until it melted. The melted mixture was then cooled and solidified rapidly in an ice bath under rigorous stirring. The final solid mass was crushed, pulverized, and sieved. Such a technique was subsequently employed with some modification by Goldberg et al and Chiou and Riegelman. The solidified masses were often found to require storage of 1 or more days in a desiccator at ambient temperatures for hardening and ease of powdering. Some systems, such as griseofulvin and citric acid, were found to harden more rapidly if kept at 37◦ or higher temperatures.
The melting point of a binary system is dependent upon its composition, i.e., the selection of the carrier and the weight fraction of the drug in the system7.
A modification of the process involves spray congealing from a modified spray drier onto cold metal surface. Decomposition should be avoided and is affected by fusion time and rate of cooling. Another modification of the above method, wherein SD(s) of troglitazone- polyvinyl pyrrolidone (PVP) k 30 have been prepared by closed melting point method. This method involves controlled mixing of water content to physical mixtures of troglitazone PVPK30 by storing at various equilibrium relative humidity levels (adsorption method) or by adding water directly (charging method) and then mixer is heated. This method is reported to produce solid dispersion with 0% apparent crystallinity9.
Solvent Evaporation Method
The solvent based process uses organic solvent to dissolve and intimately disperse the drug and carrier molecule. Identification of a common solvent for both drug and carrier can be problematic, and complete solvent removal from the product can be a lengthy process.
Moreover subtle alterations in the concentrations used for solvent evaporation may lead to large changes in the product performance. In addition large volumes of solvents are generally required which can give rise to toxicological problems. This method has been used for a long time in the preparation of solid solutions or mixed crystals of organic or inorganic compounds9.
They are prepared by dissolving a physical mixture of two solid components in a common solvent, followed by evaporation of the solvent. Many investigators studied SD of meloxicam10, naproxen11, nimesulide12, carbamezipine13 and celecoxib14 using solvent evaporation technique. These findings suggest that the above-mentioned technique can be employed successfully for improvement and stability of solid dispersions of poorly water soluble drugs. The main advantage of the solvent method is that thermal decomposition of drugs or carriers can be prevented because of the low temperature required for the evaporation of organic solvents. However, some disadvantages associated with this method are the higher cost of preparation, the difficulty in completely removing liquid solvent, the possible adverse effect of the supposedly negligible amount of the solvent on the chemical stability of the drug, the selection of a common volatile solvent, and the difficulty of reproducing crystal forms. In addition, a supersaturation of the solute in the solid system cannot be attained except in a system showing highly viscous properties, as is discussed later. It must be emphasized that the suitability of the solvent method to prepare simple eutectics or partial solid solutions remains to be studied further because their final physical properties may be quite different from those obtained by the melting method7.
EMINENT PROPERTIES OF SOLID DISPERSIONS
Solid dispersions are promising drug delivery forms which offer the possibility to disperse a hydrophobic drug in a hydrophilic matrix and thereby improve the dissolution behavior and the bioavailability of the drug. Management of the drug release profile using solid dispersions is achieved by manipulation of the carrier and solid dispersion particles properties. The various aspects such as composition and molecular weight of carrier, drug crystallinity, particle porosity and wettability, when successfully controlled, can produce improvements in bioavailability8, 15.
Higher Porosity of Drug Particles
Particles in solid dispersions have been found to have a higher degree of porosity. The increase in porosity depends on the properties of carriers used, for instance, solid dispersions containing linear polymers produce larger and more porous particles than those containing reticular polymers and, therefore, result in a higher dissolution rate and hence bioavailability5,8.
Reduced Dug Particle Size
Molecular dispersions, as solid dispersions, represent the last state on particle size reduction, and after carrier dissolution the drug is molecularly dispersed in the dissolution medium. Solid dispersions apply this principle to drug release by creating a mixture of a poorly water soluble drug and highly soluble carriers. A high surface area is formed, resulting in an increased dissolution rate and, consequently, improved bioavailability5.
Improved Wettability
A strong contribution to the enhancement of drug solubility is related to the drug wettability improvement verified in solid dispersions. It was observed that even carriers without any surface activity, such as urea improved drug wettability. Carriers with surface activity, such as cholic acid and bile salts, when used, can significantly increase the wettability properties of drugs. The powder surface composition is expected to play an important role in the wetting process, as it influences the overall hydrophobicity of the powder. In particular, high surface coverage of hydrophobic drug is assumed to give poor wetting properties with large contact angles. The amount of drug at the powder surface is further believed to significantly influence dissolution and physical drug stability. The importance of contact angles and wettability on dissolution rate is discussed in several studies5, 15.
Drugs in Amorphous State
It is well known that utilizing the amorphous form of a drug is a useful approach to improve the dissolution behaviour and bioavailability of poorly water-soluble active pharmaceutical ingredients. Poorly water soluble crystalline drugs, when in the amorphous state tend to have higher solubility. The enhancement of drug release can usually be achieved using the drug in its amorphous state, because no energy is required to break up the crystal lattice during the dissolution process. In solid dispersions, drugs are presented as supersaturated solutions after system dissolution, and it is speculated that, if drugs precipitate, it is as a metastable polymorphic form with higher solubility than the most stable crystal form. For drugs with low crystal energy (low melting temperature or heat of fusion), the amorphous composition is primarily dictated by the difference in melting temperature between drug and carrier. For drugs with high crystal energy, higher amorphous compositions can be obtained by choosing carriers, which exhibit specific interactions with them5.
LIMITATIONS OF SOLID DISPERSION SYSTEM
Despite extensive expertise with solid dispersions, there are some problems which limit the commercial application of solid dispersions. One of the primary reasons is the poor predictability of solid dispersion behaviour due to a lack of a basic understanding of their material properties. There is the possibility that during processing (mechanical stress) or storage (temperature and humidity stress) the amorphous state may undergo crystallization. The effect of moisture on the storage stability of amorphous pharmaceuticals is also of a vital concern, because it may increase drug mobility and promote drug crystallization. Moreover, most of the polymers used in solid dispersions can absorb moisture, which may result in the phase separation, crystal growth or conversion from the amorphous to the crystalline state or from a metastable crystalline form to a more stable structure during storage. This may result in decreased solubility and dissolution rate.
Other problems limiting the commercial application of solid dispersion involve its method of preparation, its formulation in dosage form and the scale up of manufacturing processes5, 15,16.
CARRIERS
Many water-soluble excipients are employed as carriers of solid solutions/dispersions.
Poloxamers
The poloxamers are a group of surface active compounds widely used in the pharmaceutical industry.Poloxamers are described as block polymers of the type aba, consisting of a central, hydrophobic block of polypropylene oxide, which is edged by two hydrophilic blocks of polyethylene oxide. The polymers are derived from the sequential polymerization of propylene oxide and ethylene oxide. A general formula is shown above in figure. Due to the possibility to combine blocks of different molecular weights, the properties of the resulting polymers vary in a wide range. Generally, these are waxy, white granules of free-flowing nature and are practically odorless and tasteless. Aqueous solutions of pluronic in presence of acids, alkalis, and metal ions are very stable. The poloxamers are readily soluble in aqueous, polar and non-polar organic solvents and due to this fact they have established themselves as a preferred molecule in the formulation techniques17,18.
Polyethylene Glycol (PEG)
Polyethylene glycols (PEG) are polymers of ethylene oxide, with a molecular weight (MW) usually falling in the range 200±300 000. For the manufacture of solid dispersions and solutions, PEGs with molecular weights of 1500±20 000 are usually employed. As the MW increases, so does the viscosity. They are most commonly used because of their good solubility in water and in many organic solvents, low melting points (under 65°C), ability to solubilize some compounds and improvement of compound wettability. The relatively low melting point is advantageous for the manufacture of solid dispersions by the melting method.
The drug/carrier ratio in a solid dispersion is one of the main influences on the performance of a solid dispersion. If the percentage of the drug is too high, it will form small crystals within the dispersion rather than remaining molecularly dispersed. On the other hand, if the percentage of the carrier is very high, this can lead to the complete absence of crystallinity of the drug and thereby enormous increases in the solubility and release rate of the drug19.
Polyvinylpyrrolidone (PVP)
Polymerization of vinylpyrrolidone leads to polyvinylpyrrolidone (PVP) of molecular weights ranging from 2500 to 3 000 000. These can be classified according to the K value, which is calculated using Fikentscher's equation. Table 2 provides an overview of the relationship between the K value and the approximate molecular weight of PVP. The glass transition temperature of a given PVP is dependent not only on its MW but also on the moisture content. In general, the glass transition temperature (Tg) is high. For example, PVPK25 has a Tg of 1550C. For this reason PVPs have only limited application for the preparation of solid dispersions by the hot melt method. Due to their good solubility in a wide variety of organic solvents, they are particularly suitable for the preparation of solid dispersions by the solvent method. Similarly to the PEGs, PVPs have good water solubility and can improve the wettability of the dispersed compound in many cases. The chain length of the PVP has a very significant influence on the dissolution rate of the dispersed drug from the solid dispersion. The aqueous solubility of the PVPs becomes poorer with increasing chain length and a further disadvantage of the high MW PVPs is their much higher viscosity at a given concentration (Table 1)
|
K value |
Approximate molecular weight |
|
12 |
2500 |
|
15 |
8000 |
|
17 |
10000 |
|
25 |
30000 |
|
30 |
50000 |
|
60 |
400000 |
|
90 |
1000000 |
|
120 |
3000000 |
Table 1 K values of PVP and the corresponding molecular weights
Similarly to PEG, solid dispersions prepared with high proportions of PVP tend to exhibit a higher drug solubility and release rate than those with high proportions of drug. For example, in case of Albendazole, it has been shown that an increase in the %PVP in the dispersion leads to an increase in the release rate. Most studies of PVP solid dispersions reported in the literature have used PVPs of MW2500±50 000 (K12 to K30)20, 21.
Polyvinyl Alcohol (PVA), Crospovidone (PVP-CL), Polvinylpyrrolidone-Polyvinylacetate Copolymer (PVPPVA)
All three polymers belong to the polyvinyl group. Whereas polyvinylalcohol (PVA) and vinylpyrrolidone/vinylacetate (PVP-PVA) copolymers are both water soluble, crospovidone swells when dispersed in water. When solid dispersions of nifedipine were prepared with carrier mixtures consisting of nicotinamide and PVP, hydroxypropylmethylcellulose (HPMC) or PVA in a drug/nicotinamide/ polymer ratio of 1:3:1, those prepared with PVA dissolved 20 times as fast as the drug alone. However, the other carriers, HPMC and PVP, yielded even better results. The use of PVA/PVP copolymers as carriers in solid dispersions has been shown to lead an enormous increases in the drug release rate. Studies with the cytostatic drug HO-221 showed that the PVA/PVP solid dispersion not only dissolved 25 times faster than the drug powder, but also enhanced the bioavailability in beagles by a factor of 3.5.
Even though crospovidone does not dissolve in water, it can also be used as a carrier to improve drug release rates. For example, a 1:2 ratio of furosemide to crospovidone led to an increase in the dissolution rate by a factor of 5.8 in comparison with either the drug powder or a physical mixture of furosemide with crospovidone. The mechanism of the increase in the release rate of furosemide proved to be the presentation of the drug in the amorphous form in the dispersion, as shown by X-ray diffraction studies5.
Cellulose Derivatives
Celluloses are naturally occurring polysaccharides that are ubiquitous in the plant kingdom. They consist of high molecular weight unbranched chains, in which the saccharide units are linked by b-1, 4-glycoside bonds. By appropriate alkylation, the cellulose can be derivatized to form methyl-(MC), hydroxypropyl- (HPC), hydroxypropylmethyl-(HPMC) and many other semi-synthetic celluloses. Since each glucose unit has three hydroxyl groups that can be derivatized, the average substitution grade (SG) cannot exceed three, unless of course the hydroxyl groups on the substituents themselves (e.g. in the case of HPMC) are also derivatized. A further possibility for derivatization is the esterification of the cellulose to form compounds such as cellulose acetate phthalate (CAP) and hydroxypropylmethylcellulose phthalate (HPMCP).
Hydroxypropylmethylcellulose (HPMC)
HPMCs are mixed ethers of cellulose, in which 16.5±30% of the hydroxyl groups are methylated and 4±32% are derivatized with hydroxypropyl groups. The molecular weight of the HPMCs ranges from about 10 000 to 1 500 000 and they are soluble in water and mixtures of ethanol with dichloromethane and methanol with dichloromethane. Studies with albendazole, a poorly soluble weak base with incomplete bioavailability, showed that the release rate and the bioavailability in beagles could be improved through preparation of a solid dispersion in HPMC. It was further demonstrated that HPMC was able to inhibit the recrystallization of the albendazole, and that a further improvement in release characteristics could be achieved when a carrier mixture consisting of HPMC and HPMCP was employed. Other drugs which exhibit faster release from solid dispersion in HPMC include the poorly soluble weak acids nilvadipine and benidipine23,24.
Hydroxypropylcellulose (HPC)
It exhibits good solubility in a range of solvents, including water (up till 408oC), ethanol, methanol and chloroform. The average MW of the HPCs ranges from 37 000 (Type SSL) to 1 150 000 (Type H). Yuasa et al. carried out extensive studies of the influence of the chain length and proportion of HPC in the solid dispersion on the release behavior of flurbiprofen. The release rate improved as the proportion of HPC was increased and when lower MW HPCs were used as the carrier25.
Carboxymethylethylcellulose (CMEC)
CMEC also belongs to the cellulose ethers, but unlike many of the others it is resistant to dissolution under gastric (acidic) conditions. It dissolves readily at pH values above 5±6, with lowest dissolution pH being dependent on the grade of the CMEC. CMECs also dissolve readily in acetone, isopropanol 70%, ethanol 60% and 1:1 mixtures of dichloromethane and ethanol. Amorphous solid dispersions of nifedipine and spironolactone show enormous increases in the dissolution rate of the drug at pH values of 6.8. Likewise, the bioavailability of the test substance MFB-1041 could be substantially improved in beagles26.
CHARACTERIZATION OF SOLID DISPERSIONS
The methods that have been used to characterize solid dispersions are summarized in Table 2. Among these, the most important methods are thermo analytical, X-ray diffraction, infrared spectroscopy and measurement of the release rate of the drug. In addition to characterizing the solid dispersion, these methods can be used to differentiate between solid solutions (molecularly dispersed drug), solid dispersions in which drug is only partly molecularly dispersed and physical mixtures of drug and carrier. Due to the complex composition of these preparations, it is often difficult to differentiate precisely between molecularly dispersed and not molecularly dispersed systems and different analytical methods may yield disparate results. It is usually assumed that dispersions in which no crystallinity can be detected are molecularly dispersed and the absence of crystallinity is used as a criterion to differentiate between solid solutions and solid dispersions (Table 2)5.
|
Methods for the characterization of solid dispersions |
|
Dissolution testing Thermoanalytical methods : differential thermoanalysis and hot stage microscopy Calorimetric analysis of the solution or melting enthalpy for calculation of entropy change X- ray diffraction Spectroscopic methods, e.g. IR spectroscopy Microscopic methods including polarization microscopy and scanning electron microscopy |
Table 2
CONCLUSION
The enhancement of oral bioavailability of poorly water soluble drugs remains one of the most challenging aspects of drug development. Most of the promising newer chemical entities are poorly water soluble drugs, which may present a lack of therapeutic effect, because of their low bioavailability. Solid dispersions are one of the most attractive processes to improve drug’s poor water solubility. Various solubility enhancers like water-soluble carriers, co solvents, surfactants and superdisintegrants via solid dispersion approach (fusion method and solvent evaporation method) aids in solubility enhancement. These significantly help to improve the bioavailability and bioequivalence.
REFERENCES
1. Yu LX, et al. A Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions. Pharm res 2002; 19: 921-925.
2. Pouton CW. Formulation of poorly water-soluble drugs for oral administration: Physicochemical and physiological issues and the lipid formulation classification system Eur J Pharm Sci 2006; 29: 278–287.
3. Urbanetz NA. Stabilization of solid dispersions of nimodipine and polyethylene glycol 2000. E J Pharm Sci 2006; 28: 67–76.
4. Noyes A.A, Whitney W.R. The rate of solution of solid substances in their own solutions. J. Am. Chem. Soc. 1897; 19: 930-934.
5. Leuner C, Dressman J. Improving Drug Solubility for Oral Delivery Using Solid Dispersion. Eur J Pharm and Biopharm 2000; 50: 47-60.
6. K. Sekiguchi, Obi N. Studies on absorption of eutectic mixtures. I. A comparison of the behaviour of eutectic mixtures of sulphathiazole and that of ordinary sulphathiazole in man, Chem. Pharm. Bull 1961; 9: 866-872.
7. Chiou WL, Riegelman S. Pharmaceutical Applications of Solid Dispersion Systems. J Pharm Sci 1971; 60: 1281-1302.
8. Vasconceleos T, et al. Solid Dispersion as Strategy to Improve Oral bioavailability of poor Water Soluble Drugs. Drug Dis Today 2007; 12: 1068-1075.
9. Sharma DK, Joshi SB. Solubility enhancement strategies for poorly water-soluble drugs in solid dispersions: A Review. Asian J of Pharmaceutics 2007; 1(1)1-19.
10. Chowdary K P R and R. Hymavathi. Enhancementof dissolution rate of meloxicam. Indian J. Pharm Sci 2001; 150-154.
11. Rao M G, et al. Preparation and evaluation of solid dispersions of naproxen. Indian J. Pharm. Sci. 2005; 67(1): 26-29.
12. Jain R K, et al. Studies on solid dispersions of nimesulide with pregelatinized starch. Biosciences, biotechnology research Asia 2006; 3(1a): 151-153.
13. Rane Y, et al. Effect of Hydrophilic Swellable Polymers on Dissolution Enhancement of Carbamazepine Solid Dispersions Studied Using Response Surface Methodology. AAPS PharmSciTech 2007; 8 (2): 427-430
14. Dixit R P, Nagarsenker M S.In vitro and In vivo advantage of celecoxib surface solid dispersion and dosage form development. Indian J. Pharm.Sci.May-June 2007: 370-377.
15. Dahlberg C, et al. Surface composition and contact angle relationships for differently prepared solid dispersions Eur J Pharm and Biopharm 2008; 70(2): 478-485.
16. Serajuddin ATM. Solid Dispersion of Poorly Water Soluble Drugs: Early Promises, Subsequent Problems, and Recent Breakthrough. J Pharm Sci 1999; 88:1058-1066.
17. BASF – Expertise in health and nutrition No. 3 1999.
18. Karmarkar A. B. Poloxamers and their applications. www.pharmainfo.net November 27, 2008.
19. Price JC. Polyethylene glycol, in: A. Wade, P.J. Weller (Eds.), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association/The Pharmaceutical Press, Washington, DC/London, 1994; pp. 355-361.
20. Walking WD. Povidone, in: A. Wade, P.J. Weller (Eds.), Handbook of Pharmaceutical Excipitients, American Pharmaceutical Association/The Pharmaceutical Press, Washington, DC/London, 1994; pp. 392-399.
21. S. Torrado, et al. Preparation, dissolution and characterization of albendazole solid dispersions, Int. J Pharm. 1996; 140: 247-250.
22. R.J. Harwood, J.L. Johnson. Hydroxypropylmethylcellulose, in: A.Wade, P.J. Weller (Eds.), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association/The Pharmaceutical Press,Washington, DC/London, 1994; pp. 229-232.
23. Kohri N, et al. Improving the oral bioavailability of albendazole in rabbits by the solid dispersion technique, J Pharm Pharmacol. 1999; 51: 159-164.
24. Harwood RJ, Johnson JL, Hydroxypropylcellulose, in: A. Wade, P.J. Weller (Eds.), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association/The Pharmaceutical Press, Washington, DC/London, 1994; pp. 223-228.
25. Kai T, et al. Oral absorption improvement of poorly soluble drug using solid dispersion technique, Chem. Pharm. Bull. 1996; 44: 568-571.
Received on 09.04.2009 Modified on 15.05.2009
Accepted on 22.05.2009 © RJPT All right reserved
Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 220-224